Modern Crop Protection Compounds 2nd 2011

Edited by
Wolfgang Krämer, Ulrich Schirmer,

Peter Jeschke, and Matthias Witschel
Modern Crop Protection Compounds

Further reading
Wiley-VCH (ed.) Ziska, Lewis H./Dukes, Jeffrey
Ullmann’s Agrochemicals Weed Biology and Climate

Change
2 Volumes
2007 2010
ISBN: 978-3-527-31604-5 ISBN: 978-0-8138-1417-9
Walters, Dale Tadros, T.F. (ed.)
Plant Defense Colloids in Agrochemicals

Warding off Attack by Pathogens, Colloids and Interface Science
Herbivores and Parasitic Plants
Volume 5 of the ‘‘Colloids and Interface
2010
Science Series”
ISBN: 978-1-4051-7589-0
2009
ISBN: 978-3-527-31465-2
Edited by Wolfgang Krämer, Ulrich Schirmer, Peter Jeschke,
and Matthias Witschel
Second, Revised and Enlarged Edition
Volume 1
The Editors All books published by Wiley-VCH are
carefully produced. Nevertheless, authors,
Dr. Wolfgang Krämer editors, and publisher do not warrant the
Rosenkranz 25 information contained in these books,
51399 Burscheid including this book, to be free of errors.
Germany Readers are advised to keep in mind that
statements, data, illustrations, procedural
details or other items may inadvertently be
Dr.Ulrich Schirmer
inaccurate.
Berghalde 79
69126 Heidelberg
Germany Library of Congress Card No.: applied for
Dr. Peter Jeschke British Library Cataloguing-in-Publication

Bayer CropScience AG Data
BCS AG-R-PC-PCC, Bldg. 6510 A catalogue record for this book is available
Alfred-Nobel-Str. 50 from the British Library.
40789 Monheim am Rhein
Germany Bibliographic information published by the
Deutsche Nationalbibliothek
Dr. Matthias Witschel The Deutsche Nationalbibliothek
BASF SE lists this publication in the Deutsche
GVA/HC-B009 Nationalbibliografie; detailed bibliographic
67056 Ludwigshafen data are available on the Internet at
Germany <http://dnb.d-nb.de>.
© 2012 Wiley-VCH Verlag & Co. KGaA,

Boschstr. 12, 69469 Weinheim,
Germany
All rights reserved (including those of

translation into other languages). No part
of this book may be reproduced in any
form – by photoprinting, microfilm, or any
other means – nor transmitted or translated
into a machine language without written
permission from the publishers. Registered
names, trademarks, etc. used in this book,
even when not specifically marked as such,
are not to be considered unprotected by law.
Composition Laserwords Private Ltd.,

Chennai
Printing and Binding betz-druck GmbH,
Darmstadt
Cover Design Formgeber, Eppelheim
Printed in the Federal Republic of Germany

Printed on acid-free paper
Print ISBN: 978-3-527-32965-6

XXIII
Preface to the Second Edition
Many of the challenges for humanity defined in the United Nations ‘‘millennium
goals,’’ such as population growth, energy sustainability and public health, can
only be mastered by further significant improvements in agriculture. A major
contribution will have to come from better crops by breeding and/or genetic
modification, and in the future the protection of these highly valuable crops by the
use of agrochemicals will be even more important than it is today.
In this Second Edition of Modern Crop Protection Compounds, we discuss the
new developments in chemical crop protection and their contribution to improved
agriculture, as exemplified by the most recently commercialized agrochemicals.
In order to achieve the necessary progress in chemical crop protection, we are
faced with several major challenges. Currently, the loss of active ingredients due to
new regulatory requirements is higher than the number of new active ingredients
being introduced into the market. These new hurdles also dramatically increase the
attrition rate of new candidates under development, and thereby reduce the output
of new agrochemicals furthermore. In all indications, the diminished number of
available active ingredients in the market results in fewer options for resistance
management. Furthermore, the observed dramatic increases in the resistance of
fungi, weeds and insects to agrochemicals often leaves only a very few solutions to
the problem for the farmer and, in many cases, no solution at all. This trend will
accelerate as fewer different agrochemicals become available.
During the past few decades, a major consolidation in the agrochemical market
has taken place, whereby the number of companies conducting research in all
agrochemical indications has been reduced from over 20 during the 1980s to about
five today; moreover, further consolidation, especially also in the area of generics, is
highly probable. One reason for this trend is the increasing costs involved to bring
a new agrochemical to the market. Such increases in development costs result in
higher projected market values necessary for project decisions to be made, which in
turn will result in fewer available agrochemicals, especially for many of the smaller
markets.
The changing environmental conditions will also influence future agriculture,
due to the expected increases in drought, flooding, and salination. More intensive
farming on less available land will bring additional challenges and require the
provision of special crop protection solutions.
XXIV Preface to the Second Edition
It is, therefore, extremely important that crop protection research adds new and
better agrochemicals to the farmers’ toolboxes. The aim of this book is to stim-
ulate this process, by bringing together knowledge gained from modern biology,
biochemistry and chemistry, and by discussing the rationale in the invention and
development of modern crop protection compounds.
Each of the main sections, ‘‘Herbicides’’, ‘‘Fungicides’’, and ‘‘Insecticides’’, is
introduced by a contribution from the authors of the respective Resistance Action
Committee, reflecting the common responsibilities of the crop-protection industry
to maintain the efficacy of agrochemicals and to support sustainable agriculture
and public health. These introductory chapters allow us to mention those older
compounds that are not described in detail because they are dealt with in extenso in
standard books, such as Chemistry of Plant Protection (Springer, Berlin, Heidelberg,
New York, Tokyo) and Chemistry of Pesticides (John Wiley and Sons).
Our general target for ‘‘new’’ crop protection compounds has been to include
those agrochemicals that have come to the market during the past 20 years, between
1990 and 2010.
This book would not have been realized without the support of all the major
agrochemical companies and their research and development divisions, nor without
all the highly committed authors from these companies and the universities. We
are fully appreciative of the tremendous workload involved in preparing the
manuscripts.
We are sure that the readers will enjoy this book and use it as a compendium on
plant protection research, in much the same way that we ourselves have experienced
research on crop protection, as stimulating, enjoyable, and also challenging.
Note
The authors have named the products/compounds preferably by their common

names. Although, occasionally, registered trademarks are cited their use is not free
for everyone. In view of the number of trademarks involved, it was not possible to
indicate each particular case in each table and contribution. We accept no liability
for this.
December 2011 Wolfgang Krämer

Ulrich Schirmer
Peter Jeschke
Matthias Witschel
V
Contents
Volume 1
Preface XXIII
List of Contributors XXV
I Herbicides 1
Overview 3
Matthias Witschel
1 HRAC Classification of Herbicides and Resistance Development 5

Hubert Menne and Helmut Köcher
1.1 Introduction 5
1.2 HRAC Classification System of Herbicides 7
1.3 Herbicide Resistance 7
1.3.1 Biochemistry of Herbicide Resistance 13
1.3.1.1 Target-Site Resistance 14
1.3.1.2 Non-Target-Site Resistance by Enhanced Metabolic Detoxification 21
1.3.1.3 Non-Target-Site Resistance by Altered Herbicide Distribution 24
1.3.1.4 Multiple Resistance 25
References 26
2 Acetohydroxyacid Synthase Inhibitors (AHAS/ALS) 29
2.1 Biochemistry of the Target and Resistance 29

Steven Gutteridge and Mark E. Thompson
2.1.1 Acetohydroxyacid Synthase (AHAS) 29

2.1.2 Higher-Order Subunit Structure 33
2.1.3 Herbicides That Target AHAS 35
2.1.4 Binding Site for AHAS-Inhibiting Herbicides 36
2.1.5 Molecular Basis for Resistance to AHAS Inhibitors 41
2.1.6 Resistance to AHAS-Inhibiting Herbicides in Weeds 43
VI Contents
2.1.7 Engineered Resistance to AHAS-Inhibiting Herbicides in Crops 46

Acknowledgments 46
References 47
2.2 Newer Sulfonylureas 50

Oswald Ort
2.2.1 Introduction 50
2.2.1.1 History and Development 52
2.2.1.2 Synthesis 56
2.2.2 Agricultural Utility 56
2.2.2.1 Cereals 57
2.2.2.2 Rice 67
2.2.2.3 Maize 75
2.2.2.4 Other Crops 78
2.2.3 Sulfonylurea Herbicides: Metabolic Fate and Behavior in the Soil 81
2.2.4 Concluding Remarks 83
Acknowledgments 83
References 83
2.3 Imidazolinone Herbicides 88

Dale L. Shaner, Mark Stidham, Bijay Singh, and Siyuan Tan
2.3.1 Overview 88
2.3.2 History of Discovery 90
2.3.3 Physico-chemical Properties 91
2.3.4 Structural Features of Herbicidal Imidazolinones 92
2.3.5 Imidazolinones: The Mode of Action 93
2.3.6 Imidazolinone-Tolerant Crops 94
2.3.7 Imidazolinones: Mechanisms of Selectivity 95
2.3.8 Commercial Uses of the Imidazolinone Herbicides 96
2.3.9 Conclusion 98
References 98
2.4 Triazolopyrimidines 99
Timothy C. Johnson, Richard K. Mann, Paul R. Schmitzer,
Roger E. Gast, and Gerrit J. deBoer
2.4.2 N-Triazolo[1,5-c]Pyrimidine Sulfonanilide 100
2.4.2.2 Biology 100
2.4.2.3 Cloransulam-Methyl and Diclosulam Crop Utility 102
2.4.2.4 Mechanism of Crop Selectivity 103
2.4.2.5 Environmental Degradation, Ecotoxicology, and Toxicology 104
2.4.3 N-Triazolo[1,5-c]Pyrimidine Sulfonamides 105
Contents VII

2.4.3.2 Biology 107
2.4.3.3 Penoxsulam Crop Utility 109
2.4.3.4 Penoxsulam: Mechanism of Crop Selectivity 109
2.4.3.5 Penoxsulam: Environmental Degradation, Ecotoxicology, and
Toxicology 110
2.4.4 N-Triazolo[1,5-a]Pyrimidine Sulfonamides 111
2.4.4.2 Biology 111
2.4.4.3 Pyroxsulam: Crop Utility 112
2.4.4.4 Pyroxsulam: Mechanism of Crop Selectivity 112
2.4.4.5 Pyroxsulam: Environmental Degradation, Ecotoxicology, and
Toxicology 113
2.4.5 AHAS Inhibition 114
2.4.6 Conclusions 115
References 115
2.5 Pyrimidinylcarboxylates and Sulfonanilides 117

Takumi Yoshimura, Ryo Hanai, and Tsutomu Shimizu

2.5.2 Discovery of the PCs Herbicides 118
2.5.3 Structure–Activity Relationships of PCs Herbicides 120
2.5.3.1 Effects of Benzene Ring Substituents in the O-Pyrimidinylsalicylic
Acids 122
2.5.3.2 Effect of a Bridge Atom in the Pyrimidinylsalicylates 123
2.5.3.3 Pyrimidinylglycolates 123
2.5.4 ‘‘Pyrithiobac-Sodium’’: Cotton Herbicide 125
2.5.4.1 Discovery 125
2.5.4.2 Biology 126
2.5.5 ‘‘Bispyribac-Sodium’’: Herbicide in Direct-Seeded Rice 128
2.5.5.2 Biology 128
2.5.6 ‘‘Pyriminobac-Methyl’’: Rice Herbicide 129
2.5.6.2 Biology 131
2.5.7 Mode of Action of the PCs Herbicides 132
2.5.8 Mode of Selectivity of the PCs Herbicides in Crops 133
2.5.9 Discovery of the Sulfonanilides 134
2.5.10 Structure–Activity Relationships 135
2.5.10.1 Effect of the Sulfonamide Moiety in the Sulfonanilides 136
2.5.10.2 Effects of the Bridge Moiety in the Sulfonanilides 136
2.5.10.3 Effects of Benzene Ring Substitution in the
Sulfonanilides 137
2.5.11 ‘‘Pyrimisulfan’’: Rice Herbicide 139
VIII Contents
2.5.11.1 Biology 139

Abbreviations 140
References 140
2.6 Sulfonylaminocarbonyl-Triazolinones 142

Klaus-Helmut Müller, Ernst-Rudolf F. Gesing, and Hans-Joachim Santel

2.6.2 Discovery of the Lead Structure 143
2.6.3 Optimization of the Lead Structure 143
2.6.4 Discovery of Thiencarbazone-Methyl (TCM) 146
2.6.5 Synthesis 148
2.6.5.1 Sulfonyl Components 149
2.6.5.2 Triazolinone Synthesis 150
2.6.6 Biology 151
References 158
3 Protoporphyrinogen IX Oxidase Inhibitors 163

George Theodoridis, Rex Liebl, and Cyrill Zagar
3.1 Introduction 163

3.2 Historical Development 165
3.2.1 Diphenyl Ether 165
3.2.2 Phenyl Ring Attached to Heterocycle 166
3.2.3 Phenyl Tetrahydrophthalimide 168
3.3 Non-Classical Protox Chemistries 171
3.3.1 N-Phenyl Heterocycles: New Heterocyclic Systems 172
3.3.2 Phenoxyphenyl and Benzyloxyphenyl Attached to Heterocycle 176
3.3.3 Benzoheterocyclic Attached to Heterocycle 178
3.3.4 Benzyl Attached to Heterocycle 181
3.3.5 Replacement of Phenyl Ring with Pyrazole 182
3.4 Recent Developments 182
3.5 Toxicology 190
3.6 Summary 190
References 191
4 Herbicides with Bleaching Properties 197
4.1 Phytoene Desaturase Inhibitors 197

Gerhard Hamprecht and Matthias Witschel

4.1.2 Carotenoid Biosynthesis and Phytotoxic Effects of Bleaching
Herbicides 197
4.1.2.1 Targets for Bleaching Herbicides 197
Contents IX
4.1.2.2 Carotenoids: Properties and Functions 198

4.1.2.3 Carotenoid Biosynthesis in Higher Plants 199
4.1.3 Primary Targets 201
4.1.3.1 Inhibition of PDS and ζ -Carotene Desaturase 201
4.1.3.2 Inhibition of Lycopene Cyclase (LCC) 201
4.1.3.3 Genetic Engineering of Herbicide Resistance by Modification of the
Carotenogenic Pathway 203
4.1.4 Chemical Structure and Activities of PDS Inhibitors 203
4.1.4.1 Enzyme Activity, Physical Data, and Acute Oral Toxicity of
Commercial PDS Herbicides 203
4.1.4.2 Phenoxybenzamides 203
4.1.4.3 Phenoxypyridinecarbonamides 206
4.1.4.4 Phenoxypyridine Ethers 207
4.1.4.5 Phenylfuranones 207
4.1.4.6 Phenylpyridazinones 209
4.1.4.7 Phenylpyridinones 210
4.1.4.8 Phenylpyrrolidinones 210
4.1.4.9 Phenyltetrahydropyrimidinones 211
4.1.4.10 Structural Overlay for Diaryl Heterocycle PDS Inhibitors, and Newer
Developments 211
4.1.4.11 Models of the Active Site: Structural Requirements 212
4.1.5 Biology and Use Pattern 217
4.1.6 Major Synthetic Routes for PSD Inhibitors 218
References 222
4.2 Hydroxyphenylpyruvate Dioxygenase (HPPD): The Herbicide

Target 225
Timothy R. Hawkes
4.2.1 Herbicidal Mode of Action 225

4.2.2 Selectivity 228
4.2.3 Structure and Mechanism 229
4.2.4 Inhibition 232
References 234
4.3 Triketones 235

Andrew J.F. Edmunds and James A. Morris

4.3.2 Discovery 236
4.3.3 Mode of Action 237
4.3.4 Synthesis of Triketones 237
4.3.5 Structure–Activity Relationships (SARs) 239
4.3.6 Review of the Patent Literature 241
4.3.7 Commercialized Triketone Herbicides 249
X Contents
4.3.7.1 Sulcotrione 249

4.3.7.2 Mesotrione 250
4.3.7.3 Tembotrione and Tefuryltrione 254
4.3.7.4 Benzobicyclon 256
4.3.8 Summary 258
References 258
4.4 Hydroxyphenylpyruvate Dioxygenase (HPPD) Inhibitors:

Heterocycles 262
Andreas van Almsick

4.4.2 Market Products 263
4.4.2.1 Pyrazolynate (Pyrazolate) 263
4.4.2.2 Pyrazoxyfen 266
4.4.2.3 Benzofenap 267
4.4.2.4 Isoxaflutole 269
4.4.2.5 Topramezone 271
4.4.2.6 Pyrasulfotole 272
References 275
5 New Auxin Mimics and Herbicides 277
5.1 The Molecular Mode of Action of Auxin Herbicides 277

Terence A. Walsh and Paul R. Schmitzer

5.1.2 TIR1/AFB Proteins as Auxin Herbicide Receptors 277
5.1.3 The AFB5 Class of Picolinate Auxin Herbicide Receptors 281
5.1.4 TIR1/AFB Auxin Receptors in Other Plants 282
5.1.5 Are There Other Auxin Herbicide Receptors? 282
5.1.6 Downstream Effects of Auxin Herbicides 283
5.1.7 Weed Selectivities at the Site of Auxin Action 283
5.1.8 Field Resistance to Auxin Herbicides 285
References 286
5.2 Aminopyralid 287

Robert A. Masters, William C. Lo, and Roger E. Gast

5.2.2 Aminopyralid Synthesis 288
5.2.3 Biology 288
5.2.4 Aminopyralid Utility 290
5.2.5 Aminopyralid Mechanism of Crop Selectivity 291
5.2.6 Aminopyralid Environmental Degradation 292
Contents XI
5.2.6.1 Animal Toxicity 293

5.2.6.2 Carcinogenicity/Teratogenicity 294
References 294
5.3 Pyrimidine Carboxylic Acids, Aminocyclopyrachlor 295

Jon S. Claus and Bruce L. Finkelstein

5.3.2 Discovery of Aminocyclopyrachlor 295
5.3.3 Herbicidal Activity and the Properties of Aminocyclopyrachlor 300
5.3.4 Mode of Action and Site of Action 302
5.3.5 Resistance Management 303
References 304
6 Herbicides Disturbing the Synthesis of Very Long-Chain Fatty

Acids 305
6.1 Inhibitors of the Synthesis of Very Long-Chain Fatty Acids

(VLCFAs) 305
Peter Babczinski
6.1.1 Biochemistry 305

6.1.1.1 Introduction 305
6.1.1.2 HRAC Classification 305
6.1.1.3 Biosynthesis of VLCFAs 306
References 314
6.2 Chemistry and Biology of Oxyacetamides, Tetrazolinones,

and Triazolinones 316
Yukiyoshi Watanabe

6.2.2 Mefenacet and Flufenacet (Oxyacetamides) 316
6.2.3 Fentrazamide (Tetrazolinone) 318
6.2.4 Ipfencarbazon (Triazolinone) 322
Acknowledgments 324
References 325
6.3 Isoxazolines 326

Masao Nakatani, Takumi Yoshimura, Ryo Hanai, Yoshitaka Tanetani,
and Tsutomu Shimizu

6.3.2 Chemistry of Pyroxasulfone 326
XII Contents
6.3.3 Biological Activities of Pyroxasulfone 328

6.3.4 Biological Activities of Fenoxasulfone 330
6.3.5 Mode of Action of Pyroxasulfone 330
6.3.6 Mode of Action of Fenoxasulfone 334
Abbreviations 335
References 335
7 Inhibitors of Cellulose Biosynthesis 339

Hansjörg Dietrich and Bernd Laber

7.1.1 Cellulose Biosynthesis 339
7.2 Cellulose Biosynthesis Inhibitors from Different Chemical
Substance Classes 344
7.2.1 Nitriles 345
7.2.1.1 Chemistry of Benzonitriles 345
7.2.1.2 Biological Activity of Benzonitriles 346
7.2.2 Benzamides 348
7.2.2.1 Chemistry of Benzamides 348
7.2.2.2 Biology of Isoxaben 349
7.2.3 Triazolocarboxamides 351
7.2.3.1 Chemistry of Triazolocarboxamides 351
7.2.3.2 Biology of Triazolocarboxamides: Flupoxam 352
7.2.4 Alkylazines 353
7.2.4.1 Chemistry of Alkylazines 353
7.2.4.2 Biology of Alkylazines 354
7.2.5 Thiatriazines 356
7.2.5.1 Chemistry of Thiatriazines 356
7.2.5.2 Biology of CGA 325615 357
7.2.6 N-Aryl Lactams 359
7.2.6.1 Chemistry of N-Aryl Lactams 359
7.2.6.2 Biology of N-Aryl Lactams 359
7.2.7 Coumarins 360
7.2.8 Properties of Commercialized Inhibitors of Cellulose
Biosynthesis 361
7.3 Cellulose Biosynthesis Inhibitors from Natural Sources 361
7.3.1 Thaxtomins 361
7.3.2 Inthomycins 364
7.3.3 Epopromycins 365
References 365
8 Safeners for Herbicides 371

Chris Rosinger, Klaus Bartsch, and Wolfgang Schulte

8.2 Overview of Selected Safeners 372
Contents XIII
8.2.1 Dichloroacetamide Safeners 372

8.2.1.1 Benoxacor 375
8.2.1.2 Dichlormid 377
8.2.1.3 Furilazole 378
8.2.2 Oxime Ethers 379
8.2.3 Cloquintocet-Mexyl 379
8.2.4 Mefenypr-Diethyl 380
8.2.5 Isoxadifen-Ethyl 382
8.2.6 Cyprosulfamide 383
8.3 Mechanisms of Herbicide Safener Action 385
8.3.1 Effects of Safeners on Herbicide Metabolism 385
8.3.2 Gene Induction and Signaling Pathways 387
8.3.3 Influence on Herbicide Uptake 388
8.3.4 Influence on Herbicide Translocation 388
8.4 Mode of Action of Safeners in Agricultural Practice 389
8.4.1 1,8-Naphthalic Anhydride (NA), Flurazole, and
Fluxofenim 389
8.4.2 Dichloroacetamides 390
8.4.3 Fenclorim 391
8.4.4 Fenchlorazole-Ethyl, Cloquintocet-Mexyl 391
8.4.5 Mefenpyr-Diethyl 392
8.4.6 Isoxadifen-Ethyl 393
8.4.7 Cyprosulfamide 394
8.5 Concluding Remarks 394
References 395
9 Genetically Modified Herbicide-Resistant Crops 399
9.1 Overview 399

Susan J. Martino-Catt, Paul C.C. Feng, and Stephen R. Padgette

9.1.2 Mechanisms for Engineering Herbicide Resistance 400
9.1.2.1 Detoxification of Herbicides 400
9.1.2.2 Expression of an Insensitive Herbicide Target 401
9.1.3 Commercialized Herbicide-Resistant Crops 401
9.1.3.1 Herbicide-Resistant Soybeans 401
9.1.3.2 Herbicide-Resistant Cotton 402
9.1.3.3 Herbicide-Resistant Corn 403
9.1.3.4 Herbicide-Resistant Canola 404
References 406
XIV Contents
9.2 Inhibitors of 5-Enolpyruvyl Shikimate 3-Phosphate Synthase

(EPSPS) 406
Paul C.C. Feng, Susan Martino-Catt, and Stephen R. Padgette

9.2.2 Factors That Impact Glyphosate Efficacy 407
9.2.2.1 Foliar Absorption 407
9.2.2.2 Systemic Translocation 409
9.2.3 Development of Glyphosate-Resistant Crops 411
9.2.3.1 Alternative Mechanisms for Engineering Glyphosate Resistance 411
9.2.3.2 Disease Control Benefits of Glyphosate-Resistant Crops 412
9.2.4 Effects of CP4 Expression on Plant Resistance 414
9.2.4.1 Roundup Ready Cotton 415
9.2.4.2 Roundup Ready Corn 416
9.2.4.3 Roundup Ready Soybean 416
9.2.5 Stacking Traits in Roundup Ready Crops 416
9.2.5.1 Engineering Crop Resistance to Dicamba 417
9.2.5.2 Engineering Crop Resistance to Glufosinate 418
References 420
9.3 Glutamine Synthetase Inhibitors 423

Günter Donn

9.3.2 Role of Glutamine Synthetase in Plant Nitrogen Metabolism 424
9.3.3 Phosphinothricin, a Potent GS Inhibitor 426
9.3.4 Discovery of the Herbicidal Activity of Phosphinothricin 427
9.3.5 Mode of Glutamine Synthetase Inhibition 428
9.3.6 Physiology of the Herbicidal Activity of Phosphinothricin 429
9.3.6.1 Herbicidal Symptoms of Phosphinothricin 429
9.3.6.2 Physiological Effects of GS Inhibition in Plants 429
9.3.6.3 Modulation of Herbicidal Activity of Glufosinate by Environmental
Conditions 430
9.3.6.4 Uptake and Translocation of Glufosinate-Ammonium 430
9.3.7 Use of Phosphinothricin-Containing Herbicides in Agriculture and
Horticulture 431
9.3.8 Attempts to Generate Crop Selectivity for Glufosinate 431
9.3.8.1 Genetic Approaches to Generate Glufosinate-Selectivity in Crops:
Target-Based Approaches 431
9.3.8.2 Crop Selectivity by Expression of Phosphinothricin Acetyl
Transferase 432
9.3.8.3 Bar and Pat Genes in Plant Breeding 433
9.3.9 The Use of N-Acetyl-Phosphinothricin as a Proherbicide 434
References 435
Contents XV
10 Microtubulin Assembly Inhibitors (Pyridines) 439

Darin W. Lickfeldt, Denise P. Cudworth, Daniel D. Loughner,
and Lowell D. Markley

10.2 Biology of the Microtubulin Assembly Inhibitors (Pyridines) 439
10.2.1 Dithiopyr 439
10.2.2 Thiazopyr 440
10.3 Environmental Fate of Microtubulin Assembly Inhibitors
(Pyridines) 441
10.3.1 Dithiopyr 441
10.3.2 Thiazopyr 441
10.4 Toxicology of Microtubulin Assembly Inhibitors
(Pyridines) 441
10.5 Mode of Action of Microtubulin Assembly Inhibitors
(Pyridines) 442
10.6 Synthesis of Dithiopyr and Thiazopyr 443
References 445
11 Acetyl-CoA Carboxylase Inhibitors 447

Jean Wenger, Thierry Niderman, and Chris Mathews

11.2 Biochemistry 447
11.2.1 Overview 447
11.2.2 Mode of Action of ACCase Inhibitors 450
11.2.3 Resistance 453
11.2.4 Detection of Resistance 454
11.3 Chemistry of ACCase Inhibitors 455
11.3.1 Aryloxyphenoxypropionates (AOPPs or fops) 455
11.3.2 Cyclohexanediones (CHDs or dims) 455
11.3.3 Aryl-1,3-Diones (DENs) 460
11.3.3.1 Discovery of 2-Aryl-1,3-Diones 460
11.3.3.2 Syntheses 461
11.4 Structure–Activity Relationships 465
11.4.1 Procide Structure–Activity Relationships 466
11.4.2 Dione Structure–Activity Relationships 466
11.4.3 Phenyl Structure–Activity Relationships 467
11.4.3.1 2,4,6-Trisubstituted Phenyl Structure–Activity Relationships 467
11.4.3.2 2,5-Disubstituted Phenyl Structure–Activity Relationships 468
11.5 Pinoxaden 469
11.5.1 Characteristics 469
11.5.2 Technical Synthesis 470
11.5.3 Biology 470
11.5.4 Metabolism and Selectivity 471
11.6 Summary and Outlook 473
XVI Contents
Acknowledgments 474
References 474
12 Photosynthesis Inhibitors: Regulatory Aspects, Re-Registration

in Europe, Market Trends, and New Products 479
Martyn Griffiths, Karl-Wilhelm Münks, and Klaus-Helmut Müller

12.2 The Re-Registration Process in the European Union 482
12.3 Main Changes in Guidelines Regarding EU Re-Registration 488
12.3.1 Good Laboratory Practice 488
12.3.2 Physical and Chemical Properties of Active Substances 489
12.3.3 Storage Stability 489
12.3.4 Physical and Chemical Characteristics of Preparation 489
12.3.5 Operator Exposure Data Requirements 490
12.3.6 Residue Data Requirements 490
12.3.7 Estimation of Dietary Intakes of Pesticides Residues 490
12.3.8 Fate and Behavior of Agricultural Pesticides in the Environment 491
12.3.8.1 Concentration of Chemical in the Relevant Environmental
Compartment 491
12.3.8.2 Bioavailability of the Chemical 491
12.3.8.3 Nature of the System or Organism 492
12.3.9 Specific Guidance Regarding Water Limits According to Annexes
of the Authorizations Directive 492
12.3.10 Ecotoxicology Requirements 492
12.3.10.1 EPPO Risk-Assessment Schemes 493
12.3.10.2 Buffer Zones and LERAPs 494
12.3.10.3 Honeybee Risk Assessment 494
12.3.10.4 Risk to Nontarget Arthropods 494
12.3.10.5 Risk for Soil Nontarget Microorganisms 494
12.4 New Regulations in Europe 494
12.4.1 MRL Regulation 494
12.4.2 New PPP Regulation (to Replace Directive 91/414) 495
12.4.2.1 Dossier 495
12.4.2.2 Efficacy 495
12.4.2.3 Metabolites 496
12.4.2.4 Composition 496
12.4.2.5 Methods of analysis 496
12.4.2.6 Impact on human health 496
12.4.2.7 Fate and behavior in the environment 497
12.4.2.8 Ecotoxicology 497
12.4.2.9 Residue definition 498
12.4.2.10 Fate and behavior concerning groundwater 498
12.4.2.11 Candidate for substitution 498
12.4.2.12 Low-risk active substances 498
Contents XVII
12.5 Situation of PS II Inhibitors in the EC Markets 499

12.6 Current Market Share of PS II Compound Groups 500
12.7 A New Herbicide for Corn and Sugarcane: Amicarbazone –
BAY MKH 3586 510
12.7.2 Physico-Chemical Properties of Amicarbazone 511
12.7.3 Discovery of the Active Ingredient 511
12.7.4 Synthesis 514
12.7.4.1 Final Product 514
12.7.5 Biological Behavior 515
12.7.6 Metabolites 516
12.8 Conclusions 517
References 517
13 New Aspects of Plant Regulators 523

Hans Ulrich Haas

13.2 Plant Growth Regulators 523
13.3 PGRs in Modern Agriculture 526
13.3.1 Growth Inhibition 526
13.3.2 Fruiting and Growth 528
13.3.3 Fruit Storage and Ripening 528
13.3.4 Sprout Inhibition 529
13.3.5 Stress Defense 530
13.4 Conclusions and Developments 532
References 532
Volume 2
II Fungicides 535
Overview 537
Peter Jeschke
14 FRAC Mode of Action Classification and Resistance Risk of

Fungicides 539
Karl-Heinz Kuck, Andy Leadbeater, and Ulrich Gisi
15 Fungicides Acting on Oxidative Phosphorylation 559
15.1 The Biochemistry of Oxidative Phosphorylation: A Multiplicity of

Targets for Crop Protection Chemistry 559
Fergus Earley
XVIII Contents
15.2 Strobilurins and Other Complex III Inhibitors 584

Hubert Sauter
15.3 Succinate Dehydrogenase Inhibitors 627

Joachim Rheinheimer, Heiko Rieck, and Pierre-Yves Coqueron
15.3.1 Succinate Dehydrogenase Inhibitors: Anilides 627

Joachim Rheinheimer
15.3.2 Succinate Dehydrogenase Inhibitors: Pyridinyl-Ethyl Benzamide 639

Heiko Rieck and Pierre-Yves Coqueron
15.4 Uncouplers of Oxidative Phosphorylation 645

William G. Whittingham
15.5 NADH Inhibitors (Complex I) 670

Harald Walter
16 Fungicides Acting on Amino Acids and Protein Synthesis 693
16.1 Natural Compounds Used in Agriculture Interfering in Protein

Synthesis of Fungi and Bacteria 693
Heinrich Buchenauer and Frank Walker
16.2 Anilinopyrimidines: Methionine Biosynthesis Inhibitors 706

Ulrich Gisi and Urs Müller
17 Fungicides Acting on Signal Transduction 715
17.1 Mode of Action 715

Andrew Corran
17.2 Chemistry and Biology of Fludioxonil, Fenpiclonil, and

Quinoxyfen 721
Gertrude Knauf-Beiter and Ronald Zeun
18 Fungicides Acting on Mitosis and Cell Division 739
18.1 Zoxamide: An Antitubulin Fungicide for the Control of Oomycete

Pathogens 739
David H. Young
18.2 Pencycuron: A Phenylurea Fungicide for Rhizoctonia solani 748

Isao Ueyama and Yoshio Kurahashi
Contents XIX
19 Sterol Biosynthesis Inhibitors 761

Karl-Heinz Kuck, Klaus Stenzel, and Jean-Pierre Vors
20 Carboxylic Acid Amide (CAA) Fungicides 807

Ulrich Gisi, Clemens Lamberth, Andreas Mehl, and Thomas Seitz
21 Fluopicolide: A New Anti-Oomycetes Fungicide? 831

Valérie Toquin, Marie-Pascale Latorse, and Roland Beffa
22 Melanin Synthesis in the Cell Wall 839

Michael Schindler, Haruko Sawada, and Klaus Tietjen
23 Fungicides with Unknown Mode of Action 865

Stefan Hillebrand, Jean-Luc Zundel, and Klaus Tietjen
24 Recently Introduced Powdery Mildew Fungicides 887

Jochen Dietz
25 Newest Aspects of Nucleic Acid Synthesis Inhibitors:

Metalaxyl-M 901
Urs Müller and Ulrich Gisi
26 Host Defense Inducers 909

Valerie Toquin, Catherine Sirven, Lutz Assmann, and Haruko Sawada
Volume 3
III Insecticides 929
Overview 931
Peter Jeschke
27 IRAC: Insecticide Resistance, and Mode of Action Classification of

Insecticides 935
Ralf Nauen, Alfred Elbert, Alan Mccaffery, Russell Slater,
and Thomas C. Sparks
28 Insect Molting and Metamorphosis 957
28.1 Bisacylhydrazines: Novel Chemistry for Insect Control 957

Tarlochan Singh Dhadialla and Ronald Ross Jr
28.2 Pyriproxyfen: A New Juvenoid 983

Makoto Hatakoshi
XX Contents
29 Chitin Synthesis 999
29.1 Chitin Synthesis and Inhibitors 999

Joel J. Sheets
29.2 Mite Growth Inhibitors: Clofentezine, Hexythiazox, Etoxazole 1012

Thomas Bretschneider and Ralf Nauen
30 Midgut–Transgenic Crops Expressing Bacillus thuringiensis

Cry Proteins 1029
Jeroen Van Rie and Stefan Jansens
31 Metabolic Processes 1059
31.1 Inhibitors of Oxidative Phosphorylation 1059

Josef Ehrenfreund
31.2 Inhibitors of Oxidative Phosphorylation via Disruption of the Proton

Gradient 1070
David Kuhn and Nigel Armes
31.3 Inhibitors of Mitochondrial Electron Transport: Acaricides and

Insecticides 1078
Thomas C. Sparks and Carl V. DeAmicis
31.4 Inhibitors of Lipid Synthesis: Acetyl-CoA-Carboxylase Inhibitors 1108

Thomas Bretschneider, Reiner Fischer, and Ralf Nauen
32 Nervous System 1127
32.1 Nicotinic Acetylcholine Receptor Agonists: Target and Selectivity

Aspects 1127
Peter Jeschke and Ralf Nauen
32.2 Chemical Structural Features of Commercialized Neonicotinoids 1165

Peter Jeschke
32.2.1 Open-Chain Compounds 1169

Peter Jeschke
32.2.2 Five-Membered Compounds: Imidacloprid and Thiacloprid 1189

Peter Jeschke and Koichi Moriya
32.2.3 Six-Membered Heterocycles: Thiamethoxam and AKD 1022 1203

Peter Maienfisch
Contents XXI
32.3 Sulfoxaflor 1226

Thomas C. Sparks, Michael R. Loso, Gerald B. Watson, Jonathan M.
Babcock, Vincent Kramer, Yuanming Zhu, Benjamin Nugent,
and James Thomas
32.4 Spinosad and Spinetoram, a New Semi-Synthetic Spinosyn 1238

Gary D. Crouse, James E. Dripps, Thomas C. Sparks, Gerald B. Watson,
and Clive Waldron
32.5 Sodium Channel-Blocking Insecticides 1257
32.5.1 Sodium Channel-Blocking Insecticides: Indoxacarb 1257

Stephen F. McCann, Daniel Cordova, John T. Andaloro, and
George P. Lahm
32.5.2 Semicarbazone Insecticides: Metaflumizone 1273

David Kuhn, K. Takagi, Tomokazu Hino, and Nigel Armes
32.6 Ligand-Gated Chloride Channel Antagonists (Fiproles) 1283

Vincent L. Salgado, Stefan Schnatterer, and Keith A. Holmes
32.7 Chloride Channel Activators/New Natural Products: Avermectins and

Milbemycins 1305
Thomas Pitterna
33 New Unknown Modes of Action 1327
33.1 Selective Feeding Blockers: Pymetrozine, Flonicamid, and

Pyrifluquinazon 1327
Peter Maienfisch
33.2 Acaricides with Undefined Mode of Action: Bifenazate, Cyflumetofen,

and Amidoflumet 1346
Mark A. Dekeyser
33.3 Pyridalyl: Discovery, Insecticidal Activity, and Mode of Action 1358

Shigeru Saito and Noriyasu Sakamoto
33.4 Recent Nematicides 1367

Olivier Loiseleur, Brigitte Slaats, and Peter Maienfisch
34 Insecticides Affecting Calcium Homeostasis 1389
34.1 Ryanodine Receptor Modulators: Diamides 1389

Hiroshi Hamaguchi, Takashi Hirooka, and Takao Masaki
XXII Contents
34.2 Flubendiamide 1396

Hiroshi Hamaguchi and Takashi Hirooka
34.3 Anthranilic Diamide Insecticides: Chlorantraniliprole and

Cyantraniliprole 1409
George P. Lahm, Daniel Cordova, James D. Barry, John T. Andaloro,
I. Billy Annan, Paula C. Marcon, Hector E. Portillo,
Thomas M. Stevenson, and Thomas P. Selby
Index of Common Names 1427
Subject Index 1449

XXV
List of Contributors
John T. Andaloro Jonathan M. Babcock

DuPont Crop Protection Dow AgroSciences
Stine-Haskell Research Center 9330 Zionsville Road
1090 Elkton Road Indianapolis, IN 46268
Newark, DE 19711 USA
USA
Peter Babczinski
I. Billy Annan Gartenstrasse 31 b
DuPont Crop Protection 40764 Langenfeld
Stine-Haskell Research Center Germany
1090 Elkton Road
Newark, DE 19711 James D. Barry
USA DuPont Crop Protection
Stine-Haskell Research Center
Nigel Armes 1090 Elkton Road
BASF Corporation Newark, DE 19711
Agricultural Solutions USA
26 Davis Drive
Research Triangle Park Klaus Bartsch
Raleigh, NC 27709 Bayer CropScience AG
USA Industriepark Höchst - H872
65926 Frankfurt am Main
Lutz Assmann Germany
Bayer CropScience AG
Alfred-Nobel-Str. 50 Roland Beffa
40789 Monheim am Rhein Bayer CropScience AG
Germany Industriepark-Hoechst-H872
Germany
XXVI List of Contributors
Thomas Bretschneider Gary D. Crouse

Bayer CropScience AG Dow AgroSciences LLC
Research Pest Control Chemistry 9330 Zionsville Road
Alfred-Nobel-Str. 50 Indianapolis, IN 46268
40789 Monheim USA
Germany
Denise P. Cudworth
Heinrich Buchenauer Dow AgroSciences LLC
Bayer CropScience AG 9330 Zionsville Road
Research Pest Control Chemistry Indianapolis, IN 46268
Alfred-Nobel-Str. 50 USA
40789 Monheim
Germany Carl V. DeAmicis
Dow AgroSciences LLC
Jon S. Claus Process Research
DuPont Crop Protection 9330 Zionsville Road
Stine-Haskell Research Center Indianapolis, IN 46268
P. O. Box 30 USA
1090 Elkton Road
Newark, DE 19714 Gerrit J. deBoer
USA Dow AgroSciences
Discovery Research
Pierre-Yves Coqueron 9330 Zionsville Road
Bayer S.A.S./ Indianapolis, IN 46268
Bayer CropScience AG USA
Chemistry Fungicides
Building: D1 Mark A. Dekeyser
BP 99163 Chemtura Canada Co./Cie.
69263 Lyon Cedex 09 Guelph Technology Centre
France 120 Huron Street
Guelph
Daniel Cordova Ontario N1E 5L7
DuPont Crop Protection Canada
Stine-Haskell Research Center
1090 Elkton Road Hansjörg Dietrich
Newark, DE 19711 Bayer CropScience AG
USA Industriepark Höchst - H872
Andrew Corran Germany
Syngenta
Jealott’s Hill International
Research Centre
Bracknell
Berkshire RG42 6EY
UK
List of Contributors XXVII
Jochen Dietz Alfred Elbert

BASF SE Formerly Bayer CropScience AG
Global Research Crop Protection Biology Herbicides
GVA/FO - A30 Sachsenring 35
67056 Ludwigshafen 65719 Hofheim
Germany Germany
Günter Donn Paul C.C. Feng

Formerly Bayer CropScience AG Monsanto Co.
Biology Herbicides 800 N. Lindbergh Blvd
Industriepark Höchst - H872 St Louis, MO 63167
65926 Frankfurt am Main USA
Germany
Bruce L. Finkelstein
James E. Dripps DuPont Crop Protection
Dow AgroSciences LLC Stine-Haskell Research Center
9330 Zionsville Road P. O. Box 30
Indianapolis, IN 46268 1090 Elkton Road
USA Newark, DE 19714
USA
Fergus Earley
Syngenta Reiner Fischer
Jealott’s Hill Research Centre, Bayer CropScience AG
Bracknell Research Pest Control
Berkshire RG42 6EY Alfred-Nobel-Straße 50
UK 40789 Monheim am Rhein
Germany
Andrew J.F. Edmunds
Syngenta Crop Protection Roger E. Gast
Münchwilen AG Dow AgroSciences LLC
Schaffhauserstrasse Discovery Research
4332 Stein 9330 Zionsville Road
Switzerland Indianapolis, IN 46268
USA
Josef Ehrenfreund
Syngenta Crop Protection AG Ernst-Rudolf F. Gesing
WRO–1060.144 Bayer CropScience AG
P. O. Box Research
4002 Basel Bldg. 6550, 1.11
Switzerland Alfred-Nobel-Straße 50
Germany
XXVIII List of Contributors
Ulrich Gisi Gerhard Hamprecht1

Syngenta Crop Protection BASF SE GVA/HC-B009
Muenchwilen AG 67056 Ludwigshafen
Schaffhauserstrasse Germany
4332 Stein
Switzerland Ryo Hanai
Kumiai Chemical Industry
Toshio Goto Co. Ltd
Formerly Bayer CropScience KK 4-26, Ikenohata 1-chome, Taito-ku
Yuki Research Center Tokyo 110-8782
Japan Japan
Martyn Griffiths Makoto Hatakoshi

Bayer S.A.S Sumitomo Chemical Co. Ltd
Bayer CropScience 2-1 Takatsukasa 4-chome
Takarazuka
European Regulatory Affairs
Hyogo 665-8555
16, rue Jean-Marie Leclair,
Japan
CS 90106
69266 Lyon Cedex 09
Timothy R. Hawkes
France
Syngenta Ltd
Jealott’s Hill International
Steven Gutteridge
Research Centre
DuPont Crop Protection
Bracknell
Stine-Haskell Research Center Berkshire RG42 6EY
1090 Elkton Road UK
Newark, DE 19711
USA Stefan Hillebrand
Bayer CropScience
Hans Ulrich Haas Alfred-Nobel-Str. 50
Syngenta Crop Protection 40789 Monheim
Münchwilen AG Germany
Schaffhauserstrasse
4332 Stein Tomokazu Hino
Switzerland BASF Corporation
Agricultural Solutions
Hiroshi Hamaguchi 26 Davis Drive
Nihon Nohyaku Co. Ltd Research Triangle Park
R&D Division Raleigh, NC 27709
5th Floor, Eitaro Building USA
1-2-5 Nihonbashi, Chuo-Ku
Tokyo 103-8236
Japan
1
Retired
List of Contributors XXIX
Takashi Hirooka Helmut Köcher

Nihon Nohyaku Co. Ltd Bayer CropScience AG
Production Division Industriepark-Höchst - H872
8th Floor, Eitaro Building 65926 Frankfurt am Main
1-2-5 Nihonbashi, Chuo-Ku Germany
Tokyo 103-8236
Japan Vincent J. Kramer
Dow AgroSciences
Keith A. Holmes 9330 Zionsville Road
BASF Corporation Indianapolis, IN 46268
Agricultural Solutions USA
26 Davis Drive
Research Triangle Park Karl-Heinz Kuck
Raleigh, NC 27709 Pastor-Löh-Str. 30a
USA 40764 Langenfeld
Germany
Stefan Jansens
Bayer BioScience N. V. David Kuhn
Technologiepark 38 BASF Corporation
9052 Gent Agricultural Solutions
Belgium 26 Davis Drive
Research Triangle Park
Peter Jeschke Raleigh, NC 27709
Bayer CropScience AG USA
BCS AG-R-PC-PCC
Building 6510 Yoshio Kurahashi2
Alfred-Nobel-Str. 50 Bayer CropScience
40789 Monheim am Rhein K.K. 1-6-5, Marunouchi
Germany Chiyoda-ku
Tokyo
Timothy C. Johnson Japan
Dow AgroSciences
Discovery Research Bernd Laber
9330 Zionsville Road Bayer CropScience AG
Indianapolis, IN 46268 Industriepark-Höchst - H872
USA 65926 Frankfurt am Main
Germany
Gertrude Knauf-Beiter
Syngenta Crop Protection AG
Research Biology
WST-540.2.65
4332 Stein
Switzerland
2
Retired
XXX List of Contributors
George P. Lahm William C. Lo

DuPont Crop Protection Dow AgroSciences LLC
Stine-Haskell Research Center Discovery Research
1090 Elkton Road 9330 Zionsville Road
Newark, DE 19711 Indianapolis, IN 46268
USA USA
Clemens Lamberth Olivier Loiseleur

Syngenta Crop Protection Syngenta Crop Protection
WST-820.1.09 Münchwilen AG
Schaffhauserstr. 101 Crop Protection Research
4332 Stein Schaffhauserstr
Switzerland 4332 Stein
Switzerland
Marie-Pascale Latorse
Bayer SAS, Bayer CropScience Michael R. Loso
Research Center La Dargoire Dow AgroSciences
14-20 Impasse Pierre Baizet 9330 Zionsville Road
69009 Lyon Indianapolis, IN 46268
France USA
Andy Leadbeater Daniel D. Loughner

Syngenta Crop Protection Dow AgroSciences LLC
Schwarzwaldallee 9330 Zionsville Road
4002 Basel Indianapolis, IN 46268
Switzerland USA
Darin W. Lickfeldt Peter Maienfisch

Dow AgroSciences LLC Syngenta Crop Protection
9330 Zionsville Road Muenchwilen AG
Indianapolis, IN 46268 Crop Protection Research
USA Schaffhauserstrasse
4332 Stein
Rex Liebl Switzerland
BASF Corporation
26 Davis Drive Richard K. Mann
Research Triangle Park Dow AgroSciences
Raleigh, NC 27709 Discovery Research
USA 9330 Zionsville Road
Indianapolis, IN 46268
USA
List of Contributors XXXI
Paula C. Marcon Alan Mccaffery

DuPont Crop Protection Syngenta Crop Protection
Stine-Haskell Research Center Jealott’s Hill International
1090 Elkton Road Research Centre
Newark, DE 19711 Bracknell
USA Berkshire RG42 6EY
UK
Lowell D. Markley
Dow AgroSciences LLC Stephen F. McCann
9330 Zionsville Road DuPont Crop Protection
Indianapolis, IN 46268 Stine-Haskell Research Center
USA Newark, DE 19711
USA
Takao Masaki
Nihon Nohyaku Co. Ltd Hubert Menne
R&D Division Bayer CropScience AG
Research Center Industriepark Höchst - H872
345 Oyamada-Cho 65926 Frankfurt am Main
Kawachinagano Osaka Germany
586-0094
Japan Andreas Mehl
Susan J. Martino-Catt BCS AG-R-DC-DCM
Monsanto Co. Alfred-Nobel-Str. 50
800 N. Lindbergh Blvd 40789 Monheim am Rhein
St Louis, MO 63167 Germany
USA
Koichi Moriya
Robert A. Masters Bayer CropScience K.K.
Dow AgroSciences LLC Production Support Department
Discovery Research Industrial Operation & QHSE
9330 Zionsville Road Division
Indianapolis, IN 46268 2061, Shinden Tsukiji
USA Hofu City
Yamaguchi 747-0825
Chris Mathews Japan
Syngenta Jealott’s Hill
International Research Centre James A. Morris
Bracknell Syngenta Ltd
Berkshire RG42 6EY Jealott’s Hill International
UK Research Centre
Bracknell
Berkshire RG42 6EY
UK
XXXII List of Contributors
Klaus-Helmut Müller Benjamin M. Nugent

Bayer CropScience AG Dow AgroSciences
Research 9330 Zionsville Road
Bldg. 6510, 2.37 Indianapolis, IN 46268
Alfred-Nobel-Straße 50 USA
Germany Oswald Ort
Urs Müller Research Building 6550
Drosselstrasse 6 Alfred-Nobel-Straße 50
4142 Münchenstein 40789 Monheim
Switzerland Germany
Karl-Wilhelm Münks Stephen R. Padgette

Bayer CropScience AG Monsanto Co.
Strategy & Business Management 800 N. Lindbergh Blvd
Project Management St Louis, MO 63167
Bldg 6100, C 3.82 USA
Alfred-Nobel-Straße 50
40789 Monheim am Rhein Thomas Pitterna
Germany Syngenta Crop Protection
Münchwilen AG
Masao Nakatani WST–820.2.09
K-I Chemical Research Institute Schaffhauserstrasse
Co., Ltd 4332 Stein
408-1, Shioshinden Switzerland
Iwata, Shizuoka 437-1213
Japan Hector E. Portillo
DuPont Crop Protection
Ralf Nauen Stine-Haskell Research Center
Bayer CropScience AG 1090 Elkton Road
BCS AG-R-PC-PCC Newark, DE 19711
Bldg. 6220 USA
Alfred-Nobel-Str. 50
40789 Monheim am Rhein Joachim Rheinheimer
Germany BASF SE
GVA–A 30
Thierry Niderman 67056 Ludwigshafen
Formerly of Syngenta Crop Germany
Protection AG
Schwarzwaldallee 215 Chris Rosinger
P.O. Box Bayer CropScience AG
4002 Basel Industriepark Höchst - H872
Switzerland 65926 Frankfurt am Main
Germany
List of Contributors XXXIII
Heiko Rieck Vincent L. Salgado

Bayer CropScience AG BASF Corporation
Alfred-Nobel-Str. 50 Agricultural Solutions
40789 Monheim 26 Davis Drive
Germany Research Triangle Park
Raleigh, NC 27709
Ronald Ross Jr USA
9330 Zionsville Road Hubert Sauter
Indianapolis, IN 46268 Consultant
USA Alte Reichenbacher Straße 113
72270 Baiersbronn
Shigeru Saito Germany
Sumimoto Chemical Co. Ltd
Health & Crop Sciences Research Haruko Sawada
Laboratory Bayer CropScience AG
2-1, Takatsukasa 4-chome Reseach-DC-DCM
Takarazuka.Hyogo Alfred-Nobel-Str. 50
665-8555 40789 Monheim
Japan Germany
Hans-Joachim Santel Michael Schindler

Bayer CropScience AG Bayer CropScience AG
Development Research Building 6500
Agronomic Development Alfred-Nobel-Str. 50
Herbicides 40789 Monheim
Bldg. 6100, A 2.19 Germany
Alfred Nobel Straße 50
40789 Monheim am Rhein Paul R. Schmitzer
Germany Dow AgroSciences
Discovery Research
Noriyasu Sakamoto 9330 Zionsville Road
Sumimoto Chemical Co. Ltd Indianapolis, IN 46268
Health & Crop Sciences Research USA
Laboratory
2-1, Takatsukasa 4-chome Stefan Schnatterer
Takarazuka.Hyogo Bayer CropScience AG
665-8555 Research Chemistry Frankfurt
Japan Frankfurt Industriepark Hoechst,
Bldg G 836, L118
Germany
XXXIV List of Contributors
Wolfgang Schulte Bijay Singh

Bayer CropScience AG BASF Corporation
Industriepark-Höchst - H872 26 Davis Drive, Research
65926 Frankfurt am Main Triangle Park
Germany Raleigh, NC 27709
USA
Thomas Seitz
Bayer CropScience Tarlochan Singh Dhadialla
BCS AG-R-DC-DCM Dow AgroSciences LLC
Alfred-Nobel-Str. 50 9330 Zionsville Road
40789 Monheim am Rhein Indianapolis, IN 46268
Germany USA
Thomas P. Selby Catherine Sirven

DuPont Crop Protection Bayer CropScience SA
Stine-Haskell Research Center Department of Biology
1090 Elkton Road 14–20 rue Pierre Baizet
Newark, DE 19711 69009 Lyon
USA France
Dale L. Shaner Brigitte Slaats

United States Department of Syngenta Crop Protection
Agriculture Muenchwilen AG
Agricultural Research Service Crop Protection Research
2150 Centre Avenue Schaffhauserstr.
Bldg D, Suite 320 4332 Stein
Fort Collins, CO 80526-8119 Switzerland
USA
Russell Slater
Joel J. Sheets Syngenta Crop Protection
Dow AgroSciences LLC Werk Stein
Biochemistry/Molecular Biology 4332 Stein
9330 Zionsville Road Switzerland
USA Thomas C. Sparks
Tsutomu Shimizu 9330 Zionsville Road
Kumiai Chemical Industry Indianapolis, IN 46268
Co. Ltd USA
4-26, Ikenohata 1-chome, Taito-ku
Tokyo 110-8782 Klaus Stenzel
Japan Bayer CropScience AG
Alfred-Nobel-Str. 50
40789 Monheim
Germany
List of Contributors XXXV
Thomas M. Stevenson James D. Thomas

DuPont Crop Protection Dow AgroSciences
Stine-Haskell Research Center 9330 Zionsville Road
1090 Elkton Road Indianapolis, IN 46268
Newark, DE 19711 USA
USA
Mark E. Thompson
Mark Stidham Stine-Haskell Research Center
Trius Therapeutics 1090 Elkton Road
11749 Windcrest Lane Newark, DE 19711
San Diego, CA 92128 USA
USA
Klaus Tietjen
K. Takagi Bayer CropScience AG
BASF Corporation Reseach-DC-DCM
Agricultural Solutions Alfred-Nobel-Str. 50
26 Davis Drive 40789 Monheim
Research Triangle Park Germany
Raleigh, NC 27709
USA Valérie Toquin
Bayer SAS, Bayer CropScience
Siyuan Tan La Dargoire Research Center
BASF Corporation 14 Impasse Pierre Baizet
26 Davis Drive 69009 Lyon
Research Triangle Park France
Raleigh, NC 27709
USA Isao Ueyama3
Bayer CropScience K.K.
Yoshitaka Tanetani 1-6-5, Marunouchi
Kumiai Chemical Industry Chiyoda-ku
Co. Ltd Tokyo 100-8262
4-26, Ikenohata 1-chome, Taito-ku Japan
Tokyo 110-8782
Japan Andreas van Almsick
George Theodoridis Industriepark Höchst - G836
The College of New Jersey 65926 Frankfurt am Main
Department of Chemistry Germany
2000 Penning Road
Ewing, NJ 08618
USA
3
Retired
XXXVI List of Contributors
Jeroen Van Rie Yukiyoshi Watanabe

Bayer BioScience N. V. Bayer CropScience K.K.
Technologiepark 38 6-5, Marunouchi 1-chome
9052 Gent Chiyoda-ku
Belgium Tokyo 100-8262
Japan
Jean-Pierre Vors
Bayer SAS Gerald B. Watson
CRLD Dow AgroSciences
14 Impasse Pierre Baizet 9330 Zionsville Road
BP99163 Indianapolis, IN 46268
69263 Lyon Cedex 09 USA
France
Jean Wenger
Frank Walker Formerly of Syngenta Crop
Universität Hohenheim Protection AG
Institut für Phytomedizin Schwarzwaldallee 215
Otto-Sander-Str. 5 P.O. Box
70599 Stuttgart 4002 Basel
Germany Switzerland
Harald Walter William G. Whittingham

Syngenta Crop Protection Syngenta Ltd
Muenchwilen AG Jealott’s Hill International
Schaffhauserstrasse Research Centre
4332 Stein Bracknell
Switzerland Berkshire RG42 6EY
UK
Clive Waldron
Michigan State University Matthias Witschel
Department of Microbiology & BASF SE GVA/HC-B009
Molecular Genetics 67056 Ludwigshafen
6179 Biomedical and Physical Germany
Sciences Building
East Lansing, MI 48824-4320 Takumi Yoshimura
USA K-I Chemical Research
Institute Co. Ltd
Terence A. Walsh 408-1, Shioshinden
Dow AgroSciences Iwata, Shizuoka 437-1213
Discovery Research Japan
9330 Zionsville Road
USA
List of Contributors XXXVII
David H. Young Yuanming Zhu

Dow AgroSciences LLC Dow AgroSciences
Discovery Research 9330 Zionsville Road
9330 Zionsville Road Indianapolis, IN 46268
Indianapolis, IN 46268 USA
USA
Jean-Luc Zundel
Cyrill Zagar Formerly Bayer CropScience SA
BASF Corporation La Dargoire Research Centre
26 Davis Drive 14–20 rue Pierre Baizet
Research Triangle Park BP 9163
Raleigh, NC 27709 69263 Lyon Cedex 09
USA France
Ronald Zeun
Syngenta Crop Protection AG
Research Biology
WST-540.E67
4332 Stein
Switzerland
Edited by

Further reading

Change
2 Volumes
2007 2010
ISBN: 978-3-527-31604-5 ISBN: 978-0-8138-1417-9

2010
Science Series”
ISBN: 978-1-4051-7589-0
2009
ISBN: 978-3-527-31465-2
Volume 2
Dr.Ulrich Schirmer
inaccurate.
Berghalde 79
69126 Heidelberg


Germany


Chennai
Darmstadt

Print ISBN: 978-3-527-32965-6

V
Contents
Volume 1
Preface XIX
List of Contributors XXI
I Herbicides 1
Overview 3
Matthias Witschel



Oswald Ort



VI Contents



Target 225
Timothy R. Hawkes
4.3 Triketones 235


Heterocycles 262
Andreas van Almsick



6 Herbicides Disturbing the Synthesis of Very Long-Chain

Fatty Acids 305

(VLCFAs) 305
Peter Babczinski

Yukiyoshi Watanabe

and Tsutomu Shimizu
Contents VII


9.1 Overview 399


(EPSPS) 406

Günter Donn




Hans Ulrich Haas
Volume 2
II Fungicides 535
Overview 537
Peter Jeschke

Fungicides 539
14.1 History of Fungicide Use 539

14.2 Fungicides: Importance of Individual Modes of Action 540
VIII Contents
14.3 Fungicide Resistance 543

14.3.1 Mechanisms and Occurrence of Resistance 543
14.3.2 The Fungicide Resistance Action Committee (FRAC) 545
14.3.3 Resistance Risk Assessment 546
14.3.4 Resistance Management and Risk Modifiers 546
14.4 Fungicide Classes and Modes of Action 547
References 556

Fergus Earley

15.1.2 Components of the Mitochondrial Electron-Transport Chains 562
15.1.2.1 Complex I and Its Inhibitors 562
15.1.2.2 Complex III (Cytochrome bc1 Complex) and Its Inhibitors 564
15.1.2.3 Complex IV 569
15.1.2.4 Succinate Dehydrogenase (Complex II) and Its Inhibitors 570
15.1.2.5 Alternative Electron-Transport Chains 572
15.1.3 Energy Conservation 573
15.1.3.1 F1 F0 ATP Synthase and Its Inhibitors 574
15.1.3.2 Inhibitors of the Mitochondrial ADP/ATP Carrier 575
15.1.4 Concluding Remarks 578
References 579

Hubert Sauter

15.2.2 Evolution of Strobilurins as Agricultural Fungicides 587
15.2.3 Structure–Activity Relationships of Strobilurins 595
15.2.3.1 Interplay of Target Activity and Biokinetic Behavior 595
15.2.3.2 Target Activity 597
15.2.3.3 Transportation and Distribution 603
15.2.3.4 Metabolic Degradation Rates 607
15.2.3.5 Summary of Strobilurin Structure–Activity Relationships 608
15.2.4 Beneficial Influences on Plant Physiology and Crop Yield 609
15.2.5 Insecticidal and Acaricidal Activity 610
15.2.6 Fungal Resistance 611
15.2.7 Other Complex III Inhibitors 613
15.2.7.1 Azolones 614
15.2.7.2 N-(N , N -Dimethylaminosulfonyl)azoles 615
15.2.7.3 Ametoctradin 617
Contents IX
15.2.8 Synthesis Routes 618

Acknowledgments 621
References 621


Joachim Rheinheimer

15.3.1.2 Active Ingredients 631
15.3.1.3 Research Activities and Patent Situation 632
15.3.1.5 Biological Activity and Application 633
15.3.1.6 Structure–Activity Relationships 634
15.3.1.7 Resistance 635
15.3.1.8 Metabolism 635
15.3.1.9 Discussion 636
Acknowledgments 637
References 637
15.3.2 Succinate Dehydrogenase Inhibitors: Pyridinyl-Ethyl

Benzamide 639

15.3.2.2 Origin of the Chemical Structure 639
15.3.2.3 Influence of Structural Elements on the Biological
Spectrum 641
15.3.2.4 Synthesis of Fluopyram 641
15.3.2.5 Biological Activity and Application Rates 644
15.3.2.6 Conclusions 645
Acknowledgments 645
References 645


15.4.2 Mechanism of Action of Uncouplers 646
15.4.3 Selectivity and Toxicity 648
15.4.4 Resistance 650
15.4.5 Physico-Chemical Properties of Protonophoric Uncouplers 651
15.4.6 Chemical Uncouplers 653
15.4.7 Dinitrophenols 659
X Contents
15.4.8 Arylhydrazones, Including Ferimzone 660

15.4.9 Diarylamines, Including Fluazinam 662
References 664

Harald Walter

15.5.2 The Aminoalkylpyrimidines 671
15.5.2.1 The Competitors’ Contributions in the Aminoalkylpyrimidine
Area 675
15.5.2.2 Summary: Aminoalkylpyrimidines 677
15.5.3 Arylacetic Acid Amides Derived from 4-Aminopyridines and
4-Aminoquinolines 677
15.5.4 Phenylacetic Acid Amides Derived from Aminopyrimidines and
Aminoquinazolines 681
15.5.5 Arylsulfonyl Acid Amides Derived from
Heteroarylmethylamines 683
15.5.6 Other Leads in the Area of Complex I Inhibitors 686
15.5.7 Conclusions and Summary 687
References 687


16.1.2 General Mechanisms of Protein Biosynthesis 693
16.1.3 Blasticidin S 694
16.1.4 Kasugamycin 696
16.1.5 Mildiomycin 698
16.1.6 Cycloheximide 699
16.1.7 Streptomycin 700
References 703


16.2.2 Chemistry of the Anilinopyrimidines 706
16.2.3 Biological Activity 709
16.2.5 Mode of Action and Mechanism of Resistance 711
16.2.6 Degradation and Metabolism 713
References 713
Contents XI

Andrew Corran
17.1.1 Phenylpyrroles and Dicarboximides 715

17.1.2 Quinoxyfen 719
References 720

Quinoxyfen 721
17.2.1 Phenylpyrroles: Fenpiclonil and Fludioxonil 721

17.2.1.1 Chemistry 721
17.2.1.2 Biology 726
17.2.2 Quinoxyfen 731
17.2.2.1 Chemistry 731
17.2.2.2 Biology 732
References 733

Pathogens 739
David H. Young

18.1.2 Mechanism of Action 739
18.1.3 Analysis of the Benzamide Binding Site Using Radioligand Binding
Assays 740
18.1.4 Cross-Resistance Relationships between Zoxamide, Carbendazim, and
Diethofencarb 742
18.1.6 Synthesis of Zoxamide 744
18.1.7 Resistance Risk 745
18.1.8 Metabolism and Toxicology 746
18.1.9 Biology and Use in Agriculture 746
References 747


18.2.1.1 Overview of the Compound 748
18.2.1.2 Pencycuron: Background to the Development 749
XII Contents
18.2.2 Chemistry 750

18.2.2.1 Developmental History 750
18.2.3 Chemical Synthesis and Physico-Chemical Properties 755
18.2.3.1 Preparation of Pencycuron 755
18.2.3.2 Physico-Chemical Properties of Pencycuron 756
18.2.4 Mode of Action and Biology 756
18.2.4.1 Mode of Action 756
18.2.4.2 Biology 757
18.2.4.3 Sensitivity to Several Anastomosis Groups (AGs) of Rhizoctonia
solani 757
18.2.5 Toxicology, Ecotoxicology, and Metabolism 758
18.2.5.1 Toxicology and Ecotoxicology 758
18.2.5.2 Metabolism of Pencycuron 759
References 760

19.1 Sterol Biosynthesis Inhibitor SBI Fungicides in Agriculture 761

19.1.1 Market Importance of SBI Fungicides 763
19.1.2 Biochemical Targets of SBI Fungicides 763
19.1.3 SBI Classes 764
19.2 SBI Class I: DMI Fungicides 767
19.2.1 Piperazines, Pyridines, Pyrimidines, and Imidazoles 770
19.2.1.1 Pefurazoate 773
19.2.1.2 Oxpoconazole 774
19.2.2 Triazoles 776
19.2.2.1 Triazoles Launched Before 1990 776
19.2.2.2 Triazole Fungicides Launched Since 1990 776
19.3 SBI Class II: Amines 793
19.3.1 Morpholines and Piperidines 793
19.3.2 Biochemical Targets of Amines 793
19.3.3 Spiroxamine: The First of the Spiroketalamines 795
19.4 SBI Class III: Hydroxyanilides 796
19.4.1 Fenhexamid: The First of the Hydroxyanilides 796
19.4.2 Biochemical Target of Fenhexamid 798
19.4.3 Biology 798
19.5 SBI Class IV: Squalene Epoxidase Inhibitors 801
References 801


20.2 Chemistry of the Carboxylic Acid Amides 809
Contents XIII
20.2.1 Cinnamic Acid Amides 809

20.2.1.1 Dimethomorph 809
20.2.1.2 Flumorph 810
20.2.1.3 Pyrimorph 811
20.2.2 Amino Acid Amides 812
20.2.2.1 Iprovalicarb 812
20.2.2.2 Benthiavalicarb 814
20.2.2.3 Valifenalate 816
20.2.2.4 Aminosulfones (Experimental Compounds) 817
20.2.2.5 N-Sulfonyl Amino Acid Amides (Experimental Compounds) 818
20.2.3 Mandelic Acid Amides 818
20.2.3.1 Mandipropamid 818
20.2.3.2 Glyoxylic Acid Derivatives (Experimental Compounds) 822
20.3 Biological Activity of Carboxylic Acid Amides 824
20.4 Mode of Action and Mechanism of Resistance of the CAA
Fungicides 825
References 828


21.2 Chemical and Physical Properties 831
21.3 Toxicology/Ecotoxicology 832
21.4 Spectrum of Activity 833
21.4.1 Effect on Zoospores and Mycelial Growth of P. infestans 833
21.5 Effect of Fluopicolide on Spectrin-Like Protein Distribution 834
21.5.1 Characterization of Spectrin-Like Proteins in P. infestans by
Bioanalysis 835
21.6 Conclusion 837
References 838

22.1 Biological Occurrence and Function of Melanin in Fungi 839

22.2 Overview: Fungicides Inhibiting DHN Melanin
Biosynthesis 842
22.3 Biology of Scytalone Dehydratase Inhibitors 842
22.4 Biochemical Reaction Mechanism of Scytalone Dehydratase and
Structure-Based Inhibitor Design 850
22.4.1 X-Ray Structures and the Active Site of Scytalone
Dehydratase 850
22.4.2 Computational Investigations of the Enzyme Mechanism 854
22.4.3 Comparison of Inhibitor Structures in the SD Binding
Niche 855
XIV Contents
22.4.4 Complementary Information by Site-Directed

Mutations 857
22.5 Chemistry and Stereochemistry of Carpropamid 857
22.6 Resistance Problems and Successful Management in Japan 858
22.7 Final Remarks 860
References 861


23.2 Cymoxanil 865
23.3 Fosetyl-Aluminum 869
23.4 Flusulfamide 873
23.5 Diclomezine 875
23.6 Triazoxide 877
23.7 Tebufloquin 879
References 882

Jochen Dietz

24.2 Cyflufenamid 887
24.2.1 Discovery 887
24.2.2 Cross-Resistance and Mode of Action 889
24.2.3 Manufacturing Process 890
24.2.4 Fungicidal Profile 890
24.2.5 Registration, Products, Formulations, and Crops 890
24.3 Metrafenone 891
24.3.4 Registration, Products, Formulation, and Crops 893
24.4 Pyriofenone 893
24.5 Proquinazid 893
24.5.1 Discovery 894
24.5.5 Registration, Products, Formulation, and Crops 896
24.6 Flutianil 896
Contents XV

Acknowledgments 898
References 898

Metalaxyl-M 901

25.2 Chemistry of Metalaxyl-M/Mefenoxam 901
25.3 Biological Activity 904
25.4 Mode of Action and Mechanism of Resistance 904
25.5 Degradation and Metabolism of the Two Enantiomers 906
References 907


26.2 General Mechanism of Induced Resistance 910
26.2.1 Induced Resistance 910
26.2.2 Systemic Signals in Systemic Acquired Resistance 911
26.2.3 Signal Perception and Transduction 912
26.2.4 Biochemical Changes in Systemic Acquired Resistance 912
26.2.5 Priming in Systemic Acquired Resistance 913
26.3 Market Products 913
26.3.1 Probenazole 913
26.3.1.1 Synthesis of Probenazole 914
26.3.1.2 Systemic Acquired Resistance Induction by Probenazole 914
26.3.2 Acibenzolar-S-Methyl 915
26.3.2.1 Synthesis of Acibenzolar-S-Methyl 915
26.3.2.2 Systemic Acquired Resistance Induction by
Acibenzolar-S-Methyl 916
26.3.3 Tiadinil 917
26.3.3.1 Synthesis of Tiadinil 917
26.3.3.2 Systemic Acquired Resistance by Tiadinil 918
26.3.4 Isotianil 919
26.3.4.1 Synthesis of Isotianil 919
26.3.4.2 Systemic Acquired Resistance by Isotianil 919
26.3.4.3 Gene Expression Profiling Experiments 920
References 926
XVI Contents
Volume 3
Overview 931
Peter Jeschke

Insecticides 935


Makoto Hatakoshi

Joel J. Sheets

30 Midgut–Transgenic Crops Expressing Bacillus thuringiensis

Cry Proteins 1029

Josef Ehrenfreund

Gradient 1070

Insecticides 1078
Contents XVII


Aspects 1127

Peter Jeschke

Peter Jeschke


Peter Maienfisch

and James Thomas

and Clive Waldron

George P. Lahm



Milbemycins 1305
Thomas Pitterna
XVIII Contents

Peter Maienfisch

Mark A. Dekeyser





Subject Index 1449

Edited by

Further reading

Change
2 Volumes
2007 2010
ISBN: 978-3-527-31604-5 ISBN: 978-0-8138-1417-9

2010
Science Series”
ISBN: 978-1-4051-7589-0
2009
ISBN: 978-3-527-31465-2
Volume 3
Dr.Ulrich Schirmer
inaccurate.
Berghalde 79
69126 Heidelberg


Germany


Chennai
Darmstadt

Print ISBN: 978-3-527-32965-6

V
Contents
Volume 1
Preface XXIII
List of Contributors XXV
I Herbicides 1
Overview 3
Matthias Witschel



Oswald Ort



VI Contents



Target 225
Timothy R. Hawkes
4.3 Triketones 235


Heterocycles 262
Andreas van Almsick



6 Herbicides Disturbing the Synthesis of Very Long-Chain

Fatty Acids 305

(VLCFAs) 305
Peter Babczinski

Yukiyoshi Watanabe

and Tsutomu Shimizu
Contents VII


9.1 Overview 399


(EPSPS) 406

Günter Donn




Hans Ulrich Haas
Volume 2
II Fungicides 535
Overview 537
Peter Jeschke

Fungicides 539
VIII Contents

Fergus Earley

Hubert Sauter


Joachim Rheinheimer
15.3.2 Succinate Dehydrogenase Inhibitors: Pyridinyl-Ethyl Benzamide 639



Harald Walter



Andrew Corran

Quinoxyfen 721
Contents IX

Pathogens 739
David H. Young







Jochen Dietz

Metalaxyl-M 901

Volume 3
Overview 931
Peter Jeschke
X Contents

Insecticides 935

27.2 Objectives of the IRAC 935
27.3 Structure and Organization of the IRAC 936
27.3.1 Project and Functional Teams 936
27.3.2 Country Groups 936
27.4 Activities 937
27.4.1 Resistance-Monitoring Methods 937
27.4.2 IRAC and the Regulatory Requirements of Resistance
Management 938
27.4.3 Education and Communication 938
27.4.4 Resistance Database Managed by Michigan State University and
Supported by IRAC 939
27.4.5 The Mode of Action Classification Scheme 939
27.5 Principles of Resistance 940
27.5.1 Mode of Action, Target-Site Resistance, and Cross-Resistance 940
27.5.2 Non-Target-Site Resistance Mechanisms 940
27.6 The Mode of Action (MoA) Classification Scheme v7.0, August
2010 941
27.6.1 Rules for Inclusion of a Compound in the MoA List 941
27.6.2 Organophosphates and Carbamates 950
27.6.3 Pyrethroids 951
27.7 Effective IRM Strategies and Approved Principles 952
27.8 Future Market Trends 953
References 954


28.1.1.1 Physiological and Molecular Basis of Insect Molting Hormone
Action 957
28.1.2 Discovery and Structures of Commercialized Bisacylhydrazine
Insecticides 960
28.1.3 Synthesis of Commercial Bisacylhydrazines 960
28.1.4 Structure–Activity Relationship of Ecdysteroids and
Bisacylhydrazines 962
Contents XI
28.1.4.1 Structure–Activity Relationship (SAR) of the

Ecdysteroids 964
28.1.4.2 Structure–Activity Relationship (SAR) of the
Bisacylhydrazines 964
28.1.5 Mode of Action of BAH Insecticides 966
28.1.5.1 Whole-Organism Effects 972
28.1.5.2 Basis for Selective Insect Toxicity of Bisacylhydrazine
Insecticides 973
28.1.6 Spectrum of Activity of Commercial Bisacylhydrazine
Insecticides 974
28.1.6.1 Tebufenozide (MIMIC™ ; CONFIRM™ ; ROMDAN™ ; RH-5992),
Methoxyfenozide (RUNNER™ ; INTREPID™ ; PRODIGY™ ; FALCON™ ;
® ®
RH-2485), and Chromafenozide (MATRIC ; KILLAT ; ANS-118;
CM-001) 975
28.1.6.2 Halofenozide (MACH 2™ ; RH-0345) 975
28.1.7 Ecotoxicological and Mammalian Reduced-Risk Profiles 975
28.1.8 Resistance Mechanisms and Resistance Potential 977
28.1.9 Fufenozide: A New Bisacylhydrazine in Development 978
28.1.10 Other Chemistries and Potential for New Ecdysone Agonist
Insecticides 979
28.1.11 Conclusions and Future Prospects of Ecdysone Agonist
Chemistries 980
Acknowledgments 980
References 980

Makoto Hatakoshi

28.2.2 History of Juvenoid Research 984
28.2.3 Process of Pyriproxyfen Research 985
28.2.4 Activity of Optical Isomers 987
28.2.5 Mechanism of Action 987
28.2.6 Biological Activity 990
28.2.6.1 Laboratory Evaluations 990
28.2.6.2 Field Evaluations 991
28.2.6.3 Resistance 995
28.2.7 Synthesis 995
28.2.8 Physico-Chemical Properties and Formulation 995
28.2.8.1 Physico-Chemical Properties 995
28.2.8.2 Stability 996
28.2.8.3 Formulation 996
28.2.9 Toxicology 996
References 997
XII Contents

Joel J. Sheets

29.1.2 Chitin Structure and Biosynthesis 1000
29.1.2.1 Chitin Synthase 1000
29.1.3 Inhibitors of Chitin Synthesis 1002
29.1.3.1 Benzoylphenyl Ureas 1003
29.1.3.2 Other Chitin Synthesis Inhibitors 1004
29.1.4 The Future of Chitin Synthesis Inhibitors for Crop Protection 1005
Acknowledgments 1006
References 1007


29.2.2 Tetrazines (Clofentezine, Diflovidazin = Flutenzine) 1013
29.2.2.1 Biology and Biochemistry 1014
29.2.3 Thiazolidinones (Hexythiazox) 1018
29.2.4 Oxazolines (Etoxazole) 1020
References 1026
30 Midgut–Transgenic Crops Expressing Bacillus thuringiensis Cry
Proteins 1029

30.2 Plant Engineering 1029
30.3 Insecticidal Crystal Proteins from B. thuringiensis 1030
30.4 Bt Plants 1032
30.5 Insect Resistance to Bt 1039
30.6 Resistance Management with Bt Plants 1040
30.7 Safety of Bt Plants 1043
References 1050

Josef Ehrenfreund

Contents XIII
31.1.2 Mitochondrial ATP Synthase as a Target for Insecticides and

Acaricides 1059
31.1.3 Diafenthiuron: Mode of Action 1061
31.1.4 Diafenthiuron: Discovery, Structure–Activity Relationships, and
Production Process Chemistry 1063
31.1.5 Diafenthiuron: Mammalian Toxicology and Ecotoxicology 1066
31.1.6 Diafenthiuron: Biological Activity and Significance for Crop
Protection 1066
References 1068

Gradient 1070

31.2.2 Biochemical Mode of Action 1072
31.2.4 Pest Species and Markets 1073
31.2.5 Chlorfenapyr Resistance 1076
References 1077

Insecticides 1078

31.3.2 Complex I Inhibitors 1078
31.3.2.1 Fenpyroximate 1079
31.3.2.2 Pyridaben 1085
31.3.2.3 Fenazaquin 1086
31.3.2.4 Tebufenpyrad 1086
31.3.2.5 Tolfenpyrad 1087
31.3.2.6 Pyrimidifen 1087
31.3.2.7 Flufenerim 1090
31.3.2.8 Experimental MET Complex I Compounds 1092
31.3.3 Complex II Inhibitors 1092
31.3.3.1 Cyenopyrafen 1092
31.3.3.2 Cyflumetofen 1093
31.3.3.3 Experimental and Other MET Complex II Compounds 1093
31.3.4 Complex III Inhibitors 1095
31.3.4.1 Acequinocyl 1095
31.3.4.2 Fluacrypyrim and β-Methoxyacrylate/Strobilurin-Based
Compounds 1096
31.3.4.3 Hydramethylnon 1097
XIV Contents
31.3.4.4 Bifenazate 1097

31.3.5 Metabolism 1097
31.3.5.1 MET-I Inhibitors 1097
31.3.5.2 MET-II Inhibitors 1100
31.3.5.3 MET-III Inhibitors 1100
31.3.6 Resistance and Resistance Mechanisms 1100
31.3.7 The Future for MET Acaricides and Insecticides 1102
References 1103
31.4 Inhibitors of Lipid Synthesis: Acetyl-CoA-Carboxylase

Inhibitors 1108

31.4.2 The Cyclic Ketoenols, Spirodiclofen, and Spiromesifen: A New
Generation of ACCase Inhibitors 1108
31.4.3 Synthesis of Spirodiclofen and Spiromesifen 1111
31.4.4 Biology and Mode of Action 1114
31.4.5 Development, Registration, and Integrated Pest Management
Suitability of Envidor™ and Oberon™ 1116
31.4.6 Discovery of Spirotetramat 1118
31.4.7 Synthesis of Spirotetramat 1120
31.4.8 Biology and Mode of Action of Spirotetramat 1122
31.4.9 Development and IPM Suitability of Movento™ 1123
References 1125

Aspects 1127

32.1.2 Structure of the Nicotinic Acetylcholine Receptor 1132
32.1.2.1 Agonist-Binding Sites 1135
32.1.3 Insect nAChRs 1137
32.1.3.1 Consideration of AChBP versus nAChR α-Subunit 1138
32.1.3.2 Interaction of Loop D of the α7 nAChR with Neonicotinoids 1139
32.1.4 Nicotinic Pharmacophore Models 1140
32.1.5 Mode of Action in Insects 1142
32.1.5.1 Nereistoxin and Analogs 1142
32.1.5.2 Neonicotinoids 1143
32.1.5.3 Spinosyns and Semi-Synthetic Analogs (Spinosoids) 1144
Contents XV
32.1.6 Selectivity for Insect versus Vertebrate nAChRs 1145

32.1.6.1 Neonicotinoids 1145
32.1.6.2 Spinosyns 1147
32.1.7 Insect Selectivity in Recombinant nAChRs 1147
32.1.8 Whole-Cell Voltage–Clamp of Native Neuron Preparations 1149
32.1.8.1 Correlation between Electrophysiology and Radioligand-Binding
Studies 1151
32.1.8.2 nAChR Agonists versus Antagonists 1153
32.1.9 Molecular Features of a nAChR Agonists 1154
References 1157

Peter Jeschke

32.2.2 The Term Neonicotinoid 1166
References 1168

Peter Jeschke

32.2.1.2 Nitenpyram 1169
32.2.1.3 Acetamiprid 1171
32.2.1.4 Clothianidin 1175
32.2.1.5 Dinotefuran 1181
32.2.1.6 Open-Chain Compounds versus Ring Systems 1184
References 1186


32.2.2.2 Imidacloprid 1189
32.2.2.3 Thiacloprid 1196
References 1200

Peter Maienfisch

32.2.3.2 History of Six-Membered Neonicotinoids 1203
32.2.3.3 Biological Activity and Structure–Activity Relationship 1207
32.2.3.4 Synthesis 1208
XVI Contents
32.2.3.5 Hydrolytic Stability of the Six-Membered Nitroimino-

Heterocycle 1210
32.2.3.6 AKD-1022 1210
32.2.3.7 Thiamethoxam (CGA 293’343) 1211
References 1222

and James Thomas

32.3.2 Chemistry, Structure–Activity Relationships, and Toxicology 1226
32.3.2.1 Synthesis 1226
32.3.2.3 Physico-Chemical Properties of Sulfoxaflor 1228
32.3.3 Mammalian and Environmental Toxicology 1230
32.3.3.1 Mammalian Toxicology 1230
32.3.3.2 Environmental Toxicology: Bees 1230
32.3.3.3 Environmental Toxicology: Other Nontarget Organisms 1231
32.3.5 Biology and Field Use Patterns 1233
32.3.5.1 Biology 1233
32.3.5.2 Field Use Patterns 1233
32.3.6 Efficacy on Resistant Pests 1233
References 1236

and Clive Waldron

32.4.2 Biological Activity and Primary Uses of Spinosad 1239
32.4.3 Mode of Action of Spinosyns 1240
32.4.4 Spinosyn Analogs 1241
32.4.4.1 Core-Modified Analogs (Aglycone) 1241
32.4.4.2 Modifications Involving the C17 Sugar 1244
32.4.4.3 Modifications Involving the C9 Sugar: Rhamnose Derivatives 1245
32.4.5 Spinetoram 1247
32.4.6 Biosynthesis and Genetics of the Spinosyns 1249
32.4.7 Metabolism and Penetration of the Spinosyns 1252
32.4.8 Summary 1253
References 1254
Contents XVII

George P. Lahm
32.5.1.1 History and Discovery of the Sodium Channel Blockers 1257

32.5.1.2 Discovery of Indoxacarb 1260
32.5.1.3 Insecticidal Activity and Properties of Indoxacarb 1263
32.5.1.4 Indoxacarb: Mode of Action 1265
References 1271


32.5.2.2 Discovery 1273
32.5.2.3 Insecticidal Activity 1280
32.5.2.5 Cross-Resistance Potential 1281
References 1282

32.6.1 Discovery and Development of Fipronil and Other Fiproles 1283

32.6.2.1 Discovery of the GABA Receptor as an Insecticide Target
Site 1285
32.6.2.2 Cloning the Insect GABA Receptor and Resistance Mutations 1286
32.6.2.3 Ligand-Gated Chloride Channel Structure and Classification 1288
32.6.2.4 Mechanism of Blockade 1290
32.6.2.5 Structure of the Binding Site 1292
32.6.2.6 Mechanism of Insect Selectivity of Fipronil 1293
32.6.3.1 Chemistry and Synthesis of Fiproles and Intermediates 1293
32.6.4 Biological Properties 1296
32.6.4.1 General 1296
32.6.4.2 Biological Spectrum 1297
32.6.4.3 Soil Applications 1297
32.6.4.4 Seed Treatment 1298
32.6.4.5 Use in Crop Baiting Systems 1298
32.6.4.6 Urban Pest Control Applications 1299
XVIII Contents
32.6.4.7 Turf and Ornamental Applications 1299

32.6.4.8 Animal and Human Health Uses 1300
32.6.4.9 Resistance and Its Management 1300
References 1301

Milbemycins 1305
Thomas Pitterna

32.7.3 Discovery and Chemistry of Avermectins 1308
32.7.4 Discovery and Chemistry of Milbemycins 1312
32.7.5 Acaricidal and Insecticidal Activity 1315
32.7.6 Safety and Bioavailability 1319
32.7.7 Use in Agriculture 1322
References 1324

Peter Maienfisch

33.1.2 Pymetrozine 1327
33.1.2.1 Discovery 1327
33.1.2.2 Pyridine Azomethines: Structure–Activity Relationships 1328
33.1.2.3 Synthesis of Pymetrozine 1328
33.1.2.4 Chemical and Physical Properties of Pymetrozine 1328
33.1.2.5 Mode of Action and Resistance 1331
33.1.2.6 Biological Activity and Use Recommendations 1333
33.1.2.7 Safety Profile 1333
33.1.3 Flonicamid 1333
33.1.3.1 Discovery of Flonicamid and the Trifluoromethylnicotinamide
Insecticides 1335
33.1.3.2 Trifluoromethylnicotinamides: Structure–Activity
Relationships 1335
33.1.3.3 Synthesis of Flonicamid 1335
33.1.3.4 Chemical and Physical Properties of Flonicamid 1335
33.1.3.5 Mode of Action of Flonicamid 1335
33.1.3.6 Biological Activity and Use Recommendations 1338
33.1.3.7 Safety Profile of Flonicamid 1339
33.1.4 Pyrifluquinazon 1340
33.1.4.1 Discovery of Pyrifluquinazon 1340
Contents XIX
33.1.4.2 Structure–Activity Relationship 1340

33.1.4.3 Synthesis of Pyrifluquinazon 1340
33.1.4.4 Chemical and Physical Properties of Pyrifluquinazon 1342
33.1.4.6 Biological Activity and Safety Profile of Pyrifluquinazon 1343
References 1344

Mark A. Dekeyser

33.2.2 Bifenazate 1347
33.2.2.1 Discovery and Structure–Activity Relationship 1347
33.2.2.3 Ecobiology 1350
33.2.2.4 Registration Status 1351
33.2.2.5 Resistance Behavior 1352
33.2.3 Cyflumetofen 1352
33.2.3.1 Discovery 1353
33.2.3.3 Ecobiology 1354
33.2.3.4 Registration Status 1354
33.2.3.5 Resistance Behavior 1354
33.2.4 Amidoflumet 1354
33.2.4.1 Discovery and Development 1355
References 1357


33.3.2.1 Lead Generation 1360
33.3.2.2 Optimization of the Lead Compound to Pyridalyl 1360
33.3.2.3 Physico-Chemical Properties 1360
33.3.3 Biological Aspects 1362
33.3.3.1 Insecticidal Activity and Uses 1362
33.3.4 Commercial Status 1366
References 1366
XX Contents


33.4.2 Nematicide Chemical Classes and Products 1368
33.4.2.1 Fumigants 1369
33.4.2.2 Carbamates and Organophosphates 1370
33.4.2.3 Abamectin 1374
33.4.3 Experimental Nematicides 1376
33.4.3.1 Spirotetramat 1376
33.4.3.2 Fluensulfone 1377
33.4.3.3 Benclothiaz 1383
References 1384


34.1.2 Insecticides Affecting Calcium Homeostasis 1389
34.1.4 Selectivity and Binding Site 1393
References 1395


34.2.2 History of the Invention 1397
34.2.3.1 Challenge of Chemistry 1399
34.2.3.2 Structure–Activity Relationship 1399
34.2.3.3 X-Ray Structural Analysis 1401
34.2.4 Biological Profiles 1401
34.2.4.1 Activity against Lepidopterous Pests 1401
34.2.4.2 Fast-Acting Activity and Persistence 1402
34.2.4.3 Cross-Resistance 1406
34.2.5 Toxicity towards Beneficial Arthropods 1406
34.2.6 Toxicological Properties 1407
References 1408
Contents XXI


34.3.2 Discovery of Anthranilic Diamides 1410
®
34.3.3 Discovery of Chlorantraniliprole (Rynaxypyr ) 1411
™
34.3.4 Discovery of Cyantraniliprole (Cyazypyr ) 1413
34.3.5 Chemical Synthesis 1413
®
34.3.7 Biological Profile of Chlorantraniliprole (Rynaxypyr ) 1418
34.3.7.1 Insecticidal Potency and Attributes 1418
34.3.7.2 Mammalian and Environmental Safety 1420
34.3.7.3 Insecticide Resistance Management 1421
34.3.8 Biological Profile of Cyantraniliprole (Cyazypyr™) 1421
References 1422
Subject Index 1449

1
Part I
Herbicides
Modern Crop Protection Compounds, Second Edition. Edited by Wolfgang Krämer,

Ulrich Schirmer, Peter Jeschke, and Matthias Witschel.
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA.
3
Overview
Matthias Witschel
This section on Herbicides reflects not only changes in herbicide markets worldwide,
but also changes in the importance of different herbicide classes and modes of
action (MoA) for the market, as well as for research and development, that have
occurred since the publication of the First Edition of this book.
Following the broad introduction of herbicide-tolerant crops during the late
1990s – in particular of glyphosate-tolerant soybean and corn – the value of the
selective herbicide market fell for several years. Since then, an increasing num-
ber of herbicide-resistant weed species have been observed – especially against
glyphosate, but also against other herbicides – including many inhibitors of ACCase
(acetyl-CoA carboxylase) and AHAS/ALS (acetohydroxyacid synthase/acetolactate
synthase). This has resulted in an increasing demand for new active ingredients
and new mixture concepts for resistance management. Following the downsizing
in herbicide research capacities within the industry, and a resultant lack of new
candidates in the development pipelines, the recent positive outlook for the herbi-
cide market has resulted in a renewed focus on herbicide research, which is often
referred to as the ‘‘herbicide renaissance’’.
In spite of the high demand for innovations in the field, only limited progress has
been made in the identification and commercialization of new MoA in the herbicide
area, with HPPD (p-hydroxyphenylpyruvate dioxygenase) being the newest MoA,
introduced into the market during the 1980s. Hence, the focus of this Second
Edition will also be on innovations in the older MoA areas.
An analysis of the names provisionally approved by ISO of new chemical entities
developed since 2000 shows that the herbicide area has clearly been dominated
by inhibitors of AHAS/ALS (10 entries), followed by inhibitors of HPPD (five),
ACCase (three), VLCFA (very long-chain fatty acid synthesis; three) and PPO
(protoporphyrinogen oxidase; three). The targets of the remaining new entities
were auxin receptors, cellulose biosynthesis and PDS (phytoene desaturase), with
one entry each. This distribution reflects a trend towards systemic, post-emergence
herbicides that allow economic, weed-specific applications later in the season, when
it is clearer which species need to be controlled.

4 Overview
At the same time, the increasing weed resistance to post-emergence herbicides,

the trend towards greater numbers of weed flushes, and the growers’ demands
for more flexible application timing and less complex weed control solutions has
also led, in recent years, to a refocus on pre-emergence, as well as pre-planting,
herbicides.
This volume starts with the most recent developments in herbicide resistance
(Chapter 1), which is of key strategic importance for herbicide research. The
subsequent Chapters 2.1 to 2.6 discuss the continuing development of highly
potent inhibitors of AHAS, which still dominate the new introductions in spite
of increasing resistance issues, and of their use in herbicide-tolerant crops. PPO
(Chapter 3) and ‘‘bleaching’’ herbicides targeting PDS (Chapter 4.2) and HPPD
(Chapters 4.3 and 4.4) also show some interesting new developments. In the
field of auxin mimics (Chapters 5.1–5.3), better understandings of the target
processes are described, as well as new structure types. In the area of VLCFA
inhibitors (Chapters 6.1–6.3), the identification of isoxazolines as a new structure
type within the MoA has initiated a renewed interest, as have triazines for cellulose
biosynthesis inhibitors (Chapter 7) and ketoenols for ACCase inhibitors (Chapter
11). Even though no new chemistry has been introduced for the inhibitors of
EPSPS (5-enolypyruvyl shikimate-3-phosphate synthase; Chapters 9.1 and 9.2),
glutamate synthase (Chapter 9.3), microtubule assembly inhibitors (Chapter 10) or
photosynthesis inhibitors (Chapter 12), there are many new developments for the
first two areas, due to their importance in herbicide tolerance, and new regulatory
issues are of high relevance for the last two areas. New structures have been
identified for safeners (Chapter 8), and some new aspects in the area of plant
growth regulators (Chapter 13) will also be described.
The developments in herbicide chemistry that have been made since the publica-
tion of the First Edition of this book – and, in particular, the lack of fundamentally
new MoAs – can best be understood in the context of a sudden, industry-wide
de-emphasis of herbicide discovery research, resulting from the rapid penetration
of several important markets by herbicide-tolerant crops. Now that the limitations
of this strategy are becoming apparent, a renewed emphasis can be anticipated on
the identification of new MoAs, alongside continued evolution in known MoAs.
5
1
HRAC Classification of Herbicides and
Resistance Development
1.1
Introduction
The use of herbicides has caused significant changes in the production systems of all
major crops globally. The highly effective chemical control of weeds has replaced
manual, animal, and mechanical weed control, has increased the productivity,
and has also enabled the development of larger farm sizes and an improved
subsistence for farmers. However, herbicides have not resulted in the extinction of
weeds; rather, they cause – together with other influencing factors – a continuous
selection of plants to occur which are able both to survive and to reproduce. As
a consequence, these plants with such survival properties are able to become
dominant and to become distributed over increasingly large areas.
The first cases of herbicide resistance were reported around 1970, since then
the resistance of both mono- and dicotyledonous weeds to herbicides has become
an increasing problem worldwide. At the end of 2010, the International Survey
of Herbicide-Resistant Weeds recorded 348 herbicide-resistant biotypes with 194
weed species – 114 dicotyledonous and 80 monocotyledonous (I. Heap, 2010, per-
sonal communication, http://www.weedscience.com). The relatively steady increase
in the number of new cases of resistance since 1980 accounts for the increasing
importance of herbicide resistance in weeds in the major agricultural regions
(Figure 1.1).
During the period between 1970 and 1990, most documented cases of resistance
concerned the triazines. The introduction of new herbicides with different sites of
action (SoA) resulted in a shift, so that more recently both acetolactate synthase
(ALS) and acetyl-CoA carboxylase (ACCase)-resistant weeds have been reported
(Figure 1.2). Additionally, the rapid adoption of glyphosate-resistant crops in North
and South America, and the use of glyphosate as a pre-sowing treatment in differ-
ent cropping systems, have resulted in increasing cases of glyphosate resistance
(I. Heap, 2010, personal communication, http://www.weedscience.com). The proba-
bility of resistance developing towards glyphosate had been expressed as being likely
but underestimated, though less frequently in comparison with most other SoA
classes [1].

6 1 HRAC Classification of Herbicides and Resistance Development
350
300
Number of resistant biotypes
250
200
150
100
50
0
1950 1960 1970 1980 1990 2000 2010
Year
Figure 1.1 The recent chronological increase in the

number of herbicide-resistant weeds worldwide. I. Heap
(2010), personal communication; http://www.weedscience.
com.
120
ACCase Inhibitors
ALS Inhibitors
100 Dinitroanilines
Number of resistant biotypes
Triazines
Ureas, Amides
80 Bypyridiliums
Synthetic Auxins
Glycines
60
40
20
0
1950 1955 1960 1965 1970 1975 1980 1985 1990 1995 2000 2005 2010
Year
Figure 1.2 The recent chronological increases in the num-

bers of herbicide-resistant weeds for the different herbi-
cide classes. I. Heap (2010), personal communication;
http://www.weedscience.com.
1.2
HRAC Classification System of Herbicides
The Herbicide Resistance Action Committee (HRAC) is an international body

founded by the agrochemical industry as part of the Global Crop Protection
Federation (GCPF) organization. The aims of the HRAC are to support cooperative
approaches to the management of herbicide resistance by fostering understanding,
cooperation, and communication between industry, government, and farmers.
The global HRAC group proposed a classification system for herbicides according
to their target sites, their SoA, the similarity of induced symptoms, or their chemical
classes (Table 1.1). This system proved to be the most comprehensive classification
system of herbicides globally, although with the Weed Science Society of America
(WSSA) Code System and Australian Code System two similar classification
systems had been developed at an earlier stage for regional needs. The use of
different numbers and letters in the different classification systems very often led
to confusion and misunderstanding on the global level. Therefore, it was considered
that one common global system would be highly desirable for all users, and would
also provide a better understanding of the differences between molecular classes.
In particular, all single systems should support and advise all users of herbicides;
moreover, such advice should be descriptive and also state how the individual active
compounds must be applied in order to achieve the best results in terms of weed
control.
The classification system describes not only the chemical family belonging
to a specific SoA but also all compounds (via their common names) belonging
to each family. This is shown in Table 1.2 for the SoA such as ‘‘Inhibition of
dihydropteroate (DHP) synthase,’’ ‘‘Microtubule assembly inhibition,’’ ‘‘Inhibition
of mitosis/microtubule organization,’’ ‘‘Inhibition of very long-chain fatty acid
synthases (VLCFAs; Inhibition of cell division),’’ and ‘‘Inhibition of cell wall
(cellulose) synthesis,’’ as examples. (Note: these are not mentioned in other
chapters of this book; for a more detailed table, see www.hracglobal.com). The
scheme ‘‘The World of Herbicides,’’ which is available under this internet address,
also lists all the chemical structures of the various herbicides belonging to the
different chemical families.
1.3
Herbicide Resistance
Among the weed population, herbicide resistance is a natural phenomenon that

occurs at a low frequency and which has evolved over millions of years. Herbicide
applications select only for these weeds in a population, but do not cause resistance
nor alter the plant genetics. Increasing problems with herbicide-resistant weed
populations have occurred predominantly in countries with intensive agriculture
cropping systems. The reliance on a few available weed management tools, coupled
with a disregard of the principles of Integrated Weed Management (IWM), are
closely related to changes in the weed population community. Changes in the
8
Table 1.1 HRAC classification system in comparison to Weed Science Society of America (WSSA) and Australian code system. Adapted from Refs [2–4].
Site of action Chemical family HRAC WSSA Australian

group groupa groupa
Inhibition of acetyl CoA carboxylase (ACCase) Aryloxyphenoxy-propionate, cyclohexanedione, A 1 A

phenylpyrazoline
Inhibition of acetolactate synthase (ALS) Sulfonylurea, imidazolinone, triazolopyrimidine, B 2 B
(acetohydroxyacid synthase; AHAS) pyrimidinyl(thio)benzoate,
sulfonylaminocarbonyl-triazolinone
Inhibition of photosynthesis at PS II Triazine, triazinone, triazolinone, uracil, pyridazinone, C1 5 C
phenyl-carbamate
Inhibition of photosynthesis at PS II Urea, amide C2 7 C
Inhibition of photosynthesis at PS II Nitrile, benzothiadiazinone, phenyl-pyridazine C3 6 C
Photosystem I-electron diversion Bipyridylium D 22 L
Inhibition of protoporphyrinogen oxidase (PPO) Diphenylether, phenylpyrazole, N-phenylphthalimide, E 14 G
thiadiazole, oxadiazole, triazolinone, oxazolidinedione,
pyrimidindione, otherd
Inhibition of the phytoene desaturase (PDS) Pyridazinone, pyridinecarboxamide, otherd F1 12 F
1 HRAC Classification of Herbicides and Resistance Development
Inhibition of 4-hydroxyphenyl-pyruvate-dioxygenase Triketone, isoxazole, pyrazole, otherd F2 27 H

(4-HPPD)
Inhibition of carotenoid biosynthesis (unknown target) Triazole, diphenylether, urea (also C2) F3 11 Q
Inhibition of 1-deoxy-d-xylose 5-phosphate synthase Isoxazolidinone F4 13 Q
(DOXP synthase)
Inhibition of EPSP synthase Glycine G 9 M
Inhibition of glutamine synthetase Phosphinic acid H 10 N
Inhibition of dihydropteroate (DHP) synthase Carbamate I 18 R
Inhibition of microtubule assembly Dinitroaniline, phosphoroamidate, pyridine, benzamide, K1 3 D
benzoic acid
Inhibition of mitosis/microtubule organization Carbamate K2 23 E
Arylaminopropionic acid K2 25 Z
Inhibition of VLCFAs (inhibition of cell division) Chloroacetamide, acetamide, oxyacetamide, tetrazolinone, K3 15 K
isoxazolineb, otherd
Inhibition of cell wall (cellulose) synthesis Nitrile L 20 O
Benzamide L 21 O
Triazolocarboxamide L 28
Alkylazine L 29c
Uncoupling (membrane disruption) Dinitrophenol M 24
Inhibition of lipid synthesis – not ACCase Thiocarbamate, phosphorodithioate N 8 J
inhibition Benzofuran N 16 J
Chloro-carbonic-acid N J
Action like indole acetic acid (synthetic auxins) Phenoxy-carboxylic-acid, benzoic acid, pyridine carboxylic O 4 I
acid, quinoline carboxylic acid, and otherd
Inhibition of auxin transport Phthalamate, semicarbazone P 19 P
UnknownNote: While the mode of action of Pyrazolium Z 26
herbicides in Group Z is unknown, it is likely that
they differ in mode of action between themselves
and from other groups.
Organoarsenical Z 17 Z
Otherd Z 26
a
Not all chemical classes are classified.
b
Proposed.
c
Proposed by WSSA.
d
Includes additional molecules which belong to the same SoA (e.g., PPO) but have no special chemical family such as ‘‘thiadiazole or oxazolidinedione.’’
VLCFA, very long-chain fatty acid synthase.
1.3 Herbicide Resistance
9
Table 1.2 Selected groups of the HRAC classification system

with examples of the active ingredients, which are not men-
tioned in following chapters. Adapted from Refs [2–4].
Site of action Chemical family Active HRAC WSSA Australian

ingredient group groupa groupa
Inhibition of Carbamate Asulam I 18 R

dihydropteroate
(DHP) synthase
Microtubule Dinitroaniline Benefin=benfluralin K1 3 D
assembly Butralin
inhibition Dinitramine
Ethalfluralin
Oryzalin
Pendimethalin
Trifluralin
Phosphoroamidate Amiprophosmethyl
Butamiphos
Pyridine Dithiopyr
Thiazopyr
Benzamide Propyzamide=
pronamide tebutam
Benzoic acid DCPA=chlorthal-
dimethyl
Inhibition of Carbamate chlorpropham K2 23 E
mitosis / propham
microtubule carbetamide
organisation
Arylaminopropionic acid Flamprop-m K2 25 Z
Inhibition of Chloroacetamide Acetochlor K3 15 K
VLCFAs Alachlor
(inhibition of Butachlor
cell division) Dimethachlor
Dimethanamid
Metazachlor
Metolachlor
Pethoxamid
Pretilachlor
Propachlor
Propisochlor
Thenylchlor
Acetamide Diphenamid
Napropamide
Naproanilide
Oxyacetamide Flufenacet
Mefenacet
Tetrazolinone Fentrazamide
Ipfencarbazone
Table 1.2 (continued)
Site of action Chemical family Active HRAC WSSA Australian

ingredient group groupa groupa
Isoxazolineb Pyroxasulfone
Other Anilofos
Cafenstrole
Piperophos
Inhibition of cell Nitrile Dichlobenil L 20 O
wall (cellulose) Chlorthiamid
synthesis
Benzamide Isoxaben 21
Triazolocarboxamide Flupoxam 28
Alkylazine Indaziflam 29c
Triaziflam
a
Not all chemical classes are classified.
b
Proposed.
c
Proposed by Weed Science Society of America (WSSA).
VLCFA, very long-chain fatty acid synthase.
farming environment – and, specifically, the economic pressure on farming – are

key factors that force farmers to change their practices to those that encourage
resistance development.
The limitation in cropping systems, a lack of rotation of herbicide chemistry,
or SoA, a limitation in weed control techniques, and the reduction of dose rates
represent some of the major drivers for the selection of herbicide resistances.
Regular country-based surveys often make it clear that farmers are aware of the
problems and their causes. In fact, a survey conducted in Germany in 2004 showed
that 94% of farmers were aware that the repeated use of the same herbicide, and
89% that a reduction in dose rates, would cause the development of herbicide
resistance. However, 86% of the farmers were forced to reduce their costs and thus
did not have available a wide range of weed management techniques (BCS, 2004,
Internal communication).
As mentioned above, the planting of herbicide-resistant crops worldwide, which
increased from 1.7 mio ha−1 in 1996 to about 134 mio ha−1 in 2009, has
changed the farmers’ weed control tactics completely [5]. These systems have
provided farmers with favorable economic advantages, simplicity of a better weed
management system, as well as more cropping flexibility. In Canada, the adoption of
herbicide-resistant cropping systems reached 93% of the canola production in 2009.
Moreover, with a total of 21.4 mio ha (an increase of 5.6 mio ha, or 35%), Brazil has
in recent years become the second largest grower of biotech crops worldwide [5, 6].
The reliance on one herbicide has reduced the number of applications, and also
the number of SoA used. In 2006, glyphosate was applied to 96% (87% in 2004,
62% in 2000, 25% in 1996) of the entire herbicide applied acreage of soybeans
(98% of the total area) in the US. No other herbicide was applied to more than 7%
(2004) of the applied acreage (four herbicides with >10% in 2000) [7].
The continued use of herbicide-resistant cropping systems, with an over-reliance
on single weed management techniques, selects for weeds that have already evolved
resistance to the applied herbicide. Additionally, among the plant population
specific weed species that previously were less frequent but naturally resistant
can become dominant and, therefore, more difficult to control. It was suspected
that a weed population shift would have a greater impact on the cropping system
than would the selection of resistant weeds [8, 9]. The results of recent studies
have suggested that resistance in weeds and shifts in weed populations occur more
quickly than might have been expected [10]. Indeed, statistical observations have
shown that the use, dose rates, and application frequency have already changed.
For example, in the USA in 1996, glyphosate was applied on average to soybeans
1.2 times at 841 g a.i. ha−1 ; this was subsequently increased to 1.4 applications (at
1065 g a.i. ha−1 ) in 2000, and to 1.7 applications (at 1569 g a.i. ha−1 ) in 2006 [7].
Similar trends can be observed for corn and cotton and also for soybeans in other
countries such as Argentina and Brazil.
In the past, intensive soil cultivation techniques and stubble burning were
commonly used control techniques in agricultural areas. However, the increasing
limitation or banning of stubble burning caused an increasing weed coverage, an
increasing soil seed bank, and the development of herbicide resistance in many
agricultural regions. Several different investigations have highlighted the fact that
the burning of straw drastically decreased weed densities, an example being the
number of water plants (Echinochloa spp.) in comparison to the incorporation of
rice straw into the soil [11]. Australian farmers, in particular, have sought alternative
weed control techniques during their harvest operations, because of the limited
choice of chemical solutions available during the growing season. The method used
to bale of straw, such as a trailing baler attached to the harvester, or the physical
destruction of weed seeds during the harvesting operation (‘‘Rotomill’’), represent
additional possibilities [12].
During recent years, the economic pressure placed on farmers to produce at
the lowest costs, coupled with changing environmental influences such as soil
erosion or water availability, have led to the adoption of no-till practices. Typically,
soybean farmers in the USA increased their planted areas with no-till from
45.3% in 2006 to almost 50% in 2009 [13], and the use of no-till is expected to
further increase globally. In most cases, the shift to a no-till system causes an
over-reliance on herbicides, and the price erosion of herbicides during the past
few years has played a significant role in the adoption of no-till practices. Recent
surveys have shown that farmers are aware that no-till practices increase herbicide
costs and also herbicide resistance – in particular, to glyphosate. Nevertheless, the
acreage for no-till is expected to continue increasing, especially in areas where the
level of no-till approach is currently low [14]. However, growers with increasing
herbicide-resistance problems are planning to reduce the use of no-till where no
other herbicide option is no longer available.
The results of simulation studies have shown that the risk of adopting no-till,
as well as the development of herbicide resistances, can be reduced by alternating
between minimum and no-tillage systems, or by alternating between nonselective
herbicides for pre-sowing weed control [15]. The most efficient weed control strategy
for conserving susceptibility in no-tillage systems was the ‘‘double knockdown’’
pre-sowing application scheme of glyphosate and paraquat in sequence.
One of the most effective tools in the management of herbicide resistance and
weed density is to include a diverse crop rotation practice. Weed species are typically
associated with crops, and crop rotations determine their specific weed population
over time [16]. A high diversity provides the farmer with more opportunities and
more flexibility with respect to the growing conditions, tillage practices and planting
time, the selection of crop cultivars, the rotation of herbicides with different SoA,
the variation of application timings of herbicides across the years to a specific weed
emergence period, and/or the inclusion of nonchemical management techniques
[17]. Consequently, these practices provide farmers with opportunities either to
prevent or to slow down the selection and development of herbicide resistance.
Selected resistance can persist among field populations for many years, with the
populations remaining stable until resistant weed seeds disappear from the soil
seed banks, which occurs very seldom. Investigations with triazine-resistant weed
populations have shown that resistant weed seeds remained in the soil, despite
changes in crop rotation and an absence of triazine herbicides (J. Gasquez, 2003,
personal communication). Similar results were obtained from studies in which
the effect of management practices on ACCase-resistant Alopecurus myosuroides
was evaluated in the field [18]. In this case, the percentage of resistant plants
remained unchanged during a three-year period, even without the application of
ACCase inhibitors. The density of blackgrass plants decreased, especially when
spring crops were part of the crop rotation.
Neither cropping systems nor the single weed management tactic can be applied
to solve specific weed problems on a long-term basis. The use of all possible
practices to prevent and to manage herbicide resistances in an integrated fashion
should be the long-term goal for agricultural production.
The continuous application of herbicides leads to the selection of rare genotypes
of weeds that are resistant to the herbicide and, eventually, at the same time are
already cross-resistant to other herbicides which have not been used previously (as
noted above). These genotypes may already exist in a weed population in very low
frequency, before the introduction the selecting herbicide.
More comprehensive overviews of herbicide resistance in general, including
additional examples that are not described in this book, are available elsewhere (see
Refs [19–22]).
1.3.1
Biochemistry of Herbicide Resistance
Resistance to herbicides can be based on one of the following biochemical mecha-

nisms [20, 22]:
• Target-site resistance: This is due to a reduced (or even lost) ability of the herbicide
to bind to its target protein. The effect usually relates to an enzyme with a crucial
function in a metabolic pathway, or to a component of an electron-transport
system. Target-site resistance may also be caused by an overexpression of the
target enzyme (via gene amplification or changes in a gene promoter).
• Non-target-site resistance: This is caused by mechanisms that reduce the amount
of herbicidal active compound reaching the target site. One important mechanism
is an enhanced metabolic detoxification of the herbicide in the weed, which leads
to insufficient amounts of the active substance reaching the target site. A reduced
uptake and translocation, or sequestration of the herbicide, may also result in an
insufficient herbicide transport to the target site.
• Cross-resistance: In this case, a single resistance mechanism causes resistance to
several herbicides. The term target-site cross-resistance is used when the herbicides
bind to the same target site, whereas non-target-site cross-resistance is due to a
single non-target-site mechanism (e.g., enhanced metabolic detoxification) that
entails resistance across herbicides with different SoA.
• Multiple resistance: In this situation, two or more resistance mechanisms are
present within individual plants, or within a plant population.
1.3.1.1 Target-Site Resistance

Cases analyzed to date have shown that herbicide resistance is very frequently
based on a target-site mutation. Within the past 40 years, weed species have
developed target-site resistance to most known herbicide chemistries; those of
major importance are discussed in the following subsections.
1.3.1.1.1 Inhibitors of Photosystem II (PS II) Early reports on the resistance of

weeds to photosystem (PS) II inhibitors of the triazine group first appeared around
1970. Since then, triazine resistance has been reported for numerous – mainly
dicotyledonous – weed species.
Investigations into the mechanism of resistance to triazines have revealed that,
in most cases, such resistance is due to a mutation that results in a modification
of the target site; this is known to be the Qb site of the D1 protein in the PS II
reaction center. The triazine herbicides bind to this site, thereby inhibiting the
photosynthetic electron flow. In resistant mutants, the triazine binding is markedly
reduced; for example, the concentration of atrazine required to achieve a 50%
inhibition of photosynthetic electron flow in isolated chloroplasts of Chenopodium
album was at least 430-fold higher for chloroplasts from an atrazine-resistant
mutant than for those from wild-type plants [23].
In many cases, the mutants of weed species with target-site resistance to triazines
showed a lower growth rate and ecological fitness than the susceptible wild-type,
when analyzed in the absence of a triazine herbicide as selection agent. The
quantum yield of CO2 reduction in resistant biotypes was decreased; furthermore,
electron transfer between the primary and secondary quinones in the PS II reaction
center was slowed. The latter effect may have been the cause of an increased
susceptibility to photoinhibition in the resistant biotypes [24, 25].
The D1 protein is encoded by the chloroplast psbA gene, which is highly conserved
among higher plants, algae, and cyanobacteria [26]. In almost all investigated
cases of the resistance of field-grown weed species to triazines, resistance was
attributed to a mutation in the psbA gene with a resultant Ser264 → Gly change
in the herbicide-binding niche of the D1 protein. Consequently, this resistance
is usually maternally inherited. Although herbicides of the phenylurea group are
also inhibitors of the PS II system, a cross-resistance of atrazine-resistant mutants
with a Ser264 → Gly change has not been observed with phenylureas. It has been
proposed that the binding sites of triazines and phenylureas are not identical, but
rather overlap [27–29], with Ser264 providing a hydrogen bond to atrazine or other
herbicides of the triazine group. The substitution of Ser264 by glycine removes
this bond, which is important for binding the triazines. According to the concept
of overlapping binding sites, hydrogen bonding to Ser264 is not important for
phenylureas, due to a different binding geometry; consequently, the binding of
phenylurea will not be affected by the Ser264 → Gly mutation.
In 1999, Masabni and Zandstra described a mutant of Portulaca oleracea with
a resistance pattern to PS II inhibitors, but which was different from most
triazine-resistant weeds [30]. In fact, the mutant was not only resistant to the
phenylureas linuron and diuron, but was also cross-resistant to atrazine and other
triazines. Sequencing of the D1 protein revealed that, in the resistant biotype, Ser264
had been replaced by Thr, and not by Gly, this being the first report of a Ser264 →
Thr mutation on a whole-plant level. It was suggested that the Ser → Thr mutation
had modified the conformation of the herbicide-binding niche at the D1 protein,
in such a way that the binding of phenylureas and triazines was also reduced.
An additional novel mutant was identified when field accessions of P. annua
with a known resistance to PS II inhibitors, collected in Western Oregon, were
analyzed after amplification of the herbicide-binding region (933 bp fragment) of
the chloroplast psbA gene, using the polymerase chain reaction (PCR). A sequence
analysis of the fragment from a mutant which showed resistance to diuron and
metribuzin (resistance factors of between 10 and 20) revealed a substitution from
Val219 to Ile in the D1 protein encoded by the psbA gene. This amino acid
substitution had previously been identified following the mutagenesis of laboratory
cultures of algae and cell cultures of Chenopodium rubrum. The finding of a
Val219 → Ile substitution in P. annua, however, was the first reported case of
a weed species with resistance to PS II inhibitors in the field, that was due to a
psbA mutation other than at position 264. As noted above, the electron-transfer
processes in the PS II reaction center of weeds with a mutation at position 264 were
slowed, and the ecological fitness of the mutants reduced. In contrast, no effect on
electron transfer in the PS II reaction center was found for the P. annua mutant
with the Val219 → Ile change, and it was supposed that this mutant (in ecological
terms) may be as fit as its wild-type counterpart [31]. The same mutation was also
found among Kochia scoparia and Amaranthus powelli populations [32, 33]. Further
mutations from a selection of non-triazine PS II inhibitors have evolved in Senecio
vulgaris from Asn266 to threonine [34], in C. album from Ala251 to Val [35], and
Capsella bursa-pastoris from Phe255 to Ile [36]. All of these populations remained
sensitive to triazine herbicides, including atrazine and simazine.
1.3.1.1.2 Inhibitors of Acetyl-CoA Carboxylase (ACCase) The enzyme ACC

catalyzes the carboxylation of acetyl-CoA, which results in the formation of
malonyl-CoA. In plastids, this reaction is the initial step of de novo fatty acid
biosynthesis and is, therefore, of crucial importance in plant metabolism.
Species of the Poaceae family (grasses) have in their plastids a homomeric,
multifunctional form of ACCase with the following domains: biotin carboxy
carrier protein (BCCP); biotin carboxylase (BC); and carboxyltransferase (CT).
Other monocotyledonous species examined to date, as well as most dicotyledonous
species, have in their plastids a heteromeric, multisubunit type of ACCase with
the BCCP, BC, and CT domains encoded on separate subunits. In addition,
all dicotyledons and monocotyledons (including the Poaceae) have a cytosolic
ACCase, which belongs to the homomeric type. The ACCase-inhibiting herbicides
inhibit only the plastidic homomeric ACCase in grasses (Poaceae), but have no
inhibitory effect on the plastidic heteromeric form of other monocotyledonous
and dicotyledonous species, nor the homomeric ACCase in the cytosol. Therefore,
while these herbicides will have a selectively lethal effect only on grass species,
they are tolerated by other monocotyledonous and by dicotyledonous species. To
date, three different chemical groups of ACCase inhibitor have been identified: (1)
the aryloxyphenoxypropionates (APPs); (2) the cyclohexanediones (CHDs), which
have been developed during the past 15–20 years to provide a very important
herbicide family with a selective action on a broad spectrum of grass weed species;
and (3) the phenylpyrazoline (PPZ) group.
Until now, a target-site resistance of biotypes to ACCase inhibitors has been
confirmed for several grass weed species of economic importance. The earliest
cases of target-site based resistance were reported for biotypes of Lolium multiflorum
from Oregon, USA [37], and for Lolium rigidum from Australia [38].
ACCase prepared from the resistant L. multiflorum biotype, which had been
selected by the field use of diclofop, was inhibited by the APPs diclofop, haloxyfop,
and quizalofop with IC50 -values (the herbicide concentration required for 50%
enzyme inhibition) that were 28-, 9-, and 10-fold higher than for ACCase from a
susceptible biotype. There was no cross-resistance to the CHD herbicides sethoxy-
dim or clethodim [39]. ACCase resistance was subsequently also confirmed for
L. multiflorum biotypes from other countries. In a resistant biotype selected by
diclofop in Normandy, the resistance factor (ratio of the IC50 for ACCase from the
resistant to the IC50 for ACCase from the susceptible biotype) was 19 for diclofop,
and 5 for haloxyfop, but only 2 for the CHDs clethodim and sethoxydim [40].
Interestingly, a different ACCase resistance pattern was found for the resistant
L. multiflorum biotype Yorks A2, although field selection was apparently also mainly
by diclofop. The resistance factors were 3 and 9, respectively, for the APPs diclofop
and fluazifop, but 20 for the CHD herbicide cycloxydim [41].
The first biotypes of L. rigidum with target-site resistance to ACCase inhibitors
were identified during the early 1990s in Australia. Selection either with an APP
or with a CHD herbicide resulted in target-site cross-resistance to both herbicide

groups. However, regardless of whether the selection was by an APP or a CHD
compound, the level of resistance in these biotypes was higher towards APP than
towards CHD herbicides. The ACCase resistance factors were 30 to 85 for diclofop,
>10 to 216 for haloxyfop, and 1 to 8 for sethoxydim [38, 42, 43].
Biotypes with target-site-based resistance to ACCase inhibitors were also selected
in wild oat species (Avena fatua, Avena sterilis). The resistance patterns were found
to be variable; for example, the resistance factors for ACCase from the Canadian A.
fatua biotype UM1 were 105 for sethoxydim, 10 for tralkoxydim, and 10 for diclofop
and fenoxaprop, whereas for the A. fatua biotype UM33 from Canada the ratios
were 10.5 for fenoxaprop, 1.2 for diclofop, 5 for sethoxydim, and 1.7 for tralkoxydim.
It was proposed that this effect was due to different point mutations, each being
associated with a characteristic resistance pattern [44]. However, another reason
might be the frequency of homozygote and heterozygote resistant and susceptible
plants within a tested population.
During studies of resistance conducted with Alopecurus myosuroides populations
from the UK, two biotypes – Oxford A1 and Notts A1 – were identified which
were highly resistant to fenoxaprop, diclofop, fluazifop, and sethoxydim due to
an insensitive ACCase. Genetic studies revealed that the target-site resistance in
the two A. myosuroides biotypes was monogenic and nuclear inherited, with the
resistant allele showing complete dominance [45].
Target-site based resistance to ACCase has also been reported for several other
grass weeds, including two biotypes of Setaria viridis from Manitoba, Canada.
One of these (UM8) confers high levels of ACCase insensitivity to fenoxaprop
and sethoxydim, while the ACCase of biotype UM 131 was highly insensitive to
sethoxydim, but only moderately to fenoxaprop (for a review, see Ref. [43]). Biotypes
of Setaria faberi and Digitaria sanguinalis, obtained in a vegetable cropping system in
Wisconsin, USA, each had an ACCase which was highly insensitive to sethoxydim
but only moderately insensitive to clethodim and fluazifop [46]. Based on the
patterns of target-site-based cross-resistance of weeds to APP and CHD herbicides,
it was postulated that the two classes of ACCase inhibitor do not bind in an identical
manner to the target site (‘‘overlapping binding sites’’), and that different point
mutations at the target enzyme accounted for the variable resistance patterns.
Molecular investigations with chloroplastic ACCase from wheat indicated, first,
that a 400-amino-acid region in the CT domain was involved in insensitivity to both
APP and CHD herbicides [47]. Subsequent follow-up studies with a chloroplastic
ACCase of L. rigidum showed that the resistance to ACCase inhibitors was due
to a point mutation, which resulted in an isoleucine to leucine change in the CT
domain of the enzyme [48]. Previously, Tal and Rubin investigated the molecular
basis of ACCase resistance in a L. rigidum biotype from Israel, with resistance to
CHD and APP herbicides [49]. Following the amplification (by PCR) of a 276 bp
DNA encoding the CT domain of ACCase, the substitution of a single isoleucine
by leucine was also found in this resistant biotype. The results of inheritance
studies conducted by the same authors suggested that the alteration of ACCase in
L. rigidum was governed by a single nuclear codominant gene.
It was shown that a point mutation resulting in an isoleucine to leucine

substitution within the chloroplastic ACCase CT domain is also responsible for
the target-site resistance of A. fatua [50], A. myosuroides [51], and S. viridis [52].
The mutant leucine ACCase allele in the Setaria species was characterized to
be dominant, and no negative effect was detected on ACCase function of the
mutant. It was suggested that the change in ACCase conformation caused by the
isoleucine to leucine mutation was only minor, yet sufficient to prevent (or at least
strongly reduce) the herbicide binding to the enzyme. Brown et al. [51] also pointed
to the very interesting fact that the leucine found in the plastidic homomeric
ACCase of mutated resistant grass weeds is also found in the heteromeric plastidic
enzyme of non-grass species, and also in the cytosolic homomeric enzymes
that are ‘‘naturally’’ resistant to these herbicides. Hence, the selective action of
ACCase-inhibiting herbicides appears to reside at this enzyme site [43].
Further studies conducted in France by Délye and coworkers, with A. myosuroides
accessions from different sites, shed more light on the molecular basis of the dif-
ferent resistance patterns to ACCase inhibitors, thus providing further support
to overlapping binding sites for APP and CHD herbicides at the ACCase en-
zyme [53, 54]. Meanwhile, different point mutations were identified in different
grass weed species that gave rise to insensitive ACCase due to the exchange of
one amino acid: in addition to the Ile → Leu (position 1781), the Trp → Cys
(pos. 1999), Trp → Cys (pos. 2027), Ile → Asn (pos. 2041), Ile → Val (pos. 2041),
Asp → Gly (pos. 2078), Cys → Arg (pos. 2088), and Gly → Ala (pos. 2096) could be
identified ([55, 56]; for a review, see Ref. [22]). The Ile → Leu mutation (pos. 1781)
is the most common, and results in a resistance to mainly all ACCase herbicides.
However, the determination of cross-resistance patterns and resistance levels for
the different mutations cannot be generalized, and these differ between grass weed
species. Both, high-level and low-level resistance to the individual ACCase herbi-
cides is given for the different mutations based on evaluation criteria, including the
herbicide used and the dose rate, the homozygosity/heterozygosity of mutations,
and the presence of non-target-site resistance mechanisms.
Recently, the PCR amplification and sequencing of plastidic ACCase domains
involved in herbicide resistance have been employed to screen a spectrum of
29 grass species for target-site-based resistance to ACCase inhibitors by direct
comparison of the sequences of plastidic ACCase around the critical codons [57].
In P. annua and Festuca rubra, it was found that a leucine residue occurred at
position 1781, while the wild types of all other grass species had an isoleucine in
this position. P. annua and F. rubra are already known (based on enzyme inhibition
assays) to possess a plastidic ACCase that is markedly less susceptible to ACCase
inhibitors than the ACCase of other grass species. Thus, the leucine in position
1781 can clearly be regarded as the basis, or a substantial part, of the natural
inherent tolerance of both species to ACCase-inhibiting herbicides.
A different mechanism of target-site resistance to ACCase inhibitors that should
be mentioned here was identified in a Sorghum halepense biotype from Virginia,
USA, which had been selected in the field by quizalofop applications. The resistance
level of this biotype in vivo was relatively low, with resistance factors [based on ED50
(effective dose) values] ranging between 2.5 and 10 for quizalofop, sethoxydim, and
fluazifop. No difference was found between the herbicide susceptibility of ACCase
from the resistant biotype and a susceptible standard. However, the specific activity
of ACCase in the resistant biotype was found to be two- to threefold greater
than in susceptible plants. These results suggested that an overproduction of
ACCase was the mechanism that conferred a moderate level of resistance to these
herbicides. Owing to the enzyme overproduction the resistant biotype was able,
presumably, to sustain a level of malonyl-CoA production necessary for survival of
herbicide treatment. To date, however, this has been the only reported case for this
mechanism in a naturally occurring biotype [58].
1.3.1.1.3 Inhibitors of Acetolactate Synthase (ALS/AHAS) The enzyme ALS plays

an essential role in branched-chain amino acid biosynthesis in plants. In the
pathway leading to valine and leucine, ALS catalyzes the formation of 2-acetolactate
from two pyruvate molecules, and in the pathway to isoleucine the formation of
2-acetohydroxybutyrate from 2-ketobutyrate and pyruvate. Due to this double func-
tion, the enzyme is also referred to (with a more general term) as acetohydroxyacid
synthase. ALS is inhibited by several groups of herbicides, mainly the sulfonylureas
(SUs), imidazolinones (IMIs), triazolopyrimidines (TPs), pyrimidinylthiobenzoates
(PTBs), and sulfonylaminocarbonyltriazolinones (SCTs) (see Chapter 2.1).
Resistant biotypes which were being reported during the early 1990s were
selected by chlorsulfuron or metsulfuron-methyl in wheat-growing areas, or by
sulfometuron-methyl in non-crop areas. While the resistance of L. rigidum to
ALS-inhibitors was attributed to an enhanced herbicide metabolism [59], it was
shown, for Lolium perenne and dicotyledonous species such as Stellaria media,
Kochia scoparia, Salsola iberica, and Lactuca serriola, that resistant biotypes had a
mutated ALS with a reduced susceptibility to ALS-inhibiting herbicides [60–62].
The IC50 -values for the SUs, which were determined in vitro with ALS isolated from
S. media, S. iberica, and L. perenne, were increased by four- to 50-fold in the resistant
biotypes. Smaller increases, from about two- to sevenfold, were determined in the
same biotypes for the IMI herbicide, imazapyr [62].
Later, ALS-inhibitors were developed for selective use in rice, and this led to
the selection of resistant rice weed biotypes. A biotype of Monochoria vaginalis,
discovered in Korea, showed high levels of cross-resistance to bensulfuron-methyl,
pyrazosulfuron-ethyl, and flumetsulam. The resistance factors determined for ALS
in vitro were 158 to bensulfuron-methyl and 58 to flumetsulam, but only 1.6 to
imazaquin [63]. In rice fields in Japan, a biotype of Scirpus juncoides was selected
which exhibited a high degree of resistance to imazasulfuron (resistance factor of
271, calculated from ED50 -values for growth inhibition). Inhibition studies with
isolated ALS revealed an IC50 of 15 nm for the enzyme from susceptible plants, but
of more than 3000 nm for ALS isolated from the resistant biotype; this suggested
that the resistance was due to an altered ALS enzyme [64].
It appears that a reduced sensitivity of the target enzyme is the predominant
cause of resistance to ALS inhibitors, and that resistance is conferred by a single,
dominant, or at least partial dominant, nuclear-encoded gene. The results of
molecular studies revealed that resistance is caused by single substitution of one of

seven highly conserved amino acids in the ALS enzyme. These are the following,
with 22 known resistance substitutions (amino acid number standardized to
the Arabidopsis thaliana sequence): Pro197, Ala122, and Ala205, located at the
amino-terminal end, Asp376, and Trp574, Ser653, and Gly654, located near the
carboxy-terminal end [65, 66]. For more details, see also Chapter 2.1.
When, in the ALS of a Lactuca serriola biotype, which was highly resistant
to SUs and moderately resistant to IMIs, Pro197 was substituted by His, the
pyruvate-binding domain on the ALS enzyme was found not to be altered by the
mutation [67]. From K. scoparia it was reported that several substitutions of Pro197
by another amino acid (Thr, Arg, Leu, Gln, Ser, Ala) would confer resistance to
SUs [68]. In the same species, it was found later that the substitution of Trp574 by
Leu would also cause resistance to SUs and, in addition, a cross-resistance to IMIs
[69]. The latter substitution was also detected in resistant biotypes of several other
dicotyledonous weed species.
In a biotype of Amaranthus retroflexus from Israel, resistance was caused by a
change of Pro197 to Leu. This biotype exhibited cross-resistance to SUs, IMIs,
TPs, and also to pyrithiobac-sodium in vivo and on the ALS enzyme level [70]. In
mutations of Amaranthus rudis, Ser653 was found to be exchanged by Thr or Asn;
such mutants were only resistant to IMIs [71].
It was concluded from the multiplicity of amino acid substitutions carried out,
that the herbicide-binding site of the ALS can tolerate substitutions of each of the
seven conserved amino acids, without causing any major consequences to normal
catalytic functions. It was, therefore, speculated that the herbicide-binding site and
the active site of ALS are different, despite probably their being in close proximity.
In the absence of herbicide selection, the weed biotypes with mutated ALS showed,
in most cases, no reduction (or only a negligible reduction) of fitness (for reviews,
see Refs [65, 72]), whereas others [73] found for the Trp574 → Leu substitution
in Amaranthus powellii a substantial fitness cost. A review of the fitness costs of
herbicide resistance alleles was recently produced [74].
1.3.1.1.4 Glyphosate Today, glyphosate has become the most important herbi-
cide worldwide, and is widely used as nonselective herbicide in different indications,
and also as a selective herbicide in transgenic crops. The introduction of trans-
genic crops in 1996 changed the use pattern of the compound and the weed
management system, as discussed above [6]. Glyphosate inhibits the chloroplast
enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which catalyzes
the reaction of shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) to
form 5-enolpyruvylshikimate-3-phosphate (EPSP). The inhibition of EPSPS activity
disrupts the shikimate pathway and aromatic amino acid production, which finally
causes the plant to be destroyed.
Since the introduction of glyphosate in 1974, there have been no reports of
evolved glyphosate-resistant weeds during a 20-year period of intensive use [75].
Even in 1997, following the introduction of transgenic crops, it was not believed
that glyphosate resistance in weeds would ever become a major problem [76]. Due
to the loss of diversity in weed management systems, however, the simplicity and
flexibility of this technology was changed, such that resistance to glyphosate has
emerged and has been confirmed in at least 20 weed species in 16 countries (I. Heap,
2010, personal communication, http://www.weedscience.com). Both, target-site and
non-target-site resistance mechanisms have evolved in different weed species. In
resistant accessions of Eleusine indica from Malaysia, this was found to have resulted
from point mutations of the target enzyme EPSPS. By using PCR amplification
and the sequence analysis of an EPSPS fragment, an exchange of Pro106 by Ser
was found in two resistant accessions, and an exchange of Pro106 by Thr in a
third resistant accession [77, 78]. This mutation Pro106, with exchanges by Ser,
Thr, and Ala, was also found in different L. rigidum and L. multiflorum biotypes
from different locations in Australia, USA, Chile, and South Africa (for a review,
see Ref. [79]). In contrast to other target-site mutations (see ACCase and ALS),
the amino-acid substitution at position Pro106 resulted in a modest degree of
glyphosate resistance of 2- to 15-fold in most cases [79]. Recent investigations have
led to the identification of just over a 160-fold EPSPS gene amplification, resulting
in an increased EPSPS overexpression in glyphosate-resistant Amaranthus palmeri
biotypes. The gene amplification was not due to genome duplication, however. The
results of these studies showed that the increasing number of copies conferred an
increasing glyphosate resistance level in plants [80].
1.3.1.2 Non-Target-Site Resistance by Enhanced Metabolic Detoxification

Typically, crop and weed species dispose of the enzyme systems that catalyze
the metabolic conversion of herbicides such that the metabolites, which usually
are more polar than the parent compound, are either nonphytotoxic at all or have
a reduced phytotoxic potential. Among the various enzyme systems involved in
metabolic herbicide detoxification, two are of particular importance in weeds and
crops:
• The cytochrome-P450 monooxygenase system: This system catalyzes oxidative
transformations of the herbicide molecule (e.g., hydroxylations and oxidative
dealkylations). In fact, the system is a member of a large enzyme family that
consists of multiple cytochrome-P450 monooxygenases with diverse substrate
specificities.
• Glutathione-S -transferase (GST): This family of enzymes catalyzes conjugation
reactions that result in the nucleophilic displacement of aryloxy moieties, chlo-
rine, or other substituents by the tripeptide glutathione (GSH). The GSTs also
occur in various isoforms that differ in their catalytic properties.
The herbicide tolerance of crop species has been found to be based frequently
on differential rates of metabolic herbicide detoxification in crop and weed species.
Whilst the rates of herbicide detoxification among weed species are too low to pre-
vent the binding of a lethal herbicide dosage at the target site, the tolerant crop is able
metabolically to detoxify the herbicide at such a high rate that binding of the herbi-
cide at the target site in sufficient amounts to cause irreversible herbicidal effects will
be prevented. If weed biotypes with an improved ability for herbicide detoxification,
comparable to the tolerant crop species, occur in a population they will survive
herbicide application and will thus be selected. These enzyme system-based resis-
tance mechanisms can affect herbicides from different SoA, and (potentially) cause
unexpected cross-resistances to herbicides that have not been used.
To date, several weed biotypes have been described for which herbicide resistance
was related to an enhanced metabolic herbicide detoxification. Indeed, several cases
have been reported for L. rigidum. An early report from Christopher et al. stated that
the excised shoots of biotype SLR 31 from Australia, which was resistant to diclofop,
exhibited a cross-resistance to the SUs chlorsulfuron, metsulfuron-methyl, and tria-
sulfuron [81]. Although the metabolite pattern of chlorsulfuron was identical in the
resistant biotype and a susceptible standard, the resistant biotype metabolized the
herbicide more rapidly. The pathway of chlorsulfuron detoxification in L. rigidum
was similar to that described for wheat, with ring hydroxylation being followed by
glucose conjugation. The time course of chlorsulfuron metabolism in the L. rigidum
biotype SR 4/84 (resistant to diclofop and cross-resistant to chlorsulfuron) was an-
alyzed separately in shoots and roots. The half-life of chlorsulfuron in susceptible
plants was longer in the roots (13 h) than in the shoots (4 h), and was reduced in the
resistant biotype to 3 h and 1 h, respectively. Detoxification of the herbicide by ring
hydroxylation, most likely catalyzed by a cytochrome-P450-dependent monooxy-
genase, with subsequent glucose conjugation, was enhanced in the resistant
biotype [59].
Two other L. rigidum biotypes from Australia (WLR2 and VLR69) developed
metabolism-based resistance to PS II inhibitors. In this case, WLR2 was obtained
from a field with selection pressure by atrazine and amitrole, but never by pheny-
lureas, while VLR69 was obtained from a field with selection pressure by diuron and
atrazine. Both biotypes were resistant to triazines and, despite the field selection
by atrazine, resistance was more pronounced to the structurally related simazine.
Furthermore, both biotypes were resistant to chlorotoluron, though only VLR69 had
previously been exposed to phenylureas. The results of analytical studies revealed
that, in both resistant biotypes, the metabolism of chlorotoluron and simazine was
enhanced, and that the main route of their metabolism was via N-dealkylation
reactions. This type of reaction, coupled to the fact that herbicide metabolism
was inhibited by 1-aminobenzotriazole (ABT; an inhibitor of cytochrome-P450
monooxygenases) suggested an increased activity of cytochrome-P450 monooxyge-
nases in the resistant biotypes [82, 83]. The mechanism of phenylurea resistance
of L. rigidum biotypes from Spain has been studied [84]. A biotype (R3) selected in
the field by applications of diclofop plus isoproturon or plus chlorotoluron had in
vivo resistance factors [ED50 R (resistant)/ED50 S (susceptible)] of about 9.3 and 5.5
to chlorotoluron and isoproturon, respectively, and was also resistant to a broad
spectrum of other phenylureas. Metabolism studies with chlorotoluron, in the
absence and presence of the cyochrome-P450 monooxygenase inhibitor 1-ABT,
suggested that resistance was due to an enhanced ability to degrade the molecule
to nontoxic ring-alkylhydroxylated intermediates suitable for follow-up conjugation
reactions. Thus, several biotypes of L. multiflorum from the UK, with resistance to
diclofop, have been analyzed [41]. While one biotype had an insensitive ACCase, the
resistance of three other biotypes could be attributed to an enhanced metabolism

of this herbicide.
The resistance of the grass weed Phalaris minor to isoproturon, and of the di-
cotyledonous weed species Abutilon theophrasti to atrazine, has also been attributed
to an enhanced metabolism. Here, GST was noted as the enzyme responsible for
atrazine detoxification in A. theophrasti [85], whereas in P. minor the cytochrome
P450 monooxygenase was most likely involved in the enhanced detoxification of
isoproturon [86].
An increasing occurrence of the resistance of A. myosuroides to herbicides in sev-
eral European countries has prompted investigations into resistance mechanisms
in this species. Aside from target-site-based resistance, cases of resistance due to an
enhanced herbicide metabolism have also been reported. Two biotypes – Peldon
A1 and Lincs. E1 – with in vivo resistance factors to isoproturon of 28 and 2.6,
respectively, were shown to metabolize this herbicide faster than a susceptible
standard, with the rate of metabolism being higher in Peldon than in Lincs. The
addition of the cytochrome-P450 monooxygenase inhibitor 1-ABT lowered the rate
of chlorotoluron metabolism, and correspondingly increased phytotoxicity; this
suggested an involvement of the cytochrome-P450 monooxygenase system in the
detoxification of the herbicide. However, the major detoxification reaction in these
biotypes appeared to be the formation of a hydroxymethylphenyl metabolite [87].
The same biotypes, Peldon A1 and Lincs. E1, are also resistant to the graminicide
fenoxaprop, which is used for the selective control of A. myosuroides and other
grassy weeds in cereals (mainly wheat). On a whole-plant level, Lincs. E1 was
more resistant than Peldon A1. The selectivity of this herbicide has been attributed
to a rapid detoxification via GST-catalyzed conjugation in the cereal species. In
both resistant A. myosuroides biotypes, the GST activities toward fenoxaprop were
shown to be increased to a similar degree, when compared with a susceptible
biotype. This was due to an increased expression of a constitutive GST, and to the
expression of two novel GST isoenzymes. Furthermore, GSH levels were increased
in the resistant biotypes, in Peldon more than in Lincs. These data pointed to
an involvement of GST activity and GSH levels in the resistance to fenoxaprop,
although a lack of correlation to the whole-plant resistance of these biotypes did
not permit definite conclusions to be drawn [88]. Recently, a range of European A.
myosuroides biotypes with resistance to fenoxaprop has been investigated [89], and
several of these biotypes – notably one from Belgium – were shown to detoxify the
herbicide at an increased rate. The biotype from Belgium also had the highest GST
activity towards the unspecific substrate chloro-dinitrobenzene (CDNB), although
GST activity towards the herbicide was not tested.
Studies on the mode of inheritance of metabolic herbicide resistance in A.
myosuroides and L. rigidum postulated that more than one gene is involved in
cytochrome-P450 metabolism-based resistance in weed biotypes [90–92]. The occur-
rence of an enhanced metabolic detoxification can be associated with an ecological
cost expressed in a reduction of the vegetative biomass and reproduction rate [74].
In contrast to the above-described cases, the herbicide propanil is
detoxified in rice and weed species by the action of an aryl acylamidase
(aryl-acylamine amidohydrolase). A high activity of this enzyme in rice confers

crop tolerance. In Colombia, a biotype of Echinochloa colona was found that is
resistant to propanil; subsequent enzyme tests with extracts from this biotype
revealed an almost threefold higher activity of aryl acylamidase in the resistant than
in a susceptible biotype. Based on these findings, it was concluded that resistance
of the E. colona biotype is related to an enhanced propanil detoxification [93].
1.3.1.3 Non-Target-Site Resistance by Altered Herbicide Distribution

Cases of non-target-site resistance by altered herbicide distribution have been
reported for two important herbicides, paraquat and glyphosate.
The intensive use of paraquat has resulted in an evolution of resistance in various
weed species. Subsequently, intensive investigations into the resistance mecha-
nisms involved was mainly carried out using resistant biotypes from Hordeum
spp. and Conyza spp., and an altered distribution of the herbicide in the resistant
weeds was suggested as the cause – or at least the partial cause – of resistance.
In resistant Conyza canadensis, it was supposed that a paraquat-inducible protein
might function by carrying paraquat to a metabolically inactive compartment,
either the cell wall or the vacuole. This sequestration process would prevent suf-
ficient amounts of the herbicide from entering the chloroplasts, which is the
cellular site of paraquat action. Inhibitors of membrane transport systems, such
as N,N-dicyclohexylcarbodiimide (DCCD), caused a delay in the recovery of the
photosynthetic functions of a paraquat-resistant biotype, when administered after
the herbicide. The results of these transport inhibitor experiments supported the
involvement of a membrane transporter in paraquat resistance [94].
Translocation studies with two paraquat-resistant biotypes of Hordeum leporinum
revealed that the basipetal transport of paraquat in resistant H. leporinum was
much reduced compared to susceptible plants. It was concluded, therefore, that a
resistance to paraquat was the result of a reduced herbicide translocation out of
the treated leaves [95]. It might be supposed that, also in this species, herbicide
sequestration into the leaf vacuoles may have been the primary cause for the altered
long-distance transport.
The high efficiency of glyphosate as a potent herbicide is based on its ability
to translocate within the plant via xylem and phloem to the apical and root
meristems, as well as to the reproductive organs of perennial plants. Independent
populations of L. rigidum with resistance to glyphosate have been reported from
different locations in Australia. One of these, with an approximately 10-fold
in vivo resistance to glyphosate, was used to conduct intensive investigations
into the mechanism of resistance. Neither a modification of the target enzyme
EPSPS, nor of herbicide metabolism, contributed to the resistance in this case.
However, translocation studies following foliar application revealed that, in the
resistant biotype, glyphosate accumulated preferentially in the leaf tips, whereas in
susceptible plants the accumulation was greater in the leaf bases and roots. These
results suggested a shift of glyphosate transport in the resistant plants, from the
phloem to the xylem system. Thus, it was speculated that the resistant biotype
might have lost an efficiency to load glyphosate into the symplast, such that more
of the herbicide would remain in the apoplast and be translocated acropetally with
the transpiration stream. Consequently, the concentration of glyphosate in the
plastids of the sensitive meristematic tissues at the shoot base and in the roots
would be reduced [96]. Meanwhile, a reduced glyphosate translocation within the
plants and to the roots was confirmed for different C. canadensis and L. rigidum
biotypes from different countries (for reviews, see Refs [79, 97]). It was speculated,
that the membrane transporters were responsible for pumping the herbicide either
into vacuoles or out of the chloroplast, such that the herbicide was unable to reach
the target site [97].
1.3.1.4 Multiple Resistance

As defined above, multiple resistance means that more than one resistance mecha-
nism occurs in a weed population or an individual plant. This can either mean that
both target site-based and non-target-site-based mechanisms occur in the same
biotype, or that a biotype is resistant to herbicides with different mechanisms of
action. Multiple resistance can result in the resistance of a weed biotype to a very
broad range of herbicide chemistries. Multiple resistance has been reported for sev-
eral weed species, notably Lolium rigidum, Alopecurus myosuroides, Kochia scoparia,
Conyza canadensis, and Amaranthus rudis. Such multiple resistance developed to
a major extent especially in the Australian biotypes of L. rigidum, most likely as a
result of agricultural conditions paired with biological characteristics of this weed
(cross-pollinating species with a high genetic variability and seed production, and
high plant numbers per area).
Multiple resistance can develop by selection with a single herbicide, or by selection
with several herbicides that are used either sequentially or simultaneously. More-
over, cross-pollinating species may become multiple resistant when two individuals,
each with a different resistance mechanism, undergo hybridization. An example of
the selection of multiple resistance by a single herbicide (the ALS inhibitor chlor-
sulfuron) is the L. rigidum biotype WLR1. As the main mechanism of resistance,
this biotype had an ALS with reduced sensitivity to chlorsulfuron, sulfometuron
and imazamethabenz, and as additional mechanism an enhanced metabolism of
chlorsulfuron [98]. Extreme cases of multiple resistance, due to an application
history of many herbicides, were reported from Australia for several L. rigidum
biotypes. For example, biotype VLR69 possessed the following mechanisms: An
enhanced metabolism of ACCase-inhibiting herbicides; a resistant form of the
ACCase enzyme; an enhanced metabolism of the ALS-inhibitor chlorsulfuron; and
also a resistant form of the ALS enzyme in 5% of the population [43].
The selection of multiple resistance following the sequential use of different
herbicides has been described for a biotype of K. scoparia from North America. In
this case, many years of triazine usage resulted in the selection of a biotype with
target-site resistance of the D1 protein in PS II. Following the subsequent use of
ALS inhibitors, a point mutation in the gene encoding for ALS was selected in
addition, which made this biotype target-site-resistant also to SUs and IMIs [69].
Some Lolium populations from Australia and South Africa have shown both
target-site as well as a reduced translocation to glyphosate [79]. Further examples
of weed species and biotypes with multiple resistance mechanisms have been
described in various reviews, and also in the database of the International
Survey of Herbicide-Resistant Weeds (I. Heap, 2010, personal communication,
http://www.weedscience.com) [19, 20, 22].
Clearly, multiple resistance leads to complex patterns of broad herbicide resis-
tance, particularly in cross-pollinating weed species. This places a serious restriction
on the remaining options for chemical weed control in agricultural practice.
References
1. Heap, I. and LeBaron, H. (2001) in 11. Bird, J.A., Eagle, A.J., Horwath, W.R.,
Herbicide Resistance and World Grains Hair, M.W., Zilbert, E.E., and Kessel, C.
(eds S.B. Powles and D.L. Shaner), CRC (2002) Calif. Agric., 2, 69–75.
Press, Boca Raton, FL, pp. 1–22. 12. Walsh, M. (2003) Aust. Farm J., 3,
2. Herbicide Resistance Action Committee 40–41.
(HRAC) (2010) Classification of Her- 13. Horowitz, J., Ebel, R., and Ueda, K.,
bicides According to Mode of Action. USDA (2010) Econ. Info. Bull., 70,
Available at: http://www.hracglobal.com. 1–22.
3. Weed Science Society of America 14. D’Emden, F.H., Llewellyn, R.S., and
(WSSA) (2011) Resistance, WSSA Burton, M.P. (2003) Tech. Forec. Soc., 3,
Classification of Herbicide Resis- 40–41.
tance Mechanism of Action, pp. 1–6; 15. Neve, P., Diggle, A.J., Smith, F.P.,
http://www.wssa.net. and Powles, S.B. (2003) Weed Res., 43,
4. CropLife Australia (2010) Resis- 418–427.
tance Management, Herbicides, 2010 16. Hyvönen, T. and Salonen, J. (2002) Plant
Herbicide Mode of Action Table; Ecol., 154, 73–91.
17. Valverde, B.E. (2003) in Weed Manage-
http://www.croplifeaustralia.org.au, pp.
ment for Developing Countries (ed. R.
1–4, update 2010.
Labrada), FAO, Rome.
5. James, C. (2009) Executive Summary.
18. Chauvel, B., Guillemin, J.P., Colbach,
Global Status of Commercialized
N., and Gasquez, J. (2001) Crop Prot.,
Biotech/GM Crops: 2009. Brief 41.
20, 127–137.
The International Service for the Ac-
19. Powles, S.B. and Holtrum, J.A.M. (eds)
quisition of Agri-biotech Applications;
(1994) Herbicide Resistance in Plants:
http://www.ISAAA.org.
Biology and Biochemistry, Lewis Publish-
6. Duke, S.O. and Powles, S.B. (2009)
ers, S. Boca Raton, Ann Arbor, London,
AgBioForum, 12, 346–357. Tokyo.
7. National Agricultural Statistics 20. Powles, S. B. and Shaner, D. L. (eds)
Service Agricultural Chemical (2001) Herbicide Resistance and World
Usage–1996/2000/2004/2006 Field Grains, CRC Press, Boca Raton, FL.
Crops Summary, U.S. Department of 21. Gressel, J. (2002) Molecular Weed Bi-
Agriculture, Washington, DC, and ology, Taylor & Francis, London, New
Agricultural Chemical Use Data Base, York.
http://www.nass.usda.gov. 22. Powles, S.B. and Yu, Q. (2010) Annu.
8. Duke, S.O. (1999) Proceedings of the Rev. Plant Biol., 61, 317–347.
Workshop of Ecological Effects of Pest 23. Böger, P. (1983) Biol. Z., 13 (6),
Resistance Genes in Managed Ecosys- 170–177.
tems, Bethesda, MD. 24. Ort, D.R., Ahrens, W.H., Martin, B., and
9. Owen, M.D.K. (1997) Proc. Brighton Crop Stoller, E.W. (1983) Plant Physiol., 72,
Prot. Conf., 3, 955–963. 925–930.
10. Owen, M.D.K. (2008) Pest Manage. Sci., 25. Sundby, C., Chow, W.S., and Anderson,
64, 377–387. J.M. (1993) Plant Physiol., 103, 105–113.
References 27
26. Zurawski, G., Bohnet, H., Whitfeld, P., 44. Devine, M.D. (1997) Pestic. Sci., 51,
and Bottomley, W. (1982) Proc. Natl 259–264.
Acad. Sci. USA, 79, 7699–7703. 45. Moss, S.R., Cocker, K.M., Brown, A.C.,
27. Trebst, A. (1991) in Herbicide Resis- Hall, L., and Field, L.M. (2003) Pest
tance in Weeds and Crops (eds J.C. Manage. Sci., 59, 190–201.
Caseley, G.W. Cussans, and R.K., Atkin), 46. Volenberg, D. and Stoltenberg, D. (2002)
Butterworth-Heinemann, Oxford, pp. Weed Res., 42, 342–350.
145–164. 47. Nikolskaya, T., Zagnitko, O.,
28. Trebst, A. (1996) in Molecular Genetics Tevzadze, G., Haselkorn, R., and
and Evolution of Pesticide Resistance, ACS Gornicki, P. (1999) Proc. Natl Acad.
Symposium Series, Vol. 645 (ed. T.M. Sci. USA, 96, 14647–14651.
Brown), American Chemical Society, 48. Zagnitko, O., Jelenska, J., Tevzadze, G.,
Washington, DC, pp. 44–51. Haselkorn, R., and Gornicki, P.
29. Oettmeier, W. (1999) Cell. Mol. Life Sci., (2001) Proc. Natl Acad. Sci. USA, 98,
55, 1255–1277. 6617–6622.
30. Masabni, J.G. and Zandstra, B.H. (1999) 49. Tal, A. and Rubin, B. (2004) Pest Man-
Weed Sci., 47, 393–400. age. Sci., 60, 1013–1018.
31. Mengistu, L.W., Mueller-Warrant, G.W., 50. Christoffers, M.J., Berg, M.L., and
Liston, A., and Barker, R.E. (2000) Pest Messersmith, C.G. (2002) Genome, 45,
Manage. Sci., 56, 209–217. 1049–1056.
32. Dumont, M. and Tardif, F.J. (2002) 51. Brown, A.C., Moss, S.R., Wilson, Z.A.,
Weed Sci. Soc. Am. Abstr., 42, 49–50. and Field, L.M. (2002) Pestic. Biochem.
33. Mengistu, L.W., Christoffers, M.J., and
Physiol., 72, 160–168.
Lym, R.G. (2005) Pest Manage. Sci., 61,
52. Délye, C., Wang, T., and Darmency, H.
1035–1042.
(2002) Planta, 214, 421–427.
34. Park, K.W. and Mallory-Smith, C.A.
53. Délye, C., Straub, C., Matéjicek, A., and
(2006) Pest Manage. Sci., 62, 880–885.
Michel, S. (2003) Pest Manage. Sci., 60,
35. Mechant, E., De Marez, T., Hermann,
35–41.
O., Olsson, R., and Bulcke, R. (2008) J.
54. Délye, C., Zhang, X.-Q., Michel, S.,
Plant Dis. Prot. Spec., 21, 37–40.
Matéjicek, A., and Powles, S.B. (2005)
36. Perez-Jones, A., Intanon, S., and
Plant Physiol., 137, 794–806.
Mallory-Smith, C. (2009) Weed Sci.,
55. Delye, C. (2005) Weed Sci., 53, 728–746.
57, 574–578.
37. Stanger, C.E. and Appleby, A.P. (1989) 56. Delye, C., Matejicek, A., and Michel, S.
Weed Sci., 37, 350–352. (2008) Pest Manage. Sci., 64, 1179–1186.
38. Holtum, J.A.M. and Powles, S.B. (1991) 57. Délye, C. and Michel, S. (2005) Weed
Brighton Crop Prot. Conf. – Weeds, 3, Res., 45, 323–330.
1071–1078. 58. Bradley, K.W., Wu, J., Hatzios, K.K., and
39. Gronwald, J.W., Eberlein, C.V., Betts, Hagood, E.S. Jr (2001) Weed Sci., 49,
K.J., Baerg, R.J., Ehlke, N.J., and Wyse, 477–484.
D.L. (1992) Pestic. Biochem. Physiol., 44, 59. Cotterman, J.C. and Saari, L.L. (1992)
126–139. Pestic. Biochem. Physiol., 43, 182–192.
40. De Prado, R., González-Gutiérrez, J., 60. Mallory-Smith, C.A., Thill, D.C., and
Menéndez, J., Gasquez, J., Gronwald, Dial, M.J. (1990) Weed Technol., 4,
J.W., and Giménez-Espinosa, R. (2000) 163–168.
Weed Sci., 48, 311–318. 61. Saari, L.L., Cotterman, J.C., and
41. Cocker, K.M., Northcroft, D.S., Primiani, M.M. (1990) Plant Physiol.,
Coleman, J.O.D., and Moss, S.R. (2001) 93, 55–61.
Pest Manage. Sci., 57, 587–597. 62. Saari, L.L., Cotterman, J.C., Smith,
42. Tardif, F.J., Holtum, J.A.M., and Powles, W.F., and Primiani, M.M. (1992) Pestic.
S.B. (1993) Planta, 190, 176–181. Biochem. Physiol., 42, 110–118.
43. Powles, S.B. and Preston, C. (1995) The 63. Hwang, I.T., Lee, K.H., Park, S.H., Lee,
Herbicide Resistance Action Committee B.H., Hong, K.S., and Han, S.S. (2001)
Monograph Number 2. Pestic. Biochem. Physiol., 71, 69–76.
64. Tanaka, Y. (2003) Pestic. Biochem. Phys- 81. Christopher, J.T., Powles, S.B.,
iol., 77, 147–153. Liljegreen, D.R., and Holtum, J.A.M.
65. Tranel, P.J. and Wright, T.R. (2002) (1991) Plant Physiol., 95, 1036–1043.
Weed Sci., 50, 700–712. 82. Burnet, M.W.M., Loveys, B.R., Holtum,
66. Tranel, P.J., Wright, T.R., and J.A.M., and Powles, S.B. (1993) Pestic.
Heap, I.M. (2010) ALS Mutations Biochem. Physiol., 46, 207–218.
from Herbicide-Resistant Weeds; 83. Burnet, M.W.M., Loveys, B.R., Holtum,
http://www.weedscience.org. J.A.M., and Powles, S.B. (1993) Planta,
67. Guttieri, M.J., Eberlein, C.V., 190, 182–189.
Mallory-Smith, C.A., Thill, D.C., and 84. De Prado, R., De Prado, J.L., and
Hoffmann, D.L. (1992) Weed Sci., 40, Menendez, J. (1997) Pestic. Biochem.
670–676. Physiol., 57, 126–136.
68. Guttieri, M.J., Eberlein, C.V., and Thill,
85. Anderson, M.P. and Gronwald, J.W.
D.C. (1995) Weed Sci., 43, 175–178.
(1991) Plant Physiol., 96, 104–109.
69. Foes, M.J., Liu, L., Vigue, G., Stoller,
86. Singh, S., Kirkwood, R.C., and
E.W., Wax, L.M., and Tranel, P.J. (1999)
Marshall, G. (1998) Pestic. Biochem.
Weed Sci., 47, 20–27.
Physiol., 59, 143–153.
70. Sibony, M., Michel, A., Haas, H.U.,
Rubin, B., and Hurle, K. (2001) Weed 87. Hall, L.M., Moss, S.R., and Powles,
Res., 41, 509–522. S.B. (1995) Pestic. Biochem. Physiol., 53,
71. Patzold, W.L. and Tranel, P.J. (2001) 180–192.
Proc. North Cent., Weed Sci. Soc., 56, 67. 88. Cummins, I., Moss, S., Cole, D.J.,
72. Holt, J.S. and Thill, D.C. (1994) in Her- and Edwards, R. (1997) Pestic. Sci., 51,
bicide Resistance in Plants: Biology and 244–250.
Biochemistry (eds S.B. Powles, J.A.M. 89. Cocker, K.M., Moss, S.R., and Coleman,
Holtrum), Lewis Publishers, Boca Ra- J.O.D. (1999) Pestic. Biochem. Physiol.,
ton, Ann Arbor, London, Tokyo, pp. 65, 169–180.
299–316. 90. Letouzé, A. and Gasquez, J. (2001)
73. Tardiff, F.J., Rajcan, I., and Costea, M. Theor. Appl. Genet., 103, 288–296.
(2006) New Phytol., 169, 251–264. 91. Chauvel, B. (1991) Polymorphisme
74. Vila-Aiub, M.M., Neve, P., and Powles, génétique et sélection de résistance
S.B. (2009) New Phytol., 184, 751–767. aux urées substituées chez Alopecu-
75. Dyer, W.E. (1994) in Herbicide Resistance rus myosuroides Huds. PhD thesis.
in Plants: Biology and Biochemistry (eds University of Paris-Orsay.
S.B. Powles and J.A.M. Holtrum), Lewis 92. Preston, C. (2003) Weed Sci., 51, 4–12.
Publishers, Boca Raton, Ann Arbor, 93. Leah, J.M., Caseley, J.C., Riches, C.R.,
London, Tokyo, pp. 229–242. and Valverde, B. (1994) Pestic. Sci., 42,
76. Bradshaw, L.D., Padgette, S.R., Kimball, 281–289.
S.L., and Wells, B.H. (1997) Weed Tech- 94. Halász, K., Sóos, V., Jóri, B., Rácz, I.,
nol., 11, 189–198. Lásztity, D., and Szigeti, Z. (2002) Acta
77. Baerson, S.R., Rodriguez, D.J., Tran, Biol. Szeged., 46, 23–24.
M., Feng, Y., Biest, N.A., and Dill, G.M.
95. Preston, C., Soar, C.J., Hidayat, I.,
(2002) Plant Physiol., 129, 1265–1275.
Greenfield, K.M., and Powles, S.B.
78. Ng, C.H., Wickneswari, R., Salmijah, S.,
(2005) Weed Res., 45, 289–295.
Teng, Y.T., and Ismail, B.S. (2003) Weed
96. Lorraine-Colwill, D.F., Powles, S.B.,
Res., 43, 108–115.
Hawkes, T.R., Hollinshead, P.H.,
79. Preston, C., Wakelin, A.M., Dolman,
F.C., Botamam, Y., and Boutsalis, P. Warner, S.A.J., and Preston, C. (2003)
(2009) Weed Sci., 57, 435–441. Pestic. Biochem. Physiol., 74, 62–72.
80. Gaines, T.A., Zhang, W., Wang, 97. Shaner, D.L. (2009) Weed Sci., 57,
D., Bukun, B., Chrisholm, S.T., 118–123.
Shaner, D.L., Nissen, S.J. et al. 98. Christopher, J.T., Powles, S.B., and
(2010) Proc. Natl Acad. Sci. USA, 107, Holtum, J.A.M. (1992) Plant Physiol.,
1029–1034. 100, 1909–1913.
29
2
Acetohydroxyacid Synthase Inhibitors (AHAS/ALS)
2.1
Biochemistry of the Target and Resistance
2.1.1
Acetohydroxyacid Synthase (AHAS)
The first committed step in the biosynthetic pathway of the branched-chain amino
acids (bcaas) is catalyzed by the enzyme acetohydroxyacid synthase (AHAS; EC
2.2.1.6), which is also referred to as acetolactate synthase (ALS). As depicted in
Figure 2.1.1, the pathway leading to valine and leucine begins with the condensation
of two molecules of pyruvate accompanied by the loss of carbon dioxide to give
(S)-2-acetolactate. A parallel reaction leading to isoleucine involves the condensation
of pyruvate with 2-ketobutyrate to afford (S)-2-aceto-2-hydroxybutyrate after the loss
of carbon dioxide. In the past, many authors have used the acronym ALS to refer
to the enzyme that catalyzes the reaction of Figure 2.1.1. However, ALS as a term
implies that the enzyme only initiates the pathway leading to valine and leucine
through the formation of (S)-2-acetolactate, whereas the designation AHAS better
describes all products of catalysis, including those involving the other keto-acid
substrate, 2-ketobutyrate. Both reactions catalyzed by AHAS require the cofactors
thiamine diphosphate (ThDP) and flavin adenine dinucleotide (FAD), and a divalent
metal ion (most commonly Mg2+ ) is also implicated. Several excellent reviews of
AHAS have been produced that describe the biochemistry, genetics, inhibition,
and active site modeling of the enzyme [1–6].
AHAS is present in bacteria, fungi, and plants. Many of the early kinetic,
mechanistic, and structural studies were carried out with AHAS that had been
isolated and purified from enteric bacteria such as Escherichia coli and Salmonella
typhimurium. Eukaryotic AHAS has proven more difficult to isolate and purify
because of its reduced stability. Whilst three AHAS isoenzymes – AHAS I, II, and
III – have been characterized in bacteria, only one isoenzyme is known in fungi
and plants.
The mechanism of the AHAS reaction is shown in Figure 2.1.2 [7, 8]. The first step
involves removal of the proton attached to the 2-carbon atom of the thiazolium ring
of ThDP to form an ylide. This ionization is followed by addition of the thiazolium

30 2 Acetohydroxyacid Synthase Inhibitors (AHAS/ALS)
H3C COO−
Threonine
pyruvate
O O
H3C
COO− H3C COO−
2-ketobutyrate pyruvate
Acetohydroxyacid synthase (AHAS)

CO2 or CO2
Acetolactate synthase (ALS)
O O
H3C
H3C CH3 CH3
HO COO− HO COO−
(S )-2-aceto-2-hydroxybutyrate (S )-2-acetolactate
3 steps 3 steps 6 steps
COO− COO−
H3C CH3 COO−
H3C NH3+ NH3+
CH3 CH3 H3C NH3+
Isoleucine Valine Leucine
Figure 2.1.1 Branched-chain amino acid biosynthetic pathway.
2-carbanion to the carbonyl group of pyruvate to give lactyl-ThDP, which loses

carbon dioxide to generate the intermediate, hydroxyethyl-thiamine diphosphate
(HEThDP). The deprotonation is the only common step for all ThDP-dependent
enzymes, and crystallographic studies combined with site-directed mutagenesis
have identified a highly conserved glutamate residue as a key feature assisting
ThDP-mediated catalysis [9]. Previous nuclear magnetic resonance (NMR) spec-
troscopic analyses of ThDP-ylide formation in yeast pyruvate decarboxylase (PDC)
have provided convincing evidence that interaction of the glutamate with the
1 -nitrogen atom of the ThDP pyrimidine ring activates the 4 -amino group to
facilitate removal of the thiazolium ring C-2 proton in an intramolecular process
[10]. The exact details of the AHAS mechanism have not been completely resolved,
especially with respect to the formation of the reactive ylide in the first step, and
whether it is a discrete or concerted process.
O
−
H3C COO
4′-N
Step 1 Me N NH2
−
E-ThDP C-2 S
Deprotonation N
at thiazole C-2
1′ C N
H2 +
Me
OP2O6−3
E-ThDP-ylide
(S )-2-aceto-2-hydroxybutyrate (AHB)
or
(S )-2-acetolactate (AL)
Step 5 Step 2
OH −
RCH2 COO CH3
−
Me N Me N HO COO
HO CH3
S
N S N
C N C N
H2 + H2 +
NH2 NH2
Me
Me
OP2O6−3 OP2O6−3
E-AHA-HEThDP E-Lactyl-ThDP
O Step 3
Step 4
−CO2
RCH2 COO −
R = H, pyruvate
R = CH3, 2-ketobutyrate
CH3
Me N CH3
HO Me N HO −
N S
C N N S
H2 + C N
NH2 H2 +
Me NH2
OP2O6−3 Me
OP2O6−3
E-HEThDP E-HEThDP
Figure 2.1.2 Mechanism of the AHAS-catalyzed reaction.
Following formation of the HEThDP intermediate, the enzyme is then primed

to react with either a second molecule of pyruvate or with 2-ketobutyrate to produce
acetolactate (AL) or acetohydroxybutyrate (AHB), respectively. In-depth studies
of the kinetics of this reaction have been conducted on E. coli AHAS III [11],
and a comparison of the results obtained with earlier data for AHAS I and II
has indicated that all three bacterial isoenzymes catalyze similar mechanisms.
However, AHAS II and III show a much more pronounced preference for reaction
with 2-ketobutyrate in step 4, where the substrate specificity is an intrinsic property
of the enzyme and is unaffected by pH or feedback inhibitors such as valine [12].
Recent studies using NMR to detect covalent reaction intermediates has enabled
the calculation of microscopic rate constants for both wild-type and mutant AHAS
II from E. coli. The results of these studies showed that addition of the ThDP C-2
anion to pyruvate is the rate-limiting step, and that all other steps in the reaction
are comparatively fast [8, 13]. Subsequent steps in the reaction involving HEThDP
and the carboligation of the second keto acid substrate and product release have
been investigated using molecular dynamics (MD) simulations [14], and results
have shown that the HEThDP anion could act as the acid-base necessary to drive
these partial reactions. Such a mechanism would avoid implicating other amino
acid residues that might play a direct role in the chemistry, other than acting to
stabilize the various reaction intermediates.
The dependence of AL formation on pyruvate concentration in all three bacterial
AHAS isoenzymes obeys Michaelis–Menten kinetics. Such behavior implies that
there is an irreversible step between the addition of the first and second pyruvate
molecules [11, 15, 16]. The results of steady-state experiments have confirmed
that the rate-determining and product-determining steps in the mechanism are
different. The observation that a wide range of substrate concentrations, whether
pyruvate or ketobutyrate, changes in pH, or the presence of feedback inhibitors do
not affect the specificity of the enzyme, supports the idea that the first steps in the
mechanism, preceding binding of the second substrate, are rate-determining [11].
Modulation of these first steps would be expected to affect the turnover rate of the
enzyme, without necessarily affecting product partitioning.
The role of FAD in the AHAS reaction is not fully understood, since no oxidation
or reduction of the cofactor during the catalytic cycle is apparent. Although several
hypotheses have been put forth, no experimental evidence has yet been obtained
to conclusively support any single explanation. One possibility is that FAD plays
a structural role only, and is simply an evolutionary remnant from a pyruvate
oxidase (POX)-like ancestor [17]. The divalent metal ion does not play a direct role
in the reaction, but rather serves to anchor the ThDP molecule to the protein by
coordinating the diphosphate group and certain amino acid side chains [18].
The functional holoenzyme from bacterial sources are heterotetrameric and
composed of two types of subunit: (i) two large, identical catalytic subunits (CSUs)
of approximately 60 kDa, which contain all of the catalytic machinery; and (ii)
two small, identical regulatory subunits (RSUs) of molecular weight 9–17 kDa
[15, 19, 20]. Whilst the CSU of the bacterial enzyme alone possesses low or no
activity, reconstitution in vitro with the RSU restores full enzymatic activity [21].
The genes ilvBN, ilvGM, and ilvIH, which code for E. coli catalytic and RSU pairs
of AHAS I, II, and III, respectively, have been cloned and sequenced [22–25].
The use of recombinant approaches has enabled the production of significant
quantities of each isoenzyme with subsequent purification to near homogeneity.
While AHAS I and III of E. coli are regulated by feedback inhibition from the
bcaas (especially valine), isoenzyme II is insensitive. The binding sites for the bcaa
feedback modulators are located in the RSUs [26].
The yeast AHAS gene, ilv2, shares significant amino acid sequence homology
with that of the E. coli AHAS CSU [27]. The expression of ilv2 in E. coli produced
a subunit with considerably diminished enzymatic activity after isolation and

purification [28]. Production of the recombinant ilv6 gene that codes for the yeast
AHAS RSU in E. coli was found to substantially enhance the catalytic activity of the
yeast enzyme and confer sensitivity to valine inhibition, thereby providing the first
evidence for the existence of a smaller eukaryotic RSU [29]. Studies of the bacterial
isoenzymes, where recombinant small subunits have been used to reconstitute
the activities of the various CSUs, has provided insights into how interchangeable
the RSUs are in restoring full activity to the holoenzyme and sensitivity to bcaa
binding [30].
Early studies conducted in plants showed that AHAS is a nuclear-encoded,
chloroplast-localized enzyme [31, 32]. The first genes from higher plants encoding
AHAS were isolated from Arabidopsis thaliana and Nicotiana tabacum, using a
yeast AHAS gene as a heterologous hybridization probe [33]. Comparison of the
DNA and amino acid sequences from the two plants showed approximately 70%
identity at the nucleotide level, and 85% homology of the putative mature proteins.
Alignment of the plant sequences with those from E. coli and yeast showed many
regions of high homology interspersed with regions of divergence. One region of
low homology between the plant proteins occurred in the first 85 amino acids,
which were presumed to comprise an N-terminal chloroplast transit peptide. The
isolation and sequencing of the mature CSU from chloroplasts has positioned the
site more precisely, and shown that the transit peptide for plant enzymes comprises
about 50–60 amino acids of the N-terminus of the full-length subunit.
The first isolation of a plant AHAS RSU was reported by Hershey et al. [34], when
a cDNA clone from Nicotiana plumbaginifolia was used to produce the peptide
in E. coli. Based on homology with various bacterial AHAS small subunits, and
the observation of enhanced enzymatic activity when reconstituted with the large
subunit of AHAS from either N. plumbaginifolia or A. thaliana, it was concluded
that a competent RSU had been isolated. Identification of the RSU of A. thaliana
was subsequently reported by Lee and Duggleby [21], who showed that in vitro
reconstitution not only enhanced the activity of the CSU but also conferred
sensitivity to all three bcaas.
2.1.2
Higher-Order Subunit Structure
Several early structural models of an AHAS CSU were proffered based on homology
to other known ThDP-dependent enzymes that had yielded to three-dimensional
(3-D) structural determination, such as POX from Lactobacillus plantarum. Carefully
planned site-directed mutagenesis studies assisted in testing and defining the
functional roles of amino acids in these models. Many of the features of these
early models were borne out by the first X-ray crystal structure at 2.6 Å resolution
of the dimeric catalytic subunit of AHAS from S. cerevisiae [18, 35]. Pang et al.
thus confirmed that AHAS shares many structural features in common with other
ThDP-dependent enzymes. Figure 2.1.3 depicts one of the monomers of the AHAS
protein and the organization of the α-, β-, and γ-domains; the positions of the
α
domain
γ β
domain domain
Figure 2.1.3 Structure of a single monomer γ-domain of the partner subunit has been
of the yeast AHAS catalytic subunit. ThDP added to more clearly illustrate the position
and FAD cofactors are represented as stick of the enzyme active site at the interface of
models. Mg2+ anchored to the ThDP is the dimer. Modified with permission from
shown as a green sphere. ThDP of the Ref. [18]; © 1985, Elsevier.
cofactors, ThDP, FAD, and Mg2+ are clearly defined. In this representation, the
second ThDP molecule has been included to show the location of the enzyme
active site, which is formed by the interface of the α-domain of one monomer and
the γ-domain of the second monomer. The crystal structure shows that the active
site without bound substrate or inhibitor is quite accessible to solvent. The two
rings of ThDP are held in a bent (‘‘V’’) conformation by interactions with Met525,
Met555, Tyr113, Gly523, and Ala551 residues, and by two hydrogen bonds [7]. The
cofactor is further anchored to the γ-domain through its phosphate groups, which
interact with the Mg2+ ion that is coordinated to several amino acids. The fact
that other divalent cations can substitute for magnesium, with little impact on the
overall reaction [36], suggests that the involvement of magnesium in the chemistry
is minimal. The FAD cofactor is most closely associated with, and binds with high
affinity to, the β-domain; typically, it is secured through numerous hydrogen bonds
and van der Waals interactions in an extended planar conformation [7, 28]. While
the crystal structure sheds no light on any functional role for FAD in the AHAS
reaction, it does indicate that the cofactor appears to be too far removed from ThDP
(and substrates) to be a direct participant.
Early insights into understanding how bcaa feedback inhibition might influence
turnover through the RSU in plant AHAS were based on sequence and structural
homology to proteins, the structures of which had yielded to crystallographic
analysis. A common feature shared among all the AHAS RSUs is the presence of
an ACT domain that forms when the N termini of each subunit come together
as a dimer so as to create a site where small ligands can bind. The ACT family
of proteins are involved in a variety of regulatory functions, such as threonine
deaminase sensitivity to valine or isoleucine, and phosphoglycerate dehydrogenase
with sensitivity to serine. An unusual feature of the plant RSU polypeptide is a
sequence duplication similar to that found in the regulatory domain of threonine

deaminase [21]. The plant monomer, with its repeat of regulatory sequences, folds
to form the functional state equivalent to the regulatory dimer in the case of the
bacterial enzyme. By using the structure of threonine deaminase as template,
it was surmised that the plant RSU would be likely to adopt a similar domain
organization. Furthermore, as the repeat sequences in the plant subunit are only
about 40% identical with each other it was reasoned that, when folded, nonidentical
ligand binding sites would emerge. The resulting structural model of the putative
nonidentical ligand binding sites could explain certain features of bcaa inhibition,
such as the synergy between valine and leucine.
Following the solution of the ilvH RSU from E. coli, further progress has been
made in providing an understanding of which features of the subunit might influ-
ence the turnover and modulation of the holoenzyme [37]. The interchangeability of
the RSUs inducing an enhanced activity on the different isoenzymes suggests that
there is a common site on the larger catalytic dimer where the RSUs must reside
[30]. Although many RSU mutants have been investigated, it remains unclear as to
how the binding of the bcaas is sensed by the catalytic protomer. It seems certain
that inhibition by the bcaas does not result from inducing the RSU to dissociate
from the CSU; rather, the loss of turnover is most likely due to the conformation
of the CSU adopting a more relaxed state, similar to what occurs in the absence of
the smaller subunit. Further insights into the interactions between the RSUs and
CSUs await the solution of the complete holoenzyme structure.
2.1.3
Herbicides That Target AHAS
The discovery that different types of synthetic small organic compound inhibit
AHAS and cause plant death has contributed significantly to the attention garnered
by this enzyme. The first class of herbicides known to inhibit AHAS was the sul-
fonylureas (SUs) [38, 39]. The first commercial example of a SU was chlorsulfuron,
which was introduced by DuPont in 1982 under the trade name Glean®. This prod-
uct provided a highly effective control of dicotyledonous weeds in post-emergence
applications, with excellent selectivity towards wheat [40]. Almost simultaneously,
the research group at American Cyanamid discovered a structurally distinct family
of herbicides, the imidazolinones (IMIs), which were also shown to inhibit the
AHAS enzyme [41, 42]. Since then, three additional classes of AHAS-inhibiting
herbicides have been discovered and commercial products introduced: triazolopy-
rimidines (TPs) from Dow AgroSciences [43]; pyrimidinyl (thio)benzoates from
Kumiai [44, 45]; and sulfonylaminocarbonyltriazolinones from Bayer CropScience
[46]. By many measures, AHAS is a superb herbicidal target, as inhibitors of
different chemical types have proven to be among the most successful and widely
used herbicides. Indeed, to date more than 50 active ingredients have been com-
mercialized, satisfying a global annual market which is worth more than US $3.0
billion and which each year continues to increase, even in the face of greater resis-
tance among major weed species. The tremendous success of the AHAS-inhibiting
herbicides can be attributed to the compounds’ generally high bioefficacy, as well

as their low field application rates, selectivity to many agronomically important
crops, favorable environmental profiles, and ultra-low mammalian toxicity [47].
Before the mechanism of SU or imidazaolinone action was fully understood,
early mode-of-action studies on the SUs had revealed that the treatment of plants
with chlorsulfuron resulted in a rapid cessation of cell division [48]. A further key
observation was that the growth inhibitory effect on S. typhimurium due to the SU
herbicide, sulfometuron methyl, could be reversed by supplementing the growth
medium with isoleucine – a fact which pointed towards the bcaa pathway being the
disrupted biological process. The mode of action of SUs in bacteria was confirmed by
LaRossa and Schloss, who showed that the AHAS activity in extracts from wild-type
S. typhimurium could be completely inhibited by sulfometuron methyl [49]. These
findings were subsequently confirmed in plants by Ray, who reported IC50 -values
for the chlorsulfuron-mediated inhibition of the AHAS enzyme from several plant
species that ranged from 18.5 nM (in wheat) to 35.9 nM (in Johnsongrass) [50].
Subsequently, Chaleff and Mauvais, by using tissue cell cultures, isolated tobacco
mutants that were resistant to both chlorsulfuron and sulfometuron methyl, and
showed conclusively that inhibition of the AHAS enzyme was indeed the mode
of herbicidal action in plants [51]. At about the same time, Shaner et al. reported
that the phytotoxic effects of three IMI herbicides on corn tissue culture could
be reversed by the addition of valine, leucine, and isoleucine. These authors also
showed that IMIs were potent inhibitors of the AHAS enzyme from Zea mays, with
Ki values ranging from 1.7 to 12 μM [41].
2.1.4
Binding Site for AHAS-Inhibiting Herbicides
The inhibition of AHAS by herbicidal compounds is a time-dependent process that

is complex and not well understood [4, 52]. As bacterial AHAS II is most similar
to the enzyme in higher plants with respect to its sensitivity to various herbicides,
most enzymological studies on the effects of synthetic small molecule inhibitors
have been conducted using that particular variant [17, 53]. Early experiments
had shown that sulfometuron methyl exhibited a slow, tight-binding inhibition
of AHAS II from S. typhimurium, with an initial apparent Ki of 1.7 μM (50 mM
pyruvate), followed by a time-dependent increase in potency to a final Ki of 82 nM
(at equilibrium). Although sulfometuron methyl is known to bind to AHAS II in the
absence of pyruvate, it only forms the reversible, tight-binding complex observed
under turnover conditions, and is competitive with pyruvate at both initial and final
inhibition levels. Thus, while pyruvate is required for the slowly reversible form of
inhibition, it competes with sulfometuron methyl for binding to the enzyme. This
observation has been explained in the context of the AHAS reaction mechanism
by assuming that sulfometuron methyl binds most tightly to the enzyme following
addition of the ThDP-ylide to the first molecule of pyruvate and its decarboxylation
(Figure 2.1.2; steps 1–3) [54]. Evidence in support of this hypothesis was obtained
from chemical quench experiments performed with AHAS II using 14 C-labeled
pyruvate and ThDP. The results showed that the level of HEThDP obtained by
quenching steady-state reaction mixtures would be increased in the presence
of sulfometuron methyl. Thus, while the SU virtually eliminated the enzymatic
reaction, it increased the level of the HEThDP intermediate by inhibiting the
binding and condensation of the second molecule of pyruvate [55].
The IMI herbicides also exhibit complex interactions with AHAS. For example,
when enzyme activity was measured over an extended period in the presence of
various concentrations of imazapyr, the inhibition was increased with time. This
suggested that an equilibrium between the herbicide and AHAS was reached only
slowly, an effect that is characteristic of tight-binding inhibitors [56]. In contrast
to the SUs, the results of substrate–inhibitor studies with IMIs suggested that
inhibition by imazapyr would be uncompetitive with respect to pyruvate, and
implied that the synthetic molecule would bind to AHAS only after the formation
of a ternary enzyme–pyruvate–ThDP complex [57]. The fact that noncompetitive
binding has also been reported for the IMIs underscores the complexity of the
kinetics of AHAS inhibition [54], and may reflect the fact that the IMI molecule
binds further from the active site than does the SU molecule.
At an early stage, the lack of any obvious structural similarities among the AHAS
inhibitors and the substrates or intermediates in the reaction catalyzed by AHAS
suggested that the herbicides might not actually occupy the active site of the enzyme
and this, coupled with the differences in binding kinetics, led to speculation that
the various classes of herbicides bind to different, albeit overlapping, sites in the
enzyme [4]. Based on the similarities between AHAS and POX, and the fact that
the former enzyme requires FAD, even though the reaction it catalyzes does not
involve oxidation or reduction, Schloss et al. proposed that the SUs bind at a
site that is distinct from the active site, this being an evolutionary vestige of the
ubiquinone-binding site of the POX enzyme. This proposal was further supported
by the finding that ubiquinone-0 and ubiquinone-5 were reasonable competitive
inhibitors of SU binding [17].
Without a detailed 3-D model to work from, early attempts at elucidating
the herbicide-binding site of AHAS were based on the similarity of AHAS to
other ThDP-dependent enzymes for which X-ray crystallographic data existed. For
example, a herbicide-binding site structural model was postulated on the basis of
homology between AHAS and POX, and an IMI molecule positioned in the binding
pocket, by using structure–activity information [58]. A significant milestone that
greatly advanced the state of knowledge of herbicide binding was the report from
Pang et al. which described the crystal structure at 2.8 Å resolution of yeast AHAS
with bound chlorimuron ethyl, a commercial SU herbicide that is a potent inhibitor
of the enzyme [59]. This crystal structure showed that the overall features of the
AHAS•SU complex are quite similar to those of the free enzyme. The location
of ThDP at the interface of the α-domain of one monomer and the γ-domain
of a second monomer defines the enzyme active site that remains essentially
unchanged vis-à-vis the free enzyme. Chlorimuron ethyl is positioned near FAD,
which is in the same general location as in the free enzyme. However, the flavin
ring of FAD has been displaced by several angstroms, thus avoiding unfavorable
steric interactions with the herbicide molecule.
One noteworthy difference between the crystal structures of the AHAS•SU
complex and the free enzyme is that the volume of the protein in the region in
which the active site and the herbicide-binding site are located has been reduced.
A second, more significant, difference is that a new substrate access channel has
been formed in the AHAS•SU complex as a result of the ordering of two relatively
short sequences of amino acids near the active and herbicide-binding sites. As a
result, ThDP exposure is substantially reduced, with only the C-2 position of the
thiazolium ring readily accessible to solvent. Numerous hydrophobic interactions
between chlorimuron ethyl and amino acid residues along with four hydrogen
bonds to the molecule’s ‘‘bridge’’ (–SO2 NHCONH– moiety) anchor the SU in
the substrate access channel in such a way that the herbicide completely blocks
access to the enzyme active site. The authors showed, through molecular modeling,
that a cavity in the herbicide-binding site that is normally occupied by a single
water molecule will accommodate the reaction intermediate, HEThDP, with no
unfavorable interactions and a stabilizing hydrogen bond to the 4 -amino group of
the ThDP [59]. This structural feature is consistent with the hypothesis that SUs
bind most tightly to the AHAS enzyme following addition of the first pyruvate
molecule to ThDP and the displacement of carbon dioxide.
The crystal structures of four additional SUs bound to yeast AHAS were sub-
sequently reported by McCourt et al. [60]. The chemical structure of chlorsulfuron
and the key contact points with various amino acids in the binding site are shown
in Figure 2.1.4. While the conformations of all four bound SUs were similar, it
was possible to relate certain differences of the structural features of the molecules
to their respective binding affinities. For example, previous structure–activity
studies had demonstrated the importance of the substituent being located in the
ortho-position of the phenyl ring adjacent to the SU bridge in order to achieve
optimal herbicidal activity [39] (in the case of chlorsulfuron, this substituent is
‘‘chloro’’). The crystal structures show that the chlorine atom of chlorsulfuron does
not fit as tightly into the binding site as the carboxylic ester ortho groups of the other
three SUs. This observation could account for the 39-fold lower binding affinity
of chlorsulfuron for yeast AHAS, and would be consistent with the earlier finding
that the size of the ortho group is the most important attribute in determining the
potency of the SU-mediated inhibition of the enzyme [61]. The four SUs employed
in these studies differed somewhat in the nature of the extensive hydrophobic
interactions that each made with the highly conserved amino acid side chains
lining the substrate access channel. Mutation of several of the residues is known
to disrupt such interactions, and has been shown previously to confer resistance to
the four SUs, although considerable variability in resistance was evident [62]. It was
pointed out that, of the 13 amino acid residues which made contact with the SUs,
11 were highly conserved across bacterial, fungal, and plant AHAS sequences, and
this in turn suggested that they play important roles in the AHAS reaction.
Some 22 years after the introduction of the first commercial AHAS-inhibiting
herbicide, a preliminary crystal structure at 3.0 Å resolution of the AHAS catalytic
W586 W586
M582 M582
V583 V583
M354 G116 K251′ M354 G116 K251′
R380 F201′ A117′ R380 F201′

A117′
P192′ P192′
V191′ V191′
FAD FAD
D379 D379
A195′ A195′
(a) A200′ A200′
O O
O Pro 192′ O
C2M FAD
Val 191′ C3 C6 Met 354
NH 3.49
HN 3.86 3.87 NH
N Phe 201′
CH3 O
3.73 3.86 S
H3C
3.75 3.54 CH3
O
H2O NH
Asp 379 3.83(B) 3.77 (AC1), 3.81 (B,AC2)
O
HN O− O CH3
S Met 582
3.88(A)
O N 3.85(B)
O O CH3
3.84 S NH C NH N
Ala 195′ CH3
O O N CH3
HN 3.58 3.01
CH3 3.80 H3C
CH3 Val 583
O 3.04
(A)
HN O
Ala 200′ NH
2.99 2.92 O
H2O 16 contacts
H2N NH2
+ NH
+ Gly 116′ NH (3.48-3.88)
Lys 251′ NH2
HN NH
NH
Arg 380 O Trp 586
O
NH
(b)
O
Figure 2.1.4 Chlorsulfuron in the to nearby amino acids. Hydrophobic con-

herbicide-binding site of yeast AHAS. (a) The tacts are broken black lines and hydrogen
herbicide and nearby amino acids are shown bonds are broken blue lines. Prime numbers
as ball-and-stick models. The isoalloxazine denote residues from the other monomer.
ring of FAD is shown as a stick model; (b) Reproduced with permission from Ref. [60];
Key contact distances from chlorsulfuron © 1991, American Chemical Society.
subunit from a plant, A. thaliana, was reported by Pang et al. [63]. This was followed
two years later by crystal structures of the same enzyme complexed with five SUs
and one IMI at 2.5 and 2.8 Å resolution, respectively [64]. The latter achievement,
by McCourt et al., represented the first reported X-ray crystal structures of plant
protein–herbicide complexes. The overall structural features of the AHAS•SU and
E383 E383
R199′ D376 M200′ D376
K381 R199′
P197′ P197′ K381
D257′ D257′
G654 K256′ G654
K256′
S653 G655 G655
S653
Q260′ Q260′
W574 P652 W574 P652

M124′ M124′
Y579 F578 Y579 F578
(a) (b)
O
CH2CH3
O
O CH
SO2 3
H HN
HN N N Cl CH3
N
N CH3
O N
COOH
OMe
Chlorimuron ethyl (R )-Imazaquin
Figure 2.1.5 Herbicides bound to Arabidop- surfaces. Prime numbers denote residues
sis thaliana AHAS, showing blockage of the from the other monomer. Reproduced with
channel leading to the enzyme active site. permission from Ref. [64]; © 1992, National
The herbicides are shown as stick models, Academy of Sciences, USA.
and the residues lining the channel are gray
AHAS·IMI enzymes are similar, and include a region consisting of short amino
acid sequences in the vicinity of the active site that was hypothesized to become
more ordered in the presence of the inhibitors so as to form the substrate access
channel, in analogy to yeast AHAS. In addition, the conformations of the SU
molecules bound to the plant AHAS were seen to be similar to those in yeast.
Chlorimuron ethyl, bound in the AHAS substrate access channel, is shown in
Figure 2.1.5a. Here, the phenyl ring is located at the entrance to the channel,
with both the ortho-carboxylic ester group and the SU bridge pointing towards the
enzyme active site. The pyrimidine ring is barely visible, and is inserted deeper
into the active site. Key interactions with several conserved amino acid residues are
apparent, including Trp574, Pro197, and one that is not present in yeast, Ser653.
In Figure 2.1.5b, the IMI imazaquin is shown positioned in the AHAS substrate
access channel, with the IMI ring directed towards the enzyme active site, as is the
carboxylate substituent in the 3-position of the quinoline ring. Although racemic
imazaquin was used in the crystallization, only the (R)-enantiomer was observed
bound to the enzyme; this is consistent with the known higher herbicidal efficacy
of this isomer versus the (S)-enantiomer [57].
The crystal structures provide excellent insight into the known higher AHAS
binding affinity of SUs compared to IMIs. For example, the apparent Ki values for
the inhibition of AHAS from A. thaliana for chlorimuron ethyl and chlorsulfuron
are 10.8 and 54.6 nM, respectively, while that for imazaquin is 3 μM [65]. These
differences can be attributed to the significantly greater number of van der Waal
contacts and hydrogen bonds between the SUs and the enzyme versus imazaquin,
and also by the fact that the SUs are buried deeper in the access channel and
positioned closer to ThDP at the active site. While many of the residues interacting
with the SUs and imazaquin are the same, there are six that make contact only
with SUs and two that interact only with imazaquin. Thus, the crystal structures
confirmed earlier supposes that the two classes of herbicides occupy partially
overlapping, but different, binding sites. Two recent structures of the plant enzyme
with bound SUs that are monosubstituted only on the heterocycle closest to ThDP,
reveal that the cofactor is bound as the HEThDP intermediate [66].
2.1.5
Molecular Basis for Resistance to AHAS Inhibitors
Several plants and cultured plant cells that are resistant to AHAS-inhibiting
herbicides have been created by using both conventional mutation breeding and
tissue culture cell selection. Mutants resistant to chlorsulfuron and sulfometuron
methyl were first isolated from cultured cells of Nicotiana tabacum that had been
grown in the presence of one of the herbicides [67]. Crosses of fertile plants
from several isolates established that resistance resulted from single dominant or
semidominant nuclear mutation, and that the isolates were cross-resistant to both
compounds. The cosegregation of resistance to the herbicides demonstrated that
both resulted from the same, or at least closely linked, mutations. Tobacco plants
regenerated from the sulfometuron methyl-derived mutant cell lines showed a
resistance to high concentrations of chlorsulfuron.
Cloned yeast and bacterial genes were used to investigate the molecular basis
of resistance to the SU herbicides [68]. Spontaneous mutations that conferred
resistance to sulfometuron methyl were obtained in cloned genes for AHAS from
S. cerevisiae and E. coli. The yeast mutant, Pro192Ser, resulted in reduced levels of
enzyme activity, a reduced sensitivity to sulfometuron methyl, and an unaltered
resistance to feedback inhibition from valine. The bacterial mutant, Ala26Val,
resulted in unaltered levels of enzyme activity, a greatly reduced sensitivity to
sulfometuron methyl, and a slightly reduced sensitivity to valine.
In yeast AHAS, spontaneous mutations at ten separate sites have each been
shown to confer resistance to SUs [69, 70]. The X-ray crystal structure of yeast
AHAS bound with chlorimuron ethyl revealed that nine of those residues make
direct contact with the herbicidal molecule [59]. Subsequently, the effects of several
mutations on SU sensitivity were studied in the context of molecular interactions
in the herbicide active site. For example, the crystal structure showed that the
indole ring of Trp586 is involved in aromatic π-orbital stacking interactions with
the pyrimidine ring of chlorimuron ethyl. The mutation Trp586Leu in yeast AHAS
is known to result in a more than 6000-fold reduction in sensitivity to chlorimuron

ethyl, which can be understood in terms of the total disruption of the aromatic ring
interactions.
A systematic investigations was conducted in which 10 active mutants of yeast
AHAS were constructed by mutagenesis, and the resultant enzymes evaluated for
their resistance to six SU and three IMI herbicides. The results were interpreted in
terms of the herbicide-binding site that was revealed by the X-ray crystal structure
of AHAS from S. cerevisiae [62]. All 10 mutants were resistant to some degree to the
six SUs, although the levels of resistance spanned a range of almost 104 , and there
was also considerable variability in several mutants. Although, the most consistent
and highest levels of resistance were observed with Trp586Leu, the Pro192Ser
mutant also displayed relatively high levels of resistance to all six SUs; moreover,
the crystal structure of yeast AHAS supported this observation, in that Pro192
interacts with the phenyl rings of all of the bound herbicides. Eight of the mutants
were resistant to the IMI, imazethapyr, though several of these were only barely
affected and Asp379Asn was more sensitive than the wild-type enzyme. As shown
schematically in Figure 2.1.6, the positions in AHAS from various sources where
mutations are known to confer resistance to one or more herbicides are distributed
across the three domains of the protein [4, 7, 71]. At some sites, virtually any amino
acid substitution will confer resistance, whilst at other sites only a few substitutions
are permitted.
Genes that specify herbicide-resistant forms of the AHAS enzyme were isolated
from mutant N. tabacum, sequenced, and characterized [72]. It was shown that
a single amino acid change, Pro196Gln, in one of two distinct tobacco AHAS
genes conferred resistance to SU herbicides. In the other tobacco AHAS gene, a
Transit seq α-domain β-domain γ- domain
1 100 200 300 400 500 600
A. thaliana
A122 P197 W574 S653
M124 R199
S. cerevisiae
G116 P192 K251 M354 V583
A117 A200 D379 W586
F590
E. coli II
A26 V99 A108 M460
W464
Figure 2.1.6 AHAS mutations conferring where spontaneous or induced mutations

herbicide resistance. The arrows point to result in a herbicide-insensitive enzyme.
positions in the sequences of AHAS from Colors designate substitutions occurring in
plant (A. thaliana), yeast (S. cerevisiae), and more than one species.
bacterial (E. coli, isoenzyme II) sources,
double mutation of Pro196Ala and Trp573Leu resulted in significantly enhanced

resistance to SUs. Transgenic plants carrying these mutant genes are highly
resistant to chlorsulfuron treatments. Hattori et al. showed that a single mutation
in plant AHAS can confer resistance to multiple classes of AHAS-inhibiting
herbicides [73, 74]. Thus, tobacco lines transformed with a plant AHAS gene
specifying a single mutation, Trp557Leu, were strongly resistant to SUs, TPs, and
IMIs. The tryptophan residue, as well as the Pro, is conserved in virtually all
wild-type AHAS proteins (Figure 2.1.6).
The enhanced resistance of the Pro and Trp double mutation is based on
their location in the functional dimeric state of the enzyme. As described above,
the active site is composed of residues of the α-domain of one subunit and
residues of the γ-domain of a second subunit. The relative locations in the plant
enzyme of the N-terminal Pro in the α-domain and the Trp of the γ-domain at
different positions of the same active site access channel are shown in Figure 2.1.7.
Mutations at these two positions results in the loss of two regions of interaction
with the phenyl and pyrimidine rings of the SU molecule and, thus, a loss of
inhibition. It is not surprising that similar mutations in these two positions, when
introduced into other plant AHAS genes, impart high levels of resistance to the
SUs when the gene is introduced back into the same plant [75]. The Pro and
Trp dual mutation in the CSU of soybean AHAS translates well, and provides
the plant with a broad tolerance to commercial SU herbicides, as well as to other
classes of AHAS inhibitor. This highly resistant variant of the enzyme (HRA,
highly resistant ALS), in combination with a glyphosate tolerance trait (glyphosate
acetyl transferase; see e.g., Ref. [76]), forms the basis of the Optimum-GAT®
concept.
2.1.6
Resistance to AHAS-Inhibiting Herbicides in Weeds
Certain characteristics of the AHAS target have played roles in the development
of weeds that are resistant to herbicides which inhibit the enzyme: (i) target-based
resistance is inherited as a single, semidominant trait that is carried on a nuclear
gene; (ii) AHAS is the single site of action; (iii) there are multiple sites in AHAS
that can be mutated to confer resistance; and (iv) those mutations do not play a
critical role in catalysis thus the enzyme possesses full activity. All of these factors,
when combined, lead to resistant weeds that are both fit and vigorous [77]. Certain
characteristics of the molecules themselves have also contributed to resistance
development, such as their potency and the relatively long soil residual of some of
the early products. The first examples of resistance to chlorsulfuron that occurred
in the field were identified during 1987 in the United States. The fields which
contained the resistant weeds had been treated continuously with chlorsulfuron for
five years, but by 1992 numerous examples of weeds were found that had developed
resistance to the AHAS-inhibiting herbicides. Many additional observations of
weeds resistant to AHAS-targeted herbicides have since been reported, such that
today there are 107 known weed species with a confirmed resistance to one or more
γ-domain
α-domain
α-domain
γ-domain
(a)
Pro
Trp
(b)
Figure 2.1.7 The catalytic dimer of Ara- (b) A stereo diagram of the SU binding site
bidopsis thaliana AHAS (1YI1.pdb), showing occupied by tribenuron methyl, relative to
the interaction of the α-domain with the the ThDP cofactor and the conserved Pro
γ-domain of the second subunit. (a) The and Trp residues.
location of ThDP as a space-filled molecule;
of the five chemical classes of these compounds [78]. By 1998, the AHAS-inhibiting
herbicides had surpassed all other classes of herbicides in terms of the number of
weed species for which at least one resistant population had been reported. Further
information on the resistance of weeds to AHAS-inhibiting herbicides is provided
in some excellent reviews (e.g., see Refs [76, 79, 80]).
More recently, the results of various studies have confirmed that AHAS
resistance-conferring mutations can have subtle effects on plant growth and de-
velopment, but do not consistently reduce plant fitness. For example, the catalytic
efficiencies of AHAS enzymes isolated from both resistant and susceptible biotypes
of L. serriola, K. scoparia, S. iberica, S. media, and L. perenne have been shown to
be virtually identical [79, 81]. In weed biotypes where the mechanism of evolved
resistance has been confirmed, the majority has been due to a reduced sensitivity
of AHAS to the herbicide. One exception here is Lolium rigidum, which first de-
veloped a metabolism-based resistance to the ACCase inhibitor, diclofop-methyl,
and then showed a metabolism-based cross-resistance to SUs and other classes of
herbicide [82]. The identities of specific mutations in weed species that had evolved
resistance to AHAS inhibitors were first determined by Guttieri et al. [83]. In this
case, a Pro173His mutation was identified in resistant L. serriola, and a Pro173Thr
mutation was identified in K. scoparia. Mutations of five amino acid residues are
known to be involved in causing resistant weed species: Ala122, Pro197, Ala205,
Trp574, and Ser653 (A. thaliana numbering) [84]. These five amino acid residues
are highly conserved across all known plant AHAS sequences [80]. Multiple substi-
tutions have been identified for Pro197 with resistance primarily to SUs and, to a
lesser extent, to TPs and IMIs. The mutation, Pro197Thr, results in resistance to at
least one herbicide from all five classes of AHAS inhibitors in Chrysanthemum coro-
narium, although the levels are only moderate for IMIs and TPs. The substitution
Pro197Leu confers high levels of resistance to four classes of AHAS inhibitor in
Amaranthus retroflexus, while six different amino acid substitutions in Pro197 have
been linked to resistance in K. scoparia alone. Substitutions of Ala122 or Ser653
result in a resistance to IMI herbicides, but not to SU herbicides. The mutation
Trp574Leu confers resistance to several plant species, with the levels of resistance
all being high against IMIs, SUs, and TPs. No other evolved Trp574 mutations
have been reported. A similar study of homozygous populations of L. rigidum,
which carry mutations at the conserved Pro or Trp positions of AHAS, allowed an
investigation of the effects of mutations on both enzyme functionality and plant
growth [85].
The patterns of mutation that confer an evolved resistance to AHAS-inhibiting
herbicides are quite understandable in light of the results of previously conducted
site-directed mutagenesis studies, and of the X-ray crystal structures of AHAS from
yeast and A. thaliana. In Figure 2.1.5, for example, it can be seen that Trp574
is located strategically at the opening of the herbicide-binding site, and interacts
extensively with both chlorimuron ethyl and imazaquin. A leucine substitution will
alter many of those interactions and also modify the shape of the substrate access
channel [64]. It can also be seen in Figure 2.1.5 that the Pro197 residue contacts
the phenyl ring of chlorimuron ethyl, but is further removed from imazaquin. This
structural feature explains why almost any Pro197 replacement will hinder SU
access to the channel and confer resistance, whilst only the most bulky amino acid
substitutions will displace IMIs. Conversely, Ser653 lies in close proximity to the
quinoline ring of imazaquin, so that any replacement with a bulky amino acid would
be expected to interfere with the compound’s binding in the channel and confer
resistance, though this residue does not interact as strongly with chlorimuron ethyl.
Although there is a wide variability in cross-resistance to the different classes of
AHAS-inhibiting herbicides, resistance to one compound within a particular class
does not necessarily guarantee cross-resistance to all members of that class. This
is particularly true of the SUs, for which differential resistance has been reported
in several species [86–89].
2.1.7
Engineered Resistance to AHAS-Inhibiting Herbicides in Crops
Despite the evolution of natural resistance to SUs, these products – along with
other classes of AHAS-targeted herbicides – remain among the most efficacious
and widely used weed control agents worldwide [80], accounting for approximately
16% of the total herbicide market in 2007 [90]. Their commercial longevity has
been further assured by the planned introduction of crops with an engineered
resistance to SUs. The concept of using a mutant variant of AHAS in plants
to achieve resistance to AHAS-directed herbicides has been established in the
marketplace for some time; examples include the Clearfield® trait in corn, which
provides tolerance to IMI herbicides, and STS® soybeans, which are tolerant to
a specific SU. Consequently, in order to achieve a broader resistance to most
commercial AHAS herbicides – and thus a wider application – different versions
of the enzyme, engineered specifically to offer these characteristics, have been
introduced into major crops by transgenic means. As noted above, the HRA double
mutant is insensitive to almost all AHAS inhibitors, and in transgenic plants
provides excellent tolerance to commercial AHAS herbicides. In combination with
other herbicide tolerance traits, such as glyphosate, another powerful dimension is
now available for managing invasive and noxious weeds.
Clearly, the ability to increase natural resistance to any herbicide – and, in
particular, to a class of herbicides that is so effective – represents a major concern.
In cases where resistance to AHAS inhibitors has been selected, this has typically
occurred after five to eight years of the repeated (if not continuous) use of
herbicides with such a mode of action. Resistance has generally not been selected
where AHAS-inhibiting herbicides have been used as part of an integrated program
[91]. Resistance can be effectively managed by following several well-documented
best practices, such as rotating herbicides or by using mixtures of herbicides with
different modes of action across the same spectrum of weeds [79, 92–94]. Within the
past few years, many new active ingredients have been introduced to target AHAS,
along with innovations in delivery methods. Today, with the advent of homogeneous
blends, customized mixtures of two or more different granular herbicides are
possible [95, 96]. In the case of a combined approach, the judicious use of
glyphosate along with AHAS inhibitors may provide a powerful tool for managing
resistance to both classes of herbicide. By integrating stacked technologies such as
Optimum-GAT® into well-planned weed management programs, growers should
be able to use the environmentally friendly AHAS-inhibiting herbicides for many
years to come to achieve an effective, broad-spectrum weed control.
Acknowledgments
The authors are grateful to Drs Hugh Brown, Josephine Cotterman, Ronald
Duggleby, Jerry Green, Jennifer McCourt, Robert Pasteris, and Leonard Saari for
their critical review of the manuscript, to Drs Ya-Jun Zheng and John Andreassi
References 47
for assistance with Figures 2.1.3–2.1.7, to Mr Thomas Dougherty for access to the
global sales figures for AHAS-inhibiting herbicides, and to Ms Debbie Carman and
Ms Susan Titter for assistance with the literature references.
References
1. Umbarger, H.E. (1978) Annu. Rev. 15. Grimminger, H. and Umbarger, H.E.
Biochem., 47, 533–606. (1979) J. Bacteriol., 137, 846–853.
2. Chipman, D.M., Barak, Z., and Schloss, 16. Schloss, J.V., Van Dyk, D.E., Vasta, J.F.,
J.V. (1998) Biochim. Biophys. Acta, 1385, and Kutny, R.M. (1985) Biochemistry, 24,
401–419. 4952–4959.
3. Duggleby, R.G. and Pang, S.S. (2000) 17. Schloss, J.V., Ciskanik, L.M., and Van
J. Biochem. Mol. Biol., 33, 1–36. Dyk, D.E. (1988) Nature, 331, 360–362.
4. Duggleby, R.G., Guddat, L.W., and 18. Pang, S.S., Duggleby, R.G., and Guddat,
Pang, S.S. (2004) Thiamine: Catalytic L.W. (2002) J. Mol. Biol., 317, 249–262.
Mechanisms in Normal and Disease States, 19. Eoyang, L. and Silverman, P.M. (1984)
vol. 11, Marcel Dekker, New York, pp. J. Bacteriol., 157, 184–189.
251–274. 20. Ibdah, M., Bar-Ilan, A., Livnah, O.,
5. Duggleby, R.G. (2006) Amino Acids, 31, Schloss, J.V., Barak, Z., and Chipman,
73–210. D.M. (1996) Biochemistry, 35,
6. Duggleby, R.G., McCourt, J.A., and 16282–16291.
Guddat, L.W. (2008) Plant Physiol. 21. Lee, T.-Y. and Duggleby, R.G. (2001)
Biochem., 46, 309–324. Biochemistry, 40, 6836–6844.
7. Pang, S.S., Duggleby, R.G., Schowen, 22. Squires, C.H., DeFelice, M.,
R.L., and Guddat, L.W. (2004) J. Biol.
Devereux, J., and Calvo, J.M. (1983)
Chem., 279, 2242–2253.
Nucleic Acids Res., 11, 5299–5313.
8. McCourt, J.A. and Duggleby, R.G. (2005)
23. Lawther, R.P., Calhoun, D.H., Adams,
Trends Biochem. Sci., 30, 222–225.
C.W., Hauser, C.A., Gray, J., and
9. Wikner, C., Meshalkina, L., Nilsson,
Hatfield, G.W. (1981) Proc. Natl Acad.
U., Nikkola, M., Lindqvist, Y.,
Sci. USA, 78, 922–925.
Sundstrom, M., and Schneider,
24. Wek, R.C., Hauser, C.A., and Hatfield,
G. (1994) J. Biol. Chem., 269,
G.W. (1985) Nucleic Acids Res., 13,
32144–32150.
3995–4010.
10. Kern, D., Kern, G., Neef, H.,
Tittmann, K., Killenberg-Jabs, M., 25. Friden, P., Donegan, J., Mullen, J.,
Wikner, C., Schneider, G., and Hübner, Tsui, P., Freundlich, M., Eoyang, L.,
G. (1997) Science, 275, 67–70. Weber, R., and Silverman, P.M. (1985)
11. Gollop, N., Damri, B., Barak, Z., and Nucleic Acids Res., 13, 3979–3993.
Chipman, D.M. (1989) Biochemistry, 28, 26. Mendel, S., Vinogradov, M.,
6310–6317. Vyazmensky, M., Chipman, D.M.,
12. Barak, Z., Chipman, D.M., and and Barak, Z. (2003) J. Mol. Biol., 325,
Gollop, N. (1987) J. Bacteriol., 169, 275–284.
3750–3756. 27. Falco, S.C. and Dumas, K.S. (1985)
13. Tittmann, K., Golbik, R., Uhlemann, K., Genetics, 109, 21–35.
Khailova, L., Schneider, G., Patel, M., 28. Poulsen, C. and Stougaard, P. (1989)
Jordan, F., Chipman, D.M., Duggleby, Eur. J. Biochem., 185, 433–439.
R.G., and Hübner, G. (2003) Biochem- 29. Pang, S.S. and Duggleby, R.G. (1999)
istry, 42, 7885–7891. Biochemistry, 38, 5222–5231.
14. Jana, G., Jimenez, V., Villa-Freixa, J., 30. Vyazmensky, M., Zherdev, Y.,
Prat-Resina, X., Delgado, E., and Slutzker, A., Belenky, I., Kryokov, O.,
Alderete, J. (2010) Proteins - Struct. Barak, Z., and Chipman, D.M. (2009)
Funct. Bioinform., 78, 1774–1788. Biochemistry, 48, 8731–8737.
31. Miflin, B.J. (1974) Plant Physiol., 54, 48. Ray, T.B. (1982) Pestic. Biochem. Physiol.,
550–555. 17, 10–17.
32. Jones, A.V., Young, R.M., and Leto, K. 49. LaRossa, R.A. and Schloss, J.V. (1984)
(1985) Plant Physiol., 77, S293. J. Biol. Chem., 259, 8753–8757.
33. Mazur, B.J., Chui, C.-F., and Smith, J.K. 50. Ray, T.B. (1984) Plant Physiol., 75,
(1987) Plant Physiol., 85, 1110–1117. 827–831.
34. Hershey, H.P., Schwartz, L.J., Gale, J.P., 51. Chaleff, R.S. and Mauvais, C.J. (1984)
and Abell, L.M. (1999) Plant Mol. Biol., Science, 224, 1443–1445.
40, 795–806. 52. Kishore, G.M. and Shah, D.M. (1988)
35. Pang, S.S., Guddat, L.W., and Duggleby, Annu. Rev. Biochem., 57, 627–663.
R.G. (2001) Acta Crystallogr., D, 57, 53. Schloss, J.V. and Van Dyk, D.E. (1988)
1321–1323. Methods Enzymol., 166, 445–454.
36. Tse, J.M. and Schloss, J.V. (1993) Bio- 54. Schloss, J.V. and Aulabaugh, A. (1990)
chemistry, 32, 1039–10403. in Biosynthesis of Branched Chain Amino
37. Kaplun, A., Vyazmansky, M., Acids (eds Z. Barak, D.M. Chipman, and
Zherdev, Y., Belenky, I., Slutzker, J.V. Schloss), VCH Verlagsgesellschaft,
A., Mendel, S., Barak, Z., Weinheim, pp. 329–356.
and Chipman, D.M. (2006) 55. Schloss, J.V. and Ciskanik, L.M. (1985)
J. Mol. Biol., 357, 951–963. Biochemistry, 24, 3357 (abstract).
38. Beyer, E.M., Duffy, M.J., Hay, J.V., and 56. Muhitch, M.J., Shaner, D.L., and
Schlueter, D.D. (1988) Herbicides: Chem- Stidham, M.A. (1987) Plant Physiol.,
istry, Degradation, and Mode of Action,
83, 451–456.
Marcel Dekker, New York, pp. 117–189.
57. Stidham, M.A. and Singh, B.K. (1991) in
39. Levitt, G. (1991) in Synthesis and Chem-
The Imidazolinone Herbicides (eds D.L.
istry of Agrochemicals II (eds D.R. Baker,
Shaner and S.L. O’Connor), CRC Press,
J.G. Fenyes, and W.K. Moberg), Ameri-
Boca Raton, FL, pp. 71–90.
can Chemical Society, Washington, DC,
58. Ott, K.-H., Kwagh, J.-G., Stockton, G.W.,
pp. 16–31.
Sidorov, V., and Kakefuda, G. (1996)
40. Sweetser, P.B., Schow, G.S., and
J. Mol. Biol., 263, 359–368.
Hutchison, J.M. (1982) Pestic. Biochem.
59. Pang, S.S., Guddat, L.W., and Duggleby,
Physiol., 17, 18–23.
R.G. (2003) J. Biol. Chem., 278,
41. Shaner, D.L., Anderson, P.C., and
7639–7644.
Stidham, M.A. (1984) Plant Physiol., 76,
545–546. 60. McCourt, J.A., Pang, S.S., Guddat, L.W.,
42. Shaner, D.L. and O’Connor, S.L. (eds) and Duggleby, R.G. (2005) Biochemistry,
(1991) The Imidazolinone Herbicides, 44, 2330–2338.
CRC Press, Boca Raton, FL. 61. Andrea, T.A., Artz, S.P., Ray, T.B.,
43. Kleschick, W.A., Gerwick, B.C., Carson, and Pasteris, R.J. (1992) in Rational
C.M., Monte, W.T., and Snider, Approaches to Structure, Activity, and
S.W. (1992) J. Agric. Food Chem., 40, Ecotoxicology of Agrochemicals (eds W.
1083–1085. Draber and T. Fujita), CRC Press, Boca
44. Shimizu, T. (1997) J. Pestic. Sci., 22, Raton, FL, pp. 373–395.
245–256. 62. Duggleby, R.G., Pang, S.S., Yu, H., and
45. Shimizu, T., Nakayama, I., Guddat, L.W. (2003) Eur. J. Biochem.,
Nagayama, K., Miyazawa, T., and Nezu, 270, 2895–2904.
Y. (2002) in Herbicide Classes in Develop- 63. Pang, S.S., Guddat, L.W., and Duggleby,
ment (eds P. Böger, K. Wakabayashi, and R.G. (2004) Acta Crystallogr., D, 60,
K. Hirai), Springer-Verlag, Berlin, pp. 153–155.
1–41. 64. McCourt, J.A., Pang, S.S., King-Scott, J.,
46. Pontzen, R. (2002) Pflanz.-Nachr. Bayer, Guddat, L.W., and Duggleby, R.G.
55, 37–52. (2006) Proc. Natl Acad. Sci. USA, 103,
47. Russell, M.H., Saladini, J.L., and 569–573.
Lichtner, F. (2002) Pestic. Outlook, 13, 65. Chang, A.K. and Duggleby, R.G. (1998)
166–173. Biochem. J., 333, 765–777.
References 49
66. Wang, J.G., Lee, P.K., Dong, Y.H., Pang, 80. Tranel, P.J. and Wright, T.R. (2002)
S.S., and Duggleby, R.G. (2009) FEBS J., Weed Sci., 50, 700–712.
276, 1282–1290. 81. Holt, J.S. and Thill, D.C. (1994) in Her-
67. Chaleff, R.S. and Ray, T.B. (1984) Sci- bicide Resistance in Plants: Biology and
ence, 223, 1148–1151. Biochemistry (eds S.B. Powles and J.A.M.
68. Yadav, N., McDevitt, R.E., Bernard, S., Holtum), CRC Press, Boca Raton, FL,
and Falco, S.C. (1986) Proc. Natl Acad. pp. 299–316.
Sci. USA, 83, 4418–4422. 82. Christopher, J.T., Powles, S.B., and
69. Falco, S.C., McDevitt, R.E., Chui, C.-F., Holtum, J.A.M. (1992) Plant Physiol.,
Hartnett, M.E., Knowlton, S., Mauvais, 100, 1909–1913.
C.J., Smith, J.K., and Mazur, B.J. (1989) 83. Guttieri, M.J., Eberlein, C.A.,
Dev. Ind. Microbiol., 30, 187–194. Mallory-Smith, D.C., Thill, D.C., and
70. Bedbrook, J.R., Chaleff, R.S., Falco, S.C., Hoffman, D.L. (1992) Weed Sci., 40,
Mazur, B.J., Somerville, C.R., and Yadav, 670–677.
N.S. (1991) U.S. Patent 5,013,659. 84. Tranel, P.J., Wright, T.R., and
71. Mazur, B.J. and Falco, S.C. (1989) Annu. Heap, I.M. ALS Mutations from
Rev. Plant Physiol. Plant Mol. Biol., 40, Herbicide-resistant Weeds. (Online
441–470. Internet), http://www.weedscience.com
72. Lee, K.Y., Townsend, J., Tepperman, J., (accessed 10 February 2006).
Black, M., Chui, C.F., Mazur, B., 85. Yu, Q., Han, H., Vila-Aiub, M.M., and
Dunsmuir, P., and Bedbrook, J. (1988) Powles, S.B. (2010) J. Exp. Bot., 61,
EMBO J., 7, 1241–1248. 3925–3934.
73. Cole, D.J. and Rodgers, M.W. (2000) 86. Devine, M.D., Marles, M.A.S., and Hall,
L.M. (1991) Pestic. Sci., 31, 273–280.
in Herbicides and Their Mechanisms of
87. Hart, S.E., Saunders, J.W., and
Action (eds A. Cobb and R.C. Kirkwood),
Penner, D. (1993) Weed Sci., 41,
Sheffield Academic Press, Sheffield, pp.
317–324.
239–278.
88. Saari, L.L., Cotterman, J.C., Smith,
74. Hattori, J., Brown, D., Mourad, G.,
W.F., and Primiani, M.M. (1992) Pestic.
Labbé, H., Ouellet, T., Sunohara, G.,
Biochem. Physiol., 42, 110–118.
Rutledge, R., King, J., and Miki, B.
89. Sibony, M., Michel, A., Haas, H.U.,
(1995) Mol. Gen. Genet., 246, 419–425.
Rubin, B., and Hurle, K. (2001) Weed
75. Green, J.M., Hale, T., Pagano, M.A.,
Res., 41, 509–522.
Andreassi, J., and Gutteridge, S. (2009)
90. McDougall, P. (2008) Global Herbicide
Weed Sci., 57, 142–148. Product Summary.
76. Green, J.M. (2007) Weed Technol., 21, 91. Shaner, D.L., Feist, D.A., and Retzinger,
547–558. E.J. (1997) Pestic. Sci., 51, 367–370.
77. Hartnett, M.E., Chui, C.-F., Mauvais, 92. Shaner, D.L. and Heap, I. (2002) in
C.J., McDevitt, R.E., Knowlton, S., Agrochemical Resistance, ACS Sym-
Smith, J.K., Falco, S.C., and Mazur, posium Series, Vol. 808, American
B.J. (1990) in Managing Resistance to Chemical Society, Washington, DC, pp.
Agrochemicals (eds M.B. Green, H.M. 161–167.
Le Baron, and W. K. Moberg), American 93. Shaner, D.L. (1999) J. Weed Sci. Technol.,
Chemical Society, Washington, DC, pp. 44, 405–411.
459–473. 94. Preston, C. and Mallory-Smith, C.A.
78. Heap, I.M. The International Survey (2001) in Herbicide Resistance and
of Herbicide Resistant Weeds. (Online World Grains (eds S.B. Powles and
Internet), http://www.weedscience.com D.L. Shaner), CRC Press, Boca Raton,
(accessed 22 November 2010). FL, pp. 23–60.
79. Saari, L.L., Cotterman, J.C., and Thill, 95. Geigle, W.L. and Gleich, S.I. (2001) US
D.C. (1994) in Herbicide Resistance Patent 6,270,025.
in Plants (eds S.B. Powles, J.A.M. 96. Green, J.M., Hazel, C.B., Forney, D.R.,
Holtum), CRC Press, Boca Raton, FL, and Pugh, L.M. (2008) Pest. Manage.
pp. 83–139. Sci., 64, 332–339.
2.2
Newer Sulfonylureas
Oswald Ort
2.2.1
Introduction
Most commercial sulfonylurea herbicides are characterized by the typical sul-

fonylated urea bridge connecting a nitrogen-containing heterocycle with an ortho-
substituted aryl or heteroaryl moiety (Figure 2.2.1). To date, sulfonylurea herbicides
have been developed and commercialized worldwide in over 80 countries, for use
with all major agronomic crops, and for many specialty uses, including range-
land/pasture, forestry, and vegetation management applications.
Herbicidal sulfonylureas have a unique mode of action, in that they interfere with
a key enzyme required for plant cell growth known as acetohydroxyacid synthase
(AHAS; EC 2.2.1.6) [1–3] (see Section 2.1.1). AHAS is the enzyme responsible for
the synthesis of the branched-chain amino acids valine, leucine, and isoleucine.
The inhibition of this enzyme disrupts the plant’s ability to manufacture proteins,
with such disruption leading subsequently to the cessation of all cell division and
eventual death of the plant.
The visible signs of herbicide action following the post-emergent application
of sulfonylurea herbicides are an almost immediate arrest of growth, followed by
leaf yellowing (chlorosis), a stimulation of anthocyanin production (leading to the
typical reddish coloration of weed leaves) and, finally, to progressive shoot death.
Depending on the weed species and environmental conditions, plant death will
usually occur between 7 and 20 days after herbicide application.
Since the initial discovery of the first sulfonylurea herbicides by George Levitt
at Du Pont in 1975, this compound class has attracted – and, indeed, continues to
O
R-(X) SO2 N C N Het
H H
typical sulfonylurea bridge
O
OCH3 CH3 Cl OCH3
O N O N
SO2 N C N SO2 N C N N
H H H H
N N
CH3 CH3
Sulfometuron-methyl (DuPont) Chlorsulfuron (DuPont)
Figure 2.2.1 First-generation sulfonylurea herbicides.

attract – very high interest, as well as encouraging much activity within the domain
of agrochemical research. Such continued research interest can largely be attributed
to the fact that the herbicidal activity levels demonstrated by this class of compounds
remain unsurpassed today, with the most active compounds capable of controlling
undesired vegetation at application rates below 10 g a.i. ha−1 (= 1 mg a.i. m−2 ). This,
in turn, then contributes to a reduction in environmental burden by the replacement
of older, higher application-rate herbicides, and also provides an attractive return
on investment to both the farmer and the producing company. In addition, the
favorable environmental properties and low acute mammalian toxicology shown by
the sulfonylureas usually provide a large margin of safety with regards to ecological
and toxicological effects (cf. Tables 2.2.1 and 2.2.2).
The aim of this chapter is to provide an overview of the sulfonylurea herbicides
that either have been introduced to the market since 1995, or are currently in
their later stages of development. These include flupyrsulfuron-methyl-sodium,
sulfosulfuron, iodosulfuron-methyl-sodium, mesosulfuron-methyl, tritosulfuron,
monosulfuron, and monosulfuron-ester for use in cereals; ethoxysulfuron, az-
imsulfuron, cyclosulfamuron, flucetosulfuron, orthosulfamuron, propyrisulfuron,
and metazosulfuron in rice; foramsulfuron in maize; oxasulfuron in soybeans; and
trifloxysulfuron-sodium in sugarcane and cotton.
Table 2.2.1 Acute toxicity of the sulfonylureas in birds and

water-borne organisms (LD50 , LC50 , or EC50 ).
Compound Birdsa (LD50 ; Fish (LC50 ; 96 h, Daphnia magna (EC50 ;

mg kg−1 ) mg l –1 ) 48 h, mg l –1 )
Azimsulfuron >2250 >154b 941

Cyclosulfamuron 1880 8 9
Ethoxysulfuron >2000 78.4 307
Flucetosulfuron NA >10c >10
Flupyrsulfuron >2250 470 721
Foramsulfuron >2000 >100 >100
Iodosulfuron >2000 >100 >100
Mesosulfuron >2000 >100 >100
Orthosulfamuron >2000 122b 105
Oxasulfuron >2250 >100 >89.4
Sulfosulfuron >2250 >91 >96
Trifloxysulfuron >2000 >120c >108
Tritosulfuron >2000 >100 >100
a
Mallard duck/Bobwhite quail.
b
Oncorhynchus mykiss.
c
Cyprinus carprio.
LD50 : lethal dose, 50% in mg kg−1 ; LC50 : lethal concentration, 50% in mg l−1 ;
EC50 : half maximal effective concentration in mg l−1 ; NA: Not available.
Table 2.2.2 Acute toxicity of the sulfonylureas in the rat (LD50 or LC50 ).
Compound Oral (LD50 ; Dermal (LD50 ; Inhalation (LC50 ;

mg kg –1 ) mg kg –1 ) mg l –1 )
Azimsulfuron >5000 >2000 >5.94 × 10 –3

Cyclosulfamuron >5000 >4000a >5.2
Ethoxysulfuron >3270 >4000 3.6
Flucetosulfuron >5000 >2000 >5.0
Flupyrsulfuron >5000 >2000 >5.8
Foramsulfuron >5000 >2000 >5.0
Iodosulfuron 2678 2000 2.8
Mesosulfuron 5000 5000 1.3
Orthosulfamuron 5000 5000 2.2
Oxasulfuron 5000 2000 5.1
Sulfosulfuron 5000 5000 3.0
Trifloxysulfuron 5000 2000 5.0
Tritosulfuron 4700 2000 5.4
a
Rabbit.
2.2.1.1 History and Development

Since George Levitt’s landmark discovery of herbicidal sulfonylurea herbicides at
Du Pont in 1975, many hundreds of patents have been granted to Du Pont and, in
addition, to over 20 other agrochemical companies.
Currently, numerous reviews are available concerning sulfonylurea herbicides,
with that of George Levitt (which includes an original description of his investiga-
tions) being particularly recommended [4], as it covers the literature up to 1991.
Another, earlier standard text for enthusiasts of the sulfonylureas is that of Beyer
et al. [5], while a more recent review is available in Ref. [6].
The first generation of crop-selective sulfonylurea herbicides, such as chlorsul-
furon and metsulfuron-methyl, was found to be mainly active against broadleaf
weeds. At that time, it was thought that this herbicide class would be specifically
applicable for controlling broadleaf weed species in a wide range of crops. How-
ever, this view changed with the appearance of second-generation sulfonylureas
bearing pyridylsulfonamide moieties, such as nicosulfuron and rimsulfuron, for
use in maize. These compounds were active against not only broadleaf weeds but
also a broad spectrum of grasses. A further important breakthrough was achieved
with the advent of the third-generation sulfonylurea herbicides, which included
‘‘grasskiller experts’’ such as mesosulfuron, ‘‘blackgrass specialists’’ such as flupyr-
sulfuron, and cross-spectrum compounds such as iodosulfuron (cf. Table 2.2.6)
and foramsulfuron (cf. Table 2.2.21).
An overview of the sulfonylurea herbicides introduced prior to 1995 is provided in
Table 2.2.3. Before descriptions of the newer sulfonylureas are provided, and their
uses detailed, a brief overview of the most commonly applied synthesis methods
that can be used in their production is provided in the following subsection.
Table 2.2.3 Sulfonylurea herbicides introduced before 1995 (in alphabetical order).
Chemical structure Main crop Common name Application Reference(s)

(company) rates
(g a.i. ha –1 )
OCH3 Cereals Amidosulfuron 30–60 [6, 7]

O N (Bayer Crop
CH3 SO2 N SO2 N C N Science)
H H N
CH3
OCH3
O Rice Bensulfuron- 20–75 [8]
OCH3 OCH3 methyl (Du Pont)
O N
CH2 SO2 N C N
H H N
OCH3
O Soybeans Chlorimuron- 8–13 [9]

OCH2CH3 OCH3 ethyl (Du Pont)
O N
SO2 N C N
H H N
Cl
Cl OCH3 Cereals Chlorsulfuron 9–25 [10]

O N (Du Pont)
SO2 N C N N
H H N
CH3
OCH3 Rice Cinosulfuron 20–40 [6]

O OCH3 (Syngenta)
O
N
SO2 N C N N
H H N
OCH3
O Oilseed rape Ethametsulfuron- 15–20 [6, 11]

OCH3 NHCH3 methyl (Du Pont)
O N
SO2 N C N N
H H N
OCH2CH3
CF3 OCH3 Turf, Flazasulfuron 25–100 [6, 12]

O N vegetation (Ishihara)
SO2 N C N management
N H H N
OCH3
(continued overleaf)
Table 2.2.3 (continued)

(company) rates
(g a.i. ha –1 )
O Maize, turf Halosulfuron 18–35 [6, 13]

OCH3 OCH3 (Nissan)
Cl O N
SO2 N C N
N N H H N
CH3 OCH3
Cl OCH3 Rice, turf Imazosulfuron 50–100 [6]

O N (Takeda)
N
SO2 N C N
N H H N
OCH3
O Cereals, rice Metsulfuron- 3–8 [6, 14]

OCH3 OCH3 vegetation methyl (Du Pont) 14–168
O N management
SO2 N C N N
H H N
CH3
O Maize Nicosulfuron (Du 35–70 [15, 16]

N(CH3)2 OCH3 Pont/Ishihara)
O N
SO2 N C N
N H H N
OCH3
O Maize Primisulfuron- 20–40 [17]

OCH3 OCF2H methyl (Syngenta)
O N
SO2 N C N
H H N
OCF2H
CF3 Cereals, Prosulfuron 20–40 [6, 18]

OCH3 maize (Syngenta)
O
N
SO2 N C N N
H H N
CH3

(company) rates
(g a.i. ha –1 )
O Rice Pyrazosulfuron- 15–30 [6]

OCH2CH3 OCH3 ethyl (Nissan)
O N
SO2 N C N
N N H H N
CH3 OCH3
SO2CH2CH3 OCH3 Maize Rimsulfuron (Du 5–35 [19]

O N Pont)
SO2 N C N
N H H N
OCH3
O Vegetation Sulfometuron- 26–420 [20]
OCH3 CH3 management methyl (Du Pont)
O N
SO2 N C N
H H N
CH3
O Cereals, Thifensulfuron- 2–30 [21]

OCH3 OCH3 maize, methyl (Du Pont)
O N soybeans
S
SO2 N C N N
H H N
CH3
Cl Cereals Triasulfuron 10–30 [22]

O OCH3 (Syngenta)
O
N
SO2 N C N N
H H N
CH3
O Cereals Tribenuron-methyl 9–18 [6, 23]

OCH3 OCH3 (Du Pont)
O N
SO2 N C N N
H
CH3 N
CH3
O Sugar beet Triflusulfuron- 15–30 [6, 24]

OCH3 N(CH3)2 methyl (Du Pont)
O N
SO2 N C N N
H H N
CH3 OCH2CF3
2.2.1.2 Synthesis
The five synthetic approaches shown in Scheme 2.2.1 have been employed most
widely in the construction of the typical sulfonylurea bridge [25, 26].
Of the methods listed in Scheme 2.2.1, method 1 is the most commonly used
for the commercial production of sulfonylureas. The reaction is both high-yielding
and highly atom efficient, providing advantages for downstream waste process-
ing. In addition, the required sulfonylisocyanates are readily accessed from the
corresponding sulfonamides by reaction with phosgene under several conditions.
Method 2 has the advantages of saving two reaction steps to prepare the sulfonyliso-
cyanate and allowing the reaction to proceed in one pot. This method is particularly
useful in cases where the sulfonylisocyanate is difficult to isolate, or where for-
mation of a saccharin as byproduct is problematic. While methods 3 and 4 result
in good conversions into the targeted products, they suffer from the undesirable
production of phenol as a byproduct. This can be overcome by employing alkoxy
N-heterocyclylcarbamates in the presence of Al(CH3 )3 , leading to the generation of
more innocuous alcoholic byproducts.
2.2.2
Agricultural Utility
In 2009, the value of the global crop protection market (excluding seeds and
biotechnology) amounted to US$37 860 mio, with herbicides accounting for slightly
less than half (US$ 17 527 mio) [27]. The herbicide subtotal is spread relatively
O
Ar/Het SO2 N C N Het
M H
O
1) Ar/Het SO2 N C O + H2N Het Ar/Het SO2 N C N Het
H H
O
2) Ar/Het SO2 Cl + O C N− M+ + H2N Het Ar/Het SO2 N C N Het
H H
O O
3) Ar/Het SO2 N C OPh + H2N Het Ar/Het SO2 N C N Het
H H H
O O
+ DBU
4) Ar/Het SO2 NH2 + PhO C N Het Ar/Het SO2 N C N Het
H H H
O + Al(CH3)3 O
5) Ar/Het SO2 NH2 + RO C N Het Ar/Het SO2 N C N Het
H H H
Scheme 2.2.1 Basic construction routes to the sulfonylurea bridge.

evenly over the three main crops: cereals, maize, and soybeans (20%, 16%, and 14%,
respectively); rice (8%) and others (42%; including nonselective/noncrop use). The
main markets for herbicides are in North America and Europe (Bayer CropScience,
internal communication). An overview of the global acreage and production of the
world’s major arable crops is provided in Table 2.2.4.
In the following subsections, an overview (split by crop segment) is provided of
the new sulfonylurea herbicides that have either been introduced since 1995 or are
currently in their later stages of development.
2.2.2.1 Cereals
Cereals (wheat, barley, sorghum, oats, rye, triticale, and millet) are the most
important of the arable crops (Table 2.2.4). In 2009, global cereal production was
approximately 982 mio tonnes on 380 mio ha of land, with wheat (Triticum aestivum)
being the most important cereal grain, accounting for more than two-thirds of the
total production (Table 2.2.5).
Geographically, the largest cereal production areas are in regions with temperate
conditions, such as Europe, North America, and the cooler parts of Australia and
China.
A significant proportion of the cereal fields worldwide is infested with grass
weeds. Indeed, more than 40 major grass weed species can be found in cereal
fields, and these are often highly competitive with the crop, causing substantial
losses in both yield and quality. As noted above, sulfonylurea herbicides have
been used in cereals, mainly as herbicides against broadleaf weeds, since their
introduction during in the early 1980s. Tank-mixtures with these first-generation
sulfonylureas, or spraying programs involving different grass weed herbicides,
remained the most widely employed chemical control strategy up to the late
1990s. Subsequently, the introduction of a new generation of sulfonylurea her-
bicides, such as flupyrsulfuron-methyl-sodium, iodosulfuron-methyl-sodium, or
Table 2.2.4 Major crops of the world, average 2008–2009.
Crop Land area (mio ha) Production (mio tonnes)a
Cerealsb 381 990

Rice, Paddy 160 682
Maize (grain) 160 822
Soybeans 98 226
Sugarcane 24 1709
Rapeseed 31 60
Sunflower seed 24 34
Potatoes 18 328
Sugar beets 4 225
a
Metric tonnes
b
Wheat, rye, oats, triticale, barley, sorghum, millet.
Data from FAO (Ref. [28]).
Table 2.2.5 Major cereala crops of the world, average 2008–2009.
Crop Land area (mio ha) Production (mio tonnes)b
Wheat 224 683

Barley 55 153
Oats 11 24
Rye 7 18
Triticale 4 14
Sorghum 44 64
Millet 36 34
a
(w/o Buckwheat; Canary seed; Cereals, nes; Fonio; Mixed grain; Quinoa).
b
Metric tonnes.
Data from FAO (Ref. [28]).
mesosulfuron-methyl, with their broad-spectrum grass and broadleaf performance,

provided the farmer with a single, innovative, and easy one-pass solution which
saved both time and costs (Table 2.2.6). Each of the compounds listed in Table 2.2.6
are described in greater detail in the following subsections.
2.2.2.1.1 Flupyrsulfuron-Methyl-Sodium Flupyrsulfuron-methyl-sodium (DPX-

KE459) (Table 2.2.7) [29] is a post-emergent cereal herbicide designed for the
control of problem grass weeds, such as Alopecurus myosuroides and Apera
spica-venti, as well as a wide range of broadleaf weeds. The application rates are
8–10 g a.i. ha−1 . It is commercialized by Du Pont [30] under the trade name
‘‘Lexus® 50DF’’ as a stand-alone product, or as ‘‘Lexus® Class’’ in a 1 : 2 ratio
in combination with carfentrazone-ethyl, and was launched in 1997. At the
10 g a.i. ha−1 rate, the following broadleaf weeds are well controlled: Chenopodium
album, Lamium purpureum, Matricaria spp., Polygonum aviculare, P. convolvulus,
Senecio vulgaris, and Sinapis arvensis.
Ciral® is a combination of flupyrsulfuron-methyl-sodium (33.3%) and
metsulfuron-methyl (16.7%) used for the control of Alopecurus myosuroides, Apera
spica-venti, Poa annua, and also annual broadleaf weeds (except Galium aparine)
such as Thlaspi arvense, Capsella bursa-pastoris, Galeopsis spp., Matricaria spp.,
Papaver rhoeas, Centaurea cyanus, Brassica napus, Viola arvensis, Lamium spp.,
Myosotis arvensis, and Stellaria media. Ciral® is a flexible product that can be
applied in autumn and spring in wheat and in flax at a dose rate of 25 g ha−1 [31]
of formulated product containing 8 g a.i. ha−1 of flupyrsulfuron-methyl-sodium.
2.2.2.1.2 Sulfosulfuron Sulfosulfuron (MON 37500) (Table 2.2.8) [32] is a

post-emergent herbicide for the control of grass (especially Bromus sp.) and
broadleaf weeds in cereal crops at rates of 10–35 g a.i. ha−1 . Sulfosulfuron
was jointly developed [33, 34] by Monsanto Company and Takeda Chemical
Industries, and launched in 1997. As barley and oats are both sensitive
Table 2.2.6 Cereal sulfonylurea herbicides in order of market introduction.
Chemical structure Common name Agricultural Application

(company, utility rate
launch year) (g a.i. ha –1 )
O Flupyrsulfuron- Grass weeds 8–10

OCH3 OCH3 methyl-sodium and select
O N (Du Pont, 1997) broadleaf
SO2 N C N weeds
N + H N
Na
F3C OCH3
SO2C2H5 OCH3 Sulfosulfuron Grass weeds 10–35
O N (Takeda/ and broadleaf
N Monsanto, 1997) weeds
SO2 N C N
N H H N
OCH3
O Iodosulfuron- Broadleaf and 5–10 +

OCH3 OCH3
O methyl-sodium grass weeds safener
N
(Bayer Mefenpyr
SO2 N C N N
+ H CropScience,
Na N
1999)
I CH3
O Mesosulfuron- Grass weeds 6–15 +

OCH3 OCH3 methyl (Bayer and select safener
O N CropScience, broadleaf Mefenpyr
SO2 N C N 2001) weeds
H H N
CH3SO2 N OCH3
H
CF3 OCH3 Tritosulfuron Broadleaf 30–50

O
N (BASF, 2004) weeds
SO2 N C N N
H H N
CF3
to applications of sulfosulfuron, its use in these crops is not recommended. At rates

of 20–30 g a.i. ha−1 the following weeds are controlled with at least 85% efficiency:
Elymus repens, Apera spica-venti, Agrostis stolonifera, Avena fatua (North America),
Bromus commutatus, B. japonicus, B. mollis, B. rigidus, B. secalinus, B. sterilis, B.
tectorum, Poa bulbosa and Poa trivialis, Ambrosia artemisiifolia, Amsinckia lycopsoides,
Atriplex patula, Brassica nigra, Capsella bursa-pastoris, Claytonia per, Descurainia
pinnata, D. sophia, Fumaria officinalis, Galium aparine, Helianthus sp., Matricaria
chamomilla, M. inodora, Polygonum aviculare, P. persicaria, Sinapis arvensis, Sisym-
brium altissimum, Stellaria media, Thlaspi arvense, and Viola arvensis. In Europe,
®
sulfosulfuron is commercially available under the trade name ‘‘Monitor .’’
Table 2.2.7 Physico-chemical properties of flupyrsulfuron-methyl-sodium.
O
OCH3 OCH3
O N
SO2 N C N
N + H N
Na
F3C OCH3
Common name (ISO) Flupyrsulfuron-methyl-sodium

CAS-No. 144740-54-5
Code numbers DPX-KE459
Melting point Not determined (decomposition at 165–170 ◦ C)
Vapor pressure (Pa at 20 ◦ C) <1 × 10 –9
Dissociation constant (pKa at 20 ◦ C) 4.94 (93.4%)
–1 ◦
Solubility in water (g l at 20 C) 0.06 (93.4%) (pH 5)
0.61 (pH 6)
Instability of the solution (pH 7)
Solubility in organic Acetone 3.1 (93.4%)
solvents (g l –1 at 20 ◦ C) Acetonitrile 4.3
Benzene 0.028
Dichloromethane 0.60
Hexane <0.001
Methanol 5.0
n-Octanol 0.19
Partition coefficient (log Pow ) 9.17 (pH 5)
in octanol–water (at 20 ◦ C) 1.16 (93.4%) (pH 6)
2.2.2.1.3 Iodosulfuron-Methyl-Sodium At a rate of 2.5–10 g a.i. ha−1 , iodo-

sulfuron-methyl-sodium (AE F115008) (Table 2.2.9) [35] controls more than 50
different broadleaf weed species, including some very competitive weeds that
cause a substantial reduction of cereal productivity, for example, Galium aparine,
Matricaria chamomilla, Stellaria media, Raphanus ssp., Cirsium arvense, and
Lamium ssp. While the application of iodosulfuron-methyl-sodium at the lower
end of the suggested use-rate is usually sufficient to control broadleaf weeds,
a higher rate is needed for consistent grass weed control. Major grass weeds
controlled with a 7.5–10 g a.i. ha−1 dose rate applied at the three-leaf stage up to
the end of tillering are Agrostis gigantea, Apera spica-venti, Lolium multiflorum, L.
perenne, L. persicum, L. rigidum, Phalaris brachystachys, P. canariensis, P. paradoxa,
Poa annua, and P. trivialis.
When introduced in 1999, iodosulfuron-methyl-sodium was the first ‘‘safened’’
sulfonylurea herbicide available commercially [36, 37], and has been marketed
Table 2.2.8 Physico-chemical properties of sulfosulfuron.
SO2C2H5 OCH3
O N
N
SO2 N C N
N H H N
OCH3
Common name (ISO) Sulfosulfuron

CAS-No. 141776-32-1
Code numbers MON 37500
TKM 19
Melting point (◦ C) 201.1–201.7
Vapor pressure (Pa at 20 ◦ C) 3.05 × 10 –8
◦
Dissociation constant (pKa at 20 C) 3.51
–1 ◦
Solubility in water (g l at 20 C) 0.018 (pH 5)
1.627 (pH 7)
0.482 (pH 9)
Solubility in organic solvents Acetone 0.71
(g l –1 at 20 ◦ C) Ethyl acetate 1.01
n-Heptane <0.001
Methanol 0.33
Xylene 0.16
in octanol–water (at 20 ◦ C) –0.77 (pH 7)
–1.44 (pH 9)
by Bayer CropScience for use both in cereals and maize. A ‘‘safener,’’ such as
mefenpyr-diethyl (cf. Figure 2.2.2) is a chemical that, when applied to crop plants,
reduces the injury caused by herbicides to an acceptable level. Ideally, a safener
does not reduce activity against the target weeds. When a series of experiments
was conducted to compare the behavior of iodosulfuron-methyl-sodium with and
without mefenpyr-diethyl as safener, the results suggested that the safener acted
via a specific catalytic enhancement of herbicide degradation in cereals, but not in
O
H3C OC2H5
OC2H5
N
N
Cl O Figure 2.2.2 Cereal safener mefenpyr-diethyl
Cl (AE F107892).
Table 2.2.9 Physico-chemical properties of iodosulfuron-methyl-sodium.
O
OCH3 OCH3
O N
SO2 N C N N
+ H
Na N
I CH3
Common name (ISO) Iodosulfuron-methyl-sodium

CAS-No. 144550-36-7
Code numbers AE F115008
◦
Melting point ( C) 148–152
◦
Vapor pressure (Pa at 20 C) 2.6 × 10 –9
6.7 × 10 –9a
Dissociation constant (pKa at 20 ◦ C) 3.22 (under strong acidic conditions–
pH 2 – formation of iodosulfuron-methyl)
Solubility in water (g l –1 at 20 ◦ C) 0.16 (pH 5)
25.0 (pH 7)
65.0 (pH 9)
Solubility in organic solvents Acetonitrile 52
(g l –1 at 20 ◦ C) Ethyl acetate 23
n-Heptane 0.001
Methanol 12
2-Propanol 4.4
Toluene 2.1
Partition coefficient (log Pow ) in 1.96 (pH 4)
octanol–water (at 20 ◦ C) 1.22 (pH 9)
a
At 25 ◦ C.
target weeds such as wild oat. The subject of safeners is covered in greater detail
in Chapter 8.
In cereals, iodosulfuron-methyl-sodium is available commercially under the
trade name ‘‘Hussar,’’ as a straight product in a 1 : 3 ratio with mefenpyr-diethyl
as safener. The compound is also sold in various combinations with other mixing
partners, such as ‘‘Hussar® OF’’ (+ fenoxaprop-P-ethyl + mefenpyr-diethyl),
‘‘Sekator®’’/‘‘Chekker®’’ (+ amidosulfuron + mefenpyr-diethyl), ‘‘Cossack®’’ (+
mesosulfuron + mefenpyr-diethyl), and ‘‘Atlantis®’’ (+ mesosulfuron + mefenpyr-
diethyl) (cf. Table 2.2.10).
2.2.2.1.4 Mesosulfuron-Methyl Mesosulfuron-methyl (AE F130060) (Table 2.2.11)
[38] was the second safened sulfonylurea herbicide for cereal crops to be
commercialized, having been introduced by Bayer CropScience in 2001
[39, 40]. Its strength is broad-spectrum post-emergence grass weed control.
Table 2.2.10 Iodosulfuron-based products, formulations, and composition.
Iodosulfuron- Formulation Iodo- Meso- Fenoxa- Amido- Mefenpyr-

based products typea sulfuron sulfuron prop-ethyl sulfuron diethyl
® ® ®
Hussar , Husar , Huzar , WG 50b – – – 150b
® ® ®
Huszar , Al Fares , Wipe
® ® ®
Sekator , Grodyl Ultra WG 12.5b – – 50b 125b
®
Sekator OD OD 25c – – 100c 250c
® ®
Chekker , Hoestar Super WG 12.5b – – 125b 125b
®
Hussar OF Evolution SC 8c – 64c – 24c
®
Hussar OD OD 100c – – – 300c
Atlantis WG WG 6b 30b – – 90b
®
Pacifica WG 10b 30b – – 90b
® ®
Archipel , Cossack , WG 30b 30b – – 90b
® ®
Chevalier , Hussar maxx
a
WG: water-dispersible granules; OD: oil dispersion; SC: suspension concentrate.
b
Units: g a.i. kg−1 .
c
Units: g a.i. l−1 .
Mesosulfuron-methyl, at a dose rate of 4.5–15 g a.i. ha−1 , reliably controls 24

different grass weed species from 12 different families. Among the commercially
important grass weed species, it provides good control of Agrostis spp., Alopecurus
myosuroides, Apera spica-venti, Avena spp. Lolium spp., Phalaris brachystachis,
P. minor, P. paradoxa, Poa annua, Poa trivialis, Pucciniella spp., and Sclerochloa
kengiana. Additionally, mesosulfuron-methyl will control, or at least have a strong
suppressive effect on, some very persistent grass weed species, such as Bromus
catharticus, B. diandrus, B. erectus, B. japonicus, B. mollis, B. tectorum, B. secalinus,
B. sterilis, and Vulpia spp.
The compound is applied on soft and durum wheat, triticale, and rye, together
with mefenpyr-diethyl as safener (Figure 2.2.2) as the straight products ‘‘Atlantis®
OF’’, ‘‘Silverado®,’’ and ‘‘Osprey®,’’ or in combination with iodosulfuron-methyl-
sodium (‘‘Atlantis® WG,’’ ‘‘Cossack®,’’ ‘‘Pacifica®’’), diflufenican and propoxy-
carbazone-sodium (Table 2.2.12).
‘‘Atlantis® WG’’ is positioned in market segments where grass weeds are
the main target, whereas ‘‘Cossack®’’ is a cross-spectrum product that is ac-
tive against both grasses and a large number of important broadleaf weeds.
Mesosulfuron-methyl belongs to the group of modern OnePass® products. It acts
predominantly via the leaves of treated weeds; however, highly susceptible grasses,
such as Apera and Alopecurus, can also be successfully controlled by the uptake of
mesosulfuron-methyl via the soil and roots.
Table 2.2.11 Physico-chemical properties of mesosulfuron-methyl.
O
OCH3 OCH3
O N
SO2 N C N
H H N
CH3SO2 N OCH3
H
Common name (ISO) Mesosulfuron-methyl

CAS-No. 208465-21-8
◦
Melting point ( C) 195.4 (98.7% purity)
◦
Dissociation constant (pKa at 20 ◦ C) 4.35
◦
Solubility in water (g l –1 at 20 C) 0.007 (pH 5)
0.483 (pH 7)
15.39 (pH 9)
Dichloromethane 3.8
n-Hexane <0.0002
Toluene 0.013
in octanol–water (at 25 ◦ C) –0.48 (pH 7)
–2.06 (pH 9)
The safener mefenpyr-diethyl, as with iodosulfuron-methyl-sodium, selectively

accelerates the degradation of the active ingredient to non phytotoxic compounds
in cereals, but not in weeds.
2.2.2.1.5 Tritosulfuron Tritosulfuron (BAS-635) (Table 2.2.13) [41] is a

broad-spectrum post-emergent dicotyledonous herbicide mainly for use in cereals,
rice, maize, and turf, with application rates of 40–75 g a.i. ha−1 . In cereals, it was
commercialized by BASF in 2004 under the trade name ‘‘Biathlon®’’ [42] as a
water-dispersible (WG) formulation containing 714 g kg−1 tritosulfuron, and is
applied at a rate of 50 g a.i. ha−1 . The following weeds are well controlled: Thlaspi
arvense, Mercurialis annua, Urtica urens, Cirsium arvense, Veronica hederifolia,
Chenopodium spp., Sinapis arvensis, Capsella bursa-pastoris, Galeopsis tetrahit,
Matricaria spp., Galium aparine, Polygonum spp., Centaurea cyanus, Lamium spp.,
Myosotis arvensis, Stellaria media, Vicia spp., Convolvulus arvensis, Sonchus arvensis,
Brassica napus. Tritosulfuron acts mainly through the treated leaves, and not via
Table 2.2.12 Mesosulfuron-based products, formulations, and composition.
Mesosulfuron- Formulation Mesosulfuron- Iodo-sulfuron- Diflu- Propoxy- Mefenpyr-

based products typea methyl methyl sodium fenican carbazone diethyl
®
Atlantis OD OD 30b – – – 90b
®
Atlantis WG 30c 6c – – 90c
®
Pacifica WG 30c 10c – – 90c
®
Archipel , WG 30c 30c – – 90c
®
Cossack ,
®
Chevalier ,
®
Hussar maxx
®
Silverado WG 20c – – – 120c
®
Osprey WG 45c – – – 90c
®
Alister OD 9b 3b 150b – 27b
®
Othello OD 7.5b 2.5b 50b – 22.5b
®
Olympus flex WG 45c – – 67.5c 90c
a
OD: oil dispersion; WG: water-dispersible granules.
b
c
the soil. The compound has the advantage of having a short soil half-life, which
allows re-cropping after 60 days, without plowing [43, 44].
Tritosulfuron is selective in the following cereal crops: wheat, rye, barley, triticale,
oat, durum wheat, and spelt. The application window of tritosulfuron in all winter
and summer cereals ranges from vegetation start up to growth stage (GS) 39. Sold
in maize as ‘‘Tooler®,’’ it can be applied from GS 12 to GS 18.
2.2.2.1.6 Cereals Development Candidates Two compounds, monosulfuron and

methiopyrsulfuron (NPC-C9908), developed by research groups in China, are
currently marketed there. Outside mainland China, only limited public knowledge
is available concerning these compounds.
Monosulfuron (CAS-No.: 155860-63-2) (Figure 2.2.3) is a new herbicide for the
control of weeds in wheat (Triticum aestivum) and millet (Panicum miliaceum and
Setaria italica), with application rates ranging from 15 to 60 g a.i. ha−1 . The molecule
was discovered at Nankai University in 1993 [45], and recently registered in China.
Monosulfuron provides an effective control of various broadleaf and grass weeds,
such as Leptochloa chinensis, Amaranthus retroflexus, Chenopodium album, Abu-
tilon theophrasti, Xanthium sibiricum Patrin., Portulaca oleracea, Acalypha australis,
Solanum nigrum, Digitaria sanguinalis, Descurainia sophia, Echinochloa phyllopogon,
Eriochloa villosa, and Puccinellia distans. Further properties and environmental data
Table 2.2.13 Physico-chemical properties of tritosulfuron.
CF3 OCH3
O
N
SO2 N C N N
H H N
CF3
Common name (ISO) Tritosulfuron

CAS-No. 142469-14-5
Code numbers BAS-635
◦
◦
◦
Solubility in water (g l –1
at 20 C) <0.001 (pH 1.7)
0.04 (pH 7.0)
78.32 (pH 10.2)
Solubility in organic solvents Acetone –
(g l –1 at 20 ◦ C) Acetonitrile –
Ethyl acetate 83.0
n-Heptane <0.001
Methanol 23
Toluene 4.2
Partition coefficient (log Pow ) in octanol–water (at 2.85 (pH 4)
20 ◦ C) 0.62 (pH 7)
–2.38 (pH 10)
NO2 CH3 Figure 2.2.3 Monosulfuron.

O N
SO2 N C N
H H N
of monosulfuron have been described in several reports by Fan et al. [46] and Wang
et al. [47, 48].
Methiopyrsulfuron (CAS-No.: 441050-97-1) (Figure 2.2.4) is a novel sulfonylurea
herbicide [49, 50] discovered by the Hunan Branch of the National Pesticide R&D
South Center, Changsha, China. It is reported to be effective in controlling various
broadleaf weeds such as Polygonum bungeanum, Acalypha australis, Amaranthus
retroflexus, Chenopodium album, as well as some grasses in wheat, at a dosage of
30–45 g a.i. ha−1 [51].
Monosulfuron-ester (NK94827; CAS-No : 175076-90-1) (Figure 2.2.5) [52] is
another new herbicide for the control of weeds in wheat (Triticum aestivum) and
millet (Panicum miliaceum and Setaria italica), with an effective application rate
O Figure 2.2.4 Methiopyrsulfuron.

OCH3 OCH3
O N
SO2 N C N
H H
N
SCH3
O Figure 2.2.5 Monosulfuron-ester.

OCH3 CH3
O N
SO2 N C N
H H
N
of 22.5 g a.i. ha−1 . Like monosulfuron, this molecule was also discovered by the
research team of Li Zheng-Ming at Nankai University, and is currently entering
industrialization in China. Monosulfuron-ester provides an effective control of
various broadleaf weeds, such as Descurainia sophia, Capsella spp., and Chenopodium
spp. [53]. The crystal structures of monosulfuron and monosulfuron-ester in
complex with Arabidopsis thaliana AHAS monosulfuron were reported recently by
Wang et al. [54].
2.2.2.2 Rice
Approximately 60% of the global population, particularly in Asia, rely on rice (Oryza
sativa) as a major food source. Rice is grown mainly in the humid and subhumid
tropics of the Far East, with rice production on approximately 161 mio ha totaling
679 mio tonnes in 2009. Although the two major producers, China and India, were
responsible for growing more than half of the global total [28], in terms of value
Japan is the largest rice market, with over 35% of the total market value.
It is estimated that, on average, weed infestation in tropical rice areas accounts for
10–20% of yield loss, although some studies have shown that certain ‘‘problem’’
weed species, such as red rice (O. sativa ssp.) and barnyardgrass (Echinochloa
crus-galli), can cause even higher losses. Red rice (the term ‘‘red rice’’ is used
synonymously for weedy rice because its grains frequently have a red pigmented
pericarp) is in the same genus and species as cultivated conventional rice, which
makes it very difficult to eliminate in rice fields [55, 56]. Fisher and Ramirez [57]
found that a 5% density of red rice decreased conventional rice yields by up to 40%.
Typically, herbicides in rice are used in combinations of active ingredients and, as
labor is becoming more expensive, the trend is towards single-application products
which usually contain sulfonylureas as the main active ingredient (Table 2.2.14).
Details of the compounds listed in Table 2.2.14. are provided in each of the
following subsections.
2.2.2.2.1 Ethoxysulfuron Ethoxysulfuron (HOE 095404) [58] is a very flexible her-

bicide for the control of broadleaf and sedge weed species (Table 2.2.15). Although
Table 2.2.14 Rice sulfonylurea herbicides in order of market introduction.

(company, launch utility rate
year) (g a.i. ha –1 )
O C2H5 OCH3 Ethoxysulfuron Annual and 6–60

O N (Bayer perennial
O SO2 N C N CropScience, 1996) broadleaf
H H N and sedge
OCH3 weeds
Azimsulfuron (Du Annual and 6–25
N N CH3
Pont, 1996) perennial
N
N OCH3 broadleaf
O N and sedge
SO2 N C N weeds
N N H H N
CH3 OCH3
O Cyclosulfamuron Annual and 10–60

OCH3 (BASF, 1997) perennial
O N broadleaf
N SO2 N C N and sedge
H H H N weeds
OCH3
O Flucetosulfuron Annual and 15–60
CH2OCH3 (KRICT/LG perennial
O Chem., 2004) broadleaf
CHFCH3 OCH3 and sedge
N O N weeds
SO2 N C N
H H N
OCH3
O Orthosulfamuron Broadleaf, 40–75

N(CH3)2 OCH3 (Isagro, 2007) grass, and
O N sedge weeds
N SO2 N C N
H H H N
OCH3
Cl OCH3 Propyrisulfuron Annual and 70–90
O N (Sumitomo, 2011a ) perennial
N
SO2 N C N expected) broadleaf
N H H N weeds and
N OCH3 sedges
CH3
a
Expected launch year.
Table 2.2.15 Physico-chemical properties of ethoxysulfuron.
O C2H5 OCH3
O N
O SO2 N C N
H H N
OCH3
Common name (ISO) Ethoxysulfuron
CAS-No. 126801-58-9
Code numbers Hoe 095404

◦

–1 ◦
1.353 (pH 7)
9.628 (pH 9)

n-Hexane 0.006
Methanol 7.7
Polyethylene glycol 22.5
Toluene 2.5

–1.22 (pH 9)
rice is the main use crop, the compound can also be applied to cereals and sugar-
cane [59]. Selectivity is achieved due to a differential metabolism in the target crops
to that in the weeds [60]. With an application rate of 15–60 g a.i. ha−1 , a wide range
of important annual and perennial rice weeds can be controlled, such as Cyperus
spp., Aeschynomene spp., Eleocharis spp., Sagittaria spp., Scirpus spp., Amannia
spp., Lindernia spp., Ludwigia spp., and Monochoria vaginalis. Ethoxysulfuron is
fully selective in all types of seeded rice (dry-drilled, pre-germinated wet-seeded,
pre-germinated water-seeded) and all types of transplanted rice. The selectivity is
not influenced by the rice growth stage at application time, nor by the water man-
agement or other environmental factors. Ethoxysulfuron was introduced in rice
in 1996 (in Vietnam), and has been marketed by Bayer CropScience as a straight
product under the trade name ‘‘Sunrice® WG’’ and as suspension concentrate (SC)
formulated products in combination with anilofos as ‘‘Riceguard®,’’ ‘‘Benefiter®,’’
‘‘Sunrice® Super’’, and ‘‘Sunrice® Plus’’ (Table 2.2.16).
Table 2.2.16 Ethoxysulfuron-based products, formulations, and composition.
Ethoxysulfuron- Formulation Ethoxy- Anilofos Fenoxaprop- Safener

based products typea sulfuron ethyl
® ® ®
Gladium , Grazie , Hero , WG 600b – – –
® ®
Skol , Sunrice
® ®
Sunrice , Sunstar WG 150b – – –
® ®
Ricestar , Ricestar Xtra, OD 20c – 69c 75c
® ®
Tiller Gold, Turob
®
Sunrice Plus SC 15c 300c – –
a
WG: water-dispersible granules; OD: oil dispersion; SC: suspension concentrate.
b
c
2.2.2.2.2 Azimsulfuron Azimsulfuron (DPX-A8947) [61] is a new rice herbicide

introduced in 1996 by Du Pont [62] for the control of broadleaf weeds (including
hard-to-control perennials) (Table 2.2.17). At application rates of 8–20 g a.i. ha−1 ,
it provides superior weed control, including E. crus-galli, when compared to
the first-generation sulfonylurea bensulfuron at 50–75 g a.i. ha−1 . Azimsulfuron
is targeted to replace or supplement bensulfuron in some applications. In
Japan, in planted rice, azimsulfuron is used as a pre-mixture with bensulfuron
(6 + 30 g a.i. ha−1 ) to boost the activity against perennial weeds. Good control has
also been reported of other members of the Echinochloa family, such as E. hispidula,
E. oryzicola, and E. oryzoides. Other weeds controlled include Alisma lanceolatum, A.
plantago-aquatica, Butomus umbellatus, Cyperus difformis, Scirpus maritimus, S. mu-
cronatus, S. supinus, Heteranthera limosa, Potamogeton nodosus, Ammannia coccinea,
A. robusta, Bergia capensis, and Lindernia dubia.
® ®
Azimsulfuron is sold under the trade names ‘‘Gulliver ’’ and ‘‘Azin .’’
2.2.2.2.3 Cyclosulfamuron Cyclosulfamuron (AC 322,140) herbicide was

launched in 1997 by American Cyanamid, and is marketed by BASF for the
control of a wide range of broadleaf weeds and sedge species in rice, wheat,
and barley [63] (Table 2.2.18). Rice weeds controlled with greater 90% efficiency
at an application rate of 45–60 g a.i. ha−1 include Cyperus serotinus, C. difformis,
Elatine triandra, Eleocharis congesta, E. kuroguwai, Lindernia annua, L. procumbens,
Monochoria vaginalis, Rotala indica, Sagittaria pygmaea, S. trifolia, and Scirpus
juncoides. Selectivity in the rice paddy is achieved due to various factors, including
rapid metabolic degradation of the herbicide in rice shoots, placement of rice
seedlings during transplanting, and the compound’s soil binding properties,
which retains cyclosulfamuron in the upper soil layer of the paddy [64].
In rice, cyclosulfamuron is available commercially under the trade name ‘‘Ichiy-
onmaru’’ and ‘‘Saviour.’’ In combinations with daimuron and cafenstrole it is sold
Table 2.2.17 Physico-chemical properties of azimsulfuron.
N N CH3
N
N OCH3
O N
SO2 N C N
N N H H N
CH3 OCH3
Common name (ISO) Azimsulfuron

CAS-No. 120162-55-2
Code numbers DPX-A8947
◦
Melting point ( C) 170
◦
1.050 (pH 7)
6.536 (pH 9)
(g l –1 at 25 ◦ C) Acetonitrile 13.9
Ethyl acetate 13.0
n-Hexane <0.2
Methanol 2.1
Toluene 1.8
octanol–water (at 25 ◦ C) –1.37 (pH 7)
–2.08 (pH 9)
as ‘‘Nebiros,’’ and in combination with pentoxazone as ‘‘Utopia.’’ ‘‘Shakariki’’ is

the trade name for the mixture with esprocarb.
At application rates of 25–50 g a.i. ha−1 , cyclosulfamuron can also be used in
cereal crops for the pre- and post-emergent control of several important broadleaf
weeds, such as Veronica persica, V. hederifolia, Galium aparine, Matricaria spp., and
Polygonum convolvulus [64].
Cyclosulfamuron cannot be synthesized by any of the general methods depicted
in Scheme 2.2.1 From the methods reported in the patent literature, Brady et al.
[65] describe a straightforward reaction of 2-amino-4,6-dimethoxypyrimidine with
chlorosulfonylisocyanate (CSI) at 0 ◦ C with a mixture of 2-aminophenyl cyclopropyl
ketone and triethylamine to yield 70% of the desired herbicide (Scheme 2.2.2). The
synthesis of cyclosulfamuron and its intermediate products was also reported in
2005, by Tan [66].
Table 2.2.18 Physico-chemical properties of cyclosulfamuron.
O
OCH3
O N
N SO2 N C N
H H H N
OCH3
Common name (ISO) Cyclosulfamuron

CAS-No. 136849-15-5
Code numbers AC 322,140
◦
◦
Vapor pressure (Pa at 20 C) <2.2 × 10 –5
–1 ◦
0.003 (pH 6)
0.006 (pH 7)
0.032 (pH 8)
n-Hexane <0.001
Methanol 1.5
Toluene 1
1.69 (pH 6)
1.41 (pH 7)
0.70 (pH 8)
O O
OCH3 OCH3
N O N
N(C2H5)3
NH2 + Cl SO2 N C O + H2N N SO2 N C N
H H H N
N
OCH3 OCH3
Scheme 2.2.2 Chlorosulfonylisocyanate (CSI) route to cyclosulfamuron.
2.2.2.2.4 Flucetosulfuron Details of flucetosulfuron (LGC-42153) [67] were pre-

sented at the BCPC Conference in 2003 by the research group from LG Life
Sciences Ltd and KRICT [68, 69], and was commercialized in 2004 (Table 2.2.19).
It can be used for the control of broadleaf weeds, some grass weeds, and also
sedges in rice and cereal crops. In rice, flucetosulfuron provides excellent control of
Table 2.2.19 Physico-chemical properties of flucetosulfuron.
O
CH2OCH3
O
CHFCH3 OCH3
N O N
SO2 N C N
H H N
OCH3
Common name (ISO) Flucetosulfuron

CAS-No. 412928-75-7
Code numbers LGC-42153
◦
◦
Vapor pressure (Pa at 25 C) <1.86 × 10 –5
–1 ◦
Dimethylformamide 265.0
Dimethylsulfoxide 211.7
n-Hexane 0.006
Methanol 3.8
in octanol–water (at 21 ◦ C)
E. crus-galli, which is usually not controlled by other commercial rice sulfonylurea

products. In addition, the following weeds are controlled at an application rate of
10–20 g a.i. ha−1 : Alisma spp., Ammannia coccinea, Cyperus difformis, Fimbristylis
spp., Lindernia spp., Monochoria vaginalis, Rorippa silvestri, Rotala indica, Scirpus
juncoides, S. mucronatus, and S. maritimus. At a higher rate of 20–30 g a.i. ha−1 ,
a greater than 90% control of Aeschymene indica, Butomus umbellatus, Eleocharis
kuroguwai, Sagittaria pygmaea, S. trifolia, and Sparganium erectum is achieved by
flucetosulfuron, with a high crop safety margin when applied to soil or foliage in
direct-seeded or transplanted rice.
In cereal crops, flucetosulfuron at a rate of 20–30 g a.i. ha−1 shows excellent activ-
ity against Galium aparine and other broadleaf weeds, such as Capsella bursa-pastoris,
Galeopsis tetrahit, Lamium purpureum, Matricaria spp., Myosotis arvensis, Papaver
rhoeas, Raphanus raphanistrum, Senecio vulgaris, Sinapis arvensis, Stellaria media,
and Thlaspi arvense, while being safe to use in wheat and barley at up to three times
the recommended application rate.
2.2.2.2.5 Orthosulfamuron Orthosulfamuron (IR-5878) (Table 2.2.20) [70, 71], is

a broad-spectrum pre- and post-emergent rice herbicide developed by Isagro for
the control of annual broadleaf and sedge weeds.
The compound has been marketed since 2007 under the trade names ‘‘Kelion® 50
WG,’’ ‘‘Strada® WG,’’ and ‘‘Pivot® 50 WG’’ for early and middle post-emergence
application against susceptible weeds and sedges, such as Abutilon theophrasti,
Alisma plantago, Alisma lanceolatum, Cyperus difformis, Cyperus serotinus, Cyperus
ferax, Cyperus iria, Echinochloa crus-galli (red biotype), Heteranthera reniformis,
Heteranthera rotundifolia, Heteranthera limosa, Lindernia dubia, Schoenoplectus mu-
cronatus, Bolboschoenus maritimus, Sagittaria sagittifolia, and Scirpus supinus at a
usage rate of 40–75 g a.i. ha−1 [72].
Table 2.2.20 Physico-chemical properties of orthosulfamuron.
O
N(CH3)2 OCH3
O N
N SO2 N C N
H H H N
OCH3
Common name (ISO) Orthosulfamuron
CAS-No. 213464-77-8
Code numbers IR5878

◦
Dissociation constant (pKa at 20 ◦ C) Predicted to have five overlapping dissociation

constants: −1.4; 0.7; 3.5; 9.6; 11.5
Solubility in water (g l –1 at 20 ◦ C) 0.026 (pH 4, buffer)

0.63 (pH 7, buffer)
39 (pH 8.5, buffer)
Solubility in organic solvents Acetone 20

Dichloromethane 56
n-Heptane 0.00023
Methanol 8.3
Xylene 0.13

octanol–watera 1.3 (pH 7)
<0.3 (pH 10)
a
Temperature not reported.
2.2.2.2.6 Rice Development Candidates In the case of rice, there are currently two
compounds, propyrisulfuron (TH 547, CAS-No.: 570415-88-2) from the research
group at Sumika-Takeda, and metazosulfuron (NC620, CAS-No.: 868680-84-6)
from Nissan, in the development phase.
Propyrisulfuron (Figure 2.2.6) is a new sulfonylurea under development by Sum-
itomo, and will be marketed as solo product and in combination with pyraclonil.
The structure is related to the imazosulfuron class [73–75]. The compound will
be introduced in 2011, and is considered to be a new-generation sulfonylurea for
the control of annual and perennial broadleaf weeds and sedges, especially against
acetolactate synthase (ALS)-resistant weed biotypes. At rates of 70 g a.i. ha−1 , propy-
risulfuron will control Cyperus serotinus, C. difformis, Elatine triandra, Eleocharis
congesta, E. kuroguwai, Lindernia annua, L. procumbens, Monochoria vaginalis, Rotala
indica, Sagittaria pygmaea, S. trifolia, and Scirpus juncoides. At a higher application
rate of 90 g a.i. ha−1 , it gives total weed control, including Echinochloa spp.
Metazosulfuron (Figure 2.2.7) [76] is a new development candidate from the
pyrazosulfuron class which has been developed by Nissan for the Japanese and
South Korean markets. This compound is expected to be introduced in 2012 or
2013.
2.2.2.3 Maize
Approximately 817 mio tonnes of maize were produced worldwide in 2009, on
more than 160 mio ha of land [28]. Maize (Zea mays), occupies third place in world
production as a source of food, forage, and processed products for industry. The
main producing countries are the USA, China, and Brazil, which together account
for approximately two-thirds of the global production. Maize is most commonly
Cl OCH3
O N
N
SO2 N C N
N H H
N
N OCH3
CH3
Figure 2.2.6 Propyrisulfuron.
H 3C
O
O
N OCH3
H3 C O N
SO2 N C N
N N H H
N
CH3 OCH3
Figure 2.2.7 Metazosulfuron (NC620).
grown for animal feed use, although it is a dietary staple in some areas such as
Mexico and other Latin American countries.
Maize, with its shallow root system, is particularly prone to competition by
other plants during its early growth stages. While older generations of maize
herbicides are used predominantly as pre-emergent herbicides (e.g., atrazine from
the triazines class), today there are modern, post-emergent sulfonylurea products
available to the farmer for cost-effective and time-flexible weed control. The latest
compound to be introduced after 1995 is discussed in the following subsection (see
also Table 2.2.21).
2.2.2.3.1 Foramsulfuron Foramsulfuron (AE F130360) [77] is a post-emergence

sulfonylurea herbicide used for the control of major grass species and cer-
tain broadleaf weeds in maize (Table 2.2.22). It is applied with the safener
isoxadifen-ethyl (AE F122006) (Figure 2.2.8), and in some products in combination
with small quantities iodosulfuron-methyl-sodium [78].
Introduced in 2001 and subsequently commercialized by Bayer CropScience,
the three-way mixture of foramsulfuron with iodosulfuron-methyl-sodium and
isoxadifen-ethyl is used for post-emergent weed control in maize. Foramsulfuron,
at a dose rate of 30–45 g a.i. ha−1 , offers a minimum of 90% weed control on most
grassy weeds, such as Echinochloa crus-galli, Setaria spp., Agropyron repens, Apera
spica-venti, Alopecurus myosuroides, Lolium multiflorum, Panicum dichotomiflorum,
Poa annua, and Sorghum halepense, and a wide selection of broadleaf weed species,
such as Abutilon theophrasti, Amaranthus spp., Galinsoga parviflora, Lamium pur-
pureum, Solanum nigrum, and Stellaria media [79]. The addition of 1–2 g a.i. ha−1
of iodosulfuron-methyl-sodium improves the level of weed control on broadleaf
weed species, including Chenopodium album, Galium aparine, Fallopia convolvulus,
Ipomoea spp., Polygonum aviculare, P. lapathifolium, Sonchus arvensis, and Xanthium
strumarium.
The basis of selectivity of foramsulfuron in the presence of the safener
isoxadifen-ethyl is a more rapid rate of metabolic detoxification in maize compared
with target weeds, in which little or no degradation of the parent sulfonylurea
Table 2.2.21 Maize sulfonylurea herbicides.

O Foramsulfuron Grass and 30–45 +

N(CH3)2 OCH3 (Bayer broadleaf safener
O N CropScience, weeds isoxadifen
SO2 N C N 2001)
O H H N
N OCH3
H H
Table 2.2.22 Physico-chemical properties of foramsulfuron.
O
N(CH3)2 OCH3
O N
SO2 N C N
O H H N
N OCH3
H H
Common name (ISO) Foramsulfuron

CAS-No. 173159-57-4
◦
Melting point ( C) 194.5 (98.4 % w/w)
◦
1.3 × 10 –10a
Dissociation constant (pKa at 21.5 ◦ C) 4.6
◦
Solubility in water (g l –1 at 20 C) 0.037 (pH 5)
3.293 (pH 7)
94.577 (pH 8)
(g l –1 at 20 ◦ C) Acetonitrile 1.111
1,2-Dichloroethane 0.185
Ethyl acetate 0.362
n-Heptane <0.010
Methanol 1.660
p-Xylene <0.010
octanol–water (at 20 ◦ C) 0.60 (pH 5.5–5.7)
–0.78 (pH 7)
–1.97 (pH 9)
a
At 25 ◦ C.
Figure 2.2.8 Maize safener isoxadifen-ethyl (AE F122006).

O
OC2H5
O N
occurs [80]. Three main routes of metabolism have been established in maize: a
hydrolytic cleavage of the sulfonylurea bridge; a deformylation of the amino group;
and oxidative metabolism of the dimethoxypyrimidine ring.
Foramsulfuron is commercialized with the safener isoxadifen-ethyl under the
trade names ‘‘Option®’’ and ‘‘Equip®,’’ whereas in combination with iodosulfuron-
® ® ®
methyl-sodium the ternary mixture is sold as ‘‘MaisTer ,’’ ‘‘Mester ,’’ ‘‘Fortuna ’’
or ‘‘Equip® Plus,’’ and ‘‘Option® 360’’ (Table 2.2.23). The combination of the two
herbicides probably make ‘‘MaisTer®’’ and ‘‘Mester®’’ the widest-spectrum maize
herbicides used in Europe today.
2.2.2.4 Other Crops

Soybeans are the number one oilseed crop worldwide, with a total of 222 mio tonnes
of soybean being produced in 2009. Relatively few countries produce soybeans:
the USA accounts for more than 40% of the world production, with Brazil,
Argentina, and China together accounting for an additional 55%. Within Europe,
the main producer countries are Italy, Russia, and the Ukraine. In the USA, Brazil,
and Argentina, the most widely planted soybeans are genetically modified (GM)
varieties, which are tolerant against the herbicide glyphosate.
Table 2.2.23 Foramsulfuron-based products, formulations, and composition.
Foramsulfuron-based products Formulation Foramsulfuron Iodosulfuron Isoxadifen-

typea ethyl
®
Option WG 70 350b – 350b
® ® ®
MaisTer , Mester , Fortuna WG 61 300b 10b 300b
®
Equip OD 05 22.5c – 22.5c
® ®
Equip Plus, Option 360 WG 62 300b 20b 300b
a
WG: water-dispersible granules; OD: oil dispersion.
b
c
Table 2.2.24 Other sulfonylurea herbicides for use in soybeans, cotton, and sugarcane.
Chemical structure Common name Crop Agricultural Application

Oxasulfuron Soybeans Broadleaf 45–90

O O
(Syngenta, 1996) weeds
O CH3
O N
SO2 N C N
H H N
CH3
O CH2CF3 OCH3 Trifloxysulfuron- Sugarcane, Sedges and 5–23

O N sodium (Syngenta, cotton, turf broadleaf
SO2 N C N 2001) weeds
N + H
Na N
OCH3
Sugarcane and cotton also represent important crops that benefit from newer
sulfonylurea herbicides. The most recent compounds, introduced after 1995, are
listed in Table 2.2.24.
2.2.2.4.1 Oxasulfuron Oxasulfuron (CGA 277476) (Table 2.2.25) [81] was
launched in 1996 by Syngenta as a pre-emergent and post-emergent herbicide.
At application rates of 66–92 g a.i. ha−1 , it provides a greater than 80% control
of Abutilon theophrasti, Xanthium strumarium, Amaranthus spp., Ambrosia
artemisiifolia, A. trifida, Bidens pilosa, Cyperus esculentus, Polygonum pensylvanicum,
Sorghum bicolor, Echinochloa crus-galli, Helianthus annuus, Sesbania exaltata, and
Ipomoea spp. [82] in soybeans. The observed selectivity is due to the compound’s
rapid metabolism in the target crop.
2.2.2.4.2 Trifloxysulfuron-Sodium Trifloxysulfuron-sodium (CGA 362622)
(Table 2.2.26) [83] is a post-emergence herbicide commercialized by Syngenta in
2001 for use in all major cotton and sugarcane production areas [84].
Table 2.2.25 Physico-chemical properties of oxasulfuron.
O O
O CH3
O N
SO2 N C N
H H N
CH3
Common name (ISO) Oxasulfuron

CAS-No. 144651-06-9
Code numbers CGA 277476
Melting point (◦ C) 158 (decomposition)
Vapor pressure (Pa at 25 ◦ C) <2 × 10 –6
Dissociation constant (pKa ) 5.10
–1 ◦
Solubility in water (g l at 25 C) 0.052 (pH 5.1)
0.063 (pH 5.0, buffer solution)
n-Hexane 0.0022
Toluene 0.32
octanol–water (at 20 ◦ C) –0.81(pH 7)
–2.2 (pH 9)
a
Temperature not reported.
Table 2.2.26 Physico-chemical properties of trifloxysulfuron-sodium.
O CH2CF3 OCH3
O N
SO2 N C N
N + H
Na N
OCH3
Common name (ISO) Trifloxysulfuron-sodium

CAS-No. 199119-58-9
Code numbers CGA 362622
Vapor pressure (Pa at 20 ◦ C) <1.3 × 10 –6
◦
Dissociation constant (pKa at 20 C) 4.76
5.016 (pH 7)
25.7 (pH 7.4)
n-Hexane <0.001
Methanol 50.0
Octanol 4.4
Toluene <0.001
Acetone 17.0
octanol–water (at 25 ◦ C) –0.43 (pH 7)
In cotton, it is formulated as a WG 75, and can be applied in post-emergent

fashion at 5–7.5 g a.i. ha−1 in conventional or GM cotton, but at higher rates of
10–15 g a.i. ha−1 if post-emergent directed. At the lower rates, the following weeds
are controlled: Acanthospermum hispidum, Ambrosia artemisiifolia, Bidens pilosa,
Senna obtusifolia, Cassia occidentalis, Chenopodium album, Euphorbia heterophylla,
Ipomoea spp., Melochia corchorifolia, Mollugo vertillata, Sesbania exalta, Trianthema
portulacastrum, and Xanthium strumarium. With post-directed sprays and higher
dosages, additional control is achieved of Ageratum conyzoides, Amaranthus hy-
bridus, A. palmeri, Cyperus esculentus, and Tridax procumbens. The application of
trifloxysulfuron-sodium may be made after cotton (picker-type varieties only) has
reached a minimum of five true leaves, with applications continuing until 60 days
before harvest. Due to a reduced crop tolerance, the product is not recommended
as a post-emergent over-the-top spray on stripper-type cotton varieties.
For cotton and sugarcane, trifloxysulfuron-sodium is marketed under the trade
name ‘‘Envoke®’’ as a straight product. In cotton, it is used in combination with
prometryn as ‘‘Suprend®,’’ and in sugarcane in combination with ametryn as
‘‘Krismat®.’’ In sugarcane, ‘‘Envoke®’’ can be used for a maximum of three
applications pre-spiking, post-emergence over-the-top, and/or post-emergence
directed at a total rate of 78 g a.i. ha−1 per season. The product may be applied to
sugarcane at a plant height of 45–60 cm up to 100 days before harvest. At a dose rate
of 16 g a.i. ha−1 , the following weeds are controlled with greater than 85% efficacy:
Alternanthera philoxeroides, Acanthospermum hispidum, Panicum adspersum, Mollugo
vertillata, Xanthium strumarium, Cassia occidentalis, Gnaphalium pensylvanicum, Eu-
patorium cappilliforium, Desmodium tortuosum, Trianthema portulacastrum, Sesbania
exaltata, Rottboellia cochinchinensis, Chenopodium album, Ipomoea spp., Cyperus
esculentus, C. rotundus, Amaranthus spp., Ambrosia artemisiifolia, Melochia corchori-
folia, Senna obtusifolia, Bidens bipinnata, Linaia canadensis, Abutilon theophrasti, and
Euphorbia heterophylla.
2.2.3
Sulfonylurea Herbicides: Metabolic Fate and Behavior in the Soil
Although abundant knowledge exists regarding the metabolic fate of sulfonylurea

herbicides, especially with regards to animal data to support product registrations,
most such information has not been made publicly available. For those readers
interested in acquiring further information on plant metabolism and crop selectiv-
ity, the review of Brown et al. [85] is recommended. A second excellent review on
the metabolic fate of sulfonylurea herbicides is available in Part 1 of the Metabolic
Pathways of Agrochemicals series [86].
Two major pathways of sulfonylurea degradation have been identified in the soil
[87], namely chemical hydrolysis and microbial degradation. The breakdown of the
sulfonylureas in sterile soils is attributed solely to chemical hydrolysis, whereas
their breakdown in nonsterile soils involves a combination of microbial degradation
and chemical hydrolysis. The relative importance of microbial degradation can then
be calculated from the differential rate.
The main soil degradation pathways of sulfonylurea herbicides are cleavage of
the sulfonylurea bridge, O- and N-dealkylation reactions, aryl and aliphatic hy-
droxylation reactions, dehalogenation, and ester hydrolysis. It is beyond the scope
of this chapter to discuss each of these in detail for all of the above-mentioned
new sulfonylureas. Instead, mesosulfuron-methyl will be taken as a general illus-
tration of the commonly found soil degradation pathways established within the
sulfonylurea family.
Mesosulfuron-methyl is degraded in soil and water via hydrolysis and
O-demethylation reactions. The metabolites are also readily degraded to nonex-
tractable residues (NERs) and CO2 [88]. During soil metabolism studies with ra-
diolabeled mesosulfuron-methyl, the major metabolites found (which represented
more than 10% of the applied radioactivity) were mesosulfuron acid AE F154851,
pyrimidinyl urea AE F099095, and aminopyrimidine AE F092944 (Scheme 2.2.3).
O-Demethylation of the methyl ether in the pyrimidine moiety to yield hydrox-
ypyrimidine derivative AE F160459 proved to be of minor relevance in soil. Other
minor soil metabolites were the sulfonylurea AE F160460, the sulfonamide AE
F140584, and the saccharin derivative AE F147447. Carbon dioxide and unidentified
NERs bound to the soil matrix were the final products of degradation in the soil.
O
OCH3 OCH3
O N
SO2 N C N
H H
82
N
CH3SO2 N OCH3
H
AE F130060 O
O OH OCH3
OCH3 OH O N
O N SO2 N C N
SO2 N C N H H N
H H N CH3SO2 N OCH3
CH3SO2 N OCH3 H
H AE F160459 AE F154851
O O
OCH3 OH OCH3 OCH3
O N O N
SO2 NH2 SO2 N C N H2N C N
H H N H N
2 Acetohydroxyacid Synthase Inhibitors (AHAS/ALS)
CH3SO2 N CH3SO2 N OCH3 OCH3

H AE F140584 H AE F099095
AE F160460
O
OCH3
NH N
H2N
SO2
N
OCH3
CH3SO2 N
H CO2 + non extractable residues (NER) AE F092944
AE F147447
Scheme 2.2.3 Metabolic pathway of mesosulfuron-methyl in soil under aerobic and anaerobic conditions. Compounds marked in
bold type constituted >10% of the applied radioactivity.
References 83
2.2.4
Concluding Remarks
Since 1995, a total of 14 new sulfonylurea herbicides for the treatment of all major
crops has been commercialized, and three new compounds from this class are
currently in their late development stages. These, together with the 20 sulfonylurea
products available commercially prior to 1995, provide a remarkable figure that
clearly outnumbers any other herbicidal class in modern crop protection. The
reason for this dominance is a combination of the environmental friendliness of
the products, their versatility as regards applicable crops and timing flexibility,
and also their cost/benefit performance. It remains to be seen whether the market
can accept yet further innovations from this class, and whether resistant weed
development will one day become an issue, despite hitherto successful resistance
strategies employed by the agro-industry.
In conclusion, it is fascinating to witness the development that began with
George Levitt’s pioneering studies at Du Pont over 30 years ago. In Gulliver’s
Travels (Voyage to Brobdingnag, Ch. 6), the Irish author Jonathan Swift (1667–1745)
wrote that:
‘‘Whoever could make two ears of corn or two blades of grass grow upon
a spot of ground where only one grew before, would deserve better of
mankind, and do more essential service to his country than the whole race
of politicians put together.’’
It is against this background that the achievements of George Levitt and all other
colleagues involved in the world’s agrochemical industry should be viewed.
Acknowledgments
The author is pleased to acknowledge Drs Darren Mansfield, Klaus-Helmut

Mueller, Graham Holmwood, Arno Schulz, and Shinichi Shirakura for their
critical review of the manuscript and many helpful suggestions. Thanks are also
given to Mrs Tong Lin, Mr Tetsuya Murata, and Dr Tomoki Tsuchiya for providing
translations of the Chinese and Japanese publications.
References
1. Chaleff, R.S. and Mauvais, C.J. (1984) 3. Delfourne, D., Bastide, J., Badon, R.,
Acetolactate synthase is the site of action Rachon, A., and Genix, P. (1994) Speci-
of two sulfonylurea herbicides in plants. ficity of plant acetohydroxyacid synthase:
Science, 224, 1443–1444. formation of products and inhibition by
2. LaRossa, R.A. and Schloss, J.V. (1984) herbicides. Plant Physiol. Biochem., 32,
The sulfonylurea herbicide sulfometuron 473–477.
methyl is an extremely potent and selec- 4. Levitt, G. (1991) Discovery of the sul-
tive inhibitor of acetolactate synthase in fonylurea herbicides, in Synthesis and
Salmonella typhimurium. J. Biol. Chem., Chemistry of Agrochemicals II, ACS
259, 8753–8757. Symposium Series, Vol. 443 (eds
D.R. Baker, J.G. Fenyes, and W.K. II, ACS Symposium Series, (eds D.R.
Moberg), American Chemical Society, Baker, J.G. Fenyes, and W.K. Moberg),
pp. 16–31. American Chemical Society, vol. 443,
5. Beyer, E.M., Duffy, M.J., Hay, J.V., and pp. 107–119.
Schlueter, D.D. (1987) Sulfonylureas, in 13. Suzuki, K., Nawamaki, T., Watanabe,
Herbicides – Chemistry, Degradation and S., Yamamoto, S., Sato, T., and
Mode of Action, vol. 3 (eds P.C. Kearney Morimoto, K. (1991) NC 319 - a new
and D.D. Kaufman), Marcel Dekker, herbicide for control of broad-leaved
New York, pp. 117–189. weeds and Cyperus spp. in corn. Proc.
6. Brown, H.M. and Cotterman, J.C. Brighton Crop Prot. Conf. – Weeds, 1,
(1994) Recent Advances in Sulfonylurea 31–37.
Herbicides, in Herbicides Inhibiting 14. Doig, R.I., Carraro, G.A., and McKinley,
Branched-chain Amino Acid Biosynthesis N.D. (1983) DPX-T6376 - a new broad
(eds W. Ebing (editor-in-chief), J. Stetter spectrum cereal herbicide. Proceedings
(volume editor)), Chemistry of Plant of the 10th International Congress of
Protection, Springer, Berlin, Heidelberg, Plant Protection, British Crop Protection
Vol. 10, pp. 47–81. Council, Croydon, UK, pp. 324–331.
7. D’Souza, D.S.M., Black, I.A., and 15. Murai, S., Haga, T., Fujikawa, K.,
Hewson, R.T. (1993) Amidosulfuron – Sakashita, N., and Kimura, F. (1991) A
a new sulfonylurea for the control of novel sulfonylurea herbicide for corn, in
Galium aparine and other broadleaf Synthesis and Chemistry of Agrochemicals
weeds in cereals. Proc. Brighton Crop II, ACS Symposium Series, (eds D.R.
Prot. Conf. – Weeds, 2, 567–572. Baker, J.G. Fenyes, and W.K. Moberg),
8. (a) Yuyama, T., Takeda, S., American Chemical Society, vol. 443, pp.
Watanabe, H., Asami, T., Peudpaichit, 98–106.
S., Malassa, J.L., and Heiss, P. (1983) 16. Kimura, F., Haga, T., Sakashita, N.,
Proceedings of the 9th Asian Pacific Murai, S., and Fujikawa, K. (1989)
Weed Science Society Congress, Manilla, SL-950, a novel sulfonylurea herbi-
Philippines, November/December, 1983, cide for corn. Proc. Brighton Crop Prot.
pp. 554–559; (b) Takeda, S.S., Erbes, Conf. – Weeds, 1, 29–34.
D.L., Sweetser, P.B., Hay, J.V., and 17. Maurer, W., Gerber, H.R., and
Yuyama, T. (1986) Mode of herbicidal Rufener, J. (1987) CGA 136’872: a
and selective action of DPX-F5384 be- new post-emergence herbicide for the
tween rice and weeds. Weed Res. (Jpn), selective control of Sorghum spp. and
31, 157–163. Elymus repens in maize. Proc. Brighton
9. Russell, M.H., Saladini, J.L., and Crop Prot. Conf. – Weeds, 1, 41–48.
Lichtner, F. (2002) Sulfonylurea her- 18. Schulte, M., Kreuz, K., Nelgen, N.,
bicides. Pestic. Outlook, 13 (4), 166–173. Hudetz, M., and Meyer, W. (1993) CGA
10. Palm, H.L., Riggleman, J.D., and 152,005 - a new herbicide for control of
Allison, D.A. (1980) Worldwide review broadleaved weeds in European maize.
of the new cereal herbicide - DPX 4189. Proc. Brighton Crop Prot. Conf. – Weeds,
Proc. Brighton Crop Prot. Conf. – Weeds, 1, 53–59.
1, 1–6. 19. Palm, H.L., Liang, P.H., Fuesler,
11. Hutchison, J.M., Peter, C.J., Amuti, T.P., Leek, G.L., Strachan, S.D.,
K.S., Hageman, L.H., Roy, G.A., and Wittenbach, V.A., and Swinchatt, M.L.
Stichbury, R. (1987) DPX-A7881: a new (1989) New low-rate sulfonylureas for
herbicide for oilseed rape. Proc. Brighton post-emergence weed control in corn.
Crop Prot. Conf. – Weeds, 1, 63–67. Proc. Brighton Crop Prot. Conf. – Weeds,
12. Haga, T., Tsujii, Y., Hayashi, K., 1, 23–28.
Kimura, F., Sakashita, N., and Fujikawa, 20. Harding, R.H., Chumley, C.B.,
K. (1991) Trifluoromethylpyridines as Cook, G.E., and Holmes, F.A. (1981)
building blocks for new agrochemicals. DPX-5648 – a new herbicide for control
Discovery of a new turf herbicide, in of johnsongrass and many other weeds.
Synthesis and Chemistry of Agrochemicals Proc. West. Soc. Weed Sci., 34, 120–121.
References 85
21. Sionis, S.D., Drobny, H.G., Lefebvre, P., 9 1992, http://ec.europa.eu/food/

and Upstone, M.E. (1985) DPX-M6316 – plant/protection/evaluation/newactive/
a new sulfonylurea cereal herbicide. list2_flupyrsulfi_en.pdf (accessed
Proc. Brighton Crop Prot. Conf. – Weeds, November 2010).
1, 49–54. 30. Teaney, S.R., Armstrong, L., Bentley, K.,
22. Amrhein, J. and Gerber, H.R. (1985) Cotterman, D., Leep, D., Liang, P.H.,
CGA 131’036: a new herbicide for Powley, C., Summers, J., Cranwell, S.,
broadleaved weed control in cereals. Lichtner, F., and Stichbury, R. (1995)
Proc. Brighton Crop Prot. Conf. – Weeds, DPX-KE459 - a new sulfonylurea for
1, 55–62. postemergence grass and broadleaf weed
23. Ferguson, D.T., Schehl, S.E., Hageman, control in cereals. Proc. Brighton Crop
L.H., Lepone, G.E., and Carraro, G.A. Prot. Conf. – Weeds, 1, 49–56.
(1985) DPX-L5300 - a new cereal 31. Lechner, M. (2002) Ciral® – Ein neues
herbicide. Proc. Brighton Crop Prot. Nachauflaufherbizid zur Bekämpfung
Conf. – Weeds, 1, 43–48. von Ungräsern und Unkräutern
24. Peeples, K.A., Moon, M.P., Lichtner, im Getreide. Mitt. Biol. Bundesanst.
F.T., Wittenbach, V.A., Carski, T.H., Land-Forstwirtsch., 390, 243–244.
Woodward, M.D., Graham, K., and 32. Ishida, Y. Ohta, K., and Yoshikawa, H.
Reinke, H. (1991) DPX 66037 - a new (1992) Preparation of sulfonylurea her-
low-rate sulfonylurea for postemer- bicides. European Patent Application EP
gence weed control in sugar beet and 477808 A1, April 1 1992.
fodder beet. Proc. Brighton Crop Prot. 33. Parrish, S.K., Kaufmann, J.E., Croon,
Conf. – Weeds, 1, 25–30. K.A., Ishida, Y., Ohta, K., and Itoh, S.
25. Gee, S.K. and Hay, J.V. (1994) Re- (1995) MON 37500: A new selective her-
cent developments in the chemistry bicide to control annual and perennial
of sulfonylurea herbicides, in Her- weeds in wheat. Proc. Brighton Crop Prot.
bicides Inhibiting Branched-chain Conf. – Weeds, 1, 57–63.
Amino Acid Biosynthesis (eds W. Ebing 34. Gibson, G. and de Kerchove, G. (1999)
(editor-in-chief), J. Stetter (volume ed- Timing-related yield increases in winter
itor)), Chemistry of Plant Protection, wheat after applications of MON37500
Springer, Berlin, Heidelberg, Vol. 10, herbicide to control barren brome (Bro-
pp. 15–46. mus sterilis). Proc. Brighton Crop Prot.
26. Hay, J.V. (1990) Chemistry of sul- Conf. – Weeds, 1, 87–92.
fonylurea herbicides. Pestic. Sci., 29, 35. Ort, O., Bauer, K., and Bieringer, H.
247–261. (1992) Preparation of [(arylsul-
27. Croplife International (2010) Facts and fonyl)ureido]azines as herbicides and
Figures – The Status of Global Agri- plant growth regulators. PCT Int. Appl.
culture, http://www.croplife.org/files/ WO 9213845 A1, August 20 1992.
documentspublished/1/en-us/PUB-BR/ 36. Hacker, E., Bieringer, H., Willms, L.,
5743_PUB-BR_2010_11_23_Facts_ Ort, O., Koecher, H., and Kehne,
and_figures_-_The_status_of_global_ H. (1999) Iodosulfuron plus
agriculture_(2010).pdf. (accessed mefenpyr-diethyl - a new foliar herbi-
November 2010). cide for weed control in cereals. Proc.
28. UN Food & Agriculture Organisa- Brighton Crop Prot. Conf. – Weeds, 1,
tion (FAO) (2010) FAOSTAT Data, 15–22.
http://faostat.fao.org. (accessed November 37. Hacker, E., Bieringer, H., Willms, L.,
2010). Roesch, W., Koecher, H., and Wolf, R.
29. Andrea, T.A. and Liang, P.H.T. (1992) (2000) Mefenpyr-diethyl – A safener
Preparation of Me 2-[[[[(4,6-dimethoxy- for fenoxaprop-P-ethyl and iodosul-
2-primidinyl)amino]carbonyl]amino] furon in cereals. Z. PflKrankh. PflSchutz,
sulfonyl]-6-trifluoromethyl-3- Sonderh., XVII, 493–500.
pyridinecarboxylate and salts as 38. Lorenz, K., Willms, L., Bauer, K., and
herbicides. European Patent Appli- Bieringer, H. (1995) Phenylsulfonyl
cation EP 502740 A1, September ureas, process for producing the same
and their use as herbicides and plant 48. Wang, J., Ma, N., Wang, B., Wang, S.,
growth regulators. PCT Int. Appl. WO Song, H., and Li, Z. (2006) Synthesis,
9510507 A1, April 20 1995. crystal structure and biological activ-
39. Hacker, E., Bieringer, H., Willms, L., ity of N-(4-methylpyrimidin-2-yl)-N -2-
Lorenz, K., Koecher, H., Huff, H.P., (nitrophenylsulfonyl)urea and its dock-
Borrod, G., and Brusche, R. (2001) ing with yeast AHAS. Chin. J. Org.
Mesosulfuron-methyl – a new active Chem., 26, 648–652.
ingredient for grass weed control in 49. Ou, X., Lei, M., Huang, M., Wang, Y.,
cereals. Proc. Brighton Crop Prot. Conf. – Wang, X., and Fan, D. (2003) Ef-
Weeds, 1, 43–48. fects of novel sulfonylurea herbicide
40. Atlantis® (2005) Pflanz.-Nachr. Bayer, 58 HNPC-C9908 on growth of green algae
(2), 165–299, ISSN 0340-1723. Chlorella pyrenoidosa. Nongyaoxue Xuebao
41. Mayer, H., Hamprecht, G., Westphalen, (Chin. J. Pestic. Sci.), 5 (3), 16–23.
K.-O., Walter, H., Gerber, M., 50. Huang, M., Huang, L., Chen, C., Zhao,
Grossmann, K., and Rademacher, W. L., Lei, M., Wu, T., and Yu, S. (2000)
(1992) Herbicidal N-[(1,3,5-triazin-2-yl) Faming Zhuanli Shenqing Gongkai
aminocarbonyl]benzenesulfonamides Shuomingshu. Chinese Patent Ap-
and their preparation Ger. Offen., plication CN 2000-113423, May 11
DE 4038430 (Publ. 04.06.1992), PCT 2000.
WO92/09608 (Publ. 11.06.1992). 51. Pang, H., Yang, J., Huang, M., Ren, Y.,
42. Dombo, P., Brix, H.D., Landes, M., and and Yin, D. (2007) Synthesis and her-
Nuyken, W. (2002) Der neue Herbizid- bicidal activity of 2-[[[[(4-methoxy-6-
wirkstoff Tritosulfuron – Charakter- methylthio-2-pyrimidinyl)amino]carbonyl]
isierung und Einsatz im Ackerbau. Mitt.
amino]sulfonyl]benzoic acid methyl
Biol. Bundesanst. Land-Forstwirtsch., 390,
ester. Nongyao, 46 (2), 86–88.
473–474.
52. Wang, B., Ma, N., Wang, J., Ma, Y.,
43. Schönhammer, A., Pörksen, N.,
Li, Z., and Leng, X. (2004) Synthesis
Freitag, J., and Nuyken, W. (2002)
and dimeric crystal structure of sul-
Biathlon –das erste Tritosulfuron-haltige
fonylurea compound N-[2-(4-methyl)
Herbizid zur Unkrautbekämpfung
pyrimidinyl]-N -2-methoxycarbonyl-
in monokotylen Kulturen. Mitt. Biol.
benzene sulfonylurea. Chin. J. Struct.
Bundesanst. Land-Forstwirtsch., 390,
Chem., 23, 783–787.
241–242.
53. Weng, H., Guo, Q., Wei, Y., Guo, L.,
44. Dressel, J. and Beigel, C. (2001) Estima-
tion of standardized transformation rates and Cheng, L. (2008) Weed control and
of a pesticide and its four soil metabo- safety study of monosulfuron-ester 10%
lites from field dissipation studies for WP on spring wheat fields. Xiandai
use in environmental fate modeling. Nongyao, 7 (5), 49–51.
BCPC Symposium Proceedings. 78 - 54. Wang, J., Lee, P., Dong, Y., Pang, S.,
Pesticide Behaviour in Soils and Water, Duggleby, R., Li, Z., and Guddat, L.
pp. 119–126. (2009) Crystal structures of two novel
45. Li, Z., Jia, G., Wang, L. et al. (1993) sulfonylurea herbicides in complex with
Faming Zhuanli Shenqing Gongkai Arabidopsis thaliana acetohydroxyacid
Shuomingshu (1994). Chinese Patent synthase. FEBS J., 276 (5), 1282–1290.
Application CN 93-101976, February 27 55. Diarra, A., Smith, R.J., and Talbert, R.E.
1993. Jr (1985) Interference of red rice (Oryza
46. Fan, Z., Ai, Y., Qian, C., and Li, Z. sativa) with rice (O. sativa). Weed Sci.,
(2005) Herbicide activity of monosul- 33, 644–649.
furon and its mode of action. J. Environ. 56. Ferrero A. (2003) Weedy rice, bio-
Sci. (China), 17 (3), 399–403. logical features and control, in Weed
47. Wang, M., Kou, J., Ju, G., and Liu, B. Management for Developing Countries
(2008) Applied research of a new sul- (Addendum 1), FAO Plant Production
fonylurea herbicide monosulfuron. and Protection Papers – 120 Add.1 (ed.
Nongyao, 47 (6), 412–414, 422. R. Labrada), ISBN: 9251050198.
References 87
57. Fisher, A. and Ramirez, A. (1993) Red 66. Tan, X., Wang, D., and Yu, H. (2005)
rice (Oryza sativa): competition studies Sheng Wu Gong Cheng, 22 (2), 47–48.
for management decisions. Int. J. Pest 67. Koo, S.J., Cho, J.H., Kim, J.S., Kang,
Manage., 39 (2), 133–138. S.H., Kang, K.G., Kim, D.W., Chang,
58. Kehne, H., Willms, L., Bauer, K., H.S., Ko, Y.K., and Ryu, J.W. (2002)
Bieringer, H., and Buerstell H. (1989) Preparation of herbicidally active
Heterocyclic 2-alkoxyphenoxy- pyridylsulfonyl ureas. PCT Interna-
sulfonylureas and their use as herbi- tional Application WO 2002030921 A1,
cides or as plant growth regulators. April 18 2002.
European Patent Application EP 342569 68. Kim, D.S., Koo, S.J., Lee, J.N., Hwang,
A1, November 23 1989. K.H., Kim, K.G., Kang, K.G., Hwang,
59. Hacker, E., Bauer, K., Bieringer, H., K.S., Joe, G.H., Cho, J.H., and Kim,
Kehne, H., and Willms, L. (1995) HOE D.W. (2003) Flucetosulfuron: a new sul-
095404: a new sulfonylurea herbicide fonylurea herbicide. Proceedings of the
for use in cereals, rice and sugarcane. BCPC International Congress – Crop
Proc. Brighton Crop Prot. Conf. – Weeds, Science & Technology, November
1, 73–78. 10–12, Glasgow, UK, British Crop
60. Hess, M. and Rose, E. (1995) HOE Protection Council, Bracknell, UK, vol.
095404: a new herbicide for broadleaf 1, pp. 87–92.
weed and sedge control in rice. Proc. 69. Kim, D.S., Lee, J.N., Hwang, K.H.,
Brighton Crop Prot. Conf. – Weeds, 2, Kang, K.G., Kim, T.Y., and Koo, S.J.
(2003) Flucetosulfuron: a new tool to
763–768.
control Galium aparine and broadleaf
61. Levitt, G. (1988) Tetrazole-containing
weeds in cereal crops. Proceedings of
sulfonylureas, their herbicidal composi-
the BCPC International Congress – Crop
tions, and their use in weed control. US
Science & Technology, November
Patent 4,746,353 A, May 24 1988.
10–12, Glasgow, UK, British Crop Pro-
62. Marquez, T., Joshi, M.M., Pappas Fader,
tection Council, Bracknell, UK, vol. 2,
T., and Massasso, W. (1995) Az-
pp. 941–946.
imsulfuron (DPX-A8947)-a new
70. Bettarini, F., Massimini, S., Meazza,
sulfonylurea for post-emergence con-
G., Zanardi, G., Portoso, D., and
trol of Echinochloa species, broadleaf
Signorini, E. (1998) PCT International
and sedge weeds for southern Euro-
Application WO 9840361 A1, September
pean rice production. Proc. Brighton 17 1998.
Crop Prot. Conf. – Weeds, 1, 65–72, 71. Federal Register (2007) Orthosulfamuron;
http://www.fao.org/ag/AGP/agpp/Pesticid/ Pesticide Tolerance, 72 (39), 8928–8931;
Specs/docs/Pdf/new/azimsu05.pdf http://www.epa.gov/opprd001/factsheets/
(accessed November 2010). orthosulfamuron.htm (accessed November
63. Condon, M.E., Brady, T.E., Feist, D., 2010).
Malefyt, T., Marc, P., Quakenbush, 72. ISAGRO S.p.A. (2010) Orthosulfamuron:
L.S., Rodaway, S.J., Shaner, D.L., and Technical Bulletin, May; http://www.
Tecle, B. (1993) AC 322,140 – a new isagro.com/images/stories/file/PDF/
broad-spectrum herbicide for selective Orthosulfamuron%20Technical%20Bulletin
weed control in rice and cereals. Proc. %20may2010.pdf (accessed November
Brighton Crop Prot. Conf. – Weeds, 1, 2010).
41–46. 73. Tanaka, Y., Kajiwara, Y., Noguchi, M.,
64. Rodaway, S.J., Tecle, B., and Shaner, Kajiwara, T., and Tabuchi, T. (2003)
D.L. (1993) Mechanisms of selectiv- Fused heterocyclic sulfonylurea com-
ity of AC 322,140 in paddy rice, wheat pound, herbicide containing the same,
and barley. Proc. Brighton Crop Prot. and method of controlling weed with
Conf. – Weeds, 1, 239–246. the same. PCT International Application
65. Brady, T.E., Condon, M.E., and Marc, WO 03061388, July 31 2003.
P.A. (1991) US Patent 5,009,699, April 74. Tanaka, Y., Kajiwara, Y., and
23 1991. Nishiyama, T. (2005) Composition of
herbicide. US Patent JP 2005-126415, programs. Proc. Brighton Crop Prot.

May 19 2005. Conf. – Weeds, 1, 79–85.
75. Kagaku, S. (2010) ‘‘i-nouryoku day- 83. Foery, W. (1997) Sulfonylurea salts as
ori’’ No. 72, http://www.i-nouryoku. herbicides. PCT International Appli-
com/index.html) (accessed November cation WO 9741112 A1, November 06
2010). 1997.
76. Kita, H., Tamada, Y., Nakaya, Y.,
84. Howard, S., Hudetz, M., and Allard,
Yano, T., and Saeki, M. (2005) Prepara-
J.-L. (2001) Trifloxysulfuron-sodium: a
tion of pyrazole sulfonylurea compounds
containing pyrimidine moiety as herbi- new post emergence herbicide for use
cide. PCT International Application WO in cotton and sugarcane. Proc. Brighton
2005103044 A1, November 03 2005. Crop Prot. Conf. – Weeds, 1, 29–34.
77. Schnabel, G., Willms, L., Bauer, K., 85. Brown, H.M., Fuesler, T.P., Ray, T.B.,
and Bieringer, H. (1995) Acylated and Strachan, S.D. (1991) Pesticide
Aminophenylsulphonylureas, process Chemistry: Advances in International
for their preparation and their use as Research, Development and Legislation,
herbicides and plant-growth regulators. in Proceedings of the International IUPAC
PCT International Application WO Congress of Pesticide Chemistry, 7th edn,
9529899, November 09 1995. (ed. H. Frehse), Wiley-VCH Verlag
78. Collins, B., Drexler, D., Maerkl, M., GmbH, Weinheim, pp. 257–266.
Hacker, E., Hagemeister, H., Pallett,
86. Brown, H.M., Gaddamidi, V., and
K.E., and Effertz, C. (2001) Foramsul-
Lee, P.W. (1998) in Metabolic Pathway
furon – a new foliar herbicide for weed
of Agrochemicals, Part 1: Herbicides
control in corn (maize). Proc. Brighton
Crop Prot. Conf. – Weeds, 1, 35–42. and Plant Growth Regulators (eds
79. Bunting, J.A., Sprague, C.L., and T.R. Roberts (editor-in-chief) D.H.
Riechers, D.E. (2005) Incorporating Hutson, P.W. Lee, P.H. Nicholls, and
foramsulfuron into annual weed control J.R. Plimmer (contributing editors)),
systems for corn. Weed Technol., 19 (1), The Royal Society of Chemistry Infor-
160–167. mation Services, pp. 451–473, ISBN:
80. Bunting, J.A., Sprague, C.L., and 0-85404-494-9.
Riechers, D.E. (2004) Physiological 87. Brown, H.M. (1990) Mode of action,
basis for tolerance of corn hybrids to crop selectivity, and soil relations of the
foramsulfuron. Weed Technol., 52 (5), sulfonylurea herbicides. Pestic. Sci., 29,
711–717.
263–281.
81. Meyer, W. (1992) Sulfonylureas as her-
88. Gildemeister, H. and Schäfer, D.
bicides. European Patent Application EP
(2005) Behaviour of the herbicide
496701 A1, July 29 1992.
82. Brooks, R.L., Zoschke, A., and Porpiglia, mesosulfuron-methyl in the environ-
P.J. (1995) CGA-277476: a short resid- ment. Pflanz.-Nachr. Bayer, 58 (2),
ual herbicide for soybean weed control 195–214. ISSN: 0340-1723.
2.3
Imidazolinone Herbicides
2.3.1
Overview
The imidazolinone herbicides (Figure 2.3.1) comprise a family of six compounds

that were discovered and initially developed by American Cyanamid Company, with
O
R1 Common name
R1
H Imazapyr
OH
CH3 Imazapic
N
N CH2CH3 Imazethapyr
HN CH2OCH3 Imazamox
O
O O O
H3C CH3 CH3
O O OH
+ N
N H3C N
N
HN HN HN
O O O
Imazamethabenz-methyl Imazaquin
Figure 2.3.1 Structure of commercialized imidazolinones.
subsequent development continuing with the BASF Corporation after acquisition in

2000. Comprehensive and detailed information on these compounds is available in
the book The Imidazolinone Herbicides [1], written by the research groups responsible
for their discovery and development. The imidazolinone herbicides as a class are
broad spectrum, and are active both pre- and post-emergence. They are absorbed
and moved through both the xylem and phloem, eventually accumulating in the
meristematic tissues. The activity of the imidazolinone herbicides is characterized
by a rapid cessation of growth, followed by plant death days or weeks after
treatment. The selectivity of these herbicides is based most often on their metabolic
inactivation, except for selection-developed target site-based resistance.
The synthesis methodology for numerous imidazolinones has been described
in the patent literature [2–6]. The details of a simple, one-step method are shown
in Figure 2.3.2 [7].
Imidazolinones are generally formulated as the amine salts. However, perhaps
because of their high potency, broad spectrum activity and high water solubility,
they have been coformulated with many other herbicides.
O O
t -BuOK OK
OMe + H2N CONH2
toluene, N
OMe 80°C N
N
O HN
O
Figure 2.3.2 Synthesis method for imidazolinones.

2.3.2
History of Discovery
The imidazolinone herbicides were discovered through a long process of obser-

vation, exploration, and optimization, the details of which have been presented
in great detail elsewhere [2, 3]. The initial lead molecule (1 in Figure 2.3.3) was
synthesized during the 1950s by an American Cyanamid Medical Division chemist
working on anticonvulsants. However, on its arrival some years later at the Agricul-
tural Division for random screening it showed herbicidal activity, at an application
rate of 4 kg ha−1 , that warranted additional synthesis effort. Although the mode of
action was not known, nor even investigated at the time, several years after the
discovery of the imidazolinones this original phthalimide was shown to inhibit
the enzyme acetohydroxyacid synthase (AHAS), which was involved primarily
in the biosynthesis of the branched-chain amino acids valine, leucine, and
isoleucine.
Although initial modifications of 1 failed to improve its herbicidal activity,
derivative 2 showed interesting plant growth-regulant activity similar to that of
gibberellic acid (Figure 2.3.3) [4, 5]. This new compound, when further optimized
for plant growth regulation, resulted in compound 3.
Associated investigations to enable the production of field trial samples resulted in
a tricyclic compound and, in the spirit of comprehensive exploration (and thorough
patent coverage), the same reaction was attempted on the original herbicide
lead compound, which resulted in 4 (see Figure 2.3.4). The latter compound
showed broad-spectrum herbicidal activity, while continued exploration in the series
resulted in the first imidazolinone herbicide 5 (Figure 2.3.4), which had a markedly
O O O
CH3 O CH3O
N NH2 N NH2 N NH2
CH3 CH3 O
O H3C Cl O H3C O
Cl
1 2 3
Figure 2.3.3 Early lead compounds that led to the imidazolinones.
O O
N CH3OH OH
CH3ONa N
N O
5 HN
4
O
Figure 2.3.4 Synthesis of the first imidazolinone lead compound.

improved herbicidal spectrum and potency with some selectivity in rice. As studies
continued in the program, the eventual result was compound 6, an isomeric
mixture of imazamethabenz-methyl, a wheat-selective herbicide (Figure 2.3.5).
A quantum leap in both the herbicidal potency and spectrum occurred when the
benzene ring was replaced with a pyridine ring, such that the resulting compound
had pre- and post-emergence activities at doses ranging from 10 to 100 g ha−1 in
greenhouse tests. Further exploration of this new series showed that the picolinic
acid and isonicotinic acid had far less herbicide activity than the nicotinic acid. In
addition, a high activity would be maintained only in derivatives with substituents
at the 5- and 6-position of the pyridine ring. Thus, unlike the other major classes
of AHAS-inhibiting herbicides, the imidazolinones possessed a relatively narrow
structure–activity pattern [8].
2.3.3
Physico-chemical Properties
The imidazolinone salts have a high water solubility, ranging from over 57%
(for the imazapyr isopropylamine salt) to 17% (for the imazaquin ammonium
salt). Imazapyr has two sites for protonation, namely the imidazolinone secondary
nitrogen and the carboxylic acid substituent on the pyridine ring. The ionization
constants are similar for the pyridine imidazolinone herbicides; for imazapyr,
pK1 is 1.9 and pK2 is 3.6 (Figure 2.3.6). A third ionization on the primary
imidazolinone nitrogen occurs at pH ∼ 11 (Figure 2.3.6) [9]. pK2 is important
for concentrating the herbicide inside the cell through a weak acid-trapping
mechanism. Outside the cell, in the apoplast, a relatively low pH allows a substantial
proportion of the imidazolinone to exist in an uncharged state, with sufficient
lipophilicity to passively cross the cell membranes. Once inside the cell, the pH is
O O
H3C CH3 CH3
O O
+
N H3C N
HN HN
O O
6
Figure 2.3.5 Structure of imazamethabenz methyl.
O O O
O
pK1 OH pK2
O− pK3 O−
OH
H+ N N N N
N N N N
HN HN N−
HN
O O O
O
Figure 2.3.6 Ionization states of imidazolinones.

much higher such that the charged form will predominate, effectively trapping the
herbicide inside the cell [10].
2.3.4
Structural Features of Herbicidal Imidazolinones
The structural features of imidazolinones that are important for target site and
herbicide activity have been summarized [11–14]. The orientation of the imidazoli-
none ring ortho to the acid equivalent is critical; derivatives of the acid equivalent
are herbicidally active if the derivative can be converted to the acid either in the soil
or in the plant. Likewise, tricyclic derivatives such as 4 are pro-herbicides that must
be transformed to the active acid imidazolinone form.
The commercial herbicides are a mixture of R- and S-isomers at the chiral center
where the methyl and isopropyl substituents are placed, though the (R)-isomer is
more potent both as an enzyme inhibitor and as a herbicide. Substituents other
than methyl and isopropyl result in substantially weaker enzyme inhibitors and
herbicides [14].
The aromatic ring component illustrates the relative contributions of enzyme
inhibition and physico-chemical properties towards the herbicidal activity. Thus,
imidazolinones with a benzene component are approximately 10-fold more potent
than the corresponding pyridine derivatives as enzyme inhibitors but, paradoxically,
are less potent as herbicides.
The primary factor that governs the biological activity of the imidazolinones,
besides the inhibition of AHAS, is their ability to translocate to the meristematic
tissues. AHAS – the target site for these herbicides – functions primarily in rapidly
dividing tissue, but its activity decreases rapidly as the tissues mature [15]. Thus,
any differences in herbicidal activity among the six commercially available imida-
zolinones arise from variations in plant absorption and translocation properties.
Imazaquin, which is used primarily via soil-application, is the most lipophilic of
all commercially available herbicides, and is the most readily absorbed by roots
and translocated to the shoots [16, 17]. Imazapyr and imazamox, in contrast,
are the least lipophilic and are most active when applied to the foliage [16, 17].
Imazethapyr and imazapic have lipophilicity characteristics located between these
two extremes.
The differences in herbicidal activity among these analogs appear to be related
to their ability to be trapped in the phloem. The imidazolinones are absorbed into
phloem via an ion-trapping mechanism (see Section 2.3.3); thus, they are all able
to penetrate the phloem and subsequently be carried to the meristematic tissues.
The concentration of herbicide that actually reaches the meristems, however, is
a function of how rapidly the chemicals diffuse out of the phloem as it moves
through the plant. Imazaquin diffuses away from the phloem more readily than
imazamox or imazapyr, mainly because of its lipophilicity; consequently, it does
not far in the phloem and therefore has a limited post-emergent activity compared
to imazamox or imazapyr. As noted above, the benzene imidazolinones are less
herbicidally active than the pyridine imidazolinones, despite the former analogs
being more potent inhibitors of AHAS. The benzene imidazolinones are more
lipophilic than the pyridine imidazolinones, and hence are not trapped also in the
phloem.
It is possible that other factors might also govern the herbicidal activity of the
imidazolinones. For example, the position of the nitrogen in the pyridine ring in
relation to the carboxylic and imidazolinone ring substitution is critical, although
the inhibition of AHAS is unaffected by the relative position of the nitrogen in
the imidazolinone ring in relation to the substitutions [16]. The cellular uptake of
imidazolinones is affected by the relative position of the nitrogen to the carboxylic
acid moiety. Hawkes et al. have shown that an effective cellular absorption occurs
only when the ring nitrogen is ortho to the imidazolinone ring and meta to the
carboxylic acid [18]. The reason for this differential uptake is not known, but if an
imidazolinone is neither absorbed nor translocated well within the plant it cannot
show herbicidal characteristics.
2.3.5
Imidazolinones: The Mode of Action
Although plant growth ceases soon after application of the imidazolinone

herbicides, the whole plant may not die for another two to three weeks. Following
imidazolinone herbicide application, the meristematic tissues will initially exhibit
chlorosis and necrosis, followed by a slow necrosis of the mature tissues. The
physiological changes that result from herbicide treatment include changes in
metabolite concentrations [19, 20], a reduction of assimilate transport [21, 22],
and the inhibition of both DNA synthesis [23, 24] and cell division [23, 25].
These physiological effects in plants result from an inhibition of AHAS. The
fact that supplementation of the treated plants with branched-chain amino acids
reverses the herbicidal effects [21] suggests that starvation of branched-chain
amino acids is the primary cause of plant death [26]. Microarray analyses in
which wild-type Arabidopsis was compared with an imidazolinone-tolerant mutant,
csr1-2D , that contained an imidazolinone-resistant AHAS, showed that a number
of genes had been upregulated and downregulated following the application of
imazapyr to the wild-type organism, but that no changes had occurred in the
imidazolinone-tolerant line [27]. These results confirmed AHAS as the only target
site for the imidazolinone herbicides.
In in vitro AHAS assays, the degree of enzyme inhibition (monitored as I50 )
for various commercial imidazolinone herbicides was shown to vary between
0.1 and 10 μM, depending on the assay conditions [28]. Under in vivo conditions,
the binding of imidazolinones appears to cause an irreversible loss of AHAS activity
[29], with the level of extractable AHAS activity after treatment with a lethal dose
of an imidazolinone herbicide being reduced by more than 80%. This inhibitory
effect can be discerned within an hour of treatment, with the loss of AHAS activity
being proportional to the concentration of inhibitor in the plant tissue. Several
possible explanations have been proposed for the loss of extractable AHAS activity
in imidazolinone-treated plants:
• The imidazolinones may interact with the enzyme in such a way in vivo that
the herbicide does not easily separate from the enzyme during the extraction
procedure.
• The herbicide causes a change in the protein structure such that it becomes
enzymatically inactive.
• The inhibitor-bound enzyme is easily degraded by proteases (though this last
possibility has now been ruled out on the basis of immunoassay studies; Bijay
Singh, unpublished results).
The binding of imazethapyr with AHAS appears to stabilize the AHAS protein in
relation to other proteins, which are degraded following herbicide treatment [30].
2.3.6
Imidazolinone-Tolerant Crops
Despite the many desirable herbicidal properties of the imidazolinones, the emer-
gence of imidazolinone-tolerant crops was first noted during the early 1980s, at
exactly the time when a wide variety of imidazolinone herbicides had been discov-
ered and were undergoing development for their commercialization. Indeed, this
is probably the first time when selection for a herbicide-tolerant crop was started
so early during the development of a class of herbicides. Following the successful
production of imidazolinone-tolerant maize plants through tissue culture selection
and regeneration [31], subsequent investigations confirmed that the resistance of
the whole plant was a semi-dominant trait that resulted from an alteration in
the gene encoding AHAS. The results of these early studies not only proved that
imidazolinone-tolerant crops could be selected, but also led to the discovery of
the site of action for this class of herbicides, and to the development of other
imidazolinone-tolerant crops.
Although plants tolerant to imidazolinones have been produced by both trans-
genic and nontransgenic mechanisms, all of the imidazolinone-tolerant crops
currently sold were developed using nontransgenic methods. Following the intro-
duction of the first imidazolinone-tolerant crop (maize) in 1992, five additional
imidazolinone-tolerant crops (canola, lentil, rice, wheat, and sunflower) have be-
come available on a commercial basis [32], all of which are marketed under the
trade names of Clearfield® or Clearfield® Plus.
The imidazolinone-tolerance traits in different crops were developed by em-
ploying a variety of methods that included tissue culture selection (maize), pollen
mutagenesis (maize), microspore selection (canola), seed mutagenesis (lentil, sun-
flower, wheat, rice), and the incorporation of resistance trait from a weedy relative
(sunflower). Details of these methods have been reviewed previously [32, 33]. In
all cases, the basis of tolerance is due to the presence of an altered form of AHAS
that is resistant to inhibition by imidazolinones. A single base-pair change in the
gene encoding the large subunit of AHAS, and the consequent single amino acid
change in the mature protein, results in an imidazolinone-resistant enzyme. Sev-

eral different mutations in the gene encoding the large subunit of AHAS have been
identified that confer tolerance to imidazolinones [32, 34]. Specifically, the amino
acid changes identified in different imidazolinone-tolerant crops are Ala122Thr or
Ala205Val (sunflower; amino acid number in reference to AHAS sequence from
Arabidopsis thaliana), Trp574Leu (maize and canola), Ser653Asn (maize, canola,
wheat, and rice), and Gly654Glu (rice). The amino acid changes that confer tol-
erance to imidazolinones are distributed over the entire primary structure of the
AHAS protein. However, these amino acids reside in a pocket of the folded protein
in the quaternary structure of the enzyme [35, 36].
Among the imidazolinone family, four different molecules – imazapyr, imaza-
pic, imazethapyr, and imazamox – have now been registered for weed control in
various imidazolinone-tolerant crops in different regions of the world. Typically,
the herbicides can be applied either alone or in combination with other imidazoli-
nones, or with other classes of herbicides to provide a broad spectrum, season-long
weed control. A combination of different imidazolinone-tolerance traits and mul-
tiple herbicide options provides an effective weed management tool for farmers
worldwide.
2.3.7
Imidazolinones: Mechanisms of Selectivity
The crop selectivity of the imidazolinones is dependent primarily on the dif-

ferential metabolism of the herbicide between the crops and targeted weeds.
For the 5 -substituted imidazolinones (i.e., imazethapyr, imazamox, imazapic,
imazamethabenz-methyl), detoxification of the herbicides occurs via a mixed-
function oxidase that hydroxylates the 5 substituent, followed by conjugation of
the metabolite to glucose through the hydroxyl group [37] (Figure 2.3.7). Both,
imazapyr and imazaquin are metabolized via a different route, in which there is
condensation between the carboxylic acid on the aromatic ring to nitrogen in the
imidazolinone ring, followed by cleavage of the imidazolinone ring (Figure 2.3.7).
The half-life of imidazolinones in naturally tolerant crops is less than 24 h [38].
Imidazolinone-resistant crops contain a selected mutation in the AHAS gene which
encodes an enzyme that no longer binds these herbicides, although metabolism
may play a role in determining the level of tolerance of the resistant crop.
The weed spectrum of each imidazolinone is governed by the relative rate of
metabolism in the individual weed species. Imazethapyr controls many broadleaf
weeds and some grasses, but has limited activity on legumes and many composites.
Imazamox, in contrast, has a much better activity than imazethapyr on grasses.
Imazapyr controls the broadest spectrum of weeds of all the imidazolinones,
although it is not as active on legumes and composites. The reason for these
differences is due to the ability of weeds to metabolize the herbicides. The
half-life of imazethapyr in many grasses is less than 24 h because they can rapidly
hydroxylate the 5’-ethyl substituent of imazethapyr [38]. However, most grasses
are unable to rapidly hydroxylate the 5’-methoxyethyl substituent on imazamox
O O
H3C
O OH OH
N N
N N
10 HN
7 HN
O O O
O
O N
O
N N
N NH2 14 N O
HO OH H
11 O
N
N
8 HN O
O
O NH2
N
H
N O
O 12 OH
GLUCOSE OH O
N OH
N
9 HN O
N
O 13
OH
Figure 2.3.7 General routes of the metabolism of imidazolinones [36].
[38]. Legumes and many composites can hydroxylate the 5’ substituent of both
imazethapyr and imazamox. Since imazapyr is unsubstituted on the pyridine
ring, most weed species cannot metabolize the herbicide. However, legumes and
some composites show a slow metabolism of imazapyr via the condensation/ring
cleavage mechanism described above [38].
2.3.8
Commercial Uses of the Imidazolinone Herbicides
Currently, six imidazolinones are commercially available, all of which have an

extremely low toxicity or are nontoxic to mammals, birds, invertebrates, and fish
[37]. The crops for which these herbicides are registered, and whether or not they
are applied to foliage or to the soil, is determined by the structure of the chemical
(Table 2.3.1). When applied to the foliage of plants, a nonionic surfactant or oil
adjuvant is required for maximum activity. The addition of either urea or another
form of nitrogen can also increase herbicidal activity.
• Imazamethabenz methyl is strictly applied post-emergent to most major varieties

of wheat (spring and winter), barley (spring and winter) and rye, as well as to
some varieties of winter triticale and sunflower and safflower.
Table 2.3.1 Registered uses of imidazolinone herbicides in the United States.
Imidazolinone Application Crop Imidazolinone-

resistant crop
Imazamethabenz Foliar Barley, wheat, sunflower –

methyl
Imazethapyr Foliar and Edible beans, peas, soybean, lentils, Maize, rice
soil alfalfa, peanuts, clover, birdsfoot
trefoil, crown vetch, lupine,
switchgrass, wheatgrass, little
bluestem, orchardgrass, western
wheatgrass, big bluestem,
canarygrass
Imazamox Foliar Soybeans, chicory, peas, edible Canola, wheat,
beans, alfalfa, clover sunflowers, lentils, rice
Imazapyr Foliar and Forest lands, wetlands, noncrop Maize
soil areas, roadsides, bahiagrass,
Bermudagrass
Imazaquin Foliar and Soybeans, yucca, hosta, –
soil Bermudagrass, centipedegrass,
mondograss, pachysandra, St
Augustinegrass, zoysiagrass,
Liriope, crape myrtle, Gardenia,
Indian hawthorn, wax-myrtle,
dwarf yaupon, Holly, Fraser
photinia, Pfitzer juniper
Imazapic Foliar and Peanuts, sugarcane, pastures, –
soil rangeland, ornamental turf, ditch
banks, conservation reserve
program land, noncrop areas
• Imazamox is used post-emergent in leguminous crops, including soybeans,

alfalfa, and edible beans, as well as in imidazolinone-resistant wheat, sunflower,
rice, lentil, and canola.
• Imazaquin is primarily a soil-applied herbicide that is used in soybeans, es-
tablished Bermudagrass, centipedegrass, St Augustinegrass, zoysiagrass, and
selected landscape ornamentals.
• Imazethapyr is used both post-emergent and pre-emergent in soybeans, edible
beans, alfalfa, peanut, and imidazolinone-resistant maize, rice, lentil, and canola.
• Imazapic is also applied both to the foliage and the soil in peanuts, rangeland,
sugarcane, and imidazolinone-resistant canola, maize, wheat, and rice.
• Imazapyr controls the broadest spectrum of weeds of the imidazolinones, but has
selectivity on many coniferous species as well as date and oil palms. It is used for
weed control and site preparation in pines and date and oil palms. It is also used
in noncrop sites for the control of weedy vegetation and/or the maintenance of
bare ground, as well as in imidazolinone-resistant maize and sunflower.
2.3.9
Conclusion
The imidazolinone herbicides have been – and continue to be – highly successful

products. The ability to mix-and-match different imidazolinones, in order to take
advantage of their differing weed control spectrum and pre- and post-emergent ac-
tivities, has proved invaluable when designing products for imidazolinone-resistant
crops throughout the world. Although the number of imidazolinone analogs that
have been commercialized is extremely small compared to other AHAS-inhibiting
herbicides, these compounds fill vital niches in many weed management programs.
References
1. Shaner, D.L. and O’Connor, S.L. (eds) 9. Ladner, D.W. (1991) Structure–activity
(1991) The Imidazolinone Herbicides, relationships among the imidazoli-
CRC Press, Inc., Boca Raton, FL. none herbicides, in The Imidazolinone
2. Los, M. (1996) Preparation of imi- Herbicides (eds D.L. Shaner and S.L.
dazolinyl benzoic acids. US Patent O’Connor), CRC Press, Inc., Boca
4,608,437. Raton, FL, pp. 31–52.
3. Los, M. (1987) Herbicidal 10. Van Ellis, M.R. and Shaner, D.L. (1988)
2-(2-Imidazolin-2-yl) fluoroalkoxy-, Pestic. Sci., 23, 25–34.
alkenyloxy- and alknyloxypyridines. US 11. Los, M. (1991) Discovery of the
Patent 4,647,301. imidazolinone herbicides, in The Im-
4. Los, M., Ladner, D.W., and Cross, B. idazolinone Herbicides (eds D.L. Shaner
(1987) (2-Imidazolin-2-yl)thieno- and and S.L. O’Connor), CRC Press, Inc.,
-furo[2,3-b] and -[3,2-b]pyridines and in- Boca Raton, FL, pp. 7–14.
termediates for the preparation thereof, 12. Los, M., Kust, C.A., Lamb, G., and
and use of said compounds as herbicidal Diehl, R.E. (1986) HortScience, 15,
agents. US Patent 4,650,514. 22–28.
5. Los, M. (1988) Herbicidal 2-(2 13. Suttle, J.C. and Schreiner, D.R. (1982)
Imidazolin-2-yl)fluoroalkoxy-, alkenyloxy- J. Plant Growth Regul., 1, 139–145.
and alkynyloxyquinolines. US Patent 14. Ladner, D.W. (1990) Pestic. Sci., 29,
4,772,311. 317–325.
6. Los, M., Ladner, D.W., and Cross, B. 15. Stidham, M.A. and Singh, B.K. (1991)
(1988) (2-Imidazolin-2-yl)thieno- and Imidazolinone-acetohydroxyacid syn-
-furo[2,3-b]pyridines and use of said thase interactions, in The Imidazolinone
compounds as herbicidal agents. US Herbicides (eds D.L. Shaner and S.L.
Patent 4,752,323. O’Connor), CRC Press, Inc., Boca
7. Ciba-Geigy (1986) 2-Imidazolinyl- Raton, FL, pp. 71–90.
pyridine- and -quinolinecarboxylic acid 16. Wepplo, P.J. (1991) Chemical and
production by reaction of pyridine or physical properties of the imidazoli-
quinoline-2,3-dicarboxylic Acid Esters none herbicides, in The Imidazolinone
with a 2-Amino-alkanoic Acid Amide. Herbicides (eds D.L. Shaner and S.L.
EP Patent 233-150A. O’Connor), CRC Press, Inc., Boca
8. Los, M. (1987) Synthesis and biology Raton, FL, pp. 15–30.
of the imidazolinone herbicides, in 17. Little, D.L., Shaner, D.L., Ladner, D.W.,
Pesticide Science and Biotechnology (eds Tecle, B., and Ilnicki, R.D. (1994) Pestic.
R. Greenhalgh and T.R. Roberts), Black- Sci., 41, 161–169.
well Scientific Publications, Oxford, 18. Hawkes, T.R. (1989) Monog. Br. Crop
pp. 35–42. Prot. Counc., 42, 131–138.
19. Rhodes, D., Hogan, A.L., Deal, L., 29. Shaner, D.L., Singh, B.L., and Stidham,
Jamieson, G.C., and Haworth, P. (1987) M.A. (1990) J. Agric. Food Chem., 38,
Plant Physiol., 84, 775–780. 1279–1282.
20. Singh, B.K. and Shaner, D.L. (1995) 30. Shaner, D.L. and Singh, B.K. (1991)
Plant Cell, 7, 935–944. Plant Physiol., 97, 1339–1341.
21. Shaner, D.L. and Singh, B.K. (1992) 31. Anderson, P.C. and Georgeson, M.
How does inhibition of amino acid (1989) Genome, 31, 994–999.
biosynthesis kill plants, in Biosynthesis 32. Tan, S., Evans, R.R., Dahmer, M.L.,
and Molecular Regulation of Amino Acids Singh, B.K., and Shaner, D.L. (2005)
in Plants (eds B.K. Singh, H.E. Flores, Pest Manage. Sci., 61, 246–257.
and J.C., Shannon), American Society 33. Shaner, D.L., Bascomb, N.F., and Smith,
of Plant Physiologists, Rockville, MD, W. (1996) Imidazolinone-resistant crops:
pp. 174–183. selection, characterization, and man-
22. Kim, S. and Van den Born, W.H. (1996) agement, in Herbicide-Resistant Crops
Pestic. Biochem. Physiol., 56, 141–148. (ed. S.O. Duke), Lewis Publishers, Boca
23. Rost, T.L., Gladish, D., Steffen, J., and Raton, FL, pp. 143–157.
Robbins, J. (1990) J. Plant Growth Regul., 34. Tranel, P.J. and Wright, T.R. (2002)
9, 227–232. Weed Sci., 50, 700–712.
24. Shaner, D.L. (1989) Sites of action of 35. Ott, K.H., Kwagh, J.G., Stockton, G.W.,
herbicides in amino acid metabolism: Sidorov, V., and Kakefuda, G. (1996)
primary and secondary physiological J. Mol. Biol., 263, 359–368.
effects, in Plant Nitrogen Metabolism 36. McCourt, J.A., Pang, S.S., King-Scott,
(eds J.E. Poulton, J.T. Romeo, and J., Guddat, L.W., and Duggleby, R.G.
E.E. Conn), Plenum Press, New York, (2006) Proc. Natl Acad. Sci. USA, 103,
pp. 227–261. 569–573.
25. Pillmoor, J.B. and Caseley, J.C. (1987) 37. Shaner, D.L. (2003) Imidazolinone her-
Pestic. Biochem. Physiol., 27, 340–349. bicides, in Encyclopedia of Agrochemicals
26. Shaner, D.L. and Singh, B.K. (1993) (ed. J. Plimmer), John Wiley & Sons,
Plant Physiol., 103, 1221–1226. Inc., New York, pp. 769–784.
27. Manabe, Y., Tinker, N., Colville, A., and 38. Shaner, D.L. and Tecle, B. (2001)
Miki, B. (2007) Plant Cell Physiol., 48, Designing herbicide tolerance based
1340–1358. on metabolic alteration: the challenges
28. Shaner, D.L. and Singh, B.K. (1997) and the future, in Pesticide Biotransfor-
Acetohydroxyacid synthase inhibitors, in mation in Plants and Microorganisms,
Herbicide Activity: Toxicology, Biochem- ACS Symposium Series, Vol. 777 (eds
istry and Molecular Biology (eds R.M. J.C. Hall, R.E. Hoagland, and R.M.
Roe, J.D. Burton, and R.J. Kuhr), IOS Zablotowicz), American Chemical Soci-
Press, Washington, DC, pp. 69–110. ety, Washington, DC, pp. 353–374.
2.4
Triazolopyrimidines
Timothy C. Johnson, Richard K. Mann, Paul R. Schmitzer, Roger E. Gast,

and Gerrit J. deBoer
2.4.1
Introduction
Since their discovery during the early 1980s, the triazolopyrimidine sulfonamides
and related compounds have undergone extensive investigation. Following the
discovery of the initial lead compound while examining the bioisosteric relation-
ships with the sulfonylureas [1], further investigations of the structure–activity
relationships (SARs) relating to this lead compound led eventually to the
triazolo[1,5-a]pyrimidine sulfonanilides, and the development of flumetsulam
(1) and metosulam (2) (see Table 2.4.1). Initially, flumetsulam was developed
for use in maize and soybeans, and metosulam for use in maize and cereals.
Since these discoveries, additional investigations have led to the development
of diclosulam (3) and cloransulam-methyl (4) for broadleaf weed (BW) control
in soybeans, and of florasulam (5) for BW control in cereals. The research
efforts into the new N-aryl-triazoloazinyl sulfonamides, which include the
triazolo[1,5-a]pyridine, the triazolo[1,5-a]pyrazine, N-triazolo[1,5-c]pyrimidine, and
N-triazolo[1,5-a]pyrimidine sulfonamides, that led in turn to the discovery of
penoxsulam (6) and pyroxsulam (7), have been reviewed [2]. Penoxsulam was
developed for broadleaf, grass, and sedge weed control in rice, while pyroxsulam
is currently being developed for broadleaf and grass weed (GW) control in wheat.
Advantageously, in addition to their wide-ranging utility in crops, these molecules
also possess a favorable mammalian toxicity profile [3].
2.4.2
N-Triazolo[1,5-c]Pyrimidine Sulfonanilide
2.4.2.1 Synthesis
Synthetic routes leading to triazolo[1,5-c]pyrimidine sulfonanilides have been
reviewed [4]. Scheme 2.4.1 shows a general synthetic route to the triazolo[1,5-
c]pyrimidine sulfonanilides [5], whereby an appropriately substituted 4-hydrazino-
2-methylthiopyrimidine is reacted with carbon disulfide, followed by benzyl
chloride, to afford 3-benzylthio-5-methylthio-1,2,4-triazolo[4,3-c]pyrimidine (8). The
latter compound is then treated with sodium methoxide to afford 2-benzylthio-
5-methoxy-1,2,4-triazolo[1,5-c]pyrimidine (9). The benzyl sulfide 9 is oxidized to the
sulfonyl chloride 10 by treatment with chlorine and water, after which the sulfonyl
chlorides are reacted with N-trimethylsilylanilines in the presence of a catalytic
amount of dimethylsulfoxide (DMSO), or with anilines in the presence of pyridine
and a catalytic amount of DMSO, to afford the desired sulfonanilides 11.
2.4.2.2 Biology
Unless otherwise noted, the in vivo greenhouse screening data presented in the
following sections is a tabulation of post-emergence foliar applied results, and are
expressed as ‘‘percentage in growth reduction’’ (GR) for treated plants compared to
untreated plants, where the rate identified provides the level of weed control or crop
injury. The BW activity is given as an average percentage reduction in growth at a
given concentration, as indicated, over five to eight BWs chosen from the following:
Xanthium strumarium, Datura stramonium, Chenopodium album, Helianthus spp.,
Ipomoea spp., Amaranthus retroflexus, Abutilon theophrasti, Veronica heteraefolia,
Ipomoea hederacea, Stellaria media, and Polygonum convolvulus. The GW activity is
averaged over five weeds chosen from the following: Alopecurus spp., Echinochloa
Table 2.4.1 Commercial triazolopyrimidine sulfonamides.
Chemical structure Common name Launch date Major crops
F Flumetsulam 1994 Barley, corn, oats,

O N N peanut, rye,
1 N S soybean, sugar
H O N N cane, triticale,
F wheat
Metosulam 1994 Corn, barley, rye,
Cl O
triticale, wheat
O N N
2 N S
H O N N O
Cl
O Diclosulam 1997 Peanut, tree

Cl crops, soybean,
O N N
3 N sugar cane
N S
H O N F
Cl
O Cloransulam- 1997 Soybean

O O
methyl
O N N
4 N
N S
H O N F
Cl
O Florasulam 1999 Corn, citrus,

F barley, forest,
O N N N grassland, millet,
5 N S oat, rye, triticale,
H O N
turf, wheat
F F
F Penoxsulam 2004 Aquatics, barley,
F grape, rice, rye,
sorghum, turf,
O O triticale, tree
O N N nuts, wheat
6 N
S N
O H N
F F O
F
Pyroxsulam 2008 Rye, triticale,
O O
wheat
N O N N
7 S N
O H N N O
F F
F
R2 R2 R O
R1 H 1
N NH2 R
N N
a, b c N N
N SBn
N N N N
1 N
R
S S SBn
R2
8 9
R O R O
N O N
N N e N N
S NHAr SO2Cl
N O N
R1 R1
R2 R2
11 10
Scheme 2.4.1 (a) CS2 , dioxane, Et3 N; (b) BnCl;

(c) NaOMe, MeOH, ethyl acrylate; (d) Cl2 , H2 O;
(e) ArNHSi(Me)3 , DMSO (catalytic), CH3 CN or ArNH2 ,
pyridine, DMSO (catalytic), CH3 CN.
crus-galli, Setaria fabarii, Sorghum halapense, Digitaria Sanguinalis, and Avena fatua,
and is expressed in a manner similar to that for BWs.
The general SARs for triazolo[1,5-c]pyrimidine sulfonanilides (11) have been
described [4]. The SAR identified compounds with alkoxy in the 5-position (11, O–R)
and halogen or alkoxy in the 7- and 8-positions (11, R1 and R2 , respectively) as
having the highest levels of activity. Further investigation identified compounds
with halogen in the 7-position as having good levels of activity on BWs and
selectivity to soybeans. In addition, compounds with halogen in the 8-position were
identified as having good activity on BWs with selectivity to wheat.
2.4.2.3 Cloransulam-Methyl and Diclosulam Crop Utility

Cloransulam-methyl and diclosulam are members of the triazolo[1,5-c]pyrimidine
sulfonanilide family of AHAS (acetohydroxyacid synthase) -inhibiting herbi-
cides. Both compounds show excellent crop selectivity, broad-spectrum BW
control, and low toxicity. The herbicidal utility of cloransulam-methyl in soybeans
was first presented in 1994 [6, 7] and further described in 1995 [8, 9] and 1996
[10–12]. Diclosulam was first described for use in soybeans and peanuts in 1997
[13], with additional descriptions in 1998 [14] and 1999 [15–17].
Cloransulam-methyl was commercialized in the United States under the trade
name FirstRate® (a trademark of Dow AgroSciences LLC) herbicide for the control
of annual BWs and certain perennial sedges in soybeans. Applications can be made
preplant surface, preplant incorporated, pre-emergence, and post-emergence for

the control of BW species. Post-emergence applications of cloransulam-methyl at
17.5 g a.i. ha−1 or soil-applied treatments at rates of 35–44 g a.i. ha−1 provide control
of a large number of soybean-relevant weeds. However, cloransulam-methyl does
not provide any control of annual and perennial GWs or certain BWs such as
Solanum spp. [18].
Diclosulam is registered in the United States and in Latin America for use
in peanuts and soybeans. Applications can be made preplant surface, preplant
incorporated, and pre-emergence at rates of 17.5–26 g a.i. ha−1 for the control of a
large number of BW species. Diclosulam does not provide any control of annual
and perennial GWs, or of certain BWs such as Solanum spp.
2.4.2.3.1 Florasulam Crop Utility Florasulam (5) provides excellent post-

emergence selectivity in turf and small grain cereal crops such as wheat, barley,
oats, rye, and triticale [19, 20]. The European and North American cereal markets
are of primary commercial interest for florasulam, due to its specialized spectrum
of BW control. Florasulam is highly active on economically important species in the
compositae, caryophyllaceae, cruciferae, rubiacea, and leguminosae plant families,
at a typical use rate of 5 g a.i. ha−1 [21]. Due to the relatively short half-life in soil,
however, only post-emergence applications are used in commercial practice [22].
2.4.2.4 Mechanism of Crop Selectivity
2.4.2.4.1 Cloransulam-Methyl and Diclosulam Mechanism of Crop Selectivity The

metabolism of triazolopyrimidine sulfonanilides (1–4) in plants has been reviewed
[23, 24]. It has been shown that both diclosulam (3) and cloransulam-methyl (4)
are rapidly metabolized in soybeans by facile conjugation with homoglutathione,
which displaces the 7-fluoro substituent (Figure 2.4.1) [25]. This mechanism was
found to only occur in soybeans for 3 and 4. Subsequent oxidation at the 4-position
of the aniline ring occurs rapidly in maize for 3 and 4. In wheat, 4 undergoes
Cl OEt Cl OEt
N N N N N N
NHSO2 NHSO2
Cl N F Cl N
homoGSH
Diclosulam (3) Soybeans
Homoglutathione
CO2Me OEt CO2Me OEt
N N N N N N
NHSO2 NHSO2
Cl N F Cl N
homoGSH
Cloransulam-methyl (4)
Figure 2.4.1 Metabolism of diclosulam (3) and

cloransulam-methyl (4) in soybeans (Glycine max).
Table 2.4.2 Herbicidal activity and metabolism of florasulam (5).
Species GR50 (g ha –1 ) t1/2 a (h)
Triticum aestivum >>32 2.4

Galium aparine L. <<2.5 >48 (202.4)b
Galeopsis tetrahit L. <2.5 19.8
Polygonum papathifolium <2.5 43.6
a
The time required for plants to metabolize 50% of the applied compound.
b
Extrapolated value.
F OMe HO F OMe
N N N N N N
NHSO2 Wheat Glucose
NHSO2
N N Conjugate
F F
F F
Florasulam (5)
Figure 2.4.2 Metabolism of florasulam (5) in wheat (Triticum aestivum).

O-dealkylation of the 5-ethoxy, followed by glucose conjugation, while 3 undergoes
oxidation at the 4-position of the aniline ring [25].
2.4.2.4.2 Florasulam Mechanism of Crop Selectivity The selectivity of florasulam

to wheat, and the high level of herbicidal activity on important weeds, are related
primarily to difference in the rates of metabolism [26]. Florasulam has a half-life
in wheat of 2.4 h, compared to half-lives in Galeopsis tetrahit L., Polygonum papathi-
folium, and Galium aparine L. of 19.8, 43.6, and >48 h, respectively (Table 2.4.2). In
wheat, florasulam has been shown to undergo rapid metabolism at the 4-position
of the phenyl ring to give the 4-hydroxy metabolite which, in turn, is conjugated
to glucose (Figure 2.4.2). In contrast, a slow metabolism is observed in Galeopsis
tetrahit L. and Polygonum papathifolium with minimal degradation of florasulam
observed in Galium aparine L., even at 48 h after treatment. Similar differences in
the rate of metabolism in wheat compared to BWs accounted for the sensitivity of
BWs to closely related analogs [24].
2.4.2.5 Environmental Degradation, Ecotoxicology, and Toxicology
2.4.2.5.1 Cloransulam-Methyl and Diclosulam Environmental Degradation, Ecotoxi-

cology, and Toxicology Metabolism in aerobic soils represents a significant dissipa-
tion mechanism for both cloransulam-methyl and diclosulam. The analysis of soil
samples from bare-ground applications of cloransulam-methyl and diclosulam gave
half-life ranges of 3 to 11 days and 13 to 43 days, respectively [27, 28]. The two factors
that most influenced the soil degradation rates were the organic matter content
and soil temperature. Dealkylation of the 5-ethoxy on the triazolopyrimidine ring
to form the associated 5-hydroxytriazolopyrimidine is a metabolic manipulation
shared by both compounds. The shared aminosulfonyl triazolopyrimidine (12)

was a metabolite identified in soil degradation studies for both compounds
[29, 30], and both underwent additional, unique metabolic manipulations in the soil.
Unextractable residues accounted for a significant amount of the final metabolite
distribution for both cloransulam-methyl and diclosulam. Photolysis in water has
also been shown to be a significant avenue of degradation for cloransulam-methyl,
with a half-life of <1 h [31]. However, diclosulam does not show any significant
degradation due to photolysis (half-life >100 days).
O
O N N N
H2N S
O N F
12
Both, cloransulam-methyl and diclosulam have low acute toxicology profiles, and
no indication of any chronic toxicology issues. Although both compounds show a
slight toxicity to Daphnia, they are considered practically nontoxic to birds, insects,
aquatic organisms, and earthworms.
2.4.2.5.2 Florasulam Environmental Degradation, Ecotoxicology, and Toxicology

Whilst florasulam dissipates primarily through microbial degradation [22],
other patterns of degradation or dissipation contribute minimally to its loss in
the agricultural field environment. As florasulam degrades in the soil, several
metabolites of the herbicide are formed. The primary soil metabolite, the
5-hydroxytriazolo[1,5-c]pyrimidine sulfonanilide analog of 5 (5-OH), has been
shown to have limited plant activity (a factor of >100-fold) relative to the parent,
whereas the other metabolites are inactive. The field half-life of florasulam in soil
ranges from 2 to 18 days. Although the soil pH and the texture and level of organic
matter influence the rate of florasulam degradation, it is the temperature that has
the greatest impact on the soil half-life. In natural sediment and surface water in
the dark at 20 ◦ C, florasulam is degraded to the 5-OH metabolite with a half-life of
9–29 days. Under anaerobic conditions, the half-life was approximately 13 days,
whilst in water the aqueous photolytic half-life was 4.9 days.
The overall toxicological profile of florasulam is very favorable. It is not acutely
toxic, it does not pose any inhalation hazard, and nor is it a skin sensitizer. No
evidence has been obtained of any mutagenic or carcinogenic potential, and it has
shown no teratogenic effects in either rats or rabbits. A variety of investigations have
indicated that florasulam does not represent any reproductive hazard or concern.
2.4.3
N-Triazolo[1,5-c]Pyrimidine Sulfonamides
The N-triazolo[1,5-c]pyrimidine sulfonamides and related compounds differ from

their sulfonanilide counterparts by the orientation of the linkage between the
triazoloazine and the aryl or heteroaryl ring. However, in most cases the synthesis
of sulfonamides is similar to that of the sulfonanilides, as the target molecules
are formed by the reaction of a sulfonyl chloride and an amine in the final step.
Notably, the synthesis of arylsulfonyl chlorides accounts for much of the diversity
in these molecules [32–36].
2.4.3.1 Synthesis
Scheme 2.4.2 outlines a straightforward and general route for the synthesis of
N-triazolo[1,5-c]pyrimidine sulfonamides (13) [32, 35]. In this case, 4-hydrazino-
2-methylthiopyrimidines (14) are reacted with cyanogen bromide to give the 3-
amino-5-methylthiotriazolo[4,3-c]pyrimidines (15), usually as the hydrogen
bromide salt. Treatment of 15 with sodium methoxide affords the 2-amino-5-
methoxytriazolo[1,5-c]pyrimidine ring system (16). The sulfonamides (13) are
prepared by reacting 16 with arylsulfonyl chlorides 17 in the presence of pyridine
and a catalytic amount of DMSO.
Several substituted benzene and pyridine sulfonyl chlorides from which to
prepare sulfonamides have been investigated. However, with respect to crop
selectivity, the majority of interest has focused on 2,6-disubstituted benzenesulfonyl
chlorides and 2,4-disubstituted pyridine-3-sulfonyl chlorides. A general method for
the preparation of a variety of benzene and pyridine sulfonyl chlorides is via
ortho-directed metalation [32, 37]. The sulfonyl chlorides (17) can be prepared
directly from the aryl lithium species by reacting with sulfur dioxide, followed by
sulfuryl chloride (Scheme 2.4.3). Alternatively, reaction of the aryl lithium species
with a disulfide (most commonly propyl disulfide) provides an alkyl aryl sulfide
R2 R2 R O
H
R1 N NH2 R1 N
a b N N N
N NH2
N N N N
R1 N
S S NH2
R2
14 15 16
R O
X
O Q
N N N
Q = N or CH N S
N
R1 O
Y
R2 13
Scheme 2.4.2 (a) BrCN, i-PrOH; (b) NaOR, ethyl acrylate;

(c) ArSO2 Cl, pyridine, DMSO (catalytic), CH3 CN.
R1 R1
SO2Cl
a, b, c
Q R2 Q R2
19 17
a, d e
R1
S R
Q = N or CH
Q R2
18
Scheme 2.4.3 (a) Excess 19, BuLi, tetramethylethylenedi-

amine (TMEDA), i-Pr2 NH, tetrahydrofuran (THF) or Et2 O;
(b) SO2 , Et2 O; (c) SO2 Cl2 ; (d) (n-PrS)2 ; (e) Cl2 , H2 O,
HOAc.
(18: R = n-Pr) which can be converted to the sulfonyl chloride using chlorine and
water. The latter method is commonly used when further manipulation on the aryl
ring is required.
2.4.3.2 Biology
The structure–activity trends for triazolo[1,5-c]pyrimidine sulfonamides (13) have
been studied extensively [32–36]. The herbicidal activities for 13, with various
substitutions on the triazolo[1,5-c]pyrimidine ring and 2,6-disubstitutions on the
aryl ring, are summarized in Table 2.4.3. Analogs with substitution in the 8-position
of the triazolopyrimidine ring (R2 ) are more active than those with substitution in
the 7-position (R1 ). The 8-methoxy analog has the best levels of activity on both GWs
and BWs. Halogen substitutions in the 8-position provide good levels of activity on
broadleaf species, with somewhat reduced levels of activity on grass species. High
levels of activity are achieved with 2,6-disubstitutions on the phenyl ring, especially
when one of the substituents is methoxy. The highest levels of activity are achieved
when both the 2- and 6-positions are methoxy, although good levels of activity are
achieved with a variety of substituents in the 6-position when there is a methoxy
in the 2-position. For substitutions on the pyridine ring, good levels of activity
are achieved when at least one of the substituents is methoxy. The best level of
activity on both grass and broadleaf species is gained with the dimethoxy analog
(13, Q = N, X = Y = OMe, R = Me, R2 = OMe). The 4-methoxy analog (13, Q = N,
X = CF3 , Y = OMe) has very little activity on either GWs or BWs.
Several 2-trifluoromethylphenyl analogs of 13 have been prepared with various
alkoxy and substituted alkoxy groups in the 6-position of the phenyl ring, and
some of these molecules have demonstrated trends for selectivity toward rice
(Oryza sativa) with activity also on barnyardgrass (Echinochloa crus-galli) [32, 36].
Table 2.4.3 Herbicidal activity for analogs of 13 (R = Me).
X Y Q R2 R1 Average GR80 Average GR80

BW (ppm) GW (ppm)
Cl Cl CH OMe H 3 11
Cl Cl CH OEt H 216 >500
Cl Cl CH Me H <15 >500
Cl Cl CH Cl H 1 15
Cl Cl CH H OMe >1000 >1000
OMe CF3 CH OMe H <0.2 1
OMe OMe CH OMe H 0.5 <0.1
OMe F CH OMe H 1 3
OMe Me CH OMe H 1 3
OMe CO2 Me CH OMe H 10 2
OMe Cl N OMe H 12 15
OMe OMe N OMe H <1 2
OMe CF3 N OMe H 4 16
OEt CF3 N OMe H 2 64
CF3 OMe N OMe H >250 >250
Table 2.4.4 Herbicidal activity on transplanted paddy rice

and weeds for analogs of 13 (Q = CH, R = Me, R1 = H,
R2 = OMe).
X Y Oryza Echinochloa Monochoria Scirpus Cyperus

sativa crus-galli vaginalis juncoides difformis
GR20 GR80 GR80 GR80 GR80
(g a.i. ha –1 ) (g a.i. ha –1 ) (g a.i. ha –1 ) (g a.i. ha –1 ) (g a.i. ha –1 )
CF3 OCH2 CH2 F 14 10 5.2 9 8

CF3 OCH2 OMe 124 16 4 15 18
CF3 OCH2 CF3 51 19 5 21 36
CF3 OCH2 CF2 H 75 12 <2 12 14
CF3 OCH(CH2 F)2 140 14 1 9 31
Details of the herbicidal activity observed on rice and key rice weeds when the
compounds were applied in the greenhouse to one- to three-leaf (lf) rice and
key rice weeds, either as a water-injected treatment or as a post-emergence foliar
treatment, are listed in Tables 2.4.4 and 2.4.5, respectively. Of particular note here
are the 2,2-difluoroethoxyphenyl (13, Q = CH, X = CF3 , R = Me, R2 = OMe) and
2-fluoroethoxyphenyl (13, Q = CH, X = CF3 , R = Me, R2 = OMe) analogs, both of
which showed high levels of activity on all weed species, particularly barnyardgrass,
and with good selectivity towards rice.
Table 2.4.5 Herbicidal activity on direct-seeded rice and

weeds as a post-emergence foliar application for 13 (Q = CH,
R = Me, R1 = H, R2 = OMe).
X Y Oryza sativa Echinochloa crus- Scirpus juncoides

GR20 (g a.i. ha –1 ) galli GR80 (g a.i. ha –1 ) GR80 (g a.i. ha –1 )
CF3 O(CH2 )2 F 4 1 3
CF3 OCH2 OMe >70 12 5
CF3 OCH2 CF3 >70 9 –
CF3 OCH2 CF2 H >140 20 30
CF3 OCH(CH2 F)2 >70 10 –
2.4.3.3 Penoxsulam Crop Utility

Based on the above greenhouse-based results, several 2-trifluoromethyl-6-
alkoxyphenyl analogs of 13 were tested in key rice-growing countries between
1997 to 1999, to characterize their activity. Among these analogs, the 2,2-
difluoroethoxyphenyl analog of 13 (6) was identified as having good rice tolerance,
broad spectrum weed control (Echinochloa spp. and many key broadleaf and
sedge weeds), and providing good residual weed control, depending on the rates
applied. Other analogs tested were not selected for a number of reasons, such as
being too injurious to rice, providing poor weed control, or having short residual
activity, when compared to 6. Additionally, it was discovered that 6 could be
coapplied with the grass herbicide cyhalofop-butyl, which cannot be tank-mixed
with commercially available AHAS or auxin mode of action herbicides without
antagonizing the control of Echinochloa spp. Based on the ability to meet many
of the commercial rice herbicide needs (crop tolerance, broad-spectrum weed
control, residual weed control activity, and tank-mix ability) in transplanted rice,
direct-seeded rice, and water-seeded rice in over 25 rice countries, 6 was identified
for development as a new rice herbicide with the code number DE-638 and the
common name ‘‘penoxsulam’’ [38–46]. Additional crops and uses for which
penoxsulam is currently registered (other than rice) are listed in Table 2.4.1.
Additional crop uses are still being explored due to the excellent post-emergence
burndown and pre-emergence residual activity provided by penoxsulam.
2.4.3.4 Penoxsulam: Mechanism of Crop Selectivity

Metabolism studies conducted on 6 have shown that the O-dealkylation of one
heterocycle methoxy group occurred in rice (Figure 2.4.3) [47]. A comparison
of the metabolic degradation rates and activity on indica rice, japonica rice,
and barnyardgrass for 6 indicated that degradation rates would explain the major
differences observed in activity (Table 2.4.6). Other factors, such as the site of uptake
and transport, both of which are modulated by the plant structure and metabolism,
may contribute to the additional rice selectivity observed for penoxsulam.
F F
OMe O OH O
F F
N N N Rice N N N
NHSO2 NHSO2
O-Dealkylation
N N
F3C F3C
OMe OMe
Penoxsulam (6)
Figure 2.4.3 Metabolism of 6 in Rice (Oryza sativa).
Table 2.4.6 Herbicidal activity and metabolism of 6.
Species GR80 (ppm) t1/2 a (h)
Indica rice >250 14.4

Japonica rice >250 38.4
Echinochloa crus-galli 0.24 106
a
Time required to metabolize 50% of the applied compound.
2.4.3.5 Penoxsulam: Environmental Degradation, Ecotoxicology, and Toxicology

The dissipation of penoxsulam occurs primarily through microbial degradation,
although other patterns of degradation or dissipation (e.g., photolysis, volatility,
leaching, and chemical hydrolysis) also contribute to the compound’s loss in
the agricultural field environment. In the soil, penoxsulam is degraded to form
several metabolites. The primary soil metabolite, 5-hydroxytriazolo[1,5-c]pyrimidine
sulfonamide analog of 6, has been shown to have very limited plant activity
(a factor of >100×) relative to the parent, while other metabolites are inactive. The
average half-life of penoxsulam under field conditions was 6.5 (range 4–10) days
under flooded water-seeded rice conditions, and 14.6 (range 13–16) days under
nonflooded, dry-seeded rice conditions. The soil pH and the texture and level of
organic matter also influence the rate of degradation; typically, the average half-life
for aerobic aquatic conditions was 25 (range 11–34) days, while that for anaerobic
conditions was 7 (range 5–11) days. The major route of degradation in water was
shown to be via photolysis (the half-life in water from photolysis was 2 days, in
summer sunlight, at 40◦ N latitude).
Penoxsulam has a toxicological profile similar to that of other triazolopyrimidine
sulfonamides, with no indication of any acute or chronic toxicity issues to mam-
malian or nontarget organisms such as fish, fresh-water invertebrates, honey bees,
earthworms, and beneficial arthropods.
2.4.4
N-Triazolo[1,5-a]Pyrimidine Sulfonamides
2.4.4.1 Synthesis
A general route for the synthesis of N-triazolo[1,5-a]pyrimidine sulfonamides (20)
is outlined in Scheme 2.4.4 [48]. Here, the 2-amino-4,6-dimethoxypyrimidine (21)
is reacted with ethoxycarbonyl isothiocyante followed by hydroxylamine in the
presence of a base to give 23. The latter compound is then reacted with a variety
of substituted benzene and pyridine sulfonyl chlorides (17, Scheme 2.4.3) in the
presence of a base and a catalytic amount of DMSO to produce 20.
2.4.4.2 Biology
Structure–activity trends for the analogs of 20 have been reported previously
[1]. In the case of substitutions on the triazolo[1,5-a]pyrimidine ring, a better
herbicidal activity was achieved when the 5- or 7-position was substituted with
methoxy than with alkyl, halogen, or halo alkyl. Recent efforts have shown that,
when both the 5- and 7-position are substituted with methoxy, superior levels
of herbicidal activity are achieved. With respect to the phenyl ring, it has been
shown previously that 2,6-disubstitutions have a superior herbicidal activity over
other substitution patterns. Many recent studies have focused on 2,6-disubstituted
phenyl and 2,4-disubstituted 3-pyridyl analogs of 20 with a 5,7-dimethoxy substi-
tution on the triazolo[1,5-a]pyrimidine ring [48]. The general trends in activity on
GWs, BWs, blackgrass (Alopecurus myosuroides) and wheat (Triticum aestivum) for
phenyl and pyridyl analogs of 20 are summarized in Table 2.4.7. Good levels of
herbicidal activity were achieved on both GWs and BWs with 2,6-substitutions
on the phenyl ring (20, Q = CH) and, in particular, when one of the substituents
was methoxy. Superior levels of activity on grass species were achieved with the
OMe
OMe OMe
a N S b
N N N
NH2
MeO MeO N N N CO2Et
N NH2 H H MeO N N
21 22 23
OMe X
Q = N or CH N O Q
N
N S
MeO N N O
Y
20
Scheme 2.4.4 (a) SCNCO2 Et; (b) HONH2 , Et(i-Pr)2 N;

(c) ArSO2 Cl, pyridine, DMSO (catalytic).
Table 2.4.7 Herbicidal activity for analogs of 20.
X Y Q Average Average Alopecurus Triticum

GR80 GR80 myosuroides aestivum
BW (ppm) GW (ppm) GR80 (ppm) GR20 (ppm)
Cl Cl CH 4 4 4 <1
OMe OMe CH 8 8 4 <1
OMe CF3 CH 15 1 <1 <1
O(CH2 )F CF3 CH 2 16 27 13
OCH2 CF2 H CF3 CH 2 30 28 >250
OCH2 CF3 CF3 CH 4 62 240 46
OMe CF3 N 2 2 2 2
OMe CF2 CF3 N >250 125 164 3
OMe I N 31 8 1 <1
OEt CF3 N 15 62 10 62
2-methoxy-6-trifluormethylphenyl analog of 20 (Q = CH, X = OMe, Y = CF3 ), al-

though these analogs were found to cause significant injury to wheat. With higher
alkoxy substitutions (e.g., 20, Q = CH, X = OMe, Y = OCH2 CH2 F), the activity on
blackgrass was seen to decrease. With 2-methoxy-4-trifluoromethyl substitution on
the pyridyl ring of 20 (Q = N, X = OMe, Y = CF3 ), an excellent activity was achieved
on both GWs and BWs. The replacement of methoxy with ethoxy (20, Q = N,
X = OEt, Y = CF3 ) resulted in a loss of herbicidal activity which was more signif-
icant on grass than broadleaf species. Good levels of activity on blackgrass are
observed for 4-trifluoromethylpyridyl analogs of 20 (Q = N, X = OMe, Y = CF3 ),
and this analog was found to show a trend for wheat selectivity. Based on GW and
BW control in field studies, this analog was identified for development as a new
herbicide for wheat, with the common name pyroxsulam.
2.4.4.3 Pyroxsulam: Crop Utility

Pyroxsulam requires the addition of a safener to achieve commercial levels of
post-emergence selectivity in small-grain cereal crops, which is the only commer-
cially developed crop market. Commercial selectivity is limited to wheat, rye, and
triticale varieties. Pyroxsulam is broadly active on annual GWs and BWs, with some
activity on certain perennial weed species. Although most of its activity is derived
from foliar application, pyroxsulam does have some ability to provide soil residual
control of emerging weeds. Pyroxsulam is broadly registered worldwide, and is
used to control many economically important GW and BW species in the global
cereals markets [49].
2.4.4.4 Pyroxsulam: Mechanism of Crop Selectivity

Metabolism studies have been conducted on 7 and the closely related
triazolo[1,5-c]pyrimidine analog 24; the metabolites of both compounds that were
identified in wheat (Triticum aestivum) are shown in Figure 2.4.4. In this case,
the O-dealkylation of one heterocycle methoxy group was shown to occur with
7 in wheat (Figure 2.4.4) [50], whereas both methoxy groups on the heterocycle
of 24 underwent O-dealkylation. The metabolism rates and activity on wheat and
blackgrass (Alopecurus myosuroides) for 5, 7, and 24 are compared in Table 2.4.8. The
order of ranking was 5 > 24 > 7 with respect to rate of metabolism in wheat, and
7 > 5 > 24 with respect to activity on wheat. The slower rate of metabolism in
wheat, along with the higher levels of activity, most likely account for the injury
observed when 7 was not used in conjunction with a herbicide safener, such as
cloquintocet. In fact, cloquintocet was found to increase the rate of O-dealkylation
in wheat, without affecting the rate of metabolism in blackgrass or changing the
pathway of metabolism [50]. However, 7 was significantly more active on blackgrass
than either 5 or 24, with a rate of metabolism in blackgrass comparable to that of 24.
2.4.4.5 Pyroxsulam: Environmental Degradation, Ecotoxicology, and Toxicology

Under aerobic conditions, laboratory studies have shown that pyroxsulam
degrades rapidly in soil, with an average laboratory half-life of 4 days at 20 ◦ C
across 20 different soils from Europe, the United States, and Canada. The
principal metabolites are 7-hydroxytriazolo[1,5-a]pyrimidine sulfonamide (25),
5-hydroxytriazolo[1,5-a]pyrimidine sulfonamide (26), 7-hydroxy-6-chlorotriazolo
CF3 OMe
CF3 OMe
N N
SO2NH N N
N N SO2NH
N OMe
N N N OH
OMe
Wheat OMe
Pyroxsulam (7)
OMe O-Demethylation
CF3
CF3 OH
N N N
SO2NH N N N
N N SO2NH
N N
OMe OMe
24 OMe OH
Figure 2.4.4 Metabolites identified for pyroxsulam (7) and 24 in wheat (Triticum aestivum).
Table 2.4.8 Herbicidal activity and metabolism of 5, 7, and 24.
Herbicide Triticum aestivum Alopecurus myosuroides
GR20 (ppm) t1/2 (h)a GR80 (ppm) t1/2 (h)a
Florasulam (5) 7.8 2.4 15.6 NA

24 12.2 5.7 4.4 51.6
Pyroxsulam (7) 2.9 14 0.31 46
a
Time required for plants to metabolize 50% of the applied compound.
CF3 OH CF3 OMe

N N N N
SO2NH SO2NH
N N N OMe N N N OH
OMe OMe
25 26
CF3 OH OH
CF3
N N Cl
N N
SO2NH SO2NH
N N N OMe N N N OH
OMe OMe
27 28
CF3
SO3H
N
OMe
29
Figure 2.4.5 Soil metabolites of pyroxsulam (7).
[1,5-a]pyrimidine sulfonamide (27), 5,7-dihydroxytriazolo[1,5-a]pyrimidine sulfon-

amide (28), and the corresponding sulfonic acid of pyroxsulam (29) (Figure 2.4.5).
All soil metabolites have little or no phytotoxicity compared to pyroxsulam, which
is degraded at a moderate rate under anaerobic conditions, with a half-life of
47 days determined on a single soil. Pyroxsulam does not photodegrade at a
measurable rate on soil surfaces. Moreover, studies have indicated that the overall
toxicological profile of pyroxsulam is very favorable, and similar to that of other
triazolopyrimidine sulfonamides.
2.4.5
AHAS Inhibition
Since the initial discovery of the triazolopyrimidines, the inhibition of AHAS activ-
ity has routinely been measured by utilizing partially purified extracts of etiolated
barley, as described previously [4]. All of the registered triazolopyrimidine herbi-
cides show a potent inhibition of etiolated barley AHAS (Table 2.4.9), regardless of
the orientation of the triazolopyrimidine ring, the substituents on the triazolopy-
rimidine or aryl rings, or the orientation of the amide and sulfone linkage between
the rings.
Although flumetsulam (1) provides the weakest AHAS inhibition of the com-
mercially available triazolopyrimidines, this level of inhibition is consistent with
inhibition values reported previously for a set of triazolo[1,5-a]pyrimidine sulfo-
nanilides [51]. The other triazolo[1,5-a]pyrimidine sulfonanilides, metosulam (2),
References 115
Table 2.4.9 AHAS inhibition of triazolopyrimidine herbicides.
Herbicide AHAS IC50 a (nM)
Flumetsulam (1) 230

Metosulam (2) 1.0
Diclosulam (3) 3.0
Cloransulam-methyl (4) 4.5
Florasulam (5) 11.5
Penoxsulam (6) 4.5
Pyroxsulam (7) 0.5
a
Concentration required to provide 50% inhibition of AHAS activity.
provides a more potent inhibition than flumetsulam, due to the additional substitu-
tion on the triazolopyrimidine ring and to the greater lipophilicity of the trisubsti-
tuted phenyl ring. The three triazolo[1,5-c]pyrimidine sulfonanilides – diclosulam
(3), cloransulam-methyl (4) and florasulam (5) – all provide a potent inhibition
of AHAS activity. As noted previously [4], the triazolo[1,5-c]pyrimidine sulfon-
amides are potent inhibitors of AHAS. Penoxsulam (6), the only commercial
triazolo[1,5-c]pyrimidine sulfonamide, provides AHAS inhibition similar to that
of 3 and 4. The recently commercialized N-triazolo[1,5-a]pyrimidine sulfonamide
herbicide, pyroxsulam (7), is also a potent inhibitor of AHAS activity with an IC50
value similar to that of 2.
2.4.6
Conclusions
In recent years, the triazolopyrimidine class of AHAS inhibitors has grown to

include a number of fused triazole ring systems containing a bridgehead nitrogen.
The class members have each demonstrated a control of grass, broadleaf, and sedge
weeds in a number of agronomically important crops. The discovery of molecules
with crop safety and favorable environmental profiles has led to the development
of seven new herbicides for use in many crops, including corn, peanuts, soybeans,
wheat, barley, oats, rye, triticale, rice, sugarcane, sorghum, and turf.
References
1. Kleschick, W.A., Costales, M.J., Dunbar, in Modern Crop Protection

J.E., Meikle, R.W., Monte, W.T., Compounds, vol. 2 (eds W. Kramer
Pearson, N.R., Snider, S.W., and and U. Schrimer), Wiley-VCH
Vinogradoff, A.P. (1990) Pestic. Sci., Verlag GmbH, Weinheim,
29, 341. pp. 93–113.
2. Johnson, T.C., Mann, R.K., 3. Jabusch, T.W. and Tjeerdema, R.S.
Schmitzer, P., Gast, R., and (2008) Rev. Environ. Contam. Toxicol.,
deBoer, G. (2007) Triazolopyrimidines, 193, 31–52.
4. Kleschick, W.A. (1994) Triazolopy- 19. Thompson, A.R., McReath, A.M.,

rimidine sulfonanilides and related Carson, C.M., Ehr, R.J., and deBoer,
compounds, in Herbicides Inhibiting G.J. (1999) Proc. Brighton Crop Prot.
Branched Chain Amino Acid Biosynthesis, Conf., 1, 73–80.
vol. 10 (ed. J. Stetter), Spinger-Verlag, 20. Lepiece, D., Rijckaert, G., and
pp. 119–143. Thompson, A. (2000) Med. Fac. Univ.
5. Van Heertum, J.C., Gerwick, B.C., Gent, 65 (2a), 141–149.
Kleschick, W.A., and Johnson, T.C. 21. Daniau, P. and Prove, P. (2001)
(1992) US Patent 5,163,995. Phytoma, 534, 49–51.
6. Hunter, J.J., Schultz, M.E., Mann, R.K., 22. Jackson, R., Ghosh, D., and Paterson, G.
Cordes, R.C., and Lassiter, R.B. (1994) (2000) Pest Manage. Sci., 56 (12),
Proc. North Cent. Weed Sci. Soc., 49, 1065–1072.
124. 23. Gerwick, B.C., deBoer, G.J., and
7. Jachetta, J.J., Van Heertum, J.C., and Schmitzer, P.R. (1994) in Herbicides
Gerwick, B.C. (1994) Proc. North Cent. Inhibiting Branched Chain Amino Acid
Weed Sci. Soc., 49, 123–124. Biosynthesis, vol. 10 (ed. J. Stetter),
8. Jachetta, J.J., Van Heertum, J.C., Spinger-Verlag, pp. 145–160.
Gerwick, B.C., and Barrentine, J.L. 24. Owen, W.J. and deBoer, G.J. (1994)
(1995) Proc. South. Weed Sci. Soc., 48, in Eighth International Congress of
199. Pesticide Chemistry: Options 2000, ACS
9. Hunter, J.J., Langston, V.B., Grant, D.L., Conference Proceedings Series (eds
N.N. Ragsdale, P.C. Kearney, and J.R.
McCormick, R.W., Barrentine, J.L., and
Plimmer), American Chemical Society,
Braxton, L.B. (1995) Proc. South. Weed
Washington, DC, pp. 257–268.
Sci. Soc., 48, 201.
25. Owen, W.J. (2000) in Metabolism of
10. Braxton, L.B., Barrentine, J.L., Dorich,
Agrochemicals in Plants (ed. T. Roberts),
R.A., Geselius, T.C., Grant, D.L.,
John Wiley & Sons, Ltd, London, pp.
Langston, V.B., Redding, K.D., and
240–249.
Richburg, J.S. (1996) Proc. South. Weed
26. deBoer, G.J., Ehr, R.J., and
Sci. Soc., 49, 170–171.
Thornburgh, S. (2006) Pest. Manage.
11. Choate, J.H., Wilcut, J.W., and York,
Sci., 62, 316–324.
A.C. (1996) Proc. South. Weed Sci. Soc.,
27. van Wesenbeek, I.J., Zabik, J.M., Wolt,
49, 193.
D.W., Bormett, G.A., and Roberts,
12. Stabler, G.F., Murdock, E.C., Keeton, A., D.W. (1997) J. Agric. Food Chem., 45,
and Isgett, T.D. (1996) Proc. South. Weed 3299–3307.
Sci. Soc., 49, 18. 28. Zabic, J.M., van Wesenbeeck, I.J.,
13. Sheppard, B.R., Braxton, R.L., Peacock, A.L., Kennard, L.M., and
Barrentine, J.L., Geselius, T.C., Grant, Roberts, D.W. (2001) J. Agric. Food
D.L., Langston, V.B., Redding, K.D., Chem., 49, 3284–3290.
Richburg, J.S., and Roby, D.B. (1997) 29. Wolt, J.D., Smith, J.K., Sims, J.K., and
Proc. South. Weed Sci. Soc., 50, 161. Duebelbeis, D.O. (1996) J. Agric. Food
14. Arnold, J.C., Shaw, D.R., and Bennett, Chem., 44, 324–332.
A.C. (1998) Proc. South. Weed Sci. Soc., 30. Yoder, R.N., Huskin, M.A., Kennard,
51, 2. L.M., and Zabik, J.M. (2000) J. Agric.
15. Bailey, W.A., Wilcut, J.W., Jordan, D.L., Food Chem., 48, 4335–4340.
Swann, C.W., and Langston, V.B. (1999) 31. Krieger, M., Yoder, R., Stafford, L.,
Weed Technol., 13, 450–456. Batzer, F., Smith, J., Cook, W., and
16. Bailey, W.A., Wilcut, J.W., Jordan, D.L., Lewer, P. (2000) Book of Abstracts,
Swann, C.W., and Langston, V.B. (1999) 219th ACS National Meeting, San
Weed Technol., 13, 771–776. Francisco.
17. Shaw, D.R., Bennett, A.C., and Grant, 32. Johnson, T.C., Ehr, R.J., Johnston, R.D.,
D.L. (1999) Weed Technol., 13, 791–798. Kleschick, W.A., Martin, T.P., Pobanz,
18. Nelson, K.A. and Renner, K.A. (1998) M.A., Van Heertum, J.V., and Mann,
Weed Technol., 12, 293–299. R.K., (1999) US Patent 5,858,924.
33. Johnson, T.C., Ehr, R.J., Martin, T.P., the 20th Asian-Pacific Weed Science
Pobanz, M.A., Van Heertum, J.V., and Society, Vietnam, pp. 289–294.
Mann, R.K. (2000) US Patent 6,130,335. 42. Min, Y.K., Mann, R.K., and Korean, J.
34. Johnson, T.C., Ehr, R.J., Martin, T.P., (2004) Weed Sci., 24 (3), 192–198.
Pobanz, M.A., Van Heertum, J.V., and 43. Min, Y.K. and Mann, R.K. (2004) Korean
Mann, R.K. (2001) US Patent 6,303,814. J. Weed Sci., 24 (3), 199–205.
35. Johnson, T.C., Ehr, R.J., Johnston, R.D., 44. Shiraishi, I. (2005) J. Pestic. Sci., 30 (3),
Kleschick, W.A., Martin, T.P., Pobanz, 265–268.
M.A., Van Heertum, J.V., and Mann, 45. Wang, C.L., Lee, M.S., Li, Y.W., Yao,
R.K. (1999) US Patent 6,005,108. Z.W., Shieh, J.N., Mann, R.K., and
36. Johnson, T.C., Ehr, R.J., Kleschick, Huang, Y.H. (2004) 15th International
W.A., Pobanz, M.A., Van Heertum, Plant Protection Congress, Abstracts,
J.V., and Mann, R.K. (1999) US Patent Beijing, China, p. 598.
5,965,490. 46. Mann, R.K., Huang, Y.H., Larelle, D.,
Mavrotas, C., Min, Y.K., Morell, M.,
37. Smith, M.G., Pobanz, M.A., Roth, G.A.,
Nonino, H., and Shiraishi, I. (2003)
and Gonzales, M.A. (2002) US Patent
Proceedings of the 3rd International
6,462,240.
Temperate Rice Conference, Punta
38. Larelle, D., Mann, R., Cavanna, S.,
del Este, Uruguay, March 1–13, 2003.
Bernes, R., Duriatti, A., and
Abstract WD055, p. 68.
Mavrotas, C. (2003) Proceedings of
47. Deboer, G.J. and Thornburgh, S. (2005)
the International Congress British Crop
Abstract Agro 062, 229th ACS National
Protection Conference – Crop Science Meeting, San Diego, CA.
Technology, Glasgow, UK, Vol. 1, pp. 48. Johnson, T.C., Pobanz, M.A., Van
75–80. Heertum, J.V., Ouse, D.G., Arndt, K.E.,
39. Mann, R.K., Lassiter, R.B., Haack, and Walker, D.K. (2003) US Patent
A.E., Langston, V.B., Simpson, D.M., 6,559,101.
Richburg, J.S., Wright, T.R., Gast, R.E., 49. Wells, G.S. (2008) Proceedings of the
and Nolting, S.P. (2003) Proc. Weed Sci. 16th Australian Weeds Conference,
Soc. Am., 43, 40. North Queensland, Australia, pp.
40. Mann, R.K., Haack, A.E., Langston, 297–299.
V.B., Lassiter, R.B., and Richburg, J.S. 50. deBoer, G.J., Thornburgh, S., Gilbert, J.,
(2005) Proc. Weed Sci. Soc. Am., 45, 308. and Gast, J. (2011) Pest Manage. Sci., 67
41. Mann, R.K., Mavrotas, C., Huang, (3), 279–286.
Y.H., Larelle, D., Patil, V., Min, Y.K., 51. Gerwick, B.C., Subramanian, M.V.,
Shiraishi, I., Nguyen, L., Nonino, H.L., Loney-Gallant, V.I., and Chandler, D.P.
and Morell, M. (2005) Proceedings of (1990) Pestic. Sci., 29, 357.
2.5
Pyrimidinylcarboxylates and Sulfonanilides
2.5.1
Introduction
The pyrimidinylsalicylates (PSs) constitute a class of acetolactate synthase

(ALS)-inhibiting herbicides that was discovered during the late 1990s at the
Kumiai Chemical Industry and Ihara Chemical Industry. The biological activities
of these compounds are equally potent as those of the sulfonylureas (SUs).
Further investigations led to the development of the pyrimidinylglycolates, which

are experimental herbicides. As both types of compound include the carboxyl
moiety in their chemical structure, they are referred to as pyrimidinyl carboxy (PC)
herbicides.
The sulfonanilide herbicides were redesigned from the PCs by replacing the
oxy-bridge with a carbon-bridge, and the carboxyl group with a sulfonamide group.
Details of the sulfonanilides, which were found to have good herbicidal activities
against various paddy weeds, were first reported in 2000 [1].
2.5.2
Discovery of the PCs Herbicides
The discovery of the PC herbicides began with attempts to synthesize new

herbicides that incorporated a dimethoxypyrimidine [2]. During the subsequent
synthetic and bioassay projects, phenoxyphenoxypyrimidine 1 was found to demon-
strate potent herbicidal activity, with symptoms similar to those of Hill reaction
inhibitors. In elaborating the structure to develop more systemic herbicides, a
carboxylate group was introduced to produce compound 2 [3]. Although the ethyl
ester 2 was inactive, its ‘‘regio-isomer’’ 3 exhibited a moderate activity against
broadleaf weeds, with both pre-emergent and post-emergent treatments [4]. The
symptoms observed in plants following treatment with 3 were similar to those
seen after treatment with SUs (which are ALS-inhibiting herbicides) and unlike
those of the Hill reaction inhibitor 1. By removing the second phenoxy group from
3, the resulting O-PS 4 was found to exhibit a highly potent herbicidal activity,
characteristic of ALS inhibition (Figure 2.5.1).
Using the skeletal structure of 4 as a new lead compound, various derivatives
were synthesized to optimize the herbicidal activity. Among conventional sub-
stituents on the benzene ring, the carboxylate group ortho to the pyrimidinyloxy
group was essential for potentiating activity. A pyrimidine ring was better than any
F3C OCH3 CO2C2H5

F3C O
OCH3
O O N
N O N
OCH3 N
2 OCH3
1
O CO2CH3 CO2CH3
OCH3 OCH3
2 N N
O O
N N
3 4
Cl Cl
Figure 2.5.1 Structural modification pathway towards the lead compound 4.

other nitrogen heterocycle; however, the most favorable substitution pattern was
the 4,6-dimethoxypyrimidin-2-yl, 5 which, at an application rate of 1 kg a.i. ha−1 ,
controlled various grass- and broadleaf weed species both pre- and post-emergence,
with phytotoxic symptoms similar to those of the SUs. Unfortunately, the safety
margin of 5 for crops was narrow and unacceptable as a selective herbicide, despite
a marked increase in herbicidal activity (Figure 2.5.2) [5]. Thus, the ALS inhibitory
activities of 4 and 5 were assessed. As shown in Table 2.5.1, the free acid of 5
was potent in terms of the I50 (nM) of ALS inhibitory activity; this compound
exhibited a much higher ALS inhibitory activity than imazapyr, a representative of
the imidazolinone (IMI) group of herbicides [6].
The results of this study of ALS inhibition showed the PCs to be a novel class of
ALS-inhibiting herbicides, differing from both the SUs and the IMIs. Although the
PCs and SUs are structurally unrelated, they possess common structural parts of a
weakly acidic proton and an N-containing heterocyclic ring. On a two-dimensional
hexagonal grid template, the common parts in both molecules overlap (Figure 2.5.3)
[7]. This suggested that a weakly acidic proton and an N-containing heterocyclic
ring, appropriately located in a molecule, are both requisites for the inhibition
of ALS. Further modifications based on this hypothesis led to the highly active
pyrimidinylglycolates, in which a carboxylic and a pyrimidinyloxy group were not
directly connected with a benzene ring (Figure 2.5.4) [8].
CO2CH3
CO2CH3 CO2CH3
OCH3 OCH3
N N
O O
N Bridge atom N-hetero
N
Cl ring
OCH3
4 5
Figure 2.5.2 Optimization from the lead compound 4.
Table 2.5.1 Acetolactate synthase (ALS)a inhibitory activity

(I50 )b of the acids of compounds 4 and 5.
Compound I50 (nM)
4 (COOH) 4600
5 (COOH) 250
Imazapyr 9100
Chlorsulfuron 27
a
ALS prepared from etiolated pea seedlings.
b
I50 , molar concentration required for 50% inhibition of the ALS
activity.
Cl Cl
O O
C S
O
O S N O N N N
H H
N O N N
O O
Pyrithiobac Chlorsulfuron
Figure 2.5.3 Comparison of pyrithiobac and chlorsulfuron on hexagonal templates.
Pyrimidinyl carboxylate(PCs)
X
CO2CH3
OCH3
N
W
Hypothetical lead N
OCH3
Modulater
CO2CH3
N
OCH3
Acid N
W
OCH3 N
N OCH3
W
N
OCH3 CO2CH3
R OCH3
N
W
N
OCH3
Figure 2.5.4 Pyrimidinylcarboxylate inhibitors of acetolactate synthase (ALS) activity.
2.5.3
Structure–Activity Relationships of PCs Herbicides
The first PCs compound was prepared using the 4,6-dimethoxy-2-methylsulfonyl-

pyrimidine (DMSP) 6 and the salicylic acid ester in dimethylformamide (DMF).
The sulfonyl compound 6 proved to be a very efficient intermediate for the synthesis
of 2-substituted pyrimidines [9], as the methylsulfonyl group is easily replaced by
nucleophilic reagents such as 7 and 8 (Figure 2.5.5) [8, 10, 11]. This method was
generally employed to synthesize numerous analogs, the aim being to develop new
X
CO2R3
Ar X
CO2R3
W H
7 Ar OCH3
N
OCH3 W Ar:Aromatic ring
N
CH3SO2 N X:halogen,alkyl,alkoxy,etc.
N OCH3
OCH3 W:O,S,NH
6 COOH
DMSP
Pyrimidinylating agent OCH3
C N R1:Phenyl,Phenylalkyl
COOH R1 O R2:H,alkyl
N
R2 OCH3
R1 C 8
OH
R2
Figure 2.5.5 Synthesis via 2-methanesulfonylpyrimidinyl intermediate 6.

2.5 Pyrimidinylcarboxylates and Sulfonanilides
121
CO2CH3 X CO2H
1. Effect of benzene
OCH3 OCH3 substituents (X)
N N
O W 2. Effect of bridge atom
N N exchange (W)
5 OCH3 9 OCH3
Figure 2.5.6 Optimization for both herbicidal activities and safety to crops.
herbicides with not only a high potency but also an enhanced crop safety. Structural
modifications were first made with the skeletal structure 9 (Figure 2.5.6) [4].
2.5.3.1 Effects of Benzene Ring Substituents in the O-Pyrimidinylsalicylic Acids

The herbicidal activity of the PSs varied with the structure and position of the ring
substituents (X) (Table 2.5.2), with the position-specific effect of ring substituents
first being examined. For Cl derivatives, the 6-Cl was clearly more potent than
3-Cl and 5-Cl (Table 2.5.2) [5, 12]; however, in other cases, such as the methyl-
and fluoro-derivatives, a similar positional pattern was shown, and the sequence of
effects of the ring substituents in position was 6 > H (unsubstituted) > 3 > 5>>4.
Thus, only substitution at the 6-position proved to be favorable for enhancing
Table 2.5.2 Post-emergence herbicidal and ALS inhibitory

activities of the dimethoxypyrimidinyl salicylic acids.a
X 6
CO2H
5 OCH3
N
4 O
3
N
OCH3
X pI50 b Echc Digd Pole Amaf Cheg Cyph
3-Cl 6.3 2 1 5 5 1 5
4-Cl 4 0 0 0 0 0 0
5-Cl 5.4 0 0 2 5 3 4
6-Cl 7.6 5 5 5 5 5 5
H 6.6 5 4 5 5 5 4
a
Applied at a dosage of 250 g a.i. ha−1 , and assessed with six grades from 0
(no effect) to 5 (complete kill).
b
pI50, ALS inhibitory activity (−log I50 ).
c
Echinochloa crus-galli L.
d
Digitaria adscendens.
e
Polygonum nodosum.
f
Amaranthus retroflexus.
g
Chenopodium album.
h
Cyperus iria.
the herbicidal activity of the unsubstituted compound. Among the 6-substituted

derivatives, the halogen, methyl, acetyl, phenyl, CF3 , and lower alkoxy derivatives
exhibited extremely high activity at both the pre- and post-emergent applications.
Although these compounds were capable of controlling weeds completely at a dose
of 250 g a.i. ha−1 , their phytotoxicity towards the crops could not be improved to
achieve practical use [5].
2.5.3.2 Effect of a Bridge Atom in the Pyrimidinylsalicylates

After fixing the 6-substituent as Cl, the effect of the bridge between the two rings was
examined (Table 2.5.3). The S-bridge derivative showed excellent herbicidal activity,
the CH2 -bridge derivative was moderately active, but the NH-bridge and SO-bridge
derivatives exhibited poor activity, even at an application rate of 250 g a.i. ha−1 . In
an in vitro study of the ALS inhibition, the S-bridge and SO-bridge derivatives
showed inhibitory activities comparable to that of the O-bridge derivative, whereas
those of the NH-bridge and CH2 -bridge derivatives were decreased [5].
2.5.3.3 Pyrimidinylglycolates
As discussed in Section 2.5.2, the pyrimidinylglycolates shown in Figure 2.5.4,
in which the carboxylic and pyrimidinyloxy groups were not directly connected
with a benzene ring, also have high herbicidal activity [8, 10]. An attempt was
made to examine the favorable distances among important substructures, namely
a carboxylic group, a pyrimidine, and a benzene ring (Figure 2.5.7). Initially,
Table 2.5.3 Herbicidal activities of 6-Cl pyrimidinylsalicylic

acid analogs in which the O-bridge is modified.a
Cl
CO2H
OCH3
N
W
N
OCH3
W Post-emergenceb Pre-emergenceb
Ech Dig Pol Ama Ech Dig Pol Ama
O 5 4 5 5 4 5 2 4
S 5 3 5 5 5 5 5 5
NH2 0 0 0 3 0 0 0 2
SO 3 0 3 5 1 3 1 5
CH2 4 4 4 5 4 4 5 5
a
Applied at a dosage of 250 g a.i. ha−1 , and assessed with six grades from 0
(no effect) to 5 (complete kill).
b
Ech, Echinochloa crus-galli L; Dig, Digitaria adscendens; Pol, Polygonum
nodosum; Ama, Amaranthus retroflexus.
CH2CO2CH3
OCH3
N
O
N
OCH3 Inactive
10
CH2CO2CH3
Hypothetical lead OCH3
N
CH2O
Modulater N Inactive
11 OCH3
Acid
OCH3
N
W
N
OCH3 CO2CH3 Active
ALS
OCH3
N inhibitor
12 O
Lead
N compound
OCH3
Figure 2.5.7 The discovery of a new lead compound.
compounds 10 and 11 were synthesized, because the distances between the

carboxylic group and pyrimidine ring in these compounds were considered to be
close to that in the PSs with strong herbicidal activity [13], but these proved to be
totally inactive. Subsequently, compound 12, with the carboxylic group placed at the
α-position of the pyrimidinyloxy group, demonstrated encouraging ALS inhibitory
and herbicidal activities. Starting from 12, structural modifications were made
as shown in Figure 2.5.8 to discern the effects of the side chains (R1 , R2 ), ester
residues (R), bridge atoms (W), and pyrimidine substituents (X, Y) on herbicidal
activity. As a result, the bridge atom (W) was fixed as oxygen, and the pyrimidine
substituents (X, Y) as methoxy, in subsequent examinations, and these options led
to the most active compounds.
The herbicidal activities were seen to vary with the substituents R1 , R2 , and
R (Table 2.5.4). The α-hydrogen atom is, probably, essential for the herbicidal
activity because the disubstituted compound (R1 = Ph, R2 = CH3 ) completely lost
its activity. In contrast, two compounds (R1 = PhCH2 , R2 = H) and (R1 = PhC2 H4 ,
R2 = H) extended by methylene length(s) in R1 almost retained the herbicidal
activities of the starting compound 12 (R1 = Ph, R2 = H). Furthermore, the phenyl
group in 12 was replaced by straight alkyl chains, where the optimal length of the
alkyl chain R1 was around C3 but with a lower herbicidal activity. If the phenyl
group in 12 is replaced by α-branching alkyl groups, tert-alkyl groups are more
2) Effect of ester
1) Effect of side chain

R1 CO2R
CO2CH3 C X
OCH3 R2 N
N W
O N
N Y
12 OCH3
4) Effect of
3) Effect of bridge atom
hetero ring
Figure 2.5.8 Structural modifications from lead compound 12.
Table 2.5.4 Herbicidal efficacy of the pyrimidinylglycolates.
R1 CO2R
C OCH3
R2 N
O
N
OCH3
R1 R2 R Pre-emergency (ED90 a ) Post-emergence (ED90 a )
Echb Polc Echb Polc
Ph H H B A A B
PhCH2 H H A A A A
PhC2 H4 H H B A A B
Ph CH3 H C C C C
Ph H C2 H5 C C A C
PhCH(CH3 ) H H A A A A
tert- C4 H9 H H A A A A
a
ED90 (1 kg a.i. ha−1 ): A, 1 or less; B, 1–4; C, 4 or more.
b
Echinochloa crus-galli L.
c
Polygonum nodosum.
active than sec-alkyl groups, but again with a lower herbicidal activity than 12. The
free acid is more active than the esters (e.g., R = ethyl).
2.5.4
‘‘Pyrithiobac-Sodium’’: Cotton Herbicide
2.5.4.1 Discovery
The effect of the 6-substituents in the thiosalicylate moiety was fine-tuned
(Table 2.5.5). Halogen and thioalkyl derivatives demonstrated a good post-emergent
Table 2.5.5 Post-emergence herbicidal activities of

6-substituted pyrimidinylthiosalicylic acids.a
X
CO2H
OCH3
N
S
N
OCH3
X Cropsb Weedsc
Zea Gly Gos Ech Abu Ipo Xan
F 7 10 3 3 6 7 5
Cl 9 8 0 4 9 6 7
Br 9 7 0 1 7 4 6
I 10 7 0 2 9 6 8
CH3 8 6 1 0 4 0 4
CF3 8 5 0 0 6 2 2
COCH3 10 4 1 10 9 8 4
OCH3 9 9 0 2 5 1 6
OC3 H7 -i 2 4 1 0 2 0 0
SCH3 9 8 4 0 10 10 9
NO2 3 6 2 1 6 3 1
a
Applied at a dosage of 16 g a.i. ha−1 , and assessed with 11 grades from 0 (no effect) to 10 (complete
kill).
b
Crops: Zea, Zea mays; Gly, Glycine max; Gos, Gossypium hirsutum.
c
Weeds: Ech, Echinochloa crus-galli L; Abu, Abutilon theophrasti; Ipo, Ipomoea lacunosa L; Xan,
Xanthium strumarium L.
herbicidal activity against broadleaf weeds, but were weak on Echinochloa crus-galli,
whereas the acetyl derivative showed good herbicidal activity both against Abutilon
theophrasti and Echinochloa crus-galli. This suggested that hydrophobic substituents
such as halogens would be favorable for killing broadleaf weeds, whereas the
hydrophilic properties of the acetyl group would have a positive effect on activity
against grass weeds [5, 12]. Based on the good safety margin of the sodium salt of
13 (pyrithiobac-sodium), this compound was selected for development as a cotton
herbicide against broadleaf weeds such as Abutilon theophrasti and Ipomoea lacunosa
(Figure 2.5.9) [14].
2.5.4.2 Biology
The herbicide pyrithiobac-sodium is used to control a wide range of weeds in
cotton [12, 14, 15]; notably, it provides excellent control of troublesome weeds
such as Ipomoea spp., Xanthium strumarium, Amaranthus spp., Abutilon theophrasti,
Sida spinosa, Sesbania exaltata, and Sorghum halepense. Pyrithiobac-sodium can
Cl
CO2Na
M.p., 233.8–234.2 C° (decomp.); V.p., 4.80 ×10−9 Pa (25 C°);
OCH3
N Kow: logP = 0.6 (pH 5); −0.84 (pH 7)
S Solubility: In water, 264 g I−1 (pH 5); 705 g I−1 (pH 7); 690 g I−1 (pH 9);
In methanol, 270 g mI−1; In n-hexane, 10 mg mI−1 (all in 20 C°)
N
Acute oral: LD50 for male rats, 3300 mg kg−1; LD50 for female rats,
OCH3 3200 mg kg−1
Eye irritation for rabbits, Irritating
13 TLm (96 h) for bluegill sunfish, > 930 ppm; TLm (96 h) for
rainbow trout, > 1000 ppm; TLm (48 h) for daphnia, > 1100 ppm
“Pyrithiobac-sodium” Staple®
1996 market introduction (USA)
H3CO OCH3
N N
M.p., 223–224 C°; V.p., 5.05×10−9 Pa (25 C°); Kow,
O logP = −1.03 (23C°)
CO2Na Solubility: In water, 73.3 g I−1; In methanol, 26.3 g/100l;
In n-hexane, 3.56 mg I−1 (all in 25 C°)
OCH3 Acute oral: LD50 for male rat, 4111 mg kg−1; LD50 for female rats,
N 2635 mg kg−1 Eye irritation for rabbits, Slightly irritating
O TLm (96 h) for carp, > 1000 ppm; TLm (96 h) for rainbow
14 trout, >100 ppm; TLm (48 h) for daphnia, >100 ppm
N
OCH3
“Bispyribac-sodium” Nominee®
1997 market introduction
CH3 M.p.: Tech. 105 C°; E–isomer, 107–109 C°; Z–isomer, 70 C°

V.p.: E–isomer, 3.5×10−5 Pa (25 C°); Z–isomer, 2.681×10−5 Pa
(25 C°)
CH3O~N C Kow: logP of E–isomer = 2.98 (21.5 C°); logP of Z–isomer = 2.70
CO2CH3 (20.6 C°)
Solubility: E–isomer in water, 9.25 mg mI−1; E–isomer in methanol,
15 OCH3 14.6 g mI−1; Z–isomer in water, 175 mg mI−1; Z–isomer in methanol,
N 14.0 g I−1 (all in 20 C°)
O Acute oral: LD50 for rats, > 5000 mg kg−1
N Eye irritation for rabbits, Slightly irritating
OCH3 TLm (96 h) for carp, 30.9 ppm; TLm (96 h) for rainbow trout,
21.2 ppm;
“Pyriminobac methyl” Prosper® TLm (24 h) for daphnia, > 20 ppm
Figure 2.5.9 The physico-chemical and toxicological charac-

teristics of commercialized PC herbicides.
be applied either pre- or post-emergently, with soil or foliar treatment at 35

to 105 g a.i. ha−1 providing an excellent control of weeds. Adjuvants such as
nonionic surfactants, or some petroleum-based adjuvant oils, play an important
role in achieving consistent performance on several weed species when applied
post-emergent. A good safety margin for cotton, at rates that are effective on weeds,
has been observed with post- and pre-emergence treatment with pyrithiobac-sodium
in both the greenhouse and the field.
2.5.5
‘‘Bispyribac-Sodium’’: Herbicide in Direct-Seeded Rice
2.5.5.1 Discovery
Previous investigations with the PCs showed that substitution at the 6-position of
the salicylate moiety was preferable for both herbicidal and ALS inhibitory activities.
Some PCs demonstrated a strong activity against various weeds, even at rate of
about 10 g a.i. ha−1 , but rice injury was severe. When structurally modifying the
6-substitutent on the benzene ring, compounds with halogen, alkyl, or alkoxy group
failed to improve rice safety. With a bulky substituent, such as a phenoxy group,
the herbicidal activity was somewhat decreased, but rice injury was significantly
alleviated [16, 17]. Starting from the 6-phenoxy compound as a basic structure,
various substituted phenoxy compounds were prepared, although unfortunately
no compound provided simultaneous acceptable rice safety and strong herbicidal
activity. As the severe rice injury was attributed to the hydrophobic property of the
phenoxy group, more hydrophilic substituents of the heterocyclyl-oxy groups were
introduced in its place. Among five- or six-membered hetero-rings, pyrimidinyloxy
groups exhibited the most suitable performances as a rice herbicide, in both aspects
of activity and rice safety. PCs with a 2 or 4-(substituted)pyrimidinyloxy group as
a 6-substituent on the benzene ring were, furthermore, synthesized and evaluated
(Table 2.5.6).
In comparison with the unsubstituted compound (R1 = R2 = H), it was,
consequently, revealed that the introduction of substituents into the 4 and 6
positions of the pyrimidine was favorable for improving rice safety, without
decreasing herbicidal activity. In particular, the 4,6-dimethoxy compound, being
a bis-pyrimidinyl compound, showed both a remarkable improvement in rice
safety and excellent activity against Echinochloa spp. and broadleaf weeds.
Finally, the sodium salt of 2,6-bis[(4,6-dimethoxypyrimidin-2-yl)oxy]benzoic acid
(14, bispyribac-sodium) was selected for commercial development as a herbicide
on direct-seeded rice (Figure 2.5.9) [18].
2.5.5.2 Biology
Bispyribac-sodium is a post-emergent herbicide which is used to control a wide
range of weeds, and has excellent selectivity on direct-seeded Indica-type rice
[18, 19]. The low application rate of bispyribac-sodium (15–60 g a.i. ha−1 with
surfactant) has provided outstanding efficacy on Echinochloa spp., and it can
be applied from the one- to seven-leaf stage of the weed. Moreover, it can
also be used to control other troublesome weeds, including Ischaemum rugosum,
Aeschynomene indica, Brachiaria spp., Cyperus spp., Scirpus spp., Polygonum spp.,
Sagittaria spp., Commelina spp., and Sesbania exaltata. Adjuvants, such as nonionic
surfactants, silicon-type adjuvants, or crop oil concentrates play an important role
in enhancing the activity and achieving a consistent performance of this compound.
Bispyribac-sodium has a high selectivity between Indica-type rice and Echinochloa
oryzicola by foliar application under dry-seeded conditions, which suggests that it
could be used against a wide range of growth stages of Echinochloa spp., without
Table 2.5.6 Effect of substituents Rn on the pyrimidine ring

of bispyrimidinyloxybenzoic acids on herbicidal activity and
rice phytotoxicity at post-emergence application.a
R1 CO2H OCH3
N N
W O O
Z N
R2 OCH3
R1 R2 Z W Phytotoxicityb Herbicidal activityc
Ory Ech Pol Ama Xan
H H N CH 8 4 8 6 0
Cl CH3 N CH 3 4 7 9 2
Cl OCH3 N CH 5 9 8 9 9
CH3 CH3 N CH 5 8 8 8 6
CH3 OCH3 N CH 4 9 9 9 8
OCH3 OCH3 N CH 1 10 9 10 9
H H N CCl 7 4 8 7 2
OCH3 OCH3 CH N 5 8 9 8 8
a
Applied at dosage of 16 g a.i. ha−1 , and assessed with 11 grades from 0 (no effect) to 10 (complete
kill).
b
Ory, Oryza sativa.
c
Weeds: Ech, Echinochloa crus-galli L; Pol, Polygonum nodosum; Ama, Amaranthus retroflexus; Xan,
Xanthium strumarium L.
causing rice crop injury. On the other hand, bispyribac-sodium applied at a rate
of 150 g a.i. ha−1 and pre-mixed with a nonionic surfactant, reduced the vegetative
growth of weeds such as Imperata cylindrica, Digitaria adscendens, Miscanthus
sinensis, and Artemisia princes [20]. Such growth reduction persisted for 50 days
after application of the compound when applied 5–10 days after mowing (at
10–20 cm plant height). Bispyribac-sodium also controlled a wide range of weed
species such as Murdannia keisak, Solidago altissima, Paspalum distichum, and
Pueraria lobata that grew in rice levees or on highway and railroad right-of-ways.
The results obtained indicated that bispyribac-sodium could reduce the frequency
of mowing in paddy rice levees, and also on highway and railroad right-of-ways.
2.5.6
‘‘Pyriminobac-Methyl’’: Rice Herbicide
2.5.6.1 Discovery
Since, when the pyriminobac-methyl project was initiated, ALS inhibitory and
low-dose herbicides (including the SUs) were not commercially available for the
effective control of Echinochloa spp. in transplanted rice, attention was focused on
developing low-dose herbicides that would be particularly effective in controlling
Echinochloa spp. in the paddy rice. Previous studies with PSs had provided the
following findings: (i) substitution at the 6-position of the salicylate moiety was
preferable for herbicidal and ALS inhibitory activities; (ii) electron-withdrawing
groups contributed to the ALS activity; and (iii) hydrophilic groups led to better
activities against grass weeds than broad-leaf weeds. Consequently, the 6-acyl
compounds proved to be especially interesting as the acyl groups were both
hydrophilic and electron-withdrawing. Although compound 16 showed excellent
control of Echinochloa spp., it caused unacceptable phytotoxicity to rice. Nonetheless,
the herbicidal profile of 16 satisfied the minimum requirements as a prototype for
a herbicide effective against Echinochloa spp. [21].
In order to reduce rice injury, while retaining the herbicidal activity of 16, an
attempt was made to introduce an oxyimino group so as to produce a hypothetical
bio-isosteric analog of 16. The methoxyimino group has a similar [σp ] (acyl group:
σp = 0.4, methoxyimino group: σp = 0.3) and a steric similarity to a carbonyl
group, and the hydrophilicity of the oxyimino moiety can be varied by alkylation
and acylation. Consequently, extensive synthetic modifications were made to the
6-alkyl moiety (R1 ), the alkoxyimino moiety (R2 ), and the ester moiety (R3 ) of 17
(Figure 2.5.10).
The structure–activity relationships of the synthesized compounds were studied
by examining their herbicidal activities against Echinochloa oryzicola in paddy
rice at various growth stages, including pre-emergence (Table 2.5.7). Compounds
with R1 = CH3 , R3 = CH3 , and R2 = alkyl provided the best selectivity/activity
relationship, but compounds with R3 > CH3 had a reduced herbicidal activity at a
higher growth stage [21, 22].
According to a study of the mode of action (Table 2.5.8), the ALS inhibitory
activities of pyriminobac-methyl against both Echinochloa oryzicola and rice
were almost identical, and about 1000-fold lower than that of the carboxylic
acid (18). The metabolic transformation of pyriminobac-methyl into the
acid (which is considered to be the metabolically activated form as an ALS
inhibitor) was also enhanced, particularly in Echinochloa oryzicola, though
no enhancement occurred in rice (Tables 2.5.7 and 2.5.8) [22]. Ultimately,
methyl 6-[1-(methoxyimino)ethyl]-2-[(4,6-dimethoxypyrimidin-2-yl)oxy]-benzoate
(pyriminobac-methyl) (15) was selected as a candidate for commercialization as a
novel barnyard-grass killer, with an excellent selectivity for rice [22].
O R1
C CH3 C N ~ OR2
CO2CH3 CO2CH3
OCH3 OCH3
N N
O O
N N
16 OCH3 17 OCH3
Figure 2.5.10 Modification to an oxyimino group from an acyl group.

Table 2.5.7 Herbicidal activities of the

6-alkoxyiminosalicylate analogs against barnyardgrass.
R1
C N~OR2
CO2R3
OCH3
N
O
N
OCH3
R1 R2 R3 Pre-emergence Selectivity
Phytotoxicitya Herbicidal activityb ED10 /ED90

ED10 (Orya ) ED90 (Echb )
H CH3 CH3 63 16 4
CH3 CH3 CH3 250 16 16
C2 H5 CH3 CH3 63 63 1
C3 H7 CH3 CH3 63 >1000 <1/16
CH3 H CH3 4 16 1/4
CH3 C2 H5 CH3 250 16 16
CH3 C3 H7 CH3 250 16 16
CH3 C3 H7 -i CH3 63 16 4
CH3 C4 H9 CH3 63 16 4
CH3 CH3 C2 H5 63 63 1
CH3 CH3 H <4 16 <1/4
a
Active ingredient amounts (g ha−1 ) required for less than 10% phytotoxicity of Oryza sativa (Ory).
b
Active ingredient amounts (g ha−1 ) required for more than 90% control of Echinochloa oryzicola
(Ech).
2.5.6.2 Biology
Pyriminobac-methyl is a selective herbicide with outstanding efficacy on Echinochloa
spp. in paddy rice [22–24]. This compound has a specific efficacy against Echinochloa
spp. over a wide range of growth stages, from pre- to late post-emergence,
with an excellent crop safety in rice. At 30–60 g a.i. ha−1 , the application rate of
pyriminobac-methyl was very low compared to rates recommended for molinate
and thiobencarb. Pyriminobac-methyl has shown excellent safety on all 11 varieties
of water-seeded rice tested, and can be applied at any growth stage of the rice.
Moreover, there were no observed significant differences in susceptibility to
pyriminobac-methyl among the rice varieties tested. Pyriminobac-methyl can be
used either alone or mixed with other rice herbicides such as bensulfuron-methyl
or bentazon. Under flooded conditions in the greenhouse, the residual activity
of pyriminobac-methyl at 30 g a.i. ha−1 was much superior that of thiobencarb, at
Table 2.5.8 ALS inhibitory activity, herbicidal activity, and

phytotoxicity of 6-methoxyiminosalycylate and its acid.
Compound ALS I50 a (μM) Phytotoxicityb Herbicidal activityc
Rice Barnyardgrass ED10 (Ory) ED90 (Ech)
15 59 47 6.3 25
18d 0.018 0.016 <0.4 0.4
a
Concentration required for 50% inhibition.
b
Maximum effective dosage (g a.i. ha−1 ) for less than 10% phytotoxicity against transplanted Oryza
sativa (Ory) at 3 cm in depth.
c
Minimum effective dosage (g a.i. ha−1 ) for required more than 90% control against Echinochloa
oryzicola (Ech) in pre-emergence.
d
Free acid of compound 15.
CH3
CH3O~N=C
COOH
OCH3
N
O
18 N
OCH3
3 kg a.i. ha−1 . Pyriminobac-methyl, when applied at a rate of 120 g a.i. ha−1 , also
controlled perennial weeds such as Sagittaria trifolia [25].
2.5.7
Mode of Action of the PCs Herbicides
The growth inhibition of rice seedlings and chlorella by the PCs were alleviated
by the simultaneous application of three branched-chain amino acids [6]. The
PCs, including pyrithiobac, bispyribac, and pyriminobac, strongly inhibited ALS
in various plant species at concentrations in the nanomolar range [26]. The SUs
[27] and the triazolopyrimidines (TPs) [28] inhibited plant ALS activity in the
mixed-type with respect to pyruvate in the steady-state analysis, while the IMIs
inhibited in uncompetitive manner [29]. The following kinetic results have been
demonstrated [30, 31]: in etiolated pea seedlings, pyrithiobac and bispyribac each
inhibited the ALS activity of mixed-type with respect to pyruvate by means of a
40 min steady-state analysis. This inhibition pattern was identical to that of the SU
herbicide, chlorsulfuron, but differed from that of the IMI herbicide, imazapyr,
which inhibited the enzyme in an uncompetitive manner. The inhibition pattern
of pyrithiobac for the ALS of Pseudomonas aeruginosa was noncompetitive with
respect to pyruvate, as was that of chlorsulfuron, whereas imazapyr inhibited the
enzyme in uncompetitive manner [32]. The inhibition patterns of these inhibitors
differed from those of the feedback inhibitors, the inhibition patterns of which
were partially competitive. The low ALS activity of etiolated pea seedlings, which
lost its sensitivity to the feedback inhibition, was potently inhibited by the PCs.
Taken together, these results indicated that the binding sites of the inhibitors on the
enzyme differed from those of the feedback inhibitors. Imazapyr has been shown
to compete with a SU, sulfometuron-methyl, in the binding to ALS of Salmonella
typhimurium [33]. In recent studies, chlorsulfuron was shown to compete with
bispyribac in binding to the ALS of etiolated pea seedlings, and this competition
was more potent than that of pyrithiobac [31]. The binding site of the PCs on ALS is
located on the allosteric site in a wide sense, close to the catalytic center. It appears
likely that both the SUs and TPs share the binding site with the PCs, whereas the
IMIs bind to a site that is somewhat distinct from, but which overlaps, that of the
SUs, the TPs, and the PSs. These sites are not located on the regulatory subunit;
rather, they are considered to be within the vestige of the ubiquinone binding site
on the catalytic subunit [33] that lost its role in the enzymatic reaction during the
evolutionary process.
Despite their reversible nature to the inhibition of ALS, the SUs and IMIs
are slow-binding inhibitors of plant ALS [34, 35], that inactivate the enzyme
irreversibly after reaching the final steady inhibitions. An irreversible inactivation
of the enzyme has been demonstrated in both the presence [36] and absence
[37] of pyruvate. Pyrithiobac and bispyribac each inhibited the ALS of etiolated
pea seedlings with slow-binding properties, with pyrithiobac demonstrating a
mixed-type pattern with respect to pyruvate in the initial inhibition. The inhibition
constants in the initial inhibition by pyrithiobac and bispyribac were approximately
20-fold larger than those in the final steady state. The maximal first-order rate
constant (k1 , 0.069 min−1 ) for transition from the initial to the final steady-state
inhibition of pyrithiobac [30] was almost identical to the rate constants of the SU
and IMI. However, the dissociation constant of bispyribac to the ALS of etiolated
pea seedlings after reaching the final steady inhibition was almost identical with
the inhibition constant in the initial inhibition [26].
2.5.8
Mode of Selectivity of the PCs Herbicides in Crops
Despite the high selectivity of pyrithiobac for cotton and bispyribac for rice,
there were no differences in the sensitivities of ALSs to pyrithiobac between
cotton and other plants, and to bispyribac between rice and other plants. The
selectivities of pyrithiobac and bispyribac must be determined by other factors. In
the case of pyrithiobac, no reports have been made on its selectivity for cotton;
however, oxidative demethylation of the 4,6-dimethoxy moiety of the pyrimidinyl
group has been shown to account for the tolerance of tall morning-glory to
this herbicide [38]. Thus, the same mechanism is assumed to be involved in
pyrithiobac’ selectivity between cotton and other sensitive plants. For bispyribac,
translocation of the compound mainly accounts for its selectivity between rice
and barnyardgrass (unpublished data). As desmethylbispyribac was detected in a
rice plant treated with bispyribac [39], and the application of cytochrome P-450
inhibitors, such as 1-aminobenzotriazol and piperonyl butoxide, led to a reduction
in the selectivity of bispyribac for Indica-type rice (unpublished data), oxidative
detoxification metabolism – as for pyrithiobac – is presumed to be another factor
in the selectivity of bispyribac for the rice plant.
One of the methyl ester compounds of the PC (5 in Figure 2.5.2), which
has the same herbicidal potency as its free acid, barely inhibited the activity of
ALS when separated from the esterase, but inhibited the ALS activity with equal
potency as the free acid when the esterase was added to the reaction mixture
[40]. Thus, whilst the active forms of the ester compounds appear to be their
free acids, pyriminobac-methyl inhibited ALS less potently than its free acid,
even in the presence of esterase. Pyriminobac-methyl was barely hydrolyzed by
an esterase in the soluble fractions of both rice and barnyardgrass, but was
hydrolyzed by the microsomal fraction of barnyardgrass (unpublished data). The
free acid of pyriminobac-methyl was also detected in barnyardgrass treated with
this compound, but not in rice [41]. Taken together, these results indicate that
the selectivity of pyriminobac-methyl between rice and barnyardgrass depends on
differences in the substrate specificity of the enzyme, and whether esterase activity
is present in the plant membrane fraction.
2.5.9
Discovery of the Sulfonanilides
The discovery of the herbicidal class of sulfonanilides is outlined in detail in

Ref. [42]. During investigations into PCs it was confirmed that, in order for the
herbicide to exhibit a high ALS inhibitory activity and a high herbicidal activity, it
was necessary that a hydrophobic group, a pyrimidine ring, and an acidic group
be placed in a certain configuration via a suitable linkage group. Because the
carboxyl group of PCs has a comparable high acidity, these compounds are often
distinctly susceptible to the effects of water movement and, consequently, will have
an unstable herbicidal activity during pre-emergence application under flooded
conditions. Furthermore, due to the presence of a characteristic oxy-bridge, they
easily undergo metabolic degradation. In the past, attempts have been made to
change the oxy-bridge to a carbon-bridge in order to secure an adequate residual
activity, and also to exchange the acidic group for a sulfonamide group so as to
provide a broader weed spectrum [1]. Following the introduction of these changes,
compound 19 was redesigned from PS herbicides and prepared via synthesis
(Figure 2.5.11).
Subsequent pot tests of the herbicidal activity of compound 20 under flooded
conditions showed the effective dose (ED90 ) values for Echinochloa crus-galli, Mono-
choria vaginalis, and Scirpus juncoides to vary from 250 g a.i. ha−1 to 1.0 kg a.i. ha−1 .
Further, compound 21, which was synthesized by substituting the methylsulfonyl
group with a chloromethylsulfonyl group, showed a marked increase in herbici-
dal activity, particularly against Monochoria sp. and Scirpus sp., with ED90 values
ranging from 16 to 63 g a.i. ha−1 (Figure 2.5.12). As the logP of compound 21 was
O
R1
X O S
COOH R2
OCH3 NH
OCH3
N
W N
N
OCH3 O N
9 19
OCH3
PS herbicides 2′-pyrimidinecarbonyl sulfonanilides
Figure 2.5.11 Conversion of a pyrimidinylsalicylate (PS) her-

bicide to 2 -pyrimidinecarbonyl sulfonanilide.
O O
O S O S
NH NH
OCH3 OCH3
N N
logP = 2.04
O N N pK a = 6.55
O
OCH3 OCH3 S w = 7.1 ppm
20 21
Or: Oryza sativa, Eo: Echinochloa oryzicola

g a.i. ha−1 (Or :ED20, Eo, Mo, Sc : ED90) Mo: Monochoria vaginalis, Sc: Scirpus juncoides
16 16
63 63
250 250
1000 1000
Or Eo Mo Sc Or Eo Mo Sc
Figure 2.5.12 The biological activity of 2 -pyrimidinecarbonyl

sulfonanilides as a rice herbicide.
2.04 and the pKa 6.55 (i.e., it was less acidic than the carboxyl compound), this
compound was considered to have suitable physico-chemical properties to serve as
a rice herbicide.
2.5.10
Structure–Activity Relationships
The general synthetic route of sulfonanilide derivatives is shown in Figure 2.5.13.

Intermediate i was first synthesized by reacting o-nitrophenylacetonitrile with
DMSP, and then oxidized, dehydrocyanated and reduced to produce the anilines
iii and v. The reaction of sulfonylchloride with these anilines led to the creation
OCH3
N
CH3SO2
N
OCH3 R2
R2 6 NaH
R2
NO2 NO2 OCH3
NO2 OCH3
5 1) mCPBA/CHCl3 N
DMF N
4 CH2CN 2) NaOH/H2O
3 N O N
NC
ii OCH3
OCH3
i
R2 NHSO2R1 R2 NHSO2R1
Fe, cat. NH2 R2
OCH3 OCH3 OCH3
AcOH R1SO2Cl
N N Reduction N
AcOEt-
O N N N
H 2O O HO
OCH3 OCH3 OCH3
iii iv vi
reduction
R1SO2Cl
R2
NH2 OCH3
N
R2 6 NHSO2R1
HO N 5 OCH3
OCH3 N
v 4
3 N
X
vii OCH3
Figure 2.5.13 Synthetic route of sulfonanilide derivatives

having a pyrimidinyl-containing group at the 2 -position.
of the various sulfonanilides, iv and vi. The substituent of the bridge moiety was
then exchanged by using compound vi as the starting material. Following synthesis
of these sulfonanilides, their herbicidal activities against Echinochloa oryzicola, M.
vaginalis, and S. juncoides were evaluated.
2.5.10.1 Effect of the Sulfonamide Moiety in the Sulfonanilides

The haloalkyl group (ClCH2 , FCH2 , F2 CH, CF3 ) and the cyanomethyl group show
comparatively high weed control activity. In particular, sulfonanilides with an F2 CH
group and a CF3 group demonstrate remarkably high herbicidal activities against
M. vaginalis and S. juncoides. The presence of the F2 CH group results in a superior
herbicidal activity against E. oryzicola.
2.5.10.2 Effects of the Bridge Moiety in the Sulfonanilides

Details of the correlation between the bridge moiety and biological activity are
listed in Table 2.5.9. Most sulfoanilides were found to have only a weak herbicidal
activity against E. oryzicola, except for the methoxy compound which had moderate
activity, with an ED90 of 63 g a.i. ha−1 . In contrast, almost all groups showed a
high biological activity against M. vaginalis and S. juncoides. Although the acetyloxy,
ethylthio, and carbonyl compounds each showed high herbicidal activities in pot
Table 2.5.9 Substituted trifluoromethanesulfonanilides with

pre-emergence herbicidal activity.
NHSO2CF3
OCH3
N
X N
OCH3
X ED20 a ED90 b
Ory Ech Mon Sci
=O 63 1000 16 16
OH 63 1000 4 4
OCH3 63 63 16 63
OSiMe3 63 1000 4 4
OCOCH3 63 >1000 16 4
ON=CHCH3 63 1000 63 16
Cl 63 1000 16 4
NMe2 250 >1000 63 16
SH 250 >1000 63 63
SC2 H5 63 1000 16 4
SOC2 H5 1000 >1000 1000 63
SO2 C2 H5 1000 >1000 250 250
a
Active ingredient amounts (g ha−1 ) required for less than 10% phytotoxicity
of Oryza sativa (Ory).
b
Active ingredient amounts (g ha−1 ) required for more than 90% control of
Echinochloa oryzicola (Ech), Monochoria vaginalis (Mon), and Scirpus juncoides
(Sci).
tests, they showed low ALS inhibitory activities in vitro (unpublished data). Thus,
it appeared that these compounds would exhibit herbicidal activity only after their
in vivo conversion to an active ingredient, with the hydroxyl and trimethylsilyloxy
compounds showing the highest activities. Moreover, as the high activity of the
trimethylsilyloxy compound was thought to result from its easy conversion to
the hydroxyl compound, the latter was deemed responsible for the herbicidal
activity.
2.5.10.3 Effects of Benzene Ring Substitution in the Sulfonanilides

On the basis that difluoromethanesulfonanilides appeared to be better herbicides,
due to their more balanced activity against a variety of weeds (including E. oryzicola),
a variety of studies on the effects of substituents on the benzene ring was initiated,
with attention focused on difluoromethyl compounds. The structure–activity rela-
tionship of substituents on the benzene ring is shown in Table 2.5.10, where the
Table 2.5.10 Substituted difluoromethanesulfonanilides with

pre-emergence herbicidal activity.
R2 NHSO2CHF2
OCH3
N
HO N
OCH3
R2 ED20 a ED90 b
Ory Ech Mon Sci
H 63 250 4 16
3-F 16 63 16 16
3-Cl 1000 >1000 64 250
4-Cl 1000 >1000 1000 >1000
4-OCH3 63 >1000 250 >1000
5-Cl 63 250 63 63
5-CH3 16 1000 16 16
5-OCH3 16 16 16 63
6-F 16 250 4 4
6-CH3 16 16 4 16
6-C2 H5 63 16 16 4
6-C3 H7 63 16 16 63
6-CH2 OCH3 63 16 4 4
6-OCH3 4 16 16 63
6-OC2 H5 16 63 16 16
6-OC3 H7 63 250 63 250
6-OC6 H5 1000 >1000 250 >1000
6-SCH3 63 250 63 63
a
Active ingredient amounts (g ha−1 ) required for less than 10% phytotoxicity
of Oryza sativa (Ory).
b
Active ingredient amounts (g ha−1 ) required for more than 90% control of
Echinochloa oryzicola (Ech), Monochoria vaginalis (Mon), and Scirpus juncoides
(Sci).
results of substitution by halogen, alkyl, and alkoxy groups at respective positions

of the benzene ring are summarized. The effects of the substitution position on
the enhancement of activity were most evident at the 6-position, but the effects
decreased in the order of 6 > H (unsubstituted) > 5, 3 >> 4. This sequence of
effects of the position of ring substituents was similar to the sequence of effects in
pyrimidinyl salicylate herbicides [5].
Among the 6-substituted derivatives, the herbicidal spectrum and selectivity were
changed depending on the substituent. However, in particular, it was noted that
the alkyl compounds tended to enhance both herbicidal activity against E. oryzicola
and selectivity between rice plants and paddy weeds. Among the halogenated
compounds, the 6-fluoro compound showed a higher herbicidal activity than did
the unsubstituted compound. In the case of alkylated or alkoxylated compounds,
herbicidal activity against M. vaginalis and S. juncoides was similar or slightly
lower than that of the unsubstituted compound, but herbicidal activity against
E. oryzicola was enhanced. The propyl and ethoxy compounds both showed high
activity, whereas the propoxy and phenoxy compounds showed reductions in
activity. The size and/or length of the substituents at the 6-position were crucial for
herbicidal activity. Alkoxy compounds caused severe rice plant injuries, whereas
alkyl compounds were relatively safe towards rice plants. On the basis of these
findings, alkyl groups were considered to be favorable and consequently a variety of
compounds were synthesized and their herbicidal activities evaluated. Ultimately,
a compound with a methoxymethyl group showed most promise, with a good
balance of enhanced herbicidal activity, weed control spectrum, and safety towards
rice plants [43].
2.5.11
‘‘Pyrimisulfan’’: Rice Herbicide
As noted above, for the novel series of sulfonanilides with a pyrimidinyl-containing

group at the 2 -position, the structure–activity relationships were probed by substi-
tution of the sulfonyl group, bridge, and benzene ring. Among the sulfonamides,
the F2 CH compound showed a comparatively high activity and a broad spectrum
to control weeds, including E. oryzicola. The most preferable substitution position
on the benzene ring was the 6-position, while the lower alkyl group showed a high
herbicidal activity and a broad spectrum of weed control. Among the compounds
tested, the methoxymethyl group provided the greatest herbicidal efficacy, whilst
in terms of the bridge moiety the hydroxyl group proved superior.
Among the compounds examined, 2 -[(4,6-dimethoxypyrimidin-2-yl)(hydroxy)-
methyl]-1,1-difluoro-6 -(methoxymethyl)methanesulfon-anilide (pyrimisulfan; 22)
showed excellent pre-emergence herbicidal activity of a broad spectrum against
grass, sedge, and broadleaf weeds, without causing injury to rice plants
(Figure 2.5.14).
2.5.11.1 Biology
Pyrimisulfan is a herbicide used to control a wide range of weeds, with selec-
tivity on transplanted and water-seeded rice [42, 43]. The low application rate of
50–75 g a.i. ha−1 has provided outstanding efficacy on almost all major weeds of
Japanese paddy fields, such as Echinochloa spp., S. juncoides, M. vaginalis, Lindernia
spp., with only one single active ingredient. In addition, pyrimisulfan can control
other troublesome weeds, including Sagittaria trifolia, Cyperus nippnicus, Cyperus
planiculmis, Eleocharis kuroguwai, and SU-resistant weeds, which have presented
serious problems in recent years. Pyrimisulfan has a wide application window
from pre- to post-emergence of the weed, it is very easy to use, and it has suffi-
cient rice safety and residual activity. As is common to all herbicides, however,
CH2OCH3
NHSO2CHF2 Appearance: White Granular Crystal
OCH3 M.p.: 98.8°C log Pow: 2.01 (pH 5)
N Water Solubility: 114 ppm (pH 5)
Acute Oral (Rat) LD50: 1000-2000 mg kg−1
Eye Irritation (Rabbit): Minimal irritant
HO N TLm (96 h) for Carp: >127 mg l−1
OCH3 TLm (48 h) for Daphnia: >122 mg l−1
22
“pyrimisulfan” Best Partner®
Figure 2.5.14 Commercialized sulfonanilide herbicide. TLm,

LC50 in fish acute toxicity study.
pyrimisulfan may cause phytotoxicity and hence a reduced efficacy depending on

the paddy field conditions. In order to optimize its performance, the compound
has been incorporated into a new controlled-release formulation technology. In
Japan, pyrimisulfan alone is available commercially under the trade name ‘‘Best
® ®
Partner ’’; it is also sold as ‘‘My Way ’’ in combination with oxaziclomefone, as
® ®
‘‘Yaiba ’’ in combination with fentrazamide, and as ‘‘Musou ’’ in combination
®
with mefenacet. Best Partner , the first one-shot herbicide for rice to contain
only one active ingredient, has shown excellent pre-emergence herbicidal activity
against broad-spectrum paddy weeds, without causing injury to the rice plants.
Abbreviations
ALS acetolactate synthase

SU sulfonylurea
IMI imidazolinone
PS pyrimidinylsalicylate
PC pyrimidinylcarboxy(late)
TP triazolopyrimidine sulfonamide
DMSP 4,6-dimethoxy-2-methylsulfonylpyrimidine
References
1. Yoshimura, T., Nakatani, M., 4. Nezu, Y., Miyazaki, M., Sugiyama, K.,
Tamaru, M., Danjo, T., Ono, Y., Wada, N., Kajiwara, I., and
and Yanagisawa, K. (2000) Miyazawa, T. (1996) Pestic. Sci., 47,
PCT Int. Appl. 115–124.
WO 0006553. 5. Nezu, Y., Wada, N., Saitoh, Y.,
2. Nezu, Y., Miyazaki, M., Sugiyama, K., Takahashi, S., and Miyazawa, T. (1996)
and Kajiwara, I. (1996) Pestic. Sci., 47, J. Pestic. Sci., 21, 293–303.
103–113. 6. Shimizu, T., Nakayama, I., Nakao, T.,
3. Shimizu, T., Nakayama, I., Nezu, Y., and Abe, H. (1994) J. Pestic.
Nagayama, K., Miyazawa, T., and Sci., 19, 59–67.
Nezu, Y. (2002) Herbicide Classes in 7. Nezu, Y. (2003) Development of Agro-
Development, Springer, Berlin, chemicals in Japan, Pesticide Science
pp. 1–41. Society of Japan, pp. 279–290.
References 141
8. Takabe, F., Kaku, K., Wada, N., 23. Hanai, R., Kawano, K., Shigematsu, S.,
Takeuchi, A., and Shigematsu, S. (1994) and Tamaru, M. (1993) Proc. Brighton
Abstracts of Papers, 19th Annual Meet- Crop Prot. Conf. Weeds, 1, 47–52.
ing of the Pesticide Science Society of 24. Tamaru, M., Kawamura, N., Sato, M.,
Japan, p. 55. Tachikawa, S., Yoshida, R., and
9. Nezu, Y., Nagata, T., Itoh, S., and Takabe, F., (1991) EP Patent 435170.
Masuda, K. (1990) JP2-85262. 25. Hanai, R., Ohno, S., and Ogawa, Y.
10. Yokota, S., Wada, N., Hanai, R., and (2004) J. Weed Sci. Technol., 49 (Suppl.),
Shimizu, T. (1995) Abstracts of Papers, 206–207.
20th Annual Meeting of the Pesticide 26. Shimizu, T. (1997) J. Pestic. Sci., 22,
Science Society of Japan, p. 78. 245–256.
11. Hirai, K., Uchida, A., and Ohno, R. 27. Durner, J., Gailus, V., and Böer, P.
(2002) Herbicide Classes in Development, (1991) Plant Physiol., 95, 1144–1149.
Springer, Berlin, pp. 202–210. 28. Subramanian, M.V. and Gerwick, B.C.
12. Nezu, Y., Wada, N., Yoshida, F., (1989) ACS Symp. Ser., 389, 277–288.
Miyazawa, T., Shimizu, T., and Fujita, 29. Shaner, D.L., Anderson, P.C., and
T. (1998) Pestic. Sci., 52, 343–353. Stidham, M.A. (1984) Plant Physiol., 76,
13. (a) Kaku, K., Kusano, S., Toyokawa, Y., 545–546.
Miyazawa, T., and Yoshida, R. (1989) 30. Shimizu, T., Nakayama, I., Wada, N.,
JP1-301668 ; (b) Kaku, K., Wada, N., Nakao, T., and Abe, H. (1994) J. Pestic.
Shigematsu, K., and Takeuchi, A. (1989) Sci., 19, 257–266.
JP2-85262. 31. Shimizu, T., Yamashita, K., Kato,
14. Takahashi, S., Shigematsu, S., H., Hashimoto, N., Abe, H., and
Morita, A., Nezu, Y., Claus, J.S., and Nakayama, I. (1995) Abstracts of Papers,
Williams, C.S. (1991) Proc. Brighton Crop 20th Annual Meeting of the Pesticide
Prot. Conf. Weeds, 1, 57–62. Science Society of Japan, p. 136.
15. Saito, Y., Wada, N., Kusano, S., 32. Shimizu, T., Nakayama, I., Nakao,
Miyazawa, T., Takahashi, S., T., Yamashita, K., Nagayama, K., and
Toyokawa, Y., and Kajiwara, Y. (1990) Abe, H. (1993) Abstracts of Papers, 18th
US Patent 4,932,999. Annual Meeting of the Pesticide Science
16. Watanabe, O., Yokoyama, M., Fujita, S., Society of Japan, p. 76.
Miyazaki, T., and Wada, N. (2003) J. 33. Schloss, J.V., Ciskanik, L.M., and Van
Weed Sci. Technol., 48, 24–30. Dyk, D.E. (1988) Nature, 331, 360–362.
17. Yokoyama, M., Watanabe, O., 34. Muhitch, M.J., Shaner, D.L., and
Yanagisawa, K., Ogawa, Y., Wada, N., Stidham, M.A. (1987) Plant Physiol.,
and Shigematsu, S. (1994) J. Weed Sci. 83, 451–456.
Technol., 42 (Suppl.), 32–33. 35. Hawkes, T.R. (1989) Br. Crop. Prot.
18. Yokoyama, M., Watanabe, O., Council Monogr., 42, 131–138.
Kawano, K., Shigematsu, S., and Wada, 36. Hawkes, T.R. and Thomas, S.E. (1990)
N. (1993) Proc. Brighton Crop Prot. Conf. Biosynthesis of Branched Chain Amino
Weeds, 1, 61–66. Acids, Balaban Publishers, Weinheim,
19. Wada, N., Kusano, S., and Toyokawa, Y. pp. 373–389.
(1990) US Patent 4,906,285. 37. Ortega, F., Bastide, J., and Hawkes,
20. Tachikawa, S., Miyazawa, T., and T.R. (1996) Pestic. Biochem. Physiol., 56,
Sadohara, H. (1997) Proceedings of the 231–242.
16th Asian-Pacific Weed Science Society 38. Sunderland, S., Burton, J.D., Coble,
Conference, Vol. 2A, pp. 114–117. H.D., and Maness, E.P. (1995) Weed Sci.,
21. Tamaru, M., Takehi, T., Matsuzawa, N., 43, 21–27.
and Hanai, R. (1996) Pestic. Sci., 47, 39. Matsushita, H., Hukai, Y., Unai, T.,
327–335. Ishikawa, K., and Yusa, Y. (1994) Ab-
22. Tamaru, M., Inoue, J., Hanai, R., and stracts of Papers, 19th Annual Meeting
Tachikawa, S. (1997) J. Agric. Food of the Pesticide Science Society of Japan,
Chem., 45, 2777–2783. p. 127.
40. Nakayama, I., Shimizu, T., Nakao, T., Annual Meeting of the Pesticide Science
and Abe, H. (1993) The Abstracts of Society of Japan, p. 106.
Papers, 18th Annual Meeting of the 42. Yoshimura, T., Nakatani, M.,
Asakura, S., Hanai, R.,
Pesticide Science Society of Japan, p.
and Hiraoka, M. (2011)
77. J. Pestic. Sci., 36, 212–220.
41. Mizutani, H., Shinba, K., Asano, Y., and 43. Hanai, R. (2008) Jpn. J. Agric. Technol.,
Yusa, Y. (1998) Abstracts of Papers, 23rd 52 (10), 53–58 (in Japanese).
2.6
Sulfonylaminocarbonyl-Triazolinones
2.6.1
Introduction
The first examples of the new herbicidal class of sulfonylaminocarbonyl-

triazolinones (SACTs) were reported in 1989 [1]. Following intensive chemical op-
timization, three representatives were developed and commercialized for selective
weed control in cereals and corn. In 2000, flucarbazone-sodium (1) (Figure 2.6.1)
®
was introduced into the Canadian market under the trade name Everest for the
control of wild oats (Avena fatua) and green foxtail (Setaria viridis) in spring wheat
(Triticum aestivum) and durum wheat (Triticum durum).
Propoxycarbazone-sodium (2) (Figure 2.6.2), which was first launched in Kenya
in 2000, is now registered in the major cereal-producing European countries as
® ®
Attribut , and in the United States as Olympus . It is especially effective against
brome (Bromus spp.), loose silky-bent (Apera spica-venti), common couchgrass
(Elymus repens), blackgrass (Alopecurus myosuroides), jointed goatgrass (Aegilops
cylindrica), and several broadleaf weeds from the mustard family.
Thiencarbazone-methyl (TCM) (3) (Figure 2.6.3) obtained its first registration
2008 in Romania, and is now registered in wheat and corn producing countries such
OCF3 O
O
−
SO2 N C N N CH3
+ N
Na
OCH3
® ®
Figure 2.6.1 Compound 1, flucarbazone-sodium (Everest , Vulcano ).
COOCH3 O
O
− CH3
SO2 N C N N
+ N
Na O-C3H7-n
® ®
Figure 2.6.2 Compound 2, propoxycarbazone-sodium (Attribut , Olympus ).
COOCH3 O
O
CH3
SO2 N C N N
S H
N
CH3 O CH3
® ® ®
Figure 2.6.3 Compound 3, thiencarbazone methyl (TCM) (Adengo , Corvus , Capreno ,
® ®
Maister Power, Velocity m3, Celsius ).
as Canada, United States, Argentina, Ukraine, Belarus, and Hungary. Registrations

in other countries are pending. For optimal crop safety in corn, TCM is either
applied in combination with the leaf-active safener isoxadifen-ethyl, or with the
recently developed safener cyprosulfamide. For wheat, TCM is applied with the
safener mefenpyr-diethyl. The tradenames for various mixtures of TCM with
® ® ®
other herbicides are Adengo , Corvus , Capreno Maister Power for corn and
®
Velocity m3 for cereals.
®
In late 2009, a registration in US was received for Celsius , a post-emergence her-
bicide for application to warm-season turf, containing TCM + iodosulfuron-sodium
+ dicamba.
All of these new sulfonylaminocarbonyl-triazolinones are inhibitors of the ace-
tolactate synthase (ALS) enzyme (also known as aceto hydroxy acid synthase;
AHAS), and are classified in group B by the Herbicide Resistance Action Commit-
tee (HRAC). The physico-chemical properties of compounds 1 to 3 are listed in
Table 2.6.1.
2.6.2
Discovery of the Lead Structure
The discovery of the herbicidal class of SACTs is outlined in detail in Ref. [2].
Starting from the concept of seeking new applications for the Nylon 6 intermediate
ε-caprolactam, the bicyclic triazolinone (4) was synthesized during the late 1970s
as a possible intermediate for potential fungicides (Scheme 2.6.1) [3].
Amongst many other derivatives (by NH-acylation, sulfonylation, alkylation,
arylation), a sulfonylaminocarbonyl-triazolinone with the internal code no. BAY
DAM 4493 was synthesized in 1985 (Figure 2.6.4). This compound demonstrated
not only activity against rice blast (Pyricularia oryzae), but also phytotoxic symptoms
at application rates of 500 g a.i. ha−1 .
About two years later, this compound was identified in an in vitro assay as an
unusual ALS inhibitor [4–6], and became the ‘‘starting signal’’ for a major synthesis
program.
2.6.3
Optimization of the Lead Structure
Although all efforts to improve the herbicidal activity of BAY DAM 4493 by variation
of the seven-membered ring proved to be unsuccessful, a ring contraction to the
144
Table 2.6.1 Physico-chemical properties of flucarbazone-sodium, propoxycarbazone-sodium, and thiencarbazone.
Flucarbazone-sodium 1 Propoxycarbazone-sodium 2 Thiencarbazone methyl 3
CAS-No. 181274-17-9 181274-15-7 317815-83-1

Code numbers BAY MKH 6562 BAY MKH 6561 BAY GSE 18636
Melting point (◦ C) 200 (with decomposition) 230–240 (under decomposition) 205
Vapor pressure Cannot be determined directly due to its Cannot be determined directly due to its Cannot be determined directly due to
extremely low value. From experimental extremely low value. From the its extremely low value. Above 133 ◦ C a
results obtained for 70 ◦ C a value of experimental results obtained for 70 ◦ C a vapor pressure could be measured.
<4 × 10 –8 Pa can be estimated as an upper value of <9 × 10 –8 Pa can be estimated as Calculated values:
limit. This limit would correspond to an upper limit. This limit would 8.8 × 10 –14 Pa (20 ◦ C)
<1 × 10−9 Pa at 20 ◦ C. correspond to a value of <1 × 10−8 Pa at 3.7 × 10 –13 Pa (25 ◦ C)
20 ◦ C. 2.3 × 10 –10 Pa (50 ◦ C)
Dissociation constant (at The free acid produced by protonation The free acid produced by protonation pKa = 3.0
20 ◦ C) under acidic conditions has a pKa of 1.9. under acidic conditions has a pKa of 2.1.
Solubility in water (at 20 ◦ C) Water solubility is 44 g l –1 in unbuffered Unbuffered water and buffered between 72 mg l –1 (unbuffered)
aqueous solutions in the range pH 4–9. pH 7 and 9: 42 g l –1 . Solubility is not 172 mg l –1 (pH 4)
Solubility is not influenced by pH in the influenced by pH in the range pH 7–9. 436 mg l –1 (pH 7)
range pH 4–9. Solubility at pH 4.5: 2.9 g l –1 417 mg l –1 (pH 9)
Solubilities in organic Dimethylsulfoxide: 250 Dimethylsulfoxide: 190 Dimethylsulfoxide: 29.15
solvents (g l –1 at 20 ◦ C) Poly(ethylene glycol): 48 Poly(ethylene glycol): 5.2 Ethyl acetate: 2.19
Acetonitrile: 6.4 Acetonitrile: 0.9 Acetone: 9.54
2-Propanol: 0.27 2-Propanol: <0.1 Ethanol: 0.23
Xylene: <0.1 Xylene: <0.1 Toluene: 0.91
Partition coefficient log POW –2.85 (unbuffered) –2.60 (unbuffered) Not determined
(octanol–water) (20 ◦ C) –0.89 (pH 4.0) –0.30 (pH 4.0) –0.13 (pH 4.0), (24 ◦ C)
–1.84 (pH 7.0) –1.55 (pH 7.0) –1.98 (pH 7.0), (24 ◦ C)
–1.88 (pH 9.0) –1.59 (pH 9.0) –2.14 (pH 9.0), (23 ◦ C)
O
H O
N HN N O
O N (O) SO2 N C N N
n
R′ N
e-caprolactam 4 R
Scheme 2.6.1 Concept of preparing the first sulfonylaminocarbonyl-triazolinones, starting

with Nylon 6 intermediate ε-caprolactam.
COOCH3 O
O
SO2 NH C N N
N
BAY DAM 4493
Figure 2.6.4 Structure of lead structure BAY DAM 4493 (internal code no.).
COOCH3 O
O
SO2 N C N N
H (CH2)n
N
Figure 2.6.5 Structure of bicyclic sulfonylaminocarbonyl-triazolinones.
five- and six-membered analogs (n = 3, 4) and ring enlargement (n = 5, 6, 7) up to the

13-membered ring (n = 11) led to a significant reduction in the herbicidal potency
(Figure 2.6.5) (W. Daum, Central Research, Bayer AG, Uerdingen, Germany,
unpublished results) [1, 7]. Similar results were obtained upon the introduction
of oxygen into the ring (Figure 2.6.6) (K. König and K.-H. Müller, Bayer AG,
Leverkusen, Germany, unpublished results).
In contrast to this observation, the introduction of monocyclic triazolinones
resulted in a dramatic enhancement of the herbicidal activity. Subsequently, in a
systematic manner, hundreds of previously known and new intermediates were
prepared and transformed into SACTs. To this end, many new synthetic procedures
have been developed such that, to date, derivatives of the type shown in Table 2.6.2
have been synthesized and their characteristics reported. The major breakthrough
here proved to be the introduction of oxygen-bound residues, either on nitrogen
and/or on carbon (Figure 2.6.7).
Great interest was aroused in the SACTs on the basis of their biological profile.
Whereas, during the early 1990s the use of most known ALS inhibitors such as
sulfonylureas was focused on dicotyledons, the new class of SACTs was shown
to exhibit a generally high activity against grassy weeds, often with rather good
dicotyledon activity, as well as selectivity in cereals.
Following initial outdoor trials in 1991 with the N-ethoxy derivative BAY MKH
4340 (internal code) (Figure 2.6.8), during the following two years a further seven
compounds were tested in parallel in the form of their sodium salts in Europe, the
United States, and Canada (Figure 2.6.9). The special biological spectrum of these
R O R O
O O
SO2 N C N N SO2 N C N N
H H
N N
O O
R = COOCH3, Br, CF3, OC2H5
Figure 2.6.6 Structure of bicyclic sulfonylaminocarbonyl-triazolinones with oxygen in the

alkylen group.
Table 2.6.2 Synthesized derivatives of monocyclic

triazolinones and the relevant references.
A O O
H N 1 2
N (Cyclo)Alkyl H N N NR R
N N
A A
Reference(s) Reference(s)
A = (cyclo)alkyl [1, 8] [1, 8]

S-R3 [9, 10] [9]
Hal [11] [11]
NR4 R5 [8] [8]
O O O
1 1
H N N O R H N N (Cyclo)Alkyl H N N O R
N N N
(Cyclo)Alkyl O R2 O R2
Ref. [8] Ref. [8, 12, 71] Ref. [14]
Figure 2.6.7 Structure of monocyclic triazolinones substituted by oxygen residues [71].
materials – especially against difficult-to-control monocotyledonous weeds – and

their selectivity in cereals led to the development of two compounds which later
became known as flucarbazone-sodium (1) and propoxycarbazone-sodium (2).
2.6.4
Discovery of Thiencarbazone-Methyl (TCM)
During the optimization studies that led to the development of 1 and 2, several
derivatives with heterocyclic sulfonyl moieties, such as thiophene, were prepared
(Figure 2.6.10) [8, 9, 11–14]. However, in contrast to the phenyl analogs, the
herbicidal performance of these compounds proved to be rather disappointing.
COOCH3 O
O
O-C2H5
SO2 N C N N
H N
C2H5
BAY MKH 4340
Figure 2.6.8 Structure of BAY MKH 4340 (internal code no.), the first sulfonylamino-
carbonyl-triazolinone tested in field trials.
COOCH3 (O)nCF3
O O
O O
− CH3
N CH3
−
SO2 N C N SO2 N C N N
+ N + N
Na Na
S-C2H5, O-C3H7-n, O-CH3, O-C2H5
O-C3H7-i n = 0, 1
Figure 2.6.9 Structure of seven field products including (1) and (2) tested side-by-side in
cereals in Europe and North America 1992/1993.
O
O
1
SO2 N C N N R
S H
N
COOCH3 R2
Figure 2.6.10 General structure of sulfonylaminocarbonyl-triazolinones with a monosubsti-

tuted 3-sulfamoylthiophene moiety showing weak herbicidal activity.
Surprisingly, however, the introduction of a second substituent (e.g., alkyl), either

in position 4 or in both positions, caused the activity to be raised to an interesting
level (Figure 2.6.11) [15, 16]. This, in turn, led to an evaluation of the biological
behavior of several compounds of this type in field trials (Figure 2.6.12). A further
improvement in the biological profile with regards to the weed spectrum and plant
compatibility was achieved by synthesizing isomeric compounds with the alkyl
and ester groups in reversed positions (Figure 2.6.12) [17, 18], such that the top
compound, TCM (3), was selected for further development.
Despite the compounds possessing a rather exciting weed spectrum in cereals and
corn, their selectivity was rather critical in the initial trial series, and consequently
much time was spent seeking a commercially available safener. In June 2002, the
compound development was favored during the early phase of field testing by the
founding of Bayer CropScience, as this allowed the herbicide from the former Plant
Protection Division of Bayer AG and the safener portfolio from the former Aventis
CropScience AG, to be brought together.
It was a further fortunate circumstance that a new multicrop safener (cyprosul-
famide; 5) from a new chemical class was at that time investigated in early field
testing such that, in combination with the existing safener isoxadifen-ethyl (6) and
alkyl1 O alkyl1 O
O O
1 1
SO2 N C N N R SO2 N C N N R
S H S H
N N
COO-alkyl2 R2 alkyl2 R2
Figure 2.6.11 General structure of sulfonylaminocarbonyl-triazolinones with

2,4-disubstituted 3-sulfamoylthiophene moieties having improved herbicidal potency.
alkyl1 O COO-alkyl2 O
O O
CH3 1
SO2 N C N N improvement SO2 N C N N R
S H S H
N N
COOCH3 O CH3, OC2H5 alkyl1 O CH3, OC2H5
alkyl = CH3, C2H5, n -C3H7

1
alkyl = CH3, C2H5 R = CH3, cyclopropyl
1 1
alkyl2 = CH3, C2H5
Figure 2.6.12 Optimization of the best substitution pattern leading to TCM (3) on the
basis of outdoor trials.
O OCH3 COOC2H5
O O COOC2H5
S H5C2OOC
N N
H H
N N H3C N
O
Cl
O
Cl
5 6 7
Figure 2.6.13 Chemical structure of safener cyprosulfamide (5), isoxadifen-ethyl (6), and
mefenpyr-diethyl (7).
mefenpyr-diethyl (7) (Figure 2.6.13), the full herbicidal potential of TCM (3) could
be identified and made accessible for commercial selective weed control.
2.6.5
Synthesis
Flucarbazone, propoxycarbazone, and TCM (3) can be prepared by using a variety

of methods:
1) Sulfonyl isocyanate addition (Scheme 2.6.2) [1, 17, 19].

2) The phenylurethane route (Scheme 2.6.3) [8, 17].
3) The cyanate route (Scheme 2.6.4) [17, 20].
For reasons of stability, flucarbazone and propoxycarbazone are formulated as

their sodium salts, with several synthetic procedures having described the salt
formation [21, 22].
X O X O
O
CH3
SO2 NCO HN N SO2 NH C N N CH3
Y Y
N N
Z O R Z O R
Flucarbazone: X = OCF3, Y = CH=CH, Z = H, R = CH3
Propoxycarbazone: X = COOCH3, Y = CH=CH, Z = H, R = C3H7-n
Thiencarbazone-methyl: X = COOCH3, Y = S, Z = CH3, R = CH3
Scheme 2.6.2 Preparation of flucarbazone, propoxycarbazone and TCM (3) by sulfonyl

isocyanate addition.
O
O
O C Cl HN N CH3
N
O R
base −HCl
X O X O
O O
N CH3
1) base CH3
SO2 - NH2 O C N SO2 NH C N N
Y 2) HCl Y
N N
Z O R Z O R
Scheme 2.6.3 Preparation of flucarbazone, propoxycarbazone and TCM (3) by the

phenylurethane route.
2.6.5.1 Sulfonyl Components

The sulfonyl component of propoxycarbazone-sodium (2) is an integral part of sev-
eral commercially available sulfonylureas.1) Technical procedures exist for sulfonyl
isocyanate preparation starting from saccharin (Scheme 2.6.5) [23, 24]. The sulfonyl
moiety (8) of flucarbazone-sodium (1) can be prepared by a classical Meerwein
reaction from the aniline (9) prepared according to Refs [25, 26] (Scheme 2.6.6).
Another method is based on sulfochlorination and the different solubilities of the
isomeric sulfonamides (10a) and (10b) (Scheme 2.6.7) [27].
The new sulfonyl intermediate (11) for the TCM synthesis is accessible in a
similar Meerwein reaction as for (8) from the known amino thiophene (12) [17, 18].
The latter compound can be obtained in high yields according to published protocols
1) Sulfometuron-methyl, Metsulfuron-me- HNPC-C9908 (Hunan Branch of

thyl, Tribenuron-methyl, Ethametsulfuron- the National Pesticide R&D South
methyl (all from E. I. Du Pont de Nemour Center, Changsha, P.R. of China) and
and Company), Primisulfuron-methyl Monosulfuron-ester NK94827 (Nankai
(Syngenta Corporation), and the exper- University, Nankai, P.R. of China).
imental herbicides Methiopyrsulfuron
X O X O
O
SO2-Cl NaOCN HN N CH3 SO2 NH C N N
CH3
Y Y
N N
Z O R Z O R
Scheme 2.6.4 Preparation of flucarbazone, propoxycarbazone and TCM (3) by the cyanate
route.
O
COOCH3 COOCH3
NH
CH3OH Cat. n -C4H9-NCO
SO2 SO2NH2 SO2-NCO
H+ Phosgene
Saccharin
Scheme 2.6.5 Conversion of saccharin to 2-(methoxycarbonyl)phenylsulfonyl isocyanate.
O-CF3 1. Nitration
O-CF3 O-CF3
Fractional
Ref. [25] distillation
NH2
2. Reduction
H2 N OCF3
Approx. 10 : 1 Meerwein
reaction
NH2
O-CF3 H2N OCF3 OCF3 9
1. Nitration O-CF3
Ref. [26]
NH2
2. Reduction Dechlorination SO2Cl
Cl Cl Cl Cl Cl Cl
8
Scheme 2.6.6 Preparation of flucarbazone-sodium intermediate (9) by two different nitra-

tion/reduction procedures and conversion to sulfochloride (8).
[17, 18, 28, 29], whereby oxime formation and Wolff–Semmler-type aromatization
can be combined in a one-pot reaction [30–32]. A recently reported method allows
the synthesis of (12 × HCl) in a two-step process [33], and for the sulfonyl isocyanate
formation a base-free procedure was developed (Scheme 2.6.8) [34].
2.6.5.2 Triazolinone Synthesis

The flucarbazone-sodium and TCM intermediate (13) has long been known [35],
but for reasons of safety it became necessary to develop alternative procedures of
production (Scheme 2.6.9):
O-CF3 O-CF3 O-CF3 O-CF3

Bromination 1. Sulfochlorination
SO2NH2
2. Ammonia
Br Br Br SO2NH2
O-CF3 O-CF3
H2 / Pd/ C
SO2NH2
SO2NH2
10a 10b
Approx. 10 - 15 %
Scheme 2.6.7 Preparation of flucarbazone-sodium intermediate (10a) by sulfochlorination.
1) Iminoester route (‘‘tin pathway’’) (Scheme 2.6.10) [36–39]. Here, the iminoester
(15) reacts with carbazate (14) to give (16), which cyclizes under basic conditions
to the triazolinones (17).
2) Iminothioester route (Scheme 2.6.11) [40, 41]. The iminothioester (18) is much
more easily prepared than the iminoester (15).
3) Methylation of NH-triazolinones (20) (Scheme 2.6.12) [42, 43]. The methylation
of (20), prepared via imidoester (19) [44], takes place exclusively at the amidic
nitrogen.
Various patents have been registered which describe alternative methods for the
formation of NH-triazolinones (20) (Scheme 2.6.13) [43, 45–47].
2.6.6
Biology
Flucarbazone-sodium (1) was discovered and developed by the former Plant Pro-
tection Division of Bayer AG (now Bayer CropScience AG) [48–50]. Since 2002, it
has been commercialized by Arysta LifeScience in Canada, the USA, Mexico, and
®
The Peoples Republic of China under the trade name Everest [51], and in Chile
®
as Vulcano .
Flucarbazone-sodium is a selective herbicide for the control of wild oats (Avena
fatua) [52], green foxtails (Setaria viridis), Italian ryegrass (Lolium multiflorum),
windgrass (Apera spica-venti and A. interrupta), and Bromus species such as cheat-
grass (Bromus secalinus) and Japanese brome (Bromus japonicus) in spring, durum,
and winter wheat [48–51].
In addition to these grasses, numerous broadleaf weeds are controlled, such as
redroot pigweed (Amaranthus retroflexus), wild mustard (Brassica kaber), stinkweed
(Thlaspi arvense), shepherd’s purse (Capsella bursa-pastoris), green smartweed (Poly-
gonum scabrum Moench.), and volunteer canola (Brassica napus). For a broader spec-
®
trum control of broadleaf weeds, Everest may be mixed with broadleaf herbicides
such as 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4-chlorophenoxyacetic
acid (MCPA) amine or ester, bromoxynil, dicamba, fluroxpyr, or sulfonylureas
152
Refs [30-32]
COOCH3 COOCH3 COOCH3 COOCH3 COOCH3

Base Base H2NOH
COOCH3 COOCH3 O NOH NH2
SH Cl S S HCl S S
(× HCl) × HCl
CH3 CH3 CH3 CH3 CH3
12 × HCl
C
COOCH3
NaOCH3, HCOONH4 SO2Cl2
NH2
Ref. [33] S Ref. [33]
CH3
COOCH3 COOCH3 COOCH3 COOCH3 COOCH3

Base Ref. [34]
NH2 NH2 SO2Cl SO2NH2 SO2-NCO
S S S S S
× HCl
CH3 CH3 CH3 CH3 CH3
12 × HCl 12 11
Scheme 2.6.8 Preparation of several intermediates of the TCM sulfonyl moiety.

O O
H N CH2N2
N CH3 H N N CH3
N − N2 N
H O O-CH3
13
Scheme 2.6.9 Published procedure for triazolinone building block (13) of flucarbazone-
sodium (1) and TCM (3) by the addition of diazomethane.
O
COOalkyl
H N COOalkyl CH3 H N
N
H N
NH CH3 N CH3
N N R = CH3, C3H7-n
NH2
R O O R O R O R
14 15 16 17
Scheme 2.6.10 Preparation of alkoxy triazolinones (17) using iminoester intermediates (15).
H CH3 CH3
N N
H3C NCS R OH
S O R H 3C S O R
18
O
CH3 COOalkyl
H N COOalkyl N H N H N CH3
NH CH3 N
N N R = CH3, C3H7-n
NH2 H 3C S O R
O R O R
14 18 16 17
Scheme 2.6.11 Preparation of alkoxy triazolinones (17) using iminothioester intermediates

(18).
like metsulfuron-methyl, triasulfuron, tribenuron-methyl, chlorsulfuron,

thifensulfuron-methyl, or prosulfuron [51]. Currently, application rates of
15–20 g a.i. ha−1 are registered in Canada, and 21–42 g a.i. ha−1 in the USA.
Propoxycarbazone-sodium (2) is a new selective herbicide for grass control in
®
wheat, rye, and triticale [53]. In Europe, it is registered as Attribut , and applied
−1
at rates of 28–70 g a.i. ha . It acts predominantly against important grasses such
as Bromus species [54, 55], blackgrass (Alopecurus myosuroides) [56, 57], and loose
silky-bent (Apera spica-venti) [57]. It is applied post-emergence in spring at a core
use rate of 42 g a.i. ha−1 . Compared with single applications, a split application
or sequences following a standard autumn treatment are more favorable, giving
good to very good grass control. The following Bromus species can be controlled
effectively [54, 55]: field brome (B. arvensis), meadow brome (B. commutatus),
Japanese brome (B. japonicus), soft brome (B. mollis), rye brome (B. secalinus), barren
brome (B. sterilis), and drooping brome (B. tectorum). Propoxycarbazone-sodium is
taken up via the leaves and, particularly, via the roots. Especially on light soils, and
under moist conditions, it controls couchgrass (Elymus repens) at a commercially
Br-CN R-OH / R-ONa

− NaBr
O O
COOalkyl
H N COOalkyl H H N H N
N NH2 H N N H N CH3
NH2 N N N
R O O R O R O R O R
14 19 20 R = CH3, C3H7-n 17
Scheme 2.6.12 Preparation of alkoxy triazolinones (17) by methylation of desmethyl

triazolinones (20).
O O
O H N
R-OH R O NH N2H4x H2O N H
Cl COOR NaSCN R-O C NCS
− H2S, − H2O N
S O R − R-OH O R
20
R = CH3, C3H7-n
Scheme 2.6.13 Preparation of desmethyl triazolinones (20).
acceptable level, but on heavy soils higher rates of application are recommended.
Owing to its systemic mobility, it also kills the rhizomes of E. repens [58].
In the USA, propoxycarbazone-sodium is registered for use in spring, winter,
and durum wheat, with application rates of 30–45 g a.i. ha−1 , and is marketed
®
under the trade name Olympus . Bromus control is the primary target and, as in
Europe, all important species, including cheatgrass (B. secalinus) and downy brome
(B. tectorum), are well controlled [59]. Besides brome, the following grasses can be
economically reduced or suppressed [59]: loose silky-bent (Apera spica-venti), wild
oats (Avena fatua), littleseed canarygrass (Phalaris minor) [60], and paradoxagrass
(P. paradoxa). The suppression of jointed goatgrass (Aegilops cylindrica) can be
achieved by two sequential treatments of 30 g a.i. ha−1 in autumn and spring [61].
Besides grasses, broadleaf species belonging to the mustard complex such as
Sisymbrium sp., Brassica sp., Descurainia sp., Chorispora sp., Camelina sp., Capsella
sp., and Thlaspi sp. [59] represent further target weeds for this herbicide.
Propoxycarbazone-sodium (2) can be applied either straight or in tank mixtures
with other herbicides such as triasulfuron, metsulfuron-methyl, chlorsulfuron,
thifensulfuron-methyl, prosulfuron, carfentrazone, dicamba, bromoxynil, clopy-
ralid, MCPA amine or ester, 2,4-D amine or ester, metribuzin, or fluroxypyr.
The ‘‘second-generation SACT’’ herbicide (3) offers more diverse utility
for weed control than the first-generation post-emergence wheat herbicides
flucarbazone-sodium (1) and propoxycarbazone-sodium (2). TCM (3) can be used
both pre- and post-emergence [62] and, in combination with appropriate safener
technology, it can also be applied selectively to wheat and corn [63]. Although its
uses in other crops are currently still under investigation, this may result in the
development of additional TCM-based herbicides.
TCM (3) is a cross-spectrum herbicide with flexible use characteristics and a

performance which meets the requirements for reliable weed control in any geo-
graphical area of the world where any of the target crops are produced. Control
includes a wide spectrum of grass weeds and broadleaf weeds. During product
development, the control of 80–100% of 67 weed species after pre-emergence ap-
plication, and 92 weed species after post-emergence application, were documented
simply by using TCM with a safener. The weed-control spectrum of the commercial
products in which TCM is combined with other herbicides is even wider, and in-
cludes important grasses such as wild oat (Avena fatua), barnyardgrass (Echinochloa
sp.), foxtails (Seteria sp.), millets (Panicum sp.), as well as ragweeds (Ambrosia
sp.), pigweeds (Amaranthus sp.), morning glories (Ipomoea sp.), black nightshade
(Solanum nigrum), lambsquarters (Chenopodium album), and difficult-to-control
species such as wild buckwheat (Polygonum convolvulus).
TCM is fully systemic in treated weeds, with acropetal and basipetal translocation.
Application is possible either to the soil or on the foliage of target weeds, with
uptake occurring via the root system and through the leaves that have intercepted
the herbicide spray. Consequently, sprays are both possible and useful for weed
control at different timings, and can be applied prior to crop planting, after seeding,
and also before emergence (pre-emergence) and after (post-emergence) the crop
and weeds are established. In addition to immediate activity, TCM also offers
residual activity which, depending on the use rate, soil type, and environmental
conditions, can persist for several weeks.
For corn protection, the novel safener cyprosulfamide (5) was codeveloped with
TCM (3) [64–66], where the primary target crop is maize, field corn grown for
grain, or silage. The application window of the herbicide for corn closes when the
crop has fully expanded its sixth leaf (crop development stage 16 or V6).
The maximum seasonal use rate of TCM is 45 g a.i. ha−1 , while the maximum
dose for pre-emergence to early post-emergence applications is 45 g a.i. ha−1 . If
applied as a single treatment, the maximum seasonal use rate must be applied
pre-emergence or early post-emergence, before the second leaf of corn is fully
expanded [62]. Between the three- and six-leaf stages of corn, the maximum use
rate is limited to 15 g a.i. ha−1 . In post-emergence applications, a tank mixture of
TCM (3) with a methylated seed oil adjuvant is recommended to achieve the most
consistent weed control. Furthermore, split applications starting with a 30 g a.i. ha−1
rate pre-emergence, followed by a 15 g a.i. ha−1 post-emergence, have proved to be
useful [62].
® ®
Commercial corn herbicides currently in use include Adengo (Europe)/Corvus
(US) for preplant burndown up to 30 days prior to planting, pre-emergence, and
early post-emergence (up to V2) weed control. In this product, TCM (3) is combined
with the 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) -inhibiting herbicide isox-
aflutole (21) (Figure 2.6.14), and with the novel safener cyprosulfamide (5) [67, 68].
®
For post-emergence use, the product Capreno , which combines the herbicides
TCM (3), tembotrione (22) (Figure 2.6.14), and the safener isoxadifen-ethyl (6), was
introduced into corn weed control in 2010 in the USA [68–70]. Important weed
®
control features of Capreno include supplementation of the efficacy of glyphosate
O SO2CH3 O O Cl H3C O SO2CH3
N O CF3 N
O CF3 OH N
SO2CH3 OH CF3
H3C
21 22 23
Figure 2.6.14 Chemical structure of TCM mixing partners isoxaflutole (21), tembotrione
(22), and pyrasulfotole (23) (4-HPPD-inhibiting herbicides).
and glufosinate in herbicide-tolerant types of corn, resistance management, and

the addition of a residual control component to the foliar active nonselective
®
mixpartners. In conventional corn, Capreno can be used effectively in weed control
spray programs as an herbicide for early to mid post-emergence applications (V1
through V5, 1–5 leaf collars) after a pre-emergence treatment.
To summarize, in corn TCM can be used for the management of weeds before
planting in reduced or conventional tillage systems for clean-up of the field, and
from the time of planting of the crop up to the stage characterized by the complete
® ®
development of the sixth leaf on the corn plant. TCM in Adengo /Corvus and
®
Capreno covers the timing from field preparation through seeding up to the
development of the fully expanded second leaf of corn. At weed control timings
starting from the full expansion of the second leaf up to the full expansion of
®
the sixth leaf on the crop, TCM can be applied as Capreno in the US or as a
novel TCM-containing product to be launched in 2011/2012 in conventional corn.
At any use timing, TCM offers the control of weeds that already exist at the time
1 Pre plant Pre-emergence Spike Post-emergence

2 00 09 10 11 12 16
4 45 g a.i. ha 15 g a.i. ha
5 45 g a.i. ha
1: Application timing of herbicide

2: Crop development stage of maize largely equal to number of leaves
3. Illustration of crop at time of application
4. Use rate of active ingredient per application timing
5. Use rate of active ingredient per application timing
Figure 2.6.15 Application timing and application rate of thiencarbazone-methyl (3).

of application and, depending on factors such as the use rate, soil moisture, and
temperature, a residual control for up to several weeks. Despite its residual control
TCM has shown minimum risk for rotation crops following on after corn. The
use rates and application timings for TCM in corn are shown schematically in
Figure 2.6.15.
®
In Canadian wheat, a TCM-containing herbicide Velocity m3 [a mixture with
pyrasulfotole (23; Figure 2.6.14) plus bromoxynil, safened by (7)] at a TCM dose of
5 g a.i. ha−1 , can be used to control wild oats (Avena fatua), green- and yellow foxtail
(Setaria spp.) and barnyardgrass (Echinochloa C. galli), as well as a significant range
of broadleaf weeds [63, 64].
®
For landscape weed management in warm-season turf, the herbicide Celsius ,
which contains TCM, iodosulfuron-sodium and dicamba, was developed. This al-
lows the control of 150 weed species, with TCM providing not only a residual
control but also a control of weeds already present at the time of applica-
tion. The use and weed control targets for SACTs in crops are summarized in
Figure 2.6.16.
Sulfonylaminocarbonyl-triazolinones
SACTs
Flucarbazone-sodium 1 Propoxycarbazone - sodium 2
Wheat Cereals
post post
AVEFA, SETSS, LOLSS, BL BROSS, ALOMY, AGRRE, BL
Thiencarbazone-methyl 3
?
Cereals Corn Turf Possible other crops

post pre/post post
GR + BL GR + BL GR + BL + sedges GR + BL + sedges
Post: Post-emergence application = foliar spray; Pre: Pre - emergence application = soil applied spray
AGRRE: Elymus repens, quackgrass; ALOMY: Alopecurus myosuroides, blackgrass;
AVEFA: Avena fatua, wild oat; BROSS: Bromus species, brome species; LOLSS: Lolium
species, ryegrass BL: Dicotyledonous weeds, broadleaves; GR: Monocotyledonous weeds, grasses;
Sedges: Cyperacea, sedges
Figure 2.6.16 Herbicidal characteristics of flucarbazone-sodium (1), propoxycarbazone-

methyl (2), and thiencarbazone-methyl (3).
2.6.7
Conclusions
The SACTs represent a new class of ALS inhibitor that was discovered in 1987 at the
former Plant Protection Division of Bayer AG. During the 1990s, two compounds
were developed for selective grass control in cereals: (i) flucarbazone-sodium,
which acts predominantly against green foxtail and wild oats, and is regis-
® ®
tered in the USA and Canada as Everest and in Chile as Vulcano ; and
(ii) propoxycarbazone-sodium, which is a brome specialist for use both in Eu-
® ®
rope (Attribut ) and the USA (Olympus ). Besides other grasses, such as loose
silky-bent and blackgrass, the rhizomes and the aerial parts of couchgrass can also
be controlled with these compounds which, in addition, are highly active against
several broadleaf weeds from the mustard family.
TCM (3) is a new SACT which was first registered in 2008. In combination
with suitable safeners such as cyprosulfamide (5) from a new chemical class, or
isoxadifen-ethyl (6) and mefenpyr-diethyl (7), the selective application of TCM is
possible in wheat, and especially in corn. The intention is to market TCM in
various mixtures with other herbicides, thereby offering flexible use characteristics
from pre-plant burndown to pre-emergence and post-emergence applications.
TCM is fully systemic in treated weeds, and offers a residual activity depending
on its use rate and the environmental conditions encountered. The combination
of TCM with various 4-HPPD-inhibiting herbicides offers broad grass and dicot
control, and also minimizes the risk of resistance development by combining
different modes of action. Even without other herbicides, an 80–100% control
of 67 weed species (pre-emergence) and 92 weed species (post-emergence) was
observed with TCM. During late 2009, a registration in the USA was received for
®
Celcius , a post-emergence herbicide for warm-season turf, which contains TCM
+ iodosulfuron-sodium + dicamba. Uses of TCM in other crops are currently
under investigation and development.
References
1. Daum, W., Müller, K.-H., Schwamborn, 5. Babczinski, P. (1988) Annual Report

M., Babczinski, P., Santel, H.-J., 1988, Internal Report, Bayer AG, Lev-
Schmidt, R.R., and Strang, H. (1989) erkusen, Germany.
Sulfonylaminocarbonyltriazolinone. 6. Babczinski, P. (2002) Pflanz.-Nachr., 55
EP Patent 341489, (Prio: 09. 05. 1988), (1), 5–14.
Bayer AG, Leverkusen, Germany. 7. Daum, W. (1989) 4,5-Alkanylen-1,2,
2. Müller, K.-H. (2002) Pflanz.-Nachr., 55
4-triazol-5-in-3-on – potentielle Fungicide
(1), 15–28.
und Herbicide, Internal Report, Bayer
3. Daum, W. (1984) Derivatisierung von
2,5,6,7,8,9-Hexahydro-3H-triazolo[4, AG, Uerdingen, Germany.
3a]azepin-3-on, Internal Report, Bayer 8. Müller, K.-H., Babczinski, P., Santel,
AG, Uerdingen, Germany. H.-J., and Schmidt, R.R. (1991) Sulfony-
4. Babczinski, P. (1987) Unusual ALS laminocarbonyltriazolinone, EP Patent
Inhibitors, Internal Report, Bayer AG, 422469, (Prio: 12. 10. 1989), Bayer AG,
Leverkusen, Germany. Leverkusen, Germany.
References 159
9. Müller, K.-H., Babczinski, P., Santel, Dollinger, M. (1998) Thienylsulpho-

H.-J., and Schmidt, R.R. (1991) nylamino(thio)carbonyl compounds. WO
Sulfonylaminocarbonyl-triazolinones Patent 1998/24787, (Prio: 04. 12. 1996),
having substituents attached through Bayer AG, Leverkusen, Germany.
sulfur. EP Patent 431291, (Prio: 03. 11. 17. Gesing, E.R.F., Kluth, J., and
1989), Bayer AG, Leverkusen, Germany. Müller, K.-H. (2001) Substituted
10. Müller, K.-H., Kirsten, R., Gesing, Thiene-3-yl-sulfonylamino(thio)carbonyl-
E.R.F., Kluth, J., Findeisen, K., Jansen, triazolin(thi)ones. WO Patent
J.R., König, K., Riebel, H.-J., Bielefeldt, 2001/05788, (Prio: 15. 07. 1999), Bayer
D., Dollinger, M., Santel, H.-J., and AG, Leverkusen, Germany.
Stenzel, K. (1996) Herbicidal or fungici- 18. Gesing, E.-R., Drewes, M.W.,
dal sulfonylaminocarbonyltriazolinones Dahmen, P., Feucht, D., and Pontzen,
with halogenated alk(en)ylthio sub- R. (2003) Substituted thien-3-yl-
stituents. WO Patent 1996/27591, (Prio: sulfonylamino(thio)carbonyl-
08. 03. 1995), Bayer AG, Leverkusen, triazolin(thi)ones. WO Patent
Germany. 2003/037086, (Prio: 02. 11. 2001),
11. Müller, K.-H., Babczinski, P., Santel, Bayer AG, Leverkusen, Germany.
H.-J., and Schmidt, R.R. (1991) 19. Prasad, V.A. and Jelich, K. (2000)
Halogenierte Sulfonylaminocarbonyl- Process for the manufacture of sul-
triazolinone. EP Patent 425948, (Prio: fonylaminocarbonyltriazolinones in the
03. 11. 1989), Bayer AG, Leverkusen, presence of xylene as solvent. US Patent
Germany.
6,160,125, (Prio: 27. 12. 1999), Bayer
12. Müller, K.-H., König, K., Kluth, J.,
Corporation, Pittsburgh, PA.
Lürssen, K., Santel, H.-J., and Schmidt,
20. Kluth, J. and Müller, K.-H. (1995)
R.R. (1992) Sulfonylaminocarbonyl-
Process for the preparation of sulfony-
triazolinones with oxygen-bound
laminocarbonyltriazolinones. EP Patent
substituents. EP Patent 507171, (Prio:
659746, (Prio: 21. 12. 1993), Bayer AG,
04. 04. 1991), Bayer AG, Leverkusen,
Leverkusen, Germany.
Germany.
21. Prasad, V.A., Kulkarny, S.V.,
13. Haas, W., Müller, K.-H., König, K.,
Rivadeneira, E., and Desai, V.C. (2000)
Santel, H.-J., Lürssen, K., and Schmidt,
Process for the manufacture of sulfony-
R.R. (1993) Sulfonylaminocarbonyltri-
laminocarbonyltriazolinones and salts
azolinones with two through oxygen
bonded substituents. EP Patent 534266, thereof. US Patent 6,147,221, (Prio: 27.
(Prio: 25. 09. 1991), Bayer AG, Lev- 12. 1999), Bayer Corporation, Pittsburgh,
erkusen, Germany. PA.
14. Müller, K.-H., König, K., Santel, H.-J., 22. Prasad, V.A. and Jelich, K. (2000)
Dollinger, M., and Stenzel, K. (1996) Process for the manufacture of sul-
Sulfonylaminocarbonyltriazolinones fonylaminocarbonyltriazolinones and
with substituents bound by oxygen and salts thereof under pH controlled con-
sulphur. WO Patent 1996/11188, (Prio: ditions. US Patent 6,147,222, (Prio: 27.
05. 10. 1994), Bayer AG, Leverkusen, 12. 1999), Bayer Corporation, Pittsburgh,
Germany. PA.
15. Müller, K.-H., Drewes, M.W., Findeisen, 23. Levitt, G. (1981)
K., Gesing, E.R.F., Jansen, J.R., Kirsten, 2-Isocyanatosulfonylbenzoic acid es-
R., Kluth, J., Philipp, U., Riebel, ters and preparation thereof. EP Patent
H.-J., Dollinger, M., and Santel, H.-J. 34431, (Prio: 06. 02. 1980), E. I. Du Pont
(1997) Substituted sulphonylamino de Nemour and Company, Wilmington,
(thio)carbonyl compounds as herbicides. Del.
WO Patent 1997/16449, (Prio: 02. 11. 24. Levitt, G. (1982) Substituted benzene-
1995), Bayer AG, Leverkusen, Germany. sulfonyl isocyanates and preparation
16. Müller, K.-H., Gesing, E.R.F., Drewes, thereof. EP Patent 46626, (Prio: 30. 05.
M., Jansen, J.R., Kirsten, R., Kluth, 1978), E. I. Du Pont de Nemour and
J., König, K., Philipp, U., and Company, Wilmington, Del.
25. Olah, G.A., Yamato, T., Hashimoto, 35. Arndt, F., Loewe, L., and Tarlan-Akön,
T., Shih, J.G., Trivedi, N., Singh, B.P., A. (1948) Rev. Faculté Sci. Univ. Istanbul,
Piteau, M., and Olah, J.A. (1987) J. Am. 13A, 127–146.
Chem. Soc., 109, 3708–3713. 36. Alleston, D.L. and Davies, A.G. (1962)
26. Lantzsch, R. and Marhold, A. (1998) J. Chem. Soc., 2050–2054.
Process for the preparation of 37. Sakai, S., Niimi, H., Kobayashi, Y., and
2-trifluoromethoxy-aniline. EP Patent Ishii, Y. (1977) Bull. Chem. Soc. Jpn, 50,
820981, (Prio: 26. 07. 1996), Bayer AG, 3271–3275.
Leverkusen, Germany. 38. Wroblowsky, H.-J. and König, K. (1996)
27. Lantzsch, R., Marhold, A., and Process for the preparation of alkoxy-
Kysela, E. (1997) Method of preparing triazolinones. EP Patent 703225, (Prio:
2-trifluoromethoxy benzene sulfon- 23. 09. 1994), Bayer AG, Leverkusen,
amide. WO Patent 1997/19056, (Prio: Germany.
21. 11. 1995), Bayer AG, Leverkusen, 39. Wroblowsky, H.-J. and König, K., (1996)
Germany. Process for the preparation of alcoxy-
28. Confalone, P.N., Pizzolato, G., Rouge, triazolinones. EP Patent 703226, (Prio:
M., and Uskokovic, M.R. (1978) 23. 09. 1994), Bayer AG, Leverkusen,
3-Amino-thiophen-4-carbonsäuren Germany.
und deren Derivate, Verfahren zu 40. Wroblowsky, H.-J., König, K., Kluth, J.,
deren Herstellung und diese enthal- and Müller, K.-H. (1996) Process for
tende pharmazeutische Mittel. DE the preparation of alkoxytriazolinones.
EP Patent 703224, (Prio: 23. 09. 1994),
Patent 2,737,738, (Prio: 23. 08. 1976), F.
Bayer AG, Leverkusen, Germany.
Hoffmann-La Roche & Co AG, Basel,
41. Jackman, D.E. (2001) Process for manu-
Switzerland.
facturing substituted triazolinones. US
29. Confalone, P.N., Pizzolato, G.,
Patent 6,222,045, (Prio: 20. 09. 2000),
Uskokovic, M.R., and Rouge, M.
Bayer Corporation, Pittsburgh, PA.
(1982) Novel thiophene derivatives. US
42. Wroblowsky, H.-J. and König, K. (1996)
Patent 4,317,915, (Prio: 23. 08. 1976),
Process for the preparation of alkoxytri-
Hoffmann-La Roche Inc., Nutley, N.J.,
azoles. EP Patent 708095, (Prio: 23. 09.
USA.
1994), Bayer AG, Leverkusen, Germany.
30. Barker, J.M. and Huddleston, P.R.
43. Kulkarni, S.V., Prasad, V.A., Desai, V.C.,
(1989) Process for preparing thiophene
Rivadeneira, E., and Jelich, K. (2001)
derivatives. EP Patent 298542, (Prio: 07. Process for the manufacture of substi-
07. 1987), Shell Int. Res. Maatschappij tuted triazolinones. US Patent 6,197,971,
B.V., Den Haag, Netherland. (Prio: 27. 12. 1999), Bayer Corporation,
31. Henderson, S.A., O’Connor, J., Rendina, Pittsburgh, PA.
A.R., and Savage, G.P. (1995) Aust. J. 44. Lopyrev, V.A., Kurochkina, V.N., Titova,
Chem., 48, 1907–1916. I.A., and Voronkov, M.G. (1989) Russ.
32. Barker, J.M., Huddleston, P.R., and Chem. Bull., 38 (10), 2174–2175.
Wood, M.L. (2002) Synth. Commun., 32, 45. Conrad, M., Lantzsch, R., Desai, V.C.,
2565–2568. and Kulkarni, S.V. (1999) Process for
33. Geller, Th. (2006) Method for preparing alkoxytriazolinones. US Patent
the production of substituted 5,917,050, (Prio: 11. 02. 1998), Bayer
2-alkoxycarbonyl-3-aminothiophenes. Corporation, Pittsburgh, PA, USA, Bayer
WO Patent 2006/072375, (Prio: 29. 12. AG, Leverkusen, Germany.
2004), Bayer CropScience AG, Mon- 46. Kulkarni, S.V. (2000) Process for man-
heim, Germany. ufacturing N-alkoxy (or aryloxy)carbonyl
34. Geller, Th. and Stölting, J. (2006) isothiocyanate derivatives using
Method for the production of substi- N,N-dialkylarylamine as catalyst. US
tuted thiophenesulfonyl isocyanates. WO Patent 6,066,754, (Prio: 10. 06. 1999),
Patent 2006/072376, (Prio: 29. 12. 2004), Bayer Corporation, Pittsburgh, PA.
Bayer CropScience AG, Monheim, 47. Kulkarni, S.V. and Desai, V.C.
Germany. (2001) Process for manufacture of
References 161
N-alkoxy (or aryloxy)carbonyl isoth- P. (2001) Proc. Brighton Crop Prot. Conf.
iocyanate derivatives in the presence – Weeds, 2, 487–492.
of N,N-dialkylarylamine catalyst and 62. Philbook, B.D. and Santel, H.-J. (2007)
aqueous solvent. US Patent 6,184,412, Thiencarbazone-methyl: a new molecule
(Prio: 10. 06. 1999), Bayer Corporation, for pre- and postemergence weed
Pittsburgh, PA. control in corn. North Central Weed
48. Santel, H.J., Bowden, B.A., Sorensen, Science Society, Champaign, IL, Ab-
V.M., and Müller, K.H. (1999) Proc. stract 62, December, 2007. (Proceedings
Brighton Conf. – Weeds, 1, 23–28. Editor: R.G. Hartzler; Technical Editor:
49. Santel, H.J., Bowden, B.A., Sorensen, A.N. Hartzler) [CD-ROM Computer
V.M., Mueller, K.H., and Reynolds, J. File].
(1999) Proc. West. Soc. Weed Sci., 52, 63. Feucht, D., Dahmen, P., Drewes, M.W.,
124. Pontzen, R., Gesing, E.-R., Schwarz,
50. Brenchley, R., Rasmussen, D.E., Santel, H.-G., and Müller, K.-H. (2003) Selec-
H.J., Sorensen, V.M., Bowden, B.A., tive Herbicides based on substituted
Bulman, P.G.M., Feindel, D.E., Gibb, B., Thien-3-yl-sulfonylamino(thio)carbonyl-
and Tomolak, B.M. (1999) Proc. West. triazolin(thi)ones and Safeners. WO
Soc. Weed Sci., 52, 125. Patent 2003/026427, (Prio: 21. 09. 2003),
51. Everest 70 WDG Label (2008) Bayer CropScience AG, Monheim,
Arysta LifeScience North America Germany.
Corp., San Francisco, EPA Regis- 64. Feucht, D., Dahmen, P., Drewes, M.W.,
tration No. 66330-49, EPA Est. No.
Pontzen, R., and Gesing, E.R.F. (2003)
554-ND-002, Label Number 20444-D,
Herbicides containing substituted
http://www.cdms.net/LDat/ld48U005.pdf.
Thien-3-yl-sulfonylamino(thio)carbonyl-
52. Kirkland, K.J., Johnson, E.N., and
triazolin(thi)one, WO Patent
Stevenson, F.C. (2001) Weed Technol.,
2003/026426, (Prio: 21. 09. 2001), Bayer
15, 48–55.
CropScience AG, Monheim, Germany.
53. Feucht, D., Müller, K.-H., Wellmann,
65. Dollinger, M., Santel, H.-J., Gesing,
A., and Santel, H.J. (1999) Proc. Brighton
E.R., and Hacker, E. (2005) Novel Her-
Crop Prot. Conf. – Weeds, 1, 53–58.
bicides based on substituted Thien-
54. Amann, A. and Wellmann, A. (2001)
3-yl-sulphonylamino(thio)carbonyl-
Proc. Brighton Crop Prot. Conf. – Weeds,
triazolin(thi)ones and 4-HPPD-
2, 469–474.
55. Amann, A. (2002) Pflanz.-Nachr., 55 (1), Inhibitors. WO Patent 2005/087004,
87–100. (Prio: 05. 03. 2004), Bayer CropScience
56. Godley, N.P. and Bubb, G.W. (2001) AG, Monheim, Germany.
Proc. Brighton Crop Prot. Conf. – Weeds, 66. Patel, S., Hannemann, Th., and Weick,
2, 633–638. T. (2007) Aqueous Herbicidal Composi-
57. Wellmann, A. and Feucht, D. (2002) tion based on Concentrate comprising
Pflanz.-Nachr., 55 (1), 67–86. Herbicides and Safener, WO Patent
58. Amann, A. and Wellmann, A., (2002) 2007/057107, (Prio: 17. 11. 2005), Bayer
Wageningen, 2002 Proceedings 12th CropScience AG, Monheim, Germany.
EWRS (European Weed Research Soci- 67. Santel, H.J. and Philbrook, B.D. (2008)
ety) Symposium, Papendal, Netherlands, Thiencarbazone-methyl & isoxaflutole:
June 24–27, 2002, pp. 188–189. a new herbicide premixture for pre-
59. Scoggan, A.C., Santel, H.J., Wollam, emergence weed control in corn (Zea
J.W., and Rudolph, R.D. (1999) Proc. mays). Weed Science Society of America,
Brighton Crop Prot. Conf. – Weeds, 1, Lawrence, KS, Abstract 117, February
93–98. 2008 [On line Abstract File].
60. Bell, C.E. (1999) Proc. Brighton Crop 68. Hicks, Ch., Philbrook, B.,
Prot. Conf. – Weeds, 1, 211–216. and Bloomberg, J. (2010)
61. Santel, H.J., Anderson, J.E., Brenchley, Thiencarbazone-methyl Combinations
R.G., Cagle, J.E., Scoggan, A.C., for pre and postemergence weed control
Wellmann, A., Stahlman, P., and Geier, in western corn. Western Society of
Weed Science, Waikoloa, HI, Abstract (Thiencarbazone-methyl + Tembotrione

171, March 2010. + Isoxadifen-ethyl): A new herbicide
69. Simkins, G., Lamore, D., Hora, J., for grass and broadleaf weed control in
Philbrook, B., and Bloomberg, J. (2009) corn. North East Weed Science Society,
CAPRENO (Thiencarbazone-methyl +
TM
Boston, MA, January 2010.
Tembotrione + Isoxadifen-ethyl): a new 71. Müller, K.-H., Kirsten, R., Gesing,
herbicide for grass and broadleaf weed E.R.F., Kluth, J., Findeisen, K., Jansen,
control in corn. North Central Weed
J.R., König, K., Riebel, H.-J., Schallner,
Science Society, Champaign, IL, Ab-
O., Wroblowsky, H.-J., Dollinger, M.,
stract, 122, December 2009 (Proceedings
Santel, H.-J., and Stenzel, K. (1996)
Editor: Hartzler, R.G.; Technical Editor:
Hartzler, A.N.) [CD-ROM Computer Herbicidal or fungicidal sulfony-
File]. laminocarbonyltriazolinones with
70. Mahoney, M., Lamore, D., Hora, halogenated alk(en)oxy substituents.
J., Simkins, G., Philbrook, B., and WO Patent 1996/27590, (Prio: 08. 03.
Bloomberg, J. (2010) CAPRENOTM 1995), Bayer AG, Leverkusen, Germany.
163
3
Protoporphyrinogen IX Oxidase Inhibitors
3.1
Introduction
Rarely is an area of agrochemical research encountered with both the chemical

diversity and the very specific and often conflicting structure–activity relationship
(SAR) rules as is the case with the herbicidal protoporphyrinogen oxidase (Protox)
inhibitors. It was this incredible array of possibilities that lured every single
agrochemical organization during the 1980s and 1990s in the United States,
Europe, and Asia into initiating a research effort, in the hope of identifying the next
‘‘blockbuster herbicide.’’ Unfortunately, many Protox areas that were initially seen
as having unlimited potential soon turned into dead ends, with only a handful of
commercial products achieving any significant market share. Part of the difficulty
in exploiting the Protox area of herbicide chemistry was the fact that, even though
it was relatively easy to identify chemistries with good biological activity, it was
much more difficult to find clear crop selectivity, either on pre-emergently or
post-emergently applied materials.
The lack of clear selectivity of a number of commercially significant row crops
was overcome following the discovery of several highly active and selective Protox
herbicides such as the postemergence soybean selective herbicide fomesafen 7
® ® ®
(Flex , Flexstar , Reflex ) [1, 2], introduced in 1983 by ICI Plant Protection
Division, and the soybean-selective pre-emergence herbicides F5231 (14) [3–5] and
® ® ®
sulfentrazone (15; Authority , Boral , Capaz ) [6–8], as introduced by FMC in
® ® ®
1991, and saflufenacil (99; Sharpen , Heat , Treevix ) [9, 10], as introduced by
BASF in 2009 for pre-emergence use in corn, soybean, and wheat.
Research investigations into Protox herbicides peaked during the early 1990s
[11] but diminished soon thereafter as the use of glyphosate-resistant crops
gained an increased market share. Glyphosate [N-(phosphonomethyl)glycine] is
a broad-spectrum, post-emergence, systemic herbicide that has been used exten-
sively over the past 30 years. This intense and prolonged employment of glyphosate
has resulted in documented resistance to its use in several weed populations [12]
which, in turn, has stimulated new interest in Protox-inhibiting herbicides.

164 3 Protoporphyrinogen IX Oxidase Inhibitors
The mode of action of Protox herbicides, which has been extensively reviewed
[13–19], is via an inhibition of the enzyme protoporphyrinogen oxidase, the last
common enzyme to both heme and chlorophyll biosynthesis [20–25]. This enzyme
catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX by molecular
oxygen. Inhibition of the Protox enzyme results in an accumulation of the enzyme
product protoporphyrin IX, but not the substrate, via a complex process that has not
been entirely elucidated. In the presence of light, protoporphyrin IX generates large
HO2C CO2H
O
CO2H
HO2C
NH2 HO2C
+ CO2H
CO2H
N N
H H
HO2C N H
O H NH2 HO2C N HN
CO2H
NH2
CO2H
CO2H
Uroporphyrinogen III
Chlorophylls CO2H
CO2H
N N
H H
H
N N HN
NH
N HN
CO2H
CO2H
Coproporphyrinogen III
CO2H CO2H
Protoporphyrinogen
oxidase
Protoporphyrin IX
Protox
Inhibitors Non-enzymatic
N oxidation
NH
H Protoporphyrin IX
H
N HN
Light / O2
Lipid peroxidation
CO2H CO2H
Protoporphyrinogen IX Plant death
Figure 3.1 Chlorophyll biosynthetic pathway.

amounts of singlet oxygen, which results in the peroxidation of the unsaturated

bonds of the fatty acids found in cell membranes (Figure 3.1). The end result of
this peroxidation process is the loss of membrane integrity and leakage, pigment
breakdown, and necrosis of the leaf that results in the death of the plant. This
is a relatively fast process, with leaf symptoms such as a flaccid wet appearance
observed within hours of plant exposure to the Protox herbicide under sunlight.
In this chapter, the recent developments and challenges in the field of
Protox-inhibiting agrochemicals are discussed, and the agrochemicals are placed
in the context of research conducted in this area of chemistry during the past four
to five decades.
3.2
Historical Development
The diphenyl ether nitrofen 1 [26], introduced in 1963 by Rohm and Haas (now
® ®
Dow AgroSciences); the oxadiazolinone oxadiazon 2 [27, 28] (Explorer , Herbstar ,
® ®
Romax , Ronstar ), introduced in 1968 by Rhone-Poulenc; and the tetrahydroph-
thalimide chlorophthalim 3 [29], introduced in 1972 by Mitsubishi, represent the
earliest examples of Protox herbicides (Figure 3.2). Though all three classes are
chemically quite different, they share a common mode of action – namely, an
inhibition of the Protox enzyme – though this was not known until the late 1980s.
Each of these chemistries incorporated intensive investigations during the 1960s,
1970s, and 1980s, and this resulted in a large number of diverse chemistries from
which many useful commercial products were obtained.
3.2.1
Diphenyl Ether
Following the discovery of the herbicidal activity of nitrofen 1 in 1963, intense

research by several agrochemical companies resulted in a vast number of highly
active and diverse chemistries [30, 31]. As mentioned earlier, the diphenyl ether
chemistry represents the first class of Protox herbicides. Replacement of the
aromatic 4-chloro group with a trifluoromethyl, as is the case with oxyfluorfen
®
5 (Goal ) [32], resulted in a significant improvement in biological activity, and
2-chloro-4-(trifluoromethyl)benzene became the dominant substitution pattern for
the second generation of diphenyl ethers (Figure 3.3), eventually replacing products
® ®
such as nitrofen 1 and bifenox 4 (Foxpro , Modown ) [33]. As can be seen from
Figure 3.3, 2-chloro-1-(3-substituted-4-nitrophenoxy)-4-(trifluoromethyl)benzene
became the most successful diphenyl ether chemistry scaffold, with four significant
products launched in fewer than 10 years. In general, diphenyl ether herbicides
® ®
such as oxyfluorfen 5 (Goal ), acifluorfen-sodium 6 (Blazer ) [34, 35], fomesafen
® ® ® ®
7 (Flex , Flexstar , Reflex ) [1, 2], and lactofen 8 (Cobra ) [36] are more effective
when applied post-emergently, and are also more effective for the control of
broadleaf than of grass weeds.
Cl O O
Cl
Cl NO2 Cl N O Cl N
O
N
O O
1 2 3
Nitrofen 1963 Oxadiazon 1968 Chlorophthalim 1972
Figure 3.2 Chemical structures of three early examples of Protox inhibitors.
Cl R1 Cl R2
Cl O NO2 CF3 O NO2
Entry R1 Herbicide Entry R2 Herbicide
1 H nitrofen 5 OEt oxyfluorfen

4 CO2Me bifenox 6 CO2Na acifluorfen-sodium
7 CONHSO2CH3 fomesafen
8 CO2CH(CH3)CO2Et lactofen
Figure 3.3 Evolution of diphenyl ether herbicides.
Although the 1980s and early 1990s were a period of intense research in diphenyl
ether chemistry, the main products described above had all been introduced
by 1987. By 1996, sales of diphenyl ethers had peaked at US$ 381 million,
steadily declining to US$ 200 million by 2001 [37]. This decline was due to the
introduction of more effective herbicides, as well as to the increasing adoption of
herbicide-tolerant crops. In spite of this decline, the investigations into diphenyl
ether chemistry were continued, and this resulted in the third generation of
diphenyl ether herbicides in which either the nitrophenyl ring was replaced by a
variety of fused benzoheterocycles such as benzotriazole [38], benzoisoxazole [39],
and indolin-2(3H)-ones [40], or the 2-chloro-4-(trifluoromethyl)benzene group was
replaced by a heterocyclic ring such as pyrazole [41].
Although the extensive research investments made by many companies in this
third generation of diphenyl ether chemistry resulted in a large number of active
molecules, no successful commercial product resulted from these efforts.
3.2.2
Phenyl Ring Attached to Heterocycle
During the 1960s, several discoveries were made that would have a signifi-
cant impact on the understanding of the SAR of Protox herbicides. The first
such breakthrough was the discovery of the importance of the 2,4-dihalo-5-
substitutedphenyl substitution pattern. Rhone-Poulenc first introduced the 3-(2,4-
dichlorophenyl)-1,3,4-oxadiazol-2(3H)-one 9 in 1965 [42]. Further structure–activity
Cl O O Cl O
Cl N O Cl N Cl N
N
O O O
9 3 10
1965 Rhone-Poulenc Chlorophthalim 1980 Mitsubishi
1972 Mitsubishi
F O
Cl O F O
Cl N
Cl N O Cl N
N O O
O O
2 11 12
Oxadiazon 1976 DuPont S-23142
1968 Rhone-Poulenc 1982 Sumitomo
Figure 3.4 Incorporation of the 2,4-dihalo-5-alkoxy aromatic

pattern of oxadiazon into new phenyl tetrahydrophthalimide
ring systems.
optimization at the phenyl ring soon led to the discovery in 1968 of the
2,4-dichloro-5-isopropoxyphenyl substitution pattern of the herbicide oxadiazon
2 [43, 44]. The 2,4-dihalo-5-substituted pattern at the aromatic ring would become
the basis for much of the 2,4,5-trisubstituted phenyl tetrahydrophthalimide 10 [45]
-based research that followed in the Protox area of chemistry.
Another breakthrough discovery was the boost in biological activity that occurred
when fluorine at the 2-phenyl position was replaced by chlorine. In 1976, DuPont
introduced the first example of a 2-fluoro-4-chlorophenyl tetrahydrophthalimide
Protox inhibitor 11 [46] (see Figure 3.4). The dramatic increase in biological activity
caused by fluorine being present in the 2 position of the phenyl ring would, during
the 1980s, influence many investigations in the Protox area, such as the discovery
of the 4-chloro-2-fluorophenyl tetrahydrophthalimide herbicide S-23142 (12) [47].
The herbicide oxadiazon 2 is used for the pre-emergence control of annual
broadleaf weeds and grasses and bindweed, and for the post-emergence control
of annual broadleaf weeds in ornamentals such as carnations and roses, as well
as in fruit trees, vines, cotton, rice, and turf. It requires high application rates of
1 kg a.i. ha−1 for weed control in rice, and up to 4 kg a.i. ha−1 for pre-emergence
weed control in vines and orchards [27, 28]. The high rates of pre-emergence
application, the limited selectivity in a number of row crops, and the introduction
of newer, more effective herbicides all have served to limit the commercial use
® ®
of oxadiazon 2. Later, Rhone-Poulenc introduced oxadiargyl 13 (Raft , Topstar )
[48, 49] (Figure 3.5), a compound related to oxadiazon, for the control of broadleaf
weeds, grass, and annual sedge in transplanted rice.
During the 1980s, a number of chemical changes made to the 1,3,4-oxadiazol-
2(3H)-one heterocyclic resulted in several significant improvements in the pre- and
post-emergence biological efficacy and crop selectivity of Protox herbicides. A de-
tailed discussion of the various classes of phenyl heterocycles that were introduced
Cl O Figure 3.5 Chemical structure of oxadiargyl.
Cl N O
N
O
13
Oxadiargyl
F O Cl O F O
F F
Cl N N Cl N N Cl N N
F F
N N F N EtO2C N
HN HN
SO2CH2CH3 SO2CH3 Cl
14 15 16
F5231 Sulfentrazone Carfentrazone-ethyl
Figure 3.6 Chemical structure of F5231, sulfentrazone, and carfentrazone-ethyl.
several decades ago is beyond the scope of this chapter, but they have been reviewed
previously [30]. The introduction in 1985 of the 5-aminosulfonyl group into the
phenyl ring of 2,4,5-trisubstituted phenyltetrazolinones was one such significant
change. F5231 (14) [5], a molecule which was under development consideration
by FMC during the late 1980s, was the first Protox inhibitor to provide excellent
pre-emergence broadleaf control and clear selectivity at low application rates on
a number of crops such as soybean, rice, corn, and wheat. FMC later replaced
F5231 14 with the phenyl triazolinone sulfentrazone 15 for soybean, sugarcane,
and other crops [6–8]. Sulfentrazone 15 provides pre-emergence control of several
broadleaf weeds, as well as a number of selected grass weeds, for the soybean
market. A few years later, FMC introduced a second commercial phenyltriazoli-
®
none, the post-emergence cereal and corn herbicide carfentrazone-ethyl 16 (Aim ,
® ®
Affinity , Aurora ; Figure 3.6) [50, 51]. At low application rates of 20–35 g a.i. ha−1 ,
carfentrazone-ethyl 16 provides excellent control of weeds in commercially impor-
tant cereal crops, including bedstraw, speedwell, morning glory, kochia, spurge,
and deadnettle [52].
In addition to the oxadiazolinone, tetrazolinone, and triazolinone heterocyclic
ring systems, other five-membered ring systems investigated during the 1980s
included pyrazoles, such as fluazolate 17 [53] from Monsanto; pyraflufen-ethyl 18
®
(Ecopart ) [54, 55], introduced in 1993 by Nihon Noyaku as a post-emergence
broadleaf herbicide in cereals; and fused triazolinone rings such as azafenidin 19
[56, 57] from DuPont (Figure 3.7).
3.2.3
Phenyl Tetrahydrophthalimide
The phenyl tetrahydrophthalimides, which represent the third class of early Protox
herbicides, were introduced during the early 1970s, after the diphenyl ether and
F Br
CH3
Cl
N N CH
3
O
O
17
Fluazolate
F Cl Cl O
O
CHF2 N
Cl Cl N
N N CH N
3
O O
EtO2C 18 19
Pyraflufen-ethyl Azafenidin
Figure 3.7 Chemical structure of fluazolate, pyraflufen-ethyl, and azafenidin.
O F O
Cl N Cl N
O O
O
3 R
Chlorophthalim 12 R = H S-23142 1982 Sumitomo

1972 Mitsubishi 20 R = CH3 S-23121 1982 Sumitomo
Figure 3.8 Chemical structures of chlorophthalim, S-23142, and S-23121.
1,3,4-oxadiazol-2(3H)-one chemistries. Following the introduction by Mitsubishi

of chlorophthalim 3 [29] in 1972, incorporation of the 2,4,5-trisubstitutedphenyl
pattern during the 1980s resulted in the synthesis of highly active molecules such
as S-23142 (12) [47] and S-23121 (20) [47, 58] (Figure 3.8).
Between 1980 and 2000 the tetrahydrophthalimide area of chemistry generated
a great deal of interest, with hundreds of patents issued by a wide range of
agrochemical companies [31]. Once the fluorine group at the 2-phenyl position
and the chlorine group at the 4-phenyl position had been established as the
optimum substituents for the aromatic ring, the 5 position of the phenyl ring and
the tetrahydrophthalimide heterocycle became the targets of intense research. A
wide variety of oxygen 21, sulfur 22, amino 23, and carbonyl 24 derivatives at the
5 position of the aromatic ring were introduced [31] (Figure 3.9), some of which
®
became commercially available, including flumiclorac-pentyl 25 (Resource ) [59]
®
and cinidon-ethyl 26 (Lotus ) [60] (Figure 3.10).
The phenyl tetrahydrophthalimides are primarily post-emergence herbicides
used to control broadleaf weeds, although they will show pre-emergence activity at
F O F O
Cl N Cl N
R O O R S O
21 22
R = Propargyl (S 23142), alkyls, allyl, benzyl, R = Alkyls, allyl, benzyl, acetates
acetates
F O
Cl N
R O
F O F O
Cl N Cl N
R N O R O
H O
23 24
R = CF3SO2-, acetate, alkyl, acetyl R = AlkylO, benzyl, AlkylNH
Figure 3.9 Derivatization of aromatic position 5 of phenyl tetrahydrophthalimide.
F O
O
Cl N
Cl N
O O Cl
O O
O
O O
25 26
Flumiclorac-pentyl Cinidon-ethyl
1988 Sumitomo 1992 BASF
Figure 3.10 Chemical structures of flumiclorac-pentyl and cinidon-ethyl.
higher rates of application. Flumiclorac-pentyl is a post-emergence herbicide used

to control broadleaf weeds such as cocklebur, common lambsquarters, jimsonweed,
Amaranthus, prickly sida, and velvetleaf in soybean and corn at 25–50 g a.i. ha−1 .
Cinidon-ethyl is used for the post-emergence control of annual broadleaf weeds,
particularly bedstraw, deadnettle, and speedwell in winter and spring small grain
cereals, at a rate of 50 g a.i. ha−1 .
In addition to the phenyl tetrahydrophthalimides discussed here, several sig-
nificant Protox herbicides in which a phenyl ring is attached to a fused thiadi-
azole[3,4a] pyridazine or to an oxazolidinedione ring have been reported. Two
examples of phenyl thiadiazole[3,4a] pyridazine systems are fluthiacet-methyl 27
F
F S
Cl N N
Cl N
N N
S S N S O
O
O
O O
27 O 28
Fluthiacet-methyl
F S
F
N
Cl N
Cl N N
N S
S O
S N O
O
O
O O
29 30
NCI-876-648
Figure 3.11 Rearrangement of phenyl thiadiazole [3,4a] pyri-

dazine to phenyl triazolopyridazines.
F O Figure 3.12 Chemical structure of pentoxazone.

O
Cl N
O O
31
Pentoxazone
®
(Appeal , CadetTM ) [61] and NCI-876-648 29 [62], both of which are said to act
as ‘‘pro-herbicides,’’ because they are converted in the plant to the corresponding
phenyl triazolopyridazines [63] (Figure 3.11).
The phenyl oxazolidinedione chemistry is best exemplified by pentoxazone 31
®
(Wechser ) [64, 65] (Figure 3.12), as discovered by Sagami for the pre-emergence
control of weeds such as barnyard grass in rice at application rates of
200–450 g a.i. ha−1 .
3.3
Non-Classical Protox Chemistries
During the 1990s, a number of chemistries were introduced that did not
conform to the established SARs of previous chemistries, such as the diphenyl
ethers and the 2,4-dihalo-5-substitutedphenyl heterocycles. The SARs of
2-fluoro-4-chloro-5-substituted phenyl heterocycles are shown in Figure 3.13
[5, 66]; these newer developments impacted on both the aromatic and the
heterocyclic portions of N-phenyl heterocycles.
Electron-withdrawing
Substitution decreases lipophilic group -- fluorine best
activity
Electron-withdrawing F O
lipophilic group -- Cl
chlorine best N
R
Best overall activity and limited

crop selectivity when R = OCH2CCH
Best broadleaf weed activity and

excellent multicrop selectivity
when R = NHSO2ET
Figure 3.13 Structure–activity relationships of the two aro-

matic ring of 2,4,5-trisubstituted phenyl heterocyclic systems.
3.3.1
N-Phenyl Heterocycles: New Heterocyclic Systems
A large number of heterocyclic systems, which usually are attached to aromatic rings
via a nitrogen or carbon atom, have been introduced during the past 15 years. Some
of these heterocyclic rings, such as oxadiazolinone [27, 28], tetrahydrophthalimide
[29], tetrazolinone [5], triazolinone [6–8, 50], pyrazole [53–55], and oxazolidinedione
[64], have already been discussed. At about the same time, phenylpyridines [67, 68]
were synthesized by employing a Suzuki coupling reaction, and found to exhibit
similarly high levels of herbicidal activity. Extensive details of these heterocyclic
systems, their properties, and their synthesis have been reviewed [30, 31].
Of the several dozen new heterocyclic systems introduced between 1990 and
2005, the system that clearly had the greatest impact in terms of a significant
increase in biological activity was 6-trifluoromethyl-2,4(1H,3H)-pyrimidinedione
ring – commonly referred to as uracil – as introduced initially by Hoffman-La
Roche and Uniroyal [69, 70] (Table 3.1).
Replacement of the tetrahydrophthalimide and other heterocyclic rings, such as
the triazolinone 32 with the uracil 33 ring, resulted in a significant improvement
in biological activity, particularly when applied pre-emergently [71] (Table 3.2).
Based on this significant improvement in herbicidal activity, the uracil ring has
subsequently become a ‘‘standard’’ ring in any N-aryl heterocycle Protox-related
patent application. Initial SAR studies of the uracil nitrogen position showed that
methyl and amino both resulted in optimum activity [71], whereas increasing the
size and length of the R group in compound 34 resulted in a significant reduction
in biological activity. It is interesting to note that both the lipophilic methyl group
(in 35) and the hydrophilic amino group (in 36) are equally active (Table 3.3).
Four examples of molecules that contained the uracil or closely related pyridazi-
none ring, and which reached an advanced stage of development, are benzfendizone
Table 3.1 Introduction of six-membered heterocyclic ring systems during the 1990s.
Cl, F
Cl Heterocycle
Ring system Heterocycle Heterocycle Ring system
Oxadiazolinones N O
N
O O
CH3
Tetrahydrophthalimides N N Uracil
N
O CF3
O
O O
Triazolinones N N CHF2 1990 N CF3 Pyridazinone
N N
O Cl
Tetrazolinones Pyridine
N N CF3
N N F N
Br
Pyrazoles CF3
N N
N O
Oxazolidinediones
O
® ®
41 [72, 73], butafenacil 42 (Inspire , Rebin ) [74], flufenpyr-ethyl 43 [75], and
SYN523 115 [76] which, although originally discovered by Sumitomo, had sub-
sequently been investigated as broad-spectrum herbicide by both Sumitomo and
Syngenta (Figure 3.14). Benzfendizone is a post-emergence herbicide that provides
good control of grass and broadleaf weeds in tree fruits and vines, acts as a cotton
defoliant, and has applications in total vegetation control. The 6-trifluoromethyl
group in the uracil ring is essential for biological activity; its replacement with a
methyl group resulted in a complete loss of activity.
Table 3.2 Comparison of biological activity of triazolinone and uracil

heterocycles.
F O
F O CH3
Cl N N CHF2 N
N Cl N CF3
O
O O
32 33
Pre-emergent biological activity ED85 (g a.i. ha –1 )

Weed species Compound 32 Compound 33
Morning glory 395 22

Johnson grass 300 10
ED85 , effective dose required to kill 85% of weeds.
Table 3.3 Effects of uracil N-substituents on herbicidal activity of analogs

of compound 40.
F O R
N
Cl N CF3
O O O
34
Compound R Velvet leaf Morning glory Green foxtail
35 CH3 3 3 3
36 NH2 3 3 3
37 CH2 CH3 8 17 3
38 CH2 OCH3 18 52 44
39 CH2 C6 H5 958 >1000 307
40 CH2 CH2 CH3 >1000 >10000 >1000
The uracil heterocycles are readily prepared in high yields from the correspond-
ing arylisocyanates 44, and from ethyl trifluoromethylaminocrotonate 45 in the
presence of a base [77]. The uracil heterocycle is then directly N-methylated with
methyl iodide in a one-pot reaction (Scheme 3.1). The uracil ring is stable to treat-
ment with strong acids such as HBr and weak bases such as potassium carbonate;
O
O O
N
O N CF3 O O
N
O
O Cl N CF3
O
O
O
41 42
Benzfendizone Butafenacil
F O F O
N
Cl N CF3 Cl N CF3
N
O O O
O
O O O
N
43 O
115
Flufenpyr-ethyl SYN 523
Figure 3.14 Chemical structures of benzfendizone, butafenacil, flufenpyr-ethyl, and SYN523.
H2 N O
(1) NaH N
O N C O + CF3 O O N CF3
O (2) MeI
O
44 45 46
(1) HBr / acetic acid

or BBr3 / CH2Cl2 O
(2) K2CO3
O
O
N
O N CF3
Cl
O
O 47
O
O 41
Scheme 3.1 Synthesis of the uracil heterocyclic ring of benzfendizone.
both of these reagents are used in the further derivatization of the intermediate 46
with the benzyl chloride 47 to produce benzfendizone 41.
A less familiar ring system, but one that was part of a molecule selected for
advanced testing, was the 5-methyl-6-oxo-4-(trifluoromethyl)-1-(6H)-pyridazinyl
ring system of flufenpyr-ethyl 43. The pyridazinyl heterocycle can be prepared
from the reaction of 4-chloro-2-fluoro-5-hydroxyphenyl hydrazine 48 and
O
F Br
CF3 F
Br H O
Cl NHNH2 49 Cl N CF3
N CO2H
HO HO
48 50 CO2H
51
Base
F CO2H
H
Cl N CF3
N OH
HO
52
EtCO2H
F O F O
ClCH2CO2Et
Cl N CF3 Cl N CF3
N N
O K2CO3, DMF HO
EtO2C 43 53
Flufenpyr-ethyl
Scheme 3.2 Synthesis of the pyridazinyl heterocyclic ring of flufenpyr-ethyl.
1,1-dibromo-3,3,3-trifluoroacetone 49 to give the corresponding hydrazone 50

which, when reacted with methyl malonic acid 51 in the presence of a base, provides
the intermediate 52. Acid-catalyzed ring closure of 52, followed by O-alkylation
of 53 with ethyl chloroacetate, results in the synthesis of flufenpyr-ethyl 43 [78]
(Scheme 3.2).
3.3.2
Phenoxyphenyl and Benzyloxyphenyl Attached to Heterocycle
Extensive modeling and quantitative structure–activity relationship (QSAR) studies

of Protox herbicides have been reported [14, 79, 80]. Earlier, it was postulated that
Protox herbicides acted by mimicking the Protox substrate, protoporphyrinogen
IX [81] (Figure 3.15), an observation which resulted in the discovery of the
three-ring 2,4-dihalo-5-phenoxyphenyl propionate heteroaryl herbicide 54, and later
the heterocyclic phenoxymethylphenoxy propionate chemistry 56. These proved to
be significant improvements over the 4-chlorobenzyloxyphenyl heterocycles 55,
with both 54 and 56 being shown as highly potent classes of Protox herbicides.
Subsequently, molecular modeling studies demonstrated a good overlap between
the diphenyl ether aromatic rings and protogen [82], as well as between a set of
imide-type Protox inhibitors and protogen [83]. Whilst these studies were important
in advancing the hypothesis that the diphenyl ethers mimicked protogen, they were
of limited practical value in that they failed to reveal any new chemical structures.
F O
CHF2
N
Cl b Na
N
O
c
54
O
O
O
b a Cl
NH N O
H a
b N
H
N HN d c
c O
O
55
CO2H CO2H
O
Protoporphyrinogen IX a
N
c
O O
d
Cl
O O
56
Figure 3.15 Phenoxyphenoxy and benzyloxyphenyl as three-ring mimics of tetrapyrroles.
One class of Protox inhibitors that redefined the accepted SARs and QSARs
of the aromatic 4 position was the substituted benzyloxyphenyl heteroaryl area.
As discussed above, both SAR and QSAR studies of the phenyl ring of Protox
herbicides had demonstrated the need for halogens in the 2 and 4 positions of
the phenyl ring, with the exception of the 4-chlorobenzyloxy group such as that of
4-chlorobenzyloxyphenyl tetrahydrophthalimide outlier 55 (see Figure 3.15), and
as reported by Ohta and coworkers in 1980 [84]. Chlorine at the para position of the
benzyloxy was reported to provide optimum biological activity.
Further QSAR studies conducted by Fujita and Nakayama [85] rationalized the
high activity of this outlier 4-chlorobenzyloxy group by stating that the unexpected
activity of the 4-benzyloxy ring was due to additional interactions of this group with
the target enzyme. Given the strict steric and electronic requirements of groups
at the 4 position of the phenyl ring, with chlorine as the optimum group, Fujita’s
explanation for the unexpected activity of a bulky electron-donating group such
as the 4-benzyloxyphenyl is highly unlikely. In general, the presence of outliers
in SAR or QSAR analyses indicates an unusual property of that group – such
as a switch in the nature of the binding of the outlier in the enzyme site – and
an opportunity for a major breakthrough. It was speculated that the activity of
the 4-benzyloxy outlier was potentially due to a shift in the binding mode for
phenyl rings attached to heterocycles containing two flanking carbonyl rings,
such as tetrahydrophthalimides and the newer uracil rings [66, 73]. Based on this
new binding mode, the benzyloxy group could mimic the lipophilic portion of
protoporphyrinogen IX, ring B, or the hydrophilic portion of protoporphyrinogen
IX, ring D. Based on this working hypothesis, a series of compounds were prepared
that contained an oxypropionate side chain, in addition to chlorine, in the benzyloxy
group [73]. These studies resulted in benzfendizone, a highly active broad-spectrum
post-emergence herbicide.
3.3.3
Benzoheterocyclic Attached to Heterocycle
As discussed above, extensive studies of the 2, 4, and 5 positions of the phenyl

ring of Protox inhibitors revealed very specific electronic, lipophilic, and steric
requirements for chemical groups at these positions. Thus, it was rather surprising
when it was shown possible to obtain highly active molecules by linking the
4 and 5 or the 5 and 6 positions of the phenyl ring to yield a wide variety of
benzoheterocycles, such as those shown in Figures 3.16 and 3.17.
Linking the 4 and 5 or 5 and 6 positions of the phenyl ring of Protox inhibitors to
give a new heterocyclic ring resulted in two new classes of Protox herbicides, both
with increased biological efficacy and new SARs. In the first instance, previous
SAR studies of 2,4,5-trisubstitutedphenyl heterocycles have shown that position
2 of the phenyl ring required a halogen group for optimum biological activity,
with chlorine and fluorine generating the highest overall activity. Position 4 of
the phenyl ring required a hydrophobic, electronegative group such as halogen
for optimum activity, with chlorine and bromine resulting in the best activity.
Electron-donating groups such as methoxy resulted in significant loss of biological
activity. The benzoxazinone SAR does not fit these rules, with compound 58 being
far more active than its open-chain analog 57 [86, 87] (Table 3.4).
Incorporating the benzoxazinone ring into Protox herbicides resulted in several
® ®
commercial molecules, such as flumioxazin 59 (Sumisoya , Valor ) [88] and
thidiazimin 60 [89] (Figure 3.16). Other heteroaryl rings include the quinolin-2-one
61 [87] and benzimidazole 62 [90].
The second class of benzoheteroaryl Protox herbicides are obtained when aro-
matic positions 5 and 6 are linked together to form a variety of 3-(4,6-substituted
benzoheterocyclyl) rings, which can be attached to a wide range of heterocycles
(Figure 3.17). The 3-(4,6-substituted benzoheterocyclyl) ring system represents a
highly active area of Protox inhibitors, particularly when applied pre-emergently.
The benzodioxolane uracil 63 provides complete control of pigweed, wild mustard,
velvetleaf, green foxtail, and johnsongrass at rates as low as 10 g a.i. ha−1 when
applied pre-emergently [77]. Other rings include benzoisoxazolone 64 [77] and the
corn and rice benzimidazole F7967, compound 65, a new pre-emergence herbicide
F S N
O N N
N
F O O
60 F
O N Benzoxazinone O
Thidiazimin F
N O N N N
O N
O F
59 61
Benzoxazinone
Flumioxazin Quinolin-2-one
X
heterocycle
N F
Linking of 4 and 5 positions O
of aromatic ring CF3 F
N N N
N F
Y4 2 X 62
5 1 Benzimidazole
z heterocycle
6
Figure 3.16 Benzoheterocycles resulting from link-

ing aromatic positions 4 and 5 of phenyl heterocyclic
Protox inhibitors.
from FMC [91]. In pre-emergence applications, under greenhouse conditions,
compound 65 controlled at application rates as low as 10–30 g a.i. ha−1 , a number
of broadleaf weeds (e.g., velvetleaf, morning glory, pigweed, bindweed, nightshade,
kochia, and chickweed) and grass weeds such as crabgrass, foxtails, johnsongrass,
and shattercane.
The SARs of these 3-(4,6-substituted benzoheterocyclyl) heterocycle herbicides
differ from the more traditional 2,4-dihalo-5-substitutedphenyl heterocycles dis-
cussed earlier. As shown in Table 3.5, the introduction of a methoxy group at
position 6 of compound 66 resulted in a dramatic loss of biological activity, the re-
sulting compound 67 being more than fivefold less active than 66. Linking together
the aromatic positions 5 and 6 into a benzodioxolane ring resulted in compound
63, which was more active than either compound 66 or 67 [77].
Substituents at the 6 position of the benzoheterocyclic ring had a dramatic effect
on the weed spectrum and crop selectivity of these compounds when applied
pre-emergently. First (as shown in Figure 3.18 in the case of compound 68),
a fluorine at position 6 of the 2,3-dihydrobenzofuran ring gives compound 69,
which has excellent corn selectivity and control of broadleaf weeds (velvetleaf, wild
mustard, and pigweed) at 10–30 g a.i. ha−1 . Next, replacement of the 6-fluoro group
with a chlorine resulted in a molecule (70) which has good grass weed control
(barnyardgrass, green foxtail, and johnsongrass) at 10–30 g a.i. ha−1 . Finally,
compound 71, with a hydrogen substituent at the 6 position, resulted in
Y4 2 X
5 1
z heterocycle
6
Linking of 5 and 6 positions

of aromatic ring
O
F O Y X
N N
Cl N CF3 Cl N CF3
heterocycle
O O O O O O
N
63
64
Benzodioxolane
Benzoisoxazolone
F O
N
Cl N CF3
N NH O
CF3
65
Benzimidazole
F7967
Figure 3.17 Benzoheterocycles resulting from link-

ing aromatic positions 5 and 6 of phenyl heterocyclic
Protox inhibitors.
Table 3.4 Comparison of biological activity of open- versus fused-ring analogs.
O F O F
O O
O N N O N N
O O
57 58
Compound Velvet leaf Morning glory
57 2000 >4000
58 62.5 125

Table 3.5 Comparison of biological activity of open- versus fused-ring analogs.
F O F O F O
N N N
Cl N CF3 Cl N CF3 Cl N CF3
O O O O O O O O
63 66 67
Compound Velvet leaf Green foxtail
63 3 4
66 6 9
67 32 143
Cl X
O
O N N
O
O CF3
68
Cl F Cl Cl Cl
O O O
O N N O N N O N N
O O O
O CF3 O CF3 O CF3
69 70 71
Corn selectivity and broadleaf weed Grass weed control at Broadleaf and grass weed
control at 10-30 g a.i. ha-1 10-30 g a.i. ha-1 control at 10 g a.i. ha-1
Figure 3.18 Effect on biological activity of substituents at

position 6 of the 2,3-dihydrobenzofuran ring.
broad-spectrum control of both grass and broadleaf weeds at 10 g a.i. ha+–1.

Neither compound 70 nor 71 provided the same degree of corn selectivity as
compound 69 [77].
3.3.4
Benzyl Attached to Heterocycle
This very interesting chemistry class of Protox-inhibiting herbicides, which has

attracted less attention than other Protox herbicides, is the only class to use a benzyl
Cl F Cl O
O
Cl
N N
O N N
O CF3
O CF3
Cl
66 72
Figure 3.19 Substitution patterns of phenyl and benzyl uracils.
ring instead of a phenyl ring and, in so doing, has redefined the structure–activity
of the aromatic ring. Previous SAR studies of the benzyl uracil series resulted
in compound 72, with a 2,3,5-trisubstitution pattern of the phenyl ring; this was
significantly different from that of 3-phenyl-6-trifluoromethyluracils, where the
optimum substitution pattern has substituents at the 2,4, and 5 positions of the
phenyl ring, as in compound 66 [92] (Figure 3.19).
3.3.5
Replacement of Phenyl Ring with Pyrazole
In this section, the unusual replacement of the phenyl portion of Protox herbicides
with a pyrazole ring to give pyrazogyl 78 [93], a rice herbicide initially discovered
by Aventis (now Bayer CropScience) is discussed.
Several examples of Protox inhibitors have been described in which the phenyl
ring has been replaced with pyridine [94, 95] – a fairly common bioisosteric
move – while preserving the 2,4-dihalo-5-substituted pattern in the heteroaromatic.
Pyrazogyl 78 is unusual in that it involves several changes, such as the na-
ture and placement of substituents, the size of the ring, and the replacement
of phenyl with a heterocycle. Pyrazogyl 78 can be prepared in several steps
from 2-hydrazino-4,5,6,7-tetrahydropyrazo[4,5-a]pyridine 73 and ethoxymethylen-
emalononitrile 74, followed by bis-chlorination of the pyrazole rings in 75,
N-methylation of 76, and finally, N-propargylation of 77 [93] (Scheme 3.3).
3.4
Recent Developments
Although, several reviews of Protox herbicides are available, covering the period
between the 1960s and 2002 [13–15, 30, 78, 96], only those Protox-related investiga-
tions conducted between 2003 and 2010 will be discussed at this point. Following
the momentous volume of research into all aspects of Protox herbicides – their
chemistry, biology, biochemistry – between 1970 and 1990, the number of investi-
gations in this area of herbicide chemistry has slowed significantly in recent years.
Although corporations have continued to invest in Protox research, and several new
chemical structures have recently been introduced, none of these new chemical
structures has been shown to differ significantly from those discussed previously.
The crystal structure of the mitochondrial protoporphyrinogen IX oxidase enzyme
obtained from tobacco, and complexed with phenyl pyrazole Protox inhibitors, was
NC
NC OEt
NH2 NH2Cl
74 NC SO2Cl2 NC
NH2NH N N
N N EtOH
N N N N N N
Cl
73 Reflux 75 76
CH3 H3C
N Cl NH Cl
NC Br NC
N N
N N N Base N N N
Cl Cl
78 77
Pyrazogyl
Scheme 3.3 Synthesis of pyrazogyl.
first reported in 2004 [97]. As discussed in Section 3.1, the membrane-embedded

Protox enzyme is the target of the Protox herbicides. It was also noted in Section 3.2
that molecular modeling studies of Protox inhibitors identified a good overlap
between the diphenyl ether aromatic rings and protoporphyrinogen IX (protogen)
[82], and between a set of imide-type Protox inhibitors and protogen [83]. A report
on the protoporphyrinogen IX oxidase crystal structure (following collaboration be-
tween the Max-Plank Institute, Bayer CropScience and Proteros) discussed how the
active site architecture suggested a specific substrate-binding mode that would be
compatible with a rare, six-electron oxidation. It was also proposed that the pyrazole
ring of 4-bromo-3-(5 carboxy-4 -chloro-2 -fluorophenyl)-1-methyl-5-trifluoromethyl
pyrazole 79 would match ring A, and the phenyl ring would match ring B, of
protoporphyrinogen IX (Figure 3.20).
In terms of recent patent activity related to Protox inhibitors, a series of
N-substituted phenyl isothiazolone Protox herbicides have been prepared in or-
der to investigate the potential of the isothiazolone heterocycle ring to act as
a bioisostere for comparable tetrahydrophthalimides such as compound 80 [98]
(Figure 3.21). The 2-(4-chloro-3-isopropoxycarbonyl)phenyl isothiazole-1,1-dioxide
(83) was found to be the most active in the isothiazolone series, as measured
by the inhibition of protoporphyrinogen IX oxidase isolated from corn, as well
as by growth inhibition, chlorophyll decrease, and peroxidative destruction of cell
membranes of green microalga Scenedesmus acutus [99]. Compound 83 was more
active than either compound 81 or 82, but about 100-fold weaker than the reference
compound 80.
Also reported in 2004 were details of the synthesis and structure–activity of a
number of 2-fluoro-4-chloro-5-substitutedphenyl pyrrole Protox herbicides, such as
compound 84 [100]. This interesting pyrrole class of chemistry further extends the
B A
NH N Protoporphyrinogen IX oxidase
H
H
N HN Protoporphyrin IX
C D
F Br
CF3
CO2H CO2H Cl
N N
Protoporphyrinogen IX O−
O 79
Figure 3.20 Protox inhibitor 4-bromo-3-(5 -

carboxy-4 -chloro-2 -fluorophenyl)-1-methyl-5-trifluoromethyl
pyrazole used in protoporphyrinogen IX oxidase binding
studies.
O
Cl
N
O
S
O
81
O
Cl
O
N Cl
O
N
O O O
S
O O
80
82
O
Cl
N
O
O S
O O
83
Figure 3.21 Isothiazolone bioisostere for tetrahydrophthalimide Protox inhibitors.
structure–activity of the 2-fluoro-4-chloro-5-substitutedphenyl pyrazoles fluazolate

17 and pyraflufen-ethyl 18, as discussed in Section 2.2 (Figure 3.22).
Compound 84 was extensively field tested in cereals and soybeans between
1999 and 2002 in France, Italy, and the United States. The post-emergence field
application of 84 at 50 g a.i. ha−1 demonstrated broadleaf weed control, with soy-
bean tolerance, of morning glory, redroot pigweed, and prickly sida. Soybean
plants eventually outgrew initial injury at seven days after application. Field test-
ing on winter wheat provided >80% control of a number of broadleaf weeds,
including cleavers, at application rates of 50–60 g a.i. ha−1 . The pyrazole 84
can be prepared in several steps, starting from the 1,3-dipolar cycloaddition of
X F Cl
F CH3
Y
Cl
Cl N
N N CH CH3
3 O Cl
R O
O O 84
R X Y Compound
CO2CH(CH3)2 Br CH3 Fluazolate 17

OCH2CO2Et Cl OCHF2 Pyraflufen-ethyl 18
Figure 3.22 Phenyl pyrrole Protox inhibitors.
O
O CF3
O
F N+ F
N CO2H
CF3 CF3
Cl 86 Cl
O N
HO O
85 Ac2O 87
(1) NCS / DMF
(2) K2CO3 / CH3OH

H2O
O
O Br
F Cl
O F Cl
Cl CF3
CF3
O Cl
O O N Base
N
O Cl CH3 HO Cl
84 88
Scheme 3.4 Synthesis of phenyl pyrrole Protox inhibitors.
2-trifluoromethyl-3-methyl-1,3-oxazolium-5-olate 86 to 2-chloro-4-fluoro-5-ethynyl-
phenol 85, followed by chlorination of the resulting pyrrole compound 87, and
reaction of 88 with the corresponding bromo acetate [100] (Scheme 3.4).
Another area related to fluazolate 17 and pyraflufen-ethyl 18 chemistry is a
series of 2,4,5,6-tetrasubstituted phenylpyrazoles 89 from Ishihara Sangyo Kaisha
[101]. These compounds differ from previous phenylpyrazoles in that they have
substituents at the 6 position of the phenyl ring; an example of one such compound
is shown in Figure 3.23. The pre-emergence application of compound 89 provided
100% control at 63 g a.i. ha−1 of barnyardgrass, crabgrass, green foxtail, redroot
pigweed, prickly sida, and velvetleaf. Soybean was reported to have 20% injury for
compound 89 at this rate of application.
F Figure 3.23 Chemical structure of tetrasubstituted phenyl

Cl
Cl pyrazoles.
O
CHF2
O N N
NH CH3
O
89
F Cl F Cl
Cl Cl
N N
O N O N
90 NC O
N 91
S-275
Figure 3.24 Chemical structure of

2-fluoro-4-chloro-5-isoxazolinylmethoxy tetrahydroindazole.
F Figure 3.25 Chemical structure of 2-fluoro-4-chloro-5-

O
Cl alkoxy phenyl imidazolididine triketone Protox inhibitors.
N N
O
O O
92
Several 2-phenyl-4,5,6,7-tetrahydro-2H-indazoles with a number of isoxazolinyl-

methoxy groups at the 5 position of the aromatic ring, such as compound 91, were
introduced by the Korea Research Institute of Chemical Technology [102, 103] as
paddy rice herbicides (Figure 3.24). These compounds have the general structure
of S-275 (compound 90) from Sumitomo [104]. Introduction of the isoxazolinyl-
methoxy groups at the 5 position of the aromatic ring is said to provide good
broadleaf control with good tolerance by transplanted rice seedlings.
Herbicidal activity on a number of weeds, such as hairy beggarsticks, black
nightshade, and knotweed, was reported in 2004 for a series of 2,4,5-imidazolididine
triketones, such as compound 92 [105] (Figure 3.25).
A series of four- and five-membered benzoheterocycle uracils derived from tying
back the 4 and 5, as well as the 2 and 3, aromatic positions were disclosed. The
benzoheterocycles obtained from linking aromatic positions 4 and 5 were developed
by Bayer in 2003 [106]. The differentiating feature between these benzoheterocyclic
uracils and earlier types discussed in Section 3.1 is the replacement of the N-methyl
group with an amino group in the uracil heterocycle, as exemplified by compounds
93 and 94 (Figure 3.26).
Ishihara Sangyo Kaisha disclosed a series of benzoheterocycles derived from
linking the 2 and 3 aromatic positions, such as compounds 95 [107] and 96 [108]
(Figure 3.27).
Further derivatization at the 5 position of the phenyl ring of 2,4,5-trisubsituted
phenyl heterocycles has resulted in several new Protox herbicide patents. Ishihara
F O NH2 F O NH2
N N
O N CF3 S N CF3
N O O N O
O
EtO2C NC
93 94
Figure 3.26 Benzoheteroaryl N-aminouracil derivatives.
F O CH3
N F O CH3
Cl N CF3 N
Cl N CF3
O N O
O O
Ph
95 96
Figure 3.27 Benzoheterocycle uracils.
Sangyo Kaisha introduced a number of benzohydrazide derivatives such as com-

pound 97 [109] for use as herbicides, desiccants, and defoliants. Both the pre- and
post-emergence control of a number of weeds such as redroot pigweed, velvetleaf,
sicklepod, ivyleaf morning glory, and cocklebur was demonstrated at application
rates as low as 63 g a.i. ha−1 . BASF reported the following new chemistries: the
benzoic acid derivatives compound 98, with good post-emergence activity in redroot
pigweed and common lambsquarter, as well as potential use as cotton desiccants or
defoliants [110], and saflufenacil, compound 99 [9, 10, 111]; aminosulfonylamino
phenyl uracil derivatives, compound 100 [112]; and benzosulfonamides, compound
101 [113] (Figure 3.28). Saflufenacil 99 was commercialized in 2009 for pre-plant
foliar burndown and selective pre-emergence dicot weed control in multiple crops,
including corn, wheat, rice, and soybean. The rapid, P-450 based N-dealkylation
of the sulfamoylcarboxamide side chain contributes to the unusual broad crop
selectivity of saflufenacil, especially in grass crops such as corn, wheat, and rice.
Bayer introduced 2-aryl-1,2,4, triazine-3,5-diones with the 2,4-dihalo-5-aminoal-
kylsulfonylphenyl, such as compounds 102 [114] and 103 [115], the aromatic
substitution pattern reminiscent of sulfentrazone 15 (Figure 3.29).
In addition, in 2003 Bayer introduced phenyluracil derivatives with heteroaryl-
methyleneoxy groups at the 5 position of the phenyl ring, as in compound 104 [116],
and N-(thiocarbonylaminophenyl)uracils such as compound 105 [117] (Figure 3.30).
Isagro claimed good pre-emergence and post-emergence weed control at rates
as low as 15 g a.i. ha−1 for a number of Protox inhibitors with a wide variety of
groups in the five aromatic position of 2,4-dihalo-5-substituted uracils, such as
compounds 107 and 108 [118] (Figure 3.31). Among the weeds controlled were
bedstraw, barnyardgrass, redroot pigweed, prickly sida, and velvetleaf, with crop
selectivity in rice, wheat, barley, corn, and soybean.
The research groups at Central South University and Hunan Research Institute
of Chemical Industry in Changsha, Hunan, China have reported the herbicidal
F O
F O N
N Cl N CF3
Cl N CF3
O O O
H O
N O O
O
N O S N
H
97 N 98
F O O F O
N N N
Cl N CF3 NC N CF3 Cl N CF3
H H
N O HN O H N SO2 O
N SO2 O SO2 N
99 N O
100 101
Figure 3.28 Protox inhibitors with diverse groups at the aromatic meta position.
F O
N F O
Cl N O N
Cl Cl N CF3
N Cl
N O
Cl S O N O O
O Cl S O
O
102 103
F O F O
N N
Cl N CF3 Cl N CF3
O O O
O O S N O
O N H
S
104 N
105
activity of a number of isoindoline-1,3-diones molecules such as compound 109, and

compared their biological activity to that of flumioxazin 59 (Figure 3.32). Compound
109 provided >80% control at 75 g a.i. ha−1 in both pre- and post-emergence
treatments against broadleaf weeds such as velvetleaf, common lambsquarter, and
redroot pigweed, and against grass weeds such as large crabgrass, barnyardgrass,
and green foxtail. Compound 109 was reported to be safe on cotton and corn
at a rate of 150 g a.i. ha−1 when applied pre-emergently, and it also provided
good wheat safety when applied post-emergently at 7.5–30 g a.i. ha−1 . The IC50
(inhibitory concentration, in g a.i. ha−1 , to obtain 50% growth inhibition) values
for the post-emergence control of velvetleaf and crabgrass were given for 109
(IC50 = 3.6 and 4.8, respectively), and compared to those of flumioxazin (IC50 = 1.0
F O F O
N
N
Cl N CF3
Cl N CF3
O
N N O
O O N
N N
N
107 108
O F O F
O O
O N N O N N
O O
59 109 F
Flumioxazin
Cl F Cl F
X N
O N Y O N N
O O N
N
119
116, X-Y = -CO-N(CH3)-
117, X-Y = -N=N-
118, X-Y = -CO-CH2-
Figure 3.32 Chemical structure of benzoxazine

isoindoline-1,3-diones and further Protox inhibitors
with a bicyclic heterocyclic ring.
and 2.5) [119]. The research groups at Shenyang Institute of Chemical Engineering,
Nankai University, and Lanzhou University have extensively explored further Protox
inhibitors with a bicyclic heterocyclic ring, such as benzotriazinones 116 [120–122],
quinazolinediones 117 [123], isoquinolinediones 118 [124], and pyrazolotriazinones
119 [125–127].
Bencarbazone 114 [128] is a recently developed Protox inhibitor triazolinone
herbicide from Arysta LifeScience for the post-emergence control of broadleaf
weeds in cereals and corn. It provides a good control of bedstraw, velvetleaf,
redroot pigweed, common lambsquarter, and speedwell at application rates of
20–30 g a.i. ha−1 . Bencarbazone 114 has many of the features associated with
Protox herbicides, particularly those of the Protox herbicide sulfentrazone. The
most striking chemical feature of bencarbazone 114 is the replacement of the
phenyl 4-chloro group with a thioamide group.
Bencarbazone 114 can be prepared in several steps from the nucleophilic
displacement reaction of 2,4,5-trifluorobenzonitrile 110 with 4-methyl-3-
trifluoromethyl-1,2,4-triazolin-5-one 111 to give 1-(4-cyano-2,5-difluorophenyl)-
HN N CH3
F N
CF3 F O F O
EtSO2NH
111
NC F N N CH3 CH3
NC NC N N
K2CO3 N K2CO3 N
F F CF3 HN O CF3
DMSO DMSO
110 112 S
O 113
F O H2S
S
N N CH3
H2N N
HN O CF3
S
O
114
Bencarbazone
Scheme 3.5 Chemical structure and synthesis of bencarbazone.
4-methyl-3-trifluoromethyl-1,2,4-triazolin-5-one 112. The reaction of compound

112 with ethanesulfonamide in the presence of a base such as potassium carbonate
gives compound 113 which, on reaction with hydrogen sulfide, gives bencarbazone
114 [129] (Scheme 3.5).
3.5
Toxicology
The toxicology of Protox inhibitors has been discussed previously [15, 130]. The ad-
dition of high doses of the Protox-inhibiting herbicides fomesafen, oxyfluorfen and
oxadiazon to the diet of mice was shown to increase the porphyrin contents of the
liver, bile, and feces. Porphyrin accumulation induced by high-dose, short-term her-
bicide treatment was reversible, however, and within days of treatment withdrawal
the porphyrin levels had returned to normal. Based on these findings – namely, the
high dose required to elicit an effect and the reversible nature of that effect – it was
concluded that the toxicological risk resulting from exposure to Protox-inhibiting
herbicides is small [130].
3.6
Summary
Protox-inhibiting herbicides continue to be an area of interest to agrochemical

companies, with the majority of effort focused on fine-tuning the 5 position of the
aromatic ring of N-phenyluracil to gain both a particular crop/weed/application
method as well as a proprietary position.
References 191
In addition to the Protox herbicide activity reported in the patent literature,

there is continued interest in understanding the SARs of Protox inhibitors
[131–133]. Research efforts continue to be devoted to the development of Pro-
tox inhibitor-resistant crops [134]. In 1999, Syngenta announced its discovery of a
novel gene technology, under the trademark Acuron™, that provides crops with
tolerance to Protox inhibitors.
Finally, weed shifts observed in genetically modified crops, caused by the
development of weed resistance to the widely used herbicide glyphosate, will
offer market opportunities for herbicides with other modes of action, such as the
Protox-inhibiting herbicides.
References
1. Cartwright, D. and Collins, D.J. (1981) Wyle, M.J. (1991) Proc. Brighton Crop
US Patent 4,285,723. Prot. Conf. – Weeds, 1, 77.
2. Colby, S.R., Barnes, J.W., Sampson, 9. Zagar, C., Witschel, M., and Landes, A.
T.A., Shoham, J.L., and Osborn, D.J. (2004) WO 2004080183.
(1983) 10th International Congress 10. Liebl, R., Walter, H., Bowe, S., Holt,
of Plant Protection, BCPC, Croydon, T., and Westberg, D. (2008) Weed Sci.
England, Vol. 1, pp. 295–302. Soc. Am. Conf., 48, 120.
3. Theodoridis, G. (1989) US Patent 11. Iwataki, I. (1999) in Peroxidizing
4,868,321. Herbicides (eds P. Böger and K.
4. Theodoridis, G., Hotzman, F.W., Wakabayashi), Springer-Verlag, Berlin,
Scherer, L.W., Smith, B.A., Tymonko, pp. 73–89.
J.M., and Wyle, M.J. (1990) Pestic. Sci., 12. Nandula, V.K., Reddy, K.N., Duke,
30 (3), 259–274. S.O., and Poston, D.H. (2005) Outlooks
5. Theodoridis, G., Hotzman, F.W., Pest Manage., 16 (4), 183–187.
Scherer, L.W., Smith, B.A., Tymonko, 13. Matsumoto, H. (2002) in Herbi-
J.M., and Wyle, M.J. (1992) in Syn- cide Classes in Development (eds P.
thesis and Chemistry of Agrochemicals Böger, K. Wakabayashi, and K. Hirai),
III, ACS Symposium Series, Vol. 504 Springer-Verlag, Berlin, pp. 151–161.
(eds D.R. Baker, J.G. Fenyes, and J.J. 14. Wakabayashi, K. and Böger, P. (1999)
Steffens), American Chemical Society, in Peroxidizing Herbicides (eds P. Böger
Washington, DC, pp. 122–133. and K. Wakabayashi), Springer-Verlag,
6. Theodoridis, G. (1989) US Patent Berlin, pp. 163–190.
4,818,275. 15. Dayan, F.E. and Duke, S.O. (1997) in
7. Theodoridis, G., Baum, J.S., Hotzman, Herbicide Activity: Toxicology, Biochem-
F.W., Manfredi, M.C., Maravetz, L.L., istry and Molecular Biology (eds R.M.
Lyga, J.W., Tymonko, J.M., Wilson, Roe, J.D. Burton, and R.J. Kuhr), IOS
K.R., Poss, K.M., and Wyle, M.J. Press, Amsterdam, pp. 11–35.
(1992) in Synthesis and Chemistry of 16. Scalla, R. and Matringe, M. (1994) Rev.
Agrochemicals III, ACS Symposium Weed Sci., 6, 103–132.
Series, Vol. 504 (eds D.R. Baker, J.G. 17. Jacobs, J.M. and Jacobs, N.J. (1994)
Fenyes, and J.J. Steffens), American Am. Chem. Soc. Symp. Ser, 559,
Chemical Society, Washington, DC, pp. 105–119.
134–146. 18. Scalla, R., Matringe, M., Camadro,
8. Van Saun, W.A., Bahr, J.T., Crosby, J.M., and Labbe, P. (1990) Z. Natur-
G.A., Fore, Z.A., Guscar, H.L., forsch., 45 (5), 503–511.
Harnish, W.N., Hooten, R.S., Marquez, 19. Duke, S.O., Becerril, J.M., Sherman,
M.S., Parrish, D.S., Theodoridis, G., T.D., Lydon, J., and Matsumoto, H.
Tymonko, J.M., Wilson, K.R., and (1990) Pestic. Sci., 30 (4), 367–378.
20. Matringe, M. and Scalla, R. (1988) Pest. (1995) in Synthesis and Chemistry of
Biochem. Physiol., 32 (2), 164–172. Agrochemicals IV, ACS Symposium
21. Matringe, M. and Scalla, R. (1988) Series, Vol. 584 (eds D.R. Baker, J.G.
Plant Physiol., 86 (2), 619–622. Fenyes, and G.S. Basarab), American
22. Witkowski, D.A. and Halling, B.P. Chemical Society, Washington, DC,
(1988) Plant Physiol., 87 (3), 632–637. pp. 122–135.
23. Lydon, J. and Duke, S.O. (1988) Pestic. 39. Wepplo, P., Birk, J.H., Lavanish, J.M.,
Biochem. Physiol., 31 (1), 74–83. Manfredi, M., and Nielsen, D.R. (1995)
24. Matringe, M., Camadro, J.M., Labbe, in Synthesis and Chemistry of Agro-
P., and Scalla, R. (1989) Biochem. J., chemicals IV, ACS Symposium Series,
260 (1), 231–235. Vol. 584 (eds D.R. Baker, J.G. Fenyes,
25. Matringe, M., Camadro, J.M., Labbe, and G.S. Basarab), American Chem-
P., and Scalla, R. (1989) FEBS Lett., 245 ical Society, Washington, DC, pp.
(1–2), 35–38. 149–160.
26. Wilson, H.F. (1963) US Patent 40. Karp, G.M., Condon, M.E., Arthen,
3,080,225. F.J., Birk, J.H., Marc, P.A., Hunt, D.A.,
27. Metivier, J. and Boesch, R. (1968) US Lavanish, J.M., and Schwindeman,
Patent 3,385,862. J.A. (1995) in Synthesis and Chemistry
28. Burgaud, L., Deloraine, J., Guillot, M., of Agrochemicals IV, ACS Symposium
and Riottot, M. (1970) Proceedings of the Series, Vol. 584 (eds D.R. Baker, J.G.
10th British Weed Control Conference, Fenyes, and G.S. Basarab), American
Brighton, UK, British Crop Protection
Chemical Society, Washington, DC,
Council, Vol. 2, pp. 745–751.
pp. 136–148.
29. Matsui, K., Kasugai, H., Matsuya, K.,
41. Moedritzer, K., Allgood, S.G.,
and Aizawa, H. (1972) FR 2119703.
Charumilind, P., Clark, R.D., Gaede,
30. Anderson, R.J., Norris, A.E., and Hess,
B.J., Kurtzweil, M.L., Mischke, D.A.,
F.D. (1994) in Porphyric Pesticides:
Parlow, J.J., Rogers, M.D., Singh,
Chemistry, Toxicology, and Pharmaceu-
R.K., Stikes, G.L., and Webber, R.K.
tical Applications, ACS Symposium
(1992) in Synthesis and Chemistry of
Series, Vol. 559 (eds S.O. Duke and
Agrochemicals III, ACS Symposium
C.A. Rebeiz), American Chemical
Series, Vol. 504 (eds D.R. Baker, J.G.
Society, Washington, DC, pp. 18–33.
Fenyes, and J.J. Steffens), American
31. Hirai, K. (1999) in Peroxidizing
Herbicides (eds P. Böger and K. Chemical Society, Washington, DC, pp.
Wakabayashi), Springer-Verlag, Berlin, 147–160.
pp. 15–70. 42. Boesch, R. and Metivier, J. (1965) FR
32. Yih, R.Y. and Swithenbank, C. (1975) J. 1394774.
Agric. Food Chem., 23 (3), 592–593. 43. Boesch, R. and Metivier, J. (1968) GB
33. Theissen, R.J. (1972) US Patent 1110500.
3,652,645. 44. Blind, A., Cassal, J.M., and Boesch, R.
34. Bayer, H.O., Swithenbank, C., and Yih, (1971) DE 2039397.
R.Y. (1975) US Patent 3,928,416. 45. Wakabayashi, O., Matsuya, K., Ota, H.,
35. Johnson, W.O., Kollman, G.E., Jikihara, T., and Susuki, S. (1980) DE
Swithenbank, C., and Yih, R.Y. (1978) 3013162.
Agric. Food Chem., 26 (1), 285–286. 46. Goddard, S.J. (1976) US Patent
36. Johnson, W.O. (1980) EP 20052. 536,322.
37. Phillips McDougall AgriService (2002) 47. Nagano, E., Hashimoto, S., Yoshida,
Market Report. Vineyard Business Cen- R., Matsumoto, H., Oshio, H., and
tre, Saughland, Pathhead, Midlothian Kamoshita, K. (1982) EP 61741.
EH37 5XP, UK. 48. Dickmann, R., Melgarejo, J., Loubiere,
38. Condon, M.E., Alvarado, S.I., Arthen, P., and Montagnon, M. (1997) Proc.
F.J., Birk, J.H., Brady, T.E., Crews, Brighton Crop Prot. Conf. – Weeds, 1,
A.D., Marc, P.A., Karp, G.M., Lavanish, 51–57.
J.M., Nielsen, D.R., and Lies, T.A. 49. Boesch, R. (1972) DE 2227012.
References 193
50. Poss, K.M. (1992) US Patent H., Unai, T., Yamaguchi, M., and
5,125,958. Abe, H. (1995) Plant Cell Physiol., 36,
51. Theodoridis, G., Bahr, J.T., Davidson, 625–632.
B.L., Hart, S.E., Hotzman, F.W., Poss, 64. Hirai, K., Futikami, T., Murata, A.,
K.M., and Tutt, S.F. (1995) in Syn- Hirose, H., and Yokota, M. (1989) US
thesis and Chemistry of Agrochemicals Patent 4,818,272.
IV, ACS Symposium Series, Vol. 584 65. Hirai, K., Yano, T., Ugal, S.,
(eds D.R. Baker, J.G. Fenyes, and G.S. Yoshimura, T., and Hori, M. (2001)
Basarab), American Chemical Society, J. Pestic. Sci., 26 (2), 194–202.
Washington, DC, pp. 90–99. 66. Theodoridis, G. (1997) Pestic. Sci., 50,
52. Van Saun, W.A., Bahr, J.T., 283–290.
Bourdouxhe, L.J., Gargantiel, F.J., 67. Schaefer, P., Hamprecht, G.,
Hotzman, F.W., Shires, S.W., Sladen, Heistracher, E., Koenig, H., Klintz, R.,
N.A., Tutt, S.F., and Wilson, K.R. Muenster, P., Rang, H., Westphalen,
(1993) Proc. Brighton Crop Prot. Conf. – K.-O., Gerber, M., and Walter, H.
Weeds, 1, 19–28. (1998) US Patent 5,783,522.
53. Prosch, S.D., Ciha, A.J., Grogna, R., 68. Schaefer, P., Hamprecht, G., Puhl,
Hamper, B.C., Feucht, D., and Dreist, M., Westphalen, K.-O., and Zagar, C.
M. (1997) Proc. Brighton Crop Prot. (2003) Chimia, 57 (11), 715–719.
Conf. – Weeds, 1, 45–50. 69. Wenger, J., Winternitz, P., and Zeller,
54. Miura, Y., Ohnishi, M., Mabuchi, T.,
M. (1988) WO 8810254.
and Yanai, I. (1993) Proc. Brighton Crop
70. Bell, A. (1990) US Patent 4,943,309.
Prot. Conf. – Weeds, 1, 35–40.
71. Theodoridis, G., Bahr, J.T., Crawford,
55. Miura, Y., Takaishi, H., Ohnishi, M.,
S., Dugan, B., Hotzman, F.W.,
and Tsubata, K. (2003) Yuki Gosei
Maravetz, L.L., Sehgel, S., and Suarez,
Kagaku Kyokaishi, 61 (1), 4–15.
D.P. (2002) in Synthesis and Chemistry
56. Wolf, A.D. (1979) US Patent
of Agrochemicals VI, ACS Symposium
4,139,364.
57. Shapiro, R., DiCosimo, R., Hennessey,
Fenyes, G.P. Lahm, T.P. Selby, and
S.M., Stieglitz, B., Campopiano, O.,
T.M. Stevenson), American Chemical
and Chiang, G.C. (2001) Org. Process
Res. Dev., 5 (6), 593–598.
58. Nagano, E., Hashimoto, S., Yoshida, 72. Theodoridis, G. (1994) US Patent
R., Matsumoto, H., and Kamoshita, K. 5,344,812.
(1984) US Patent 4,484,941. 73. Theodoridis, G., Bahr, J.T., Hotzman,
59. Nagano, E., Hashimoto, S., Yoshida, F.W., Sehgel, S., and Suarez, D.P.
R., Matsumoto, H., and Kamoshita, K. (2000) Crop Prot., 19, 533–535.
(1988) US Patent 4,770,695. 74. Kunz, W., Siegrist, U., and Baumeister,
60. Grossmann, K. and Schiffer, H. (1999) P. (1995) WO 9532952.
Pestic. Sci., 55 (7), 687–695. 75. Katayama, T., Kawamura, S.,
61. Miyazawa, T., Kawano, K., Shigematsu, Sanemitsu, Y., and Mine, Y. (1997)
S., Yamaguchi, M., Matsunari, K., WO 9707104.
Porpiglia, P., and Gutbrod, K.G. (1993) 76. Tohyama, Y. and Sanemitsu, Y. (2003)
Proc. Brighton Crop Prot. Conf. – Weeds, US Patent 6,537,948.
1, 23–28. 77. Theodoridis, G. (1998) US Patent
62. Satow, J., Fukuda, K., Itoh, K., and 5,798,316.
Nawamaki, T. (1995) in Synthesis and 78. Furukawa, T. (1999) EP 943610.
Chemistry of Agrochemicals IV, ACS 79. Nandihalli, U. and Duke, S.O. (1994)
Symposium Series, Vol. 584 (eds in Porphyric Pesticides: Chemistry, Toxi-
D.R. Baker, J.G. Fenyes, and G.S. cology, and Pharmaceutical Applications,
Basarab), American Chemical Society, ACS Symposium Series, Vol. 559 (eds
Washington, DC, pp. 100–113. S.O. Duke and C.A. Rebeiz), American
63. Shimizu, T., Hashimoto, N., Chemical Society, Washington, DC, pp.
Nakayama, I., Nakao, T., Mizutani, 133–146.
80. Dayan, F.E., Reddy, K.N., and Duke, J.G. Fenyes, G.S. Basarab, and D.A.
S.O. (1999) in Peroxidizing Herbicides Hunt), American Chemical Society,
(eds P. Böger and K. Wakabayashi), Washington, DC, pp. 67–78.
Springer-Verlag, Berlin, pp. 141–161. 93. Dorfmeister, G., Franke, H., Geisler, J.,
81. Theodoridis, G., Poss, K.M., and Hartfiel, U., Bohner, J., and Rees, R.
Hotzman, F.W. (1995) in Synthesis (1994) WO 09408999.
and Chemistry of Agrochemicals IV, 94. Kunz, W., Nebel, K., and Wenger, J.
ACS Symposium Series, Vol. 584 (eds (1999) WO 9952892.
D.R. Baker, J.G. Fenyes, and G.S. 95. Nebel, K., Kunz, W., and Wenger, J.
Basarab), American Chemical Society, (1999) WO 9952893.
Washington, DC, pp. 78–89. 96. Matsumoto, H. (2002) in Herbi-
82. Nandihalli, U., Duke, M.V., and Duke, cide Classes in Development (eds P.
S.O. (1992) Pestic. Biochem. Physiol., 43 Böger, K. Wakabayashi, and K. Hirai),
(3), 193–211. Springer-Verlag, Berlin, pp. 255–289.
83. Uraguchi, R., Sato, Y., Nakayama, A., 97. Koch, M., Breithaupt, C., Kiefersauer,
Sukekawa, M., Iwataki, I., Böger, P., R., Freigang, J., Huber, R., and
and Wakabayashi, K. (1997) J. Pestic. Messerschmidt, A. (2004) EMBO J.,
Sci., 22 (4), 314–320. 23, 1720–1728.
84. Ohta, H., Jikihara, T., Wakabayashi, K., 98. Yamada, O., Yanagi, M., Futatsuya, F.,
and Fujita, T. (1980) Pestic. Biochem. and Kobayashi, K. (1981) GB 2071100.
Physiol., 14 (2), 153–160. 99. Miyamoto, Y., Ikeda, Y., and
85. Fujita, T. and Nakayama, A. (1999) in
Wakabayashi, K. (2003) J. Pestic. Sci.,
Peroxidizing Herbicides (eds P. Böger
28, 293–300.
and K. Wakabayashi) Springer-Verlag,
100. Meazza, G., Bettarini, F., La Porta, P.,
Berlin, pp. 92–139.
Piccardi, P., Signorini, E., Portoso, D.,
86. Theodoridis, G., Baum, J.S., Chang,
and Fornara, L. (2004) Pest Manage.
J.H., Crawford, S.D., Hotzman, F.W.,
Sci., 60 (12), 1178–1188.
Lyga, J.W., Maravetz, L.L., Suarez, D.P.,
101. Shimoharada, H., Tsukamoto, M.,
and Hatterman-Valenti, H. (1998) in
Kikugawa, H., and Kitahara, Y. (2005)
Synthesis and Chemistry of Agrochemi-
US Patent Application Publication
cals V, ACS Symposium Series, Vol.
2005/0245399.
686 (eds D.R. Baker, J.G. Fenyes, G.S.
102. Hwang, I.T., Kim, H.R., Jeon, D.J.,
Basarab, and D.A. Hunt), American
Chemical Society, Washington, DC, pp. Hong, K.S., Song, J.H., Chung, C.K.,
55–66. and Cho, K.Y. (2005) Pest Manage. Sci.,
87. Lyga, J.W., Chang, J.H., Theodoridis, 61 (5), 483–490.
G., and Baum, J.S. (1999) Pestic. Sci., 103. Hwang, I.T., Hong, K.S., Choi, J.S.,
55, 281–287. Kim, H.R., Jeon, D.J., and Cho, K.Y.
88. Nagano, E., Haga, T., Sato, R., and (2004) Pestic. Biochem. Physiol., 80 (2),
Morita, K. (1987) US Patent 4,640,707. 123–130.
89. Ganzer, M., Franke, W., Dorfmeister, 104. Nagano, E., Takemoto, I., Fukushima,
G., Johann, G., Arndt, F., and Rees, R. M., Yoshida, R., and Matsumoto, H.
(1989) EP 311135. (1984) GB 2127410.
90. Crawford, S.D., Maravetz, L.L., and 105. Li, B., Xu, J., and Man, Y. (2004) CN
Theodoridis, G. (1997) US Patent 1515560.
5,661,108. 106. Schallner, O., Hoischen, D., Drewes,
91. Crawford, S.D., Maravetz, L.L., M.W., Dahmen, P., Feucht, D., and
Theodoridis, G., and Dugan, B. (2000) Pontzen, R. (2003) WO 2003006461.
US Patent 6,077,812. 107. Tsukamoto, M., Gupta, S., Wu, S.-Y.,
92. Konz, M.J., Wendt, H.R., Cullen, T.G., Ying, B.-P., and Pulman, D.A. (2003)
Tenhuisen, K.L., and Fryszman, O.M. US Patent 6,573,218.
(1998) in Synthesis and Chemistry of 108. Tsukamoto, M., Kikugawa, H., and
Agrochemicals V, ACS Symposium Sano, M. (2004) US Patent Application
Series, Vol. 686 (eds D.R. Baker, 2004157738.
References 195
109. Tsukamoto, M. and Read, M. (2004) Congress: Crop Science and Tech-
US Patent 6,770,597. nology, Glasgow, United Kingdom,
110. Puhl, M., Hamprecht, G., Reinhard, November 10–12, 2003, Vol. 1, pp.
R., Sagasser, I., Seitz, W., Zagar, C., 619–622.
Witschel, M., and Landes, A. (2004) 123. Li, B. and Xu, J. (2002) CN 1355164.
WO 2004009561. 124. Li, B., Wu, H., Cui, D., Yu, H., Xu, J.,
111. Grossmann, K., Niggeweg, R., and Yang, H. (2005) CN1687061.
Christiansen, N., Looser, R., and 125. Yang, H., Li, H., Zou, X., Zhu, Y., Hu,
Ehrhardt, T. (2010) Weed Sci., 58, F., Niu, Z., Liu, B., Li, Y., Liu, P., and
1–9. Song, X. (2008) CN 101215289.
112. Reinhard, R., Hamprecht, G., Puhl, 126. Lei, B., Li, J., Lu, J., Du, J., Liu, H., and
M., Sagasser, I., Seitz, W., Zagar, C., Yao, X. (2009) J. Agric. Food Chem., 57
Witschel, M., and Landes, A. (2004) (20), 9593–9598.
WO 2004007467. 127. Li, H., Zhu, Y., Song, X., Hu, F.,
113. Hamprecht, G., Puhl, M., Reinhard, R., Liu, B., Li, Y., Niu, Z., Liu, P., Wang,
Seitz, W., Zagar, C., Witschel, M., and Z., Song, H., Zou, X., and Yang, H.
Landes, A. (2004) WO 2004089914. (2008) J. Agric. Food Chem., 56 (20),
114. Linker, K.-H., Andree, R., Hoischen, 9535–9542.
D., Schwarz, H.-G., Kluth, J., Drewes, 128. Wroblowsky, H.-J. and Thomas, R.
M.W., Feucht, D., and Pontzen, R. (1997) WO 9733876.
(2004) DE 10255416. 129. Linker, K.-H., Findeisen, K., Andree,
115. Andree, R., Drewes, M.W., Dahmen, R., Drewes, M.-W., Lender, A.,
P., Feucht, D., Pontzen, R., and Loesel, Schallner, O., Haas, W., Santel, H.-J.,
P. (2003) WO 2003043994. and Dollinger, M. (2002) US Patent
116. Schwarz, H.-G., Andree, R., Hoischen, 6,451,736.
D., Linker, K.-H., Drewes, M.W., 130. Krijt, J., Vokurka, M., Sanitrák, J.,
Dahmen, P., Feucht, D., and Pontzen, and Janousek, V. (1994) in Porphyric
R. (2003) WO 2003099009. Pesticides: Chemistry, Toxicology, and
117. Schwarz, H.-G., Andree, R., Pharmaceutical Applications, ACS Sym-
Hoischen, D., Kluth, J., Linker, posium Series, Vol. 559 (eds S.O. Duke
K.-H., Vidal-Ferran, A., Drewes, M.W., and C.A. Rebeiz), American Chem-
Dahmen, P., Feucht, D., and Pontzen, ical Society, Washington, DC, pp.
R. (2003) WO 2003093244. 247–254.
118. Meazza, G., Paravidino, P., Bettarini, 131. Sung, N.-D., Song, J.-H., and Park,
F., Fornara, L. (2004) WO 2004056785. K.-Y. (2004) Han’guk Eungyong,
119. Huang, M.-Z., Huang, K.-L., Ren, Sangmyong Hwahakhoeji, 47 (4),
Y.-G., Lei, M.-X., Huang, L., Hou, 414–421.
Z.-K., Liu, A.-P., and Ou, X.-M. (2005) 132. Wan, J., Zhang, L., Yang, G., and
J. Agric. Food Chem., 53, 7908–7914. Zhan, C.-G. (2004) J. Chem. Inf. Com-
120. Li, B. and Hsu, A.C.-T. (2001) US put. Sci., 44, 2099–2105.
Patent 6,303,542. 133. Zhang, L., Wan, J., and Yang, G. (2004)
121. Xu, J. and Li, B. (2002) CN 1345722. Bioorg. Med. Chem., 12, 6183–6191.
122. Li, B. and Yang, H.Z. (2003) Congress 134. Li, X. and Nicholl, D. (2005) Pest
Proceedings – BCPC International Manage. Sci., 61 (3), 277–285.
197
4
Herbicides with Bleaching Properties
4.1
Phytoene Desaturase Inhibitors
Gerhard Hamprecht1 and Matthias Witschel
4.1.1
Introduction
Herbicidal activity through inhibition of the enzyme phytoene desaturase (PDS)

is easily recognized by the striking whitening effect on the tissues of newly
grown plant leaves in sunlight. Ultimately, these symptoms led to such herbicides
being classified as ‘‘bleaching herbicides,’’ based on their ability to interfere with
the biosynthesis of photosynthetic pigments, chlorophylls, or carotenoids [1–3].
Although norflurazon (as the oldest representative of the class) was first introduced
by Sandoz as a spin-off of phenylpyridazinone chemistry (see Section 4.1.4.6)
during the late 1960s, it was almost two decades before the mode of action (MoA) of
these compounds – that is, the inhibition of PDS and consequently of carotenoid
biosynthesis – was fully realized. Since then, due to the low application rates of the
compounds, their lack of resistance in the field – which could only be introduced
genetically [4, 5] – and their favorable mammalian toxicity, industrial investigations
have been focused on this new MoA, and this in turn had led to the development
of several potent herbicides for use in modern agriculture.
4.1.2
Carotenoid Biosynthesis and Phytotoxic Effects of Bleaching Herbicides
4.1.2.1 Targets for Bleaching Herbicides

The bleaching of leaves is most likely the result of photo-oxidative events generated
within the plant cell or chloroplast, that lead to the destruction of plant pigments or
to a direct inhibition of pigment biosynthesis, whereby carotenoid and chlorophyll
formation is blocked [6].
In the case of carotenoid biosynthesis, plastoquinone is involved as an electron
acceptor (this is also encountered in photosynthetic electron transport) [2]. One
important precursor in the synthesis of plastoquinone, which also serves as
1
Retired
198 4 Herbicides with Bleaching Properties
a cofactor for the PDS enzyme, is homogentisic acid, which is formed from
4-hydroxyphenyl-pyruvate by the action of the enzyme 4-hydroxyphenylpyruvate
dioxygenase (HPPD) [7, 8]. The inhibition of plastoquinone biosynthesis through
HPPD blockade will, therefore, result in herbicidal and bleaching phytotoxicity
symptoms similar to those of PDS inhibition [7, 9]. However, HPPD inhibition
induces a reduced growth and chlorosis, which can be antagonized by homogentisic
acid. Additionally, the synthesis of α-tocopherol – a scavenger of activated singlet
oxygen – is blocked, leading ultimately to the oxidation of the D1 protein chain
with the nonheme iron of the photosystem II (PS II) reaction center, oxidative
tissue damage, and bleaching [7]. Details of the discovery of HPPD herbicides and
the structural requirements for herbicidal diketones have been described [10, 11]
(see also Chapter 4.2). In addition to being inhibitors of both PDS and HPPD,
other herbicides have became known for their bleaching properties, including
amitrole (an ‘‘old-timer’’ herbicide first applied during the 1950s) and clomazone,
both of which inhibit an early step in carotenoid biosynthesis [7, 12, 13].
4.1.2.2 Carotenoids: Properties and Functions

Carotenoids are constituents of the photosynthetic reaction centers and the
light-harvesting complex (LHC) of the antennae [14], where they serve as redox
intermediates in the electron-transfer processes of PS II [15] and also as accessory
pigments in light harvesting [6, 16].
The reaction centers are rich in β-carotene, and in some plant species may also
contain α-carotene. In contrast, the peripheral LHC contains several xanthophylls,
including lutein, violaxanthin, and neoxanthin [6].
Carotenes play a vital role in protecting the chloroplast against photo-oxidative
damage. At high light intensities, the chlorophyll molecules are exposed to more
light than they can direct into electron transport; this leads to chlorophyll fluo-
rescence being employed as one means of energy offtake [16], and intersystem
crossing of the excited singlet chlorophyll to a longer-lived triplet state as a second
means [6]. This triplet-state chlorophyll can use its energy to convert molecular
oxygen into the highly active and destructive singlet oxygen (1 O2 ), which in turn
will cause the destruction of lipids, membranes, nucleic acids, and even of whole
tissues. As a result, the degradation of chlorophyll – depending on the intensity of
illumination – leads to typical bleaching symptoms in plants and a decline in their
photosynthetic activity. Generally, only the newly formed green leaves are affected,
and these fade away as a result of the bleaching. Carotenoids protect against such
photosensitized damage by directly quenching the excitation energy of triplet-state
chlorophyll. The carotenoid molecule can also quench any 1 O2 build-up, by pro-
ducing carotenoid triplets that decay harmlessly, so as to develop heat rather than
toxic products [6, 16].
Besides their function as light collectors and photoprotectors, carotenoids also
have important effects as membrane-stabilizing agents in chloroplasts. The xan-
thophyll violaxanthin and its enzymatic de-epoxidation products, antheraxanthin
and zeaxanthin, partition between the LHCs of PS I and PS II and the lipid phase
of the thylakoid membranes. This brings about a decrease in membrane fluidity,
an increase in membrane thermostability, and a lowered susceptibility to lipid

peroxidation [17].
4.1.2.3 Carotenoid Biosynthesis in Higher Plants
4.1.2.3.1 The Biosynthetic Pathway The carotenoids of higher plants, algae, and
fungi are C40 tetraterpenes that are biosynthesized by the well-known isoprenoid
pathway [1, 6, 7, 9, 18, 19]. The early steps of the process, which involve formation
of the C5 isoprenoid units and the subsequent synthesis of prenyldiphosphate
intermediates, are common to all classes of terpenoids.
4.1.2.3.2 Early Steps and Formation of Phytoene The first specific precursor for
terpenoids in the cytoplasma is the C6 molecule mevalonic acid (MVA), which is
constructed via the classical acetate/mevalonate pathway and converted by a series
of phosphorylating and decarboxylation reactions into C5 isopentenyldiphosphate
(IPP), the universal building block for chain elongation up to C20 . In the chloro-
plasts, the biosynthesis of IPP starts from glyceraldehyde-3-phosphate and pyruvate
to give 1-deoxy-d-xylulose-5-phosphate (DOXP) via the non-mevalonate pathway
as a recently detected alternative IPP route [20]. The reaction is catalyzed by the
enzyme DOXP synthase, which can be inhibited by a metabolite of the herbicide
clomazone [13, 21].
Following the 1,3-allylic isomerization of IPP to dimethylallyl pyrophosphate
(DMAPP), catalyzed by the enzyme IPP isomerase, a further IPP unit is added
to yield C10 geranylpyrophosphate (GPP). The subsequent addition of a second
or third molecule of IPP leads to the formation of C15 farnesyl pyrophosphate
(FPP) and the C20 geranylgeranyl pyrophosphate (GGPP). The chain elongation is
a head-to-tail condensation process, which forms carbon–carbon bonds between
C-4 of IPP and C-1 of the allylic substrate.
4.1.2.3.3 The Specific Carotene Pathway The stages unique to carotenoid biosyn-
thesis start with the formation of the C40 phytoene (7,8,11,12,7 ,8 ,11 ,12 -octahydro
- , -carotene) from two molecules of GGPP via the C40 intermediate prephy-
toene pyrophosphate (PPPP), from which phytoene with its central double bond
is directly derived (Figure 4.1.1). Phytoene, which is colorless, is formed by the
head-to-head condensation of two molecules of GGPP (all-trans) and obtained
in all photosynthetic organisms as the 15-cis-phytoene [22]. The condensation is
catalyzed by the enzymes PPPP synthase and phytoene synthase. Desaturation
starts from the symmetrical phytoene on both of its identical halves to give, in a
first step, phytofluene as an intermediate and then ζ -carotene, catalyzed by the
enzyme PDS. Further desaturation of the latter occurs by a stepwise sequence
of reactions to form neurosporene and the maximally desaturated lycopene. At
each stage, two anti-hydrogen atoms from adjacent functions are lost by oxidation
to extend the chromophore by two double bonds. Starting with three conjugated
double bonds in phytoene, the final product – lycopene – has 11 such bonds. The
other enzyme involved is ζ -carotene desaturase (ZDS), which catalyzes a closely
similar desaturation to PDS (Figure 4.1.1) [23].
IPP
IPP- PPPP
Isomerase Prenyl transferase synthase
DMAPP GPP FPP GGPP PPPP
Phytoene-
IPP IPP IPP synthase
Phytoene
Phytofluene Phytoene-
desaturase
z-Carotene
z-Carotene-
Neurosporene desaturase
Lycopene
e-Cyclase b-Cyclase
d- Carotene g - Carotene
e-Cyclase
b-Cyclase b-Cyclase
a-Carotene b-Carotene
Lutein Zeaxanthin
Antheraxanthin
Violaxanthin
DMAPP dimethylallyl pyrophosphate; FPP farnesyl pyrophosphate; GPP geranyl

pyrophosphate; GGPP geranylgeranyl pyrophosphate; IPP isopentenyl
pyrophosphate; PPPP prephytoene pyrophosphate.
Figure 4.1.1 Pathway of carotene biosynthesis from IPP to α- and β-carotene.
4.1.2.3.4 Cyclization Lycopene is the starting building block for the cyclization
reactions to the final α- and β-carotenes via their intermediates δ-carotene (with one
ε-ionone ring) and γ -carotene (with one β-ionone ring), respectively. Two different
enzymes are responsible for the β- and ε-cyclizations, referred to as lycopene β- and
ε-cyclase, respectively [24, 25].
In contrast to the 15-cis phytoene, the colored, fully desaturated carotenoids
present in photosynthetic tissues are usually in the (all-ε) all-trans form, for example,
β-carotene or lutein. By hydroxylation of carotenes with molecular oxygen, and in

the presence of NADPH-dependent mixed-function oxygenase, hydroxy groups are
introduced; in fact, epoxidation represents another path for further derivatization,
although little is presently known about the epoxidase involved [6]. Typical repre-
sentatives are xanthophylls containing a hydroxy group at C-3 in the β- or ε-ring,
violaxanthin (5,6,5 ,6 -diepoxy-5,6,5 ,6 -tetrahydro-β,β-carotene-3,3 -diol) or zeaxan-
thin (β,β-carotene-3,3 -diol). The importance of the violaxanthin–zeaxanthin cycle
for both high rates of photosynthesis and energy dissipation has been described
[6, 26].
4.1.2.3.5 Isolated Enzymes Carotenoid biosynthesis takes place in a

membrane-bound multienzyme complex, which makes the system difficult to
isolate and to purify the enzymes involved. Owing to their sensitivity to detergents
and low abundance, only a few such enzymes have been purified from plant
tissue; many others had to be heterologously expressed in such a way that
high-pressure cell-breaking resulted in a soluble and enzymatically active form
[19, 27]. As an example, PDS was cloned and expressed in recombinant Escherichia
coli. To prepare the enzyme, the E. coli cells were disrupted by passing them
through a French press; the cell suspension was then centrifuged and the soluble
supernatant fraction used for enzymatic assays with HPLC recording, or recording
via optical absorption spectra [28].
4.1.3
Primary Targets
4.1.3.1 Inhibition of PDS and ζ -Carotene Desaturase

Owing to the similarity of desaturation reactions catalyzed by PDS or ZDS,
differentiation in plants is not easy to detect, with most herbicidal inhibitors
probably inhibiting both enzymes, although to different extents [7]. If a strong
inhibition of PDS has taken place with an accumulation of phytoene, then the
compound’s ability to inhibit ZDS cannot be seen. The commercially available
products that primarily inhibit PDS are listed in Figure 4.1.2 [7, 9, 29–31]. Cell-free
studies, exemplified by norflurazon and fluridone, have shown them to act as
reversible noncompetitive inhibitors of PDS [29]. Other PDS active structures are
shown in Table 4.1.2 and are described in Section 4.1.4.10.
Direct interaction with the enzyme ZDS was shown for the dihydropyrone
LS-80707 and the pyrimidine SAN 380H [9]. Later, the compound RH 1965 and
substituted 4-phenyl-3-benzylthio-4H-1,2,4-triazoles were reported also to inhibit
ζ -carotene desaturation [32, 33].
4.1.3.2 Inhibition of Lycopene Cyclase (LCC)

Amitrole (3-amino-1H-1,2,4-triazole) has been known to lead to some lycopene
accumulation in vivo at a temperature-dependent rate, but it is not considered
primarily to be an LCC inhibitor and may indirectly inhibit an early step
Inhibitors of phytoene desaturase (PDS)
O F
O
N Cl O
H
O F N
N Cl
F 3C N
F3C
CF3
Diflufenican Flurochloridone Fluridone
O O Cl
H
N NHCH3 N
F3 C O N
O N
F 3C O
F3C MeHN F
Flurtamone Norflurazon Picolinafen
Inhibitors of z - carotene desaturase (ZDS)
O O N
Cl N
CO2C2H5 O
N N
N SCH2 CF3
O F3C N
N N F
H
LS-80707 SAN 380H RH 1965 Ref. 31
(cis)
Inhibitors of lycopene cyclase (LCC)
Cl S N H3C O N
CPTA MPTA
Figure 4.1.2 Structure of commercial herbicides and

some herbicidically active compounds that inhibit differ-
ent enzymes in the biosynthetic pathway leading to the
carotenoids.
in carotenoid biosynthesis [7, 12, 34]. The only specific, more recently
identified inhibitors are substituted diethylamines such as N,N-diethyl-N-
[2-(4-chloro- phenylthio)ethyl]amine) (CPTA) and N,N-diethyl-N-[2-(4-methyl-
phenoxy)ethyl]amine (MPTA), which appear to inhibit both β- and ε-cyclases
(Figure 4.1.1) [7]. The MoA of these inhibitors is via the noncompetitive inhibition
of lycopene cyclase versus lycopene [35].
In 2001, potent diethylamines were identified as a new type of LCC inhibitor
structure [34] which, although having been shown as very effective in seedling
tests, have not yet demonstrated sufficient activity to warrant their development as
herbicides.
4.1.3.3 Genetic Engineering of Herbicide Resistance by Modification

of the Carotenogenic Pathway
The availability of numerous carotenogenic genes makes it possible to modify
and engineer the carotenoid biosynthetic pathways in both microorganisms and
plants. The unicellular cyanobacteria represent convenient tools for the generation
of mutants with a herbicide-resistant PDSs [3–5, 8]. Subsequently, various lines
of resistant mutants of Synechococcus have been selected against norflurazon
which show not only a resistance factor of up to 70 but, in most cases, also a
cross-resistance to other PDS herbicides [8].
4.1.4
Chemical Structure and Activities of PDS Inhibitors
4.1.4.1 Enzyme Activity, Physical Data, and Acute Oral Toxicity

of Commercial PDS Herbicides
During recent years, structural evolution, detailed quantitative and qualitative
structure–activity studies have been performed with a range of chemically different
PDS inhibitors. The earlier studies, conducted up until about 1990, have been
reviewed in Ref. [29], whereas reports produced up until the late 1990s have been
reviewed elsewhere [36].
The in vivo inhibitory concentration (IC)50 -values of commercial herbicides for
the inhibition of carotenoid biosynthesis, according to Ref. [28], are listed in
Table 4.1.1. This assay is easier to conduct than the earlier radioactive approach
with unicellular cyanobacteria [37], and produced three cases of differing results.
Such variation may have been caused by differences in target site sensitivity, uptake,
and translocation effects, or by metabolism of the herbicides in the treated bacteria
cells in the early test assays. Both physical and acute oral toxicity data of such
commercial herbicides are listed in Table 4.1.1 [38, 39].
4.1.4.2 Phenoxybenzamides
The removal of a p-nitro group from peroxidative diphenyl ethers drastically
reduced their peroxidative activity, while increasing the inhibition of carotenoid
biosynthesis, provided that a substituted formamide substituent was present in
the meta-position (1, Figure 4.1.3); notably, both the o- and p-derivatives were
inactive (for a review, see Ref. [29]). The lipophilicity of the phenoxy ring and the
chain length of the alkyl group of up to five carbon atoms was shown to increase
the enzymic activity, while branching resulted in a loss of activity. The QSAR
equations of the effect of the carbonamide substituent have been calculated [54].
Although no commercial product has yet been developed, some structurally related
diphenylethers have been patented by Bayer [55].
204
Table 4.1.1 IC50 values and physico-chemical and oral toxicity data for commercial herbicides for carotenoid biosynthesis inhibition.
Structure IC50 (mol L−1 ) log P (pH 7.5) Vapor pressure (mbar) M (g mol−1 ) mp (◦ C) LD50 rats (mg kg−1 )
O F 3.40 × 10−8 4.90 4.25 × 10−8 394.3 159−161 >2000
N
H F
N O
4 Herbicides with Bleaching Properties
CF3
Diflufenican
O 2.02 × 10−6 3.36 4.40 × 10−6 312.1 41 (eutectic) 4000

Cl
N
Cl
F3C
Flurochloridone
2.93 × 10−7 1.87 1.30 × 10−7 329.3 154−155 >10000
N
F3C
Fluridone
O 8.21 × 10−7 3.22 4.20 × 10−7 333.3 152–155 500
O
F3C MeHN
Flurtamone
O Cl 5.18 × 10−7 2.45 3.86 × 10−8 303.7 174–180 >5000
N NHCH3
N
F3C
Norflurazon
8.98 × 10−8 5.37 1.66 × 10−12 376.3 107 >5000

H
N
F3C O N
O F
Picolinafen
F 1.75 × 10−6 4.28 1.10 × 10−7 355.3 75 >5000

H
N
F3C O
O
Beflubutamid
Enzyme values obtained from BASF Agricultural Research, other values taken from The Pesticide Manual [36] and SRC PhysProp Database [37].
4.1 Phytoene Desaturase Inhibitors
205
Figure 4.1.3 Phenoxybenzamide S 3422 (1).

NHalk
O
O
1 alk = C2H5
S 3422
F
O O
N N 0,1
H
F R3
N O R2 N O
R4
CF3
R1
2 3
Diflufenican Substitution pattern of
phenoxynicotinamides
Figure 4.1.4 Diflufenican (2).
4.1.4.3 Phenoxypyridinecarbonamides
Phenoxypyridinecarbonamides are surprisingly flexible, when the pyridine ring
is substituted (for reviews, see Refs [29, 36]). The first active pattern consisted
of nicotinamides with a 2-phenoxy substitution (3; Figure 4.1.4). In the latter
case, the m-position (R1 ) was important, with 3-CF3 and 3-Cl being the most
active, while a double substitution led to a decrease in activity. Whereas, small
substituents R2 such as H or CH3 provided good herbicidal activity, Br or Cl were
both weaker. In the amide moiety, N-phenyl and N-benzyl derivatives showed
comparable activity, while ethylene as a spacer greatly decreased the in vitro activity,
and the thioamides were slightly less active. Substitution of the amide hydrogen
(R3 ) by alkyl led to a decrease in activity that was parallel to their length. A
single substitution of the N-phenyl ring (R4 ) resulted in a loss of activity, with the
exception of the 4-F-moiety; the 2,4-difluoro derivative showed comparable activity
to the unsubstituted compound. Most SAR contributions were derived from the
laboratory of May & Baker, where diflufenican (2; Figure 4.1.4) was first identified
and later developed by Rhône-Poulenc [56, 57].
The research team at the Shell laboratories later discovered a gap in the di-
flufenican patent, the 2,6-isomer 5, which showed great promise and led in 2001,
following its acquisition by American Cyanamid (and later BASF), to the marketing
of picolinafen (4) (Figure 4.1.5) [58]. The discovery of the 2,6-pyridine cluster
marked the beginning of a considerable number of follow-up patents to secure
the new lead [36]. It could be shown that lower alkyl amino groups (R2 = CH3 )
may substitute the 4-F- or 2,4-difluoroanilide moiety, while R1 must be H or CH3 .
In addition, R3 –R5 is best with H or F. The 6-phenoxy unit could be replaced
X R4
R3 R5
H
N NR1R2
F3 C O N Y N
O F 5 O
4
F3C
Picolinafen
H
S N
O N
O F
6
Figure 4.1.5 Picolinafen (4).
by 4-oxypyridine, 5-oxypyrazole [36], and 3-oxythiophene 6 (Figure 4.1.5) [59]. The

latter all require, again, a substituent (X) meta to the ether bridge (Y), and while the
pyridine ether gives similar activity with Cl, a CF3 group would be necessary for
the phenoxy, 5-oxy-pyrazole, and 3-oxy-thiophene unit.
With the finding that the amide moiety could be totally replaced by aryl or hetaryl
ethers, and also directly substituted by pyrazol, several new combinations became
possible, which in time led to the discovery of the phenoxypyridine ethers (see
Section 4.1.4.4).
4.1.4.4 Phenoxypyridine Ethers

In the 6-phenoxy moiety of this lead compound (8) a CF3 substituent X proved
best for herbicidal activity, and this was also the case for 5-pyrazole-oxy and
3-thiophene-oxy substitution (see Ref. [36] and Table 4.1.2). In the 4-pyridyl-oxy
group, m-Cl and m-difluoromethoxy proved to be good choices of substituent
since, when 2,6-bisaryloxy-pyridines were synthesized, one phenyl group could
be replaced by benzyl. Only a highly lipophilic aliphatic substituent R4 , such
as trifluoromethylthiopropyl 14 (n = 1), could compete with the (hetero)-aryloxy
compounds. In general, the best substituents for R1 and R3 were H and F, whereas
the substituent for R2 may be H, CH3 , and CH3 O; in the case of 16 (R1 = R3 = F)
this brought about a rise in activity. Interestingly, the activity in structures 13, 15,
and 16 was retained even when R4 is linked directly to the pyridine.
4.1.4.5 Phenylfuranones
The oldest-known phenylfuranone, difunon (17; Figure 4.1.6), was shown to be a
rigid structure in which replacement of the 4-phenyl group by n-butyl, cyclohexyl,
and of the 3-CN group by carbonamide or an ester, resulted in a loss of activity
(for a review, see Ref. [29]). Only the 3-position of the phenyl ring was tolerant of
substitution, such as –SCH3 , –OCH3 , –C6 H5 , and –CF3 , which in some weeds
led to a rise in activity. The compound, however, was never commercialized.
The other representative, flurtamone (18; Figure 4.1.6) has only the 4-phenyl
group and a basic side chain in common with difunon, while the remaining
substituents varied considerably. The best substituent of position 2 proved to be
Table 4.1.2 Structural evolution of phenoxypyridine ethers since 1994.
R2
R1 R3
H
N
F3C O N X Y N [O] R4
n = 0,1
O 8
7 F
Picolinafen
No. Ref. Year
9 60 1994
Cl
N N
Cl O N O
10 CN 61 1996
F3C O N O CF3
11 OMe 62 1998
CF3
N
O N O N
12 63 1999
N N
HF2CO O N O OCHF2
13 F3C 64 2001
N
N O N N N CF3
14 65 2001
N
F3C O N O(CH2)3SCF3
OMe
F3C
15 66 2003
S
O N N N CF3
16 F3C 67 2003
F F
S
O N N N CF3
Figure 4.1.6 Difunon (17) and flurtamone

O
NC (18).
O O O
N F3C MeHN
17 18
Difunon Flurtamone
phenyl; surprisingly, this could be replaced by C1 –C3 -alkyl, but the branching was
unfavorable.
The 4-phenyl ring was shown to require m-substitution by CF3 , whereas a
decreasing lipophilicity showed a lower degree of inhibition of PDS (for a review,
see Ref. [29]).
4.1.4.6 Phenylpyridazinones
The pyridazinones were found to be very flexible and, depending on the position and
substitution, to show a different MoA. While the cluster of the early chloridazon (19)
is responsible for photosynthesis inhibition [68], BAS 10501W (20) inhibited fatty
acid desaturation, altering the ratio of 18 : 2/18 : 3 fatty acids in plant membranes
[29, 69], while norflurazon (21; Figure 4.1.7) finally was shown to inhibit PDS. Such
an inhibitory effect went along with a CF3 -substituent R3 in the phenyl moiety and
small alkylamino groups R2 in structure 22. Longer chains or branching lowered
the activity. While position 4 of the heterocycle (R1 ) requires electron-withdrawing
substituents, R2 at C-5 must be connected with electron-donating substituents,
enabling electrons to be shifted towards the heterocycle in order to increase their
activity (for a review, see Ref. [29]).
Subsequent QSAR studies were performed with 2-phenylpyridazinones sub-
stituted at position 3 of the phenyl ring (R3 ), where lipophilicity exerted a very
strong effect on activity, counteracted by electronic properties. Steric factors did
not show any influence [29, 70]. The results were subsequently confirmed by
new m-substituted derivatives [71]. Replacing the CF3 -group by fluorophenoxy or a
O Cl O Cl O Cl O R1
N NH2 N N(CH3)2 N NHCH3 N R2

N N N N
F3C R3
19 20 21 22
Chloridazon BAS 10501W Norflurazon
Figure 4.1.7 Phenylpyridazinones.
Figure 4.1.8 Fluridone (23).

O
N
F3C
23
Fluridone
fluorophenylalkyl side chain led to a superior activity, in spite of their much larger
size. When Cl in R1 was substituted later by a m-CF3 -phenyl group, while R2 was
retained as CH3 NH, an early member of the diaryl heterocycle PDS inhibitor type
with strong herbicidal activity was identified (see Section 4.1.4.10).
4.1.4.7 Phenylpyridinones
The pyridinone structure of fluridone (23; Figure 4.1.8) is biologically rather
inflexible, so that the thiopyridinone and higher N-alkyl derivatives showed only
minimal activity (for a review, see Ref. [29]). The m-substitution of one phenyl
ring by the highly lipophilic CF3 group is necessary, while exchange by Cl or
CO2 H decreased the activity. QSAR equations with whole-cell data confirmed the
lipophilic and inductive effects; however, the in vitro results correlated only to π
[72]. To leave the pyridinone cluster, other contributors omitted the C=O group
and continued with a series of 2,4-diphenylpyrimidines.
Although the hetero ring system of fluridone and flurtamone are completely dif-
ferent, their three-dimensional structures and projection to a common overlay gave
rise to the concept of the ‘‘diaryl heterocyclic PDS inhibitors’’ (see Section 4.1.4.10)
[36].
4.1.4.8 Phenylpyrrolidinones
The most prominent representative of the phenylpyrrolidinones 25 is flurochlori-
done (24; Figure 4.1.9). Again, an electron-withdrawing lipophilic substituent R1 is
required in the 3-phenyl position, such as 3-CF3 or SCF3 , whereas CN or SO1−2 CF3
were somewhat weaker (see Refs [29, 36]). The activity ended with NO2 , NH2 , or
C=O as substituents, which are no longer lipophilic and instead are more prone to
hydrogen bridging, with exception of the NO2 group. Surprisingly, a high activity
could be conserved by replacing Cl in R2 with methyl and ethyl carbonamide. For
reasons of activity, however, the chain length of R3 is restricted to two, and the
5-position (R4 ) must be unsubstituted. Flurochloridone has two asymmetric carbons
O O Figure 4.1.9 Flurochloridone (24).

Cl R2
N N
CH2Cl R3
F 3C R1 R4
24 25
Flurochloridone
Figure 4.1.10 Phenyltetrahydropyrimidi-

nones.
O O
N N
N N
R1 X
F3C
26 27
NTN-28621
in the pyrrolidinone ring, and 3,4-trans stereochemistry provides a better herbicidal

activity than does the cis form. During the early 1990s, the 3-Cl was replaced by
phenyl carrying an m-CF3 group or halides in the 3–5-positions while varying R1
and R3 . Among these, 1-(3-isopropylphenyl)-3-phenyl-4-ethyl-2-pyrrolidinone was
one of the herbicidally most active. When R2 = Cl was omitted, it was essential
that R3 became CF3 . The lipophilic CF3 in R1 was also replaced by phenoxy units
with different substituents, or ring-anellated with the adjacent o-position into a
2 ,2 -difluordioxol-2,3-benzo ring.
4.1.4.9 Phenyltetrahydropyrimidinones
A prerequisite for high herbicidal activity in the phenyltetrahydropyrimidinones
(27) is the m-CF3 -substitution in one phenyl group (Figure 4.1.10). The substitution
of R1 by an electron-withdrawing group shows the same biological ranking as in
the other compound classes discussed above (see Ref. [29]). Based on the ring
size of the heterocycle, a six-membered ring with X = CHCH3 as optimum would
show the best results, whereas five- or seven-membered cyclic ureas would be less
effective. Based on their three-dimensional structure, there is a certain similarity
between the phenyl-pyridinones (Section 4.1.4.7) and the saturated NTN-28621
(26; Figure 4.1.10) which, with its CH–CH3 group, indeed imitated the N–CH3
group of fluridone and thus became a precursor for the compounds described in
Section 4.1.4.10 [36].
4.1.4.10 Structural Overlay for Diaryl Heterocycle PDS Inhibitors,

and Newer Developments
The structural overlay of flurtamone, fluridone, and NTN-28621 led to a new pyra-
zolone 28 and pyridine 29, many pyrimidines 30, and also some 1,2,4-triazines 31
with the joint possession of 1,3-connected phenyl groups (‘‘1,3-diaryl-heterocycle’’)
(Figure 4.1.11) [36]. A pyrimidine 30 with R1 = CH3 O, R2 = H, X = 3-CF3 , and Y = F
has been reported with application rates as low as 63 g a.i. ha−1 . Other substituents
for R1 are NMe2 , CH3 , and CO2 Et, while R2 was H. In another series, R1 was kept
O O O
N
O N
N
F3C MeHN F3C F 3C
23 26
18
Flurtamone Fluridone NTN-28 621
N Y F
F
N N N
N N
N N
O
F 3C SMe X N
F2CHO R1 R2 Cl EtHN
28 29 30 31
R1 = H, OCH3, NMe2, CH3, CO2Et

R2 = H, OH, SMe
X = 2-CH3, 2-Cl, 3-CF3
Y = H, F, Cl, CF3
Figure 4.1.11 Structural overlay of diaryl heterocycle PDS

inhibitors and newer developments.
constant with hydrogen, while R2 was allowed to vary from OH to MeS, and X was
2-CH3 , 2-Cl, and 3-CF3 .
Along this line, Table 4.1.3 represents recent developments from which 32–37
pursue substituted hetarylethers with 35 integrating its ether bridge into a het-
erocycle. Compounds 38–40 constitute classical pyrimidines, while pyrimidines
41 and 42 are purely aliphatically substituted. Notably, both also inhibit ZDS. The
compounds 44 and 45 may be viewed as substituted phenylpyrrolidinones, and the
ketomorpholine 43, the carbonamide 46, and the carbamate 47 represent new PDS
leads. The same holds for the pyrazolethers 36 and 37.
The 2-acylimino-3-phenylthiazolines which were discovered by Sumitomo re-
sulted in the development candidate S-3085 (56) [49]. However, this compound was
ultimately discontinued due to insufficient crop safety and too-high application
rates.
4.1.4.11 Models of the Active Site: Structural Requirements

To date, very few reports have been made on models of the PDS herbicide binding
site. The early QSAR equations correlated molecular properties such as the σ , π,
and steric parameters of one lead with its enzyme activity, and good results
were obtained with regards to the nature and position of substituents, or when
optimizing the length of a side chain to obtain an approximate impression of the
Table 4.1.3 Diaryl heterocycle PDS inhibitors and recent developments.
Number Type Ref. Year IC50 (μM) Number Type Ref. Year IC50 (μM) dose
dose
Hetarylethers
32 OMe [40] 17–140 g ha –1 40 CF3 [41] 10–15 g ha –1

1998 N 2002
N N
N N
F3C N
O N N
CF3
DPX-MY926
O [40] N N [42]
33 OMe 41
H
8.6 × 10 –7
1998 N 2002 ZDS 4.2 × 10 –6
N
N Cl
CF3 O
Cl
34 O [43] 0.75 × 10 –9 42 [42] 7.9 × 10 –6

N NH-C2H5
C 1999 2002 ZDS 6.5 × 10 –5
H N
N
O S O
F3C
213
214
Number Type Ref. Year IC50 (μM) Number Type Ref. Year IC50 (μM)
dose dose
35 N [43] 0.35 × 10 –8 Saturated heterocycles

1999
O
F3C
36 [44] 9.6 × 10 –7 43 [45] 1.7 × 10 –7

H H
N 2004 O N Me 1995
O OMe [46]
F3C O 2001
O
F3C N N (2R), (5S)
37 O [47] 44 F [48] 1 × 10 –9
H
O N F 2004 O 2001
F3C Br N
N
N
H
F
C2H5
KPP-856
F
56 [49] 2.1 × 10 –7 45 O [48] 4.8 × 10 –7
N 2007 2001
S O N
F3C F
N
C2H5
S-3085 F
O
F
Pyrimidines Aliphatic scaffolds
38 [50] 1.4 × 10 –6 46 O [51]

2001 O 1999
N
N H
Cl F
NH CF3
HO
O Beflubutamid
39 [52] 5–10 g ha –1 47 [53] 1.3 × 10 –7
CF3 2002 2003
O
N O
O N N O N
H
N
CF3
CF3
215
electronic and steric prerequisites. An activity prediction of structurally diverse

molecules would not be possible, however. It should also be borne in mind that
the QSAR does not take into account any biological uptake, nor stability against
light and water at different pH-values. Neither does it consider the metabolism of
a molecule in the plant, when activity in the field is desired.
An initial hypothetical binding site model was proposed during studies of the
substituted 3(2H)-furanones [73]. Later, a steric model of the binding site of the
PDS enzyme was developed by the superposition of five commercial, structurally
diverse inhibitors which were assumed to bind in the same manner [45]. In this
case, a conformational analysis was carried out with the aid of three molecular
mechanics programs in order to investigate three common regions in an orthogonal
view: region X (with the phenyl ring preferentially substituted by the lipophilic CF3
group); Y (central heterocyclic ring with an amide, vinylogous amide CO group), in
which steric and electronic requirements appear to be relatively well defined; and
region Z, which appears to be sterically more tolerant. This model was similar to
that shown in Figure 4.1.12, when A, B, and C are represented by X, Y, and Z. The
model was employed to predict the likely levels of activity of some analogs of the
6-ketomorpholine 43, and could be used to show that the inhibitory activity resides
almost exclusively with the (2R),(5S) form.
In 1999, another contribution described the PDS-active structures 34 and 35,
showing an overall similarity to the leads mentioned earlier [43]. In computa-
tional studies with SYBYL and a pharmacophore mapping approach, putative
receptor-bound conformations of benzoxazole and benzothiazoles that differed
from these were obtained, thus supporting a coplanar geometry over a tilted con-
formation. In addition to previous findings, a functional group at the enzyme
Additional
Phenyl ring Central substituents
A heterocycle C
B
O O O O
ArO Ar Cl Ar Ar Ar N Ar Ar N Ar
N N
N N N
ArO NHMe N
SCH3 OH
3a 5a 21a 23a 29a 30a
O O
Ar Ar Ar N Cl
O
NHCH3 Cl
18a 24a
Figure 4.1.12 Common structural elements of PDS inhibitors. Modified from Ref. [7].
opposite to the central ring acting as either a hydrogen bond donor or an acceptor
was suggested for interaction with the sp2 -hybridized nitrogen of the benzoxazole
or the benzothiazole heteroatoms. The herbicide binding site thus generated would
be large and sufficiently defined to fit most of the other inhibitors.
When comparing compounds with a modified central ring, it was concluded that
the optimum inhibition of PDS would be further reflected by a similar diagonal
length from the negatively charged regions across the central heterocycle B carrying
one or two substituted phenyl rings A and C (Figure 4.1.12) [8]. A region of a total of
six C- and hetero-atoms spans from one end of the substituted central heterocycle,
with C=O and N=C groups to the opposite site. In the nonplanar ketomorpholine
43, the optimum length was only five atoms with the 5-methyl group in the
(5S)-form. The stereospecific inhibitory property of certain substituents of this
heterocycle represent another indication for the spatial requirement of this model.
Notably, most PDS inhibitors fit this description quite well, although some
exceptions must be noted. Norflurazon (21) is highly active and does not contain
a ring C, whereas in beflubutamid (46; Table 4.1.3) and in structure 47, an
oxycarbonamide and oxyalkanecarbamate moiety (respectively) replace the central
ring B by constituting a polar scaffold for rings A and C to interact with the binding
niche. Hence, in structures 41 and 42 only one central ring, without any aromatic
substituents, is left.
Finally, the question was raised as to whether inhibitors of PDS and ZDS could
be modeled as analogs of plastoquinone, because of their competitive behavior with
the latter [74].
4.1.5
Biology and Use Pattern
Diflufenican, which initially was introduced by May & Baker Ltd (now Bayer
CropScience), is applied at 125–250 g a.i. ha−1 either pre- or early post-emergence
in autumn-sown wheat and barley, for the control of broadleaved weeds [38].
Degradation proceeds via the metabolites 2-(3-trifluormethylphenoxy)nicotinamide
and 2-(3-trifluormethylphenoxy)nicotinic acid to bound residues and CO2 . The
dissipation time (DT)50 may vary from 15 to 30 weeks, depending on the soil type
and the water content.
Fluorochloridone was first introduced by the Stauffer Chemical Company (now
Syngenta AG), although the production rights were acquired by Agan Chem-
ical Manufacturers Ltd in 2002. Fluorochloridone is applied pre-emergence at
250–750 g a.i. ha−1 to control weeds in winter wheat and winter rye, sunflowers,
and potatoes. It is degraded in the soil under laboratory conditions, mostly forming
CO2 and a bound residue. The DT50 in the field is 9 to 70 days.
Fluridone was introduced by Eli Lilly & Company (now Dow Agrosciences), and
later sold to SePRO. It is used as an aquatic herbicide to control most submerged
and emerged aquatic plants in ponds, lakes, reservoirs, and irrigation ditches. The
application rates are 45–90 ppb in ponds and 10–90 ppb in lakes. As an upland crop,
only cotton has been found to be selective for fluridone. In aquatic environments,
the agent is degraded mainly via photolytic processes, although microorganisms

and aquatic vegetation are also factors. The DT50 -values in water under anaerobic
conditions is about nine months, and under aerobic conditions about 20 days.
Flurtamone was introduced by the Chevron Chemical Co. and later acquired
by Rhône-Poulenc Agrochemical (now Bayer CropScience). It is incorporated
either pre-plant or pre-emergence, or may be applied post-emergence to control
broadleaved and some grass weeds in small grains, peanuts, cotton, peas, and
sunflowers at 250–375 g a.i. ha−1 . The main metabolite is trifluoromethylbenzoic
acid and, at 10 months after its application, no residues could be detected. The field
DT50 is 46–65 days.
Norflurazon, which was introduced by Sandoz Ag (now Syngenta AG), is used
at 0.5–2 kg a.i. ha−1 for the pre-emergence control of grasses and sedges as well
as broadleaved weeds in cotton, soya beans, and peanuts, and at 1.5–4 kg a.i. ha−1
for the treatment of nuts, citrus, vines, pomefruit, stone fruit, ornamentals, hops,
and industrial vegetation management. Norflurazon dissipates in the soil by
photodegradation and volatilization, with a DT50 -value of 6–9 months.
Picolinafen was discovered by Shell International Research, and later acquired
by the American Cyanamid Company (now BASF AG). It is applied at between
50 and 100 g a.i. ha−1 as a cereal herbicide for the post-emergence control of
broadleaved weeds, and marketed in mixtures with other cereal herbicides such
as pendimethalin, isoproturon, and 2-methyl-4-chlorophenoxyacetic acid (MCPA).
It shows strong synergistic properties, for instance with pendimethalin, to also
control grasses [75], and it is further registered for use in lupines. Its metabolism
proceeds via a rapid hydrolytic cleavage of the amide bond. Picolinafen is degraded
photochemically in the environment; the DT50 -value is one month.
Beflubutamid was developed by Ube Industries Ltd, and is now sold by Stähler. It
is used in a mixture with isoproturon or Ioxynil and methylchlorophenoxypropionic
acid (MCPP) for both pre- and early post-emergence control of broadleaved weeds
and some grasses in wheat and barley at 170–255 g a.i. ha−1 . The soil DT50 -value
is 5.4 days; the main metabolite is the corresponding butanoic acid, which is itself
rapidly degraded in soil.
An overview of the currently available commercial PDS herbicides is provided in
Table 4.1.4.
The engineering of resistance opens the possibility to obtain tolerant plants, and
thereby to increase the crop spectrum beyond the scope described above [3, 82].
Thus, it has proved possible not only to confer resistance to tobacco plants, but also
to increase the carotenoid content in tomato fruits, rapeseed, and rice [19].
4.1.6
Major Synthetic Routes for PSD Inhibitors
The major synthetic routes of the PSD inhibitors, which are described below, are
depicted in Scheme 4.1.1
Table 4.1.4 Summary of application data of commercial PDS herbicides.
Chemical structure Tradename (year) ISO name Code No. Dose (g ha−1 ) Applic. Ref.
Company method Target crops
F Fenikan Diflufenican 125–250 Pre, [56]

O Tigrex (1987) MB-38544 post Cereals
N May & Baker
H Bayer CropScience
O F
N
CF3
Racer (1985) Flurochloridone 250–750 Pre [76]

O
Cl R-40244 Cereals, cotton,
N Stauffer Chemical sunflowers, potatoes
Cl Syngenta
F3C
Sonar (1977) Fluridone 500–2000 Pre, [68]

EL171 Elanco post Cotton [77]
O
SePRO aquatic herbicide
45–90 ppb
N
F3C
Bacara (1997) Flurtamone 250–375 Pre, [78]
O RE-40885 Chevron post Cereals,
Bayer CropScience peanuts, peas,
cotton, sunflowers
O
F3C MeHN
219
220
Chemical structure Tradename (year) ISO name Code No. Dose (g ha−1 ) Applic. Ref.
Company method Target crops
O Solicam Norflurazon 500–2000 Pre [79]

Cl
Zorial (1968) H 9789 Cotton, peanuts, [80]
N NHMe Sandoz soybeans, orchard

N Syngenta AG
F3C
Pico (2001) Picolinafen 50–100 Post [58]
H AC 90001 Cereals, lupines
N
F3C O N ACC
O BASF AG
F
Herbaflex (2003) Beflubutamid 170–255 Pre, early [81]
F
H UBH-820 post Wheat, barley
N Ube under
F3C O
development
O
Synthesis of diflufenican O F
CO2H
OH N
CO2H NaOCH3 O 1. SOCl2 H
N F
N O
2. F
N Cl CF3
CF3 H2N
48 F CF3
Diflufenican (2)
Synthesis of flurochloridone
O Cl O Cl CuCl O
O Cl Cl
NH2 Bu2NH
N Cl N Cl N
Cl Cl H toluene Cl
F3C
F3C F3C F3C
49 Flurochloridone (24)
Synthesis of fluridone
O HCO2Et O MeNH2HCl
F3C NaOMe F3C
50 51 HO OH O
O F3C
HN NH2·AcOH Mel
F3C N
HCONH2 NaH
Me
52 N
H Fluridone (23)
Synthesis of flurtamone
EtOOC O
CN NaOMe O (1) Br2, AcOH
F3C (2) methylation O

F 3C CN F3C MeHN
53
Flurtamone (18)
Synthesis of norflurazon
O Cl O Cl O Cl
NH2 (1) EtOH MeNH2
N HO Cl N Cl N NHCH3
H O (2) Ac2O, AcOH N N
F3C F3 C F3C
54 Norflurazon (21)
Synthesis of picolinafen
H2SO4 F NH2
H 48 H
N N
Cl N CCl3 NEt3 Cl N F3C O N
N O O
F F
Picolinafen (4)
Synthesis of beflubutamid
Br K2CO3 F benzylamine F
F OH COOEt H
NaOMe N
F3 C O COOEt F3C O
F3C O
55
Beflubutamid (46)
Scheme 4.1.1 Major synthetic routes for diflufenican (2),

flurochloridone (24), fluridone (23), flurtamone (18), norflu-
razon (21), picolinafen (4), and beflubutamid (46).
Diflufenican is synthesized via the nucleophilic substitution of 2-chloronicotinic

acid with 3-hydroxybenzotrifluoride and further reaction with thionyl chloride and
2,4-difluoroaniline to the final product [56].
Flurochloridone is prepared via a copper chloride-catalyzed cyclocondensation of
N-allyl-(3-trifluormethylphenyl)dichloroacetamide 49 [36].
Fluridone is accessible in two ways. First, 1-(3-trifluormethylphenyl)-3-phenyl-2-
propanone (50) is reacted with ethyl formate in the presence of a base to yield
the diformyl derivative 51; this is then cyclized with methylamine to the final
product. Alternatively, 50 is condensed with formamidine acetate in the presence
of formamide to the 4(1H)-pyridone intermediate 52 and then methylated to
produce fluridone [36].
Flurtamone is prepared by the cyclization of 4-phenyl-2-(3-trifluormethylphenyl)-
3-oxobutyronitrile (53) with bromine in the presence of acetic acid and methylation
to the final heterocycle [36].
Norflurazone production is based on the condensation of 3-trifluormethylphenyl-
hydrazine and mucochloric acid, followed by cyclization with acetic anhydride to
give 4,5-dichloro-2-(3-trifluormethylphenyl)pyridazin-3-one (54). The nucleophilic
substitution of 54 with methylamine yields norflurazon [36].
Picolinafen is built on the partial hydrolysis of 2-chloro-6-trichlormethylpyridine,
reaction with 4-fluoroaniline and subsequent nucleophilic substitution with
3-hydroxybenzotrifluoride [83].
Beflubutamid is synthesized from ethyl 2-(4-fluoro-3-trifluormethylphenoxy)
butanoate 55 and benzylamine in the presence of a base [36].
References
1. Sandmann, G. and Böger, P. (1989) in Elstner), Marcel Dekker, New York,

Target Sites of Herbicide Action (eds P. pp. 271–330.
Böger and G. Sandmann), CRC Press, 8. Sandmann, G. (2002) in Herbicide
Boca Raton, FL, pp. 26–44. Classes in Development (eds P. Böger,
2. Wakabayashi, K. and Böger, P. (2002) K. Wakabayashi, and K. Hirai),
Pest. Manage. Sci., 58, 1149–1154. Springer-Verlag, Berlin, Heidelberg,
3. Arias, R.S., Dayan, F.E., Michel, A., pp. 43–57.
Howell, J., and Scheffler, B.E. (2006) 9. Böger, P. and Sandmann, G. (1998) Pes-
Plant Biotechnol. J., 4, 263–273. tic. Outlook, 9, 29–35.
4. Windhövel, U., Geiges, B., Sandmann, 10. Lee, D.L., Prisbylla, M.P., Cromartie,
G., and Böger, P. (1994) Plant Physiol., T.H., Dagarin, D.P., Howard, S.W.,
104, 119–125. McLean Provan, W., Ellis, M.K., Fraser,
5. Windhövel, U., Sandmann, G., and T., and Mutter, L.C. (1997) Weed Sci.,
Böger, P. (1997) Pestic. Biochem. Physiol., 45, 601–609.
57, 68–78. 11. Lee, D.L., Knudsen, C.G., Michaely,
6. Young, A.J. (1991) in Herbicides (eds W.J., Chin, H.-L., Nguyen, N.H., Carter,
N.R. Baker and M.P. Percival), Elsevier C.G., Cromartie, T.H., Lake, B.H.,
Science Publishers B.V., Amsterdam, Shribbs, J.M., and Fraser, T. (1998)
pp. 132–171. Pestic. Sci., 54, 377–384.
7. Fedtke, C. and Duke, S.O. (2005) in 12. Agnolucci, L., Dalla Vecchia, F., Barbato,
Plant Toxicology (eds B. Hock and E.F. R., Tassani, V., Casadoro, G., and
References 223
Rascio, N. (1996) J. Plant Physiol., 147, 31. Kowalczyk-Schröder, S. and Sandmann,

493–502. G. (1992) Pestic. Biochem. Physiol., 42,
13. Mueller, C., Schwender, J., Zeidler, J., 7–12.
and Lichtenthaler, H.K. (2000) Biochem. 32. Burdge, E.L. (2000) Pest Manage. Sci.,
Soc. Trans., 28, 792–793. 56, 245–248.
14. Siefermann-Harms, D. (1987) Physiol. 33. Yamada, N., Kusano, D., and Kuwano,
Plant, 69, 561–568. E. (2002) Biosci. Biotechnol. Biochem., 66
15. Tracewell, C.A., Vrettos, J.S., Bautista, (8), 1671–1676.
J.A., Frank, H.A., and Brudvig, G.W. 34. Fedtke, C., Depka, B., Schallner, O.,
(2001) Arch. Biochem. Biophys., 385, Tietken, K., Trebst, A., Wollweber,
61–69. D., and Wroblowsky, H.-J. (2001) Pest
Manage. Sci., 57, 278–282.
16. Cogdell, R. (1988) in Plant Pigments
35. Schnurr, G., Böger, P., and Sandmann,
(eds T.W. Goodwin and T. Walworth),
G. (1998) J. Pestic. Sci. (Jpn), 23,
Academic Press, London, pp. 183–230.
113–116.
17. Havaux, M. (1998) Trends Plant Sci., 3,
36. Hirai, K., Uchida, A., and Ohno, R.
147–151.
(2002) in Herbicide Classes in De-
18. Bramley, P.M. and Pallett, K.E. (1993) velopment: Mode of Action, Targets,
Proc. Brighton Crop Prot. Conf. – Weeds, Engineering, Chemistry (eds P. Böger,
2, 713–722. K. Wakabayashi, and K. Hirai),
19. Sandmann, G. (2001) Arch. Biochem. Springer-Verlag, Berlin, Heidelberg,
Biophys., 385, 4–12. pp. 213–221.
20. Lichtenthaler, H.K., Rohmer, M., and 37. Sandmann, G. (1993) in Target Assays
Schwender, J. (1997) Physiol. Plant, 101, for Modern Herbicides and Related Phy-
643–652. totoxic Compounds (eds P. Böger and
21. Ferhatoglu, Y. and Barrett, M. (2006) G. Sandmann), Lewis Publishers, Boca
Pestic. Biochem. Physiol., 85, 7–14. Raton, FL, pp. 15–20.
22. Britton, G. (1979) Z. Naturforsch., 34c, 38. Tomlin, C.D.S. (ed.) (2003) The Pesti-
979–985. cide Manual, 13th edn, BCPC - Alton,
23. Breitenbach, J., Fernández-Gonzáles, B., Hampshire.
Vioque, A., and Sandmann, G. (1998) 39. SRC PhysProp Database; http://
Plant Mol. Biol., 36, 725–732. esc.syrres.com (accessed 10 May 2011).
24. Cunningham, F.X. Jr, Pogson, B., Sun, 40. South, M.S., Yakuboski, T.L., Miller,
Z., McDonald, K.A., DellaPenna, D., M.J., Marzabadi, M., Corey, S.,
and Gantt, E. (1996) Plant Cell, 8, Molyneaux, J., South, S.A., Curtis, J.,
1613–1626. Dukesherer, D., Massey, S., Kunng,
25. Pogson, B., McDonald, K.A., Truong, F.-A., Chupp, J., Bryant, R., Moedritzer,
K., Woodward, S., Mayonado, D., and
M., Britton, G., and DellaPenna, D.
Mahoney, M. (1998) in Synthesis and
(1996) Plant Cell, 8, 1627–1639.
Chemistry of Agrochemicals V, ACS Sym-
26. Ort, D.R. (2001) Plant Physiol., 125,
posium Series, Vol. 686 (eds D.R. Baker,
29–32.
J.G. Fenyes, G.S. Basarab, and D.A.
27. Sandmann, G. (1997) Pure Appl. Chem.,
Hunt), American Chemical Society,
69, 2163–2168. Washington, D.C., pp. 107–119.
28. Sandmann, G., Schneider, C., and 41. Stevenson, T.M., Selby, T.P., Koether,
Böger, P. (1996) Z. Naturforsch., 51c, G.M., Drumm, J.E., Meng, X.J.,
534–538. Moon, M.P., Coats, R.A., Thieu, T.V.,
29. Sandmann, G. and Böger, P. (1992) in Casalnuovo, A.E., and Shapiro, R. (2002)
Rational Approaches to Structure, Activity in Synthesis and Chemistry of Agrochem-
and Ecotoxicology of Agrochemicals (eds icals VI, ACS Symposium Series, Vol.
W. Draber and T. Fujita), CRC Press, 800 (eds D.R. Baker, J.G. Fenyes, G.R.
Inc., Boca Raton, FL, pp. 357–371. Lahm, T.P. Selby, and T.M. Stevenson),
30. Sandmann, G., Linden, H., and Böger, American Chemical Society, Washing-
P. (1989) Z. Naturforsch., 44c, 787–790. ton, D.C., pp. 85–95.
42. Breitenbach, J., Böger, P., and 54. Sandmann, G. (1986) in Pesticide Science
Sandmann, G. (2002) Pestic. Biochem. and Biotechnology (eds R. Greenhalgh
Physiol., 73, 104–109. and T.R. Roberts), Blackwell Scientific,
43. Laber, B., Usonow, G., Wiecko, E., Oxford, pp. 43–48.
Franke, W., Franke, H., and Köhn, 55. Helmke, H., Hoffmann, M.G., Auler,
A. (1999) Pestic. Biochem. Physiol., 63, L.A., Kehne, H., Hills, M., and Feucht,
173–184. D. (2006) Bayer CropScience, WO
44. Ohno, R., Watanabe, A., Matsukawa, 06/103 003.
T., Ueda, T., Sakurai, H., Hori, M., and 56. Cramp, M.C., Gilmour, J., and Parnell,
Hirai, K. (2004) J. Pestic. Sci., 29, 15–26; E.W. (1982) May & Baker Limited,
enzyme value obtained from BASF European Patent EP 53011.
Agricultural Research. 57. Cramp, M.C., Gilmour, J., Hatton, L.R.,
45. Mitchel, G. (1995) in Synthesis and Hewett, R.H., Nolan, C.J., and Parnell,
Chemistry of Agrochemicals IV, ACS Sym- E.W. (1987) Pestic. Sci., 18, 15–28.
posium Series, Vol. 584 (eds D.R. Baker, 58. Foster, C.J., Gilkersen, T., and Stocker,
J.G. Fenyes, and G.S. Basarab), Ameri- R. (1991) Shell International Research,
can Chemical Society, Washington, D.C., European Patent EP 447004.
pp. 161–170. 59. Kleemann, A., Karp, G.M., and Condon,
46. Sandmann, G. and Mitchel, G. (2001) J. M.E. (1999) US Patent US 5,869,426,
Agric. Food Chem., 49, 138–141. American Cyanamid Co.
47. Ohno, R., Watanabe, A., Nagaoka, 60. Kleemann, A. (1994) Shell International
Research, WO 94/22833.
M., Ueda, T., Sakurai, H., Hori, M.,
61. Kanno, H., Kanda, Y., Shimizu, S.,
and Hirai, K. (2004) J. Pestic. Sci., 29,
Kubota, Y., Sato, T., Arahira, M., and
96–104.
Kagaku, K. (1996) European Patent EP
48. Ogawa, H., Yamada, I., Arai, K.,
692 474.
Hirase, K., Moriyasu, K., Schneider,
62. Baltruschat, H.S., Kleemann, A.,
C., Sandmann, G., Böger, P., and
Maier, T., Scheiblich, S., Pont, J.L.,
Wakabayashi, K. (2001) Pest Manage.
and Höllmüller, T. (1998) American
Sci., 57, 33–40.
Cyanamid Co., WO 98/04 548.
49. Sanemitsu, Y., Kawamura, S., Satoh,
63. Baltruschat, H.S., Pont, J.L. Maier, T.,
J., Katayama, T., and Hashimoto, S.
and Scheiblich, S. (1999) American
(2007) Synthesis and Chemistry of Agro-
Cyanamid Co., US 5,922,738.
chemicals VII, ACS Symposium Series, 64. Maier, T., Kleemann, A., Scheiblich,
Vol. 948, American Chemical Society, S., and Siegfried, H. (2001) BASF AG,
Washington, D.C., pp. 197–207. European Patent EP 1 101 764.
50. Sandmann, G. (2001) Pestic. Biochem. 65. Scheiblich, S., Maier, T., and
Physiol., 70, 86–91. Baltruschat, H. (2001) BASF AG, WO
51. Takamura, S., Okada, T., Fukuda, S., 01/36 410.
Akiyoshi, Y., Hoshide, F., Funaki, E., 66. Hofmann, M., Parra, L.R., von Deyn,
and Sakai, S. (1999) Proc. Brighton Crop W., Baumann, E., Kordes, M., Misslitz,
Prot. Conf. – Weeds, 1, 41–52. U., Witschel, M., Zagar, C., and Landes,
52. Selby, T.P., Drumm, J.E., Coats, R.A., A. (2003) BASF AG, WO 03/022 831.
Coppo, F.T., Gee, S.K., Hay, J.V., 67. Hofmann, M., Parra, L.R., von Deyn,
Pasteris, R.J., and Stevenson, T.M. W., Baumann, E., Kordes, M., Misslitz,
(2002) in Synthesis and Chemistry of U., Witschel, M., Zagar, C., and Landes,
Agrochemicals VI, ACS Symposium A. (2003) BASF AG, WO 03/022 843.
Series, Vol. 800 (eds D.R. Baker, J.G. 68. Hock, B., Fedtke, C., and Schmidt, R.R.
Fenyes, G.P. Lahm, T.P. Selby, and T.M. (1995) Herbizide: Entwicklung, Anwen-
Stevenson), American Chemical Society, dung, Wirkungen, Nebenwirkungen, Georg
Washington, DC, pp. 74–84. Thieme Verlag, Stuttgart, New York,
53. Ohki, S., Miller-Sulger, R., Wakabayashi, p. 124.
K., Pfleiderer, W., and Böger, P. (2003) 69. St John, J.B., Schirmer, U., Rittig, F.R.,
J. Agric. Food Chem., 51, 3049–3055. and Bleiholder, H. (1984) in Pesticide
4.2 Hydroxyphenylpyruvate Dioxygenase (HPPD): The Herbicide Target 225
Synthesis Through Rational Approaches, 74. Breitenbach, J., Zhu, C., and Sandmann,
ACS Symposium Series, Vol. 255 (eds G. (2001) J. Agric. Food Chem., 49,
P.S. Magee, G.K. Kohn, and J.J. Menn), 5270–5272.
American Chemical Society, Washing- 75. Gardon, M., Gosselin, N., Grosjean, O.,
ton, DC, pp. 145–162. and Grollier, T. (2002) La Défense des
70. Sandmann, G. and Böger, P. (1982) Z. Végétaux, 550, 56–60.
76. Teach, E.G. (1976) Stauffer Chemical
Naturforsch., 37c, 1092–1094.
Co., DE 2 612 731.
71. Wriede, U., Freund, W., Hamprecht,
77. Taylor, H.M. and Lilly, E. (1976) DE 2
G., Parg, A., Würzer, B., and Meyer, 537 753.
N. (1990) in (eds H. Frehse, E. 78. Ward, C.E. (1984) Chevron Research, DE
Kesseler-Schmitz, and S. Conway), Book 3 422 346.
of Abstracts 7th International Congress 79. Ebner, C. and Schuler, M. (1972) San-
of Pesticide Chemistry, vol. 1, IUPAC, doz Ltd, US Patent 3, 644, 355.
Hamburg, p. 68. 80. Ebner, C. and Schuler, M. (1974) San-
72. Sandmann, G., Kowalczyk-Schröder, doz Ltd,US Patent 3, 834, 889.
S., Taylor, H.M., and Böger, P. (1992) 81. Takematsu, T., Takeuchi, Y., Takenaka,
Pestic. Biochem. Physiol., 42, 1–6. M., Takamura, S., and Matsushita, A.
73. Ward, C.E., Lo, W.C., Pomidor, P.B., (1987) Ube Industries, European Patent
EP 239 414.
Tisdell, F.E., Ho, A.W.W., Chiu, C.L.,
82. Arias, R., Netherland, M.D., Scheffler,
Tuck, D.M., Bernardo, C.R., Fong, P.J.,
B.E., Puri, A., and Dayan, F.E. (2005)
Omid, A., and Buteau, K.A. (1987) in
Herbicide-resistant crops from biotech-
Synthesis and Chemistry of Agrochemicals, nology. Pest Manage. Sci., 61 (Special
ACS Symposium Series, Vol. 355 (eds Issue), 258–268.
D.A. Baker, J.G. Fenyes, W.K. Moberg, 83. Knell, M., Brink, M., Wevers, J.H., and
and B. Cross), American Chemical Heinz, W. (1999) BASF AG, European
Society, Washington, DC, pp. 65–73. Patent EP 899 262.
4.2
Hydroxyphenylpyruvate Dioxygenase (HPPD): The Herbicide Target
Timothy R. Hawkes
4.2.1
Herbicidal Mode of Action
The corn herbicide sulcotrione and destosyl pyrazolate (DTP), the active hydrolysis
product of the rice herbicides pyrazolate and pyrazoxyfen (Figure 4.2.1), were known
[1, 2] as ‘‘bleaching herbicides’’ long before their hydroxyphenylpyruvate dioxyge-
nase (HPPD)-based mode of action was recognized. The loss of chlorophyll and an
accumulation of phytoene suggested possible sites of action in protochlorophyllide
biosynthesis, or at the phytoene desaturase (PDS) step in carotenoid biosynthesis
[3]. However, these polar acids neither resemble typical PDS inhibitor herbicide
types [4], nor inhibit PDS in vitro [5]. The clue to their true mode of action came from
the results of toxicological studies which indicated that rats fed with experimental
benzoyl cyclohexane-1,3-dione (CHD) herbicides such as nitisinone (Figure 4.2.1)
exhibited increased levels of tyrosine in the blood, and of p-hydroxyphenylpyruvate
(HPP) in the urine. These findings suggested the presence of a block in the
HPP O HGA
O
O2 CO2 O−
OH
O−
Fe(II)
(a) HO O HO
F
sulcotrione Cl DTP F DKN
O MeO2S
O O Cl F
Cl O
N OH
O SO2Me N
O N
(1) (2) (3)
mesotrione nitisinone
−
O− O O O−
O O O N+ O O O N+ O O N +
F
O O O [Cl, CF3] O
SO2Me F
F
(b) (4) (5) (6)
Figure 4.2.1 (a) The HPPD reaction; (b) The structures of some HPPD inhibitors.
catabolic degradation of tyrosine, and further investigative studies [6–8] confirmed

nitisinone to be a potent inhibitor of mammalian HPPD, the enzyme that cat-
alyzes the oxidative decarboxylation and rearrangement of HPP to homogentisic
acid (HGA). The discovery that CHDs also inhibit HPPD in plants, and the evi-
dence firmly linking this to their herbicidal effect, was disclosed shortly afterwards
[8, 9]. Thus, HGA was found to be a specific antidote for HPPD inhibitor-induced
bleaching [8, 9] and a phytoene-accumulating Arabidopsis mutant, PDS1, which
exhibited a homozygous lethal bleaching phenotype that was rescuable by HGA,
was mapped to a lesion in the plant HPPD gene [10]. The subsequent expression
of heterologous HPPDs in transgenic plants resulted in a specific tolerance to
‘‘HPPD-inhibitor’’ herbicides [11].
HPPD catalyzes an early step in the tyrosine degradation pathway [12] that is
widely distributed in nature [13] and as in animals, the treatment of plants with
inhibitors causes a significant accumulation of tyrosine [8, 14]. HPP, derived from
the transamination of tyrosine, is first converted into HGA via HPPD; the HGA is
then oxidized via HGA oxidase to 4-maleylacetoacetate, which is further degraded
via 4-maleylacetoacetate isomerase and 4-fumarylacetoacetate lyase to fumarate
and acetoacetate. In microbes, the pathway can provide assimilable carbon from
tyrosine and phenylalanine; alternatively, HGA itself can oxidize to pyomelanin,
which fulfils a variety of functions in certain bacteria [15]. In higher mammals,
defects in the tyrosine degradation pathway cause hereditary diseases [12] such
as tyrosinemia type I, where a lesion in 4-fumarylacetoacetate lyase causes a
build-up of toxic and hepatocarcinogenic keto acids. Nitisinone, which blocks
HPPD and thereby prevents toxin accumulation [7], is currently a Food and Drugs
Administration (FDA)-approved treatment for tyrosinemia type I; moreover, it
may also be used to ameliorate other diseases arising from defects in tyrosine
degradation [12, 16].
The unique phytotoxicity of HPPD inhibitors arises from the fact that, in photo-
synthetic organisms, HGA is not merely an intermediate in tyrosine degradation,
but is also an intermediate in the biosynthesis of plastoquinone (PQ) and of
tocopherols [17, 18]. For this, the HGA is decarboxylated and prenylated by HGA
solanesyltranferase (HST) and HGA phytyltransferase (HPT), both of which are
located in the inner plastid envelope, to 2-methyl-6-solanesyl-1,4-benzoquinone
(MSBQ) and 2-methyl-6-phytyl-1,4-benzoquinone (MPBQ), respectively. These in-
termediates are then further methylated (by a single enzyme, MSBQ/MPBQ
methyltransferase, common to both pathways) to yield, respectively, PQ or, in the
case of the tocopherol pathway, 2,3-dimethyl-6-phytyl-1,4-benzoquinone. The latter
gives rise to γ - and α-tocopherol (the dominant tocopherol in leaves) via further
steps of cyclization and methylation. In higher plants, such as Arabidopsis, a single
HPPD gene [10] which is expressed only in the cytosol [19] fulfils the need for both
tyrosine degradation and pigment biosynthesis.
PQ is an essential electron acceptor in the PDS reaction of carotenoid biosynthesis
[10, 20]. HPPD herbicides that block PQ biosynthesis cause stunting and bleaching
symptoms [8, 9] similar to the effects of herbicides such as norflurazon, that directly
inhibit PDS by competing with PQ [4]. The PQ levels in plants treated with HPPD
herbicides decrease [8] before the onset of bleaching and phytoene accumulation
[14]. Both types of herbicide prevent the synthesis of carotenoids, which serve as
accessory light-harvesting pigments in the light-harvesting antenna structure, as
well as precursors of abscisic acid, although their main role is in photo-protection
[4]. The extended conjugated double-bond system of carotenoids makes them
effective quenchers of high-energy triplet states of chlorophyll that otherwise would
generate singlet oxygen. β-carotene, when bound to the D2 protein, is also a
structural part of the photosystem II (PS II) core. The herbicide-induced depletion
of carotenoids is associated with a light-dependent generation of singlet oxygen,
which damages lipids and proteins and causes disassembly of the photosynthetic
complex and the release of free chlorophyll. Free chlorophyll is photodynamically
photodestructive and generates singlet oxygen, which leads in turn to further leaf
pigment destruction and the white bleaching characteristics of PDS and HPPD
herbicides. Both types of herbicide are most effective in newly developing tissues
that emerge bleached, presumably as the consequence of a failure to properly
assemble photosynthetic units in the absence of carotenoids [21]. Tissue damage
is slower in mature tissue as it depends on both light intensity and carotenoid
turnover. HPPD inhibitors are more effective when applied post-emergence than
are PDS inhibitors, and they exert greater effects on growth whereas the PDS
inhibitors cause more damage to mature leaves. These differences most likely
arise from differences in translocation. Yet, HPPD inhibitors also cause some
phytotoxic effects that are distinct from those of the PDS inhibitors. In mature
cotyledons, for example, the effect of sulcotrione on both PS II quantum yield
and pigment content appeared intermediate between that of the PDS inhibitor
fluridone and the PS II herbicide, diuron. Thus, it was suggested that a direct
inhibition of electron transport from PS II, due to a depletion of PQ, would also
contribute to sulcotrione-based phytotoxicity [22]. The synergy between PS II and
HPPD inhibitor herbicides may further support this notion. The control of several
weeds by atrazine, including that of certain resistant biotypes, is synergized by the
addition of mesotrione; in addition, a significantly faster rate of tissue necrosis
is observed with the mixture [23, 24]. This could be due to improved competitive
binding of PS II herbicides when the PQ level is depleted. However, there is also
evidence that PS II synergy effects might be mediated via the depletion of tocopherol
since, under high light and when electron transport from PS II is blocked (as by
PS II herbicides), the PS II P680 reaction center becomes over-reduced and the
chlorophyll partitions towards the triplet state. If unquenched, this would lead
to the generation of singlet oxygen, causing damage to the adjacent D1 protein,
PS II disassembly and chlorophyll release. Evidence from studies conducted in
Chlamydomonas [25] has suggested that tocopherol has a key role in PS II center
quenching. Thus, with careful poising of the concentration of HPPD inhibitor,
it was possible partly to inhibit photosynthesis in a culture of Chlamydomonas
such that, at low light levels, PQ did not limit the photosynthetic rate and
the tocopherol levels were only 50% diminished. On transfer to high light the
tocopherol pool diminished sharply and, within less than 3 h, PS II was inactivated
and the D1 protein had virtually disappeared. These effects were either prevented
or slowed by a direct addition of short-chain cell-permeable analogs of tocopherol
or diphenylamine, another direct chemical quencher of singlet oxygen. On the
other hand, various tocopherol-deficient mutants of Arabidopsis, vte2 (HPT), vte1
(tocopherol cyclase), and vte3-1 (a MSBQ/MPBQ point mutant deficient in α- and
γ -tocopherols) exhibit quite normal phenotypes [18, 26]. Only under rather drastic
conditions of short exposure to high light and low temperatures could bleaching and
lipid damage be induced. Under normal conditions, other protective mechanisms
such as the xanthophyll cycle (which HPPD inhibitors would effectively block)
and non-photochemical energy dissipation must compensate adequately for the
lack of tocopherols. Overall, it seems that the depletion of tocopherol [14] could
only contribute significantly to the phytotoxic effects of HPPD herbicides under
photo-inhibitory conditions. Nevertheless, it may underpin the synergy between PS
II and HPPD inhibitors where the two effects – of generating singlet oxygen and
removing the means of protection – may combine to cause more rapid damage.
4.2.2
Selectivity
Clearly, in order to be useful as herbicides in crops, HPPD inhibitors must

be selective. To date, commercially available HPPD inhibitor herbicides have
been mainly for use in corn (sulcotrione [1], mesotrione [27], tembotrione [28],
topramezone [29], and isoxaflutole [30]), although HPPD inhibitors are also used
in rice (pyrazolate, pyrazoxyfen [2]) and wheat (pyrasulfotole [31]), and are also
being developed for sugarcane (bicyclopyrone [32]). Pyrazolate, pyrazoxyfen, and
isoxaflutole are ‘‘pro-herbicides,’’ with the former being detosylated [2] and the
latter, (5-cyclopropylisoxazol-4-yl 2 mesyl-4-trifluoromethylphenyl ketone), quickly

hydrolyzed non-enzymically to the corresponding diketonitrile (DKN) [14]. The
corn-safety of DKN depends on its metabolism to an inactive benzoic acid derivative
[14], and cytochrome P450-based differential metabolism also serves as a major
basis for the corn-safety of topramezone [29], mesotrione [33], and tembotrione,
which is used in combination with the safener isoxadifen-ethyl [28]. Inherent
selectivity at the level of the enzyme is also an important factor. For example,
consistent with its broadleaved weed spectrum, mesotrione is a much more potent
inhibitor of Arabidopis HPPD (Kd ∼15 pM) than of wheat HPPD (Kd ∼5 nM).
Allowing for differences in the Km for HPP, this translates to an at least 100-fold
difference in the inherent potency of mesotrione between these examplars of
a broadleaved weed and grass-derived HPPD. Accordingly, transgenic tobacco
expressing wheat HPPD is highly resistant to mesotrione [33], whereas tobacco
likewise expressing Arabidopsis HPPD is not (T.R. Hawkes, unpublished data).
Where natural mechanisms of crop selectivity are inadequate, such genetic
engineering might provide an alternative route. However, as yet no transgenic
HPPD-herbicide-resistant crops have been commercialized, despite many details
having been reported in the patent and academic literature [11, 34]. Analogous to the
engineering of enolpyruvylshikimate-3-phosphate (EPSPS)-based crop-tolerance to
glyphosate [35], the mechanisms include increased expression as well as the
mutation and selection of HPPDs with improved tolerance. Novel here are
examples where crop tolerance is enhanced: (i) by increasing the concentration
of the substrate, HPP, through the coexpression of prephenate dehydrogenase;
or (ii) the bypassing of HPPD via the expression of a three-enzyme algal pathway
that provides an alternative route to HGA [11].
Aside from crop-safety, HPPD herbicides also need to be selected for properties
that minimize the potential for side effects in mammals. Thus, the pharmacokinet-
ics of, for example, nitisinone – which was selected for use as a drug to maintain
a long-term block on tyrosine degradation in humans – contrasts with those of
the herbicide mesotrione, which exerts only weak and transitory effects on blood
tyrosine levels [36]. The recently resolved crystal structures of rat and Arabidopsis
HPPDs with inhibitors bound [37] promises to further facilitate the design of HPPD
herbicides that can further discriminate between plant and mammalian HPPDs.
4.2.3
Structure and Mechanism
HPPD has a subunit polypeptide mass of 40–50 kDa, and is typically a tetramer in
bacteria or a dimer in eukaryotes (including mammals and plants) [12], although
the HPPD recently characterized from Chlamydomonas is tetrameric [38]. X-ray
structures (P. fluorescens, Arabidopsis thaliana, Zea mays, S. avermitilis, and rat) [37,
39–41] have indicated that the Fe and its associated inhibitor/substrate-binding
site is structurally well conserved. This region is located within the C-terminal
region of the protein that folds as a discrete domain. At the primary sequence
level, plant proteins appear somewhat distinct because they include a 15-amino
acid insertion, although at a structural level the core active-site region remains
similar to that in HPPDs from other phyla. As in all non-heme Fe(II) oxygenases,
this core consists of an active-site Fe(II) coordinated by a triad of two histidine
residues and one carboxylate [42]. In HPPD, the overall peptide fold, as well as
the disposition of these three residues through the primary sequence, is similar
to that of extradiol dioxygenases [12, 40]. Indeed, it is suggested that, like its
sister enzyme hydroxymandelate synthase (HMS) [15], HPPD exemplifies a 2-keto
acid-type dioxygenase that arose by convergent evolution from an extradiol type. In
all HPPDs the Fe(II) is located at the center of a cavity, between 8 and 14 Å wide,
that is formed by an eight-stranded twisted half-open β-barrel. The three residues
coordinating the Fe are located on three of these strands, while the surrounding
cavity environment is almost entirely conserved and dominated by hydrophobic
amino acid residues within rigid secondary structural elements.
As a non-heme Fe(II)-containing dioxygenase, HPPD is a member of a wider
group that couple the oxidation of a substrate by dioxygen to the oxidative de-
carboxylation of an α-keto acid (commonly α-ketoglutarate) [12, 15]. Of the four
electrons required to reduce dioxygen, two are derived from oxidative decarboxyla-
tion and the other two from the substrate itself. In the case of HPPD (Figure 4.2.1),
the 2-keto acid (pyruvyl) group is not a separate substrate but rather a side chain of
the phenyl ring substrate. The latter is first decarboxylated to a carboxymethylene
which then detaches and migrates to the adjacent carbon, while the phenyl ring
is hydroxylated at the original point of attachment. The current understanding
of the catalytic mechanism derives from a combination of structural, spectro-
scopic, and kinetic information and, as most of the proposed intermediates are
too short-lived to be observed directly, also from theoretical considerations. The
results of steady-state kinetic studies [12, 43] have suggested that HPP binds first,
and that CO2 is the first product to be released. Thus, in an analogous reaction to
the α-ketoglutarate dioxygenases, HPP binds initially as a bidentate ligand of the
Fe(II) [12] to form an enzyme–Fe(II)–HPP complex. Fe(II) is relatively unreactive
in the resting enzyme, and it is the binding of the keto acid moiety that primes
the Fe(II) to react with dioxygen [15, 44]. The binding of dioxygen is endergonic,
and the reactive Fe(III)–O2 species thus formed is predicted to be short-lived. The
withdrawal of electrons into the Fe-bound dioxygen facilitates a nucleophilic attack
on the α-carbonyl carbon, which results in decarboxylation and the generation of a
theoretical short-lived Fe(II)-peracid species that heterolytically disproportionates
to yield the oxo-Fe(IV) electrophile intermediate which is believed to be common
to all keto acid dioxygenase reactions [12, 15].
Proposals for the subsequent steps are varied, with some proposing that the
strongly electrophilic oxo-Fe(IV) species abstracts two electrons from the aromatic
ring to generate an arenium cation or oxide [12]. Alternatively [45], it can be argued
that with two negatively charge ligands in the iron coordination shell, the transfer
of a second electron from the ring to the iron would be hindered and that a single
electron process would be more likely. The single-electron process [45] depicted in
Figure 4.2.2 yields a radical sigma complex (that could also generate arene oxide
via nonproductive side reactions), the C–C bond of which cleaves homolytically
HO HO
HGA HPP
H
OH2
H 2O OH2
Fe(II)
O O O2
O −
O
Fe(II) − HO
HO O O
H2O Fe(II)
O
O O HO HO −
−
Fe(II) O O O− O
HO O Fe(III)
O
O O
Fe(III) O− CO2
O O
O Fe(II)
O O−
Fe(IV)
Figure 4.2.2 Proposed intermediates in the HPPD reaction. Based on Ref. [45].
to yield a highly unstable biradical species that then decays to form the new C–C
bond (the side-chain migration step) attached to a ketone intermediate. Finally,
re-aromatization and tautomerization would yield the enzyme-bound HGA.
Of these various putative intermediates, an experimental basis has been provided
for only a few. The initial Fe(II):HPP enzyme complex has been isolated under
anaerobic conditions, and shown to exist as a mixture of five- and six-coordinate
Fe species [46]; Fe(II):inhibitor enzyme complexes are structurally similar
[12, 41]. There is direct evidence for the oxo-Fe(IV) species as an intermediate in
the catalytic reactions of α-ketoglutarate dioxygenases [15] and, consistent with the
involvement of this powerful oxidant, HPPD hydroxylates the alternative substrate
(4-hydroxyphenyl) thiopyruvate on sulfur rather than on the ring [47]. Similarly,
HPPD can readily be mutated [48] towards HMS activity, where hydroxylation
occurs at the benzylic carbon rather than on the ring. It appears less easy to mutate
HMS enzymes towards HPPD activity. Further mutant forms of HPPD make yet
alternative products from HPP such as quinolacetic acid (which is responsible
for the hereditary disease Hawkinsinurea) and oxepinone [16]. These products
could derive from either an arene epoxide or an arenium cation, which suggests
that such an epoxide or cation species might be an intermediate of the normal
catalytic pathway. Overall, it seems likely that all of these reactions – whether
normal or mutant – share a common pathway as far as an oxo-Fe(IV) species
and then diverge, with HGA-formation likely to be the most demanding of a
precise active-site geometry. The HPPD:HGA product complex has been identified
through a combination of fluoresence-monitored stopped-flow and rapid chemical
quench studies. In this case, the release of HGA is associated with a solvent
deuterium isotope effect, and appears to have a major rate-limiting effect on the
catalytic cycle [49, 50].
4.2.4
Inhibition
X-ray crystallography has provided considerable insight into how inhibitors bind.
In the S. avermitilis HPPD–nitisinone complex, Fe is five/six-coordinate with
bidentate chelation from the 5 and 7 oxygens of the inhibitor, and with water
weakly occupying the sixth position [41]. The inhibitor binding shifts a C-terminal
helix to provide one of two phenylalanines that sandwich the phenyl ring of the
inhibitor in a π-stacking interaction. No other energetically significant interactions
between the inhibitor and enzyme surfaces are evident, other than the exclusion of
waters through space-filling van der Waals contacts. Nitisinone, mesotrione, and
DKN bind significantly only to the Fe(II) forms of the carrot, Arabidopsis, and S.
avermitilis enzymes [49, 51, 52], and the so-formed inhibitor complexes are highly
stable, unreactive to oxygen, and (similar to the anaerobic Fe(II)–enzyme–HPP
complex) weakly colored due to charge-transfer transitions [12, 52]. Both, magnetic
and nonmagnetic circular dichroism spectroscopy, combined with calculations
based on density function theory, have indicated that nitisinone interacts with
the Fe(II) somewhat more weakly than does the substrate [53]. Consequently, it
is considered that the π-stacking interaction of electron-deficient aromatic rings
with the enzyme phenylalanines must make a major contribution to the inhibitor
binding. It is possible that similar π-stacking interactions may help to drive the
catalysis by electronically stabilizing the putative arenium cation [12] or the phenyl
radical intermediate (see Figure 4.2.2).
Both, spectroscopic and kinetic studies [12, 53] have partly unraveled the steps
involved in inhibitor binding. The di- and triketone inhibitors are present in
solution as an equilibrium between different tautomeric forms; at pH 7.0 the
exocyclic enol(ate) of nitisinone predominates, whereas the keto form of HPP is
the substrate [54]. The results of stopped-flow studies have indicated that, under
anaerobic conditions at 5 ◦ C, nitisinone binds rapidly to S. avermitilis HPPD to
initially form a weak non-chromophoric complex that isomerizes (8 s−1 ) to a first
chromophoric complex that exhibits a further slow (0.76 s−1 ) chromophoric shift to
a final tightly bound complex. A solvent deuterium isotope effect of ∼3.0 suggests
that a proton shift is involved in the formation of the latter. Mesotrione binding to
Arabidopsis HPPD has been shown to follow a similar pattern [49].
Whatever the exact details of the binding process, it is clear that HPPD herbicides
are remarkably potent as inhibitors of HPPD, and that this underpins their
effectiveness. Indeed, they are so potent and they equilibrate so slowly with the
enzyme and substrate that the dissociation constants cannot be evaluated by
using conventional steady-state assay methods. Thus, at this point only values
based on the few reported estimates of the constants governing formation (kon )
and dissociation (koff ) of enzyme–inhibitor complexes are cited (Table 4.2.1).
These have been estimated on the basis of time-dependent changes in enzyme
activity, inhibitor binding, and rates of radiolabeled inhibitor exchange [6, 33, 34,
51, 55]. At 25 ◦ C, the apparent kon values (condensing what is really at least a
three-step binding process) obtained at nonsaturating concentrations of inhibitors,
Table 4.2.1 Estimated inhibition constants of the HPPD inhibitors.
Inhibitor (Figure 4.2.1) HPPD kon (M –1 s –1 ) koff (s –1 ) Kd (pM) Reference
(3) Carrot 1.5 × 104 9 × 10 –5 6000 [41]

(3) P. fluorescens 1.6 × 104 1.8 × 10 –4 11000 [27]
(3) Wheat 6.9 × 104 6.2 × 10 –5 900 [27]
(3) Arabidopsis 1.8 × 105 8.3 × 10 –6 46 [27]
(4) Arabidopsis 2.3 × 105 3.3 × 10 –6 14 [26]
(4) Wheat 1.8 × 105 1.0 × 10 –3 5500 [26]
(5) [Cl] Arabidopsis 3.0 × 105 1.2 × 10 –6 4 [27]
(5) [Cl] P. fluorescens – >2 × 10 –4 – [27]
(5) [Cl] Wheat 3.0 × 105 4.2 × 10 –6 12 [27]
(5) [CF3 ] Rat 1.5 × 104 1.9 × 10 –6 125 [45]
(6) Rat 9.9 × 104 3.2 × 10 –6 32 [6]
(1) Rat 3.3 × 10-4 1.9 × 10 –5 575 [6]
fall within the range 1E4−3E5 M−1 s−1 , while the half-times for dissociation of the
so-formed enzyme–inhibitor complexes range from hours to days. Thus, HPPD
inhibitor–herbicides exhibit Ki values in the picomolar range and, indeed, have
been described as effectively irreversible. However – perhaps surprisingly in view
of the fact that the active-site regions of HPPD are so highly conserved – inhibition
is not always so potent, and there are large species-dependent differences in
inhibitor Kd and koff values of up to several hundred-fold. For example, syncarpic
acid structures such as (5) of Figure 4.2.1 can be two or more orders of magnitude
weaker as inhibitors of the Pseudomonas HPPD than of the enzyme from Arabidopsis;
mesotrione is a much more potent (Ki ∼15 pM) inhibitor of Arabidopsis HPPD than
it is of the enzyme from wheat (Ki ∼5 nM). Similarly, an inhibitor derived from an
enzyme-screening campaign was reported to be some three orders of magnitude
less potent an inhibitor of mammalian than of Arabidopsis HPPD [37]. Such
differences in intrinsic potency are sufficient to determine important biological
differences in the herbicide weed spectrum, the potential for tyrosinemia, and
the comparative utility of different HPPD transgenes in herbicide-tolerant crops.
Hundred-fold and greater differences in Kd (12 kJ mol−1 ) may originate from the
sum of structural and flexibility differences that are too subtle to discern on the basis
of X-ray structural comparisons. It is difficult to know which kinetic parameter to
take as being the most predictive of biological activity. ‘‘Stickiness’’ (koff ) may be
key to maintaining a persistent blockade of the pathway since, once inhibited,
HPPD may then remain inhibited for days (until new enzyme is synthesized),
and this may be important to achieve good herbicidal activity. Interestingly, it
appears that inhibition requires the binding of only a single inhibitor molecule
per HPPD dimer; while only a single DKN molecule was bound per dimer of
carrot HPPD; this ‘‘half-site’’ binding was, nevertheless, associated with complete
enzyme inhibition [51].
References
1. Beraud, J.M., Claument, J., and 18. Eckardt, N.A. (2003) Plant Cell, 15,
Monturey, A. (1991) Proc. Brighton 2233–2235.
Crop Prot. Conf. – Weeds, 1, 51–55. 19. Garcia, I., Rodgers, M., Pepin, R.,
2. Matsumoto, H. (2005) Am. Chem. Soc. Hsieh, T.-F., and Matringe, M. (1999)
Symp. Ser., 892, 161–171. Plant Physiol., 119 (4), 1507–1516.
3. Mayonado, D.J., Hatzios, K.K., Orcutt, 20. Mayer, M.P., Beyer, P., and Kleinig, H.
D.M., and Wilson, H.P. (1989) Pest. (1990) Pestic. Biochem. Physiol., 34,
Biochem. Physiol., 35, 138–145. 111–117.
4. Sandmann, G. (2002) in Herbicide 21. Masamoto, K., Hisatomi, S.-I.,
Classes in Development (eds P. Boger, Sakurai, I., Gombos, Z., and Wada,
K. Wakabayashi, and K. Hirai), H. (2004) Plant Cell Physiol., 45 (9),
Springer-Verlag, Berlin, Heidelberg, 1325–1329.
pp. 221–229. 22. Kim, J.-S., Kim, T.-J., Kwon, O.K., and
5. Sandmann, G., Boger, P., and Kumita, I. Cho, K.Y. (2002) Photosynthetica, 40 (4),
(1990) Pestic. Sci., 30, 353–355. 541–545.
6. Ellis, M.K., Whitfield, A.C., Gowans, 23. Armel, G.R., Hall, G.J., Wilson, H.P.,
L.A., Auton, T.R., Provan, W.M., Lock, and Cullen, N. (2005) Weed Sci., 53 (2),
E.A., and Smith, L.L. (1995) Toxicol. 202–211.
Appl. Pharmacol., 133 (1), 12–19. 24. Woodyard, A.J., Hugie, J.A., and
7. Lindstedt, S., Holme, E., Lock, E.A., Reichers, D.E. (2009) Weed Sci., 57,
Hjamarson, O., and Strandvik, B. (1992) 369–378.
Lancet, 340, 813–817. 25. Trebst, A., Depka, B., Jaeger, J., and
8. Prisbylla, M.P., Onisko, B.C., Shribbs, Oettmeier, W. (2004) Pest Manage. Sci.,
J.M., Adams, D.O., Liu, Y., Ellis, M.K., 60 (7), 669–674.
Hawkes, T.R., and Mutter, L.C. (1993) 26. Havaux, M., Eymery, F., Porfirova, S.,
Proc. Brighton Crop Prot. Conf. – Weeds, Reay, P., and Dormann, P. (2005) Plant
2, 731–738. Cell, 17, 3451–3469.
9. Schulz, A., Ort, O., Beyer, P., and 27. Mitchell, G., Bartlett, D.W., Fraser,
Kleinig, H. (1993) FEBS Lett., 318 (2), T.E.M., Hawkes, T.R., Holt, D.C.,
162–166. Townson, J.K., and Wichert, R.A. (2001)
10. Norris, S.R., Shen, X., and Pest Manage. Sci., 57 (2), 120–128.
DellaPenna, D. (1998) Plant Physiol., 28. Schulte, W. and Kocher, H. (2009) Bayer
117 (4), 1317–1323. CropSci. J., 62, 35–51.
11. Matringe, M., Sailland, A., Pelissier, B., 29. Grossman, K. and Ehrhardt, T. (2007)
Rolland, A., and Zink, O. (2005) Pest Pest Manage. Sci, 63, 429–439.
Manage. Sci., 61 (3), 269–276. 30. Luscombe, B.M., Pallett, K.E., Loubiere,
12. Moran, G.R. (2005) Arch. Biochem. P., Millet, J.C., Melgarejo, J., and Vrabel,
Biophys, 433, 117–128. T.E. (1995) Proc. Brighton Crop Prot.
13. Fernandez-Canon, J.M. and Penalva, Conf. – Weeds, 1, 35–42.
M.A. (1995) J. Biol. Chem., 270, 31. Schmitt, M.H., van Almsick, A., and
21199–21205. Willms, L. (2008) Pflanz.-Nachr. Bayer,
14. Pallett, K.E., Little, J.P., Sheekey, M., 61, 7–14.
and Veerasekaran, P. (1998) Pest. 32. Edmunds, A.J.F., Michel, A., Mitchell,
Biochem. Physiol, 62 (2), 113–124. G., and Zelaya, I. (2010) Bicyclopyrone:
15. He, P. and Moran, G. (2009) Curr. Opin. chemistry transforming weed control.
Chem. Biol., 13, 443–450. Lecture presented at the 12th IUPAC
16. Brownlee, J.M., Heinz, B., Bates, J., and International Congress on Pesticide
Moran, G.R. (2010) Biochemistry, 49, Chemistry, Melbourne, Australia, July
7218–7226. 4–8, 2010.
17. Schultz, G., Soll, J., Filder, E., and 33. Hawkes, T.R., Holt, D.C., Andrews,
Schulze-Siebert, D. (1985) Physiol. Plant, C.J., Thomas, P.G., Langford, M.P.,
64, 123–129. Hollingworth, S., and Mitchell, G. (2001)
4.3 Triketones 235
Proc. Brighton Crop Prot. Conf. – Weeds, 44. Johnson-Winters, K., Purpero, V.M.,
2, 563–568. Kavan, M., Nelson, T., and Moran, G.R.
34. Warner, S.A.J., Hawkes, T.R., and (2003) Biochemistry, 42, 2072–2080.
Andrews, C.J. (2002) PCT Patent Appli- 45. Borowski, T., Bassan, A., and Siegbahn,
cation WO 02/46387. P.E.M. (2004) Biochemistry, 43 (38),
35. Barry, G., Kishore, G., Padgette, S., 12331–11234.
Taylor, M., Kloacz, K., Weldon, M., 46. Neidig, M.L., Kavana, M., Moran, G.,
Re, D., Eichholtz, D., Fincher, K., and and Solomon, E.I. (2004) J. Am. Chem.
Hallas, L. (1992) in Biosynthesis and
Soc., 126, 4486–4487.
Molecular Regulation of Amino Acids in
47. Pascal, R.A. Jr, Oliver, M.A., and Chen,
Plants, (eds B.K. Singh, H.E. Floras,
Y.C.J. (1985) Biochemistry, 24 (13),
and J.C. Shannon), American Society of
3158–3165.
Plant Physiologists, Rockville, MD, pp.
139–145. 48. O’Hare, H.M., Huang, F., Holding, A.,
36. Hall, M.G., Wilks, M.F., Provan, W.Mc., Choroba, O.W., and Spencer, J.B. (2006)
Eksborg, S., and Lumholtz, B. (2001) FEBS Lett., 580, 3445–3450.
Br. J. Clin. Pharmacol., 52, 169–177. 49. Purpero, V.M. and Moran, G.R. (2006)
37. Yang, C., Pflugrath, J.W., Camper, Biochemistry, 45, 6044–6055.
D.L., Foster, M.L., Pernich, D.J., and 50. Johnson-Winters, K., Purpero, V.M.,
Walsh, T.A. (2004) Biochemistry, 43 (32), Kavan, M., and Moran, G.M. (2005)
10414–10423. Biochemistry, 44, 7189–7199.
38. Galvez-Valdivieso, G., Pienda, M., 51. Garcia, I., Job, D., and Matringe, M.
and Aguilar, M. (2010) J. Phycol., 46, (2000) Biochemistry, 39 (25), 7501–7507.
297–308. 52. Kavana, M. and Moran, G. (2003) Bio-
39. Fritze, I.M., Linden, L., Freigang, chemistry, 42, 10238–10245.
J., Auerbach, G., Huber, R., and 53. Neidig, M.L., Decker, A., Kavana, M.,
Steinbacher, S. (2004) Plant Physiol., Moran, G.R., and Solomon, E.I. (2005)
134 (4), 1388–1400. Biochem. Biophys. Res. Commun., 338 (1),
40. Serre, L., Sailland, A., Sy, D., 206–214.
Boudec, P., Rolland, A., Pebay-Peyroula,
54. Lindstedt, S. and Rundgren, M. (1982)
E., and Cohen-Addad, C. (1999) Struc-
Biochim. Biophys. Acta, 704 (1), 66–74.
ture, 7 (8), 977–988.
55. Ellis, M.K., Whitfield, A.C., Gowans,
41. Brownlee, J.M., Johnson-Winters,
D.H.T., Harrison, G.R., and Moran, L.A., Auton, T.R., Provan, W.M., Lock,
G. (2004) Biochemistry, 43, 6370–6377. E.A., Lee, D.L., and Smith, L.L. (1996)
42. Hegg, E.L. Jr and Que, L. (1997) Eur. J. Chem. Res. Toxicol., 9 (1), 24–27.
Biochem., 250, 625–629. 56. Wu, C.S., Huang, J.L., Sun, Y.S., and
43. Rundgren, M. (1977) J. Biol. Chem., 252 Yang, D.Y. (2002) J. Med. Chem., 45,
(14), 5094–5099. 2222–2228.
4.3
Triketones
4.3.1
Introduction
The aim of this chapter is to provide an insight into the discovery of the trike-
tone class of herbicides, and their continuing development. A very qualitative
picture of structure–activity relationships (SARs) will be discussed and currently
commercialized triketones, in terms of their use, weed spectrum, crop selectivity,

environmental and toxicological profiles, and manufacture will be described. Also
included is an overview of the activities of the major companies in the field during
the past three decades, focusing on compounds that are likely to be brought to the
market, or which were putatively close to development.
4.3.2
Discovery
In 1977, at the Western Research Center in California, the research team at Stauffer
(a former legacy company of Syngenta) noticed that relatively few weeds grew
under the bottle brush plant Callistemon citrinus. An analysis of soil samples where
C. citrinus was growing revealed that the plants were in fact excreting a herbicide,
leptospermone (1) [1]. This natural product had previously been isolated from the
volatile oils of Australian myrtacious plants [2]. Pure samples of leptospermone (1)
showed unique bleaching symptoms on several weed species, albeit at relatively
high application rates (5 kg ha−1 ). This herbicidal activity was patented in 1980 [3].
Independent of this discovery, in 1982 a research team from the same company
was investigating the preparation of novel acetyl-CoA carboxylase inhibitors, based
on the typical cyclohexanedione structure known for this class. As the first targeted
compound (2; prepared as shown in Figure 4.3.1) had demonstrated a degree of
herbicidal activity, an attempt was made to prepare a phenyl analog in similar
manner. This led not to the expected product (3) but rather to the triketone (4),
a compound which was devoid of herbicidal activity, but (luckily!) in safener
screens showed antidotal effects in soya for thiocarbamate herbicides. However,
following a further round of synthesis optimization, this compound (5) – with
O
N
O O O
NH2
O O
O O O O
O
O 2
N O
O
NH2
Leptospermone 1
O O
3
O
O Optimization O O Rn O
O rounds O O X
Cl Cl
O
O O O Rm
Generic structure of
4 5 6 herbicidal triketones
Figure 4.3.1 Discovery of the triketone herbicides.

4.3 Triketones 237
an ortho-chloro substituent – was found to demonstrate reasonable herbicidal

activity. Moreover, it exhibited the same unique bleaching symptomatology as
observed for leptospermone (1; Figure 4.3.1) [4]. Further optimization showed that
removal of the methyl groups at the 5-position of the cyclohexanedione moiety
(compound 6) resulted in a significantly enhanced herbicidal activity against a wide
range of broadleaved weeds, with good corn tolerance, when applied both pre- and
post-emergence at rates of about 2 kg ha−1 . Subsequently, the first patent was filed
[5], and the discovery of the benzoylcyclohexanedione herbicides had been made.
These events, and the generic structure of herbicidal triketones, are summarized
in Figure 4.3.1.
4.3.3
Mode of Action
As discussed in detail in Chapter 4.2, triketones exert their herbicidal mode

of action via the inhibition of the enzyme 4-hydroxyphenylpyruvate dioxygenase
(HPPD) (see Chapter 4.2). Triketones are not the only herbicide class to have
this mode of action; indeed, it has been shown retrospectively that apparently
structurally non-related heterocyclic commercial herbicides such as isoxaflutole
(7, Balance® and Merlin®), and the rice herbicides pyrazolate (8, Sanbird®)
and benzobicyclon (9, Show-Ace®) also cause these bleaching symptoms via the
same mode of action. A common feature of these herbicides, however, following
metabolic activation to the active metabolites (7 ) [6], (8 ) [7], and (9 ) [8], is the
presence of an acidic 1,3-dicarbonyl moiety, which is also present in triketones
(Figure 4.3.2). Triketones and related herbicides mimic the α-keto acid group of the
HPPD substrate hydroxyphenyl pyruvate, and competitively bind to the iron at the
active site of the enzyme, causing its inhibition. Homology models of all structure
types bound to this enzyme [9], as well as the crystal structures of pyrazoles [10]
and triketones [11] bound to the same enzyme, have been reported.
4.3.4
Synthesis of Triketones
The majority of triketone herbicides (13) reported to date have been synthesized via
the O-acylation of a cyclic 1,3-dione (10) with an activated aroyl acid (11), followed
by an O- to C-acyl rearrangement of the O-acyl intermediate (12) in the presence
of a catalyst (Scheme 4.3.1). The O-acylation is generally achieved using an acid
chloride in the presence of a base, although other reagents such as dicyclohexylcar-
bodiimide (DCC) [12], N-methyl-2-chloropyridinium iodide (Mukaiyama coupling
agent) [13] and 2-chloro-1,3-dimethylimidazolinium chloride [14] have been used
in triketone synthesis. Typical catalysts used for the rearrangement include cyanide
[15], aluminum trichloride [16], 1,2,4-triazole [17], potassium fluoride [18], and
azide salts [19], with the cyanide source (including acetone cyanohydrin) -induced
O–C-rearrangement having generally been the method of choice. Although the
reaction may be carried out in stepwise fashion (i.e., the isolation of 12), one-pot
Isoxaflutole (7)
SO2Me SO2Me
O O
CF3 CF3 OH
O
N 7′
N
Pyrazolate (8)
Cl Cl X
O O O Rm O
Cl O S Cl OH OH
O
N N O
N N Rn
8′
Benzobicyclon (9) 13
Cl
Cl Generic structure
O
O MeO2S of herbicidal
S OH
MeO2S triketones
O
O
9′
Figure 4.3.2 Commercial herbicides with HPPD mode of action.
O O
O O
Z X O X Rn O
Rn O-Acylation Rearrangement
Rn X
Rm Rm O
O
O Rm
10 11 12 13
one-pot variations
Scheme 4.3.1 Catalyzed O- to C-acyl rearrangement in the preparation of triketones.
variations have been developed in many cases by choice of the correct solvent
[20]. There is also an isolated report of direct C-acylation (10) with an aroyl acid
chloride (11, Z = Cl) using potassium carbonate in acetonitrile [21]. Alternatively,
triketones can be obtained directly by the acylation of 10 with the appropriate
benzoyl cyanide (11, Z = CN) [22].
Other syntheses that have been developed for the preparation of triketones
include activating the dione portion (to give compound 14) and coupling this with
an aroyl acid (15) in the presence of a Lewis acid catalyst [23]. This is followed by
O-acyl rearrangement, or by a palladium-catalyzed carbonylation of an aroyl halide
(16) in the presence of a dione (10) [24], with subsequent rearrangement of the
O-acyl product (12) formed to create the triketone (13) (Scheme 4.3.2).
The synthesis of cyclcohexane diones with various substitution patterns can be
readily achieved by the two general routes shown in Scheme 4.3.3 [25, 26].
4.3 Triketones 239
O ZnCl2/Di-
isopropylethylamine
LG HO X Toluene
Rn
Rm
O O
14 15 O X
Rearrangement
Rn
LG = Leaving Group, e.g. Br
Rm
O
Hal X
O 12
O
Rn Rn O
Rm LiCl/Et3N/PdCl2(PPh3)2 X
Toluene
O
O
10 16 Rm
13
Scheme 4.3.2 Alternative strategies for triketone synthesis.
R1 O R1 O R1 O
O R Base R2 O Base R2
R2
R3 R3 R3
R5 R4 O R4 R O
R5 5
R4 O
R1 O 1. 2 eq. LDA R1 E O
R2 2. E+ R2
R3 R3
R4 R O R4 R O
5 5
Scheme 4.3.3 Synthesis of substituted cyclohexane-1,3-diones.
The synthesis of the benzoyl portions of triketones cannot be so generalized,

and specific syntheses have been developed for developmental and commercialized
compounds.
4.3.5
Structure–Activity Relationships (SARs)
The triketones can be separated into two parts for analysis of the SARs, namely
the benzoyl and dione moieties. Each part can be examined independently, as they
appear to play distinct and different roles in the overall expression of herbicidal
activity [1]. Apart from the necessity for an ortho-substituent on the phenyl ring, it
was established that 2,4,- or 2,3,4-benzoic acid substitution patterns were required
for optimal activity, with the 2,5-pattern(s) being the least effective. After more
than 30 years of HPPD research, this original optimal substitution pattern still
seems to be valid, based on the analysis of published patents. A correlation was
found between the pKa of triketones (which can be viewed as vinylogous benzoic
acids) and herbicidal activity [27], with a pKa of <6 being required for activity,
as this will affect not only binding to the iron at the active site of the enzyme
but also transport and translocation within the plant. As the pKa will be affected
by substituents on the aromatic ring, those which generally reduce the electron
density of the aromatic ring lead to compounds with a reduced pKa and improved
herbicidal activity. A survey of the patent literature and reported SAR studies
[4, 28] has suggested that small ortho electron-withdrawing substituents, such as
Cl, NO2 , and CF3 , are particularly favored. An ortho-methyl substituent is also
tolerated as long as the total electron density of the aromatic ring is kept low. The
para substituent is generally also an electron-withdrawing moiety, particularly halo,
haloalkyl, and alkylsulfonyl, with some restraints on size according to published
data [4]. Zeneca (now Syngenta) arrived at several compounds with these types of
substitution pattern, such as sulcotrione (17) and mesotrione (18; Scheme 4.3.4),
at an early stage in triketone research. Both compounds have since reached the
marketplace (see Section 4.3.6).
At the meta position, a multitude of functionalities have been reported to lead to
herbicidally active compounds. However, one problem that often leads to reduced
potency is the presence of an electron-withdrawing substituent at the meta position,
combined with a potential leaving group at the 2-position (e.g., nitro or chloro),
as this may give rise to dihydroxanthenones (19) that are known to be much less
herbicidally active (Scheme 4.3.5) [4].
Two strategies have been generally used to remedy this situation. The first
strategy has been to use meta substituents such as alkoxy or thioalkyl; these are
reasonably electron-donating at their ortho positions, thus hindering the formation
of 19, but are inductively electron-withdrawing at the carbonyl, thus increasing
overall acidity [4]. The second, more frequently used, strategy is to have a small
non-leaving group at the ortho position (such as methyl), while having substituents
at the 3,4-positions which make the aromatic ring overall electron-deficient. Many
fused ring types have been reported in the patent literature, and it would appear
that para,meta-fused ring systems are generally more favored than ortho,meta-fused
systems.
The effect of adding substituents to the cyclohexanedione ring is to block the
site(s) of metabolism by plants [28, 29]. This results in a greater herbicidal activity,
O O
O O
Cl NO2
O O
SO2Me SO2Me
Scheme 4.3.4 Commercial triketone her-
Sulcotrione (17) Mesotrione (18)
bicides.
4.3 Triketones 241
O O O O Scheme 4.3.5 Dihydroxanthenone

formation in triketones.
Z O Z
OH X
Y Y
13 19
as the plants have much more difficulty in detoxifying the molecule. The results
of studies using model compounds have indicated that the principal routes of
metabolism of the benzoylcyclohexanediones in plants are hydroxylation at the
4-position of the cyclohexanedione (if this position is blocked, then hydroxylation
takes place at the chemically equivalent 6-position), and hydrolytic cleavage of the
benzoyl group. It has been shown that placing two methyl groups at the cyclohex-
anedione 4-position slows the rate of both of these metabolic processes in plants.
As the sites for hydroxylation are sequentially blocked, an increase in overall activity
against grasses is observed. Some of the most active triketones known contain the
2,2,4,4-tetramethyl-cyclohexane-1,3,5-trione moiety, also found in leptospermone
(1; Figure 4.3.1) [28]. However, reducing the potential for metabolism has other
consequences, such as a reduced corn selectivity and a dramatic increase in soil
persistence [1]. To compensate for this effect, several important diones have reg-
ularly appeared in the patent literature, which have strained bicyclic rings and/or
heterocyclic atoms (Scheme 4.3.6, compounds 20–23), and thus (at least putatively)
would be more easily metabolized.
4.3.6
Review of the Patent Literature
Some of the important and typical structural types patented by a variety of

companies are discussed in this section. It should be noted that the generic
structures are simplified, and thus are not necessarily those that appeared in the
referenced patents.
Following their initial discoveries, Stauffer (later Zeneca, now Syngenta) patented
their findings extensively, and thus were able to gain good intellectual property
advantage over their competitors, particularly in terms of important 2,4- and
2,3,4-substituted benzoic acid types (e.g., compounds 17 and 18, respectively 24
[22b] and 25 [30]), as well as important triketones with more substituted cyclohexane
diones [e.g., compounds 13a, X = C(O)] [5, 30] and heteroatom-containing diones
(e.g., compounds 13a, X = O; Figure 4.3.3) [31].
Stauffer (now Syngenta) were also granted a patent which covered cyclohexane
diones coupled to heteroaroyl acids (e.g., compounds 26–28; Figure 4.3.4) with very
O O O O
N
O
N
O Scheme 4.3.6 Diones with putatively
O O O
preferred balance of activity and soil
20 21 22 23
metabolism.
O O O
Rn O O O
X Cl NO2
O O O
1 Rm
SO2Me SO2Me
Rn = e.g. H, COOR, Sulcotrione (17) Mesotrione (18)
Alkyl
n = e.g. 0, 1, 2, 3, 4
R6 R1 O O
O O
R7 O Cl Cl
X R2
R8 R3 O O O O
R9 O
R4
R5 SO2Me SO2Et
13a 24 25
X, e.g. O, C(=O)
R6, R7, R8, R9 e.g. Methyl
R1, e.g. OH, SAr
R2, e.g. Halo, Alkyl, NO2, CF3,
R3, R4, R5, e.g Halo, SO2Alkyl,
CF3, OAlkyl
Figure 4.3.3 Typical Zeneca patents.
O O O
X1 O O O
X2 NO2 Cl
X
Het-Ar O O S O N
X3 N
O O Cl Cl
X1-X3 = O,S.
Het-Ar, e.g. subs. 5-and 6-Ring 26 27 28
and fused heterocycles but not
pyridine or pyrimidine.
X e.g. (CH2)3, opt. subst and/or
interupted by C=O, -O-
Figure 4.3.4 Zeneca heteroaroyl triketones.
broad scope [32], which made patenting rather difficult for companies following
Zeneca.
Pyridines and pyrimidines were patented separately, to complete an impressive
array of protection for the heterocyclic triketones [33]. Nevertheless, after the first
patent had appeared regarding this novel substance class, most of the major
companies began programs in the field. There were basically two strategies: some
companies searched for novel diones that were, at the time, beyond the scope of the
Zeneca published patents, while other companies searched for novel aromatic acids.
For example, Sandoz (now Syngenta) concentrated on the search for novel diones,
4.3 Triketones 243
and several compounds containing bicyclo[3.2.1]octane-2.4-dione, such as (29)

[34, 35] and (30) [36], as well as the oxazinedione types (31) [37, 38] proved to be im-
portant compounds for use in corn (Figure 4.3.5). A collaboration between Sandoz
and SDS Biotech has also led to the identification of proform triketones containing
bicyclo[3.2.1]octane-2.4-diones for use in rice, such as benzobicyclon (9) [39].
Nippon Soda also initially investigated the dione portion of triketones, and
patented many compounds containing bicyclo[4.1.0]heptane-2,4-diones, such as
32–35 (Figure 4.3.6) [40]. Nippon Soda also apparently were very interested in
triketones with a nicotinoyl acid moiety, based on the number of applications filed
in this area (compounds 34, 36, and 37; Figure 4.3.6) [40, 41]. Nissan had noticed
the similarity of the triketones to the pyrazole-type herbicides such as pyrazolate (8),
and had secured intellectual property freedom in this area by patenting pyrazoles
with the optimally substituted aroyl acids discussed previously [42]. Some important
triketones containing novel trisubstituted acids were also first patented by Nissan
(compounds 38–40; Figure 4.3.7) [43].
BASF initially attempted to conquer some intellectual property by using propri-
etary diones from their DIMS chemistry (41 and 42; Figure 4.3.8) [44]. However,
O O O S
O O O
O
NO2 O NO2
Cl
N
O O O O
O
SO2Me SO2Me Cl
SO2Me
29 30 31 Benzobicyclon (9)
Figure 4.3.5 Typical Sandoz compounds.
O O O
O O O
NO2 NO2
N O N
O O O N
SO2Me CF3 SO2Me

32 33 34
O O O
O O O
O
O O O N O N
SO2Me SO2Me SO2Me

35 36 37
Figure 4.3.6 Nippon Soda types.

O O O
O O O
Cl
O O
O O
O CO2Me
SO2Me SO2Me SO2Me

38 39 40
Figure 4.3.7 Nissan triketones.
O O O
O O O
S S NO2
O
O O O O
N
SO2Me SO2Me S
41 42 43 O
O
O O O
O O O
O
O O
O O O N
N
S N S O O
44 O 45 O 46
O O
O O O
O O O
O
O
O N O S O O O
S S
47
O
48 O 49 O S
O O
O O O
O O O
O Cl Cl
O O O
O S O O S O O S O
O S
50 O 51 52 O
Figure 4.3.8 BASF triketones.
after probably realizing that large substituents at the 5-position of cyclohexane-

diones are not optimal for herbicidal activity, they switched their attention to the
search for novel acids. Particularly prominent acids from BASF that have appeared
in the patent literature are the saccharins (e.g., 43–45; Figure 4.3.8) [45] and other
fused 3,4-aroyl acids such as 46 and 47; Figure 4.3.8) [46], and especially those in
which the alkylsulfonyl group is incorporated into a fused ring at the 3,4-positions
(compounds 48–52; Figure 4.3.8) [47, 48].
4.3 Triketones 245
BASF also explored patent-free examples of triketones with novel meta sub-
stituents, particularly acids containing heterocyclic rings at this position (e.g.,
53; Figure 4.3.9). The 4,5-dihydro-isoxazole-containing pyrazole corn herbicide
topramezone (54, Impact®, Clio®) [48, 49] has resulted from these studies
(Figure 4.3.9).
With regard to triketones of this structure type, Aventis (now Bayer) patented
substituted 4,5-dihydro-isoxazole compounds [12] prior to BASF [50], and two
compounds from Bayer (55 and 56; Figure 4.3.9) have frequently appeared in
mixture patents with safeners and other herbicides for use in corn [51].
Idemitsu also concentrated their efforts on new acids, with emphasis placed on
those in which the alkyl sulfonyl substituent at the 4-position was joined into a
ring at the 3-position (typical Idemitsu types 57–61 are shown in Figure 4.3.10)
[52]. Although a patent was received for compounds of this type with a 2-chloro
substituent (e.g., 59), Idemitsu were also forced to switch to more complicated
substituted heteroaromatic systems following the publication of interfering patents
from Zeneca [53], or to pyrazoles [54]. Idemitsu also appeared to have a pyrazole
compound (generic structure 61; Figure 4.3.10) in development for use in corn,
based on some published mixture patents [55].
Du Pont started relatively late in the triketone field, and directed their efforts
towards novel fused bicyclic acids. As a result of investigations in this area,
the broadleaf weed cereal herbicide ketospiradox (62, proposed common name;
Figure 4.3.10) was discovered [56]. However, for some reason unknown to the
authors ketospiradox has failed to reach the marketplace.
Ishihara, inspired by the earlier studies of Hokko [57], identified some new
benzoyl analogs with cyclic acetal meta substituents (63 and 64; Figure 4.3.11),
R9 R O OH
R10 8
O O N O
X R1 O N
N O N O N O
R11 O
R12O R4
R5
R3 R2 R7 R6 O S Cl O S
O O
BASF 53 Topramezone (54)
O
R5 O
O
X O O
Cl
Y R2 Ra Rb Cl
A R1 N O N O
Z
Rc O
O O
N O
O S CN
R4 R3
O O S O
O
Aventis (Now bayer) 55 56
Figure 4.3.9 BASF and Bayer meta-heterocyclic-substituted triketones.

O O O
O O O
Cl
N O
O O O
S S
O O S O
O O O
57 58 59
O Me, Et O-K+
O N OH, OSO2nPr, OCH2 O
N
O O O
O O O
O S S
O O O
O S
O
60 61 Ketospiradox (62)
Figure 4.3.10 Idemitsu and Du Pont triketones.
O O Figure 4.3.11 Ishihara rice

O O O triketones.
Cl Cl O
O
O O O O O
SO2Me SO2Me
63 64
that were claimed to have good pre-emergent activity in flooded rice paddy fields,
without damaging the rice seedlings [58].
Recently, Bayer have published – mainly after the successful merger with
Aventis–a multitude of patents [51], in which they have explored in more detail the
effect of several novel meta substituents on the biological activity of triketones, es-
pecially those with substituted 3-alkoxyalky-2-chloro-4-alkylsulfonyl substituents in
the aromatic ring. These studies have resulted in the post-emergence corn herbicide
tembotrione (65; Figure 4.3.12) [59]. Another very similar commercialized product
that has arisen from this class is tefuryltrione (66; Figure 4.3.12) [60], which has
been registered for use in paddy rice. Thus, it appears that the nature of the alkoxy
substituent at the meta position of the benzoyl moiety can be used to tune the weed
spectrum and crop selectivity. Tembotrione (65) and tefuryltrione (66) are not only
similar to one another, but are clearly also very similar to compounds previously
patented by Nissan (see Figure 4.3.7, 40) and the Ishihara rice compounds shown
in Figure 4.3.11, respectively.
DOW have invested virtually all of their effort in the field of pyrazoles, and has
recently produced several reviews in this area (see Chapter 4.4). Although very few
4.3 Triketones 247
O Figure 4.3.12 Bayer

O
O O commercialized triketones.
Cl CF3 Cl O
O O
O O
SO2Me SO2Me
Tembotrione (65) Tefuryltrione (66)
triketone patents have appeared, they referred to meta-amino substituents, such as

compounds 67 and 68 (Figure 4.3.13) [61].
Despite the broad granted scope of the initial Zeneca heteroaroyl triketone patent
[32], the pyridyl triketones were not further pursued by Zeneca, neither (behind the
earlier Stauffer claims [34]) by Sandoz [26b] nor by Ciba-Geigy [62] (all now Syn-
genta). Nippon Soda too also left gaps in their patents in claiming pyridyls [40, 41].
All of this was exploited then once more by Novartis (now Syngenta), with a series
of patents and publications involving novel pyridyl acid-containing triketones being
published [11b, 63a, 63b]. Indeed, on the basis of both mixture [64] and process
[23, 35, 65] patents, it appears that Syngenta have identified some very interesting
new compounds for use in corn (69 and 70) and cereals (71 and 72; Figure 4.3.14).
In fact, Syngenta is currently developing compound 70, which is referred to com-
monly as bicyclopyrone. This active ingredient is reported to provide season-long,
broad-spectrum weed control, including that of large-seeded dicotyledons such as
Ambrosia trifida (giant ragweed), Xanthium strumarium (common Cocklebur), Ipo-
moea sp. (morning glory), and difficult-to-control grasses including Eriochloa villosa
(woolly cupgrass), and Panicum sp. (millets) in corn and sugarcane, with excellent
crop selectivity (no safener required). Bicyclopyrone not only allows application
flexibility from pre-planting to post-emergence, but also functions under different
O O
O O
Cl
O N O N
SO2Me SO2Me
67 68 Figure 4.3.13 Dow triketones.
O O O O O O
O O O O
O O
N
O N O N O N O O
CF3 CF3 CF3 CF3
69 70 71 72
Bicyclopyrone
Figure 4.3.14 Syngenta pyridyl triketones.

environmental conditions and cropping practices [11b]. The use of bicyclopyrone

in other crops is currently under investigation.
One particularly interesting point here is that compounds 70 and 71, which
contain larger ortho substituents than the usual patented types (e.g., Cl or Me), are
tolerated at the enzyme site, and that the picolinic acid (72) has no ortho substituent,
which suggests that the lone pair on the pyridyl nitrogen can act as such.
The research team at Bayer has since also disclosed information relating to
6-substituted 1,2,4-triazole nicotinic acids (73–75; Figure 4.3.15) [66], with activity
in corn, wheat, and soya having been claimed for these compounds.
Syngenta have continued their interest in the nicotinoyl area by fusing a second
heterocyclic ring to the nicotinoyl ring (Figure 4.3.16). It is claimed that fused bicylic
heteroaryl systems, in the form of triazolopyridine triketones, display interesting
activity with a range of classic diones (76–78; Figure 4.3.17) [67].
Following the discovery of the triazolopyridines, a number of published Syn-
genta patent applications have disclosed various other fused heterocyclic systems,
containing naphthyridines and naphthyridinones as novel acid scaffolds [68–70].
A broad weed spectrum is claimed for these compounds, which suggests that
R4 R5 O
R3 R6
R2 R7
O O O O O O O O
Examples
O O O O
N N N O CF3 N
N N R1 N N N N N N
N N N N
73 74 75
Figure 4.3.15 Bayer triazole pyridine triketones.
O O R O O R
R4 R4
R3 N R3 N
X X Figure 4.3.16 Syngenta
O R5 O R5
fused bicyclic heteroaryl
R2 R1 R6 R2 R1 R6
systems.
O O N X1 O O N N O O N N
R4 X2
R3 N N N
X
O R5 O CF3 O OMe
R2 R1 R6 76 77
O O N
N
N
O CF3
78
Figure 4.3.17 Syngenta triazolopyridine triketones.

4.3 Triketones 249
the enzyme accommodates these fused heteroaryl systems favorably. A series of

published Kumiai patent applications have disclosed pyridones, oxopyrazines and,
most recently, fused pyridone triketones, all of which suggests that the area of
fused pyridyl heterocycles could be of potential interest [71–73].
Despite the long history and wide-ranging existing chemistries designed as
HPPD inhibitors, these new classes show that is still room for new and important
discoveries that may take this mode of action to another level in terms of potency
and weed spectrum.
4.3.7
Commercialized Triketone Herbicides
4.3.7.1 Sulcotrione
The first triketone herbicide to be commercialized was sulcotrione (17), which was
discovered by Stauffer (a legacy company of Syngenta) and first registered for use
in 1993 [74]. The product is sold under the trade name of Mikado® in Europe, and
had sales in 2009 of US$ 55 million [75]. In 2000, sulcotrione was sold by Syngenta
to Bayer, in order to satisfy conditions imposed by the European Commission in
connection with the merger of Novartis Agribusiness and Zeneca Agrochemicals to
form Syngenta. Sulcotrione is used for the post-emergence control of (particularly)
broadleaf weeds and some grass weeds (Digitaria sanguinalis, Echinochloa crus-galli,
and Panicum miliaceum) in corn, with application rates ranging from 300 to
450 g ha –1 . Bayer has now obtained registration for sulcotrione in the USA and has
thus expanded its market presence; however, the compound will always have to
compete with mesotrione for market share. Sulcotrione is readily absorbed through
the leaves and also via the roots; details of its metabolism in soil are shown in
Figure 4.3.18 [76]. Soil half-lives of sulcotrione (as the parent compound) ranging
O HO O O
O O OH O
Cl Cl Cl
O O O
SO2Me SO2Me SO2Me

79
Sulcotrione (17)
O O
Cl HO Cl
SO2Me SO2Me
80 81
Figure 4.3.18 Metabolism of sulcotrione in the soil.

1. Cl2/FeCl3/I2
2. Na2SO3
3. NaOH/H2O
O Cl O Cl Cl
HO O
O Cl O HO O Δ
Cl S S S S
O NaO O O
83 84
O2/HBr/Co(OAc)2
O H 2O
O Cl Cl
Cl DMF/Dioxan O Cl SOCl2 O OH
S S
O O O O O
SO2Me 85 81
Sulcotrione (17) H2/Raney Ni

O NaOH/H2O OH
50°C/60Atm.
O OH
82
Scheme 4.3.7 A possible technical synthesis of sulcotrione.
from 15 to 74 days have been measured; consequently, the compound causes no

threat to cereals, which are the usual rotational crops to corn in Europe [77].
The details of a possible technical synthesis of sulcotrione that provided a yield
of 59–62%, starting from resorcinol (82) and p-toluenesulfonyl chloride (83), is
shown in Scheme 4.3.7 [78].
Selected physical chemical, toxicological, and environmental properties of sul-
cotrione are listed in Table 4.3.1.
4.3.7.2 Mesotrione
Mesotrione (18) is a second-generation triketone product that was developed by
Zeneca (now Syngenta) as Callisto® for both pre-emergence and post-emergence
control of all the important broadleaved weeds in corn, together with some of
the annual grass weeds (Digitaria and Echinochloa species), that are important
in European corn production [1]. The typical usage rates of mesotrione range
from 100 to 225 g ha−1 when applied pre-emergence, and from 70 to 150 g ha−1
for post-emergence applications. Broadleaved weeds controlled include Xanthium
strumarium, Abutilon theophrasti, Ambrosia trifida, together with Chenopodium,
Amaranthus, and Polygonum sp.
The selectivity of corn is based on its ability to rapidly metabolize (detoxify)
mesotrione into inactive metabolites (86) and (87) (Figure 4.3.19), a process that is
mediated by cytochrome P450 enzymes in both corn and weeds. In corn, the detoxi-
fication process is so rapid that mesotrione is not translocated away from the directly
treated area. In contrast, the cytochrome P450 enzymes in susceptible weeds are
Table 4.3.1 Selected physical, chemical, toxicological, and environmental properties of commercial triketones.
Compound Sulcotrionez Mesotrione Benzobicyclon
O O SPh
O O O
Structure Cl NO2 Cl
O O O
17 SO2Me 18 SO2Me 9 SO2Me
Major product names Mikado®. Callisto®: Other products (mixtures): Show-Ace® Other products
Camix®, Calaris®, Lexar® Lumax® (mixtures); Focus Shot®
(+ pentoxazone) Kusakonto®
(+ pretilachlor) Smart®’
(+ benzofenap + fentrazamide)
Melting point (◦ C) 139 165 187.3
Vapor pressure (mPa) 5 × 10−3 (25 ◦ C) 5.69 × 10−3 (20 ◦ C) < 5.6 × 10−2 (25 ◦ C)
Kow logP <0 0.11 3.1
Solubility in water (mg l –1 ) 165 (25 ◦ C) 15 (25 ◦ C); 2.2 (20 ◦ C) (pH 4.8) 0.00052 (20 ◦ C)
Stability in water Stable to hydrolysis Stable to hydrolysis Rapidly hydrolyzed
pKa 3.13 3.12 –
Rat: (acute oral) LD50 (mg kg –1 ) >5000 >5000 >5000
Birds: dietary LC50 for bobwhite >5620 >2000 2250
quail (mg kg –1 )
Fish: LC50 (96 h) for rainbow trout 227 >120 LC50 (48 h) for carp >10 ppm
(mg l –1 )
Algae (Selenastrum capricornutum) EC50 (96 h) 1.2 mg l –1 EC50 (72 h) 4.5 mg l –1 EC50 (72 h) >1 ppm
Bees LD50 (oral and contact) (μg per >200 >9 >200
4.3 Triketones
bee)
Koc 44 (high pH) to 940 (low pH) 19 (pH 7.7) to 387 (soil pH 4.6) –
251
Soil degradation time (DT50 ; days) 1–11 4–31.5 –
(continued overleaf )
Compound Tembotrione Tefuryltrione

252
O O
O O
Structure Cl O
Cl CF3
O O
O O
65 SO2Me 66 SO2Me
Major product names LAUDIS® OD (tembotrione with BODYGUARD® (mixture with

isoxadifen as safener) SOBERAN® (Brazil fentrazamide) POSSIBLE® (mixture
only, tembotrione with with mefenacet) GET-STAR® (mixture
isoxadifen) CAPRENO® (thiencarbazone with pyraclonil) A-ONE® (mixture
+ tembotrione + isoxadifen) with oxaziclomefone)
Melting point (◦ C) 123 113.7–115.4

Vapor pressure (mPa) 1.1 × 10−5 (25 ◦ C) < 1.0 × 10−5 (20 ◦ C)
Kow logP –1.09 1.9
Solubility in water (g l –1 ) 28.3 (20 ◦ C) 64.2
Stability in water Stable Stable to hydrolysis
pKa 3.13 3.2
Rat: (acute oral) LD50 (mg kg –1 ) >2000 >2500
Birds: dietary LC50 for bobwhite >1788 Japanese quail: male 1625; female
quail (mg kg –1 ) >2000
Fish: LC50 (96 h) for rainbow trout >100 Carp >100
(mg l –1 )
Algae (Pseudokirchneriella EC50 (72 h) 0.38 mg l –1 EbC50 3 mg l−1 ; ErC50 5.3 mg l−1
subcapitata)
Bees LD50 (μg per bee) Oral 92.8 Oral >100; contact >95.78
Koc 33 (high pH) to 130 (low pH) 108–1226
Soil degradation time (DT50 ; days) 2–10 (Lab test in container) Volcanic ash LiC
44, Diluvial CL 62; (Field) Volcanic ash
LiC 39, Diluvial CL 14
4.3 Triketones 253
O
O
NO2
Mesotrione (18)
O
Oxidation SO2Me
O O
HO NO2 HO NH2
Conjugates Conjugates
SO2Me SO2Me
MNBA (86) AMBA (87)
Figure 4.3.19 Mesotrione in planta metabolism.
able to metabolize mesotrione only slowly, and this allows an extensive translocation
throughout the weed (uptake occurs through the leaves, roots, and shoots) and
permits the inhibition of HPPD [1]. It has also been suggested that a secondary
effect contributing to corn selectivity might be based on a slower foliar uptake
of mesotrione by corn than by weed species. The results of recent studies have
suggested that the selectivity of sulcotrione might also be rationalized by similar
arguments [79]. During extensive field tests in the USA and Europe, no corn
injury has been observed with pre-emergence applications of mesotrione, while
injury averages ≤3% for post-emergence applications. In contrast, soybean is
extremely sensitive, developing bleaching symptoms when treated with mesotrione
post-emergence at application rates as low as 4 g ha−1 . There is, however, no risk of
carry-over in rotational soybean crops, due to the rapid degradation of mesotrione
in soil.
To complete its treatment spectrum, mesotrione is also available in mixtures
with other herbicides (notable gaps are pre-emergent grass control and Setaria
sp. in general). Some important brand products are Lexar® and Lumax® (various
mixtures of mesotrione, S-metolachlor, and atrazine or, alternatively, terbuthylazine
in countries/regions where atrazine use is prohibited). These mixtures are used as
pre-emergence broad-spectrum weed control products in corn (one-shot treatment).
The products have a high S-metolachlor content for areas where Setaria species are
a major problem. Some formulations of Lumax® also contain the S-metolachlor
safener, benoxacor. Camix® (mesotrione plus S-metolachlor) has been developed
to provide broad-spectrum pre-emergence control of broadleaf and grass weeds
in corn, where triazine herbicides are not permitted or desired, while Calaris®
(mesotrione plus terbuthylazine) is used as an early post-emergence weed control
herbicide in corn (dicotyledons and some grass weeds) for countries where atrazine
use is forbidden. Mixtures with inhibitors of photosystem II (PS II), such as
atrazine and terbuthylazine are highly synergistic [80], which is a consequence of

the complementary mode of action of triketones and PS II inhibitors.
Since its introduction into the USA in 2001, mesotrione has been a major success,
with sales of mesotrione-based products steadily increasing (US$ 485 million in
2009) [75], such that it is now regularly among the five best-selling herbicides
worldwide. The synthesis of mesotrione can be achieved in similar fashion to that
of sulcotrione. A possible technical synthesis of the required benzoic acid (86)
starting from (88) is shown in Scheme 4.3.8 [81, 82].
Selected physical chemical, toxicological, and environmental properties of
mesotrione are listed in Table 4.3.1.
4.3.7.3 Tembotrione and Tefuryltrione

Tembotrione (65) is a new active ingredient from Bayer CropScience that was
originally discovered in 1997 within the laboratories of Hoechst Schering AgrEvo
GmbH. It is used for the control of broadleaved and grass weeds in corn. Application
rates of 75–100 g a.i. ha−1 provide a reliable cross-spectrum weed control, including
key grass species such as foxtails and woolly cupgrass. It is also effective on the
difficult-to-control broadleaved species, including glyphosate-, acetolactate synthase
(ALS)-, and dicamba-resistant weeds. Tank mixing with other active ingredients
such as atrazine offers an even better graminicidal action, due to synergistic effects
similar to those described for mesotrione. All formulations include the safener
isoxadifen-ethyl, which ensures an excellent crop safety in all corn varieties.
Tembotrione (65) remains active in the soil throughout the growing season,
offering a residual control of grass and broadleaf weeds up until canopy closure.
Its short soil half-life (DT50 2–10 days) means that it does not limit crop rotation
opportunities in soybeans or other crops in the following season. The metabolism of
tembotrione in soil follows a similar pattern to that of other commercial triketones,
such as sulcotrione and mesotrione, in that the acid X is a major metabolite, though
other significant metabolites (non-herbicidally active) have been identified (e.g., A
NO2 NO2 O O NO2

HO
O Cl Na2SO3 O OH Cl HO O OH
Cl S S S
O O NaO O O O
88
Heat
O
O
Cl NO2
O OH
O S
O O
SO2Me
Mesotrione (18) 86
Scheme 4.3.8 A possible technical synthesis of a key mesotrione intermediate.

4.3 Triketones 255
O
O
Cl CF3
O
O
Tembotrione (65)
SO2Me
O O
HO Cl HO Cl CF3 HO Cl CF3
OH O O
SO2Me SO2Me SO2Me
X (A)
O Cl CF3
Further O
degradation products,
(B)
CO2, etc.
SO2Me
Scheme 4.3.9 Soil degradation of tembotrione (65).
and B; Scheme 4.3.9) that do not follow the typical soil degradation pathway for
triketone herbicides.
Commercial formulations of tembotrione (65) are available under the tradenames
Laudis® and Soberan® (only for Brazil). Some other characteristics of tembotrione
(65) are listed in Table 4.3.1. Bayer has provided several reviews detailing the
discovery of tembotrione [83], its biological performance [84], mode of action [85],
environmental and human safety aspects [86], environmental fate [87], and dietary
risk assessment [88].
Tembotrione (65) and tefuryltrione (66) differ only in the nature of the alkyl
group attached in the 3-position to the benzylic alcohol of the benzoic acid. As
such, there is a clear synergy in the manufacture of these two active ingredients.
The synthesis of these types of molecule is shown in Scheme 4.3.10 [89] . An
alternative synthesis of these compounds has been reported from Syngenta [62b].
Tefuryltrione (66) is a novel early to mid post-emergence herbicide for
broad-spectrum weed control in rice produced by Bayer CropScience which, in
collaboration with Zen-Noh and Hokko Chemical Industry, has recently been
introduced into the Japanese market [90]. At application rates of 300 g a.i. ha−1 ,
tefuryltrione controls a wide spectrum of both annual and perennial broadleaf
weeds and sedges, including those of the Cyperus microiria family. It also
offers an application window similar to that of sulfonylurea herbicides and,
importantly, it shows excellent efficacy against sulfonylurea-resistant weeds. Due
to a weakness against Echinochloa, however, a mixture with a graminicide is
necessary. Consequently, Bayer Crop Science is introducing Bodyguard®, as a
Cl Cl O Cl
AlCl3
NaSCH3 CH3COCl
Cl HMPA SCH3 ClCH2CH2Cl

SCH3
97% 87%
O Cl 1) NaOClaq, Dioxan O Cl
2) HClaq HClg
H2O2, Na2WO4
HO
AcOH 88% MeOH
48% SO2CH3 SO2CH3 98%
O Cl O Cl 1) ROH, KOtBu O Cl
NBS 2) NaOH, THF R
O O Br HO O
CCl4
SO2CH3 65% SO2CH3 SO2CH3
O-acylation, O O Cl
rearrangement R R = CH2CF3 = Tembotrione (65)
O R= = Tefuryltrione (66)
O O
SO2CH3
Scheme 4.3.10 Synthesis of tembotrione (65) and tefuryl-

trione (66). HMPA, hexamethylphosphoramide; NBS,
N-bromosuccinimide.
two-way mixture of fentrazamide (a cell-division inhibitor) and tefuryltrione as a

sulfonylurea one-shot solution into the Japanese rice market.
Selected physical chemical, toxicological, and environmental properties of
Tefuryltrione are listed in Table 4.3.1 [91] (data kindly supplied by Dr Andreas
van Almsick, Bayer CropScience).
4.3.7.4 Benzobicyclon
Benzobicyclon (9, Show-Ace®; Table 4.3.1) is a new triketone niche product
herbicide (sales of <US$ 30 million in 2009) [75] that has been developed for
the control of broadleaf weeds (especially sulfonylurea-resistant weeds such as
Lind sp., Lindernia attenuata, and Monochoria vaginalis) and some important
grasses (e.g., Scirpus juncoides, which is sulfonylurea-resistant, Echinochloa oryzicola,
and other Echinchloa sp.) in paddy rice [91]. An inspection of the compound’s
structure shows that it contains the sulcotrione acid moiety combined with a
bicyclo[3.2.1]octane-2,4-dione, which in turn has been further elaborated to a
pro-herbicide (the attachment of a hydrolytically labile phenylsulfide group to the
vinylogous acid hydroxyl moiety). The latter imparts some positional selectivity to
rice by decreasing the water-solubility of the molecule. The phenylsulfide moiety
is either slowly hydrolyzed in water, or is metabolized in the plant to generate the
active principle (9 ; see Figure 4.3.2).
4.3 Triketones 257
Benzobicyclon is reported to be very selective in rice and is environmentally

friendly due to its low water-solubility (3000-fold less than sulcotrione) and low
fish toxicity (LD50 (48 h) carp >10 ppm) [91]. Low water-solubility is important for
paddy rice herbicides, as the volume of herbicide-containing water flowing out of
the paddy field will be minimized.
The development of benzobicyclon arose from a joint venture between SDS
Biotech and Sandoz Crop Protection (now Syngenta). However, the presence of
Sandoz’s bicyclo[3.2.1]octane-2,4-dione moiety (BIOD, 20; Scheme 4.3.9) posed a
major problem in the development of this compound, as the initial synthesis of
benzobicyclon was cost-prohibitive, particularly the synthesis of BIOD. However,
following extensive process developments the research team at SDS were able to
reduce the synthesis of BIOD to four steps [91, 92]. Another critical breakthrough
in the technical synthesis was the finding that an aluminum trichloride-mediated
C-acylation could be achieved in high yield directly from BIOD and the acid
CH2=O
NHEt2/H2O
100 °C
Step 2
OH O O
89 90 91 92
H2SO5/H2O Step 1
Step 3 1. H2O2/AcOH
2. MeOH/H+
O OH O Cl O NaOMe
toluene O
Cl Cl reflux O
O
O Step 4
SO2Me SO2Me 20
93
81 85
AlCl3
ClCH2CH2Cl
58°C
Step 5
Cl 1. SOCl2 Cl
O 2. PhSNa O
OH Et3N/CHCl3 S
MeO2S MeO2S
O Step 6 O
9′ Benzobicyclon (9)
Overall yield steps 1-6 = 45%
Scheme 4.3.11 The industrial synthesis of benzobicyclon.

chloride (85), since the commercial use of a cyanide-catalyzed O- to C-acylation

was prohibited by competitor patents [15]. Details of the industrial synthesis of
benzobicyclon are shown in Scheme 4.3.11 [91, 92].
Selected physical chemical, toxicological, and environmental properties of Ben-
zobicyclon are listed in Table 4.3.1.
4.3.8
Summary
Since their discovery during the early 1980s, the triketone herbicides have been
studied extensively over almost three decades. In view of this, it may be surprising
that only five commercial products have appeared to date. However, as has been
noted in this chapter, other triketone products are due to appear commercially
(e.g., bicyclopyrone), and related compounds with this mode of action (see also
Chapter 4.4) are likely to play an important role in weed control over the coming
years.
References
1. Mitchell, G., Bartlett, G., Fraser, T.E., 9. Kakidani, H. and Hirai, K. (2003) J. Pes-
Hawkes, T.R., Holt, D.C., Townson, J.K., tic. Sci., 28, 409–415.
and Wichert, R.A. (2001) Pest Manage. 10. Yang, C., Pflugrath, J.W., Camper,
Sci., 57, 120–128. D.L., Foster, M.L., Pernich, D.J., and
2. Hellyer, R.O. (1968) Aust. J. Chem., 21, Walsh, T.A. (2004) Biochemistry, 43,
2825–2828. 10414–10423.
3. Gray, R.A., Rusay, R.J., and Tseng, C.K. 11. (a) Brownlee, J.M., Johnson-Winters,
(1980) Stauffer, now Syngenta AG, US K., Harrison, D.H.T., and Moran, G.R.
Patent 4,202,840. (2004) Biochemistry, 43, 6370–6377;
4. Lee, D.L., Knudsen, C.G., Michaelay, (b) Edmunds, A.J.F., Michel, A.,
W.J., Chin, H., Nguyen, N.H., Carter, Mitchell, G., and Zelaya, I. (2010) Bi-
C.G., Cromartie, T.H., Lake, B.H., cyclopytrone: chemistry transforming
Shribbs, J.M., and Fraser, T.E. (1988) weed control. Lecture presented at the
Pestic. Sci., 54, 377–384. 12th IUPAC International Congress
5. Michaely, J.W. and Kraatz, G.W. (1983) on Pesticide Chemistry, Melbourne,
Stauffer, now Syngenta AG, European Australia, July 4–8, 2010.
Patent EP 90262. 12. Willms, L., van Almsick, A., Bieringer,
6. Pallett, K.E., Cramp, S.M., Little, J.P., H., Auler, T., and Thurwachter, F.
Veerasekaran, P., Crudace, A.J., and (2001) Aventis, now Bayer CropScience,
Slater, A.E. (2001) Pest Manage. Sci., 57, WO 01/007422.
133–142. 13. Zhang, W. and Pugh, G. (2001) Tetrahe-
7. Matsumoto, H., Mizutani, M., dron Lett., 42, 5617–5620.
Yamaguchi, T., and Kadotani, J. (2002) 14. Isobe, T. and Ishikawa, T. (1999) J. Org.
Weed Biol. Manage., 2, 39–45. Chem., 64, 6984–6988.
8. Sekino, K., Wang, J., Nakano, T., 15. (a) Chin, H.M. (1988) Zeneca, now
Nakahara, S., Asami, T., Koyanagi, H., Syngenta AG, US Patent 4,781,751;
Yamada, Y., and Yoshida, S. (2001) (b) Knudsen, C.G. (1989) Zeneca, now
Abstracts of 26th Annual Meeting of Syngenta AG, US Patent 4,838,932 (c)
the Pesticide Science Society of Japan, Nguyen, N.H. (1991) Stauffer, now Syn-
Tokyo, p. 97. genta AG, US Patent 4,997,473 (d) Bay,
References 259
E. (1988) Stauffer, now Syngenta AG, 29. Tarr, J.B. (2000) Book of Abstracts,
US Patent 4,774,360. 219th ACS National Meeting, San
16. Akhrem, A., Lakhvich, F.A., Budai, S.I., Francisco, March 26–30, 2000.
Khlebnicova, T.S., and Petrusevich, I.I. 30. Lee, D.L. (1993) ICI Americas, now Syn-
(1978) Synthesis, 12, 925–927. genta, WO 93/03009.
17. Brown, P., Stephen, M., Bentley, T.W., 31. (a) Carter, C.G. (1986) Stauffer, now
and Jones, R.O. (1999) Zeneca, now Syngenta AG, EP 186118; (b) Michaely,
Syngenta AG, WO 99/28282. W.J. and Kraatz, G.W. (1985) Stauffer,
18. Wojtkowski, P.W. (2003) E.I. Du now Syngenta AG, European Patent EP
Pont de Nemours and Co., US Patent 137963.
20030232984. 32. Barton, J.E.D., Cartwright, D., Carter,
19. Coppola, K. (2001) Syngenta AG, WO C.G., Cox, J.M., Lee, D.L., Mitchell, G.,
01/010806. Walker, F.H., and Woolard, F.X. (1988)
20. See for example; Beaudegnies, R., ICI PLC UK and ICI Americas Inc.,
Edmunds, A.J.F., Luethy, C., Hall, R.G., now Syngenta AG, European Patent EP
Wendeborn, S., and Schaetzer, J. (2004) 283261.
Syngenta AG, WO 04/058712. 33. Lee, D.L., Walker, F.H., Woolard, F.X.,
21. Brown, S.M., Rawlinson, H., and and Carter, C.G. (1989) Stauffer, now
Wiffen, J.W. (1996) Zeneca, now Syn- Syngenta AG, European Patent EP
genta, WO 96/22957. 316491; (b) Carter, C.G. (1987) Stauffer,
22. (a) Inoue, T., Takata, M., Suzuki, T., now Syngenta, US Patent 4,708,732.
34. Lee, S.F. (1989) Sandoz AG, now Syn-
and Kenji, I. (1993) Nippon Soda,
genta AG, European Patent EP 338992.
WO 93/20035; (b) Michaely, W.J.
35. Schneider, H., Luethy, C., and
and Kraatz, G.W. (1988) Stauffer, now
Edmunds, A.J.F. (2003) Syngenta AG,
Syngenta, US Patent 4,780,127.
European Patent EP 1352901.
23. Jackson, D.A., Edmunds, A.J.F.,
36. Rueegg, W. (2000) Novartis AG, now
Bowden, M.C., and Brockbank, B. (2005)
Syngenta AG, WO 00/000029.
Syngenta AG, WO 05/105745.
24. Kudis, S., Misslitz, U., Baumann, E.,
genta AG, WO 92/07837.
Von Deyn, W., and Langemann, K.
(2002) BASF AG, WO 02/016305.
genta AG, European Patent EP 394889.
25. Rubinov, D.B., Rubinova, I.L., Akhrem,
39. Komatsubara, K., Sato, T., Mikami, K.,
A.A., and Aphanasy, A. (1999) Chem. Yamada, J., and Sato, M. (1994) SDS
Rev., 99, 1047–1065. Biotech, JP 06025144.
26. (a) Berry, N.M., Darey, M.C.P., and 40. (a) Adachi, H., Tanaka, K., Kawana, T.,
Harwood, L.M. (1986) Synthesis, 6, and Hosaka, H. (1994) Nippon Soda,
476–480; (b) Anderson, R.J., Grina, J., US Patent 5,294,598; (b) Adachi, H.,
Kuhnen, F., Lee, S.F., Luehr, G.W., Aihara, T., Tanaka, K., Kawana, T.,
Schneider, H., and Seckinger, K. (1989) Yadama, S., and Hosaka, H. (1993)
Sandoz AG, now Syngenta AG, DE Nippon Soda, WO 93/01171.
3902818. 41. (a) Sagae, T., Yamaguchi, M., Adachi,
27. Lee, D.L., Prisbylla, M.P., Cromartie, H., Tomida, K., Takahashi, A., and
T.H., Dagarin, D.P., Howard, S.W., Kawana, T. (1996) Nippon Soda, WO
Provan, W.M., Ellis, M., Fraser, T.E., 96/14285; (b) Ueda, A., Suga, S.,
and Mutter, L.C. (1997) Weed Sci., 45, Adachi, H., Tomita, K., Yamagishi, H.,
601–609. and Hosaka, H. (1992) Nippon Soda,
28. Lee, D.L., Knudsen, C.G., Michaely, JP 04029973; (c) Ueda, A., Suga, S.,
W.J., Tarr, J.B., Chin, H.-L., Nguyen, Tomita, K., and Hosaka, H. (1991)
N.H., Carter, C.G., Cromartie, T.H., Nippon Soda, JP 03052862.
Lake, B.H., Shribbs, J.M., Howard, 42. Baba, M., Tanaka, N., Tsunoda, T.,
S., Hanser, S., and Dagarin, D. (2001) Oya, E., Igai, T., Nawamaki, T.,
ACS Symp. Ser., 774 (Agrochemical and Watanabe, S. (1989) Nissan, JP
Discovery), 8–19. 01052759; (b) Tanaka, N., Oya, E.,
and Baba, M. (1989) Nissan, European (2001) BASF AG, WO 01/040199; (b)
Patent EP 344775. BASF had previously filed a case which
43. (a) Tsunoda, T., Tanaka, N., Oya, claiming 2-(meta-heterocyclylbenzoyl)-1,
E., Baba, M., Igai, T., Suzuki, K., 3-cyclohexanediones. See von Deyn, W.,
Nawamaki, T., and Watanabe, S. (1990) Hill, R., Kardorff, U., Engel, S., Otten,
Nissan, JP 02000222; (b) Tsunoda, T., M., Vossen, M., Plath, P., Rang, H.,
Tanaka, N., Ooya, E., Baba, M., Suzuki, Harreus, A., Rohl, F., Misslitz, U.,
K., Nawamaki, T., and Watanabe, S. Walter, H., and Westphalen, K.-O.
(1989) Nissan, JP 01143851. (1998) BASF AG, WO 96/26200.
44. Kast, J., von Deyn, W., Nuebling, C., 51. (a) Krause, H.-P., Kocur, J., de
Walter, H., Gerber, M., and Westphalen, Una Martinez, J., Bickers, U.,
K.O. (1995) BASF AG, European Patent Hacker, E., and Schnabel, G. (2001)
EP 666254. Aventis, now Bayer CropScience, WO
45. (a) Misslitz, U., Baumann, E., von 01/097614; (b) Bieringer, H., van
Deyn, W., Kudis, S., Langemann, K., Almsick, A., Hacker, E., and Willms, L.
Mayer, G., Neidlein, U., Witschel, M., (2001) Aventis, now Bayer CropScience,
Gotz, R., Rack, M., Plath, P., Otten, M., WO 01/028341; (c) van Almsick, A.,
Westphalen, K.-O., and Walter, H. Willms, L., Hacker, E., and Bieringer,
(2000) BASF AG, WO 00/53590; (b) H. (2002) Bayer CropScience, WO
Walter, H., Plath, P., Kardorff, U., 02/085121; (d) Ziemer, F., van Almsick,
Witschel, M., Hill, R., von Deyn, W., A., Willms, L., Auler, T., Bieringer, H.,
Engel, S., Otten, M., Misslitz, U., and Hacker, E., and Rosinger, C. (2002)
Westphalen, K.-O. (1998) BASF AG, Bayer CropScience, WO 02/085120.
WO 98/40366; (c) Plath, P., Rang, 52. (a) Nasuno, I., Shibata, M., Sakamoto,
H., Westphalen, K.-O., Gerber, M., M., and Koike, K. (1994) Idemitsu, WO
and Walter, H. (1996) BASF AG, DE 94/08988; (b) Tomita, S., Saito, M.,
4427996; (d) Plath, P., Kardorff, U., von Sekiguchi, H., and Okawa, S. (2002)
Deyn, W., Engel, S., Kast, J., Rang, H., Idemitsu, JP 2002114776; (c) Saitou, M.,
Koenig, H., Gerber, M., Walter, H., and Sekiguchi, H., and Ogawa, S. (2000)
Westphalen, K.-O. (1996) BASF AG, DE Idemitsu, WO 01/074802; (d) Saitou,
4427995. M., Sekiguchi, H., and Ogawa, S. (2000)
46. Mayer, G., Misslitz, U., Baumann, E., Idemitsu, WO 00/069853; (e) Saitou,
von Deyn, W., Kudis, S., Hofmann, M., Sekiguchi, H., and Ogawa, S. (2000)
M., Volk, T., Witschel, M., Zagar, C., Idemitsu, WO 00/020408.
Landes, A., and Langemann, K. (2002) 53. No inventor data available (1987) Stauf-
BASF AG, WO 02/048121. fer, now Syngenta AG, JP 62298584.
47. Otten, M., von Deyn, W., Engel, S., 54. (a) Nasuno, I., Sakamoto, M., and
Hill, R., Kardorff, U., Vossen, M., Plath, Nakamura, K. (1996) Idemitsu, WO
P., Walter, H., Westphalen, K.-O., and 96/25412; (b) Nakamura, K., Koike, K.,
Misslitz, U. (1997) BASF AG, WO Sakamoto, M., and Nasuno, I. (2002)
97/30986. Idemitsu, US Patent 20020016262.
48. von Deyn, W., Hill, R., Kardorff, U., 55. (a) Koike, K., Abe, S., Kubota, M.,
Baumann, E., Engel, S., Mayer, G., and Takashima, Y. (2004) Idemitsu,
Witschel, M., Rack, M., Goetz, N., JP 2004043397; (b) Nasuno, I. and
Gebhardt, J., Misslitz, U., Walter, H., Kazuyoshi, K. (2004) Idemitsu, JP
Westphalen, K.-O., Otten, M., and 2004018416.
Rheinheimer, J. (1998) BASF AG, WO 56. Tseng, C.-P. (1998) E. I. Du Pont de
98/31681. Nemours & Co., WO 98/28291.
49. Boerboom, C. (2006) Wisconsin Crop 57. (a) Morita, T., Shimozono, T., Hirayama,
Manager, 13, 10–11. K., Ishikawa, H., Yoshizawa, H., and
50. (a) Kudis, S., Baumann, E., von Yoshihara, M. (1995) Hokko Chem
Deyn, W., Langemann, K., Mayer, Ind Co., JP 07206808; (b) Morita,
G., Misslitz, U., Neidlein, U., Witschel, T., Oono, T., Kido, Y., Maehara, S.,
M., Westphalen, K.-O., and Walter, H. Ishikawa, H., Yoshizawa, H., and
References 261
Hirokazu, H. (1994) Yamamura, Hi- C. (2009) Bayer CropScience, WO

roshi, (Hokko Chem. Ind. Co.), JP 09/018925.
06271562. 67. Lüthy, C., Hall, R.G., Edmunds, A.J.F.,
58. (a) Nakamura, Y. and Sano, M. Riley, S., and Diggelmann, M. (2008)
(2005) Ishihara Sangyo Kaisha, WO Syngenta AG, WO 08/006540.
05/118530; (b) Nakamura, Y., Palmer, 68. Mitchell, G., Salmon, R., Bacon, D.P.,
C.J., Kikugawa, H., and Sano, M. (2001) Aspinall, I.H., Briggs, E., Avery, A.J.,
Ishihara Sangyo Kaisha, WO 01/014303. Morris, J.A., and Russell, C.J. (2009)
59. Gullickson, G. Bayer’s New Poste- Syngenta AG, WO 09/115788.
mergence Corn Herbicide Slated 69. Mitchell, G., Salmon, R., and Morris,
for 2009, Agriculture Online, J.A. (2010) Syngenta AG, WO
http://www.agriculture.com/crops/corn/ 10/112826.
technology/Bayers-new-postemergence-corn- 70. Mitchell, G., Salmon, R., and Morris,
herbicide-slated-for-2009_139-ar5. J.A. (2010) Syngenta AG, WO
60. (a) Endo, K., Ito, S., Seishi, N., and 10/116122.
Nakajima, T. (2005) Bayer CropScience, 71. Takabe, F., Fukumoto, S., Kajiki, R.,
JP 2005306813; (b) Endo, K., Ito, S., Sohei, A., Uneo, R., Kobayashi, M.,
and Mukaida, H. (2004) Bayer Crop- Takahashi, S., Yonekura, N., Mitsunari,
Science, WO 04/105482. R., and Hanai, T. (2007) Kumiai, Ihara
61. Benko, Z.L. and Turner, J.A. (1998) Dow AG, WO 07/088876.
Agrosciences LLC, WO 98/42648. 72. Tamai, R., Ito, M., Kobayashi, M., and
62. Brunner, H.G. (1990) Ciba-Geigy AG, Mitsunari, T. (2007) Kumiai, Ihara AG,
now Syngenta AG, European Patent EP European Patent EP 174934.
353187, (1991) US Patent 4,995,902. 73. Funyu, A., Hirano, Y., Kobayashi, M.,
63. (a) Edmunds, A.J.F., Seckinger, K., Mitsunari, T., and Takabe, F. (2010)
Luethy, C., Kunz, W., De Mesmaeker, Kumiai, Ihara AG, WO 10/089993.
A., and Schaetzer, J. (2000) Novartis, 74. Beraud, M., Claument, J.J., and
now Syngenta AG, WO 00/015615; Montury, A. (1993) ICIA0051, A new
(b) Luethy, C., Edmunds, A.J.F., herbicide for the control of annual
Beaudegnies, R., Wendeborn, S., and weeds in maize. Proceedings of the
Schaetzer, J. (2005) Syngenta AG, WO Brighton Crop Protection Conference -
05/058831; (c) Luethy, C., Edmunds, Weeds, Farnham, Surrey, UK, pp.
A.J.F., Beaudegnies, R., Wendeborn, 51–56.
S., Schaetzer, J., and Lutz, W. (2005) 75. Phillips McDougal AgriService (2010)
Syngenta AG, WO 05/058830; (d) Products Section, 2009 Market, Novem-
Beaudegnies, R., Edmunds, A.J.F., ber, 2010.
Fraser, T.E., Hall, R.G., Hawkes, 76. Rouchaud, J., Neus, O., Bulcke, R.,
T.R., Mitchell, G., Schaetzer, J., Cools, K., and Eelen, H. (1998) Bull.
Wendeborn, S., and Wibley, J. (2009) Environ. Contam. Toxicol., 61, 669–676.
Bioorg. Med. Chem., 17, 4134–4152. 77. Purnell, T.J. (1991) The technical prop-
64. (a) Rueegg, W.T. (2004) Syngenta AG, erties of ICIA0051, a new herbicide for
DE 102004012192; (b) Rueegg, W.T. maize and sugarcane. Proc. Annu. Congr.
(2003) Syngenta AG, WO 03/047343; (c) S. Afr. Sugar. Technol. Assoc., 65, 30–32.
Rueegg, W.T. (2001) Syngenta AG, 78. Guo, S., Yang, F., and Zhang, L. (2001)
WO 01/054501; (d) Rueegg, W.T. Nongyao, 40, 20–21.
(2003) Syngenta AG, WO 03/047344; 79. Armel, G.R., Mayonado, D.J., Hatzios,
(e) Rueegg, W.T. (2003) Syngenta AG, K.K., and Wilson, H.P. (2004) Weed
WO 03/047342. Technol., 18, 211–214.
65. Jackson, D.A., Bowden, M.C., Edmunds, 80. Lake, B.H. and Purnell, T.J. (1995)
A.J.F., and Brockbank, B. (2005) Syn- Zeneca, now Syngenta AG, WO
genta AG, WO 05/105718. 95/28839.
66. van Almsick, A., Narabu, S., Sato, Y., 81. Beilstein, E III Band. 11, p. 685.
Domon, K., Araki, K., Shirakura, S., 82. Brown, R.W. (1990) ICI, now Syngenta
Ukawa, S., Ichihara, T., and Ueno, AG, WO 90/06301.
83. van Almsick, A., Benet-Buchholz, J., 90. van Almsick, A. and Willms, L. (2010)
Olenik, B., and Willms, L. (2009) Bayer Discovery and chemistry of tefuryltri-
CropSci. J., 62, 5–16. one, a new HPPD herbicide for rice
84. Santel, H.-J. (2009) Bayer CropSci. J., 62, production. Poster presented at the 12th
95–108. IUPAC International Congress on Pes-
85. Schulte, W. and Köcher, H. (2009) Bayer ticide Chemistry, Melbourne, Australia,
CropSci. J., 62, 35. July 4–8, 2010.
86. Leake, C., Diot, R., Glass, H., Newby, S., 91. (a) Komatsubara, K. (2005) Fain
Semino, G., Tarara, G., and Wegmann, Kemikaru, 34, 38–48; (b) Sekino, K.,
T. (2009) Bayer CropSci. J., 62, 53–62. Komatsubara, K., Koyanagi, H., and
87. Tarara, G., Fliege, R., Desmarteau, D., Yamada, Y. (2004) Shokubutsu no Seicho
Kley, C., and Peters, B. (2009) Bayer Chosetsu, 39, 23–32.
CropSci. J., 62, 63–78. 92. (a) Komatsuhara, K., Sano, K.,
88. Kelly, I., Leake, C., Diot, R., and Tabuchi, T., Iwasawa, J., and Kishi,
Lemke, V. (2009) Bayer CropSci. J., J.H. (1998) SDS Biotech Corp., JP
62, 79–94. 10265441; (b) Kishi, H., Tabuchi,
89. van Almsick, A., Willms, L., Auler, T., T., and Komatsuhara, K. (2002) SDS
Bieringer, H., Auler, T., and Rosinger, Biotech Corp., JP 2002003467; (c)
C. (2002) Hoechst Schering AgrEvo Komatsubara, K., Sano, M., Tabuchi,
GmbH, now Bayer CropScience, US T., Iwasawa, J., and Kishi, H. (1998)
Patent 6,376,429. SDS Biotech Corp., JP 10265432.
4.4
Hydroxyphenylpyruvate Dioxygenase (HPPD) Inhibitors: Heterocycles
Andreas van Almsick
4.4.1
Introduction
As noted previously in Chapter 4.2, all known inhibitors of the enzyme hydrox-
yphenylpyruvate dioxygenase (HPPD) are chelating agents. In order for these
compounds to exhibit not only in vitro but also in vivo activities, then additional
requirements are necessary such as uptake, transport, and metabolic stability in
plants (especially in weeds). Currently, the available commercial compounds, and
the long list of reported HPPD inhibitory agents, with the general structure 1
(Figure 4.4.1), fulfill all of these needs [1].
O R1 Q= O R6
O O
R2
Q , N , R8
(R5)n OH
N
R3 O CN
R7
R4
1 R1-4 = H, alkyl, haloalkyl, halogen, etc.

R5-6 = H, alkyl, etc.
R7 = alkyl
R8 = alkyl, cycloalkyl
Figure 4.4.1 Markush structure of many HPPD inhibitors.

4.4 Hydroxyphenylpyruvate Dioxygenase (HPPD) Inhibitors: Heterocycles 263
Whilst many Q moieties (see Figure 4.4.1) exist, 1,3-cylohexanediones, pyra-

zolones, and diketonitriles (DKNs) are the most important examples. It is essential
to know that all compounds of the general structure 1 could exist in different
tautomeric forms, as shown in Figure 4.4.2 for Q = 1,3-cyclohexanedione.
Regarding the substitution pattern of 1, the 2,4-disubstitution and the 2,3,4-
trisubstitutions are particularly important (see Chapter 4, Section 4.3.5). In vitro
activity is strongly connected with a substitution in the 2-position (R1 = H).
Whilst compounds of the general structure 1 are active in vitro – and, therefore,
are drugs – different prodrugs are also known for each Q moiety (see Chapter 4.3,
Figure 4.3.2).
For simplification, the HPPD inhibitors in which Q differs from 1,3-cyclo-
hexanedione and its prodrugs are summarized here as heterocycles. The hetero-
cyclic HPPD-inhibitor pyrazolynate (6; Figure 4.4.3) was the first such product to
be launched commercially.
4.4.2
Market Products
4.4.2.1 Pyrazolynate (Pyrazolate)

In 1980, the launch of pyrazolynate (pyrazolate) (6; Figure 4.4.3) by Sankyo Co.,
Ltd in Japan, signified the first HPPD compound to enter the herbicide market
worldwide, even though at the time the actual target site of the compound
was unknown. Although, some two years earlier, Sankyo had presented details
of the company’s activity in this area at the Fourth International Congress of
Pesticide Chemistry in Zurich, Switzerland [2], they had already patented the main
O O R1
R2
O R3
2 R4
OH O R1 O OH R1 O O R1
R2 R2 R2
O R3 O R3 OH R3
R 4 R4 R4
3 4 5
Figure 4.4.2 Tautomeric forms of HPPD inhibitors of type 1,3-cyclohexanedione.

H3C O Cl Figure 4.4.3 Structure of pyrazolynate (6).
N
N Cl
O
H3C
O S
O
CH3
6
pyrazolynate
compounds earlier, in 1974 [3]. Interestingly, this had all occurred without any
knowledge of the compound’s precise mode of action. Pyrazolynate and two analogs
had been classified previously as protoporphyrinogen oxidase (Protox) inhibitors
[4]; nonetheless, although pyrazolynate is not a ‘‘new’’ compound, it has been
included in this review since it is relatively unknown outside of Japan.
®
The herbicide with the trade name Sanbird (pyrazolate) is able to control
both annual and perennial weeds in paddy fields [5] with application rates of
®
3–4 kg ha−1 . As a very selective herbicide in rice, Sanbird proved to be a good
innovation for the Japanese rice market, and the product achieved peak sales
in Japan in 1986 with a total coverage of 650 000 ha (28.6% market share).1)
Unfortunately, this share declined with the introduction of sulfonylurea herbicides
®
such as bensulfuron-methyl in 1987 such that, by 2005, Sanbird was used on only
101 200 ha in Japan (5.9% market share) [6].
Pyrazolynate is in fact a ‘‘prodrug’’ that, in itself, is not herbicidally active.
It has a low solubility in water (0.056 mg l−1 at 25 ◦ C), and in solution it is
hydrolyzed to produce p-toluenesulfonic acid (7) and 4-(2,4-dichlorobenzoyl)-1,3-
dimethyl-5-hydroxypyrazole (8), which is the herbicidal entity of pyrazolate [7–9]
(Scheme 4.4.1). The half-lives of pyrazolynate in water at 25 ◦ C are: 52.7 h at pH 3;
17.5 h at pH 1; 25.0 h at pH 7; and 4.3 h at pH 9 [10]. In soil, a degradation time
(DT50 ) of 8–10 days has been observed [1a].
The synthesis of pyrazolynate is shown schematically in Schemes 4.4.2 and
4.4.3. Typically, 1,3-dimethyl-5-pyrazolon (9) and 2,4-dichlorobenzoyl chloride
(10) react in the presence of calcium hydroxide in isopropanol to give 4-(2,4-
dichlorobenzoyl)-1,3-dimethyl-5-hydroxypyrazole (8) [3]. 4-Methylbenzenesulfonyl
chloride (11) is then added to a solution of 8 and triethylamine in benzene [3].
With regards to the current state of this synthesis, two points should be
highlighted. First, instead of benzene other solvents such as toluene are now used.
Second, for the formation of substituted 4-benzoyl-1-alkyl-5-hydroxyxpyrazole such
1) The Japan Association for Advancement of

Phyto-Regulators collected data.
H3C O Cl H3C O Cl
N H3C SO3H + N
N H 2O N
O Cl OH Cl
H3C O H 3C
S
O
CH3
6 7 8
Scheme 4.4.1 The prodrug pyrazolynate.
H3C O Cl H3C O Cl
N + Cl
OH N
N Ca(OH)2 N
Cl OH Cl
CH3 H3C
iPrOH
9 10 8
Scheme 4.4.2 Synthesis of the drug of pyrazolynate.
H3C O Cl
H3C O Cl
NEt3 N
N + CH3 SO2Cl N
benzene O Cl
N Cl H 3C
OH O S
H3C
O
8 11 6 CH3
Scheme 4.4.3 Synthesis of pyrazolynate.
as 8, other routes have also been identified. Of these routes, the most popular is
shown in Scheme 4.4.4, using compound 8 as an example [11].
The current commercial availability of both 1,3-dimethyl-5-pyrazolon (9) and
2,4-dichlorobenzoic acid permits a more cost-effective, limited-steps synthesis
of pyrazolynate to be conducted. Nevertheless, owing to the high application
rate of pyrazolynate (3–4 kg ha−1 ), the treatment costs are still very high al-
though, in theory, the rate could be reduced by using 4-(2,4-dichlorobenzoyl)-1,
3-dimethyl-5-hydroxypyrazole (8) instead of the prodrug. Another important factor
for the Japanese rice market is that a herbicide should provide season-long weed
control. Whilst this is not possible with the more polar and more water-soluble
H3C Cl H3C
O
NEt3
O Cl
N + Cl N
OH N
N CH3CN O
CH3 Cl H3C
Cl
9 10 12
H3C O Cl
[(CH3)2C(OH)CN]
N
NEt3 N Cl
OH
H3C
CH3CN
8
Scheme 4.4.4 Alternative route for the synthesis of the drug of pyrazolynate.
H3C O Cl Figure 4.4.4 Structure of pyrazoxyfen (13).
N
N Cl
O
H3 C
O
13
pyrazoxyfen
drug 8, it can be achieved by using the prodrug pyrazolynate. The effect is similar
to that of a slow-release drug formulation, where the active ingredient is released
over a long period of time, such that it is present at levels which are lethal to the
weeds over a much longer period of time.
4.4.2.2 Pyrazoxyfen
Pyrazoxyfen (13; Figure 4.4.4), a very close analog of pyrazolynate, was launched
by Ishihara Sangyo Kaisha Ltd in 1985 for the Japanese rice market. The herbicide,
which was patented in 1977 [12] and first reported in 1984 by F. Kimura [13], is
®
marketed under the trade name Paicer . It has a broad weed-control spectrum
under flooded-field conditions, including activity against many annual and peren-
nial weeds, with a typical application rate of 3 kg ha−1 . It is very selective towards
transplanted rice, and also to direct-seeded rice at temperatures below 35 ◦ C. At
higher temperatures, however, temporary crop damage may occur [13].
O H3 C O Cl
H3C O Cl
Br K2CO3
+ N
N
CH3COC2H5 N Cl
N Cl O
OH H 3C
H3C
refluxing O
8 14 13
Scheme 4.4.5 Synthesis of pyrazoxyfen.
®
Paicer reached peak sales in Japan in 1988, with 45 000 ha coverage in Japan
(2.2% market share),1 but in 2005 it was used on only 6911 ha (0.4% market share)
[6]. As the second product to reach the Japanese market for the same segment as
®
pyrazoynate, and with the same mode of action, Paicer was – and remains –
much less successful.
In order to synthesize pyrazoxyfen (13), 2-bromoacetophenone 14 is added
to a solution of 8 and anhydrous potassium carbonate in methyl ethyl ketone
(Scheme 4.4.5) [12].
The main difference between pyrazoxyfen and pyrazolynate is in the selected pro-
drug system. In plants, both herbicides are metabolized to 4-(2,4-dichlorobenzoyl)-
1,3-dimethyl-5-hydroxypyrazole (8). Although pyrazolynate is only slightly soluble
in water, once dissolved it is rapidly hydrolyzed to the herbicidally active metabolite
[10]. In contrast, pyrazoxyfen shows considerable stability in aqueous solutions [14].
4.4.2.3 Benzofenap
As a third compound of this series, benzofenap (15; Figure 4.4.5) was launched in
1987 by Mitsubishi Petrochemical Co. Ltd (now Mitsubishi Chemical Corp.) and
commercialized by Rhône-Poulenc Yuka Agro KK, as a joint venture of Mitsubishi
Chemical Corp. and Rhône-Poulenc Agro (now part of Bayer CropScience), for the
®
rice market. Interestingly, benzofenap is applied not only in Japan, as Yukawide ,
®
but also in Australia, as Taipan . The new herbicide was patented in 1982 [15] and
first reported in 1991 [16].
®
Yakawide reached its peak sales in 1998 in Japan, with 180 000 ha coverage (10%
market share),1 but in it was used on only 62 000 ha (3.6% market share) [6]. As the
third product with an HPPD mode of action aimed at the Japanese rice market,
® ®
Yakawide proved to be much more successful than its closest analog, Paicer .
The main differences between benzofenap and pyrazoxyfen are the addi-
tional methyl groups on the biologically active metabolite 4-(2,4-dichloro-3-methyl-
benzoyl)-1,3-dimethyl-5-hydroxypyrazole (16) (Figure 4.4.6), and the prodrug moi-
ety 4 -methylacetophenone. These variations result in different environmental
behaviors and different herbicidal activities [13, 16]. For example, the half-life in
paddy field soil was increased from 4 to 15 days for pyrazoxyfen, and to 38 days
for benzofenap. Although the application rate of 3 kg ha−1 is equally high as for
other rice herbicides, benzofenap provides a longer period of weed control of up
to 50 days, compared to 21–35 days with pyrazoxyfen. Importantly, benzofenap is
also a more crop-selective herbicide. An additional advantage of benzofenap over
pyrazoxyfen is that it is not temperature-dependent, with no phytotoxicity being
observed even at higher temperatures.
None of the three HPPD rice herbicides is able to control all annual and perennial
weeds in rice, and consequently they require mixture partners, especially to fill
the gaps in their efficacy, such as for barnyard grasses or Cyperus spp. The most
common mixture partners are butachlor, pretilachlor, thiobencarb [17], piperophos
[13], pyribaticarb, and bromobutide [16].
H3C O Cl
CH3
N
N Cl
O
H3C
O
CH3
15
benzofenap Figure 4.4.5 Structure of benzofenap (15).
H3C O Cl H3C O Cl
CH3
N N
N Cl N Cl
OH OH
H3C H3C
16 8
Figure 4.4.6 Biologically active metabolites, 16 and 18, of

benzofenap and pyrazoxyfen, respectively.
O SO2CH3
O
N CF3
17
isoxaflutole Figure 4.4.7 Structure of isoxaflutole (17).

4.4.2.4 Isoxaflutole
With the introduction of isoxaflutole (IFT) (17; Figure 4.4.7), a variety of new
crops – including corn and sugarcane – were brought into the focus of the
HPPD-inhibitor-type heterocycles. Although IFT was not the first HPPD compound
to be used for corn (this honor fell to sulcotrione, in 1990), it was the first to be
used for pre-emergence application. Having been reported in 1995 by Luscombe
et al. [18], IFT was first patented in 1991 [19] by Rhône-Poulenc Agriculture Limited
(now Bayer CropScience).
® ® ®
The IFT herbicide, with the tradenames Merlin , Balance and Provence
(among others), was first launched in 1996 in South America for the control of
broadleaf weeds and grasses in corn and sugarcane. In corn, IFT is a selective
pre-emergence herbicide, with applications generally being made in spring during
post-sowing/pre-emergence of the crop. However, it is also possible to apply
IFT at early pre-plant stage up to three weeks before planting of the crop.
The application rate of 100 g ha−1 is very low compared to other conventional
pre-emergence herbicides for corn (e.g., S-metolachlor, at 0.8–1.6 kg ha−1 ). Based
on the newly developed safener technology, cyprosulfamide, the application window
for IFT can now be extended to early post-emergence uses, up to the three-leaf
stage.
Common mixture partners in corn are thiencarbazone-methyl, flufenacet,
aclonifen, terbuthylazine, and atrazine to complete the weed spectrum. For
example, in sugarcane IFT is used to control annual grasses and some key an-
nual broadleaf weeds, with application being made either pre- or post-emergence
(normally, pre-emergence is the preferred option). The application rate of 140 g ha−1
is still very low compared to other pre-emergence products, however [20].2)
In sugarcane, IFT is used preferably in spray sequences, although it may also
be tank-mixed with triazine products to complete a spectrum that is targeted
specifically against broad-seeded weeds. In some countries, IFT is also registered
for weed control in other crops such as chick peas, poppy seed, and occasionally
also in nurseries.
IFT is a much more complex compound than the three above-described rice
compounds, and consequently it requires a longer route of synthesis [19, 21]
(Scheme 4.4.6). One possible educt is 2-chloro-4-trifluoromethylbenzoic acid
sodium salt (18) to obtain 2-methylthio-4-trifluoromethylbenzoic acid (19); further
treatment with hydrogen peroxide and acetic anhydride in acetic acid then yields
2-methylsulfonyl-4-trifluoromethylbenzoic acid (20). With thionyl chloride, the cor-
responding benzoyl chloride 21 is available, which will be transformed into t-butyl
2-(2-methylsulfonyl-4-trifluoromethylbenzoyl)-3-cyclopropyl-3-oxopropionate (22)
via the magnesium enolate of t-butyl 3-cyclopropyl-3-oxopropionate in methanol.
In order to remove the t-butyl carboxylate group, 22 is refluxed in toluene in the
presence of toluenesulfonic acid. The so-formed 1-(2-methylsulfonyl-4-trifluoro-
methylphenyl)-3-cyclopropylpropan-1,3-dione (23) is then used to obtain 1-(2-
methylsulfonyl-4-trifluoromethylphenyl)-3-cyclopropyl-2-ethoxymethylenepropan-
2) Isoxaflutole – Herbicide for broadleaf weed Technical Information, Bayer CropScience

and grass control in maize and sugar cane, AG, Alfred-Nobel-Str. 50, 40789 Monheim.
O Cl O SCH3
CH3SNa
Na+ O HO
N-methylpyrrolidone
CF3 90 °C, 2 h CF3
18 19
O SO2CH3 O SO2CH3
H2O 2 SOCl2
HO Cl
(CH3CO)2O/CH3CO2H refluxing
CF3 CF3
20 21
O O O SO2CH3 O O SO2CH3
CO2t-Bu
pTsOH
Mg toluene
O Ot-Bu CF3 CF3
CCl4 refluxing
CH3OH 22 23
O O SO2CH3 O SO2CH3
HC(OC2H5)3 H2NOHXHCl
(CH3CO)2O CH3CO2Na O
EtO CF3 N CF3
refluxing
24 17
Scheme 4.4.6 Synthesis of isoxaflutole (17).
1,3-dione (24) in a mixture of triethylorthoformate and acetic anhydride.

Finally, the addition of sodium acetate and hydroxylamine hydrochloride yields
IFT (17).
As noted above, isoxazoles such as IFT are prodrugs and hence are not suf-
ficiently persistent in plants to inhibit the HPPD enzyme. Rather, it is the
primary metabolite of IFT – the so-called DKN {3-cyclopropyl-2-[2-(methylsulfonyl)-
4-(trifluoromethyl)benzoyl]-3-oxopropanenitrile} (25) – that is the herbicidally ac-
tive entity. In both soil and in plants, IFT undergoes a rapid conversion into
DKN [22]. In aqueous solutions, the temperature and pH can each influence the
chemical hydrolysis of IFT to DKN; notably, hydrolysis is increased with rising pH
and temperature. For example, at 295 K and pH 9.3 the rate of degradation was
O SO2CH3 O O SO2CH3
O
N base
CF3 CF3
N
17 25
Scheme 4.4.7 Conversion of isoxaflutole (IFT) to diketonitrile (DKN) under basic

conditions.
100-fold faster than at pH 3.8 (Scheme 4.4.7) [23]. The DT50 of IFT in water is 11
days at pH 5, 20 h at pH 7, and 3 h at pH 9.2)
In soil, the DT50 of IFT is also very low, and ranges between 12 h and three
days under laboratory conditions. Such degradation is, however, highly dependent
on several factors that include the temperature, pH, moisture content, and soil
type [24]. Moreover, the half-life of IFT in aqueous sterile solution is higher than
in soil at the same temperature and pH, thus confirming the catalytic effect of
soil first reported by Taylor-Lovell et al. [25]. When used under normal agricultural
conditions, the rate of DKN formation will be affected by the quantity and frequency
of rainfall. The log P of IFT is 2.19 and the water solubility 6.2 mg l−1 , compared
to values for DKN of 0.4 and 326 mg l−1 , respectively. Such properties restrict the
mobility of IFT, causing it to be retained at the soil surface from where it can be
taken up by surface-germinating weed seeds. DKN, which has a laboratory DT50 of
20–30 days, is more mobile and consequently is taken up by the roots. In addition
to influencing the soil behavior of IFT and DKN, the greater lipophilicity of IFT
leads to a greater uptake by seed, shoot, and root tissues. In both plants and soil,
DKN is converted into the herbicidally inactive benzoic acid 26 (Scheme 4.4.8), with
the degradation occurring more rapidly in corn than in susceptible weed species.
In particular, this contributes to the mechanism of selectivity, in association with a
deeper sowing depth of the crop [24].
4.4.2.5 Topramezone
The launch of topramezone (27; Figure 4.4.8) for the post-application corn market
®
was undertaken in 2006 under the tradenames Impact in USA and Canada, and
O O SO2CH3 O SO2CH3
HO
soil, plant
CF3 CF3
N
25 26
Scheme 4.4.8 Decomposition of diketonitrile (DKN) to

2-methylsulfonyl-4-trifluoromethylbenzoic acid (20).
Figure 4.4.8 Structure of topramezone (27).

O CH3 N O
N
N SO2CH3
OH
H3 C
27
topramezone
®
Clio in Germany and Austria. The compound is based on a BASF patent dating
back to 1995 [26].
In 2005, BASF granted the rights to develop, register, and commercialize
topramezone in North America to Amvac Chemical, while the rights in Japan have
been granted to Nippon Soda. The new corn compound will be marketed in Latin
America and Europe [27] only by BASF.
Topramezone is targeted at the post-emergence control of grasses and major
broadleaf weeds in corn crops worldwide. This would mean that it could be differ-
entiated from sulcotrione and mesotrione in that it shows a better cross-spectrum
activity, similar to IFT, and thus would not be limited mainly to broadleaf weed
control.
®
Clio is a 336 g l−1 suspension concentrate (SC) formulation with recommended
application rates of 50–75 g ha−1 of topramezone [28]. In order to complete the
active spectrum, however, topramezone may be tank-mixed with a herbicide such
as dimethenamid, or a sulfonylurea such as nicosulfuron. Triazines or dicamba
may also be recommended to fill the activity gaps against broadleaved weeds.
Despite being classed as a pyrazolone (like pyrazolynate, pyrazoxyfen and ben-
zofenap), topramezone does not have a protective group and is, therefore, not
a prodrug. The presence of a 4,5-dihydroisoxazol group in the 3-position of the
benzoyl moiety is also worthy of mention.
Several different synthetic routes for topramezone have been reported, one
of which is shown in Scheme 4.4.9 [26, 29]. Starting with 3-nitro-o-xylene
(28), 3-(2-methyl-6-nitrophenyl)-4,5-dihydroisoxazole (31) is synthesized via the
benzaldehyde oxime 29. Subsequent reduction of the nitro group, replace-
ment of the corresponding amino group by methyl sulfide, bromination to
3-[3-bromo-2-methyl-6-(methylthio)phenyl]-4,5-dihydroisoxazole (34), followed by
oxidation, affords the sulfone 35. Finally, topramezone (27) is available by the con-
version of 35 with 1-methyl-5-hydroxypyrazol in the presence of carbon monoxide
and a suitable palladium catalyst. This represents an alternative process to those
described in Schemes 4.4.2 and 4.4.4.
4.4.2.6 Pyrasulfotole
During the Analyst & Investor Days in Lyon on 5–6 September 2005, Bayer
CropScience announced the development of a new pyrazolone for the cereals
market, termed pyrasulfotole (36; Figure 4.4.9) [30]. The compound had been
initially patented in 2000 [31] by Aventis CropScience (now Bayer CropScience).
OH OH
CH3 CH3 N CH3 N
CH3 nBuONO NCS
H Cl
KOCH3 CH3CN
NO2 NO2 NO2
DMF
28 29 30
CH3 N O CH3 N O
CH2=CH2
H2
NaHCO3 PT/C
NO2 NH2
CH3CO2C2H5
toluene/H2O
31 32
CH3 N O CH3 N O
Br2 Br
t-BuONO
Cu conc. H2SO4
SCH3 SCH3
S2(CH3)2
33 34
CH3 N O N OH
O CH3 N O
N
H2O2 Br CH3
N
Na2WO4 · 2 H2O
SO2CH3
CO N SO2CH3
OH
CH3CO2H [(C6H5)3P]2PdCl2 H3C
35 K2CO3
27
1,4-dioxane
Scheme 4.4.9 Synthesis of topramezone (27).
Pyrasulfotole for the post-cereals market was launched in 2008 under the trade-
® ® ®
names of Huskie in the USA, Infinity in Canada, and Velocity in Australia
(the product is a mixture of pyrasulfotole, bromoxynil, and mefenpyr-diethyl). It is
intended that the same product will be launched in 2011 in South Africa under the
®
trade name Resolve . Further products with pyrasulfotole are to be launched un-
®
der the tradenames Precept [pyrasulfotole, 2-methyl-4-chlorophenoxyacetic acid
® ®
(MCPA), and mefenpyr-diethyl], Wolverine and Tundra (fenoxaprop, pyrasul-
®
fotole, bromoxynil, and mefenpyr-diethyl), and Velocity M3 (thiencarbazone,
pyrasulfotole, bromoxynil, and mefenpyr-diethyl).
H3C O SO2CH3 Figure 4.4.9 Structure of pyrasulfotole (36).
N
N CF3
OH
H3C
36
pyrasulfotole
As pyrasulfotole is the first HPPD compound to be applied for cereals, it

clearly has a new mode of action for this crop. In addition, it is an innovative
tool for resistance management, with excellent post-emergence use on all types
of wheat, barley, and triticale. Pyrasulfotole has the ability to control a wide
range of broadleaved weeds, such as chickweed (Stellaria media), lamb’s quarters
(Chenopodium album), nightshades (Solanum spp.), pigweeds (Amaranthus spp.),
and velvetleaf (Abutilon theophrasti). Application rates of 25–50 g a.i. ha−1 provide a
reliable weed control, especially in mixtures with bromoxynil (dicotyledonous) and
fenoxaprop (grasses).
Crop tolerance is guaranteed through inclusion of the safener mefenpyr-diethyl,
which ensures excellent crop safety in all cereal varieties [32]. The synergistic
interaction of pyrasulfotole with inhibitors of PS II (such as bromoxynil) results
in products with unique properties against weed species that, otherwise, would be
difficult to control. All such products control a broad spectrum of dicotyledonous
weeds that are resistant to either acetolactate synthase (ALS) or PS II inhibitors, as
well as to inhibitors of auxin and carotenoid biosynthesis, and they have not yet
shown any cross-resistance [33].
Interestingly, pyrasulfotole employs the same benzoic acid 20 as IFT, as well as
the well-known 1,3-dimethyl-5-pyrazolon (9). Moreover, like topramezone, it is not
a prodrug.
4.4.3
Conclusions
It is clear that HPPD inhibitors of the heterocycle type are represented in rice,
corn, sugarcane and, also in the cereals market. Moreover, even when the three
compounds applied to rice are deemed to have passed their ‘‘commercial peak,’’ it
is likely that the HPPD inhibitors will become highly successful, especially when
applied to corn.
Although most of the compounds described in this chapter are prodrugs, this
approach is not a prerequisite for HPPD inhibitors, as noted for the cases of
topramezone and pyrasulfotole. Notably, the prodrug concepts used are chemically
quite different, with those compounds applied to rice leaving heir prodrug moieties
as ‘‘waste’’ in the environment, while IFT is converted by opening of the isoxazole
ring, without altering the molecular mass. Similarly, although the HPPD inhibitors
share the features of a relatively higher log P-value, and thus a poor water solubility,
References 275
the prodrugs are metabolized to active metabolites with much lower log P-values
and an increased water solubility.
Other more important differences observed between these compounds involve
their application rates, with the three that are applied to rice being used on the
kilogram scale, while IFT and topramezone are applied at 100 g ha−1 , or less.
In chemical terms, the compounds described here are quite similar, with the
exception of topramezone. The substitution patterns of the benzoyl moieties bear
some resemblance, despite there being major differences between Cl, CH3 , CF3 , or
SO2 CH3 . Benzofenap and, in particular, topramezone also show that substitution
is not only permitted in the 3-position, but is also clearly important for good
biological activity. Also important are the different Q-moieties although, as noted
above, both pyrazolone and DKN may serve as chelating agents for Fe(II).
It is also fascinating to observe the quite different biological activities achieved
with the variations described here. Undoubtedly, the future will reveal the ‘‘end’’
of the ‘‘HPPD story’’ with regards to the nature of the crop, the application rate of
the compound, and its activity profile.
References
1. (a) Tomlin, C.D.S. (ed.) (2000) The 6. Ministry of Agriculture,

Pesticide Manual, 12th edn, British Forestry and Fisheries, Japan (2005)
Crop Protection Council, pp. 602, 793, Statistical Yearbook. Available at:
797, and 848; (b) Patent examples: http://www.maff.go.jp/e/tokei/kikaku/
Michaely, W.J. and Kraatz, G.W. nenji_e/nenji_index.html.
(1983) Stauffer Chemical Company, 7. Kawakubo, K., Shindo, M., and
EP 0090262 ; (c) Benkö, L.Z., Turner, Knonotsune, T. (1979) Plant Physiol.,
J.A., Weimer, M.R., Garvin, G.M., 64, 774.
Jackson, J.L., Shinkle, S.L., and Webster, 8. Matsui, T., Konotsune, T., Kawakubo,
J.D. (1998) DOW Agrosciences LLC, K., and Ishida, M. (1983) in Pesticide
WO 98/42678; (d) von Deyn, W., Hill, Chemistry: Human Welfare and the
R.L., Kardorff, U., Engel, S., Otten, Environment, vol. 1 (eds J. Miyamoto and
M., Vossen, M., Plath, P., Rang, H., P.C. Kearney), Pergamon Press, Oxford,
Harreus, A., and Koenig, H. (1996) pp. 327–332.
BASF, WO 96/26193. 9. Sandmann, G., Reck, H., and Boeger, P.
2. Konotsune, T., Kawakubo, K., and (1984) J. Agric. Food Chem., 32, 868.
Yanai, T. (1978) Advances in Pesticide 10. Yamaoka, K., Nakagawa, M., and Ishida,
Sciences, (ed. H. Geissbuhler), Pergamon M. (1987) J. Pestic. Sci., 12, 209–212.
Press, Oxford, New York, Part 2, pp. 11. For example: Gee, S.K., Hanagan, M.A.,
94–98. Hong, W., Kucharczyk, R., and Pont, D.
3. Konotsune, T. and Kawakubo, K. (1975) (1997) WO 97/08164.
Sankyo Co. Ltd, JP 50126830, (1975) DE 12. Nishiyama, R., Kimura, F., Haga, T.,
2513750, (1975) GB 1463473. Sakashita, N., and Nishikawa, T.
4. Cole, D., Pallett, K., and Rodgers, M. (1978) Ishihara Syngyo Kaisha Ltd,
(2000) Discovering new modes of ac- GB 2002375.
tion for herbicides and the impact of 13. Kimura, F. (1984) Jpn. Pestic. Inform.,
genomics. Pestic. Outlook, 12, 223–229. 45, 24.
5. Ishida, M., Matsui, T., Yanai, T., 14. Hiroshi, M. (2005) ACS Symp. Ser., 892,
Kawakubo, K., Honma, T., Tanizawa, 161–171.
K., Nakagawa, M., and Okudaira, H. 15. Koho, K.T. (1980) Mitsubishi Petrochem-
(1984) Sankyo Kenkyusho Nenpo, 36, 44. ical Co., Ltd, JP 57072903.
16. Ikeda, K. and Goh, A. (1991) Jpn. Pestic. 26. von Deyn, W., Hill, R.L., Kardorff, U.,
Inform., 59, 1991. Engel, S., Otten, M., Plath, P., Rang, H.,
17. Miyahara, M. (1986) Jpn. Pestic. Inform., Harreus, A., König, H., Walter, H., and
49, 15. Westphalen, K.-O. (1996) BASF AG, WO
18. Luscombe, B.M., Pallett, K., 96/26206.
Loubiere, P., Millet, J.C., Melgarejo, 27. AGR World Crop Protection News
J., and Vrabel, T.E. (1995) Proc. Brighton (2005) BASF licenses maize herbicide.
Crop Prot. Conf. – Weeds, 1, 35. No. 427, p. 20.
19. Cain, P.A., Cramp, S.M., Little, G.M., 28. Schönhammer, A., Freitag, J., and
and Luscombe, B.M. (1991) Rhône- Koch, H. (2006) J. Plant Dis. Protect., 20,
Poulenc Agricultutes Ltd, EP 0527036. 1023–1031.
20. Luscombe, B.M. and Pallett, K.E. (1996) 29. Rheinheimer, J., von Deyn, W.,
Pestic. Outlook, 7, 29–32. Gebhardt, J., Rack, M., Lochtman, R.,
21. Bernard, D. and Viauvy, A. (1999) Götz, N., Keil, M., Witschel, M., Hagen,
Rhône-Poulenc Agricultutes Ltd, WO H., Misslitz, U., and Baumann, E.
99/02489. (1999) BASF AG, WO 99/58509.
22. Pallett, K.E., Little, J.P., Veerasekaran, 30. Garthoff, B. (2005) Innovation Driv-
P., and Viviani, F. (1997) Pestic. Sci., 50, ing Future Growth, Analyst & Investor
83. Days, 05.09.–06.09.2005, Lyon, Bayer
23. Beltran, E., Fenet, H., Cooper, J.F., and CropScience AG.
Coste, C.M. (2000) J. Agric. Food Chem., 31. Schmitt, M., van Almsick, A., Preuss, R.,
48, 4399. Willms, L., Auler, T., Bieringer, H.,
24. Pallett, K.E., Cramp, S.M., Little, J.P., and Thuerwaechter, F. (2001) Aventis
Veerasekaran, P., Crudace, A.J., and CropScience GmbH, WO 2001/074785.
Slater, A.E. (2001) Pest Manage. Sci., 57, 32. Schmitt, M.H., van Almsick, A., and
133. Willms, L. (2008) Pflanz.-Nachr. Bayer,
25. Taylor-Lovell, S., Sims, G.K., Wax, L.M., 61, 7–14 (English Edition).
and Hassett, J.J. (2000) Environ. Sci. 33. Menne, H. (2008) Pflanz.-Nachr. Bayer,
Technol., 34, 3186. 61, 107–120 (English Edition).
277
5
New Auxin Mimics and Herbicides
5.1
The Molecular Mode of Action of Auxin Herbicides
5.1.1
Introduction
Auxin herbicides comprise a remarkable set of chemistries that have profound

morphological and physiological effects on the growth of mainly (but not exclu-
sively) broadleaf weeds. Currently, about 20 commercially available compounds are
classified as auxin herbicides, in addition to many more experimental compounds.
These all consist of a planar aryl group with an attached carboxylic acid functionality
[1], and can be subcategorized in a variety of ways (Figure 5.1.1). The first commer-
cial auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), was introduced over 60
years ago, and since that time in almost every decade new herbicide introductions
have been made that exhibit this mode of action (Figure 5.1.1). The latest addition,
aminopyralid, was launched in 2005, and further innovations appear likely in the
near future, based on new chemistries [2] and auxin herbicide-resistant crops
[3, 4]. Thus, the auxinic mode of action has been one of the most robust, widely
used and prolific of all herbicidal modes of action. Despite this long and august
pedigree, knowledge of the molecular interactions and the precise target site of
auxin herbicides has lagged behind that of much ‘‘younger’’ modes of action. Weed
scientists have taken comfort in describing the mode of action as ‘‘mimicking that
of the natural auxin, indole-3-acetic acid (IAA).’’ However, for many years this has
been relatively uninformative at the molecular level as the receptor sites for IAA
were, until recently, unknown.
5.1.2
TIR1/AFB Proteins as Auxin Herbicide Receptors
Insights into the molecular mode of action of auxin (IAA) and auxin herbicides
have benefited from the extensive use of 2,4-D as a convenient surrogate for IAA
in genetic and biochemical studies of auxin action [5]. As an endogenous plant

278 5 New Auxin Mimics and Herbicides
H 3 NH2
IAA N
Picloram Cl Cl
Natural plant
hormone (1963)
0.07-1.2 Cl N COOH
COOH
1 Cl Cl Cl
2,4-D Clopyralid
(1948) (1975)
0.28-2.24 O COOH 0.15-0.56 Cl N COOH
NH2
Triclopyr Cl Cl Aminopyralid Cl
(1979) (2005)
0.28-10 Cl N O COOH 0.05-0.12 Cl N COOH
NH2
NH2 Cl
Cl Cl N
Fluroxypyr Aminocyclopyrachlor
(1985*) (2010) N COOH
0.14-0.56 F N O COOH 0.08-0.11
2 Cl 4
Cl
Dicamba Quinclorac
Cl COOH
(1967) (1992) N Cl
0.07-2.24 O 0.28-0.56 COOH
Figure 5.1.1 Chemical structures of some important com-

mercial auxin herbicides. The year of US registration and
typical use rates (kg ha−1 ) are shown. 1, Aryloxyacetates; 2,
benzoate; 3, pyridine/pyrimidine carboxylates; 4, quinoline
carboxylate. *European registration (registered in the US in
1998).
hormone, IAA is subject to complex and intensive regulation via endogenous

synthesis, transport, localization, derivatization, and degradation [6], whereas 2,4-D
acts as a longlived xenobiotic reagent that avoids many of these homeostatic
mechanisms. This is the basis of the powerful herbicidal activity of these types
of compound as so-called ‘‘auxin agonists.’’ By using 2,4-D and other auxinic
compounds, a number of studies have uncovered a series of auxin-resistant
mutants using forward genetic screens of mutagenized populations of the model
plant/weed Arabidopsis thaliana [5, 7, 8]. The mutant loci were progressively
identified via positional cloning techniques [9]. Many of the dominant mutations
proved to be in transcriptional repressors and activators, consistent with the role
of auxin in rapid gene induction, whereas many of the recessive mutations were
in proteins associated with the ubiquitin (Ub)-mediated proteolytic machinery that
targets intracellular proteins for degradation via the proteasome [10].
These and other forward and reverse genetic studies led to a model in which
auxins promote the specific degradation of short half-life transcriptional repressors
(Aux/IAA proteins) [11] that bind to and prevent the action of transcriptional
activators of auxin-responsive genes known as auxin response factor (ARF) proteins
[12]. This enables a rapid and specific activation of auxin-responsive genes, which
in turn gives rise to the many and varied downstream effects of auxins. The cellular
complex that tags proteins for proteosomal degradation is the E3 Ub-protein ligase.
A majority of plant E3 Ub-ligases are comprised of three protein components,
Skp1, Cul1, and an F-box protein (termed the SCF complex). The exact site of
the interaction of auxins to promote Aux/IAA protein degradation was unclear
until two landmark reports were made in 2005 [13, 14]. These confirmed the site
of auxin interaction to be, surprisingly, the F-box protein component (TIR1) of
the SCF complex that recognizes the Aux/IAA protein targeted for degradation
(Figure 5.1.2). This unique signaling mechanism revealed by the 65-year-old
herbicide, 2,4-D was new to biology, and in a subsequent tour-de-force, the crystal
structure of IAA (and 2,4-D), in complex with the TIR1 receptor and the interaction
domain of the Aux/IAA protein, was determined [15]. Thus, the venerable auxin
Auxin herbicide treatment
2,4-D
TIR1/AFB
Aux/IAA Ub Aux/IAA ARF Auxin-responsive genes repressed
E2
SCFTIR1 F-box Ub-ligase
Complex Auxin-response element
(E3 Ub-ligase) ASK1 RBX + Herbicide: Auxin-responsive
genes activated
CUL1 ARF
RUb
Aux/IAA Ub Ub Ub
Ethylene + cyanide ACS
ABA NCED
ROS etc.
Epinasty
26S Growth Inhibition Plant
proteasome Degraded Death
Senescence
Aux/IAA
Phytotoxicity
Figure 5.1.2 A model for the action of Ub-ligase. This tags the Aux/IAA repressor
auxin herbicides such as 2,4-D on plants. protein for degradation by the 26S protea-
In the untreated state, auxin-responsive some. The ARF is released from repression
genes are repressed by the dimerization and binds to the auxin-response element of
of short-lived Aux/IAA proteins with ARF auxin-responsive genes, allowing their tran-
transcription factors. In the presence of her- scriptional activation. Auxin-regulated genes
bicide, auxin herbicides bind to TIR1/AFB include key enzymes for ethylene and ABA
receptor proteins in the nucleus, occupy- biosynthesis. The unregulated production
ing the IAA binding pocket. This promotes of these plant hormones promotes a cas-
an interaction of the receptors with a tar- cade of phytotoxic events that results in
get Aux/IAA transcriptional repressors to the physiological and morphological con-
form a ternary complex. The resulting E3 sequences associated with auxin herbicide
Ub-ligase (SCFTIR1/AFB ) complex ubiquiti- symptomatology.
nates the bound Aux/IAA protein via an E2
herbicides joined more recently discovered herbicides in having their ligand/protein

target site interactions elucidated in molecular detail.
The intricate and elegant biology surrounding IAA perception and signal trans-
duction has been described in detail in several recent reviews [16–19], and it is
worthwhile considering the implications from an herbicide biology perspective.
Figure 5.1.2 illustrates the principal upstream components of the signaling cascade
that occurs in the plant nucleus. In this case, 2,4-D acts as an auxin agonist by
binding to the same site as IAA on the TIR1 receptor protein to promote the asso-
ciation of TIR1 with the Aux/IAA protein targeted for Ub tagging and degradation.
This interaction is unusual, as the auxin small molecule occupies a deep pocket
in the protein and interacts with both the TIR1 receptor and the Aux/IAA protein,
providing so-called ‘‘molecular glue’’ [15] at the bottom of the binding cavity (and
without inducing significant protein conformational changes). Thus, in the ternary
complex the herbicide is effectively sealed within the binding pocket. A surprising
new small molecule player in the auxin interaction is also revealed within the
structure: a molecule of inositol hexakisphosphate (IP6) is positioned within the
TIR1 protein, and acts as a structural component at a conserved site close to
the binding pocket. It remains to be seen if the role of IP6 is purely structural, or if
it has a more dynamic function in modulating auxin affinity and signaling.
The main biochemical and structural elements of auxin perception have been
elucidated to date by studies of the Arabidopsis TIR1 protein. To date, three close
homologs of the TIR1 receptor encoded in the Arabidopsis genome have been
identified. These are referred to as Auxin F-Box (AFB) proteins, that share 60–70%
polypeptide sequence identity to TIR1 [20]. Reverse genetic studies have shown that
plants with stacked mutant alleles in TIR1, AFB1, AFB2, and AFB3 have high levels
of auxin and 2,4-D resistance. This combination of mutations also has significant
deleterious consequences on plant growth and development, consistent with the
critical need for functional auxin perception [20, 21]. Recent evidence suggests that
the TIR1/AFB receptors are not entirely functionally redundant with respect to
auxin and herbicide responses [21]. The Arabidopsis tir1 and afb2 mutants exhibit re-
sistance to 2,4-D in root length inhibition assays (tir1 > afb2), whereas afb1 and afb3
single mutants have no appreciable 2,4-D resistance. The reintroduction of AFB1
and AFB2 proteins under control of the TIR1 promoter does not rescue the tir1 mu-
tant phenotype, which also indicates that the receptors are functionally distinct [21].
In addition to redundancy in the TIR1/AFB proteins, there is additional genetic
redundancy in the Aux/IAA transcriptional repressors that comprise the third
integral component of IAA and auxin herbicide signal perception. As there are
29 Aux/IAA proteins encoded in the Arabidopsis genome [11, 22], there are many
potential ternary complex combinations between the herbicide, TIR1/AFB proteins,
and Aux/IAA proteins. Different Aux/IAA proteins could have differing affinities
for TIR1/AFB:herbicide complexes and invoke different downstream responses.
For example, biochemical assays have indicated that both TIR1 and AFB2 have
auxin-induced interactions with Aux/IAA proteins IAA3 and IAA7, whereas AFB1
and AFB3 have negligible interactions, at least under the reported assay conditions
[21]. The affinity of 2,4-D in biochemical binding assays with the TIR1/IAA7
complex appears to be approximately 100-fold less than that of the natural ligand
IAA, whereas the affinity of 1-naphthylacetic acid (NAA) is about 10-fold less than
that of IAA [13, 14]. However, these in vitro assays may not accurately reflect in vivo
herbicidal affinities, as other cellular components involved with the highly regulated
Ub proteolysis machinery may affect the in vivo binding, stability, and turnover
of the herbicide/SCFTIR1/AFB complex. Nevertheless, the lower apparent affinity of
2,4-D for the TIR1 receptor relative to IAA may be effectively counteracted by its
intracellular accumulation via physico-chemical properties and cellular longevity.
2,4-D is recognized by the auxin permease influx pump, but not by the AUX1
family of auxin efflux pumps; consequently, this may also lead to an intracellular
hyperaccumulation of 2,4-D [23], promoting herbicidal activity.
5.1.3
The AFB5 Class of Picolinate Auxin Herbicide Receptors
The Arabidopsis genome encodes two other AFB-type proteins, AFB4 and AFB5, that
have approximately 45% polypeptide sequence identity to TIR1/AFB1-3. Mutant al-
leles of AFB5 were identified from a screen of mutagenized Arabidopsis seedlings to
find mutants that were resistant to an experimental picolinate-type auxin herbicide
but remained sensitive to 2,4-D [24]. The afb5 mutants were found to have a robust
resistance to picloram and aminopyralid, but were not resistant to 2,4-D, dicamba,
or fluroxypyr. They were also slightly hypersensitive to IAA, which suggests that
picolinate auxin herbicides primarily function through a different subclass of auxin
receptors than does 2,4-D. The AFB4 and AFB5 proteins have certain differences
to the TIR1/AFB1-3 proteins characterized via 2,4-D-resistant mutants. Both AFB4
and AFB5 possess an additional 45-residue serine-rich N-terminal extension of
unknown function. In addition, the residues within the binding pocket of TIR1,
which are highly conserved in the AFB1-3 family, contain several substitutions in
equivalent residues of AFB5 (and AFB4). These include the replacement of Ser438
of TIR1 that forms a critical salt bridge with the carboxyl group of 2,4-D and IAA
[15] with an alanine residue in AFB5 that is unable to form this type of bond. There
are also differences in the Aux/IAA protein contact residues between TIR1/AFB1-3
and AFB5. The afb5 mutants show no major morphological phenotype in the
absence of herbicide, in contrast to many 2,4-D-resistant Arabidopsis mutants.
This indicates that the AFB5 picolinate auxin receptor does not play a vital role
in IAA perception (consistent with the alterations in the binding site). This leaves
open the question as to the natural endogenous role and ligand of AFB5. Is there
a plant-derived signaling molecule that is mimicked by picolinate auxins?
Mutants in another protein component involved in SCF complex interactions,
SGT1b, also confer selective resistance to picloram equivalent to that of afb5
mutants. Mutations in SGT1b have been noted as enhancing 2,4-D resistance in the
tir1 mutant background [25], but the resistance of sgt1b plants to picloram is more
profound [24]. SGT1b mutants also confer resistance to the phytotoxin coronatine
that recently has been found to be perceived by a similar mechanism to auxins via
an F-box protein jasmonate receptor, COI1 [26]. SGT1 is an essential eukaryotic
protein which originally was characterized by its role in chromosome segregation

in yeast. It has since been shown to be involved in a variety of important cellular
processes, acting in particular as a ‘‘client adaptor’’ for the molecular chaperone
HSP90 in association with the Skp1 component of the SCF complex [27]. In plants,
SGT1 has a key role in disease immunity via interactions with RAR1 and HSP90
[28]. More recently, a role has emerged for auxin in plant disease resistance [29] and,
SGT1b may be a factor that connects the auxin response and disease-resistance
systems via an association with SCF complexes. It remains to be seen if the
picolinate auxin herbicide receptor AFB5 is also involved in these interactions.
5.1.4
TIR1/AFB Auxin Receptors in Other Plants
The TIR1/AFB receptor family has been well characterized in Arabidopsis, and con-
sists of three subclades: TIR1/AFB1; AFB2/AFB3; and AFB4/AFB5. An analysis of
reported plant genome sequences has indicated that these families are maintained
deep into the evolution of vascular plants, prior to the separation of monocots and
dicots and angiosperms and gymnosperms [21]. A fourth subclade (AFB6) can also
be distinguished in a wide variety of plants, but is not represented in the Brassi-
caceae dicots or the Poaceae grasses [21]. Four functional AFB homologs can also
be found in the moss Physcomitrella genome [30]. Thus, it can be expected that all
weed genomes contain a similar diversity of auxin receptors (as well as associated
Aux/IAA proteins, etc.). There appears to be no major phylogenetic distinction be-
tween the AFBs of grass and broadleaf species, which suggests that the basic mecha-
nism of auxin perception is similar [31]. Thus, the tolerance of most grasses to auxin
herbicides may lie in the different downstream physiological and morphological
consequences of auxinic stimulation [31–33]. Clearly, much deeper investigations
are required if these differences are to be elucidated at the molecular level.
5.1.5
Are There Other Auxin Herbicide Receptors?
From the accumulated molecular genetic and biochemical evidence, it appears that
the major morphological and phytotoxic effects of auxin herbicides are exerted
via nuclear TIR1/AFB receptors and the resulting indiscriminate induction of
auxin-responsive genes and downstream processes. Good evidence for this is the
observation that loss-of-function mutants in AFB5 show no significant injury at the
whole-plant level to field application rates of picloram and aminopyralid [24]. Many
early studies invoked the possible involvement of auxin-binding protein (ABP)
as an auxin herbicide receptor. ABP was initially identified biochemically as an
IAA-binding protein in maize by employing affinity purification, and subsequent
studies have shown it to be encoded by a single essential gene in the Arabidopsis
genome [34]. ABP localizes to both the plasma membrane and the endoplasmic
reticulum, and appears to have an important role in the cell cycle, cell expansion,
and early cell division by transducing an extracellular IAA signal via an as-yet
uncharacterized pathway which is initiated at the plasma membrane [35].
To date, no ABP mutants have been identified from many deep genetic screens
conducted using auxin herbicide probes. Similarly, in biochemical binding studies,
many potent commercial and experimental auxin herbicides have either low or no
affinity for ABP (R. Napier and T. Walsh, unpublished results). Certain cellular
effects of IAA (and herbicidal auxins) have been associated with rapid (<5 min) ion
fluxes and cell expansion that may be sensed at the plasma membrane, perhaps
by ABP, and are independent of TIR1/AFB [21]. However, auxin (and auxin
herbicide) symptoms in classic bioassays typically have a lag time of 10–15 min,
consistent with TIR1/AFB-modulated gene activation [36]. Thus, there is at present
no compelling evidence that ABP plays a role in the primary events of auxin
herbicide perception and eventual plant intoxication.
5.1.6
Downstream Effects of Auxin Herbicides
The upstream site of auxin herbicide perception, and the relatively short signal
pathway to rapid gene induction, now seems clearer. To date, many accumulated
studies have detailed the various downstream physiological effects of auxin her-
bicides that ultimately are responsible for weed growth inhibition and death [33].
Principal among these are the overproduction of the plant hormones ethylene and
abscisic acid (ABA) [32, 37]. The presence of excessive ethylene leads to tissue
epinasty and senescence, while the potentially toxic metabolite cyanide is also
released as a side product of the formative reaction for ethylene [38, 39]. The
presence of excessive ABA results in stomatal closure (with resulting consequences
on photosynthetic processes), growth inhibition, and senescence [33, 39]. These
downstream effects can now begin to be integrated into models incorporating
the upstream sensing mechanism involving the perception of auxin herbicides in
the nucleus by TIR1/AFB proteins and the rapid induction of auxin-responsive
genes (Figure 5.1.2). For example, early auxin-responsive genes include those
for 1-aminocyclopropane-1-carboxylate synthases (ACSs) that are responsible for
generating the precursor to ethylene [40, 41], and for the 9-cis-epoxycarotenoid
dioxygenases (NCEDs) that catalyze a controlling step in ABA biosynthesis [39]. It
is possible that certain weed species may suffer different consequences and time
courses of symptom development, based on variations in the panoply of herbicide
auxin-induced effects. However, it remains to be seen if specific herbicidal auxins,
SCFTIR1/AFB complexes and/or Aux/IAA target proteins are responsible for the
activation of certain genes and physiological responses in certain weed species,
or if such responses are relatively nonspecific consequences of herbicide auxin
treatment across all susceptible plant species.
5.1.7
Weed Selectivities at the Site of Auxin Action
In the past, because their gross downstream morphological effects appear similar,
the chemical classes of auxin herbicides have generally been grouped together, as
have the general classes of genes induced by auxin treatment [42–44]. Nevertheless,
subtle differences can be observed in the effects of different auxin treatments,
an example being the effects of the picolinate auxin herbicide picloram, the
phenoxyacetate 2,4-D, and the benzoate dicamba on various plant cells, tissues,
and whole plants. These effects may perhaps be attributable to differential receptor
triggering, as well as to differences in mobility and the distribution of the herbicides
within the plant. Many auxin herbicides exhibit distinct broadleaf weed selectivities,
a property which has led these materials frequently to be marketed as mixtures.
Whilst some of these selectivities have been ascribed to differential herbicide
metabolism, others seem to have been due to differences in perception at the target
site, or in immediate downstream physiological responses [32]. The Cruciferae are
relatively insensitive to pyridine auxins, and particularly to clopyralid, which is
used for broadleaf weed control in oilseed rape [45], whereas phenoxyacetate auxins
are used widely to control crucifers in cereals. Quinmerac, a quinoline carboxylate
auxin, is also selective to oilseed rape. The benzoate auxin dicamba also has a
relatively weak activity on the Brassicaceae, enabling its use in forage rape and kale,
while the selectivity of clopyralid and quinmerac extends to sugar beet, a member
of the Chenopodiaceae. These modes of selectivity seem to be primarily at the site
of action rather than via differential metabolism [46, 47].
Auxin herbicides generally have low or no herbicidal activity on grass crops and
weeds [32, 48] yet, despite many years of investigation, the principal reasons for
this are not completely clear. There is evidence for a more rapid metabolism of
auxin herbicides in grasses, but this does not appear to account completely for the
insensitivity [32]. IAA perception is clearly conserved and vital for monocot growth
and development, and the major gene families responsible for auxin signaling are
present in monocots [31]. Auxin herbicides do cause injury to grass crops at certain
growth stages, elevated application rates or under environmental stresses. This may
lead to developmental abnormalities resulting in ‘‘plant-leaning,’’ incomplete leaf
unfurling (‘‘rat-tailing’’ or ‘‘buggy-whipping’’ in maize, ‘‘onion-leafing’’ in wheat),
stalk brittleness or deformed brace roots in maize, and seed head deformation in
wheat and barley. There is some evidence for a differential intrinsic phytotoxicity
across various auxin herbicides in maize, that may be due to differences in target
site sensitivity [49]. Clearly, the overall weed-killing effects of auxin herbicides are
less profound on grasses than on broadleaf weeds. However, further studies are
warranted to determine if this is due to reduced affinities of herbicidal auxins at
grass TIR1/AFB receptors, or to reduced/altered downstream physiological and
morphological responses to auxinic stimulation in grass cell types. Alternatively,
grasses may have a greater tolerance towards the various downstream effects of
auxin stimulation.
Quinclorac is a unique auxin herbicide that is used for grass weed control, most
notably for the control of Echinochloa species in rice, as well as for broadleaf control
[50]. Although the effect of quinclorac on susceptible broadleaf weeds is similar to
that of other auxin herbicides, the injury to susceptible grass weeds is considerably
different from that elicited by high doses of other auxin herbicides. Quinclorac
causes a rapid cessation of growth via meristematic intoxication, followed by leaf
yellowing and eventual plant death, with little or no growth abnormalities or

developmental effects typical of other auxin herbicides [50]. The unique effect of
quinclorac on susceptible grasses has been explained by downstream biochemical
responses leading to an overproduction of ethylene, the associated release of
cyanide, and a build-up of reactive oxygen species (ROS) [33]. As insensitive species
do not hyperproduce ethylene and cyanide, this suggests that any selectivity lies
upstream of these processes. Whilst the site of chemical perception for quinclorac
in grasses has yet to be determined, the question remains as to whether the unique
grass activity is mediated by TIR1/AFB-type receptors, or by a new or related
signaling pathway. Chemical genetic studies conducted in a model grass species
should be valuable for elucidating these details.
5.1.8
Field Resistance to Auxin Herbicides
Despite over 60 years of widespread use, only 28 auxin-resistant weed species have
been documented to date, most of which are restricted to areas of under 50 ha
[51]. This relatively modest number over time may be in part due to the frequent
use of auxin herbicides in mixtures with other herbicidal modes of action. The
redundancy in receptor components, and the observed phenotypic fitness penalty
in plants with mutations in IAA perception, may also combat rapid resistance
development. Some documented resistant biotypes appear to have been due to
an increased metabolism of the applied herbicide, but others do not [32, 48]. An
increased knowledge of herbicidal auxin signaling components may now offer
candidate genes to be interrogated for potential resistance-conferring mutations
in auxin herbicide-resistant biotypes. For example, the recessive picloram-specific
resistance documented in yellow starthistle [52] may be due to mutations in an
AFB5 homolog, while dominant resistance, as in wild mustard and Kochia [53, 54],
might possibly arise from mutations in key Aux/IAA or ARF proteins.
5.1.9
Conclusions
IAA perception in plants is exquisitely fine-tuned to respond to cell type, develop-

ment stage, and to both physiological and environmental cues. The complexities of
the central role of IAA in plant growth and development, and its interconnections
with other plant hormone signaling pathways, continue to emerge [6, 16, 18].
However, from an herbicidal perspective, the strong agonist effect of exogenous
swamping of the system with a long-lived xenobiotic chemistry may obliterate
much of this fine control, leading to the profound herbicidal effects of auxin
herbicides. The discovery of the TIR1/AFB auxin receptors, and of their unique
role in auxin perception and signaling, has provided a long-awaited breakthrough
in understanding the mode of action of auxin herbicides. This immediately gives
rise to further intriguing routes of inquiry on the complex mode of action of
auxin herbicides with regards to monocot selectivity, differential broadleaf activity,
and chemical/target specificity. Undoubtedly, significant headway will be achieved

by translating the genetic and biochemical studies pioneered in the model plant
Arabidopsis into crop and weed species (both broadleaf and grasses), by using the
diverse chemical suite of auxin herbicides.
References
1. Ferro, N., Bredow, T., Jacobsen, H.J., 12. Guilfoyle, T.J. and Hagen, G. (2007)
and Reinard, T. (2010) Chem. Rev., 110, Curr. Opin. Plant Biol., 10, 453–460.
4690–4708. 13. Dharmasiri, N., Dharmasiri, S., and
2. Finkelstein, B.L., Armel, G.R., Estelle, M. (2005) Nature, 435, 441–445.
Bolgunas, S.A., Clark, D.A., Claus, 14. Kepinski, S. and Leyser, O. (2005)
J.S., Crosswicks, R.J., Hirata, C.M., Nature, 435, 446–451.
Hollingshaus, G.J., Koeppe, M.K., 15. Tan, X., Calderon-Villalobos, L.I.A.,
Rardon, P.L., Wittenbach, V.A., and Sharon, M., Zheng, C., Robinson,
Woodward, M.D. (2008) Abstracts of C.V., Estelle, M., and Zheng, N. (2007)
Papers, 236th ACS National Meet- Nature, 446, 640–645.
ing, Philadelphia, PA, August 17–21, 16. Mockaitis, K. and Estelle, M. (2008)
AGRO-019. Annu. Rev. Cell Dev. Biol., 24, 55–80.
3. Wright, T.R., Shan, G.M., Walsh, 17. De Rybel, B., Audenaert, D., Beeckman,
T.A., Lira, J.M., Cui, C., Song, P., T., and Kepinski, S. (2009) ACS Chem.
Zhuang, M.B., Arnold, N.L., Lin, G.F., Biol., 4, 987–998.
Yau, K., Russell, S.M., Cicchillo, R.M., 18. Calderon-Villalobos, L.I., Tan, X., Zheng,
Peterson, M.A., Simpson, D.M., Zhou, N., and Estelle, M. (2010) Cold Spring
N., Ponsamuel, J., and Zhang, Z.Y. Harbor Perspect. Biol., 2, a005546.
(2010) Proc. Natl Acad. Sci. USA, 107, 19. Santner, A. and Estelle, M. (2010) Plant
20240–20245. J., 61, 1029–1040.
4. Behrens, M.R., Mutlu, N., Chakraborty, 20. Dharmasiri, N., Dharmasiri, S., Weijers,
S., Dumitru, R., Jiang, W.Z., LaVallee, D., Lechner, E., Yamada, M., Hobbie,
B.J., Herman, P.L., Clemente, T.E., L., Ehrismann, J.S., Juergens, G., and
and Weeks, D.P. (2007) Science, 316, Estelle, M. (2005) Dev. Cell, 9, 109–119.
1185–1188. 21. Parry, G., Calderon-Villalobos, L.I.,
5. Estelle, M.A. and Somerville, C. (1987) Prigge, M., Peret, B., Dharmasiri, S.,
Mol. Gen. Genet., 206, 200–206. Itoh, H., Lechner, E., Gray, W.M.,
6. Zhao, Y. (2010) Annu. Rev. Plant Biol., Bennett, M., and Estelle, M. (2009) Proc.
61, 49–64. Natl Acad. Sci. USA, 106, 22540–22545.
7. Woodward, A.W. and Bartel, B. (2005) 22. Dreher, K.A., Brown, J., Saw, R.E., and
Ann. Bot., 95, 707–735. Callis, J. (2006) Plant Cell, 18, 699–714.
8. Leyser, O. (1997) Physiol. Plant., 100, 23. Delbarre, A., Muller, P., Imhoff, V., and
407–414. Guern, J. (1996) Planta, 198, 532–541.
9. Lukowitz, W., Gillmor, C.S., and 24. Walsh, T.A., Neal, R., Merlo, A.O.,
Scheible, W.-R. (2000) Plant Physiol., Honma, M., Hicks, G.R., Wolff, K.,
123, 795–806. Matsumura, W., and Davies, J.P. (2006)
10. Ward, S.P. and Estelle, M. (2001) J. Plant Physiol., 142, 542–552.
Plant Growth Regul., 20, 265–273. 25. Gray, W.M., Muskett, P.R., Chuang,
11. Overvoorde, P.J., Okushima, Y., Alonso, H.W., and Parker, J.E. (2003) Plant Cell,
J.M., Chan, A., Chang, C., Ecker, 15, 1310–1319.
J.R., Hughes, B., Liu, A., Onodera, 26. Sheard, L.B., Tan, X., Mao, H., Withers,
C., Quach, H., Smith, A., Yu, G., and J., Ben-Nissan, G., Hinds, T.R.,
Theologis, A. (2005) Plant Cell, 17, Kobayashi, Y., Hsu, F.-F., Sharon,
3282–3300. M., Browse, J., He, S.Y., Rizo, J., Howe,
G.A., and Zheng, N. (2010) Nature, 468, 42. Pufky, J., Qiu, Y., Rao, M.V.,
400–405. Hurban, P., and Jones, A.M. (2003)
27. Catlett, M.G. and Kaplan, K.B. (2006) J. Funct. Integr. Genomics, 3, 135–143.
Biol. Chem., 281, 33739–33748. 43. Paponov, I.A., Paponov, M., Teale, W.,
28. Shirasu, K. (2009) Annu. Rev. Plant Biol., Menges, M., Chakrabortee, S., Murray,
60, 139–164. J.A.H., and Palme, K. (2008) Mol. Plant,
29. Kazan, K. and Manners, J.M. (2009) 1, 321–337.
Trends Plant Sci., 14, 373–382. 44. Raghavan, C., Ong, E.K., Dalling, M.J.,
30. Prigge, M.J., Lavy, M., Ashton, N.W., and Stevenson, T.W. (2006) Funct.
and Estelle, M. (2010) Curr. Biol., 20, Integr. Genomics, 6, 60–70.
1907–1912. 45. Hall, J.C. and Van den Born, W.H.
31. McSteen, P. (2010) Cold Spring Harbor (1988) Weed Sci., 36, 9–14.
Perspect. Biol., 2, a001479. 46. Grossmann, K. and Scheltrup, F. (1998)
32. Sterling, T.M. and Hall, J.C. (1997) Rev. Pestic. Sci., 52, 111–118.
47. Thompson, L.M.L. and Cobb, A.H.
Toxicol. (Amsterdam), 1, 111–141.
(1987) Proceedings of the British Crop
33. Grossmann, K. (2010) Pest Manage. Sci.,
Protection Conference - Weeds, pp.
66, 113–120.
1097–1104.
34. Napier, R.M., David, K.M., and
48. Kelley, K.B. and Riechers, D.E. (2007)
Perrot-Rechenmann, C. (2002) Plant
Pestic. Biochem. Physiol., 89, 1–11.
Mol. Biol., 49, 339–348.
49. Vettakkorumakankav, N.N., Deshpande,
35. Tromas, A., Paponov, I., and
S., Walsh, T.A., and Hall, J.C. (2002)
Perrot-Rechenmann, C. (2010) Trends
Weed Sci., 50, 713–720.
Plant Sci., 15, 436–446. 50. Grossmann, K. (1998) Weed Sci., 46,
36. Badescu, G.O. and Napier, R.M. (2006) 707–716.
Trends Plant Sci., 11, 217–223. 51. Heap, I. (2011) The International Sur-
37. Grossmann, K. (2003) J. Plant Growth vey of Herbicide Resistant Weeds,
Regul., 22, 109–122. http://www.weedscience.com (accessed
38. Grossmann, K. (1996) Physiol. Plant., 97, December 2010).
772–775. 52. Sabba, R.P., Ray, I.M., Lownds, N.,
39. Kraft, M., Kuglitsch, R., Kwiatkowski, J., and Sterling, T.M. (2003) J. Hered., 94,
Frank, M., and Grossmann, K. (2007) J. 523–527.
Exp. Bot., 58, 1497–1503. 53. Jugulam, M., McLean, M.D., and Hall,
40. Wei, Y.D., Zheng, H.-G., and Hall, J.C. J.C. (2005) Weed Sci., 53, 417–423.
(2000) Pest Manage. Sci., 56, 377–387. 54. Preston, C., Belles, D.S., Westra, P.H.,
41. Grossmann, K. and Hansen, H. (2001) Nissen, S.J., and Ward, S.M. (2009)
Physiol. Plant., 113, 9–14. Weed Sci., 57, 43–47.
5.2
Aminopyralid
5.2.1
Introduction
Aminopyralid (4-amino-3,6-dichloro-2-pyridinecarboxylic acid) is the active ingre-

dient in several herbicide products formulated by Dow AgroSciences, a subsidiary
of The Dow Chemical Company. This herbicide is a member of the pyridine
carboxylic acid herbicide family that includes picloram, clopyralid, fluroxypyr, and
triclopyr. Picloram, one of the first herbicides in this family, was discovered in 1960
and launched in the USA in 1963.
The herbicidal effects of aminopyralid were discovered in 1998. Aminopyralid
controls a variety of broadleaf weeds, including noxious, poisonous, and invasive
plants, in rangeland, pasture, and industrial vegetation management sites. When
left uncontrolled, these weeds have many direct and secondary negative effects.
These adverse effects include the creation of weed-dominated plant communities
that reduce the diversity of desirable plant, animal, and insect species [1, 2]. Such
weeds also reduce rangeland and pasture productivity, increase livestock production
costs, reduce grassland carrying capacity, increase soil erosion, and degrade the
aesthetic qualities of the land. In industrial management sites, such as roadside,
energy transmission, and railroad rights-of-way, weeds disrupt energy distribution,
impede transportation, and create safety hazards. Collectively, these plant impacts
burden local economies and have adverse societal impacts.
Aminopyralid was first registered in the US in 2005, since then many aminopy-
ralid containing products have been commercialized in over 50 countries. De-
pending on the country, aminopyralid is approved for use in weed management
programs in rangeland, permanent grass pastures, industrial vegetation manage-
ment areas, natural areas, small grains (wheat, durum, barley, rye, and triticale),
oilseed rape, and oil palm and rubber plantations.
5.2.2
Aminopyralid Synthesis
Aminopyralid (1) may be synthesized via the electrolysis of picloram [3], as depicted
in Scheme 5.2.1
5.2.3
Biology
Aminopyralid is a systemic, ambi-mobile growth regulator herbicide that is rapidly

absorbed by the plant’s roots and leaves. The herbicide exhibits auxin-like symp-
toms and characteristics as it moves throughout the plant. It deregulates plant
growth metabolic pathways by binding at protein receptor sites normally utilized
by naturally occurring plant hormones. As the compound translocates and accu-
mulates in the meristematic tissues, it causes uneven cell division and growth,
NH2 NH2
Cl Cl Cl
electrolysis
OH OH
Cl N Cl N
O O
Picloram 1, Aminopyralid
Scheme 5.2.1 Synthesis of aminopyralid (1) via the electrolysis of picloram.

and this results in the death of susceptible plant species. Aminopyralid provides a
broad spectrum of broadleaf weed control.
Within hours or days of application, aminopyralid causes symptoms such as
thickened, curved, and twisted stems and leaves, cupping and crinkling of the leaves,
stem cracking, narrow leaves with callus tissue, hardened growth on stems, enlarged
roots, and proliferated growth. Most susceptible annual plants are controlled within
four to eight weeks after application; however, the control of perennial herbaceous
broadleaf plants or woody plants may take two months or more after application.
Aminopyralid, when applied at 5 to 120 g a.e. ha−1 provides post-emergence
control of many broadleaf and some semi-woody weeds. Depending on the rate of
application and the weed species targeted, aminopyralid can also provide residual or
pre-emergence control of some germinating weed seeds and emerging seedlings.
Aminopyralid is selective to grasses at typical use rates, and most annual and
perennial grasses are not adversely affected by post-emergence applications in field
situations.
There is a difference in the unit activity and level of phytoxicity achieved between
aminopyralid and picloram. Across most broadleaf species, aminopyralid is more
active than picloram when comparing amounts of each herbicide required to control
plants. Typically, aminopyralid provided 90% control of several broadleaf plants at
lower rates than picloram in experiments conducted in controlled environments
(Table 5.2.1).
Aminopyralid is effective at very low rates when compared to currently registered
herbicides with the same mode of action, including 2,4-D, clopyralid, triclopyr,
picloram, and dicamba. For example, aminopyralid controlled both Canada thistle
(Cirsium arvense) and Russian knapweed (Acroptilon repens) on several rangeland
sites at lower usage rates than did either picloram or clopyralid [4–6]. Despite the
greater efficacy of aminopyralid in Canada thistle, the absorption and translocation
of clopyralid in the plant was higher, which suggests that aminopyralid may exert a
Table 5.2.1 Application rates (g a.e. ha –1 ) of aminopyralid

and picloram required to provide 90% control of selected
plant species 28 days after post-emergence application in
greenhouse experiments.
Plant species Aminopyralid Picloram
Amaranthus spinosus 49 105

Ambrosia artemisifolia 11 117
Centaurea maculosa 20 105
Daucus carota 61 85
Rumex obtusifolius 6 31
Senna obtusifolia 7 33
Sida rhombifolia 30 58
Solanum carolinense 11 54
Solanum viarum 9 29
greater biological activity at the protein receptor sites than clopyralid [7]. Aminopy-
ralid, when applied at 60 g a.e. ha−1 controlled tropical soda apple (Solanum viarum),
both established plants and seedlings that emerged after application, in subtropical
grasslands; more over, the level of control was similar to that of picloram, at
140 g a.e. ha−1 [8].
5.2.4
Aminopyralid Utility
Aminopyralid products provide selective, broad-spectrum control of broadleaf

herbaceous and woody plants. Consequently, a range of products has been de-
veloped for use in rangeland, pastures, rights-of-way, noncropland, natural areas,
small grains, oilseed rape, and oil palm and rubber plantations. Aminopyralid
application alone and/or in premixes with other herbicides can be recommended,
depending on the target species to be controlled. In addition to providing the
control of many broadleaf weeds, foliar-applied aminopyralid can also provide
residual control, thus reducing the need for re-treatment, depending on the rate of
application and the target weeds.
Aminopyralid represents an option for land managers to control invasive plants
and other broadleaf weeds that reduce forage production on pastures [9], de-
grade wildlife habitat, and cause economic losses. Aminopyralid offers excellent
season-long control of broadleaf herbaceous plants at low rates of between 50
and 120 g a.i. ha−1 . Aminopyralid can be used to control several plants, includ-
ing Russian knapweed, musk thistle (Carduus nutans), plumeless thistle (Carduus
acanthoides), Canada thistle, spotted knapweed (Centaurea stoebe), yellow starthistle
(Centaurea solstitialis), and tropical soda apple, with tolerance by established grasses.
Aminopyralid is also available in premix formulations with 2,4-D, fluroxypyr, tri-
clopyr, and metsulfuron-methyl. In these premixes, aminopyralid contributes to
the control of a broad spectrum of broadleaf weeds and woody plants, including
species in the genera Eupatorium, Sida, Solidago, Symphoricarpos, Urtica, Verbena,
Daucus, and Vernonia.
Aminopyralid can be applied to rangeland or established permanent grass
pastures and industrial vegetation management areas as either an aerial or ground
broadcast treatment, as a spot application, or as a high-volume foliar application.
Long-term weed control with aminopyralid is most effective where grass and
other desirable vegetation recover to better compete with invasive plants after
aminopyralid application. The benefits of weed control achieved with aminopyralid
may be optimized and extended when integrated with other vegetation management
practices (i.e., prescribed fire, grazing management, fertilization, planting improved
temperate and tropical grass and legume forages, mechanical controls, biological
controls, etc.) that promote the recovery of desirable vegetation. Aminopyralid
has been used to control weeds and to improve perennial grass establishment on
degraded rangeland sites in California [10]. Likewise, the benefits of aminopyralid in
terms of Canada thistle control, the removal of undesirable species, and the increase
in native grass cover has helped to improve restored prairie plant communities [11].
Aminopyralid can be applied as a post-emergence foliar application to small

grains. In wheat, the usage rates for aminopyralid range from 4 to 10 g a.e. ha−1 , ap-
plied post-emergence, from the three-leaf crop growth stage up to early jointing. The
herbicide provides excellent post-emergence control, as well as residual activity on
several difficult-to-control broadleaf weeds, including Centaurea spp., Polygonum
convolvulus, Polygonum aviculare, Papaver rhoeas [including acetolactate synthase
(ALS)-resistant and 2,4-D-tolerant biotypes], Silybum marianum, and Chrysanthe-
mum segetum. In order to provide a complete weed control spectrum, aminopyralid
has been mainly commercialized in formulation mixtures with other wheat herbi-
cides, such as fluroxypyr, florasulam, pyroxsulam, and metsulfuron-methyl.
In oil palm and rubber plantations, aminopyralid + glyphosate is applied as a
post-emergence treatment around the base of the trees for the broad-spectrum con-
trol of broadleaf and grass weeds, including Ageratum conyzoides, Asystasia intrusa,
Hedyotis verticillata, Mikania cordata, and Paspalum conjugatum. Aminopyralid +
glyphosate treatments are also applied as directed sprays to manage unwanted
vegetation and to minimize injury to desirable plants growing in close proximity to
weeds.
5.2.5
Aminopyralid Mechanism of Crop Selectivity
The mechanism of grass crop tolerance is not solely the result of the plant’s
metabolism of aminopyralid. Rather, it is likely that grass tolerance results from
an inability of the herbicides to bind effectively to protein receptor sites where they
would otherwise bind and exert an herbicidal effect.
The metabolism of aminopyralid has been investigated in three perennial grass
species, namely big bluestem (Andropogon gerardii), perennial ryegrass (Lolium
perenne), and Guinea grass (Panicum maximum), and one annual grass, wheat
(Triticum aestivum). When aminopyralid was applied at 360 g a.e. ha−1 to the
perennial grasses, and at 60 and 120 g a.e. ha−1 to wheat, the residues identified
in the fresh grass and grass hay samples and in the wheat forage, straw and
grain samples, consisted of aminopyralid and its glucose conjugates, with the latter
tending to be sequestered in the plant cell wall fractions.
Aminopyralid offers a high level of tolerance on a wide range of C3 and C4 forage
grasses. Most established forage grasses are tolerant to aminopyralid applied
at rates up to 240 g a.e. ha−1 , which is twice the global maximum usage rate.
Grasses evaluated included many in the genera Agropyron, Andropogon, Bouteloua,
Brachiaria, Bromus, Buchloe, Cynodon, Dactylis, Digitaria, Eragrostis, Festuca sp.,
Lolium sp., Panicum, Paspalum, Phleum, Poa, and Sorghastrum.
Most varieties of wheat, durum, barley, and triticale are tolerant to aminopyralid
when applied at rates of 10 g a.e. ha−1 or less post-emergence and at the correct
growth stage. Although visible foliar injury is rare, under certain stress conditions
the typical symptom observed is a splaying of the grass tillers. Although injury levels
can become marginal at application rates higher than 10 g a.e. ha−1 , the greatest fac-
tor impacting on injury is the cereal growth stage. The safest interval during which
to apply aminopyralid is BBCH (Biologische Bundesanstalt, Bundessortenamt and

CHemical industry) stages 13 to 31.
5.2.6
Aminopyralid Environmental Degradation
In the US, aminopyralid was accepted for evaluation in October 2004, under the
Reduced Risk Pesticide program of the US Environmental Protection Agency (US
EPA), based on factors related to the management of noxious and invasive weeds in
rangeland, pastures, and noncropland, when compared to registered products. The
factors considered included risk assessments for toxicological, ecotoxicological, and
environmental fate effects, in addition to other factors such as:
• The equivalent or superior post-emergence and residual control of a broad
spectrum of difficult-to-control noxious and invasive broadleaf or semi-woody
plants compared to currently registered herbicides.
• A reduction in environmental herbicide loading in rangeland and pastures,
industrial vegetation management areas, and natural areas by offering the
control of many key species at application rates that were substantially lower than
the labeled usage rates for many currently registered herbicides.
• Residual weed control activity to reduce the need for re-treatment.
• The ability to fit into integrated weed-management programs.
• The tolerance of C3 and C4 perennial grasses.
The primary route of aminopyralid breakdown in soil is via aerobic microbial

degradation, during which only non-phytotoxic materials are produced. By con-
trast, under anaerobic conditions, as might occur in flooded soils, the microbial
degradation will be slow and not represent a significant route of degradation. On
the soil surface, aminopyralid is not expected to degrade significantly by photolysis.
As a condition of registration, the US EPA requested a repeat of the soil aerobic
metabolism laboratory studies, including an assessment of the degradation kinetics
of seven soil types. The aminopyralid half-lives in the soils ranged from 14.7 to 143
days (average 72.6 days). The degradation of aminopyralid in field experiments was
faster and more consistent than in the laboratory, with an average half-life of 34.5
days (at eight North American sites) and 25 days (at four European sites).
In surface water, the primary route of degradation of aminopyralid is via
photolysis, with a photolytic half-life measured in laboratory studies of 0.6 days.
The rate of photolysis is reduced by any factor that attenuates sunlight, such as
increased water turbidity and the depth of the water column. During photolysis,
two major degradation products – oxamic acid and malonamic acid – are formed,
but neither was considered to be of concern to environmental or human health. In
aquatic sediments, aminopyralid is stable to hydrolysis and degrades very slowly
under anaerobic conditions.
When ingested by mammals, aminopyralid is not broken down and is excreted in
the urine and feces within three days of ingestion. In a metabolism study conducted
in rats, aminopyralid was found to be excreted unchanged, indicating an absence
of metabolism. The repeated administration of aminopyralid also indicated a lack

of bioaccumulation in the tissues [14].
Based on this lack of metabolism of aminopyralid, any manure and/or slurry
obtained from animals fed aminopyralid-treated grass, hay, or silage may contain
residues that could be phytotoxic to susceptible plants. Consequently, such slurry or
manure should be applied to crops as specified on the product labels, and managed
according to the labeled precautions and restrictions.
The adsorption/desorption of a herbicide has a major influence on dissipation
processes in soil. That aminopyralid is adsorbed only weakly to soils means that it
may have a degree of soil mobility, though the potential for aminopyralid movement
is mitigated by its low usage rates (5 to 120 g a.e. ha−1 ). When experiments were
conducted to compare the soil adsorption of aminopyralid to that of picloram and
clopyralid, aminopyralid was shown to bind more tightly than clopyralid to six of
the eight soils tested [12], while the soil sorption of aminopyralid was greater than
that of picloram [13].
The residue concentrations of aminopyralid are not reduced in plant materials
or manure during the thermophilic or curing phases of the composting process.
Although the degradation of residues may occur, the plant biomass dry matter
decline occurs at a similar rate (or even faster), such that the aminopyralid
concentrations are not altered or may even increase slightly, as the compost dries
during its preparation.
Based on conservative and protective assumptions regarding the toxicity of,
and potential exposures to, aminopyralid, adverse effects are unlikely to become
apparent in agricultural workers or the general public, even at the maximum
application rate [14]. Previously, two possible routes of consumer exposure have
been assessed in a US EPA report: (i) dietary exposure to aminopyralid via the food
and drinking water; and (ii) incidental exposure to aminopyralid of children eating
grass or soil from treated camping grounds. The US EPA concluded that ‘‘ . . .
there is reasonable certainty that no harm will come from aggregate [cumulative]
exposure to aminopyralid residues’’ [15].
5.2.6.1 Animal Toxicity

Aminopyralid has been determined to have a very low order of toxicity in many
organisms:
• It is essentially nontoxic to avian species: the acute exposure of bobwhite quail
resulted in a lethal dose (LD)50 >2250 mg kg−1 , whilst dietary studies with
bobwhite quail and mallard duck showed the lethal concentration (LC)50 to be
>5000 mg kg−1 .
• Aminopyralid is also practically nontoxic to fish: in acute toxicity tests the
96 h LC50 values for sunfish, rainbow trout, and sheepshead minnow were all
>100 mg l−1 .
• Aminopyralid is practically nontoxic (or slightly toxic) to aquatic invertebrates, as
noted in acute toxicity tests reviewed by the US EPA. For Daphnia magna, a 48 h
effective concentration (EC)50 of >98.6 mg l−1 was reported (slightly toxic), while
for estuarine marine invertebrates, the 48 h EC50 was >89 mg l−1 (slightly toxic)
for Eastern oyster, and the LC50 was >100 mg l−1 (practically nontoxic) for Mysid
shrimp.
• Aminopyralid is practically nontoxic to mammals, based on an oral LD50 of
>5000 mg kg−1 in rats, mice, rabbits, and dog.
5.2.6.2 Carcinogenicity/Teratogenicity
With regards to possible carcinogenicity/teratogenicity, aminopyralid did not cause
any cancer or birth defects in laboratory animals. In fact, aminopyralid is classified
by the US EPA as ‘‘not likely’’ to be carcinogenic to humans, which is the least
carcinogenic category. No increases were identified in the incidence of any tumors
in studies with rats or mice.
Thus, the overall toxicological profile of aminopyralid is very favorable. It is
not acutely toxic, does not pose any inhalation hazard, and neither is it a skin
sensitizer. No evidence of mutagenic or carcinogenic potential was obtained from
any study, and it showed no teratogenic effects in either rats or rabbits. Additional
investigations confirmed that aminopyralid is not a reproductive hazard or concern.
5.2.7
Conclusions
Aminopyralid is a low-use rate herbicide with a very favorable ecotoxicology and

toxicology profile. Aminopyralid can be used to control broadleaf weeds that include
noxious, poisonous, and invasive plants in rangeland, pasture, and industrial vege-
tation management sites. Its use fits well into vegetation management programs,
based on its broad utility in controlling herbaceous and woody plants. Further devel-
opment activities have shown aminopyralid bring significant value to weed-control
programs in cereals, oilseed rape, and oil palm and rubber plantations.
References
1. Masters, R.A. and Sheley, R.L. (2001) J. 6. Enloe, S.F., Kyser, G.B., Dewey, S.A.,
Range Manage., 54, 502–517. Peterson, V.F., and DiTomaso, J.M.
2. Clark, J.K. and Duncan, C.L. (2005) As- (2008) Invasive Plant Sci. Manage., 1,
sessing the Economic, Environmental and 385–389.
Societal Losses from Invasive Plants on 7. Bekir, B., Gaines, T.A., Nissen, S.J.,
Rangeland and Wildlands, Weed Science Westra, P., Brunk, G., Shaner, D.L.,
Society of America, Champaign, IL. Sleugh, B.B., and Peterson, V.F. (2009)
3. Krumel, K.L., Bott, C.J., Gullo, M.F., Weed Sci., 57, 10–15.
Hull, J.W. Jr, and Scortichini, C.L. 8. Ferrell, J.A., Mullahey, J.J., Langeland,
(2001) WO 01/51684 A1. K.A., and Kline, W.N. (2006) Weed
4. Enloe, S.F., Lym, R.G., Wilson, R., Technol., 20, 453–457.
Westra, P., Nissen, S.J., Beck, G., 9. Brinkworth, L.A., Egerton, S.A., Bailey,
Moechnig, M., Peterson, V.P., Masters, A.D., and Bernhard, U. (2005) Proc.
R.A., and Halstvedt, M. (2007) Weed BCPC Int. Congr. Crop Sci. Technol., 1,
Technol., 21, 890–894. 43–48.
5. Samuel, L.W. and Lym, R.G. (2008) 10. Wilson, R.G., Orloff, S.B., Lancaster,
Invasive Plant Sci. Manage., 1, 265–278. D.L., Kirby, D.W., and Carlson, H.L.
(2010) Invasive Plant Sci. Manage., 3, Assessment-Final Report. SERA TR

81–92. 052-04-04a, Report dated June 28, 2007,
11. Almquist, T.L. and Lym, R.G. (2010) In- http://www.fs.fed.us/foresthealth/pesticide/
vasive Plant Sci. Manage., 3, 155–168. risk.shtml.
12. Fast, B.J., Ferrell, J.A., Macdonald, G.E., 15. U.S. Environmental Protection
Krutz, L.J., and Kline, W.N. (2010) Weed Agency/Office of Prevention (2005)
Sci., 58, 484–489.
Pesticides Environmental Protec-
13. Bukun, B., Shaner, D.L., Nissen, S.,
tion and Toxic Substances – Pes-
Westra, P., and Brunk, G. (2010) Weed
ticide Fact Sheet: Aminopyralid,
Sci., 58, 473–477.
14. Syracuse Environmental Research http://www.epa.gov/opprd001/factsheets/
Associates, Inc. (2007) Aminopyralid aminopyralid.pdf (accessed July 14,
Human Health and Ecological Risk 2011).
5.3
Pyrimidine Carboxylic Acids, Aminocyclopyrachlor
5.3.1
Introduction
While the existence of auxin-mimic plant regulators has been recognized [1] for
many years, the pyrimidine carboxylic acids – as represented by aminocyclopy-
rachlor (Figure 5.3.1) – represent a new subclass. In this chapter, the discovery,
and physical and biological properties, of aminocyclopyrachlor will be discussed.
5.3.2
Discovery of Aminocyclopyrachlor
The project which led to the discovery of aminocyclopyrachlor was an offshoot of

the research program at DuPont in anthranilamide insecticides (see Chapter 34.3).
That program, which had resulted in the discovery of Rynaxypyr® and Cyazypyr™
insecticides, had progressed to a point where there was a good understanding of the
structure–activity relationships (SARs) of the compounds. With Rynaxypyr® having
already been identified, the search was continued for compounds which, in addition
to providing excellent an control of lepidopteran insects, would also provide control
hemipteran species (these efforts led ultimately to the discovery of Cyazypyr™).
One of the avenues explored to accomplish this goal was the preparation of
compounds possessing a better systemic movement within the plant. It has been
well established that there is an optimum range of lipophilicity values for a
Cl OH
H2N
O
N N
Figure 5.3.1 Aminocyclopyrachlor.

CH3 R R3
Br
HN O HN O
H N H N
N N
N N N
Cl Cl
O O
Cl CH3 N R1 N R2 N
Rynaxypyr® Pyrimidine analogs
Figure 5.3.2 Rynaxypyr® and pyrimidine analogs.
compound in order to achieve movement [2]. Lipophilicity is generally measured

via the octanol/water partition coefficient (log Kow ) which, for Rynaxypyr®, is
2.86; notably, a compound with a slightly lower value should have a better plant
systemicity. Consequently, a number of approaches were explored, one being to
replace the phenyl ring of an anthranilamide with a heterocycle, whereby the
presence of additional heteroatoms would produce compounds with a higher
water solubility. One heterocycle that would satisfy the SAR requirements of
the anthranilamides would be a pyrimidine substituted with a carboxamide in
the 4-position, an acylated amine in the 5-position, and with a substituent in the
6-position, as shown in Figure 5.3.2. (According to the SAR, a substituent in the
2-position could improve the activity, but would not be required.) On this basis, a
series of molecules of that structure were prepared, as shown in Scheme 5.3.1.
O O
O O NCCO2Et
H3C O
H 3C O Zn(acac)2 O
H2N
O
NH
CH3 O CH3 O
R1 NH2
N O MeI
N O
TMG OH Li2CO3 O
R1 N R1
N
O O
R R3
CH3 HN O
1. TFA
NH2 H N
N N
N N
2. DPPA/Et3N O Cl
R1 N O
t -BuOH R1 N CH3 N
3. TFA O
1
R1 = H, Me, Et, c -Pr, t -Bu
Scheme 5.3.1 Synthesis of pyrimidine analogs of Rynaxypyr®.

NH2 O Figure 5.3.3 Synthetic intermediate with herbicidal activity.

H3C
O
N N
For this synthesis, t-butylacetoacetate was first condensed with ethyl cyanofor-
mate, and the resulting enamine condensed with a series of amidines in the pres-
ence of 1,1,3,3-tetramethylguanidine (TMG) to afford pyrimidine 4,5-dicarboxylate
mono esters [3]. Methylation of the carboxylic acid was achieved by treatment
with methyl iodide and lithium carbonate in dimethylformamide (DMF). The
t-butyl ester could be selectively cleaved with trifluoroacetic acid (TFA), while
the resulting acid then underwent a Curtius rearrangement when treated with
diphenylphosporyl azide [4]. Removal of the protecting group with TFA yielded
the key 5-aminopyrimidine-4-carboxylic acid esters (1). These compounds were
transformed into Rynaxypyr® analogs by a straightforward sequence.
Although the insecticidal activity of the pyrimidine anthranilamide analogs
proved to be modest, when the intermediates of Formula1 were screened for
herbicidal activity, the compound where R1 is cyclopropyl (Figure 5.3.3) uniquely
showed modest activity. The activity observed was more pre- than post-emergent,
with similar levels of activity on both grass and broadleaf weeds.
As a result, investigations were undertaken into the preparation of analogs of
this structure. The cyclopropyl group proved to be the most active substituent in
the 2-position and a methyl group in the 6-position proved to be optimal. However,
none of the compounds was more active than the original structure (these data are
summarized in Figure 5.3.4).
Despite the project almost being terminated at this point, the research group
continued to synthesize a 5-bromopyrimdine-4-carboxylic acid, which could be
prepared in a one-step reaction from commercially available mucobromic acid (see
Scheme 5.3.2) [5]. Interestingly, although this compound lacked a substituent in
the 6-position, it still retained a degree of herbicidal activity. Spurred on by this
H and Me Active
Me = Et>>H=Pr=i -Pr R3
HN O
R2 R4 Variety of esters active
O
N N
R1
c -Pr>SEt= OEt>>OMe>i -Pr>t -Bu =Et= Me
Figure 5.3.4 SAR of 5-aminopyrimidine 4-carboxylic acid esters.

O NH Br OH Br O
Br NH2
MeI
OH O O
O N N Li2CO3 N N
Br TMG
H
Scheme 5.3.2 Synthesis of 5-bromo-2-cyclopropylpyrimidine-4-carboxylic acid methyl ester.
NH2 O Br O
isoamyl
H3 C H3C
nitrite
O O
N N CuBr2 N N
Scheme 5.3.3 Synthesis of 5-bromo-2-cyclopropyl-6-

methylpyrimidine-4-carboxylic acid methyl ester.
observation, the next step was to prepare a compound with a methyl group in the
6-position by the nonaqueous diazotization [6] of the original hit compound in the
presence of copper (II) bromide, as shown in Scheme 5.3.3. This compound (2)
provided a boost in activity over the previous structures, importantly a change was
observed in the compound’s spectrum of herbicidal activity, to predominantly a
post-emergent control of broadleaf weeds.
Post-emergent broadleaf control is considered to be a ‘‘hallmark’’ of the synthetic
auxin herbicides. As the structure of compound 2 bore some resemblance to
that of the pyridine auxins (e.g., picloram), it was hypothesized that it would be
possible to boost the activity by moving the amino group of the hit compound from
the 5-position to the 6-position, while inserting a halogen atom in the 5-position
(Figure 5.3.5).
Cl O Br O
H2N H3C
OH O
N N N
Cl
Cl
Picloram 2
Br O
NH2 O
H3C H2N
O
O
N N
N N
Figure 5.3.5 Relationship of compound 2 to picloram.

NH
O O Br O
O NH2 O O
O O Br2 O
O O TMG HN N HOAc HN N
Br O Br O
POCl3 Cl NH3 H2N
O O
N N microwave N N
Scheme 5.3.4 Synthesis of 6-amino-5-bromo-2-

cyclopropylpyrimidine-4-carboxylic acid ethyl ester.
O Cl O
O O
O NCS O
HN N DMF HN N
Cl O Cl O
POCl3 Cl NH3 H2N
O O
N N microwave N N
Scheme 5.3.5 Synthesis of 6-amino-5-chloro-2-

cyclopropylpyrimidine-4-carboxylic acid ethyl ester.
The condensation of diethyl oxalylacetate with cyclopropanecarboxamidine gave

the pyrimidinone in low yield, while bromination followed by chlorination and
reaction with ammonia provided the target structure (Scheme 5.3.4). Again, a
boost in herbicidal activity was observed, such that a spectrum of broadleaf weeds
could be controlled post-emergence at greenhouse application rates of 31 to
125 g ha−1 .
The next step was to replace the bromine with a chlorine atom (Scheme 5.3.5),
whereupon further greenhouse testing demonstrated a substantial boost in activity,
with post-emergent broadleaf weed control observed at rates of 16–31 g ha−1 in
initial tests. The prepared compound was the methyl ester of aminocyclopyrachlor,
while the free carboxylic acid was prepared at a later stage.
5.3.3
Herbicidal Activity and the Properties of Aminocyclopyrachlor
Aminocyclopyrachlor is a member of a new class of synthetic auxin herbicides that

was discovered and registered in the United States in non-crop and turf for the
control of annual and perennial broadleaves and brush weeds. The unique feature
of aminocyclopyrachlor is its low usage rate and the broad spectrum of species
controlled. Moreover, it is especially active on most noxious and invasive weeds, and
will undoubtedly become an important new tool for the control of weeds that are
resistant to herbicides such as glyphosate, and/or which are acetolactate synthase
(ALS) inhibitor-resistant [7]. Currently, aminocyclopyrachlor is under development
in many countries for broadleaf, brush, and tree control in noncrop and range
and pasture. It is also being developed for use in forestry, orchards, oil palm, and
sugarcane, with crop registrations anticipated to start in 2012.
Aminocyclopyrachlor is currently registered in the United States for:
• Noncrop areas such as: private, public and military lands, highways, airports,
railroads, utility rights of way, farmyards, fence rows, nonirrigation ditches,
pipelines, tank farms, natural areas such as wildlife openings and habitats,
recreation areas, campgrounds, trailheads, and trails.
• Turf and residential lawns including sites such as: sod farms, industrial sites,
institutional, golf courses, parks, cemeteries, and athletic fields [8].
The physico-chemical, toxicological, and environmental properties of aminocy-

clopyrachlor are listed in Table 5.3.1.
Aminocyclopyrachlor is highly active on most broadleaf weeds and brush, and
has a low mammalian and environmental impact due to its minimal toxicity
and low to moderate effects on nontarget plants and organisms (see Tables 5.3.1
and 5.3.2). Although mainly a broadleaf herbicide, aminocyclopyrachlor does
have important activity on some weedy monocots, such as Japanese Stiltgrass
(Microstegium vimineum) and Cogongrass (Imperata cylindrica), offering control
while being selective to most turf, roadside, range, and pasture grasses.
Aminocyclopyrachlor is a synthetic auxin herbicide that blocks the growth of
plants by interfering with the hormonal balance necessary for normal shoot and
root development. The herbicide has unique features, and acts via a distinctive
mechanism that targets a family of auxin receptor complexes. Two biochemical
processes are impacted: (i) a set of proteins that is essential for gene repression
are upregulated; and (ii) the expression of genes involved in maintaining the
correct response to the hormone are no longer under tight control. The effects
of aminocyclopyrachlor on transcript response are more intense and more pro-
tracted than those of other molecules that exhibit a similar mode of action. This
unique set of binding properties, coupled to an ability to regulate genes, leads to
aminocyclopyrachlor being one of the most active – if not the most active – of
the auxin herbicides on a per gram basis, as well as providing a broader spectrum
of control when compared side by side with other commercial products with
the same mode of action. Examples of important weeds controlled include: leafy
Table 5.3.1 Properties of aminocyclopyrachlor.
Physical properties
Physical state Solid amorphous

Molecular weight 213.62
Color White
Odor Mild fruity
Melting point (◦ C) 140.5 ± 0.1
Density (g ml –1 ) 1.4732 ± 0.0777
Solubility in water at 20 ◦ C (g l –1 ) 3.13 at pH 4; 4.2 at pH 7; 3.87 at pH 9
Vapor pressure (Pa; 20◦ C) 6.92 × 10 –6
Octanol/water partition coefficient (Kow ) –1.12 (pH 4 at 20 ◦ C), −2.48 (pH 7 at 20 ◦ C)
Soil sorption coefficient (Koc ) 28 (average value)
Stability Stable (metal ions, normal and elevated
temperature)
Oxidizing/reducing activity No oxidizing/reducing properties
Flammability Nonflammable solid
Explodability The test substance was not found to be
sensitive to thermal, friction, or impact stimuli
Storage stability Stable based on accelerated storage
Corrosion characteristics Noncorrosive
Half-life in water; pH 4, 7, and 9: Stable at all three pH-values
Shallow water photolysis
DT50 0.3 days
DT90 1.1 days
Acute toxicity technical
Oral LD50 (mg kg –1 ) >5000
Dermal LD50 (mg kg –1 ) >5000
Inhalation LC50 (mg l –1 ) >5.4
Eye irritation Mild irritant
Skin irritation Nonirritant
Skin sensitization Nonsensitizer
spurge (Euphorbia esula), mesquite (Prosopis juliflora), huisache (Acacia farnesiana),

field bindweed (Convolvulus arvensis), Canada thistle (Cirsium arvense), glyphosate
resistant fleabane/marestail (Conyza spp.), kochia (Kochia scoparia), box elder (Acer
negundo), hackberry (Celtis occidentalis), and knapweeds (Centaurea spp.).
Aminocyclopyrachlor has been shown as nonvolatile in both laboratory and field
tests [9]. Moreover, with its low usage rate and relatively sharp dose response for
an auxin herbicide, the risk of any off-site movement is less than for many other
commercial products in terms of drift, soil, or water movement from the treated
area. Field trials with aminocyclopyrachlor have demonstrated sharp borders that
are indicative of a lack of off-site movement, negligible volatility, and a sharp dose
response.
In water, aminocyclopyrachlor undergoes a rapid photodegradation, with a half
life of 0.3 days. In compost studies about 40% of the aminocyclopyrachlor was
Table 5.3.2 Details of aminocyclopyrachlor ecotoxicology.
Test description Ecotoxicology results
Avian oral LD50 – bobwhitea >2075/>2250 mg a.i. kg –1

Avian dietary LD50 – bobwhitea >1177 mg a.i. kg –1
Avian dietary LD50 – mallarda >2423 mg a.i. kg –1
Avian reproduction (bobwhite) NOECa 100.9 mg a.i. kg –1
Avian reproduction (mallard) NOECa 126.7 mg a.i. kg –1
Freshwater fish toxicity – trout LC50 >122/14 mg a.i. l –1
Freshwater fish toxicity – bluegill LC50 >120 mg a.i. l –1
Acute toxicity, Daphnia EC50 43/23 mg a.i. l –1
Oyster shell deposition (estuarine) EC50 >118 mg a.i. l –1
Mysid shrimp EC50 (estuarine) >122 mg a.i. l –1
Sheepshead minnow (estuarine) LC50 >129 mg a.i. l –1
Daphnia life-cycle NOEC 6 mg a.i. l –1
Fish early life stage (trout) NOEC 11 mg a.i. l –1
a
Values expressed per kg body weight.
NOEC, no observed effect concentration.
shown to have degraded by 59 days [10]. Following ingestion by animals, aminocy-

clopyrachlor is able to pass through the digestive tract as the unchanged compound.
It is excreted at levels that may cause injury to sensitive plants (a similar situation
occurs with the pyridine carboxylic acid synthetic auxins). The most important
physical properties of aminocyclopyrachlor are summarized in Table 5.3.1.
Following its application to plants, amincyclopyrachlor is rapidly absorbed and
translocated to the growing points in both the roots and shoots, via xylem and
phloem, so as to provide control of the whole plant. In some plants, the death of
the growing point can occur so rapidly that very little uptake is observed, whereas
in other plants that are less sensitive, large amounts of amincyclopyrachlor are ab-
sorbed and translocated to the growing points above and below the treated area [11].
Aminocyclopyrachlor controls weeds and brush by both root and shoot uptake.
A dry granule application of the compound to turf and pastures has demonstrated
an excellent control of perennial weeds such as dandelion (Taraxacum officinale)
and wild violets (Viola papilionacea), that are well established with little or no foliar
contact. Commercial control has also been demonstrated by basal applications to
trees and brush when the diameter is less than 12 cm, and also by cut-stump and
stem-injection applications.
5.3.4
Mode of Action and Site of Action
The response of plants to aminocyclopyrachlor treatment was studied at the molec-

ular level by using a microarray analysis of changes to the mRNA transcripts of
Arabidopsis thaliana (Brassicaceae). The study involved growing wild-type Arabidop-

sis plants on solid media in six-well plates in a growth chamber at 23 ◦ C, subjected
to an 18 h day cycle.
After having grown the seeds for eight days, the plants were treated with various
auxinic compounds at 5 ppm concentrations. Plants treated with aminocyclopy-
rachlor, along with untreated controls and others treated with indole acetic acid
(IAA; a natural auxin), were examined. The results showed the general response of
plants to aminocyclopyrachlor to be similar to produced by other auxinics such as
2,4-D and picloram. In addition, many of the components of the auxin signaling
transduction pathway showed major changes at the mRNA transcript level.
However, a more careful analysis of the responses of the genes to individual
auxinics showed that a distinctive subset of pathway components was implicated,
depending on which treatment had been applied. Typically, aminocyclopyrachlor
was found to produce a distinct and enhanced pattern of response among mem-
bers of the auxin-induced transcription factor and the auxin-responsive family of
proteins when compared to picloram or 2,4-D treatments. This confirmed that the
unique herbicidal action of aminocyclopyrachlor is due not only to its superior
physico-chemical properties but that, following its absorption into the transport
(phloem/xylem) system, the effects of aminocyclopyrachlor were extended to a
rapid modulation of the components of the complex auxin transduction pathway.
This, in turn, would interfere with the fine control of the development of root and
shoot architecture, in contrast to the effects caused by other auxin mimics.
5.3.5
Resistance Management
The application of aminocyclopyrachlor provides a good control of many

weeds that are resistant to other modes of actions, such as the inhibition of
5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (e.g., glyphosate), ALS, and
photosystem II (PS II). Despite a number of weeds being reported as resistant to the
auxin mode of action, the cross-resistance of such weeds to aminocyclopyrachlor
has not yet been investigated. Nevertheless, when aminocyclopyrachlor is applied
in the field, care must be taken to follow the labeled directions with regards to
resistance management, in order to prevent weeds from developing resistance to
this compound.
5.3.6
Conclusions
Aminocyclopyrachlor, a new class of auxin herbicide, demonstrates unique prop-

erties with regards to the wide spectrum of species controlled, its low rate of usage,
minimal mammalian toxicity, favorable environmental properties, and wide appli-
cation control methods. Moreover, it has a favorable stewardship profile in terms
of its volatility, which includes a sharp dose response to limit off-site damage, and
a rapid photolysis in water. Although, currently, aminocyclopyrachlor is registered
for noncrop uses such as turf, industrial weed control, rights-of-way, and roadsides,
its registration is pending on pasture, rangeland in the United States. Registration
is also currently under consideration for pastures, rangelands, oil palm, sugarcane,
and tree fruits in other countries.
References
1. Haas, H.U. (2008) New aspects of plant 7. Claus, J., Turner, R., Armel, G.,
growth regulators, in Modern Crop Pro- and Holliday, M. (2008) DuPont
tection Compounds (eds W. Krämer Aminocyclopyrachlor (proposed common
and U. Schirmer), Wiley-VCH name) (DPX-MAT28/KJM44) herbicide
Verlag GmbH, Weinheim, pp. for use in turf, IWC, bare-ground, and
401–409. brush markets. Proceedings of the 5th
2. Kleier, D.A. (1994) Phloem mobility of International Weed Science Congress,
xenobiotics. V. Structural requirements 1:654, Vancouver, Canada, June 22–27,
for phloem-systemic pesticides. Pestic. 2008, p. 654.
Sci., 42 (1), 1–11. 8. E. I. du Pont de Nemours and Com-
3. Veronese, A.C., Callegari, R., Morelli, pany (2010) DuPont™ METHOD™
C.F., and Vicentini, C.B. (1997) A facile 50SG Herbicide label (SL-1605 090110
synthesis of pyrazole, isoxazole and 08-30-10).
pyrimidine ortho-dicarboxylic acid 9. Strachan, S.D., Casini, M.S., Heldreth,
K.M., Scocas, J.A., Nissen, S.J., Bukun,
derivatives via β-enaminoketoesters.
B., Lindenmayer, R.B., Shaner, D.L.,
Tetrahedron, 53 (42), 14497–14506.
Westra, P., and Brunk, G. (2010) Va-
4. Shioiri, T., Ninomiya, K., and
por movement of synthetic auxin
Yamada, S. (1972) Diphenylphospho-
herbicides: aminocyclopyrachlor,
ryl azide: new convenient reagent for
aminocyclopyrachlor-methyl ester,
a modified Curtius reaction and for
dicamba, and aminopyralid. Weed Sci.,
peptide synthesis. J. Am. Chem. Soc., 94
58 (3), 103–108.
(17), 6203–6205. 10. Michel, F.C., Muñoz-Castaneda, S.V.,
5. (a) Budesinsky, Z. (1949) Derivatives Hurak, D., Pentz, A., Nanita, S.C.,
of 2-methylpyrimidine. Collect. Czech. and Baker, R. (2010) Biodegradation
Chem. Commun., 14, 223–235; (b) of aminocyclopyrachlor and clopyralid
Kunckell, F. and Zumbusch, L. (1902) during yard trimmings composting.
On the effect of mucobromo and mu- Proceedings of the 7th International
cochloric acid on benzamidine. Ber. Conference ORBIT 2010, Heraklion,
Dtsch Chem. Ges., 35, 3164–3168. Crete, June 29–July 3, 2010.
6. Beck, J.R., Gajewski, R.P., Lynch, 11. Bell, J., Stevens, R., Prather, T.S., and
M.P., and Wright, F.L. (1987) Burke, I.C. (2009) Absorption and
Nonaqueous diazotization of translocation of aminocyclopyrachlor
5-amino-1-aryl-1H-pyrazole-4-carboxylate in rush skeletonweed, prickly lettuce,
esters. J. Heterocycl. Chem., 24 (1), and yellow starthistle. Proc. Western Soc.
267–270. Weed Sci., 61, 96.
305
6
Herbicides Disturbing the Synthesis of Very Long-Chain
Fatty Acids
6.1
Inhibitors of the Synthesis of Very Long-Chain Fatty Acids (VLCFAs)
Peter Babczinski
6.1.1
Biochemistry
6.1.1.1 Introduction
The classical herbicides from the chloroacetamide/chloroacetanilide and thiocar-
bamate groups, as well as newer herbicide classes such as oxyacetamides and
tetrazolinones, are characterized by an excellent efficacy against many major an-
nual grass weeds and certain (mostly small-seeded) dicotyledonous weeds. The
chloro-/oxyacetamides demonstrate pre- and post-emergent activities and longlast-
ing weed control, with both compounds inhibiting early plant development by
disturbing cellular and biochemical level functions in meristematic cells. As a
consequence, the induced morphological and physiological symptoms are very
similar, with those compounds taken up via the soil providing a strong effect
on meristem-bearing cell division in the root and shoot tips, but not damaging
the pre-existing tissues. Ultimately, the complete arrest of cell division results in
a cessation of growth and distortion of the elongated tissues, leading to plant
death.
6.1.1.2 HRAC Classification

According to the similarity of their induced physiological symptoms, the Herbicide
Resistance Action Committee (HRAC) has classified these herbicides into the
action groups K3 and N. The K3 herbicides (chloroacetamides, oxyacetamides) were
described – according to an elder definition – as inhibitors of cell division, whereas
group N herbicides (thiocarbamates) were earlier classified as inhibitors of lipid
synthesis [not acetyl-coenzyme A carboxylase (ACCase) inhibition].
According to more recently conducted research, common to all members of
the group K3 herbicides [chloroacetamides, oxyacetamides, tetrazolinones, sul-
fonylisoxazolines (expectedly in analogy to tetrazolinones) and others], and common
to all members of the group N herbicides (thiocarbamates and others), is their

306 6 Herbicides Disturbing the Synthesis of Very Long-Chain Fatty Acids
capability to inhibit the biosynthesis of nonsphingolipid very long-chain fatty

acids (VLCFAs; >18-carbon chain length) in plants. (Such inhibition is described
in Refs [1–4] for chloro-/oxyacetamides and functionally related structures such
as tetrazolinones and others, and in Refs [5–10] for thiocarbamates and related
compounds.) Based on the similar behavior/symptoms of these compounds, a
common biochemical mode of action was proposed as early as 1987 [11]. In the
case of thiocarbamates this occurred after conversion to their respective sulfoxides
(respectively sulfones) in the plant.
6.1.1.3 Biosynthesis of VLCFAs
6.1.1.3.1 Biological Function of VLCFAs in Plants Within the eukaryotic mem-

brane, the VLCFAs serve as essential biological components of sphingolipids [12],
and are synthesized via the action of the fatty acid-elongating enzymes of the ELO
(elongase) gene family [13, 14]. Sphingolipids also have a role in the creation of
lipid rafts, which are essential for effective eukaryotic cell function [15].
In plants, other VLCFAs serve as constituents of cuticular waxes [16] and as
seed storage triacylglycerols [17]. These VLCFAs serve as an energy storage system
in seeds, as building blocks in membranes, and as components of the protective
barrier at the plant–environment interface [18]. In addition, the VLCFAs play a role
in reproductive organ development (pollen–stigma recognition [19, 20]), and are
thought also to be involved in the promotion of the hypersensitive response (HR,
[21]), amongst others. These VLCFAs are synthesized via a plant-specific biosyn-
thetic pathway that involves VLCFA elongase (VLCFAE)-type elongating enzymes
which, in contrast to the ELO-type elongases, are inhibited by the K3 and N class
herbicides.
6.1.1.3.2 Nonsphingolipid VLCFA Biosynthesis in Plants In plants, nonsphin-

golipid VLCFAs are synthesized by the action of a membrane-bound, multienzyme
acyl-CoA elongase (VLCFAE; EC 2.3.1.119) system containing four distinct enzymes
(Figure 6.1.1) [22].
The synthesis involves the sequential addition of a C2-unit from malonyl-CoA
to a fatty acid acceptor via a four-step reaction that is analogous to de novo fatty
acid synthesis in the plastid. The first step is the condensation of an acyl-CoA
primer (fatty acids >18 carbon long) with malonyl-CoA to form 3-ketoacyl-CoA
(via 3-ketoacyl-CoA synthase; KCS or condensing enzyme), followed by reduc-
tion to 3-hydroxyacyl-CoA (via 3-ketoacyl-CoA reductase; KCR), dehydratation to
2-enoyl-CoA (via 3-hydroxyacyl-CoA dehydratase, HCD), and a second reduction
(via 2,3-trans-enoyl-CoA reductase; ECR) to form a longer chain acyl-CoA. The
substrates of acyl elongation are esterified to CoA as opposed to acyl carrier protein
(ACP), as occurs in the case of plastidic fatty acid synthase [24].
Whilst the KCS enzyme catalyzes the rate-limiting step of the cascade and
determines the substrate and tissue specificities of fatty acid elongation, the other
three enzymes are considered to be constitutive, to exhibit a broad substrate
specificity, and to be common to all tissues with VLCFA biosynthetic capacity
6.1 Inhibitors of the Synthesis of Very Long-Chain Fatty Acids (VLCFAs) 307
O Acetyl-CoA
+ HCO3− Acetyl-CoA Carboxylase
SCoA
Malonyl-CoA
3-Ketoacyl-CoA Synthase
CO2 + HSCoA (Condensing enzyme)
O O
SCoA
NAD(P)H
3-Ketoacyl-CoA Reductase
NAD(P)+
OH O
SCoA
3-Hydroxyacyl-CoA Dehydratase
H2O
SCoA
NAD(P)H
2,3-trans -Enoyl-CoA Reductase
NAD(P)+
SCoA
Figure 6.1.1 The four-step reaction sequence for elongation

of fatty acids in the endoplasmic reticulum. Modified from
Ref. [23].
[25, 26]. In this regard, ELO-type elongases share KCR-, HCD-, and ECR-type
enzymes with VLCFAE activities.
Recently, several genes have been identified as encoding 3-ketoacyl-CoA synthase
(condensing enzyme). The first such gene, FAE1, was isolated from the Arabidopsis
thaliana fae1 mutant and characterized as expressing gene activity for seed-specific
VLCFAs present in storage lipids (KCS1 gene) [27]. In total, in the Arabidopsis
genome, 21 FAE-like genes were identified (KCS1 to KCS21) [3, 18, 28, 29], eight
of which have been shown meanwhile to encode proteins with VLCFA-producing
activity [2, 18, 30, 31]. Gene KCS10 – also referred to as the FDH (fiddle head)
gene – plays a role in the target identification of both K3 and N herbicides (see
Section 6.1.1.3.3).
6.1.1.3.3 Mode of Action of VLCFA-Inhibitors The mode of action of K3 herbicides

has been reported based on the results of physiological and biochemical studies
conducted with chloroacetamide herbicides and functionally related compounds
[1]. The findings have proposed an involvement of the inhibition of VLCFA
biosynthesis through a reaction that involves covalent binding between herbicide
and a cysteine residue in the reactive site of the target enzyme. The exact target site
could not sufficiently be clarified by these investigations, however.
Recent genomics studies with genes encoding VLCFA-elongases from Arabidopsis
and heterologous expression in Saccharomyces have supported the physiological
and biochemical arguments for the molecular target of class K3 and also class
N herbicides [2, 3]; furthermore, they have identified the condensing reaction of
VLCFA elongase as the activity that is inhibited by these molecules. The common
eukaryotic ELO elongases are not inhibited by the herbicides [2].
Historically, chloro-/oxyacetamides were characterized as inhibitors of the germi-
nation and development of young tissues in plants, while the existing tissues were
unaffected (for a review, see Ref. [32]). The characteristic morphological aberrations
observed in shoot, root, and coleoptiles (see Ref. [33]) were assigned to inhibitions
of both cell enlargement and cell mitosis as a basis for their phytotoxicity [34–36].
Subsequently, numerous (partly contradictory) effects on these physiological and
biochemical mechanisms have been reported [37], with many such investigations
with chloro-/oxyacetamides being focused on fatty acid metabolism. A role for
acetyl-CoA was originally proposed as early as 1956, but ultimately lipid biosynthesis
was shown to be impaired by an inhibition of the incorporation of radiolabeled
acetyl- and malonyl-CoA precursors in lipids [38, 39].
Previously, phytotoxic chloroacetamides have been shown to reduce the amounts
of VLCFAs in both the plasma membrane [40] and in epicuticular waxes [16].
Chloroacetamides demonstrated a linear relationship between the severe inhibition
of growth and an inhibition of the incorporation of [14 C]oleic acid into VLCFAs
in Scenedesmus acutus [41]. In higher plants, the incorporation of [14 C]stearic acid
or malonyl-CoA into VLCFAs was inhibited by chloroacetamides, but the latter
had no influence on the formation of fatty acids up to C18 [42]. Acyl elongation
with 20 : 0-CoA and 18 : 0-CoA primer substrates was inhibited by the active
(S)-enantiomer of metolachlor but not by the (R)-isomer [1, 43].1) An inhibition of
VLCFA formation was also observed in metazachlor-resistant mutant (Mz-1) cells
of S. acutus [43] in the absence of the herbicide. Thus, the phytotoxic action of
chloroacetamide herbicides was assumed most likely to be caused by an inhibition
of VLCFA synthesis.
1) Currently, commercially available S-meto- synthesis, which reduces its field applica-
lachlor is produced via an enantioselective tion rate by approximately 50% [44].
On preincubation, the inhibition of 20 : 0-CoA elongation was shown to increase

in both time- and temperature-dependent fashion, which in turn indicated that
the formation of an enzyme–inhibitor complex is indeed an irreversible chemical
reaction [45]. The enzyme–inhibitor link is formed by the nucleophilic attack of
an enzyme to an electrophilic inhibitor (or inhibitor fragment). Whilst chloroac-
etamides bind covalently to cysteines in vitro [46], condensing enzymes contain one
essential, highly reactive cysteine, which binds the acyl primer substrate covalently
before the condensing reaction; subsequent mutagenesis studies demonstrated the
enzymatic similarity of the fatty acid elongase [47]. Based on a peptide mapping
analysis of the covalent binding between chloroacetamide and purified recombi-
nant chalcone synthase or stilbene synthase (two plant polyketide synthases), the
active-site cysteine residue in these condensing enzymes was concluded recently
to be the primary common target of the herbicides [48].
The results of the above investigations imply: (i) a high affinity of the condensing
elongation enzyme towards its inhibitors; (ii) an increase in the inhibition of the
elongation step with the decrease of acyl-CoA substrate concentration; and (iii) a
tight binding of inhibitors with the target enzyme [1, 45]. The inhibition is due
to a nucleophilic attack of the elongase-condensing enzyme. Whilst the inhibitors
should have an electrophilic C-atom, in the chloro- or oxyacetamides an active
methylene may be formed by the leaving chloro moiety or the oxo-heterocycle.
Tetrazolinones (e.g., fentrazamide) may bind with a target enzyme through a
nucleophilic addition to the carbamoyl moiety, thus eliminating the tetrazolinone
moiety (Figure 6.1.2).
Similar mechanisms that provide an electrophilic C-atom made available by
a leaving group might act in the case of sulfonylisoxazolines (pyroxasulfone,
fenoxasulfone), sulfonyltriazoles (cafenstrole) and carbamoyltriazolinones (ipfen-
carbazone), and others. A nucleophilic interaction of the elongase-condensing
enzyme with these inhibitors is assumed to be an inhibitor–enzyme-binding
mechanism [1].
Despite the above investigations being conducted, the exact molecular site of
action remained unknown, as one of the four VLCFA elongating activities could
not be defined as a target of the K3 and N class herbicides. Despite strong
support from cysteine binding considerations and evidences (see above), the
proposed condensing KCS reaction of the elongase activity remained to be proven
experimentally as the actual target.
Progress in this area was made, however, when a study was conducted to analyze
the phenotype and gene expression of K3 and N herbicide-treated Arabidopsis plants
[3]. By using low herbicide concentrations, the treatments were able to phenocopy
the known (transposon-induced) Arabidopsis mutant fiddlehead,2) which exhibits
fused organs and a typical fiddlehead-like inflorescence caused by epidermal cell
fusion (Figure 6.1.3) [49, 50]. The fiddlehead gene is a member of the 21-gene family
in Arabidopsis (see Section 6.1.1.3.2), and codes for a putative VLCFA elongase (gene
2) Named after the similarity of the form of its

fused inflorescences with a violin’s head.
O Cl O S Enz
H
N + S
.. Enz N + HCl
O CH3 O CH3
Chloroacetamides (Alachlor)
N N H N N
O + S Enz OH + Enz S
S ..
O S
O
Oxyacetamides (Mefenacet)
Cl O O Cl O O
H
N + S N NH + Enz S N
N N .. Enz
N N N N
Carbamoyltetrazolinones (Fentrazamide)
Figure 6.1.2 Assumed nucleophilic interaction of VLCFAE

(‘‘Enz’’) with herbicide structures. The arrows point to the
electrophilic part of the herbicide which will react with the
nucleophilic thiol-group of the cysteine residue of the con-
densing enzyme leading to the elimination of a correspond-
ing leaving group. Redrawn from Ref. [1].
KCS10, also termed FDH; [18, 50, 51]) which is absent (defective?) in the mutant.
This may serve as strong support for the original conclusions based on biochemical
studies, which considered the inhibition of (fiddlehead-like) VLCFA elongases by K3
and N group herbicides [1, 6]. The phenotypes created by the K3 and N herbicide
treatments differed in their durations of plant destructions: the N-induced fiddlehead
phenotypes were stable throughout the experiment under the conditions applied,
whereas K3 treatment under identical conditions led to transient phenotypes which
returned to normal after a few days. Besides Arabidopsis, K3 and N herbicide
treatment phenocopied fiddlehead-like organ fusions in other dicotyledonous and
also monocotyledonous plant species.
Additional support was provided by the gene-expression profiling of
herbicide-treated plant material, carried out by conducting DNA-chip hybridization
experiments [3]. When a commercially available Arabidopsis gene array containing
oligonucleotide probes of 8247 genes (approximately one-third of the total number
of A. thaliana genes) was used, the K3 and N class herbicide treatments altered the
expression of the 150–200 genes that were monitored by a factor of greater than 3.
The elimination of unspecifically stress-inducible genes (by unrelated herbicides)
resulted in only 11 genes that could be induced specifically by the K3 -class agent
flufenacet and the N-class benfuresate. Of these genes, three are known to be
connected with desiccation stress, and one gene codes for acyl-CoA reductase
(also known as male-sterility connected protein; see Pollen–stigma recognition
in Section 6.1.1.3.1), which is assumed to convert VLCFA-CoA esters into free
Figure 6.1.3 Phenotypes of control, fiddlehead mu-

tant, and flufenacet-treated Arabidopsis thaliana. (a) A.
thaliana, wild type; (b) A. thaliana, fiddlehead mutant; (c)
A. thaliana, flufenacet-treated (100 g ha−1 ). Reproduced
with permission from Ref. [52].
(a)
(b)
(c)
fatty alcohols (Section 6.1.1.3.1; precursors of wax esters [53, 54]). Following the
subtraction of genes coding for unspecific wounding responses [55], ‘‘ . . . the
amazingly small number of three genes remain as specifically affected’’ [3] by
flufenacet and benfuresate. One of these genes is directly involved in VLCFA
biosynthesis (KCR; see Section 6.1.1.3.2).3)
Further progress and proof of the mode of action was derived from cloning
experiments conducted with Arabidopsis VLCFA elongase genes [2]. Both, putative
3) This is not necessarily the herbicidal target proof of target, but rather as evidence for
itself. An increased or suppressed gene a gene being directly connected with the
expression should not be interpreted as actual target.
and known VLCFA elongases (KCS genes) from A. thaliana were cloned [with
leaf and inflorescence complementary DNA (cDNA) used as the PCR template],
heterologously expressed (using pYES2 vector and a galactose-inducible promoter).
and characterized in Saccharomyces cerevisiae. Expressing strains were used to exam-
ine the spectrum of VLCFAs [by gas chromatography/mass spectrometry (GC/MS)
analysis of their respective methyl esters] produced in the absence/presence of var-
ious K3 and N group herbicides. Of the 21 known genes in the Arabidopsis genome
encoding VLCFA elongases (VLCFAEs), six were found to be enzymatically active in
S. cerevisiae, using endogenous or externally supplied substrates.4) Elongation prod-
ucts comprising saturated and monounsaturated fatty acids of 20–30 carbon atoms
in length (and absent in the S. cerevisiae cells transformed with an empty vector5) )
were detected, depending on the respective expressed elongase. The six VLCFAEs
revealed differential inhibitory sensitivity to various K3 herbicides; in some cases,
none (napropamide) or even all six elongases tested were inhibited (flufenacet,
allidochlor).6) These findings identified the first step in VLCFA biosynthesis (KCS,
condensing enzyme) as the biochemical target of the K3 class herbicides.7,8) The
inhibition was concentration-dependent, with pI50 values of up to 7.1.9)
Surprisingly, none of the N class herbicides tested on heterologously expressed
VLCFAEs in yeast was significantly active [2], despite being shown earlier as
active inhibitors of VLCFA biosynthesis in plants [6, 7] and as inducers of the
fiddlehead phenotype [3]. This might be explained by a possible inability of the
transgenic yeast system to bioactivate the thiocarbamates to the corresponding
sulfoxides [58], or of ethofumesate and similar compounds to the corresponding
semiacetals [59].
Pyroxasulfone, a newly developed wheat and corn herbicide, was recently de-
scribed as a new K3 class compound in studies which demonstrated its inhibitory
effects on the biosynthesis of VLCFAs [4]. When using cultured rice cells, besides
a build-up of fatty acid precursors, VLCFA biosynthesis was drastically reduced
by pyroxasulfone (as analyzed by measurement of fatty acid methyl esters by GC
and GC/MS). In rice plants, specifically elongation steps from C18 : 0 to C20 : 0
4) The FDH VLCFAE exhibited no biochemi- broad range of VLCFAEs including FDH,
cal activity when expressed in yeast [2]. It is each with a specific affinity. Thus, the
presumed, that FDH needs a special fatty symptoms caused by a certain herbicide
acid substrate like a C18-ω-dicarboxylic may depend on the inhibition of several
acid (K. Tietjen, personal communication) different elongases [3].
[52]. Such a substrate does not occur in 7) If one of the three following elongase
yeast. FDH is expressed in meristem cells enzymes (KCR, HCD, ECR), which are
only, fitting the fiddlehead phenotype, constitutively expressed also in yeast and
which is meristem cell-linked. which act unspecifically both in VLCFA
5) With the exception of the C26 fatty acid, and ELO elongase processes, would have
which is a component of wild-type S. cere- been inhibited, then all inhibitors tested
visiae sphingolipids and which is not a should have been equally active (which was
product of the cloned plant genes but rather not the case).
of the unrelated endogenous ELO-gene in 8) Additional effects of fentrazamide were de-
yeast. ELO elongase was unaffected by K3 scribed recently [56, 57].
herbicides (see Section 6.1.1.3.1). 9) This is not a true enzyme pI50 value as
6) Proposedly, due to VLCFAE sequence sim- it was determined in a complex cellular
ilarity, in plants the herbicides inhibit a system.
up to C26 : 0 to C28 : 0, in addition to C18 : 1 and C20 : 1 to their corresponding

homologs, were found to be inhibited (as measured by the incorporation of
radioactive malonyl-CoA into alkyl-CoA thioesters). When using the blastp search
with the amino acid sequences of Arabidopsis VLCFAEs, 23 putative VLCFAE
candidates were identified in rice, of which 15 were considered to be VLCFAEs. By
employing microarray and real time RT-PCR analysis, two of these were suspected
to play important roles in the VLCFA biosynthesis within shoot formation and cell
proliferation, and are likely targets for pyroxasulfone. The detoxification of pyroxa-
sulfone via the action of the enzyme glutathione-S-transferase (GST) is thought to
reflect the observed corn selectivity.
6.1.1.3.4 Resistance Despite their having experienced over 50 years of

widespread and repeated use in agriculture, resistance to the K3 and N class
herbicides in the field has occurred on only extremely rare occasions. According to
Ref. [60], four weeds which were resistant to K3 class herbicides, and eight which
were resistant to N class herbicides, were listed in 2010 (the first cases occurred in
1982). In all of these cases, resistance was reported to be based on an enhanced
metabolism (covering compounds of up to seven different modes of action), and
none on an altered VLCFAE target site.
According to Ref. [1], as a probable assumption, target-site (cysteine) mutation in
VLCFAEs would abolish herbicide binding but would also be lethal for the plant;
consequently, resistant weed mutants would not occur. Yet, the same would happen
if K3 and N class herbicides were to attack additional targets of the condensing type
in other pathways. In this case, target resistance would require genetic modification
in several enzymes, but this would be an unlikely event [1].
Resistance against chloroacetamides was reported for Lolium rigidum, with
resistance ratios of about 7 in barley and wheat (in Australia) [61], and against
thiocarbamates for Echinochloa crus-galli in Chinese rice fields [62]. Furthermore,
prominent findings of resistance against N class herbicides were reported for
Avena fatua in cereals and other croplands in the USA and Canada [60]. In Europe,
until recently only one case of K3 /N class herbicide resistance had been identified
(Alopecurus myosuroides against flufenacet in wheat, multiple metabolic resistance,
Germany, 2007) [60].
The crop tolerance of K3 and N class herbicides is attributed to a rapid detoxi-
fication by GSTs [37, 46, 63]. In this regard, the molecular mechanisms of both
tolerance and phytotoxicity are assumed to be based on essentially the same nucle-
ophilic reaction (S-alkylation) of the herbicides, with a cysteine residue either as
the active-site component in VLCFA elongase (see Section 6.1.1.3.3) or as part of
the glutathione molecule. A direct proof of the role of GSTs in providing protection
against K3 herbicides is that the expression of corn GST (isoform IV) in tobacco
provides resistance against, for example, metolachlor [64]. By overexpressing the
respective corn GST gene in transgenic tobacco, a three- to fourfold metabolic
resistance compared to wild-type was achieved.
Other resistance mechanisms, such as O-demethylation described for chloroac-
etamides [65], may also be involved in field metabolic resistance.
As an alternative to Ref. [1] (see above), based on the results of VLCFAE gene
expression experiments (Section 6.1.1.3.3), a lack of altered target site resistance
against K3 and N class herbicides in the field may be explained by the obvious
sequence similarity of the plant VLCFA elongases. As noted for the Arabidopsis
cloning experiments [2], the herbicides most likely inhibit a broader range of
elongases (each with different functions in the plant), and this would also be
expected to be the case from the fiddlehead mutant investigations [3]. The different
phenotypes caused by a certain herbicide (or by K3 versus N class herbicides) may
result from an inhibition of several different elongases. Presumably, in order to
become an effective compound, it may become a prerequisite to inhibit a broad
spectrum of different elongases with essential functions in the plant. Therefore, the
inhibition of several different elongases may explain the rarity (perhaps even a lack)
of any target resistance to K3 /N class herbicides, because the genetic modification of
several enzymes would be necessary in a mutant to establish target site resistance.
Clearly, the occurrence of such an event would be extremely unlikely.
References
1. Böger, P., Matthes, B., and Schmalfuß, 13. Toke, D.A. and Martin, C.E. (1996) J.
J. (2000) Pest Manage. Sci., 56, 497–508. Biol. Chem., 271, 18413–18422.
2. Trenkamp, S., Martin, W., and Tietjen, 14. Oh, C.S., Toke, D.A., Mandala, S., and
K. (2004) Proc. Natl Acad. Sci. USA, 101, Martin, C.E. (1997) J. Biol. Chem., 272,
11903–11908. 17376–17384.
3. Lechelt-Kunze, C., Meissner, R.C., 15. Grennan, A.K. (2007) Plant Physiol., 143,
Drewes, M., and Tietjen, K. (2003) Pest 1083–1085.
Manage. Sci., 59, 847–856. 16. Ebert, E. and Ramsteiner, K. (1984)
4. Tanetani, Y., Kaku, K., Kawai, K., Weed Res., 24, 383–389.
Fujioka, T., and Shimizu, T. (2009) 17. Fehling, E., Murphy, D.J., and
Pestic. Biochem. Physiol., 95, 47–55. Mukherjee, K.D. (1990) Plant Physiol.,
5. Kolattukudy, P. and Brown, L. (1974) 94, 492–498.
Plant Physiol., 53, 903–906. 18. Joubes, J., Raffaele, S., Bourdenx, B.,
Garcia, C., Laroche-Traineau, J., Moreau,
6. Abulnaja, K.O. and Harwood, J.L. (1991)
P., Domergue, F., and Lessire, R. (2008)
Phytochemistry, 30, 1445–1447.
Plant Mol. Biol., 67, 547–566.
7. Abulnaja, K.O., Tighe, C.R., and
19. Preuss, D., Lemieux, B., Yen, G.,
Harwood, J.L. (1992) Phytochemistry,
and Davis, R.W. (1993) Genes Dev., 7,
31, 1155–1159.
974–985.
8. Barrett, P.B. and Harwood, J.L. (1993) 20. Hülskamp, M., Kopczak, S.D., Horejsi,
Proceedings of the Brighton Crop T.F., Kihl, B.K., and Pruitt, R.E. (1995)
Protection Conference – Weeds, pp. Plant J., 8, 703–714.
183–188. 21. Raffaele, S., Vailleau, F., Leger, A.,
9. Barrett, P.B., Howells, T., Kloppenstein, Joubes, J., Miersch, O., Huard, C., Blee,
W.E., and Harwood, J.L. (1994) Biochem. E., Mongrand, S., Domergue, F., and
Soc. Trans., 22, 260S. Roby, D. (2008) Plant Cell, 20, 752–767.
10. Schmutz, A., Buchala, A.J., and Ryser, 22. Cassagne, C., Lessire, R., Bessoule, J.J.,
U. (1996) Plant Physiol., 110, 403–411. Moreau, P., Creach, A., Schneider, F.,
11. Fürst, E.P. (1987) Weed Technol., 1, and Sturbois, B. (1994) Prog. Lipid Res.,
270–277. 33, 55–69.
12. Cahoon, E.B. and Lynch, D.V. (1991) 23. Böger, P. and Matthes, B. (2002) in
Plant Physiol., 95, 58–68. Herbicide Classes in Development (eds P.
References 315
Böger, K. Wakabayashi, and K. Hirai), 43. Schmalfuß, J., Matthes, B., Mayer, P.,
Springer-Verlag, Berlin, Heidelberg, pp. and Böger, P. (1998) Z. Naturforsch., 53c,
115–137. 995–1003.
24. Harwood, J.L. (ed.) (1998) Plant Lipid 44. Blaser, H.U. and Spindler, F. (1997)
Biosynthesis, Cambridge University Chimia, 51, 297–299.
Press, pp. 185–220. 45. Schmalfuß, J., Matthes, B., Knuth, K.,
25. Fehling, E. and Mukherjee, K.D. (1991) and Böger, P. (2000) Pestic. Biochem.
Biochim. Biophys. Acta, 1082, 239–246. Physiol., 67, 25–35.
26. Millar, A.A. and Kunst, L. (1997) Plant 46. Leavitt, J.R.C. and Penner, D. (1979) J.
J., 12, 121–131. Agric. Food Chem., 27, 533–536.
27. James Jr., D.W. and Dooner, H.K. 47. Ghanevati, M. and Jaworski, J.G. (2001)
Biochim. Biophys. Acta, 1530, 77–85.
(1990) Theor. Appl. Genet., 80, 241–245.
48. Eckermann, C., Matthes, B., Nimtz, M.,
28. Kunst, L. and Samuels, A.L. (2003) Prog.
Reiser, V., Lederer, B., Böger, P., and
Lipid Res., 42, 51–80.
Schröder, J. (2003) Phytochemistry, 64,
29. Costaglioli, P., Joubes, J., Garcia, C.,
1045–1054.
Stef, M., Arveiler, B., Lessire, R., and
49. Lolle, S.J., Cheung, A.Y., and Sussex,
Garbay, B. (2005) Biochim. Biophys. Acta, I.M. (1992) Dev. Biol., 152, 383–392.
1734, 247–258. 50. Yephremov, A., Wismann, E., Huijser,
30. Blacklock, B.J. and Jaworski, J.G. (2006) P., Huijser, C., Wellesen, K., and
Biochem. Biophys. Res. Commun., 346, Saedler, H. (1999) Plant Cell, 11,
583–590. 2187–2201.
31. Paul, S., Gable, K., Beaudoin, F., 51. Pruitt, R.E., Vielle-Calzada, J.-P.,
Cahoon, E., Jaworski, J., Napier, J.A., Ploense, S.E., Grossniklaus, U., and
and Dunn, T.M. (2006) J. Biol. Chem., Lolle, S.J. (2000) Proc. Natl Acad. Sci.
281, 9018–9029. USA, 97, 1311–1316.
32. Hamm, P.C. (1974) Weed Sci., 22, 52. Trenkamp, S. (2003) Pflanzliche
541–545. Fettsäure-Elongasen als Wirkort von
33. Fuerst, E.P. (1987) Weed Technol., 1, Herbiziden. Dissertation, University of
270–277. Düsseldorf, Düsseldorf, p. 49.
34. Deal, L.M. and Hess, F.D. (1980) Weed 53. Aarts, M.G., Hodge, R., Kalantidis, K.,
Sci., 28, 468–475. Florack, D., Wilson, Z.A., Mulligan, B.J.,
35. Hess, F.D., Holmsen, J.D., and Fedtke, Stiekema, W.J., Scott, R., and Pereira, A.
C. (1980) Weed Res., 30, 21–27. (1997) Plant J., 12, 615–623.
36. Fedtke, C. (1991) Pestic. Sci., 33, 54. Metz, J.G., Pollard, M.R., Anderson, L.,
421–426. Hayes, T.R., and Lassner, M.W. (2000)
37. Hock, B., Fedtke, C., and Schmidt, R.R. Plant Physiol., 122, 635–644.
55. Cheong, Y.H., Chang, H.S., Gupta, R.,
(1995) Herbizide; Entwicklung, Anwen-
Wang, X., Zhu, T., and Luan, S. (2002)
dungen, Wirkungen, Nebenwirkungen,
Georg Thieme Verlag, Stuttgart, New
56. Lim, S.J., Sunohara, Y., and Matsumoto,
York, pp. 224–234.
H. (2007) J. Pestic. Sci., 32, 249–254.
38. Jaworski, E.G. (1956) Science (Washing-
57. Ito, S., Ueno, C., and Goto, T. (2008) J.
ton), 123, 847–848. Pestic. Sci., 33, 228–233.
39. Mann, J.D. and Pu, M. (1968) Weed Sci., 58. Kern, A.J., Jackson, L.L., and Dyer, W.E.
16, 197–198. (1997) Pestic. Sci., 51, 21–26.
40. Matthes, B. and Böger, P. (2002) Z. 59. Kawahigashi, H., Hirose, S., Hayashi,
Naturforsch., 57c, 843–852. E., Ohkawa, H., and Ohkawa, Y. (2003)
41. Couderchet, M., Schmalfuß, J., and Pestic. Biochem. Physiol., 74, 139–147.
Böger, P. (1998) Pestic. Sci., 52, 60. Heap, I.M. (2010) International Sur-
381–387. vey of Herbicide-Resistant Weeds,
42. Matthes, B., Schmalfuß, J., and http://www.weedscience.org.
Böger, P. (1998) Z. Naturforsch., 53c, 61. Burnet, M.W.M., Barr, A.R., and Powles,
1004–1011. S.B. (1994) Weed Sci., 42, 153–157.
62. Huang, B.Q. and Gressel, J. (1997) (1997) in Regulation of Enzymatic Systems
Resist. Pestic. Manage., 9, 5–7. Detoxifying Xenobiotics in Plants (ed K.K.
63. Bieseler, B., Fedtke, C., Neuefeind, T., Hatzios), Kluwer Academic Publishers,
Etzel, W., Prade, L., and Reinemer, Dordrecht, pp. 313–323.
P. (1997) Pflanz. Nachr. Bayer, 50, 65. Moreland, D.E., Corbin, F.T.,
117–140. Fleischmann, T.J., and McFarland,
64. Jepson, I., Holt, D.C., Roussel, V., F.E. (1995) Pestic. Biochem. Physiol., 52,
Wright, S.Y., and Greenland, A.S.J. 98–108.
6.2
Chemistry and Biology of Oxyacetamides, Tetrazolinones, and Triazolinones
Yukiyoshi Watanabe
6.2.1
Introduction
Herbicidal substance classes that are considered to inhibit the biosynthesis of

very long-chain fatty acids (VLCFAs) include, for example, chloroacetamides [1],
thiophosphates [2], oxyacetamides [3], and carbamoyl-heterocycles [4]. Since the
Monsanto Company introduced their first chloroacetamide (alachlor) to the mar-
ket in 1969, a variety of herbicidal compounds that are thought to inhibit VLCFA
biosynthesis have been commercialized. In this chapter, the development processes
of representatives of these products are described, including two heteroaryloxyac-
etamides (mefenacet and flufenacet) and a carbamoyl tetrazolinone (fentrazamide),
marketed by Bayer CropScience AG, as well as a carbamoyl-triazolinone (ipfencar-
bazon) that currently is under development at Hokko Chemical Industry.
6.2.2
Mefenacet and Flufenacet (Oxyacetamides)
The fortunate discovery of the first herbicidal product of the oxyacetamide

class – mefenacet – began in 1976 when Bayer initiated the research of a novel
herbicide based on the oxyacetamide phosphates 1 (Figure 6.2.1). These com-
pounds, which were highly active on lowland weeds and showed good safety
towards rice, had been first disclosed by Nihon Tokushu Nouyaku Seizo in 1975
[5]. The first stage of the variations, which involved a substitution of the phosphate
moiety of 1 with various heterocycles containing sulfur, led to an initial compound
with herbicidal properties that made it eminently suitable for use in rice paddy
fields. Indeed, following its subsequent commercialization as mefenacet, the com-
pound has now been used for the past 20 years, in Japan and other countries, as a
leading paddy herbicide.
In parallel with the commercialization of mefenacet, Bayer continued to explore
the potential of this chemical class as herbicides, in particular for those that can
be used in upland fields. A series of structure–activity relationship (SAR) studies
performed on mefenacet indicated that:
6.2 Chemistry and Biology of Oxyacetamides, Tetrazolinones, and Triazolinones 317
O
O N
P N N
S O S O
O O
1 Mefenacet
N N
F N
S O
F
F O F
Flufenacet
Figure 6.2.1 Optimization from the lead compound 1.
• For the phosphate moiety of 1, a monocyclic heterocycle (thiazole, oxazole, thiadia-

zole, and oxadiazole rings) was preferable to a bicyclic heterocycle (benzothiazole
and benzoxazole rings) to provide a high herbicidal activity. In particular, a
1,3,4-thiazole ring substituted with a CF3 group provided the highest activity.
• The amide moiety of 1 was associated with both herbicidal activity and selectivity.
When plant compatibility was assessed for corn, the presence of a phenyl
group substituted with a halogen provided a high herbicidal activity and good
safety towards corn. A final selection was accomplished, based on the results of
pre-emergence studies, that a combination of an isopropyl group and a phenyl
group substituted with fluorine at the 4-postion showed very good efficacy against
grasses and very good compatibility to corn and soybeans. Thus, flufenacet [6]
was discovered (Figure 6.2.1).
Mefenacet, which has been used as a leading paddy herbicide for 20 years in Japan
and other countries, shows high activity against barnyard grass, and is also active
against other grass weeds (Cyperus difformis, Scirpus juncoides) and some broadleaf
weeds (Lindernia pyxidaria, Monochoria vaginalis). It controls barnyard grass of the
zero- to three-leaf stages at application rates of 1–1.2 kg ha−1 , and also shows a long
residual activity and high safety towards transplanted rice. The mixture partners
of mefenacet include, for example, sulfonylureas such as bensulfuron-methyl
(Zark®), pyrazosulfuron-ethyl (Act®), and imazosulfuron (Magma®).
Flufenacet, on the other hand, is used as an upland herbicide primarily to
treat corn, soybeans, and cereals. It shows high activity against grass weeds
(Echinochloa crusgalli, Digitaria sanguinalis, Setaria viridis, Panicum miliaceum,
Alopecurus myosuroides) and some broadleaf weeds (Amaranthus retroflexus,
Chenopodium album, Galium aparine, Galinsoga parviflora) at an application
rate of 125–250 g ha−1 , together with a high safety towards not only broadleaf
crops (soybeans, cotton, sunflowers) but also gramineous crops (corn, cereals)
[7]. The mixture partners of flufenacet include, for example, photosystem II
(PS II) inhibitors such as metribuzin (Axiom®), and 4-hydroxyphenylpyruvate

dioxygenase (4-HPPD) inhibitors such as isoxaflutole (Radius®).
The synthetic pathways employed for the production of mefenacet and
flufenacet are shown in Figure 6.2.2. Mefenacet is obtained by reacting 2-
chlorobenzothiazole 2 and 2-hydroxy-[N-(methyl)-N-phenyl]acetamide 3 in 45%
sodium hydroxide solution [8]. Flufenacet is obtained by reacting 2-methylsulfonyl-
5-trifluoromethyl-1,3,4-thiadiazole 4 and 2-hydroxy-[N-(4-fluorophenyl)-N-isopro-
pyl] acetamide 5 in the presence of 25% sodium hydroxide in toluene [9].
Following the disclosure of mefenacet and flufenacet, several patents regarding
this chemical class were disclosed, including an analog of mefenacet 6 from the
Korea Research Institute of Chemical Technology [10] and an analog of flufenacet
7 from BASF [11] (Figure 6.2.3). To date, however, none of these compounds has
been commercialized.
The physico-chemical properties and toxicological data relating to mefenacet and
flufenacet are listed in Tables 6.2.1 and 6.2.2, respectively.
6.2.3
Fentrazamide (Tetrazolinone)
The investigation of herbicidal carbamoyl heterocycles was pioneered by Uniroyal

Chemical during the mid-1980s, with the first practical result being obtained in
N
N N NaOH
N
+ HO S O
S Cl O O
2 3 Mefenacet
N N O
N N
F N NaOH
S S + HO F N
F S O
F O O F
F F O F
4 5 Flufenacet
Figure 6.2.2 Synthesis of mefenacet and flufenacet.
F N N
N F N
O S O
F
O F O
F F F
6 (WO99047491) [10] 7 (JP2001-192371) [11]

Korea research institute BASF
of chemical technology
Figure 6.2.3 Analogs of mefenacet and flufenacet.

Table 6.2.1 The physico-chemical properties of mefenacet and flufenacet.
Physico-chemical property Data
Mefenacet Flufenacet
CAS number 73250-68-7 142459-58-3

Code number FOE1976 BAY FOE5043
Melting point (◦ C) 134.8 76–79
Vapor pressure 6.4 × 10 –4 mPa (20 ◦ C) 9 × 10 –2 mPa (20 ◦ C)
Dissociation constant (at 20 ◦ C) No acidic and no basic in No acidic and no basic in
aqueous solution aqueous solution
Solubility in water (mg l –1 at 20 ◦ C) 4 (pH 7.0) 56 (pH 4.0)
56 (pH 7.0)
54 (pH 9.0)
Solubility in organic solvents Dichloromethane: >200 Acetone: >200
(g l –1 at 20 ◦ C) Toluene: 20–50 Dichloromethane: >200
Isopropanol: 5–10 Dimethylformamide: >200
n-Hexane: 0.1–1.0 Dimethylsulfoxide: >200
Toluene: >200
Isopropanol: 170
n-Octanol: 88
Polyethylene glycol: 74
n-Hexane: 8.7
Partition coefficient (log Pow ) in 3.23 3.20
octanol-water (20 ◦ C)
Table 6.2.2 Toxicological data of mefenacet and flufenacet.
Test system Toxicological data
Mefenacet Flufenacet
Rat oral LD50 (mg kg –1 ) >5000 (♂,♀) 1617 (♂)

589 (♀)
Rat dermal LD50 (mg kg –1 ) >5000 (♂,♀) >2000 (♂,♀)
Rat inhalation LC50 (mg m –3 ) >94 500 (♂,♀) >3740 (♂,♀)
Rabbit skin and eye irritation Nonirritant Nonirritant
Guinea pig skin sensitization Nonsensitizing Nonsensitizing (Buechler method)
Slightly sensitizing (Maximization
method)
Mutagenicity in vitro and in vivo Nonmutagenic Nonmutagenic
Teratogenicity (rat and rabbit) Nonteratogenic Nonteratogenic
O Cl
O O
O N
S N N
N N N
O N
N N
Cafenstrole Fentrazamide
Figure 6.2.4 Carbamoyl-heterocycles.
1987 when the Chugai Pharmaceutical Co. launched cafenstrole, a carbamoyl

triazole herbicide for barnyard grass control in rice [12] (Figure 6.2.4).
In 1991, Bayer CropScience focused their attention on the carbamoyl tetrazoli-
nones that had been first introduced by Uniroyal Chemical in 1986 [13], and began
to prepare derivatives of these compounds. As the results of preliminary green-
house tests had indicated a potential of these agents as grass killers in paddy fields,
the aim of the research was subsequently narrowed to their use in transplanted rice.
In SAR studies, the presence of a phenyl group rather than an alkyl or benzyl group
at the 4-position of the tetrazolinone ring was found to increase safety to rice, while
the introduction of a substituent (notably a chloro-group) at the 2-position of the
phenyl ring enhanced herbicidal activity against barnyard grass, yet maintaining a
high safety towards rice. With regards to the carbamoyl moiety, the presence of an
N-cyclohexyl-N-ethylcarbamoyl group produced the best results both in terms of
herbicidal activity and safety to rice, while the increased or decreased total carbon
number of the N-cyclohexyl-N-ethylcarbamoyl group from 8 reduced the activity
against barnyard grass or safety to rice. Eventually, these findings led to the develop-
ment of 4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-4,5-dihydro-5-oxo-1H-tetrazole-1
carboxamide, which was referred to as ‘‘fentrazamide’’ [14] (Figure 6.2.4).
Fentrazamide is highly effective in the control of annual grass weeds (Echinochloa
spp., Cyperus difformis, Scirpus juncoides) and broadleaf weeds (Lindernia pyx-
idaria, Monochoria vaginalis). It controls barnyard grass of zero- to three-leaf
stage at application rates of 200–300 g ha−1 . This compound is also character-
ized by a long-term residual activity and shows excellent safety towards both
transplanted rice and seeded paddy rice. Indeed, based on these properties fen-
trazamide became the first paddy herbicide to be used as simultaneous treatment
at the time of transplanting. The mixture partners of fentrazamide in one-shot
paddy herbicides include sulfonylureas such as bensulfuron-methyl (Innova®) or
pyrazosulfuron-ethyl (Doublestar®), and 4-HPPD inhibitors such as benzobicyclon
(Smart®). In south-east Asian countries, fentrazamide is used as a mixture with
propanil (Lecspro®) to treat direct-seeded rice.
The synthetic pathway employed in the production of fentrazamide is shown
in Figure 6.2.5. The reaction of 2-chlorophenyl isocyanate 8 and sodium azide in
the presence of a catalytic amount of aluminum trichloride in dimethylformamide
produces 1-(2-chlorophenyl)-5(4H)-tetrazolinone 9 in high yields [15]. The tetra-
zolinone 9 is then mixed with N-cyclohexyl-N-ethylcarbamoyl chloride 10 in the
Cl N
Cl
O O
Cl
Cl O 10
NaN3 N N N
AlCl3(cat.) N NH K2CO3 N N
NCO DMAP(cat.)
N N
8 9 Fentrazamide
Figure 6.2.5 Synthesis of fentrazamide.
presence of potassium carbonate and a catalytic amount of 1-dimethylaminopyridne

(DMAP) in toluene to afford fentrazamide in high yields [16].
Following the disclosure of fentrazamide, several companies entered the her-
bicidal tetrazolinone chemistry race, and as a consequence many compounds of
this class were prepared. The main agents included carbamoyl tetrazolinones with
either a heterocycle group [17], a cycloalkyl group [18], or a heterocyclemethyloxy
group [19] at the 1-position of the tetrazolinone ring, thiocarbamoyl tetrazoli-
nones [20], carbamoyl tetrazolinones with tetrahydro-2H-pyran-4-yl group in the
carbamoyl moiety [21], and hydrazine carbamoyl tetrazolinones [22] (Figure 6.2.6).
To date, none of these compounds has achieved commercial status.
The physico-chemical properties and toxicological data relating to fentrazamide
are listed in Tables 6.2.3 and 6.2.4, respectively.
Cl
Cl O O O O
N O O O
N
N N N N N N N
N
N N N N N N N
N N
EP695748(1996)[17] EP733625(1996)[18] JPH11-180965(1999)[19]

Bayer Bayer Nissan
Cl Cl
O S O O O O
N
N N N N N N N N N
N N N N N N
JPH9-100272(1997)[20] WO9835961(1998)[21] WO9851683(1998)[22]

Bayer Du Pont Du pont
Figure 6.2.6 Herbicidal tetrazolinones developed after fentrazamide.

Table 6.2.3 The physico-chemical properties of fentrazamide.
Physico-chemical property Data
CAS number 158237-07-1

Code number BAY YRC2388
Melting point (◦ C) 79
Vapor pressure 5 × 10 –5 mPa (20 ◦ C)
Dissociation constant (at 20 ◦ C) No acidic and no basic in
aqueous solution
Solubility in water (mg l –1 at 20 ◦ C) 2.3 (pH 4.0)
2.3 (pH 7.0)
2.3 (pH 9.0)
Solubility in organic solvents (g l –1 at Dichloromethane: >250
20 ◦ C) Xylene: >250
Isopropanol: 32
n-Heptane: 2.1
Partition coefficient (log Pow ) in 3.60
octanol-water (20 ◦ C)
Table 6.2.4 Toxicological data of fentrazamide.
Test system Toxicological data
Rat oral LD50 (mg kg –1 ) >5000 (♂,♀)

Rat dermal LD50 (mg kg –1 ) >5000 (♂,♀)
Rat inhalation LC50 (mg m –3 ) >5000 (♂,♀)
Rabbit skin and eye irritation Nonirritant
Guinea pig skin sensitization Nonsensitizing
Mutagenicity in vitro and in vivo Nonmutagenic
Teratogenicity (rat and rabbit) Nonteratogenic
6.2.4
Ipfencarbazon (Triazolinone)
In 1990 – a year before fentrazamide appeared on the market – a patent was

disclosed for herbicidal carbamoyl triazolinones that had been filed by Hokko
Chemical Industry [23]. These carbamoyl triazolinones have been reported to have
qualitatively and quantitatively similar behavior to the carbamoyl tetrazolinones
such as fentrazamide. Subsequent SAR studies revealed that a combination of
the triazolinone part with a halogen-substituted phenyl group at the 1-position
and nonsubstitution at the 3-position, and of the carbamoyl moiety with a lower
alkyl group and a halogen-substituted phenyl group, produced a high herbicidal
activity and good safety towards rice. As a result of extensive studies, ipfencarbazon
Cl Cl Figure 6.2.7 Ipfencarbazon.

O O
N N N
N F
(Figure 6.2.7) was selected as the most promising candidate for new paddy
herbicide.
Ipfencarbazon is highly active against barnyard grass, and also active against
other annual grass weeds (Cyperus difformis, Scirpus juncoides) and some broadleaf
weeds (Lindernia pyxidaria, Monochoria vaginalis). It controls the zero- to three-leaf
stages of barnyard grass at an application rate of 125–250 g ha−1 , and has been
confirmed to have a longlasting efficacy with satisfactory safety to rice. It can be used
as a paddy herbicide that can be applied simultaneously with rice transplanting,
similar to fentrazamide. Currently, ipfencarbazon is undergoing development as
the mixture partner of sulfonylureas or 4-HPPD inhibitors as a one-shot application
paddy herbicide.
The synthetic route for ipfencarbazon is shown in Figure 6.2.8. The reaction of
2,4-dichlorophenylhydrazine hydrochloride 11 and glyoxylic acid 12 gives hydrazone
carboxylic acid 13, which is then reacted with diphenylphosphoryl azide 14 to
OCHCOOH Cl Cl
Cl Cl
12
N N
NHNH2 H OH
O
11 13
O
Cl N
F
Cl Cl
O O
(PhO)2P(O)N3 F
Cl Cl O
14 16 N N N
N F
N NH
N
15 F
Ipfencarbazon
Figure 6.2.8 Synthesis of ipfencarbazon.

Cl Cl
O O O O
N N N N N N N
F N F
N
F F
JP2000-63379 [25] JP2000-72755 [26]

Hokko Chemical Hokko Chemical
Figure 6.2.9 Herbicidal triazolinones developed from ipfencarbazon.
afford 1-(2,4-dichlorophenyl)-1,2,4-triazol-5-one 15. Ipfencarbazon is formed, in

high yields, by a reaction of compound 15 and N-isopropyl-N-(2,4-difluorophenyl)
carbamoyl chloride 16 [24].
Following the disclosure of ipfencarbazon, Hokko Chemical Industry – the sole
company involved in carbamoyl triazolinone chemistry – filed additional patents
for 1-pyridyl triazolinone derivatives [25] and 1-alkyl triazolinone derivatives [26]
(Figure 6.2.9). At the time of writing, ipfencarbazon appears to be the only
carbamoyl triazolinone selected for commercialization.
6.2.5
Conclusions
Based on their high herbicidal activity with consequent low application doses,
excellent crop compatibility and long resistance-free periods, a variety of VLCFA
synthesis inhibitors have been developed over the past few decades to provide
weed control in corn, soybean, cereal, or rice. With the recent progress achieved
in both biochemical and genetic research into the pathways of VLCFA synthesis,
however, it is inevitable that new chemical classes, with core structures that are
completely different to the conventional VLCFA synthesis inhibitors, will emerge in
the future. The combination of these novel compounds with current products such
as mefenacet, flufenacet, and fentrazamide, supported by the development of new
products such as ipfencarbazon, will be expected to make valuable contributions to
the weed management of crop farming, and thereby to increase productivity.
Acknowledgments
This chapter is based partly on the chapter 8. ‘‘Inhibition of Cell Division (Oxy-
acetamides, Tetrazolinones)’’ with the authors Toshio Goto, Akihiko Yanagi, and
Yukiyoshi Watanabe in the first edition of Modern Crop Protection Compounds, I
thank my former colleagues for their contribution in that book.
References 325
References
1. (a) Olin, J.F. and Mo, B. (1969) US 11. Newton, T.W. and Baltruschat, H.S.
Patent US3,442,945; (b) Vogel, C. and (2001) Jpn. Kokai Tokkyo Koho JP 2001-
Aebi, R. (1974) US Patent US3,937,730; 192371.
(c) Takematsu, T., Kato, S., Ishizaki, 12. Takeuchi, M., Yasuda, M., and
M., and Suyama, T. (1989) US Patent Kanzaki, M. (1989) Eur. Pat. Appl.
US4,802,907; (d) Eicken, K., Rohr, EP332133.
W., and Zeeh, B. Wurzer, B. (1978) 13. Covey, R.A., Forbes, P.J., and Bell, A.R.
Jpn. Kokai Tokkyo Koho JP S53-53651; (1985) Eur. Pat. Appl. EP146279.
(e) Moser, H. and Vogel, C. (1983) Jpn. 14. (a) Yanagi, A., Watanabe, Y., Narabu,
Kokai Tokkyo Koho JP S58-79964; (f) S., Ito, S., and Goto, T. (2002) J. Pestic.
Seckinger, K., Kühnen, F., and Milzner, Sci., 27, 199–209; (b) Goto, T., Ito, S.,
K. (1983) Jpn. Kokai Tokkyo Koho JP Yanagi, A., Watanabe, Y., and Yasui, K.
S58-148868; (g) Kato, S., Takematsu, (2002) Weed Biol. Manage., 2, 18–24.
T., Okamoto, H., and Ogasawara, 15. Yanagi, A., Watanabe, Y., and Narabu, S.
M. (1986) Jpn. Kokai Tokkyo Koho (1995) Eur. Pat. Appl. EP638561.
JP S61-293956. 16. Yanagi, A., Watanabe, Y., and Narabu, S.
2. (a) Werner, T. (1974) US Patent (1995) Eur. Pat. Appl. EP646577.
US3,833,600; (b) Patel, N.R. (1984) 17. Goto, T., Moriya, K., Maurer, F., Ito, S.,
US Patent US4,453,965. Wada, K., Ukawa, K., Watanabe, R., Ito,
3. (a) Förster, H., Hofer, W., Mues, V., A., and Minegishi, N. (1996) Eur. Pat.
Appl. EP695748.
Eue, L., and Schmidt, R.R. (1989) US
18. Goto, T., Kitagawa, Y., Ito, S.,
Patent US4,833,243; (b) Förster, H.,
Shibuya, K., Yamaoka, T., Ueno, C.,
Andree, R., Santel, H.J., Schmidt, R.R.,
and Kyo, Y. (1996) Eur. Pat. Appl.
and Strang, H. (1990) Jpn. Kokai Tokkyo
EP733625.
Koho JP H02-45475.
19. Morimoto, K., Oonari, M., Furusawa,
4. (a) Takeuchi, M., Yasuda, M., and
H., Teraji, H., Nishio, K., Nawamaki, T.,
Kanzaki, M. (1990) Jpn. Kokai Tokkyo
Nakadaira, K., and Oki, S. (1999) Jpn.
Koho JP H02-1481; (b) Goto, T., Ito, S.,
Kokai Tokkyo Koho JP H11-180965.
Watanabe, Y., Narabu, S., and Yanagi,
20. Goto, T., Ito, S., Minegishi, N.,
A. (1994) Jpn. Kokai Tokkyo Koho JP
Narabu, S., Watanabe, Y., and Yanagi,
H06-306061. A. (1997) Jpn. Kokai Tokkyo Koho JP
5. Aya, M., Saito, J., Kume, T., and H9-100272.
Yasui, K. (1977) Jpn. Kokai Tokkyo 21. Murali Dhar, T.G. (1998) PCT Int. Appl.
Koho JP S52-51027. WO9835961.
6. Förster, H., Schmidt, R.R., Santel, H.J., 22. Baik, K.H. (1998) PCT Int. Appl.
and Andree, R. (1997) Pflanz.-Nachr. WO9851683.
Bayer, 50, 105–116. 23. Morita, K., Kido, T., Hirayama, K.,
7. Deege, R., Förster, H., Schmidt, R.R., Okita, H., Ohno, T., Watanabe, Y.,
Thielert, W., Tice, M.A., Aagesen, G.J., and Onoe, M. (1998) PCT Int. Appl.
Bloomberg, J.R., and Santel, H.J. (1995) WO98038176.
Proc. Brighton Crop Prot. Conf. – Weeds, 24. Okita, H. and Kido, T. (2004) Jpn. Kokai
43–48. Tokkyo Koho JP 2004-175756.
8. Thomas, S. (1982) Jpn. Kokai Tokkyo 25. Morita, T., Ono, T., Kido, Y.,
Koho JP S57-93969. Hirayama, K., Okita, H., Watanabe, Y.,
9. Vidyanatha, P.A., Jacqueline, A.M., and Onoe, M. (2000) Jpn. Kokai Tokkyo
Daniel, W.M., and Klaus, J. (1999) Jpn. Koho JP 2000-063379.
Kokai Tokkyo Koho JP H11-246539. 26. Morita, T., Ono, T., Kido, Y.,
10. Kim, B.T., Park, N.K, Hong, K.S., Park, Hirayama, K., Okita, H., Watanabe, Y.,
J.E., and Kwon, Y.W. (1999) PCT Int. and Onoe, M. (2000) Jpn. Kokai Tokkyo
Appl. WO95047491. Koho JP 2000-072755.
6.3
Isoxazolines
and Tsutomu Shimizu
6.3.1
Introduction
The chemical compounds of the new class of isoxazoline herbicides were first
disclosed in 2002 by Kumiai Chemical Industry and Ihara Chemical Industry
[1]. These demonstrate excellent efficacy against a wide range of grass and
broadleaf weeds with both pre- and post-emergence activities, in addition to
longlasting weed control [2]. Following their intensive chemical optimization,
two representatives of the class – pyroxasulfone and fenoxasulfone – are currently
undergoing development (Figure 6.3.1). The intention is that pyroxasulfone
{3-[5-(difluoromethoxy)-1-methyl-3-(trifluoromethyl) pyrazol-4-ylmethylsulfonyl]-4,
5-dihydro-5,5-dimethyl-1,2-oxazole} will be introduced as a selective herbicide
for cereal in the Australian market and corn, soybean and wheat in the
United States market in 2012, while fenoxasulfone {3-[(2,5-dichoro-4-ethoxy
benzyl)sulfonyl]-4,5-dihydro-5,5-dimethyl-1,2-oxazole} is currently undergoing
development as a selective rice herbicide in Japan. Both of these isoxazoline
herbicides are potent inhibitors of the biosynthesis of very long-chain fatty acids
(VLCFAs), and have been classified as the group K3 herbicides by the Herbicide
Resistance Action Committee (HRAC) [3].
6.3.2
Chemistry of Pyroxasulfone
Thiocarbamate herbicides such as thiobencarb 1 (n = 0) are known as ‘‘fatty acid

and lipid biosynthesis inhibitors’’, and classified as group N herbicides by the HRAC
[4]. It has been suggested that oxidative metabolites of these herbicides, such as
thiocarbamate sulfoxides 1 (n = 1), might be the active form [5]. To stabilize the
active compounds 1 (n = 1), replacement of the carbamate moiety of the sulfoxide
to the heterocyclic ring 2 was designed to achieve a higher herbicidal activity as
compared to the classical thiocarbamates. In such heterorings, the isoxazoline ring
F F
F Cl
N
O
N
S S
O N O O O N
O O O Cl
F
F
Pyroxasulfone Fenoxasulfone
Figure 6.3.1 Chemical structures of pyroxasulfone and fenoxasulfone.

was noted because several bioactive compounds with such a ring, including 3 [6]
and 4 [7], have been reported in many patents. Moreover, the isoxazoline ring
could be synthesized easily by 1,3-dipolar cycloaddition with nitrile oxides and
olefins. Consequently, the 3-benzylsulfonylisoxazoline derivatives 5 were designed
and synthesized (Figure 6.3.2).
The structure–activity relationship (SAR) of the substituent on the isoxazoline
ring indicated that 5,5-dimethylisoxazoline was the most suitable herbicide for
pre-emergence applications in upland fields. Substitution at the 2-position of the
benzyl group was considered favorable to enhance the herbicidal activity, and the
2-difluoromethoxy compound showed good herbicidal efficacy but poor selectivity
against corn. On examining the physico-chemical properties of the isoxazolines
for selective pre-emergence herbicides, the pyrazol-4-yl groups were shown to
have better properties, notably the Kd value. These pyrazole compounds also
showed particularly good herbicidal activity and high safety towards corn and soy-
bean. In particular, the 5-difluoromethoxy-1-methyl-3-trifluoromethylpyrazol-4-yl
compound (pyroxasulfone 6) [1] showed excellent performance in field trials.
X
N Cl
S Het S
O (O)n (O)n
1 2
O
Me
R3 N
O
O O R4
N R
Xn Q
4
3
R7 R8
R6 (Het)Ar
R5 S
O N
(O)n
F F
F
N
N
S
O N
O O O
F
F
Pyroxasulfone 6
Figure 6.3.2 The strategy of drug design.

F F F F
F F
N N
NH NaOH
N N
S S + O S
O NH2 O N
F F O O
F HBr salt F
7 8 9
F F F F
F F
N N
mCPBA
N N
S S
O N O N
O O O O
F F
F F
10 Pyroxasulfone (6)
Figure 6.3.3 Synthesis of pyroxasulfone.
The synthetic route for pyroxasulfone is shown in Figure 6.3.3. Initially,

the isothiourea derivative 7 is hydrolyzed and then reacted with 5,5-dimethyl-
3-ethanesulfonylisoxazoline 9 to obtain the sulfide compound 10. Pyroxasulfone 6
is then obtained by oxidizing the sulfide 10 with m-chloroperbenzoic acid [8].
Following the disclosure of pyroxasulfone and related patents from the Kumiai
group, several patents relating to the isoxazolines have been disclosed from
Syngenta (11, 12, 14, 15, 16) [9–13], Bayer CropScience (13) [14], Chinese Academy
of Agricultural Sciences (17) [15], and DuPont (18) [16] (Figure 6.3.4). In addition,
many other patents have been disclosed, including those for compounds in which
other heterocyclic rings have been replaced by using the same strategy from Otsuka
Chemical (18) [17], Bayer CropScience (19, 21–23) [18–22], and Syngenta (20)
(Figure 6.3.5).
6.3.3
Biological Activities of Pyroxasulfone
Pyroxasulfone has high potential as both a selective pre- and post-emergence her-
bicide for use on corn, soybean, cereals, and cotton, among several other crops
[23–25]. It is used at a low application rate when compared to other currently
registered soil-applied herbicides; typical application rates are 125–250 g a.i. ha−1
for corn and soybean, and 50–100 g a.i. ha−1 for cereals. Pyroxasulfone is active over
a wide range of weed species, including annual and perennial grasses such as Dig-
itaria sanguinali, Echinochloa crus-galli, and Setaria spp., as well as many broadleaf
weeds such as Amaranthus spp., Chenopodium album, Datura stramonium, Kochia
scoparia, Sida spinosa, and Tribulus terrestris. It also has activity on difficult-to-control
F F F F
F F F
F F F
N N
N N
S S S
O O N O
N O O O N O O N O O F
F H F
F F
10 (WO2006024820) 11 (WO2006037945) 12 (WO2007003294)
Syngenta Syngenta Bayer cropscience
F F F
F F
Br Br F F N
N N N
N N
S S N S N
O O O N
N O O O N O O O O
F
F F
13 (WO2007071900) 14 (WO2007096576) 15 (WO2008074991)
Syngenta Syngenta Syngenta
F F
Cl F
N
S N N S N
O O N
N O O O O O
F
F
F
16 (CN101362753)
17 (WO2009158258)
Chinese academy of
DuPont
agricultural sciences
Figure 6.3.4 Analogs of pyroxasulfone.
F F
F
Cl N
S N Cl Br S
N
S S S
F
N O O N O O N O O O F
F
18 (JP2003096059) 19 (WO2003037878) 20 (WO2006123088)
Otsuka Chemical Bayer CropScience Bayer CropScience
F
F F F
N
S O O
N
S S S
N O F N O O F N O O O
F
F
21 (WO2009068170) 22 (WO2009129953) 23 (WO2009129954)
Bayer CropScience Bayer CropScience Bayer CropScience
Figure 6.3.5 Replacement of other heterocyclic rings.

weeds, such as Sorghum bicolor, Panicum texanum, Panicum miliaceum, Brachiaria

platyphylla, and Lolium spp., as compared to other pre-emergence grass herbicides
[23,26–28]. Pyroxasulfone has provided effective controls of trifluralin-resistant
Lolium rigidum in Australia [29], and of glyphosate-resistant Amaranthus rudis in
the United States. In addition, it has a favorable residual activity profile which,
together with its excellent crop tolerance, allows its application to be extended from
the very early pre-plant stage through post-emergence [30]. The intention is that
pyroxasulfone alone will be available commercially as a corn, soybean and wheat
herbicide under the trade name Zidua® in the United States, and as a cereal
herbicide under the trade name Sakura® in Australia from 2012. In combination
with flumioxazin, it will also be commercialized as a corn and soybean herbicide
under the trade name Fierce® in the United States from 2012.
6.3.4
Biological Activities of Fenoxasulfone
Fenoxasulfone is used as a pre-emergence or early post-emergence herbicide on

transplanted rice to control many annual grass weeds, as well as some broadleaf
and sedge weeds. When applied at 200 g a.i. ha−1 , fenoxasulfone provides good
selectivity for rice, and an outstanding efficacy on major weeds in Japanese
paddy fields, such as Echinochloa spp., Monochoria vaginalis, Monochoria korsakowii,
Lindernia spp., Cyperus difformis, and Cyperus serotinus. In particular, it provides
an effective control of Echinochloa oryzicola, from pre-emergence to the three-leaf
stage, with a long residual activity [31]. Fenoxasulfone has unique physico-chemical
properties (notably an extremely low water solubility) when compared to other rice
herbicides that are used under flooded conditions. Consequently, a stable efficacy in
the long term can be maintained, even under overflow conditions caused by heavy
rain [31]. Although the registration of this herbicide is scheduled for 2014, certain
combinations – for example, fenoxasulfone and pyrimisulfan – are currently being
developed as one shot-herbicides for use on rice in Japan.
6.3.5
Mode of Action of Pyroxasulfone
Pyroxasulfone potently inhibits the growth of shoots of Barnyard millet and Italian
ryegrass although, in addition to these weedy species, it has also been shown
that rice is sensitive to this herbicide. The pyroxasulfone-sensitive plants displayed
symptoms similar to those exhibited in the presence of the group K3 herbicides,
such as metolachlor. Unlike rice shoots, however, cultured rice cells showed only a
slight sensitivity to pyroxasulfone [3]. The plasma membrane of plant cells contains
VLCFAs in the form of sphingolipids [32]; hence, although the inhibition of VLCFA
biosynthesis would lead to a reduction in sphingolipid levels, this would most likely
not prove lethal to cultured plant cells. Consequently, cultured rice cells might
represent a useful model for examining the effects of pyroxasulfone on the de novo
biosynthesis of VLCFAs, without the need for radioactive precursors.
700
626
pyroxasulfone 10−7 M
pyroxasulfone10−6 M
Relative fatty acid content (%)
200
100
0
C14:0 C15:0 C16:0 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C22:0 C22:1 C24:0
Figure 6.3.6 Inhibitory effects of pyroxa- the basis of the fatty acid content of cultured
sulfone on the biosynthesis of VLCFAs in rice cells grown in the absence of pyroxa-
cultured rice cells. The relative fatty acid sulfone. Each data set is expressed as the
content (% of control) in cultured rice cells mean ± SE of three or four independent ex-
treated with pyroxasulfone was calculated on periments.
For this purpose, an investigation was made of changes in the fatty acid content
of cultured rice cells treated with pyroxasulfone. A marked decrease in the level
of VLCFAs was observed, whereas the levels of long-chain fatty acids, which
serve as precursors of VLCFAs, were increased (Figure 6.3.6). Furthermore, the
enzyme activity of the very long-chain fatty acid elongase (VLCFAE) in etiolated
rice seedlings and etiolated Italian ryegrass seedlings was potently inhibited
by pyroxasulfone [3]. Taken together, these data clearly suggest not only that
pyroxasulfone is a potent inhibitor of VLCFA biosynthesis, but also that the
potency of the VLCFAE inhibition differed between pyroxasulfone-tolerant plants
(wheat and corn) and pyroxasulfone-susceptible plants (rice, Italian ryegrass, and
Barnyard millet) (Figure 6.3.7).
In Arabidopsis, it has been shown that multiple VLCFAEs catalyze multiple
elongation steps in the biosynthesis of VLCFAs [33]. By analogy, therefore, it might
be anticipated that many types of VLCFAE are involved in the same pathway in
rice. A BLASTP search using the amino acid sequences of some VLCFAEs, such
as FAE1 from Arabidopsis, led to the identification of 23 VLCFAE candidates in rice
[3], of which 14 were considered to be VLCFAEs (Figure 6.3.8). Oligo microarray
and real-time RT-PCR analysis revealed that, among these candidates, the Q5Z6S3
(Uniplot ID number) gene was strongly expressed in rice shoots, whereas the
Q6F365 (Uniplot ID number) gene was strongly expressed in rice roots. Whereas,
the rice shoots contained elevated levels of C28:0 and C30:0 VLCFAs, the cultured
rice cells contained lower levels of these VLCFAs; consequently, Q5Z6S3 might
play an important role in the biosynthesis of C28:0 and C30:0 VLCFAs during
shoot formation in rice. These rice VLCFAEs, as well as Arabidopsis VLCFAEs, are
assumed to be targets for pyroxasulfone.
C24:0→C26:0 elongation
Inhibition of VLCFAEs (%)

100
Rice
80 Italian ryegrass
Corn
60 Wheat
40
20
0
10−8 10−7 10−6 10−5
Pyroxasulfone concentration (M)
Figure 6.3.7 Comparison of the inhibition from C24:0 to C26:0 of etiolated seedlings
of VLCFAE activity by pyroxasulfone among of rice, wheat, and corn was, respectively,
rice, wheat, and corn. Each data set is ex- 11.2, 3.5, and 4.4 pmol per 30 min per 20 μl
pressed as the mean ± SD of four indepen- of enzyme suspension.
dent experiments. The elongation activity
At4g34520 (FAE1)
Q688V9
Q10QW1
Q10P61
Q6Z6C7
Q69X62
Q6F365
At5g43760
Q2R1V7
Q7XEM4
Q8H7Z0
Q5Z6S3
Q5Z7C8
Q5Z7E7
Q6Z2K7
Q10PV5
Figure 6.3.8 Phylogenetic tree for Arabidopsis VLCFAEs and

putative rice VLCFAEs based on amino acid sequences. The
putative rice VLCFAEs are described by Uniplot ID, and the
two Arabidopsis VLCFAEs are described by gene name.
To date, eight of 21 putative Arabidopsis VLCFAEs have been characterized

[33–39]. For this, transgenic yeast cells were prepared that had been transformed
with the Arabidopsis FAE1 gene, which is one of the eight characterized VLCFAEs
[38]. In a microsome preparation from the transformed yeast cells, pyroxasul-
fone potently inhibited the VLCFAE activity of the recombinant FAE1 enzyme,
which catalyzes the elongation step from C18:1 to C20:1 (Figure 6.3.9a). Moreover,
Inhibition of VLCFAEs (%) 100
80
60
40
Pre-incubation 2 min
20
Pre-incubation 10 min
0
10−8 10−7 10−6 10−5
(a) Pyroxasulfone concentration (M)
100
80
60
40
20
0
0 5 10 15 20 25 30
(b) Pre-incubation period (min)
Figure 6.3.9 Inhibitory effects of pyrox- of VLCFAE activity of FAE1, which catalyzes
asulfone on the VLCFAE activity of re- the elongation step from C18:1 to C20:1. The
combinant Arabidopsis FAE1. Each data VLCFAE activity of recombinant FAE1 was
set was expressed as the mean ± SD of 2.7 nmol mg protein−1 per 90 min; (b) Inhi-
three or four independent experiments. (a) bition of FAE1 by 10−6 M pyroxasulfone after
Pyroxasulfone-mediated potency of inhibition different pre-incubation periods.
pyroxasulfone inhibited the VLCFAE activity of FAE1 in a time-dependent man-

ner, indicating that the inhibition of FAE1 by pyroxasulfone might be irreversible
(Figure 6.3.9b). The pseudo-first-order rate constant (ki ) for the irreversible inhibi-
tion of FAE1 by pyroxasulfone was calculated as 3.2 × 105 M−1 min−1 . On the basis
of these results, the inhibition of FAE1 by pyroxasulfone was assumed to be due
to the nucleophilic reaction of the SH group of the cysteine residue in the active
center of VLCFAEs with pyroxasulfone, which is consistent with former hypotheses
[40–43].
An attempt was also made to construct an assay system for recombinant
rice VLCFAEs using yeast cells. However, the fact that the rice VLCFAE genes
introduced were only slightly expressed, or were not expressed at all in the
transformed yeast cells, suggested that it would be difficult to construct such an
assay system for rice VLCFAEs in yeast cells. Hence, a further attempt was made
to construct the assay in cultured rice cells. To test this method the Q6F365 gene
was chosen; this is rarely expressed in cultured rice cells, but is strongly expressed
in rice roots, according to the results of gene expression profiling using microarray
[3]. The transformed cultured rice cells were cultivated, after which the expression
of the introduced genes, the VLCFA content and the VLCFAE activity in these
cultured rice cells, were each evaluated [44]. Pyroxasulfone was shown to inhibit

100
80
60
40
C18:0→C20:0
20
C20:0→C22:0
0
10−7 10−6 10−5
(a) Pyroxasulfone concentration (M)
100
80
60
40
20
0
0 5 10 15 20 25 30
(b) Pre-incubation period (min)
Figure 6.3.10 Inhibitory effects of pyroxa- pyroxasulfone. The VLCFAE activity of recom-
sulfone on the VLCFAE activity of recombinant rice Q6F365, which catalyzes the elon-
binant Q6F365 from rice. Each data set gation steps from C18:0 to C20:0 and from
is expressed as the mean ± SD of three C20:0 to C22:0, was 93.2 and 14.1 pmol mg
or four independent experiments. (a) Po- protein−1 per 30 min, respectively; (b) In-
tency of inhibition of the VLCFAE activ- hibition of recombinant Q6F365 by 10−5 M
ity of Q6F365 under the conditions of pyroxasulfone after different pre-incubation
10 min pre-incubation of microsomes with periods.
the VLCFAE activity of the recombinant Q6F365 enzyme, which catalyzes the
elongation steps from C18:0 to C20:0 and from C20:0 to C22:0 (Figure 6.3.10a). It
also inhibited such VLCFAE activity in time-independent manner (Figure 6.3.10b).
The manner of inhibition of Q6F365 differed from that of FAE1, which suggested
that the inhibition mechanism of Q6F365 by pyroxasulfone would, most likely, be
reversible. Considering that the inhibition of microsomal VLCFAEs of other plants
by pyroxasulfone is time-independent [44], and similar to that of recombinant
Q6F365, most plant VLCFAEs would be presumed to be inhibited by pyroxasulfone
in time-independent manner. Taken together, these results confirmed the
identification of a new mechanism of inhibition of VLCFAEs by pyroxasulfone.
6.3.6
Mode of Action of Fenoxasulfone
Fenoxasulfone treatment was found to decrease VLCFAs such as C20:0, C20:1,

C22:0, C24:0, C24:1, and C26:0 in Barnyard millet cultured cells, and to increase
References 335
long-chain fatty acids and medium-chain fatty acids such as C18:0 and C15:0, which
are precursors of VLCFAs. In particular, the accumulation of C15:0 was remarkable
[45]. These results revealed that fenoxasulfone inhibits the biosynthesis of plant
VLCFAs, which are incorporated into the plasma membrane of the cultured cells.
The VLCFAE activity of recombinant FAE1, which catalyzes the elongation step
from C18:1 to C20:1, was potently inhibited by fenoxasulfone in time-dependent
manner. Thus, similar to pyroxasulfone, the inhibition mechanism of FAE1 by
fenoxasulfone was assumed to be irreversible. Fenoxasulfone, however, potently
inhibited the microsomal VLCFAE activity of Barnyard millet, which catalyzes the
elongation step from C24:0 to C26:0, in time-independent manner, as was shown
for the inhibition of VLCFAEs by pyroxasulfone [45].
In general, VLCFAE-inhibiting herbicides contain a highly electrophilic carbon
atom in their chemical structure. Accordingly, the mechanism of VLCFAE inhibi-
tion has been assumed to occur via a nucleophilic reaction of the SH group of the
cysteine residue in the active center of the VLCFAE with the herbicides. Although
such a mechanism of inhibition was seen to be time-dependent, both fenoxasulfone
and pyroxasulfone inhibited the VLCFAE activity of plants in time-independent
manner, which inferred that the inhibition mechanism would most likely be
reversible. Consequently, it was suggested that fenoxasulfone and pyroxasulfone
might not react with the SH group of the cysteine residue in the active center of
the VLCFAE. It should be possible, however, to confirm the mechanism by which
fenoxasulfone and pyroxasulfone inhibit the VLCFAE by monitoring the kinetics
of enzyme–herbicide binding.
Abbreviations
VLCFA Very long-chain fatty acid

VLCFAE Very long-chain fatty acid elongase
HRAC Herbicide Resistance Action Committee
References
1. Nakatani, M., Kugo, R., Miyazaki, M., Kobayashi, M. (2008) 4th Pan Pacific
Kaku, K., Fujinami, M., Ueno, R., and Conference on Pesticide Science.
Takahashi, S. (2002) PCT Int. Appl. WO 3. Tanetani, Y., Kaku, K., Kawai, K.,
2002062770. Fujioka, T., and Shimizu, T. (2009)
2. (a) Porpiglia, P.J., Nakatani, M., Ueno, Pestic. Biochem. Physiol., 95, 47–55.
4. http://www.hracglobal.com/Portals/5/
R., and Yamaji, Y. (2005) Weed Sci-
moaposter.pdf.
ence Society of America 45th Meeting;
5. Ashton, F.M. and Crafts, A.S. (1981)
(b) Nakatani, M., Ito, M., Kaku, K.,
Mode of Action of Herbicides, 2nd edn,
Miyazaki, M., Ueno, R., Kawasaki, H., John Wiley & Sons, Inc., New York.
and Takahashi, S. (2006) 11th IUPAC 6. (a) Debuitsudo, M. and Bipin, P.
International Congress of Pesticide (1993) Jpn. Kokai Tokkyo Koho JP
Chemistry; (c) Ito, M., Nakatani1, H05-178844; (b) Morita, T., Ono, T.,
M., Yamaji, Y., Honda, H., and Masumizu, T., Kido, Y., Yoshizawa, H.,
and Watanabe, Y. (1997) Jpn. Kokai Dittgen, J., Martelletti, A., and
Tokkyo Koho JP H09–143171. Dietrich, H. (2009) PCT Int. Appl.
7. (a) Von Deyn, W., Hill, R.L., Kardorff, WO2009129953.
U., Engel, S., Otten, M., Vossen, M., 22. Kehne, H., Dietrich, H., Feucht, D.,
Plath, P., Rang, H., Harreus, A., Koenig, Schmutzler, D., Haeuser-Hahn, I.,
H., Walter, H., Westphalen, K., and Paulitz, C., Dittgen, J., Martelletti, A.,
Misslitz, U. (1996) PCT Int. Appl. and Rosinger, C. (2009) PCT Int. Appl.
WO96026206; (b) Von Deyn, W., HIll, WO2009129954.
R.L., Kardorff, U., Baumann, E., Engel, 23. Yamaji, Y., Ueno, R., Takahashi, S.,
S., Mayer, G., Witschel, M., Rack, M., Honda, H., and Nakatani, M. (2005) J.
Goetz, N., Gebhardt, J., Misslitz, U., Weed Sci. Technol., 50 (Suppl.), 54–55.
Walter, H., Westphalen, K., Otten, M.,
24. Kobayashi, M., Honda, H., Yamaji, Y.,
and Rheinheimer, J. (1998) PCT Int.
and Hanai, R. (2007) J. Weed Sci. Tech-
Appl. WO98031681.
nol., 52 (Suppl.), 68–69 (in Japanese).
8. Nakatani, M., Ito, M., and Miyazaki, M.
25. Hillger, D.E., Gibson, K.D., and Weller,
(2004) PCT Int. Appl. WO2004013106.
S.C. (2005) North Cent. Weed Sci. Proc.,
9. Plant, A., Boehmer, J.E., Black, J., and
Sparks, T.D. (2006) PCT Int. Appl. 60, 57.
WO2006024820. 26. Steele, G.L., Porpiglia, P.J., and
10. Plant, A., Boehmer, J.E., and Peace, A.L. Chandler, J.M. (2005) Weed Technol.,
(2006) PCT Int. Appl. WO2006037945. 19, 866–869.
11. Black, J., Boehmer, J.E., Stal, E.J.T., 27. Geier, P.W., Stahlman, P.W., and
Kiewicz, A.M., and Plant, A. (2007) PCT Frihauf, J.C. (2006) Weed Technol.,
Int. Appl. WO2007071900. 20, 622–626.
12. Boehmer, J.E. and Mclachlan, M.M.W. 28. King, S.R. and Garcia, J.O. (2008) Weed
(2007) PCT Int. Appl. WO2007096576. Technol., 22, 420–424.
13. Boehmer, J.E. and Mclachlan, M.M.W. 29. Walsh, M.J., Fowler, T.M., Crowe, B.,
(2008) PCT Int. Appl. WO2008074991. Clarke, M., Ambe, T., and Powles, S.B.
14. Helmke, H., Schaper, W., Auler, T., (2011) Weed Technol., 25, 30–37.
Kehne, H., Hills, M., and Feucht, D., 30. Kurtz, M.E., Yamaji, Y., and DeLoach,
(2007) PCT Int. Appl. WO2007003294. Y.T. (2009) Weed Sci. Soc. Am. Proc.,
15. (a) Ning, J., Chen, Z., Zhao, Q., Mei, 457.
X., Song, Q., Ma, H., Yang, G., Zhang, 31. Takahashi, Y., Fujinami, M., Hanai, R.,
T., and Li, Y. (2008) Chinese Pat. Appl. Ito, M., and Nakatani, M. (2010) The
CN101362753; (b) Ma, H., Li, Y., Zhao, Abstracts of Papers, 35th Annual Meet-
Q., Zhang, T., Xie, R., Mei, X., and ing of the Pesticide Science Society of
Ning, J. (2010) J. Agric. Food. Chem., 58, Japan, p. 88 (in Japanese).
4356–4360.
32. Matthes, B. and Böger, P. (2002) Z.
16. Smith, B.T., Selby, T.P., Stevenson,
Naturforsch., 57c, 843–852.
T.M., Clark, D.A., and Taggi, A.E. (2009)
33. Joubes, J., Raffaele, S., Bourdenx, B.,
PCT Int. Appl. WO2009158258.
Garcia, C., Laroche-Traineau, J., Moreau,
17. Manabe, H., Ishii, N., and Akasaka, T.
P., Domergue, F., and Lessire, R. (2008)
(2003) Jpn. Kokai Tokkyo Koho JP
2003-96059. Plant Mol. Biol., 67, 547–566.
18. Gesing, E.R., Drewes, M.W., Dahmen, 34. Millar, A.A., Clemens, S., Zachgo, S.,
P., Feucht, D., and Pontzen, R. (2003) Giblin, E.M., and Taylor, D.C. (1999)
PCT Int. Appl. WO2003037878. Plant Cell, 11, 825–838.
19. Elliott, A.C., Hughes, P., and Plant, A. 35. Ghanevati, M. and Jaworski, J.G. (2001)
(2006) PCT Int. Appl. WO2006123088. Biochim. Biophys. Acta, 15, 77–85.
20. Dietrich, H., Martelletti, A., Rosinger, 36. Ghanevati, M. and Jaworski, J.G. (2002)
C., Dittgen, J., and Feucht, D. (2009) Eur. J. Biochem., 269, 3531–3539.
PCT Int. Appl. WO2009068170. 37. Lechelt-Kunze, C., Meissner, R.C.,
21. Rosinger, C., Feucht, D., Drews, M., and Tietjen, K. (2003) Pest
Schmutzler, D., Haeuser-Hahn, I., Manage. Sci., 59, 847–856.
References 337
38. Trenkamp, S., Martin, W., and Tietjen, Wakabayashi, and K. Hirai), Springer
K. (2004) Proc. Natl Acad. Sci. USA, 101, Publishing, Berlin, Heidelberg,
11903–11908. pp. 115–137.
39. Blacklock, B.J. and Jaworski, J.G. (2006) 43. Eckermann, C., Matthes, B., Nimtz, M.,
Biochem. Biophys. Res. Commun., 346, Reiser, V., Lederer, B., Böger, P., and
583–590. Schroder, J. (2003) Phytochemistry, 64,
40. Böger, P. (2003) J. Pestic. Sci., 28, 1045–1054.
324–329.
44. Tanetani, Y., Fujioka, T., Kaku, K., and
41. Böger, P., Matthes, B., and Schmalfu β,
Shimizu, T. (2011) J. Pestic. Sci., 36,
J. (2000) Pest. Manage. Sci., 56,
221–228.
497–508.
42. Böger, P. and Matthes, B. (2002) in 45. Tanetani, Y., Fujioka, T., Horita, J.,
Herbicide Classes in Development- Kaku, K., and Shimizu, T. J. Pestic. Sci.,
Mode of Action, Targets, Genetic Engi- 36 (in press). d.o.i. 10.1584/jpestics.
neering, Chemistry (eds P. Böger, K. G10-97.
339
7
Inhibitors of Cellulose Biosynthesis
7.1
Introduction
Plant cell walls represent the most abundant source of terrestrial biomass on Earth.
Each year, an estimated 200 billion tons of CO2 is converted to plant biomass, with
the bulk of the carbon being incorporated into plant cell walls. The plant cell wall
is a highly organized composite of many different polysaccharides, proteins, and
aromatic substances. It lends the cell stability, determines its shape and size, and
also plays a central role in the mechanical protection of the plant and its defense
against pathogens.
Cellulose is the major component of cell walls in plants and the most abundant
polysaccharide, accounting for 15–30% of the dry mass of all primary cell walls
and an even larger percentage of secondary walls. Cellulose biosynthesis in higher
plants is essential to cell growth and division as well as tissue formation and
differentiation. Any inhibitor targeting cellulose biosynthesis would severely impair
plant growth and development and would, therefore, be of considerable interest as
a herbicide.
7.1.1
Cellulose Biosynthesis
As cellulose biosynthesis has been reviewed frequently during recent years [1–11],
only a brief overview of the biochemistry and molecular genetics of the process will
be included at this point. Nonetheless, this should provide a sufficient understand-
ing of the mechanism(s) of action of the various cellulose biosynthesis inhibitors
(CBIs) outlined in the following sections.
In higher plants, following cell division, a cellulose-containing primary cell wall
develops between the two daughter cells, generally as a thin and flexible layer that
is extended as the cell continues to grow. In some cell types, when the cell has
become fully grown, a secondary wall is constructed by the successive encrustation
and deposition of cellulose and other components, between the plant cell and the
primary wall. However, whereas the primary wall structure is the same in almost

340 7 Inhibitors of Cellulose Biosynthesis
all cell types and species, cell-type and species-related differences are typical of the
secondary cell wall.
In both primary and secondary cell walls, the major polysaccharide present is
cellulose, a simple polymer of unbranched β-1,4-linked glucan chains. The suc-
cessive glucose residues are inverted 180◦ , thereby forming a flat ribbon in which
the repeating unit is cellobiose. The individual cellulose chains are organized into
crystalline microfibrils, which (on average) are assemblies of 36 β-(1 → 4)-d-glucan
chains, hydrogen-bonded to one another along their length. The number of 36
glucan chains per microfibril is compatible with the size of microfibrils isolated
from primary cell walls (3.5–4 nm) [12]. However, other data are more consistent
with the packing of 18 chains or even less per microfibril [13, 14]. Thus, although
each β-(1 → 4)-d-glucan chain may be several thousand units long, the individual
chains begin and end at different places within the microfibril. This allows the
microfibrils to reach lengths of hundreds of micrometers and to contain thousands
of individual glucan chains, all of which are arranged parallel to each other so
that their reducing ends all point in the same direction. The cellulose microfibrils,
which are interlocked together by crosslinking glycans (hemicelluloses), contain
significant amounts of aromatic substances and structural proteins.
Cellulose is synthesized in the plasma membrane by enzymatic supramolecular
structures known as rosettes. Electron microscopy studies have revealed that the
rosettes are hexagonal structures of 25–30 nm diameter, each composed of six
subunits organized with a sixfold symmetry. In the preferred model, the subunit
contains six catalytic cellulose synthase subunits (CesA proteins), each producing
a single β-(1 → 4)-d-glucan chain. Thus, although it is assumed that each rosette
synthesizes a microfibril that contains 36 glucan chains, the actual number of
catalyzing subunits per rosette remains the subject of controversy and has never
been determined experimentally.
The catalytic cellulose synthase subunits are encoded by CesA genes. All higher
plants investigated to date are known to contain families of CesA genes. For
example, the Arabidopsis genome contains 10 different CesA genes [15], which have
been the most extensively studied to date, whereas 12 and 18 CesA genes have
been identified in Zea mays and Populus trichocarpa, respectively [16, 17]. Electron
microscopy and immunocytochemistry studies have shown cellulose synthase to
be localized to the plasma membrane, where it appears to exist as an integral
membrane protein. Based on the amino acid sequences deduced from the CesA
genes, a hypothetical three-dimensional (3-D) structure has been proposed [15].
In this model, the enzyme has eight putative transmembrane helices that form a
pore in the plasma membrane through which the growing glucan chain passes to
reach the newly forming cell wall. The catalytic site faces the cytoplasmic side of
the plasma membrane. The amino terminus, which also resides in the cytoplasm,
contains a conserved Zn-binding domain that might play a role in the association
of the catalytic subunits.
The results of genetic studies have revealed that, in Arabidopsis thaliana, three
genes (AtCesA1, 3, and 6) are required for cellulose biosynthesis in primary walls
[18], while three different CesA genes (AtCesA4, 7, and 8) are required for cellulose
biosynthesis in the secondary walls [19]. However, it remains controversial as to

exactly how many different CesA gene products are required for the assembly of
a rosette, and whether the different CesA isoforms are functionally redundant. In
one model [20], it was proposed that each rosette subunit was composed of three
CesA heterodimers, and that a dimeric structure of cellulose synthase would help to
overcome the reorientation problem of cellulose biosynthesis. If cellulose chains are
elongated by the addition of single sugar molecules, the β-1,4-linkage in cellulose
requires that each glucose residue must be flipped almost 180◦ with respect to its
neighbors. Hence, to construct the chain would require either the growing glucan
chain or the cellulose synthase to rotate by 180◦ . The two sugar-binding sites present
in a dimer would eliminate the need for reorientation, but each rosette subunit
would then produce only three β-(1 → 4)-d-glucan chains, yielding microfibrils
that are composed of 18 glucan chains – in conflict with the proposed number
of 36 glucan chains per microfibril. Recent estimates of microfibril size, however,
have suggested that 18 glucan chains could produce a 3.5 nm-diameter fibril, which
was the size initially estimated for a 36-chain microfibril [13, 14]. Alternatively, two
catalytic sites per CesA protein would also eliminate the problem of reorientation,
although experimental data relating to the 3-D structure of glycosyltransferases
clearly argue for a single active site [21, 22]. Finally, the addition of previously
formed disaccharides to the growing chain would also be possible without a need
for rotation. Unfortunately, there is at present no experimental evidence available
to support either of these latter models; it also remains largely unknown as to how
many – and how many identical and different CesA polypeptide chains – would
be required to form a rosette subunit, and if there were any accessory proteins
associated with them. The recent purification to homogeneity of the cellulose
synthase complex involved in secondary cell wall biosynthesis in Arabidopsis has
revealed the presence of no proteins other than CesA4, 7, and 8 [23].
Much of the present knowledge of cellulose synthesis has been derived from
the identification of mutants with cellulose deficiencies in Arabidopsis thaliana. The
molecular genetic characterization of these mutants has shown that the respective
genetic lesions often affect not only the CesA genes, but also on occasion the other
enzymes involved in carbohydrate metabolism. Details of the respective mutations
and the affected genes are summarized in Table 7.1.
Regardless of the precise number of chains that form a microfibril, the pack-
ing of β-(1 → 4)-d-glucan chains into a crystalline structure most likely occurs
spontaneously, and is not assisted by proteins [10].
Each β-(1 → 4)-d-glucan chain is constructed from the activated sugar donor
UDP-glucose, which in turn is either synthesized by the cytosolic enzyme
UDP-glucose pyrophosphorylase from UTP and glucose-1-phosphate, or by
sucrose synthase from sucrose and UDP. In the past, several isoforms of sucrose
synthase have been reported, with a membrane-associated form (which has been
localized at sites where the rate of cellulose synthesis is high) being proposed to
channel UDP-glucose directly to cellulose synthases [42]. The direct association
of sucrose synthase with cellulose synthases has never been demonstrated
experimentally, however.
Table 7.1 Arabidopsis thaliana mutants deficient in cellulose biosynthesis.
Mutanta Phenotype Gene mutated/amino Reference

acid exchangeda
cev1 Constitutive expression of stress CesA3/G617E [24]

response genes, reduced cellulose
content in roots
cyt1 Lethal, dramatic decrease in GTP:mannose-1-P [25]
cellulose content, callose guanylyltransferase
accumulation, incomplete cell
walls; characteristic aspects of the
phenotype can be mimicked with
the inhibitor of N-glycosylation,
tunicamycin
eli1-1 Reduced cellulose synthesis, CesA3/S301F [26]
reduced root length
eli1-2 Reduced cellulose synthesis, CesA3/A522V [26]
reduced root length
fra5 Reduction of fiber cell-wall CesA7/P557T [27]
thickness and cellulose content
fra6 Reduction of fiber cell-wall CesA8/R362K [27]
thickness and cellulose content
irx1 Reduced cellulose in secondary cell CesA8/D683N [28]
walls, collapsed xylem walls
irx3 Reduced cellulose in secondary cell CesA7 [29]
walls, collapsed xylem walls; loss of
function allele due to premature
stop codon
irx5 Reduced cellulose in secondary cell CesA4 [19]
walls, collapsed xylem walls;
loss-of-function allele due to
premature stop codon
ixr1-1 Isoxaben (4) resistance, CesA3/G998D [30]
crossresistant to thiazolidinone
herbicides, sensitive to dichlobenil
(1) and triazofenamide (10)
ixr1-2 Isoxaben (4) resistance, CesA3/T942I [30]
crossresistant to thiazolidinone
herbicides, sensitive to dichlobenil
(1) and triazofenamide (10)
ixr2 Isoxaben (4) resistance CesA6 (allelic to prc1) / [31]
R1064W
knf Loss of function allele due to α-Glucosidase I [32]
premature stop codon
kor Reduced cellulose in primary walls, Membrane-bound [33]
hypocotyl elongation defect, endo-1,4-β-D-glucanase
dwarfism
Mutanta Phenotype Gene mutated/amino Reference

acid exchangeda
lew2-1 Drought and salt stress tolerance, CesA8/W217stop [34]

accumulation of proline and
abscisic acid
lew2-2 Drought and salt stress tolerance, CesA8/L792F [34]
accumulation of proline and
abscisic acid
prc1 Cellulose deficiency, decreased cell CesA6 [35]
elongation, gapped cell walls; loss
of function alleles due to premature
stop codons
reb1 Reduced root elongation, bulging UDP-D-glucose [36]
of root epidermal cells; the mutant 4-epimerase
phenotype can be suppressed by
d-galactose
rsw1-1 Reduction in cellulose synthesis, CesA1/A549V [37]
accumulation of a lipid-linked
noncrystalline β-1,4-glucan,
disassembly of rosettes, defective
microfibril alignment
rsw1-2 Radially swollen embryos with CesA1/G631S [32]
greatly reduced levels of cellulose
rsw1-20 Dramatic distortion of seedling CesA1/D780N [38]
morphology, dwarfism
rsw1-45 Dramatic distortion of seedling CesA1/E779K [38]
morphology, dwarfism
rsw2 Reduced cellulose production, Membrane-bound [39]
accumulation of a noncrystalline endo-1,4-β-d-glucanase
β-1,4-glucan (allelic to kor)
rsw5 Reduced cellulose production CesA3 (allelic to [40]
eli1)/P1056S
rsw10 Root and hypocotyl swelling in Ribose-5-phosphate [41]
seedlings, no phenotype in aerial isomerase
parts of mature plants; the mutant
phenotype can be suppressed by
uridine
a
Abbreviations: cev = constitutive expression of VSP1; cyt = cytokinesis defective; eli = ectopic lignin;
fra = fragile fiber; irx = irregular xylem; ixr = isoxaben-resistant; knf = knopf; kor = korrigan;
lew = leaf wilting; prc = procuste; reb = root epidermal bulger; rsw = radially swollen.
Irrespective of whether UDP-glucose is used directly by the cellulose syn-

thase catalytic subunits to elongate cellulose, or whether the formation of
disaccharides is interconnected with elongation, cellulose biosynthesis must be
initiated, and this may require a primer. Peng et al. [43] have recently shown that
sitosterol-β-glucoside can serve as a primer in vitro, and that sterol cellodextrins are
produced by crude membranes from cotton fibers in the presence of UDP-glucose.

This concept was supported by the observation that UDP-glucose:sterol glucosyl-
transferase, an enzyme which is responsible for the synthesis of sterol-β-glucosides,
has been found associated with the plasma membrane [44]. Furthermore, mem-
brane preparations of yeast cells expressing the cotton CesA1 gene (the Arabidopsis
ortholog is CesA8) were shown to catalyze the same reaction, providing evidence
that the CesA protein carried out this reaction. Such a synthetic mechanism might
explain the requirement for multiple CesA isoforms: some may be required for
sterol cellodextrin primer formation, catalyzing sitosterol-β-glucoside elongation,
while others could function in the elongation of the primer [2]. Since sterol attach-
ment occurs at the reducing end of the glucose moiety, the nonreducing end will be
free for the further addition of glucose residues, in accordance with the majority of
reported data [12, 22, 45], and consistent with the mode of elongation of chitin [46].
At a certain point, the elongation of the growing cellulose chains and microfibrils
must be terminated, although how this is achieved is presently unknown. It has
been suggested that the endo-glucanase KORRIGAN (KOR) might be responsible
for chain termination. However, alternative functions for KOR have also been
proposed, such as cleaving glucan residues from a sitosterol-linked primer that is
then incorporated into the growing cellulose chain [43] or the recycling of the sterol
glucoside primers [47].
The cellulose microfibrils must be deposited in a very specific orientation in
order for them to provide the strength required in cell walls. In growing cells,
the microfibrils are usually deposited in parallel arrays, transverse to the direction
of growth. A large body of evidence has indicated that cortical microtubules are
in some way involved in the ordered deposition of cellulose microfibrils [5] and,
indeed, microscopic evidence obtained for different plant species has indicated
that the orientation of cellulose microfibrils in both primary and secondary walls
is often parallel to the underlying cortical microtubule arrays. It has also been
shown that the rosettes in the plasma membrane move at constant rates in linear
tracks that coincide with the track of cortical microtubules [48], and suggested
that the microtubules guide the cellulose synthase complexes in the plasma
membrane. As movement of the rosettes has been observed beyond the ends
of microtubules, however, it would appear that they are not responsible for the
motile force. Instead, the energy for rosette movement through the plane of the
plasma membrane is believed to be provided by cellulose chain elongation [49].
Nonetheless, microtubules are still required for the correct distribution of rosettes
in the secondary cell wall of developing root xylem cells [50].
7.2
Cellulose Biosynthesis Inhibitors from Different Chemical Substance Classes
Cellulose biosynthesis serves as the target of several different chemically unrelated

herbicides, including dichlobenil (1), isoxaben (4), flupoxam (9), indaziflam (18),
and CGA 325615 (19). All of these herbicides have been demonstrated to act as
7.2 Cellulose Biosynthesis Inhibitors from Different Chemical Substance Classes 345
CBIs, principally by measuring their inhibitory effects on 14 C-glucose incorporation

into crystalline cellulose. Their specific target(s) in cellulose biosynthesis, however,
is (are) almost always unknown, which is not surprising bearing in mind the
present somewhat limited understanding of cellulose biosynthesis and deposition
[11]. A summary of the available data for the respective herbicides is provided below
(see Section 7.2.8), together with data relating to herbicidal inhibitors of cellulose
biosynthesis that have not yet achieved commercial status.
7.2.1
Nitriles
7.2.1.1 Chemistry of Benzonitriles

The herbicidal properties of 2,6-dichlorobenzonitrile, commonly named dichlobenil
(1), were first discovered in the mid-1950s by Philips-Duphar in the Netherlands
[51, 52]. The chemical variability of nitrile-type CBIs appears to be quite limited, as
shown in Figure 7.1.
As a substituent at positions X and Y, chlorine is a particularly effective halo-
gen, while methyl and ethyl are interesting as alkyl groups. Occupation of the
second ortho-position by any of these substituents, as mentioned for Y, is benefi-
cial to the activity of the compounds of the general formula I (Figure 7.1). This
also applies to compounds in which this ortho-position is occupied by a chlorine
substituent, a methyl or an ethyl group. For example, the dichlobenil (1) deriva-
tives 2-chloro-6-methyl-benzonitrile and 2,4,6-trichloro-benzonitrile have also been
described as potent inhibitors of germination [52].
In 1961, the British company Shell Research Ltd filed a patent on herbicidal
thiobenzamides [53], specifically claiming chlorthiamid (2) (Figure 7.2). This
compound, which has been commercialized under the trade name Prefix™, can be
regarded as pro-herbicide, as it is converted into the active agent dichlobenil (1) in
N N
C C X, Y = Hal, NO2, (C1-C5)Alkyl
Cl Cl X
n=0-4
Yn
1 I
Dichlobenil General formula
Figure 7.1 Structure of dichlobenil (1) and general formula I of nitrile-type CBIs.
S NH2
Cl Cl
2
Chlorthiamid Figure 7.2 Structure of chlorthiamid (2).
CH3 CHCl2 CH=O

Cl2 H2O
Cl Cl Cl Cl
Toluene
H2NOH
N
S NH2
C CH=NOH
H2S Heat
Cl Cl Cl Cl Cl Cl
2 1
Scheme 7.1 Synthesis of dichlobenil (1) and chlorthiamid (2).
soil [54]. Dichlobenil (1) can readily be synthesized in four reaction steps, starting
from toluene [55], as shown in Scheme 7.1; the subsequent treatment of 1 with
hydrogen sulfide produces chlorthiamid (2).
7.2.1.2 Biological Activity of Benzonitriles

Dichlobenil is a pre-emergence herbicide for the control of annual and many
perennial weeds in woody ornamentals, fruit orchards, vineyards, forest plantations,
and public green areas at application rates of between 2700 and 5400 g a.i.
ha−1 , while for total weed control in noncrop areas, rates of up to 8100 g a.i.
ha−1 are applied. The selectivity of dichlobenil can be ascribed to the fact that
the active ingredient is relatively volatile and escapes rapidly from the soil; this
effectively decreases its concentration in the upper soil layer and limits its downward
movement into the soil. This combination results in a limited uptake by more deeply
rooted crop plants. The inhibition of cellulose biosynthesis by dichlobenil, as
inferred from the inhibition of 14 C-glucose incorporation into crystalline cellulose,
was first described more than 35 years ago [56], and later confirmed in a variety
of other plants. Early observations of the effects of dichlobenil in higher plants
described dwarfed plant phenotypes [57], the inhibition of cell wall formation
and cytokinesis [58], and a disruption of the ability of cell plates to flatten out
and become rigid [59]. Treatment with dichlobenil has also been shown to cause
changes in the number of intact rosettes at the plasma membranes of wheat [60],
and to cause cessation of the CesA-yellow fluorescent protein (CesA::YFP) fusion
protein mobility at the plasma membrane of A. thaliana [61]. A common theme
between these different observations seems to be that dichlobenil inhibits cellulose
synthesis by disrupting the ordered deposition of crystalline cellulose rather than
inhibiting polymerization of the β-(1 → 4)-d-glucan chains per se [62].
In contrast to dichlobenil, the structurally related nitrile herbicides bromoxynil
® ®
(Buctril ) and ioxynil (Totril ) do not exhibit CBI activity, but are inhibitors of
electron transport at photosystem II (PS II).
Cl + N Figure 7.3 2,6-Dichloro-phenylazide (3), a photoreactive

N analog of dichlobenil (1).
N
Cl
3
2,6-dichloro-phenylazide
In 1987, Delmer et al. [63] showed that 2,6-dichloro-phenylazide (3) (Figure 7.3),
a photoreactive analog of 1, specifically labeled an 18 kDa polypeptide in treated
cotton fibers and a 12 kDa polypeptide in treated suspension-cultured tomato cells.
However, because it was not associated with the membrane fraction, and was too
small to be cellulose synthase, this polypeptide was suggested to function as a
regulatory protein for β-glucan synthesis in plants. Unfortunately, as no follow-up
studies were conducted the potential role of these proteins in cellulose biosynthesis
remained unclear.
Interestingly, in 2008 Rajangam et al. [62] identified the hybrid aspen (Populus
tremula × tremuloides) PttMAP20 gene to be highly expressed during secondary cell
wall formation in xylem cells. The expression of PttMAP20 was tightly coregulated
with expression of the PttCesA1, PttCesA3, and PttCesA9 genes. PttMAP20 encodes
a soluble, microtubule-associated 20.8 kDa polypeptide that reacted specifically
with the photoreactive dichlobenil-analog 3, similar to the 18 kDa protein in cotton
fibers. Dichlobenil (1) was shown to have a competing effect on labeling by 3,
but did not influence binding of the PttMAP20 protein onto the microtubules.
The overexpression of PttMAP20 in A. thaliana resulted in a helical growth of
rosette leaves and stunted root growth – phenotypes which are frequently observed
in plants with altered microtubule dynamics. The results of these investigations
suggested, for the first time, the existence of a direct link between a specific
microtubule-associated protein and cellulose biosynthesis. However, as binding of
1 fails to prevent the binding of PttMAP20 to microtubules, another important – but
unknown – interaction of PttMAP20 during cellulose biosynthesis was proposed
to be affected.
A completely different mechanism of action of dichlobenil (1) can be inferred
from the studies of Peng et al. [43], who showed that dichlobenil inhibits the
synthesis of sitosterol-β-glucoside in vivo in developing cotton fibers. The addition
of sitosterol-β-glucoside to the cotton culture medium reversed the effect of 1 on
cellulose synthesis. In vitro, both sitosterol-β-glucoside and sitosterol-cellodextrin
synthesis was inhibited, but inhibition was effective only if the fibers were pretreated
with 1; no effect occurred if 1 was added directly to the isolated membranes. As a
link between sitosterol-β-glucoside synthesis and microtubule-associated proteins
is not evident, the mechanism of action of dichlobenil seems still to be far from
being understood, and additional biochemical investigations are urgently required
in this respect. Unfortunately, molecular genetics studies have been of minimal
assistance, as the only dichlobenil-resistant mutant reported to date [64] has not
been characterized on the molecular level.
7.2.2
Benzamides
7.2.2.1 Chemistry of Benzamides

In 1984, novel 2,6-dimethoxy-benzamide chemistry, developed by Eli Lilly &
Company, reached the herbicide marketplace. Isoxaben (4) (Figure 7.4), which was
discovered in 1979, is used for the pre-emergence weed control of germinating
broadleaved weeds in, for example, cereals, turf, fruits, and tree cultivars.
The 2,6-dimethoxybenzamides can be prepared as shown in Scheme 7.2 [65],
where the treatment of methyl 2-ethylbutanoate (5) with methyl iodide produces
methyl 2-ethyl-2-methylbutanoate (6). The further reaction of 6 with acetonitrile
CH3
O O
N H
H3C N
H3C O
O
H3C CH3
4
Isoxaben Figure 7.4 Chemical structure of isoxaben (4).
O O O
H3C H3C N
CH3 1. CH3I CH3 2. CH3CN
H3 C O H3C O H3C
Base
H 3C H3C H3C
5 6 7
H2NOH·HCl
N O
H3C NH2
H3C
H3C
8
Toluene
CH3 Heat Cl O
O O
N H O O
H 3C N H3C CH3
H 3C O
O
H3 C 2,6-dimethoxy-
CH3
benzoyl chloride
4
Scheme 7.2 Synthesis of isoxaben (4).

H3C
O
H O N
N Het R Het = N N
S
O R R
O
H3C R'
R= X
II X XH
X
General formula X XH
R'
X X = O or S
X R' = Me, Et
Figure 7.5 Herbicidal active 2,6-dimethoxybenzamides of the general formula II.
in the presence of a strong base leads to 4-ethyl-4-methyl-3-oxohexane nitrile (7),

which can be cyclized with hydroxylamine to form the 5-amino-isoxazole (8). A
final acylation of the latter compound with 2,6-dimethoxy-benzoyl chloride results
in isoxaben (4).
The syntheses and herbicidal activities of various heteroalkyl-substituted ben-
zamides of the general formula II have been described by Brinkmeyer et al. [66]
(Figure 7.5). As a result of structure–activity relationship (SAR) investigations, the
following conclusions were reached: (i) the 2,6-dimethoxy-benzoyl moiety leads
to the greatest activity; (ii) the amide linkage is important for activity; (iii) two of
the most active heterocycles (Het) are isoxazole and 1,3,4-thiadiazole; and (iv) the
substituted alkyl group (R) is necessary for activity, and some flexibility is allowed
in this region of the molecule.
7.2.2.2 Biology of Isoxaben

Isoxaben is used primarily for the pre-emergence control of annual broadleaf weeds
in winter cereals, fruit, vines, turf, and ornamentals. Its herbicidal properties were
first reported in 1982 [67], and it was introduced into the market in 1984. It is
phytotoxic towards dicotyledonous plants, whereas most monocotyledonous species
are tolerant. Isoxaben has been shown to specifically inhibit the incorporation of
14
C-glucose into crystalline cellulose [68, 69], with IC50 -values in the nanomolar
range, thus exhibiting a potency approximately 40-fold greater than dichlobenil.
Isoxaben has also been reported to inhibit cell plate completion in root tips [70].
Nicotiana tabacum BY-2 cells, when treated with isoxaben, produced incomplete
thin cell plates that were reduced in both callose and cellulose content [71]. Based on
these observations, it was originally proposed that isoxaben acted on a second site
in cellulose biosynthesis, upstream of dichlobenil, possibly inhibiting the synthesis
of UDP-glucose from sucrose [57]. However, based on the results of the molecular
genetics studies summarized below, the inhibition of UDP-glucose synthesis by
isoxaben seems rather unlikely.
Two isoxaben-resistance loci (originally termed ixrA and ixrB, but since renamed
ixr1 and ixr2) were described in Arabidopsis as early as 1989 [72] and 1990 [69].
Homozygous ixr1–1 and ixr1–2 mutant plants are 300- and 90-fold, respectively,
more resistant to isoxaben than are wild-type plants [72]. The ixr1 mutations appear
to have a direct effect on the herbicide target, because the resistant cell lines
show no alterations in uptake or detoxification of the herbicide [73]. Yet, since the
molecular genetics of Arabidopsis has become an indispensable tool for the study
of practically all aspects of plant biology, these mutants have attracted considerable
attention from research groups investigating cellulose biosynthesis.
Isolation of the ixr1 gene by map-based cloning [30], revealed that it encodes
the AtCesA3 isoform of cellulose synthase. The two known ixr1 mutant alleles
contain point mutations: glycine (G) 998 is replaced with aspartic acid (D), and
threonine (T) 942 is replaced with isoleucine (I), respectively (Table 7.1). Inter-
estingly, the ixr1-mutants are crossresistant to the structurally dissimilar thiazo-
lidinone carbamate herbicide 5-tert-butylcarbamoyloxy-3-(3-trifluoromethyl)phenyl-
1,3-thiazolidin-4-one (21) (Figure 7.11) [30], but sensitive to dichlobenil (1) and tri-
azofenamide (10) [64]. Currently, it is unclear why these mutations render the
enzyme resistant, because they occur quite distant from the proposed active site,
but close to the center of a predicted transmembrane helix (ixr1–1) and in a short ex-
tracellular loop that connects two proposed transmembrane helices (ixr1–2) [12, 74].
The second isoxaben-resistance locus, ixr2, has also been cloned and shown to
encode another cellulose synthase isoform, AtCesA6 [31]. The ixr2-1 allele contains
a point mutation that replaces arginine (R) 1064 with tryptophan (W) (Table 7.1)
at the end of the last predicted membrane-spanning domain. The CesA6 gene was
previously identified, by using knockout mutations, to cause a cellulose-deficient
short hypocotyl phenotype (prc1). All six prc1 alleles sequenced contained premature
stop codons and therefore were complete loss-of-function alleles [35].
At first sight, the existence of two isoxaben-resistance loci, ixr1 and ixr2, would
suggest redundant roles for the corresponding proteins. However, the strong
phenotype observed for dark-grown hypocotyls of prc1 suggests that CesA3 either
is absent in this organ, or is present but unable to compensate for the absence
of CesA6. Since available evidence suggests that CesA3 is present in dark-grown
hypocotyls, the functions of CesA3 and CesA6 apparently are not redundant.
Therefore, Desprez et al. [31] proposed that CesA3 and CesA6 are active as a
complex, and that isoxaben might bind at the interface between the two proteins.
Furthermore, based on a detailed comparison of the phenotypes of light- and
dark-grown wild-type, ixr2, and prc1 seedlings and their response to isoxaben
treatment, other CesA-isoforms – most likely CesA2 and CesA5 – were proposed
to be additional targets for isoxaben [31].
Confocal microscopy of expanding Arabidopsis hypocotyl cells expressing a
CesA6::YFP fusion protein in a CesA6-null mutation background (prc1-1) allowed
an observation of the movement of CesA complexes in the plasma membrane,
and their functional association with cortical microtubules [48]. On the addition of
isoxaben, most of the plasma membrane CesA6::YFP was lost within 20 min – a
situation which was in striking contrast to the dichlobenil-induced cessation of the
CesA mobility at the plasma membrane, as observed by DeBolt et al. [61]. Both,
the different responses of CesA complexes in the plasma membrane to isoxaben
and dichlobenil treatment, and the dichlobenil-sensitivity of the isoxaben-resistant
ixr1-mutants, strongly suggest that the two herbicides affect different targets
within cellulose biosynthesis. However, it remains unclear precisely how, and
where, isoxaben interferes with the synthesis of cellulose.
7.2.3
Triazolocarboxamides
7.2.3.1 Chemistry of Triazolocarboxamides

The triazolocarboxamide flupoxam (9) (Figure 7.6) was discovered and patented
by the Kureha Corporation during the mid-1980s [75], although the compound’s
herbicidal properties were not reported until 1991 [76]. Triazofenamide (10) is a
second member of this chemical class.
A laboratory synthesis of flupoxam (9) is shown in Scheme 7.3 [55, 75]. Following
benzylether formation between 2-chloro-5-nitro-benzyl chloride (11) and pentaflu-
orinated n-propanol (Step 1), the aromatic nitro group was reduced (Step 2) and the
resultant aniline 12 diazotated to form the diazonium salt, 13. An addition reaction
with 2-phenyl-oxazolidin-5-one (14) then produces the heterocyclic hydrazone 15.
Finally, ring transformation of 15 in presence of ammonium hydroxide leads to
9. The closely related compound triazofenamide (10) can be prepared in a similar
manner, starting from 3-methylaniline.
Herbicidal triazolocarboxamides, comprising specific halo-substituted alkenyl
and carbocyclic moieties in the alkoxymethylen side chain, have been described by
Bayer AG [77].
The research group from Sankyo AgroScience reported details of the synthe-
sis and investigation of pyrazole derivatives, namely diaryl pyrazolecarboxylates
and carboxamides [78]. Some of these compounds – in particular, methyl
4-chloro-1-(2,5-difluorophenyl)-5-(4-fluorophenyl)-pyrazole-3-carboxylate – demon-
strated notable pre-emergent herbicidal activity against a variety of weeds. The same
Cl F F
CH3
O F
F F
N N
N N
N N
NH2 NH2
O O
9 10
Flupoxam Triazofenamide
Figure 7.6 Chemical structures of flupoxam (9) and triazofenamide (10).

F F
Cl Cl HO F Cl F F Cl F F
1. F F O F O F
F F F F
+
2. H2NNH2 · H2O
N Pd NH2
+
O O N Cl
N
11 12 13
Cl F F
Cl F F
O F
O F
F F N
F F NH4OH
O O
NH
N N
N 14
N N O
NH2
O
O Ac2O
9 15 N-benzoylglycine
Scheme 7.3 Synthesis of flupoxam (9).
group also described a series of structurally related imidazoles, but these failed to
demonstrate any herbicidal activity.
7.2.3.2 Biology of Triazolocarboxamides: Flupoxam

Flupoxam was jointly developed by Kureha and Monsanto for the European
cereal market for both the pre- and early post-emergence control of annual
broadleaved weeds in winter wheat and winter barley. It was launched in 1993
by Synexus as Ovation™, a herbicide in which flupoxam was combined with
isoproturon (though this product appears to have been withdrawn from the market
in 1995). Flupoxam is active at an application rate of approximately 100 g a.i.
ha−1 [76]. Mode of action studies with flupoxam showed that it was not a mitotic
disrupter [79], but rather a CBI which caused radical changes in the cell wall
structure and composition of developing cotton fibers, inducing a phenotype
similar to that induced by isoxaben, but distinctly different from dichlobenil [80].
Although flupoxam and isoxaben have very similar effects on cotton fibers, the
two compounds presumably have different binding sites or different modes of
action, since Arabidopsis mutants resistant to isoxaben were not crossresistant to
flupoxam [80].
It is worthy of mention at this point that the closely related esters, such as methyl
1-(2-chlorophenyl)-5-phenyl-1H-1,2,4-triazole-3-carboxylate, have been described as
‘‘safeners’’, which have the ability to protect crops against the phytotoxic activities
of herbicides [81].
7.2.4
Alkylazines
7.2.4.1 Chemistry of Alkylazines

Alkylazines emerged from triazine herbicides on structural variation of the N-alkyl
side chains. The first hints of a new underlying mode of action were identified in
1987, when Omokawa et al. [82] reported differential physiological properties upon
the introduction of optically active N-α-methylbenzyl moieties to sym-triazines.
In this case, the (S)-enantiomers showed leaf-burning symptoms, which were typi-
cal of PS II inhibitors, whereas the (R)-enantiomers exhibited a light-independent
growth inhibition [83].
Prolongation of the N-α-methylbenzyl side chain and modification of the substi-
tution pattern led to triaziflam (16), a broadleaf herbicide which has been developed
by Idemitsu Kosan [84]. A related structure is seen in AE F150944 (17), an exper-
imental herbicide reported by Bayer AG [85]. Both compounds contain a chiral
C-atom in the side chain (Figure 7.7).. Such longer-chain alkylazines (Type II in
Figure 7.9) have been optimized for post-emergence broadleaf weed control in both
cereals and turf.
A specific characteristic of the alkylazine class is their intrinsic potential as non-
selective pre-emergence herbicides. A particularly strong non-selective alkylazine
is indaziflam (18) (Figure 7.8), which is obtained by the introduction of chiral
bicyclic amine moieties to the triazine core fragment. Indaziflam proved to be a
major breakthrough in herbicide chemistry with regards to its application rate and
spectrum of weed control [86, 87].
F F
H3C CH3 H3C CH3
H3 C
CH3 N N N N
H 3C O
* N N NH2 * N N NH2
H H
CH3 16 17
Triaziflam AE F150944
Figure 7.7 Chemical structures of triaziflam (16) and the experimental herbicide
AE F150944 (17).
F CH3
S CH3
N N
R N N NH2
H
H3C 18
Indaziflam Figure 7.8 Chemical structure of indaziflam (18).
R1
R2 N N
Type I (R4)n Typical features
N N NH2
H R1 = H, Alkyl, Haloalkyl
R2 = Me, Et, cycloAlkyl

R1
R3 = Me, Et
(R4)n R2 N N
Type II R4 = Me, OMe, Hal
X
N N NH2
H X = O, CH2, CH2CH2
m = 0, 1
R1 n=0-3
m
( ) R3
X N N
Type III
N N NH2
(R4)n H
Figure 7.9 Basic scaffolds of alkylazines described by types I, II, and III.
In summary, alkylazines can be categorized into three subclasses, depending on

the nature of the alkylamino side chain (Figure 7.9). Type I alkylazines include the
N-α-methylbenzyl compounds, type II have longer side chains (notably, compounds
of type II, in which X is equivalent to a bond (N-α-methyl phenethylamines), are
comparatively weak CBIs), while type III have a bicyclic alkylamino side chain. The
typical structural elements of the alkylazines are summarized in Figure 7.9.
The free amino group is sensitive to further modification. However,
N-acyl-substituted derivatives have been reported [88] that appear to act as
pro-herbicides. Further variations of these standard motifs are allowed, and have
been described in various patents; such derivatives refer to modifications of the
side chain [89], cyclopropyl derivatives [90], or thienyl compounds [91, 92].
Two general approaches (Routes A and B) can be applied for the synthesis of
alkylazines (Scheme 7.4). Both routes start from the appropriate alkylamines (III),
which are usually applied as their hydrochloride salts. Route A consists of a two-step
process in which N-cyanoguanidine is first reacted with III to give the correspond-
ing N-alkyl biguanidide intermediates (IV). Subsequent cyclization with an ester
(V) under basic conditions [93], or via aluminum complexes [94], leads to the pro-
duction of alkylazines of types I–III. Route B describes the one-step conversion of
alkylamines (III) with readily available 4-substituted 2-amino-6-chloro-1,3,5-triazine
(VI) building blocks [95].
7.2.4.2 Biology of Alkylazines

Triaziflam (16) has been developed for use in cereals and on turf, and is effective
in low doses and also has a long persistence. The physiological profile of triaziflam
NH
N
N NH2
H
R2 NH NH
N-cyano-guanidine · HCl
R3 N N NH2
Route A heat H H
IV
R2 R1
NaOMe
R3 NH2 · HCl OMe
R1 O
III V
N N
Cl N NH2 R1
Route B VI R2 N N
Base R3 N N NH2
H
Type I - III
Scheme 7.4 Synthetic approaches toward alkylazines according to routes A and B.
combines the characteristics of PS II electron-transport inhibitors and antimicro-

tubular agents. Grossmann et al. [93] showed that the less active (S)-enantiomer of
triaziflam preferentially inhibited PS II electron transport, while the more potent
(R)-enantiomer caused a decline in cellulose deposition in the cell walls. Mode
of action studies with triaziflam confirmed the inhibition of both, PS II electron
transport and cellulose biosynthesis. Whilst racemic triaziflam inhibited the Hill
reaction of isolated chloroplasts, with a pI50 -value of 6.5, it was found to be a
much more potent CBI, affecting the incorporation of radiolabeled glucose into
crystalline cellulose in Sorghum halepense cell suspension cultures with a pI50 -value
of 9.7 (B. Laber and H. Dietrich, Bayer CropScience AG, unpublished results).
Clearly, the herbicidal effects of triaziflam at the recommended dose rates are due
to cellulose biosynthesis inhibition.
AE F150944 (17) was intended for use in the pre-emergence control of a wide
range of mono- and dicotyledonous weeds, causing both emergence inhibition
and growth suppression. It also showed post-emergence effects in some species,
causing the cessation of plant growth and eventual necrosis or discoloration.
When the physiological effects of AE F150944 were investigated by Kiedaisch
et al. [96], it was shown to effectively and specifically inhibit the incorporation of
14
C-glucose into crystalline cellulose during primary wall synthesis in suspension
cultures of S. halepense (pI50 = 9.8), and also during primary and secondary wall
synthesis in cultured cells of Zinnia elegans (pI50 = 7.4 and 8.4, respectively).
The results of freeze-fracture electron microscopy studies revealed a depletion of
cellulose-synthesizing rosettes in the plasma membrane of Z. elegans tracheary
elements treated with AE F150944. Interestingly, however, AE F150944 did not

inhibit cellulose synthesis the bacterium Acetobacter xylinum and the cellular slime
mold Dictyostelium discoideum, both of which synthesize cellulose via linear arrays
of membrane proteins rather than via rosettes.
In 2010, Bayer CropScience received the first registration worldwide for its
herbicidal active ingredient indaziflam (18) in the USA. Initially, indaziflam
will be targeted at the turf segment market, under the trade name Specticle™,
with subsequent planned entries into the ornamental and industrial vegetation
management segments. Indaziflam is also currently undergoing development as a
new base herbicide in crops such as fruits and vines, nuts, citrus, olives and sugar
cane. This new active ingredient will be formulated as a suspension concentrate and
branded as Alion™ for use in these crops. Indaziflam controls a broad spectrum
of weeds, and provides excellent longlasting efficacy at low application rates. These
characteristics make indaziflam an environmentally compatible and pioneering
active ingredient in the global herbicides market.
In contrast to triaziflam (16), which is marketed as a racemic mixture, indaziflam
comprises an enantiomerically pure (1R,2S)-2,3-dihydro-2,6-dimethyl-1H-inden-1-
yl side chain. Unpublished Bayer CropScience data have revealed that 18 is an
extremely potent CBI, affecting the incorporation of 14 C-glucose into crystalline
cellulose with a pI50 -value of 9.4 in S. halepense cell suspension cultures (B. Laber
and H. Dietrich, Bayer CropScience AG, unpublished results). In addition, 18 has
only a minor effect on photosynthetic electron transport.
Thus, to date the alkylazines represent the most potent CBIs discovered, their
inhibitory activity being three orders of magnitude higher than that of nitriles such
as dichlobenil (pI50 = 6.0), or of benzamides such as isoxaben (pI50 = 6.5).
7.2.5
Thiatriazines
7.2.5.1 Chemistry of Thiatriazines

For many years, the 1λ4 ,2,4,6-thiatriazines were considered as chemical curiosities
and had failed to attract any synthetic interest, until the research team at Ciba-Geigy
showed that some compounds based on this scaffold demonstrated clear herbicidal
activities [97]. The underlying chemistry has been developed by the research
group of Prof. Haake at Marburg University, while investigating sulfodiimines.
From a structural point of view, the 1λ4 ,2,4,6-thiatriazines might be considered as
sulfur analogs (−SR = versus − CR =) of well-known sym-triazines, though their
biological behavior is quite different. The experimental herbicide CGA 325615 (19)
is a well-described compound of this class (Figure 7.10) [98].
One particular feature of the 1λ4 ,2,4,6-thiatriazines is that the sulfur atom in the
ring is tetragonal, and points out of the average plane formed by the five other atoms.
As a result, exo-cyclic substituents of the sulfur atom are in a pseudo-axial position.
In cases where the 3- and 5-substituents are different from each other, the sulfur
atom forms an asymmetric center. As this stereocenter is configurationally stable,
enantiomers of CGA 325615 (19) can be separated by using chiral chromatography.
NH2 F Figure 7.10 The experimental 1λ4 ,2,4,6-thiatriazine-type

F F herbicide CGA 325615 (19).
N N
S
N O F
F
19
CGA 325615
Cl Cl
ClAlR2
N N N N
S − 78 °C S
Cl N Cl R N Cl
20 VII R = Alkyl
Base Nu1H Nu1H = NH3, RNH2, R2NH
Nu2H = AlkylOH, AlkylSH,

Nu2 Cl ArylOH, ArylSH
Nu2H
N N N N
S S
R N Nu1 Base R N Nu1
IX VIII
Scheme 7.5 Synthesis of 3,5-substituted S-alkyl-1λ4 ,2,4,6-thiatriazines (IX).
Interestingly, only one enantiomer has shown herbicidal activity. No racemization

of the pure isomer has been observed, even after prolonged storage at room
temperature [98].
In addition to the rather limited sulfodiimine-based preparation method, flex-
ible synthetic routes starting from 2,3,5-trichloro-1λ4 ,2,4,6-thiatriazine (20) have
been developed which provide access to a greater array of compounds [97, 98].
For example, 1-alkylated 1λ4 ,2,4,6-thiatriazines VII can be obtained by the conver-
sion of 20 with chlorodialkylaluminum reagents (ClAlR2 , R = alkyl). Subsequent
treatment with nucleophiles (Nu = amines, alcohols, phenols, or thiophenes) pro-
duces the di- and trisubstituted 1λ4 ,2,4,6-thiatriazines VIII and IX, respectively
(Scheme 7.5).
Variations of the three substituents of the heterocycle have led to following SAR.
An amino function is preferred to attain good weed control (IX with Nu1 = NH2 ),
while the herbicidal activity is greatly increased when Nu2 is altered from a
phenoxy to a pentafluorophenoxy group, while keeping the other substituents
constant. Depending on the sulfur substituents, the overall herbicidal activity,
weed spectrum, and crop selectivity can be modulated [98].
7.2.5.2 Biology of CGA 325615

Following the post-emergence application of 1λ4 ,2,4,6-thiatriazines, such as
CGA 325615, susceptible grass species developed fragile culms and were impaired
in their growth and development of the root system. Dicotyledonous plants

exhibited a characteristic swelling of the stem base, splitting of the stems, and
rapid wilting. A microscopic examination of suspension cultures of plant cells
revealed a characteristic expansion of the cells, with an up to threefold increase in
diameter after a few days of exposure to nanomolar concentrations of CGA 325615.
CGA 325615 specifically inhibits the synthesis of crystalline cellulose in cultured
soybean cells (pI50 = 8.3), and also of a variety of other plants tested [99]. In devel-
oping cotton fibers, the compound also causes the accumulation of a noncrystalline
form of β-1,4-glucan. Glucan accumulation is not induced by the treatment of
developing cotton fibers with dichlobenil (1), but has been reported to occur in the
rsw1 (AtCesA1) mutant of Arabidopsis [100]. CGA 325615 also causes rosette disinte-
gration and the accumulation of CesA proteins, both in the plasma membrane and
in tight association with the cell wall-localized β-1,4-glucan, and of a protein that
reacts with an antibody against a tomato membrane-associated cellulase [99]. This
type of cellulase is encoded in Arabidopsis by the gene locus korrigan (kor) which,
when mutated, leads to altered cell wall structures that contain less cellulose [33].
CGA 325615 and dichlobenil also differ in their effects on sitosterol-β-glucoside
and sitosterol-cellodextrin synthesis, in that both processes are inhibited in vitro by
dichlobenil but neither is inhibited by CGA 325615 [43].
The inhibition of crystalline cellulose synthesis, concomitant β-1,4-glucan ac-
cumulation, plus rosette disintegration may indicate that the CesA subunits are
still functional in the presence of CGA 325615, but cannot assemble crystalline
microfibrils. Rosette assembly was proposed to be initiated by a redox-regulated
dimerization of CesA proteins [101], and H2 O2 was suggested to function as an
important signal in this process. The conserved region at the N terminus of plant
CesA proteins contains two putative zinc fingers. An analysis of cotton fiber CesA1
and CesA2 in the yeast two-hybrid system indicated that these domains interacted
via intermolecular disulfide bond formation to yield either homo- or heterodimers.
Dimerization occurred under oxidative conditions, and was promoted by H2 O2 . The
role of H2 O2 as a redox regulator in cell-wall biosynthesis is supported by the ob-
servation that the production of H2 O2 was greatly increased during secondary wall
formation in cotton fibers, and coincided with increases in CesA mRNA and protein
expression. Furthermore, secondary cell-wall development can be inhibited by the
depletion of endogenous H2 O2 [102]. CGA 325615 was shown to affect the oxidative
state of the zinc finger domains of CesA proteins, as the addition of increasing
concentrations of H2 O2 to CGA 325615-treated Arabidopsis seedlings significantly
alleviated the CGA 325615-induced phenotype. This effect of CGA 325615 was
specific because H2 O2 did not attenuate the phenotype obtained after dichlobenil
treatment [101]. Taken together, these observations support the concept that CGA
325615 somehow inhibits the dimerization of CesA proteins, and thereby prevents
assembly of the hexameric rosettes.
This model is further supported by studies in which confocal microscopy was
used to observe the behavior of green fluorescent protein (GFP)-labeled CesA3 in
untreated and CGA 325615-treated Arabidopsis cells. Treatment with CGA 325615
did not affect microtubules labeled with either GFP::α-tubulin6 or a GFP fusion to
the Arabidopsis microtubule end binding protein1A. Likewise, it did not affect actin
filaments marked by an N-terminal GFP fusion to the second actin-binding domain
of Arabidopsis fimbrin 1, and nor did it alter the morphology of the Golgi apparatus
or other organelles observed with electron microscopy. However, as visualized
by time-lapse imaging, treatment with CGA 325615 resulted in the clearance of
cellulose synthase complexes from the plasma membrane and their internalization
into intracellular compartments [103].
7.2.6
N-Aryl Lactams
7.2.6.1 Chemistry of N-Aryl Lactams

In 1994, the research group at Zeneca Agrochemicals identified a group of novel
phenylheterocyclyl (thio)carbamate herbicides [104], a well-described representa-
tive of which is the active 5-tert-butylcarbamoyloxy-3-(3-trifluoromethyl)phenyl-1,
3-thiazolidin-4-one (21) (Figure 7.11) [105]. The synthesis of 21 via 3-(3-trifluoro-
methyl)phenyl-1,3-thiazolidin-4-one (22) is shown in Scheme 7.6.
Analogs of 21 with different substituents on the aromatic ring, were prepared
from the appropriate anilines, using the same method. Similarly, carbamate deriva-
tives with N-substituents different from tert-butyl can be prepared by reaction of
the 5-hydroxy-thiazolidinone intermediate 23 with various isocyanates. Alterna-
tively, N,N-dialkylated carbamates of generic structure X were prepared by the
carbamoylation of 23 with N-dialkylated carbamoyl chlorides [104].
A systematic SAR study around this agrophore revealed that a considerable degree
of structural variation is tolerated, without total loss of herbicidal activity [106]. An
optimum activity was observed when the central heterocyclic thiazolidinone ring
was replaced by either a pyrrolidinone or an oxazolidinone ring, and when the
tert-butylcarbamoyloxy substituent was replaced by a sterically hindered N-linked
amide unit. Herbicidal activity is maintained when the carbamate function is
replaced by a wide range of alternative groups, with aryl-linked amides and ureas
providing the most potent activity. Oxazolidinone N-linked amides were found to
be the most potent herbicides.
7.2.6.2 Biology of N-Aryl Lactams

The thiazolidinone carbamate 21 demonstrated potential for the pre-emergence
control of grasses and small-seeded broadleaved weeds in soybean. It inhibits the
growth and lateral branching of the roots of agar-grown seedlings of suscepti-
ble species, and also inhibits the growth of, for example, Zea mays and Daucus
O
O H
N N
F CH3
S
F F O CH
H3 C 3
Figure 7.11 Structure of the thiazolidinone carba-
21
mate 21.
HSCH2COOH, (CH2O)n O
F F
NH2 N
F PTSA (cat), toluene, heat F
F F S
22
3-trifluoromethyl-aniline
1. SO2Cl2 2. H2O, K2CO3
O
H3CC-N=C=O O
O H
N N OH
F CH3 N
S NEt3 F
F F O
H3C CH3 F F
S
21 23
R
Cl N
R R = alkyl
O
R O
N O
F N
S R
F F O Base
Scheme 7.6 Preparation of the thiazolidinone carbamate 21 and analogs X.
carota plant cells in liquid culture. Consistent with the selective herbicidal activity
observed, the compound was shown to be a potent inhibitor of 3 H-glucose incor-
poration into the acid-insoluble cell-wall fraction of seedling roots of Z. mays and
Setaria viridis, but was only relatively weakly active on Glycine max and Ipomoea
hederacea. Thus, 21 exerts its herbicidal effect, either directly or indirectly, through
an inhibition of the biosynthesis of cellulose and cellulose-like polysaccharides, in a
manner similar to dichlobenil and isoxaben. Mutant lines of A. thaliana selected for
resistance to isoxaben were crossresistant to the thiazolidinone carbamate 21 [105].
7.2.7
Coumarins
The inhibitory effects elicited by coumarin and its analogs on seed germination and
plant growth were first described over 50 years ago [107]. The coumarin derivative
morlin (24) (Figure 7.12) was identified among a screen of several thousand small
molecules that cause altered cell morphology resulting in a swollen root phenotype
in Arabidopsis [108].
Live-cell fluorescence imaging of CesA6::YFP and GFP-labeled microtubules
showed that morlin acts on the cortical microtubules and alters the movement
of CesA6 in the plasma membrane. At high concentrations, morlin inhibited the
7.3 Cellulose Biosynthesis Inhibitors from Natural Sources 361
H3C O O O Figure 7.12 Chemical structure of morlin (24).
CH3
24
Morlin
incorporation of 14 C-glucose into acid-insoluble cell wall material only slightly.

Therefore, the primary effect of morlin is, most likely, on the cytoskeleton [108].
7.2.8
Properties of Commercialized Inhibitors of Cellulose Biosynthesis
The selected biochemical, agrochemical, physico-chemical, environmental, and

toxicological properties of commercialized CBIs such as dichlobenil (1), isoxaben
(4), and indaziflam (18) are summarized in Table 7.2. Further data relating to the
experimental CBI herbicides and compounds that have been withdrawn from the
market are available in Sections 7.2–7.3 (and references therein).
7.3
Cellulose Biosynthesis Inhibitors from Natural Sources
7.3.1
Thaxtomins
The thaxtomins represent a group of cyclic dipeptide phytotoxins produced by

Streptomyces scabies and other species in the genus Streptomyces, that cause scab
disease in potato and several other root crops. Thaxtomin A (25) (Figure 7.13), the
predominant member of this group, has moderate to good pre- and post-emergence
herbicidal activity, mainly on dicotyledonous species.
Plants treated with thaxtomin A displayed symptoms which were remarkably
similar to those caused by dichlobenil or isoxaben [111]. Furthermore, thaxtomin A
specifically inhibited the incorporation of 14 C-glucose into the acid-insoluble
cell-wall material of Arabidopsis seedlings, and was thereby confirmed as a CBI.
Arabidopsis mutants resistant to thaxtomin A (txr1) carry a point mutation in the
NO2
O
H3C
CH3
N
N H C N
H 3 OH
OH
O
25
Thaxtomin A Figure 7.13 Structure of thaxtomin A (25).
362
Table 7.2 Selected properties of commercialized CBI herbicides dichlobenil (1), isoxaben (4), and indaziflam (18)a.
Compound Dichlobenil (1) Isoxaben (4) Indaziflam (18)
Structure Cl CH3 F CH3

N
O O
N H S CH3
H3C N N N
Cl
H3C O R N N NH2
O H
7 Inhibitors of Cellulose Biosynthesis
H3C CH3
H3C
Trade names Barrier™, Casoron™, Flexidor™, Gallery™ Alion™, Specticle™

Norosac™, Sibenil™
Chemical class Nitriles Benzamides Alkylazines
WSSA MoA group 20 21 29
pI50 -value 6.0 6.5 9.4
Use rate (g a.i. ha−1 ) 2700–8100b 50–125c, e 35–70e, f
≤1000d, e 73–95b, g
Formulation types GR, WP GR, SC WPf , SCg
Koc 400 351 <1000
Soil half-life 1–6 months 3–4 months 9–66 days
Melting point (◦ C) 144–145 176–179 183–184
Solubility in water 21h 1.4 2.8
(mg l−1 at 20 ◦ C)
KOW logP 2.7 3.9i 2.8j
Compound Dichlobenil (1) Isoxaben (4) Indaziflam (18)
Vapor pressure (Pa at 1.4 × 10−1 5.5 × 10−7 6.8 × 10−8

25 ◦ C)
Henry’s law constant 1.0 × 10−5k 1.29 × 10−9k 2.69 × 10−6
(Pa·m3 mol –1 at 20 ◦ C)
LD50 rat oral (mg kg−1 ) >2000 >10 000 >2000
LD50 rabbit dermal >2000 >2000 >2000
(mg kg−1 )
a
The pI50 -values obtained from Bayer CropScience Research [96]; other values for dichlobenil (1) and isoxaben (4) taken from The Pesticide
Manual [109]; for indaziflam (18) from the Environmental Protection Agency (EPA) Pesticides Fact Sheet Database [110].
b
Pre-emergent.
c
Use in cereals.
d
Other uses.
e
Pre- to early post-emergent.
f
Given for Specticle.
g
Given for Alion™.
h
At 25 ◦ C.
i
At pH 5.
j
At pH 4 to 9.
k
Calculated.
7.3 Cellulose Biosynthesis Inhibitors from Natural Sources
363
txr gene; this results in the replacement of an arginine codon at amino acid 98 with
a stop codon, which truncates the 19 C-terminal amino acids from the 116-amino
acid cytoplasmic polypeptide. The txr gene has homologs in many eukaryotic
genomes, but its function is unknown [112]. Treatment with thaxtomin A strongly
compromised the mobility of CesA3- and CesA6-GFP fusion proteins, depleted
rosettes from the plasma membrane, and also caused an accumulation of these
particles in a small microtubule-associated compartment, but lacked any significant
effects on the cortical microtubule array [113].
The isolation, chemical structure elucidation and total synthesis, biosynthesis,
transformation by microorganisms and biological activity of the thaxtomins have
been reviewed by King and Calhoun [114].
The research group at BASF has also disclosed a series of piperazines with
herbicidal activity, derived from the thaxtomin motif [115–117].
7.3.2
Inthomycins
The natural product phthoxazolin A (26) (Figure 7.14) was first isolated from
Streptomyces species by Omura in 1990 [118].
The identical compound, alternatively named inthomycin A [119] and C22T
[120], was isolated later independently by two different research groups from
Streptomyces sp. strain Gö2 and S. griseoaurantiacus, respectively, together with
the isomers inthomycin B and inthomycin C. Subsequently, naturally occurring
hydroxylated derivatives were reported [121]. Inthomycins have been shown to
be specific CBIs. In contrast to other CBIs, phthoxazolin A also inhibits cellu-
lose biosynthesis in cellulose-producing bacteria and certain lower fungi which
contain cellulose in their cell walls [118, 122]. No growth inhibition has been ob-
served against several non-cellulose-containing Gram-positive and Gram-negative
bacteria, such as Bacillus and Escherichia species [118]. Herbicidal activity has
been demonstrated with radish seedlings and Abutilon theophrasti by treatment
with phthoxazolin A after emergence [123]. Furthermore, phthoxazolin A was
shown recently to inhibit prostate cancer cell growth in vitro by modulating
tumor–stromal cell interactions. Its efficacy in vivo, however, could not be evalu-
ated due to its high toxicity in mice [124]. The latter finding contradicts previous
reports of the low toxicity of phthoxazolin A following oral administration to
mice [118].
O OH O
NH2
H3C H3C CH3
26
Figure 7.14 Structure of phthoxazolin A
Phthoxazolin A (inthomycin A) (26).
References 365
A unified synthetic route to the inthomycin family has been described by Webb
et al. [125], based on the Stille coupling between a common oxazole vinyl iodide
and stereodefined stannyl-diene units.
7.3.3
Epopromycins
Epopromycin A and B are natural CBIs produced by Streptomyces species which

have demonstrated some herbicidal activity in the greenhouse [126]. The absolute
stereochemistry of (+)-epopromycin B (27) (Figure 7.15) was determined by a
combination of total synthesis and determination of its CBI effect on tomato
suspension culture cells (pI50 = 5.1) [127].
H3C
CH3
O O
H
H3C N S OH
N S R
H
CH3 O O
OH
27
(+)-epopromycin B
Figure 7.15 Structure of (+)-epopromycin B (27).
An additional enantio- and stereocontrolled route to (+)-epopromycin B is based

on a cinchona alkaloid-catalyzed Baylis–Hillman reaction [128].
References
1. Williamson, R.E., Burn, J.E., and 9. Taylor, N.G. (2008) New Phytol., 178,
Hocart, C.H. (2001) Cell. Mol. Life Sci., 239–252.
58, 1475–1490. 10. Guerriero, G., Fugelstad, J., and
2. Doblin, M.S., Kurek, I., Jacob-Wilk, Bulone, V. (2010) J. Integr. Plant Biol.,
D., and Delmer, D.P. (2002) Plant Cell 52, 161–175.
Physiol., 43, 1407–1420. 11. Acebes, J.L., Encina, A., Garcı́a-Angulo,
3. Reiter, W.D. (2002) Curr. Opin. Plant P., Alonso-Simón, A., Mélida, H.,
Biol., 5, 536–542. and Álvarez, J.M. (2010) in Cellulose:
4. Saxena, I.M. and Brown, R.M. Jr (2005) Structure and Properties, Derivatives and
Ann. Bot., 96, 9–21. Industrial Uses (eds A. Lejeune and T.
5. Somerville, C. (2006) Annu. Rev. Cell Deprez), Nova Science Publishers, Inc.,
Dev. Biol., 22, 53–78. New York, pp. 39–73.
6. Finaev, D. (2007) Biol. Plant., 51, 12. Delmer, D.P. (1999) Annu. Rev. Plant
407–413. Physiol. Plant Mol. Biol., 50, 245–276.
7. Mutwil, M., Debolt, S., and Persson, 13. Ha, M.A., Apperley, D., Evans, B.,
S. (2008) Curr. Opin. Plant Biol., 11, Huxham, I.M., Jardine, W.G., Viëtor,
252–257. R.J., Reis, D., Vian, B., and Jarvis, M.C.
8. Saxena, I.M. and Brown, R.M. Jr (1998) Plant J., 16, 183–190.
(2008) Adv. Plant Biochem. Mol. Biol., 1, 14. Thimm, J.C., Burritt, D.J., Sims, I.M.,
135–160. Newman, R.H., Ducker, W.A., and
Melton, L.D. (2002) Physiol. Plant., 116, 31. Desprez, T., Vernhettes, S., Fagard, M.,
164–171. Refregier, G., Desnos, T., Aletti, E., Py,
15. Richmond, T. (2000) Genome Biol., 1, N., Pelletier, S., and Höfte, H. (2002)
1–6. Plant Physiol., 128, 482–490.
16. Appenzeller, L., Doblin, M., Barreiro, 32. Gillmor, C.S., Poindexter, P., Lorieau,
R., Wang, H., Niu, X., Kollipara, K., J., Palcic, M.M., and Somerville, C.
Carrigan, L., Tomes, D., Chapman, M., (2002) J. Cell Biol., 156, 1003–1013.
and Dhugga, K.S. (2004) Cellulose, 11, 33. Nicol, F., His, I., Jauneau, A.,
287–299. Vernhettes, S., Canut, H., and Höfte,
17. Djerbi, S., Lindskog, M., Arvestad, H. (1998) EMBO J., 17, 5563–5576.
L., Sterky, F., and Teeri, T.T. (2005) 34. Chen, Z., Hong, X., Zhang, H., Wang,
Planta, 221, 739–746.
Y., Li, X., Zhu, J.K., and Gong, Z.
18. Desprez, T., Juraniec, M., Crowell,
(2005) Plant J., 43, 273–283.
E.F., Jouy, H., Pochylova, Z., Parcy,
35. Fagard, M., Desnos, T., Desprez, T.,
F., Höfte, H., Gonneau, M., and
Goubet, F., Refregier, G., Mouille, G.,
Vernhettes, S. (2007) Proc. Natl Acad.
McCann, M., Rayon, C., Vernhettes,
Sci. USA, 104, 15572–15577.
19. Taylor, N.G., Howells, R.M., Huttly, S., and Höfte, H. (2000) Plant Cell, 12,
A.K., Vickers, K., and Turner, S.R. 2409–2424.
(2003) Proc. Natl Acad. Sci. USA, 100, 36. Seifert, G.J., Barber, C., Wells, B.,
1450–1455. Dolan, L., and Roberts, K. (2002) Curr.
20. Perrin, R.M. (2001) Curr. Biol., 11, Biol., 12, 1840–1845.
R213–R216. 37. Arioli, T., Peng, L., Betzner, A.S., Burn,
21. Charnock, S.J. and Davies, G.J. (1999) J., Wittke, W., Herth, W., Camilleri,
Biochemistry, 38, 6380–6385. C., Höfte, H., Plazinski, J., Birch, R.,
22. Charnock, S.J., Henrissat, B., and Cork, A., Glover, J., Redmond, J., and
Davies, G.J. (2001) Plant Physiol., 125, Williamson, R.E. (1998) Science, 279,
527–531. 717–720.
23. Atanassov, I.I., Pittman, J.K., and 38. Beeckman, T., Przemeck, G.K.H.,
Turner, S.R. (2009) J. Biol. Chem., 284, Stamatiou, G., Lau, R., Terryn, N.,
3833–3841. De Rycke, R., Inze, D., and Berleth, T.
24. Ellis, C., Karafyllidis, I., Wasternack, (2002) Plant Physiol., 130, 1883–1893.
C., and Turner, J.G. (2002 Plant Cell, 39. Lane, D.R., Wiedemeier, A., Peng, L.C.,
14, 1557–1566. Höfte, H., Vernhettes, S., Desprez, T.,
25. Lukowitz, W., Nickle, T.C., Meinke, Hocart, C.H., Birch, R.J., Baskin, T.I.,
D.W., Last, R.L., Conklin, P.L., and Burn, J.E., Arioli, T., Betzner, A.S., and
Somerville, C.R. (2001) Proc. Natl Acad. Williamson, R.E. (2001) Plant Physiol.,
Sci. USA, 98, 2262–2267.
126, 278–288.
26. Cano-Delgado, A., Penfield, S., Smith,
40. Wang, J., Howles, P.A., Cork, A.H.,
C., Catley, M., and Bevan, M. (2003)
Birch, R.J., and Williamson, R.E. (2006)
Plant J., 34, 351–362.
27. Zhong, R., Morrison, W.H. III,
41. Howles, P.A., Birch, R.J., Collings,
Freshour, G.D., Hahn, M.G., and
Ye, Z.H. (2003) Plant Physiol., 132, D.A., Gebbie, L.K., Hurley, U.A.,
786–795. Hocart, C.H., Arioli, T., and
28. Taylor, N.G., Laurie, S., and Turner, Williamson, R.E. (2006) Plant J., 48,
S.R. (2000) Plant Cell, 12, 2529–2540. 606–618.
29. Taylor, N.G., Scheible, W.R., Cutler, 42. Amor, Y., Haigler, C.H., Johnson,
S., Somerville, C.R., and Turner, S.R. S., Wainscott, M., and Delmer, D.P.
(1999) Plant Cell, 11, 769–779. (1995) Proc. Natl Acad. Sci. USA, 92,
30. Scheible, W.R., Eshed, R., Richmond, 9353–9357.
T., Delmer, D., and Somerville, C. 43. Peng, L., Kawagoe, Y., Hogan, P.,
(2001) Proc. Natl Acad. Sci. USA, 98, and Delmer, D. (2002) Science, 295,
10079–10084. 147–150.
References 367
44. Elbein, A.D., Forsee, W.T., Schultz, 63. Delmer, D.P., Read, S.M., and Cooper,
J.C., and Laine, R.A. (1975) Lipids, 10, G. (1987) Plant Physiol., 84, 415–420.
427–436. 64. Heim, D.R., Larrinua, I.M., Murdoch,
45. Koyama, M., Helbert, W., Imai, M.G., and Roberts, J.L. (1998) Pestic.
T., Sugiyama, J., and Henrissat, B. Biochem. Physiol., 59, 163–168.
(1997) Proc. Natl Acad. Sci. USA, 94, 65. Burow, K.W. Jr (1987) US Patent
9091–9095. 4,636,243, Eli Lilly & Company.
46. Imai, T., Watanabe, T., Yui, T., and 66. Brinkmeyer, R.S., Terando, N.H.,
Sugiyama, J. (2003) Biochem. J., 374, and Waldrep, T.W. (1991) in Synthe-
755–760. sis and Chemistry of Agrochemicals II,
47. Robert, S., Mouille, G., and Höfte, H. ACS Symposium Series, Vol. 443 (eds
(2004) Cellulose, 11, 351–364. D.R. Baker, J.G. Fenyes, and W.K.
48. Paredez, A.R., Somerville, C.R., and Moberg), American Chemical Society,
Ehrhardt, D.W. (2006) Science, 312, Washington, DC, pp. 158–169.
1491–1495. 67. Huggenberger, F., Jennings, E.A.,
49. Lloyd, C.W. (1984) Int. Rev. Cytol., 86, Ryan, P.J., and Burow, K.W. (1982)
1–51. Proc. Brighton Crop Prot. Conf. – Weeds,
50. Gardiner, J.C., Taylor, N.G., and 1, 47–52.
Turner, S.R. (2003) Plant Cell, 15, 68. Levebvre, A., Maizonnier, D., Gaudry,
1740–1748. J.C., Clair, D., and Scalla, R. (1987)
51. Koopman, H. and Daams, J. (1960) Na- Weed Res., 27, 125–134.
ture, 186, 89–90.
69. Heim, D.R., Roberts, J.L., Pike, P.D.,
52. Koopman, H. and Daams, J. (1962)
and Larrinua, I.M. (1990) Plant Physiol.,
US Patent 3,027,248, North American
92, 858–861.
Philips Company, Inc.
70. Roberts, J.L. (1990) Plant Physiol., 93,
53. Yates, J. (1965) GB Patent 987 253,
S–107.
Shell Research Ltd.
71. Durso, N.A. and Vaughn, K.C. (1997)
54. Beynon, K.I. and Wright, A.N. (1968) J.
Sci. Food Agric., 19, 718–722.
72. Heim, D.R., Roberts, J.L., Pike, P.D.,
55. Unger, T.A. (1996) Pesticides Synthesis
and Larrinua, I.M. (1989) Plant Physiol.,
Handbook, Noyes Publications, New
90, 146–150.
York.
73. Heim, D.R., Skomp, J.R., Waldron,
56. Hogetsu, T., Shibaoka, H., and
Shimokoriyama, M. (1974) Plant Cell C., and Larrinua, I.M. (1991) Pestic.
Physiol., 15, 389–393. Biochem. Physiol., 39, 93–99.
57. Sabba, R.P. and Vaughn, K.C. (1999) 74. Brown, R.M., Saxena, I.M., and
Weed Sci., 47, 757–763. Kudlicka, K. (1996) Trends Plant Sci., 1,
58. Meyer, Y. and Herth, W. (1978) Planta, 149–156.
142, 253–262. 75. Shida, T., Arabori, H., Watanabe, T.,
59. Vaughn, K.C., Hoffman, J.C., Hahn, Kubota, Y., Ichinose, I., Kanda, Y.,
M.G., and Staehelin, L.A. (1996) Proto- Yamazaki, S., and Shinkawa, H. (1988)
plasma, 194, 117–132. EP Patent 0 282 303, Kureha Kagaku
60. Herth, W. (1987) Naturwissenschaften, Kogyo Kabushiki Kaisha.
74, 556–557. 76. O’Keeffe, M.G., Klevorn, T.B., Chida,
61. DeBolt, S., Gutierrez, R., Ehrhardt, T., and Shida, T. (1991) Proc. Brighton
D.W., and Somerville, C. (2007) Plant Crop Prot. Conf. – Weeds, 1, 63–68.
Physiol., 145, 334–338. 77. Bretschneider, T., Andres, P., Lui, N.,
62. Rajangam, A.S., Kumar, M., Aspeborg, Dollinger, M., and Santel, H.-J. (1994)
H., Guerriero, G., Arvestad, L., Pansri, DE Patent 4320076, Bayer AG.
P., Brown, C.J., Hober, S., Blomqvist, 78. Kudo, N., Furuta, S., Taniguchi, M.,
K., Divne, C., Ezcurra, I., Mellerowicz, Endo, T., and Sato, K. (1999) Chem.
E., Sundberg, B., Bulone, V., and Pharm. Bull., 47, 857–868.
Teeri, T.T. (2008) Plant Physiol., 148, 79. Hoffman, J.C. and Vaughn, K.C. (1996)
80. Vaughn, K.C. and Turley, R.B. (2001) 93. Grossmann, K., Tresch, S., and
Protoplasma, 216, 80–93. Plath, P. (2001) Z. Naturforsch. C,
81. Böhner, B. and Moser, H. (1989) EP 56, 559–569.
Patent 0 310 555, Ciba Geigy AG. 94. Ford, M. (2009) WO Patent
82. Omokawa, H., Ichizen, N., and 2009/077059, Bayer CropScience AG.
Takematsu, T. (1987) Agric. Biol. 95. Zindel, J., Hollander, J., Minn, K., and
Chem., 51, 2563–2568. Willms, L. (2000) WO Patent 2000/002
83. Omokawa, H. (2002) in Herbicide 868, Hoechst Schering AgrEvo GmbH.
Classes in Development: Mode of Action, 96. Kiedaisch, B.M., Blanton, R.L., and
Targets, Genetic Engineering (eds P. Haigler, C.H. (2003) Planta, 217,
Böger, K. Wakabayasi, and K. Hirai), 922–930.
97. Stoller, A., Haake, M., and Zondler,
Springer-Verlag, Berlin, Heidelberg,
H. (1994) WO Patent 96/01814 A1,
New York, pp. 291–318.
Ciba-Geigy AG.
84. Nishii, M., Kobayashi, I., Uemura, M.,
98. Stoller, A., Kreuz, A., Haake, M.,
and Takematsu, T. (1990) WO Patent
and Wenger, J. (2003) Chimia, 57,
90/09378, Idemitsu Kosan Company
725–730.
Ltd. 99. Peng, L., Xiang, F., Roberts, E.,
85. Riebel, H.-J., Lehr, S., Stelzer, U., Kawagoe, Y., Greve, L.C., Kreuz, K.,
Watanabe, Y., and Dollinger, M. (1998) and Delmer, D.P. (2001) Plant Physiol.,
WO Patent 98/15537, Bayer AG. 126, 981–992.
86. Lorenz, K., Minn, K., Willms, L., 100. Arioli, T., Burn, J.E., Betzner, A.S., and
Bauer, K., Bieringer, H., and Rosinger, Williamson, R.E. (1998) Trends Plant
C.H. (1997) WO Patent 97/31904, Sci., 3, 165–166.
Hoechst Schering Agrevo GmbH. 101. Kurek, I., Kawagoe, Y., Jacob-Wilk,
87. Ahrens, H., Dietrich, H., Minn, K., D., Doblin, M., and Delmer, D.
Auler, T., Bieringer, H., Hills, M., (2002) Proc. Natl Acad. Sci. USA,
Kehne, H., and Menne, H. (2004) WO 99, 11109–11114.
Patent 2004/069814, Bayer CropScience 102. Potikha, T.S., Collins, C.C., Johnson,
AG. D.I., Delmer, D.P., and Levine, A.
88. Minn, K., Dietrich, H., Auler, T., (1999) Plant Physiol., 119, 849–858.
Hills, M., Kehne, H., and Feucht, D. 103. Crowell, E.F., Bischoff, V., Desprez,
(2007) WO Patent 2007/118589, Bayer T., Rolland, A., Stierhof, Y.D.,
CropScience AG. Schumacher, K., Gonneau, M., Höfte,
89. Hollander, J., Willms, L., Zindel, J., H., and Vernhettes, S. (2009) Plant
Minn, K., Giencke, W., Rosinger, C., Cell, 21, 1141–1154.
Auler, T., and Bieringer, H. (2000) WO 104. Kay, I.T., Barton, J.E.D., Collins, D.J.,
Kowalczyk, B., Mitchell, G., Shribbs,
Patent 00/00480, Hoechst Schering
J.N., Cox, J.M., Barnes, N.J., and
Agrevo GmbH.
Smith, S.C. (1994) WO Patent 94/013
90. Ahrens, H., Dietrich, H., Auler, T.,
652, Zeneca Agrochemicals.
Hills, M., Kehne, H., Feucht, D.,
105. Sharples, K.R., Hawkes, T.R., Mitchell,
Hermann, S., Kather, K., and Lehr, S.
G., Edwards, L.S., Langford, M.P.,
(2008) WO Patent 2008/074403, Bayer Langton, D.W., Rogers, K.M.,
CropScience AG. Townson, J.K., and Wang, Y. (1998)
91. Kather, K., Lehr, S., Riebel, H.-J., Voigt, Pestic. Sci., 54, 368–376.
K., Drewes, M.W., Feucht, D., Pontzen, 106. Mitchell, G., Barnes, N.J., Cox, J.M.,
R., and Wetcholowsky, I. (2000) DE Matthews, I.R., Parry, D.R., Pearson,
Patent 19921883, Bayer AG. D.P.J., and Smith, S.C. (2002) in Syn-
92. Angermann, A., Dietrich, H., Giencke, thesis and Chemistry of Agrochemicals
W., Auler, T., Kehne, H., Hills, M., VI, ACS Symposium Series, Vol. 800
Feucht, D., Lehr, S., and Kather, K. (eds D.R. Baker, J.G. Fenyes, and W.K.
(2006) DE Patent 10 2004 037 428, Moberg), American Chemical Society,
Bayer CropScience GmbH. Washington, DC, pp. 18–29.
References 369
107. Bernhard, R.A. (1959) Bot. Gaz., 121, 117. Parra Rapado, L., Hupe, E., Witschel,
17–21. M., Seitz, T., Simon, A., Reinhard, R.,
108. DeBolt, S., Gutierrez, R., Ehrhardt, Sievernich, B., Großmann, K., Erhardt,
D.W., Melo, C.V., Ross, L., Cutler, T., Newton, T.W., Stelzer, F., Qu, T.,
S.R., Somerville, C., and Bonetta, D. Moberg, W.K., Song, D., Rack, M.,
(2007) Proc. Natl Acad. Sci. USA, 104, Frassetto, T., Kreuz, K., and Major, J.
5854–5859. (2010) WO Patent 2010/012649, BASF
109. Tomlin, C.S.D. (ed.) (2009) The Pes- AG.
ticide Manual, 15th edn, British Crop 118. Omura, S., Tanaka, Y., Kanaya, I.,
Protection Council (BCPC), Alton
Shinose, M., and Takahashi, Y. (1990)
(Hampshire).
J. Antibiot., 43, 1034–1036.
110. U.S. Environmental Protection
119. Henkel, T. and Zeeck, A. (1991) Liebigs
Agency (2010) Pesticides Fact Sheets,
Ann. Chem., 367–373.
http://www.epa.gov/opprd001/factsheets/
indaziflam.html. 120. Legendre, F., Maturano, M.D., Etienne,
111. King, R.R., Lawrence, C.H., and Gray, G., Klaebe, A., and Tiraby, G. (1995)
J.A. (2001) J. Agric. Food Chem., 49, J. Antibiot., 48, 341–343.
2298–2301. 121. Shiomi, K., Arai, N., Shinose, M.,
112. Scheible, W.R., Fry, B., Kochevenko, Takahashi, Y., Yoshida, H., Iwabuti,
A., Schindelasch, D., Zimmerli, J., Tanaka, Y., and Omura, S. (1995)
L., Somerville, S., Loria, R., and J. Antibiot., 48, 714–719.
Somerville, C.R. (2003) Plant Cell, 122. Tanaka, Y., Kanaya, I., Takahashi, Y.,
15, 1781–1794. Shinose, M., Tanaka, H., and Omura,
113. Bischoff, V., Cookson, S.J., Wu, S., and S. (1993) J. Antibiot., 46, 1208–1213.
Scheible, W.R. (2009) J. Exp. Bot., 60, 123. Omura, S. (1992) Gene, 115, 141–149.
955–965. 124. Kawada, M., Inoue, H., Usami, I.,
114. King, R.R. and Calhoun, L.A. (2009) and Ikeda, D. (2009) Cancer Sci., 100,
Phytochemistry, 70, 833–841. 150–157.
115. Hupe, E., Zagar, C., Witschel, M., 125. Webb, M.R., Addie, M.S., Crawforth,
Kühn, T., Moberg, W.K., Parra Rapado, C.M., Dale, J.W., Franci, X., Pizzonero,
L., Stelzer, F., Vescovi, A., Puhl, M., Donald, C., and Taylor, R.J.K.
M., Reinhard, R., Sievernich, B.,
(2008) Tetrahedron, 64, 4778–4791.
Grossmann, K., Ehrhardt, T., and Rack,
126. Tsuchiya, K., Kobayashi, S., Nishikiori,
M. (2007) WO Patent 2007/077201,
T., Nakagawa, T., and Tatsuta, K.
BASF AG.
(1997) J. Antibiot., 50, 261–263.
116. Hupe, E., Zagar, C., Witschel, M.,
Kühn, T., Moberg, W.K., Parra Rapado, 127. Dobler, M.R. (2001) Tetrahedron Lett.,
L., Stelzer, F., Vescovi, A., Reinhard, 42, 215–218.
R., Sievernich, B., Grossmann, K., 128. Iwabuchi, Y., Sugihara, T., Esumi, T.,
and Erhardt, T. (2007) WO Patent and Hatakeyama, S. (2001) Tetrahedron
2007/077247, BASF AG. Lett., 42, 7867–7871.
371
8
Safeners for Herbicides
8.1
Introduction
Herbicide safeners, which are also referred to as herbicide antidotes or protectants,

fulfill an important role in crop protection. Safeners are chemicals that protect
crop plants from unacceptable injury caused by herbicides. Either by placement
on the crop seed or by way of a physiological selectivity mechanism, safeners
in commercial use do not negatively impact the weed control of the herbicide.
Although many herbicides have been developed for use without a safener, some
of the strongest and most broad-spectrum herbicides tend towards borderline crop
selectivity, which may completely preclude their use in a particular crop, or at
least limit the usage rates or the crop varieties that can be safely treated. It is for
such situations that safeners have been developed. With the subject of safeners
having been detailed in several books and reviews over the past 20 years [1–3], the
intention here is not only to focus on recently developed commercial safeners, but
also to describe some of the ‘‘older’’ compounds still in wide commercial use.
The story of herbicide safeners began in 1947 with an accidental observation
by Otto Hoffmann, at the Gulf Oil Company. On a hot summer afternoon,
Hoffmann noticed that his greenhouse tomato plants had suffered injury that
he presumed was caused by 2,4-D vapor drift, but that those plants treated
with 2,4,6-trichlorophenoxyacetic acid showed no symptoms of such injury [4].
Hoffmann immediately recognized the potential use of such an effect, and be-
gan investigations into compounds that could protect crops against herbicide
injury.
One fundamental problem for safener discovery and development is to identify
safeners that do not also antagonize weed control. The fruits of the Gulf Oil Com-
pany research (as reported by Hoffmann in 1969) was 1,8-naphthalic anhydride
(NA), which functions best as a seed treatment, whereby the antagonism of weed
control is not an issue. To the present authors’ knowledge, about 15 further safeners
have been commercialized during the years since NA was introduced, although
several of the early safeners have since been superseded and/or withdrawn. This
subsequent period of safener commercialization may be informally split into three

372 8 Safeners for Herbicides
phases: (i) mainly seed treatment safeners; (ii) pre-emergence tank-mix safeners;
and (iii) post-emergence tank-mix safeners. The chemical structures and usage of
these safeners are listed in Table 8.1. In all cases, the crops are monocotyledons
(maize, sorghum, rice, and cereals such as wheat and barley), and the effect of one
such safener, mefenpyr-diethyl, is shown in Figure 8.1. Although, to date, no com-
parable safeners have been commercialized for broadleaved crops, the ‘‘extender’’
dietholate is currently used by FMC to help protect cotton against the herbicide
clomazone (US patent application 20050009702). In addition, several compounds
(daimuron, cumyluron, and dimepiperate) that generally are considered to be her-
bicidal have been included in some products, principally because they reduce crop
injury from another herbicidal component. These are of relevance, particularly in
rice and the structures are also shown in Table 8.1; however, because they are
relatively old compounds they will not be included at this point.
In order to ensure maximum crop safety, safeners that are applied in mixture
with the herbicides need to act more quickly than the herbicide injury develops.
The mechanism of action of safeners has attracted much scientific attention, and
will be detailed in Section 8.3.
Safeners, like pesticides, must be registered before use. However, the regulatory
situation for safeners is complex, in particular when considered on a global basis.
For example, whereas several European countries require for safeners full data
packages like those for pesticides, safeners did not previously fall under Annex I of
the European Union pesticide directive 91/414. However, from mid 2011 directive
91/414 has been replaced by a regulation 1107/2009 under which new safeners will
be treated just like active substances, and must be approved at European Union
(EU) level. In the USA, safeners are treated under inert legislation as opposed to
pesticide legislation. However, full data sets (like those for active ingredients) are
actually required for evaluation by the Environmental Protection Agency (EPA) to
establish a residue limit for the federal food, drug, and cosmetic act. In Canada and
Australia, safeners are now treated legally as pesticides, whereas in other parts of
the world safeners are legally treated as formulation additives.
8.2
Overview of Selected Safeners
8.2.1
Dichloroacetamide Safeners
This class contains several important commercial safeners, as well as a range

that were reported (and patented) but not launched. They are all of relatively
low molecular weight (≤ 300 Da), and have the –N–C(O)–CHCl2 substituent in
common. Although, the first member of this class was commercialized during the
early 1970s, three compounds are still of considerable commercial importance,
namely benoxacor, dichlormid, and furilazole.
Table 8.1 Structures of commercial safeners.
Common name Chemical structure Application/

(development codes) crop/herbicides
1,8-Naphthalic O O O Seed treatment

anhydride (NA) Protect® Maize
Thiocarbamates
Cyometrinil N O Seed treatment

(CGA 43089) CN Sorghum
Concep I® CN Thiocarbamates/
chloracetamides
Oxabetrinil (CGA 92194) N O O Seed treatment

Concep II® Sorghum
Superseded Concep I® CN O Thiocarbamates/
chloracetamides
Fluxofenim (CGA N O O Seed treatment

Cl
133205) Sorghum
CF3 O
Concep III® Thiocarbamates/
Superseded Concep II® chloracetamides
Flurazole (MON4606) CF3 N Seed treatment

Cl
Screen® Sorghum
O S Chloracetamides
Benoxacor (CGA O Spray Pre, PPI

154281) Maize
N Chloracetamides
Cl
O
Cl
Dichlormid (R25788) Spray Pre, PPI

Cl
Maize
N Cl Thiocarba-
mates/chloracetamides
O
Furilazole (MON13900) Cl Spray pre-emergence

Cl Maize
O Chloracetamides
O
N (Acetochlor)
O
Common name Chemical structure Application/

(development codes) crop/herbicides
AD-67 MON4660 Cl Spray pre-emergence

O N Maize
Cl Chloracetamides
O (Acetochlor)
Fenclorim (CGA Cl Spray pre-emergence

123407) N Rice
Pretilachlor
N
Cl
Daimuron (K223, H Water application

SK-23) H N post-emergence
N Rice
O
Sulfonylureas
Cumyluron (JC-940) Cl Water application

H post-emergence
H N
N Rice
Sulfonylureas
O
Dimepiperate O Water application

(MY-93) post-emergence
N Rice
S
Sulfonylureas
Cloquintocet-mexyl N Spray post-emergence

O
(CGA 185072) Cereals
O
O C5H11 Clodinafop-propargyl
Cl
Fenchlorazole-ethyl O Spray post-emergence

(AE F070542) Cl Cereals
N O
Fenoxaprop-ethyl
N N
Cl
Cl Cl
Cl
Mefenpyr-diethyl Cl O Spray post-emergence

(AE F107892) N Cereals
O
Cl N ACCase and
sulfonylureas
O
O
Isoxadifen-ethyl (AE O Spray post-emergence

F122006) Maize/Rice
O ACCase and
N sulfonylureas
O
Cyprosulfamide (AE CH3 Spray pre-emergence or

0001789) O O O post-emergence
O
N S N Maize
H H HPPD and ALS
O
inhibitors
PPI, Pre-plant incorporated.
(a) (b) (c)
Figure 8.1 Post-emergence safening of wheat by

mefenpyr-diethyl against mesosulfuron-methyl. (a) Un-
treated; (b) Mesosulfuron-methyl-treated at 60 g a.i. ha−1 ; (c)
mesosulfuron-treated at 60 g a.i. ha−1 plus mefenpyr-diethyl
at 30 g a.i. ha−1 .
8.2.1.1 Benoxacor
Commercially, benoxacor is one of the most important members of this class of
safeners. It is included in products containing metolachlor as racemate or as the
single isomer S-metolachlor [subsequently ‘‘(S)-metolachlor’’ indicates that both
compounds are being referred to]. These products are principally used in maize
pre-plant, pre-plant incorporated, and pre-emergence. Benoxacor was developed
under the code CGA 154281 by Ciba-Geigy AG (now Syngenta), and first reported
NO2 NO2
O NaBr, NaHCO3
+ Cl
OH TBMAC O
H2O, toluene
O
H2 (200psi), Pt/C
60 °C
i
PrOH, toluene
Cl
O
O Cl
Cl Cl H
N
N Cl
NaOH, toluene
O
O
Scheme 8.1 Synthesis of benoxacor.
Table 8.2 Product examples containing benoxacor.
Herbicide(s) Tradename examples
S-Metolachlor Dual II magnum®, Cinch®

S-Metolachlor + atrazine Bicep II magnum®, Cinch® ATZ, Cinch® ATZ lite,
Metolachlor + atrazine Stalwart® Xtra
Metolachlor Stalwart®, Parallel® Me-Too-Lachlor II®
S-Metolachlor + mesotrione Camix®
S-Metolachlor + mesotrione + atrazine Lexar®, Lumax®
in 1988 [5]. It was specifically claimed in the US patent US4601745 (filed 18 March
1985), but a priority date of 12 December 1983 relates back to general claims for
the structure class (EP 149974). The synthesis of benoxacor, as disclosed in WO
2001090088, involves a three-step process (Scheme 8.1).
Today, numerous products that contain benoxacor and (S)-metolachlor, with and
without further herbicide components (Table 8.2). As the patents for benoxacor
and metolachlor have both expired, this list also contains several products from
generic producers.
The use of benoxacor with (S)-metolachlor is particularly necessary under stress
conditions for maize. Injury to corn from (S)-metolachlor is greater under cool
or wet soil conditions [6–8], where both the availability of the herbicide may be
increased and the ability of maize to metabolize metolachlor reduced [9]. Benoxacor
and metolachlor have similar physico-chemical properties that influence their
behavior in soil (Table 8.3); this tends to ensure that the safener and herbicide are
taken up together, hence providing safening under a variety of weather conditions.
Products such as Dual-II-magnum® and Cinch® are also labeled for use on
sorghum seed treated with fluxofenim or flurazole. This is a notable example of
safeners used in sequence so as to obtain optimal crop safety. The data listed in
Table 8.4 indicate that benoxacor has a favorable toxicological profile.
Table 8.3 Physico-chemical properties of benoxacor

and metolachlor.
Property Benoxacor Metolachlor
Log P 2.6 2.9

Koc 42–176 121–309
Table 8.4 Toxicological and soil degradation data

for benoxacor.
Property Value/characteristic
Rat, oral LD50 (mg kg−1 ) >5000

Rat, inhalation LD50 (mg m−3 ) >2000
Rabbit: skin and eye irritation Not irritant
Guinea pig: skin sensitizing Slightly sensitizing
DT50 in soil Rapid, ca. 5 days
O O
Cl NaOH, 30 °C Cl
Cl + HN Continuous
N
Cl process Cl
Scheme 8.2 One-step synthesis of dichlormid.
8.2.1.2 Dichlormid
Of the safeners covered separately in this chapter, dichlormid is the oldest still in
use. It was developed under the code number R25788 by Stauffer (now Syngenta),
first reported in 1972 [10], and is used to safen maize against injury from acetochlor.
Current products include Surpass®, TopNotch®, Volley®, and Confidence®;
Stalwart C® is a metolachlor product that contains dichlormid instead of benoxacor.
Dichlormid is also present in several acetochlor products that also contain atrazine
(e.g., Confidence Xtra®, Keystone®, Volley® ATZ).
The simple one-step synthesis of dichlormid (claimed in US 4278799) is shown
in Scheme 8.2.
As described for benoxacor, dichlormid also has similar physico-chemical prop-
erties to those of the herbicide components, allowing for similar plant uptake
profiles for good safening potential. Further extensive coverage of dichlormid can
be found in Crop Safeners for Herbicides [1].
8.2.1.3 Furilazole
Furilazole was developed by Monsanto Co. under the code number MON13900,
and first reported in 1991 [11]. It was claimed that furilazole can safen many
herbicides from diverse classes, although detailed efficacy was presented only for
the combination with the sulfonylurea herbicide halosulfuron-methyl (NC-319).
Since its launch in 1995, furilazole has been marketed with halosulfuron-methyl
in products such as Battalion® and Permit®, which are used both pre- and
post-emergence in corn and sorghum. It is also used in pre-emergence maize
products containing acetochlor (e.g., Degree®, Degree Extra®, Harness®, and
Guardian®).
It should be noted that acetochlor can be safened by several dichloroacetamide
safeners other than dichlormid and furilazole. For example, the product Acenit®
contains the safener AD67 (MON4660), which has no assigned common name (see
Table 8.1 for chemical structure).
The two-step synthesis of furilazole (claimed in patent EP 648768) is shown in
Scheme 8.3.
The toxicological profile of furilazole is quite favorable (Table 8.5).
OH
O O
O NO2
H2 (100psi)/Ni NH
+ O
t
BuOMe
40 °C
O
Cl CaO,
Cl Me2CO
Cl
O O
N
O
Cl
Cl
Scheme 8.3 Two-step synthesis of furilazole.
Table 8.5 Toxicological and soil degradation data for furilazole.

Rabbit, skin, and eye irritation Not irritant to skin/slight eye irritant
Guinea pig, skin sensitizing Not sensitizing
DT50 in soil Rather rapid, ca. 10–20 days
8.2.2
Oxime Ethers
Three oxime ethers have been commercialized by Ciba Geigy (now Syngenta) as
seed treatment safeners for sorghum, with protection being provided against both
thiocarbamate and chloroacetamide herbicides (in particular metolachlor). The first
of the oxime ethers (cyometrinil) was launched in 1978 as Concep I®, but this
was replaced in 1982 by oxabetrinil (Concep II®), which had less potential for
negative crop effects from the seed treatment. Concep II® was, in turn, superseded
by fluxofenim (Concep III®), which is still in commercial use today. In this case,
the reason for replacement was not fully clear, but was reportedly due to an
undesirable interaction of Concep II® with downy mildew disease in sorghum [1].
Fluxofenim was developed under the code CGA 133205 and first reported in 1986
[12]. The physical chemistry of fluxofenim (log P = 2.9) allows rapid uptake into
seeds at usage rates of 0.3–0.4 g a.i. kg−1 . The commercial market of fluxofenim
is, nonetheless, limited by its use only in the relatively minor crop sorghum.
The two-step synthesis of fluxofenim is shown in Scheme 8.4.
8.2.3
Cloquintocet-Mexyl
Cloquintocet-mexyl was developed under the code CGA 185072 by Ciba-Geigy (now
Syngenta), and is used post-emergence in cereals. The basic patent (EP 94349) has
a priority date of 7 May 1982. Various other country patents followed (e.g., US
4902340 and US 5102445). Cloquintocet-mexyl was first reported in 1989 [13] along-
side the acetyl coenzyme A carboxylase (ACCase) inhibitor clodinafop-propargyl,
and until now the main use of cloquintocet-mexyl remains in mixtures with
this ACCase-inhibiting herbicide. Products include Topik®, Horizon®, Discover®
(US), Celio®, Hawk®, Magestan®. The first launch was in 1991 in Switzer-
land, South Africa and Chile, with the US registration of the safener/herbicide
CF3 CF3
H2NOH*HCl
OH
O N
Cl Cl
Na, O
EtOH, Br
O
DMSO
CF3 O
O
N O
Cl
Scheme 8.4 Synthesis of fluxofenim.

Cl
O
+ Cl
O
N NaOH, toluene, NMP
OH K2CO3
80 °C
25−30 kPa
Cl
N O
O
O
Scheme 8.5 Synthesis of cloquintocet-mexyl.
Table 8.6 Toxicological data for cloquintocet-mexyl.

Rat, dermal LD50 (mg kg−1 ) >2000
Rabbit, skin, and eye irritation Not irritant
combination in 2000. The greatest safening with cloquintocet-mexyl is observed in

wheat, with less safening in barley.
In 2005, Syngenta launched Axial® for use in cereals; this contains
cloquintocet-mexyl to safen pinoxaden, an aryl-dione ACCase inhibitor. However,
the most recent use of cloquintocet-mexyl is to safen cereals against the acetolactate
synthase (ALS) inhibitor pyroxsulam from Dow AgroSciences. With global
launches starting in 2008, products include Powerflex® in the US and Broadway®
(which also contains florasulam) in Europe.
A single-step synthesis route for cloquintocet-mexyl is claimed in patent WO
2002000625 (Scheme 8.5).
Cloquintocet-mexyl has a favorable toxicological profile (Table 8.6).
In the soil, cloquintocet-mexyl degrades rapidly to the free acid (DT50 < 3 days),
with further degradation and mineralization occurring within weeks or a few
months. The parent safener and major metabolites are reported to bind strongly to
soil, and hence to have a low leaching potential.
8.2.4
Mefenypr-Diethyl
Mefenpyr-diethyl is, like cloquintocet-mexyl, used post-emergence to safen cereals.

It is used in combination with various aryloxyphenoxypropionates and sulfonylurea
Cl Cl O O
Cl Cl
+
NaNO2, HCl O
OEt
NH2 N
Cl N OEt
Cl
Et3N, KHCO3, H2O

60 °C
COOEt
Cl Cl
N
N COOEt
EtOOC
Scheme 8.6 Two-step synthesis of mefenpyr-diethyl.
herbicides in wheat, rye, triticale, and in some varieties of barley. Mefenpyr-diethyl

was developed under the code AE F107892 by AgrEvo (now Bayer CropScience),
was first reported alongside iodosulfuron-methyl in 1999 [14], and has since
replaced its predecessor fenchlorazole-ethyl. Mefenpyr-diethyl had the advantage
over fenchlorazole-ethyl of providing post-emergence selective grass weed control
not only in wheat and rye but also in spring barley. A further, very important
advantage of mefenpyr-diethyl was the property to act as a safener for a wider range
of herbicides used post-emergence in cereal crops. The priority date for patent
coverage of the pyrazoline safeners was November 1989 (WO 9107874), and the
first registration of mefenpyr-diethyl was in 1994. It is prepared via a two-step
synthesis (Scheme 8.6).
As already pointed out, mefenpyr-diethyl is a versatile safener, and has been
commercialized in combinations with several single or mixed herbicides, including
fenoxaprop-P-ethyl (e.g., Puma S®), iodosulfuron-methyl-sodium (e.g., Hussar®),
and mesosulfuron-methyl (Atlantis®). In general, the quantity of mefenpyr-diethyl
required to provide adequate safening lies between 20 and 100 g a.i. ha−1 , and there
is no set ratio between the rates of the herbicides and mefenpyr-diethyl.
At this point, it is worth mentioning some general considerations with regards
to the dose rates required for safeners. Of course, from a commercial and safety
standpoint the safener rate should be the lowest needed to obtain crop safety.
Seed treatment rates can be selected independent of the subsequent herbicide
dose. However, the maximum rate on the seed may sometimes be limited by
negative phytotoxic effects. This is exemplified well by the germination inhibition
in sorghum caused by cyometrinil, which eventually led to its replacement by
oxabetrinil. For products containing a mixture of safener and herbicide, significant
development effort is needed to define the required herbicide/safener ratio. This
ratio should be adequate to ensure crop safety and weed control at all recommended
rates. A farmer that reduces the product rate to below the minimum recommended
Table 8.7 Toxicological data for mefenpyr-diethyl.

Rabbit, skin, and eye irritation Not irritant
Mutagenicity in vitro and in vivo Nonmutagenic
on the product label runs the risk of not only inadequate weed control (due to
insufficient herbicide) but also possible crop injury due to insufficient safener.
For mefenpyr-diethyl, a wide range of products exist globally, in which this
critical herbicide/safener ratio is tuned to the specific herbicide(s) and agronomic
conditions.
Mefenpyr-diethyl has a highly favorable toxicological and ecotoxicological profile
(Table 8.7).
In the environment, mefenpyr-diethyl dissipates rapidly with a soil DT50 of <10
days. Complete mineralization occurs due to photolysis, hydrolysis, and microbial
degradation. There is no leaching risk, with concentrations of the parent compound
and soil metabolites not exceeding 0.1 ppb at 1 m soil depth in lysimeter trials.
Mefenpyr-diethyl is most probably a ‘‘pro-safener’’ – a term introduced by
Rubin in 1985 [15]. With mefenpyr-diethyl, decarboxylation occurs rapidly in plants
and soil, and it is likely that the safening activity comes from mefenpyr-ethyl.
However, the good post-emergence performance of mefenpyr-diethyl depends on
its physico-chemical characteristics (log P = 3.83 at pH 6.3, 21 ◦ C), which lead to
a better leaf uptake than from mefenpyr-ethyl. The biochemical mode of action of
mefenpyr-diethyl is described in Section 8.3.
8.2.5
Isoxadifen-Ethyl
Isoxadifen-ethyl is used post-emergence to safen maize and rice, and was developed
under the code AE F122006 by AgrEvo (now Bayer CropScience). It was first re-
ported in 2001 [16–18], and launched in the USA in 2002 in maize in combination
with foramsulfuron (Option®). Isoxadifen-ethyl is also used in combinations with
foramsulfuron plus iodosulfuron-methyl-sodium (Equip®, Maister®), while in rice
it is used with fenoxaprop-P-ethyl (Ricestar®, Starice®) and ethoxysulfuron (Tiller
Gold®). Most recently, Bayer CropScience has launched a global herbicide product
family for post-emergence use in maize based on the new 4-hydroxyphenyl pyru-
vate dioxygenase (HPPD) inhibitor tembotrione with isoxadifen-ethyl as safener
(Laudis®, Soberan®). The product, Capreno®, contains additionally the ALS in-
hibitor thiencarbazone. From this it can be seen that isoxadifen-ethyl has taken
safeners to a new level in being able to safen multiple herbicides (of various modes
HO COOEt
N Et3N
+
Cl COOEt
Et2O O N
Scheme 8.7 One-step route to isoxadifen-ethyl.
Table 8.8 Toxicological data for isoxadifen-ethyl.
Rat, oral LD50 (mg kg−1 ) 1740

of action) in multiple crops. This is emphasized still further by the interest that other
agrochemical companies have shown in this safener. Both, BASF and DuPont have
signed licensing agreements with Bayer CropScience, to use isoxadifen in products
containing dicamba (Status®), nicosulfuron (Accent Q®), and rimsulfuron plus
nicosulfuron (Steadfast Q®).
The priority date for patent coverage of the isoxazoline safeners was 16 September
1993 (DE 4331448, US 9507897, US 5516750).
The synthesis of isoxadifen-ethyl claimed in WO 1995007897 is via a one-step
route (Scheme 8.7).
Isoxadifen-ethyl has a favorable toxicological profile according to the US EPA
notice of filing (Table 8.8) [19].
8.2.6
Cyprosulfamide
The most recently commercialized safener, cyprosulfamide (CSA), was developed

under the code AE 0001789 by Bayer CropScience and first reported in 2008 [20,
21], and again later in 2009 [22]. Cyprosulfamide received first regulatory approval
in Romania in 2008, with subsequent registrations in the USA and Europe in 2009.
It is used to increase the safety of maize toward isoxaflutole pre-emergence (e.g.,
in Merlin Flexx® and Balance Flexx®), and to allow the selective use in maize
of isoxaflutole plus thiencarbazone pre-emergence to early post-emergence (e.g.,
in Adengo® and Corvus®). The pre-emergence field efficacy of CSA in maize is
shown in Figure 8.2. The soil and foliar uptake of CSA helps to explain how it
can be used effectively in pre- and post-emergence products. The priority date for
patent coverage of this acyl-sulfonamide type of safener was 29 September 1997
(DE 19742951; WO 1999016744; EP 1019368; US 6251827). Both, CSA and related
structures are also covered in many subsequent mixture patents.
(a) (b)
Figure 8.2 Pre-emergence safening of isoxaflutole by cypro-

sulfamide in maize. (a) Two central rows treated with isox-
aflutole at 315 g a.i. ha−1 ; (b) Two central rows treated with
isoxaflutole at 315 g a.i. ha−1 plus cyprosulfamide at
315 g a.i. ha−1 .
Table 8.9 Toxicological data for cyprosulfamide.

O O SOCl2
O O O
chlorobenzene
OH + H2N S O O
120 °C
O OH N S
O H Cl
O
Et3N or K2CO3
NH2 acetonitrile
O
O O
N S
O H N
O
H
Scheme 8.8 Synthesis of cyprosulfamide.
CSA has a favorable toxicological and eco-toxicological profile according to the

US EPA notice of filing (Table 8.9) [23, 24].
The synthesis of CSA as disclosed in WO 2005000797 (EP 1491527) is via a
two-step procedure (Scheme 8.8).
8.3
Mechanisms of Herbicide Safener Action
When applied alone, safeners generally have little visible effect on crop or weed
species, as found for example with fenchlorazole-ethyl and mefenpyr-diethyl
[25, 26]. In contrast, fenchlorazole-ethyl exerted an immediate protective ef-
fect on wheat by preventing even a transient inhibition of leaf growth by
fenoxaprop-ethyl [27]. A similar effect was observed subsequently with combi-
nations of mefenpyr-diethyl and fenoxaprop-P-ethyl.
For many years, the mechanism by which safeners were able to reduce herbicide
injury of large-seeded grass crops such as maize, wheat, sorghum, or rice, without
effecting the control of target weeds, was not fully understood. It has become
increasingly clear, however, that almost all of these compounds function by in-
creasing the rate of herbicide metabolism [2, 28, 29] in the crops, but not in the
weeds. In plants, the biotransformation of herbicides may occur via multistep pro-
cesses that involve the activity of herbicide-degrading enzymes, such as cytochrome
P450 monooxygenases (Cyt P450s), glycosyltransferases and ATP-binding cassette
(ABC) transporters [30, 31]. Recent developments in gene expression analysis have
shown that the detoxification responses of safeners are activated by an induction
of gene expression coding for such herbicide-degrading enzymes [28, 29]. These
mechanisms of herbicide safener action are summarized in the following sections,
while evidence for and against further potential safener action mechanisms with
regards to the reduction of herbicide uptake or translocation is also discussed.
8.3.1
Effects of Safeners on Herbicide Metabolism
With few exceptions, herbicides are subject to metabolic transformations both in

weed and crop species, after they have penetrated the plant tissue and are under way
to their target site. The selective action of a commercial herbicide in a certain crop
is mostly based on a faster rate of herbicide detoxification in this crop than in the
target weeds. Selectivity can be further increased by safener-enhanced metabolism
in crops to guarantee tolerance in a wider range of varieties or under borderline
conditions. As a rule, herbicide metabolites are more polar and more water-soluble
than the herbicidal parent compound, and they exhibit reduced phytotoxicity or are
completely nonphytotoxic.
Depending on the chemical structure, herbicide metabolism may involve a
three-phase process [30, 32]. The enzymatic reactions in all three steps of this
process are known to be safener-induced [28, 29] and are detailed in the following
three sections:
• Phase I: In this phase, the herbicides are oxidized, reduced, or hydrolyzed to
introduce or expose a functional group via CytP450-mediated reactions; typically,
carboxyl esters are hydrolyzed by esterases. While the first step of herbicide
metabolism often entails a partial or total detoxification of the parent compound,
there are other cases where the herbicidally active form is generated in the
first metabolic reaction (e.g., hydrolysis of the inactive fenoxaprop-P-ethyl to the

herbicidally active free acid fenoxaprop-P) [33] followed by detoxification of the
molecule in the subsequent metabolic step.
• Phase II: This includes conjugations to endogenous molecules such as glu-
tathione (GSH) or glucose, catalyzed by glutathione-S-transferases (GSTs) or
UDP-dependent glycosyltransferases (UGTs), respectively. Amino acid conjuga-
tion is very common in plants, with extensive research having been conducted on
2,4-D [30]. The GSTs are homo- or heterodimer, multifunctional enzymes that
are located in the cytosol and catalyze the nucleophilic attack of the sulfur atom
of GSH by the electrophilic center of the herbicide substrate [34, 35]. Dozens
of GST gene sequences from several plant species have been identified [29, 36,
37]. The important role of GSH and GSTs for herbicide detoxification in plants
encompasses the regulation and transport of often GS-X-tagged herbicides for
compartmentation in the vacuole and cell wall [38]. The glucose conjugation
of herbicides in plants may result in several metabolites, including O-, S-, and
N-glucosides, glucose esters, and malonyl-glucose conjugates. The most com-
mon glucose conjugates are O-glucosides, because pesticide oxidation reactions
generate hydroxyl groups that are suitable sites for glucose conjugation [30, 39].
Simple glucose conjugates are frequently subjected to secondary conjugations
to form glucosylglucosides or 6-O-malonylglucosides. The latter metabolites
are formed by the action of malonyltransferases using malonyl-CoA as acyl
donor [40]. Further conjugations with malonate via malonyl-CoA have also been
described [41].
• Phase III: At this stage, the hydrophilic and nontoxic Phase II conjugates are
transported to the vacuole, where further catabolic reactions may occur [39,
40]. Conjugate deposition, compartmentation or sequestration are catalyzed by
several types of ABC transporter located in the tonoplast or plasmalemma
[28, 42].
The degradation of GSH conjugates in the vacuole is often observed for herbi-
cides, and this may represent a further ‘‘trapping’’ mechanism [43, 44]. Various
peptidase enzymes sequentially cleave the amino acid glycine, then glutamate,
from the GSH moiety. It is assumed that these so-called ‘‘Phase IV’’ reactions
are not safener induced. The deposition of Phase II conjugates into the cell-wall
constituents as ‘‘bound residues’’ are also assigned as Phase IV reactions.
Based on presently available research data, the herbicide safeners clearly act
in crops predominantly by enhancing herbicide metabolism to create nonphyto-
toxic degradation products. Notably, all of the safeners investigated to date have
influenced only the rate of herbicide metabolism, and not the metabolic pathway.
Hence, safeners have never altered the metabolite patterns of herbicide, but have
only led to quantitative shifts in the ratios between the phytotoxic parent compound
and the metabolites, when compared to control plants without safener application.
Such quantitative differences between plants, with and without safener treatment,
are mainly apparent within the first days of the plant being treated with the
herbicide/safener combination.
8.3.2
Gene Induction and Signaling Pathways
During recent years, evidence has emerged that most safeners act on the transcrip-
tional level by interfering with pre-existing signaling pathways involved in plant
defense and stress responses.
Hundreds of genes that encode the proteins involved in herbicide metabolism and
detoxification (e.g. GSTs, CytP450 monooxygenases, ABC transporters, multidrug
resistance proteins) are induced rapidly and become proactive within a few hours
of safener treatment [28, 40, 45]. Transcriptional gene activation occurs not only
in monocotyledonous plants, but in a similar manner also in non-safener target
dicotyledonous species such as Arabidopsis thaliana. This suggests that there is a
common molecular safener mode of action for all plants [46].
The results of gene expression profiling experiments, conducted mainly in
Arabidopsis, have indicated parallels between the oxidative stress-related oxylipin
pathway and safener signaling [29]. In response to oxidative stress, plants accu-
mulate oxidized lipids that are derived from alpha-linolenic acid – the so-called
oxylipins (cyclopentenones, phytoprostanes). It has been shown [47] that these
compounds induce the expression of genes involved in defense and detoxification
reactions in a manner similar to safeners. Thus, it is suggested that safeners
might interfere with this oxylipin-mediated signaling pathway, thereby inducing
the expression of proteins that are involved in the detoxification of xenobiotics.
There is also evidence that many safener-inducible genes share a common
as-1-like cis-element in their promoters that binds the bZIP transcription factors
of the salicylic acid (SA)-responsive TGA protein family, which are known to
regulate defense and oxidative stress signaling [48, 49]. Indeed, it has been
shown (C. Behringer, K. Bartsch, and A. Schaller, unpublished results) that a
significant portion of the safener-regulated genes are inducible by SA. The same
study also demonstrated the presence of another group of TGA/SA-independent
safener-responsive genes having W-box elements in their promoters. These genes
may be regulated by WRKY transcription factors, which are known to play a key
role in pathogen defense [50] and oxidative stress response [51].
Taken together, these observations suggest that several signaling pathways
contribute to the full safener response in plants, although the primary target of
safener signaling remains unknown.
Future investigation to acquire an understanding of safener-mediated signaling
might be accomplished via metabolite profiling techniques, by determining the
levels of plant internal signaling substances (e.g., oxylipins, SA) in response to
safener treatment in wild-type plants and signal transduction mutants.
Other important questions which remain to be clarified are the organ-, tissue-,
and cell-specific patterns of gene expression for detoxifying pathways [52, 53], and
also the relationship between metabolic pathways induced by safeners in crops and
in weeds expressing a metabolic herbicide resistance [54].
8.3.3
Influence on Herbicide Uptake
Typically, part of any investigation into the mechanism(s) of safener action will
include seeking possible interactions of the safener with a herbicide at the point
of its uptake into the crop. An examination of the relevant literature provided
a complex picture, as was also noted by Davies and Caseley [2] in an exhaustive
compilation of the safener effects on herbicide uptake for relevant herbicide/safener
combinations developed up to that time. For example, in 20% of cases herbicide
uptake was reduced by the safener, in 40% the safener had no influence on herbicide
uptake, and in the remaining 40% the herbicide uptake was stimulated. In those
cases where herbicide uptake was reduced by the safener, however, the question
remained as to whether this was the basis of the safener’s action. For example, the
finding that the root uptake of an imidazolinone herbicide AC 263222 (imazapic)
by chlorophyllous maize seedlings was reduced by 19% after dressing the seed with
NA suggested that the latter had contributed to a protective action of the safener.
However, follow-up studies showed that NA could also exhibit a protective effect
when its interaction with herbicide uptake was excluded by applying the safener
one day after the herbicide. This observation, in conjunction with contradictory
results from other studies that showed either no effect or a stimulatory effect of NA
on herbicide uptake, questioned whether any interference with herbicide uptake
might play a significant role in the safener’s mechanism of action [55, 56]. It should
be noted here that contrasting results (e.g., inhibition of, stimulation of, or no
effect on herbicide uptake) have also been reported for other herbicide/safener
combinations.
Uptake studies were also conducted with combinations of the safener
mefenpyr-diethyl and the sulfonylurea herbicides mesosulfuron-methyl and
iodosulfuron-methyl-sodium, both of which are used for selective post-emergence
weed control in wheat crop. On both occasions, the safener had no influence on
herbicide uptake [57].
In summary, based on current experience only in a very few cases was herbicide
uptake by the crop reduced in combination with a safener, and even then doubts
remained as to whether the reduction of herbicide uptake formed part of the
safener’s mechanism of action. It is concluded, therefore, that interference with
herbicide uptake by the crop has no importance as a mechanism of safener action,
although it cannot be excluded that some cases might exist where it plays an
auxiliary role.
8.3.4
Influence on Herbicide Translocation
Many modern-day herbicides, when used in combination with a safener for selective
post-emergence weed control in cereal crops, serve as inhibitors of either ALS or
ACCase. The most sensitive morphological sites of action of these herbicides are
the meristematic tissues which, during the early stages of development, are located
at the shoot base of the grass weed, as well of the gramineous crops. After foliar
spraying of these herbicides, long-distance transport to the basal meristems is
a basic requirement to achieve either a herbicidal action in grass weeds, or the
phytotoxic effects in cereal crops. It follows that, in theory, such phytotoxic effects
could be prevented if a safener were to act via a specific inhibition of herbicide
translocation from the phloem to the site of action. Although, to date, no case
is known where a safener directly interferes with the long-distance translocation
of these herbicides, there may be indirect effects on translocation due to a
safener-induced enhancement of herbicide metabolism in the leaf mesophyll, which
in turn may influence the amount of herbicide and metabolites transferred into
the long-distance transport system. As an example, following the foliar application
of 14 C-labeled fenoxaprop-ethyl to wheat, translocation of the 14 C-labeled material
was not influenced by combination with the safener fenchlorazole-ethyl soon after
application. However, after a period of three days, the percentage of translocated
14
C-labeled material was reduced in the presence of the safener. This was interpreted
as an effect of differential kinetics of herbicide metabolism, and hence differential
mobility characteristics of 14 C, in the presence and absence of the safener [27].
Indirect effects on mobility were also reported for combinations of herbicides with
safeners applied pre-emergence, or by seed dressing. In corn seedlings treated with
[14 C]metazachlor, the amount of 14 C in the developing leaves was reduced when
the seedlings had been incubated with the dichloroacetamide safener BAS 145138
(Dicyclonon). Subsequent analytical data suggested that the safener-enhanced
metabolism of metazachlor to a polar non-mobile metabolite in the adjacent
seedling tissues had reduced the amount of 14 C reaching the developing leaves [58].
In maize seedlings treated with the 14 C-labeled imidazolinone herbicide imazapic
(AC 263222), the acropetal movement from root to shoot was markedly less in
seedlings that had received a seed dressing with the safener NA. This was attributed
to the safener-enhanced formation of an immobile metabolite being retained in
the seedling root [56].
8.4
Mode of Action of Safeners in Agricultural Practice
The mode(s) of action of selected safeners used in agricultural practice will be

reviewed more precisely in the following subsections. In all of these examples,
the safener action results from an enhancement of herbicide detoxification in
crops.
8.4.1
1,8-Naphthalic Anhydride (NA), Flurazole, and Fluxofenim
These chemically diverse safeners must all be applied to the crop (maize, sorghum)
by seed dressing in order to obtain a selective safener effect. The oldest and
best-examined of these compounds is NA, which acts as a safener in combination
with several classes of herbicide. Previous studies conducted by Sweetser [59]

had suggested that the action of NA as a safener for chlorsulfuron and other
sulfonylureas in maize was due to an enhancement effect on the oxidative
detoxification of these herbicides. Later, it was shown with the sulfonylurea
compound triasulfuron, that the effect of NA as a safener for this herbicide in maize
seedlings was due to the induction of a specific cytochrome P450-monooxygenase
that catalyzes the hydroxylation of the parent compound to the detoxification
product, 5-hydroxytriasulfuron [60].
NA also showed a stimulatory effect on the oxidative metabolism of the herbicide
bentazone. In this case, microsomal preparations of etiolated shoots from maize, the
seeds of which had been treated with NA, revealed bentazone hydroxylase activity
that was not present in extracts from control seeds without safener pre-treatment
[61]. The improved tolerance of maize to the imidazolinone AC 263222 after NA
seed treatment was also related to an enhanced hydroxylation of AC 263222 by the
stimulation of a cytochrome P450 monooxygenase [56].
Gronwald et al. [62] reported that NA and flurazole each caused substantial
increases in GST activity in corn and sorghum (17- and 30-fold, respectively),
when the herbicide metolachlor was used as substrate. This was well correlated to
the protective effect of these safeners against metolachlor injury. In contrast, the
stimulation of GST activity was less than twofold when, instead of metolachlor, the
unspecific substrate 1-chloro-2,4-dinitrobenzene (CDNB) was used as substrate.
Flurazole had very similar effects in combination with the herbicide metazachlor.
In particular, in sorghum it strongly stimulated the conjugation of this herbicide
with GSH [63].
The treatment of wheat seed with fluxofenim caused a ninefold increase in GST
activity, using the herbicide dimethenamid as a substrate. This increase correlated
well with an accelerated herbicide metabolism in wheat shoots, which was observed
as a response to fluxofenim treatment [64].
8.4.2
Dichloroacetamides
Safeners of the dichloroacetamide family are usually applied in combination with

the herbicide, either pre-plant-incorporated or pre-emergence. When Ekler and
Stephenson [63] investigated the mode of interaction of the dichloroacetamide
dichlormid, BAS 145138 and MG-191 with the herbicide metazachlor in maize
and sorghum, an increase was found in the GST-catalyzed conjugation rate of
metazachlor with GSH (five- to 11-fold), as well as an increase in GSH levels. The
influence of BAS 145138 on the behavior of metazachlor in maize was also examined
by Fuerst and Lamoureux [58], who concluded that the safener protected against
metazachlor injury by accelerating the enzyme-mediated GSH conjugation of the
herbicide. Similarly, the safener benoxacor was found to induce GST isoenzymes
in maize. The increase in GST activity, when assayed with metolachlor as substrate,
was closely correlated with the protection of maize against metolachlor injury. The
resolution of total GST activity, using fast protein liquid chromatography (FPLC),
resulted in four major activities which, to different degrees, were all stimulated by
the safener, though one of these was detectable only in safener-treated plants [65].
An induction of GST activity, determined with alachlor as the herbicide substrate,
was also reported for the dichloroacetamide safener R-29148 [66].
Although this group of safeners appeared predominantly to influence the GST
system, Lamoureux and Rusness [67] reported that, in maize, the safener BAS
145138 stimulated not only the GSH conjugation but also hydroxylation of the
herbicide chlorimuron-ethyl.
8.4.3
Fenclorim
The safener fenclorim is used to prevent injury caused by the herbicide pretilachlor
in paddy rice. When Deng and Hatzios [68] analyzed GST extracts from several
rice cultivars with and without fenclorim pre-treatment, the safener increased
GST activity with pretilachlor as substrate in all cases. Moreover, the FPLC
elution patterns revealed the presence of multiple GSTs, while mass spectrometry
confirmed the formation of a pretilachlor–GSH conjugate. In addition to increasing
the activity of the constitutive GST peaks, fenclorim also induced the formation
of up to five new peaks, depending on the cultivar, which had activity towards
pretilachlor.
8.4.4
Fenchlorazole-Ethyl, Cloquintocet-Mexyl
Fenchlorazole-ethyl/fenoxaprop-ethyl and cloquintocet-mexyl/clodinafop-propargyl

were the first safener/herbicide combinations to be used for selective post-
emergence weed control in cereals. Studies of the metabolism of fenoxaprop-ethyl
revealed a more rapid decline in the levels of fenoxaprop-ethyl and the free
acid fenoxaprop in wheat, when applied in combination with the safener
fenchlorazole-ethyl. The results of further studies conducted in wheat suggested
that the safener stimulated a GST-catalyzed detoxification reaction of the free
acid fenoxaprop, and this resulted in the formation of a GSH conjugate with the
6-chloro-benzoxazolone moiety of the herbicide molecule. This effect, which was
apparent within only a few hours after plant treatment, occurred only in the cereal
crop and not in the target grass weed species. These results suggested that the
specificity of safener action is based on a differential induction of the detoxification
reaction in the crop versus the grass weed species [27, 69, 70]. Subsequently, when
multiple isoenzymes of GST were purified by Cummins et al. [71] from wheat
shoots treated with fenchlorazole-ethyl, only the safener-inducible isoenzymes
were found to catalyze the detoxification of fenoxaprop-ethyl.
The protective effect of the safener cloquintocet-mexyl against the phytotoxicity
of clodinafop-propargyl in wheat was also found to be based on an enhancement
of herbicide detoxification in this crop. Following ester hydrolysis, the free acid
clodinafop was metabolized in wheat by ring hydroxylation and ether cleavage with
subsequent conjugate formation, whereas in the grass weed species the metabolism
involved malate ester formation. Notably, the safener specifically enhanced only
herbicide metabolism in wheat, and not in grass weed species [72, 73].
8.4.5
Mefenpyr-Diethyl
In the above-mentioned combination, fenoxaprop-ethyl/fenchlorazole-ethyl, the

racemic form of the herbicide was subsequently replaced by the biologically active
optical isomer fenoxaprop-P-ethyl, and fenchlorazole-ethyl by the new safener,
mefenpyr-diethyl. Mefenpyr-diethyl alone had no phytotoxic effects, even when
applied in very high dosages, but was readily taken up by the foliage of the
cereal crop and acted systemically. When fenoxaprop-P-ethyl, either alone or in
combination with mefenpyr-diethyl, was applied to the foliage of wheat, durum
wheat, or barley, it was – after foliar penetration – in both cases rapidly hydrolyzed
to the free acid fenoxaprop-P. However, the rate of the subsequent conversion of
the herbicidally active free acid into polar nonphytotoxic products was significantly
faster in the presence than in absence of the safener [26]. The key step leading to
the detoxification of fenoxaprop-P was again (as described above for the racemate
fenoxaprop) the GST-catalyzed attack of GSH at the fenoxaprop-P molecule, which
resulted in the formation of 4-hydroxyphenoxypropanoic acid and of a GSH
conjugate of 6-chlorobenzoxazolone (Figure 8.3a). Both products were subject
N O
OH
O O O O
O O O N
Cl O
S N N
Fenoxaprop O H H N
O
S N Mesosulfuron-methyl O
H
O
GSH
O
N O OH
OH O O
SG + HO O N
Cl O O S N N
O H H N
Glutathione 4-hydroxy-phenoxy- O
conjugate propanoic acid S N O
H
O
(a) Follow-up reactions (b) Follow-up reactions
Figure 8.3 Examples of herbicide detoxification reactions

stimulated by the safener mefenpyr-diethyl. (a) Conjugation
reaction of fenoxaprop with glutathione (GSH); (b) Oxidative
demethylation of mesosulfuron-methyl.
to further transformation reactions. Notably, mefenpyr-diethyl, as with the older

safener fenchlorazole-ethyl, acted exclusively by enhancement of the detoxification
reaction, but did not alter the pathway of herbicide metabolism nor the metabolite
pattern in the crop species. Furthermore, mefenpyr-diethyl did not significantly
influence the rate of fenoxaprop-P metabolism in wild oats (Avena fatua), as an
example of a representative grass weed species; hence, it acted specifically only in
the cereal crops.
Determinations of GST activity against CDNB and fenoxaprop in barley plants
revealed that the exposure to mefenpyr-diethyl increased the conjugation rate
with the unspecific substrate CDNB about twofold, while a 12-fold increase was
determined for the conjugation rate with fenoxaprop [74]. The suggestion was that
this was due to the specific induction by mefenpyr-diethyl of GST isoenzymes with
fenoxaprop-conjugating ability. Analogous findings were previously described after
application of the safener fenchlorazole-ethyl.
As mentioned above, one major advantage of mefenpyr-diethyl is that it can
act as a safener also in combination with sulfonylurea herbicides in cereal
species. Consequently, combinations have been developed with the sulfonylureas
iodosulfuron-methyl-sodium and mesosulfuron-methyl.
Studies on the mode of safener action in wheat indicated that the safener
enhanced the metabolic degradation of both herbicides in the crop species, but did
not significantly alter their rates of degradation in the target weed species wild oats
and blackgrass (Alopecurus myosuroides) [14, 26, 75].
In wheat, in analogy to the findings for fenoxaprop-P-ethyl, mefenpyr-diethyl
influenced only the rate of metabolism of the sulfonylurea compounds, but
did not cause any changes in the metabolite pattern. However, in contrast to
fenoxaprop-P-ethyl, GSTs were found not to be involved in the metabolic detoxifi-
cation of iodosulfuron-methyl sodium or mesosulfuron-methyl. Rather, the results
of plant metabolism studies suggested that specific cytochrome P450 monooxyge-
nases are responsible for catalyzing the early detoxification reactions. A metabolite
of mesosulfuron-methyl, which appeared first after application of the herbicide
to wheat plants, was identified as methyl 2-[3-(4-hydroxy-6-methoxypyrimidin-
2-yl)ureidosulfonyl]-4-methanesulfonamidomethyl-benzoate, and was most likely
formed by a P450 monooxygenase-catalyzed oxidative demethylation of the parent
compound. The formation of this metabolite was markedly stimulated in wheat by
the safener mefenpyr-diethyl [57] (Figure 8.3b).
8.4.6
Isoxadifen-Ethyl
This compound was developed as a safener for the sulfonylurea herbicide foram-
sulfuron in maize. The results of metabolism studies revealed that isoxadifen-ethyl
also acts by an enhancement of foramsulfuron metabolism in maize, but does
not influence the rate of metabolism of this herbicide in susceptible weed species
[17, 18].
8.4.7
Cyprosulfamide
The effect of CSA on the metabolic rate of the herbicide thiencarbazone-methyl

(TCM) was investigated in maize and three representative target weeds; Se-
taria viridis (SETVI), Chenopodium album (CHEAL), and Polygonum convolvulus
L. (POLCO). This was achieved by feeding excised shoots with 14 C-TCM alone
and in combination with the safener, and then measuring the levels of parent
and metabolites one and two days later. The results showed that, even without
safener, TCM was metabolized much faster in maize than in the weeds, which
explains why TCM has a certain innate selectivity margin between the maize and
the weeds. However, the metabolism rate in maize was increased significantly by
CSA, which may account for the strong safening activity observed. In accordance
with the specificity of the safener action in maize, no enhancement of herbicide
metabolism by the safener was found in CHEAL and POLCO, and only a weak
increase was detected in SETVI, which has no practical effect on weed control.
The pattern of 14 C-TCM metabolites in maize was not changed by the safener
treatment. Thus, it was concluded that the safener does not influence the pathway
of TCM metabolism, but merely acts by an enhancement of herbicide biotrans-
formation to nonphytotoxic products. CSA is taken up by roots and leaves and
shows systemic behavior. The long-distance transport of 14 C-labeled substances
could be demonstrated in both the apoplast (xylem) and symplast (phloem) of
plants.
8.5
Concluding Remarks
Since their discovery and introduction during the 1960–1970s, herbicide safeners
have provided a valuable tool for agriculture, enabling highly effective herbicides
to be used in situations that would otherwise be impossible. Although today, this
technology competes with genetically modified organisms (GMOs) or non-GMO
herbicide-tolerant crops, safeners still underpin an important part of the herbicide
market in maize, cereals, and rice. As described in Section 8.3, studies to identify
the mechanism of safener action have also provided valuable information to help
increase the present understanding of herbicide metabolism in crops and weeds.
The fact that, currently, many of the commercial safeners are off-patent, offers a
major opportunity for generic suppliers to enter the market together with off-patent
herbicides. In contrast, recent mixture patents with new herbicides still allow
exclusive usage by the patent holder. However, the fact that the number of patents
for new safener classes has declined dramatically over the past 15 years suggests
either a diminishing research success rate or, more likely, a discontinuation of
investigations into safeners in most research-based agrochemical companies.
References 395
In the first edition of this book, this chapter concluded . . . .

Nonetheless, because crop safeners allow the use of highly active and thus
commercially competitive herbicides in situations not otherwise possible,
it is expected that safeners will continue to play a valuable role in world
agriculture for the foreseeable future . . .
Since that sentence was written in 2006, almost all important new herbicide
products launched for cereals and corn have contained safeners. It appears,
therefore, that this still remains a valid way to close the chapter!
References
1. Hatzios, K. and Hoagland, R. (eds) 15. Rubin, B., Kirino, O., and Casida, J.
(1989) Crop Safeners for Herbicides, (1985) J. Agric. Food Chem., 33,
Academic Press, San Diego, CA. 489–494.
2. Davies, J. and Caseley, J. (1999) Pestic. 16. Collins, B., Drexler, D., Merkl, M.,
Sci., 55, 1043–1058. Hacker, E., Hagemeister, H., Pallett,
3. Davies, J. (2001) Pestic. Outlook, 10–15. K., and Effertz, C. (2001) Proc. Brighton
4. Hoffman, O. (1953) Plant Physiol., 28, Crop Prot. Conf. – Weeds, 1, 35–42.
622–628. 17. Pallett, K., Veerasekaran, P., Crudace,
5. Peek, J., Collins, H., Porpiglia, P., and M., Koecher, H., and Collins, B. (2001)
Ellis, J. (1988) Abstr. Annu. Weed Sci. Proceedings of the NCWSS Confer-
Soc. Am., 28, 13. ence, Milwaukee, Wisconsin, December
6. Boldt, L. and Barrett, M. (1989) Weed 10–13, 2001, Abstract 77.
Technol., 3, 303–306. 18. Hacker, E., Bieringer, H., Willms, L.,
7. Rowe, L., Kells, J., and Penner, D. Schnabel, G., Koecher, H., Hagemeister,
H., and Steinheuer, W. (2002) Z.
(1991) Weed Sci., 39, 78–82.
Pflanzenkr. Pflanzenschutz, Sonderh.,
8. Viger, P., Eberlein, C., and Fuerst, E.
XVII, 747–756.
(1991) Weed Sci., 39, 227–231.
19. Anonymous (1999) Fed. Regist., 64 (110),
9. Viger, P., Eberlein, C., Fuerst, E., and
30997–31000.
Gronwald, J. (1991) Weed Sci., 39,
20. Philbrook, B.D. and Santel,
324–328.
H.J. (2008) Weed Sci. Soc. Am.
10. Chang, F., Bandeen, J., and Stephenson,
Meet., On line Abstract no. 116.
G. (1972) Can. J. Plant Sci., 52,
http://wssa.net/Meetings/WSSAAbstracts/
707–714.
abstractsearch.php.
11. Bussler, B., White, R., and Williams, E. 21. Santel, H.J. and Philbrook,
(1991) Proc. Brighton Crop Prot. Conf. – B.D. (2008) Weed Sci. Soc. Am.
Weeds, 1, 39–44. Meet., On line Abstract no. 117.
12. Dill, T. (1986) Abstract 39th Annual http://wssa.net/Meetings/WSSAAbstracts/
Meeting Southern Weed Science Society, abstractsearch.php.
Nashville, Tennessee, January 20–22, 22. Watteyne, K., Castillo, T., Hinz, J.,
1986. Philbrook, B., and Bloomberg, J. (2009)
13. Amrein, J., Nyffeler, A., and Rufener, J. Proc. North Cent. Weed Sci. Soc. Conf.,
(1989) Proc. Brighton Crop Prot. Conf. – 119.
Weeds, 1, 71–76. 23. Anonymous (2008) Fed. Regist., 73 (200),
14. Hacker, E., Bieringer, H., Willms, L., 60969–60974.
Ort, O., Koecher, H., and Kehne, H. 24. EPA Memorandum (2008) Document
(1999) Proc. Brighton Crop Prot. Conf. – ID: EPA-HQ-OPP-2008-0042-0004.
Weeds, 1, 15–22. http://www.federalregister.gov/articles/
2008/10/15/E8-24034/cyprosulfamide- Butterworth-Heinemann, Oxford, pp.

pesticide-tolerances#p-3. 227–261.
25. Bieringer, H., Bauer, K., Hacker, E., 40. Kreuz, K., Tommasini, R., and
Heubach, G., Leist, K., and Ebert, E. Martinoia, E. (1996) Plant Physiol.,
(1989) Proc. Brighton Crop Prot. Conf. – 111, 349–353.
Weeds, 1, 77–82. 41. Sandermann, H., Haas, M., Messner,
26. Hacker, E., Bieringer, H., Willms, L., B., Pflumacher, S., Schroder, P., and
Rösch, W., Köcher, H., and Wolf, R. Wetzel, A. (1997) in Regulation of En-
(2000) Z. Pflanzenkr. Pflanzenschutz, zymatic Systems Detoxifying Xenobiotics
Sonderh., XVII, 493–500. in Plants, NATO ASI, Series (ed. K.K.
27. Köcher, H., Büttner, B., Schmidt, E., Hatzios), Kluwer Academic Publish-
Lötzsch, K., and Schulz, A. (1989) Proc. ers, Dordrecht, The Netherlands, pp.
Brighton Crop Prot. Conf. – Weeds, 2, 211–231.
495–500. 42. Jasinski, M., Ducos, E., Martinoia, E.,
28. Zhang, Q., Xu, F.-X., Lambert, K.N., and Boutry, M. (2003) Plant Physiol.,
and Riechers, D.E. (2007) Proteomics, 7, 131, 1169–1177.
1261–1278. 43. Wolf, A.E., Dietz, K.-J., and Schröder, P.
29. Riechers, D.E., Kreuz, K., and Zhang, Q. (1996) FEBS Lett., 384, 31–34.
(2010) Plant Physiol., 153, 3–13. 44. Cole, D.J. and Edwards, R. (2000) in
30. Van Eerd, L.L., Hoagland, R.E., Metabolism of Agrochemicals in Plants
Zablotowicz, R.M., and Hall, J.C. (2003) (ed. T. Roberts), John Wiley & Sons, Ltd,
Chichester, pp. 107–154.
Weed Sci., 51, 472–495.
45. Theodoulou, F.L., Clark, I.M., He,
31. Tommasini, R., Vogt, E., Schmid, J.,
X.-L., Pallett, K.E., Cole, D.J., and
Fromenteau, M., Anrhein, N., and
Hallahan, D.L. (2003) Pest Manage.
Martinoia, E. (1997) FEBS Lett., 411,
Sci., 59, 202–214.
206–210.
46. DeRidder, B.P., Dixon, D.P., Beussman,
32. Hatzios, K.K. (1991) in Environmental
D.J., Edwards, R., and Goldsbrough,
Chemistry of Herbicides (eds R. Grover
P.B. (2002) Plant Physiol., 130,
and A.J. Cessna), CRC Press, Boca
1497–1505.
Raton, FL, pp. 141–185.
47. Loeffler, C., Berger, S., Guy, A., Durand,
33. Köcher, H., Kellner, H.M., Lötzsch, K.,
T., Bringmann, G., Dreyer, M., von Rad,
Dorn, E., and Wink, O. (1982) Proc.
U., Durner, J., and Mueller, M.J. (2005)
Brighton Crop Prot. Conf. – Weeds, 1, Plant Physiol., 137, 328–340.
341–347. 48. Fode, B., Siemsen, T., Thurow, C.,
34. Armstrong, R.N. (1994) Adv. Enzymol. Weigel, R., and Gatz, C. (2008) Plant
Relat. Areas Mol. Biol., 69, 1–44. Cell, 20, 3122–3135.
35. Marrs, K.A. (1996) Annu. Rev. Plant 49. Mueller, S., Hilbert, B., Dueckershoff,
Physiol. Plant Mol. Biol., 47, 127–158. K., Roitsch, T., Krischke, M., Mueller,
36. Edwards, R., Dixon, D.P., and Walbot, M., and Berger, S. (2008) Plant Cell, 20,
V. (2000) Trends Plant Sci., 5, 193–198. 768–785.
37. Dixon, D.P., Lapthorn, A., and 50. Pandey, S.P. and Somssich, I.E. (2009)
Edwards, R. (2002) Genome Biol., 3, Plant Physiol., 150, 1648–1655.
3004.1–3004.10. 51. Miller, G., Shulaev, V., and Mittler, R.
38. Hatzios, K.K. (2001) in Pesticide (2008) Physiol. Plant., 133, 481–489.
Biotransformation in Plants and Mi- 52. Xu, F.-X., Lagudah, E.S., Moose, S.P.,
croorganisms: Similarities and Divergences and Riechers, D.E. (2002) Plant Physiol.,
(eds J.C. Hall, R.E. Hoagland, and 130, 362–373.
R.M. Zablotowicz), American Chemical 53. Riechers, D.E., Zhang, Q., Xu, F.-X.,
Society, Washington, DC, pp. 218–239. and Vaughn, K.C. (2003) Planta, 217,
39. Lamoureux, G.L., Shimabukuro, R.H., 831–840.
and Frear, D.S. (1991) in Herbicide Re- 54. Cummins, I., Bryant, D.N., and
sistance in Weeds and Crops (eds J.C. Edwards, R. (2009) Plant Biotechnol.,
Casely, G.W. Cussans, and R.K. Atkin), 7, 807–820.
References 397
55. Davies, J., Caseley, J., and Jones, O. 66. Holt, D., Lay, V., Clarke, E., Dinsmore,
(1995) Proc. Brighton Crop Prot. Conf. – A., Jepson, J., Bright, S., and Greenland,
Weeds, 1, 275–280. A. (1995) Planta, 196, 295–302.
56. Davies, J., Caseley, J., Jones, O., Barrett, 67. Lamoureux, G. and Rusness, D. (1992)
M., and Polge, N. (1998) Pestic. Sci., 52, Pestic. Biochem. Physiol., 42, 128–139.
29–38. 68. Deng, F. and Hatzios, K. (2002) Pestic.
Biochem. Physiol., 72, 24–39.
57. Köcher, H. (2005) Pflanz.-Nachr. Bayer,
69. Yaakoby, T., Hall, J., and Stephenson,
58, 179–194.
G. (1991) Pestic. Biochem. Physiol., 41,
58. Fuerst, E. and Lamoureux, G. (1992) Pes- 296–304.
tic. Biochem. Physiol., 42, 78–87. 70. Köcher, H., Trinks, K., and Schmidt, E.
59. Sweetser, P. (1985) Proc. Brighton Crop (1993) Abstracts SCI Meeting, Interac-
Prot. Conf. – Weeds, 3, 1147–1154. tions between Pesticides in Mixtures,
60. Persans, M. and Schuler, M. (1995) London.
Plant Physiol., 109, 1483–1490. 71. Cummins, I., Cole, D., and Edwards,
61. McFadden, J., Gronwald, J., and R. (1997) Pestic. Biochem. Physiol., 59,
Eberlein, C. (1990) Biochem. Biophys. 35–49.
Res. Commun., 168, 206–213. 72. Kreuz, K., Gaudin, J., Stingelin, J., and
62. Gronwald, J., Fuerst, E., Eberlein, C., Ebert, E. (1991) Z. Naturforsch., 46c,
and Egli, M. (1987) Pestic. Biochem. 901–905.
73. Kreuz, K. (1993) Proc. Brighton Crop
Physiol., 29, 66–76.
Prot. Conf. – Weeds, 3, 1249–1258.
63. Ekler, Z. and Stephenson, G. (1989)
74. Scalla, R. and Roulet, A. (2002) Physiol.
Weed Res., 29, 181–191. Plant., 116, 336–344.
64. Riechers, D., Irzyk, G., Jones, S., and 75. Hacker, E., Bieringer, H., Willms, L.,
Fuerst, E. (1997) Plant Physiol., 114, Lorenz, K., Koecher, H., Huff, H.,
1461–1470. Borrod, G., and Brusche, R. (2001) Proc.
65. Fuerst, E., Irzyk, G., and Miller, K. Brighton Crop Prot. Conf. – Weeds, 1,
(1993) Plant Physiol., 102, 795–802. 43–48.
399
9
Genetically Modified Herbicide-Resistant Crops
9.1
Overview
9.1.1
Introduction
Herbicides are classified as either selective or broad-spectrum. Selective herbicides

can be used in-crop to control weeds without causing any significant crop damage,
whereas broad-spectrum herbicides such as glyphosate and glufosinate are limited
to pre-plant or post-directed applications. The technology to engineer herbicide
resistance has enabled the in-crop use of broad-spectrum herbicides for improved
weed control and yield.
Herbicide resistance can be generated either through the introduction of a
gene, or through a selection process. Crops generated via the introduction of a
gene are referred to here as genetically modified (GM), whilst those generated
through a selection process have been developed by identification of the desired
herbicide-resistant trait from a natural or mutagenized population of cells or
plants. In this section, whenever data are available, consideration will be given
to all herbicide-resistant crops, regardless of the process by which they were
generated.
In 2009, GM crops were cultivated on 330 million acres in 25 countries, with 62%
of those acres being accounted for by herbicide-resistant traits in soybean, corn,
canola, and cotton. This percentage was increased to 83% if herbicide-resistance
trait acres that were stacked with other biotechnology traits were included. Globally,
GM herbicide-resistant soybean, cotton, canola, and corn were grown on 171 million
(52%), 9 million (3%), 16 million (5%), and 80 million (24%) acres, respectively
(Figure 9.1.1) [1].
The growing global use of GM crops has had several positive agronomic,
economic, and environmental impacts. Since 1996, the use of GM crops has
reduced pesticide use by 8.4% (equivalent to 352×106 kg of active ingredient),
while the environmental impact quotient [2] has also been decreased by 16.3%
[3]. GM crops have also produced significant environmental benefits; in addition

400 9 Genetically Modified Herbicide-Resistant Crops
450
400
350
300
Acres (Millions)
250
200
150
100
50
0
Soybean Cotton Canola Corn
Conventional Herbicide-resistant
Figure 9.1.1 Global cultivation of conventional or GM-resistant crops in 2009.
to reduced pesticide use, increased no-till practices have led to reduction in water
run-off, greenhouse gas emissions, and have also improved the habitats for birds
and animals [4]. These benefits are even greater if one includes all herbicide
resistant crops and are expected to continue to expand with the adoption of
biotechnology crops.
9.1.2
Mechanisms for Engineering Herbicide Resistance
In general, herbicide resistance can be achieved through four primary strategies: (i)
detoxification of the herbicide to a nonphytotoxic metabolite; (ii) the expression of
an herbicide-insensitive target; (iii) overexpression of the herbicide target; and (iv)
cellular sequestration of the herbicide away from the target. Of these strategies, only
the first two have so far been used successfully to develop commercial products.
Alternative reviews on herbicide resistance are available to the interested reader
[5, 6].
9.1.2.1 Detoxification of Herbicides

This strategy has led to the commercial development of herbicide resistance
for glufosinate, glyphosate, and bromoxynil. Resistance to both glufosinate and
glyphosate are discussed in detail later in the chapter (see also Chapter 9.2). The
herbicidal activity of bromoxynil is based on the inhibition of electron transport in
photosystem II (PS II). Crops engineered with the enzyme bromoxynil nitrilase are
capable of metabolizing the herbicide to a nonphytotoxic compound [7].
Detoxification has also been utilized to engineer resistance against several other
herbicides, including various auxins such as 2,4-D and dicamba, diphenyl ethers
(DPEs) such as oxyfluorfen and acifluoren, pyridines such as thiazopyr, and
9.1 Overview 401
chloroacetanilides such as alachlor. However, to date, none of these has been

developed into a commercial product [5].
9.1.2.2 Expression of an Insensitive Herbicide Target

This strategy was used to engineer resistance against glyphosate, and also against
imidazolinones and sulfonylureas that inhibit acetolactate synthase (ALS), a key
enzyme in the biosynthesis of branched-chain amino acids. ALS-resistant crops
have been generated primarily through the selection for an herbicide-insensitive
ALS allele from natural or mutagenized cell or plant populations [5].
The expression of an herbicide-insensitive target has also been reported to provide
resistance to diclofop and sethoxydim – both of which are acetyl-CoA carboxylase
(ACCase) inhibitors – in addition to various dinitroanilines and inhibitors of phy-
toene desaturase (PDS), lycopene cyclase, and hydroxyphenylpyruvate dioxygenase
(HPPD). None of these traits is currently incorporated into commercial products,
however.
Currently, there are no commercially available herbicide-resistant crops that
function via an increased expression of the protein target, although some level of
plant resistance has been reported for glyphosate, glufosinate, some DPEs, and
inhibitors of HPPD. Similarly, the cellular sequestration of a herbicide from the
target has been reported with some DPEs, auxins, and PS I inhibitors, but none
has yet been developed commercially [5].
9.1.3
Commercialized Herbicide-Resistant Crops
This section includes data for herbicide-resistant crops generated by both selection
and biotechnology processes. The first herbicide-resistant crop to be made commer-
cially available in the United States was imidazolinone-resistant corn, introduced
in 1992, followed in 1996 by glyphosate-resistant soybean and canola. Since then,
the cultivation of herbicide-resistant crops has expanded globally, with multiple
herbicide-resistant traits available in many large-acre crops. The United States
cultivates the greatest acreage of herbicide-resistant crops and will be the primary
focus of these discussions.
9.1.3.1 Herbicide-Resistant Soybeans

Currently, three commercially available herbicide-resistant traits are present in
®
soybean, namely glyphosate resistance (Roundup Ready , RR), sulfonylurea re-
® ®
sistance (STS ), and glufosinate resistance (Liberty Link , LL). Today, herbicide
resistance accounts for over 90% of approximately 73 million total soybean acres
grown in the United States (Figure 9.1.2) [8].
The RR soybean was developed using a glyphosate-insensitive CP4-EPSPS
(5-enolpyruvyl shikimate 3-phosphate synthase). Since its introduction in 1996,
the RR soybean has increased steadily from 2% to approximately 94% of the total
soybean acreage grown in 2009 (Figure 9.1.2). The widespread adoption of RR
soybeans has resulted in significant grower and environmental benefits. Since
100
LL Soybean
STS soybean
80 RR soybean
% Total Acres
60
40
20
0
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
Year
Figure 9.1.2 Percentage of total acres of herbicide-resistant soybeans by trait.
1996, there has been about a 64% increase in the number of no-till soybean acres,
leading to decreased soil erosion, dust and pesticide run-off, and improved soil
moisture, air, and water quality [4]. The STS soybean was introduced in 1993, with
crop resistance being achieved by traditional breeding for an insensitive ALS allele.
Since its introduction in 1993, the STS soybean has accounted for 2–4% of the
total soybean acres in production (Figure 9.1.2). Glufosinate resistance in soybean
(LL) involves a bacterial gene encoding the enzyme protein of l-phosphinothricin
acetyl transferase (PAT), which confers tolerance to glufosinate ammonium. PAT
functions by detoxifying l-phosphinothricin, which is the active constituent of
glufosinate ammonium herbicides. The LL soybean was first commercialized in
2009.
9.1.3.2 Herbicide-Resistant Cotton

Currently, three commercially available herbicide resistant traits are present in cot-
ton, namely glyphosate resistance (RR), glufosinate resistance (LL), and bromoxynil
®
resistance (BXN ). In 2009, herbicide resistance traits were cultivated on 90% of
approximately nine million total cotton acres in the United States (Figure 9.1.3) [9],
an acreage which represents a combination of herbicide resistance alone or stacked
with other traits such as insect control.
RR cotton, engineered with CP4-EPSPS, was commercialized in 1997 and has
since grown from 5% to 88% of the total cotton acres in 2009 (Figure 9.1.3). RR
Flex cotton, a second-generation product, was commercialized in 2006. The new
technology enables glyphosate applications over-the-top from emergence through
seven days prior to harvest, and represents a significant improvement from the
first-generation product that limited glyphosate application through the four-leaf
stage.
9.1 Overview 403
100
LL cotton
BXN cotton
80
RR cotton
% Total Acres
60
40
20
0
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
Year
Figure 9.1.3 Percentage of total herbicide-resistant cotton acres by trait.
®
Glufosinate (Liberty™ and Basta ) inhibits the glutamine synthetase activity
that results in a toxic build-up of ammonia in plant cells. LL cotton, which was
commercialized in 2004, was derived via detoxification by PAT. In 2009, LL cotton
was grown on 2% of the total cotton acres (Figure 9.1.3).
®
Bromoxynil (Buctril ) inhibits electron transport in PS II by binding to the D1
protein. BXN cotton, achieved via detoxification and introduced in 1996, peaked at
about 7% of the total cotton acres in 1998, but has since steadily declined in use
and was last sold in 2004 (Figure 9.1.3).
As with soybean, the adoption of herbicide-resistant cotton has resulted in
significant grower and environmental benefits. The use of these traits in 2006
alone has resulted in a reduction in crop production costs of US$ 230 million.
One major effect of herbicide-resistant cotton has been the significant increase in
the adoption of no-till production. In the case of cotton in the US, this resulted in
savings of fuel and labor costs of about US$ 135 million in 2006 alone [4].
9.1.3.3 Herbicide-Resistant Corn

Currently, three commercially available herbicide-resistant traits are present in
corn, namely glyphosate resistance (RR), glufosinate resistance (LL), and imida-
®
zolinone resistance (Clearfield , CF). In 2009, herbicide-resistant traits were grown
on almost 90% of approximately 86.5 million total corn acres in the United States
(Figure 9.1.4) [9]. This total represents a combination of herbicide resistance alone
or stacked with other traits.
RR corn was commercialized in 1998, with resistance being achieved via the
expression of a glyphosate-insensitive TIPS-EPSPS, which is a maize enzyme with
two amino acid mutations that conferred glyphosate insensitivity (see Chapter
9.2, Section 9.2.3). A second-generation RR corn trait with CP4-EPSPS showed
120
100 CF corn
LL corn
% Total Acres
80 RR corn
60
40
20
0
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
Year
Figure 9.1.4 Percentage of total acres of herbicide-resistant corn by trait. 2008 and
2009 percentage exceeds 100% due to the presence of products containing multiple
herbicide-resistant traits.
improved glyphosate resistance and was introduced in 2001. Since its introduction
in 1998, RR corn has grown to approximately 80% of the total corn acres in
production for 2009 (Figure 9.1.4).
Imidazolinones (e.g., imazethapyr and imazapyr), like sulfonylureas, inhibit the
enzyme ALS. CF corn, commercialized in 1992, was developed by mutagenesis
and the selection of an imidazolinone-insensitive ALS allele. Since its introduction
in 1992, CF corn has been used up to approximately 5% of the total corn acres,
declining to less than 1% in 2009 (Figure 9.1.4).
LL corn was developed via detoxification with the PAT gene, and was introduced
commercially in 1997. LL corn has grown to about 36% of the total corn acres in
2009 (Figure 9.1.4).
®
Sethoxydim (e.g., Poast ) inhibits ACCase, which is the first committed step
in de novo fatty acid synthesis. Sethoxydim-resistant (SR) corn was achieved by
traditional breeding and selection for the herbicide-insensitive ACCase allele, and
introduced in 1996. Typically, SR corn accounted for less than 0.3% of corn
acres in any one year, and is no longer commercially available in field corn
(Figure 9.1.4).
Until 2004, the adoption of herbicide-resistant traits in corn was slower than in
other crops, due mainly to trade restrictions in export markets for GM products.
Nevertheless, the adoption of herbicide-resistant corn has resulted in significant
grower and environmental benefits. For example, the use of glyphosate and
glufosinate resistance traits in 2006 alone resulted in a reduction in crop production
costs of US$ 315 million, with numerous positive environmental attributes from
the increase in no-till acreage [4].
9.1.3.4 Herbicide-Resistant Canola

Canada cultivates 95% of the canola acres in North America, and is the focus
of this survey. The three primary commercial herbicide-resistant traits in canola
References 405
120
CF canola
100 LL canola
RR canola
% Total acres
80
60
40
20
0
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
Year
Figure 9.1.5 Percentage of total acres of herbicide-resistant canola by trait.
are glyphosate resistance (RR), glufosinate resistance (LL), and imidazolinone

resistance (CF). Two other herbicide-resistant traits that are used on a relatively
®
small number of acres are bromoxynil resistance (BXN ) and triazine tolerance
(TT). In 2009, herbicide-resistant traits were grown on over 99% of the 16.7 million
total canola acres in Canada (Figure 9.1.5) [9].
RR canola was introduced in Canada in 1996. Unlike other RR crops, RR canola
was achieved by a combination of an insensitive enzyme (CP4-EPSPS) and a
detoxification enzyme (glyphosate oxidase; GOX) that catalyzes the degradation of
glyphosate to aminomethylphosphonic acid (AMPA) [10]. Since its commercializa-
tion, RR canola has expanded from about 1% to 41% of the total Canadian canola
acres in 2009 (Figure 9.1.5).
LL canola, introduced commercially in 1995, was achieved via detoxification by
PAT. The use of LL canola has grown from about 6% to 50% of the total Canadian
canola acres in 2009 (Figure 9.1.5).
CF canola, introduced commercially in 1995, was achieved by mutagenesis and
the selection of an imidazolinone-insensitive ALS allele. CF canola was grown on
approximately 8% of Canadian canola acres in 2009.
BXN canola was used in less than 100 000 acres at its peak, and has since
been withdrawn from the market. Triazine-resistant canola, achieved through a
selection process, has been used on a limited number of acres but has never gained
popularity.
The adoption of herbicide-resistant canola has resulted in significant grower and
environmental benefits. Herbicide-resistant varieties allow farmers to plant earlier
and to control weeds better with minimal crop injury, which in turn results in
greater yields. GM canola varieties have allowed farmers to save an estimated 8.2
million gallons of fuel and earned, on average, US$ 10 more per acre. Their use
has also increased the adoption of low and no-till farming, saving millions of acres
from soil erosion [11].
References
1. James, C. (2009) Global Status of Com- 6. Gressel, J. (2002) Molecular Biology
mercialized Biotech/GM Crops: 2009, of Weed Control, Taylor and Francis,
ISAAA Brief No. 41, ISAAA, Ithaca. London, New York, pp. 219–277.
2. Kovach, J., Petzoldt, C., Degni, J., and 7. Freyssinet, G., Leroux, B., Pelissier, B.,
Tette, J. (1992) A method to mea- Lebrun, M., and Sailland, A. (1989) Serv.
sure the environmental impact of Biol. Mol. Cell Veg., 75, 49–55.
pesticides, New York’s Food and Life 8. GfK–Kynetec–AgroTrak
Sciences Bulletin, NYS Agricultural Ex- Study http://www.gfk.com/gfk-
periment Station, Cornell University, kynetec/industries/crop_protection_biotech_
Geneva, NY, p. 139. Annually updated MarketShare_AgroTrak/index.en.html
http://www.nysipm.cornell.edu/publications/ (accessed December 2010).
EIQ.html (accessed December 2010). 9. The Context Network Biotech Traits
3. Brookes, G. and Barfoot, P. (2010)
Commercialized (Global 2010 edition),
Global impact of biotech crops: environ-
http://www.contextnet.com (accessed
mental effects, 1996–2008. AgBioForum,
December 2010).
13 (1), 76–94.
10. Barry, G.F., Kishore, G.M., Padgette,
4. Sankula, S. and Blumenthal, E. (2005)
S.R., Taylor, M., Kolacz, K., Weldon, M.,
Biotechnology-derived Crops Planted
Re, D., Eichholtz, D., Fincher, K., and
in 2004 – Impacts on US Agriculture,
http://www.ncfap.org (accessed December Hallas, L. (1992) in Biosynthesis and
2010). Molecular Regulation of Amino Acids in
5. CaJacob, C.A., Feng, P.C.C., Heck, Plants (eds B.K. Singh, H.E. Flores, and
G.R., Alibhai, M.F., Sammons, R.D., J.C. Shannon), American Society of
and Padgette, S.R. (2004) in Handbook Plant Physiologists, Madison, WI, pp.
of Plant Biotechnology (eds P. Christou 139–145.
and H. Klee), John Wiley & Sons, Ltd, 11. BioteCanada http://www.biotech.ca
Chichester, pp. 353–372. (accessed December 2010).
9.2
Inhibitors of 5-Enolpyruvyl Shikimate 3-Phosphate Synthase (EPSPS)
9.2.1
Introduction
5-Enolpyruvyl shikimate 3-phosphate synthase (EPSPS) is the sixth enzyme in

the shikimate pathway that leads to the biosynthesis of aromatic amino acids,
tryptophan, tyrosine, and phenylalanine. These aromatic amino acids, along with
intermediates of the pathway, give rise to important secondary metabolites com-
monly referred to as phenylpropanoids that include phenolics, lignins, tannins, and
phytoalexins [1]. The shikimate pathway is localized in plastids, and EPSPS is a key
enzyme in regulating the flux through the pathway.
EPSPS catalyzes the transfer of phosphoenol pyruvate (PEP) to shikimate
3-phosphate (S3P) to produce 5-enolpyruvyl shikimate 3-phosphate (EPSP). This
reaction starts with the binding of S3P to EPSPS to form a binary complex, followed
by the binding of PEP to produce EPSP. Glyphosate (N-phosphonomethyl glycine)
9.2 Inhibitors of 5-Enolpyruvyl Shikimate 3-Phosphate Synthase (EPSPS) 407
is an uncompetitive inhibitor of EPSPS, and competes favorably with PEP for

binding to the EPSPS/S3P binary complex to form a ‘‘dead-end complex.’’ The
binding specificity of glyphosate to EPSPS is extremely high [2]. Thousands of
structural homologs, analogs, and derivatives have been synthesized and screened
for their ability to inhibit EPSPS, though most have shown little to no activity.
To date, glyphosate is the only commercialized molecule for which the herbicidal
activity is attributed to the inhibition of EPSPS.
Since EPSPS is found in plants, fungi, and bacteria [3], glyphosate shows a
broad spectrum of activity against most plant species. However, owing to the
absence of EPSPS in mammals, birds, or fish, glyphosate exhibits little to no
toxicity in these species [1]. Glyphosate, which was first commercialized in 1975, is
®
the active ingredient of numerous commercial formulations, including Roundup .
The mechanism of action of glyphosate is attributed the inhibition of EPSPS, which
results in a build-up of shikimate and thus a depletion of aromatic amino acids and
phenylpropanoid metabolites.
9.2.2
Factors That Impact Glyphosate Efficacy
The success of glyphosate as a herbicide goes well beyond its ability to inhibit
EPSPS, however. Glyphosate as a salt is highly water-soluble; in fact, it is one of
the most systemic herbicides and is readily translocated in the phloem, thereby
accessing difficult-to-control underground tissues such as the roots [4]. Glyphosate
is applied post-emergence to foliar tissues, and its efficacy is dependent on the
efficiency of absorption. Glyphosate exhibits little pre-plant activity as the molecule
is tightly bound to soil and not available to plants or leaching through the
soil. Glyphosate in soil is quickly metabolized by microorganisms, primarily to
aminomethylphosphonic acid (AMPA), with a half-life ranging from less than a
week to months, depending on the soil type [2].
9.2.2.1 Foliar Absorption

A variety of studies have shown consistently that foliar absorption through the
cuticle represents the greatest barrier to glyphosate efficacy [5–7]. Over the years,
glyphosate formulations have been developed to facilitate its absorption into the
plant, with proprietary formulations having been developed using surfactants to
maximize the amount and rate of absorption. Unfortunately, overly aggressive
surfactants that cause excessive local tissue injury may antagonize glyphosate
translocation [5, 8].
The standard method for studying the plant uptake of glyphosate is to apply
droplets of the radiolabeled compound to a single leaf of a plant. This method
of application is drastically different from field applications, where a formulation
after dilution to the desired volume is atomized and sprayed over-the-top of plants
through a nozzle (Figure 9.2.1). The leaf droplet method ignores any potential
variables in foliar spray interception, retention, and coverage. Furthermore, studies
have shown that the foliar absorption of glyphosate is affected by concentrations of
Spray atomization
Plant interception, retention, coverage
Foliar absorption
Systemic translocation
Efficacy
Figure 9.2.1 Schematic representation of events that char-

acterize an over-the-top spray application of glyphosate for-
mulation in planta.
glyphosate and surfactant, as well as the spray droplet size [6, 8]. The leaf droplet
method typically uses a droplet size of 1 μl, which is equivalent to a diameter
of 1200 μm. In comparison, a typical flat fan nozzle generates a broad range of
droplet sizes from less than 100 μm to more than 1000 μm, with a considerably
smaller volume median diameter of only 173 μm [9]. Studies have shown that
glyphosate uptake is improved with large size droplets; however, this is offset
by a reduced foliar retention and coverage during spray applications. Increasing
the spray volume increases the foliar coverage, but at the expense of reduced
surfactant and glyphosate concentrations. It is apparent that the plant absorption
of glyphosate is affected by numerous interdependent variables that cannot be
adequately modeled by the leaf droplet method [8].
An initial study in velvetleaf (Abuthilon theophrasti) plants employed the standard
leaf droplet method to compare glyphosate absorption among commercial formu-
lations [5]. Realizing the limitation of the leaf droplet method, a cautious start was
made by using a track sprayer for over-the-top spray application of formulations
augmented with radiolabeled glyphosate [8, 10–13]. The track sprayer method
required much greater care and preparation during experimentation, but allowed
data to be generated on the absorption of glyphosate under realistic field use rates
and parameters.
In a subsequent study, the track sprayer method was used to compare the ab-
sorption of 14 C-glyphosate among different commercial formulations in velvetleaf
[10]. The plants were sprayed with a field use rate (0.2 kg acid equivalent [a.e.]
ha−1 ) at a volume of 93 l ha−1 , using a commercially available flat-fan nozzle. A
similar spray retention was observed in plants among the three formulations,
indicating that difference in efficacy could not be attributed to differential spray
retention. Following spray application, the plants were harvested at various times
to measure the levels of glyphosate that were remaining on the foliar surfaces,
had been absorbed into the leaves, and had been translocated to the roots. With
spray application, the applied dose varied, depending on the plant size; therefore,
the absorption efficiency was expressed as a percentage of total recovered dose
from each plant. Significant differences were observed among the formulations
in the rate of glyphosate absorption (Figure 9.2.2). The most efficient formulation
Foliar absorption as 40
% recovered dose
30
20
A
10 B
C
0
0 20 40 60 80 100 120
Time (HAT)
Figure 9.2.2 Comparison of foliar absorption of

14 C-glyphosate with time (hours after treatment, HAT) in
commercial formulations A, B, and C, using an over-the-top

track spray application in young velvetleaf plants.
(Formulation A) showed a rapid absorption of glyphosate at 28% of the applied

dose by 24 hours after treatment (HAT) and plateaued thereafter. Formulation B
showed a much reduced absorption, at only 16% by 24 HAT, while formulation C
showed a slow initial absorption (13% at 24 HAT) but a more prolonged absorption
[10]. These results illustrated the subtle differences in glyphosate absorption among
commercial formulations in velvetleaf under realistic field application parameters.
Not surprisingly, glyphosate absorption is also affected by species differences in
cuticle structure and leaf morphology. Using the track sprayer method, the foliar
absorption of 14 C-glyphosate at field use rates with a flat-fan nozzle ranged from
®
20% to 36% in velvetleaf, prickly sida, kochia, and Roundup Ready (RR) corn,
which is glyphosate-resistant (GR) [10, 14]. These results indicated that studies on
foliar absorption of glyphosate must not only employ a relevant method (i.e., track
sprayer) but also be conducted in the species of interest.
9.2.2.2 Systemic Translocation

Most plant species do not metabolize glyphosate [2], with the exception of soy-
bean, which slowly metabolizes it to AMPA [15]. As a result, once glyphosate
enters the phloem, it is translocated along the sucrose gradient from source to
sink tissues [16]. The accumulation of glyphosate in sink tissues produces local
injuries that diminish the sink strength and sucrose demand, resulting in reduced
glyphosate translocation; thus, glyphosate imposes a self-limitation on its own
efficacy [17, 18]. Sink tissues can sense the effect of glyphosate within a few
hours after application, although visible injury may take 7–10 days to appear [17].
For these reasons, efficacious glyphosate formulations generally exhibit a rapid
uptake and translocation in order to avoid self-limitation [10]. At the same time,
a rapid absorption also results in favorable rainfastness for greater application
flexibility.
With the track sprayer method, the systemic translocation of absorbed glyphosate
among commercial formulations was measured in velvetleaf plant roots that had
been shielded from the spray by soil. Formulation A – the most extensively absorbed
(28%; see Figure 9.2.2) – showed a 6% translocation to roots at 24 HAT (Figure 9.2.3)
[10]. The root translocation was found to be proportional to foliar absorption and
followed the ranking of formulation A > C > B, which was also the ranking of
the formulations in terms of overall plant efficacy. These results showed that, even
with an efficient absorption, only about one-third of the applied dose was absorbed,
and only a fraction of that would be translocated to the roots at 24 HAT. As the
amount translocated was proportional to that absorbed, increasing the absorption
would increase the overall efficacy, as long as translocation was not hindered in the
process.
Initial microscopy studies in velvetleaf plants showed that the large 1 μl droplets
used in the leaf droplet method caused localized spot necrosis on the leaf [5, 19,
20], whereas smaller droplets – as encountered in spray applications – caused little
to no visible local injury [21]. The results of a recent study conducted in RR cotton
also showed that glyphosate distribution to bolls differed between an over-the-top
spray versus a manual leaf droplet application [12]. This difference was caused by
the fact that different leaves, depending on age and position, source different bolls
in plants, and consequently the glyphosate translocation was dependent on which
leaves intercepted the spray. These results further demonstrated that glyphosate
application to a single leaf may produce misleading data on the absorption and
translocation of glyphosate, with little relevance to spray applications in the field.
Nevertheless, the leaf droplet method continues to be used by most research groups
when studying herbicide absorption and translocation in plants.
12
A
10 B
Root translocation as
% recovered dose
C
8
0
0 20 40 60 80 100 120
Time (HAT)
Figure 9.2.3 Comparison of root translocation of

14 C-glyphosate with time (hours after treatment, HAT)
in commercial formulations A, B, and C following an

over-the-top spray application in young velvetleaf plants.
9.2.3
Development of Glyphosate-Resistant Crops
A major milestone occurred in 1996 with the commercial introduction of GR

soybean [22], although since that time the GR trait (RR) has been introduced
into canola, cotton, corn, alfalfa, and sugar beet, among others. Until now, all RR
crops contain a glyphosate-insensitive EPSPS derived either from the plant or from
bacteria.
The X-ray crystal structures of Escherichia coli EPSPS showed that the enzyme
consists of two domains that undergo a conformational change upon ligand binding
[23, 24]. The resulting closure of the two domains forms the catalytic pocket at
the interface [25]. Glyphosate binding appeared to be modulated by several key
amino acids which are located in close vicinity to the catalytic pocket [23] and which
are highly conserved across species. In fact, they have been used to predict the
sensitivity of the EPSPS to glyphosate [26]. The results of kinetic analyses showed
that the endogenous maize EPSPS has a Km -PEP of 27 μM with a Ki of 0.5 μM
(Table 9.2.1). When mutations were introduced at the T102I and P106S positions,
in order to produce the variant TIPS [26], the TIPS-EPSPS showed a Km of 10.6 μM
with a Ki of 58 μM. The double-mutant enzyme preserved the EPSPS function
while reducing sensitivity to glyphosate. An analysis of single mutations (T102I
or P106S) showed an increased Km or reduced affinity for glyphosate; hence, the
desired kinetic properties were obtained only in the double-mutant enzyme [26].
Bacterial sources of EPSPS were screened for insensitivity to glyphosate [27].
The EPSPS which proved to be most insensitive to glyphosate was isolated from
Agrobacterium species CP4. This enzyme showed a desirable Km -PEP (12 μM) but
a much greater Ki (2720 μM), with a Ki /Km ratio that was 41-fold higher than that
of TIPS-EPSPS [26]. CP4-EPSPS, which is kinetically superior to TIPS-EPSPS,
is currently utilized in all RR crops. The TIPS-EPSPS was utilized in the
first-generation RR corn (GA21) [28], but has since been replaced by CP4-EPSPS
in the second-generation RR corn 2 (NK603) [29].
9.2.3.1 Alternative Mechanisms for Engineering Glyphosate Resistance

Attempts were made to engineer glyphosate resistance by the increased expression
of a glyphosate-sensitive (GS) EPSPS in E. coli, petunia, and Arabidopsis [30–32].
Transgenic petunia with >20-fold increased expression of EPSPS showed a limited
Table 9.2.1 Comparison of steady-state kinetics of EPSPS

enzymes from plant and bacteria that are used to engineer
glyphosate resistance in crops.
EPSPS Km -PEP (μM) Ki (μM) Ki /Km
Maize wild-type 27.0 ± 4.0 0.50 ± 0.06 0.02

Maize TIPS 10.6 ± 1.6 58.0 ± 14 5.5
Agrobacterium CP4 12 2720 227
resistance to glyphosate, with growth being inhibited at field use rates [31]. These
results suggested that an increased expression of a sensitive EPSPS would not be
likely to generate commercial-level resistance to glyphosate in crops.
An alternative mechanism would be to use enzymes that catalyze glyphosate
detoxification. Glyphosate oxidoreductase (GOX) was cloned from the Achro-
mobacter sp. strain LBAA and subsequently shown to catalyze the degradation of
glyphosate to AMPA (Figure 9.2.4) [33]. Glyphosate resistance was observed in
plants expressing the GOX gene, but at a level which was insufficient for com-
mercialization. GOX is currently utilized in conjunction with CP4-EPSPS in RR
canola. Recent reports have indicated that AMPA may exhibit some plant toxicity
of its own right [15], which in turn makes GOX less desirable for the engineering
of glyphosate resistance.
Reports by Castle et al. [34] and Siehl et al. [35] described a glyphosate acetyl-
transferase (GAT; Figure 9.2.4) which is useful for engineering plant resistance to
glyphosate. GAT was originally cloned from Bacillus licheniformis, but conferred no
resistance to glyphosate when expressed in any host. The catalytic efficiency of GAT
was improved through 11 iterations of DNA shuffling such that the resulting gene,
when expressed in tobacco and maize, conferred resistance to field use rates of
glyphosate. A recent patent publication [36] described yet another bacterial enzyme
(glyphosate decarboxylase GDC) which was suitable for engineering glyphosate
resistance and described as having homology to decarboxylases and, presumably,
an ability to catalyze the decarboxylation of glyphosate. At present, both GAT
and GDC are reportedly under development, although neither gene has yet been
utilized in a commercial crop.
9.2.3.2 Disease Control Benefits of Glyphosate-Resistant Crops

Although, in addition to plants, EPSPS is also found in both fungi and bacteria,
glyphosate was shown previously to possess a very weak fungicidal activity (ED50
100 to >1000 mg g−1 ). Recent reports by Feng et al. [13], and Anderson and Kolmer
[37] have shown that glyphosate reduced the incidence of leaf and stripe rusts in
O H
P N + CHOCOOH
GOX HO H
O H OH
P N COOH AMPA glyoxylate
HO
OH O
Glyphosate GAT O C
P N COOH
HO
OH
N-acetyl glyphosate
Figure 9.2.4 Detoxification enzymes that may be useful for

engineering glyphosate resistance in crops. GOX, glyphosate
oxidoreductase; GAT, glyphosate acetyltransferase.
RR wheat, and also of Asian rust in RR soybean. The causative fungi of these
conditions (Puccinia triticina, P. striiformis, and Phakopsora pachyrhizi, respectively)
are obligate pathogens, and have in the past been responsible for major yield losses
in both wheat and soybean.
The results of studies conducted in RR wheat have shown that glyphosate, at a
spray dose typically recommended for weed control (i.e., 0.84 kg a.i. ha−1 ), provided
both preventive and curative activities for a period of 2–4 weeks against leaf and
stripe rusts. The disease control was shown to be minimal in formulation blanks
without glyphosate, and to correlate directly with the level of systemic glyphosate
in the leaves. Field tests conducted under natural heavy stripe rust pressure further
confirmed the activity of glyphosate [13]. Recent studies have also demonstrated
the activity of glyphosate towards P. pachyrhizi, which causes Asian soybean rust
[38]; the preliminary results have also demonstrated activities against several early
season diseases in soybeans [39].
The inhibition of EPSPS by glyphosate causes an accumulation of shikimate
in plant tissues, and this has in the past served as a molecular marker for
glyphosate injury. In contrast to conventional plants, RR soybeans – which are
engineered with a glyphosate-insensitive CP4-EPSPS – showed neither injury nor
any increase in tissue shikimate levels following glyphosate application. When
infected with P. pachyrhizi, the shikimate levels in RR soybeans remained low;
however, when infected RR soybeans were treated with glyphosate there was
significant and time-dependent accumulation of shikimate. As the source of this
shikimate could not be from RR soybeans, which are insensitive to glyphosate and
displayed no injury, the increased levels were attributed to an inhibition of EPSPS
in P. pachyrhizi. As further evidence of these findings, EPSPS from P. pachyrhizi
was cloned and its sensitivity to glyphosate confirmed. Taken together, these
results indicated that fungal EPSPS can be sensitive to glyphosate and accumulate
shikimate when exposed to glyphosate. Whilst previous results had suggested that
disease suppression was due to the direct actions of glyphosate [13], these latest
results provided further evidence that the disease-suppression activity of glyphosate
is due to an inhibition of fungal EPSPS [39]. Indeed, the results obtained suggest
that glyphosate may provide beneficial effects of disease suppression in a variety of
RR crops.
From the standpoint of engineering glyphosate resistance, either an insensitive
EPSPS or a detoxification gene should be equally feasible. However; the disease
control benefits of glyphosate would be expected to be associated mostly with
the use of an insensitive EPSPS due to the preservation of glyphosate in crops.
This has been observed in glufosinate-resistant crops, with glufosinate having
also shown fungicidal and disease control activities in glufosinate-resistant plants
[40–42]. Glufosinate showed only a two- to three-day disease control window
[40], which was much shorter than that observed with glyphosate [13]. This can
be explained simply by the fact that glufosinate-resistant plants are engineered
with phosphinothricin-N-acetyltransferase (PAT), which effectively detoxifies the
herbicidal and fungicidal activities of glufosinate. Presumably, plants engineered
with a glufosinate-insensitive glutamine synthetase would demonstrate a longer

disease control window.
9.2.4
Effects of CP4 Expression on Plant Resistance
The lack of plant metabolism and the use of a glyphosate-insensitive EPSPS

translate to a persistence of glyphosate, which continues to mobilize from the source
to the sink tissues in RR crops [26]. The sink tissues in a plant vary, depending
on the growth stage; consequently, the timing of a glyphosate spray – which is
determined by the best weed control – will impact on which sink tissues are at risk
for glyphosate injury. Past experience in developing RR crops has taught that male
reproductive tissues are not only strong sinks but are also vulnerable to glyphosate
injury [26].
Monsanto’s approach to the creation of second-generation RR traits in crops such
as corn, cotton, and soybean has been to improve upon the first-generation products
by coordinating the expression of the highly glyphosate-insensitive CP4-EPSPS
in tissues that are at-risk to glyphosate injury. Therefore, the development of
second-generation RR cotton, corn, and soybean has shifted from strong con-
stitutive viral promoters to regulatory elements with enhanced expression, both
spatial and temporal, in at-risk tissues such as the developing pollen and tape-
tum. These regulatory expression elements have been engineered as part of a
second CP4-EPSPS expression cassette in the case of second-generation RR corn
2 and Roundup Ready Flex® (RRF) cotton. These second-generation products
have shown an enhanced field performance compared to their forerunners. The
different promoters used in first- and second-generation RR crops are highlighted
in Table 9.2.2.
Table 9.2.2 Genetic elements used to engineer glyphosate
resistance in first- and second-generation Roundup
Ready crops.
Roundup Event(s) Expression cassette 1 Expression cassette 2

Ready® crop
Promoter/ Coding Promoter/ Coding
intron region intron region
RR corn GA21 Os.Act1/Os.Act1 TIPS-EPSPS None None

RR corn 2 NK603 Os.Act1/Os.Act1 CP4-EPSPS e35S/hsp70 CP4-EPSPS
RR cotton 1445 FMV CP4-EPSPS None None
RR Flex cotton MON88913 FMV/TSF1 CP4-EPSPS 35S/ACT8 CP4-EPSPS
RR soybean 40-3-2 e35S CP4-EPSPS None None
RR 2 yield soybean MON19788 FMV/TSF1 CP4-EPSPS None None
RR canola RT73 FMV CP4-EPSPS FMV GOX
RR alfalfa J101 and J163 eFMV CP4-EPSPS None None
9.2.4.1 Roundup Ready Cotton

The second-generation RRF cotton was commercialized in 2006 to provide growers
with a greater flexibility in the amount and timing of glyphosate application [43].
Both, first- and second-generation products employ CP4-EPSPS; however, RRF
cotton employs two CP4-EPSPS expression cassettes [44–47]. In particular, the
first cassette uses a chimeric promoter composed of the Arabidopsis thaliana TSF1
gene promoter that encodes the elongation factor EF-1alpha [48–50] and enhancer
sequences from the Figwort Mosaic virus 35S promoter [51], together with a
cis-acting TSF1 intron. The second cassette utilizes another chimeric promoter
composed of the ACT8 gene promoter of A. thaliana [52], combined with the
enhancer region of the cauliflower mosaic virus (CaMV) 35S promoter [53] together
with intron sequences from the ACT8 gene. These chimeric promoters provide a
strong vegetative expression from the viral enhancer elements and, at the same
time, boost expression in key male reproductive organs via the promoter elements
from TSF1 and ACT8 (Figure 9.2.5b,c). The result is that RRF cotton plants are
able to withstand glyphosate applications, with excellent boll retention throughout
the growing season (Figure 9.2.5a).
(a)
(b) (c) MP
MP
AW
AW
Figure 9.2.5 Field performance and tis- and 14-node stages; (b) Immunolocaliza-
sue expression profiles of CP4-EPSPS tion of CP4-EPSPS protein in the anther
in the first-generation (RR cotton) and wall (AW), but not in the mature pollen
second-generation (Roundup Ready Flex (MP), in event 1445 of RR cotton; (c) Strong
cotton, RRF) products. (a) Comparison CP4-EPSPS expression in both AW and
of boll retention between RRF cotton MP in event RR60, a predecessor of RRF
(right) and RR cotton (left) treated with cotton.
Roundup (2.5 kg a.e. ha−1 ) at 4, 6, 10,
9.2.4.2 Roundup Ready Corn

The expression of CP4-EPSPS by the e35S promoter led to the production of
corn plants that exhibited vegetative tolerance but poor reproductive tolerance (i.e.,
male sterility) when challenged with commercially applicable rates of glyphosate
[26, 29]. The use of rice actin 1 promoter (Os Act1) and intron elements [54]
boosted expression in key male reproductive tissues and produced male fertility.
The first-generation RR corn (GA21) employs the Os Act1 promoter and introns
with the TIPS-EPSPS gene [28]. The second-generation RR corn 2 (NK603) [29]
employs CP4-EPSPS in two expression cassettes driven by Os Act1 and e35S
promoters for high expression in both male reproductive and vegetative tissues,
thus giving rise to robust and consistent field performance.
9.2.4.3 Roundup Ready Soybean

The first-generation RR soybean event utilizes the CP4-EPSPS gene under the
transcriptional regulation of the 35S promoter [22]. RR soybeans demonstrate
excellent reproductive fertility following the application of glyphosate at labeled
rates. The second-generation product employs a chimeric promoter, which is
comprised of enhancer elements from the figwort mosaic virus 35S promoter and
the A. thaliana promoter from the TSF1 gene (35S/TSF1) in combination with the
cis-acting TSF1 intron, driving an optimized synthetic version of the CP4 EPSPS
gene [50, 55, 56]. The second-generation soybean product was commercialized in
2009 as Roundup Ready 2 Yield® (RR2Y), which will be the platform for stacking
future soybean traits [57].
9.2.5
Stacking Traits in Roundup Ready Crops
The extensive – and often exclusive – use of glyphosate for weed control in many
crops has contributed to the development of resistance in weeds. Given sufficient
and persistent selection pressure, it can be assumed that weeds have been evolv-
ing – and will continue to evolve – resistance to herbicides. To date, glyphosate
resistance has been reported in 18 biotypes of weeds worldwide [58]; nevertheless,
glyphosate is expected to remain as the dominant herbicide for weed control due
to its many attributes that include its efficacy, cost, and broad spectrum activity
[57, 59].
Currently, weed resistance management strategies have been developed to
control GR weeds, and to delay the development of resistance in weeds. A key
component of this strategy is the use of herbicides with an alternative mode of
action (MOA). Although several selective herbicides have been used effectively to
manage GR-weeds, the choices can be limited depending on the crop because, while
many of the selective herbicides have marginal crop safety and weed resistance
issues of their own.
A second approach has been to identify alternative broad-spectrum herbicides
and to genetically engineer crop safety. The decision was taken to engineer crop
resistance to dicamba and glufosinate based on numerous factors including efficacy,
weed spectrum, safety, costs, and compatibility with glyphosate. Both, dicamba
and glufosinate control a significant number of weed species with relatively few
weed resistance issues; furthermore, the ability to use dicamba, glufosinate, and
glyphosate in crop would provide options and flexibility for the control of weeds [57].
At present, other companies are pursuing a similar approach and have selected to
engineer crop resistance to other herbicides, including 2,4-dichlorophenoxyacetic
acid (2,4-D) and the inhibitors of acetolactate synthase (ALS) and hydroxyphenyl
pyruvate dioxygenase (HPPD) [60, 61].
9.2.5.1 Engineering Crop Resistance to Dicamba

Dicamba, a synthetic auxin herbicide which has been in commercial use since the
1960s, belongs to the benzoic acid class and differs structurally from 2,4-D (in the
phenoxy class). Dicamba exhibits broad-spectrum efficacy against dicotyledonous
or broadleaf plants, with limited safety for monocotyledonous or narrow-leaf
plants. Commercial formulations such as Clarity® and Banvel® (BASF Corp) are
labeled for soybean, cotton, and corn. In soy and cotton, Clarity can be applied
pre-emergence at 0.27–0.55 kg a.e. ha−1 with a 15- to 30-day delayed planting
in order to avoid crop injury. In corn, similar rates can be applied between
pre-emergence and early post-emergence; however, injury such as lodging and the
malformation of brace roots can occur in corn, depending on the rate of use, the
germplasm, and the environmental conditions.
Soybean resistance to dicamba was achieved via a deactivation mechanism
using an enzyme, dicamba mono-oxygenase (DMO), which had been isolated
from a soil microbe, Stenotrophomonas maltophilia DI6 (previously Pseudomonas
maltophilia). DMO catalyzes the O-demethylation of dicamba to 3,6-dichlorosalicylic
acid (DCSA), which is devoid of herbicidal activity (Figure 9.2.6) [57, 62]. A soybean
transformation of DMO was mediated by Agrobacterium and utilized a cassette
containing a constitutive viral promoter from peanut chlorotic streak virus [63]
with a chloroplast targeting sequence. The transformants were challenged with
a post-emergence spray of dicamba in the greenhouse at 0.55 kg a.e. ha−1 (the
estimated rate of use in the field). At subsequent generations, transgenic plants
were repeatedly challenged with pre- and post-emergence applications of dicamba.
Events that showed safety to more than twofold rates of dicamba were selected and
advanced for further field analysis [57].
The performance of a herbicide-resistant trait is ultimately measured by yield
under realistic field environments. To date, dicamba-resistant (DR) soybean events
NADH, O2
COOH COOH
Cl OCH3 Cl OH
+ H2O + HCHO
DMO
Cl Cl
dicamba 3,6-DCSA
Figure 9.2.6 Metabolic deactivation of dicamba

to 3,6-dichlorosalycilic acid (DCSA) by dicamba
mono-oxygenase (DMO).
have undergone several field tests to examine the impact of dicamba applications
on yield. The average yield across 15 locations between plots with or without
dicamba treatment in three DR soybean events is shown in Figure 9.2.7. The yield
was measured independent of weed control, as all plots were maintained weed-free
with conventional herbicides. The DR-soybean events displayed excellent safety
and, most importantly, no yield reduction from multiple, high rates of dicamba
(1.12 or 2.24 kg ha−1 , 2× or 4×) applied from pre-emergence to the V3 (three-leaf )
or R1 (reproductive) stages. The use of DR-soybeans will eliminate planting delays
following dicamba application, and allow growers the flexibility of applying dicamba
from pre-emergence to R1. It is planned that DR-soybeans will be stacked with
RR2Y soybeans to provide resistance to both dicamba and glyphosate.
9.2.5.2 Engineering Crop Resistance to Glufosinate

Glufosinate is a broad-spectrum herbicide that inhibits glutamine synthetase,
the enzyme that catalyzes the assimilation of ammonia with glutamate to form
glutamine. Crop resistance to glufosinate has been achieved by use of either the
BAR gene from Streptomyces hygroscopicus, or the PAT gene from Streptomyces
viridochromogenes. These genes produce highly homologous l-phosphinothricin
acetyltransferases that effectively deactivate glufosinate. Glufosinate-resistant crops
(Liberty Link® trait, Bayer CropScience) have been developed and commercialized,
as described previously [57]. Subsequently, the decision was taken to introduce
dicamba and glufosinate resistance into cotton, which will then be stacked with
glyphosate resistance.
For this, an Agrobacterium-mediated transformation was used to introduce a
binary cassette containing the DMO and BAR genes into cotton plants, after which
60
Average yield (bu a−1)
(N = 15 locations, SE)
No dicamba control
40 1.12 kg ha−1, Pre/V3
2.24 kg ha−1, Pre/V3
1.12 kg ha−1, Pre/V3/R1
20
0
DR-1 DR-2 DR-3
Dicamba-resistant (DR) soybean events in
field trials under weed-free conditions
Figure 9.2.7 Average soybean yield [bushels (bu) a−1 ]

across 15 locations of three dicamba-resistant (DR) events
without and with dicamba treatments at 1.12 or 2.24 kg ha−1
(2× or 4×) at pre-emergence (Pre) and V3, or 1.12 kg ha−1
at Pre, V3 and R1 growth stages. All plots were maintained
weed-free with the use of conventional herbicides.
the transgenic plants were challenged in the greenhouse with spray applications of
dicamba and glufosinate. The results from initial field trials (Figure 9.2.8) showed
excellent crop safety to multiple sprays of dicamba and glufosinate. The arrows in
Figure 9.2.8a point to injury in conventional cotton after treatment with 0.55 kg ha−1
(1×) of glufosinate (top left) or dicamba (top right). In contrast, good crop safety was
observed with dicamba- and glufosinate-resistant (DGR)-cotton plants treated with
three applications of glufosinate (2×) (Figure 9.2.8a bottom) or four applications of
dicamba (2×) (Figure 9.2.8b) during the growing season. Preliminary field results
showed that DGR-cotton would allow multiple applications of dicamba and/or
glufosinate. When stacked with RR Flex cotton, the flexibility of using dicamba,
glufosinate, and glyphosate in crop will greatly expand weed control options for the
growers.
The use of stacked herbicide-resistant traits is exemplified in the control of
palmer pigweed (Amaranthus palmeri), a species that has developed resistance
to glyphosate and which has significantly impacted upon agricultural practices
in southern USA. In this case, populations of GS and GR palmer were treated
in the greenhouse with a post-emergence application of glyphosate, dicamba, or
glufosinate. The results showed that glyphosate or dicamba at their 1× labeled
rates (840 or 560 g a.e. ha−1 , respectively) provided excellent control of GS-palmer,
whereas glufosinate was considerably less effective (Figure 9.2.9). With GR-palmer,
glyphosate control at the 1× rate was reduced to about 50%, although the dicamba
control remained excellent. Dicamba has demonstrated a similar control of other
GR-weeds, including waterhemp, horseweed, and ragweeds. These data not only
show clearly that dicamba will be an effective tool in managing many dicotyledonous
Conventional cotton DGR-cotton event

1× glufosinate 1× dicamba Four 2× dicamba applications
DGR-cotton event
Three 2× glufosinate applications
(a) (b)
Figure 9.2.8 Field performance of dicamba- application of glufosinate (1×, 0.55 kg ha−1 )
and glufosinate-resistant (DGR) and con- or dicamba (1×, 0.55 kg ha−1 ); bottom:
ventional cotton following applications of DGR-cotton following three applications of
dicamba or glufosinate. (a) Top left and 2× glufosinate; (b) DGR-cotton following
right arrows: conventional cotton following four applications of 2× dicamba.
100
% Control at 18 DAT (N = 6, SE)

−1
140 g ha
−1
280 g ha
80 −1
560 g ha
−1
840 g ha
60
40
20
0
glyphosate dicamba glufosinate glyphosate dicamba glufosinate
(1x=840 (1x=560 (1x=560 (1x=840 (1x=560 (1x=560
g ha−1) g ha−1) g ha−1) g ha−1) g ha−1) g ha−1)
GS-Palmer GR-Palmer
GS, glyphosate-sensitive; GR, glyphosate-resistant; glyphosate as in
Roundup Weathermax; dicamba as in Clarity; glufosinate as in Ignite
Figure 9.2.9 Greenhouse control of glyphosate-sensitive

(GS) or glyphosate-resistant (GR) palmer pigweed (Ama-
ranthus palmeri) with a post-emergence application of
glyphosate (1× rate at 840 g a.e. ha−1 ), dicamba (1× rate at
560 g a.e. ha−1 ), or glufosinate (1× rate at 560 g a.e. ha−1 ).
The data are averages of six replicates (with SE).
GR-weeds, but also illustrate the benefits of stacked herbicide resistance traits in
crops.
The development of glyphosate resistance in weeds has necessitated changes in
weed management strategies by incorporating herbicides with alternative MOAs. In
addition to the use of selective herbicides, crop safety is currently being engineered
in other broad-spectrum herbicides such as dicamba and glufosinate. This should
not only greatly increase the available weed control options but also reduce the
potential occurrence of weed resistance. Ultimately, the main objective is to preserve
the utility of glyphosate and to maintain the effectiveness and simplicity of weed
control in order to maximize crop yields.
References
1. Buchanan, B.B., Gruissem, W., and 3. Kishore, G.M. and Shah, D.M. (1998)
Jones, R.L. (2000) Biochemistry Molecular Annu. Rev. Biochem., 57, 627–663.
Biology of Plants, American Society of 4. Bromilow, R.H., Chamberlain, K., and
Plant Physiologists, Rockville, MD, pp. Patil, S.G. (1990) Pestic. Sci., 30, 1.
1286–1311. 5. Feng, P.C.C., Ryerse, J.S., and
2. Franz, J.E., Mao, M.K., and Silorski, Sammons, R.D. (1998) Weed Technol.,
J.A. (1997) Glyphosate: A Unique Global 12, 300–307.
Herbicide, ACS Monographs, Vol. 189, 6. Liu, S.H., Campbell, R.A., Studens, J.A.,
American Chemical Society, Washing- and Wagner, R.G. (1996) Weed Sci., 44,
ton, DC, pp. 65–101. 482–488.
References 421
7. Ramsey, R.J.L., Stephenson, G.R., and Amrhein, N., Evans, J.N.S., and Kabsch,
Hall, J.C. (2005) Pestic. Biochem. Physiol., W. (2001) Proc. Natl Acad. Sci. USA, 98,
82, 162–175. 1376–1380.
8. Feng, P.C.C., Chiu, T., Sammons, R.D., 24. Stallings, W.C., Meguid-Abdel, S.S.,
and Ryerse, J.S. (2003) Weed Sci., 51, Lim, L.W., Shieh, H.S., Dayringer, H.E.,
443–448. Leimgruber, N.K., Stegeman, R.A.,
9. Etheridge, R.E., Womac, A.R., and Anderson, K.S., Sikorski, J.A., Padgette,
Mueller, T.C. (1999) Weed Technol., 13, S.R., and Kishore, G.M. (1991) Proc.
765–770. Natl Acad. Sci. USA, 88, 5046–5050.
10. Feng, P.C.C., Sandbrink, J.J., and 25. Alibhai, M. and Stallings, W.C.
Sammons, R.D. (2000) Weed Technol., (2001) Proc. Natl Acad. Sci. USA, 98,
14, 127–132. 2944–2946.
11. Feng, P.C.C., Tran, M., Chiu, T., 26. CaJacob, C.A., Feng, P.C.C., Heck,
Sammons, R.D., Heck, G.R., and G.R., Alibhai, M.F., Sammons, R.D.,
CaJacob, C.A. (2004) Weed Sci., 52, and Padgette, S.R. (2004) in Handbook
498–505. of Plant Biotechnology (eds P. Christou
12. Feng, P.C.C. and Chiu, T. (2005) Pestic. and H. Klee), John Wiley & Sons, Ltd,
Biochem. Physiol., 82, 36–45. Chichester, pp. 353–372.
13. Feng, P.C.C., Baley, G.J., Clinton, W.P., 27. Padgette, S.R., Re, D.B., Barry, G.F.,
Bunkers, G.J., Alibhai, M.F., Paulitz, Eichholtz, D.A., Delannay, X., Fuchs,
T.C., and Kidwell, K.K. (2005) Proc. Natl R.L., Kishore, G.M., and Fraley, R.T.
Acad. Sci. USA, 48, 17290–17295.
(1996) in Herbicide-resistant Crops:
14. Feng, P.C.C., Sandbrink, J.J., and
Agricultural, Environmental, Economic,
Cowell, J.E. (2000) Abstract #40, Na-
Regulatory, and Technical Aspects (ed.
tional Meeting of Weed Science Society
S. Duke), CRC Lewis Publishers, Boca
of America, Toronto, Canada, p. 17.
Raton, FL, pp. 53–84.
15. Reddy, K.N., Rimando, A.M., and Duke,
28. Sidhu, R.S., Hammond, B.G., Fuchs,
S.O. (2004) J. Agric. Food Chem., 52,
R.L., Mutz, J.N., Holden, L.R., George,
5139–5143.
B., and Olson, T. (2000) J. Agric. Food
16. Feng, P.C.C., Chiu, T., and Sammons,
Chem., 48, 2305–2312.
R.D. (2003) Pestic. Biochem. Physiol., 77,
29. Heck, G.R., Armstrong, C.L., Astwood,
83–91.
J.D., Behr, C.F., Bookout, J.T., Brown,
17. Geiger, D.R. and Bestman, H.D. (1990)
Weed Sci., 38, 324–329. S.M., Cavato, T.A., DeBoer, D.L.,
18. Geiger, D.R., Shieh, W.J., and Fuchs, Deng, M.Y., George, C., Hillyard,
M.A. (1999) Pestic. Biochem. Physiol., 64, J.R., Hironaka, C.M., Howe, A.R.,
124–133. Jakse, E.H., Ledesma, B.E., Lee, T.C.,
19. Feng, P.C.C., Ryerse, J.S., Jones, C.R., Lirette, R.P., Mangano, M.L., Mutz,
and Sammons, R.D. (1999) Pestic. Sci., J.N., Qi, Y., Rodriguez, R.E., Sidhu,
55, 385–386. S.R., Silvanovich, A., Stoecker, M.A.,
20. Ryerse, J.S., Feng, P.C.C., and Yingling, R.A., and You, J. (2005) Crop
Sammons, R.D. (2001) Microsc. Today, 1, Sci., 44, 329–339.
22–24. 30. Klee, H.J., Muskopf, Y.M., and Gasser,
21. Ryerse, J.S., Downer, R.A., Sammons, C.S. (1987) Mol. Gen. Genet., 210,
R.D., and Feng, P.C.C. (2004) Weed Sci., 347–442.
52, 302–309. 31. Shah, D.M., Horsch, R.B., Klee, H.J.,
22. Padgette, S.R., Kolacz, K.H., Delannay, Kishore, G.M., Winter, J.A., Tumer,
X., Re, D.B., Lavallee, B.J., Tinius, C.N., N.E., Hironaka, C.M., Sanders, P.R.,
Rhodes, W.K., Otero, Y.I., Barry, G.F., Gasser, C.S., Aykent, S., Siegel, N.R.,
Eichholtz, D.A., Peschke, G.M., Nida, Rogers, S.G., and Fraley, R.T. (1986)
D.L., Taylor, N.B., and Kishore, G.M. Science, 233, 478–481.
(1995) Crop Sci., 35, 1451–1461. 32. Rogers, S.G., Brand, L.A., Holder, S.B.,
23. Schönbrunn, E., Eschenburg, S., Sharps, E.S., and Brackin, M.J. (1983)
Shuttleworth, W.A., Schloss, J.V., Appl. Environ. Microbiol., 46, 37–43.
33. Barry, G.F., Kishore, G.M., Padgette, 44. Chen, Y.S., Hubmeier, C., Tran, M.,
S.R., Taylor, M., Kolacz, K., Weldon, M., Martens, A., Cerny, R.E., Sammons,
Re, D., Eichholtz, D., Fincher, K., and R.D., and CaJacob, C. (2006) Plant
Hallas, L. (1992) in Biosynthesis and Biotechnol. J., 4, 477–487.
Molecular Regulation of Amino Acids in 45. Fincher, K.L., Flasinski, S., and
Plants (eds B.K. Singh, H.E. Flores, and Wilkinson, J.Q. (2003) Plant expression
J.C. Shannon), American Society of constructs. US Patent 6,660,911.
Plant Physiologists, Madison, WI, pp. 46. Fincher, K.L., Flasinski, S., and
139–145. Wilkinson, J.Q. (2005) Chimeric
34. Castle, L.A., Siehl, D.L., Gorton, R., cauliflower mosaic virus 35S-arabidopsis
Patten, P.A., Chen, Y.H., Bertain, S., actin 8 promoters and methods of using
Cho, H., Duck, N., Wong, J., and Liu, D. them. US Patent 6,919,495.
(2004) Science, 304, 1151–1154. 47. Fincher, K.L., Flasinski, S., and
35. Siehl, D.L., Castle, L.A., Gorton, R., Wilkinson, J.Q. (2005) Chimeric fig-
Chen, Y.H., Bertain, S., Cho, H., wort mosaic virus-elongation factor 1 α
Keenan, R., Liu, D., and Lassner, M.W. promoters and methods of using them.
(2005) Pest Manag. Sci., 61, 235–240. US Patent 6,949,696.
36. Hammer, P.E., Hinson, T.K., Duck, 48. Curie, C., Liboz, T., Gander, E.,
N.B., and Koziel, M.G. (2005) Meth- Medale, C., Bardet, C., Axelos, M.,
ods to confer herbicide resistance. and Lescure, B. (1991) Nucleic Acids Res.,
WO 2005003362. 19, 1305–1310.
49. Curie, C., Axelos, M., Bardet, C.,
37. Anderson, J.A. and Kolmer, J.A. (2005)
Atanassova, R., Chaubet, N., and
Plant Dis., 89, 1136–1142.
Lescure, B. (1993) Mol. Gen. Genet.,
38. Feng, P.C.C., Clark, C., Andrade, G.C.,
238, 428–436.
Balbi, M.C., and Caldwell, P. (2008) Pest
50. Axelos, M., Bardet, C., Liboz, T.,
Manag. Sci., 64, 353–359.
Le Van Thai, A., Curie, C., and
39. Dill, G.M., Sammons, R.D., Feng,
Lescure, B. (1989) Mol. Gen. Genet.,
P.C.C., Kohn, F., Kretzmer, K.,
219, 106–112.
Mehrsheikh, A., Bleeke, M., Honegger,
51. Richins, R., Scholthof, H., and Shepard,
J.L., Farmer, D., Wright, D., and
R. (1987) Nucleic Acids Res., 15,
Haupfear, E.A. (2010) in Glyphosate
8451–8466.
Resistance in Crops and Weeds: History,
52. An, Y.Q., McDowell, J.M., Huang, S.,
Development, and Management (ed. V.K. McKinney, E.C., Chambliss, S., and
Nandula), John Wiley & Sons, Inc., Meagher, R.B. (1996) Plant J., 10,
Hoboken, NJ, pp. 1–34. 107–121.
40. Wang, Y., Browning, M., Ruemmele, 53. Kay, R., Chan, A., Daly, M., and
B.A., Chandlee, J.M., and Kausch, A.P. McPherson, J. (1987) Science, 236,
(2003) Weed Sci., 51, 130–137. 1299–1302.
41. Liu, C.A., Zhong, H., Vargas, J., Penner, 54. McElroy, D., Zhang, W., Cao, J., and
D., and Sticklen, M.B. (1998) Weed Sci., Wu, R. (1990) Plant Cell, 2, 163–171.
46, 139–146. 55. CaJacob, C.A., Feng, P.C.C., Reiser,
42. Uchimiya, H., Iwata, M., and Nojiri, C. S.E., and Padgette, S.R. (2007) in Mod-
(1993) Biotechnology, 11, 835–836. ern Crop Protection Compounds (eds W.
43. Lloyd, M., Culpepper, S., Cerny, E., Krämer and U. Schirmer), Wiley-VCH
Coots, B., Corkern, C., Cothren, T., Verlag GmbH, Weinheim, pp. 283–316.
Croon, K., Ferreria, K., Hart, J., Hayes, 56. Richins, R., Scholthof, H., and Shepard,
B., Huber, S., Martens, A., McCloskey, R. (1987) Sequence of figwort mo-
B., Oppenhuizen, M., Patterson, M., saic virus DNA (Caulimovirus Group).
Shappley, Z., Subramani, J., Reynolds, Nucleic Acids Res., 15, 8451–8466.
D., Witten, T., York, A., and Mullinix, B. 57. Feng, P.C.C., CaJacob, C.A.,
(2004) Transgenic cotton with improved Martino-Catt, S.J., Cerny, E.R., Elmore,
resistance to glyphosate herbicide. Crop G.A., Heck, G.R., Huang, J., Kruger,
Sci., 44, 234–240. W.M., Malven, M., Miklos, J.A., and
Padgette, S.R. (2010) in Glyphosate Re- 61. Green, J.M. (2009) Evolution of
sistance in Crops and Weeds: History, glyphosate-resistant crop technology.
Development, and Management (ed. V.K. Weed Sci., 57, 108–117.
Nandula), John Wiley & Sons, Inc., 62. Herman, P.L., Behrens, M.,
Hoboken, NJ, pp. 45–66. Chakraborty, S., Chrastil, B.M., Barycki,
58. Heap, I. (2010) The International Survey J., and Weeks, D.P. (2005) J. Biol.
of Herbicide Resistant Weeds, Online Chem., 280, 24759–24767.
Internet, http://www.weedscience.com. 63. Maiti, I.B. and Shepperd, R.J. (1998)
(accessed December 2010). inventors; Promoter (FLT) for the
59. Duke, S.O. and Powles, S.B. (2008) full-length transcript of peanut chlorotic
Glyphosate: a once-in-a-century herbi- streak caulimovirus (PCLSV) and ex-
cide. Pest Manage. Sci., 64, 319–325. pression of chimeric genes in plants.
60. Duke, S.O. and Powles, S.B. (2009) University of Kentucky, assignee, 1998
Glyphosate-resistant crops and weeds: Dec. 15. US Patent 5,850,019.
now and in the future. AgBioForum, 12,
346–357.
9.3
Glutamine Synthetase Inhibitors
Günter Donn
9.3.1
Introduction
Despite the fact that the atmosphere consists of 78% of nitrogen, plants evolved
in contrast to animals under conditions where accessible nitrogen sources were
growth limiting due to the chemical inertness of the dintrogen molecule. Whereas
in animals effective pathways evolved to detoxify and to excrete surplus ammonia
as urea or ureides, plants are dependent on perfect mechanisms of ammonia
recycling. The key enzyme in plants to assimilate, reassimilate and to detoxify
ammonia is glutamine synthetase which converts ammonia and glutamate into
glutamine under consumption of ATP. Especially in photosynthetically active cells,
considerable amounts of ammonia are released in the photorespiratory C2 cycle
which have to be recycled with high efficiency to prevent the build up of high
ammonia levels that eventually are toxic or may cause the loss of the volatile NH3 .
Phytopathogenic Pseudomonas strains were the first organisms to exploit this
‘‘Achilles heel’’ of plants. For example, P. syringae pv. tabaci produces the GS
inhibitor tabtoximine-β-lactam, which enables the pathogen to colonize the host
tissue that has been killed by the toxin.
During the late 1960s/early 1970s, Streptomyces strains were discovered which
produce a tripeptide consisting of two molecules of alanine and an unusual
amino acid that contains a phosphino group. The latter compound was named
l-phosphinothricin, while the tripeptide became known as bialaphos (syn.
bilanaphos). l-Phosphinothricin was recognized as a glutamate analog and a
potent inhibitor of bacterial GS. However, during the mid-1970s it became
recognized that the natural tripeptide, as well as the amino acid l-phosphinothricin
and its synthetic racemate – glufosinate – each demonstrated great potential
as post-emergence, nonselective herbicides. Subsequently, over the past two
decades both glufosinate and the natural tripeptide have become recognized and
commercialized as valuable tools in post-emergence weed-control strategies.
Twenty years ago, it first became evident that the phosphinothricin-producing
Streptomyces strains included in their genomes highly specific acetyltransferase
genes which, following their transfer into transgenic crop plants, could
provide a perfect protection for the latter against the herbicidal activity of both
phosphinothricin and glufosinate. Consequently, these revolutionary findings
led to the use of such glutamine synthetase inhibitors as selective herbicides in
transgenic crops.
9.3.2
Role of Glutamine Synthetase in Plant Nitrogen Metabolism
Amongst the plant enzymes that use ammonia as substrate, glutamine synthetase
(GS; E.C. 6.3.1.2) has the highest affinity (Km 3–5 μm) for this nitrogen source.
Ammonia is released in plant tissues by nitrite reduction and amino acid catabolism
but the highest amount, up to 90%, originates in photosynthetic tissues from the
photorespiratory C2 cycle [1].
In photosynthetic tissues, under atmospheric conditions the oxygenase ac-
tivity of ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) leads to the
formation of 2-phosphoglycolate in the chloroplasts, which is in turn cleaved
into inorganic phosphate and glycolate. In peroxisomes, this intermediate is
oxidized by glycolate oxidase to form glyoxylate and H2 O2 . The glyoxylate is
rapidly metabolized by the enzymes glutamate-glyoxylate-aminotransferase and
serine-glyoxylate-aminotransferase, with glycine being the end product in both
cases. Subsequently, in the mitochondria, two molecules of glycine are converted
into one molecule of serine, while CO2 + NH3 are released and reassimilated in
the chloroplasts [2].
The GS enzyme utilizes both glutamate and ammonia as substrates, with the
resultant glutamine serving as the substrate for glutamate synthase (glutamine-2
oxoglutarate-aminotransferase; GOGAT), which transfers the amido group from
glutamine to 2-oxoglutarate, thereby synthesizing two molecules of glutamate [3].
The GS–GOGAT cycle enable plants to assimilate and to recycle ammonia with
high efficiency The end products of both enzymes serve as substrates for the
respective partner enzyme, as well as amino donors for the synthesis of amino
acids, purines, and pyrimidines [4] (Figure 9.3.1).
Due to the central role of GS in nitrogen metabolism, plants typically confer
several genes which each code for GS isoforms that are differentially expressed.
The plant GS enzymes consist (as do all known eukaryotic GS enzymes) of eight
subunits [5], the molecular weights of which range from 38 to 45 kDa, depending
on the species and the subcellular localization of the respective isoform. At least
one cytosolic isoform (GS1 ) and a chloroplast-specific (GS2 ) can be distinguished in
Arginine
Ureides
Tryptophan Asparagine
histidine Nucleic acids
Glutamine 2-Oxoglutarate Amino acids
NO2 NH3 GS GOGAT
N2 Glutamate Glutamate 2-Oxo acids
Photorespiration 5-Aminolevulinate
asparagine Proline
urea Arginine
Chlorophyll
Figure 9.3.1 The central role of the GS-GOGAT cycle in plant nitrogen metabolism.
most higher plants, whereas their relative abundance varies considerably between
species [6]. The expression of the gene is enhanced by a high light intensity [7]
and high sucrose levels. In some species, a root-specific isoform (GSR ) can be
distinguished, while at least one nodule-specific isoform has been discovered in
legumes [8].
Each subunit of the enzyme has an active center with a high binding affinity for
the substrates. Glutamate is activated by the enzyme via the formation of glutamyl
phosphate, and consequently this intermediate is amidated with ammonia. Both,
ATP and Mg2+ are required for such activation (Scheme 9.3.1).
GS2 -deficient barley mutants which were isolated under conditions that sup-
press photorespiration, can be grown without phenotypic aberrations under
nonphotorespiratory conditions (2% O2 , 0.7% CO2 ). However, mutants with
less than 40% of the wild-type GS2 activity show severe phytotoxic symptoms
(mainly chlorophyll destruction) when grown under normal atmospheric con-
ditions (20% O2 , 0.03% CO2 ) in full light [9]. The mutants show a significant
increase in the level of free ammonia in their leaves, depending on the light
intensity. Interestingly, under photorespiratory conditions this increase in the
ammonia level is correlated with the development of phytotoxic symptoms.
However, these symptoms were not observed under conditions where pho-
torespiration was suppressed, despite the ammonia level being elevated in both
cases [10].
These mutants demonstrate not only that GS might serve as a potential target for
herbicidal compounds, but also that photosynthetic tissues are most susceptible to
herbicidal damage caused by GS inhibitors.
Some 50 years ago, the discovered was made that certain phytopathogenic
Pseudomonas strains – namely, P. syringae pv. tabaci – release a toxic metabolite
at the site of leaf infection. This metabolite causes the formation of a chlorotic
halo at the infection site, after which the damaged tissue of the host is colonized
by the bacteria. The molecular structure of the toxic metabolite was identified [11]
and referred to as tabtoxinin-β-lactam, a strong inhibitor of plant GS [12]. In fact, if
Mg2+
L-glutamate + NH3 + ATP L-glutamine, ADP + Pi
GS
O O
ATP + NH3 + HO O−
H3N+
L-Glutamate
(Mg2+) -ADP
O− H3N+ OH O
O P
O O−
O−
H3N+
L-Glutamate-intermediate
-HOPO32−
O O
H2N O
H3N+
L-Glutamine
Scheme 9.3.1 The reaction catalyzed by glutamine synthetase (GS).
the toxin is applied to tobacco leaves it causes the same symptoms as do virulent
toxin-producing bacteria. Similar symptoms are seen to develop following the local
administration of methionine sulfoximine (MSO) which, at that time, was known
to be a powerful inhibitor of GS activity [13].
9.3.3
Phosphinothricin, a Potent GS Inhibitor
In 1972, the team of Professor Zaehner at Tübingen described a Streptomyces

strain that produced a novel compound with antibiotic properties. The antibiotic
tripeptide produced by Streptomyces viridochromogenes consisted of two alanine
residues and a novel amino acid, phosphinothricin [14] (Figure 9.3.2). Due
to its structural analogy to glutamate, Bayer et al. [14] tested and verified the
hypothesis that phosphinothricin could serve as an inhibitor of the bacterial
O NH2 O NH2
HO O C CH2 CH2 CH COOH H3C S CH2 CH2 CH COOH
NH
Glutamate Methionine sulfoximine
O
O NH2 HN C NH2
H3C P CH2 CH2 CH COOH H2C C CH2 CH2 CH COOH
OH OH
Phosphinothricin Tabtoximine ß-lactam
Figure 9.3.2 Glutamate and some analogs described as GS inhibitors.
GS enzyme, whereas the tripeptide phosphinothricyl-alanyl-alanine would not

cause any such inhibition. Nevertheless, the tripeptide was found to be 1000 to
10 000-fold more active in its growth inhibitory effects on different bacteria. Such
discrepancy in inhibitory activity was explained by the observation that whilst the
free phosphinothricin could not be taken up efficiently by bacteria, the tripeptide
could be taken up by peptide carriers and subsequently cleaved.
Independent of the ongoing research activities in Germany, a Japanese re-
search group identified the same tripeptide, produced by a Streptomyces isolate
discovered in a Japanese soil sample. The isolate was named S. hygroscopicus,
and the tripeptide referred to as bialaphos (syn. bilanaphos) [15]. In 1984, a
third phosphinothricin-producing microorganism was discovered and described as
Kitasatospora phosalacinea (syn. Streptomyces phosalacineus), and shown to produce
phosphinothricyl alanyl-leucine (phosalacine) [16, 17].
9.3.4
Discovery of the Herbicidal Activity of Phosphinothricin
During the mid-1970s, dl-phosphinothricin was synthesized at the Hoechst central

research laboratories and tested for its biological activity in the biological research
unit of the company’s Agricultural Division. Whereas, the compound had failed
to show any significant herbicidal activity when being screened for pre-emergence
herbicidal activity, it demonstrated a powerful and broad activity against almost
all weeds following foliar application in the post emergence herbicidal screening,
but did not show any selectivity in field crops. The results of subsequent field
experiments confirmed the excellent broad-spectrum weed control potential of
dl-phosphinothricin, such that the development of the compound as a nonselective
post-emergent herbicide was initiated [18].
In 1984, the ammonium salt of phosphinothricin was introduced to the market
under the common name glufosinate-ammonium, as a post-emergent herbicide
for directed-spray applications in vineyards. During the following years, the label
was extended for the use of glufosinate-ammonium in orchards and plantation

crops, and further uses were subsequently developed [19].
Following the discovery of the herbicidal activity of bialaphos in Japan by Meiji
Seika, and the demonstration of its ability as a natural product to control weed
growth after foliar application, the tripeptide was developed as a foliar nonselective
herbicide for the Japanese market, where it was introduced in 1984 under the trade
name Herbiace [20].
To date, all attempts to synthesize more potent structural analogs of glutamate
with herbicidal activity have failed, despite major efforts in herbicide research
having been dedicated to the herbicide target, GS [19].
9.3.5
Mode of Glutamine Synthetase Inhibition
In 1968, Ronzio and Meister had already developed a model for GS inhibition by
MSO [21], having postulated that MSO would inhibit GS in a two-step fashion. It
was suggested that the first step was reversible, when the inhibitor competed with
glutamate at the binding site. In the second step, the substrate analog was phospho-
rylated (Scheme 9.3.2) and then bound irreversibly to the enzyme. Manderscheid
and Wild [22] confirmed this two-step hypothesis by using l-phosphinothricin in
a series of inhibition studies with GS from wheat. It was found that the phos-
phorylated phosphinothricin would bind irreversibly to the enzyme. It was also
concluded that each subunit of the enzyme was capable of binding one molecule
of phosphinothricin.
Only the l-enantiomer of the racemic glufosinate [dl-homoalanin-4-yl(methyl)
phosphonic acid] acted as an GS inhibitor. The tripeptide bialaphos was shown
not to inhibit the GS enzyme directly; rather, after foliar uptake the tripeptide
was cleaved and the released l-phosphinothricin was then able to inhibit the GS
O O
H3C
ATP + P
HO O−
H3 N+
L-Phosphinothricin
(Mg2+) -ADP
O− H3C O O
O P P
O O−
O− +
H3N
Scheme 9.3.2 Formation of the phosphorylated inter-
L-Phosphinothricylphosphate mediates of L-phosphinothricin.
enzyme. Hence, both herbicides were considered to act in identical fashion at the
GS target site.
9.3.6
Physiology of the Herbicidal Activity of Phosphinothricin
9.3.6.1 Herbicidal Symptoms of Phosphinothricin

Following herbicide application, the first symptoms become visible after one to
three days, depending on the weed species and the climatic conditions. Typically,
chlorotic spots and necrotic zones increase rapidly; these symptoms develop either
simultaneously, as in most dicotyledonous weeds, or subsequently, as in grasses.
In grasses, an intensive chlorosis usually precedes wilting and desiccation. Usually,
the treated plants are killed within 7–10 days, although low temperatures will cause
a significant delay in the herbicidal activity. Sublethal doses or unfavorable climatic
conditions may lead to regrowth, especially of older plants.
9.3.6.2 Physiological Effects of GS Inhibition in Plants

When plants are kept in the light, an increase in free ammonia is measurable
at only 1 h after the foliar application of glufosinate, while the ammonia level is
increased 100-fold within 24 h. In contrast, when the plants are kept in constant
darkness the level of free ammonia is increased only slightly after 24 h [23]. This
observation is in agreement with the fact that the vast majority of the released
ammonia is generated in the photosynthetic C2 carbon cycle and, to smaller de-
grees, by nitrite reduction in the light or by amino acid metabolism in darkness.
It is known from both growth-chamber experiments and field observations that
glufosinate–ammonium causes rapid and intense symptoms at high light intensi-
ties, whereas under low light and in darkness the symptom development is delayed
[24]. The amino acid pools of glufosinate-treated plants undergo dramatic changes
in parallel to the ammonia accumulation. Glutamine, glutamate, asparagine, aspar-
tate, glycine, serine, and alanine are depleted shortly after glufosinate treatment,
while arginine and aromatic amino acids increase in parallel [25]. It was concluded
that the relative increase of the latter amino acids is a consequence of the depleted
de novo synthesis of the amino acids showing a rapid turnover, as well as a result
of protein breakdown – especially of those proteins that show a high turnover
rate, such as Rubisco. A decrease in the protein content of treated plants was
indeed observed [26]. Photosynthetic carbon fixation is also inhibited by glufosi-
nate within hours, whereas the photosynthetic electron transport in chloroplasts
prepared from glufosinate-treated plants was not reduced within 48 h after her-
bicide application. At high concentrations, ammonia is considered to be toxic to
plants [27], and leads to a perturbation of membrane transport processes, most
likely due to a collapse of the pH gradient that normally is maintained by the
membranes [28]. Originally, the accumulation of ammonia as the consequence
of glutamine and glutamate depletion caused by GS inhibition was considered
to be the main reason for the phytotoxicity of GS inhibitors in plants, although
not all experimental data can be explained on the basis of this hypothesis. The
results of chlorophyll fluorescence measurements on glufosinate-treated plants do

not support the hypothesis of an ammonia-induced uncoupling of photophospho-
rylation [29]. In addition, Sinapis plants maintained under nonphotorespiratory
conditions (0.1% CO2 , 2% O2 ) did not show any inhibition of photosynthesis,
despite the ammonia accumulating to levels that were strongly inhibitory under
normal atmospheric conditions that would cause photorespiration [30]. Further-
more, in detached Sinapis leaves maintained under photorespiratory conditions,
the feeding of glutamine or glutamate led to a drastic reduction in the degree of
inhibition of photosynthesis, even though the ammonia accumulation was more
pronounced than in leaves that did not receive additional glutamine or glutamate
[31]. These observations indicated that interruption of the GS-GOGAT cycle would
cause glyoxylate accumulation due to a depletion of the glutamate [32] that serves as
a substrate for glutamate–glyoxylate-aminotransferase which converts glyoxylate
to glycine. Either glyoxylate itself or the arrested glycine-serine conversion, thereby
arresting the reimport of C3 skeletons back into the Calvin cycle, may be the cause
for the rapid breakdown of photosynthetic CO2 fixation [33]. Wild and Wendler
[34] provided evidence that, at very high concentrations, glyoxylate inhibits Rubisco
directly and at lower concentrations inhibits Rubisco-activase, which would also
explain the rapid breakdown of photosynthetic carbon fixation. The latter hypothe-
sis, together with the observed rapid depletion of the pools of crucial amino acids
necessary for purine and pyrimidine synthesis, and also for protein synthesis,
explain the severe and eventually irreversible metabolic disturbance, leading to an
inhibition of photosynthetic carbon fixation, de novo protein synthesis and, finally,
to death of the plant tissue.
9.3.6.3 Modulation of Herbicidal Activity of Glufosinate by Environmental

Conditions
The high polarity and water-solubility of phosphinothricin, combined with its
insolubility in the plant’s epicuticular wax and lipid bilayers, indicate why environ-
mental factors such as air humidity and temperature have a major influence on the
compounds’ uptake and herbicidal efficacy. Typically, the uptake and translocation
of phosphinothricin are significantly higher at high air humidity [35], though such
effects are less pronounced in the formulated commercial product. The compound
is more active at temperatures above 25 ◦ C than below 10 ◦ C, while hot, humid
weather conditions coupled with high light intensities provide excellent weed con-
trol results, even for those weed species which are difficult to control under less
favorable conditions. When glufosinate is applied to plants at temperatures below
10 ◦ C, both compound translocation and the development of herbicidal symptoms
are delayed [36]; this may eventually lead to a reduced herbicidal activity under
adverse environmental conditions.
9.3.6.4 Uptake and Translocation of Glufosinate-Ammonium

More than 50% of the active ingredient that is able to penetrate into the leaf is taken
up within the first 4–6 h after foliar application, and more than 90% within 24 h
[37, 38]. Ullrich et al. [39] showed evidence of an active uptake of the compound
that was mediated by amino acid carriers. As noted above, compound uptake is
modulated by the air humidity such that, under conditions which favor rapid
symptom development (high temperatures and high light intensity), translocation
of the compound is limited; in contrast, when plants are kept in the dark after
application the active ingredient shows a considerable phloem mobility comparable
to that of glyphosate. Under field conditions, glufosinate-ammonium is regarded
as a contact herbicide with partial phloem mobility [40].
9.3.7
Use of Phosphinothricin-Containing Herbicides in Agriculture and Horticulture
The mode of action and slow rate of metabolism of phosphinothricin in plants

forms the basis of the compound’s very broad herbicidal activity and lack of
selectivity. Originally, herbicides containing this active ingredient were developed
and brought to market as nonselective post-emergence herbicides for vegetation
management in orchards, vineyards, plantation crops, reforested areas, and tree
nurseries. Selectivity can be generated, however, by a directed spraying of the
herbicide onto the weed canopy, and carefully avoiding any drift of the herbicide
onto the leaves of the respective crop. In field crops, or in horticultural indications
in vegetables and ornamentals, the crop can be protected against herbicidal
damage either by using shielded spraying or by applying the herbicide before the
crop is planted. In both cases, exposure of the crop to the active ingredient is
prevented.
Further registered applications of glufosinate-ammonium cover its use as a
harvest aid [19, 41], especially for pre-harvest leaf desiccation in potatoes and
conventional canola varieties.
9.3.8
Attempts to Generate Crop Selectivity for Glufosinate
Based on the activity of glufosinate against a broad spectrum of weeds, its unique
mode of action, its complete biodegradability, and its low toxicity against nontarget
organisms [42], attempts were initiated to explore approaches that might allow it
to be used as a selective herbicide in major field crops. In parallel to the genetic
approaches outlined in Section 9.3.8.1, special spraying devices were developed that
would allow a directed spraying between the crop rows while protecting the crop
from the sprayed herbicide. Despite the selective use of glufosinate in conventional
maize varieties with the help of a directed spraying device being first registered in
1993, the need to invest in specific application equipment has led to a considerable
limitation in the use of this system.
9.3.8.1 Genetic Approaches to Generate Glufosinate-Selectivity in Crops:

Target-Based Approaches
During the mid-1980s, various attempts were initiated to select glufosinate-tolerant
mutants by the exposure of regenerable tissue cultures from crops to inhibitory
concentrations of the herbicide. When plants were regenerated from in vitro-selected

tobacco and maize cell cultures, thus conferring a four- to eightfold increased
glufosinate tolerance, the regenerants showed only a marginally improved tolerance
to glufosinate after foliar application, which proved not to be worthwhile for use
in breeding programs (G. Donn, unpublished results). No significant changes
in GS activity were observed in the regenerants of both crops. In contrast to
these negative results, a phosphinothricin-tolerant alfalfa cell line was obtained by
creating a stepwise increase in inhibitor concentration in the culture medium [43].
The resulting mutant cell population tolerated a 20-fold increased herbicide dose,
but failed to regenerate to plants. The resistant alfalfa cell line overexpressed GS up
to 10-fold compared to the wild-type cells, due to an amplification of a GS1 gene.
The constitutive overexpression of the alfalfa GS1 gene in transgenic tobacco led
to a significant accumulation of alfalfa GS protein in the tobacco plants, which in
turn led to an up to 10-fold increased GS activity in these plants. Although these
plants were only partially tolerant against the foliar application of glufosinate [44],
they did show symptoms of leaf chlorosis after treatment with agronomical-relevant
glufosinate doses of 1–2 kg a.i. ha−1 .
In parallel, attempts were made to mutate the alfalfa GS complementary DNA
(c-DNA). Despite it being possible to complement a GS-deficient Escherichia coli mu-
tant by the alfalfa GS1 c-DNA [45], all attempts failed to generate glufosinate-tolerant
GS1 mutants in the E. coli system. Those few mutants which showed a reduced
binding affinity for glufosinate also lost their binding affinity for glutamate.
In summary, until now all attempts to generate glufosinate tolerance, either by
target overexpression or by target mutation, have been unsuccessful. This fact,
together with the observation that no target-based mutants were found in weed
populations exposed to glufosinate for two decades, provide a strong indication that
glufosinate-resistant weeds based on target-site mutations are unlikely to evolve in
the future.
9.3.8.2 Crop Selectivity by Expression of Phosphinothricin Acetyl Transferase

Some 20 years ago, both Japanese and German research groups characterized –
independently of each other – two genes belonging to the biosynthesis gene
clusters of phosphinothricin-producing Streptomyces strains. Both genes conferred
bialaphos resistance to E. coli, and consequently both were used successfully as
selectable marker genes in gene transfer experiments in crops. The bialaphos
resistance (bar) gene from S. hygroscopicus was described by Thompson et al.
[46], and has since been used widely in plant transformation experiments. The
homologous gene from S. viridochromogenes, which was described by Wohlleben
et al. [47] in 1988 as the phosphinothricin-acetyl-transferase (pat) gene, experienced
a similar widespread use as a selectable marker. These two genes, and their
gene products, were shown to share a high degree of homology; on the DNA
level they showed 85% homology, and they also coded for proteins which shared
87% homology. The biochemical properties of the two proteins (in respect of
optimal pH and temperature) and their substrate specificity were very similar
[48]. Both enzymes were shown to N-acetylate (with high specificity) not only
COOH COOH
O
H2N CH H3C C N CH
O PAT H
CH2 + H3C C S CoA CH2 + HS-CoA
CH2 Acetyl coenzyme A

CH2
O P CH3 O P CH3
− −
O O
L-Phosphinothricin N-acetyl-phosphinothricin
Scheme 9.3.3 Inactivation of L-phosphinothricin by N-acetylation.
phosphinothricin (Scheme 9.3.3) but also desmethyl-phosphinothricin, a precursor

molecule in the biosynthetic pathway of this natural substance, but not to acetylate
proteinogenic amino acids.
Due to the high substrate affinity of both enzymes, only trace amounts of the
proteins are required to protect the transgenic plants from herbicidal damage.
Typically, even if less than 0.1% of the total protein consists of Bar or Pat protein,
the respective plants can efficiently acetylate phosphinothricin on a quantitative
basis when it enters the plant cells. Moreover, these plants fail to show any
signs of GS inhibition even after the application of high doses of glufosinate, in
excess of 5- to 10-fold the field application rate. Natural evolution has provided
the phosphinothricin-producing Streptomyces strains with a perfect mechanism by
which to maintain extremely low levels of free phosphinothricin within their cells.
The enzymes responsible for this also protect the crop plants against the herbicidal
material in perfect manner when the Streptomyces gene responsible is transferred
and expressed in crops, under control of the appropriate promoters.
Because Streptomyces demonstrate a different codon usage profile compared to
higher plants, a synthetic pat gene was synthesized which coded for the same protein
but used the preferred plant codons [49]. This synthetic gene was characterized by
a GC content of 50%, whereas the natural pat gene had a GC content of 70%. The
main expected advantage of the synthetic gene was to minimize the risk of gene
silencing due to the lower GC content. Following a 20-year coexistence of both gene
versions in transgenic crops, it became evident that the natural gene did not reveal
a higher probability of pat gene silencing compared to synthetic version. Rather,
both genes allowed the breeding of glufosinate-tolerant crop varieties that express
the transgene of more than 20 generations.
9.3.8.3 Bar and Pat Genes in Plant Breeding

Both, the bar and pat genes facilitated the development of efficient gene transfer
protocols for a variety of crops. One especially useful point was that they could
be used to establish gene transfer protocols for maize [50–52] and rice [53, 54],
regardless of the transformation method used by the respective research groups.
Today, these genes remain attractive for crop transformation experiments because
they represent good selectable marker genes in vitro in order to select the transgenic
offsprings of the few transgenic cells scattered in the cultured plant tissue in media
that contain inhibitory concentrations of phosphinothricin, in the form of either
the tripeptide or glufosinate. The regenerated transgenic plants can be easily
distinguished from nontransgenic siblings by leaf application of the GS inhibitor,
which allows the transgenic regenerants to be unaffected while the nontransgenic
siblings develop severe herbicide symptoms. Both genes have enabled research
groups to develop clean gene constructs that confer solely agronomical useful
genes to crops, and thus to avoid the use of antibiotic resistance genes in plant
transformation experiments.
Since 1995, transgenic glufosinate-tolerant (Liberty Link; LL) canola varieties
have been grown commercially in Canada, while LL maize was introduced to North
American agriculture in 1997. Whereas, LL canola is grown on almost 50% of the
Canadian acreage planted with transgenic canola, less than 20% of the transgenic
maize acreage in North America is planted with LL corn. In maize, the ratio reflects
the price difference between glyphosate and glufosinate, whereas in canola the LL
varieties are high-yielding hybrid and sold under the brand name InVigor canola.
The agronomic performance of these canola varieties more than compensates for
the price difference between the two complementary herbicides used in transgenic
canola. Furthermore, the success story of transgenic canola in Canada can be
explained by the fact that, for the first time, a perfect weed control became reality
in oilseed rape. This allowed the elimination of weedy Brassicaceae and helped to
produce a high-quality oil crop that was not contaminated by glucosinolates and
erucic acid derived from the weedy relatives of canola that are barely controlled
with conventional selective canola herbicides [55].
Recently, LL varieties of cotton and soybean were introduced into North-American
agriculture [56, 57], as well as cultivars in which the Roundup Ready (RR) and LL
traits are stacked. As a consequence of the advent of Roundup-tolerant weed varieties
during the past few years [58, 59], new options to control these weed biotypes are
urgently needed. Glufosinate, with its unique mode of action, represents an option
to manage these ‘‘problem weeds.’’
9.3.9
The Use of N-Acetyl-Phosphinothricin as a Proherbicide
Whereas, N-acetyl-phosphinothricin is not deacetylated in plants, bacterial en-

zymes have been described that are capable of removing the acetate residue
from the molecule [60, 61]. Transgenic plants conferring a bacterial deacety-
lase gene under the control of a constitutive promoter demonstrate herbicidal
symptoms when sprayed with N-acetyl-phosphinothricin, and therefore transgenic
plants expressing an appropriate deacetylase gene may be eliminated selectively
in plant canopies [62]. If the deacetylase gene is linked to tissue-specific pro-
moters, then specific cells could be ablated in transgenic plants conferring
the gene construct. For example, a deacetylase gene from Stenotrophomonas
sp. linked to a tapetum-specific promoter was used successfully to generate
facultatively male sterile tobacco flowers following treatment of the plants with
References 435
(a) (b)
Figure 9.3.3 Tobacco flowers treated with

N-acetylphosphinothricin. (a) Nontransgenic control;
(b) Transgenic tobacco conferring a bacterial deacetylase
gene under the control of a tapetum-specific promoter.
N-acetyl-phosphinothricin at the flower bud stage (Figure 9.3.3). Recently male

sterile plants were obtained as well, when D-glufosinate was used as proherbicide,
which can be converted in transgenic plants expressing a modified D-amino acid
oxidase into 2-oxo-4-(methylphosphinyl)-butanoic acid [63]. This molecule is con-
verted via transamination into L-phosphinothricin which then causes the tissue
specific cell ablation.
9.3.10
Conclusions
The natural GS inhibitor phosphinothricin, as well as its synthetic racemate

glufosinate, represent broad-spectrum post-emergence herbicides that will play a
role in future agriculture, due to their unique modes of action. Because these GS
inhibitors are able to fully control weeds that have evolved resistances against other
types of herbicide, the use of phosphinothricin-containing herbicides in tolerant
crops will remain an important option for future sustainable agriculture.
References
1. Keys, A.J., Bird, J.E., Cornelius, M.J., 4. Lea, P.J. (1997) in Plant Biochemistry
Lea, P.J., Wallsgrove, R.M., and Miflin, (eds P.M. Dey and I. Harborne), Aca-
B.J. (1978) Nature, 275, 741–743. demic Press, pp. 273–313.
2. Siedow, J.N., Day, D.A., (2000) Chapter 5. McNally, S.F. and Hirel, B. (1983) Glu-
14, in Biochemistry & Molecular Biol- tamine synthetase in higher plants.
ogy of Plants, (eds Buchanan B.B. and
Physiol. Vég., 21, 761–774.
Gruisson W. Jones R.), American So-
6. Ridley, S.M. and McNally, S.F. (1985)
ciety of Plant Physiologists (2000),
Plant Sci., 39, 31–36.
Wiley & Sons (2002), 676–729, ISBN
0-943088-39-9. 7. Peterman, T.K. and Goodman, H.M.
3. Miflin, B.J. and Lea, P.J. (1980) in The (1990) Mol. Gen. Genet., 230, 145–154.
Biochemistry of Plants: Amino Acids and 8. Forde, B.G., Day, H.M., Turton, J.F.,
Derivatives, vol. 5 (ed. B.J. Miflin), Aca- Shen, W.J., Cullimore, J.V., and Oliver,
demic Press, New York, pp. 169–202. I.E. (1989) Plant Cell, 1, 391–401.
9. Wallsgrove, R.M., Turner, J.C., Hall, 27. Junk, A., in Hock, B., and Elstner,
N.P., Kendall, A.C., and Bright, S.W. E.F. (eds) (1984) Pflanzentoxikolo-
(1987) Plant Physiol., 83, 155–158. gie, Der Einfluß von Schadstoffen und
10. Lea, P.J., Ridley, S.M., and Dodge, A.D. Schadwirkungen auf Pflanzen, Bibli-
(eds) (1989) Herbicides and Herbicide ographisches Institut Mannheim, pp.
Metabolism, Cambridge University Press, 224–229, ISBN: 3411016655.
Cambridge, pp. 137–170. 28. Roberts, J.K.M. and Pang, M.K.L. (1992)
11. Uchytil, T.F. and Durbin, R.D. (1980) Plant Physiol., 100, 1571–1574.
Experentia, 36, 301–302. 29. Lacuesta, M., Munoz-Rueda, A.,
12. Thomas, M.D., Langston-Unkefer, P.J., Gonzalez-Murua, C., and Sivak, M.N.
Uchytil, T.F., and Durbin, R.D. (1983) (1992) J. Exp. Bot., 43, 159–165.
Plant Physiol., 71, 912–915. 30. Wild, A., Sauer, H., and Ruehle, W.
13. Leason, M., Cunliffe, D., Parkin, D., Lea, (1987) Z. Naturforsch., 42c, 263–269.
P.J., and Miflin, B. (1982) J. Phytochem., 31. Sauer, H., Wild, A., and Ruehle, W.
21, 855–857. (1987) Z. Naturforsch., 42c, 270–278.
14. Bayer, E., Gugel, K., Haegele, K., 32. Wild, A. and Wendler, C. (1990) Pestic.
Hagenmayer, H., Jessipow, S., Koenig, Sci., 30, 422–424.
W.A., and Zaehner, H. (1972) Helv. 33. Wendler, C., Putzer, A., and Wild, A.
Chim. Acta, 55, 224–239. (1992) J. Plant Phaysiol., 139, 666–671.
15. Ogawa, Y., Tsuruoka, T., Inouye, S., and 34. Wild, A. and Wendler, C. (1993) Z.
Niida, T. (1973) Sci. Rep. Meiji Seika Naturforsch., 48c, 369–373.
35. Coetzer, C., Al-Khatib, K., and Longhin,
Kaisha, 13, 42–53.
T.M. (2001) Weed Sci., 49, 8–13.
16. Omura, S., Hinotozawa, K., Imanura,
36. Kumaratilake, A.R. and Preston, C.
N., and Murata, M. (1984) J. Antibiot.,
(2005) Weed Sci., 53, 10–16.
37, 939–940.
37. Köcher, H. and Lötzsch, K. (1985) Proc.
17. Omura, S., Murata, M., Hanaki, H.,
Asian-Pacific Weed Sci. Soc. Conf., 10,
Hinotozawa, K., Oiwa, R., and Tanaka,
193–198.
H. (1984) J. Antibiot., 37, 829–835.
38. Steckel, G.J., Hart, S.E., and Wax, L.M.
18. Schwerdtle, F., Bieringer, H., and Finke,
(1997) Weed Sci., 45, 378–381.
M. (1981) Z. Pflanzenkr. Pflanzensch.
39. Ullrich, W.R., Ullrich-Eberius, C.I.,
Sonderheft, 9, 431–440.
and Köcher, H. (1990) Pestic. Biochem.
19. Hörlein, G. (1994) Rev. Environ. Contam. Physiol., 37, 1–11.
Toxicol., 138, 73–145. 40. Beriault, J.N., Horsman, G.P., and
20. Mase, S. (1984) Jpn. Pestic. Inform., 45, Devine, M.D. (1999) Plant Physiol., 121,
27–30. 619–628.
21. Ronzio, R.A. and Meister, A. (1968) 41. http://www.bayercropscience.com/bcsweb/
Proc. Natl Acad. Sci. USA, 59, 164–170. cropprotection.nsf/id/glufosinate_
22. Manderscheid, R. and Wild, A. (1986) J. ammonium_nsu.htm?open&l=EN&ccm=
Plant Physiol., 123, 135–142. 200020030 367–372.
23. Köcher, H. (1989) Proc. Soc. Chem. 42. Dorn, E., Goerlitz, G., Heusel, R.,
Ind. Pestic. Group Meet. Monogr., 42, and Stumpf, K. (1992) Z. Pflanzenk.
173–182. Pflanzensch. Sonderheft, 13, 459–468.
24. Köcher, H. (1983) Influence of the light 43. Donn, G., Tischer, E., Smith, J., and
factor on physiological effects of the Goodman, H. (1984) J. Mol. Appl.
herbicide phosphinothricin ammonium, Genet., 2, 621–635.
Aspects Appl. Biol., 4, 227–234. 44. Eckes, P., Schmitt, P., Daub, W., and
25. Wendler, C., Barniske, M., and Wild, A. Wengenmayer, F. (1989) Mol. Gen.
(1990) Photosynth. Res., 24, 55–61. Genet., 217, 263–268.
26. Lacuesta, M., González-Moro, B., 45. Dassarma, S., Tisher, E., and Goodman,
González-Murua, C., Aparicio-Tejo, H.M. (1986) Science, 232, 1242–1244.
T., and Monoz-Rueda, A. (1989) J. Plant 46. Thompson, C.J., Movva, N.R., Tizard, R.,
Physiol., 1234, 304–307. Crameri, R., Davies, J.E., Lauwereys, M.,
References 437
and Bottermann, J. (1987) EMBO J., 6, 54. Datta, S.K., Datta, K., Soltanifar, N.,
2519–2523. Donn, G., and Potrykus, I. (1992) Plant
47. Wohlleben, W., Arnold, W., Broer, J., Mol. Biol., 20, 619–629.
Hillmann, D., Strauch, E., and Pühler, 55. Devine, M.D. and Buth, J.L. (2001) Proc.
A. (1988) Gene, 70, 25–37. Brighton Crop Prot. Conf.–Weeds,
48. Wehrmann, A., VanVliet, A., Opsomer, British Crop Protection Council,
C., Botterman, J., and Schulz, A. (1996) Farnham, Surrey, pp. 367–372.
Nat. Biotechnol., 14, 1274–1278. 56. Culpepper, A.S., York, A.C., Robert, P.,
49. Eckes, P., Uijtewaal, B., and Donn, G. and Whitaker, J. (2009) Weed Technol.,
(1989) J. Cell. Biochem., 13D, 334. 23 (3), 356–362.
50. Gordon-Kamm, W.J., Spencer, T.M., 57. Allen, J. and Fischer, J. (2008) North
Mnangano, M.L., Adams, T.R., Centr. Weed Sci. Soc. Proc., 63, 87.
Daines, R.J., Start, W.G., O’Brian, 58. Powles, S.B. (2008) Pest Manag. Sci., 64,
J.V., Chambers, S.A., Adams, W.R. 360–365.
Jr, Willets, N.G., Rice, T.B., Mackey, 59. Webster, T.M. and Sosnoskie, L.M.
C.J., Krueger, R.W., Kausch, A.P., (2010) Weed Sci., 58, 73–79.
and Lemaux, P.G. (1990) Plant Cell, 60. Bartsch, K., Kriete, G., Broer, I., and
2, 603–618. Pühler, A. (1996) WO 9827201.
51. Golovkin, M., Abraham, M., Morocz, 61. Kriete, G., Niehaus, K., Perlik, A.M., and
S., Bottka, S., Feher, A., and Dudits, D. Pühler, A. (1996) Plant J., 9, 809–818.
(1993) Plant Sci., 90, 41–52. 62. Chen, X., Yang, W., Sivamani, E.,
52. Frame, B.R., Drayton, P.R., Bagnall, Bruneau, A.H., Wang, B., and Qu,
S.V., Lewnau, C.J., Bullock, W.P., R. (2005) Mol. Breed., 15, 339–347.
Wilson, H.M., Dunwell, J.M., 63. Hawkes, T., Pline-Srnic, W., Dale,
Thompson, J.A., and Wang, K. (1994) R., Friend, E., Hollinshead, T.,
Plant J., 6, 941–948. Howe, P., Thompson, P., Viner, R., and
53. Christou, P. (1991) Biotechnology, 9, Greenland, A. (2011) Plant Biotechnol. J.,
957–962. 9, 301–314.
439
10
Microtubulin Assembly Inhibitors (Pyridines)
Darin W. Lickfeldt, Denise P. Cudworth, Daniel D. Loughner, and Lowell D. Markley
10.1
Introduction
Herbicides that possess a microtubulin assembly inhibitor (MAI) [1, 2] mode

of action are generally applied pre-emergence to control annual grasses and
small-seeded, broadleaf weeds. Following application, they cause swellings in the
meristematic regions of the plant, such as the root tips, with susceptible plants
showing thickened or swollen hypocotyls or internodes [3]. The MAIs can be
grouped into five chemical families: the dinitroanilines; the phosphoroamidates;
the pyridines; the benzamides; and the benzoic acids (Herbicide Resistance Action
Committee class K1).
The most popular family of the MAIs is the dinitroanilines; these include
herbicides such as trifluralin, benefin, oryzalin, pendimethalin, and prodiamine.
During the 1980s, the demand for pre-emergence herbicides that were more
efficacious, colorless, and dependable at lower application rates led to the
investigation of potential compounds in the pyridine family. Currently, only two
pyridine-based herbicides – dithiopyr and thiazopyr – are marketed, and these
form the focus of this chapter. Both herbicides were initially patented by Monsanto
[4, 5] before subsequently being sold to Rohm & Haas Company in 1994.
Ultimately, in 2001, following the acquisition of the Rohm & Haas Agricultural
Chemical business by The Dow Chemical Company, both compounds became the
property of Dow AgroSciences. Both products can be used by professional growers
of turf, ornamental, perennial tree and vine, and Oryza (rice), to control a broad
range of troublesome broadleaf and grass weeds.
10.2
Biology of the Microtubulin Assembly Inhibitors (Pyridines)
10.2.1
Dithiopyr
Dithiopyr is a pre-emergence and early post-emergence herbicide that is used

primarily in turf, ornamentals, and Oryza in the United States, Canada, Japan,

440 10 Microtubulin Assembly Inhibitors (Pyridines)
China, Australia, Egypt, South Korea, Taiwan, and Puerto Rico. It is applied either
pre-emergence or post-emergence to turf at 150–560 g a.i. ha−1 , for each application.
Early post-emergence applications can be utilized to control Digitaria spp. seedlings
in their early stages and prior to the emergence of a second tiller [6]. Adjuvants
have a minimal influence on post-emergence control because translocation from
the treated leaves is minimal [7].
Another pattern for turf use is in the selective pre-emergence control of
Poa annua L. in overseeded, warm-season turf. A common practice in warm
climates is to overseed warm-season turfgrass species with cool-season turfgrass
species such as Lolium perenne L., the aim being to maintain a green color through
the winter months when warm-season grasses typically go dormant. Dithiopyr has
been proven effective for the selective control of Poa annua for four to six months
after treatment, while not causing any injury to Lolium perenne that may have been
seeded eight weeks prior to treatment.
Applications of dithiopyr to paddy-grown Oryza are targeted to control Echinochloa
spp. Dithiopyr can be formulated into several different formulations, including an
emulsion in water (EW) containing 240 g a.i. l−1 , an emulsifiable concentrate (EC)
with up to 120 g a.i. l−1 , and a wettable powder (WP) with 40% a.i. content. Granular
formulations are also available.
Dithiopyr controls key annual monocotyledonous and many dicotyledonous
species, including Digitaria spp., Poa annua L., Eleusine indica (L.) Gaertn., Oxalis
spp., Euphorbia spp., Medicago lupulina L., and Stellaria media (L.) Vill. In warmer
climates, or while seeking the control of more challenging weed species such as Eleu-
sine indica (L.) Gaertn., sequential applications of dithiopyr may be necessary [8, 9].
Most species of cool-season and warm-season turfgrasses are tolerant when
the root system is well established. However, some species (e.g., Agrostis tenuis)
and some varieties (e.g., Cynodon. dactylon × C. transvaalensis ‘‘Tifgreen’’) are not
tolerant. Dithiopyr should not be applied to new perennial turf until the root system
is well established [10]; neither should it be applied to sod within three months
of harvest. The effect of dithiopyr on the rooting of established turfgrass species
has been shown as minimal and not to differ significantly from that of most other
pre-emergence herbicides with an MAI mode of action [11, 12].
10.2.2
Thiazopyr
Thiazopyr is a pre-emergence herbicide that is currently used in noncrop areas,

trees, vines, and Oryza crops, and which has demonstrated selectivity in Medicago
spp., Gossypium spp., Arachis spp., Glycine spp., and Saccharum spp. [13–15]. It
is effective on most annual grasses and certain broadleaf weeds. Thiazopyr is
presently registered in 9 countries in Latin America, and Australia.
One key strength of thiazopyr is its long residual control of annual grass weeds
when used at its typical rate of 0.56 to 1.12 kg a.i. ha−1 . Also of note is the high level
of Cyperus spp. suppression when applied pre-emergence. The use of thiazopyr in
10.4 Toxicology of Microtubulin Assembly Inhibitors (Pyridines) 441
the United States was as a residual herbicide in permanent crops and in noncrop
areas; citrus, tree-nuts, vines, pomefruit, and stonefruit are of primary importance,
primarily for the control of Panicum maximum.
10.3
Environmental Fate of Microtubulin Assembly Inhibitors (Pyridines)
10.3.1
Dithiopyr
Although dithiopyr is strongly adsorbed to the soil (Koc average: 1638 ml g−1 ), it
can also be desorbed from soils that are low in organic matter [16]. In field studies,
the soil half-life of dithiopyr ranged from 3 to 49 days (average 17 days), with
degradation resulting mostly from microbial activity [17]. The major metabolites
detected were the diacid and two forms of a monoacid, all of which dissipated within
one year of application. Dissipation from the field soils can also occur through
volatilization. On the treated soil, dithiopyr has been shown stable to photolysis,
while leaching or run-off – even from highly permeable golf course putting
greens – has been shown as minimal [18–20].
The photolytic half-life of dithiopyr in water was 17.6 days, which indicated a
moderate rate of degradation and also a potential for degradation in surface water.
Again, the two monoacids and a diacid were the primary metabolites observed.
The potential movement of dithiopyr in water might be limited due to its poor
water-solubility and its ability to adsorb strongly to the soil particles and plants.
10.3.2
Thiazopyr
Thiazopyr is considered to be relatively immobile in soils, due to a poor

water-solubility and a high affinity for soil organic matter. In soil, the primary
routes of thiazopyr degradation are via the soil microorganisms and hydrolysis. In
soil dissipation studies conducted on various soils, the average dissipation time
(DT50 ) was 64 days (range: 8 to 150 days). The monoacid metabolite, when applied
at normal usage rates, also showed a limited mobility. The finding that, in aqueous
solutions, the DT50 was 15 days indicated that surface water contamination should
not be an issue in the application of thiazopyr.
In plants, oxygenase enzymes are involved in the metabolism of the dihydroth-
iazole ring to the sulfoxide, sulfone, hydroxyl derivative, and thiazole. Thiazopyr is
also de-esterified to its carboxylic acid.
10.4
Toxicology of Microtubulin Assembly Inhibitors (Pyridines)
In rats, dithiopyr is rapidly absorbed, extensively metabolized, and rapidly excreted

(Table 10.1) [16]. Eye irritation in rabbits was slight, while skin irritation was
Table 10.1 Toxicology of dithiopyr and thiazopyr [16]a.
Organism Administration Parameter Dithiopyr Thiazopyr
Rats/mice Acute oral LD50 (mg kg−1 ) >5000 >5000

Rat 2 years dietary NOEL (mg kg−1 per day) ≤10 ppm 0.36
Dog 1 year dietary NOEL (mg kg−1 per day) ≤0.5 ppm 0.5
Bobwhite quail Acute oral LD50 (mg kg−1 ) 2250 1913
Rainbow trout 96 h LC50 (mg l−1 ) 0.46 3.2
Honeybee Topical LD50 (μg per bee) 81 >100
Earthworm 14 days LC50 (mg kg−1 ) >1000 >1000
a
Dithiopyr and thiazopyr are nonmutagenic and nongenotoxic. The EPA toxicity class is III.
NOEL, no observed effect level.
nonirritating. Following three weeks of repeated skin exposure to dithiopyr techni-

cal, a mild but transient skin irritation and an increased liver weight were the only
adverse effects observed in rats.
The results of both pharmacokinetic and metabolism studies in the rat indicated
that thiazopyr was rapidly metabolized and excreted, and does not accumulate in
the tissues. In two studies, one with the 14 C label at the 4 position of the pyridine
ring and the other with the label at the 4 and 5 positions of the thiazole ring,
the absorption of an orally administered dose of thiazopyr was about 90%. The
metabolism of thiazopyr is essentially oxidative; vulnerable sites of the molecule
are the thiazoline ring, the isobutyric side chain, and the pyridine rings. Thiazapyr
appears to be rapidly and extensively eliminated, with small amounts of residues
remaining in the tissues and carcass. The proportion of the radiolabel remaining
in the carcass after feeding rats with 14 C-thiazoline-labeled thiazopyr was between
6.9% and 10.8%.
10.5
Mode of Action of Microtubulin Assembly Inhibitors (Pyridines)
Dithiopyr does not have a systemic action; rather, it is absorbed by the roots and,
to some degree, by the foliage of the susceptible plant. The most important site
of uptake appears to be the meristematic regions, since dithiopyr translocation is
limited and the primary site of action is in the meristematic tissues. The most
obvious symptoms of dithiopyr’s efficacy in susceptible plants is a swelling of the
meristematic regions, such as the root tips where the mitotic process is being
inhibited. This mode of action occurs via a disruption of spindle microtubule
formation in late prometaphase. Dithiopyr does not bind to tubulin, but rather to
another protein that might be a microtubule-associated protein (MAP) [1, 2]. As
the latter proteins have a function in microtubule stability, the action of dithiopyr
results in shortened microtubules that are unable to form the spindle fibers that
10.6 Synthesis of Dithiopyr and Thiazopyr 443
normally are responsible for separating the chromosomes to the poles of the cell
during mitosis. The cortical microtubules, which normally prevent isodiametric
cell expansion, are also essentially absent, and this results in the susceptible plants
developing club-shaped roots tips. Although thiazopyr also inhibits microtubule
assembly in the roots of emerging seedlings, it is not effective (unlike dithiopyr) as
an early post-emergence treatment.
To date, no cases of weed resistance to dithiopyr or thiazopyr have been reported.
In one study, although Digitaria ischaemum that was resistant to fenoxaprop-p was
controlled by dithiopyr [21], cross-resistance to other biotypes that were resistant to
the MAI mode of action could most likely occur.
10.6
Synthesis of Dithiopyr and Thiazopyr
Both, dithiopyr and thiazopyr (Figure 10.1) are pyridine-based herbicides (3) that
are accessed via pyridine, and synthesized by a similar route (Scheme 10.1) [22, 23].
The syntheses of dithiopyr (1), thiazopyr (2), and related compounds [4, 5]
begins with a Hantzsch-type, base-catalyzed intermolecular cyclization [22], to
O O
N O
S S
S O
F3C N CHF2
F3C N CHF2
Dithiopyr (1) Thiazopyr (2)
Figure 10.1 Structures of dithiopyr (1) and thiazopyr (2).
O H O
R R
O O O OH HO
R 1. base, solvent
2 F3C O H F3C N CF3
2. NH3 or NH4OH OH OH
H
5 6 7
O O
O H O
R R
R R O O
conc H2SO4 or O O base, solvent
TsOH or TFAA F3C N CHF2
F3C N CF3
H
4 3
Scheme 10.1 Synthetic route to the common pyridine intermediate 3 [4, 5].
provide the dihydropyridines 4 (R = Me or Et). Two equivalents of methyl or

ethyl trifluoroacetoacetate (5; R = Me or Et) are allowed to react with 1 equivalent
of isovaleraldehyde (6) in the presence of a base (e.g., piperidine) in a suitable
solvent at temperatures which may vary from room temperature to reflux. The
intermediate dihydroxytetrahydropyran (structure not shown) is converted into the
dihydroxypiperidine 7 by reaction with a nitrogen source, such as ammonium
hydroxide or ammonia gas. The subsequent reaction of 7 with a dehydrating
agent, such as concentrated sulfuric acid, toluenesulfonic acid, or trifluoroacetic
anhydride, provides a mixture of the 1,4-dihydropyridine 4 and its 3,4-isomer. In
the case of both 1 and 2, the major isomer is the 1,4-isomer, and this may be
cleanly isolated. The regiochemical preference for the 3,4-isomer is determined by
the choice of dehydrating agent, as well as by the group in the 4-position of the
dihydroxypiperidine 7. The dihydropyridine 4 is then allowed to react with a base,
such as 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), tributylamine, triethylamine,
O O
R R 1. 85% KOH, H O
O O 2
2. SOCI2, reflux
F3C N CHF2
3
O O
Cl Cl
F3C N CHF2
8
1. 1:1 MeOH:THF
CH3SH 25 °C, 2.5 h
2. H N OH
base 2
O O
S S O O
HO
F3C N CHF2 N O
Dithiopyr (1) H
F3C N CHF2
9
1. P2S5
2. (Me2N)3PO
S O
N O
F3C N CHF2
Thiazopyr (2)
Scheme 10.2 Syntheses of dithiopyr (1) and thiazopyr (2)

from the pivotal intermediate bis-acid chloride 8 [4, 5].
References 445
or 2,6-lutidine (either neat or in a suitable solvent) to yield the dehydrofluorinated

[23] pyridine 3, a common intermediate in the syntheses.
Saponification of the esters is accomplished with 85% potassium hydroxide in
aqueous media, providing the 3,5-diacid. The latter is then converted into the
pivotal intermediate, the bis-acid chloride 8, by reaction with neat thionyl chloride
under reflux. The acid chloride is treated with methanethiol in the presence of a
base to give dithiopyr (1) (Scheme 10.2).
Thiazopyr (2) is synthesized in similar fashion. Treatment of the bis-acid chloride
8 in methanol : tetrahydrofuran (1 : 1) at room temperature for 2.5 h affords the
5-chlorocarbonyl-3-methyl ester selectively [24], which is then allowed to react
with 2-hydroxyethyl amine to form the corresponding 2-hydroxyethyl amide 9.
The latter compound is subsequently treated with phosphorus pentasulfide and
hexamethyl phosphoramide, which results in a sulfurization and cyclization to
form the 4,5-dihydrothiazole, thiazopyr (2).
References
1. Vaughn, K.C. and Lehnen, L.P. Jr (1991) 14. Crane, S.E., Holmdal, J.A., and Murray,
Weed Sci., 39, 450–457. R.E. (1998) Southern Weed Sci. Soc. Proc.,
2. Lehnen, L.P. Jr and Vaughn, K.C. (1991) 51, 234.
Pestic. Biochem. Physiol., 40, 58–67. 15. Kuhns, L.J. and Harpster, T.L. (1997)
3. Vencill, W.K. (ed.) (2002) Herbicide Northeast. Weed Sci. Soc. Proc., 51,
Handbook, 8th edn, Weed Science 115–117.
Society of America, Lawrence, KS. 16. Tomlin, C.D.S. (ed.) (2000) The Pesti-
4. Lee, L.F. (1987) US Patent 4,692,184. cide Manual, 12th edn, British Crop
5. Sing, Y.-L.L. and Lee, L.F. (1991) US Protection Council.
Patent 4,988,384. 17. Hong, S. and Smith, A.E. (1996) J. Agric.
6. Johnson, B.J. (1997) Weed Technol., 11, Food Chem., 44, 3393–3398.
144–148. 18. Hong, S. and Smith, A.E. (1997) J. Envi-
7. Keeley, S.J., Branham, B.E., and Penner, ron. Qual., 26, 379–386.
D. (1997) Weed Sci., 45, 205–211. 19. Gupta, S. and Gajbhiye, V.T. (2002)
8. Wiecko, G. (2000) Weed Technol., 14, J. Environ. Sci. Health, Part B, 37,
686–691. 573–586.
9. Johnson, B.J. (1997) Weed Technol., 11, 20. Hong, S. and Smith, A.E. (2001) J. Envi-
693–697. ron. Sci. Health, Part B, 36, 529–543.
10. Reicher, Z.J., Weisenberger, D.V., and 21. Derr, J.F. (2002) Weed Technol., 16,
Throssell, C.S. (1999) Weed Technol., 13, 396–400.
253–256. 22. Hantzsch, A. (1882) Justus Liebigs Ann.
11. Dernoeden, P.H., Christians, N.E., Chem., 215, 1–82.
Krouse, J.M., and Roe, R.G. (1993) 23. Lee, L.F., Stikes, G.L., Molyneaux,
Agronomy J., 85, 560–563. J.M., Sing, Y.L., Chupp, J.P., and
12. Landschoot, P.J., Watschke, T.L., and Woodard, S.S. (1990) J. Org. Chem.,
Hoyland, B.F. (1993) Weed Technol., 7, 55, 2872–2877.
123–126. 24. Lee, L.F., Stikes, G.L., Sing, L.Y.L.,
13. Kuhns, L.J. and Harpster, T.L. (1998) Miller, M.L., Dolson, M.G., Normansell,
Northeast. Weed Sci. Soc. Proc., 52, J.E., and Auinbauh, S.M. (1991) Pestic.
127–129. Sci., 31, 555–568.
447
11
Acetyl-CoA Carboxylase Inhibitors
11.1
Introduction
The inhibition of acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) is one of the most

commercially important modes of action for weed control, with current annual
sales for such herbicidal compounds estimated to exceed US$ 1 billion. In grass
species, three distinct chemical classes are known to inhibit ACCase, which is the
first committed step in fatty acid biosynthesis [1]. The aryloxyphenoxypropionate
(AOPP or fop) and cyclohexanedione (CHD or dim) classes have been present in
the marketplace for more than 30 years, while pinoxaden – the only commercially
available 2-aryl-1,3-dione (DEN) – was launched in 2006. Owing to their mode of
action, these herbicides are used widely to control a broad selection of grass weeds
in dicotyledonous crops, with a smaller number commercialized for grass control in
cereals or in rice [2, 3]. However, the frequent application of these compounds has
resulted in the development of resistance to their use in several grass species [4, 5].
The ACCase herbicides, which have been well described in the literature, are
known to inhibit the carboxylate transferase (CT) function of homomeric ACCase
found in the plastids of grasses [2, 6]. Dicotyledon tolerance is based on the inherent
insensitivity of broadleaves to these herbicides, whereas in monocotyledon crops
the selectivity is usually due to higher rates of herbicide detoxification [4, 7].
In this chapter, an overview is presented of the ACCase-inhibiting herbicides,
with particular emphasis placed on the recent developments in biochemistry,
resistance mechanisms, and new synthetic approaches to the DENs.
11.2
Biochemistry
11.2.1
Overview
ACCase (EC 6.4.1.2) is a biotin-dependent carboxylase that produces malonyl-CoA

from bicarbonate as a source of carboxyl group, and ATP as a source of energy.

448 11 Acetyl-CoA Carboxylase Inhibitors
ACCase catalyzes the conversion of acetyl-CoA into malonyl-CoA through the

incorporation of a carboxyl group into the acetyl radical of acetyl-CoA. This
transcarboxylation reaction is performed following the two-step process utilized by
all biotin-dependent transcarboxylases (Scheme 11.1).
ACCase consists of three major functional domains: (1) the biotin carboxylase
(BC); (2) the biotin carboxyl-carrier (BCC); and (3) the carboxyltransferase (CT).
The enzyme complex can be classified into two structural types – the prokaryotic
form and the eukaryotic form. The prokaryote ACCase is a multisubunit enzyme
composed of four separate subunits which together form the three domains. The
eukaryote ACCase is a multidomain protein containing the three major functional
domains organized in one large polypeptide (Table 11.1).
Grasses of the Poaceae family contain two eukaryotic type ACCases – one
chloroplastic and one cytosolic – whereas almost all other plants contain the two
well-differentiated forms – a cytosolic eukaryotic-type ACCase and chloroplastic
prokaryotic-type ACCase. One exception is Brassica napus, the chloroplasts of which
HCO3− + enzyme-biotin + ATP-Mg → enzyme-biotin-CO2− + ADP-Mg + Pi [1]
Enzyme-biotin-CO2− + acceptor → acceptor-CO2− + enzyme-biotin [2]
Scheme 11.1 The ACCase reaction is the result of the co-

operation of the catalytic activities of [1] biotin carboxylase
and [2] acetyl-CoA carboxyltransferase.
Table 11.1 Summary of the different ACCases in plants.
Characteristic Chloroplast Cytoplasm
Grasses
Type Eukaryote type I, isoform 1 Eukaryote type I, isoform 2
Molecular mass (kDa) ≈240 ≈220
Native structure Homodimer Homodimer
Reaction Three catalytic domains per Three catalytic domains per
protein protein
Major role Fatty acid biosynthesis Secondary metabolite
biosynthesis
Dicotyledons
Type Prokaryote type II Eukaryote type I
Molecular mass (kDa) ≈32–80 ≈230
Native structure Multicomponent enzyme Homodimer
Reaction One catalytic domain per Three catalytic domains per
enzyme protein
Major role Fatty acid biosynthesis Secondary metabolite
biosynthesis
have been shown to contain both the multisubunit and the multifunctional ACCase
enzymes [8–10].
The multisubunit enzyme is encoded by the nuclear DNA, with the exception of
the β-subunit of CT which is encoded by a chloroplastic gene [11]. In grasses, the
chloroplastic multidomain ACCase is encoded by a nuclear gene, which is distinct
from that coding for the cytosolic multidomain ACCase.
The chloroplastic ACCase, together with fatty acid synthase, are responsible for
the synthesis of fatty acids up to C18. Palmitic acid C16:0, stearic acid C18:0, and
oleic acid C18:1, synthesized in the chloroplast, are exported to the cytoplasm, thus
contributing to the control of flux through the plant’s de novo fatty acid biosynthetic
pathway [12].
The cytoplasmic ACCase synthesizes malonyl-CoA required by several biosyn-
thetic pathways, including:
Acetyl CoA Chloroplast Cytosol

ACCase
Malonyl CoA
Fatty acid synthase
C16:0, C18:0, C18:1
C16:0-CoA, C18:0-CoA, C18:1-CoA

Acetyl CoA
ACCase Fatty acid elongation Waxes, suberin
& cutin
Malonyl CoA Endoplasmic reticulum
N-malonyl-D-amino
acids & ethylene
Flavonoids regulation
& stilbenes
Figure 11.1 Schematic drawing of the function of the two

ACCase isoforms in plants. The chloroplastic ACCase plays a
role in de novo fatty acid biosynthesis, whereas the cytosolic
ACCase provides malonyl-CoA for fatty acid elongation, syn-
thesis of secondary metabolites, N-malonylation of D-amino
acids, and ethylene regulation [13].
• the synthesis of very long-chain fatty acids, which are elongation products of the
C18 lipids;
• the synthesis of a large group, consisting of flavonoids, pigments, and stilbene
derivatives; and
• the synthesis of N-malonyl-d-amino acids.
The roles of the two ACCases in plants are summarized in Figure 11.1.
The cytoplasmic and plastidic ACCases from wheat are 2260 and 2311 amino
acids long, respectively, and their sequences are 67% identical [14]. A chloroplast-
targeting signal is present at the N terminus of the multidomain plastid ACCase
from wheat [15], maize [16], and Brassica napus [17].
11.2.2
Mode of Action of ACCase Inhibitors
The first study to demonstrate the effect of ACCase inhibitors on plant lipid
biosynthesis was reported by Hoppe in 1981 [18], and showed a strong inhibition
of the incorporation of 14 C-labeled acetate into plant lipids. It was only in late
1987 that the two independent laboratories of Burton and Focke showed that
the site of action of these inhibitors was located in ACCase. Burton et al. [19]
found that ACCase isolated from the chloroplasts of corn seedlings was inhibited
by the herbicides sethoxydim and haloxyfop, with IC50 concentrations of 2.9
and 0.5 μM, respectively, whereas the ACCase from pea chloroplasts was not
inhibited by these compounds. Focke and Lichtenthaler [20] reported that the
cyclohexane-1,3-dione derivatives cycloxydim, sethoxydim, and clethodim inhibited
fatty acid biosynthesis in a chloroplast enzyme preparation from barley when acetate
and acetyl-CoA were the substrates, but not when malonate and malonyl-CoA
were added. These results suggested that ACCase was the site of action for
these herbicides. Moreover, it was found that the ACCase from dicotyledonous
species showed almost no inhibition, which suggested that the mechanism of
selectivity between dicotyledonous and grass species was at the ACCase site of
action.
Two key reports [7, 21] established that the two types of ACCase enzyme in
plant correlated with the differential inhibition of the new herbicides represented
by two classes – the AOPPs and the CHDs – which are strong inhibitors of
the multidomain plastid ACCase found in grasses. Prokaryotic-type multisubunit
plastid ACCase is resistant to these herbicides, as are the eukaryote ACCases from
animals and yeast.
Widely used commercial herbicides, represented by AOPPs and CHDs, are
potent inhibitors of the ACCases of sensitive plants, which they kill by shutting
down fatty acid biosynthesis, which in turn leads to metabolite leakage from the
membranes and cell death [22]. Both, the AOPPs and CHDs, inhibit CT activity
[Scheme 11.1, reaction (2), thus blocking the transfer of the carboxyl group to
acetyl-CoA [23]. Both types of compound show nearly competitive inhibition with
respect to the substrate acetyl-Co A [24, 25]. This observation confirms that an
inhibitor of the CT domain is sufficient to block the function of the ACCase, and
it also establishes this domain as a valid target for the development of inhibitors
against these enzymes.
The manner in which herbicides interact with the CT domain has been revealed
over the past decade by the use of X-ray crystallography. Tong and coworkers
have made several reports indicating how the herbicides haloxyfop, clodinafop,
tepraloxydim, and ‘‘pinoxaden acid’’ 1 (Figure 11.2), bind at the active site of the
CT domain [26–29].
O O
CF3 Cl O Cl F O
OH OH
N O
N O
Haloxyfop Clodinafop
O
O
O Cl
N
N
OH N
O OH
O
1
Tepraloxydim "Pinoxaden acid"
Figure 11.2 Structures of haloxyfop, clodinafop, tepraloxy-

dim, and ‘‘pinoxaden acid.’’ Note that the commercial her-
bicides are frequently esters, rapidly hydrolyzed in planta to
these parent acids.
Although Tong’s structures relate to the yeast enzyme, which the herbicides
inhibit relatively weakly, sequence alignments have shown that the CT domain
of yeast and monocot chloroplastic ACCase have a sequence identity greater than
50% (and are almost completely conserved in the vicinity of the active site).
This high similarity suggests that, in the absence of any published structure of
a monocotyledon chloroplastic ACCase, the yeast CT domain remains a good
surrogate for studying the binding interactions of the various herbicides. This high
similarity has enabled Zhu and coworkers to build homology models of foxtail
and blackgrass ACCases, and to propose molecular mechanisms for herbicide
resistance using molecular dynamics (MD) simulations [30–32].
The crystal structures of the yeast CT domain reveal it to be homodimeric, with
the monomers bound in a ‘‘head-to-tail’’ fashion and a twofold rotational axis of
symmetry. Each dimer contains two symmetrically related active sites, which sit at
the interface between the two dimers (Figure 11.3).
Figure 11.3 Crystal structure of the yeast CT domain

in complex with haloxyfop (PDB code 1UYR). The two
monomers are colored orange and blue. The two haloxy-
fop molecules are shown in ‘‘spacefill’’ representation. The
dimer is viewed down the twofold rotational axis.
A comparison of the crystal structures of the CT domain–herbicide complexes

confirms the results of earlier kinetic studies [25] which indicated that the
herbicides act by blocking the binding of the acetyl-CoA substrate to the active site.
The overlapping binding modes of CoA, haloxyfop, and tepraloxydim are shown
in Figure 11.4.
Haloxyfop
Coenzyme A
Tepraloxydim
Figure 11.4 Overlay of the binding modes much larger overlap for the true substrate
of coenzyme A (orange carbons), tepraloxy- acetyl-coenzyme A, in which the terminal
dim (pink carbons), and haloxyfop (gray thiol group is acetylated. This figure was
carbons) at the CT domain active site. Al- obtained by superimposing the coordinates
though the overlap between coenzyme A and from crystal structures 1OD2, 3K8X, and
haloxyfop is small, the structures suggest a 1UYR.
A comparison of the haloxyfop and tepraloxydim structures reveals both sim-

ilarities and differences in their binding modes. Both compounds are acidic
(with pKa -values of 2.9 and 4.58, respectively) [33], while their oxy-anions share a
common binding site, with the enolate oxygen of tepraloxydim and a carboxylate
oxygen of haloxyfop both accepting hydrogen bonds from Ala1627 and Ile1735.
The methyl group in haloxyfop is located within 0.7 Å of that in the ethyl group of
tepraloxydim, and these two groups share the same site, which includes Leu1705,
equivalent to one of the mutation sites found in some resistant weed species (see
below). The oxime moiety of tepraloxydim partly overlays with the central phenyl
ring of haloxyfop. Apart from these similarities, the two herbicides probe very
different regions of the active site. One notable feature of haloxyfop (and diclofop)
binding is that it causes significant conformational changes in the active site. In
particular, the side chains Tyr1738 and Phe1956 move in order to generate a large
hydrophobic pocket, into which the pyridinyl ring of haloxyfop sits.
Very recently, Tong and coworkers reported details of the crystal structure of
the free acid of pinoxaden complexed with the CT domain of yeast ACCase [29].
Although, at the time of writing, the coordinates are not yet available from the
Protein Data Bank, Tong et al. have compared the binding mode of ‘‘pinoxaden
acid’’ 1 with tepraloxydim and haloxyfop. The pyrazoline ring of 1 is comparable to
the CHD ring of tepraloxydim, with both moieties being acidic, cyclic 1,3-diones.
The two oxygen atoms of both structures are tethered to the enzyme by hydrogen
bonds in the same manner, while one of the ethyl groups of 1 makes the same
interactions as the ethyl group of tepraloxydim and fills the same pocket. The
phenyl ring of 1 overlaps with the planar oxime moiety of tepraloxydim, and
the seven-membered ring of 1 extends into the region of space occupied by the
tetrahydropyran ring of tepraloxydim.
11.2.3
Resistance
The frequent use of ACCase-inhibiting herbicides has resulted in the evolution of

resistance to such compounds in a number of agronomically important weeds. To
date, 37 resistant species [5] have been reported, including Lolium rigidum, Alopecu-
rus myosuroides, Avena fatua, Setaria viridis, and Eleusine indica. The mechanisms
of resistance to ACCase-inhibiting herbicides can be divided into two categories –
ACCase-related and metabolism-based. Whilst metabolism-based resistance tends
to be polygenic and involve cytochrome P450 and glutathione S-transferase (GST)
enzymes [34, 35], ACCase-related resistance is due to an alteration of the target
enzyme, which makes it less sensitive to inhibition (as reviewed by Devine [4],
Délye [36], and Powles and Yu [37]). Mutations at seven different ACCase codon
positions have been linked to target site-based resistance. A single point mutation
leading to the substitution of an isoleucine (Ile) by a leucine (Leu) residue at
position 1781 within the CT binding domain of plastidic ACCase in Alopecurus
myosuroides (blackgrass) has been found to confer resistance to most CHDs and
AOPPs [38]. A homologous mutation is responsible for target site resistance in
three other grass weeds, Lolium sp. (rye-grass) [39, 40], Avena fatua L. (wild-oat) [41],
and Setaria viridis (green foxtail) [42]. The mutations leading to an Ile→asparagine
(Asn) exchange at position 2041, of tryptophan (Trp) in position 2027 to cysteine
(Cys), as well as glycine (Gly) to alanine (Ala) in 2096, affect mainly the AOPPs
in blackgrass and in ryegrass [43, 44]. In blackgrass and ryegrass, an aspartic acid
(Asp) to Gly mutation at position 2078 leads to resistance on APPs, CHDs, and
DEN herbicides [43, 45]. The Cys to arginine (Arg) change at position 2088, as
identified in Avena and Lolium species, confers broad resistance to all ACCase
herbicides tested, whilst the W1999C mutation affects fenoxaprop-P-ethyl but not
clodinafop-propargyl efficacy in Avena sterilis [46]. Thus, resistance conferred by
target-site mutation appears to be weed-, herbicide-, and dose-specific.
Three-dimensional models of homodimeric ACCase were reconstructed to pro-
vide a detailed evaluation of the effects of amino acid substitutions at positions
1781, 2027, 2041, 2078, and 2096 in black-grass ACCase upon herbicide binding
[43]. The process employed homology models based on crystal structures of the
yeast free ACCase CT domain as templates [26]. All five amino acids were shown
to be located within the active site cavity of the ACCase CT domain, and only
the substitution at position 2041 interfered directly with herbicide binding. It was
proposed that the other four mutations caused resistance either by hampering
access of the inhibitor to its binding site, or by altering the spatial shape of the
herbicide binding site [47].
11.2.4
Detection of Resistance
To date, the detection and management of resistance has predominantly been

carried out with the use of bioassays. These are based essentially on the comparative
growth of seedlings or plants of suspected resistant and sensitive weed biotypes
when subjected to different herbicide treatments [48–50]. Although such bioassays
are simple, they do not differentiate between target site and metabolic resistance
mechanisms; for example, ACCase-based resistance is expressed in pollen, whereas
metabolism-based is not [50, 51]. The main I1781L mutation leading to resistance
can occur only by the substitution of an adenine (A) with thymine (T), or cytosine
(C) at the first position in the cognate codon. As a result, it was possible to develop a
polymerase chain reaction (PCR)-based allele-specific amplification assay to detect
the I1781L mutation in the plastidic ACCase of L. rigidum and A. myosuroides plants,
thus providing a quick and efficient method to monitor a key resistance mechanism
to ACCase inhibitors in these species [39, 52]. Kaundun and Windass [53] described
an alternative derived Cleaved Amplified Sequence (dCAPS) method [54] that can
be used on several grass weeds, and which offers the additional advantage of an easy
discrimination between homozygous and heterozygous L1781 mutation-bearing
plants. However, DNA-based assays can only reveal known target-site resistance
mutations and not metabolism, which appears to be the predominant mechanisms
conferring resistance to the ACCase herbicides [55].
11.3
Chemistry of ACCase Inhibitors
Three classes of ACCase inhibitors have been commercialized, AOPPs (or fops),
CHDs (or dims), and a single example of the DEN class. Since 2000, most of the
chemical research conducted with inhibitors of the ACCases has centered on the
DEN class.
11.3.1
Aryloxyphenoxypropionates (AOPPs or fops)
The discovery of diclofop-methyl as a selective post-emergence herbicide during

the 1970s allowed for the control of grass weeds in field crops [56]. Subsequent
introductions have offered improved biological performances, in terms of potency,
weed spectrum, and/or crop selectivity. Details of the major currently commercially
available AOPPs are listed in Table 11.2.
Although most commercially available AOPPs are sold as esters (which confers
an improved foliar uptake), the corresponding carboxylic acid is considered to be
the true active ingredient. In the case of the AOPPs, foliar activity is known to
reside largely in the (R)-enantiomer [57–59].
Owing to their mode of action, selectivity in broadleaved crops is generally
excellent. By contrast, selectivity in cereals is more challenging, and the AOPP is fre-
quently sold in mixture with a safener such as mefenpyr-ethyl or cloquintocet-mexyl
(see Chapter 8).
Metamifop, a recent introduction for the control of grass weeds in rice, is unusual
in being an amide rather than an ester. Whilst the amide has been reported to
be active against isolated ACCase enzyme [60], it is not clear if hydrolysis to the
corresponding acid is required for full expression of its herbicide activity in the field.
11.3.2
Cyclohexanediones (CHDs or dims)
The discovery of alloxydim at Nippon Soda, and its launch in 1980, triggered interest
in the CHDs as herbicidal inhibitors of ACCase [61]. The systematic variation of
substituents around the CHD ring, coupled with changes to the oxime, led to
compounds with a superior biological performance, and alloxydim has now been
discontinued. Details of current commercially available CHD herbicides are listed
in Table 11.3.
For the most part, CHD herbicides such as sethoxydim and tepraloxydim are
used in broadleaf crops such as soya, cotton, oilseed rape, and sugar beet. In
contrast to the AOPPs, CHD herbicides do not appear to respond well to safeners,
and the achievement of an acceptable selectivity in monocotyledonous crops has
proven difficult, with only tralkoxydim sold for use with cereals, and profoxydim
for rice. More recently, cell culture techniques developed at the University of
Minnesota [62] have been used to identify corn cell lines which are more than
456
Table 11.2 Selected Commercial AOPP Herbicides.
Name Structure Original Year of introduction Crop use

manufacturer
Diclofop-Methyl O Hoechst 1975 Cereals

Cl Cl O
O
11 Acetyl-CoA Carboxylase Inhibitors
O
Fluazifop-Butyl O Ishihara ICI 1980 Broad- leaved Crops
CF3 O
O
N O
Fenoxaprop-Ethyl Hoechst 1984 Cereals and rice

O
Cl O
N O
O O
Quizalofop-Ethyl Nissan 1985 Broad- leaved crops
O
Cl N O
O
N O
Haloxyfop-Methyl O Dow 1986 Broad- leaved crops
CF3 Cl O
O
N O
Clodinafop-Propargyl Ciba Geigy 1991 Cereals
O
Cl F O
O
N O
Cyhalofop-Butyl Dow 1996 Rice
O
NC F O
O
Metamifop F Dongbu 2008 Rice

O
Hannong
Cl O
N N
O O
11.3 Chemistry of ACCase Inhibitors
457
458
Table 11.3 Commercial CHD Herbicides.
Name Structure Original Year of introduction Crop use

manufacturer
Alloxydim O Nippon 1977 Broad-leaved crops

O Soda(discontinued)
N
OH
11 Acetyl-CoA Carboxylase Inhibitors
CO2Me
Sethoxydim O Nippon 1981 Broad- leaved crops

SodaBASF
O
N
S OH
O
O
Tralkoxydim N ICI 1986 Cereals
OH
Cycloxydim O BASF 1987 Broad-leaved crops
O
N
OH
S
Clethodim O Chevron 1987 Broad- leaved crops
O Cl
N
S OH
Butroxydim O ICI 1995 Broad-leaved crops

O
N
OH
Profoxydim Cl BASF 1999 Rice

O
O
N O
OH
Tepraloxydim O Nippon 2001 Broad-leaved crops

O Cl SodaBASF,
N Mitsui
OH
O
11.3 Chemistry of ACCase Inhibitors
459
40-fold tolerant to sethoxydim, while a process of back-crossing into elite lines has
afforded sethoxydim-tolerant corn, which was first marketed during the mid-1990s.
Although this technology has until now enjoyed only limited success, it remains of
potential interest as a non-genetically modified organism (GMO) approach to the
development of herbicide-tolerant corn.
11.3.3
Aryl-1,3-Diones (DENs)
11.3.3.1 Discovery of 2-Aryl-1,3-Diones

The first biocidal DENs were reported at Union Carbide during the 1970s by
Wheeler and coworkers [63, 64], who described both pre- and post-emergence
herbicidal effects, as well as miticidal activity, against Tetranychus urticae for 1,3-
diones such as 2 and 3, as well as for ester 4 and carbonate derivatives (Figure 11.5).
O O
Cl
O
OH O
OH O
2 3 4
Figure 11.5 Representative 2-aryl-1,3-diones first reported by

Wheeler and coworkers at Union Carbide.
More than 10 years later, Fischer and coworkers discovered 2-aryl-indolizine-2,
4-diones with moderate herbicidal and miticidal activities [65], and reported that
compound 5 would inhibit the plastidic ACCase of grasses [66]. Almost simulta-
neously, both Cederbaum [67] and Fischer [68] claimed the herbicidal activity of
2-mesityl-tetrahydropyrazolo-1,3-diones 6 (Figure 11.6).
O Cl O
O
N N N
Cl O
N N
OH O
OH
O
5 6 Pinoxaden
Figure 11.6 Representative structures of indolizinediones

and tetrahydropyrazolodiones from the patent literature, to-
gether with pinoxaden.
Since then, more than 100 patent applications covering the herbicidal activity
of DENs have been made, with pinoxaden having been commercialized as a
broad-spectrum, post-emergence graminicide for use in wheat and barley [69].
11.3.3.2 Syntheses
Wheeler and coworkers adopted two distinct strategies for the synthesis of
2-aryl-1,3-cyclohexane-diones such as 7 and 8 [70, 71]. In the first approach
(Scheme 11.2), the coupling of a preformed dione with a suitably functionalized aro-
matic ring was carried out. Aromatic halides bearing strongly electron-withdrawing
substituents allowed for SN Ar coupling to afford compounds such as 7. By con-
trast, the photolysis of 2-diazo-1,3-diones in electron-rich aromatic solvents afforded
additional products, such as 8, albeit in modest yield. The second approach involved
the condensation of phenylacetonitriles with glutarate esters or anhydrides; the
resulting product 9 was then hydrolyzed, decarboxylated, and cyclized to afford the
cyclohexane-1,3-diones (Scheme 11.3).
These twin strategies of ring synthesis from an arylacetic acid derivative, or by
the coupling of a preformed dione with an aromatic ring, remain the methods of
choice today, albeit with some modifications to the original procedures.
Cl NO2
O Cl NO2
Cl O
Cl
K2CO3, DMF, Δ
O Cl
OH
Diazotransfer 7
O
N2 hn O
O
OH
8
Scheme 11.2 Initial syntheses of 2-aryl-1,3-diones, as reported by Haines [70].
O R1 R2 O
O R1 R2 O
NC EtO OEt
EtO
NaOEt, EtOH, Δ
CN
9
1. HCl, Δ
2. EtOH, H+, Ph-H
O O R1 R2 O
NaH, PhCH3
EtO
R2
OH
R1
Scheme 11.3 Preparation of 2-aryl-1,3-diones from phenylacetonitriles [71].

Arylacetic acid derivatives are particularly useful starting materials for the
synthesis of heterocyclic diones, and have been used widely in the synthesis of
tetronic and tetramic acids (Scheme 11.4) [67, 72, 73].
O
SOCl2
O
HO
PhCH3 Cl
NH2.HCl
Et3N, THF
CO2Me
MeO
O
O H
KOt-Bu N
MeO DMA
CO2Me
N
O MeO
H
Scheme 11.4 Representative synthesis of an aryl-tetramic acid [72].
In a variation to this procedure, aryl malonic acid derivatives can be

converted into cyclic pyrazoline-3,5-diones by condensation with hydrazines.
Chlorocarbonylketenes such as 10, which were first described by Nakanashi [74],
represent highly reactive equivalents of phenyl malonates, and react under mild
conditions with hydrazines to give desired cyclic pyrazoline-3,5-dione, such as 11
(Scheme 11.5) [75].
H
N
O N O
SOCl2 H
HO2C Cl N
Methylcyclohexane, Et3N, Et2O
Δ O N
CO2H OH
10 11
Scheme 11.5 Synthesis and use of chlorocarbonylketenes in

the preparation of 4-arylpyrazoline-3,5-diones [68].
The coupling of a 1,3-dione, or a derivative thereof, with a suitable aromatic

ring has developed beyond Wheeler’s original methodology, and several routes
have now been developed, allowing for convergent syntheses to a diverse set of
2-aryl-2,3-diones.
The direct coupling of 1,3-diones with aryl halides under conditions similar to
those reported by Buchwald and Hartwig has been described (Scheme 11.6), but this
methodology appears restricted to aryl halides bearing only one ortho-substituent,
and therefore suffers from limited utility [76–79].
O O
+ Pd(OAc)2, X-Phos, K3PO4

Br
O DME, 160°C, microwave
OH Cl
Cl
Scheme 11.6 Representative palladium-catalyzed crosscou-

pling of aryl halides with cyclic 1,3-diones [76].
A more general method, albeit with only moderate yields obtained typically,
involves a Suzuki-like coupling reaction of a phenylboronic acid and an iodo-
nium ylide 12, as shown in Scheme 11.7. The ylide is readily prepared from
a cyclic-1,3-dione and (diacetoxy)iodobenzene in aqueous ethanol (Scheme 11.7)
[80–83].
O O O
Ph
PhI(OAc)2, I Ar-B(OH)2,
LiOH.H2O Pd(OAc)2
O EtOH / H2O O LiOH.H2O, O

O O O
DME
12
Scheme 11.7 Representative synthesis of a 2-aryl-1,3-dione

from palladium-catalyzed crosscoupling of an arylboronic
acid and an iodonium ylide [83].
Alternatively, the coupling of aryllead triacetates, readily prepared from aryl-

boronic acids [84–86], and cyclic diones under mild conditions permits even highly
hindered aryl-1,3-diones to be prepared (Scheme 11.8).
Cl Cl
O
O
+ DMAP
(AcO)3Pb
O Ph-CH3 / CHCl3
OH
Scheme 11.8 Representative synthesis of a 2-aryl-1,3-dione

through the coupling of aryllead triacetates with
cyclic-1,3-diones [76].
R R
X X
O O O
Y Y
D O-alkylation D z Claisen D z
E E Rearrangement E
G O G O G O
13
Scheme 11.9 The synthesis of 2-aryl-1,3-diones via a Claisen rearrangement of

enol-ethers.
In addition to the routes described above, alternative syntheses of DENs have been
developed that offer access to further derivatives. In one approach (Scheme 11.9),
a Claisen rearrangement has been utilized to construct the key aryl–dione bond.
The rearrangement was effected by heating a solution of an enol ether 13 in
1,2-dimethoxyethane, diglyme, or triglyme at temperatures of 180–250 ◦ C under
microwave irradiation. The reaction generally worked well for furans, oxazoles,
thiazoles, selenazoles, and thiophenes, but failed for phenyl and for six-membered
heteroaromatic rings [87–89]. Synthesis of the desired enol ether starting materials
was readily accomplished by the reaction of cyclic-1,3-dione with an alkyl halide
under basic conditions, or with a suitable alcohol under Mitsunobu conditions.
Alternatively, the enol ether could be prepared by first converting the dione into a
vinyl halide such as 14, and reacting this with a suitable alcohol. An example of
this approach is illustrated in Scheme 11.10.
Cl Cl
NaH, THF S S
Triglyme, Δ Cl
S Cl
O
H O N
H N
H
N
O
HO O OH
O O
H
H O H
14
Scheme 11.10 Synthesis of 2-aryl-1,3-diones by Claisen rearrangement [89].
In a further approach, aryl Grignard reagents have been added to furfuraldehyde,

and the resulting furfuryl alcohol 15 rearranged under acidic conditions [90] to give
cyclopentenones 16, which may be oxidized to the corresponding dione 17 in high
yield. These cyclopentenediones are versatile intermediates, as they react readily
with dienes [including furan (see Scheme 11.11) and 1,3-dipoles] to afford novel
bicyclic and tricyclic diones [91–94].
O
1. Mg, THF H3O+
Br 2. Furfuraldehyde,
O
THF OH OH
15 16
Jones
oxidation
O O
O Furan, MgI2
H H2, Pd-C H
MeOH
O O O
O O
H H
17
Scheme 11.11 Representative synthesis of 2-mesityl

cyclopentene-1,3-dione, 14, and its use as a dienophile [91].
11.4
Structure–activity data relating to the herbicidal activities of the DENs have

been reported by Muehlebach and coworkers at Syngenta [95, 96]. Additional
structure–activity relationships (SARs) may be inferred from data contained within
the large number of patent applications that have been made during the past 10
years. The DENs can be separated into three parts for an analysis of the SARs,
namely, the dione, the phenyl moiety, and the group, Q (Figure 11.7). Each part
can be examined separately, as they appear to play distinct and different roles in the
overall expression of the herbicidal activity. By contrast, the relationship between
structure and crop selectivity is much less obvious, and cannot be assigned solely
to one of these contributory fragments.
Q
O V,W,X,Y
D
E
G O
Figure 11.7 Generic structure of 2-aryl-1,3-diones for con-

sideration of structure–activity relationships. Compounds
where Q = H are the true active ingredient; additional
analogs show herbicidal activity only if Q is converted to H
in planta.
11.4.1
Procide Structure–Activity Relationships
A key feature of 2-aryl-cyclic-1,3-dione is the presence of, or an ability to generate

in vivo, an acidic proton, Q = H, as this is believed to be the true active ingredient.
Chemically stable derivatives of the dione, where Q is an alkyl group, show only
weak activity at best, while less-stable derivatives (e.g., when Q is acyl or sulfonyl)
retain much of the activity of the dione, and simply act as pro-pesticides, with rapid
conversion to the dione in planta. The formation of pro-pesticides provides a way of
fine-tuning physical properties to allow for the optimization of herbicidal effects,
for example, by improving uptake into leaves [96].
11.4.2
Dione Structure–Activity Relationships
Five- and six-membered cyclic 1,3-diones show the highest levels of activity.
Typically, these diones have a pKa in the range of 3.5 to 5.5. High levels of herbicidal
activity on grasses have been found with many types of dione such as tetramic
acids, cyclopentanediones, CHDs, or oxazine-3,5-diones. These rings may be fused
to a second ring, and certain tricyclic rings also show good activity. The diversity of
cyclic 1,3-diones capable of demonstrating high levels of herbicidal activity, when
combined with optimized aryl moieties (vide infra), is shown in Figure 11.8.
O OH OH OH
MeO
Aryl Aryl Aryl N Aryl
O N N
OH O O O
O O O O
H
Aryl Aryl Aryl Aryl
N
N O
O OH OH
OH H OH
O O O O
Aryl Aryl Aryl Aryl
N
O O N
OH N OH OH OH
Figure 11.8 Representative structures of cyclic-1,3-diones

which show significant herbicidal activity, when combined
with optimized aryl moieties. Variation of the cyclic dione
affects not only herbicidal activity, but also crop selectivity.
11.4.3
Phenyl Structure–Activity Relationships
Two distinct SARs are emerging for the phenyl moiety (Figure 11.9). From a
consideration of Tong’s reported crystal structures [26–29], it is assumed that
key hydrogen bond interactions from Ala1627 and Ile1735 will likely hold the
enolate of all cyclic 1,3-diones in a common orientation. The observation that
substituted phenyl and heteroaromatic rings are tolerated in either position X or
position Y suggests that the binding of these ACCase inhibitors can therefore
induce significant changes in the shape of the active site. Further support for
this hypothesis derives from the fact that it is possible to replace the phenyl
ring attached to the dione with a five-membered heteroaromatic system, since
once again this should position the terminal aromatic ring in a slightly different
orientation (Figure 11.10).
Q V X Q V
O O
D D Y
E W E
G O G O
SAR for 2,4,6- SAR for 2,5-

substitution substitution
pattern pattern
Figure 11.9 Preferred substitution patterns for 2-aryl-1,3-dione herbicides.
Cl
O O S
O
Cl
D D D N
E E E
G O G O Cl G O
Figure 11.10 Significant structural variation of the aryl moi-

ety is tolerated, suggesting that the active site may flex to
accommodate different structural classes.
No information has been reported on the SARs for the five-membered hetero-
cycles, although compounds such as those shown in Figure 11.11 and described
in the patent literature have shown excellent levels of herbicide activity, implying a
tight binding to the target enzyme [97, 98].
11.4.3.1 2,4,6-Trisubstituted Phenyl Structure–Activity Relationships

The overview given here reflects mainly the results obtained from studies with
4-aryl-pyrazolidin-3,5-diones [95], which were optimized towards activity and
O O
S
N
N N Cl
N
Cl N
OH OH
Figure 11.11 Representative five-membered heterocyclic diones. From Refs [97] and [98].
O
Q V X
O
N
D N
O O
E W
G O O
SAR for 2,4,6- Pinoxaden
substitution pattern
Figure 11.12 Optimization of the 2,4,6-substitution pattern.

The cereal herbicide pinoxaden has this SAR pattern. Note
also that pinoxaden is a pro-pesticide; the pivalate group is
hydrolyzed in planta.
selectivity in small-grain cereals, during the research program that led to pinoxaden
(Figure 11.12).
The presence of at least one ortho-substituent was essential for activity, with
small alkyl moieties preferred. An ethyl group in both ortho-positions, V and W, led
to an increased activity, while a 2,6-diethyl-4-methyl substitution pattern boosted
the graminicidal activity still further. The level of activity could be improved by
replacing one of the ortho-ethyl groups with an ethynyl or a methoxy-substituent,
although these two functionalities induced a higher level of phytotoxicity in cereals.
Interestingly, 2,6-dimethoxy derivatives were found to be almost inactive.
Position X may be functionalized. Compared to methyl, a phenyl or substituted
phenyl group led to a broader spectrum, but also to phytotoxicity on cereals.
In parallel, some activity on broadleaf weeds was observed. These biaryl diones
proved to be extremely potent inhibitors of ACCase, and while the strong activity
against grass weeds could be explained by an inhibition of chloroplastic ACCase,
the broadleaf activity might be accounted for by additional inhibition of cytosolic
ACCase.
Recent patent applications have also indicated that substituted phenoxy and
heteroaryl phenoxy moieties are also tolerated at position X [99].
11.4.3.2 2,5-Disubstituted Phenyl Structure–Activity Relationships

Whilst simple 2,5-dialkyl-substitued phenyl rings show only a weak herbicidal
activity, replacement of the alkyl substituent at position Y with a substituted
11.5 Pinoxaden 469
phenyl, phenoxy, heteroaryl, or heteroaryloxy moiety significantly increases the

activity against grass weeds [84, 86, 100–104].
Substitution of the terminal phenyl, or heteroaryl, with electron-withdrawing
groups such as halogen, cyano, nitro or trifluoromethyl confers greater potency,
but can lead to unacceptable crop damage.
The favored 2-substituents, V, are as for the 2,4,6-trisubstituted phenyl, and
include methyl, ethyl, n-propyl, and cyclopropyl; notably, 2-halo analogs also retain
some activity.
To date, despite the high levels of herbicide activity observed, none of the DENs
bearing a 2,5-substitution pattern has been developed commercially.
11.5
Pinoxaden
11.5.1
Characteristics
Pinoxaden, a new graminicide for cereal crops, has been developed by Syngenta
[105]. The physico-chemical and toxicological data of pinoxaden are summarized
in Table 11.4.
Table 11.4 Properties of pinoxaden.
Physico-chemical properties
Common name Pinoxaden
Company code NOA 407855
Melting point 121 ◦ C
Partition coefficient octanol–water Log POW = 3.2
Solubility 200 mg l−1 in water
Vapor pressure 4.6 × 10−7 Pa
Toxicological profile
Rat: Acute oral LD50 (mg kg−1 ) >5000
Rat: Acute dermal LD50 (mg kg−1 ) >2000
Rat: Inhalation LC50 (mg l−1 ) 5.2
Skin and eye irritation Irritant
Ecotoxicological environmental profile
Birds: acute LD50 (mg kg−1 body weight) >2250: negligible risk to birds
Earthworms: LC50 (mg kg−1 dry soil) >1000: nontoxic
Bees: LD50 (μg per bee; contact) >100: safe to bees
Aquatic organisms No risk to algae, fish, and Daphnia
Nontarget flora and fauna No risk to dicotyledonous plants; no
adverse effects against beneficial
arthropods
11.5.2
Technical Synthesis
The synthesis of pinoxaden is based on a thermal condensation of a cyclic

hydrazine 18 and an aryl malonamide 19, itself prepared in a three-step procedure
from 2,6-diethyl-toluidine (Scheme 11.12). The aryl-malononitrile 20 is hydrolyzed
to the aryl-malonamide 19 in concentrated sulfuric acid, and then condensed
with [1,4,5]oxadiazepane to give the ‘‘pinoxaden acid’’ 1; the latter can be readily
converted into its enol ester, pinoxaden, under standard conditions [106].
NC CN
NaNO2, H2O NaOt-Bu, xylene NC
H2N 48% HBr, Δ Br PdCl2, PCy3, Δ CN
20
H2SO4
H2O
O NH
.2HCl O
O O NH
Cl O
18 H2N
N N
N DMAP, Et3N, THF N Et3N, xylene, Δ H2N O
O O OH
O
O
Pinoxaden 1 19
Scheme 11.12 Synthesis of pinoxaden.
11.5.3
Biology
Pinoxaden is applied post-emergence at rates of 30–60 g a.i. ha−1 [105]. Interplay of

the active compound with a safener, cloquintocet-mexyl, has proved to be essential
in order to maximize the tolerance of the crop to pinoxaden [105]. Methyl oleate, as
adjuvant, enhances the level of activity without impairing the crop safety [107].
Pinoxaden is applied flexibly, from the two-leaf stage up to the flag leaf stage of
grasses, and has a weed spectrum that covers a wide range of key annual grass
species such as Alopecurus myosuroides (blackgrass), Apera spica venti (silky bent
grass), Avena spp. (wild oats), Lolium spp. (ryegrass), Phalaris spp. (canary grass),
Setaria spp. (foxtails), and other monocotyledonous weed species commonly found
in cereals [105].
In an uptake experiment, over 90% of radiolabeled pinoxaden was incorporated
into the crops within 5 h when treatment solutions were applied in droplets to
11.5 Pinoxaden 471
the adaxial leaf surface of two-leaved plants of barley, winter wheat, or durum
wheat. After 24 h, about 20% had been translocated out of the treated leaf by a
basipetal movement below the treated area (G.J. Hall, personal communication).
Cloquintocet has no effect on either the absorption or movement of the herbicide
within the crop.
While active against certain ACCase-resistant biotypes – both target site- and
metabolic-resistant – pinoxaden is not active on all of these [105].
11.5.4
Metabolism and Selectivity
The total radioactive residues in winter wheat treated in autumn applications

under outdoor conditions declined rapidly in forage, from 6.7 mg kg−1 on day 1 to
0.3 mg kg−1 at 14 days after treatment (DAT). Ultimately, the total residues in grain,
husks, and straw at maturity were low. Details of the major detected metabolites
and a proposed metabolic pathway are shown in Scheme 11.13 (as extracted from
data sets submitted to Registration Authorities).
Pinoxaden is hydrolyzed within a very short time to ‘‘pinoxaden acid’’ (1), which
is rapidly hydroxylated to the major metabolite 21 found in plants. This benzylic
alcohol is further oxidized to a large extent to the acid 22 or glycosylated, and
further conjugated. The primary metabolite in soils, 23, is also found in plants
at lower levels, and is further hydroxylated to the diol 24. All of the metabolites
except 1 were inactive when tested on plastidic wheat ACC in vitro, and showed no
O O O
OH
N N N
N N N
O O O OH O O
Pinoxaden 1 23
CO2H
O O OH O OH
OH
N N N
N N N
O OH O OH O O
22 21 24
Scheme 11.13 Metabolism pathway for pinoxaden in planta.

phytotoxic effects on emerged grasses and cereals in greenhouse trials, even when
applied at higher rates.
The effect of the safener cloquintocet-mexyl on the biokinetics and metabolism
of pinoxaden in barley (cv. Manitou), winter wheat (cv. Soisson), durum wheat
(cv. Colssea), Avena fatua and Lolium rigidum, was investigated using the radiola-
beled herbicide (G.J. Hall, P. Carter, A. Burridge, unpublished data). Pinoxaden
was applied at a rate equivalent to 90 g ha−1 with the adjuvant A12127 used at 0.5%.
Leaves were treated with 20 × 0.2 ml droplets containing 14 C-labeled pinoxaden at
4000 dps. Treatments were made with or without cloquintocet-mexyl (S) added at
25% the rate of the herbicide (H). In this case, safening is achieved by enhancing
the metabolism of pinoxaden within the crop (Table 11.5). The safener mainly
triggers hydroxylation of the methyl group of 1 to afford the alcohol 21 in all cereal
crops, but does not seem to affect the hydroxylation of 1 to 23. Cloquintocet has no
relevant effect on the metabolism of pinoxaden in the grass weed Lolium rigidum,
or in Avena fatua.
Table 11.5 Crop metabolism of pinoxaden by barley, winter

wheat, durum wheat and key cereal weeds. The table shows
the percentage of each component found in planta follow-
ing treatment with herbicide alone (H), or with herbicide and
cloquintocet-mexyl (H + S)
Barley Winter wheat Durum wheat Lolium rigidum Avena fatua
H+S H H+S H H+S H H+S H H+S H
Pinoxaden 1.5 2.1 0 0 0.8 0.3 0 0 0 0

Compound 1 32.3 48.3 36 81.6 48.8 79.1 84.2 86.6 84.7 90.4
Total 33.8 50.4 36 81.6 49.7 79.4 84.2 86.6 84.7 90.4
Compound 21 38.3 25.5 59.8 13.8 35.6 14.7 3.8 3.5 11.1 7.1
Compound 23 8.3 8.7 4.2 4.1 5.8 3.1 4.2 3.5 3.2 2.5
Others 19.6 15.4 0 0.5 9 2.8 7.8 6.4 1 0
All metabolites 66.2 49.6 59.8 18.4 50.4 20.6 15.8 13.4 15.3 9.6
The soil metabolism of pinoxaden is summarized in Scheme 11.14. Pinox-

aden is hydrolyzed very rapidly in soil to ‘‘pinoxaden acid’’ 1 under aerobic,
aerobic–anaerobic, and sterile–aerobic conditions. Compound 1 is acidic, with a
pKa of 3.82, and an aqueous solubility of 380 g l−1 at pH 7, but is rapidly hydrox-
ylated to the neutral alcohol 23, which is much less soluble (aqueous solubility
0.370 g l−1 ). The half-life of 1 varies from 1.8 to 6.1 days, depending on the type of
soil, whereas 23 degrades with a half-life of 6.2 to 37 days (as extracted from data
sets submitted to Registration Authorities). The rapid metabolism of pinoxaden
prevents its leaching through the soil profile.
Diverse minor metabolites accounted for less than 5% of the applied radioactivity.
The bound residues reached a maximum of 49% after 14–30 days. The only
identified volatile metabolite was carbon dioxide, demonstrating mineralization.
O O O
OH
N N N
N N N
O O O OH O O
O
1 23
Pinoxaden
Minor metabolites Minor metabolites

bound residues
carbon dioxide
Scheme 11.14 Soil metabolism of pinoxaden.
Up to 47.6% of the applied radioactivity was mineralized after 100 days in laboratory
soil metabolism studies. Finally, minimal dissipation has been observed in soils.
11.6
Summary and Outlook
Three classes of commercial herbicides – the AOPPs, the CHDs, and the newly
discovered DEN derivatives – have been shown to inhibit the CT function of the
eukaryotic ACCase found in the plastids of grasses [2, 6].
Progress in elucidating the function of ACCase inhibitors has largely contributed
to an understanding and differentiation of the resistance mechanisms involved
[4, 36, 37]. During recent years, considerable effort has been undertaken to
identify the mode of action of herbicides on ACCase at the molecular level.
Point mutations in the chloroplastic ACCase CT domain of different monocots,
Alopecurus myosuroides, Avena fatua, Lolium rigidum, and Setaria viridis have been
correlated with resistant phenotypes [38–46], and fine mapping of these mutations
in the sequence has led to the identification of those amino acids which are
important for herbicide action in the CT domain. At the same time, the CT domain
of the protein, spanning the domain where the point mutations have been found,
has been crystallized [26–29].
Taken together, these approaches have allowed predictions to be made of the
herbicide binding to the CT domain. In particular, it has been possible to determine
amino acid changes responsible for herbicide resistance to AOPP and/or CHD
analogs, and to localize the amino acids that are directly involved in the binding of
herbicides, but only for this domain.
Not all ACCase inhibitors are affected by resistance to the same extent, with some
compounds retaining significant activity against many of the resistant biotypes
[37]. However, with increasing information becoming available on the structure

of ACCase, and details of the binding of various inhibitors emerging, it should
become possible to further rationalize these observations.
Despite the existence of herbicidal ACCase inhibitors having first been recognized
during the 1970s, their importance for weed control remains high. Indeed, the
commercial success of pinoxaden highlights the continued value of this mode of
action to the selective control of grass weeds in cereal crops.
Acknowledgments
The authors sincerely thank their many colleagues at Syngenta who have con-
tributed to the research of inhibitors of ACCase over recent years. Special thanks
are due to Sophie Oliver, Shiv Shankhar Kaundun, Russell Viner, and James Scutt
for their help in the preparation of this manuscript.
References
1. Harwood, J. (1999) Pestic. Outlook, 10, 11. Konishi, T., Shinohara, S., Yamada, K.,
154–115. and Sasaki, Y. (1996) Plant Cell Physiol.,
2. Hirai, K. (2002) in Herbicide Classes 37, 117–122.
in Development (eds P. Böger, 12. Ohlrogge, J.B. and Jaworski, J.G. (1984)
K. Wakabayashi, and K. Hirai), Annu. Rev. Plant Physiol., 35, 155–189.
Springer-Verlag, Berlin, Heidelberg, 13. Fa Yang, S. and Hoffmann, N.E. (1984)
pp. 234–238. Annu. Rev. Plant Physiol., 35, 155–189.
3. Hock, B., Fedtke, C., and Schmidt, R.R. 14. Nikolskaya, T., Zagnitko, O.,
(1995) Herbizide, G. Thieme Verlag, Tevzadze, G., Haselkorn, R., and
Stuttgart. Gornicki, P. (1999) Proc. Natl Acad. Sci.
4. Devine, M.D. (2002) in Herbicide USA, 96, 14647–14651.
Classes in Development (eds P. Böger, 15. Gornicki, P., Faris, J., King, I.,
K. Wakabayashi, and K. Hirai), Podkowinski, J., Gill, B., and
Springer-Verlag, Berlin, Heidelberg,
Haselkorn, R. (1997) Proc. Natl Acad.
pp. 103–113.
Sci. USA, 94, 14179–14185.
5. Heap, I.M. International
16. Egli, M., Lutz, S., Somers, D., and
Survey of Resistant Weeds,
Gengenbach, B. (1995) Plant Physiol.,
http://www.weedresearch.com (accessed
108, 1299–1300.
May 2011).
6. Nicolau, B.J., Ohlrogge, J.B., and 17. Schulte, W., Topfer, R., Stracke, R.,
Wurtele, E.S. (2003) Arch. Biochem. Schell, J., and Martini, N. (1997) Proc.
Biophys., 414, 211–222. Natl Acad. Sci. USA, 94, 3465–3470.
7. Alban, C., Balder, P., and Douce, R. 18. Hoppe, H.H. (1981) Z. Pflanzenphysiol.,
(1994) Biochem. J., 300, 557–565. 102, 189–197.
8. Incledon, B.J. and Hall, J.C. (1997) 19. Burton, J.D., Gronwald, J.W., Somers,
Pest. Biochem. Physiol., 57, 255–271. D.A., Conelly, J.A., Gengenbach, B.G.,
9. Sasaki, Y. and Nagano, Y. (2004) and Wyse, D.L. (1987) Biochem. Bio-
BioSci. Biotechnol. Biochem., 68, phys. Res. Commun., 148, 1039–1044.
1175–1184. 20. Focke, M. and Lichtenthaler, H.R.
10. Reverdatto, S., Beilinson, V., and (1987) Z. Naturforsch., 42c, 1361–1363.
Neilsen, N.C. (1999) Plant Cell Physiol., 21. Konishi, T. and Sasaki, Y. (1994) Proc.
119, 961–968. Natl Acad. Sci. USA, 91, 3598–3601.
References 475
22. Devine, M.D. and Shimabukuro, 39. Délye, C., Matéjicek, A., and
R.H. (1994) in Herbicide Resistance Gasquez, J. (2002) Pest Manage. Sci.,
in Plants (eds S.B. Powles and J.A.M. 58, 474–478.
Holtum), CRC Press, Boca Raton, FL, 40. Zagnitko, O., Jelenska, J., Tevzadze, G.,
pp. 141–169. Haselkorn, R., and Gornicki, P.
23. Rendina, A.R., Craig-Kennard, A.C., (2001) Proc. Natl Acad. Sci. USA,
Beaudoin, J.D., and Breen, M.K. (1990) 98, 6617–6622.
J. Agric. Food Chem., 38, 1282–1287. 41. Christoffers, M.J., Berg, M.L., and
24. Rendina, A.R., Beaudoin, J.D., Messersmith, C.G. (2002) Genome, 45,
Craig-Kennard, A.C., and Breen, M.K. 1049–1056.
(2003) Proc. Brighton Crop Protect. 42. Délye, C., Wang, T., and Darmency, H.
Conf. – Weeds, 163–172. (2002) Planta, 214, 421–427.
25. Burton, J.D., Gronwald, J.W., Keith, 43. Délye, C., Zhang, X.-Q., Michel, S.,
R.A., Somers, D.A., Gengenbach, B.G., Matéjicek, A., and Powles, S.B. (2005)
and Wyse, D.L. (1991) Pestic. Biochem. Plant Physiol., 137, 794–806.
Physiol., 39, 100–109. 44. Délye, C., Zhang, X.-Q., Chalopin, C.,
26. Zhang, H., Yang, Z., Shen, Y., Michel, S., and Powles, S.B. (2003)
and Tong, L. (2003) Science, 299, Plant Physiol., 132, 1716–1723.
2064–2067. 45. Kaundun, S.S. (2010) Pest Manage. Sci.,
27. Zhang, H., Tweel, B., and Tong, L. 66, 1249–1256.
(2004) Proc. Natl Acad. Sci. USA, 101, 46. Liu, W., Harrison, D.K., Chalupska, D.,
5910–5915.
Gornicki, P., O’Donnell, C.C., Adkins,
28. Xiang, S., Callaghan, M.M., Watson,
S.W., Haselkorn, R., and Williams,
K.G., and Tong, L. (2009) Proc. Natl
R.R. (2007) Proc. Natl Acad. Sci. USA,
Acad. Sci. USA, 106, 20723–20727.
104, 3627–3632.
29. Yu, L.P.C., Kim, Y.S., and Tong, L.
47. Délye, C., Calmes, C.E., and Matéjicek,
(2010) Proc. Natl Acad. Sci. USA., 107
A. (2002) Theor. Appl. Genet., 104,
(51), 22072–22077.
1114–1120.
30. Zhu, X.L., Hao, G.F., Zhan, C.G., and
48. Boutsalis, P. (1998) WO Patent
Yang, G.F. (2009) J. Chem. Inf. Model.,
WO98/55860.
49, 1936–1943.
49. Boutsalis, P. (2001) Weed Technol., 15
31. Zhu, X.L., Zhang, L., Chen, Q.,
(2), 257–263.
Wan, J., and Yang, G.F. (2006)
J. Chem. Inf. Model., 46, 1819–1826. 50. Letsouzé, A. and Gasquez, J. (2000)
32. Zhu, X.L., Yang, W.C., Yu, N.X., Yang, Weed Res., 40, 151–162.
S.G., and Yang, G.F. (2011) J. Mol. 51. Richter, J. and Powles, S.B. (1993)
Model. 17, 495–503. Plant Physiol., 102, 1037–1041.
33. Tomlin, C. (ed.) (2006) The Pesticide 52. Balgheim, N., Wagner, J., Hurle, K.,
Manual, 14th edn, British Crop Protec- Ruiz-Santaella, P., and De Prado, R.
tion Council (BCPC), p. 1350. ISBN: (2005) Congress Proceedings – BPCP
1-901396-14-2. International Congress: Crop Science
34. Van Eerd, L.L., Hoagland, R.E., and Technology, Glasgow, UK, Vol. 1,
Zablotowicz, R.M., and Hall, J.C. pp. 157–162.
(2003) Weed Sci., 51, 472–495. 53. Kaundun, S.S. and Windass, J.D.
35. Kreuz, K., Tommasini, R., and (2006) Weed Res., 46, 34–39.
Martinoia, E. (1996) Plant Physiol., 54. Faris, J., Sirikhachornkit, A.,
111, 349–353. Haselkorn, R., Gill, B., and
36. Délye, C. (2005) Weed Sci., 53, Gornicki, P. (2001) Mol. Biol. Evol.,
728–746. 18, 1720–1733.
37. Powles, S. and Yu, Q. (2010) Annu. 55. Petit, C., Bay, G., Pernin, F., and
Rev. Plant Biol., 61, 317–347. Délye, C. (2010) Pest Manage. Sci., 66,
38. Délye, C., Calmes, C.E., and 168–177.
Matéjicek, A. (2002) Theor. Appl. Genet., 56. Chow, P.N.P. (1978) Weed Sci., 26,
104, 1114–1120. 352–358.
57. Gerwick, B.C., Jackson, L.A., 73. Ruther, M., Hagemnaa, H.,
Handly, J., Gray, N.R., and Russell, Schneider, U., Dollinger, P.,
J.W. (1988) Weed Sci., 36, 453–456. Dahmen, M., Wachendorff-Neumann,
58. Walker, K.A., Ridley, S.M., Lewis, T., U., Fisher, R., Graff, A.,
and Harwood, J.L. (1988) Biochem. J., Bretschneider, T., Erdelen, C., Drewes,
254, 307–310. M.W., Feucht, D., and Lieb, F. (2001)
59. Nestler, H.J. and Bieringer, H. (1980) WO Patent WO2001/074770.
Z. Naturforsch. B, 35B (3), 366–371. 74. Nakanashi, S. and Butler, S. (1975)
60. Kim, T.J., Chang, H.S., Kim, J.S., Org. Prep. Proc., 7, 155–158.
Hwang, I.T., Hong, K.S., Kim, D.W., 75. Kruger, B.-W., Fischer, R., Bertram,
Cho, K.Y., Myung, E.J., and Chung, H.-J., Bretschneider, T., Bohm, S.,
B.J. (2003) Proceedings of BCPC In- Krebs, A., Schenke, T., Santel, H.-J.,
ternational Congress, Glasgow, pp. Luerssen, K., Schmidt, R.R., Erdelen,
833–838. C., Wachendorff-Neumann, U., and
61. Sawaki, M., Iwataki, I., Odawara, H., Stendel, W. (1992) Patent DE4109208.
Yoshihiko, H., Ishikawa, H., and 76. Mathews, C.J., Hotson, M.B., Dowling,
Odawara, K. (1975) Patent JP50046644. A.J., Scutt, J.N., and Govenkar, M.
62. Parker, W.B., Marshall, L.C., Burton, (2008) WO Patent WO2008/145336.
J.D., Somers, D.A., Wyse, D.L., 77. Palucki, M. and Buchwald, S.L. (1997)
Gronwald, J.W., and Gengenbach, J. Am. Chem. Soc., 119, 11108.
B.G. (1999) Proc. Natl Acad. Sci. USA, 78. Kawatsura, M. and Hartwig, J.F. (1999)
87, 7175–7179.
J. Am. Chem. Soc., 121, 1473.
63. Wheeler, T.N. (1979) Patent
79. Fox, J.M., Huang, X., Chieffi, A., and
DE2813341.
Buchwald, S.L. (2000) J. Am. Chem.
64. Sousa, A.A., Durden, J.A. Jr, and
Soc., 122, 1360.
Stephen, J.F. (1973) J. Econ. Entomol.,
80. Schank, K. and Lick, C. (1983) Synthe-
66 (2), 584–586.
sis, (5), 392–395.
65. Fischer, R., Krebs, A., Albrecht,
81. Zhu, Q., Wu, J., Fathi, R., and Yang, Z.
M., Santel, H.J., Schmidt, R.R.,
(2002) Org. Lett., 4 (19), 3333–3336.
Luerssen, K., Hagemann, H.,
82. Mathews, C.J., Finney, J., Robinson, L.,
Becker, B., Schaller, K., and Stendel,
Tyte, M., Muehlebach, M., and
W. (1990) Patent EP0355599.
Wenger, J. (2008) WO Patent
66. Babenzinski, P. and Fischer, R. (1991)
Pestic. Sci., 33, 455–466. WO2008/110307.
67. Cederbaum, F., Brunner, H.G., 83. Muehlebach, M., Lutz, W., Wenger, J.,
and Boeger, M. (1992) WO Patent Finney, J., Mathews, C.J., and
WO92/16510. Fawke, D. (2008) WO Patent
68. Krüger, B.W., Fischer, R., Bertram, WO2008/110308.
H.J., Bretschneider, T., Böhm, 84. Mathews, C.J., Hotson, M.B., Dowling,
S., Krebs, A., Schenke, T., Santel, A.J., Scutt, J.N., Govenkar, M., and
H.J., and Lürssen, K. (1992) Patent Challinor, L. (2008) WO Patent
DE4109208. WO2008/145336.
69. Muehlebach, M., Glock, J., Maetzke, 85. Morgan, J. and Pinney, T. (1990) J.
Th., and Stoller, A. (1999) Patent Chem. Soc. Perkin Trans. 1, 715–720.
WO99/47525. 86. Muehlebach, M., Mathews, C.J., Scutt,
70. Haines, R.G. (1979) US Patent J.N., and Govenkar, M., (2008) WO
4,175,135. Patent WO2008/071405.
71. Wheeler, T.N. (1979) J. Org. Chem., 44 87. Jeanmart, S.A.M., Taylor, J.B.,
(26), 4906–4912. Mathews, C.J., and Smith, S. (2009)
72. Fischer, R., Lehr, S., Drewes, M., WO Patent WO2009/015877.
Feucht, D., Malsam, O., Bojack, G., 88. Jeanmart, S.A.M., Mathews,
Arnold, C., Auler, T., Hills, M.J., C.J., Smith, S., Taylor, J.B., and
Kehne, H., and Rosinger, C. (2006) Govenkar, M. (2009) WO Patent
WO Patent WO2006/056282. WO2009/000533.
References 477
89. Jeanmart, S.A.M., Taylor, J.B., Tyte, M., WO Patent WO2010/089210 and WO
Mathews, C.J., and Smith, S. (2009) Patent WO2010/089211.
WO Patent WO2009/030450. 100. Finney, C.J.J., Scutt, J.N., Robinson,
90. Saito, K. and Yamchika, H. (1983) US L., and Delaney, J.S. (2010) WO Patent
Patent 4,371,711. WO2010/102848.
91. Tyte, M., Mathews, C.J., Hall, G., 101. Mathews, C.J., Finney, J., Scutt, J.N.,
Whittingham, W.G., Wailes, S.J., Scutt, Robinson, L., and Delaney, J.S. (2010)
J.N., Jeanmart, S.A.M., and Viner, R. WO Patent WO2010/081755.
(2009) WO Patent WO2009/019005. 102. Mathews, C.J., Finney, J., Robinson, L.,
92. Tyte, M., Jeanmart, S.A.M., Mathews, Tyte, M., Muehlebach, M., and
C.J., and Robinson, L. (2009) WO Wenger, J. (2008) WO Patent
Patent WO2009/019015. WO2008/110307.
93. Mathews, C.J., Finney, J., Scutt, J.N., 103. Tyte, M., Jeanmart, S.A.M., Mathews,
Robinson, L., and Delaney, J.S. (2010) C.J., and Robinson, L. (2009) Patent
WO Patent WO2010/102848. WO2009/019015.
94. Mathews, C.J., Scutt, J.N., Robinson, 104. Lieb, F., Fischer, R., Graff, A.,
L., and Tyte, M. (2010) WO Patent Schneider, U., Breitschneider, T.,
WO2010/057880. Erdelen, C., Andersch, W.,
95. Muehlebach, M., Boeger, M., Drewes, M.W., Dollinger, M., and
Cederbaum, F., Cornes, D., Friedmann, Wetcholowsky, I. (1999) WO Patent
A.A., Glock, J., Niederman, T., WO99/48869.
Stoller, A., and Wagner, T. (2009) 105. Hofer, U., Muehlebach, M., Hole, S.,
Bioorg. Med. Chem., 17, 4241–4256. and Zoschke, A. (2006) J. Plant Dis.
96. Muehlebach, M. et al. (2007) in Pesti- Prot., XX (Special Issue), 989–995.
cide Chemistry, Crop Protection, Public 106. Mühlebach, M., Brunner, H.,
Health, Environmental Safety (eds H. Cederbaum, F., Maetzke, T.,
Ohkawa, H. Miyagawa, and P.W. Mutti, R., Schnyder, A., Stoller, A.,
Lee), Wiley-VCH Verlag GmbH, pp. Wendeborn, S., Wenger, J.,
101–110. Boutsalis, P., Corens, D.,
97. Fischer, R., Ullmann, A., Friedmann, A., Glock, J., Hofer, U.,
Trautwein, A., Drewes, M., Hole, S., Niedermann, T., and
Erdelen, C., Dahmen, P., Feucht, D., Quadranti, M. (2007) in Pesticide
Pontzen, R., Kuck, K.-H., and Chemistry, Crop Protection, Public
Wachendorff-Neumann, U. (2002) Health, Environmental Safety (eds H.
WO Patent WO2002/088098. Ohkawa, H. Miyagawa, and P.W.
98. Stevenson, T.M., Marshall, E.A., Lee), Wiley-VCH Verlag GmbH,
and Taggi, A.E. (2009) WO Patent pp. 101–110.
WO2009/086041. 107. Glock, J., Friedmann, A., and
99. Mathews, C.J., Finney, J., Scutt, J.N., Cornes, D. (2001) WO Patent
Robinson, L., and Delaney, J.S. (2010) WO2001/017352.
479
12
Photosynthesis Inhibitors: Regulatory Aspects, Re-Registration
in Europe, Market Trends, and New Products
12.1
Introduction
Herbicides acting as inhibitors of photosynthesis by blocking electron transport

in photosystem II (PS II) belong to the eldest classes of plant-protection agents.
Whilst these compounds are still of market relevance, especially in developing
countries, they are no longer within the ‘‘focus’’ of modern herbicide research due
to their high application rates in response to the high enzyme concentrations for
photosynthesis in plants, and for their cross-resistance behavior.
Photosynthesis inhibitors may be divided into compound classes of: (i) triazines,
triazinones, triazolinones (the newest members; see amicarbazone in Section 12.7),
uracils, and phenylcarbamates belonging to the C1 group of the Herbicide Resis-
tance Action Committee (HRAC) classification scheme; (ii) the arylureas and
amides belonging to the C2 group; and (iii) the nitriles, benzothiadiazinones, and
phenylpyridazines of the C3 group of the HRAC classification [1]. Photosynthesis,
which takes place in the chloroplasts, was first recognized as the principle of the
‘‘assimilation of carbon dioxide’’ by plants in the mid-nineteenth century, though
the individual reaction steps were not evaluated and understood until the inves-
tigations of Hill in 1937, which were continued during the 1950s [2, 3], mainly
via investigations with these inhibitors, in particular with ureas [4–7], triazines
[8–10], and triazinones [11–13], between 1956 and 1975. In 1961, Calvin, at the
University of California, had won the Nobel Prize in chemistry ‘‘. . . for his research
on the carbon dioxide assimilation in plants’’ and investigations into the light–dark
reactions of photosynthesis [14] and the synthesis of carbohydrates from CO2 .
More modern studies, which involved protein structural chemistry and investiga-
tions using X-radiography – to describe not only the binding niches but also the
inhibitors which would bind within such niches – began only with the ubichinon
binding pocket in photosynthesis. Such studies led, in 1988, to the Nobel Prize for
Chemistry being awarded to H. Deisenhofer, R. Huber, and H. Michel [15].
The Hill reaction (Scheme 12.1) permits a quantitative determination of the in-
hibitory properties of photosynthesis blockers on chloroplast systems by monitoring
O2 evolution (oxygen electrode, Warburg manometer) and, as a consequence, the

480 12 Photosynthesis Inhibitors
H2O + A AH2 + 1/2 O2
Scheme 12.1 The Hill reaction.

50% inhibitory concentration of a photosynthesis inhibitor in the Hill reaction. The
values thus obtained (in their negative logarithm form as pI50 values) can then be
used in quantitative structure–activity relationship (QSAR) studies regarding the
in vitro activity (‘‘QSAR; Hansch approach’’ [16–20]). The pI50 -values obtained can
also be compared with those obtained in greenhouse trials, in order to evaluate
biochemical versus biological activities which include also transport, membrane,
and metabolism effects (Absorption, Distribution, Metabolism, Excretion; ADME).
Consequently, QSAR – as a method for improving the biological activities in synthe-
sis programs aimed at developing new crop protection compounds – was broadly
investigated and initially approved in a variety of research programs involving
photosynthesis [21, 22].
The 1,3,5-triazines were developed first at the Geigy laboratories [23, 24],
with simazine (1955) being the first representative of the group, followed
by atrazine (Geigy, 1958), propazine (Geigy, 1960), trietazine (Geigy, 1960),
terbuthylazine (Geigy, 1966), and cyanazine (Shell, 1971) from the 2,6-diamino-
4-chloro-1,3,5-triazines, prometryne (Geigy, 1962), ametryne (Geigy, 1964),
desmetryne (Geigy, 1964), and terbutryne (Geigy, 1966) from the 2,6-diamino-4-
methylmercapto-1,3,5-triazines, and terbumeton (Geigy, 1966) from the
2,6-diamino-4-methoxy-1,3,5-triazines.
The 1,2,4-triazinones [25], metamitron (Bayer, 1975), and metribuzin (Bayer,
Du Pont, 1971) and the 1,3,5-triazine-2,4(1H,3H)-dione [26] hexazinone (Du Pont,
1975), resulted first from the resynthesis of university publications or analog
synthesis using uracils as a starting concept.
The uracils [27], bromacil (Du Pont, 1952), lenacil (Du Pont, 1974), and terbacil
(Du Pont, 1966), were developed by Du Pont, whereas the pyridazinones [28,
29], pyrazon (BASF, 1962), evolved from research investigations at BASF and
Sandoz. The phenylcarbamates [30] desmedipham and phenmedipham, which
were developed by Schering AG, are also included in the C1 group of the HRAC
classification (Figure 12.1).
The herbicidal effect of aryl- and hetarylurea, which was studied systematically
starting from the first observations in 1946, was improved between 1951 [31]
and 1973 [32]. Indeed, from such chemistry have today been derived the com-
pounds chloroxuron (Ciba, 1960), dimefuron (Hoechst, 1969), diuron (Du Pont,
1954), ethidimuron (Bayer, 1973), fenuron (Du Pont, 1957), fluometuron (Ciba,
1960), isoproturon (Hoechst, 1974), linuron (Hoechst, 1960), methabenzthiazuron
(Bayer, 1968), metobromuron (Ciba, 1963), metoxuron (Sandoz, 1968), monolin-
uron (Hoechst, 1958), neburon (Du Pont, 1957), siduron (Du Pont, 1964), and
tebuthiuron (Elanco, 1973), all of which are in current use.
The amides propanile [33] and pentanochlor [34], which also belong to the C2
group of the HRAC classification, fulfill the general formula for photosynthesis
inhibitors, as they each bear a CONH group (Figure 12.2).
X O O
N N R NH2
N N N
R1 R3
N (CH3)2N N N
N N O N R1
H
R2 CH3
1,3,5-triazines 1,3,5-triazindiones 1,2,4-triazinones
O O
H H
X R3 R4 N O N O
Ar N R R1
N
N R2 O O O
NHR N
R1
Pyridazinones Uracils Phenylcarbamates
Figure 12.1 PS II inhibitors, C1 group.
R2
H
N N (a) R1 = CH3, R2 = H, CH3
R1
R4 (b) R1 = CH3, R2 = OCH3
O
R3
Arylureas
R
R4 R = alkyl
N O R3, R4 = halogen, alkyl
R3 H
Amides
OH O
X X CH(CH3)2
X = I, Br N
SO2
N
CN H
Nitriles Bentazone
OR
(a) R = COSC8H17
N (b) R = H
Cl N
Phenylpyridazines
Figure 12.2 PS II inhibitors, C2 and C3 groups.
The photosynthesis inhibitors of the C3 group of the HRAC classification – the

nitriles [35] bromofenoxim, bromoxynil, ioxynil, as well as the benzothiadiazinone
bentazone [36] and the phenylpyridazines [28] pyridate, and pyridafol – have
completely different structures, without a CONH group (Figure 12.2).
Whereas, in 1980, photosynthesis inhibitors represented almost 50% of the
market share for all herbicides [37], the situation has recently undergone a
significant change, not only due to the introduction of the new acetolactate synthase
(ALS) inhibitors such as the sulfonylureas (SUs), hydroxyphenylpyruvate dioxyge-
nase (HPPD) inhibitors, and genetically modified crops that are resistant against
5-enolpyruvylshikimate-3-phosphate (EPSP) synthase and glutamine synthetase
(GS) inhibitors, but also through significant changes in the current re-registration
requirements, especially within Europe.
12.2
The Re-Registration Process in the European Union
The registration of agrochemicals falls under the national laws of all countries
worldwide where plant-protection compounds are used. These national laws reg-
ulate the data requirements for active compounds, as well as for the formulations
and mixtures, the risk-assessment process, and requirements for labeling the mar-
keted plant-protection product. At an early stage in the history of agrochemicals,
those companies that were inventing, developing, and marketing plant-protection
compounds and products – as well as the general public – were seeking a har-
monization of the data requirements and risk assessment for registration. Some
examples of supranational harmonization activities are listed in Tables 12.1–12.4.
Additionally, global harmonization endeavors are undertaken by the Food and
Agriculture Organization (FAO) of the United Nations and the World Health
Organization (WHO). The FAO supports harmonization efforts, for example,
through the information system ‘‘Prior Informed Consent’’ (PIC). In this system, an
exchange of certain hazardous pesticides and industrial chemicals in international
trade takes place between the member authorities. Previously, the members will
have agreed on an international code of conduct on the distribution and use
of pesticides, and also on guidelines related to the development and evaluation
of data considered in the registration process. Further, WHO organizes Joint
Meetings of their members, together with the WHO on Pesticide Residues (JMPR)
to define and organize the maximum residue level (MRL) Database on Pesticides,
in which the maximum pesticide residue levels are documented. The WHO has
developed the pesticides evaluation scheme ‘‘WHOPES,’’ in which it establishes
and publishes specifications for technical material and related formulations of
public health pesticides. Typically, the WHO reviews the safety reports, issues,
guidelines for the laboratory and field evaluation of insecticides and repellents,
and provides recommendations on equipment and application manuals. It also
publishes health criteria (EHC) monographs on chemicals/pesticides; examples
are the WHO Classification of Pesticides by Hazard, and the WHO/FAO Pesticide
Datasheets (IPCS Inchem) [38].
The Organisation for Economic Co-operation and Development (OECD)
published a vision document [on the occasion of the 14th meeting of the Working
Group on Pesticides (WGP) held in Paris on 5–6 November 2002]. The document
included statements on achievements to-date in the international harmonization
of the regulatory approaches for agricultural pesticides (chemical and biological),
Table 12.1 Supranational harmonization activities in EC, US, and NAFTA.
Political Responsible authority Legislation Object of registration Time to registration

Union/country
EC/countries EU Commission, through the Directives like Directive 91/414/EEC, national Active ingredients Average of at least 5
of the EC European Food Safety Authority laws like COPR, COP(A)R, PPPR (a.i.) regulated by yr until Annex I
(EFSA). National authorities of Deutsches Pflanzenschutzgesetz, etc. EEC Directives inclusion for a new
the different countries New Regulation 1107/2009 into force, 14 (adopted to national active substance
December 2009 to be adopted by 14 June laws). Products according EC
2011, replacing the Directive 91/414/EEC regulated by national 91/414/EEC, detailed
laws registration times
according 1107/2009
EEC >3 yr, different
further regulations,
for example, new
data requirements
have to be set
USA/States EPA Federal Insecticide, Fungicide, and a.i. and products. Up to 3–4 yr
Rodenticide Act (FIFRA); Food Quality States may register a
Protection Act (FQPA; 1996) new end use product
or an additional use
of a federally
registered pesticide
product under
specific conditions
NAFTA Technical Working Group of Common data submissions for a.i. and products Subject to national
Pesticides (TWG). US-EPA, manufacturers – electronic harmonization timelines
Canadian Pest Management Joint reviews
12.2 The Re-Registration Process in the European Union
Regulatory Agency (PMRA), a Eliminating trade problems related to

consortium of Mexican differences in MRL (maximum residue limits)
483
agencies (CICOPLAFEST)
484
Table 12.2 Supranational harmonization activities in Central and South America.
Political Union/country Responsible authority Legislation Object of Time to

registration registration
Central America
Belize, Costa Rica, El Technical Regional Harmonized registration data Products Up to 2 yr
Salvador, Honduras, Pesticide Working and labeling requirements
12 Photosynthesis Inhibitors
Mexico, Nicaragua, Panama Group OIRSA FAO specifications and Codex

Alimentarius
Data exchange on efficacy
within region
South America
(a) Andean Community National authorities Common Pesticide Registration Products Up to 2 yr
Bolivia, Columbia, Ecuador, Manual (July 2002)
Peru, Venezuela ‘‘Norma Andina para el
(b) Mercosur Registro y Control de
Argentina, Brazil, Paraguay Plaguicidas Quimicos de Uso’’
and Uruguay, Chile, and Agricola-Decision 436.
Bolivia Comision Andina. Gaceta
Oficial del Acuerdo de
Cartagena
Ano XIV-No. 347
Lima, 17 June 1998 (based on
FAO principles ‘‘International
Code of conduct for the
distribution and use of
Pesticides’’
Table 12.3 Supranational harmonization activities in Asia.
Political Responsible Legislation Object of Time to

Union/country authority registration registration
Asia
(a) Japan MAFF National specific data a.i. and Up to 4 yr
requirements and products
test protocols
(b) P. R. National Harmonization of a.i. and Up to 2 yr
China/Vietnam MRLs products
(c) South Korea, National National efforts in a.i. and Up to 2 yr
other harmonization products
through the Regional
Network on
Pesticides in Asia
and the Pacific
(RENPAP)
India National National data a.i. and Up to 2 yr,
CIBRC generated for Indian products including late
Registration v/s data fixation of
needs of most MRL by
developing countries Ministry of
match very well Health
Australia National National comparable a.i. and Up to 2 yr
to EU requirements products
New Zealand National National comparable A.i. and Up to 2 yr
to EU requirements products
and in the use of work-sharing arrangements in examining and reporting on

data submissions (dossiers) provided by industry, as well as the use of country
evaluations (monographs) to support applications for their registration or the
reregistration or to support the establishment of MRLs or import tolerances for
particular active substances. The OECD also published a statement of their vision
for the next 10 years, including details of the specific objectives, milestones to be
reached along the way, and the indicators and measures of success to be used to
record and document progress achieved [39].
By working together, OECD governments and industry are ‘‘sharing the burden’’
of testing and assessing high-production-volume chemicals, pesticides and, most
recently, new chemicals. The OECD programs on harmonization are leading
to an exchange of documents used in re-registration and registration in OECD
countries, which began in 1992, by: (i) comparing pesticide data reviews; (ii)
elucidating OECD databases on pesticide and biocide review schedules; (iii) issuing
guidance on the preparation of dossiers and monographs; (iv) undertaking joint
reviews on new compounds, such as Project ‘‘Cornelia’’ on Bayer’s corn herbicide
foramsulfuron [a Joint review between US-EPA, Canadian Pest Management
Table 12.4 Supranational harmonization activities in Africa.
Political Union/country Responsible Legislation Object of Time to

authority registration registration
Africa CILSS – a.i. and Up to 2 yr

CSP (Comité Sahélien des products
Pesticides) (Chad, Mali,
Burkina Faso, Niger,
Mauritania, Senegal,
Cape Verde, Gambia and
Guinea-Bissau)
SADC (Southern African – South Africa; a.i. and Up to 2 yr
Development Registration products
Community) Angola, Act 36/1947
Botswana, Congo (DR), and
Lesotho, Malawi, Agricultural
Mauritius, Mozambique, Remedies
Namibia, Seychelles, Registration
South Africa, Swaziland, Procedure
Tanzania, Zambia, and Policy
Zimbabwe Document
Regulatory Agency (PMRA) and German BvL (2000–2002)]; (v) surveying the
best practices in the regulation of pesticides in 12 OECD countries; and (vi)
recommending the electronic protocols used for data submission. Progress in
the harmonization of data requirements and test guidelines may also be achieved
through surveying test guideline program (TGP) priorities for pesticides, minimum
data requirements for establishing MRLs and import tolerances, guidance notes
for the analysis and evaluation of chronic toxicity and carcinogenicity studies, and
so on. The OECD vision stated that, by the end of 2014, the regulatory system for
agricultural pesticides will have been harmonized to the extent that country data
reviews (monographs) for pesticides prepared in the OECD format on a national
or regional basis (e.g., EU or NAFTA) can be used to support independent risk
assessments and regulatory decisions made in other regions or countries.
In such a harmonization process, the European Commission (EC) enacted in
1991 the ‘‘Council Directive of 15 July 1991 concerning the placing of plant protection
products on the market’’ (91/414/EEC).
In this Directive, the EC regulates the registration and reregistration of active
ingredients and products for all countries in the EU. This Directive came into force
on 26 July 1993, and must be implemented by national laws in all countries in the
EU, for example, in the UK by the Plant Protection Products Regulations 2003.
The main elements of the Directive were as follows:
• To harmonize the overall arrangements for authorization of plant protection
products within the European Union.
This has been achieved by harmonizing the process for considering the safety
of active substances at a European Community level by establishing agreed criteria
for considering the safety of those products. Product authorization remains the
responsibility of individual Member States.
The Directive provides for the establishment of a positive list of active substances
(Annex I) that have been shown to be without unacceptable risk to humans or the
environment.
New and existing active substances can be initially included to Annex I of the
Directive for a period of 10 years when they successfully pass the EC’s review
program.
Member States can only authorize the marketing and use of plant protection products
after an active substance is listed in Annex I, except where transitional arrangements
apply, for example, National Provisional Authorisations (NPAs).
Before an active substance can be considered for inclusion in Annex I of Directive
91/414/EEC, companies must submit a complete data package (dossier) on both
the active substance and at least one plant-protection product containing that active
substance. The data required are:
• The identification of an active substance and plant-protection product.

• Descriptions of their physical and chemical properties.
• Their effects on target pests.
• A comprehensive file of study reports to allow for a risk assessment to be made
of any possible effects on workers, consumers, the environment, and nontarget
plants and animals.
Detailed lists of the data required to be evaluated to satisfy inclusion in Annex

I of the Directive, or the authorization of a plant-protection product are set out
in the Directive (Annexes II and III). Annex II data relate to the active substance,
and Annex III to the plant-protection product. These data are submitted to one or
more Member States for evaluation. A report of the evaluation is submitted to the
European Food Safety Authority (EFSA). Following peer review of the report, the
EFSA makes a recommendation to the European Commission on whether Annex
I inclusion is acceptable. This recommendation is then discussed by all Member
States in the framework of the Standing Committee on the Food Chain and Animal
Health (SCFCAH; previously the Standing Committee on Plant Health; SCPH).
Where necessary, the Scientific Panel is consulted before the SCFCAH can deliver
an opinion on whether an active substance should be included in Annex I of
91/414/EEC.
All member states are obliged to the ‘‘Uniform Principles.’’
The ‘‘Uniform Principles’’ (Annex VI of Directive 91/414/EEC) establishing
common criteria for evaluating products at a national level were published on 27
September 1997 (OJ L265, p. 87). Application of the Uniform Principles ensures
that authorizations issued in all Member States are assessed to the same standards.
The Directive states that all active ingredients should be reviewed periodically
within 10 years.
This applies to all old agrochemical compounds (substances) used in a country

of the EU prior to 1991, or before a country became a member of the EU
(Reregistration). Thus, all old photosynthesis inhibitors, for example, needed
to be reviewed and the manufacturers had to apply for registration (listing on
Annex I) by submitting dossiers prepared under the Directive 91/414/EEC [40].
A similar reregistration process was set from the US-EPA for all compounds on
the market before 1984 in the US. ‘‘In the light of the experience gained from
the application of Directive 91/414/EEC and of recent scientific and technical
developments,’’ as the European Parliament and the Council of European Union
stated in Regulation (EC) No 1107/2009 published on 24 November 2009 stated
[41]. The Directive 91/414/EEC was replaced by a new regulation including active
substances, safeners or synergists intended for the uses of protecting plants or
plant products (this includes also post-harvest uses) against all harmful organisms,
influencing the life process of plants (growth regulators, protection against heat,
salt, frost, etc.), destroying undesired plants or part of plants on walls, roads,
and places, including also coformulants and adjuvants such as rapeseed oil.
The Regulation shall apply also to microorganisms usable as crop-protection
agents.
12.3
Main Changes in Guidelines Regarding EU Re-Registration
The following main changes have also to be applied on the preparation of registra-
tion data for such compounds, which have been registered in different countries
based on dossiers regulated under the national laws before the reregistration
procedure was enforced by the Directive 91/414/EEC:
12.3.1
Good Laboratory Practice
Good laboratory practice (GLP) is concerned with the organizational process

conditions under which studies are planned, performed, monitored, and recorded.
GLP ensures that the way in which studies are conducted is adequately standardized
and they are of a sufficiently high quality to produce reliable results that can, with
confidence, be compared with results obtained by others carrying out the same work
and applying the same general principles. Internationally accepted GLP guidelines,
as drawn up by the OECD, provided a reference point for later EU legislation
[42]. The respective Directive, as applied to both active ingredients and formulated
products, came into effect on 30 June 1988. The subsequent ‘‘Authorizations’’
Directive, 91/414/EEC, and others extended the scope of GLP, by requiring GLP
compliance for all safety studies, and also GEP (good experimental practice) for
efficacy studies, whether conducted in the field or laboratory and whether using a
formulated product or the active ingredient.
12.3.2
Physical and Chemical Properties of Active Substances
A declaration of toxicological, ecotoxicological, or environmental significant im-

purities is needed. Especially hazardous chemicals, such as nitrosamines, must
be declared and are regulated by a maximum admissible concentration. In this
example, the total nitrosamine content of a pesticide formulation must not exceed
1 mg kg−1 of the active substance present.
The use and declaration of analytical methods must be in correspondence with
the ‘‘Technical Material and Preparations: Guidance’’ for generating and reporting
methods of analysis in support of the pre- and post-registration data requirements
for Annex II (part A, Section 4) and Annex III (part A, Section 5). FAO guidelines
(guidance on FAO specifications) and copies of specifications are available from
http://www.fao.org/ag/AGP/AGPP/Pesticid/Specs/Pdf/Manual_update%202006.pdf
and GCPF (formally GIFAP) guidelines. Information on CIPAC can be obtained from
http://www.cipac.org.
These guidelines apply to all studies started after 1 October 1999.
Where an FAO specification is available for an active substance in a preparation,
the tolerance limits must meet those in the FAO specification. However, where
there is no appropriate FAO specification, tolerances must meet limits as accepted
by the FAO Group of Experts [43, 44].
Where an active substance is present as an ester or a salt, the active substance
content must be expressed as the amount of the ester or salt present (as the
technical material), with a statement declaring the amount of the active principle.
The methods used for the determination of physical properties should be in
accordance with the requirements of EC Directive 94/37/1.
12.3.3
Storage Stability
The data submitted must support the proposed shelf-life of the preparation. It is
normally expected that a preparation should have a shelf-life of at least two years;
only where the shelf-life would be less than two years should the label include a
‘‘Use by . . .’’ date or other precautionary phrase.
Where a loss of ≥5% of active substance occurs during storage, the fate of the
active substance must be addressed and the breakdown products identified [45].
12.3.4
Physical and Chemical Characteristics of Preparation
The physical and chemical characteristics of preparations via parameters (e.g.,

explosive properties, oxidizing properties, flashpoint and other indications of
flammability, acidity/alkalinity and pH, surface tension, density, wettability, sus-
pensibility, dilution stability, dry and wet sieve test, particle size distribution, and
other properties of the formulation), and the corresponding methods utilized to
determine such data, must be determined and reported in detail [46].
Specific new tests on viscosity and surface tension are guided by the Commission
Directive 98/98/EC of 15 December 1998.
12.3.5
Operator Exposure Data Requirements
New regulations to protect the applicant of the plant-protection products were

brought into force, regulating data requirements, experimental details for the
measurement, and model calculations [47, 48].
12.3.6
Residue Data Requirements
The guidance documents embrace also the following aspects:

• Metabolism and distribution in plants.
• General recommendations for the design, preparation, and realization of residue
trials.
• Testing of plant-protection products in rotational crops.
• Comparability, extrapolation, group tolerances, and data requirements.
• Calculation of maximum residue levels and safety intervals (see also Ref. [49]).
Details of the data requirements for residue trials can be found in Annex II of
Directive 91/414/EC.
12.3.7
Estimation of Dietary Intakes of Pesticides Residues
Estimates of pesticide intake need to be made to compare potential consumer

dietary exposure with acceptable dietary intakes derived from toxicological studies.
At its most basic level, if estimates of long- and short-term intake are less than
the acceptable daily intake (ADI) and the acute reference dose (ARfD), respectively,
then the risks to the consumer may be regarded as acceptable.
The Guidelines and Criteria for the Preparation and Presentation of Complete
Dossiers and Summary Dossiers for the inclusion of Active Substances in Annex
I of Directive 91/414/EEC (Article 5.3 and 8.2) (Document 1663/VI/94) require
that an estimate is made regarding the theoretical intakes of pesticide residues by
consumers. Consumer risk assessment is a vital part of the approval process, and
it is in the applicant’s interest to estimate potential intakes as intake estimates can
assist in assessing whether further information is required.
Two intake calculation models are now available: one for short-term (acute) intake
calculations; and another for long-term (chronic) intake calculations. These two
models present updates of the previous versions, with new adult, vegetarian, elderly,
and more detailed child consumption data incorporated. As Excel spreadsheets,
they are designed to be more user friendly than the previous versions, and are
available with accompanying guidance notes.
12.3.8
Fate and Behavior of Agricultural Pesticides in the Environment
The data provided by applicants must permit an assessment to be made of the fate
and behavior of the pesticide in the environment. This information is subsequently
used to assess the risk to nontarget species (soil or aquatic organisms, plants, etc.)
and following crops that will be exposed to the pesticide formulation, its active
substance(s), and the metabolites, transformation, and degradation products of the
active substance(s). The information provided should therefore be sufficient:
• To predict the distribution, fate, and behavior of the pesticide in the environment,
as well as the time courses involved. That is, to estimate the concentrations in
soil, water, and air, and to assess how these concentrations compare with any
recognized limits or standards.
• In conjunction with other data, to identify measures necessary to minimize the
contamination of the environment and any impact on nontarget species.
• In conjunction with other considerations, to permit a decision to be made as to
whether the pesticide can be approved, and the uses for which it can be approved.
• In conjunction with other data, to classify the product as to risk.
• To specify relevant risk and safety phrases for the protection of the environment,
which are to be included on any labels.
As indicated above, the nature and amount of data required for pesticide approval
depend on the properties and use of each active substance. A stepwise, tiered or
triggered approach allows an efficient selection of tests essential to each individual
contamination risk analysis. The environmental exposure to a pesticide depends
primarily on the following factors.
12.3.8.1 Concentration of Chemical in the Relevant Environmental Compartment

The highest concentrations usually occur during and just after application. Follow-
ing application, the concentration of residues declines due to:
• degradation
• movement into other compartments
• dilution.
Degradation can include such processes as hydrolysis, photolysis, and microbial

metabolism. Movement reduces the concentration in the treated compartment but
may transport residues to untreated compartments – for example, from the plant
surface to the soil, or from the soil to water.
12.3.8.2 Bioavailability of the Chemical

For substances entering surface waters, the availability of a chemical to organisms
is related primarily to its concentration in the aqueous phase. When strongly
adsorbed to sediment or soil, availability will often be significantly reduced. Under
some circumstances, it would also be necessary to consider exposure of organisms
via the food chain or via the atmosphere.
12.3.8.3 Nature of the System or Organism

Exposure assessment for an organism requires information on such aspects as:
• Does it live in the treated area, or in an area to which the pesticide could be
transported?
• Does it actively consume treated crops or become exposed via the dermal or
inhalation routes, and so on?
In order to assess the risk of contamination of the environment, or the exposure
of nontarget organisms, the potential of the pesticide for movement through the en-
vironment must be addressed. For pesticides used in, on, or over soils, a study of the
pesticide’s breakdown in soil is required. Similarly, for pesticides intended for use
in or near water, or whose entry into water cannot be ruled out, information must be
supplied on mobility in soil and on the fate in the aquatic environment, including
natural water/sediment systems. Such requirements and any trigger values for
performing different types of study are detailed in the data requirements guidance.
12.3.9
Specific Guidance Regarding Water Limits According to Annexes of the
Authorizations Directive
Account must be taken of fate and behavior in relation to groundwater as well as

to surface water.
The behavior of any environmentally significant metabolites, and of any trans-
formation and degradation products of the pesticide, with significant potential to
contaminate the water or soil and to cause harm to nontarget organisms, must also
be investigated. The EC Authorizations Directive stipulates that this requirement
applies to those products which are formed from the pesticide active substance and
which occur at levels above 10% of the added pesticide. It may be necessary to inves-
tigate such products formed at levels <10% where they are known to have significant
effects on target and/or nontarget organisms. The Annexes of the Directive and EU
guidance documents should be referred to for more detailed guidance on this point.
Unless otherwise stated, the term pesticide in this document will be deemed to
include both the active substance and any significant metabolites, transformation
and degradation products. However, in the context of the 0.1 μg l−1 drinking water
limit being applied to groundwater, it is possible to make the case (with appropriate
supporting data) that metabolites, transformation, and degradation products are
‘‘not relevant,’’ and this limit does not then normally apply [50, 51].
12.3.10
Ecotoxicology Requirements
The areas that need to be addressed are:

• risk to birds and other terrestrial vertebrates;
• risk to aquatic life;
• risk to honeybees;
• risk to nontarget arthropods;

• risk to earthworms;
• risk to soil microbial processes;
• risk to other soil macroinvertebrates (see above);
• risk to other nontarget organisms (flora and fauna);
• risk to biological methods of sewage treatment (see above).
The guidance deals with each of these issues in turn. It addresses the basic data
requirements, and highlights the appropriate risk assessment schemes and other
sources of information that can be used in producing a good risk assessment.
There may also be other references and information that can be used in support
of the risk assessment. Notably, the use of such material should be scientifically
justified.
12.3.10.1 EPPO Risk-Assessment Schemes

The European and Mediterranean Plant Protection Organisation (EPPO) have
produced several schemes that can be used to assess the risk to nontarget organ-
isms. These schemes aim to provide a basis for undertaking an appropriate risk
assessment.
• Depending on the proposed use, pattern data are required on the acute, dietary,
and reproductive toxicity of an active substance and/or product to birds. Further
details of when such studies are required are outlined in Annex II Section 8.1
and Annex III 10.1 and 10.3 of Directive 96/12/EC.
• Data are always required on the acute toxicity of an active substance to two fish
species, to Daphnia magna, and to algae.
If the active substance is an herbicide, then data are also required on an
additional species of an alga as well as an aquatic plant. Full details of the
appropriate studies are provided in Section 8.2 Directive 96/12/EC and the Aquatic
Guidance Document.
Data are also required on the toxicity of the plant-protection product. Further
details on when these data are required can be found in the Aquatic Guidance
Document, as well as Section 10.2.1 of Directive 96/12/EC.
• Depending on the persistence of the active substance in the water phase of a
sediment water study, toxicity data are required to address the possible chronic
risk of an active substance. Guidance on when these data are needed and on
appropriate studies is provided in Sections 8.2 and 10.2.4 of Directive 96/12/EC,
as well as in the Aquatic Guidance Document.
• Depending on the partitioning and persistence of an active substance in the
sediment phase of natural water sediment study, data may be required on its
toxicity towards sediment-dwelling invertebrates. Details of when this study is
required and the choice of test method are given in the Aquatic Guidance
Document. Information is also given in Section 8.2.7 of Directive 96/12/EC.
• Data are required on the bioconcentration potential of an active substance when
the log POW is >3. Further details are given in Section 8.2.3 Directive 96/12/EC,
together with the Aquatic Guidance Document.
12.3.10.2 Buffer Zones and LERAPs

In certain instances, it may be necessary for the product to have a buffer zone
restriction added to the label in order to protect aquatic life.
12.3.10.3 Honeybee Risk Assessment

• Acute oral and contact toxicity tests are required in conjunction with a hazard
quotient. Where the hazard quotient is greater than 50, further testing may be
required. Details of the types of tests and calculation of the hazard quotient are
given in Sections 8.3 and 10.4 of Directive 96/12/EC. Guidance is also given in
the Terrestrial Guidance Document.
• An appropriate risk assessment is required where the hazard quotient is >50;
further testing may be required (see above).
• In certain cases, a bee brood feeding test may also be required. Reference should
be made to Section 8.3.2 of Directive 96/12/EC and the Terrestrial Guidance
Document.
12.3.10.4 Risk to Nontarget Arthropods

The risk to nontarget arthropods must be addressed, except where use is in
situations where there is no exposure. Details of when the tests are not required
are given in Section 8.3.2 of Directive 96/12/EC.
Initially, laboratory tests are undertaken, with further higher tier testing, for
example, extended laboratory tests are required if effects of >30% are seen. Tests
are usually undertaken with a representative formulation of the active substance.
Details of the tests required are given in Sections 8.3.2 and 10.5 of Directive
96/12/EC.
12.3.10.5 Risk for Soil Nontarget Microorganisms

Key guidance on the risk assessment for soil nontarget microorganisms is given in
the Guidance Document on Terrestrial Ecotoxicology. The European Commission
Working Document 2021/VI/98 describes the same risk assessment procedure as
it is applied to soil nontarget macroorganisms (earthworms, beetles, etc.)
12.4
New Regulations in Europe
12.4.1
MRL Regulation
A new MRL regulation for the European Union has been established (no. 396/2005),
which was fully adopted on 1 September 2008 [52]. The key features of the document
are:
• One EU MRL for a substance in one commodity, replacing the individual MRLs
set previously by each member state.
• This EU MRL will be approved after an evaluation by EFSA, voted in the

SCFCAH, and it then undergoes scrutiny by the European Parliament before
being published.
• For every new use or product there must be an EU MRL before the authorization
certificate can be granted.
• Several foods will be subject to MRLs for the first time.
• It provides for MRL controls to be extended to animal feeds in the future.
• A default MRL of 0.01 mg kg−1 (set as a limit of determination) will apply to
those commodities where no specific MRL is set, unless a different default level
is agreed, or until such time as an MRL is set on the basis of the evaluation of
data.
12.4.2
New PPP Regulation (to Replace Directive 91/414)
With the new Regulation (EC) No. 1107/2009, requirements and conditions for
approval of active substances (including safeners and synergists) were set based on
new approval criteria valid for all countries belonging to the European Union. This
procedure and criteria for the approval are (Annex II):
Evaluation
• Joint evaluation by the rapporteur Member State and the Authority (EFSA)
cooperating with the applicant.
• Evaluation based on scientific principles and with expert advice.
• Evaluation can be a permanent process according to Article 21 where, in the light
of new scientific and technical knowledge, the Commission considers that there
are indications that the substance no longer satisfies the approval criteria.
Submission
• Admissibility of the submitted dossier
Criteria
12.4.2.1 Dossier
• Information necessary to establish values for ADI, acceptable operator exposure
level (AOEL), and ARfD.
• Residue definitions in food and feed including succeeding crops, processed foods
(e.g., wine).
• Information which permits the definition of the MRL.
12.4.2.2 Efficacy
• Information on effectiveness at least for one representative use under good plant
protection practice. Good plant protection practice means a practice whereby
the treatments with plant-protection products applied to given plants or plant
products, in conformity with the conditions of their authorized uses, are selected,
dosed, and timed to ensure acceptable efficacy with the minimum quantity
necessary, taking due account of local conditions and of the possibilities for
cultural and biological control.
12.4.2.3 Metabolites
• Information on metabolites permitting the definition of relevant metabolites
(of toxicological, ecotoxicological, or environmental relevance). A metabolite is
deemed relevant if there is a reason to assume that it has intrinsic properties
comparable to the parent substance regarding biological target activity or its prop-
erties to organisms, or it has certain toxicological properties that are considered
unacceptable.
12.4.2.4 Composition
• Information on the composition of the active substance, safener and synergist,
including the minimum degree of purity, the identity and maximum content
of impurities, the content of isomers/diastereoisomers and additives, and the
content of impurities of toxicological, ecotoxicological, or environmental concern
within acceptable limits.
12.4.2.5 Methods of analysis

• Information on analysis methods of the active substance and determination
methods of impurities of toxicological, ecotoxicological, or environmental con-
cern, or other impurities which are present in quantities greater than 1 g kg−1 in
the active substance.
• Information on methods of residue analysis for the active ingredient (a.i.) and
relevant metabolites.
• Methods to be carried out in accordance with the Uniform Principles.
12.4.2.6 Impact on human health

• Safety margin of at least 100 for the establishment of relevant ADI, AOEL,
and ARfD values. Increased safety margins for effects judged of particular
significance, such as developmental neurotoxic or immunotoxic effects, may be
required.
• Verification of nonmutagenic classification 1A or 1B.
• Verification of noncarcinogenic classification 1A or 1B (unless the exposure
under realistic proposed conditions of use is negligible, such as use in a closed
system).
• Verification of nontoxic for reproduction to be classified in category 1A and 1B
(unless the exposure under realistic proposed conditions of use is negligible,
such as use in a closed system).
• Verification of nonendocrine-disrupting properties that may cause adverse effects
in humans (unless the exposure under realistic proposed conditions of use is
negligible, such as use in a closed system).
• Definition of specific scientific criteria for the determination of endocrine-

disrupting properties: a proposal must be made by the Commission to the
SCFCAH by 14 December 2013. Substances that are, or have to be, classified as
carcinogenic category 2 and toxic for reproduction category 2, or substances that
are or have to be classified toxic for reproduction category 2 and have toxic effects
on the endocrine organs, shall to be considered to have endocrine-disrupting
properties under an interim definition until the new definition is in force after
2014.
12.4.2.7 Fate and behavior in the environment

• Cut-off criteria:
– Classification as a persistent organic pollutant (POP): A POP is a substance

having a DT50 value in water greater than two months, or its DT50 in soil
or in sediment is greater than six months, and which has a bioaccumulation
factor in aquatic species >5000 or a partition coefficient in n-octanol/water
greater than 5, or has high bioaccumulation in other nontarget species, or high
toxicity or ecotoxicity in nontarget species and the substance has a potential
for long-range environmental transport.
– Classification as a persistent, bioaccumulative, and toxic substance (PBT): A
PBT substance is a substance which fulfills three of the following criteria:
Persistence: cut-off values: DT50 in marine water >60 days or DT50 in fresh
or estuarine water >40 days or DT50 in marine sediment >180 days or DT50
in fresh or estuarine water sediment >120 days or DT50 in soil >120 days.
Bioaccumulation: Bioconcentration factor >2000.
Toxicity: NOEC population [53] for marine or freshwater organisms
< 0.01 mg l−1 (R 50–53) or the substance is classified as carcinogenic
(category 1A or 1B) (R45 or R49) or mutagenic (category 1A or 1B) (R46), or
have reproduction toxicological properties (category 1A, 1B, or 2) (T; R60,
R61, R62, R63) pursuant to regulation (EC) No. 1272/2008 or is classified
with other chronic toxicity pursuant to specific target organ toxicity (STOT)
RE 1 (T; R 48/23; R 48/24; R 48/25) or STOT RE 2 (Xn; R48/20; R48/21;
R48/22, or R33) (pursuant to regulation (EC) No 1272/2008, entry into force
20 January 2009 (Titles II, III, and IV for substances)) [54].
– Classification as a very persistent and very bioaccumulative substance (vPvB):
A vPvB substance is a substance which fulfills the criteria: (i) DT50 in marine
water, fresh, or estuarine water >60 days or DT50 in marine, fresh, or estuarine
water sediment >180 days or DT50 in soil >180 days; and (ii) bioconcentration
factor >5000.
12.4.2.8 Ecotoxicology
• Risk assessment on the basis of criteria described under Directive 96/12/EC
and the Guidance Document on Terrestrial Ecotoxicology including European
Commission Working Document 2021/VI/98.
• Cut off criteria:

• Endocrine-disrupting properties on nontarget organisms, unless the exposure of
nontarget organisms under the realistic proposed use conditions is negligible.
• Exposure to honeybees.
• Unacceptable acute or chronic effects on colony survival and development (i.e.,
effects on honeybee larvae or honeybee behavior).
12.4.2.9 Residue definition

• Residue definition for actives, safeners, and synergists for the purpose of risk
assessment and for enforcement purposes.
12.4.2.10 Fate and behavior concerning groundwater

• According to the ‘‘Uniform Principle’’ application, especially the maximum
admissible concentration on drinking water of 0.1 μg l−1 , set by EC Directive on
Drinking Water (2000/60/EC) [57], was a hurdle for a listing into Annex I for many
compounds belonging to the class of PS II inhibitors. Nevertheless, although
this standard value does not reflect any risk under toxicological assessment, it is
binding for all EU member states as, for example, The Pesticide Safety Directorate
(now The Chemicals Regulation Directorate) stated [57].
12.4.2.11 Candidate for substitution

• For a period not exceeding seven years a substance shall be approved as a
candidate for substitution when:
• its ADI, ARfD, or AOEL is significantly lower than those of the majority of the
approved a.i.s for this use; or
• its properties cover two cut-off criteria of a PBT substance; or
• there are reasons of concern regarding probable critical effects, such as develop-
mental neurotoxic or immunotoxic effects and high potential risk to groundwater,
even with very restrictive risk management measures such as extensive personal
protective equipment or very large buffer zones; or
• it contains a significant proportion of nonactive isomers.
12.4.2.12 Low-risk active substances

• Any substance shall not be considered as a low-risk substance having carcino-
genic, mutagenic, reproductive toxic, sensitizing, very acute or acute toxic effects
[acute toxicity estimate (ATE) >50, classification category 1 or category 2] effects,
or explosive or corrosive properties, also not substances with half-life stability
(DT50 ) >60 days in soil, a bioconcentration factor >100, or which are deemed to
be an endocrine disruptor, or have neurotoxic or immunotoxic effects.
First approval under Regulation 1107/2009 shall be valid for a duration not
longer than 10 years. Subsequently reregistration (renewal of approval) can be
12.5 Situation of PS II Inhibitors in the EC Markets 499
applied by submission of further confirmatory information that the approval

criteria are satisfied and new requirements established during the evaluation
process for a duration of up to 15 years. Candidates for substitution (to be defined
by December 2013) will be approved for only seven years, and products containing
these substances will be subject to comparative assessment by MS.
12.5
Situation of PS II Inhibitors in the EC Markets
The latest submission of data for an active substance being on the market two
years after the Directive 91/414/EEC was published, or an active substance that was
on the market before 1 May 2004 in the Czech Republic, Estonia, Cyprus, Latvia,
Lithuania, Hungary, Malta, Poland, Slovenia, and Slovakia, or 1 January 2007 in
Bulgaria and Romania, and which is not included in stages one to three of the
program of work and which is not covered by Regulation (EC) No. 1112/2002 was
implemented inter alia by COMMISSION REGULATION (EC) No. 2229/2004 of 3
December 2004, laying down further detailed rules for the implementation of the
fourth stage of the work referred to in Article 8(2) of Council Directive 91/414/EEC
at the latest by November 2005 [55].
The Directive 91/414/EEC stipulates according to Article 5 for inclusion of an
active substance in Annex I, the following shall be taken into particular account:
1) Where relevant, an ADI for human.

2) An acceptable operator exposure level, if necessary.
3) Where relevant, an estimate of its fate and distribution in the environment, as
well as its impact on nontarget species.
Under the ‘‘Uniform Principle’’ application, the maximum admissible concen-

tration on drinking water of 0.1 μg l−1 , set by EC Directive on Drinking Water
(2000/60/EC) [57], was a particular hurdle for a listing into Annex I for many
compounds belonging to the class of PS II inhibitors. Nevertheless, although this
standard value does not reflect any risk under toxicological assessment, it is binding
for all EU member states as, for example, The Pesticide Safety Directorate (now The
Chemicals Regulation Directorate) stated [56]:
Especially for the distribution in the environment the EC Directive on

Drinking Water (2000/60/EC) has set a maximum admissible concentration
of 0.1 μg l−1 for any individual pesticide in drinking water. The figure is
independent of any toxicological or environmental assessment and does
not necessarily represent risk. Nevertheless it is UK Government policy to
control the use of pesticides in such a way as to reduce the occurrence
and levels of pesticide contamination found in drinking water (Annex VI of
91/414/EEC which is 97/57/EEC requires that this 0.1 μg l−1 standard for
any individual pesticide applied to groundwater).’’
By searching under European Union, Reregistration of Plant Protection Agents,

residues in Groundwater via Google.de, approximately 21 500 citations are found,
indicating the political importance of this question caused by, for example,
the ‘‘Grundwasserrichtlinie’’ in Germany. It is of high importance in public
awareness [58].
Another politically important subject is carcinogenicity, which led to nonlisting
on Annex I under 91/414/EEC Directive of atrazine [59]:
‘‘Unlike the EU the US-EPA has reregistered Syngenta’s atrazine for use
in maize, sugarcane, sorghum, cereals, and other crops. Atrazine failed to
get reregistration in Europe in October 2002 because of suggestions that it
could be linked to increased cancer risks. The US-EPA concluded that there
have been no studies confirming increased risk.’’
Other major reasons for the nonlisting of PS II inhibitors in Annex I un-
der 91/414/EEC Directive have been: (i) changes in buffer zones listings; (ii)
withdrawals for commercial reasons; and (iii) failures to meet data submission
deadlines.
The first PS II inhibitors included in Annex I under the Directive 91/414/EEC
are detailed in Table 12.5.
Additionally, from the C1 group of PS II inhibitors the phenylcarbamates
desmedipham and phenmedipham are listed in Annex I, and from the C3 group
bromoxynil and ioxynil. The triazinones metamitron and metribuzin are included
in Annex I, along with the uracil lenacil and the pyridazinon pyrazone/chloridazon.
The urea diuron was not included in Annex I, but under a resubmission based on
regulation 33/2008, it is now listed in Annex I.
Table 12.6 describes the status of the reregistration process of PS II inhibitors in
the EU (Status November 2010) under the Directive 91/414/EEC.
Evidently, from these data, the most important groups of chemistry in PS II
inhibitors in the 1980s – that is, triazines and, to a large extent, ureas – will
no longer be used in the European Union, with some very small exceptions
(Table 12.7).
12.6
Current Market Share of PS II Compound Groups
Whereas, in 1980, the photosynthesis inhibitors belonged to the most important

herbicide classes [37], the market situation began to change, especially in Europe,
during the early 1980s, notably through the introduction of new cereal, corn,
and oilseed rape herbicides from other herbicide classes, as well as through the
re-registration process in Europe.
During recent years, the value of PS II inhibitors sold in the Europe has declined
by about 40%, from ca. ¤745 Mio in 1995 to ca. ¤441 Mio in 2004. A further decline
was recorded in 2009, when farmers in Northern and Southern Europe spent only
ca. ¤330 Mio on PS II inhibitors. At the same time, the value of the total herbicide
Table 12.5 EU-listed PS II inhibitors and specific provisions (status May 2011) [60].
Common name, IUPAC name Entry into force Expiration of Classification Specific provisions,
identification inclusion (Dir. 67/548/EEC) authorized in:a
numbers
Bentazone 3-Isopropyl-(1H)- 1 August 2001 31 December Xn; R22 Xi; R36 BE, BG, CY, DE, DK, EE, EL, ES, FI, FR,
CAS No 2,1,3-benzothiadiazin-4- Extension of the 2015 R43 R52/53 HU, IE, IT, LT, LU, LV, NL, PL, PT, RO,
25057-89-0 (3H)-one-2,2-dioxide expiry date for SE, SI, SK, UK
CIPAC No 366 inclusion Uses as herbicide (cereals, maize, rice,
(2010/77): potatoes, pea, Digitalis lanata, poppy, flax,
1 December 2010 clover, yellow lily, narcissi, flower seed
cultivation (Saponaria), pasture, grazing
land, grass seed, turf, control of Cyperus
tuberosus (several crops))
Pyridate 6-Chloro-3- 1 January 2002 31 December Xi; R38 R43 N; BE, CY, DE, EE, FI, FR, IE, IT, LU, LV, NL,
CAS No phenylpyridazin-4-yl Extension of the 2015 R50/53 PL, UK
55512-33.9 S-octyl thiocarbonate expiry date for Uses as herbicide against dicotyledonous
CIPAC No 447 inclusion weeds in cereals, fodder plants and
(2010/77): vegetables
1 December 2010
Isoproturon 3-(4-Isopropylphenyl)-1, 1 January 2003 31 December Carc. Cat. 3; AT, BE, BG, CZ, DE, ES, FR, HU, IE, IT,
CAS No 1-dimethylurea Extension of the 2015 R40 N; R50/53 LT, LU, NL, PL, PT, RO, SE, SI, SK
34123-59-6 expiry date for Uses as herbicide (maximum application
CIPAC No 336 inclusion rate 1.5 kg ha−1 , single application) against
(2010/77): weeds in cereals
1 December 2010
12.6 Current Market Share of PS II Compound Groups
501
502
Common name, IUPAC name Entry into force Expiration of Classification Specific provisions,
identification inclusion (Dir. 67/548/EEC) authorized in:a
numbers
Linuron 3-(3,4-Dichlorophenyl)- 1 January 2004 31 December Carc. Cat. 3; R40 AT, BE, BG, CY, CZ, EL, ES, FI,
CAS No 330-55-2 1-methoxy-1-methylurea 2013 Repr. Cat. 2; R61 FR, HU, IE, IT, LT, LU, MT, NL,
CIPAC No. 76 Repr. Cat. 3; R62 PL, PT, RO, SI, SK, UK
Xn; R22 Xn;
R48/22 N; R50/53
Chloridazon 5-Amino-4-chloro- 1 January 2009 31 December R43, N, R50–53 AT, BE, BG, CY, CZ, DE, EE, EL,
CAS No 2-phenylpyridazin- 2018 ES, FI, FR, HU, IE, IT, LT, LU, LV,
1698-60-8 3(2H)-one NL, PT, RO, SE, SI, SK, UK
CIPAC No. 111 Only uses as herbicide in
application max. of 2.6 kg ha−1 only
every third year on the same field
may be authorized
a
Country codes according ISO 3166-1, with two exceptions: EL (not GR) is used for Greece, and UK (not GB) is used for the United Kingdom.
AT: Austria; BE: Belgium; BG: Bulgaria; CY: Cyprus; CZ: Czech Republic; DE: Germany; DK: Denmark; EE: Estonia; EL: Greece; ES: Spain; FI: Finland; FR: France;
HU: Hungary; IE: Ireland; IT: Italy; LT: Lithuania; LU: Luxembourg; LV: Latvia; MT: Malta; NL: Netherlands; PL: Poland; PT: Portugal; RO: Romania; SE: Sweden; SI:
Slovenia; SK: Slovakia; UK: United Kingdom.
Table 12.6 Status of Registration of PS II inhibitors under EU Directive 91/414/EEC (November 2010, Source: EU Commission) [60].
Chemical A.i. Number in list Countries of Rapporteur Registration Reasons for Dossier List of uses
Family of authorization authorizationa Member status non-inclusion/ submitted by supported by
State withdrawal available data
Phenyl- Desmedipham 15/477 AT, BE, BG, CZ, Finland Included: 1 – AgrEvo GmbH Herbicide against
carbamate DE, DK, EE, EL, March 2005 (now Bayer annual dicot weeds
ES, FI, FR, HU, Expiration of CropScience AG, in sugar and fodder
IE, IT, LT, LU, inclusion: 28 Germany) beet with
NL, PL, PT, RO, February 2015 240–480 g a.i. ha−1
SE, SI, SK, UK
Phenmedipham 34/77 AT, BE, BG, CZ, Finland Included: 1 – Task Force on Herbicide against
DE, DK, EE, EL, March 2005 Phenmedipham annual dicot weeds,
ES, FI, FR, HU, Expiration of (Bayer post-emergence,
IE, IT, LT, LU, inclusion: 28 CropScience AG, with application
LV, NL, PT, RO, February 2015 United rates from 160 to
SE, SI, SK, UK Phosphorus Ltd) 960 g a.i. ha−1 in
sugar, fodder and
red beet (beetroot)
Nitrile Bromoxynil 11/87 AT, BE, CZ, DE, France Included: 1 – Rhone Poulenc Herbicide against
Bromoxynil 87.407 DK, EL, ES, FR, March 2005 Agro (now Bayer broadleaved weeds
octanoate 87.406 HU, IE, IT, LU, Expiration of CropScience AG) with application rate
Bromoxynil NL, PL, PT, RO, inclusion: 28 and Makhteshim of
heptanoate SI, SK, UK February 2015 Agan 0.3–0.45 kg a.1. ha−1
in winter/spring
cereals (barley,
wheat, oats, rye,
triticale), maize
503
504
Chemical A.i. Number in list Countries of Rapporteur Registration Reasons for Dossier List of uses
Family of authorization authorizationa Member status non-inclusion/ submitted by supported by
Ioxynil 22/86 AT, BE, CY, DE, France Included: 1 – Rhone Poulenc Herbicide against
Ioxynil octanoate 86.407 DK, EL, ES, FI, March 2005 Agro (now Bayer broadleaved weeds
FR, HU, IE, IT, Expiration of CropScience AG) with application
LU, NL, RO, UK inclusion: 28 and Makhteshim rates of
February 2015 Agan 0.3–0.45 kg a.i. ha−1
in winter/spring
cereals
Triazinone Metamitron 370/381 AT, BE, CZ, DE, United Included: 1 – Bayer CropScience Herbicide in sugar
DK, EE, EL, ES, Kingdom September AG, Germany and fodder beet
FI, FR, HU, IE, 2009 against grass weeds
IT, LU, LV, NL, Expiration of with three
PL, PT, RO inclusion: 31 applications with
August 2019 rates of
700–1400 g a.i. ha−1
Metribuzin 125/283 AT, BE, BG, CY, Germany Included: 1 – Bayer CropScience Herbicide in
CZ, DE, EE, EL, October 2007 AG, Germany potatoes with
ES, FI, FR, HU, Expiration of 350 g a.i. ha−1 per
IE, IT, LT , LU, inclusion: 30 season
LV, MT, NL, PL, September
2017
Uracil Lenacil 366/163 AT, BE, CY, Belgium Included: 1 – E. I. Du Pont de Herbicide in sugar
CZ, EL, ES, January Nemours (France) and fodder beet
FR, HU, IE, 2009 S.A.S (transferred against grass and
IT, LU, PL, Expiration to Dr Schirm AG as broadleaf weeds
PT, RO, SK, of inclusion: of 24. October with a maximum
UK 31 2000) and Hermoo rate of
December Belgium NV 0.5 kg a.i. ha−1 per
2018 season
Urea Metobromuron 558/168 – – No autho- Not supported – –
rization in anymore by the
place notifier. With the
available data no safe
use could be assessed
by the Commission.
COMMISSION
REGULATION (EC)
No 2076/2002 of 20
November 2002
Chlorotoluron 149/2008 AT, BE, BG, Spain Included: 1 – Makhteshim Agan Herbicide in
CZ, DE, ES, March 2006 cereals with
FR, HU, IT, Expiration application rate up
PL, PT, RO, of inclusion: to 2.5 kg a.i. ha−1
SI, SK, UK 28 February
2016
Metoxuron 559/219 – – No autho- Not supported – –
rization in anymore by the
place notifier. With the
available data no safe
use could be assessed
by the Commission.
COMMISSION
REGULATION (EC)
505
No 2076/2002 of 20
November 2002
506
Chemical A.i. Number in list Countries of Rapporteur Registration status Reasons for Dossier List of uses
Family of authorization authorizationa Member non-inclusion/ submitted by supported by
Diuron 2184/2008 BG, PL Denmark Included: 1 – European DiuronHerbicide against

October 2008 after Task Force (DTF)
mono and dicot
re-submission. consisting of E. I.
weeds with
Expiration of Du Pont de application rate of
inclusion: 30 Nemours and 0.5 kg a.i. ha−1 in

September 2018 Lanxess orchards (pome
Distributions fruits), vines,
GmbH professional
outdoors limited
to strip band
application
Fluometuron 343/159 BG, EL, ES Greece Initially not – Makteshim Agan Herbicide against
included by and Nufarm annual broad leaf
Decision 2008/934. GmbH & Co KG weeds and grasses
Included: 1 June in cotton with
2011 following application rates
re-submission. up to 2 kg a.i. ha−1
Expiration of per treatment
inclusion 31 May
2021
a
AT: Austria; BE: Belgium; BG: Bulgaria; CY: Cyprus; CZ: Czech Republic; DE: Germany; DK: Denmark; EE: Estonia; EL: Greece; ES: Spain; FI: Finland; FR: France;
HU: Hungary; IE: Ireland; IT: Italy; LT: Lithuania; LU: Luxembourg; LV: Latvia; MT: Malta; NL: Netherlands; PL: Poland; PT: Portugal; RO: Romania; SE: Sweden; SI:
Slovenia; SK: Slovakia; UK: United Kingdom.
Table 12.7 Withdrawn PS II Inhibitors from reregistration in EU (status November 2010, Source: EU Commission).
Chemical A.i. Registration status Reason for withdrawal/no Time of withdrawal

family in EUa authorization
Amide Propanil No authorization in – –

place
Pentanochlor No authorization in – –
place
Nitrile Bromofenoxim No authorization in No registration data submitted –
place
Triazine Ametryne No authorization in – –
place
Atrazine No authorization in Cancerogenicity Withdrawn, 2004/248/EC,
place 16 March 2004
Cyanazine No authorization in COMMISSION REGULATION –
place (EC) No 2076/2002 of 20 November
2002
Desmetryne No authorization in No registration data submitted –
place
Prometryne No authorization in No registration data submitted –
place
Propazine No authorization in No registration data submitted –
place
Simazine No authorization in Ground water concerns Withdrawn, 2004/248/EC,
place 16 March 2004
Terbumeton No authorization in No registration data submitted –
place
507
508
Chemical A.i. Registration status in EUa Reason for withdrawal/no Time of withdrawal
family authorization
Terbuthylazine AT, BE, BG, CZ, DE, EL, Voluntarily withdrawn, not –
ES, HU, IE, IT, LU, MT, included in annex I, but
NL, PL, PT, RO, SI, SK, re-submitted (decision pending)
UK
– Use allowed until – –
December 2011
Terbutryne No authorization in No registration data submitted –
place
Trietazine No authorization in No registration data submitted –
place
Triazinedione Hexazinone No authorization in No registration data submitted –
place
Uracil Bromacil No authorization in No registration data submitted –
place
Terbacil No authorization in No registration data submitted –
place
Urea Chloroxuron No authorization in No registration data submitted –
place
Dimefuron No authorization in No registration data submitted –
place
Ethidimuron (= No authorization in No registration data submitted –
Sulfodiazol) place
Fenuron No authorization in No registration data submitted –
place
Methabenzthiazuron No authorization in No further registration data –
place submitted
Metobromuron No authorization in No registration data submitted –
place
Metoxuron No authorization in No registration data submitted –
place
Monolinuron No authorization in The notifier of the substance Withdrawn, 2000/234/EC,
place withdrew its support 22 March 2000, p. 18
Neburon No authorization in No registration data submitted –
place
Siduron No authorization in No registration data submitted –
place
Tebuthiuron No authorization in No registration data submitted –
place
a
AT: Austria; BE: Belgium; BG: Bulgaria; CZ: Czech Republic; DE: Germany; EL: Greece; ES: Spain; HU: Hungary; IE: Ireland; IT: Italy; LU: Luxembourg; MT: Malta;
NL: Netherlands; PL: Poland; PT: Portugal; RO: Romania; SI: Slovenia; SK: Slovakia; UK: United Kingdom.
509
market in Europe was increased from ca. ¤2400 Mio in 1995 to ca. ¤3200 Mio in
2009 – an increase of 25%. Thus, the value share of PS II inhibitors in the total
herbicide market in Europe fell from ca. 30% in 1995 to only 10% in 2009.
Currently, 12 PS II inhibitory compounds have been granted Annex I inclusion
in Europe (see Tables 12.5 and 12.6), and many of those have special restrictions
and provisions. All others have failed Annex I inclusion for several reasons (see
Section 12.4), due mainly to commercial, groundwater, or toxicological constraints.
The expiration of Annex I inclusions are rapidly approaching for bentazone and
pyridate in 2011, and for isoproturon in 2012. In the years to come, new investments
and hurdles for the renewal of Annex I inclusions will have to be passed for all
remaining PS II inhibitors.
Although, as a result of the provisions of EU Directive 91/414, the class of
urea herbicides is widely being eliminated in Europe, perhaps more distinctively
the important class of triazine herbicides is also disappearing from the European
herbicide market, with none of the 10 representatives of the class in Europe having
been allocated to Annex I. The future of the last triazine representative with na-
tional authorizations, terbuthylazine, remains unclear. Moreover, the traditionally
most important triazine representatives – atrazine and simazine – have not passed
the EU Review Program. Yet, both substances – particularly atrazine – are still
significantly important in the US market, with atrazine sales in the US accounting
for ca. ¤130 Mio in 2009 (albeit down from ca. ¤165 Mio in 2004). This com-
pound is widely and efficiently used in the US corn market, in combination with
Roundup-Ready.
Among the remaining PS II inhibitors on the European market, the top five in
terms of sales are metamitron, isoproturon, phenmedipham, terbuthylazine, and
bentazone, which together accounted for more than 50% of the total PS II-inhibitor
sales in Europe in 2009.
Overall, the EU Review Program and the associated costs of maintaining sub-
stances in the market is leading to a significant streamlining in the number of
PS II inhibitors. In fact, the value share of the remaining PS II inhibitors in the
total European herbicide market will further decline in the years to come. Indeed,
the downward trend which has been recorded, from about 45% market share in
1995 to only about 10% in 2009, represents a further characteristic of this class of
compound, since the phase-out (which is still ongoing) will be further boosted by
the upcoming Annex I renewal process.
12.7
A New Herbicide for Corn and Sugarcane: Amicarbazone – BAY MKH 3586
12.7.1
Introduction
Amicarbazone, a new herbicide aimed at providing broad-spectrum weed control

in corn and sugarcane, is a member of the carbamoyl triazolinones, and acts as
12.7 A New Herbicide for Corn and Sugarcane: Amicarbazone – BAY MKH 3586 511
®
H3C O O Figure 12.3 Amicarbazone, BAY MKH 3586, Dinamic .
H3C N NH2
N N
H
H3C N
CH3
H3C
Table 12.8 Physico-chemical properties of amicarbazone.
Melting point 137.5 ◦ C

Vapor pressure (Pa) 1.3 × 10−6 (20 ◦ C)
3.0 × 10−6 (25 ◦ C)
Dissociation constant (20 ◦ C) Amicarbazone has no acidic or basic properties in
aqueous solutions. It is not possible to specify
dissociation constants for water.
Solubility in water (g l−1 ) (20 ◦ C) 4.6 in unbuffered and buffered solutions; solubility
not influenced by pH in the range 4–9
Volatility (Henry’s law constant at 6.8 × 10−8 Pa m3 mol−1
20 ◦ C)
Solubility in organic solvents (g l−1 ) n-Heptane: 0.07
(20 ◦ C) Xylene: 9.2
Poly(ethylene glycol) (Lutrol): 79
Dimethyl sulfoxide: >250
Dichloromethane: >250
Partition coefficient, Log POW in 1.23 (pH 7)
octanol–water (20 ◦ C): 1.14 (unbuffered)
an inhibitor of PS II. Amicarbazone was first discovered 1988 by the former Plant
Protection Division of Bayer AG (now Bayer CropScience AG), and developed under
the internal code no. BAY MKH 3586 (Figure 12.3); currently, it is commercialized
by Arysta LifeSciences.
12.7.2
Physico-Chemical Properties of Amicarbazone
The physico-chemical characteristics of amicarbazone are listed in Table 12.8.
12.7.3
Discovery of the Active Ingredient
Research is, in most cases, a continuous process that takes place in small steps. In
order to better understand the discovery of amicarbazone, the story should go back
to 1964 when Dornow reported the first examples of the hitherto unknown class of
4-amino-1,2,4-triazin-5-ones [25] (Figure 12.4). Subsequently, in 1965, the research
group at the former Farbenfabriken Bayer AG identified these compounds as
herbicides [61] and specified their mode of action as an inhibition of PS II
O Figure 12.4 1,2,4-Triazinones, gen-

eral formula.
CH3, Phenyl NH2
N
N
N X-CH3 X = S, NH-R
4-Amino-1,2,4-triazin-5-one
CH3 O O Figure 12.5

NH2 NH2 1,2,4-Triazinones, marketed
H3C
N N compounds.
H3C
N N
N S-CH3 N CH3
fi fi
1966: Metribuzin, Sencor 1971: Metamitron, Goltix
X Figure 12.6 Requirements for PS II inhibitors.
[11, 12]. The optimization process led, in 1966, to the discovery of metribuzin
[61], and five years later to metamitron [62] – two commercially very successful
herbicides for soybeans and sugar beet, respectively (Figure 12.5).
A schematic representation of the essential atoms of a herbicide binding to the
32 kDa peptide of PS II is shown in Figure 12.6; this indicates the sp2 hybrid with
X (usually O, S, or C) to be attached to a lipophilic group and the essential positive
charge.
Following the above-described concepts of the structural requirements of PS
II inhibitors [63], in 1980 a series of five-membered analogs of metamitron were
synthesized and monitored for their biological activity (W. Draber and L. Eue, Bayer
AG, Leverkusen, Germany, unpublished results) (Scheme 12.2). Unfortunately,
although these compounds demonstrated a clear herbicidal activity in vitro, they
proved to be rather inactive in vivo.
Nonetheless, with the main goal being to develop a corn herbicide, research
efforts were continued in the field of triazinones, such that N-alkyl derivatives
and various sulfur [64], oxygen [64], nitrogen [64–67], and carbon substituents
[68] were each filed for patent. As a result of these studies, in 1981, a compound
with the internal code number BAY KRA 4145 was synthesized (Scheme 12.3) and
entered into a developmental process following success in a series of intensive field
tests [69].
In an attempt to reduce the high costs of a linear synthesis for BAY KRA 4145,
a convergent approach was evaluated in which trimethylaminoguanidine was used
as an intermediate. The idea of employing a new intermediate in different ways led
to extensive chemical investigations being undertaken, including the reaction with
phosgene (Scheme 12.4). In the presence of excess phosgene, the chlorocarbonyl
O O
CH3, phenyl NH2 phenyl, cyclohexyl
N N N NH2
N N
N alkyl alkyl
Scheme 12.2 Five-membered analogs of metamitron.
O O CH2F O
alkyl, aryl NH2 alkyl, aryl alkyl FCH2 CH3
N N N
H3C
N N N
N X-alkyl N X-alkyl N N(CH3)2
X = S, O, N, single bond BAY KRA 4145
Scheme 12.3 Structure elucidation to BAY KRA 4145.
O
R
O OH O
R CH3 O CH3 COCl2 CH3
N HN HN N
N H2N N
N N(CH3)2 N N(CH3)2
N(CH3)2
x HCl
Low yield
Excess Phosgene
O O
O O
CH3 R-NH2
Cl N N CH3
Base
R-NH N N
N
N(CH3)2 N
N(CH3)2
Scheme 12.4 Trimethylaminoguanidine as intermediate.
triazolinone was generated in good yield. The subsequent reaction of chlorocarbonyl

triazolinone with various amines led to the production of carbamoyl triazolinones,
the first derivative of which was isolated in May 1986 [70]. Whilst the carbamoyl
triazolinones were found to be active in vitro as PS II inhibitors, they showed – in
contrast to the directly linked triazolinones – a higher level of herbicidal activity in
vivo. Consequently, these compounds became the starting point of a major program
of synthesis in which more than 2500 compounds of the general type shown in
Figure 12.7 were generated.
Whilst all of the synthetic variants that showed a good herbicidal activity were
described in various patents (Table 12.9), with regards to activity, selectivity, and
costs of production, BAY MKH 3586 [71, 72] was found subsequently to represent
the optimum and so was further developed for selective weed control in corn and
sugarcane.
Figure 12.7 General formula of carbamoyl

Q1 Q2 Q1, Q2 = O, S
triazolinones.
R3 R1
N N N
R1 = H, C, N, O
R4 N R2 = H, C, N, O, S, Hal
R2
Table 12.9 Synthetic variations of structure shown in Figure 12.7

and associated patents.
R1 R2 References
Alkyl N(alkyl)2 [70, 73–76]

Alkyl S-alkyl [70, 73–76]
Alkyl NH-alkyl [76, 77]
Alkyl Alkyl [76, 78]
Alkyl Halogen [76, 79]
Alkyl O-alkyl [76, 80]
NH2 Alkyl [71, 72, 76–82]
NH-alkyl Alkyl [76, 83, 84]
NH2 S-alkyl [76, 85, 86]
NH2 , NH-alkyl, N(alkyl)2 O-alkyl [76, 87]
NH2 , NH-alkyl, N(alkyl)2 NH2 , NH-alkyl, N(alkyl)2 [76, 88, 89]
12.7.4
Synthesis
12.7.4.1 Final Product

A variety of methods have been employed for the synthesis of carbamoyl tria-
zolinones [70–89]. In the case of N-amino triazolinones, a protecting group such
as a Schiff base may be helpful [71, 72, 81, 82], but otherwise – under suitable
conditions – it is possible to add isocyanates directly in a kinetically controlled
reaction to the amidic nitrogen of N-NH2 -triazolinones [71, 72, 90] (Scheme 12.5).
The synthesis of the intermediate 4-amino-3-isopropyl-1,2,4-triazol-5-one, as de-
scribed initially by Malbec et al. [91], was also possible via several other routes,
such as the hydrazinolysis of acylated carbazates [92], the cyclization of carbohy-
drazide with carboxylic acids or ortho esters [92], or the hydrazinolysis of ester
carbalkoxy-hydrazones [91, 93, 94]. Unfortunately, these procedures included no-
table disadvantages such as a low yield, a long reaction time, the formation of side
products, and a large number of synthesis steps. Consequently, a variety of new syn-
thetic methods was developed, notably the in situ preparation of carbohydrazide and
cyclization with isobutyronitrile in the presence of a suitable tin compound as a reac-
tion auxiliary [95], and the hydrazinolysis of 5-isopropyl-1,3,4-oxadiazol-2(3H)-one
[96–100] (Scheme 12.6).
O O
NH2 R-NH2
R′O N N
Base
N
alkyl - R′-OH
Cl-COOR′ Base R′ = alkyl, aryl
O O O
NH2 R-NCO NH2
HN N R-NH N N
Cat.
N N
alkyl alkyl
Ketone H+
H+/ H2O - Ketone
O R1 O
O R1
N N
HN N R-NCO R-NH
R2 N N
R2
N Cat.
N
alkyl alkyl
Cl-COOR′ Base
R′ = alkyl, aryl
O O R1 - R′-OH
N R-NH2
R′O N N
R2
N Base
alkyl
Scheme 12.5 Synthesis of N-amino-carbamoyltriazolinones.
O O
Hydrazine + NC-C3H7-i
RO OR HN N NH2
H
NH2 Sn-cat. - NH3
O O
O H2N O COCl2 HN O Hydrazine HN N NH2

Hydrazine
HO N N N
H
Scheme 12.6 Synthesis of the intermediate 4-amino-3-isopropyl-1,2,4-triazol-5-one.
12.7.5
Biological Behavior
Amicarbazone is tolerated by corn and sugarcane crops, and shows excellent activity
against many major annual dicotyledonous weeds that infest these crops.
In corn, amicarbazone can be applied up to a maximum rate of 500 g a.i. ha−1 to
the soil at either pre-plant or pre-emergence timings. In combination with other
corn herbicides [101, 102], such as isoxaflutole, the application rate can be reduced.
Amicarbazone also demonstrates contact activity on emerged weeds, with the com-
pound providing both burndown as well as residual weed control – an effect which
is particularly valuable in reduced- and zero-tillage corn production systems. The
most important weeds to be controlled by amicarbazone include velvetleaf (Abutilon
theophrasti), common lambsquarters (Chenopodium album), pigweed (Amaranthus
spp.), common cocklebur (Xanthium strumarium) and morning-glory species (Ipo-
moea spp.). In October 2005, amicarbazone was granted conditional registration
by the EPA in the United States [103]. Based on its biological spectrum and its
mode of action, amicarbazone will not only compete mainly against atrazine (in
all markets), but also replace the broadleaf region of the weed control spectrum
of alachlor, acetochlor, and metolachlor, where grasses are not the dominant
weeds [103].
In 2004, amicarbazone was introduced by Arysta LifeScience into the Brazilian
®
market, under the trade name Dinamic , to provide weed control in sugarcane. In
this case, it can be applied either pre-emergence or post-emergence at application
rates of up to 1500 g a.i. ha−1 solo, or at 700 g a.i. ha−1 in combination [104, 105] with
tebuthiuron (750 g a.i. ha−1 ) or ametryne (1500 g a.i. ha−1 ). In tank mixtures with
metribuzin (960 g a.i. ha−1 ), the application rate can be reduced to 560 g a.i. ha−1
(post-emergence) or 800 g a.i. ha−1 (pre-emergence). Besides dicotyledonous weeds
such as painted spurge (Euphorbia heterophylla) and morning glories, many an-
nual grasses such as marmeladegrass (Brachiaria plantaginea), southern sandbur
(Cenchrus echinatus), bengal commelina (Commelina benghalensis) and guineagrass
(Panicum maximum) are also controlled. More detailed information regarding the
biological profile of amicarbazone was revealed at the British Crop Protection
Conference – Weeds, in 1999 [105].
Two new formulations of amicarbazone for use in sugarcane were submitted
for registration in November 2010 in Brazil, namely Poderoso (amicarbazone +
tebuthiuron) and Zonic (amicarbazone + diuron + hexazinone).
12.7.6
Metabolites
In a corn metabolism study [106], unchanged amicarbazone and two degrada-

tion products were identified as the major components in the corn matrices
(Figure 12.8).
H3C O O H 3C O O H3C O O
NH2 H H
H3C N N N H3C N N N H 3C N N N
H H H
H3C N H3C N H3C N
CH3 CH3 CH3
H3C H3C H3C OH
Amicarbazone Desamino amicarbazone Isopropyl-2-hydroxy
desamino amicarbazone
Figure 12.8 Metabolites of amicarbazone obtained in corn.

References 517
12.8
Conclusions
Whilst it is acknowledged that PS II inhibitors represent one of the most important

classes of herbicide, it is also recognized that the market share of ‘‘ripened’’
herbicides is influenced not only by the introduction of new herbicide classes, but
also by the major changes that are frequently introduced in their re-registration
requirements. However, although such changes in the regulatory arena tend to
increase the hurdles for existing compounds, they also provide room for innovative
solutions.
Amicarbazone is the latest representative in the still economically important
group of PS inhibitors. It belongs to the chemical class of carbamoyl triazinolones,
was found and developed by Bayer AG, and will be commercialized in the US in
the corn market and in sugarcane growing countries by Arysta LifeSciences.
References
1. Schmidt, R.R. (1997) Proc. Brighton 13. Trebst, A. and Wietoska, H. (1975) Z.
Crop Prot. Conf. – Weeds, 3, Naturforsch. Teil C, 30, 499–504.
1133–1140. 14. Whitmarsh, J. and Govindjee (1999)
2. Arnon, D.I., Whatley, F.R., and Allen, The photosynthetic process, in Con-
M.B. (1957) Nature, 180, 182–185. cepts in Photobiology: Photosynthesis
3. Trebst, A. and Hauska, G. (1974) and Photomorphogenesis (eds Singhal,
Naturwissenschaften, 61, 308–316. G.S., Renger, G., Sopory, S.K., Irrgang,
4. Wessels, J.S.C. and van der Veen, K.-D., and Govindjee, Narosa Pub-
R. (1956) Biochim. Biophys. Acta, 19, lishers, New Delhi and Kluwer
548–549. Academic, Dordrecht, pp. 11–51,
5. Good, N.E. (1961) Plant Physiol., 36, http://www.life.uiuc.edu/govindjee/paper/
788–803. gov.html#100.
6. Moreland, D.E. (1967) Annu. Rev. Plant 15. Deisenhofer, J., Epp, O., Miki, K.,
Physiol., 18, 365–386.
Huber, R., and Michel, H. (1984) J.
7. Trebst, A. and Harth, E. (1974) Z.
Mol. Biol., 180, 385–398.
Naturforsch. Teil C, 29 (5-6), 232–235.
16. Hansch, C. and Fujita, T. (1964) J. Am.
8. Exer, B. (1958) Experientia, 14,
Chem. Soc., 86, 2738.
136–137.
17. Hansch, C. and Deutsch, E.W. (1966)
9. Moreland, D.E., Gentner, W.A., Hilton,
Biochim. Biophys. Acta, 126, 117–128.
J.J., and Hill, K.L. (1959) Plant Physiol.,
34, 432–435. 18. Seydel, J.K. (1985) QSAR and Strategies
10. Exer, B. (1961) Weed Res., 1, 233–244. in the Design of Bioactive Compounds,
11. Draber, W., Dickoré, K., Büchel, K.H., John Wiley & Sons, Inc., New York.
Trebst, A., and Pistorius, E. (1968) 19. Leo, A., Hansch, C., and Elkins, D.
Naturwissenschaften, 55, 446. (1971) Chem. Rev., 71, 525–616.
12. Draber, W., Dickoré, K., Büchel, 20. Hansch, C. and Klein, T.E. (1991)
K.H., Trebst, A., and Pistorius, E. Methods Enzymol., 202, 512–543.
(1969) Structure–activity correlation 21. Büchel, K.H., Draber, W., Trebst, A.,
of 1,2,4-triazinones, a new group of and Pistorius, E. (1966) Z. Naturforsch.
photosynthesis inhibitors, in Progress Teil B, 21, 243–254.
in Photosynthesis Research (ed. H. 22. Draber, W., Büchel, K.H., Timmler, H.,
Metzner), Verlag C. Lichtenstern, and Trebst, A. (1974) ACS Symp. Ser.,
Munich, Vol. 3, pp. 1789–1795. 2, 100–116.
23. Gast, A., Knüsli, E., and Gysin, H. 38. Dhuri, A. (2006) Agrochemical reg-
(1955) Experientia, 11, 107–108. istration, a global view. Lecture on
24. Gast, A., Knüsli, E., and Gysin, H. the Conference on Agrochemicals,
(1956) Experientia, 12, 146–148. Mumbai, India, January 12–13, 2006.
25. Dornow, A., Menzel, H., and Marx, P. 39. A Global Approach to the Regula-
(1964) Chem. Ber., 97, 2173–2178. tion of Agricultural Pesticides, A
26. Lin, K. (1975) Herbicidal Vision for the Future. http://www.
6-Amino-s-triazinediones. US Patent oecd.org/dataoecd/30/60/33854658.pdf.
3,902,887, (Prio: 24. 05. 1972), EI 40. European Parliament, Council (1991)
Du Pont de Nemour and Comp., Council Directive 91/414/EEC of 15
Wilmington, Del., USA. July 1991, concerning the placing of
27. Bucha, H.C., Cupery, W.E., Harrod, plant protection products on the mar-
J.E., Loux, H.M., and Ellis, L.M. (1962) ket. Official Journal of the European
Science, 137, 537–538. Union, 52, L230, 1–32. http://eur-lex.
28. Fischer, A. (1962) Weed Res., 2, europa.eu/LexUriServ/LexUriServ.do?
177–184. uri=CELEX:31991L0414:EN:HTML.
29. Reicheneder, F., Dury, K., and Fischer, 41. European Parliament, Council
A. (1961) Mittel zur Beeinflussung (2009) Official Journal of the Eu-
des Pflanzenwachstums. Patent ropean Union, 52, L281. http://eur-
DE 1 105 232, (Prio: 21. 11. 1958), lex.europa.eu/LexUriServ/LexUriServ.do?
Badische Anilin- & Soda-Fabrik AG, uri=OJ:L:2009:281:0003:0004:EN:PDF.
Ludwigshafen/Rhein, Germany. 42. EC Directive 87/18/EEC.
30. Kasebeer, H. (1971) Z. Pflanzenkr. 43. FAO (1995) Manual on the Development
Pflanzenschutz, 78, 158–174. and Use of FAO Specifications for Plant
31. Bucha, H.C. and Todd, C.W. (1951) Protection Products, 4th edn, FAO, Via
Science, 114, 493–494. delle Terme di Caracalla, Rome.
32. Hörlein, G., Langelüddeke, P., and 44. FAO (1999) Manual on the Development
Schönowsky, H. (1972) Selektive and Use of FAO Specifications for Plant
Unkrautbekämpfung mit Alkylphenyl- Protection Products, 5th edn, FAO, Via
harnstoffderivaten. Patent DE 20 39 delle Terme di Caracalla, Rome.
041, (Prio: 06. 08. 1970), Hoechst AG, 45. EC Directive 91/414 EEC.
Frankfurt, Germany. 46. European Parliament, Council (1994)
33. Schäfer, W., Wegler, R., and Eue, L. Commission Directive 94/37/EEC of
(1958) Unkrautbekämpfungsmittel. 22 July 1994 amending Council Di-
Patent DE 1 039 779, (Prio: 20. 04. rective 91/414/EEC, concerning the
1957), Farbenfabriken Bayer Aktienge- physical and chemical properties of the
sellschaft, Leverkusen, Germany. active substance. Official Journal of the
34. Dorschner, K.P., Gates, R.L., and European Union, 52, L281, 65–81.
Willard, J.R. (1963) Selektive, herbicide 47. European Parliament, Council (1991)
Mittel. Patent DE 1 160 236, (Prio: EC Directive 91/414 EEC Annex III
11. 04. 1959), FMC Corporation, New Sections 7.2.1. to 7.2.3. Official Journal
York. of the European Union, 52, L230, 27.
35. Carpenter, K., Cottrell, H.J., de Silva, 48. Predictive Operator Exposure Model
W.H., Heywood, B.J., Gleeds, W., (POEM) (SC 8001). Available at:
Rivett, K.F., and Soundy, M.L. (1964) www.pesticides.gov.uk/uploadedfiles/
Weed Res., 4, 175–195. Web.../UK_POEM1.xls.
36. Zeidler, A., Fischer, A., and Scheurer, 49. European Parliament, Council (2005)
G. (1969) Z. Naturforsch. Teil B, 24, Official Journal of the European Union,
740–744. 48, 1–16.
37. Jäger, G. (1983) in Chemistry of Pes- 50. Data Requirements Handbook
ticides (ed. K.H. Büchel, transl. by G. 28/09/04 Chapter 6.3 Environmen-
Holmwood), John Wiley & Sons, Inc., tal Fate and Behaviour, http://www.
New York, p. 338. pesticides.gov.uk/psd_pdfs/registration_
References 519
guides/data_reqs_handbook/datareqhand 60. http://ec.europa.eu/sanco_pesticides/

book.pdf. public/index.cfm.
51. EC Directive 95/36 EC of 14 July 1995 61. Westphal, K., Meiser, W., Eue, L.,
L 172 8 22.7.1995, Establishing the and Hack, H. (1968) Agent Herbicide.
Data Requirements for Environmen- Patent FR 1 519 180, (Prio: 16. 04.
tal Fate and Behaviour in Annex II 1966), Farbenfabriken Bayer Aktienge-
(Chapter 7) and Annex III (Chapter 9) sellschaft, Leverkusen, Germany.
of Directive 91/414. 62. Dickoré, K., Draber, W., and Eue,
52. European Parliament, Council (2005) L. (1972) 4-Amino-1,2,4-triazin-
Official Journal of the European Union, 5-one, Verfahren zu ihrer Herstellung
48, 1–16. und ihre Verwendung als Herbizide.
53. Health and Safety Executive, Chemi- Patent DE 2 107 757, (Prio: 18. 02.
cals Regulation Directorate Pesticides 1971), Bayer AG, Leverkusen, Ger-
(2003) Guidance on Deriving No Ob- many.
served Ecologically Adverse Effect 63. Trebst, A., Donner, W., and Draber, W.
Concentrations and Ecologically Ac- (1984) Z. Naturforsch., 39c, 405–411.
ceptable Concentrations for use in 64. Timmler, H., Wegler, R., Eue, L., and
Higher Tier Aquatic Risk Assessments, Hack, H. (1971) 1,2,4-Triazin-5-one.
http://www.pesticides.gov.uk/publications. Patent DE 1 670 912, (Prio: 18. 08.
asp?id=429. 1967), Farbenfabriken Bayer AG, Lev-
54. European Parliament, Council (2008) erkusen, Germany, first published 1968
as ZA 68 04409.
Official Journal of the European Union,
65. Dickoré, K., Sasse, K., Eue, L., and
51, 1–1355. http://eur-lex.europa.eu/
Schmidt, R.R. (1980) 6-Substituierte
LexUriServ/LexUriServ.do?uri=OJ:L:
3-Dimethylamino-4-methyl-1,2,4-triazin-
2008:353:0001:1355:EN:PDF.
5-(4H)-one, Verfahren zu ihrer Her-
55. European Parliament, Council
stellung und ihre Verwendung als
(2004) Official Journal of the Euro-
Herbizide. Patent DE 2 908 963, (Prio:
pean Union, 47, 13–63. http://eur-
07. 03. 1979), Bayer AG, Leverkusen,
lex.europa.eu/LexUriServ/LexUriServ.do?
Germany.
uri=OJ:L:2004:379:0013:0063:EN:PDF.
66. Dickoré, K., Sasse, K., Eue,
56. Environmental Fate Behaviour,
L., and Schmidt, R.R. (1980)
Data Requirements Handbook and
6-Cyclohexyl-3-dimethylamino-4-methyl-
Supplementary Guidance, PSD, 1,2,4-triazin-5-(4H)-on, Verfahren zu
pp. 86–87, under www.pesticides. seiner Herstellung und seine Verwen-
gov.uk/aa_registration.asp?id=643. (EC dung als Herbizid. Patent DE 2 908
Directive 97/57 of 21 December 1994 L 964, (Prio: 07. 03. 1979), Bayer AG,
354 16 31.12. 1994, establishing Annex Leverkusen, Germany.
VI (Uniform Principles) to Directive 67. Draber, W., Schmidt,
91/414). R.R., and Eue, L. (1982)
57. Directive 2000/60/EC of European 3-Dimethylamino-4-methyl-6-phenyl-1,2,
Parliament and Council, establishing 4-triazin-5-one, Verfahren zu ihrer
a framework for community action in Herstellung sowie ihre Verwendung als
the field of water policy., http://eur- Herbizide. Patent DE 3 035 021, (Prio:
lex.europa.eu/LexUriServ/LexUriServ.do? 17. 09. 1980), Bayer AG, Leverkusen,
uri=OJ:L/2000:327:001:082:EN:PDF. Germany.
58. http://www.bmu.de/files/pdfs/allgemein/ 68. Draber, W., Dickoré, K., and Timmler,
application/pdf/richtlinie_2006_118_eg. H. (1973) Verfahren zur Herstellung
pdf. von 1,2,4-Triazin-5-onen. Patent DE
59. Regulatory News – February (2004) 2 138 031, (Prio: 29. 07. 1971), Far-
Outlook Pest Manage., 15 (1), 4–6; benfabriken Bayer AG, Leverkusen,
http://www.researchinformation.co.uk/ Germany.
pest.php/www.researchinformation.co.uk/ 69. Kranz, E., Findeisen, K., Schmidt,
pest/sample/sample.htm R., and Eue, L. (1982) Substituierte
6-Halogen-tert-butyl-1,2,4-triazin-5-one, 10. 07. 1987), Bayer AG, Leverkusen,

Verfahren zu ihrer Herstellung sowie Germany.
ihre Verwendung als Herbizide. Patent 79. Findeisen, K., Lindig, M., Santel, H.-J.,
EP 49416, (Prio: 02. 10. 1980), Bayer Lürssen, K., and Schmidt, R.R. (1991)
AG, Leverkusen, Germany. Halogen-triazolone. Patent DE 3 920
70. Findeisen, K., Lindig, M., Santel, H.-J., 414, (Prio: 22. 06. 1989), Bayer AG,
Schmidt, R.R., Lürssen, K., and Strang, Leverkusen, Germany.
H. (1988) Substituierte Triazolinone. 80. Müller, K.-H., König, K., Kluth,
Patent EP 283876, (Prio: 24. 03. 1987), J., Lürssen, K., Santel, H.-J., and
Bayer AG, Leverkusen, Germany. Schmidt, R.R. (1992) Substituierte
71. Lindig, M., Findeisen, K., Müller, 5-Alkoxy-1,2,4-triazol-3-(thi)one. Patent
K.-H., Santel, H.-J., Schmidt, R.R., EP 477646, (Prio: 22. 09. 1990), Bayer
Strang, H., and Feucht, D. (1988) AG, Leverkusen, Germany.
Substituierte Triazolinone. Patent EP 81. Kuhnt, D., Müller, K.-H., Findeisen,
294666, (Prio: 12. 06. 1987), Bayer AG, K., König, K., Lürssen, K., Santel, H.-J.,
Leverkusen, Germany. and Schmidt, R.R. (1992) Substituierte
72. Müller, K.-H., Lindig, M., Findeisen, Triazolinone. Patent EP 511569, (Prio:
K., König, K., Lürssen, K., Santel, H.-J., 30. 04. 1991), Bayer AG, Leverkusen,
Schmidt, R.R., and Strang, H. (1990) Germany.
Substituierte Triazolinone. Patent EP 82. Kuhnt, D., Müller, K.-H., Findeisen,
370293, (Prio: 19. 11. 1988), Bayer AG, K., König, K., Lürssen, K., Santel, H.-J.,
Leverkusen, Germany. and Schmidt, R.R. (1992) Substituted
73. Findeisen, K., Lindig, M., Santel, H.-J., triazolinones with herbicide proper-
and Schmidt, R.R. (1989) Substituierte ties. Patent WO1993/04050, (Prio:
Triazolinone. Patent EP 305844, (Prio: 23. 08. 1991), Bayer AG, Leverkusen,
01. 09. 1987), Bayer AG, Leverkusen, Germany.
Germany. 83. Müller, K.H., König, K., Findeisen,
74. Findeisen, K., Kuhnt, D., Müller, K.-H., K., Santel, H.-J., Lürssen, K., Schmidt,
König, K., Lürssen, K., Santel, H.-J., R.R., and Dutzmann, S. (1990) Sub-
and Schmidt, R.R. (1992) Substituierte stituierte Triazolinone. Patent EP
Triazolinone. Patent EP 503437, (Prio: 399294, (Prio: 24. 05. 1989), Bayer AG,
14. 03. 1991), Bayer AG, Leverkusen, Leverkusen, Germany.
Germany. 84. Müller, K.-H., Findeisen, K., Kuhnt, D.,
75. Findeisen, K., Kuhnt, D., Müller, K.-H., König, K., Lürssen, K., Santel, H.-J.,
Haug, M., König, K., Lürssen, K., and Schmidt, R.R. (1992) Triazolinone.
Santel, H.-J., and Schmidt, R.R. (1992) Patent EP 505819, (Prio: 23. 03. 1991),
Substituierte Triazolinone. Patent EP Bayer AG, Leverkusen, Germany.
513621, (Prio: 14. 05. 1991), Bayer AG, 85. Iwai, S., Hatano, M., Ishikawa, Y., and
Leverkusen, Germany. Kawana, K. (1978) Triazoline deriva-
76. Findeisen, K., Linker, K.-H., Schallner, tives. Patent JP 53 135 981, (Prio:
O., Müller, K.-H., König, K., Santel, 27. 04. 1977), Nippon Soda Co., Ltd.,
H.-J., and Schmidt, R.R. (1994) Japan.
Substituierte Triazolinone. Patent 86. Müller, K.-H., Kluth, J., König, K.,
WO1994/09012, (Prio: 12. 10. 1992), Gassen, K.-R., Findeisen, K., Lindig,
Bayer AG, Leverkusen, Germany. M., Lürssen, K., Santel, H.-J., and
77. Findeisen, K., Lindig, M., Santel, H.-J., Schmidt, R.R. (1990) Substituierte
Schmidt, R.R., and Lürssen, K. (1990) 4-Amino-5-alkylthio-1,2,4-triazol-3-one.
Substituierte Triazolinone. Patent EP Patent EP 391187, (Prio: 07. 04. 1989),
398096, (Prio: 18. 05. 1989), Bayer AG, Bayer AG, Leverkusen, Germany.
Leverkusen, Germany. 87. Kluth, J., Müller, K.-H., Haas, W.,
78. Lindig, M., Dickoré, K., Findeisen, Linker, K.-H., Findeisen, K., König, K.,
K., Santel, H.-J., Schmidt, R.R., and Santel, H.-J., and Dollinger, M. (1994)
Strang, H. (1989) Substituierte Tri- Substituted triazolinones and their use
azolinone. Patent EP 298371, (Prio: as herbicides. Patent EP 625515, (Prio:
References 521
17. 05. 1993), Bayer AG, Leverkusen, the preparation of substituted oxa-
Germany. diazolones. Patent EP 872480, (Prio:
88. Müller, K.-H., Findeisen, K., Haug, 15. 04. 1997), Bayer AG, Leverkusen,
M., Heinemann, U., Kluth, J., König, Germany.
K., Santel, H.-J., Lürssen, K., and 99. Desai, V.C., Jelich, K.,
Schmidt, R.R. (1991) Substituierte Diehr, H.J., Lantzsch, and R.
4,5-Diamino-1,2,4-triazol-3-(thi)one. (1999) Process for preparing
Patent EP 415196, (Prio: 30. 08. 1989), 4-amino-1,2,4-triazolin-5-ones. Patent
Bayer AG, Leverkusen, Germany. 5,912,354 (Prio: 11. 12. 1998), Bayer
89. Kuhnt, D., Findeisen, K., Haug, Corporation, Pittsburgh, Pa, USA;
M., Kluth, J., Müller, K.-H., König, Bayer AG, Leverkusen, Germany.
K., Himmler, T., Beck, G., Santel, 100. van Laak, K., Casser, C., Jautelat, M.,
H.-J., Lürssen, K., Schmidt, R.R., and Niehoff, M. (2000) Verfahren zur
Krauskopf, B. (1992) Substituierte Herstellung von Oxadiazolonen. Patent
4,5-Diamino-1,2,4-triazol-3-(thi)one. DE 19 853 863, (Prio: 23. 11. 1998),
Patent EP 502307, (Prio: 07. 02. 1991), Bayer AG, Leverkusen, Germany.
Bayer AG, Leverkusen, Germany. 101. Dahmen, P., Thielert, W., Müller,
90. Diehr, H.-J. (1997) Process for the K.-H., and Riebel, H.-J. (1998) Selektive
preparation of substituted aminocar- Herbizide auf Basis von Carbamoyltria-
bonyltriazolinones. Patent EP 757041, zolinonen. Patent DE 19 635 060, (Prio:
(Prio: 31. 07. 1995), Bayer AG, Lev- 30. 08. 1996), Bayer AG, Leverkusen,
erkusen, Germany. Germany.
91. Malbec, F., Milcent, R., and Bure, 102. Feucht, D., Dahmen, P., Drewes,
A.M. (1984) J. Heterocycl. Chem., 21, M.-W., Pontzen, R., Kremer, M., and
1769–1774. Müller, K.-H. (2001) Carbamoyl tri-
92. Kröger, K.-F., Hummel, L., Mutscher,
azolinone based herbicides. Patent
M., and Beyer, H. (1965) Chem. Ber.,
WO2001/37652, (Prio: 19. 11. 1999),
98, 3025–3033.
Bayer AG, Leverkusen, Germany.
93. Ikizler, A. and Ün, R. (1979) Chim.
103. United States Environmental Protection
Acta Turcica, 7, 269–290.
Agency (2005) Pesticide Fact Sheet
94. Milcent, R., Vicart, P., and Bure, A.-M.
Amicarbazone, October 4th, 2005,
(1983) Eur. J. Med. Chem. – Chim.
http://www.epa.gov/opprd001/factsheets/
Ther., 18, 215–220.
amicarbazone.pdf.
95. König, K., Müller, K.-H., and Rohe, L.
104. Thielert, W., Bohne, G., and Müller,
(1990) Verfahren zur Herstellung von
K.-H. (1998) Selektive Herbizide für
4-Amino-1,2,4-triazol-5-onen. Patent EP
den Zuckerrohranbau. Patent DE 19
403889, (Prio: 21. 06. 1989), Bayer AG,
635 074, (Prio: 30. 08. 1996), Bayer AG,
Leverkusen, Germany.
96. Müller, K.-H., König, K., and Leverkusen, Germany.
Heitkämper, P. (1989) Ver- 105. Philbrook, B.D., Kremer, M., Müller,
fahren zur Herstellung von K.H., and Deege, R. (1999) Proc.
4-Amino-1,2,4-triazol-5-onen. Patent Brighton Conf. Crop Prot. – Weeds,
EP 321833, (Prio: 22. 12. 1987), Bayer 1, 29–34.
AG, Leverkusen, Germany. 106. Mathew, A.E., Nguyen, T., and
97. Diehr, H.-J., Müller, K.-H., and Murphy, J.J. (1997) Analytical residue
Lantzsch, R. (1997) Process for prepar- method for MKH 3586 in plants. Ab-
ing substituted aminotriazolinones. stract of papers American Chemical
Patent EP 759430, (Prio: 18. 08. 1995), Society, Vol. 214 (1–2), AGRO 73,
Bayer AG, Leverkusen, Germany. 214th American Chemical Society
98. Diehr, H.-J., Lantzsch, R., Applegate, National Meeting, Las Vegas, NV,
J., and Jelich, K. (1998) Process for September 7–11, 1997.
523
13
New Aspects of Plant Regulators
Hans Ulrich Haas
13.1
Introduction
Plant regulators (PRs) are compounds that have active effects on the physiology of
a plant, by improving or inhibiting the plant’s growth, influencing its flowering
and/or fruit growth, altering the maturation, reducing abiotic stress, and – in a
broad sense – changing, in a positive manner, the behavior of plants. At present,
knowledge of the mode of action of PRs, and of changes that they induce in plant
physiology, remains incomplete.
In this chapter, an overview is provided of PRs, and of their current use and
new developments, together with a summary of presently available knowledge
of the topic. Information is also provided to allow more intense analyses of
specific subjects relating to PRs to be conducted. The reference section of the
chapter includes many reviews and specialist summaries, while internet links
provide detailed and up-to-date overviews, such as chemical structures, including
the chemical names [1], chemistry, usage and environmental aspects [2–4], and
summarized overviews [5–9].
By definition, PRs are compounds of either natural or synthetic origin that are
used to control or to modify plant growth processes, without causing any apparent
phytotoxic effects at the dose(s) applied. The PRs incorporate a wide range of
chemicals, including natural plant hormones, synthetic analogs, and compounds
(Figures 13.1 and 13.2).
13.2
Plant Growth Regulators
Among the PRs, plant growth regulators (PGRs) remain the major group in
practical use; indeed, the use, mode of action and plant-internal and -external
interactions of these materials have been the subject of intense research since they
were first introduced into agriculture during the early 1930s. Since that time, a
host of experimental data, knowledge and experience relating to the PGRs has been

524
Brassinosteroids
Oliosaccharides
Uniconazole-p
Forchlorfenuron (CPPU)
2,4-DP Ethylene Paclobutrazol Karrikins
13 New Aspects of Plant Regulators
2,4,5-T Etephon Hydrogen cyanamide Strigolactones

2,4-D Abscisic acid (dormin) Flurprimidol Proteins/Peptides
Maleic hydrazide Abscisin II Inabenfide Phospholipids
Tecnazene Chlorflurenol-methyl Phthalimide Trans-2-ketones
3-CPA Daminocide Trinexapac-ethyl Trans-2-aldehydes
1930 1940 1950 1960 1970 1980 1990 2000
Indole-3-acetic acid (IAA) Kinetin Ancymidol Cyclanilide
Indole-butyric acid (IBA) Benzyladenin Mepiquat-chloride Prohexadione-calcium
2-(1-naphtyl)acetic acid (NAA) Thidiazuron Dimethipin 1-methylcyclopropene (MCP)
Gibberellin (GA) Gibberellin GA3 Chlorphonium chloride Jasmonate
Carbaryl Dikegulac-sodium Aviglycine-HCl (AVG)
4-CPA Mefluidide Monoterpenes
Chlorpropham (CIPC) Ethychlozate
Chlormequat (CCC) Flumetralin
Figure 13.1 Commercialized and new plant regulators, and the decade of their market introduction or publication.
13.2 Plant Growth Regulators 525
Indole-3-acetic acid 2,4-D-dichloro-phenoxy- Etephon Gibberellinic acid

acetic acid
O
Cl Cl OH
OH P O O
OH
O Cl OH
N O
O
Kinetin N6-Benzyladenin Abscissic acid (ABA)
H
O
N N N HO H
OH O
N O N
N N O
N O O
N N O
Figure 13.2 Chemical structures of plant growth regulator

compounds among the five main PGR categories. Auxins
(IAA, 2,4-D); ethylene (ethephon); cytokinin (kinetin, benzy-
ladenin); growth inhibitors (ABA); and gibberellins (GA3 ).
acquired, and their spectrum of usage has continued to increase. Despite such
extensive application, however, the highly complex mode of action of PGRs – even
in the case of auxins, the oldest known group of PGRs – has remained essentially
undisclosed.
Classically, there are five main categories of naturally occurring PGRs: the auxins
[indole-acetic acid (IAA), naphthalene acetic acid (NAA), indole-butyric acid (IBA),
2,4-dichlorophenoxyacetic acid), gibberellins, cytokinins (kinetin, benzyladenin,
zeatin), ethylene, and growth inhibitors such as abscisic acid (ABA)].
Auxins: The auxins were the first phytohormones to be detected, with auxinic
activity having already been observed in 1879 by J. Sachs during plant
propagation. The first such compounds (IAA) were isolated and described in
1934 [10]. Auxins are involved in fruit ripening, phototropism, rooting, apical
dominance, and cell enlargement. They are widely used as herbicides, and
demonstrate such activity due to an overdose and subsequent de-/regulation
processes in plants. A herbicidally active auxin dose leads, for example, to an
overdose of ethylene, which in turn induces epinastic growth, tissue swelling,
a stimulation of ABA biosynthesis, and leaf abscission [11].
Ethylene: Although ethylene effects were first described in 1901 [12], ethylene as
a hormone was identified only after the technique of gas chromatography had
been established during the 1960s [13]. Ethylene influences the balance of
auxins versus gibberellinic acid (GA) [11], and also inhibits cell division and
strengthens the cell walls. It is also involved in the initiation of flowering, the
breaking of dormancy, the abscission of parts of the plants, and in ripening
processes. The most frequently used ethylene-based PGR is ethephon, an
ethylene releaser, which breaks down in plant tissues to form phosphate,
chloride ions, and ethylene, which acts as the PGR [14].
526 13 New Aspects of Plant Regulators
Gibberellins: The gibberellins are involved in growth processes, including the

elongation of internodes, flowering, dormancy, and fruit morphology. Their
effects were first described in 1926 [15], when the shoot elongation of rice was
observed following the treatment of plants with culture filtrates of Fusarium
moniliforme Sheld. Subsequently, Yabuta was then able to isolate a crystalline
compound (5-n-butylpicolinic acid; fusaric acid) from the fungal culture.
The first report on GA was made in 1938 by Yabuta and Sumiki [16], while
GA3 was first described in 1955 by Brian and Hemming [17]. Today, of all
known GAs, only gibberellic acids GA3 , GA4 , and GA7 are of commercial
importance [13].
Cytokinins: The cytokinins act mainly through cell cycle regulation [18], by
stimulating cell division, preventing abscission and rooting, enhancing
germination, and preventing senescence. Kinetin was the first cytokine to be
described, in 1955 [19], while benzyladenin was identified by Strong in 1958
[20]. Both compounds remain the most commonly used cytokinins in plant
micropropagation [10]. In addition, Strong [20] described thidiazuron, which
is used mainly to induce senescence in cotton.
Growth inhibitors: Growth inhibitors such as ABA (dormin) and abscisin
II retard growth, promote abscission, and induce dormancy. They were
discovered in the 1960s [21], and have direct effects on cell division and
expansion; they also induce stomatal closure [11].
13.3
PGRs in Modern Agriculture
Today, PGRs play important roles in modern agriculture, as they are used to ensure
and enhance the quantity and quality of all parts of a crop cycle, from seed to harvest
and post-harvest. In addition to their recognized use as growth regulators, some
PGRs also serve as herbicides, as well as providing positive effects on drought and
abiotic stress tolerance, yield regulation and harvest facilitation, storage control,
and propagation (Table 13.1).
13.3.1
Growth Inhibition
Both, Rademacher and Brahm [8] and Rademacher [22] have summarized the
chemistry of growth retardants in agronomically important crops. In 1949, maleic
hydrazide (MH) was one of the first PGR products to be used for shoot length
control [23], since when this property has become the classical and core use of
growth regulators. The growth inhibitors currently used in cereals act mainly as GA
inhibitors; typical examples include chlormequat-chloride (CCC), trinexapac-ethyl,
prohexadione-Ca, mepiquat-chloride, and paclobutrazol (Figure 13.3).
Paclobutrazol and uniconazole are further examples of GA biosynthesis in-
hibitors that belong to the group of N-containing heterocyclic triazoles. These
Table 13.1 Key use areas of plant regulators in modern agri-

culture, and compounds currently in use or under develop-
ment.
Factor Example Compounds
Plant growth Dormancy breaking Ammonium nitrate

Ca-cyanamide, Ca-nitrate
Shoot growth inhibition Chlormequat-, mepiquat-chloride
Dwarfing Mepiquat-pentaborate, mefluidide
Ethylene, ethephon
Paclobutrazol, uniconazole
Trinexapac-ethyl, prohexadione-Ca
Daminocide, ancymidol, butralin
Flurprimidol, inabenfide
Maleic hydrazide, n-decanol
Shoot growth promotion Gibberellic acid
24-epi-Brassinolide
Harpin, glutamic acid
Root growth promotion Paclobutrazol, naphthalene-acetic acid
Indole-3-butyric acid,
naphthaleneacetamide
Strigolactone
Propagation Auxins, giberellins, cytokinins
Stress response Drought 1-MCP
Trinexapac-ethyl, prohexadione-Ca
Salinity Paclobutrazol
Nutrient efficacy Sodium nitrophenolate
Flower induction Ammonium thiosulfate
Fruit-thinning Ethylene, ethephon
Gibberellinic acid
Glutamic acid, forchlorfenuron
Paclobutrazol, prohexadione-Ca
Naphthylacetic acid, CMMP
Yield and fruit Ripening Fruit size Aviglycine, naphthylacetic acid
quality adjustment Sugar Benzyladenine, Cu-ethylenediamine
accumulation 1-MCP, diphenylamine
Ethylene, ethephon
MBTA-HCI
Phospholipids, LPE94
Trinexapac-ethyl, MBTA
Storage Ripening Sprout Aminoethoxyvinylglycine (AVG)
suppression Carvone, menthol, menthone
Chlorpropham (CIPC)
1,2,6-DIPN, maleic hydrazide
Ethylene, ethephon
1-Methyl cyclopropene (1-MCP)
Trans-2-aldehyde/ketones
Chlormequat-chloride Mepiquat-chloride Trinexapac-ethyl Paclobutrazol

(CCC)
O O O
Cl
Cl N
Cl
N
+ N+ O N
O Cl
N
O
Figure 13.3 Chemical structures of the plant growth regula-

tors mainly used for shoot length control.
compounds, act also as demethylation inhibitors (DMIs), though some triazole

fungicides have been reported as having growth-retardant side effects [24]. Fortu-
nately, some of these PGR activities are of practical importance, such as the use of
metconazole and tebuconazole in the treatment of oilseed rape.
One other property, which has received very little attention, is the implied effect
of these fungicides on the growth inhibition of weeds [25, 26]. The extent of growth
reduction of both the crops and of the weeds may be similar unless a specific weed
species is inhibited selectively to a greater extent than the crop.
13.3.2
Fruiting and Growth
The fruiting and growth of orchard trees is variable and dependent not only on the
climate and weather, but also on plant-specific factors such as alternation, which is
the biennial fluctuation of the fruit yield in orchards. Typically, PGRs are used in
this area to reduce and harmonize plant growth, to equalize and accelerate blossom
and fruiting seasons (e.g., defoliation and regrowth), to precondition the fruits for
harvesting, and to thin the fruits in order to achieve a better quality and fruit size, and
more equal yields over a period of several years. Currently, the GA-biosynthesis
inhibitors paclobutrazol [27] and prohexadione-Ca [28] are used to control tree
growth in orchards and in arable crops, as well as to control flower- and fruit-setting.
Compounds used as blossom thinners include ammonium thiosulfate (ATS),
endothalic acid, pelargonic acid, sulfcarbamide-1-aminomethanamide, hydrogen
tetraoxosulfate, and hydrogen cyanamide [13]. Currently, NAA is used widely for
post-bloom thinning, although an adverse side effect of the insecticide carbaryl is
also used for this purpose [29]. Benzyladenine (6-BA), as a cytokinin, has been
registered for the reduction of fruits, and also stimulates cell division in the
remaining fruits [30].
13.3.3
Fruit Storage and Ripening
One further use area of PRs is to control the storage and ripening of fruits and plant
products, such as cut flowers. Whilst ethylene is broadly utilized to induce ripening,
the opposite effect – a delayed ripening to provide an extended shelf-life – is more
Methylcyclopropene Norbornadiene Daminocide Aviglycine (AVG)

(1-MCP) (2,5-NBD)
OH O
N O
O N HO N
O N
Figure 13.4 Chemical structures of ethylene binding site

inhibitors (left), and ethylene biosynthesis inhibitors (right).
difficult to manage. Daminocide was the first commercial compound used to delay
the ripening of apple fruits [31], but was replaced by the ethylene biosynthesis
inhibitor aviglycine-HCl (AVG; Figure 13.4), which had to be applied to the fruits
on the tree before harvest. AVG is also used to reduce flower senescence and
flower bud abscission on specific ornamentals [32]. Subsequently, a new episode of
controlled ripening began with the investigation of ethylene binding site inhibitors,
such as 1-methylcyclopropene (1-MCP) and norbornadiene (2,5-NBD) (Figure 13.4),
and their development for market use [33]. Meanwhile, 1-MCP continues to be
used as a post-harvest treatment to delay the ripening of apples [34–36], and was
also recently commercialized as an abiotic stress protectant to help plants better
survive periods of heat and drought stress.
13.3.4
Sprout Inhibition
During the past few years, the sprout inhibition of potatoes has mainly been driven
by the use of propham (IPC), chlorpropham (CIPC), and MH. Whilst IPC and CIPC
(Figure 13.5) are applied after harvest at the beginning of the storage period, MH
is applied to the potato foliage when the tubers have reached a size of 40–70 mm.
Tecnazene (TCNB) was also used in potato storage, but was removed from the mar-
ket because of its long degradation time. The monoterpene S-(+)-carvone, which is
produced from caraway seeds, has been developed commercially as a competitive
product to CIPC (Figure 13.5). Recently, menthol was also commercialized for
use as a sprout suppressant. In addition to their sprout-suppressing ability, the
natural terpenes also inhibit microbial growth and prevent the rotting of treated
Chlorpropham S-(1)-Carvone Menthol Menthone

(CIPC)
O OH O
N
O
Cl O
Figure 13.5 Chemical structures of commercialized sprout suppressants.

Trans-2-aldehydes Trans-2-ketones
O O
R1 H R2 R3
Figure 13.6 Chemical structures of a new class of potential sprout suppressants.
potato tubers [37]. Coleman et al. [38] detected different activities of S-(+)-carvone,
menthone, and neomenthol, a diastereomer of menthol, with the latter two com-
pounds showing 5- to 10-fold higher activities in suppressing tuber sprouting than
S-(+)-carvone.
A recent patent on a new class of sprout suppressants [39] described
trans-2-ketones and trans-2-aldehydes (Figure 13.6), both of which have been
shown to be active as potato sprout suppressants. Trans-2-hexenal, which is known
by its ‘‘grass smell,’’ was also included in this patent.
13.3.5
Stress Defense
An important role of PR is their involvement in the defending the plants against

abiotic and biotic stress. GA biosynthesis inhibitors, such as paclobutrazol have
long been known to have stress-protectant properties [40]. Indeed, many reports
have described plants better surviving drought-, frost-, salinity-, and heat-related
stress following the application of triazoles. In addition, an improved canopy
structure, more root growth, a different rooting system, and delayed senescence
were also observed [41–44]. Schubert [45] has reported the effects of PGR on
the yield of cereals under drought stress, depending on the PGR applied. For
example, trinexapac-ethyl-treated plants displayed a much higher harvest index
and an increased thousand-kernel weight compared to those treated with CCC, or
untreated controls. Ervin and Koski [46, 47] reported a reduced evapotranspiration
in kentucky bluegrass treated with trinexapac-ethyl, while Marcum and Jiang [48]
identified similar effects on tall fescue (Festuca arundinacea S.).
In addition to the GA biosynthesis inhibitors, many other PRs have been shown
capable of helping plants to compete better with abiotic stress. For example, Zhang
et al. [49] reported a better yield of soybeans after spraying with ABA and brassi-
nolides under normal and drought conditions, while a similar positive response
of kentucky bluegrass (Poa pratensis L.) to natural PGR was described by Zhang
and Schmidt [50]. Subsequently, Both [51] and Zhang and Ervin [52] described an
enhanced drought tolerance of tall fescue and creeping bentgrass (Agrostis palustris
Huds. A.) following the application of humic acid or seaweed extract. Romera
and Alcantara [53] have summarized their recent findings of the involvement of
ethylene in regulating the stress response of plants to iron-deficiency. In this
case, ethylene was shown to alleviate the stress response, especially in plants with
iron-acquisition strategy I, acidification of the rhizosphere and subapical swelling
of the roots.
A reduction in the abiotic stress of plants has also been reported as a side effect of
quinone-outside-inhibiting (QoI)-fungicides, such as the strobilurins. A prolonged
plant greening after strobilurine treatment is widely recognized in farming practice.
Both, Wu and von Tiedemann [54, 55] observed strong antioxidative properties,
which resulted in a delayed senescence and the protection of barley and wheat
against ozone injury following application of the strobilurin azoxystrobin, and of the
triazole epoxiconazole. A report on the anti-oxidative and anti-senescence effects
of pyraclostrobin has also been produced which described physiological effects
in barley and wheat contributing to abiotic stress tolerance [56]. An increased
tolerance against pathogens outside the strobilurin core fungicidal activity, such as
an increased resistance of tobacco against the tobacco mosaic virus Pseudomonas
syringae pv. tabbaci following treatment of the plants with pyraclostrobin [57],
provided further indication of the broad physiological changes that occur in the
plants after spraying.
Beside plant-enhancement activity, natural PRs are involved in the indirect
defense mechanisms of plants against herbivores [58]. Jasmonic acid, salicylic acid
(Figure 13.7), and ethylene form part of the signaling pathways of stress defense
mechanisms. Previously, Bari and Jones [41] reviewed such findings together with
the results of newer investigations that described such effects also for ABA, auxin,
GA, cytokinins, brassinolides, and peptides. As phospholipids also impact on the
hypersensitive response and systemic acquired resistance in plants, they might also
represent a potential new class of commercial PRs [59]. The influence of polyamines
[e.g., putrescine, spermidine (Figure 13.7), spermine] on plant growth – including
cell division, germination, till fruit development, and stress response – has been
reviewed on several occasions [60–62]. Evans and Malmberg [63] furthermore
summarized the current knowledge of the interactions of polyamines to commercial
PGRs and environmental stimuli. An interaction of polyamines with phospholipids
in vesicles was described by Tadolini [64]. Polyamines may have an important role
in stabilizing membranes through a protective effect against lipid peroxidation.
The metabolic link between polyamine and ethylene synthesis led to the suggestion
of an impact of these PGRs in the abiotic and biotic interactions of the roots and
the rhizosphere [65].
Jasmonates Brassinosteroids Salycilates Phospholipids

Jasmonic acid Brassinolid Salicylic acid Spermidine
O OH
O H2N
OH OH N
HO H
OH NH2
HO O
HO H O
O
Figure 13.7 Examples of chemicals among potential new groups of PGRs.

13.4
Conclusions and Developments
New developments of commercial PRs include label extensions and mixtures of

current commercially available PGRs. The investigation, evaluation, and devel-
opment of scientifically recognized PRs for agricultural purposes is increasing
rapidly, including the use of jasmonates [66], brassinosteroids [67, 68], karrikins
[69], strigolactones [70], proteins and peptides [71], polyamines [63], phospholipids
[59], and oligosaccharides [72, 73].
References
1. Wood, A. (2011) Compendium 10. Preece, J.E. (2003) HortScience, 38 (5),

of Pesticide Common Names, 1015–1025.
Plant Growth Regulators. 11. Grossmann, K. (2003) in Chemistry of
www.alanwood.net/pesticides/class_ Crop Protection (eds G. Voss and G.
plant_growth_regulators.html. Ramos), Wiley-VCH Verlag GmbH,
2. Environmental Protection pp. 131–142.
Agency (2011) Pesticides. 12. Beyer, E. Jr, Morgan, P.W., and Yang,
http://www.epa.gov/pesticides/biopesticides/ S.F. (1984) in Advanced Plant Physiology
ingredients. (ed. M.B. Wilkins), Pitman Publishers,
3. PMEP-Extoxnet (2011) Pesticide active London, pp. 111–126.
ingredient information, Herbicides, 13. Petracek, P.D. and Silvermann, F.P.
Growth Regulators, and Desiccants. (2003) HortScience, 38 (5), 937–942.
http://pmep.cce.cornell.edu/profiles/herb- 14. Maynard, J.A. and Leopold, A.C. (1963)
growthreg/index.html. Aust. J. Chem., 16, 596–608.
4. Kegley, S.E., Hill, B.R., Orme, S., 15. Kurosawa, E. (1926) Nat. Hist. Soc.
and Choi, A.H. (2011) PAN Pesticide Formosa, 16, 213–227.
Database, Pesticide Action Network,
16. Yabuta, T. and Sumiki, Y. (1938) J.
North America (San Francisco, CA).
Agric. Chem. Soc. Jpn, 14, 1526.
www.pesticideinfo.org.
17. Brian, P. and Hemming, H. (1955) Phys-
5. Wikipedia (2011) Plant hormones.
iol. Plantarum, 8, 669–681.
http://en.wikipedia.org/wiki/Plant_
18. Tang, W., Harris, L., and Newton, R.J.
hormone.
(2004) J. Forest. Res., 15 (3), 227–232.
6. Von Sengbusch, P. (2011) Botanik
19. Miller, C.O., Skoog, F., von Saltza,
online, Phytohormones (Plant Hor-
mones) and other Growth Regulators. M.H., and Strong, M. (1955) J. Am.
http://www.biologie.uni-hamburg.de/b- Chem. Soc., 77, 1329–1334.
online/e31/31.htm. 20. Strong, F.M. (1958) Topics in Microbial
7. Davies, P.J. (2010) The Plant Hormones, Chemistry, John Wiley & Sons, Inc., New
Springer eBook. York.
8. Rademacher, W. and Brahm, L. (2010) 21. Milborrow, B. (1984) in Advanced Plant
Plant growth regulators, Ullmann’s Physiology (ed. M.B. Wilkins), Pitman
Encyclopedia of Industrial Chemistry. Publishers, London, pp. 76–110.
http://onlinelibrary.wiley.com/doi/10.1002/ 22. Rademacher, W. (2000) Annu. Rev. Plant
14356007.a20_415.pub2/otherversions. Physiol. Plant Mol. Biol., 51, 501–531.
9. Rademacher, W. (2010) in Proceedings 23. Schöne, D.L. and Hoffmann, O.L. (1949)
XIth International Symposium on Plant Science, 109, 588–590.
Bioregulators in Fruit Production (ed. 24. Fletcher, R.A., Hofstra, G., and Jian-guo,
G. Costa) ISHS, Acta Horticulturae, G. (1986) Plant Cell Physiol., 27 (2),
p. 884. 367–371.
References 533
25. Benton, J.M. and Cobb, A.H. (1995) 44. Wang, C.Y. (1985) Sci. Hortic., 26 (4),
Plant Growth Regul., 17 (2), 149–155. 293–298.
26. Hanson, B.D., Mallory-Smith, C.A., 45. Schubert, S. (2006) DLG-Mitteilungen, 3,
Brewster, B.D., Wendling, L.A., and 65–67.
Thill, D.C. (2003) Weed Technol., 17 (4), 46. Ervin, E.H. and Koski, A.J. (1998)
777–781. HortScience, 33 (7), 1200–1202.
27. Qinlan, J.D. (1980) Acta Hortic., 114, 47. Ervin, E.H. and Koski, A.J. (1991) Crop
144–151. Sci., 41, 247–250.
28. Evans, R.J., Evans, R.R., Reguski, C.L., 48. Marcum, K.B. and Jiang, H. (1997) J.
and Rademacher, W. (1999) HortScience, Turfgrass Manage., 2 (2), 13–27.
34, 1200–1201. 49. Zhang, M., Duan, L., Zhai, Z., Li, J.,
29. Greene, D.W. (2002) HortScience, 37 (3), Tian, X., Wang, B., He, Z., and Li,
477–481. Z. (2004) New directions for a di-
30. Wismer, P.T., Proctor, J.T.A., and verse planet, in Proceedings of the 4th
Elfving, D.C. (1995) J. Am. Soc. Hortic. International Crop Science Congress,
Sci., 120, 802–807. Brisbane, Australia, September 26 –
31. Edgerton, L.J. and Hoffmann, M.B. October 1. (eds T. Fischer et al.).
(1965) Proc. Am. Soc. Hortic. Sci., 57, The Regional Institute Ltd, Gos-
120–124. ford, Australia. ISBN 1-920842-21-7.
32. Environmental Protection Agency http://www.cropscience.org.au/icsc2004/.
(2011) Pesticides: Regulating pes- 50. Zhang, X. and Schmidt, R.E. (1999)
ticides, Aminoethoxyvinylglycine Crop Sci., 39, 545–551.
hydrochloride (aviglycine HCl), formerly
51. Zhang, X. and Schmidt, R.E. (2000)
designated as aminoethoxyvinyl-
Crop Sci., 40, 1344–1349.
glycine (AVG) (129104). Fact Sheet.
52. Zhang, X. and Ervin, E.H. (2004) Crop
http://www.epa.gov/oppbppd1/biopesticides/
Sci., 44 (5), 1737–1745.
ingredients/factsheets/factsheet_
53. Romera, F.J. and Alcantara, E. (1994)
129104.htm.
Plant Physiol., 105, 1133–1138.
33. Sisler, E.C. and Serek, M. (1999) Bot.
54. Wu, Y.X. and von Tiedemann, A. (2001)
Bull. Acad. Sin., 40, 1–7.
34. Serek, M., Sisler, E.C., and Reid, M.S.
55. Wu, Y.X. and von Tiedemann, A. (2002)
(1995) Acta Hortic., 394, 337–346.
Environ. Pollut., 116, 37–47.
35. Blankenship, S.M. and Unrath, C.R.
(1998) HortScience, 33, 469. 56. Jabs, T., Pfirrmann, J., Schäfer, S., Wu,
36. Sisler, E.C. and Serek, M. (2003) Plant Y.X., and Tiedemann, A. (2002) Proc.
Biol., 5, 473–480. Brighton Crop. Prot. Conf. – Pests Dis.,
37. Vokou, D., Vareltzidou, S., and 9B-4, 941–946.
Katinakis, P. (1993) Agric. Ecosyst. 57. Herms, S., Seehaus, K., Koehle, H., and
Environ., 47, 223–235. Conrath, U. (2002) Plant Physiol., 130,
38. Coleman, W.K., Lonergan, G., and 120–127.
Silk, P. (2001) Am. J. Potato Res., 78, 58. Van Poecke, R.M.P. and Dicke, M.
345–354. (2004) Plant Biol., 6 (4), 387–401.
39. Knowles, N.R. and Knowles, L.O. (2005) 59. Cowan, A.K. (2006) Plant Growth Regul.,
US Patent 6855669, published 2005. 48, 97–109.
40. Gilley, A. and Fletcher, R.A. (1997) Plant 60. Slocum, R.D., Kaur-Sawhney, R., and
Growth Regul., 21 (3), 169–175. Galston, A.W. (1984) Arch. Biochem.
41. Bari, R. and Jones, D.G. (2009) Plant Biophys., 235 (2), 283–303.
Mol. Biol., 69, 473–488. 61. Smith, T.A. (1985) Annu. Rev. Plant
42. Fletcher, R.A., Gilley, A., Sankhla, N., Physiol., 36, 117–143.
and Davis, T. (2010) in Horticultural Re- 62. Kao, C.H. (1997) Bot. Bull. Acad. Sin.,
views, vol. 24 (ed. J. Janick), pp. 55–138. 38, 141–144.
43. Jung, J., Luib, M., Sauter, H., Zeeh, B., 63. Evans, P.T. and Malmberg, R.L. (1989)
Rademacher, W., and Agronomy, J. Annu. Rev. Plant Physiol. Plant Mol.
(1987) Crop Sci., 158, 324–332. Biol., 40, 235–269.
64. Tadolini, B. (1988) Biochem. J., 249, 70. Xie, X., Yoneyama, K., and
33–36. Yoneyama, K. (2010) Annu. Rev. Phy-
65. Couee, I., Hummel, I., Sulmon, C., topathol., 48, 93–117.
Goueset, G., and El Amrani, A. (2004) 71. Environmental Protection Agency
Plant Cell Tissue Organ Cult., 76 (1), (2011) Pesticides: Regulating pesti-
1–10. cides, Harpin protein (006477). Fact
Sheet. http://www.epa.gov/opp00001/
66. Creelman, R.A. and Mullet, J.E. (1995)
biopesticides/ingredients/factsheets/
Proc. Natl Acad. Sci. USA, 92 (10),
factsheet_006477.htm.
4114–4119.
72. Albersheim, P. and Valent, B.S. (1978) J.
67. Mandava, N.B. (1988) Annu. Rev. Plant Cell Biol., 78 (3), 627–643.
Physiol. Plant Mol. Biol., 39, 23–52. 73. Darvill, A., Augur, C., Bergmann, C.,
68. Hayat, S. and Ahmad, A. (eds) (2010) Carlson, R.W., Cheong, J.J., Eberhard,
Brassinosteroids, Springer. S., Hahn, M.G., Lo, V.M., Marfa, V.,
69. Wikipedia (2011) Karrikin. Meyer, B. et al. (1992) Glycobiology, 2 (3),
http://en.wikipedia.org/wiki/Karrikin. 181–198.
535
Part II
Fungicides

537
Overview
Peter Jeschke
This volume on Fungicides embraces the chemistry, biochemistry, and biology

of new, different fungicidal compounds and compound classes, the resistance
development against various fungicidal compound classes, and the changes in the
fungicide markets worldwide. The diverse contributions also deal with changes in
the importance of the different fungicide classes and their modes of action (MoA)
for research and development. They describe new compounds and compound
classes that have been introduced to the market during the past 25 years, including
newly announced developmental products.
Chapter 14, the ‘‘FRAC Mode of Action Classification and Resistance Risk of
Fungicides’’, begins with a short introduction on the history of fungicide use,
explains the significance of individual MoA relative to their market penetration,
their mechanisms and occurrence of resistance, the importance and risk of
occurrence of practical field resistance on the management of fungicide resistance
by FRAC (Fungicide Resistance Action Committee), and its recommendations
given to farmers related to the MoA of fungicides from different chemical classes.
Practical field resistance occurred very early in the history of fungicides; however,
since the 1980s advisors from universities, regulatory authorities, farmers and
farmers’ advisors and the agrochemical industry have learned to deal with, and to
recommend practical field application advice, intervals of applications, rates, and
types of treatment, and use in mixture or alternation with other MoA to prevent
crop losses caused by resistance development.
Chapter 15.1 describes the biochemistry of uncouplers of mitochondrial oxidative
phosphorylation, and of the related compounds and compound classes acting at
different sites (e.g., Complex I, II, III and IV), reflecting the relevance of these
targets for the current fungicide market. With the introduction of Complex III
(cytochrome bc1 complex) inhibitors (Chapter 15.2) in 1996, the most important
compound class – the strobilurins, derived from the natural lead structures strobil-
urin A and the structurally closely related oudemansin A – was introduced in the
reference period to the market. At the same time it became evident that resistance
development, for example in the class of strobilurins, must not lead necessarily to
an abandonment of the marketing, research and development of such chemistry.

538 Overview
In particular, succinate dehydrogenase inhibitors (SDH, Complex II) have been

further developed as a promising class of modern broad-spectrum fungicides, to-
gether with the new innovative subgroup of pyridinyl-ethyl benzamide fungicides
(see Chapters 15.3.1 and 15.3.2).
The class of sterol biosynthesis inhibitors (SBIs; Chapter 19) was developed
during the 1970s, and the past 25 years has witnessed intensive research and
development in this area from all agrochemical companies, leading to further
important innovations, including highly specific or broad-spectrum fungicides.
The Oomycetes, as fungi, are biologically and biochemically very different from the
phytopathogenic Ascomycetes fungi, they cause much damage in crops, especially in
vine grapes, potatoes, and tomatoes. With fungicides having different MoA, such
as an antitubulin action (Chapter 18), efficacy on the perturbation of a spectrin-like
protein associated with the cytoskeleton of the fungal cell (Chapter 21), or the
chemical group of carboxylic acid amides (CAA; Chapter 20), efficient weapons
have been introduced for the control of the Oomycetes pathogens.
During the past 25 years, new MoAs have been identified, leading to fungicides
being marketed against eye spot disease in wheat and barley and botrytis (e.g.,
methionine biosynthesis inhibitors; Chapter 16.2), or by acting on the signal
transduction of fungi (Chapter 17.1) or on melanin biosynthesis in the cell wall
(Chapter 22). The latter also describes the protein X-ray structure as well as the
active site of the target scytalone dehydratase, and explains the binding pocket and
molecular modeling-based synthesis of new inhibitors, leading to novel fungicides
to treat rice blast disease.
The MoA has not been detected – or until now is not exactly known – of course,
for all newly introduced fungicides (Chapters 23 and 24). The described examples
demonstrate the need for future additional biochemical research effort, not only in
academia but also in the agrochemical industry.
Chemical innovations in stereospecific synthesis may, however, represent the key
to a chiral phenylamide fungicide based on an old (known for over 34 years) racemic
structure and a long-used MoA, the inhibition of ribosomal RNA polymerization
(Chapter 25).
Chapter 26 is focused on synthetic chemicals which have been defined by the
FRAC as ‘‘host defense inducers.’’ On a practical basis, these compounds are well
established as major countermeasures for specific diseases, notably rice blast in
north-east Asia.
This volume on Fungicides reflects the research and development of recent
decades, and provides great insight into the area through the excellent contributions
of authors from both the agrochemical industry and academia.
539
14
FRAC Mode of Action Classification and Resistance Risk
of Fungicides
14.1
History of Fungicide Use
The first fungicide to be discovered, at the beginning of the nineteenth century,

was lime-sulfur; this was introduced by William Forsyth and recommended for the
control of powdery mildew diseases of fruit trees [1]. The next milestone was the
introduction by Millardet in 1885 of Bordeaux mixture, a copper-based preparation
used to combat the newly introduced downy mildew pathogen Plasmopara viticola
in grapes [2]. The first organic fungicides – organomercury compounds – were
introduced for cereal seed treatment in the early twentieth century, and since
the 1930s organic compounds such as the dithiocarbamates and the phthalimides
have become important tools for disease control in plants. The mode of action of
these fungicides is described as ‘‘multisite,’’ as they inhibit simultaneously a range
of enzymes and cellular structures and provide a preventive protection of plants
against various diseases in a nonsystemic manner, at the surface of the plant.
The first fungicides with a specific mode of action – the benzimidazoles, car-
boxamides, and early sterol biosynthesis inhibitors (SBIs) such as triforine – were
discovered in the 1960s and early 1970s. During the late 1970s and early 1980s,
dicarboximides, phenylamides, and the new SBI classes (among them the first
triazoles) entered the market. Specific fungicides control plant pathogens more
effectively and at a much lower rate compared to multisite contact fungicides. Most,
but not all, of these compounds have systemic properties and are therefore able
to penetrate the plant tissue and to be further distributed via the xylem vessels
(apoplast) into plant parts not been reached directly by the spray application. Over-
all, specific fungicides with systemic properties were regarded as a true progress in
crop protection, because they are less likely to be removed by rain and also often are
redistributed within the plant. As a result, they allowed a considerable reduction in
not only the dose rate but also the number of applications per season. The specific
mode of action, however, became the origin of a new phenomenon – the selection
of resistant individuals in fungal populations, and the development of practical
field resistance.

540 14 FRAC Mode of Action Classification and Resistance Risk of Fungicides
14.2
Fungicides: Importance of Individual Modes of Action
To date, a total of 49 different specific fungicide and bactericide modes of action

are actually available, and have been classified in the Fungicide Resistance Action
Committee (FRAC) code lists [3], including also fungicides with unknown modes
of action (Table 14.1). In addition, numerous multisite inhibitors and plant-defense
inducers are available for the control of plant diseases worldwide. Therefore, a wide
diversity of modes of action is available for the control of plant diseases and for
an effective resistance management. However, many fungicides are available only
in a restricted number of regions and crops, because they may not be registered
everywhere or because the market size may not be big enough. This means
that, despite the large number of fungicides available, in several crop/pathogen
combinations there are only very limited options for disease control. In addition, a
new Regulation (EC) No. 1107/2009, which concerns the placing of plant protection
products (PPPs) on the market, will substitute the Directives 91/414/EEC and
79/117/EEC. The new Regulation will be applicable from the third Quarter of
2011, and will have a significant impact on the future of PPPs in the European
Union. It represents a fundamental change from science-based risk assessment
to hazard-based regulatory cut-off criteria, and will result in a further decline in
the number of effective PPPs, including some of the most important fungicides
such as mancozeb and many triazoles. Hence, there will be a reduction in the
diversity of solutions to manage diseases in crops. The FRAC has given clear
recommendations that no fewer than three – and, in the case of multispray crops
(e.g., potatoes, bananas), preferably five – different modes of action are required per
crop/disease combination in order to ensure an effective resistance management
and sustainable disease management for the future.
Table 14.1 Number of modes of action mentioned in the

FRAC code lista with a broad or narrow spectrum of activity.
Pathogen group(s) controlled Broad spectrum Narrow spectrum
Bacteria – 4
Oomycetes – 10
Powdery mildews – 4
Rice blast – 4
Take all – 1
Rhizoctonia – 2
Botrytis and related – 1
Ascomycetes/Basidiomycetes 18 –
Ascomycetes/Basidiomycetes/Oomycetes 5 –
Total 23 26
a
Specific modes of action (including fungicides with unknown mode of action [U] but excluding host
plant defense inducers [P] and multisite inhibitors [M]).
14.2 Fungicides: Importance of Individual Modes of Action 541
In addition, a use limitation of fungicides results also from a narrow spectrum

of activity (26 out of 49 modes of action; Table 14.1), affecting only one systematic
group of pathogens such as the Oomycetes or even only one single pathogen
species. For example, pencycuron and validamycin control only Rhizoctonia solani,
the causal agent of sheath blight in rice and of stem canker and black scurf in
potatoes. Furthermore, the thiophene-carboxamide derivative silthiofam specifically
controls Gaeumannomyces graminis, the take-all pathogen of wheat. As shown in
Table 14.1, there are 10 specific modes of action available for Oomycetes control,
four against rice blast (incited by Magnaporthe grisea), and four with a specific
activity against powdery mildew pathogens.
An overview of the importance of individual modes of action, and of the crops
or regions in which they are mostly used, is provided in Tables 14.2–14.4. The
total worldwide fungicide market in 2009 has been estimated at US$ 9.73 billion
[4]. A few modes of action dominate the overall fungicide market (Table 14.2):
the SBIs [including demethylation inhibitor (DMI) fungicides and amines] clearly
take a leading position, accounting for about 32% of the total fungicide sales.
Another fungicidal mode of action – the inhibition of complex III in mitochon-
drial respiration – has rapidly gained importance since 1996, to reach about 22%
market share in 2009. This fungicide group is known as quinine-outside in-
hibitor (QoI) fungicides, or (chemically less correct) as ‘‘strobilurins.’’ Most other
Table 14.2 Sales of fungicide groups as a percentage of the

global fungicide market in 2009 (total fungicide market in
2009 was US$ 9.726 billion [4]). Mode of action classification
refers to FRAC code list.
Fungicide groupa Mode of action

Group %
DMI G1 29.2
QoI C3 22.1
Dithiocarbamates (mainly mancozeb) M3 6.8
Copper and sulfur formulations M1 and M2 4.7
Phthalimides (mainly folpet, captan) M4 4.2
Benzimidazoles and thiophanates B1 4.1
Carboxamides/SDHI C2 3.5
Chloronitriles (chlorothalonil) M5 3.2
Phenylamides A1 2.9
Amines G2 2.9
MBI I1 and I2 2.4
CAA H5 2.1
Dicarboximides E3 1.9
Anilinopyrimidines D1 1.9
Others (mainly fluazinam, cymoxanil, 8.1
phosphonates, host plant inducers)
a
For explanation of group designations, see Tables 14.6–14.18.
Table 14.3 Global fungicide market in different crops in 2008 [4].
Crop %
Cereals 22
Fruits and vegetables 18
Vine 9
Soybean 8
Rice 6
Potato 6
Pome fruit 5
Maize 3
Rape 3
Other crops 9
Noncrop 11
Table 14.4 Fungicide market in different regions in 2009 [4].
Region %
Europe 45
Asia/Pacific 21
South and Latin America 20
North America 12
Middle East/Africa 2
specific modes of action shown in Table 14.2 originate from the 1960s and 1970s.
A surprisingly high proportion of the fungicide market is still taken by the multisite
fungicides (ca. 19%), such as the dithiocarbamates (mainly mancozeb), chloroni-
triles (chlorothalonil), copper and sulfur formulations, and phthalimides (mainly
folpet and captan). In addition, two more recently developed fungicide groups – the
carboxamide/succinate dehydrogenase inhibitors (SDHIs) and the carboxylic acid
amides (CAAs) – are gaining importance but have not yet reached a prominent
position.
Table 14.3 documents the dominant position of cereals within the global fungicide
market, followed by ‘‘fruits and vegetables’’ (the latter form a complex segment
composed of a multitude of smaller crops). Whilst only minor changes in the
relative importance of specific crops are expected for the near future, one single
pathogen – soybean rust (Phakopsora pachyrhizi) – has created an important and
totally new segment for fungicide use within a few years.
At the regional level, fungicide sales in (Western) Europe are outstanding because
of the dominant position of cereals. This is followed by Asia, with its important
fungicide consumption in vegetable and fruit production, and by the new market
segment soybean rust, which has generated an increasing fungicide market in

Brazil and Latin-America (Table 14.4).
14.3
Fungicide Resistance
14.3.1
Mechanisms and Occurrence of Resistance
Although some multisite fungicides have been in use for over 200 years, resistance
reports for this class of chemicals are rare and usually of low practical importance. As
shown in Table 14.5, resistance in cereal pathogens to organomercury compounds
was reported in 1964, and resistance of apple scab to dodine in 1969 [5].
The occurrence of resistance to single-site fungicides was reported during the
1970s and 1980s. Important differences exist in terms of practical implications,
however, with severe problems for product performance being noted, such as
for benzimidazoles and QoIs, in only a few cases. In other cases, the practical
consequences were of limited importance for several possible reasons:
• Low-resistance factors (e.g., for DMIs).
• Reduced fitness of resistant isolates (e.g., for dicarboximides).
• Limited commercial importance of the affected fungicide class (e.g., for anilinopy-
rimidines (APs).
• Successful resistance management (e.g., for phenylamides).
The factors described above are the result of both the intrinsic properties of
resistant isolates, and the way in which the fungicides were used. The intrinsic
properties of resistant isolates are strongly related to the biochemical mechanism
that causes a reduced sensitivity. Several types of resistance mechanisms can be
distinguished:
1) Unspecific resistance based on ATP-binding cassette (ABC) transporters (e.g.,
in Botrytis cinerea).
2) Polygenic resistance characterized by a continuous selection process (e.g., for
DMIs).
3) Monogenic resistance (mutations at target site) leading to a disruptive selection
process (e.g., for QoIs).
4) Resistance based on the metabolic detoxification of the fungicide.
The greatest impact on resistance is associated with monogenic mechanisms,
especially mutations at the target site that result in high resistance factors and low or
no fitness penalties in resistant isolates. These factors apply for the E198A/G/K and
F200Y mutation in the β-tubulin gene that confers resistance to benzimidazoles,
and the G143A mutation in the cytochrome b gene delivering QoI resistance.
The most prominent examples for polygenic resistance resulting in continuous,
stepwise selection are those connected with the SBI fungicides (DMIs and amines)
Table 14.5 Occurrence of practical resistance to fungicides

(Brent and Hollomon [6], supplemented).
Date first Fungicide/class Yearsa Crop/pathogenb Practical

observed importancec
1960 Aromatic 20 Citrus storage rots Low

hydrocarbons Penicillium spp.
1964 Organomercurials 40 Cereal leaf spot and stripe Low
Pyrenophora spp.
1969 Dodine 10 Apple scab Medium
Venturia inaequalis
1970 Benzimidazoles 2 Many pathogen species of High
Ascomycetes in many crops
1971 2-Amino-pyrimidines 2 Cucumber and cereal powdery Medium
mildews Sphaerotheca fuliginea
and Blumeria graminis
1971 Kasugamycin 6 Rice blast Magnaporthe grisea Low
1976 Phosphorothiolates 9 Rice blast Magnaporthe grisea Medium
1977 Triphenyltins 13 Sugar beet Cercospora beticola Low
1980 Phenylamides 2 Phytophthora infestans High
Plasmopara viticola and other
Oomycetes in numerous crops
1982 Dicarboximides 5 Botrytis cinerea in grapes and Medium to high
other crops
1982 DMI fungicides 7 Many pathogen species of Medium
Ascomycetes in many crops
1985 Carboxamides 15 Barley loose smut Low
Ustilago nuda
1994 CAA fungicides 2 Grape downy mildew Medium
Plasmopara viticola
1998 Phenylpyrroles 4 Botrytis cinerea in grapes and Low
Venturia inaequalis in apples
1998 QoI fungicides 2 Many pathogen species of High
Oomycetes and Ascomycetes in
many crops
2000 Anilinopyrimidines 5 Botrytis cinerea in grapes, Low to medium
Venturia inaequalis in apples
Oculimacula spp. in wheat
2001 Quinoxyfen 4 Wheat and barley Blumeria Low to medium
graminis
2002 MBI-D fungicides 6 Rice blast Magnaporthe grisea Medium
2007 SDHI fungicides 3 Botrytis cinerea in grapes Medium to high
a
Estimated numbers of years after market introduction.
b
Examples given for high-risk cases only.
c
Judged based on risk of loss of control under practical conditions.
[7, 8]. Unspecific resistance mechanisms based on the energy-dependent export

of xenobiotics by ABC-transporters are often associated with polygenic resistance.
On their own, they play only a limited role for practical field resistance because
of low resistance factors and distinct fitness penalties [9]. In contrast to herbicides
and insecticides, the metabolic detoxification of an active ingredient is of low
importance for fungicides. The only documented cases of fungicide detoxification
are those for iprobenfos, [10] fenhexamid [11] and, to a lesser degree, also for
kresoxim-methyl [12].
14.3.2
The Fungicide Resistance Action Committee (FRAC)
As a consequence of widespread resistance problems with benzimidazoles and

dicarboximides, and serious concerns for phenylamide and triazole fungicides,
resistance seminars were organized at the University of Wageningen in the
Netherlands in 1981 and 1982. Representatives of the agrochemical companies
decided to establish an intercompany group with the task of coordinating resistance
management strategies. The FRAC was founded as an organization designated to
discuss resistance problems, and to make cooperative efforts in the prevention
and management of fungicide resistance. FRAC became incorporated within
GIFAP, the International Group of National Associations of Manufacturers of
Agrochemical Products. This organization evolved later on – within an organization
called the Global Crop Protection Federation (GCPF) and then within CropLife
International, the global federation representing the plant science industry.
The purpose of the FRAC is to provide guidelines for fungicide resistance
management, to prolong the effectiveness of ‘‘at-risk’’ fungicides, and to limit crop
losses should resistance occur. In more detail, the main aims of FRAC [13] are to:
• identify existing and potential resistance problems;
• collate information on fungicide resistance and distribute it to those involved in
the research, development, distribution, registration, and use of fungicides;
• provide guidelines and advice on the use of fungicides to reduce the risk of
resistance and to manage it should it occur;
• recommend procedures for fungicide resistance studies;
• stimulate open liaison and collaboration with universities, government agencies,
advisors, extension workers, distributors, and farmers.
If molecules from different manufacturers have the same mode of action, and if
this mode of action bears at the same time a significant resistance risk, an FRAC
Working Group can be established to analyze the resistance risk and to develop and
publish common resistance management recommendations. There are currently
FRAC Working Groups for SBIs, QoIs, APs, SDHIs, and CAAs which meet
regularly and publish yearly updated reports on the resistance status and suitable
resistance management recommendations. In addition, the FRAC Banana Working
Group, composed of fungicide manufacturers and fruit companies, coordinates
resistance management recommendations for all specific fungicides used in banana
production. For older modes of action for which regular monitoring programs
are no longer performed (benzimidazoles, phenylamides, and dicarboximides),
so-called Expert Fora are available at the FRAC web site (www.frac.info) to provide
advice and collect important published literature on resistance-monitoring methods
and resistance management.
14.3.3
Resistance Risk Assessment
The overall resistance risk is the result of the interaction of numerous independent
factors. The intrinsic risk is related to all aspects of the mode of action, the biology
of the pathogen and the interactions between them, whereas the extrinsic (or
management) risk includes all aspects of how a product is used, such as the number
and interval of applications, rates and type of treatment, and whether the product is
used as a solo formulation or in mixture or alternation with other modes of action.
The intrinsic risk is composed of several elements, which have been described in
more detail in different documents [6, 14, 15]. The most important elements include
the analysis of baseline sensitivity of field isolates [16], population structure (uni- or
bimodal), cross- and multiple resistance, stability of resistance, forced selection of
resistant individuals over several generations, artificial mutagenesis and selection,
biochemical site of action (single- or multisite), molecular mechanism of resistance
(mutations in target gene), genetics and the inheritance of resistance (mono- or
polygenic resistance). Some of these elements are not easy to evaluate, especially
when no resistant isolates are available from field populations. However, when the
results generated for a new active ingredient are compared with those of known
chemical classes, the intrinsic risk can mostly be assessed quite well and classified
as low, medium, or high. According to the estimated intrinsic risk, appropriate
strategies can be defined as to how to use the product and to minimize the
management risk.
14.3.4
Resistance Management and Risk Modifiers
Based on the environmental and toxicological properties of a compound, a rather

fixed frame is given that defines the maximum fungicide dose rate and number
of applications per season and area. The assessment of resistance risk must be
made within this frame, and the number of applications may be further reduced to
adequately manage the resistance risk.
Appropriate resistance management tools must be chosen and validated for each
individual pathogen/fungicide combination and out of all available resistance risk
modifiers. The European and Mediterranean Plant Protection Organization (EPPO)
Standard on Resistance Risk Analysis [17] lists the following tools for lowering the
overall resistance risk:
• Use of good plant protection practices: Lower the disease pressure by all means of
good plant protection practices. For example, the use of less-susceptible cultivars,
crop rotation, adequate use of fertilizers, and sanitation measures lowering the
primary inoculums, such as the elimination of plant debris by plowing instead
of minimum tillage.
• Use of mixtures/alternations of fungicides: The use of mixtures of fungicide
partners without crossresistance is a validated standard tool in resistance manage-
ment, as well as the alternation of non-crossresistant fungicides that is preferably
used in longer spray schedules.
• Application frequency, timing, and dose rate: Besides the use of mixtures or
alternations, limiting the number of applications per season is the most important
standard tool in resistance management. In addition, the timing of application
(preventive or curative) and the dose rate applied are of outstanding importance.
• Negative crossresistance: The use of fungicides exhibiting negative crossresis-
tance is, in theory, a suitable way to decrease the frequency of resistant isolates.
Unfortunately, negative crossresistance is very rare in fungicide resistance.
• Sensitivity monitoring, reporting to authorities, and recording of changes in
product performance: Systematic monitoring programs that allow the obser-
vation of resistance dynamics form the basis for the development of rational
resistance management concepts. The availability of detailed sensitivity profiles
needs, in addition, discussion with authorities to improve and enforce resistance
management programs.
14.4
Fungicide Classes and Modes of Action
The classification of fungicides according to their mode of action and crossresis-

tance pattern forms the basis for resistance management under practical agronomic
conditions. If fungicides are recommended to be used in alternation or mixtures
to achieve a robust disease control and delay resistance evolution, then clear infor-
mation on the crossresistance behavior for each compound is required, because
it is not possible to recognize whether two fungicides are crossresistant simply
by comparing their chemical structures, common names, or group names. The
FRAC classification covers all important fungicides (and also some bactericides
and plant-defense inducers) that are registered worldwide. As far as is available,
the classification is based on the biochemical and/or molecular mode of resistance.
This implies that the methods used to study the mode of action and the mech-
anism of resistance may be different for each new fungicide class, because they
depend on the availability of technical possibilities during the time of the studies.
For fungicides that are used in only a few countries and crops, such studies are
often very limited or not reported in one of the common languages. Therefore,
the FRAC code list is a compromise between the need to come to a reasonable
classification and pure scientific approaches. A similar compromise is often needed
for the chemical group names, which are sometimes not derived from a systematic
chemistry approach but rather have a more general character or are based on the
first member in the group.
The FRAC Code list is based on the biochemical mode of action. Once the
mode of action or crossresistance pattern of a new fungicide becomes known,
the compound is given a unique serial number. All known modes of action are
listed in Tables 14.6 to 14.16). A similar list providing, in addition, information on
resistance risk and the required resistance management measures for each mode
of action is published at the FRAC website and updated yearly [18].
One important group of fungicides that interfere with nucleic acid synthesis are
the phenylamides, and these have maintained a strong position in the Oomycete
market, despite major resistance problems (Table 14.6). Today, fungicides that
inhibit adenosine deaminase are of limited market importance, due to pronounced
resistance problems in powdery mildew pathogens. Hymexazol is used as a soil-
and seed-treatment fungicide, and oxolinic acid for the control of bacterial diseases
such as fire blight in apples and pears caused by Erwinia amylovora.
Fungicides that interfere with β-tubulin assembly (benzimidazoles and benz-
imidazole generators, B1) have lost most of their initial importance for diseases
control due to the widespread distribution of resistance (Table 14.7). The B2
compound diethofencarb has also become less important; this initially was deve-
loped specifically to combat benzimidazole-resistant strains, as it inhibits only the
mutated isolates and thus provides a negative crossresistance to benzimidazoles.
Zoxamide is specifically active against β-tubulin assembly in the Oomycetes (this
process is not affected by B1 fungicides). Pencycuron exhibits a highly specific
fungicidal action against only one pathogen, Rhizoctonia solani.
Fungicides that inhibit fungal respiration have mostly a broad spectrum of
activity, and are used to control both the Oomycetes and the true fungi (the As-
comycetes, Deuteromycetes, and Basidiomycetes) (Table 14.8). Uncouplers (C5)
Table 14.6 Group A: Fungicides interfering with nucleic acid synthesis.
FRAC code Target site Chemical group Compounds Comments

(examples)
Target Serial
no. no.
A1 4 RNA polymerase I Phenylamides (PA Metalaxyl-M Specific for

fungicides) Oomycetes; high
resistance risk
A2 8 Adenosine- Hydroxy- Ethirimol Specific for powdery
deaminase pyrimidines mildew; medium
resistance risk
A3 32 DNA/RNA Hetero- Hymexazol Broad spectrum
synthesis (prop.) aromatics
A4 31 DNA Carboxylic Oxolinic acid Bactericide
topoisomerase acids
type II (gyrase)
Table 14.7 Group B: Fungicides interfering with mitosis and cell division.

(examples)
Target Serial
no. no.
B1 1 β-Tubulin assembly Methyl- Benomyl, Broad spectrum;

in mitosis benzimidazole carbendazim, high resistance risk
carbamates (MBC thiophanate-
fungicides) methyl
B2 10 β-Tubulin assembly N-Phenyl- Diethofencarb Negative cross
in mitosis carbamates resistance to B1
B3 22 β-Tubulin assembly Toluamides Zoxamide Specific for
in mitosis Oomycetes
B4 20 Cell division (prop.) Phenylureas Pencycuron Specific for
Rhizoctonia solani
B5 43 Delocalization of Benzamides Fluopicolide Specific for
spectrin-like Oomycetes
proteins (prop.)
and most complex II inhibitors (C2) have been available for many years. Car-
boxamides such as carboxin were for many years, restricted to the control of
Basidiomycetes, but more recently a range of new representatives of the C2 group
(the first being boscalid) has been identified that can also be used to control the
Ascomycetes. Based on their broad spectrum and long-lasting activity, the QoI
fungicides have rapidly gained an important market share since the introduction
of the first representatives (azoxystrobin and kresoxim-methyl) in 1996. However,
their use has been recently limited in certain crops (e.g. cereals, grapes) due to
the high frequency of resistant isolates in field populations. The other group C
fungicide classes are of lesser commercial importance.
In Group D, all compounds except the APs are antibiotics of microbial origin
(Table 14.9). Both, D2 and D3 show a specific action against rice blast (Magnaporthe
grisea), whereas D4 and D5 are specific bactericides that are used in parallel also in
the medical field. The AP fungicides can be used against a broad range of diseases
caused by Ascomycetes in fruits and vegetables, cereals, and bananas.
In group E, the quinoline derivative quinoxyfen controls exclusively powdery
mildews in cereals and broadleaved crops, whereas phenylpyrroles such as flu-
dioxonil are used as foliar, post-harvest and seed-treatment fungicides against a
broad range of pathogens, such as Botrytis cinerea and apple scab (Table 14.10).
Dicarboximides (E3) are still used against Botrytis cinerea and related pathogens
in a range of crops, although resistance was reported repeatedly. Resistance to
this class of fungicides seems to be variable in both frequency and location,
however.
Table 14.8 Group C: Fungicides interfering with fungal respiration.

(examples)
Target Serial
no. no.
C1 39 Complex I: NADH Pyrimidinamines Diflumetorim Broad spectrum

oxido-reductase
C2 7 Complex II: ‘‘Carboxamides,’’ Carboxin, boscalid, Mostly broad
succinate- Succinate bixafen, spectrum;
dehydrogenase dehydrogenase isopyrazam, medium to high
inhibitors (SDHI fluopyram resistance risk
fungicides)
C3 11 Complex III: ‘‘Strobilurins’’ and Azoxystrobin, Mostly broad
cytochrome bc1 related (QoI pyraclostrobin, spectrum; high
(ubiquinol oxidase) fungicides) trifloxystrobin, resistance risk
at Qo site famoxadone,
fenamidone
C4 21 Complex III: Cyano-imidazoles Cyazofamid, Specific for
cytochrome bc1 and amisulbrom Oomycetes;
(ubiquinone sulfamoyl-triazoles resistance risk
reductase) at Qi site (QiI fungicides) assumed to be
medium to high
C5 29 Uncouplers of Diverse Dinocap, Broad spectrum
oxidative fluazinam
phosphorylation ferimzone
C6 30 ATP synthase, Triphenyl tins Fentin acetate Broad spectrum
oxidative
phosphorylation
C7 38 ATP production Thiophene Silthiofam Specific for
(prop.) carboxamides Gaeumannomyces
graminis
C8 45 Complex III: Triazolo- Ametoctradin Specific for
cytochrome bc1 pyrimidylamines Oomycetes
(ubiquinone
reductase) at Qx site
(prop.)
Group F compounds interfere with lipid and membrane synthesis; they cover a
range of target sites, most of which have a putative status because the modes of
action have not been fully elucidated (Table 14.11).
Since the first introduction of amines and DMIs during the 1960s and 1970s,
the inhibition of fungal sterol biosynthesis has rapidly become the most successful
biochemical target for specific fungicides (Table 14.12). In addition to the broad
spectrum of activity, which covers most pathogens belonging to the Ascomycetes
and Basidiomycetes, a pronounced curative activity and a moderate resistance risk
Table 14.9 Group D: Fungicides interfering with amino acid and protein synthesis.

(examples)
Target Serial
no. no.
D1 9 Methionine Anilinopyrimidines Pyrimethanil, Broad spectrum

biosynthesis (AP fungicides) cyprodinil, (e.g., Botrytis,
mepanipyrim Venturia,
Oculimacula,
Sigatoka); resistance
risk medium
D2 23 Protein synthesis Enopyranuronic Blasticidin-S Rice blast
acid antibiotics
D3 24 Protein synthesis Hexopyranosyl Kasugamycin Rice blast
antibiotics
D4 25 Protein synthesis Glucopyranosyl Streptomycin Bactericide; high
antibiotics resistance risk
D5 41 Protein synthesis Tetracycline Oxytetracycline Bactericide; high
antibiotics resistance risk
Table 14.10 Group E: Fungicides interfering with signal transduction.

(examples)
Target Serial
no. no.
E1 13 Signal transduction Azanaphthalenes Quinoxyfen, Specific for powdery

(prop.) proquinazid mildews; resistance
risk medium
E2 12 MAP/histidine Phenylpyrroles Fludioxonil Broad spectrum;
kinase in osmotic (PP fungicides) resistance risk low to
signal transduction medium
(os-2, HOG1 gene)
E3 2 MAP/histidine Dicarboximides Iprodione, Mainly against
kinase in osmotic procymidone, Botrytis and related
signal transduction vinclozolin spp.; resistance risk
(os-1, Daf1 gene) medium to high
are characteristics for the DMIs (G1) and amines (G2). In contrast to the other
inhibitors of fungal sterol biosynthesis, the G3 representative fenhexamid shows
a rather narrow spectrum of activity, that is confined to Botrytis cinerea and the
related pathogens Monilinia spp. and Sclerotinia spp.
Table 14.11 Group F: Fungicides interfering with lipid and membrane synthesis.

(examples)
Target Serial
no. no.
F2 6 Methyltransferase Phosphoro- Edifenphos, Mainly for rice

in phospholipid thiolates/ pyrazophos, blast control
biosynthesis dithiolanes isoprothiolane
F3 14 Lipid peroxidation Aromatic Biphenyl, chloroneb, Broad spectrum
(prop.) hydrocarbons, etridiazole
thiadiazoles
F4 28 Cell membrane Carbamates Propamocarb Specific for
permeability, fatty Oomycetes
acids (prop.)
F6 44 Microbial disrupter Bacillus subtilis Bacillus subtilis Broad spectrum
of cell membranes lipopeptides strain QST 713
(prop.)
Table 14.12 Group: G: Fungicides interfering with sterol biosynthesis in membranes.

(examples)
Target Serial
no. no.
G1 3 C14-Demethylase in Piperazines, Prochloraz, Broad spectrum;

sterol biosynthesis pyridines, tebuconazole, more than 30
(DMI fungicides) pyrimidines, epoxiconazole, commercial
imidazoles, prothioconazole, compounds;
triazoles propiconazole, resistance risk
cyproconazole, medium
difenoconazole
G2 5 14-Reductase and Amines, Fenpropimorph, Mainly against
8 → 7 isomerase (morpholines, fenpropidin, powdery mildews,
in sterol biosynthesis piperidines, spiroxamine rusts and Sigatoka;
spiroketal- resistance risk low
amines) to medium
G3 17 3-Keto reductase Hydroxy-anilides Fenhexamid specific for Botrytis
(C4 demethylation) in and related
sterol biosynthesis pathogens;
resistance risk low
to medium
G4 18 Squalene epoxidase in Thiocarbamates, Pyributicarb, Medical fungicides
sterol biosynthesis allylamines naftifine only
Table 14.13 Group H: Fungicides interfering with cell wall biosynthesis.

(examples)
Target Serial
no. no.
H3 26 Trehalase and Glucopyranosyl Validamycin Specific for rice

inositol antibiotics sheath blight
biosynthesis (Rhizoctonia solani)
H4 19 Chitin synthase Peptidyl Polyoxin Broad spectrum;
pyrimidine resistance risk
nucleosides medium
H5 40 Cellulose Carboxylic acid Dimethomorph, Specific for
synthase amides (CAA iprovalicarb, Oomycetes;
fungicides) mandipropamid resistance risk low
to medium
Some inhibitors of fungal cell wall biosynthesis are of microbial origin, produced
by Streptomyces hygroscopicus var. limoneus (H3) and by S. cacaoi var. asoensis (H4)
(Table 14.13) [19]. Validamycin is a specific compound for the control of rice
sheath blight, whereas polyoxins inhibit a target site that potentially is present
in all Ascomycetes and Basidiomycetes. Nevertheless, the market importance of
polyoxins remains limited. The mode of action of the CAAs has recently been
elucidated and described as the inhibition of cellulose synthase [20]. Resistance to
CAAs has been detected in Plasmopara viticola, but not in Phytophthora infestans.
The inhibition of melanin synthesis in fungal cell walls is a rather specific target.
However, fungicides of group I, such as tricyclazol and carpropamid, are quite
important for control of the rice blast pathogen (Magnaporthe grisea) (Table 14.14).
The melanization of the appressorium cell wall is a pathogenicity factor of this
pathogen, as it relies on mechanical pressure during penetration of the host cuticle.
Few other pathogens, such as Colletotrichum spp. use the same mechanism of
penetration. Although, as a consequence, the efficacy of melanin biosynthesis
inhibitors is (in theory) confined to both pathogens, it proved to be successful in
practice only for the control of rice blast.
The induction of systemic acquired resistance has been studied in detail during
recent decades within a multitude of university research groups and industrial
research laboratories, and is documented in many scientific reports [21]. However,
only limited control is achieved by the induction of defense mechanisms against
®
diseases under field conditions. Acibenzolar-S-methyl (trade name Bion ) is used
in numerous crops, and protects plants against fungal, bacterial, and viral diseases
®
(Table 14.15). Probenazole (trade name Oryzemate ) and, more recently, tiadinil
are used to control rice blast (Magnaporthe grisea), but have also some antibacterial
and antiviral effects.
Table 14.14 Group I: Fungicides interfering with melanin synthesis in cell wall.

(examples)
Target Serial
no. no.
I1 16.1 Reductase in MBI-R Diverse Tricyclazole, Specific for rice blast

fungicides pyroquilon
I2 16.2 Dehydratase in Carboxamides, Carpropamid, Specific for rice
MBI-D fungicides propionamides fenoxanil blast; resistance risk
medium
MBI, melanin biosynthesis inhibitor.
Table 14.15 Group P: Host plant defense induction.

(examples)
Target Serial
no. no.
P1 P Salicylic acid BTHs Acibenzolar-S- Indirect action on

pathway methyl fungi, bacteria and
viruses through host
plant defense
P2 P Unknown Benzo-isothiazoles Probenazole
P3 P Unknown Thiadiazole- Tiadinil, isotianil
carboxamides
P4 P Unknown Diverse Laminarin
BTH, benzothiadiazole.
Despite many studies having been conducted, the biochemical mode of action
of several fungicides and bactericides remains unclear (Table 14.16). Notably,
®
two compounds – cymoxanil (trade name Curzate ) and fosetyl-Al (trade name
®
Alliette ) – have gained broad market acceptance for the control of Oomycetes for
many years. The compounds listed in Table 14.16 have been designated a serial
FRAC code number because long-term sensitivity monitoring has revealed that
there is no crossresistance to other existing fungicide groups.
The compounds listed in Table 14.17 have entered the market only recently
(except for dodine, which has been available for more than 40 years). Because their
modes of action and mechanisms of resistance are still unknown, these compounds
have been given a transient status; when more detailed information becomes
available they will be given a specific FRAC code number. Dodine resistance was
Table 14.16 Group U1: Fungicides with FRAC serial number and unknown mode of action.
FRAC code serial Chemical group Compounds Comments

no. (examples)
27 Cyanoacteamide Cymoxanil Specific for

oximes Oomycetes;
resistance risk low to
medium
33 Phosphonates Fosetyl-Al Specific for
Oomycetes
34 Phthalamic acids Teclofthalam Bactericide
35 Benzotriazines Triazoxide Narrow spectrum
36 Benzene- Flusulfamide Broad spectrum
sulfonamides
37 Pyridazinones Diclomezine Broad spectrum
42 Thiocarbamates Methasulfocarb Broad spectrum
Table 14.17 Group U2: Fungicides without FRAC serial

number and unknown mode of action.
FRAC code target no. Chemical group Compounds Comments

(examples)
U5 Thiazole Ethaboxam Specific for

carboxamides Oomycetes
U6 Phenylacetamides Cyflufenamid Specific for powdery
mildews
U8 Benzophenones Metrafenone Specific for powdery
mildews and eyespot;
resistance risk
medium
U12 Guanidines Dodine Broad spectrum;
resistance risk low to
medium (resistance
known in Venturia
inaequalis)
described by Szkolnik and Gilpatrick for apple scab, Venturia inaequalis, in 1969
[22]. The polygenic control of resistance to dodine, and a continuous sensitivity
distribution, were reported by Georgopoulos [23], while Koeller and Wilcox [24]
provided evidence that dodine-resistant Venturia isolates were occasionally less
sensitive also to other fungicide classes, such as DMIs.
Most of the fungicides with a multisite mode of action have been widely used
for decades (Table 14.18). As noted above, this applies especially to the inorganic
Table 14.18 Group M: Fungicides with multisite mode of action.
FRAC code Target no. Chemical group Compounds (examples)
M1 Inorganics Copper (different salts)

M2 Inorganics Sulfur
M3 Dithiocarbamates and related Mancozeb, maneb, metiram,
propineb, thiram
M4 Phthalimides Captan, folpet
M5 Chloronitriles Chlorothalonil
M6 Sulfamides Dichlofluanid
M7 Guanidines Guazatine, iminoctadine
M8 Triazines Anilazine
M9 Anthraquinones Dithianon
fungicides based on copper salts or sulfur, which overall are the oldest fungicides.
Multisite fungicides are generally considered as a group with a low resistance
risk, and without any signs of resistance development under field conditions over
decades. Although reports on the reduced sensitivity of pathogens to some of the
multisite compounds under laboratory conditions are available, the resistance risk
under field conditions remains quite low.
References
1. Brent, K.J. (1985) Fungicides for Crop 8. Brent, K.J. and Hollomon, D.W. (1988)
Protection: 100 Years of Progress, British in Sterol Biosynthesis Inhibitors (eds
Crop Protection Council (BCPC), Croy- D. Berg and M. Plempel), Ellis Hor-
don, UK, Vol. 31, pp. 11–22. ISSN wood, Chichester and Wiley-VCH Verlag
0306-3941. GmbH, Weinheim, pp. 332–346.
2. Viennot-Bourgin, C. (1985) Fungicides 9. Zwiers, L.H., Stergiopoulos, I., Van
for Crop Protection: 100 Years of Progress, Nistelrooy, J.G.M., and De Waard, M.A.
British Crop Protection Council (BCPC), (2002) Antimicrob. Agents Chemother., 46
Croydon, UK, Vol. 31, pp. 3–11. ISSN (12), 3900–3906.
0306-3941. 10. Uesugi, Y. and Sisler, H.D. (1978) Pestic.
3. Fungicide Resistance Action Committee Biochem. Physiol., 9, 247–254.
web site: www.frac.info. 11. Suty, A., Pontzen, R., and Stenzel,
4. Phillips McDougall AgriService K. (1999) Pflanz. Nachr. Bayer, 52,
(2009/2010) Industry Overview Markets, 149–161.
Vineyard Business Centre, Saughland, 12. Jabs, T., Cronshaw, K., and Freund, A.
Pathhead, Midlothian EH37 5XP, UK. (2001) Phytomedizin, 2, 15.
5. Szkolnik, M. and Gilpatrick, J.D. (1969) 13. www.frac.info/frac/about.
Plant Dis. Rep., 53, 861–864. 14. Gisi, U. and Staehle-Csech, U. (1988)
6. Brent, K.J. and Hollomon, D.W. (2007) Proc. Brighton Crop Prot. Conf. – Pests
Fungicide Resistance, the Assessment Dis., 2, 359–366.
of Risk, Vol. 2, FRAC Monographs. 15. European and Mediterranean Plant
Available at: www.frac.info. Protection Organization (EPPO) (2010)
7. Dekker, J. (1985) Prog. Pestic. Biochem. Standard PP1/213(2). Available at:
Toxicol., 4, 166–209. www.eppo.org.
References 557
16. Russell, P.E. (2004) Sensitivity Baselines 20. Blum, M., Waldner, M., and Gisi, U.
in Fungicide Resistance Research and (2010) Fungal Genet. Biol., 47, 499–510.
Management, Vol. 3, FRAC Monographs. 21. Ryals, J.A., Neuenschwander, U.H.,
Available at: www.frac.info. Willits, M.G., Molina, A., Steiner, H.-Y.,
17. European and Mediterranean Plant and Hunt, M.D. (1996) Plant Cell, 8,
1809–1819.
Protection Organization (EPPO) (2010)
22. Szkolnik, M. and Gilpatrick, J.D. (1969)
Guidelines PP1/213(2). Available at:
Plant Dis. Rep., 53, 861–865.
www.eppo.org.
23. Georgopoulos, S.G. (1995) The genetics
18. www.frac.info (publications). of fungicide resistance, in Modern Selec-
19. Yamaguchi, J. (1995) Antibiotics as tive Fungicides (ed. H. Lyr), Fischer, New
antifungal agents, in Modern Selec- York, pp. 39–52.
tive Fungicides (ed. H. Lyr), Fischer, 24. Köller, W. and Wilcox, W.F. (2001)
New York, pp. 415–429. Phytopathology, 91, 776–781.
559
15
Fungicides Acting on Oxidative Phosphorylation
15.1
The Biochemistry of Oxidative Phosphorylation: A Multiplicity of Targets
for Crop Protection Chemistry
Fergus Earley
15.1.1
Introduction
In all eukaryotic cells, the efficient use of carbohydrate, fat, or protein as an energy
source depends on the complete oxidation of constituent carbon atoms to carbon
dioxide. The energy available from these oxidations is conserved through the
coupled synthesis of adenosine triphosphate (ATP) from adenosine diphosphate
(ADP) and phosphate (Pi), and is used to drive the kinetic, biosynthetic, and
homeostatic processes of the cell through numerous concerted reactions involving
the hydrolysis of ATP back to ADP.
The oxidation reactions involved are catalyzed by a series of nicotinamide
adenine dinucleotide (NAD+ ) or flavin adenine dinucleotide (FAD)-dependent
dehydrogenases in the highly conserved metabolic pathways of glycolysis, fatty acid
oxidation, and the tricarboxylic acid (TCA) cycle, the latter two of which are localized
to the mitochondrion, as is the bulk of coupled ATP synthesis. Reoxidation of the
reduced cofactors (NADH and FADH2 ) requires molecular oxygen, and is carried
out by protein complexes that are integral to the inner mitochondrial membrane.
These are known collectively as the respiratory, electron transport, or cytochrome
chain. Ubiquinone (UQ), and the small soluble protein cytochrome c, act as carriers
of electrons between the complexes (Figure 15.1.1).
Before there was any understanding of the nature of the proteins involved,
the overall sequence of electron transfer between NADH and oxygen could be
divided into three sections by the use of exogenous substrates, specific inhibitors,
and by observing the oxidation state of the cytochromes. Thus, the site of action
of inhibitors and the sequence of electron transfer, NADH to UQ (inhibited by
rotenone), UQ through cytochrome b to cytochrome c (inhibited by antimycin A),
and cytochrome c through cytochrome a/a3 to oxygen (inhibited by cyanide), were
defined. It has since become clear that the proteins which support these sections

560 15 Fungicides Acting on Oxidative Phosphorylation
O2 H2O
OMM IMM Matrix
H+
H+
NADH IV ATPase
Pyruvate III
dehydrogenase NAD+ UQ ATP
I
Fatty acid
oxidation ADP + Pi
ETF ETFQO
TCA cycle UQ
Succ
UQ ATP
Fum ANC
SQO
Pyr PC
PyrT ADP
Pi
Glycolysis
Figure 15.1.1 Schematic overview of mi- and its ubiquinol-dependent oxidoreductase

tochondrial oxidative phosphorylation. A (ETFQO). NADH is reoxidized by Complex
part of the mitochondrion is represented, I (I), and reduced ubiquinone (UQ, in blue)
showing the outer mitochondrial membrane by electron transfer through Complex III (III),
(OMM), inner mitochondrial membrane cytochrome c (red circle), and Complex IV
(IMM), and crista (an invagination of the (IV). These three complexes pump protons
inner membrane). Substrates for oxidation (H+ ) across the membrane to create the
enter the mitochondrion through specific electrochemical gradient that drives ATP syn-
carrier proteins, for example, the pyruvate thesis by the F1 F0 ATP synthase (ATPase).
transporter (PyrT). Reducing equivalents Phosphate entry and ATP/ADP exchange
from fatty acyl-CoA dehydrogenases, pyruvate with the cytosol is mediated by the phos-
dehydrogenase, and the TCA cycle are deliv- phate carrier (PC) and adenine nucleotide
ered to the electron-transport chain through carrier (ANC), respectively. Succ, succinic
NADH, succinate ubiquinol oxidoreductase acid; Fum, fumaric acid; Pyr, pyruvic acid.
(SQO), electron-transfer flavoprotein (ETF),
of the electron transport chain are also physically associated in the mitochondrial
membrane.
ATP synthesis is coupled to electron transfer at each of the sections defined above;
these are referred to as ‘‘coupling sites’’ 1, 2, and 3, respectively. The mechanism of
coupling was first proposed by Mitchell, whose ‘‘Chemiosmotic hypothesis’’ is now,
in essence, universally accepted. That is, that electron transfer through each of the
coupling sites results in proton translocation from the inside to the outside of the
inner mitochondrial membrane, while the electrochemical gradient so generated
not only drives ATP synthesis but also controls the rate of electron transfer. (For
a comprehensive overview of mitochondrial oxidative phosphorylation, and the
genesis of our current understanding, the reader is referred to excellent historical
reviews [1, 2].)
Because these processes are essential to the survival of most aerobic organisms,
they have been exploited repeatedly by Nature as targets for the chemical armory
15.1 The Biochemistry of Oxidative Phosphorylation 561
of secondary metabolites used for defense or competition. Synthetic chemistry has

also successfully exploited these targets for the benefit of agriculture, as fungicides,
insecticides, and acaricides. Indeed, the present knowledge of the organization
and mechanism of oxidative phosphorylation owes much to the discovery of these
inhibitors and to the study of their action.
The inhibitors can be divided into three classes: inhibitors of electron transport;
inhibitors of phosphorylation; and ‘‘uncouplers.’’ Both, inhibitors and uncouplers
have generally been detected as antimicrobial, pesticidal, or cytotoxic agents that
rapidly affect the rate of oxygen utilization by the cell, and localized in their
action by their effects on isolated mitochondrial preparations. The site of action
of electron transport inhibitors is still initially determined from their ability to
inhibit the oxidation of exogenous substrates (NADH, succinate, cytochrome
c), and by their effect on the reduction or reoxidation of the cytochromes, in
crude mitochondrial, or submitochondrial preparations [3, 4]. The definition of an
uncoupler or phosphorylation inhibitor is more demanding, and requires the use of
intact ‘‘coupled’’ mitochondrial preparations, in which oxidative phosphorylation
can be monitored by the rate of oxygen consumption under controlled conditions
[5]. Mitochondria from animal tissues are generally preferred for these studies
over fungal mitochondria – not only because of their ease of preparation, but
because the mitochondria of fungi – like those of plants – may express alternative
electron-transfer pathways that complicate the analysis (see Section 15.1.2.5).
Numerous structurally diverse organic chemicals have been identified as highly
potent (IC50 < 100 nM) and specific inhibitors of electron transport, principally
from plant and microbial secondary metabolites, but also from the screening of
synthetic chemical libraries for fungicides and insecticides. Among the natural
producers, the myxobacteria have proved a particularly abundant source of novel
structural classes [6], although potent inhibitors have also been isolated from plants,
fungi, streptomycetes, and marine organisms. Natural products have provided the
inspiration for the commercially successful strobilurin class of fungicides [7] (see
Chapter 15.2), and even found commercial use themselves (e.g., rotenone was
registered for use as an insecticide in the USA between 1947 and 2006 [8]).
Most of the potent electron-transport inhibitors discovered to date are selective
either for the NADH–UQ oxidoreductase or the ubiquinol–cytochrome c oxidore-
ductase sections of the chain; very few classes are known to have similar potency
against succinate–ubiquinol oxidoreductase. For cytochrome c oxidase, the known
potent inhibitors are limited to small molecules or ions that form coordination
complexes with heme (e.g., CN− , N3 − , CO, PH3 ). The inhibitor selectivity between
UQ-dependent oxidoreductases is surprising, given that all inhibitors are thought
to act at UQ binding sites, although it is probably a consequence of the lack of
sequence and structural conservation between different classes of quinone-binding
proteins [9–11]. In contrast to their selectivity between different sites in the respira-
tory chain, potent inhibitors generally do not show much selectivity between their
mitochondrial targets in different species [12, 13] (although there are exceptions
[14, 15]), which reflects the evolutionary conservation of sequence and structure
of the key functional subunits of the respiratory chain complexes. This, of course,
means that toxicity to nontarget organisms is always a major consideration in the

development of respiration inhibitors for use in agriculture.
15.1.2
Components of the Mitochondrial Electron-Transport Chains
When mitochondria from bovine heart were solubilized by treatment with mild
detergents, it proved possible to separate and purify the sections of the res-
piratory chain referred to earlier as coupling sites 1, 2, and 3. Subsequently,
these sections were named Complex I (NADH–UQ oxidoreductase), Complex III
(ubiquinol–cytochrome c oxidoreductase, cytochrome bc1 complex), and Complex
IV (cytochrome c oxidase) [16]. Although these complexes have since been char-
acterized as independent entities, it is now recognized that the three complexes
coassemble with specific stoichiometry to form respiratory chain ‘‘supercomplexes’’
or ‘‘respirasomes’’ in fungal, plant, and mammalian mitochondria [17]. There is
also evidence that succinate–UQ oxidoreductase (which was purified alongside
the other complexes and named Complex II [18]) forms a tight association with
Complex III in yeast mitochondria [19].
Purification and reconstitution has revealed the full complement of proteins
necessary for oxidative phosphorylation, and also enabled an identification of
the genes encoding these proteins. The gene sequences are necessary tools for
understanding the relationship between the components of the mitochondrial
respiratory chain and their prokaryotic ancestors, and also for tracking their
divergence among the eukaryotes, both of which have helped to assign function
to some of the subunits. The gene sequences also provide the protein sequences
essential for structural analysis.
The degree of conservation, in terms of subunit composition and protein se-
quence, between mammalian respiratory chain complexes and those characterized
from fungi and other organisms, depends on the subunit and complex under
consideration (see specific sections below). In general, those subunits which are
known to have a central role in electron transport are well conserved in terms of
their protein sequence and, where known, their tertiary structure. For these sub-
units, the fact that a clear relationship to bacterial respiratory chain components
can also be seen leads to the conclusion that the mitochondrial respiratory chain
complexes have evolved and adapted from those of the symbiotic bacterial ancestor
of the mitochondrion [20]. Mitochondrial complexes have, in most cases, acquired
many additional subunits, the function(s) of which remains obscure.
15.1.2.1 Complex I and Its Inhibitors

Complex I catalyzes the two-electron oxidation of NADH, coupled to the transport
of four protons across the membrane [21], and is the largest of the respiratory chain
complexes. The form purified from bovine heart mitochondria has an aggregate
molecular weight of at least 980 kDa, and is composed of 46 different proteins [22].
Complex I has also been purified from other sources, including the filamentous
mold, Neurospora crassa, the obligate aerobic yeast, Yarrowia lipolytica, and the
Gram-negative bacterium, Escherichia coli. Bacterial Complex I is much simpler

than the mitochondrial forms, having only 14 subunits, yet it is functionally
equivalent in terms of electron-transport and proton-pumping capabilities [23], and
even retains some inhibitor sensitivities [13]. All 14 subunits of E. coli Complex I
have homologs in the mitochondrial forms; hence, these (or a subset) are considered
as the ‘‘core’’ functional subunits [24, 25]. Complex I from Y. lipolytica and N. crassa
contains 37 and at least 39 nonidentical subunits, respectively, of which all but
four, in each case, have homologs in the bovine heart enzyme [26, 27].
Analyses using electron microscopy and X-ray crystallography have shown that
Complex I from bovine heart, N. crassa, Y. lipolytica, Thermus thermophilus and
E. coli can adopt a similar L-shaped structure, which spans the inner mitochon-
drial membrane with an arm extending into the matrix compartment [28–31]
(Figure 15.1.2). A subdomain representing part of this arm can be isolated that con-
tains the flavin mononucleotide (FMN) and retains NADH dehydrogenase activity
coupled to reduction of ferricyanide (which is also an enzymic activity of intact
Complex I). This activity is sensitive to the now superseded fungicide fenaminosulf
[32, 33]. Although fenaminosulf is not a highly selective Complex I inhibitor, its
ability to inhibit ferricyanide reduction is unusual, and this defines its site of action
NADH NAD+ + H+
FMN
Fe
Fe 49kDa
PSST
Fe Fe
Fe
Fe
Matrix Fe Iγ
N2 Fe UQ
UQ
UQH2
Ia Ib
B9
ND1
ND5
Figure 15.1.2 Schematic representation of on subunits within Iγ . Center N2 is believed

the structure and function of Complex I. The to be the oxidant for ubiquinone (UQ). The
complex can be resolved into three subcom- subdomain assignments for subunits referred
plexes: Iα, Iγ (a subset of Iα) and Iβ, ar- to in the text in the context of inhibitor bind-
ranged as indicated [28]. The NADH-binding ing are indicated. Subunit PSST is also be-
site, flavin cofactor (FMN), and all of the lieved to ligate iron-sulfur center N2.
known iron-sulfur centers (Fe) are carried
as being at or close to the site of initial direct electron transfer between NADH and
FMN.
The application of electron paramagnetic resonance (EPR) spectroscopy has
shown that electron transfer from FMN to UQ involves the reduction of seven or
more iron-sulfur clusters, of which that with the highest redox potential, center
N2, is responsible for UQ reduction [34]. EPR spectroscopy has also revealed the
presence of two ubisemiquinone species, suggesting the presence of multiple
quinine-binding sites. There may also be other redox centers involved in electron
transport through the complex [23, 35–37]. With the exception of fenaminosulf,
none of the inhibitors listed in Table 15.1.1, or elsewhere in this volume, has been
distinguished in their site of action based on effects on the reduction or reoxidation
of detectable redox centers. Rather, all those that have been studied in detail seem
to act to prevent electron transfer somewhere between center N2 and UQ [13].
Several studies have been conducted in attempts to identify inhibitor-binding
sites through affinity or photoaffinity labeling, but these have not produced entirely
consistent results. Photoaffinity labels based on the structures of the unrelated
acaricides fenpyroximate and pyridaben (Table 15.1.1) predominantly labeled dif-
ferent proteins, the ND5 and PSST subunits, respectively, although each prevented
labeling by the other [39, 43]. Other studies using rotenone and acetogenin analogs
predominantly labeled another subunit, ND1 [44, 45], which was also labeled to
a minor degree by the pyridaben analog. The 49 kDa subunit is predominantly
labeled by an analog of quinazoline-type inhibitors [46], and also by a piperazine
analog of acetogenin [47]. The pattern of labeling is sensitive to the conforma-
tional state of the complex, since it is altered in the presence of NADH and other
ligands [48, 49], and some differences may be attributable to this. Another possi-
bility is that all of these subunits are involved in the construction of one or more
UQ/inhibitor-binding sites, and that the yield and site of crosslinking is determined
by the structure, half-life, and chemical reactivity of the reactive species generated
by photolysis. Yet another polypeptide (known as subunit B9 in the bovine complex)
is photolabeled by a UQ analog [49, 50]. Interestingly, both ND1 and B9 react with
dicyclohexylcarbodiimide (DCCD), the effect of which on the enzymatic activities
of Complex I is similar to those of rotenone and piericidin [51]. The 49 kDa subunit
has also been implicated in inhibitor binding because mutant forms of this protein
are resistant to a range of inhibitor classes [52–54]. Thus, polypeptides belonging
to all three subdomains of Complex I have been implicated in inhibitor binding
(Figure 15.1.2). The unambiguous assignment of inhibitor- and UQ-binding sites
will most likely require high-resolution structure determination.
Fungicidal, acaricidal, and insecticidal Complex I inhibitors are discussed in
detail in Chapters 15.5 and 31.3 of this volume.
15.1.2.2 Complex III (Cytochrome bc1 Complex) and Its Inhibitors

Complex III has been purified from many sources, and the structure resolved using
X-ray crystallography for several mitochondrial forms (for a review, see Ref. [55]).
The bovine mitochondrial form contains 11 subunits [56], one of which represents a
processing fragment of another, whilst fungal forms from Saccharomyces cerevisiae
Table 15.1.1 Selected potent inhibitors of Complex I.

Further fungicidal and acaricidal inhibitors are described in
Chapters 13.5 and 28.3, respectively.
Name Structure Use Reference
OH O
N
H
Ajudazol B O – [4]
OH O O N
O
OH
H O HO
Annonin VI O H H – [38]
O O
OH
Aurachin A OH – [13]
N+ O
O
O
O+
O O
Aureothin N − – [13]
O
O
O
Fenaminosulf N N N S OH Fungicide [32]
O
N
N N
O
Fenpyroximate O O Acaricide [39]
Myxalamide OH – [13]
PI (related to O O
N
phenalamides)
O
O N
Piericidin A OH – [40]
O
OH
O Cl
Pyridaben Insecticide, [41]
N S acaricide
N
O O
H O
Rotenone O Insecticide [40]
O
O H
H OH
Mycothiazole N N – [42]
O
O S
O S
Thiangazole N N N – [13]
N N
O S S
[57, 58] and N. crassa [59] contain nine or ten subunits. The bacterial forms are
functionally equivalent in terms of electron transfer and proton translocation, but
are composed of only three or four subunits [60]. All forms of Complex III contain
the same three highly conserved subunits, cytochrome b, the Rieske iron-sulfur
protein (ISP), and cytochrome c1 , which together carry all of the redox prosthetic
groups. The additional subunits in mitochondrial forms are largely conserved
between fungal and mammalian enzymes, but the function of most remains
obscure.
The complex catalyzes electron transfer from reduced UQ to cytochrome c,
coupled to the translocation of protons by a mechanism known as the Q cycle
[61–63]. This involves the diversion of half of the electrons available from ubiquinol
oxidation and deprotonation at a site on the outside of the inner mitochondrial
membrane (Qo site), in order to reduce and protonate UQ at a site on the inside of
the membrane (Qi site). The pathway for electron transfer across the membrane is
provided by the two heme centers (bL and bH ) of the mitochondrial gene product
cytochrome b. The remainder of the electrons from ubiquinol oxidation pass along
the chain to reduce first the Rieske ISP, then cytochrome c1 , and then cytochromec
(Figure 15.1.3).
Known potent and selective inhibitors of Complex III act at one of these
two UQ-binding sites (detailed in Table 15.1.2). Those acting at the Qi site are
distinguished by their ability to induce an oxidant-dependent ‘‘super reduction’’
of cytochrome b in purified Complex III or mitochondrial membranes [64, 65].
The inhibitor sites of action, and in some cases also detailed modes of binding,
have been confirmed by numerous crystal structures with inhibitors bound, and
such information is now assisting inhibitor design [66–68]. Although all inhibitors
acting at the Qo site bind in a mutually exclusive manner, they can be classified
according to the position that they occupy within the site. Some (e.g., stigmatellin)
cyt b, ISP, cyt c1
H+
Matrix
Qi
½ UQ UQ
bH
½ UQH2
e−
UQH2
UQ bL
UQ
Fe e−
Qo
2 H+ cyt c
Figure 15.1.3 Schematic representation of bH and bL . The bifurcated electron-transfer

the structure and function of Complex III. pathway from the Qo site is shown by blue
The three catalytic subunits, cytochrome arrows. One electron is transferred to the
b (cyt b), the Rieske iron-sulfur protein iron-sulfur center (Fe), then to the heme of
(ISP), and cytochrome c1 (cyt c1 ) are indi- cytochrome c1 , and then on to cytochrome c
cated. Two binding sites for inhibitors and (cyt c). The other electron passes through bL
ubiquinone (UQ), Qi and Qo are shown and bH to reduce ubiquinone at the Qi site.
within cytochrome b, close to the two hemes
bind closely to the ISP and cause a shift in its redox midpoint potential, whereas
others (e.g., myxothiazol, strobilurin, famoxadone) bind close to heme bL and
influence its absorption spectrum [69, 70].
Inhibitors can also be classified by the pattern of sensitivity of variant forms of
cytochrome b. Many amino acid substitutions arising through genetic mutations in
the cytochrome b gene have been discovered that give rise to inhibitor-insensitive
forms that remain functional. Most of these mutations map to the Qi and Qo
binding pockets, and distinguish between the Qi and Qo inhibitor classes. Inhibitors
acting at either site can be further subdivided by their crossresistance pattern;
thus, there are mutants that impart resistance to 2-n-heptyl-4-hydroxyquinoline
N-oxide (HQNO) but not to antimycin, and vice versa [67]. Similarly, at the Qo
site, there are several mutations that provide resistance to stigmatellin but not
to strobilurins or myxothiazol [60, 78]. The effects of many of these mutations
can be rationalized in terms of the binding interactions seen in crystal structures
(for a review, see Ref. [69]). Some of the variant forms of cytochrome b have
been shown to be responsible for pathogen resistance to drugs or agrochemical
fungicides, and the protein sequence differences appear to have little impact on
the fitness of the organism [79–81]. Crossresistance has been observed between
all seven classes of commercial fungicides acting at the Qo site [82] (see also
Chapter 14).
Particular classes of Complex III inhibitors are described in greater detail in
Chapters 15.2 and 31.3 of this volume.
Table 15.1.2 Selected potent inhibitors of complex III.

Fungicidal and acaricidal inhibitors are described in Chapters
15.2 and 31.3, respectively.
Name Structure Binding Use Reference

site
O O
Antimycin A1 O O Qi Piscicide [71]
O OH
O N O
N
O
OH
HO OH
OH
Funiculosin O Qi – [72]
HO
N
O
OH
O OH
H
Ilicicolin H Qi – [64]
O N
H
O
Atovaquone Cl Qo Anti- [73]
protozoal
O OH
Crocacin O N Qo – [74]
N O
O O O O
O
O
Haliangicin Qo – [75]
O
O O
O NH2
Myxothiazol S Qo – [76]
N
N O O
S
OH
O O
Stigmatellin O O Qo – [76]
O O
H OH
O
O
Ascochlorin Qo and – [77]
OH Qi
Cl
15.1.2.3 Complex IV
Complex IV, or cytochrome c oxidase, was the first of the mitochondrial
electron-transport complexes for which the molecular structure and internal path
of electron transfer were revealed, using X-ray crystallography. The catalytic core
of the complex consists of two subunits. Subunit II contains a binuclear copper
center (CuA ) that is directly responsible for the oxidation of cytochrome c. From
there, electrons are passed to heme a and then to the adjacent binuclear center
that consists of heme a3 and another copper ion (CuB ), all of which are held within
subunit I (Figure 15.1.4). Oxygen is bound and reduced between CuB and the iron
of heme a3 , and access paths for protons from the inside of the membrane and
for oxygen from within the membrane have been defined from several crystal
structures available for bovine and bacterial enzymes. In addition to the protons
taken up for the reduction of oxygen, the translocation of further protons across
the membrane is coupled to electron transfer by a mechanism that is not yet
understood (for reviews, see Refs [83, 84]).
Bacterial cytochrome c oxidases have three or four subunits, whereas the bovine
mitochondrial enzyme has 13 subunits, including closely related homologs of
bacterial subunits I–III (which includes those bearing the redox centers) that
are encoded by the mitochondrial genome. There is some uncertainty about the
number of subunits in fungal mitochondria; the yeast S. cerevisiae has nine or 11
subunits, depending on the method of isolation [85]. Most of the nuclear-encoded
yeast genes have homologs in mammalian mitochondrial subunits, with varying
degrees of conservation. The function of the additional subunits is uncertain, except
in so far as they have been shown to have a role in Complex IV assembly, or to
contain binding sites for regulatory ligands. There also exist isoforms of a number
H2O
Matrix
II I
O2
a3 Cu B
e−
Cu A a
cyt c
Figure 15.1.4 Schematic representation of oxygen at the center formed by heme a3

the structure and function of Complex IV. (a3) and copper (CuB ). Protons are taken up
Electrons enter from cytochrome c (cyt c) from the matrix side for the reduction of wa-
and are passed through the binuclear cop- ter and for transport across the membrane.
per center (CuA ) and heme a (a) to reduce
of these subunits, which are selectively expressed in a tissue-specific manner in

mammals [83] or under different growth conditions in fungi [85].
It seems that cytochrome c oxidase is highly resistant to inhibition by secondary
metabolites or synthetic organic chemistry, as no potent inhibitor from these
sources has been reported. This might be due to a lack of any requirement for
conformational flexibility [83], or to the inaccessibility of the redox centers or
substrate transport channels to anything but very small molecules [86]. However,
opportunities for inhibition may arise through exploiting natural control mecha-
nisms, such as the allosteric regulation by ATP [87] or phosphorylation of a highly
conserved tyrosine in subunit I [88].
15.1.2.4 Succinate Dehydrogenase (Complex II) and Its Inhibitors

Complex II is the succinate dehydrogenase of the TCA (or Krebs) cycle, and catalyzes
the oxidation of succinate to fumarate, coupled to the reduction of UQ to ubiquinol.
It forms part of a large family of related succinate quinone oxidoreductases and
quinol fumarate oxidoreductases found in bacteria and mitochondria that have
been classified based on their subunit structure, the number of cytochrome b
heme centers, and the class of quinone substrate [89, 90]. The mitochondrial
form belongs to the class that has a single b-type heme bound to one of two
membrane-spanning subunits that together support the catalytic iron protein
and flavoprotein subunits on the matrix side of the membrane. The flavoprotein
(subunit A) contains covalently bound FAD, which is the primary oxidant of
succinate. The iron protein (subunit B) contains three iron-sulfur clusters that
provide a path for the conduction of electrons to the junction with the membrane
domain (subunits C and D) (Figure 15.1.5). The mitochondrial enzyme belongs to
the same class as the succinate dehydrogenase from E. coli, the crystal structure of
which has been resolved to a resolution of 2.6 Å, and which has been considered a
model for the mitochondrial enzyme [91]. Recently, the 2.4 and 2.1 Å structures of
the porcine and chicken mitochondrial complexes have also been reported [92, 93].
Together, these structures revealed a highly conserved UQ-binding site (QP ) at the
interface between the membrane-spanning subunits and the iron protein subunit,
from which the quinone can receive electrons from the iron-sulfur center furthest
from the FAD. The b heme does not seem to be required for electron transfer to the
quinone at the QP site, since it is further from the bound UQ than the iron-sulfur
center and at the opposite side, towards the external face of the membrane. The
role of the b heme in electron transfer within the complex is uncertain [91, 92], and
it seems that much of the heme can be lost from the yeast enzyme with a less than
anticipated impact on function [94]. UQ binding at the QP site has been observed
directly in both the E. coli and porcine structures, and is supported by the results
of photoaffinity labeling studies on the bovine enzyme [95]. It seems that UQ can
bind in at least two overlapping positions within this site [11].
The porcine structure revealed another binding pocket in the membrane domain,
towards the external surface, which could form a second UQ-binding site (QD ),
located in a position analogous to a quinine-binding site in E. coli quinol fumarate
Fumarate + 2H+
FAD
A
Succinate e−
Fe
B
Fe
Matrix
Fe
UQ + 2H+
UQ
QP
UQH2
D C
QD
Figure 15.1.5 Schematic representation of provide an electron-transfer pathway (blue

the structure and function of succinate dehy- arrows) to the ubiquinone (UQ) and the
drogenase (Complex II). The diagram shows inhibitor-binding site (QP ) indicated at the
the topographical arrangement of the four interface of subunits B–D. The location of
subunits A–D. Subunit A binds the cofactor a possible second UQ binding site (QD ) is
flavin adenine dinucleotide (FAD). Subunit also indicated.
B carries three iron-sulfur clusters (Fe) that
oxidoreductase [96]. The existence of a second binding site for UQ on mitochon-

drial Complex II has also been suggested by inhibitor-binding and site-directed
mutagenesis studies [97, 98], although to date no direct evidence has been acquired
for a functional UQ interaction at this site.
Complex II is strongly inhibited by 3-nitropropionic acid, a toxic metabolite
produced by plants and fungi [99], and which is reported to act as a ‘‘suicide’’
substrate at the succinate-binding site [100]. As expected, this inhibitor was seen
to form a covalent bond with the protein close to the FAD in the chicken crystal
structure [93]. Other inhibitors are known (or are presumed) to compete with
UQ for binding at the QP site (Table 15.1.3). Atpenin A5, and the less potent
2-thenoyltrifluoroacetone, have each been observed directly at this site in various
crystal structures [11, 92], while mutations at this site impart resistance to carboxin
in Paracoccus denitrificans [101], and also in the fungi Pleurotus ostreatus [102],
Mycosphaerella graminicola, Ustilago maydis [103], and Coprinus cinereus [104]. The
latter was also shown to be resistant to the basidiomycete-selective fungicide
flutolanil. Mutations imparting carboxin resistance are found in all three subunits
that form the QP site.
Fungicidal inhibitors of Complex II are discussed in Chapter 15.3 of this volume.
Recently, the novel acaricide cyenopyrafen, which is structurally related to an earlier
insecticide [111], has been reported to act via a potent inhibition of Complex II after
hydrolysis of the t-butyl ester (Table 15.1.3) [112].
Table 15.1.3 Selected inhibitors of Complex II. Further

fungicidal inhibitors are described in Chapter 15.3.
OH O Cl
Atpenin A5 O Cl – [105]
O OH
O
Boscalid N Fungicide See Chapter 15.3

N Cl
Cl
O
Carboxin S Fungicide [106]
N
O
O
Flutolanil N O Fungicide [107]
F
F
F
O
Surangin B O O OH – [108]
OH
O
O
Active N Acaricide [109]
hydrolysis
product of N
N
cyenopyrafen
OH
OH O
H H
Sicanin Antibiotic [110]
H
O
15.1.2.5 Alternative Electron-Transport Chains

Both, fungal and plant mitochondria commonly contain alternative NADH–UQ
oxidoreductases that bypass Complex I, and a ubiquinol oxidase (usually termed
the ‘‘alternative oxidase’’) that bypasses Complexes III and IV. They are considered
here because their expression may be relevant to the sensitivity of fungal pathogens
to Complex I and Complex III inhibitors. Neither class is known to be targets
for chemical control in themselves. Although several other UQ reductases are

not described in this chapter, they provide important links from metabolism to
the electron-transport chain. These include the electron-transfer flavoprotein UQ
oxidoreductase (Figure 15.1.1), which is essential for the function of acyl-CoA
dehydrogenases [113], α-glycerophosphate dehydrogenase, which has an important
role in insect flight [114], and proline dehydrogenase, which is of particular
importance in some insect tissues [115]. These too are currently not known to be
targets of antibiotics or crop-protection chemicals.
The ‘‘alternative oxidase’’ is a nuclear gene product that is targeted to the
inner mitochondrial membrane, and which catalyzes the oxidation of UQ by
molecular oxygen, mediated by a di-iron center [116, 117]. As it does not transport
protons across the membrane, use of the alternative electron-transport chain that
it provides has a large cost in terms of energy conservation, and its expression
is therefore tightly regulated. Indeed, in many fungi its activity is low unless it
is upregulated in response to defects in the respiratory chain [118–121]. Even
when both pathways are present, there is evidence that electrons flow preferentially
through the energy-conserving Complex III and Complex IV [122], perhaps because
of electron channeling through ‘‘supercomplexes.’’
Alternative NADH–UQ oxidoreductases are also nuclear-encoded single polypep-
tide enzymes targeted to the inner mitochondrial membrane. They catalyze the
same reaction as Complex I but, again, are not energy-conserving. There are many
related forms, varying in orientation of the NADH-binding site (towards the matrix
or external face), substrate specificity and sensitivity to regulatory ligands, and
some fungi express more than one form (for a review, see Ref. [123]). They are
dependent on FAD as the only prosthetic group, and the results of enzyme kinetic
studies have suggested that the binding sites for NADH and UQ overlap [124].
Although these alternative electron-transport pathways are now well characterized
at the molecular level, their physiological role in fungi remains unclear; however,
they may protect against oxidative stress or mitigate the effects of inhibition of the
respiratory chain [125, 126]. Their presence may also affect the sensitivity of plant
pathogens to strobilurin fungicides with respect to biological spectrum and efficacy
at different growth stages [127].
15.1.3
Energy Conservation
The conservation of energy from electron transport requires not only the synthesis
of ATP within the mitochondrion, but also its export to the cytoplasm as well as
the import of substrates for oxidation and phosphorylation. Only those proteins
responsible for the synthesis of ATP and exchange of ATP for ADP across
the inner mitochondrial membrane will be considered in any detail here, as
they are known targets for antibiotics and pesticides. However, numerous other
mitochondrial transporters identified in plants, fungi, and animals may provide
future opportunities for useful chemical intervention [128, 129].
Energy conservation is also dependent on chemiosmotic coupling, a process

that is disrupted by a class of chemicals termed ‘‘uncouplers,’’ some of which
have found commercial application as herbicides, fungicides, and insecticides.
Uncouplers collapse the electrochemical gradient across the inner mitochondrial
membrane, allowing oxidation to proceed without the normal control and pre-
venting the action of the ATP synthase. The most effective are protonophores
(compounds that increase the proton permeability of the membrane), the contin-
uous ‘‘catalytic’’ action of which allows them to be highly efficient disrupters of
oxidative phosphorylation. Uncouplers are invariably cytotoxic to eukaryotic and
prokaryotic cells, unless the cell has some means to minimize its exposure, for
example, through metabolic inactivation [130] or efflux pumps [131, 132]. The
action and application of uncouplers are described in greater detail in Chapters
15.4 and 31.2, while comprehensive reviews are also available [133, 134].
15.1.3.1 F1 F0 ATP Synthase and Its Inhibitors

Historically, the ATP synthase complex was purified from bovine heart mito-
chondria at about the same time as the respiratory chain complexes. It is most
commonly referred to as the F1 F0 ATPase or ATP synthase, but is also known as
Complex V. It is composed of 15 different subunits (excluding the inhibitor protein
IF1 ) organized into two major subcomplexes, termed F1 and F0 . F1 , a globular
domain that extends on two ‘‘stalks’’ into the mitochondrial matrix, consists of five
different subunits in the stoichiometry α3 , β3 , γ1 , δ1 , and ε1 ; its structure has been
resolved using X-ray crystallography [135, 136]. The γ , δ, and ε subunits make up
the central stalk, and the six α and β subunits are arranged symmetrically around
extending α-helices of the γ subunit to form the globular head (Figure 15.1.6).
The isolated F1 domain, which is soluble and acts as an ATPase, contains three
catalytic sites, one on each of the β subunits, and behaves as a molecular motor;
ATPase activity causes the central stalk to rotate within the oligomer of the α and
β subunits [135, 137]. A glutamic acid residue on the β subunit is a site of reaction
of the inhibitor DCCD [136], and other inhibitor binding sites, including those for
aurovertin B (Table 15.1.4), the peptide antibiotic efrapeptin, and the endogenous
inhibitor protein IF1 , have been localized to this globular domain by using X-ray
ADP + Pi
b a
a
b a b Figure 15.1.6 Schematic representation
ATP of the structure and function of the mito-
chondrial F1 F0 ATP synthase (Complex V).
Rotation of the c subunits is believed to be
d, F6, OSCP driven by proton conduction through the
g, d, e membrane domain, which in turn drives ro-
Matrix
tation of the central stalk (subunits γ , δ,
b
ε) in the direction shown. This drives the
c condensation of ADP and Pi sequentially at
each of the catalytic β subunits. The loca-
tions of other subunits are indicated; their
a, A6L, e, f, g
function is discussed in the text.
crystallography [138]. The fungal natural product tentoxin (Table 15.1.4) is a specific
inhibitor of chloroplastic F1 , and binds at the interface of the α and β subunits
[139].
The membrane or F0 subcomplex is composed of seven subunit types, organized
as two domains. One domain consists of 10 copies of the membrane-spanning,
hairpin-shaped c subunit that are arranged as a barrel with the longitudinal axis
perpendicular to the membrane. The c subunits form extensive contacts with the γ
and δ subunits of the central stalk, an arrangement that has been confirmed by the
crystal structure of the yeast F1 –c10 complex [144]. The other membrane domain
probably consists of the a, b, A6L, e, f, and g proteins [145]. The F0 domain is
also believed to be a molecular motor. In the proposed mechanistic model for ATP
synthesis, proton transport through the F0 domain (driven by the chemiosmotic
gradient) causes rotation of the c subunit oligomer that, in turn, rotates the central
stalk within F1 , in a direction that promotes ATP synthesis (for a review, see Ref.
[146]). Rotation of the α and β subunits is prevented by the peripheral stalk or
‘‘stator’’ that connects the membrane domain to the top of F1 . The b subunit
also forms part of the peripheral stalk together with d, F6, and the oligomycin
sensitivity conferring protein (OSCP) [147]. Additional subunits can be found in
dimeric forms of the enzyme, and may function to promote the formation of
oligomers. The results of recent studies have suggested that an oligomerization of
the ATP synthase drives the membrane curvature that leads to the formation of the
mitochondrial cristae [148, 149].
The F0 domain is also a site of action for inhibitors, the best known of which
is oligomycin (Table 15.1.4), which acts to inhibit proton conduction [150]. The
oligomycin-binding site has been localized to the a and c subunits of the F0
domain through the characterization of resistant mutants in yeast [151, 152]. Re-
lated spiroketal macrolides, ossamycin, and venturicidin A are located to the same
binding site through their crossresistance profiles, and it is assumed that the grow-
ing family of structurally related macrolides – including apoptolidin, dunaimycins,
citovaricin, and rutamycin – all act at the same site [153–155]. In addition to its in-
hibitory effect on ATP hydrolysis in F1 , DCCD also inhibits the proton-conduction
channel in F0 through reaction with the c subunit [51]. The carbodiimide metabolite
of the acaricide and insecticide diafenthiuron (described in detail in Chapter 31.1)
has been shown to act in the same way [156].
Medicinal chemistry has also involved the successful generation of potent
inhibitors of the F1 F0 ATP synthase. Compounds 1 and 2 (Table 15.1.4) were
designed as selective inhibitors of ATP hydrolysis, and intended for the treatment
of ischemic tissue injury [141, 142, 157], whereas the antimycobacterial antibiotic
TMC 207 binds in the F0 domain [143].
15.1.3.2 Inhibitors of the Mitochondrial ADP/ATP Carrier

The exchange of intramitochondrial ATP for extramitochondrial ADP plays a
central role in mitochondrial function. The process is controlled by a specific
carrier protein that is a member of a large family of mitochondrial transporters
related by sequence and structure, all of which are encoded by nuclear genes.
Table 15.1.4 Inhibitors of the F1 F0 ATP synthase.
Name Structure Binding site Reference
O
O
O HO
Aurovertin B O F1 [140]
O O
O
O OH
O
HO
OH
OH
Oligomycin A F0 [140]
O
O
O
O
OH
N
Tentoxin N Chloroplast F1 [139]
O
O
N N
N N S O
Compound 1 Cl – [141]
N
N Cl
N
Cl
N
Compound 2 – [142]
Cl N N
N
N O
Cl
N
N O
OH
TMC 207 Br F0 [143]
Table 15.1.5 Inhibitors of ATP export from the mitochondrion.
Name Structure Reference
O OH
O O
Bongkrekic acid OH [161]
OH
O
H
Atractyloside O OH O O [161]
S
O O O
OH
H H
HO O O
S H
O O
OH O O
S Si
Silthiofam [162]
O N
The ADP/ATP carrier is a single polypeptide chain, the three-dimensional struc-

ture of which has recently been revealed using X-ray crystallography [158, 159]
(for a review, see Ref. [160]). The discovery and characterization of the carrier
owes much to the availability of the toxic plant secondary metabolite, atracty-
loside, and the bacterially produced bongkrekic acid, which have proved to be
potent and highly selective inhibitors [161] (Table 15.1.5). Atractyloside and its
analog carboxyatractyloside are highly negatively charged at physiological pH,
and cannot penetrate the inner mitochondrial membrane. Rather, they bind
to the external face of the transporter in such a way that excludes nucleotide
binding. In the crystal structure, carboxyatractyloside is bound at the bottom
of an externally facing cone-shaped depression [158]. Bongkrekic acid has not
yet been cocrystallized with the transporter, but it is thought to bind to the
internal face because binding requires it to penetrate the membrane. Its bind-
ing seems to lock the conformation of the transporter into the ADP bound
state [161].
The novel fungicide silthiofam (MON 65500, Latitude™) [162], which is now
marketed as a seed treatment for the control of ‘‘take-all’’ fungus (Gaeumannomyces
graminis var. tritici), has been shown to affect ATP export from mitochondrial
preparations in a manner that was mimicked by high concentrations of atractyloside
[163]; hence, it was proposed that silthiofam would act to inhibit the ATP/ADP
carrier. More direct studies on the effect of this compound on carrier function have
not been reported; neither have studies on its biochemical effects on mitochondria
from other organisms. As a fungicide, silthiofam is highly specific for G. graminis,

and it is not acutely toxic to mammals despite its high bioavailability [164]; this is
in contrast to the high mammalian toxicity of atractylosides and bongkrekic acid
[165, 166]. It seems that the toxicological species selectivity of this compound may
reflect the sensitivity of the biochemical target.
15.1.4
Concluding Remarks
Several recent developments have reinvigorated research into the mechanism of

oxidative phosphorylation and the interaction of the mitochondrion with its host
cell. Perhaps of most immediate interest from the perspective of crop-protection
chemistry is the increasingly successful application of X-ray crystallography in
revealing the structures of many of the components of oxidative phosphorylation.
This has dramatically improved the present understanding not only of the catalytic
mechanisms, but also of the action of inhibitors. Today, many common themes are
emerging, prominent among which are insights into the structure of UQ-binding
sites. Nature seems to have designed these to be relatively ‘‘open’’ binding pockets
that have provided disproportionate success in the discovery and design of novel
inhibitor classes.
Other developments include a growing understanding of the role of the mito-
chondrion in the pathogenesis of disease, aging, and the determination of cell fate.
It is now clear that, both inhibitors and uncouplers of oxidative phosphorylation,
have effects other than depriving the cell of ATP, and that these can play an
important role in the inhibitor/uncoupler action. Many inhibitors of respiration
increase the rate of production of reactive oxygen species in the cell, and may also
activate endogenous cell death or apoptotic pathways [167–169]. Consequently, the
defenses that the cell has developed to protect against reactive oxygen generation
in the mitochondrion may themselves become targets. Indeed, this may be one
of the roles of the alternative oxidase and NADH dehydrogenases found in many
fungal pathogens [126], and is a likely explanation for certain aspects of the con-
trol of cytochrome c oxidase activity, the proton-pumping activity of which is not
always fully coupled to electron transport [170]. These systems may act to limit the
mitochondrial membrane potential, thus limiting the degree of reduction of those
redox centers that serve as sites of superoxide production. Another mechanism for
the control of membrane potential is through ‘‘uncoupling proteins’’ which were
first recognized for their role in mammalian thermogenesis and are now known to
be expressed in fungal, plant, and animal mitochondria, where they provide proton
channels, the activity of which can be regulated by guanine nucleotides and free
fatty acids [171–173].
Chemical uncouplers may also influence cell fate other than through the depletion
of ATP [174, 175], and their action to depolarize membranes outside of the
mitochondrion should also be considered with respect to their overall effects on
the target cell or organism. For example, they have been shown to dissipate the
plasma membrane potential and to destabilize lyzosomes [176, 177].
References 579
Recently, a new role for the mitochondrion has emerged that, in turn, suggests
new targets for both medicinal and crop-protection chemistry. At the same time,
structural biology has greatly improved the present understanding of existing
targets and their inhibitors, allowing the synthetic chemist to design around
issues of species selectivity and resistance by using an abundant tool set provided
by natural products. It seems that the exploitation of mitochondrial targets will
continue to expand, providing safer and more effective crop-protection agents for
the future.
References
1. Ernster, L. and Schatz, G. (1981) J. Cell Weiss, H. (1994) Eur. J. Biochem.,

Biol., 91, 227s. 219, 691.
2. Saraste, M. (1999) Science, 283, 1488. 14. Mitani, S., Araki, S., Takii, Y.,
3. Thierbach, G. and Reichenbach, H. Ohshima, T., Matsuo, N., and Miyoshi,
(1981) Biochim. Biophys. Acta, 638, H. (2001) Pesticide Biochem. Physiol., 71,
282. 107.
4. Kunze, B., Jansen, R., Hofle, G., and 15. Degli Esposti, M., Crimi, M., and
Reichenbach, H. (2004) J. Antibiot., 57, Ghelli, A. (1994) Biochem. Soc. Trans.,
151. 22, 209.
5. Olorunsogo, O.O. and Malomo, S.O. 16. Hatefi, Y. (1985) Annu. Rev. Biochem.,
(1985) Toxicology, 35, 231. 54, 1015.
6. Weissman, K.J. and Muller, R. (2010) 17. Wittig, I. and Schägger, H. (2009)
Nat. Prod. Rep., 27, 1276. Biochim. Biophys. Acta, 1787, 672.
7. Beautement, K., Clough, J.M., de 18. Baginsky, M.L. and Hatefi, Y. (1969)
Fraine, P.J., and Godfrey, C.R.A. (1991) J. Biol. Chem., 244, 5313.
19. Bruel, C., Brasseur, R., and
Pesticide Sci., 31, 499.
Trumpower, B.L. (1996) J. Bioenerg.
8. United States Environmental Protection
Biomembr., 28, 59.
Agency (2006) Reregistration Eligi-
20. Berry, S. (2003) Biochim. Biophys. Acta,
bility Decision for Rotenone, March
1606, 57.
2007, http://www.epa.gov/oppsrrd1/
21. Galkin, A.S., Grivennikova, V.G., and
reregistration/REDs/rotenone_
Vinogradov, A.D. (1999) FEBS Lett.,
red.pdf (accessed May 20, 2011).
451, 157.
9. Fisher, N. and Rich, P.R. (2000) J. Mol.
22. Carroll, J., Fearnley, I.M.,
Biol., 296, 1153. Shannon, R.J., Hirst, J., and
10. Iwata, M., Abramson, J., Byrne, B., and Walker, J.E. (2003) Mol. Cell. Pro-
Iwata, S. (2003) Adv. Protein Chem., 63, teomics, 2, 117.
151. 23. Friedrich, T., Brors, B., Hellwig,
11. Horsefield, R., Yankovskaya, V., P., Kintscher, L., Rasmussen, T.,
Sexton, G., Whittingham, W., Scheide, D., Schulte, U., Mantele, W.,
Shiomi, K., Omura, S., Byrne, B., and Weiss, H. (2000) Biochim. Biophys.
Cecchini, G., and Iwata, S. (2006) J. Acta, 1459, 305.
Biol. Chem., 281, 7309. 24. Friedrich, T. and Scheide, D. (2000)
12. Ueki, M., Machida, K., and FEBS Lett., 479, 1.
Taniguchi, M. (2000) Cur. Opin. 25. Hirst, J., Carroll, J., Fearnley, I.M.,
Anti-Infect. Invest. Drugs, 2, 387. Shannon, R.J., and Walker, J.E. (2003)
13. Friedrich, T., Heek, P., Leif, H., Biochim. Biophys. Acta, 1604, 135.
Ohnishi, T., Forche, E., Kunze, B., 26. Abdrakhmanova, A., Zickermann, V.,
Jansen, R., Trowitzsch-Kienast, W., Bostina, M., Radermacher, M.,
Holfe, G., Reichenbach, H., and Schagger, H., Kerscher, S., and
Brandt, U. (2004) Biochim. Biophys. and Casida, J.E. (1999) Proc. Natl Acad.
Acta, 1658, 148. Sci. USA, 96, 4149.
27. Marques, I., Duarte, M., Assuncao, J., 44. Earley, F.G.P., Patel, S.D., Ragan, C.I.,
Ushakova, A.V., and Videira, A. (2005) and Attardi, G. (1987) FEBS Lett., 219,
Biochim. Biophys. Acta, 1707, 211. 108.
28. Guenebaut, V., Schlitt, A., Weiss, H., 45. Sekiguchi, K., Murai, M., and
Leonard, K., and Friedrich, T. (1998) Miyoshi, H. (2009) Biochim. Biophys.
J. Mol. Biol., 276, 105. Acta, 1787, 1106.
29. Djafarzadeh, R., Kerscher, S., 46. Murai, M., Sekiguchi, K., Nishioka, T.,
Zwicker, K., Radermacher, M., and Miyoshi, H. (2009) Biochemistry,
Lindahl, M., Schagger, H., and 48, 688.
Brandt, U. (2000) Biochim. Biophys. 47. Ichimaru, N., Murai, M., Kakutani, N.,
Acta, 1459, 230. Kako, J., Ishihara, A., Nakagawa, Y.,
30. Efremov, R.G., Baradaran, R., and Nishioka, T., Yagi, T., and Miyoshi, H.
Sazanov, L.A. (2010) Nature, 465, 441. (2008) Biochemistry, 47, 10816.
31. Hunte, C., Zickermann, V., and 48. Schuler, F. and Casida, J.E. (2001)
Brandt, U. (2010) Science, 329, 448. Biochim. Biophys. Acta, 1506, 79.
32. Mueller, W. and Schewe, T. (1977) Acta 49. Heinrich, H. and Werner, S. (1992)
Biol. Med. Ger., 36, 967. Biochemistry, 31, 11413.
33. Schewe, T., Hiebsch, C., and Halangk, 50. Heinrich, H., Azevedo, J.E., and
W. (1975) Acta Biol. Med. Ger., 34, Werner, S. (1992) Biochemistry, 31,
1767. 11420.
34. Ohnishi, T. and Salerno, J.C. (2005) 51. Hassinen, I.E. and Vuokila, P.T. (1993)
FEBS Lett., 579, 4555. Biochim. Biophys. Acta, 1144, 107.
35. Albracht, S.P.J., van der Linden, E., and 52. Kashani-Poor, N., Kerscher, S.,
Faber, B.W. (2003) Biochim. Biophys. Zickermann, V., and Brandt, U. (2001)
Acta, 1557, 41. Biochim. Biophys. Acta, 1504, 363.
36. Schulte, U., Abelmann, A., Amling, N., 53. Prieur, I., Lunardi, J., and Dupuis, A.
Brors, B., Friedrich, T., Kintscher, L., (2001) Biochim. Biophys. Acta, 1504,
Rasmussen, T., and Weiss, H. (1998) 173.
BioFactors, 8, 177. 54. Fendel, U., Tocilescu, M.A.,
37. Schulte, U., Haupt, V., Abelmann, A., Kerscher, S., and Brandt, U. (2010)
Fecke, W., Brors, B., Rasmussen, T., Biochim. Biophys. Acta, 1777, 660.
Friedrich, T., and Weiss, H. (1999) 55. Berry, E.A., Guergova-Kuras, M.,
J. Mol. Biol., 292, 569. Huang, L.S., and Crofts, A.R. (2000)
38. Londershausen, M., Leicht, W., Annu. Rev. Biochem., 69, 1005.
Lieb, F., Moeschler, H., and Weiss, H. 56. Iwata, S., Lee, J.W., Okada, K.,
(1991) Pesticide Sci., 33, 427. Lee, J.K., Iwata, M., Rasmussen,
39. Nakamaru-Ogiso, E., Sakamoto, K., B., Link, T.A., Ramaswamy, S., and
Matsuno-Yagi, A., Miyoshi, H., and Jap, B.K. (1998) Science, 281, 64.
Yagi, T. (2003) Biochemistry, 42, 746. 57. Hunte, C. (2001) FEBS Lett., 504, 126.
40. Storey, B.T. (1980) Pharmacol. Ther., 58. Brandt, U., Uribe, S., Schagger, H.,
10, 399. and Trumpower, B.L. (1994) J. Biol.
41. Lummen, P. (1998) Biochim. Biophys. Chem., 269, 12947.
Acta, 1364, 287. 59. Weiss, H. and Kolb, H.J. (1979) Eur. J.
42. Morgan, J.B., Mahdi, F., Liu, Biochem., 99, 139.
Y., Coothankandaswamy, V., 60. Trumpower, B.L. (1990) Microbiol. Rev.,
Jekabsons, M.B., Johnson, T.A., 54, 101.
Sashidhara, K.V., Crews, P., 61. Mulkidjanian, A.Y. (2005) Biochim. Bio-
Nagle, D.G., and Zhou, Y.-D. (2010) phys. Acta, 1709, 5.
Bioorg. Med. Chem., 18, 5988. 62. Osyczka, A., Moser, C.C., and
43. Schuler, F., Yano, T., Di Bernardo, S., Dutton, P.L. (2005) Trends Biochem.
Yagi, T., Yankovskaya, V., Singer, T.P., Sci., 30, 176.
References 581
63. Rich, P.R. (2004) Biochim. Biophys. 79. Fisher, N., Brown, A.C., Sexton, G.,
Acta, 1658, 165. Cook, A., Windass, J., and Meunier, B.
64. Gutierrez-Cirlos, E.B., (2004) Eur. J. Biochem., 271, 2264.
Merbitz-Zahradnik, T., and 80. Kessl, J.J., Hill, P., Lange, B.B.,
Trumpower, B.L. (2004) J. Biol. Chem., Meshnick, S.R., Meunier, B., and
279, 8708. Trumpower, B.L. (2004) J. Biol. Chem.,
65. van Ark, G., Raap, A.K., Berden, J.A., 279, 2817.
and Slater, E.C. (1981) Biochim. Bio- 81. Gisi, U., Sierotzki, H., Cook, A., and
phys. Acta, 637, 34. McCaffery, A. (2002) Pest Manage. Sci.,
66. Zhao, P.-L., Wang, L., Zhu, X.-L., 58, 859.
Huang, X., Zhan, C.-G., Wu, J.-W., and 82. Fungicide Resistance Ac-
tion Committee (2006) QoI
Yang, G.-F. (2010) J. Am. Chem. Soc.,
Fungicides Working Group,
132, 185.
http://www.frac.info/frac/index.htm
67. Gao, X., Wen, X., Esser, L., Quinn, B.,
(accessed May 20, 2011).
Yu, L., Yu, C., and Xia, D. (2003)
83. Richter, O.-M.H. and Ludwig, B. (2003)
Biochemistry, 42, 9067.
Rev. Physiol., Biochem. Pharmacol., 147,
68. Crowley, P.J., Berry, E.A., 47.
Cromartie, T., Daldal, F., 84. Brzezinski, P. and Gennis, R. (2008)
Godfrey, C.R.A., Lee, D.-W., Phillips, J. Bioenerg. Biomembr., 40, 521.
J.E., Taylor, A., and Viner, R. (2008) 85. Burke, P. and Poyton, R. (1998) J. Exp.
Bioorg. Med. Chem., 16, 10345. Biol., 201, 1163.
69. Esser, L., Quinn, B., Li, Y.-F., 86. Kornblatt, J.A. (1998) Biophys. J., 75,
Zhang, M., Elberry, M., Yu, L., Yu, 3127–3134.
C.-A., and Xia, D. (2004) J. Mol. Biol., 87. Beauvoit, B. and Rigoulet, M. (2001)
341, 281. IUBMB Life, 52, 143.
70. Crofts, A.R. (2004) Annu. Rev. Physiol., 88. Lee, I., Salomon, A.R., Ficarro,
66, 689. S., Mathes, I., Lottspeich, F.,
71. Rieske, J.S. (1980) Pharmacol. Ther., 11, Grossman, L.I., and Huttemann,
415. M. (2005) J. Biol. Chem., 280, 6094.
72. Nelson, B.D., Walter, P., and 89. Lemos, R.S., Fernandes, A.S., Pereira,
Ernster, L. (1977) Biochim. Biophys. M.M., Gomes, C.M., and Teixeira, M.
Acta, 460, 157. (2002) Biochim. Biophys. Acta, 1553,
73. Kessl, J.J., Lange, B.B., 158.
Merbitz-Zahradnik, T., Zwicker, K., 90. Lancaster, C.R.D. (2002) Biochim.
Hill, P., Meunier, B., Palsdottir, H., Biophys. Acta, 1553, 1.
Hunte, C., Meshnick, S., and 91. Horsefield, R., Iwata, S., and Byrne, B.
(2004) Curr. Protein Peptide Sci., 5,
Trumpower, B.L. (2003) J. Biol. Chem.,
107.
278, 31312.
92. Sun, F., Huo, X., Zhai, Y., Wang, A.,
74. Kunze, B., Jansen, R., Hoefle, G., and
Xu, J., Su, D., Bartlam, M., and Rao, Z.
Reichenbach, H. (1994) J. Antibiot., 47,
(2005) Cell, 121, 1043.
881.
93. Huang, L.-S., Sun, G., Cobessi, D.,
75. Fudou, R., Iizuka, T., and Wang, A.C., Shen, J.T., Tung, E.Y.,
Yamanaka, S. (2001) J. Antibiot., 54, Anderson, V.E., and Berry, E.A. (2006)
149. J. Biol. Chem., 281, 5965.
76. Reichenbach, H. and Hofle, G. (1993) 94. Oyedotun, K.S., Yau, P.F., and Lemire,
Biotechnol. Adv., 11, 219. B.D. (2004) J. Biol. Chem., 279, 9432.
77. Berry, E.A., Huang, L.-S., Lee, D.-W., 95. Lee, G.Y., He, D.-Y., Yu, L., and
Daldal, F., Nagai, K., and Minagawa, N. Yu, C.-A. (1995) J. Biol. Chem., 270,
(2010) Biochim. Biophys. Acta, 1797, 6193.
360. 96. Iverson, T.M., Luna-Chavez, C.,
78. Colson, A.M. (1993) J. Bioenerg. Cecchini, G., and Rees, D.C. (1999)
Biomembr., 25, 211. Science, 284, 1961.
97. Yankovskaya, V., Sablin, S.O., Congress of Pesticide Chemistry,

Ramsay, R.R., Singer, T.P., Melbourne, July 4-8. Abstract 0402.
Ackrell, B.A.C., Cecchini, G., and 113. Simkovic, M. and Frerman, F.E. (2004)
Miyoshi, H. (1996) J. Biol. Chem., 271, Biochem. J., 378, 633.
21020. 114. Ross, J.L., Davis, M.B., MacIntyre, R.J.,
98. Oyedotun, K.S. and Lemire, B.D. (2001) and McKechnie, S.W. (1994) Gene, 139,
J. Biol. Chem., 276, 16936. 219.
99. Anderson, R.C., Majak, W., 115. Tritsch, D., Mawlawi, H., and
Rassmussen, M.A., Callaway, Biellmann, J.-F. (1993) Biochim. Bio-
T.R., Beier, R.C., Nisbet, D.J., and phys. Acta, 1202, 77.
Allison, M.J. (2005) J. Agric. Food 116. Siedow, J.N. and Umbach, A.L. (2000)
Chem., 53, 2344. Biochim. Biophys. Acta, 1459, 432.
100. Coles, C.J., Edmondson, D.E., and 117. Berthold, D.A., Andersson, M.E., and
Singer, T.P. (1979) J. Biol. Chem., 254, Nordlund, P. (2000) Biochim. Biophys.
5161. Acta, 1460, 241.
101. Matsson, M. and Hederstedt, L. (2001) 118. Yukioka, H., Inagaki, S., Tanaka, R.,
J. Bioenerg. Biomembr., 33, 99. Katoh, K., Miki, N., Mizutani, A., and
102. Honda, Y., Matsuyama, T., Irie, T., Masuko, M. (1998) Biochim. Biophys.
Watanabe, T., and Kuwahara, M. (2000) Acta, 1442, 161.
Curr. Genet., 37, 209. 119. Tanton, L.L., Nargang, C.E.,
103. Skinner, W., Bailey, A., Renwick, A., Kessler, K.E., Li, Q., and Nargang,
Keon, J., Gurr, S., and Hargreaves, J. F.E. (2003) Fungal Genet. Biol., 39,
(1998) Curr. Genet., 34, 393. 176.
104. Ito, Y., Muraguchi, H., Seshime, Y., 120. Juarez, O., Guerra, G., Martinez, F.,
Oita, S., and Yanagi, S.O. (2004) Mol. and Pardo, J.P. (2004) Biochim. Biophys.
Genet. Genomics, 272, 328. Acta, 1658, 244.
105. Miyadera, H., Shiomi, K., Ui, H., 121. Descheneau, A.T., Cleary, I.A., and
Yamaguchi, Y., Masuma, R., Tomoda, Nargang, F.E. (2005) Genetics, 169,
H., Miyoshi, H., Osanai, A., Kita, K., 123.
and Omura, S. (2003) Proc. Natl Acad. 122. Medentsev, A.G., Arinbasarova,
Sci. USA, 100, 473. A.Y., Golovchenko, N.P., and
106. Mowery, P.C., Ackrell, B.A.C., Singer, Akimenko, V.K. (2002) FEMS Yeast
T.P., White, G.A., and Thorn, G.D. Res., 2, 519.
(1976) Biochem. Biophys. Res. Commun., 123. Kerscher, S.J. (2000) Biochim. Biophys.
71, 354. Acta, 1459, 274.
107. Motoba, K., Uchida, M., and Tada, E. 124. Eschemann, A., Galkin, A., Oettmeier,
(1988) Agric. Biol. Chem., 52, 1445. W., Brandt, U., and Kerscher, S. (2005)
108. Deng, Y. and Nicholson, R.A. (2005) J. Biol. Chem., 280, 3138.
Pesticide Biochem. Physiol., 81, 39. 125. Joseph-Horne, T., Hollomon, D.W.,
109. Murakami, H., Masuzawa, S., Takii, S., and Wood, P.M. (2001) Biochim. Bio-
and Ito, T. (2003) Patent JP2003201280, phys. Acta, 1504, 179.
p. 38. 126. Gredilla, R., Grief, J., and
110. Mogi, T., Kawakami, T., Arai, H., Osiewacz, H.D. (2006) Exp. Gerontol.,
Igarashi, Y., Matsushita, K., Mori, M., 41, 439.
Shiomi, K., Omura, S., Harada, S., and 127. Wood, P.M. and Hollomon, D.W.
Kita, K. (2009) J. Biochem., 146, 383. (2003) Pest Manage. Sci., 59, 499.
111. Joppien, H., Puttner, R., and Winter, E. 128. Picault, N., Hodges, M., Palmieri, L.,
(1983) Proceedings of the 10th Inter- and Palmieri, F. (2004) Trends Plant
national Conference of Congress Plant Sci., 9, 138.
Protection, Brighton, UK, British Crop 129. Palmieri, L., Lasorsa, F.M., Vozza,
Protection Council, Croydon, UK, Vol. A., Agrimi, G., Fiermonte, G.,
1, p. 392. Runswick, M.J., Walker, J.E., and
112. Nakahira, K., Takii, S., and Ito, T. Palmieri, F. (2000) Biochim. Biophys.
(2010) 12th IUPAC International Acta, 1459, 363.
References 583
130. Guo, Z.J., Miyoshi, H., Komyoji, T., Walker, J.E. (1996) Biochemistry, 35,
Haga, T., and Fujita, T. (1991) Biochim. 12640.
Biophys. Acta, 1056, 89. 148. Minauro-Sanmiguel, F., Wilkens, S.,
131. Lewis, K., Naroditskaya, V., and Garcia, J.J. (2005) Proc. Natl Acad.
Ferrante, A., and Fokina, I. (1994) Sci. USA, 102, 12356.
J. Bioenerg. Biomembr., 26, 639. 149. Dudkina, N.V., Heinemeyer, J.,
132. Krulwich, T.A., Quirk, P.G., and Keegstra, W., Boekema, E.J., and
Guffanti, A.A. (1990) Microbiol. Rev., Braun, H.P. (2005) FEBS Lett., 579,
54, 52. 5769.
133. Heytler, P.G. (1980) Pharmacol. Ther., 150. Devenish, R.J., Prescott, M., Boyle,
10, 461. G.M., and Nagley, P. (2000) J. Bioenerg.
134. Terada, H. (1990) Environ. Health Biomembr., 32, 507.
Perspect., 87, 213. 151. Galanis, M., Mattoon, J.R., and
135. Leslie, A.G. and Walker, J.E. (2000) Nagley, P. (1989) FEBS Lett., 249,
Philos. Trans. R. Soc. London, B, Biol. 333.
Sci., 355, 465. 152. John, U.P. and Nagley, P. (1986) FEBS
136. Gibbons, C., Montgomery, M.G., Lett., 207, 79.
Leslie, A.G., and Walker, J.E. (2000) 153. Salomon, A.R., Voehringer, D.W.,
Nat. Struct. Biol., 7, 1055. Herzenberg, L.A., and Khosla, C.
137. Yasuda, R., Noji, H., Kinosita, J., Jr, (2001) Chem. Biol., 8, 71.
and Yoshida, M. (1998) Cell, 93, 1117. 154. Canedo, L.M., Fernandez-Puentes, J.L.,
138. Gledhill, J.R. and Walker, J.E. (2005) and Baz, J.P. (2000) J. Antibiot., 53,
Biochem. J., 386, 591. 479.
139. Groth, G. (2002) Proc. Natl Acad. Sci. 155. Kirst, H.A., Larsen, S.H., Paschal, J.W.,
USA, 99, 3464. Occolowitz, J.L., Creemer, L.C., Steiner,
140. Lardy, H., Reed, P., and Lin, C.H. J.L., Lobkovsky, E., and Clardy, J.
(1975) Fed. Proc., 34, 1707. (1995) J. Antibiot., 48, 990.
141. Hamann, L.G., Ding, C.Z., Miller, A.V., 156. Ruder, F.J. and Kayser, H. (1993) Pesti-
Madsen, C.S., Wang, P., Stein, P.D., cide Biochem. Physiol., 46, 96.
Pudzianowski, A.T., Green, D.W., 157. Atwal, K.S., Wang, P., Rogers, W.L.,
Monshizadegan, H., and Atwal, K.S. Sleph, P., Monshizadegan, H.,
(2004) Bioorg. Med. Chem. Lett., 14, Ferrara, F.N., Traeger, S., Green,
1031. D.W., and Grover, G.J. (2004) J. Med.
142. Atwal, K.S., Ahmad, S., Ding, C.Z., Chem., 47, 1081.
Stein, P.D., Lloyd, J., Hamann, L.G., 158. Pebay-Peyroula, E.,
Green, D.W., Ferrara, F.N., Wang, P., Dahout-Gonzalez, C., Kahn, R.,
and Rogers, W.L. (2004) Bioorg. Med. Trezeguet, V., Lauquin, G.J.-M., and
Chem. Lett., 14, 1027. Brandolin, G. (2003) Nature, 426, 39.
143. Koul, A., Dendouga, N., Vergauwen, 159. Nury, H., Dahout-Gonzalez, C.,
K., Molenberghs, B., Vranckx, L., Trezeguet, V., Lauquin, G., Brandolin,
Willebrords, R., Ristic, Z., Lill, H., G., and Pebay-Peyroula, E. (2005) FEBS
Dorange, I., Guillemont, J., Bald, D., Lett., 579, 6031.
and Andries, K. (2007) Nat. Chem. 160. Nury, H., Dahout-Gonzalez, C.,
Biol., 3, 323. Trezeguet, V., Lauquin, G.J.M.,
144. Stock, D., Leslie, A.G.N.W., and Brandolin, G., and Pebay-Peyroula,
Walker, J.E. (1999) Science, 286, 1700. E. (2006) Annu. Rev. Biochem., 75,
145. Rubinstein, J.L., Walker, J.E., and 713–741.
Henderson, R. (2003) EMBO J., 22, 161. Stubbs, M. (1979) Pharmacol. Ther., 7,
6182. 329.
146. Berry, R.M. (2005) Curr. Biol., 15, 162. Beale, R.E., Phillion, D.P.,
R385. Headrick, J.M., O’Reilly, P., and Cox, J.
147. Collinson, I.R., Skehel, J.M., (1998) Brighton Crop Prot. Conf. – Pests
Fearnley, I.M., Runswick, M.J., and Dis., 2, 343.
163. Joseph-Horne, T., Heppner, C., 169. Muller, F.L., Roberts, A.G., Bowman,
Headrick, J., and Hollomon, D.W. M.K., and Kramer, D.M. (2003) Bio-
(2000) Pesticide Biochem. Physiol., 67, chemistry, 42, 6493.
168. 170. Kadenbach, B. (2003) Biochim. Biophys.
164. European Commission, Health & Con- Acta, 1604, 77.
sumer Protection Directorate-General, 171. Nogueira, F.T., Borecky, J., Vercesi,
Standing Committee on the Food A.E., and Arruda, P. (2005) Biosci. Rep.,
Chain and Animal Health (2003) 25, 209.
Review Report for the Active 172. Krauss, S., Zhang, C.Y., and
Substance Silthiofam, July 2003, Lowell, B.B. (2005) Nat. Rev. Mol.
http://europa.eu.int/comm/food/ Cell Biol., 6, 248.
plant/protection/evaluation/newactive/ 173. Toime, L.J. and Brand, M.D. (2010)
list1_silthiofam_en.pdf (accessed May 20,
Free Radic. Biol. Med., 49, 606.
2011).
174. Stoetzer, O.J., Pogrebniak, A.,
165. Hamouda, C., Hedhili, A., Ben Salah,
Pelka-Fleischer, R., Hasmann, M.,
N., Zhioua, M., and Amamou, M.
Hiddemann, W., and Nuessler, V.
(2004) Vet. Hum. Toxicol., 46, 144.
(2002) Biochem. Pharmacol., 63, 471.
166. Jiao, Z., Kawamura, Y., Mishima, N.,
Yang, R., Li, N., Liu, X., and Ezaki, T. 175. Linsinger, G., Wilhelm, S., Wagner, H.,
(2003) Microbiol. Immunol., 47, 915. and Hacker, G. (1999) Mol. Cell. Biol.,
(FIELD Publication Date 2003). 19, 3299.
167. Fujita, K., Tani, K., Usuki, Y., 176. Nicholls, D.G. (2006) J. Biol. Chem.,
Tanaka, T., and Taniguchi, M. (2004) J. 281, 14864.
Antibiot., 57, 511. 177. Fernandez Freire, P., Labrador, V.,
168. Lambert, A.J. and Brand, M.D. (2004) Perez Martin, J.M., and Hazen, M.J.
J. Biol. Chem., 279, 39414. (2005) Toxicology, 210, 37.
15.2
Strobilurins and Other Complex III Inhibitors
Hubert Sauter
15.2.1
Introduction
After more than 25 years of industrial research and development, strobilurins

have become one of the most important classes of crop-protection agents. With
a distributor sales value of US$ 2.6 billion in 2009 [1], they currently represent
approximately one-fourth of the world fungicide market (US$ 11.2 billion in 2009
[1]). Within fungicides, strobilurins now rank first (in commercial terms), having
surpassed the formerly leading triazoles (US$ 2.3 billion in 2009 [1]).
In this chapter, details are also provided of the five other Complex III inhibitors –
famoxadone, fenamidone, cyazofamide, amisulbrom, and ametoctradin – which,
besides strobilurins, have achieved introduction in agricultural practice.
In particular, several prominent and certain unique features are connected with
strobilurins, of which mention should be made at the outset:
• The strobilurins originate from academic natural products research that began
over 40 years ago, when their biological activity was first detected; this was followed
by the isolation and structure elucidation of oudemansin A and strobilurin A.

The unusual simplicity of the strobilurin A structure, together with its antifungal
activity, made it compelling inspiration for synthetic analogs.
• Simultaneous, yet distinctly different, processes were initiated to bring strobil-
urins from the universities to the research groups of the former ICI (now merged
into Syngenta) and BASF.
• The physiological mode of action of the strobilurins as mitochondrial respiration
inhibitors was established at a very early stage. Subsequent in vitro testing
allowed the intrinsic potency of new analogs to be evaluated, providing invaluable
guidance for a rational, and relatively rapid, optimization.
• The identification and X-ray structural determination of the exact target site –
namely, the mitochondrial bc1 complex – formed the basis for an understanding
of the inhibitor–target interaction, as well as of resistance phenomena, at the
molecular/submolecular level.
• Despite the potential for nontarget toxicity relating to the mode of action, a
careful optimization of the biological profile led to safe products.
• The extremely broad activity spectrum of strobilurins, with potential to control
all four major classes of phytopathogenic fungi (Ascomycetes, Basidiomycetes,
Deuteromycetes, and Oomycetes), is unique among commercial fungicides.
• Unusually, broad structural variations are possible without losing intrinsic
activity. This variability includes all areas of the molecule, including even the
pharmacophore.
• During the evolutionary process of lead-structure optimization, well over 50 000
strobilurin analogs were synthesized and tested by competing industrial fungicide
discovery research groups worldwide. Several compound types, and even specific
compounds, were prepared independently at almost the same time.
• This competition also led to an unprecedented international patent race, which
has produced more than 1000 patent applications, and several patent interferences
with narrow differences in priority dates.
• At present, nine different strobilurins have been introduced into the world fungi-
cide market (see Table 15.2.1 and Figure 15.2.2 below). More recently, several new
strobilurins have been announced as being developed or launched in China (e.g.,
enestroburin, SYP-1620, coumoxystrobin, pyraoxystrobin, pyrametostrobin), as
well as one in Japan (pyribencarb). A strobilurin has also been commercialized
as an acaricide (fluacrypyrim).
• Unexpected events have already led to changes in the market for strobilurins.
On the one hand, a surprisingly rapid development of resistance in pathogens,
such as powdery mildew and Septoria tritici, has limited opportunities in some
key segments. On the other hand, these losses have been compensated by
an outstanding efficacy against a new fungal disease of extreme economic
importance, the soybean rust epidemic in South America. In this case, resistance
has not yet developed, and for genetic reasons it is unlikely to develop towards
the rust pathogen (see Section 15.2.6).
• Further market opportunities have resulted from unexpected beneficial influ-
ences of strobilurins directly on the physiology of treated plants, even under
Table 15.2.1 Commercialized strobilurins and other complex III inhibitors.a
Fungicide Code Originator Current Launch Sales volume

number owner dateb (2009, US$ Mio)b
Kresoxim-methyl BAS 490 F BASF BASF 1996 130

Azoxystrobin ICI A 5504 ICI Syngenta 1997 910
Metominostrobin SSF-126 Shionogi Bayer 2000 <10
Trifloxystrobin CGA 279202 Ciba Bayer 2000 490
Picoxystrobin ZA 1963 Zeneca DuPont 2001 145
Pyraclostrobin BAS 500 F BASF BASF 2002 735
Fluoxastrobin HEC 5725 Bayer Bayer 2004 150
Dimoxystrobin BAS 505 F BASF BASF 2004 50
Orysastrobin BAS 520 F BASF BASF 2007 45
Famoxadone DPX JE874 DuPont DuPont 1997 60
Fenamidone EXP10745 Rhône-Poulenc Bayer 2001 40
Cyazofamid IKF 916 Ishihara Ishihara 2001 50
Amisulbrom NC 224 Nissan Nissan 2008 <10
a
For strobilurins introduced for the Chinese market only: see Figure 15.2.5.
b
From Ref. [1].
conditions of little or no fungal infection. This has led to unprecedented yield

increases, stress tolerance, and generally improved plant health. Such effects
offer a significant opportunity for further research to optimize plant physiology.
• All of these events have occurred during a period of far-reaching changes in
industrial crop-protection companies. Three points should be noted in this
respect: (i) several mergers and acquisitions led to the migration of strobilurin
patents and developmental or commercial products from one company to another;
(ii) molecular biology techniques were integrated into agrochemical research; and
(iii) there has been an increasing use of bioanalytical methods and biokinetic
considerations in this research. Each of these aspects might be said to have been
encouraged by the commercial and scientific interest in strobilurins.
In addition to their enormous significance in crop production, strobilurins

have an additional feature that is worthy of consideration in detail. That is, they
represent one of the most instructive examples of how modern fungicide research
can deliver tailored solutions, by combining rational and market-driven research
with an alertness for serendipity. In particular, the history of strobilurin research
demonstrates the value of detailed structure–activity considerations, among a
strategy focused on the variation of molecular structures aimed at optimizing a
biological profile. Clearly, much can be learned from this experience that is relevant
to future agrochemical optimization, and not only with regards to fungicides. The
opportunity to illustrate some of these aspects will be taken in this chapter.
15.2.2
Evolution of Strobilurins as Agricultural Fungicides
Several aspects of strobilurin fungicides, including the natural lead structures, have
been summarized previously [2–9]. The elegant pathway that led to the discovery
of azoxystrobin was described in detail in a sequence of reports from the Jealott’s
Hill group of the former ICI (later Zeneca, now Syngenta) [2–4]. A later review
appeared in 1998, which included kresoxim-methyl, SSF-126 (metominostrobin),
and an extensive survey of the families of related natural products known to that
time [5]. The final review from this group, which appeared in 2002, provided a
comprehensive overview of the chemical, biological, and ecotoxicological properties
of the six strobilurins available commercially at that time, and also of the azolones
famoxadone and fenamidone [6].
An initial review from the BASF group concentrated on the discovery of BAS 490
F (kresoxim-methyl), on its pharmacophore variations, and on its structure–activity
relationships (SARs) at the mitochondrial target level [7]. Another report focused
more on the biokinetic features, and their effects on the final biological properties
[8]. In 1999, a general review summarized the historical evolution from natural
products to commercial strobilurins up to trifloxystrobin, together with details of
the international R&D competition, including some dramatic ‘‘patent races’’ [9].
The strobilurins have also been reviewed in Russian, Chinese, and Indian journals
[10–13], while a more recent review focused on certain biological and ecological
aspects of the strobilurin fungicides [14].
The history of strobilurins began in academia when, in 1969, Musilek and
coworkers in Czechoslovakia reported the isolation of an antifungal antibiotic
from extracts of the fungus Oudemansiella mucida; although subsequently named
mucidin, the compound at that time was without any proposed structure [15].
Following this, the German research groups of T. Anke and W. Steglich obtained
strobilurin A from fermentations of the fungus Strobilurus tenacellus, and in 1977
reported its broad antifungal activity together with the respective chemical and
physical data [16]. In 1984, the same authors reviewed some rather confusing
earlier reports concerning the correct stereochemical structures of mucidin and
strobilurin A, and described the correct (E,Z,E)-structure on the basis of chemical
and spectroscopic evidence [17]. The assumed identity of strobilurin A and mucidin
was finally proven in 1986, following direct spectroscopic comparisons [18]. In
1979, the structurally closely related oudemansin A was described by the group
of Anke et al. [19], and subsequently many natural derivatives of the strobilurin
and oudemansin type were found to be produced in various organisms [5, 20].
Notably, all of these compounds had a common structural feature, namely the
(E)-β-methoxyacrylate unit, which was linked to the remainder of the molecule
in the α-position; accordingly, these materials were named β-methoxyacrylates
(MOAs). The same held true also for the cyrmenins, which were recently isolated
from myxobacteria [21].
One other structurally related group of natural antifungal substances is that
of the myxothiazols, which also contain an (E)-β-MOA moiety which, in this
instance, is β-linked. The discovery, characterization and structure elucidation of

these compounds were first performed in Germany by the groups of Reichenbach
and Höfle [22–24], which later were involved in discovering the closely related
melithiazols [25]. A full review of the naturally occurring β-MOAs is provided
elsewhere [26].
Clearly, natural evolution has led to the development of fungi that are capable
of synthesizing and excreting substances that compete against other fungi, yet are
completely insensitive to the fungicides that they themselves produce (see Section
15.2.6).
In this respect, an important ‘‘Rosetta Stone’’ came from the discovery that
strobilurin A, oudemansin A and myxothiazol A not only share a common struc-
tural feature (the (E)-β-MOA group) and inhibit mitochondrial respiration as a
physiological mode of action, but also act at a common molecular binding site,
the mitochondrial bc1 complex [27]. These findings inspired attempts by Steglich’s
group – and indeed by the fungicide industry as a whole – to design simplified but
still active synthetic strobilurin analogs [20]. An example of this occurred when the
ICI fungicide research team acquired a sample of oudemansin A from T. Anke
in June 1982. The team identified a potent, broad fungicidal activity in green-
house tests where the substance, which lacks the destabilizing triene functionality
of strobilurin A, proved to be sufficiently stable. Hence, a synthesis program
was quickly initiated, with attention focused also on strobilurin A, confirming
its correct E,Z,E-configuration and producing it in large quantities via a total
synthesis [2].
One year later, BASF commenced their own activities in the field and, in July
1983, while conducting a publicly funded joint project with several university
research groups on natural products as leads for new agrochemicals, the company
obtained strobilurin A. Although the compound demonstrated high activity against
fungi grown on artificial media in the laboratory, the results of greenhouse tests
were disappointing. This led to the hypothesis that the inherent lability of its triene
system might give rise to rapid photolytic, oxidative, or metabolic degradation
under greenhouse conditions. Consequently, attention was focused, in cooperation
with Steglich’s group, to create stabilized, more active analogs [7, 9].
Neither the ICI nor the BASF group were aware of each other’s similar research
objectives, and continued to work simultaneously but completely independently.
Therefore, it might be considered an interesting case of ‘‘evolutionary conver-
gence’’ that ICI and BASF, despite starting from slightly different points, arrived
very soon at the same molecule – a new, simple, and stabilized synthetic strobil-
urin, the so-called enol ether stilbene (also known as methoxyacrylate stilbene,
MOA-stilbene, or MOAS). MOAS differed from strobilurin A only in that the
central double bond of the original triene system was incorporated into a stabi-
lizing benzene ring. The compound showed not only the hoped-for increase in
fungicidal efficiency in glasshouse tests, but also – and somewhat surprisingly – an
even higher mitochondrial target activity. It soon became obvious that, for purposes
of further variations, this ‘‘second-generation lead structure’’ could be reasonably
S N
O
N S H N O
2
Myxothiazol A
O
O O O O
O O
Strobilurin A Oudermansin A
Central ring
Side chain
O O
Pharmacophore
O
Enol ether stilbene
Figure 15.2.1 From natural antifungals to the enol ether

stilbene as a new synthetic lead structure.
divided into three parts: the side-chain; the central linking ring; and the phar-
macophore (Figure 15.2.1). At BASF, the term ‘‘pharmacophore’’ is preferred, for
several reasons, to other commonly used terms.1)
The variations in all of the commercial strobilurins (Figure 15.2.2) can be
categorized according to this pattern. Whilst all possess the central linking ring – an
ortho-disubstituted benzene – unchanged, five (!) different pharmacophores and
nine different side chains can be identified.
Having obtained excellent greenhouse results with oudemansin, the ICI research
group, immediately aware of the potential of their new lead, began an extensive
research program and soon (in October and December 1984) filed a very broad,
apparently insurmountable, basic patent on stabilized synthetic β-MOAs of the
strobilurin-type [28]. BASF filed similar patent applications in May and December
1985 [29], but these were too late and, therefore, were without value. Thus, ICI
was still six months in the lead and in a very comfortable position for further
optimization studies.
1) The term ‘‘pharmacophore’’ is preferred to ‘‘pharmacophore’’ illustrates the final

the frequently used ‘‘toxophore’’ or other objective for the active ingredient, namely
terms such as ‘‘biophore’’ or ‘‘agrophore.’’ to be a (phyto-)pharmacon with useful
In medicinal chemistry, ‘‘pharmacophore’’ properties. In contrast, ‘‘toxophore’’
is the only well-established term for suggests toxicity, which is an undesired
a molecular subunit, including close side effect that can be minimized or
variants, which is characteristic for a eliminated during structure–activity
class of active pharmaceuticals. Moreover, optimization.
N N
O
O O O
O O CN O O O O
N N
O O NH
kresoxim-methyl azoxystrobin metominostrobin
CF3 O CF3 N O O
N
O O O O O O
N N
O O NH
trifloxystrobin picoxystrobin dimoxystrobin
N N
N O N O
O O O N
Cl N Cl F N O O O
O N
O O N N N
O O O NH
pyraclostrobin fluoxastrobin orysastrobin
Figure 15.2.2 The commercial strobilurin fungicides.
In contrast, the team at BASF was compelled to concentrate further research

outside of the ICI claims. The team’s decision to focus on pharmacophore variations
(admittedly, a risky strategy) was rewarded by the finding that oxime ethers (or
oximino esters) were also active. This discovery was covered by a patent application
filed in 1986, just two days before ICI filed on the same subject. So, the next round
of the patent race went to BASF [30, 31].
Meanwhile, the search for strobilurins with commercial potential went ahead in
both companies. From the outset, ICI focused on the concept that an optimal fungi-
cide must be ‘‘systemic’’ – in other words, the compound should be transportable
at least in the acropetal direction, moving upwards in the xylem transpiration
stream of the treated plants, and thereby providing protection in remote, untreated
areas of the plant. It was well known that a low octanol–water partition coefficient
(log POW ) of less than 5.0 (with a calculated maximum at 2.0) is one prerequisite
for good acropetal mobility. The other prerequisite is sufficient metabolic stability
inside the plant. ICI had already oriented their fungicide screening in this direction,
and thus were able to reach their strobilurin objective quite soon. Their brilliant
optimization strategy, based on sequential, rational variations of the side chain,
has been comprehensively described [2–5] (see also Refs [6, 9]). The end result was
azoxystrobin, first announced as a systemic fungicide with broad spectrum activity
in 1992 [32].
During its early optimization efforts to find a strobilurin with market potential,
BASF’s options regarding chemical structures were restricted from the patent side.
Thus, attention was focused on pharmacophore variants other than the enol ether
type, particularly on the patent-protected oxime ether (oximino ester) variants [9].
Ultimately, strobilurins bearing the oxime ether pharmacophore were found not to
have a good potential for systemicity, because the pharmacophore itself is degraded
relatively rapidly in plants via hydrolysis of the ester group (see Section 15.2.3.4).
On the other hand, compounds of this type showed high intrinsic activity, which
was further optimized with the aid of the mitochondrial target test. The compounds
also exhibited an outstanding activity against powdery mildews in several crops – a
market segment that traditionally was a main focus of BASF’s fungicide biology and
marketing. Thus, the fungicide testing system in place at that time ‘‘welcomed’’
this particular type of strobilurin, and anticipated a market potential for them.
It was no wonder, then, that kresoxim-methyl – the first strobilurin from BASF
research [33] and the first strobilurin to reach the market – was an oximino ester,
which fitted perfectly into the powdery mildew market despite not being designed
specifically for that purpose. In this light, kresoxim-methyl may be seen as a case
of serendipity. Later, during the development phase, H. Köhle and colleagues
discovered the physico-chemical and biokinetic basis for the compound’s particular
biological activity profile, namely its episystemic distribution properties (Section
15.2.3.3), which bring it into close contact with fungi such as powdery mildews that
grow on the leaf surface [8, 9, 34, 35].
Interestingly, starting with the natural lead structures was not the only way
to obtain new strobilurin fungicides. The route taken by Shionogi, which finally
led to metominostrobin, originated from a completely different, chemistry-driven
program. Starting from fungicidal carbamoyl isoxazoles 1, the group synthesized
first ring-opened analogs 2, and then aryl derivatives of type 3 with increased
fungicidal activity (Figure 15.2.3) [36] (see also Refs [5, 9]).
In this way, a new pharmacophore variant was identified – the oximino amides
(or oxime ether amides). When, at a later stage, the similarity of these compounds
with oximino ester strobilurins was realized, and it was established that they had the
same mode of action [37], a basic patent application was filed by Shionogi on this
new structural type [38]. By varying the oximino esters, BASF came independently
to the same new pharmacophore type 3, and filed a corresponding application [39];
this occurred after Shionogi’s application had been filed, but before it was laid open.
The result was that, while Shionogi obtained the basic patent, BASF was granted
a selection invention with claims for individual oximino amides not specifically
described in the Shionogi application. These included the compound that later
became the product dimoxystrobin [40], and agreements with Shionogi allowed
R
R′′ X
O
O N O O O O
R′′′
N R′′′ N
NHR′ NHR′ NH
1 2 3
Figure 15.2.3 Steps in Shionogi’s sequence of thoughts for

the synthesis of oximino amides.
BASF to undertake its commercial development. Later, Shionogi’s agrochemical

interests merged first into Aventis and then into Bayer CropScience.
Trifloxystrobin, like kresoxim-methyl, has the oximino ester pharmacophore. In
addition, its most interesting feature is the oxime ether side chain, which was found
to contribute considerably to strobilurins with particularly high intrinsic activity.
This side chain was not specifically claimed in the basic patent applications for enol
ethers, oximino esters, or oximino amides, and these gaps led later to an unusual
patent race, in which five competing companies were involved [9]. Despite some
claim interferences, the enol ethers were granted primarily to ICI [41], the oximino
esters to Ciba-Geigy [42]2) and the oximino amides to BASF [43]. The oximino ester
trifloxystrobin [44] was developed and announced by Novartis [45] and sold to Bayer
in 2000 as part of the requirements for the merger between Novartis Agribusines
and Zeneca Agrochemicals to form Syngenta.
Picoxystrobin, first announced in 2000 [46], is an outcome of ICI’s optimiza-
tion work on enol ethers. It combines two physico-chemical properties, namely
a relatively low octanol–water partition coefficient and a relatively high vapor
pressure, which give it both systemic xylem mobility and episystemic distribu-
tion properties. With regards to episystemicity, picoxystrobin closes a gap in the
biokinetic and biological properties of azoxystrobin. In 2006, picoxystrobin was
acquired by DuPont.
Pyraclostrobin, which was announced at the same Brighton Conference by BASF
[47a], contains not only a new pharmacophore type but also a new type of
side chain, with a five-membered, oxygen-linked heterocycle. For the first time,
the original carbon–carbon double bond of the enol ethers was replaced by a
nitrogen–oxygen single bond, and the pharmacophore at its center had a nitrogen
atom instead of carbon. In this case, too, the pharmacophore variant led to a
competition of rival patent applications [48]. According to its physico-chemical
characteristics, pyraclostrobin has no pronounced systemic or episystemic mobility
either in or on plants. However, its very distinct properties include a rapid
leaf uptake and translaminar movement, an extremely broad activity spectrum,
and outstanding crop safety and yield enhancements [47b, 49]. This bundle of
positive properties may – or may not – have its origin in the fact that pyraclostrobin
possesses the lowest melting point of all the presently commercialized strobilurins,
which should favor a rapid bioavailability in a molecularly dispersed form (see
Section 15.2.3.3).
Fluoxastrobin, which was announced by Bayer in 2004 [50], contains once more
a new pharmacophore type, in which the carbonyl group of the previous ester
or amide pharmacophores is inventively replaced by an oximino moiety that, in
addition, is incorporated into a six-membered dihydrodioxazine ring. However, the
skeleton of the remaining molecule, including its side chain, closely resembles
2) Studies on strobilurins with the oxime La-Roche/Dr Maag. As a consequence of

ether side chain, which led finally to mergers, acquisitions and divestments,
trifloxystrobin, began at the latest in 1988 trifloxystrobin migrated to Ciba-Geigy [42],
at a fungicide research team that was then to Novartis (in 1996), and finally to
originally associated with Hoffmann- Bayer (in 2000).
that of azoxystrobin. As with the latter compound, fluoxastrobin is obviously

successfully designed for xylem mobility [51] and exhibits broad-spectrum activity
[52]. It is also the first strobilurin to be marketed for seed treatment.
Orysastrobin [53], the latest of the four BASF strobilurins, was from the outset
designed specifically for use as a nursery box treatment of rice seedlings against rice
leaf diseases. In order to fulfill the special requirements necessary for this purpose,
care was taken to focus on strobilurins with low lipophilicity and high water
solubility, without losing too much intrinsic activity. The structure of orysastrobin
combines the relatively metabolically stable oximino amide pharmacophore with
a relatively large, but not too lipophilic, side chain. Such metabolic stability
and low lipophilicity allow the molecule to be taken up easily by the roots and
transported acropetally into the leaves. Moreover, its high water solubility leads to
the low aquatic toxicity necessary in rice applications, as accumulation in aquatic
organisms is minimized (H. Sauter, G.P. Dohmen, C. Künast, unpublished results)
[54]. The large spatial extension of the tris-oxime side chain clearly contributes to
the fact that sufficient intrinsic activity is obtained.
At this point, mention should also be made of several other active strobilurins
with interesting structures (see Figures 15.2.4 and 15.2.5).
DPX-KZ165 (Figure 15.2.4) [55] represents an inventive cyclic triazolinone phar-
macophore [56], and has the same side chain as trifloxystrobin. Compounds of that
pharmacophore type have been intensively investigated by the team at DuPont [57].
Interestingly, the triazolinone pharmacophore is in itself rigid and contributes to a
distinctly low lipophilicity; compared to the analogous enol ethers, its lipophilicity
is lowered by 1.4 log POW units. Nevertheless, their intrinsic activities as well as
fungicidal performance are considerably high, although no strobilurin of this cyclic
pharmacophore type has achieved commercial status.
Pyribencarb [58] was invented and is currently under development by Kumiai
and Ihara, preferentially against gray mold and sclerotinia rot diseases [58b].
Pyribencarb resembles the lead structure of strobilurin [58c], but in a considerably
Enol ether stilbene
O O
CF3 O
N O
O N O
N N
Cl
DPX KZ165 pyribencarb
N N
O
O NH
Figure 15.2.4 DPX-KZ165 and pyribencarb.

Cl
O O
N N
O O O O
Cl N
O NH
SYP-Z071, enestroburin SYP-1620
O O O O O O
O O O O
O O
SYP-3200, coumethoxystrobin SYP-3375, coumoxystrobin
O O
Cl
N N O O N N O N
O
O O
SYP-3343, pyraoxystrobin SYP-4155, pyrametostrobin
Figure 15.2.5 Strobilurins from the Chinese Shenyang Re-

search Institute of Chemical Industry.
different way to the above-discussed strobilurins. A two-dimensional mapping with

the enol ether stilbene (as in Figure 15.2.4) illustrates the right side of the central
bridging ring of the latter being now cut off, and the bridging ring itself shifted
two atom positions to the left. The central nitrogen atom of its pharmacophore
is unbranched, while the essential carbonyl group retains its relative position.
Biochemical studies on its mechanism of action [58c] have shown that pyribencarb
is a potent inhibitor of the mitochondrial bc1 complex in Botrytis cinerea and
Corynespora cassiicola. Compared to azoxystrobin and kresoxim-methyl, on the basis
of mitochondrial IC50 values, pyribencarb exhibits a lower activity and a higher
selectivity against several nonfungal mitochondria, whereas its activity against
mitochondria of G143A strobilurin-resistant strains of B. cinerea and C. cassiicola
is somewhat higher (lower resistance factors). These findings are discussed in
terms of differences in amino acid sequences of the binding site in different
species [58c].
During the past few years, a remarkable number of new strobilurins has emerged
from research at the Chinese Shenyang Research Institute of Chemical Industry
(SRICI) [59], generally starting with a three-year temporary registration. Most of
these are still at an experimental stage or early commercial status. At present (in
2010), they are expected to be developed primarily or exclusively for the Chinese
market.
The structures of several strobilurin fungicides that have resulting from R&D
investigations at SRICI are shown in Figure 15.2.5, and additional candidates are
already in the company’s pipeline. Enestroburin [60] and SYP 1620 [61], which
were patented together with Rohm and Haas (later merged into Dow Agrosciences),
achieved market introduction in China in 2006 and 2008, respectively [1]. Looking
to the structures, both compounds have the same framework of their side chains:
enestroburin has the enol ether pharmacophore, and SYP-1620 is an oxime ether
amide. Both are used as broad-spectrum fungicides for cereals, fruit, and vegetables
[62], and SYP-1620 is also used against rice diseases.
A new coumarin-type side chain is present in the enol ethers coumethoxystrobin
and coumoxystrobin [59c, 63]. Coumoxystrobin, which is used preferentially against
apple canker and stem rot, obtained temporary Chinese registration in 2010.
Recently, pyraoxystrobin [59d, 64] also achieved temporary registration in China
as a very broad-spectrum fungicide. The pyrazolyloxy side chain of this enol
ether strobilurin was designed [59d] according to the model of pyraclostrobin (cf.
Figure 15.2.2).
With its similar pyrazolyloxy side chain and its methoxy carbamate pharma-
cophore, pyrametostrobin [65] resembles again pyraclostrobin as the original
design model.
15.2.3
Structure–Activity Relationships of Strobilurins
Once a biologically active substance is defined and validated as a lead, the variation of
its chemical structure is seen as the starting point for hypotheses and experiments
to obtain optimum properties in respect to biological performance, ecological
and toxicological safety, acceptance by regulatory agencies and, finally, economic
potential. All these final properties have their origin in the molecular structure of
the active compound. Consequently, in a lead structure optimization process, SARs
that reflect all of these attributes are of great relevance, and should be included
at as early a stage as possible. Later – it must be emphasized – they may also
be of value for candidate selection processes. This means that structure–activity
considerations are not only helpful, but are in fact essential for initiating rational
and promising processes in crop protection R&D.
15.2.3.1 Interplay of Target Activity and Biokinetic Behavior

The activity of a fungicide and its selectivity under real-life conditions in a complex
system of fungal pathogen, plant, environment, and time is determined by a
multitude of quite different influencing factors [66]. In optimizing a lead structure,
it is useful to separate such influencing factors and the associated molecular
properties into two parts: (i) its intrinsic activity at the molecular target; and (ii) its
biokinetic behavior.
15.2.3.1.1 Target Activity A decisive factor is the activity of the respective active
substance for its molecular target in the fungus, and potentially also in nontarget
organisms. With strobilurins, intrinsic activity is determined primarily by the
binding affinity of the active substance to the QO site of the bc1 complex of the
respiratory chain (see Chapter 15.1).
The relevance of a given molecular target regarding a final fungicidal effect

may change during the life-cycle of the treated organism. As a consequence,
different development stages may exhibit different sensitivities to an individual
fungicide or a fungicide class directed to a particular molecular target. The
ATP energy-demanding – and therefore strongly respiration-dependent – spore
germination is, compared to mycelial growth, particularly sensitive to respira-
tion inhibitors such as strobilurins. Since, among eukaryotes, spore germination
occurs almost exclusively in fungi, this fact contributes on the physiological level
also to selective action against fungal pathogens [9].
The optimization of target activity is best carried out on the basis of a cell-free
biochemical test that is as near to the isolated target protein as possible. If this is not
possible, then tests that are still ‘‘near to the target’’ should be used, for instance
at the cellular level. Such tests can also often provide valuable complementary
information to that obtained from a cell-free system.
15.2.3.1.2 Biokinetic Behavior Equally important for in vivo activity under practi-
cal conditions is how much of the active substance actually succeeds in reaching its
target. Of critical importance here are the particular characteristics of the molecule
that govern its absorption, transportation, breakdown and, where appropriate, its
excretion. Together, these points help to answer a critical question: How much of
the active substance is where, and when? Although this seems a simple question,
to answer it empirically using the techniques of analytical chemistry generally
requires high expenditures, and the results available during optimization are thus
limited and approximate.
In this situation, invaluable help may derive from a more theoretical direction.
The basic physico-chemical characteristics of an individual compound, such as
melting point, lipophilic/hydrophilic partition coefficients, water solubility, and
vapor pressure, are easy to measure and usually provide a reasonably good
prediction for trends with regards to several aspects of a compound’s real biokinetic
(dynamic) behavior in one environment or another. This is particularly true if
consideration is given to correlating or ranking a series of analogous compounds
according to their physico-chemical parameters on the one hand, and their complex,
time-dependent ‘‘biological’’ effects in one and the same test on the other hand.
Thus, these well-defined and static, substance-specific parameters – if used
carefully – can help to estimate by an ‘‘educated guess’’ the time-dependent,
dynamic distribution processes in complex environmental systems, as a part of
the substances’ biokinetic behavior [66], and they usually provide reasonably good
predictions.
An important second aspect for these predictions are the velocities of deactivation
(which usually mean degradation rates) in different environments, for instance on
or in soil, plants, fungi, or other organisms, even if the data are available only in
semi-quantitative form, or through estimation.
Regarding fungicides, strobilurins provide an exceptionally instructive example
of these principles for several reasons: the extraordinarily broad variability of their
chemical structures with retention of their principal activity; their extremely broad
fungicidal activity spectra; and the fact that, in contrast to former fungicide classes,
a simple and reliable target test was available. By using this test, the influence of
structural changes on intrinsic activity became clear, and could be assessed and
understood separately from the parallel influences of the structure changes on
biokinetic properties and behavior. Some of these aspects for the commercialized
strobilurins are detailed in Table 15.2.2.
15.2.3.2 Target Activity

Measuring the binding constants of strobilurins directly at their membrane-bound
target, the bc1 complex, is not an easy task. Easier to measure is the active substance
concentration necessary for 50% inhibition of an appropriate submitochondrial
enzyme preparation (termed the IC50 ), which can be used as an effective surro-
gate [7, 8, 68] for the more fundamental – and more resource-intensive – binding
measurements. Tests carried out using mitochondrial preparations from various
species (yeast, Botrytis, maize, housefly, and rat) on 14 strobilurins and myxothia-
zole showed that the activity ranking for the compounds was fairly parallel in all
species (ranking correlation coefficients ≥0.8). Thus, for strobilurins no apprecia-
ble contribution to species selectivity has been observed, or can be expected, at the
target level [8, 68].
For the routine evaluation of SARs at the target level, at BASF an automated test
with yeast submitochondrial preparations was used. In order to ensure that the
individual results were comparable, all test series included a reference standard,
the enol ether stilbene, to which the IC50 -value obtained for a test substance was
referred (Equation 15.2.1):
F = IC50 (test substance)/IC50 (enol ether stilbene) (15.2.1)
By definition, F = 1 for the enol ether stilbene, and hence the smaller F-value, the
higher the activity. This test has proven to be an extremely useful tool, inter alia
in the identification of new pharmacophore variants. To provide an impression
of resulting SARs, Figure 15.2.6 shows a small selection of such pharmacophore
variants together with their corresponding F-values. The compounds presented all
contain the same side chain, namely that of kresoxim-methyl (for a more detailed
discussion, see Refs [7, 9]).
Notably, without any X-ray structure-based knowledge of submolecular details
of the binding characteristics at the target enzyme, a detailed analysis of pharma-
cophore structure versus target activity soon led to the central conclusion that a
hydrogen bond, coming from the target enzyme as donor, and interacting with the
carbonyl group as the acceptor in the strobilurin pharmacophore, contributes most
to binding, and seems to be essential for activity [7]. Later, the cyclic pharmacophore
of DPX-KZ 165 (bottom left-hand, Figure 15.2.6) provided additional information
concerning the docking conformation of the pharmacophore: the carbonyl group
must have an s-(E)-orientation (‘‘north west,’’ not ‘‘south,’’ as suggested in Ref. [7])
regarding the remaining molecule, as indicated in the formulas of Figure 15.2.6.
Looking to the pharmacophore of fluoxastrobin (Figure 15.2.6, bottom line, second
598
Table 15.2.2 Factors affecting strobilurin properties: intrinsic activity, physico-chemical data, metabolic degradation features, biokinetic properties, and
biological use patterns.
Mitochondrial Melting Aqueous Lipophilicity Vapor Pharmacophore Soil Daphnia Biokinetic or Typical
factor Fa point solubility (log POW ) pressure metabolic DT50 magna biological biological
Mycosph. ( ◦ C) (mg l−1 ; (Pa) stability (days) EC50 characteristics targets
fijiensis (20 ◦ C) (20 ◦ C) (48 h
μg l−1 )
Metominostrobin 20b 87–89 128 2.3 1.8 × 10−5c High 98 14 000d Root uptake, high Rice diseases
xylem mobility
Orysastrobina 9.5b 99 81 2.4 7 × 10−7 High 51–58 1200 Root uptake, high Rice diseases
xylem mobility
15 Fungicides Acting on Oxidative Phosphorylation
Kresoxim-methyl 2.2 102 2 3.4 2.3 × 10−6 Low <1–3 186 Episystemic Powdery
distribution, fast mildews
degradation
Trifloxystrobin 0.26 73 0.6 4.5 3.4 × 10−6c Low 4–10e 16 Episystemic Powdery
distribution mildews
Picoxystrobin 0.61 75 3 3.6 5.5 × 10−6 Medium 3–35e 18 Episystemic Powdery
distribution and mildews
xylem mobility
Dimoxystrobin 4.1 138–140 4 3.6 6.0 × 10−7 High 2–125 39 Long lasting, Fusarium
xylem systemic spp.
Azoxystrobin 4.9 116 6 2.5 1.1 × 10−10 Medium 7–56e 259 Xylem systemic, Very broad
translaminar, not activity
episystemic spectrum
Fluoxastrobin 2.9b 103–108 2.5 2.9 6 × 10−10 High 16–119 480 Xylem systemic, Very broad
translaminar, not activity
episystemic spectrum
Pyraclostrobin 0.27 64–65 1.9 4.0 2.6 × 10−8 Medium 2–37e 16 Rapid leaf uptake, Very broad
translaminar, activity
long lasting spectrum
a
BASF data.
b
Compound for mitochondrial testing re-synthesized at BASF.
c
At 25 ◦ C.
d
Daphnia pulex.
e
Ref. [6].
Data from Ref. [67] if not otherwise noted.
15.2 Strobilurins and Other Complex III Inhibitors
599
R R R R
O O O O
O O O
F = 0.67 9.0 1.2 O
R R R
O O O O O
N N N
O O O O
0.60 >100 35
R R R R
O O O O O O S O
N N N N
O O
2.2 0.7 1.3 190
R R R +
R
−H
O O O O O O O O
N N N N
NH NH2 −
OH O
10 63 >1000
R R R R
O N O N O N O N
O O O
O O O NH
97 6.6 1.4 123
R R R R
O N O N O O O
O N O O
N N O O NH
5.6 8.0 3.5 39
Figure 15.2.6 Strobilurins with the kresoxim-methyl side

chain and variations on the pharmacophore. Their relative
activities at the target level are given below the formulas; F
is inversely proportional to the activity (see text).
left), clearly, the ‘‘essential’’ hydrogen-bond-forming carbonyl group in the phar-

macophore can be replaced by other groups that can fulfill the same electronic and
spatial hydrogen acceptor function. Notably, replacement of the carbonyl group by
groups that are not good hydrogen-bond acceptors – such as the thiocarbonyl group
(Figure 15.2.6, third line) – leads to drastic losses of target activity. In contrast, a
Gly 143
Glu 272 S
N
H
O O
N
Figure 15.2.7 Model of the hydrogen bridge and the resistant Gly143Ala mutants. The
between Glu272 (yeast protein numbering) torsion of the pharmacophore relative to
of the bc1 complex and the carbonyl group the side chain S is adapted from the ac-
of a strobilurin pharmacophore. Adapted tive, torsionally restricted (+)-enantiomer in
from Refs [8, 44, 69]. Gly143 indicates the Ref. [73].
area of steric repulsion between strobilurins
considerable loss of activity does not occur if the ester methoxy group is replaced
with non-hydrogen-acceptor groups such as alkyl, as in the ketones of Figure 15.2.6
[7, 9].
The model of Figure 15.2.7 accords with crystallographic data from eight cocrys-
tallized strobilurin/bc1 complexes, showing that an N–H proton of Glu272 (yeast
enzyme numbering) is the hydrogen donor for the carbonyl group of strobilurins
[44, 69]. Using beef enzyme numbering, this residue corresponds to Glu271, and is
sometimes also referred to as the ‘‘amide N–H of Pro270’’ [70]. An alternative bind-
ing mode that favors the ester methoxy oxygen of the strobilurin pharmacophore
as the hydrogen acceptor of the Glu272 (yeast) proton [71], seems to be less likely,
based on the known SARs [7, 9]. This alternative binding mode is also disfavored
from a more theoretical viewpoint, considering the much higher proton acceptor
potency of carbonyl (sp2 ) oxygen versus methoxy ester (sp3 ) oxygen, in combination
with the exchangeable spatial positions of both oxygen types in strobilurin pharma-
cophores, simply by a single bond rotation. A recent report [70] has clarified this in
more detail by using sophisticated modeling studies for azoxystrobin, docking at
its bc1 binding site. The same report also showed clearly that only the biologically
active (+)-enantiomer of the two atropisomers of a torsionally restricted analog of
DPX-KZ165 [72] (cf. Ref. [73]) would fit into the bc1 target.
Also for side chain variations, clear – and in this case quantitative – SARs have
been established at the target level. In a series of oximino esters of type 4, a curve
was obtained which was in accordance with a bilinear equation (Figure 15.2.8)
[7, 9]. Similar correlations have been deduced for enol ethers, oximino amides,
crotonic esters, and methoxycarbamates (W. Grammenos, T. Grote, H. Kubinyi,
H. Sauter, unpublished results).
Thus, clearly the overall lipophilicity of the molecule – modified by the sub-
stituents X in these series of analogs – is one critical influencing factor. This may
reflect the importance of the distribution between lipophilic and hydrophilic micro-
6 O
p/50
X
O O
N
5 O
4
4
1 2 3 4 5 6 7 8 9
log POW
Figure 15.2.8 Target-level activity of a series of strobilurin

oximino esters as a function of the lipophilicity of the sub-
stituents X. pI50 is the negative logarithm of the I50 value
from the yeast mitochondria test. Each point represents a
variant of the general structure shown.
(or nano-) environments before and while the strobilurin can reach its docking
place at the target enzyme. Significant ‘‘underperformers’’ with respect to the curve
in Figure 15.2.8 arise if, on account of certain substituents or substitution patterns
X, the steric bulk of the side chain no longer permits optimal docking at the
target [7]. In contrast, if a compound or a compound series is located significantly
above the curve of Figure 15.2.8, those are obviously ‘‘outperformers’’ – which
represents a much more interesting case. Hints were found in that direction with
the oxime ether side chain – as in trifloxystrobin – and also with other variations
(W. Grammenos, T. Grote, H. Kubinyi, H. Sauter, unpublished results). Such
deductions and findings, once more, favor careful structure–activity analysis at the
target level, whenever possible, as a powerful tool in lead optimization procedures.
For purpose of comparison, data relating to the commercialized strobilurin
fungicides are listed in Table 15.2.2. The F-values for mitochondrial target activity
according to Equation 15.2.1 were determined at BASF, using a published standard
procedure with mitochondria from Mycosphaerella fijiensis. Measurements were
carried out with purchased reference substances, if not otherwise noted. For
an optimal comparability of the resulting values, all compounds were tested
simultaneously, and the tests were replicated three times; the average standard
deviation for the IC50 -values was 32% (J. Rether, T. Jabs, H. Sauter, unpublished
results). The resulting target activities extended over two orders of magnitude.
Independent of the different structures of the respective pharmacophores, the most
lipophilic compounds – trifloxystrobin and pyraclostrobin – showed the greatest
intrinsic activity, whereas the most hydrophilic compounds – metominostrobin,
orysastrobin and azoxystrobin – ranked at the lower end of the activity scale.
15.2.3.3 Transportation and Distribution

Besides target activity, the dosage transfer of a strobilurin from the point of
application to the receiving bc1 complex of the fungal target organism, and its
availability at this receptor, determine its activity in vivo.
15.2.3.3.1 The First Step In order to achieve an efficient transportation, the

active ingredient must start its journey in a molecularly dispersed state – that is,
in solution or in the gas phase. If the compound is deposited as a solid particulate
material, for instance on a leaf surface, then it is necessary to break up its amorphous
or crystalline supramolecular associations as a first step. The energy and time
necessary for that depends on the melting point: the lower the melting point, the
easier and faster molecular dispersion. Whatever the melting point, the formulation
can influence the bioavailability in the direction of faster or slower release, as
desired for a particular treatment. For example, in the case of fluoxastrobin, the
Bayer team was able to show (by using scanning electron microscopy) that, despite
its relatively high melting point (103–108 ◦ C; see Table 15.2.2), the compound when
formulated as a particular ‘‘EC 100’’ was deposited on barley and other plant leaves
as an ‘‘. . . amorphous layer without pronounced solid particulate material.’’ This
provided a deposit that was most likely already molecularly dispersed, for both rapid
and ‘‘prolonged foliar penetration and the associated systemic distribution’’ [51].
Another example (see Table 15.2.2) showed pyraclostrobin to have the lowest
melting point of the commercialized strobilurins (64–65 ◦ C). On that basis, it would
be expected that: (i) pyraclostrobin could be easily formulated in liquid form; and (ii)
this should provide a very rapid first-step bioavailability, reflected by an extremely
rapid leaf uptake and translaminar penetration after leaf treatment. Whilst both
characteristic properties of the compound were observed, the latter point was
supported also by the compound’s relatively high lipophilicity (log POW = 4.0).
The importance of formulation for good performance cannot be overemphasized.
Comparative field experiments of one and the same active substance in different
formulations (e.g., as EC versus WP) can demonstrate how a property such
as efficacy and, more importantly, the decision to proceed with one or another
substance, can be drastically influenced by a formulation.
15.2.3.3.2 The Next Steps With regards to distribution processes and their
influence on in vivo fungicidal efficacy, the question arises: What is the pattern of
concentration in space and time of the active ingredient with regard to the host
plant, and with regard to the location of the fungal pathogen and its organs in or
on the plant?
Leaf Surface Distribution via Vapor Phase: Episystemicity If the volatility of the active
substance is sufficiently high, migration can begin from its deposition on the
leaf surface, via vaporization and gas-phase transportation. For strobilurins, this
phenomenon first became prominent with kresoxim-methyl, and has been vari-
ously termed quasisystemic, episystemic, leaf-surface systemic, or – if connected
with translaminar movement – mesostemic. Experience shows that a minimum
vapor pressure of approximately 10−7 Pa is necessary [66] for a sufficient quan-

tity of substance to be produced in a molecularly dispersed gaseous state within
a reasonable time period. However, it is also clear that the volatility should
not be too high, in order to avoid a too-rapid substance dissipation. If the
molecular properties with regards to volatility and lipophilicity are appropriate,
then lateral distribution/redistribution processes can occur between the deposit
on the leaf, the boundary air layers above it, and the waxy leaf surface. Hav-
ing its origin in the point of deposit, the substance will then slowly lead to
time-dependent, concentric distribution patterns on the leaf surface. For episys-
temic strobilurins, such distribution patterns – and even transportation from one
plant to another – have been indirectly observed by monitoring remote fungicidal
effects [8, 35, 46, 74], and also observed directly by using radiolabeled materials
[6, 45, 46].
A cluster of three strobilurins – kresoxim-methyl, trifloxystrobin, and picoxys-
trobin – has shown very pronounced distribution properties of this type, with their
vapor pressures lying within an astonishingly narrow range of 2.3 to 5.5 × 10−6 Pa
(Table 15.2.2). With regards to the observable biological consequences resulting
from this, two points should be emphasized:
• Vapor-phase transportation enables the fungicide to attack and eradicate – very
efficiently – either fungi or fungal organs that grow on the leaf surface (e.g., the
mycelial mats of powdery mildews).
• In the case of protective treatments, if the substance is applied early enough,
then episystemic transportation can lead to a more or less uniform distribution
in the waxy leaf surfaces, thereby forming a protective shield against airborne
fungal spores, by continuously releasing the active ingredient.
In general, the strobilurins exhibit outstanding activities against fungal spore
germination, which usually exceeds their activity against other fungal development
stages [6, 8]. This is reflected in the tendency for strobilurins to show a better pro-
tective than curative performance in field treatments [75]. Thus, a combination of
their extremely efficient spore germination-inhibiting properties and their episys-
temic distribution into the waxy leaf layers makes the above-mentioned cluster of
episystemic strobilurins particularly well suited to protective leaf applications. The
fact that there is no metabolism in the waxes also allows for metabolically more
labile compounds (such as the oximino esters kresoxim-methyl and trifloxystrobin)
to be present and active for a long period, and thus to provide a long-term residual
activity.
Systemicity in Plants: Xylem Transportation Following leaf treatment, systemic

acropetal transportation in the apoplastic xylem stream is possible, if foliar pene-
tration of the active substance occurs, if the distribution between the lipophilic and
hydrophilic phases is appropriate, and if the substance is sufficiently metabolically
stable under such conditions. A necessary – but not sufficient – measure for the
distribution property is given by the POW . Maximum systemicity can be obtained
with log POW ≈ 2, while with log POW below 0 or above 4, no appreciable xylem
systemicity can be observed [76, 77]. In contrast to acropetal xylem mobility, phloem
(symplastic) systemicity – which allows additionally basipetal migration – requires
compounds with either some acidity or an extremely high hydrophilicity. With stro-
bilurins, however, both acidity and extremely high hydrophilicity are absent, and
no phloem mobility is to be expected. Four commercial strobilurins, successfully
designed for xylem systemicity, have log POW values between 2.5 and 3.6, and also
have sufficient metabolic stability in plants (Table 15.2.2), namely azoxystrobin (log
POW 2.5), fluoxastrobin (2.9), picoxystrobin (3.6), and dimoxystrobin (3.6). While
all of these show a pronounced xylem systemicity [6, 40, 51], picoxystrobin has,
in addition, episystemic mobility (see above) which favors a broader spectrum of
activity, including especially powdery mildews. For fluoxastrobin, a simulation of
its time-dependent systemic distribution in crop leaves was in a good accordance
with experimental data [51]. This report also showed the position of azoxystrobin,
fluoxastrobin, picoxystrobin, and trifloxystrobin in the time-continuous optimum
curve for the transpiration stream concentration factor (TSCF), as a measure of
xylem systemic accumulation, versus log POW . The report also touched implicitly
on the question of whether maximum systemicity always translates to optimum
fungicidal efficacy. Because target docking is a process of dynamic equilibrium, a
high mobility to reach a target is also connected with a high potential to dissociate
away from the target.
Root Uptake The data listed in Table 15.2.2 identify metominostrobin (Shionogi)
clearly as having the lowest log POW (2.3) of all the listed strobilurins, as well
as the highest water solubility and the lowest aquatoxicity. Together with the
high metabolic stability of the oximino amide pharmacophore, these properties
provide all of the prerequisites for root uptake, acropetal movement, residual
activity in leaves, and compatibility with aquatic environments. With regards to
hydrophilicity, it is well known that a high water solubility and a low lipophilicity
of bioactive compounds both correlate positively with a low aquatoxicity [54].
More impressively, an excellent linear correlation was found among 17 fungicidal
strobilurins by plotting their log POW values (which ranged from 1.8 to 4.8)
against their log EC50 -values for toxicity to Daphnia (r = 0.81; F = 24; S = 0.41) (H.
Sauter, G.P. Dohmen, C. Künast, unpublished results). The main consequence
of a low lipophilicity, however, is a relatively low intrinsic activity, at least in
the case of strobilurins (Figure 15.2.8 and Table 15.2.2). Consequently, unless
metominostrobin can be shown to have a broad spectrum of activity at higher
application rates, its primary biological target will be specialized, namely, a water
surface application in paddy rice against rice blast. It is, therefore, not too surprising
that metominostrobin was invented and developed in Japan.
The same molecular properties – a high water solubility and a low log POW ,
combined with high metabolic stability in plants – served as one focus of the R&D
studies conducted at BASF, the aim being to gain entrance into Asian rice fungicide
markets. The research team was also convinced that the increasingly popular use
of nursery boxes, in which rice seedlings are grown to a certain stage before being
transplanted into the field, would soon represent a major market segment for rice
fungicides. The strategy was simple and clear, with advantage being taken of the
accumulated knowledge of strobilurin SARs, which in turn led to deviations from
the routine screening procedures. The main principles were that:
• No compound with a log POW > 3.0 would be suitable for the targeted rice
market.
• The candidate must be independent of patent claims outside BASF’s rights.
• Metabolic stability in plants should be expected to be high (no oximino esters!).
• Toxicity towards aquatic organisms must be low, and must be tested early.
Although the latter restriction narrowed the chances of identifying extremely

active strobilurins, a sufficiently high mitochondrial target activity had to be main-
tained. It was also agreed at a very early stage that there should be a realistic
impression regarding the expected final biological performance of the candidates
under the conditions of practical application. During this preselection phase, con-
siderations of the expected synthetic availability and possible production costs
played no major role, because – according to the authors’ experiences – motivated
chemists can usually solve such problems with their own creative input and com-
petence. With orysastrobin, this proved to be the case once again [78], with the
above-described preselection criteria leading to an ensemble of only 300 initial
candidates (from more than 10 000) that then underwent specially designed, more
sophisticated tests, targeted in the direction of the final application. Only four com-
pounds reached a status of more serious concern for further development and, of
these, only one compound had – according to the criteria employed – the potential
to reach the targeted market. This compound, orysastrobin, was announced in
2004 [53] and marketed as an effective rice fungicide against both rice blast and
sheath blight, for use in nursery boxes and also for paddy rice application after
transplanting.
Despite the extensive efforts that have been focused on systemicity during strobil-
urin research worldwide, BASF’s most prominent strobilurin – pyraclostrobin –
has shown no appreciable systemicity such as xylem mobility or leaf surface
distribution. Whilst this ostensible disadvantage was to be expected from the com-
pound’s high lipophilicity and low vapor pressure, its low melting point gave rise
to a rapid bioavailability and, as a consequence, to a particularly rapid translaminar
distribution. Pyraclostrobin is also top-ranking with regards to its intrinsic activity
and longlasting efficacy. Moreover, it possesses outstanding plant compatibility
and crop safety and, in part as a consequence, is registered for use in more than
100 crops worldwide. In this connection, it should be noted that increasing the
hydrophilicity and systemicity of strobilurins may also increase the possibility of
incurring slight phytotoxic effects. To summarize, despite all of the rationally
derived, straightforward design procedures – which are undoubtedly necessary and
have been proven successful – the beneficial consequences of serendipity should
never be neglected.
To date, few data have been reported for enestroburin. Although the technical
product and the compound as reported in the patent [60] have been characterized
as an oil, the pure compound has been described as a ‘‘white crystalline solid’’
(melting point not disclosed). Some results from fungicide trials with enestroburin
have been reported [62] and, for comparison, the compound has been resynthesized
at BASF. Besides its obviously very low melting point, it has a high log POW (>4),
a low vapor pressure (<10−7 Pa), and considerable target activity (F ∼ = 0.4); taken
together, such properties might indicate other, perhaps biological, features.
15.2.3.4 Metabolic Degradation Rates

When considering the overall fungicidal activity and biokinetic behavior of these
fungicides, it is very important to take into account any active substance losses
through metabolic degradation. In the case of strobilurins, degradation generally
leads to deactivation, particularly if the pharmacophore is involved. At present,
very few exact degradation rates under the many different conditions in plants or
other metabolically active environments are available. However, as an approximate
and more semiquantitative measure, the individual 50% degradation time (DT50 )
ranges of the compounds for soil degradation can be used to provide an idea of
relative degradation rates in other environments, such as plants, as well as an
insight into structure–activity trends for metabolism (Table 15.2.2). Clearly, the
oximino esters (kresoxim-methyl and trifloxystrobin) are by far the most rapidly
metabolized, the initial metabolic step in both cases being a rapid hydrolysis of the
methyl ester group of the pharmacophore, resulting in an inactive carboxylic acid.
Hence, a longlasting activity cannot be expected with strobilurin oximino esters
when they are subjected to a metabolically active environment (as in a plant), and
this is one reason for their lack of xylem systemic activity.
According to the data listed in Table 15.2.2, the two enol ethers (azoxystrobin and
picoxystrobin) and the methoxycarbamate pyraclostrobin rank next, thus forming
a cluster of strobilurins with intermediate degradation rates.
The greatest metabolic stabilities are seen with the oximino amides (orysastrobin,
metominostrobin, and dimoxystrobin) and with the dihydrodioxazine (fluoxas-
trobin). This high level of stability is one of several prerequisites for applications
where longlasting fungicidal efficacy should be maintained while the active com-
pound is intensively exposed to plant metabolic processes, as might occur after root
uptake in rice or as a seed treatment in other crops.
The aim of this chapter is not to describe the fungicidal profiles of the various
strobilurins against different pathogens in detail; indeed, if their dosage is suffi-
ciently high, then their activity spectra will show considerable similarities. Rather,
the aim is to demonstrate, from a more global perspective, some guidelines of how
structural features in this class can influence the fine-tuning of biological activity
and use patterns. More detailed information regarding the biology and many
other aspects of the commercial or developmental products can be found in their
announcement papers or in the Pesticide Manual [67] and, most comprehensively,
in the periodically updated collection AGRO Projects [79].
At this point, a somewhat whimsical – but not entirely ludicrous – quantitative
structure–activity relationship (QSAR) is presented. Each of the nine commercial
strobilurins shown in Figure 15.2.2 contains, at least once, a three-atom fragment
consisting of oxygen and an imino group, in one of two different arrangements. Across
all nine molecules, this feature occurs exactly 2.00 times per molecule – on average,
so to speak! Less facetiously, the nine substances contain altogether 11 examples
of the oximino group –C=N–O– and seven examples of the group –N=C–O–,
the latter always in conjunction with heterocyclic structures. The incorporation
of so many relatively hydrophilic fragments reflects – at least to some extent – the
more or less directed approach to obtain moderately lipophilic, xylem-systemic
compounds.
With regards to the oximino group of oxime ethers, several reasons favor its
use as a building block for agrochemicals. Synthetically, starting with a carbonyl
group, its introduction into a molecule is extremely easily performed and is not
connected with any C–C bond formation. In most cases, the thermodynamically
preferred (E)-configuration can be obtained almost exclusively under acidic equilib-
rium conditions. From the viewpoint of physico-chemical properties, the group is
of intermediate polarity, and can contribute considerably to the size of a molecule
without enhancing its lipophilicity. The log POW is even lowered when a single
bond – for example, between two carbon atoms – is replaced by the oximino frag-
ment (CH=N–O–), and remains approximately constant when –C(CH)3 =N–O–
is introduced. Finally, from a biological standpoint, the latter group generally
possesses a remarkable metabolic stability.
15.2.3.5 Summary of Strobilurin Structure–Activity Relationships

A vague summary of the structure–activity findings with strobilurins is visualized
in Figure 15.2.9. This illustrates the complex intercorrelation network between
some molecular variables (left), activity variables (middle), and the main final
output properties (features) in agricultural practice (right). Positive correlations
between the variables are marked by green lines, and negative correlations by red
lines; the broken lines indicate that a correlation is relatively weak. Correlation in
this context does not mean a strict and exclusive linear relationship between the
variables; rather, it means simply that one variable has an influence on another
variable. This influence may sometimes be weak, and be valid only between
certain limits. Naturally, a correlation between two variables does not exclude
influences from other variables, as can be seen in Figure 15.2.9. For example, in
the case of the molecular variable lipophilicity, it is clear from the bilinear curve of
Figure 15.2.8 that the positive correlation with target activity is valid only at values
below log POW ≤ 5. Staying with lipophilicity, it is also clear that there is only a
weak positive connection with target binding (lipophilic areas of the target need
somewhat lipophilic side chains of the strobilurin) and with metabolic stability
(more hydrophilic substances tend to be degraded faster). It is also clear that
chemical or spatial changes in the molecular structure may exert a much more
drastic influence on target binding or metabolic stability than can lipophilicity.
Keeping that in mind, the chart in Figure 15.2.9 can serve as a navigation aid through
the complexity of strobilurin SARs, and also – cum grano salis – beyond strobilurins.
Molecular variable Activity variable
Intrinsic activity
(Mitochondrial)
Target fit
Fungicidal activity Practical
in vivo efficacy
Lipophilicity
Compatibility
(to non target organisms)
Ecotoxicity
Water solubility
Xylem systemicity
Melting point
Episystemicity
Vapor pressure
Soil absorption Leaching
Metabolic stability
Soil degradation Soil persistence
Figure 15.2.9 Structure–activity relationships: the complex network between variables.
15.2.4
Beneficial Influences on Plant Physiology and Crop Yield
It has been reported consistently from field trials that the yield enhancement
obtained after strobilurin treatments in wheat [80] and barley exceed significantly
the values that could be expected from comparative triazole treatments with similar
levels of visible fungal disease control [6, 75]. Also observed were a pronounced
‘‘greening’’ effect and delayed senescence that enabled the plants to maintain
their green leaf area until late in the season, thereby maximizing the grain-filling
period and yield. In other crops as well, and under conditions of no or very
low fungal infection, unexpected beneficial effects on yield and quality and better
stress tolerance after strobilurin treatments have been observed. As pyraclostrobin
seemed to be the most potent strobilurin in this respect, this led to its introduction
(as Headline™) for the optimization of plant health and crop yield in corn and
soybean.
These benefits, which are thought to be the result of direct influences on
physiological processes of the treated plants, are referred as to ‘‘physiological ef-
fects’’ [49a, 81], and have been most extensively studied with kresoxim-methyl
and pyraclostrobin. The effects include delayed senescence, an altered CO2 com-
pensation point, reduced stomatal aperture and water consumption, and a better
tolerance of oxidative stress. Significantly altered levels of enzyme activities, includ-
ing 1-aminocyclopropane-1-carboxylate synthase (ACC synthase), nitrate reductase,
peroxidases, and alternative oxidase (AOX), could be observed or inferred indirectly
in vivo, but in none of the cases investigated to date, directly with isolated enzymes
in vitro. The simplest, and therefore most convincing, hypothesis to explain all of
these different effects [49] is that strobilurins have a direct influence on mitochon-
drial respiration not only in fungi, but also in plants, and that this then leads to
a cascade of the various biochemical, physiological, and agricultural consequences
[49a, 81]. This theory includes the generation of nitric oxide as a fully systemic, acro-
and basipetally movable signal molecule [49b] for triggering – even remote – plant
defense reactions [49c]. This would occur even against pathogens that are not
sensitive to strobilurins, such as the tobacco mosaic virus or the bacterial wildfire
pathogen Pseudomonas syringae pv. tabaci [49d].
Mitochondria from maize leaves do in fact respond to a series of strobilurins, but
are in this case less sensitive than mitochondria from non-plant species (such as
yeast, Botrytis, rat, house fly) [68]. It should be noted that, in general, the inhibition
of mitochondrial respiration in plants (‘‘dark respiration’’) does not lead to severe
undesired influences on plant physiology.
There is a second theory to explain the beneficial yield effects, which does not
include any direct influences on plant biochemical processes, but relates exclusively
to fungicidal effects. This theory states that strobilurin treatments prevent the spore
germination of pathogenic, nonpathogenic and saprophytic fungi, and thereby stop
the elicitation of energy-demanding host-defense responses with resultant higher
crop yields [82].
In practice, the beneficial effects of strobilurins on plant health and crop yield
have been proven over many years, and under many different conditions. From a
more scientific point of view, however, in individual cases of practical relevance,
the reasons for this are still under discussion. It is certainly a challenge for
future research, and indeed it may initiate the search for new compounds with
optimized physiological effects. In that regard, the metabolic profiling of treated
plants – compared with untreated – may represent a key technique for identifying
such effects and interpreting them on the metabolome level. Metabolic profiling as
a new diagnostic method was introduced to crop science during the 1980s [83], and
has subsequently found increasing interest, progress and industrial applications
[84] in plant metabolome research [85].
15.2.5
Insecticidal and Acaricidal Activity
In the patent literature, many claims and some data can be found regarding the
insecticidal and acaricidal activities of strobilurins. To date, attempts to optimize
insecticidal performance have failed to lead to a commercial product, since it
appears that, with strobilurins, a sufficient insecticidal activity can only be obtained
if the compound has a very high lipophilicity and a very high metabolic stability.
Unfortunately, this combination of properties led to numerous compounds with
excellent insecticidal activity but unacceptably high acute mammalian toxicity, and
many candidates were abandoned at an early stage of research. It seems impossible
to separate the tight connection between insecticidal and mammalian toxicity in
this particular case, with similar problems having been reported for respiration
inhibitors of Complex I [86a].
S
O N O CF3 O
N
N O O O O
CF3 O CF3 O
fluacrypyrim HNPC-A3066
Figure 15.2.10 Fluacrypyrim and HNPC-A3066.
In contrast to insects, mites appear to be much more sensitive to strobilurins,

such that the chances of finding selective strobilurin acaricides are better than
for insecticides. Typically, mites are much smaller than insects, they generally
have a greater surface area per gram biomass, lack a thick cuticle and, possibly,
also have lower metabolic degradation capacities. Consequently, optimal acaricides
[86b] usually differ from optimal insecticides.
Currently, only one strobilurin acaricide, fluacrypyrim, is available commercially
(Figure 15.2.10) [78, 79] (see also Chapter 31.3).Whilst the acaricidal activity of
fluacrypyrim was originally discovered by BASF [87], the compound was later
developed by Nippon Soda for use in fruit crops and vegetables. It has a low
mammalian toxicity, while log POW = 4.5. Most interestingly, its vapor pressure
(2.7 × 10−6 Pa at 20 ◦ C) lies in the same narrow range as those of the episystemic
strobilurin fungicides (see Table 15.2.2); clearly, an episystemic distribution pattern
can be expected from fluacrypyrim. As adult mites (and also possibly their more
sensitive earlier developmental stages) live in close proximity to the plant surfaces,
a vapor-phase distribution should be particularly advantageous for acaricides. A
recent review of acaricides [86b] listed nine commercially available mitochondrial
respiration-inhibiting acaricides, of which seven (diafenthiuron, fluacrypyrim,
fenazaquin, fenpyroximate, pyridaben, tebufenpyrad, and chlorfenapyr) have vapor
pressures in the astonishingly narrow range of 2–12 × 10−6 Pa. Such a vapor
pressure would not only be very well suited to an episystemic distribution but also
provide a good prerequisite for a long residual activity.
Recently, the details of a new acaricidal strobilurin (code number HNPC-A3066)
have been reported by the Chinese Hunan Research Institute [88]. The chemical
structure of this material (see Figure 15.2.10) resembled that of trifloxystrobin
(Figure 15.2.2); moreover, the fact that its vapor pressure was very similar to that
of trifloxystrobin (3.4 × 10−6 Pa; see Table 15.2.2) meant that it fitted very well into
the above-mentioned narrow range typical of episystemic acaricides.
15.2.6
Fungal Resistance
In recent years, this topic has been comprehensively reviewed [6, 89, 90], not only
from molecular biology and biochemical [89a] viewpoints, but also from a more
practical aspect [89b]. It is also a permanently updated subject of the Fungicide
Resistance Action Committee (FRAC) QOI Working Group that forms part of the
FRAC, an inter-industry organization which monitors fungicidal resistance and

coordinates resistance management (www.frac.info). In the case of strobilurins,
several quite different mechanisms of resistance have been described which have
mainly laboratory significances [e.g., extrusion of the fungicide via the ATP-binding
cassette (ABC) transporters], but which are of no major importance for agricultural
applications.
Also of limited practical importance is a degree of resistance of Venturia in-
aequalis (which causes apple scab) against kresoxim-methyl, which has its origin
in the esterase-mediated metabolism of the active substance by resistant isolates
[91]. Target mutations of Phe129Leu (yeast numbering) have led only to lower
resistance factors in a few fungal species, and seem also to be of minor practical
relevance.
A critical review [90] on the role of the AOX pathway with respect to the
bypassing of Complex III, and its possible influence on the fungicidal performance
of strobilurins, came to the conclusion that, in many cases, the AOX can limit the
effectiveness of strobilurin once an infection has become established, but is unable
to interfere significantly with the action of strobilurin during germination. Hence,
preventive treatment with strobilurins is not severely affected by a circumvention of
Complex III via the AOX pathway. This conclusion corresponds to the observation
made with several pathogens in vitro on agar plates, that spore germination is 10- to
1000-fold more sensitive towards treatment with kresoxim-methyl than is mycelial
growth [8].
In contrast, the target mutation Gly143Ala (yeast numbering) is of major impor-
tance for Complex III QO site inhibitors. At the molecular/submolecular level, this
prevents docking of the active substance to its target completely [69, 89a, 92a]. This,
and four other mutations at different places in the amino acid sequence of the bc1
complex in different species, represent the main reason why strobilurin-producing
Basidiomycetes such as Strobilurus tenacellus are insensitive to their own fungicidal
metabolites [89a, 93]. It is not only strobilurins that are subject to Gly143Ala
resistance, but also other known commercial QO site inhibitors, famoxadone and
fenamidone; by contrast, the Qi inhibitor cyazofamid is not affected. The con-
sequences of a Gly143Ala mutation are high resistance factors and an almost
complete loss of fungicidal activity. In practice, for some pathogens a resistance
to strobilurins was observed quite soon, within two to three years following their
market introduction. Of great concern, however, was the first wheat powdery
mildew in cereals, followed somewhat later by Mycospaerella graminicola (Septoria
tritici). Although, at present, neither of these diseases are principally recommended
targets for strobilurin applications in practice, pyraclostrobin remains an excep-
tion and still shows some activity against Septoria tritici under field conditions
[75]. In greenhouse tests, it has also been shown to provide a good control of
Gly143Ala-mutated isolates of Pyrenophora tritici-repentis [94]. Notably, protective
treatments with azoxystrobin have remained very effective against resistant isolates
in greenhouse studies, whereas curative treatments proved to be much less effective
under similar conditions [75]. A bc1 modeling study has provided some evidence
to explain species-related differences in the effect of Gly143Ala mutations at the

enzyme level [92a].
Strobilurins have also been severely confronted with resistance problems in
other crops, an example being Plasmopara viticola in grapes. Yet, quite remarkably,
other economically important fungal diseases, such as those caused by rust
pathogens in different crops, have not been affected at all by Gly143Ala strobilurin
resistance – a situation that holds for all plant pathogenic Basidiomycetes studied
to date. This parallels the phylogenic separation of Basidiomycetes from the
more Gly143Ala resistance-prone Ascomycetes and Oomycetes, as demonstrated
in phylogenic relatedness studies conducted in several fungal species not only
at the mitochondrial cyt b gene level, but also at the nuclear level using internal
transcribed spacers (ITSs) in the ribosomal DNA [92b].
In general – and particularly among threatened applications – the FRAC recom-
mendations for strobilurin use have provided a major contribution in preventing
resistance. In principle, these recommendations include three major points: (i)
a limitation of the number of treatments per season; (ii) the application of
sufficiently high fungicide dosages; and (iii) alternations and/or mixtures with
non-cross-resistant fungicides having other modes of action.
15.2.7
Other Complex III Inhibitors
It should be noted that, despite a certain similarity in their names, neither the
azolones nor the N-(N ,N -dimethylaminosulfonyl)azoles are in any way related to
the azole fungicides of the demethylation inhibitor (DMI) type (see Chapter 19).
Data relating to the three commercially available products of this type are listed
in Tables 15.2.1 and 15.2.3.
Table 15.2.3 Selected data for the azolones, cyazofamid, amisulbrom, and ametoctradin.
Melting Aqueous Lipophili-city Vapor pressure Soil DT50 Daphnia

point ( ◦ C) solubility (log POW ) (Pa; 20 ◦ C) (days) magna
(mg l –1 ; EC50 (48 h ;
20 ◦ C) μg l−1 )
Famoxadone 141–142 0.05 4.7 6.4 × 10 –7 2–28c 12

a
Fenamidone 137 7.8 2.8 3.4 × 10 –7 7–8c 190c
b
Cyazofamid 153 0.12 3.2 1.3 × 10 –5 3–5 >140
Amisulbrom 129–130 0.11 4.4 2.8 × 10 –5 3–13 3
Ametoctradin 198 0.15 4.4 2.1 × 10 –10 1–3 >28 000
a
At 25 ◦ C.
b
At 35 ◦ C.
c
Ref. [79].
Data adapted from Ref. [67] if not otherwise noted.
15.2.7.1 Azolones
As with strobilurins, the central starting point for the azolone fungicides was again
academic research, this time conducted by the group of D. Geffken in Germany.
The research scheme, which basically was ‘‘pure chemistry’’ without a specific
biological target, led to a structure – a thioxo-oxazolidinone (Figure 15.2.11) – that
subsequently was transferred to the research team at Du Pont.
At Du Pont, having first identified the fungicidal activity of the azolones, an
optimization program was carried out [95] that led finally, ‘‘. . . after 3 years of
work and the preparation of over 700 analogs’’ [95b], to an optimized structure,
famoxadone (for patents, see Ref. [96]). Initially, in vivo SARs for famoxadone were
reported with the Oomycetes Phytophthora infestans and Plasmopara viticola [95],
after which the compound was launched in 1996 as a broad-spectrum fungicide
for the control of diseases caused by Ascomycetes and Basidiomycetes in various
crops, and particularly against downy mildew diseases caused by Oomycetes in
potato, vines, and vegetables [97].
A second commercial fungicide from this azolone group, fenamidone, was
announced in 1998 [98]. The compound had been developed initially by Rhône-
Poulenc (as part of Aventis’s agrochemical interests, later merged into Bayer
CropScience; for patents, see Ref. [99]; for QSARs of fenamidone analogs with
Agaricus campestris mitochondria, see Ref. [100]). Interestingly, only the active
S-enantiomer of the chirally active ingredient [101] has been developed and
distributed as a fungicide, in an attempt to reduce environmental loading. The
main agricultural target for fenamidone was the control of downy mildews and late
blights in a variety of crops [98, 102], as with famoxadone.
Similar to strobilurins, the azolones bind to the Qo site of the bc1 complex
[103], [104], and also form a hydrogen bridge to Glu272 (yeast numbering). Their
binding is also prevented in the Gly143Ala (yeast numbering) mutants, as shown
in Figure 15.2.11. The latter finding explains the cross-resistance of azolones
and strobilurins identified in almost all cases of practical relevance to date [105]
Gly143
NH Glu272 NH NH
N
H
O N S O N O O N S
O O N
Lead structure famoxadone S-fenamidone

from D. Geffken
Figure 15.2.11 Azolones: lead structure and commercial

products. Target interaction sites for the hydrogen bridge
binding (Glu272), and for the point mutation at Gly143 that
causes resistance, are sketched together with the famox-
adone formula.
(see also Chapter 14).In summary, although azolone positioning in the enzyme
niche does not completely overlap with the respective areas where strobilurins
are located during bc1 binding (cf. Ref. [106]), kinetic studies have demonstrated
differences between the strobilurin (MOAS) and famoxadone binding modes.
Whereas, famoxadone binds in a noncompetitive manner, MOAS is described
as having mixed competitive/noncompetitive binding in relation to ubiquinole
[103b].
Based on the common mode of action of strobilurins and azolones, the treated
fungi are particularly sensitive during their energy-demanding spore-germination
stage. In the case of Oomycetes, not only zoospore liberation and motility (both
high-energy-demanding processes) but also zoospore cellular integrity [103a, 107]
are extremely sensitive to Complex III inhibitors [6, 98, 102].
15.2.7.2 N-(N , N -Dimethylaminosulfonyl)azoles

As a group, the dimethylaminosulfonylazoles (Figure 15.2.12) exhibit a very high
fungicidal activity against Oomycetes (now termed Peronosporomycetes), with the
fungicidal activity being directed specifically against this fungal class and against
the Plasmodoriophoromycete Plasmodiophora brassicae. In addition, this group of
fungicides has two other points in common: (i) chemically, the dimethylaminosul-
fonyl moiety that is linked with an electron-poor azole ring; and (ii) biochemically,
a common mode of action with Complex III inhibitors at the Qi site, in contrast to
the strobilurins, which bind at the Qo site.
Cyazofamid was announced [108] and introduced commercially in 2001, as a
fungicide for the control of late blight and downy mildews (see Ref. [109] for
details of the mode of action). Amisulbrom [79], which has been developed by
Nissan for similar uses, entered official trials in Japan in 2003 and achieved market
introduction in 2008.
Br
Br
Cl N SO2
N N N
F3C
CN CN N
N N N
SO2NMe2 SO2NMe2 F SO2NMe2
cyazofamid CGA 52232 RP amisulbrom

(proposed common
name dimefluazole)
Figure 15.2.12 The Qi site inhibitor cyazofamid and analogous compounds.
NH2
N N
N N
ametoctradin Figure 15.2.13 Structure of ametoctradin.
Synthesis of kresoxim-methyl and dimoxystrobin

ONa
1.
1. SOCl2 A: R = H
R O O O
+
O 2. H CO2H 2. NaCN 1. H+/MeOH O O
O NC O N
2. H2NOMe
R R O
1. H+/MeOH kresoxim-methyl
B: R = Me
2. H2NOMe
O H2NMe O
O O O O
N N
O NH
Synthesis of trifloxystrobin dimoxystrobin
CF3 ONa
N
1.
Cl CF3 O
N
O + O O
O 2. H
N N
O O
trifloxystrobin
Synthesis of metominostrobin
1. n-BuLi 1. H2NOMe
O O
2. (COOMe)2 O 2. MeNH2 O O
O O N
O NH
metominostrobin
Synthesis of orysastrobin
HO
HO O O
1. HONO N N H2NOMe N
O O O O O
O O
2. Me2SO4 H+
O O N
O
N OH
H2NOH O N 1. Base MeNH2 N O
O N
+
H N O O
N N
O Cl
2. O NH
O O
N
O orysastrobin
Scheme 15.2.1 Synthesis routes to strobilurins with the oximino pharmacophores.

Although dimefluazole did not achieve commercial status, this compound and
an analog were the subject of a detailed investigation of the mode of action [110].
It was concluded that the azole moiety acts as a leaving group, after which the
sulfonyl group of the active ingredient can bind covalently to a nucleophile of the
Qi-center of Oomycetes [110]. The different submolecular target sites of cyazofamid
and Qo-site inhibitors led to lack of cross-resistance.
15.2.7.3 Ametoctradin
Ametoctradin [111] (Figure 15.2.13) has been developed by BASF to combat
downy mildew and late blight diseases caused by Oomycetes, and is now in
early market introduction in mixtures with other fungicides. Its structure, as a
triazolopyrimidine, is not at all related to other Complex III inhibitors, and its
exact binding site in the complex has not yet been established. Nevertheless,
ametoctradin shows no cross-resistance to Qo-inhibiting strobilurins or azolones.
It also lacks the essential dimethylaminosulfonyl moiety of the above-mentioned Qi
site inhibitors (cf. Figure 15.2.12). Ametoctradin’s history dates back to the 1980s,
when the first derivatives of this type of Oomycete fungicide were discovered
[112].
Synthesis of azoxystrobin
N N
1. HCOOMe
NaH NaOMe NaO Cl Cl
O O
2. Me2CO3 O O
O
O O
O
N N N N
OH
Cl O CN O
O
O O Base CN O O
O O
azoxystrobin
Synthesis of picoxystrobin
1. HCOOMe Cl F3C N
NaH HCl 1. Base O
O 2. Me2CO3 O O O O F3C N
O O
OH
O O O 2. O
picoxystrobin
Scheme 15.2.2 Synthesis routes to strobilurins with the methoxyacrylate pharmacophore.

15.2.8
Synthesis Routes
The schemes described in this section outline, for each of the above-mentioned,
commercialized fungicides, one possible route of synthesis as taken from reported
sources (mostly patents). For chemists, the schemes should provide an impression
of how individual compounds can be synthesized, and they also suggest synthetic
strategies and chemical reactions on which the technical processes are based. This
Synthesis of fluoxastrobin
O
H2HOMe t-BuOK
HO HO O Base
OMe t-BuONO
Br Br OMe
O N N
HO N
1. Base N N
KOH
O H2O HO F O
OMe N OMe 2. N OMe
HO N N F
O N N O N O N
O F F O
F
OH N N
Cl
O O
Base
Cl F N O
O N
O
fluoxastrobin
Synthesis of pyraclostrobin
EtO O
Cl 1. Cl Cl
1. Base
NH2 N
N
2. O2 N OH N N O
H
2. Cl NO2
NO2
Cl Cl
H2 1. MeOCOCl
N O N O
catalyst N 2. Me2SO4 N
NHOH Base O N
O
O
pyraclostrobin
Scheme 15.2.3 Synthesis routes to fluoxastrobin and pyraclostrobin.

is not necessarily so in each case, however, as production processes normally are

not reported publically.
For a synthetic organic chemist, the schemes are self-explanatory and require
no detailed comment. However, a few points deserve special comment. In the
strobilurins, the ortho substitution pattern at the central bridging ring favors the
use of easily accessible starting materials or intermediates in which a lactone-type
ring is cleaved to obtain pharmacophores and side chains built up in the correct
Synthesis of pyribencarb
Cl Cl
Radical KOCN
O O
chlorination CH3OH
Cl
Cl Cl
O NH2OH - HCl N
HO
O NH O NH
O O
N Cl
Cl
HCl N N
O pyribencarb
Base O NH
O
Scheme 15.2.4 Synthesis route to pyribencarb.
Synthesis of famoxadone
O OMe
O NH
OH
O N O
1. N N N N
O
O
2. PhNHNH2
AcOH
O
famoxadone
Synthesis of fenamidone
O OMe O OMe
NH
Cl2CS 1, PhNHNH2
NH2 NCS O N S
Base 2. t-BuOK, MeI
N
S-fenamidone
Scheme 15.2.5 Synthesis routes to the azolones.

ortho connection. As noted at the end of Section 15.2.3.4, the thermodynamically

favored (E)-configuration of the different oximino groups can usually be obtained
by equilibration under acidic conditions.
Synthetic routes to the Chinese strobilurins of Figure 15.2.4 follow, in principle,
Schemes 15.2.1 to 15.2.4 as used for the earlier commercial strobilurin, and are
therefore not described specifically.
The chiral starting material S-methyl phenyl glycine required in the synthesis of
fenamidone (Scheme 15.2.5) can be obtained via enzymatic resolution processes.
Synthesis of cyazofamid
H
O OH
O
CHCl2 N
H 1. SOCl2 / DMF
O
3 H2NOH / H + N+ CH=NOH
2. SCl2
MeOH / H2O O−
5
Cl Cl
N ClSO2NMe2 N
N CN N CN
K2CO3 / DMF
H SO2NMe2
6 cyazofamid
Scheme 15.2.6 Synthesis route to cyazofamid.
Synthesis of amisulbrom
Br
H2, Pd-C 47% HBr
O N DMSO
F NO2 NaOAc F N
Ac2O H toluene F
H
A
N ClSO2NMe2 N Cl2 N
NH N ClO2S N SO NMe
S N Na2CO3 S N SO2NMe2 MeOH-H2O N 2 2
2 ClCH2CH2Cl 2 ClCH2CH2Cl
B
Br
48% NaOH
A + B amisulbrom
Bu4N+ Br− F N N
toluene SO2
N N SO NMe
2 2
Scheme 15.2.7 Synthesis route to amisulbrom.

References 621
Synthesis of ametoctradin
OEt
N O N
Base
O
N NH
NH2
N NH2 N N
ametoctradin
ClSO3H N N
Scheme 15.2.8 Synthesis route to ametoctradin.
Alternatively, chirality can be introduced at a later, cyclic stage by using a chiral

reagent that resolves an intermediate hydantoin [101].
In the cyazofamid synthesis (Scheme 15.2.6), a discrete all-in-one reaction can
be used to convert 5 into 6; this involves a dehydration, two deoxygenations and a
chlorination at the imidazole ring system in a one-pot reaction [113].
Acknowledgments
The author expresses his great thanks to the many colleagues at BASF engaged in
strobilurin research, development, production, and marketing, for their outstand-
ing contributions to this fascinating and inspiring field. Particular thanks go to
William K. Moberg, for many enlightening discussions and invaluable editorial
assistance in preparing the manuscript.
References
1. Philips McDougall AgriService (2010) ACS Symposium Series, Vol. 551

Products Section – 2009 Market, pp. (eds P.A. Hedin, J.J. Menn, and R.M.
205–295. Hollingworth), American Chemical
2. Beautement, K., Clough, J.M., de Society, Washington, DC, pp. 37–53.
Fraine, P.J., and Godfrey, C.R.A. (1991) 5. Clough, J.M. and Godfrey, C.R.A.
Pestic. Sci., 31, 499–519. (1998) in Fungicidal Activity, Chemi-
3. Clough, J.M., de Fraine, P.J.,
cal and Biological Approaches to Plant
Fraser, T.E.M., and Godfrey, C.R.A.
Protection (eds D.H. Hutson and J.
Miyamoto), John Wiley & Sons, Inc.,
New York, pp. 109–148.
6. Bartlett, D.W., Clough, J.M.,
Fenyes, and J.J. Steffens), American
Chemical Society, Washington, DC, Godwin, J.R., Hall, A.A., Hamer, M.,
pp. 372–383. and Parr-Dobrzanski, B. (2002) Pest
4. Clough, J.M., Evans, D.A., Manage. Sci., 58, 649–662.
de Fraine, P.J., Fraser, T.E.M., Godfrey, 7. Sauter, H., Ammermann, E., and
C.R.A., and Youle, D. (1994) in Natural Roehl, F. (1996) in Crop Protection
and Engineered Pest Management Agents, Agents From Nature (ed. L.G. Copping),
The Royal Society of Chemistry, Cam- 23. (a) Trowitzsch, W., Reifenstahl,
bridge, pp. 50–80. G., Wray, V., and Gerth, K. (1980)
8. Sauter, H., Ammermann, E., J. Antibiot., 33, 1480–1490; (b)
Benoit, R., Brand, S., Gold, R.E., Trowitzsch, W., Höfle, G., and
Grammenos, W., Köhle, H., Sheldrick, W.S. (1981) Tetrahedron
Lorenz, G., Müller, B., Röhl, F., Lett., 22, 3829–3832.
Schirmer, U., Speakman, J.B., 24. Reichenbach, H., Gerth, K.,
Wenderoth, B., and Wingert, H. (1995) Irschick, H., Kunze, B., and Höfle, G.
in Antifungal Agents – Discovery and (1988) Trends Biotechnol., 6, 115–121.
Mode of Action (eds G.K. Dixon, L.G. 25. (a) Höfle, G., Augustiniak, H.,
Copping, and D.W. Hollomon), BIOS Behrbohm, H., Böhlendorf, B.,
Scientific Publishers, Oxford, pp. Herrmann, M., Hölscher, A.,
173–191. Jahn, T., Jansen, R., Kiffe, M.,
9. Sauter, H., Steglich, W., and Anke, T. Lautenbach, H., Schlummer, D.,
(1999) Angew. Chem. Int. Ed., 38, Söker, U., Stammermann, T.,
1328–1349. Steinmetz, H., Washausen, P.,
10. Zakharychev, V.V. and Kovalenko, L.V. and Wray, V. (1994) Scientific
(1998) Russ. Chem. Rev., 67, 535–544. Annual Reports, Gesellschaft für
11. Belan, S.R. (2003) Agrokhimiya, 11, Biotechnologische Forschung, Braun-
27–32. schweig, pp. 125–129; (b) Höfle, G.,
12. Liu, A. (2003) Jingxi Huagong Reichenbach, H., Böhlendorf, B.,
and Sasse, F. (1994) Gesellschaft
Zhongjianti, 33, 1–5.
für Biotechnologische Forschung,
13. Thind, T.S., Mohan, C., Rai, Prem, and
Braunschweig, Germany, Patent WO,
Arora, J.K. (2004) Indian Phytopathol.,
95/26,414, priority date March 25,
57, 104–106.
1994.
14. Balba, H. (2007) J. Environment. Sci.
26. Clough, J.M. (1993) Nat. Prod. Rep., 10,
Health B, 42, 441–445.
565–574.
15. Musilek, V., Cerna, J., Sasek, V.,
27. Becker, W.F., von Jagow, G., Anke, T.,
Semerzieva, M., and Vondracek, M.
and Steglich, W. (1981) FEBS Lett., 132,
(1969) Folia Microbiol., 14, 377–387.
329–333.
16. Anke, T., Oberwinkler, F., Steglich, W.,
28. Bushell, M.J., Beautement, K.,
and Schramm, G. (1977) J. Antibiot.,
Clough, J.M., DeFraine, P.,
30, 806–810. Anthony, V.M., and Godfrey, C.R.A.
17. Anke, T., Schramm, G., Schwalge, B., (1984) (ICI), Patent EP 178,826, priority
Steffan, B., and Steglich, W. (1984) dates October 19, 1984 and December
Liebigs Ann. Chem., 1984, 1616–1625. 20, 1984.
18. von Jagow, G., Gribble, G.W., and 29. (a) Schirmer, U., Karbach, S., Pommer,
Trumpower, B.L. (1986) Biochemistry, E.H., Ammermann, E., Steglich,
25, 775–780. W., Schwalge, B.A.M., and Anke, T.
19. Anke, T., Hecht, H.J., Schramm, G., 1985 (BASF): Patent EP 203,606,
and Steglich, W. (1979) J. Antibiot., 32, priority date May 30, 1985; (b)
1112–1117. Schirmer, U., Karbach, S., Pommer,
20. Anke, T. and Steglich, W. (1989) in Bi- E.H., Ammermann, E., Steglich, W.,
ologically Active Molecules: Identification, Schwalge, B.A.M., and Anke, T. (1985)
Characterization and Synthesis (ed. U.P. Patent EP 203,608, priority date May
Schlunegger), Springer-Verlag, Berlin, 30, 1985; (c) Schirmer, U., Karbach, S.,
Heidelberg, pp. 9–25. Pommer, E.H., Ammermann, E.,
21. Sasse, F., Leibold, T., Kunze, B., Steglich, W., Schwalge, B.A.M., and
Höfle, G., and Reichenbach, H. (2003) Anke, T. (1985) Patent EP 226,917,
J. Antibiot., 56, 827–831. priority date December 20, 1985; (d)
22. Gerth, K., Irschick, H., Schirmer, U., Karbach, S., Pommer,
Reichenbach, H., and Trowitzsch, W. E.H., Ammermann, E., Steglich, W.,
(1980) J. Antibiot., 33, 1474–1479. Schwalge, B.A.M., and Anke, T. (1985)
References 623
Patent EP 229,974, priority date De- (Shionogi), Patent EP 398,692, priority

cember 20, 1985. date May 17, 1989.
30. Wenderoth, B., Rentzea, C., 39. Brand, S., Ammermann, E., Lorenz, G.,
Ammermann, E., Pommer, E.H., Sauter, H., Oberdorf, K., Kardorff, U.,
Steglich, W., and Anke, T. (1986) and Künast, C. (1990) (BASF), Patent
(BASF), Patent EP 253,213, priority EP 477,631, priority date September 22,
date July 16, 1986. 1990.
31. Anthony, V.M., Clough, J.M., Godfrey, 40. Moronval, M.H. and Senechal, Y.
C.R.A., and Wiggins, T.E. (1986) (ICI), (2003) 7 ème Conference Internationale
Patent EP 254,426, priority date July sur les Maladies des Plantes (CIMA),
18, 1986. Tours, France, 2003.
32. Godwin, J.R., Anthony, V.M., 41. De Fraine, P.J. and Martin, A. (1988)
Clough, J.M., and Godfrey, C.R.A. (ICI), Patent EP-A, 370,629, priority
(1992) Proceedings of the Brighton Crop dates November 21, 1988 and March 9,
Protection Conference – Pests and Dis- 1989.
eases, BCPC, Farnham, pp. 435–442. 42. Isenring, H.P. and Weis, B. (1990)
33. Ammermann, E., Lorenz, G., (Ciba-Geigy), Patent EP 460,575, prior-
Schelberger, K., Wenderoth, B., ity date June 5, 1990.
Sauter, H., and Rentzea, C. (1992) 43. Brand, S., Kardorff, U., Kirstgen, B.,
Proceedings of the Brighton Crop Pro- Müller, B., Oberdorf, K., Sauter, H.,
tection Conference – Pests and Diseases, Lorenz, G., Ammermann, E.,
BCPC, Farnham, pp. 403–410. Künast, C., and Harreus, A. (1990)
34. Köhle, H., Gold, R.E., (BASF), Patent EP 463,488, priority
Ammermann, E., Sauter, H., and date June 27, 1990.
Röhl, F. (1994) Biochem. Soc. Trans., 44. Ziegler, H., Benet-Buchholz, J.,
22, 65S. Etzel, W., and Gayer, H. (2003)
35. Gold, R.E., Ammermann, E., Pflanz.-Nachr. Bayer, 56, 213–230.
Köhle, H., Leinhos, G.M.E., Lorenz, G., 45. Margot, P., Huggenberger, F.,
Speakman, J.B., Stark-Urnau, M., and Amrein, J., and Weiss, B. (1998) Pro-
Sauter, H. (1996) in Modern Fungicides ceedings of the Brighton Crop Protection
and Antifungal Compounds (eds H. Lyr, Conference – Pests and Diseases, BCPC,
P.E. Russell and H.D. Sisler), Intercept, Farnham, pp. 375–382.
Andover, pp. 79–92. 46. Godwin, J.R., Bartlett, D.W., Clough,
36. Hayase, Y., Kataoka, T., Masuko, M., J.M., Godfrey, C.R.A., Harrison, E.G.,
Niikawa, M., Ichinari, M., and Maund, S. (2000) Proceedings of
Takenaka, H., Takahashi, T., the Brighton Crop Protection Confer-
Hayashi, Y., and Takeda, R. (1995) in ence – Pests and Diseases, BCPC, BCPC,
Synthesis and Chemistry of Agrochemicals Farnham, pp. 533–540.
IV, ACS Symposium Series, Vol. 584 47. (a) Ammermann, E., Lorenz, G.,
(eds D.R. Baker, J.G. Fenyes, and G.S. Schelberger, K., Müller, B.,
Basarab), American Chemical Society, Kirstgen, R., and Sauter, H. (2000)
Washington, DC, pp. 343–353. Proceedings of the Brighton Crop Pro-
37. (a) Mizutani, A., Yukioda, H., tection Conference – Pests and Diseases,
Tamura, H., Miki, N., Masuko, M., BCPC, Farnham, pp. 541–548; (b)
and Takeda, R. (1995) Phytopathol- Stierl, R., Ammermann, E., Lorenz,
ogy, 85, 306–311; (b) Mizutani, A., G., and Schelberger, K. (2002) in
Miki, N., Yukioda, H., and Masuko, Modern Fungicides and Antifungal
M. (1996) in Modern Fungicides and Compounds III, Proceedings 13th Inter-
Antifungal Compounds (eds H. Lyr, P.E. national Reinhardsbrunn Symposium
Russell, and H.D. Sisler), Intercept, (eds H.-W. Dehne, U. Gisi, K.H. Kuck,
Andover, pp. 93–99. P.E. Russell, and H. Lyr), AgroConcept,
38. Hayase, Y., Kataoka, T., Takenaka, H., Bonn, pp. 49–59.
Ichinari, M., Masuko, M., 48. (a) Müller, B., Sauter, H., Röhl, F.,
Takahashi, T., and Tanimoto, N. (1989) Dötzer, R., Lorenz, G., and
Ammermann, E. (1992) (BASF), Patent 95/014,009, priority date November 19,

WO, 93/015,046, priority dates January 1993.
29, 1992, June 26, 1992, and October 57. Brown, R.J., Ashworth, B., Drumm,
5, 1992; (b) Ohnishi, H., Tajma, S., J.E., Frazier, D.A., Hanagan, M.A.,
Yamamoto, Y., and Kanno, H. (1993) Happerset, C., Koether, G.E., Robinson,
(Nihon Nohyaku), Patent EP 619,301, D.J., Sun, K.-M., and Woitkowsky, P.
priority date April 4, 1993. (2000) in Synthesis and Chemistry of
49. (a) Köhle, H., Grossmann, K., Jabs, T., Agrochemicals VI, ACS Symposium
Gerhard, M., Kaiser, W., Glaab, J., Series, Vol. 800 (eds D.R. Baker, J.G.
Conrath, U., Seehaus, K., and Herms, Fenyes, G.P. Lahm, T.P. Selby, and
S. (2002) in Modern Fungicides and T.M. Stevenson), American Chem-
Antifungal Compounds III, Proceedings ical Society, Washington, DC, pp.
13th International Reinhardsbrunn Sym- 327–339.
posium (eds H.-W. Dehne, U. Gisi, 58. (a) Ozaki, M., Fukumoto, S., Tamai, R.,
K.H. Kuck, P.E. Russell, and H. Lyr), Ikegaya, K., Yonekura, N., Kawashima,
AgroConcept, Bonn, pp. 61–74.; (b) T., Sakai, J., Muramatu, N., Takagaki,
Conrath, U., Amoroso, G., Köhle, H., M., and Nagayama, K. (1999) (Kumiai
and Sültemeyer, D.F. (2004) Plant and Ihara), Patent EP 1, 201,648, prior-
J., 38, 1015–1022; (c) Conrath, U., ity date August 05, 1999 (b) Takagaki,
Pieterse, C.M.J., and Mauch-Mani, B. M., Kataoka, S., Kida, K., Miura, I.,
(2002) Trends Plant Sci., 7, 210–216;
Fukumoto, S., and Tamai, R. (2010) J.
(d) Herms, S., Seehaus, K., Köhle, H.,
Pestic. Sci., 35, 10–14; (c) Kataoka, S.,
and Conrath, U. (2002) Plant Physiol.,
Takagaki, M., Kaku, K., and Shimizu,
130, 120–127; (e) Venancio, W.S.,
T. (2010) J. Pestic. Sci., 35, 99–106.
Rodrigues, M.A.T., Begliomini, E., and
59. (a) Huang, W., Zhao, P.-L., Liu, C.-L.,
de Souza, N.L. (2003) EUPG Ci. Exatas
Chen, Q., Liu, Z.-M., and Yang,
Terra, Ci. Agr. Eng., Ponta Grossa, 9,
G.-F. (2007) J. Agric. Food Chem., 55,
59–68.
3004–3010; (b) Zhao, P.-L., Liu, C.-L.,
50. Heinemann, U., Benet-Buchholz, J.,
Huang, W., Wang, Y.-Z., and Yang,
Etzel, W., and Schindler, M. (2004)
G.-F. (2007) J. Agric. Food Chem., 55,
Pflanz.-Nachr. Bayer, 57, 299–318.
5697–5700; (c) Liu, C.-L., Li, M., Guan,
51. Häuser-Hahn, I., Baur, P., and
Schmitt, W. (2004) Pflanz.-Nachr. A.-Y., Zhang, H., and Li, Z.-M. (2007)
Bayer, 57, 437–450. Nat. Prod. Commun., 2, 845–848; (d)
52. Dutzmann, S., Hayakawa, H., Li, M., Liu, C.-L., Li, L., Yang, H., Li,
Oshima, A., and Suty-Heinze, A. (2004) Z.-N., Zhang, H., and Li, Z.-M. (2010)
Pflanz.-Nachr. Bayer, 57, 415–435. Pest Manage. Sci., 66, 107–112.
53. Stierl, R., Grote, T., Bross, M., 60. Zhang, L.-X., Li, Z.-C., Li, Z.N.,
Washioka, S., and Schöfl, U. (2004) Zhang, H., Liu, C.-L., Li, B., and
Proceedings of the 15th International Shaber, S.H. (1998) (Rohm and Haas,
Plant Protection Congress, Beijing, Dow), Patent EP 936,213, priority date
China, 2004, p. 166 February 10, 1998. The Rohm and
54. (a) IUPAC (1993) Pure Appl. Chem., 65, Haas agrochemical business unit is
2003–2122; (b) Spacie, A., McCarty, now merged into Dow Agrosciences.
L.S., and Rand, G.M. (1993) in Funda- 61. Zhang, L.-X. and Shaber, S. (2000)
mentals of Aquatic Toxicology (ed. G.M. Patent WO, 2002/012172 (Dow Agro-
Rand),Taylor and Francis, Washington, sciences), priority date August 8,
DC, pp. 493–521; (b) Moriarty, F. 2000.
(1999) Ecotoxicology, 3rd edn, Academic 62. Zhang, L.-X. et al. (2003) Proceedings
Press, San Diego, CA, pp. 177–193. of the BCPC International Congress,
55. Walker, M.P. (2003) Chimia, 57, Glasgow, 2003, Vol. 1, p. 93
675–679. 63. Liu, C.-L., Guan, A.-Y., Zhang, H.,
56. Brown, R.J., Sun, K.-M., and Frasier, Zhang, M.-X., Li, Z.-M., Li, M., Li, L.,
D.A. (1993) (Du Pont), Patent WO, Li, Z.-N., and Hou, C.-Q. (2003) Patent
References 625
EP 169,3729, (Shenyang Res. Inst.), 76. (a) Briggs, G.G., Bromilow, R.H., and
priority date November 11, 2003. Evans, A.A. (1982) Pestic. Sci., 13,
64. Liu, C.-L., Li, M., Zhang, H., Li, L., 495–504; (b) Briggs, G.G., Bromilow,
Zhang, M.-X., Guan, A.-Y., Hou, C.-Q., R.H., Evans, A.A., and Williams, M.
Li, Z.-N., and Jia, J.-G. (2004) Patent (1983) Pestic. Sci., 14, 492–500.
WO, 2005/080,344, (Shenyang Res. 77. Jacob, F. and Neumann, S. (1987) in
Inst.), priority date February 20, 2004. Modern Selective Fungicides (ed. H. Lyr),
65. Liu, C.-L., Li, M., Li, Z.-N., Geng, Longman Scientific Technical, Harlow,
L.-W., Zhang, H., Zhou, D., Cui, D.-L., pp. 13–30.
and Luo, Y.-M. (2005) Patent WO, 78. Grote, T., Bayer, H., Müller, R.,
2006/125,370, (Shenyang Res. Inst.), Sauter, H., Kirstgen, R., Harries,
priority date May 26, 2005. V., Lorenz, G., and Ammermann, E.
66. (a) Briggs, G.G. (1981) Proceedings of (1995) Strathmann (BASF), S., Patent
the Brighton Crop Protection Confer- WO, 97/015,552 (BASF), priority date
ence – Pests and Diseases, BCPC, Farn- October 23, 1995.
ham, pp. 701–710; (b) Graham-Bryce, 79. Davies, M. and Sheik, A. (eds) (2005)
I.J. (1994) in Pesticide Synthesis Through AGRO Projects, Disease Projects Vol.
Rational Approaches, ACS Sympo- 2 – Product Profiles, PJB Publications
sium Series, Vol. 255 (eds P.S. Magee, Ltd, London (periodically updated).
G.K. Kohn, and J.J. Menn), American 80. Beck, C., Oerke, E.-C., and Dehne,
Chemical Society, Washington, DC, pp. H.-W. (2002) in Modern Fungicides and
185–207; see also: (c) Clarke, E. and
Antifungal Compounds III, Proceedings
Delaney, J. (2003) Chimia, 57, 731–734.
13th International Reinhardsbrunn Sym-
67. Tomlin, C.D.S. (ed.) (2003) The Pes-
posium (eds H.-W. Dehne, U. Gisi,
ticide Manual, 13th edn, British Crop
K.H. Kuck, P.E. Russell, and H. Lyr),
Protection Council, BCPC Publications,
AgroConcept, Bonn, pp. 41–49.
Alton.
81. Venancio, W.S., Rodrigues, M.A.T.,
68. Röhl, F. and Sauter, H. (1994) Biochem.
Begliomini, E., and de Souza, N.L.
Soc. Trans., 22, 63S.
(2003) Publ. UEPG Exact Soil Sci., Agr.
69. Link, T.A., Iwata, M., Björkman, J.,
Sci. Eng., Ponta Grossa, 9, 59–68.
van der Spoel, D., Stocker, A., and
82. Bertelsen, J.R., de Neergard, E., and
Iwata, S. (2003) in Chemistry of Crop
Sedegaard-Petersen, V. (2001) Plant
Protection (eds G. Voss and G. Ramos),
Wiley-VCH Verlag GmbH, Weinheim, Pathol., 50, 190–205.
pp. 110–127. 83. Sauter, H., Lauer, M., and Fritsch, H.
70. Zheng, Y.-J. (2005) J. Mol. Graph. (1991) in Synthesis and Chemistry of
Model., 25, 71–76. Agrochemicals II, ACS Symposium
71. Esser, L., Quinn, B., Li, Y.-F., Series, Vol. 443 (eds D.R. Baker, J.G.
Zhang, M., Elberry, M., Yu, L., Yu, Fenyes, and W.K. Moberg), American
C.-A., and Xia, D. (2004) J. Mol. Biol., Chemical Society, Washington, DC, pp.
341, 281–302. 288–299, (cf. (1988) Abstr. Pap. Am.
72. Brown, R.J., Annis, G., Casalnuovo, Chem. Soc., 195, 129).
A., Chan, D., Shapiro, R., and 84. (a) Fernie, A.R., Trethewey, R.N.,
Marshall, W.J. (2004) Tetrahedron, Krotzky, A.J., and Willmitzer, L.
60, 4361–4375. (2004) Nat. Rev. Mol. Cell Biol., 5,
73. Zheng, Y.-J. and Kleier, D.A. (2005) J. 1–7; (b) Trethewey, R.N. (2006) in
Mol. Struct. Theochem., 719, 69–74. Plant Metabolomics (eds K. Saito, R.A.
74. Ypema, H.L. and Gold, R.E. (1999) Dixon, and L. Willmitzer), Springer,
Plant Dis., 83, 4–19. Berlin, Heidelberg, pp. 327–339; (c)
75. Clark, W.S. (2005) Proceedings of the www.metanomics.de, (accessed 2011).
BCPC International Congress – Crop 85. Saito, K., Dixon, R.A., and
Science and Technology, The British Willmitzer, L. (eds) (2006) Plant
Crop Protection Council, Alton, pp. Metabolomics, Springer, Berlin, Hei-
283–290. delberg.
86. (a) Nauen, R. and Bretschneider, T. 96. Geffken, D., Rayner, D.R., and John,
(2002) Pestic. Outlook, 13, 241–246; (b) W.N. (1989) (Du Pont), Patent WO,
Dekeyser, M.A. (2005) Pest Manage. 90/012,719 priority date April 21,
Sci., 61, 103–110. 1989.
87. Kirstgen, R., Oberdorf, K., Schütz, F., 97. Joshi, M.M., and Sternberg, J.A. (1996)
Theobald, H., and Harries, V. (1995) Proceedings of the Brighton Crop Pro-
Patent WO, 96/016,047 (BASF), priority tection Conference – Pests and Diseases,
dates November 17, 1994 and July 21, BCPC, Farnham, pp. 21–26.
1995. 98. Mercer, R.T., Lacroix, G., Gouot, J.M.,
88. Liu, A., Wang, X., Chen, C., Pei, H., and Latorse, M.P. (1998) Proceedings
Mao, C., Wang, Y., He, H., Huang, L., of the Brighton Crop Protection Con-
Liu, X., Hu, Z., Ou, X., Huang, M., ference – Pests and Diseases, BCPC,
and Yao, J. (2009) Pest Manage. Sci., 65, Farnham, pp. 319–326.
229–243. 99. (a) Lacroix, G., Peignier, R., and
89. (a) Gisi, U., Sierotzky, H., Cook, A., Pepin, R. (1992) (Aventis Crop Sci-
and McCaffery, A. (2002) Pest Manage. ence), Patent EP 551,048, priority date
Sci., 58, 859–867; (b) Kuck, K.-H. and December 16, 1992; (b) Bascou, J.-P.,
Mehl, A. (2003) Pflanz.-Nachr. Bayer, Lacroix, G., Gadras, A., and Perez, J.
56, 313–325. (1994) (Aventis Crop Science), Patent
90. Wood, P.M. and Hollomon, D.W. EP 629,616, priority date June 10,
(2003) Pest Manage. Sci., 59, 499–511. 1994.
91. Jabs, T., Cronshaw, K., and Freund, A. 100. Genix, P. (2003) Pflanz.-Nachr. Bayer,
(2001) Phytomedizin, 31, 15–16. 56, 435–443.
92. (a) Fisher, N., Brown, A.C., Sexton, G., 101. Genix, P., Guesnet, J.-L., and
Cook, A., Windass, J., and Meunier, Lacroix, G. (2003) Pflanz.-Nachr. Bayer,
B. (2004) Eur. J. Biochem., 271, 56, 421–434.
2264–2271; (b) Grasso, V., Sierotzki, 102. Mercer, R.T. and Latorse, M.P. (2003)
H., Garibaldi, A., and Gisi, U. (2006) J. Pflanz.-Nachr. Bayer, 56, 465–476.
Phytopathol., 154, 110–118. 103. (a) Jordan, D.B., Livingston, R.S.,
93. Kraiczy, P., Haase, U., Gencic, S., Bisaha, J.J., Duncan, K.E., Pember,
Flindt, S., Anke, T., Brandt, U., and S.O., Picollelli, M.A., Schwartz, R.S.,
von Jagow, G. (1996) Eur. J. Biochem., Sternberg, J.A., and Tang, X.-S. (1999)
235, 54–63. Pestic. Sci., 55, 105–118; (b) Pember,
94. Stammler, G., Strobel, D., Semar, M., S.O., Fleck, L.C., Moberg, W.K., and
and Klappach, K. (2006) Fungicide Re- Walker, M.P. (2005) Arch. Biochem.
sistance: Are We Winning the Battle but Biophys., 435, 280–290.
Losing the War? Aspects of Applied 104. Neuburger, M., Beffa, R., and
Biology, Vol. 78, The Association of Villier, A. (2003) Pflanz.-Nachr. Bayer,
Applied Biologists, Wellesbourne, pp. 56, 449–464.
29–36. 105. (a) Heaney, S.P., Hall, A.A., Davies,
95. (a) Sternberg, J.A., Geffken, D., Adams, S.A., and Olaya, G. (2000) Proceed-
J.B. Jr, Jordan, D.B., Pöstages, R., ings of the Brighton Crop Protection
Sternberg, C.G., Campbell, C.L., Conference – Pests and Diseases,
Moberg, W.K., and Livingston, R.S. BCPC, Farnham, pp. 755–762; (b)
(1998) in Synthesis and Chemistry of QO I-fungicides, FRAC code list 2011,
Agrochemicals V, ACS Symposium www.frac.info, (accessed 2011).
Series, Vol. 686 (eds D.R. Baker, 106. Muller, F.L., Roberts, A.G., Bowman,
J.G. Fenyes, G.S. Basarab, and D.A. M.K., and Kramer, D.M. (2003) Bio-
Hunt), American Chemical Society, chemistry, 42, 6493–6499.
Washington, DC, pp. 216–227; (b) 107. Stierl, R., Scherer, M., Schrof, W.,
Sternberg, J.A., Geffken, D., Adams, and Butterfield, E.J. (2000) Proceed-
J.B. Jr, Pöstages, R., Sternberg, C.G., ings of the Brighton Crop Protection
Campbell, C.L., and Moberg, W.K. Conference – Pests and Diseases, BCPC,
(2001) Pest Manage. Sci., 57, 143–152. Farnham, pp. 261–266.
108. Mitani, S., Araki, S., Matsuo, N., M., Strathmann, S., Schoefl, U., and
and Camblin, P. (1998) Proceedings Stierl, R. (2004) (BASF), Patent WO,
of the Brighton Crop Protection Con- 2005/087,773, priority date March 10,
ference – Pests and Diseases, BCPC, 2004.
Farnham, pp. 351–358. 112. Eicken, K., Graf, H., Gramlich, W.,
109. Mitani, S., Araki, S., Takii, Y., Sauter, H., Rentzea, C., Pommer,
Oshima, T., Matsuo, N., and Miyoshi, E.-H., and Ammermann, E. (1983)
H. (2001) Pest. Biochem. Physiol., 71, Patent EP 112,283, priority date Octo-
107–115. ber 21, 1983.
110. Pillonel, C. (1995) Pestic. Sci., 43, 113. Jonishi, H., Kanamori, F., Kimura, T.,
107–113. Kanbayashi, S., Wakabayashi, T.,
111. (a) Tormo i Blasco, J., Blettner, C., Takenaka, A., Fukui, F., and
Müller, B., Gewehr, M., Grammenos, Horluchi, N. (1995) (Ishihara), Patent
W., Grote, T., Rheinheimer, J., Schäfer, EP 705,823, priority dates September
P., Schieweck, F., Schwögler, A., 08, 1994, October 07, 1994, October 28,
Wagner, O., Niedenbrück, M., Scherer, 1994 and February 16, 1995.
15.3
Succinate Dehydrogenase Inhibitors
15.3.1
Succinate Dehydrogenase Inhibitors: Anilides
Joachim Rheinheimer
As long ago as 1977, a general structure (Figure 15.3.1.1) had been reported for
carboxylic amides as inhibitors of the enzyme, succinate dehydrogenase (SDH),
and this structure still forms the basis of most modern SDH inhibitor molecules
[1]. At that time, approximately ten compounds of this structural class had been
identified as development candidates, or had been introduced into the market. Of
these, carboxin (1) and oxycarboxin (2) (Table 15.3.1.1) are well-known examples
that are still in use today [2].
Currently, the main use for carboxin is in seed dressing, predominantly against
Rhizoctonia spp. in cereals and other crops [3, 15], although Ustilago spp. and Tilletia
spp. can also be treated successfully. Oxycarboxin is active against rust diseases in
cereals, turf, and ornamentals [3]. Two other early compounds were shown to have
R1 R2
H
N
R3
R4n Figure 15.3.1.1 General structure for carboxylic amides as
O
succinate dehydrogenase inhibitors.
Table 15.3.1.1 Active ingredients.
Common • Company Structure

name • Year of introduction
• Melting point (◦ C)
• KOW log P
Carboxin • Uniroyal Chemical CH3

O
Co. [3]
• 1968 [4] H
N
• 91–92 [3] S
• 2.3 [3] O
1
Oxycarboxin • Uniroyal Chemical O CH3
Co. [3] 1971 [5]
H
• 119.5–121.5 [3] N
• 0.77 [3] S
O O
O
2
Benodanil • BASF [3] I
• 1974 [6]
H
• 137 [3] N
• Not available
O
3
Fenfuram • Shell (now Bayer CH3
CropScience) [3] O
H
• 1974 [7] N
• 109–110 [3]
• Not available O
4
Mepronil • Kumiai Chemical CH3
Industry Co. [3]
H
• 1981 [3] N O CH3
• 92–93 [3]
• 3.66 [3] O CH3
5
Flutolanil • Nihon Nohyaku Co. CF3
[3]
H
• 1986 [3] N O CH3
• 104–105 [3]
• 3.7 [3] O CH3
6
Furametpyr • Sumitomo [3] CH3 H3C
• 1997 [8] N O
H3C N H
• 150.2 [3] N CH3
• 2.36 [3] CH3
Cl O
7
Table 15.3.1.1 (continued)

• KOW log P
Thifluzamide • Monsanto (now Dow CF3

AgroSciences) [3] N Br
• 1997 [3] H3C H
N
• 177.9–178.6 [3] S
• 4.16 [3] O
Br O
CF3
8
Boscalid • BASF [3] Cl
• 2003 [3]
• 142.8–143.8 [3]
• 2.96 [3] N Cl
H
N
O
9
Penthiopyrad • Mitsui [3] H3C
CH3
• 2008 CF3 H3C
• 103–105 [9] N
• Not available H3C N H
N
S
O
10
Isopyrazam • Syngenta [10]
• 2010
• Not available CHF 2
N
• Not available H
H3C N
• Mixture of syn- and N
anti-epimers
O
syn
CHF 2
N
H
H3C N N
O
anti
31

• KOW log P
Bixafen • Bayer [11]

• 2011 Cl
• Not available Cl
• 3.33 [11]
CHF2
N
H3C N H
N
O
F
32
Penflufen • Bayer [12] CH3
N
• Not yet introduced
H3C N H
• 78–80 [12] N
• 3.26 [12]
F O
33
Sedaxane • Syngenta [13]
• Not yet introduced
• 111–113 (cis), CHF2
N
116–118 (trans) [13] H
• Not available H3C N N
O
34
Fluxapyroxad • BASF [14] F
• Not yet introduced F F
• 113–116 [14]
• Not available CHF2
N
H3C N H
N
O
35
similar biological properties, namely benodanil (3) and fenfuram (4), the latter of
which still in use today as a seed dressing. These early achievements spurred a wide
diversity of research activities by many companies, which resulted in the creation
of several new products or developmental candidates.
15.3.1.2 Active Ingredients

The four best-known older molecules of the carboxylic amides, along with six more
recently developed structures, are listed in Table 15.3.1.1. The next generation of
anilides consisted of the benzoic acid derivatives mepronil (5) and flutolanil (6);
these had very similar structures that differed only by the fluorination of a methyl
group. The biological spectrum of these active ingredients, which were introduced
during the 1980s by Nihon Nohyaku (flutolanil) [16, 17] and Kumiai (mepronil) [18,
19], is similar to that of the earlier compounds, with application being possible via
seed treatment, soil incorporation, or foliar spray [3].
In the case of furametpyr (7; Sumitomo Chemical Co.) [8, 20] and thifluzamide
(8; Dow AgroSciences) [21, 22], both of which were introduced during the 1990s,
the amide group is attached to a five-membered heterocycle, in similar fashion
to some of the early examples, such as fenfuram. The biology again focuses on
the pathogens characteristic for this class of compounds, especially Rhizoctonia
spp.
A structurally very similar molecule, tiadinil (11; Figure 15.3.1.2), has been
shown to possess another mode of action, as it serves as an activator of systemic
acquired resistance and induces defense gene expression [23]. Hence, it appears
that SDH inhibitors cannot be recognized on the basis of their structure alone.
The later generation of SDH-inhibiting anilides consists of boscalid (9; BASF)
[24–26] and penthiopyrad (also known as MTF 753; 10; Mitsui Chemicals) [9,
27]. Although, as far as their structures are concerned, these compounds can be
considered to be closely related to the older molecules, their biological activity is very
different. Boscalid, which was introduced to the market in 2003, was the first SDH
inhibitor to control Ascomycetes on various fruits and vegetables. Penthiopyrad
also is active against some pathogens of this group, in addition to the well-known
Rhizoctonia spp.
These unexpected fungicidal activities have widened the scope of this class of
compound by a substantial degree, and these agents are no longer to be considered
as restricted to specific pathogens. It is not surprising, therefore, that several
companies have reintensified their quest for new molecules, and this has in turn
resulted in the final five active ingredients detailed in Table 15.3.1.1. Each of these
bears a striking resemblance to one another, based on the pyrazole carboxylic
acid moiety of their structures, and differences occurring only in the lipophilic
substitution pattern within the anilide moiety.
Of this most recently developed group of products, the first to be launched have
been bixafen, for which Bayer has announced an application in cereals against
Mycosphaerella graminicola, and isopyrazam, for which Syngenta has not only
CH3
N
N H
N CI
S
O
CH3
11 Figure 15.3.1.2 Tiadinil (11).
obtained registration for its use in cereals but has also applied for it to be used in
fruit and vegetables against many pathogens. Taken together, these data suggest
that these compounds might progress into the cereal pathogen arena, notably to
combat M. graminicola.
15.3.1.3 Research Activities and Patent Situation

During recent years, interest in these molecules has risen considerably, with
increasing numbers of patents having been filed by many companies. A recent
analysis took into account only those patent applications devoted to active ingre-
dients (mixtures, formulations, and process-related documents were excluded).
By arranging the applications based on their year of publication, it has been
possible to demonstrate an impressive growth in the activities of these materials
(Figure 15.3.1.3). Indeed, the period between the early 1980s and the first five years
of the new millennium has witnessed a 10-fold increase in the number of patents
filed in this field, although this has tended to subside more recently. Such a decline
in interest can be attributed to the discovery of the latest generation compounds,
which have substantially broader biological spectra. Nonetheless, during this time
more than 15 companies – including not only most of the major players but also
many smaller competitors – have been involved in these efforts.
15.3.1.4 Synthesis
Whilst this class of molecule bears several striking structural similarities, the actual
strategy for a large-scale synthesis depends on the specific heterocycles or aromatics
involved, on their substitution pattern, and on the commercial availability of the
suitable precursors.
According to Alt et al. [22], thifluzamide (8) was prepared by starting from the
halogenated acetoacetate (12) and thioacetamide to yield the thiazole (13). This
intermediate was hydrolyzed and transformed into the chloride 14, which finally
60 Patent applications of active ingredients
50
40
30
20
10
0
1980-1985 1986-1990 1991-1995 1996-2000 2001-2005 2006-2010
Figure 15.3.1.3 Number of patent applications of active

ingredients (mixtures, formulations, and process-related doc-
uments have not been included).
gave the active ingredient 8. The necessary aniline (16) was obtained by direct
bromination of 4-trifluoromethoxyaniline (15; Scheme 15.3.1.1).
Boscalid (9) can be assembled starting from 2-chloronicotinic acid (17), the syn-
thesis of which from 3-methylpyridine has been described [28]. The corresponding
chloride 18 can then be reacted with the aniline (21) to obtain the desired product
9 [29]. The route from the boronic acid (19) via the 2-nitrobiphenyl (20) to 21
is the first example of the transfer of a palladium-catalyzed coupling reaction to
large-scale agrochemicals synthesis (Scheme 15.3.1.2).
In both examples, the heterocyclic carboxylic acid has been activated as the
chloride to be combined with the aniline in a straightforward converging synthesis.
For penthiopyrad (10), the details of a laboratory-scale synthesis have been
reported in which the branched alkyl residue on the thiophene ring was generated
from an acetyl group by the addition of methylmagnesium bromide and subsequent
reduction by triethylsilane [9].
15.3.1.5 Biological Activity and Application

The early SDH inhibitors were shown to be active against the Basidiomycetes,
notably against Rhizoctonia spp., and this also holds true for the next-generation
compounds. For example, thifluzamide is active against Rhizoctonia spp. on
rice, potatoes, peanuts, strawberry, turf, wheat, and cotton, and also against
Ustilago spp. and Tilletia caries on cereals, Sclerotium spp., Hemileia vastatrix,
S
H3C CF3
O O (1) NaOH
NH2 N
(2) SOCI2
H3C O CH3
F3C O CH3 S
CI
O
12 13
CF3
N
H3C CI
S CF3
N Br
14 O H3C H
N
S
O
Br Br O
H2N Br2 H2N CF3
8
O Br O
CF3 CF3
15 16
Scheme 15.3.1.1 Synthesis of thifluzamide (8).

N CI N CI
SOCI2
OH CI
CI
O O
17 18
N CI
H
N
CI CI CI
O2N O
CI
H2 9
Pd/C
Pd-complex O2N H2N
B(OH)2
19 20 21
Scheme 15.3.1.2 Synthesis of boscalid (9).
and others. Thifluzamide can also be used to treat Puccinia spp. in cereals and
peanuts.
This compound has both preventive and curative activity, as well as systemic
activity, with formulations for seed treatment, foliar application, seedling boxes, and
paddy fields each having been identified [30]. Compared to the early compounds
of this group, the current modes of application are apparently more diverse (some
older molecules were used only as a seed dressing). The recent discovery of boscalid
has produced a substantial broadening of the biological spectrum of this class of
compound, as it is most active against Ascomycetes, namely Botrytis spp. (vine,
fruit, vegetables), Sclerotinia spp. (fruit, vegetables, coffee, rape seed, turf), Alternaria
spp. (fruit, vegetables), Phoma spp. (rapeseed, coffee), Mycosphaerella spp. (fruit,
vegetables), Monilinia spp. (fruit), Pseudocercosporella herpotrichoides (cereals), and
others, including Rhizoctonia in some crops [26]. Penthiopyrad also shows activity
against some Ascomycetes (Podosphaera leucotricha, Venturia inaequalis, and Botrytis
cinerea) and the basidiomycete Rhizoctonia [3, 9].
According to company announcements for bixafen and isopyrazam, the most
recent active ingredients appear to have a rather broad spectrum and are targeted
explicitly at cereal diseases, notably M. graminicola. Applications to fruit and
vegetables, as well as seed treatment, have also been proposed.
15.3.1.6 Structure–Activity Relationships

Several investigations have been made into the structure–activity relationships
(SARs) of SDH inhibitors [31–37], with each of the analyses having been focused
on specific carboxylic acid moieties of the molecule. Subsequently, the influence of
substituents of the carboxylic acid, and also of the aniline, has been investigated,
based on both enzyme inhibition and biological data. As a result, some empirical
relationships have been established within each structural subclass, with the
importance of electron-withdrawing groups on the carboxylic acid, and of lipophilic
effects on the aniline, having been clearly observed. The orientation of the amide
bond has also been discussed, with the suggestion having been made that the cis
configuration of the amide bond might be important in molecules with bulky ortho
substituents [37].
Taking this into account, it becomes clear that compounds 6 to 10 all possess
electron-withdrawing groups such as trifluoromethyl or chlorine in the carboxylic
acid part of the molecule. Moreover, compared to their predecessors they also have
more lipophilic substituents in the aniline. Indeed, this holds true for the latest
five agents o be developed, with all having an identical or very similar pyrazole
carboxylic acid moiety. Of course, the complex agronomical implications cannot
be derived from these analyses, as is illustrated by the shift from basidiomycete to
ascomycete activity caused by subtle structural changes.
15.3.1.7 Resistance
Carboxin-resistant strains of M. graminicola have been shown to have an exchange
of histidine at codon 267 for either tyrosine or leucine [38]. The strains involved
in this study were generated artificially by applying ultraviolet light-mediated
mutagenesis. In resistant Ustilago maydis, a similar replacement of histidine with
leucine has been observed [39], whereas in Coprinus cinereus another variation
(N80K) has been found [40]. In field isolates of Alternaria alternata obtained from
pistachio orchards treated with a boscalid mixture, mutations from histidine 277
to either tyrosine or arginine have been observed, although additional strains with
unidentified changes were also identified in the same study [41].
Carboxin-resistant Ustilago nuda has been reported from field applications in
France and Italy [42]. This substance class has been regarded as being medium
risk for resistance development, and resistance management would clearly be
required if carboxin were to be used to combat certain vulnerable pathogens
[43]. Interestingly, some pathogenic strobilurin-resistant strains of Alternaria solani
(having the F129L mutation in complex III) have shown an increased sensitivity
towards boscalid [44].
Carboxin inhibits, to a different degree, the SDH of such diverse organisms
as fungi, bacteria, plants, and mammals, and studies with resistant mutants
have contributed to an understanding of the mechanism of action involved [45].
Additional information has been acquired from experiments with Saccharomyces
and bacteria [46–48].
15.3.1.8 Metabolism
The metabolism of this compound class normally begins with an hydroxylation
of the aromatic rings or alkyl or alkoxy groups, with cleavage of the amide bond
occurring at a later stage in most cases [49]. In fact, a total of 14 metabolites, some
of which are shown in Scheme 15.3.1.3, was detected during a study of furametpyr
(7) in mammals [50, 51]. Of these biotransformations, the most important were
CH3 H 3C
N O
H3 C N H
N CH3
CH3
Cl O
7
CH3 H3C
CH3 H3 C N O
N O H
H3C N
H3C N H N CH3
N COOH CH3
CH3 Cl O
Cl O CH3 H3C OH
N 28
22 O
H N H
N CH3
CH3
Cl O
CH3 H 3C CH3 H 3C
N 25 N
O O
H N H H N H
N COOH N CH3
CH3 CH3
Cl O CH3 H3C Cl O
N O OH
23 29
H N H
N CH3
H2C
Cl O OH
CH3 H C O O
N 3
26
H N H
N CH3 H3C
OH N O
CH3 H
Cl O H N
OH N CH3
24 H 2C
CH2 H 3C Cl OH
N O
O OH
N H 30
H N CH3
H2C
Cl O OH
27
Scheme 15.3.1.3 Metabolism of furametpyr in mammals.
N-demethylation (as seen in metabolites 23, 25, 29), the oxidation of methyl groups
(22, 26, 27, 30), and aromatic hydroxylation (28).
15.3.1.9 Discussion
The SDH inhibitors of the anilide type represent a class of fungicides that is of both
agronomical and commercial importance. Although the biological scope of these
materials has long been known, it has widened significantly during recent years.
A major factor in conducting long-term investigations with these compounds
has been their relatively benign toxicology profile, with LD50 values in excess of
1500 mg kg−1 body weight being identified for most land vertebrates [3]. Such
a finding was, perhaps, rather surprising as SDH is also present in mammals.
However, no comprehensive study has been conducted to date comparing the
References 637
inhibition of SDH in various organisms by different anilides, and relating such

findings to any observed biological effects.
One other reason for these longstanding research efforts is the limited, but
highly promising, fungicidal spectrum of the early examples, combined with a
flexible structure. Indeed, this has motivated many companies to exploit various
carbocyclic and heterocyclic systems to which numerous suitable substituents have
been attached.
Today, the understanding of the structure of the enzyme target, and of the
mechanism of action of the carboxylic amides, has increased considerably.
Likewise, the substantial number of patent applications confirms that both
scientific and commercial interest remain focused on the subject of SDH in-
hibition.
Acknowledgments
The author acknowledges the valuable comments and suggestions made by

Drs J. Dietz, T. Grote, P. Lane, and S. Strathmann, at BASF SE, Crop Protection
Division.
References
1. Büchel, K.H. (ed.) (1977) Pflanzen- 8. Oguri, Y. (1997) Agrochem. Jpn, 70,
schutz und Schädlingsbekämpfung, Georg 15–16.
Thieme Verlag, Stuttgart. 9. Yoshikawa, Y., Tomiya, K., Katsuta, H.,
2. von Schmeling, B. and Kulka, M. (1966) Kawashima, H., Takahashi, O., Inami,
Science, 152, 659–660. S., Yanase, Y., Kishi, J., Shimotori, H.,
3. Tomlin, C. (ed.) (2009) The e-Pesticide and Tomura, N. (1996) European Patent
Manual, Version 5.0, BCPC, Hamp- Application EP 737,682, p. 59 (Chem.
shire. Abstr., 126, 7982).
4. United States Environmental Protec- 10. Ehrenfreund, J., Tobler, H., and Walter,
tion Agency (2004) Reregistration H. (2004) PCT International Application
WO 04/035,589, p. 58 (Chem. Abstr.,
Eligibility Decision for Carboxin,
140, 375164).
Document EPA 738-R-04-015,
11. Dunkel, R., Rieck, H., Elbe, H.-L.,
http://www.epa.gov/oppsrrd1/REDs/
Wachendorff-Neumann, U., and Kuck,
0012red_carboxin.pdf.
K.-H. (2003) PCT International Applica-
5. United States Environmental Protec-
tion WO 03/070,705, p. 61 (Chem. Abstr.,
tion Agency (2004) Reregistration
139, 180060).
Eligibility Decision for Oxycarboxin, 12. Elbe, H.-L., Rieck, H., Dunkel, R.,
http://www.epa.gov/oppsrrd1/REDs/ Zhu-Ohlbach, Q., Mauler-Machnik, A.,
0012red_oxycarboxin.pdf. Wachendorff-Neumann, U., and Kuck,
6. Pommer, E.H., Girgensohn, B., König, K.-H. (2003) PCT International Applica-
K.H., Osieka, H., and Zeeh, B. (1974) tion WO 03/010,149, p. 98 (Chem. Abstr.,
Kemia – Kemi, 1 (9), 617–618. 138, 137308).
7. Spencer, E.Y. (1982) Guide to the Chemi- 13. Ehrenfreund, J., Tobler, H., and Walter,
cals Used in Crop Protection, Publication H. (2003) PCT International Application
1093, Agriculture Canada, Canadian WO 03/074,491, p. 40 (Chem. Abstr.,
Government Publishing Centre, Ottawa. 139, 246007).
14. Gewehr, M., Dietz, J., Grote, T., 28. Murugan, R. and Scriven, E.F.V. (2000)
Blettner, C., Grammenos, W., Hünger, J. Heterocycl. Chem., 37, 451–454.
U., Müller, B., Schieweck, F., Schwögler, 29. Eicken, K., Rang, H., Harreus, A.,
A., Lohmann, J.K., Rheinheimer, J., Goetz, N., Ammermann, E., Lorenz, G.,
Schäfer, P., Strathmann, S., and Stierl, and Strathmann, S. (1997) Ger. Offen.
R. (2006) PCT International Application DE 19,531,813, p. 21 (Chem. Abstr., 126,
WO 06/087,343, p. 57 (Chem. Abstr., 264007).
145, 271772). 30. Dow AgroSciences (2004) Thifluzamide,
15. Tomlin, C. (ed.) (1995) The Pesticide Technical Bulletin, Product Brochure,
Manual, 10th edn, Crop Protection http://www.dowagro.com.
Publications, British Crop Protection 31. White, G.A. and Thorn, G.D. (1975) Pes-
Council, Farnham. tic. Biochem. Physiol., 5, 380–395.
16. Araki, F. and Yabutani, K. (1981) Congr. 32. White, G.A. and Thorn, G.D. (1980) Pes-
Proc. Brighton Crop Prot. Conf. – Pests tic. Biochem. Physiol., 14, 26–40.
Dis., 1, 3. 33. White, G.A., Phillips, J.N., Huppatz,
17. Yabutani, K., Ikeda, K., Hatta, S., J.L., Witrzens, B., and Grant, S.J. (1986)
Harada, and T. (1978) Ger. Offen. Pestic. Biochem. Physiol., 25, 163–168.
DE 2,731,522, p. 27 (Chem. Abstr., 89, 34. White, G.A. and Georgopoulos, S.G.
6122). (1986) Pestic. Biochem. Physiol., 25,
18. Doi, S. (1981) Jpn. Pestic. Inf., 38, 188–204.
17–20. 35. White, G.A. (1987) Pestic. Biochem. Phys-
19. Chiyomaru, I., Kawada, S., and Takita, iol., 27, 249–260.
K. (1976) US Patent 3,937,840, Kumiai 36. White, G.A. (1988) Pestic. Biochem. Phys-
Chemical Industry Co. iol., 31, 129–145.
20. Mori, T., Ohsumi, T., Nakamura, S., 37. Phillips, W.G. and Rejda-Heath, J.M.
Maeda, K., Nishida, S., and Takano, (1993) Pestic. Sci., 38, 1–7.
H. (1989) European Patent Application 38. Skinner, W., Bailey, A., Renwick, A.,
EP 315,502, p. 38 (Chem. Abstr., 111, Keon, J., Gurr, S., and Hargreaves, J.
214478). (1998) Curr. Genet., 34, 393–398.
21. O’Reilly, P., Kobayashi, S., Yamane, 39. Broomfield, P.L.E. and Hargreaves, J.A.
S., Phillips, W.G., Raymond, P., and (1992) Curr. Genet., 22, 117–121.
Castanho, B. (1992) Congr. Proc. Brighton 40. Ito, Y., Muraguchi, H., Seshime, Y.,
Crop Prot. Conf. – Pests Dis., 1, 427–434. Oita, S., and Yanagi, S.O. (2004) Mol.
22. Alt, G.H., Pratt, J.K., Phillips, W.G., and Genet. Genomics, 272, 328–335.
Srouji, G.H. (1990) European Patent 41. Avenot, H.F., Sellam, A., Karaoglanidis,
Application EP 371,950, p. 27 (Chem. G., and Michailides, T.J. (2008) Phy-
Abstr., 113, 191337). topathology, 98 (6), 736–742.
23. Yasuda, M., Nakashita, H., and Yoshida, 42. Menzies, J., McLeod, R., Tosi, L., and
S. (2004) J. Pestic. Sci., 29, 46–49. Cappelli, C. (2005) Phytopathol. Mediterr.,
24. Michel, P. (2003) Phytoma, 566, 33–35. 44, 216–219.
25. Eicken, K., Goetz, N., Harreus, A., 43. Fungicide Resistance Action Com-
Ammermann, E., Lorenz, G., and Rang, mittee (FRAC) (2005) Code List 2:
H. (1993) European Patent Application Fungicides Sorted by Modes of Action,
EP 545,099, p. 60 (Chem. Abstr., 119, http://www.frac.info.
160132). 44. Pasche, J.S., Piche, L.M., and
26. BASF (2006) Cantus®, Gudmestad, N.C. (2005) Plant Dis.,
Champion® Product Brochures, 89, 269–278.
http://www.agrar.basf.de/. 45. Hederstedt, L. (2002) Biochim. Biophys.
27. Tomiya, K. and Yanase, Y. (2003) Acta, 1553, 74–83.
Congress Proceedings – BCPC In- 46. Lancaster, C.R.D. (2002) Biochim. Bio-
ternational Congress: Crop Science phys. Acta, 1553, 1–6.
& Technology, Glasgow, Vol. 1, pp. 47. Lemire, B.D. and Oyedotun, K.S. (2002)
99–104. Biochim. Biophys. Acta, 1553, 102–116.
48. Oyedotun, K.S. and Lemire, B.D. (2001) 50. Nagahori, H., Yoshino, H., Tomigahara,
J. Biol. Chem., 276, 16936–16943. Y., Isobe, N., Kaneko, H., and
49. Roberts, T.R. and Hutson, D.H. (1999) Nakatsuka, I. (2000) J. Agric. Food
Chem., 48, 5754–5759.
Metabolic Pathways of Agrochemi-
51. Nagahori, H., Yoshino, H., Tomigahara,
cals – Part 2: Insecticides and Fungicides, Y., Isobe, N., Kaneko, H., and
The Royal Society of Chemistry, Cam- Nakatsuka, I. (2000) J. Agric. Food
bridge. Chem., 48, 5760–5767.
15.3.2
Succinate Dehydrogenase Inhibitors: Pyridinyl-Ethyl Benzamide
The carboxylic amides, as outlined in Chapter 15.3.1 (see Figure 15.3.1.1), constitute
the first generation of succinate dehydrogenase (SDH) inhibitors. However, as it
appears the anilide unit is not essential for broad-spectrum fungicidal efficacy, the
chemical group of the pyridinyl-ethyl benzamides within the FRAC Group No. 7
was defined to cover fluopyram (1) (cf. Figure 15.3.2.1; Table 15.3.2.1) as the only
member of this new subclass of Complex II respiration inhibitors.
15.3.2.2 Origin of the Chemical Structure

Initially, fluopyram (1) was discovered by employing an ‘‘agrophore’’ chemi-
cal synthesis approach, which involves the combination of fragments present
F
F Cl F F
F O F
N N
H
1 Figure 15.3.2.1 Chemical structure of the

Fluopyram pyridinyl-ethyl benzamide fluopyram (1).
Table 15.3.2.1 Physico-chemical properties of technical fluopyram.
Common name (ISO) Fluopyram

CAS-No. 658066-35-4
Boiling point (◦ C) 319 (decomposition)
Vapor pressure (Pa at 20 ◦ C) 1.2 × 10−6
Relative densitya 1.53
Solubility in water (mg l−1 at 20◦ C) 16 (pH 7)
Partition coefficient (log Pow ) in octanol–water (at 20 ◦ C) 3.3 (pH 6.5)
a
At 20 ◦ C compared to water at 4 ◦ C.
in already-known active ingredients, the aim being to obtain molecules with

an increased likelihood of biological activity. In the case of fungicides, this
might mean targeting a log P of between 2.5 and 4.5 (C. Cornell, Aven-
tis CropScience, unpublished results) [1–3]. As shown in Figure 15.3.2.2, the
3-chloro-5-trifluoromethyl-2-pyridinyl residue was known to be present in a range
® ®
of known agrochemicals that included fluopicolide (2; Infinito ), (3; Froncide )
®
(fungicides), haloxyfop-P-methyl (4; Eloge ) (herbicide), and the benzoyl phenyl
®
urea, fluazuron (5; Acatak , an insecticide used in animal health).
In the case of fluopyram, a second agrophoric element – an ortho-substituted
benzamide residue (Figure 15.3.2.3) as found in benodanil (6, ortho-I atom),
mepronil (7, ortho-CH3 group), or flutolanil (8, ortho-CF3 group) – was incorporated
O2N
Cl O Cl F
Cl H
N F
N F
H
F N N O2N Cl
Cl F
F F
F F
2 3
Fluopicolide Fluazinam
F
O
O
N
H F
F O Cl N
F H
Cl O CH3
F O
O
CH3 N
N O
Cl
4 F 5
Haloxyfop-P-methyl Fluazuron
F F
Figure 15.3.2.2 Examples of agrochemicals bearing a

2-chloro-5-trifluoromethyl-2-pyridinyl residue.
O O O
I HN CH3 HN O CH3 CF3 HN O CH3
CH3 CH3
6 7 8
benodanil mepronil flutolanil
Figure 15.3.2.3 Examples of SDH inhibitors bearing an

ortho-substituted benzamide residue.
from the classical SDH inhibitor structures (see also Chapter 15.3.1; general
structure in Figure 15.3.2.1).
It is remarkable that the relatively minor changes of lengthening the linker
between the 3-chloro-5-trifluoromethyl-2-pyridinyl residue and the carboxylic amide
part of fluopicolide (2) by one methylene unit (CH2 → CH2 CH2 ), and changing the
phenyl substitution pattern from ortho–ortho substitution (R1 and R2 ) to a single
ortho-substitution (R1 ), would result in a complete shift of the biological spectrum
and mode of action (Figure 15.3.2.4).
Fluopicolide (2) shows an excellent anti-Oomycete activity, whereas fluopyram (1)
has an outstanding activity against Ascomycetes pathogens, without demonstrating
activity against Oomycetes diseases.
Thus, employing the agrophore approach led to the discovery of the pyridyl-ethyl
benzamides, the first non-aniline carboxamide derivatives.
15.3.2.3 Influence of Structural Elements on the Biological Spectrum

The influence of the differing structural elements in selected carboxylic amides,
such as fluopyram (1), fluopicolide (2), and compounds 9 and 10, on the biological
activity spectrum is detailed in Table 15.3.2.2.
The activity against basidiomycetes which, in general, is a core property of
classical SDH carboxylic amide inhibitors, is lost in the case of fluopyram, but
can be recovered when the ethyl linker is exchanged to an ortho-substituted phenyl
ring (CH2 CH2 → ortho-phenyl), as demonstrated with compound 10. However,
this modification is detrimental to the activity against the major Ascomycetes
diseases due to an unfavorable increase in the compound’s lipophilicity. Equally,
when the 3-chloro-5-trifluoromethyl-2-pyridinyl residue of fluopyram is replaced
by a corresponding 2-chloro-4-trifluoromethyl-phenyl residue, as in compound 9,
the level of its anti-Ascomycetes biological efficacy drops, without expanding the
biological spectrum.
In the past, considerable efforts have been made to fully explore this new subclass
of SDH inhibitors, and this has resulted in more than 30 patent applications.
15.3.2.4 Synthesis of Fluopyram

Fluopyram can be synthesized starting from 2,3-dichloro-5-trifluoromethyl-pyridine
(11) which undergoes a coupling reaction with ethyl cyanoacetate forming the
adduct 12. Subsequent hydrolysis of the ester function in 12 and decarboxylation
of the resulting carboxylic acid gives (3-chloro-5-trifluoro-2-pyridinyl)-acetonitrile
(13). Reduction of the cyano group in the presence of acetic anhydride leads
to the (3-chloro-5-trifluoro-2-pyridinylethyl)-acetamide (14), which is deacylated in
hydrochloric acid. Finally, coupling with ortho-trifluoromethyl benzoyl chloride
yields fluopyram (1) [4] (Scheme 15.3.2.1).
The presence of acetic anhydride during the catalytic hydrogenation of the nitrile
13 is critical to the success of this reaction. This reagent activates the cyano function
of compound 13 towards reduction, thus allowing the use of moderate hydrogen
pressures of around 8 bar. As a result of the mild conditions, dechlorination
of the 3-chloro-5-trifluoromethyl-2-pyridinyl core in 13 and/or 14 is minimized.
642
Cl O Cl O R1 O R1
Cl Cl
N N N
H H H
F N
Cl N R2 F N
F
F
F F
2 F F 1
F
fluopicolide fluopyram (when R1 = CF3)
Figure 15.3.2.4 From fluopicolide (2) to fluopyram (1).

Table 15.3.2.2 Influence of the structural elements in se-

lected carboxylic amides on biological spectrum.
Structure Activitya against Mode of action

Oomy- Ascomy- Basidio-
cetes cetes mycetes
Cl O Cl
N
H
F N
Cl
F
F
Fluopicolide (2) ++++ - - Effect on spectrin
(cell wall stability)
F
F Cl F F
F O F
N N
H
Fluopyram (1) - ++++ - SDH inhibitor
F
F Cl F F
F O F
N
H
9 - ++ - SDH inhibitor
F F
O F
N
H
Cl
N
F F
F
10 - ++ ++ SDH inhibitor
a
Activities are defined as: ++++, excellent; +++, good; ++, acceptable; -, loss of efficacy.
CN
F3 C Cl F 3C Cl F 3C Cl
COOEt, KOH HCl
CN CN
N Cl NMP, 70 °C N 115 °C N
COOEt
11 12 13
Ac2O
Pd/C, AcOH
H2, [8 bar]
F 3C Cl
O CF3 1) HCl aq. F 3C Cl
reflux O
N N
H
O CF3 N N CH3
H
1 2)
Cl 14
NaOH,THF
Scheme 15.3.2.1 Synthetic pathway for preparation of fluopyram (1).
In addition, the generated (3-chloro-5-trifluoromethyl-2-pyridinylethyl)-amine is

acylated in situ, preventing it from undergoing any undesired oligomerization in
the reaction medium.
15.3.2.5 Biological Activity and Application Rates

As outlined in Table 15.3.2.1, fluopyram shows a remarkably unique biological pro-
file: although inactive against the Oomycetes or Basidiomycetes, however, a broad
range of Ascomycetes are controlled very efficiently. Diseases controlled include:
Botrytis spp. on vine, fruit, and vegetables; Sclerotinia spp. on fruit, vegetables,
rapeseed, and soybeans; Monilinia spp. on fruit; Sphaerotheca spp. on fruit and veg-
etables; Erisyphe spp., Podosphaera spp. on fruit; Venturia spp. on fruit; Alternaria
spp. on fruit and vegetables; Phoma spp., Pyrenopeziza, Pseudocercosporella, on
rapeseed; Mycosphaerella spp. on fruit; Colletotrichum spp. on fruit and vegetables;
Rhizopus spp. on fruit; Cladosporium on fruit; and Stemphyllium on fruit.
In comparison to other commercially available SDH inhibitor, fluopyram shows
an outstanding efficacy with lower application rates. For example, Botrytis cinera is
effectively controlled with an application rate of only 250 g a.i. ha−1 , compared for
example to boscalid at 500–600 g a.i. ha−1 .
Hence, Botrytis, Monilia, Sclerotinia, powdery mildew and other diseases respon-
sible for yield and quality losses in the food chain can be controlled very efficiently.
Fluopyram can also be used in seed treatment applications, for example, against
Pyrenophora spp. (cereals).
The higher flexibility of the ethylene bridge in fluopyram, when compared to a
more rigid anilide moiety as found in boscalid (see Chapter 15.3.1), is thought to
allow for a more flexible binding mode at the active site of the target species. A dif-
ferent cross-resistance pattern of fluopyram to boscalid has also been reported [5, 6].
15.3.2.6 Conclusions
A successful and innovative ‘‘agrophore’’ approach has led to the discovery of
fluopyram as member of the new class of pyridinyl-ethyl benzamide fungicides,
with a distinct biological profile and properties. Although the mode of action of
fluopyram is SDH inhibition, the different cross-resistance pattern to other known
SDH inhibitors makes fluopyram the active ingredient of choice in conditions
where other SDH inhibitors are affected by resistance development. In order
to preserve the broad activity of fluopyram, an efficient anti-resistance strategy
is recommended: it is essential that the compound is used in strict alternation
with, and/or in, mixtures with other fungicides from other mode-of-action groups
according to the FRAC guidelines.
Products based on fluopyram will be launched from 2011 onwards by Bayer
®
CropScience, predominantly under the brand name Luna , subject to the necessary
regulatory approvals being granted.
Acknowledgments
The authors gratefully acknowledge the valuable comments and suggestions made
by Drs B. Hartmann, J.P. Vors, and D.J. Mansfield, at Bayer CropScience.
References
1. Clarke, E.D. and Delaney, J.S. (2003) Patent EP 1,674,455, (Bayer CropScience
Chimia, 57 (11), 731–734. A.-G).
2. Briggs, G., Mansfield, D., Moloney, B., 5. Avenot, H.F. and Michailides, T.J. (2009)
Gary, S., and Wegmann, T. (2005) Phytopathology, 99, S6.
Pflanzenschutz-Nachr. Bayer. (Engl. Ed.),
6. Ishii, H., Miyamoto, T., Ushio, S.,
59, 141–152.
and Kakishima, M. (2009) KSPP Fall
3. Jeschke, P. (2010) Pest Manage. Sci., 66,
10–27. Meeting and the First Japan – Kerea
4. Coqueron, P.-Y., Lhermitte, F., Joint Symposium, Jeju, October 28–31,
Perrin-Janet, G., and Dufour, P. (2006) 2009.
15.4
Uncouplers of Oxidative Phosphorylation
15.4.1
Introduction
In other chapters of this volume, descriptions have been provided of how inhibitors
of the different complexes of the mitochondrial electron transport chain can
produce potent antifungal effects.
As well as inhibiting the individual respiratory complexes, other ways also exist
in which the overall process of oxidative phosphorylation can be disrupted. One of
the most effective of these is to disconnect – or ‘‘uncouple’’ – the electron transport
chain from ATP synthesis, thus allowing respiration to continue but preventing the
conversion of metabolic energy into the ATP needed to operate cellular processes.
In its broadest sense, the term uncoupler has been used to describe any compound
that prevents the synthesis of ATP (other than by the direct inhibition of ATP
synthase) but allows electrons to be accepted from NADH and succinate. Thus,
menadione – which acts as an alternative acceptor for electrons, thereby diverting
them from the full mitochondrial pathway – has been described as an ‘‘uncoupler of
oxidative phosphorylation’’ [1]. However, for the purposes of this chapter, uncouplers
are more precisely defined as ‘‘. . .compounds that break the link between the intact
and functional electron transport chain and ATP synthase’’.
Several general reviews of uncouplers and the uncoupling process have been
published [2–10].
15.4.2
Mechanism of Action of Uncouplers
The chemiosmotic theory, which was first proposed by Peter Mitchell in 1961 and is
now generally accepted, explains the coupling of respiration and ATP synthesis in
terms of a proton gradient across the inner mitochondrial membrane [11–13]. The
complexes of the respiratory chain accept electrons (originally from NADH and
succinate) and use the energy released as they move down the potential gradient
to transport protons across the membrane (see Chapter 15.1 for a more detailed
discussion of this process). The resulting proton gradient and transmembrane
potential drives the flow of protons through ATP synthase, and hence the production
of ATP. Anything that dissipates the proton gradient across the membrane will
lead to an uncoupling of oxidative phosphorylation by removing the proton-motive
force required for the operation of ATP synthase. If this uncoupling occurs in an
uncontrolled manner it has a drastic effect, eventually causing cell death. There are
a number of ways in which uncoupling can be brought about.
Uncoupling proteins (UCPs) have been identified in many organisms [12, 14–16].
These allow a controlled flow of protons across the membrane, and are thought
to have a number of functions, including heat generation and control of the
transmembrane potential.
Various chemicals cause uncoupling by increasing the permeability of the
membrane to protons and other small ions. Detergents, which destabilize the
membrane structure, can have this effect [17]. A number of small molecules,
including some ‘‘classical’’ uncouplers, have been described as affecting the
membrane permeability transition (MPT), which causes a dramatic increase in the
ability of ions to move across the membrane [9, 18–20]. More specific effects that
increase the permeability of the membrane to protons, as caused by the peptide
gramicidin A, also lead to uncoupling [14, 21, 22].
Ionophores, such as valinomycin, can transport metal ions across the membrane;
this effectively dissipates the transmembrane potential without altering the proton
gradient. However, as the potential is the major driver for proton flow through ATP
synthase, this leads to a much reduced efficiency of coupling and results in similar
effects to dissipation of the proton gradient [3, 22, 23].
Long-chain fatty acids are also known to cause uncoupling. The mechanism
appears complex, but seems to be distinct from either simple membrane disruption
or a classical protonophoric effect, and appears to require the involvement of active
transporters [9, 14, 22, 24–27].
A final class of uncouplers are compounds that transport protons across the
membrane, leading to dissipation of the transmembrane proton gradient, and hence
removing the proton-motive force that drives ATP synthase [28–30]. These are the
only uncouplers of real significance so far as fungicide discovery is concerned.
The most common type of protonophoric uncouplers, sometimes referred to
as classical uncouplers, are lipophilic weak acids. The simplest explanation of
proton transport by these compounds is illustrated in Figure 15.4.1a. On the
intermembrane side of the membrane, where the proton concentration is high,
the compounds exist in their neutral, protonated form (HA). This is able to diffuse
through the membrane to the mitochondrial matrix, where the pH is considerably
higher. Under these conditions the proton is removed, and the resulting anion (A− )
is driven back across the membrane by the membrane potential, thus continuing
the cycle. In this way, the uncoupler both dissipates the proton gradient, by proton
transport from intermembrane space to matrix, and collapses the membrane
potential, by negative charge transfer in the opposite direction. It produces this
effect in a catalytic manner, with each molecule of uncoupler able to transfer many
protons as it repeatedly proceeds through the cycle.
A limiting factor in the efficiency of the uncoupling cycle is the ability of the
charged anionic form of the uncoupler to cross the lipophilic interior of the
Intermembrane
space
H+ H+
HA HA (high [H+])
A−
HA
HA A− HA
AHA−
Inner
mitochondrial
membrane
AHA−
HA A− HA
HA
A− A− Matrix
H+ H+ (low [H+])
(a) (b)
Figure 15.4.1 Mechanism of proton transport for weakly

acidic uncouplers. (a) Monomolecular; (b) Bimolecular.
Intermembrane
H+ space
BH+ (high [H+])
N N
BH+ B
+ H+ Inner
+
N N mitochondrial
− H+ H membrane
BH+ B
B Matrix
H+ (low [H+])
1
Figure 15.4.2 Structure of AU-1421 and general mechanism

of proton transport for a weakly basic uncoupler.
membrane bilayer. In some cases it has been shown that a bimolecular mechanism
occurs, in which a neutral molecule of the uncoupler associates with, and
facilitates the movement of, the anion, presumably by increasing the delocalization
of the negative charge [31]. This situation is illustrated diagrammatically in
Figure 15.4.1b. If mixtures of uncouplers are present, it has been suggested
that they can interact to form heterodimers to facilitate movement across the
membrane and hence act synergistically [32].
Although much less commonly observed, there have been a number of reports
of lipophilic weak bases acting as protonophoric uncouplers, for example, the
pyridine AU-1421 (1) [33]; these compounds can transport protons, as shown
in Figure 15.4.2. The weakly basic compound is protonated on the low-pH
intermembrane space side of the membrane, and the resulting cation (BH+ )
crosses the membrane, driven by the transmembrane potential. In the less-acidic
matrix, the proton is removed and the neutral uncoupler (B) can diffuse back
across the membrane to continue the cycle.
15.4.3
Selectivity and Toxicity
By virtue of their mechanism of action, protonophoric uncouplers interact in a

nonspecific manner with membranes, rather than acting at a specific binding
site. Because of this they also have the correct properties to interact with other
membranes, both in the target organism and other species. For this reason,
selectivity is an issue for many compounds with this mode of action. Indeed, one of
the first uncouplers to be commercialized over 100 years ago, dinitrocresol (DNOC)
(2), was first used as an insecticide and later as a herbicide [34]. It has also been
used as a fungicide on fruit trees and vines, and to break the winter dormancy of
fruit trees grown in warm climates [35], although it has now largely been replaced,
due to problems of toxicity.
In some cases the lack of selectivity can be beneficial. For example, although the
primary use of dinocap (3) is as a fungicide for the control of powdery mildews,
it also has acaricidal properties and is used to suppress the populations of various
mites [36]. In a similar way fluazinam (4), one of the most selective fungicidal
uncouplers, has been shown to control some mites [37].
In general, however, the lack of selectivity is undesirable and requires careful
management. As an example, dinocap has detrimental effects on some beneficial
insects, especially predatory mites [38–40]. Phytotoxicity can also be an issue on
some crops [41–44] and ornamentals [45].
One particular manifestation of a lack of selectivity is toxicity to humans. Carefully
controlled uncoupling mediated by UCPs is important for the regulation of cells,
and uncoupling has also been suggested as a potential target for the treatment
of obesity [46, 47]. However, the toxic effects of a natural weight-loss dietary
supplement have been linked to the presence of the uncoupler usnic acid (5) [48],
and a number of deaths have been attributed to the illegal use of 2,4-dinitrophenol
for weight loss [49]. Uncoupling has also been implicated in the toxic side effects
of some nonsteroidal anti-inflammatory drugs (NSAIDs) [50, 51].
A number of toxic effects of fungicidal uncouplers have been observed, but it
is not clear whether these are directly related to the uncoupling mode of action
or to other, compound-specific mechanisms. Some uncouplers have a very high
acute toxicity, which is almost certainly a result of their mode of action. This has
been exploited in the case of bromethalin (6), which is used as a rodenticide.
Another toxic effect that has been observed for several distinct structural types
of uncoupler – and hence is likely to be a direct result of this mode of action – is
edema of the central nervous system [52], for which direct interaction with the
myelin sheath membrane is most likely responsible.
Several other toxic effects have been observed for specific fungicidal uncouplers,
but these are more likely to be related to specific structural features of the
compounds rather than to the uncoupling mode of action. Fluazinam is a skin
sensitizer that can cause allergic contact dermatitis [53–55], an effect that is most
likely the result of thiol reactivity rather than uncoupling properties. A number of
toxic effects have been seen with dinocap, most notably a potent teratogenicity in
mice [56, 57], although the technical material is not teratogenic in rats or hamsters
[58, 59]. Two pure regioisomers of the active ingredient do not cause teratogenic
effects in mice [60], and the toxicity appears to be linked to a single isomer [61]
and hence is due to a more specific effect than uncoupling. The recently launched
single isomer meptyldinocap (24) has a much reduced toxicity compared to dinocap
[62–64], which again suggests that the toxic effects seen for dinocap are not due to
its uncoupling activity.
Various strategies have been employed to minimize the potential toxicity and
lack of selectivity of uncouplers. Several commercial compounds are sold as
pro-pesticides of the parent molecule, such that the active uncoupler is liberated
only after metabolism. These include the insecticide chlorfenapyr (7) and the
fungicides dinocap (3) and binapacryl (8). This approach can be highly successful
in reducing acute toxicity: dinocap has an acute oral LD50 of 265 mg kg−1 in mice,
whereas the LD50 for its active phenolic metabolite is 29 mg kg−1 [65]. In this case,
the active uncoupler is released by the action of esterases in the target organism, but
hydrolysis is slow in the acidic environment of the mammalian stomach (half-life

of 229 days at pH 5 [66]). A second successful approach is to design compounds that
can be readily metabolized in nontarget organisms. An example is fluazinam (4),
which reacts rapidly with glutathione and other thiols and is thus rapidly detoxified
in mammals (see Section 15.4.8).
15.4.4
Resistance
The development of resistance is a major issue for many classes of fungicide,

including those that inhibit respiration. However, uncouplers appear to be less
susceptible to the onset of resistance than many other fungicides. For example, in a
study of resistance development in Sphaerotheca fuliginea in cucumber greenhouses,
despite it having been used for more than 30 years, no resistance to dinocap
was observed [67]. This was in contrast to other classes of fungicide, such as
benzimidazoles, where resistance had developed over a much shorter period of
time. Even under artificial selection pressure, no resistance to dinocap was seen [68].
Although it has not been used for such an extended period, the situation for
fluazinam appears to be similar. Thus, despite being used for several seasons with
up to 10 applications per season and no mixture partners, there is no indication of
a change in sensitivity of Phytophthora infestans, despite the known ability of this
pathogen to develop resistance to other classes of fungicide [69]. Similar studies
of Botrytis cinerea in French vineyards have revealed no resistance development
after several years of use [70–72]. The same situation was reported for Botrytis in
Japan [73], although more recently there has been a report of a resistant isolate
being found after 10 years of repeated use in bean crops [74]. This isolated
report notwithstanding, uncouplers are classed by the Fungicide Resistance Action
Committee (FRAC) as code 29 with a low resistance risk [75], and they are often
used to control fungi that have developed resistance to other fungicides [76–80].
The reasons for the low resistance risk are not completely understood. As
protonophoric uncouplers do not act at a specific binding site on a protein, the
possibility of a single-point mutation at the binding site giving rise to resistance
does not exist. It is less clear why other possible resistance mechanisms, such as
metabolism or active transport, do not seem to operate. However, uncouplers are
often used to block the operation of multiple drug resistance pumps in biochemical
experiments [81–83]. It is possible, therefore, that the action of uncouplers in
depleting cellular ATP also prevents the operation of energy-dependent resistance
mechanisms. It is worth noting that the ATP-binding cassette (ABC) transporter
from Aspergillus nidulans has been shown to have some effect in reducing the
toxicity of fluazinam to this organism, although treatment with the arylhydrazone
uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) (9) reduced the
effectiveness of this efflux pump [84].
Although little resistance to uncouplers has been observed in fungi, a number
of bacteria are known that have reduced sensitivity to protonophoric uncouplers
[85–87]. The likelihood of similar resistance mechanisms developing in fungi is

unknown, but is probably small, as no such mutations have yet been observed.
15.4.5
Physico-Chemical Properties of Protonophoric Uncouplers
Detailed mathematical studies of the relationship between uncoupling potency

and physico-chemical properties have been conducted for several classes of
uncoupler, including phenols [88–96], arylhydrazones [97, 98], salicylanilides
[88, 99, 100], pyrroles [101], benzimidazoles [88], coumarins [102, 103], and
diarylamines [104–106]. Although the details of these analyses differ, there are
several physico-chemical parameters that are common to all, and therefore appear
to be the key properties required for uncoupling; these include acid strength,
lipophilicity, and often a measure of the steric bulk of substituents adjacent to the
acidic center [101].
In order to act as an efficient protonophoric uncoupler, a weakly acidic compound
must have properties that allow it – in both uncharged protonated and anionic
deprotonated forms – to enter and cross the membrane lipid bilayer. The compound
must have a suitable pKa such that on the more-acidic, intermembrane side of the
membrane a significant proportion is protonated, whereas on the less-acidic matrix
side a proportion is deprotonated. A compound that is not acidic enough may
transfer a single proton across the membrane, but will not release it in the matrix,
and hence cannot repeat the cycle. In contrast, too-strong an acid will remain
deprotonated, even in the intermembrane space. For this reason 2,4-dinitrophenol
(pKa 4.04) is a stronger uncoupler than 2-nitrophenol (pKa 7.14), while picric acid
(pKa 0.53) does not act as a protonophoric uncoupler in mitochondria, although all
three compounds have a similar lipophilicity [95].
In order to efficiently cross the membrane, uncouplers must be reasonably
lipophilic (generally logP > 2), although this property alone is not sufficient. A key
part of the action of a protonophoric uncoupler is an ability to cross the membrane
in the anionic form, and this requires the negative charge to be extensively
delocalized, or shielded from the lipid interior of the membrane in some way. For
this reason, many of the most potent uncouplers combine bulky lipophilic groups
adjacent to the ionizable proton with extended conjugated systems through which
the charge can be spread. A typical example of this is malonoben (10), one of the
most potent phenolic uncouplers known (Figure 15.4.3) [107].
On the other hand, many lipophilic carboxylic acids satisfy the logP and pKa
criteria for uncoupling, but the charge on the anion cannot be delocalized beyond
the carboxyl group; hence, they do not act as effective protonophoric uncouplers.
Those that do act in this way have structural features that allow the charge to
be delocalized, as in the anacardic acids where the adjacent phenol stabilizes the
negative charge (Figure 15.4.4a) [108]. A similar intramolecular interaction occurs
in the salicylanilide class of uncouplers, of which one of the most active is S-13
(11) (Figure 15.4.4b).
OH O− O O
− H+
+ H+
N N N− N
N N N N−
10
Figure 15.4.3 Delocalization and lipophilic shielding of charge on the anion of malonoben.
OH OH H H O OH
O O− O− O
+
−H
O O O O−
+ H+
R R R R
(a)
O O−
Cl NO2 + +
Cl N Cl N
O− O−
OH HN H
− H+ O− N OH N
O +H +
O O
Cl
11 Cl Cl
(b)
Figure 15.4.4 Delocalization of anionic charge by an adja-

cent group in (a) the anacardic acids and (b) salicylanilide
S-13.
In some cases, a bimolecular mechanism allows the anion to cross the membrane,
as described in Section 15.4.2. In this case, the negative charge is delocal-
ized across two molecules of uncoupler. Certain benzimidazoles, for example,
4,5,6,7-tetrachloro-2-trifluoromethyl benzimidazole (TTFB; 12), have been shown
to function in this way (Figure 15.4.5) [2, 4, 31].
It is difficult to formulate a simple set of rules that can accurately predict
from physico-chemical properties whether or not a compound will be an efficient
uncoupler, although some advances toward this goal have been made recently [109,
110]. However, some general guidelines for combinations of logP and pKa that
can lead to uncoupling can be devised, and these are illustrated in Figure 15.4.6,
with some selected examples marked (W. G. Whittingham, unpublished; based
on a suggestion from E. D. Clarke, T. E. M. Fraser and M. J. Robson). Thus,
the compound should be weakly acidic, with a pKa between 4 and 8, and have a
−
Cl
Cl N
Cl Cl
CF3
Cl N + Cl N Cl N
−H + TTFB
CF3 CF3 Cl
Cl H
+ −
N +H N − TTFB Cl
Cl Cl N
H
Cl Cl F 3C
N Cl
12
Cl
Figure 15.4.5 Bimolecular delocalization of negative charge, illustrated for TTFB.
0
picric acid
2
dinobuton/binapacryl*
TTFB Properties requires for potent uncoupling
4 2,4-DNP dinocap*
+ * propesticide group removed
DNOC ferimzone
p Ka
6 CCCP FCCP S-13 +

basic pKa
chlorfenapyr* fluazinam
2-NP AU-1421+
malonoben
8 fentrifanil
10
0 1 2 3 4 5 6 7 8 9
log P
Figure 15.4.6 Combination of logP and pKa required for uncoupling activity.
moderately high logP. It is worth noting that the properties of the basic uncoupler
AU-1421 (1) also fall within this range (logP 5.5, basic pKa 7.4) [33].
As already noted, this is a rather simplistic view, and the importance of charge
delocalization and steric effects should not be overlooked.
15.4.6
Chemical Uncouplers
It is clear from the above discussions that many different classes of chemistry
could, in theory, satisfy the physico-chemical requirements for uncoupling. This
is also the case in practice, with a wide range of different chemical types showing
fungicidal (and wider agrochemical) effects. A selection of the most important of
these is listed in Table 15.4.1, together with some key references.
Although all of the chemical classes shown in Table 15.4.1 have activity against
plant pathogenic fungi, from a commercial point of view three are of particular
interest, namely dinitrophenols, arylhydrazones, and diarylamines.
654
Table 15.4.1 Structures of selected fungicidal uncouplers.
Chemical Structure Trivial name Commercial Notes Key

class (major trade name) uses references
Dinitrophenols NO2 DNOC (Trifocide) Fungicide for top No longer used [34, 111]
HO fruit and vines
2 Herbicide Insecticide
NO2
O O
NO2
Dinobuton (Acrex) Powdery mildews Pro-pesticide of active [112, 113]
O acaricide phenol (dinoseb)
13
NO2
O
NO2
Binapacryl (Acricid) Powdery mildews Pro-pesticide of active [114]
O acaricide phenol (dinoseb)
8 Largely superseded
NO2
O
NO2
O 3 Dinocap (Karathane) Powdery mildews Pro-pesticide of active [114]
acaricide phenol
octyl NO2
Being replaced by
Mixture of regioisomers meptyldinocap
(see Figure 15.4.7)
Other phenols Malonoben, SF6847 Acaricide No longer used [115–118]
HO commercially
CN 10 Commonly used
CN biochemical standard
uncoupler
NO2
OH O
Salicylanilides S-13 Not commercialized Commonly used [119, 120]
N 11 biochemical standard
H
Cl uncoupler
Cl
Other amides – Not commercialized – [121]
F3C Cl
O
N
NC N
S H Cl
Arylhydrazones CCCP Not commercialized Commonly used [122–124]
CN biochemical standard
H
N Cl uncoupler
NC N 9
CN FCCP Not commercialized Commonly used [122]

H biochemical standard
N
NC N 14 uncoupler
OCF3
15.4 Uncouplers of Oxidative Phosphorylation
655
656

CN Cl – Not commercialized – [125, 126]

H
N
N
O2N Cl CF3
O Drazoxolon (Milcol) Broad-spectrum No longer used [127]

O Cl
H fungicide
N
N 15
Ferimzone (Blasin) Rice diseases See Section 15.4.8 [128–131]

N N
N 16
H
N
Dithiocarbazates PDTC-9 Not commercialized – [132–134]

S N
H
N
S N
8 H O
CF3
Diarylamines F3C NO2 Fentrifanil Acaricide – [135]
Not commercialized
17
N
H
Cl NO2
F3C O2N CF3 Fluazinam (Shirlan, Broad-spectrum See Section 15.4.9 [79, 136]
N Frowncide) fungicide
4
N Cl
H
Cl NO2
Br F3C
Br NO2 Bromethalin (Rampage) Rodenticide Pro-pesticide [137]
See Section 15.4.9
6
N
Cl NO2
NC Br
Nitrogen-heterocycles Chlorfenapyr (Pirate) Insecticide Pro-pesticide [138–140]
N CF3
7
Cl
O
Cl
Cl TTFB Not commercialized Commonly used [141]
N
biochemical standard
CF3 12
uncoupler
Cl N
H
Cl
15.4 Uncouplers of Oxidative Phosphorylation
657
658

Fluorinated sulfonamides O O Sulfluramid (Fluramim) Domestic insecticide Pro-pesticide [142, 143]

S
F3C(CF2)7 N
H
Coumarins OH OH Dicoumarol Not commercialized Commonly used [102, 144]

biochemical standard
uncoupler
O O
OO
HO
Natural products O Usnic acid Not commercialized Present in natural [145]
OH weight-loss dietary
O 5 supplement
O O
O
15.4.7
Dinitrophenols
The dinitrophenols were the first group of uncouplers to be commercialized, and

representatives of this group have found use as herbicides, insecticides/acaricides,
and fungicides. However, several have been superseded due to their toxicity and
lack of selectivity.
The most significant of the dinitrophenols still used as a fungicide is dinocap,
marketed by Dow under the trade name Karathane. This compound is sold as a
roughly 2 : 1 mix of regioisomers (18) and (19), each of which contains a mixture of
three isomers of the octyl group (Figure 15.4.7).
Dinocap is used as a powdery mildewicide on a range of crops, including vines,
fruit, vegetables, and ornamentals [146]. It also has secondary effects as a suppressor
of some mites, and a recent patent claims it to be active against whitefly [147].
To provide a good level of powdery mildew control, repeat spraying at intervals of
7–14 days is required, up to a maximum of 5–10 treatments a year, depending on
the crop [43]. Despite this type of repeated use over many years, no indication of
resistance to dinocap has been found, clearly demonstrating the low resistance risk
with the uncoupling mode of action [67].
In 2007, Dow launched meptyldinocap (24) as a replacement for dinocap.
Meptyldinocap is a single isomer; it is present as approximately 22% of dinocap.
The uncoupling and antifungal activity of meptyldinocap is equivalent to that
of dinocap, and the recommended use programs are very similar [62, 148], but
meptyldinocap has a much reduced toxicity [62–64]. The reason why dinocap and
meptyldinocap are only effective against powdery mildews is unclear, but this
is true also of the other two fungicidal dinitrophenol pro-pesticides, dinobuton
(13) and binapacryl (8), which suggests that efficacy may be due to either the
uptake or cleavage of the pro-pesticide. It is interesting to note that, as dinobuton
and binapacryl are both pro-pesticides of the commercial herbicide dinoseb, the
properties of the pro-pesticide group are clearly important for selectivity.
Although this class of chemistry has been well known for many years, in-
vestigations into related compounds continue. A recent example of this is the
arylnitrophenols, as disclosed by Valent [149].
O
O O
O
n O O
O 2N NO2
NO2 NO2
5-n n = 0-2
NO2 n 5-n NO2
18 19 24
Figure 15.4.7 Composition of commercial dinocap (18 + 19) and meptyldinocap (24).
15.4.8
Arylhydrazones, Including Ferimzone
Hydrazones prepared by the reaction of aryldiazonium salts with malononitrile

(trivially named carbonyl cyanide phenylhydrazones; CCPs) have been known
as uncouplers for many years [122]. Studies on the relationship between the
physico-chemical properties and uncoupling potency [97, 98] show the relationship
to be very similar to those for other classes of weakly acidic protonophoric
uncouplers. Thus, two of the most potent uncouplers of this type, CCCP (9) and
carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP; 14), have an acidic
pKa of 6.0 and log P values of 2.19 and 2.42, respectively [97, 98]. Although
these compounds are potent uncouplers in mitochondria from various organisms,
including mammals, plants, and insects [123], they have not found any agrochemical
application. They are, however, commonly used as biochemical standards for
uncoupling [81, 150].
Replacement of the malononitrile group with an isoxazolone group leads to
a well-known class of fungicides of which one member, drazoxolon (15), was
®
commercialized by ICI under the trade name Milcol . This compound was used to
control powdery mildews and other diseases on a range of crops and ornamentals,
and as a seed treatment for control of Pythium and Fusarium species, but has now
been replaced. The fungicidal effects of drazoxolon and analogs have been shown
to be a result of its uncoupling activity [127].
A more distantly related series of pyrimidinylhydrazones was discovered, through
random screening, to have high levels of fungicidal activity [151]. A program of
analog synthesis around the original lead culminated in the identification of
meferimzone (20) as a promising fungicide [151].
H
N N
N N
N HN N
N
20 is a mixture of (E )- and (Z )-isomers 16
Although the compound containing the (E)-isomer of the double bond is more
active when tested against fungi growing in media, there is little difference between
the isomers when tested on whole plants, presumably because isomerization of
the double bond occurs under the test conditions [128, 152]. Further investigations
resulted in the development of the pure (Z)-isomer 16, named ferimzone, as this
is more stable than the (E)-isomer [153]. This compound was first commercialized
®
by Sumitomo in 1991 under the trade name Blasin , and is used for the con-
trol of a range of diseases of rice, including Pyricularia oryzae, Helminthosporium
oryzae, and Cercospora oryzae [131]. Ferimzone has been shown to be fungistatic
rather than fungicidal, and is unusual amongst uncouplers in providing cura-
tive as well as protectant activity. It is also noteworthy that, although it does
not act as a pro-pesticide, ferimzone has a low acute toxicity compared to many
H H
N N N N
H2N N
O + N N
21 20
O
H
N NH H
H2 N NH O
N
NH2 N
NH2
Scheme 15.4.1 Synthetic routes to ferimzone.
typical uncouplers, with an acute oral LD50 in rats of > 600 mg kg−1 . It is also
environmentally friendly, with low toxicity to birds (acute oral LD50 for mallard
ducks > 292 mg kg−1 ), fish (96 h LC50 for carp 20 mg l−1 ), and bees (oral LD50
> 140 μg per bee), and short persistence in soil [degradation time (DT)50 3–14
days] [154].
Two main synthetic routes have been used for the synthesis of ferimzone
and its analogs, as shown in Scheme 15.4.1 [128, 151, 152]. Both start with
2 -methylacetophenone (21), and a new method for the industrial synthesis of this
key intermediate for ferimzone has been developed [155].
Although ferimzone is described as an uncoupler in the Pesticide Manual [154],
and is classified as such by the FRAC [75], its properties are somewhat different
to the other protonophoric uncouplers described in this chapter. Most notably,
ferimzone is nonacidic: the acidic pKa of the hydrazone is about 14.3) However,
it is weakly basic with a pKa of 4.16 (measured value; unpublished Syngenta
data); hence, it is possible that ferimzone is a rare example of a weakly basic
protonophoric uncoupler. A number of mode of action studies have been reported,
the results of which clearly indicate that ferimzone has an effect on fungal
membranes, increasing their permeability, and that this is likely to be responsible
for the compound’s antifungal activity [129, 130]. However, the precise details
of the primary mode of action remain unclear. In field trials, ferimzone shows
unexpected antibacterial and antiviral activity, which is not seen in in-vitro tests.
Further testing has shown these effects to be the result of a secondary activity
of ferimzone in inducing systemic acquired resistance in plants [156, 157]. It
seems likely, therefore, that the combination of effects on fungal membranes, with
resultant uncoupling, and the induction of plant defense responses is responsible
for the fungicidal activity of ferimzone.
3) Most acidic pKa 14.5 ± 0.5, calculated using

ACD Labs software.
15.4.9
Diarylamines, Including Fluazinam
Diarylamines have been utilized as NSAIDs for many years. One adverse side
effect of this class of drugs is liver toxicity, caused by the compounds’ uncoupling
effects [50]. The results of structure–activity studies have shown that very potent
uncoupling activity can be achieved in this chemical class [105]; consequently, these
compounds have also attracted attention as potential agrochemicals. Indeed, early
examples – including the development compound fentrifanil (17) [135] – proved to
be good acaricides [158, 159]. Unfortunately, these compounds suffered from toxic
effects, including severe acute toxicity and brain edema [52], which prevented their
commercialization. One positive spin-off from these studies was bromethalin (6)
which, despite being a pro-pesticide, showed such high acute toxicity that it has
now been commercialized as a rodenticide [137].
Further explorations by Ishihara revealed the arylaminopyridines also to be potent
uncouplers [105] and, although they possess a moderate acaricidal activity they have
been found to be better fungicides than the diarylamines. The optimization of this
class of chemistry resulted in the discovery of fluazinam (4) [160], which is not only
one of the most potent uncouplers known [106, 136] but also shows a high level
of reactivity with thiols, whereby the chlorine atom on the highly electron-deficient
phenyl ring is readily displaced [136, 161]. In fact, the potent, broad-spectrum
antifungal activity of fluazinam is most likely due to a combination of these two
modes of action [162, 163].
Fluazinam is unique among uncouplers in combining a potent, broad-spectrum
fungicidal activity with a very low mammalian toxicity. Such low toxicity is a result
of the high thiol reactivity of the compound, which leads to a rapid metabolism
and consequent detoxification in mammals [164]. A similar safening effect has
been observed in the diarylamine chemical series. Although compounds 22 and
23 are potent uncouplers with similar physico-chemical properties, 22 is highly
toxic to mice (oral LD50 0.9 mg kg−1 ) whereas 23, which contains a displaceable
chlorine atom and reacts rapidly with thiols, is much safer (oral LD50 in mice
> 100 mg kg−1 ) [164].
Cl F3C Cl F3C O2N

Cl NO2 Cl NO2 F3C CF3
N
N N Cl N Cl
H H H
Cl NO2 Cl NO2 Cl NO2
22 23 4
Fluazinam’s unique combination of properties has resulted in its worldwide

commercialization by Ishihara and Syngenta under a variety of trade names,
including Shirlan and Frowncide. Notably, it is to date by far the most commercially
important of the fungicidal uncouplers to have been discovered.
Although fluazinam is an excellent protectant fungicide, it has no systemic and
little curative activity [165, 166], but does show a degree of anti-sporulant effect
[165, 167]. Fluazinam shows a good residual activity, has excellent rainfastness [79,
165, 168, 169], and is capable of inhibiting several stages of the fungal lifecycle,
including spore germination and the formation of infection structures [167, 170].
Fluazinam also has a very potent inhibitory effect on the motility of zoospores of
Phytophthora species [168, 171].
Although fluazinam has a broad spectrum of fungicidal activity, it is less
potent on rusts and powdery mildews [79, 165] (in contrast to the dinitrophenols,
which are generally most effective against powdery mildews), and has not been
commercialized for use on cereal crops. It has, nonetheless, found wide application
in other crops since first being launched in New Zealand in 1988.
Fluazinam is extensively used in potatoes for the control of late blight (Phytoph-
thora infestans). It is especially effective against tuber blight, as the potent activity
against zoospores prevents them moving through the soil to infect the tubers [165,
168, 172–174]. In this role, fluazinam is sprayed up to 10 times per season, at 7-
to 14-day intervals, depending on the severity of the outbreak [175–178]. Despite
this intensive use as a stand-alone product, there is no indication of resistance
to fluazinam developing [69, 179], and it is especially useful for the control of
Phytophthora that has developed resistance to other fungicides [79]. It also shows
some positive effects against other potato diseases [180, 181].
Fluazinam is also useful for the control of Sclerotinia sp., notably on
peanuts [182–188] and turf [189]. It can be used against Botrytis on a wide
range of crops, especially beans [73, 190] and grapes [70–72, 191], where its
use has been shown to significantly reduce the levels of ochratoxin present
[192, 193].
As well as being used as a foliar spray, fluazinam is effective against soil-borne
diseases. It has a low mobility in soil [194, 195], and in some cases seed treatment
has proved to be more effective than soil application [196]. Fluazinam is particularly
effective for the control of brassica clubroot (Plasmodiophora brassicae) [197–203],
and can be used against root rots in various crops [178, 204–209]. It can also be
used to protect stored crops, for example, chicory [210].
In addition to its use in crops, fluazinam is employed for the treatment of
ornamental plants [211–213], and is particularly effective for the treatment of bulbs
[214, 215].
As noted above, the thiol reactivity of fluazinam results in a very low mammalian
toxicity, with an acute oral LD50 for rats of > 5000 mg kg−1 [216]. Unfortunately,
however, such reactivity creates certain issues and may lead to the skin sensitization
effects seen with fluazinam; repeated exposure to the compound can lead to the
development of an allergic contact dermatitis in sensitive individuals [53–55].
Fluazinam has a low toxicity in birds (acute oral LD50 for mallard ducks
4190 mg kg−1 ), bees (contact LD50 > 200 μg per bee), and worms (28-day LD50
> 1000 mg kg−1 ) [217]. Although fluazinam has a high toxicity towards aquatic
organisms (e.g., 96 h LC50 in rainbow trout 0.061 mg l−1 ) [217], it has a very short
persistence (half-life ca. 1 day) in aquatic systems [218]. In a microcosm study
based on realistic use rates in tulip cultivation, fluazinam was found to have little
or no adverse effect [218, 219].
O2N CF3
F3C F3C Cl Cl F3C O2N CF3

N N N
NH3 (aq) NO2
Cl NH2 KOH N Cl
H
Cl Cl Cl NO2
4
Scheme 15.4.2 Synthetic route to fluazinam.
Fluazinam is easily prepared from trifluoromethylpyridine intermediates that

are used for the synthesis of other agrochemicals, as shown in Scheme 15.4.2 [160,
220–223].
References
1. De Haan, E.J. and Charles, R. (1969) 12. Scheffler, I.E. (1999) Mitochondria,
Biochim. Biophys. Acta, 180, 417–419. Wiley-Liss, New York.
2. Neumcke, B. and Bamberg, E. (1975) 13. Garlid, K.D. (2004) in Encyclopedia
in Membranes, vol. 3, Chapter 3 (ed. D. of Biological Chemistry, vol. 1 (eds
Eisenman), Marcel Dekker, New York., W.J. Lennarz and M.D. Lane), Elsevier,
pp. 215–253. Oxford, pp. 405–412.
3. Heytler, P.G. (1981) in International 14. Skulachev, V.P. (1998) Biochim. Bio-
Encyclopedia of Pharmacology and Ther- phys. Acta, 1363, 100–124.
apeutics, vol. 107, Chapter 10 (eds M. 15. Hourton-Cabassa, C. and Moreau, F.
Erecinska and D.F. Wilson), Pergamon, (2008) Plant Cell Compart., 221–237.
Oxford., pp. 199–210. 16. Azzu, V. and Brand, M.D. (2010)
4. Terada, H. (1981) Biochim. Biophys. Trends Biochem. Sci., 35, 298–307.
Acta, 639, 225–242. 17. Bragadin, M., Moret, I., Piazza, R.,
5. McLaughlin, S. (1981) in Chemiosmotic Grasso, M., and Manente, S. (2002)
Chemosphere, 46, 219–223.
Proton Circuits in Biological Membranes
18. Nakagawa, Y. and Moore, G. (1999)
(eds V.P. Skulachev and P.C. Hinkle),
Biochem. Pharmacol., 58, 811–816.
Addison-Wesley, Reading, MA, pp.
19. Shinohara, Y., Yamamoto, T., and
601–609.
Terada, H. (2003) Maku, 28, 271–277.
6. Terada, H. (1990) Environ. Health
20. Halestrap, A.P. (2009) J. Mol. Cell.
Perspect., 87, 213–218.
Cardiol., 46, 821–831.
7. Miyoshi, H. (1992) Seibutsu Butsuri, 32, 21. Rottenberg, H. and Koeppe, R.E. II
60–65. (1989) Biochemistry, 28, 4355–4360.
8. Schwaller, M.A., Kühnel, J.M., Ferté, 22. Rottenberg, H. (1990) Biochim. Biophys.
J., and Allard, B. (1994) J. Chim. Phys. Acta, 1018, 1–17.
Physico-Chim. Biol., 91, 127–149. 23. Heytler, P.G. (1979) Methods Enzymol.,
9. Shinohara, Y. and Terada, H. (2000) 55, 462–472.
in Membrane Structure in Disease 24. Skulachev, V.P. (1991) FEBS Lett., 294,
and Drug Therapy, Chapter 7 (ed. G. 158–162.
Zimmer), Marcel Dekker, New York., 25. Wojtczak, L. and Wie ˛ckowski, M.R.
pp. 107–126. (1999) J. Bioenerg. Biomembr., 31,
10. Kadenbach, B. (2003) Biochim. Biophys. 447–455.
Acta, 1604, 77–94. 26. Schönfeld, P., Wie˛ckowski, M.R., and
11. Mitchell, P. (1961) Nature, 191, Wojtczak, L. (2000) FEBS Lett., 471,
144–148. 108–112.
References 665
27. Samartsev, V.N. (2000) Biochemistry 48. Han, D., Matsumaru, K., Rettori, D.,
(Moscow), 65, 991–1005. and Kaplowitz, N. (2004) Biochem.
28. Mitchell, P. (1961) Biochem. J., 81, 24P. Pharmacol., 67, 439–451.
29. Cunarro, J. and Weiner, M.W. (1973) 49. Miranda, E.J., McIntyre, I.M., Parker,
Nature, 245, 36–37. D.R., Gary, R.D., and Logan, B.K.
30. Cunarro, J. and Weiner, M.W. (1975) (2006) J. Anal. Toxicol., 30, 219–222.
Biochim. Biophys. Acta, 387, 234–240. 50. Masubuchi, Y., Yamada, S., and Horie,
31. Finkelstein, A. (1970) Biochim. Biophys. T. (2000) J. Pharmacol. Exp. Ther., 292,
Acta, 205, 1–6. 982–987.
32. Escher, B.I., Hunziker, R.W., and 51. Jacob, M., Bjarnason, I., Rafi, S.,
Schwarzenbach, R.P. (2001) Environ. Wrigglesworth, J., and Simpson, R.J.
Sci. Technol., 35, 3905–3914. (2001) Aliment. Pharmacol. Ther., 15,
33. Nagamune, H., Fukushima, Y., Takada, 1837–1842.
J., Yoshida, K., Unami, A., Shimooka, 52. Lock, E.A., Scales, M.D.C., and Little,
T., and Terada, H. (1993) Biochim. R.A. (1981) Toxicol. Appl. Pharmacol.,
Biophys. Acta, 1141, 231–237. 60, 121–130.
34. Tomlin, C.D.S. (ed.) (2003) The Pes- 53. Tominaga, K., Imamura, T., Nishioka,
ticide Manual, 13th edn, British Crop K., and Asagami, C. (1991) Hifu, 33
Protection Council, Alton, pp. 348–349, (Suppl. 11), 364–368.
entry 282. 54. van Ginkel, C.J.W. and Sabapathy, N.N.
35. Costa, C., Stassen, P.J.C., and (1995) Contact Dermatitis, 32, 160–162.
Mudzunga, J. (2004) Acta Hortic., 55. Bruynzeel, D.P., Tafelkruijer, J., and
636, 295–302. Wilks, M.F. (1995) Contact Dermatitis,
36. Hull, L.A., Asquith, D., and Mowery, 33, 8–11.
P.D. (1978) J. Econ. Entomol., 71, 56. Rogers, J.M., Carver, B., Gray, L.E. Jr,
880–885. Gray, J.A., and Kavlock, R.J. (1986) Ter-
37. Qu, S.C., Tashiro, N., Etoh, T., and atog. Carcinog. Mutagen., 6, 375–381.
Sadamatsu, M. (1997) Kyushu Byo- 57. Gray, L.E. Jr, Rogers, J.M., Ostby, J.S.,
gaichu Kenkyukaiho, 43, 125–129. Kavlock, R.J., and Ferrell, J.M. (1988)
38. Redl, H., Koschier, E., and Toxicol. Appl. Pharmacol., 92, 266–273.
Steinkellner, S. (1996) Mitt. 58. Gray, L.E. Jr, Rogers, J.M., Kavlock,
Klosterneuburg, 46, 1–7. R.J., Ostby, J.S., Ferrell, J.M., and Gray,
39. Sengonca, C. and Block, T. (1997) K.L. (1986) Teratog. Carcinog. Mutagen.,
Z. Pflanzenkr. Pflanzenschutz., 104, 6, 33–43.
140–146. 59. Rogers, J.M., Barbee, B., Burkhead,
40. Miles, M. and Green, E. (2002) Proc. L.M., Rushin, E.A., and Kavlock, R.J.
Brighton Crop Prot. Council Conf. – Pests (1988) Teratology, 37, 553–559.
Dis., 297–302. 60. Rogers, J.M., Gray, L.E. Jr, Carver,
41. Church, R.M. and Williams, R.R. B.D., and Kavlock, R.J. (1987) Teratog.
(1977) J. Hortic. Sci., 52, 429–436. Carcinog. Mutagen., 7, 341–346.
42. Butt, D.J., Swait, A.A.J., and Robinson, 61. Billington, R., Green, J.T., Mathieson,
J.D. (1985) Tests Agrochem. Cultivars, 6, E.A., Sivieri, F., Davies, J., Distler,
110–111. B., and Ehr, R.J. (2006) Patent WO
43. Whitehead, R. (ed.) (2006) UK Pesticide 2006/044,460.
Guide 2006, CABI Publishing, Walling- 62. Hufnagl, A.E., Distler, B., Bacci, L.,
ford, CT, p. 234, entry 180. and Valverde, P. (2007) Proceedings of
44. Shogaki, Y. and Yoshida, K. (1976) the 16th International Plant Protection
Noyaku Kensasho Hokoku, 16, 25–29. Conference, Glasgow, UK, October
45. Strider, D.L. (1980) Plant Dis., 64, 15–18, pp. 32–39.
188–190. 63. Environmental Protection Agency
46. Harper, J.A., Dickinson, K., and Brand, (2009) Pesticide Fact Sheet,
M.D. (2001) Obesity Rev., 2, 255–265. Meptyldinocap.
47. Spiegelman, B.M. (2006) Patent WO 64. Tomlin, C.D.S. (ed.) (2009) The Pes-
2006/014,529. ticide Manual, 15th edn, British Crop
Protection Council, Alton, pp. 388–391, B., and Ziogas, B.N. (2000) J. Phy-
entry 292. topathol., 148, 449–455.
65. Akhmedkhodzhaeva, K.S., Kamilov, 81. Stehmann, C. and De Waard, M.A.
G.D., and Sadritdinov, F.S. (1984) Dokl. (1995) Pestic. Sci., 45, 311–318.
Akad. Nauk. UZSSR, 3, 44–45. 82. Chapeland, F., Fritz, R., Lanen, C.,
66. Tomlin, C.D.S. (ed.) (2003) The Pes- Gredt, M., and Leroux, P. (1999) Pestic.
ticide Manual, 13th edn, British Crop Biochem. Physiol., 64, 85–100.
Protection Council, Alton, pp. 334–336, 83. Bai, J., Lai, L., Yeo, H.C., Goh, B.C.,
entry 270. and Tan, T.M.C. (2004) Int. J. Biochem.
67. Schepers, H.T.A.M. (1984) Neth. Cell. Biol., 36, 247–257.
J. Plant Pathol., 90, 165–171. 84. Andrade, A.C., Del Sorbo, G., Van
68. Gancheva, I. and Vitanov, M. (1986) Nistelrooy, J.G.M., and De Waard, M.A.
Pochvoz. Agrokhim. Rastitelna Zashtita, (2000) Microbiology, 146, 1987–1997.
21, 94–101. 85. Sedgwick, E.G., Hou, C., and Bragg,
69. Cooke, L.R., Little, G., and Wilson, P.D. (1984) Biochim. Biophys. Acta, 767,
D.G. (1998) Proc. Brighton Crop Prot. 479–492.
Conf. – Pests Dis., 517–522. 86. Lewis, K., Naroditskaya, V., Ferrante,
70. Leroux, P., Chapeland, F., Arnold, A., A., and Fokina, I. (1994) J. Bioenerg.
and Gredt, M. (1998) Phytoma, 504, Biomembr., 20, 639–646.
62–67. 87. Krulwich, T.A., Quirk, P.G., and
71. Leroux, P., Fritz, R., Debieu, D., Guffanti, A.A. (1990) Microbiol. Rev.,
Albertini, C., Lanen, C., Bach, J., 54, 52–65.
Gredt, M., and Chapeland, F. (2002) 88. Tollenaere, J.P. (1973) J. Med. Chem.,
Pest Manage. Sci., 58, 876–888. 16, 791–796.
72. Albert, J.-P., Stéva, H., Olivier, P., 89. Miyoshi, H., Nishioka, T., and Fujita,
Coulon, T., Herlemont, B., and Laveau, T. (1986) Bull. Chem. Soc. Jpn, 59,
É. (2004) Phytoma, 574, 37–40. 1099–1107.
73. Suzuki, K., Nishimura, A., Sugimoto, 90. Miyoshi, H., Nishioka, T., and Fujita,
K., and Komyoji, T. (1995) Ann. Phy- T. (1987) Biochim. Biophys. Acta, 891,
topathol. Soc. Jpn, 61, 399–404. 194–204.
74. Tamura, O. (2000) Abstracts of the 91. Miyoshi, H., Nishioka, T., and Fujita,
10th Symposium of Research Com- T. (1987) Biochim. Biophys. Acta, 891,
mittee of Fungicide Resistance, The 293–299.
Phytopathological Society of Japan, 92. Miyoshi, H. and Fujita, T. (1987)
Okoyama, April 5, 2000, pp. 7–16. Biochim. Biophys. Acta, 894, 339–345.
75. Fungicide Resistance Action Commit- 93. Miyoshi, H. and Fujita, T. (1988)
tee (2010) Code List. Biochim. Biophys. Acta, 935, 312–321.
76. Abelentsev, V.I., Savchenko, V.I., and 94. Miyoshi, H., Tsujishita, H., Tokutake,
Vishnevskaya, A.M. (1980) Zashchita N., and Fujita, T. (1990) Biochim.
Rastenii (Moscow), 23. Biophys. Acta, 1016, 99–106.
77. Abelentsev, V.I. and Savchenko, V.I. 95. Cajina-Quezada, M. and Schultz, T.W.
(1980) Khim. Sel’skom Khozyaistve, 18, (1990) Aquat. Toxicol., 17, 239–252.
31–33. 96. Argese, E., Bettiol, C., Marchetto, D.,
78. Schepers, H.T.A.M. (1984) Tagungs- De Vettori, S., Zambon, A., Miana,
bericht – Akademie der Land- P., and Ghetti, P.F. (2005) Toxicol. In
wirtschaftswissenschaften DDR, 222 Vitro, 19, 1035–1043.
(Systemic Fungicides and Antifungal 97. Baláž, Š., Šturdı́k, E., Ďurčová, E.,
Compounds), pp. 259–263. Antalı́k, M., and Sulo, P. (1986)
79. Komyoji, T., Sugimoto, K., Mitani, S., Biochim. Biophys. Acta, 851, 93–98.
Matsuo, N., and Suzuki, K. (1995) 98. Šturdı́k, E., Baláž, Š., Ďurčová, E.,
J. Pestic. Sci., 20, 129–135. Antalı́k, M., Sulo, P., Šturdı́ková, M.,
80. Kalamarakis, A.E., and Mikeš, V. (1988) Collect. Czech.
Petsikos-Panagiotarou, N., Mavroidis, Chem. Commun., 53, 1094–1101.
References 667
99. Storey, B.T., Wilson, D.F., Bracey, A., 116. Terada, H. and van Dam, K. (1975)
Rosen, S.L., and Stephenson, S. (1975) Biochim. Biophys. Acta, 387, 507–518.
FEBS Lett., 49, 338–341. 117. Terada, H. (1975) Biochim. Biophys.
100. Terada, H., Goto, S., Yamamoto, K., Acta, 387, 519–532.
Takeuchi, I., Hamada, Y., and Miyake, 118. Terada, H., Kumazawa, N., Ju-Ichi, M.,
K. (1988) Biochim. Biophys. Acta, 936, and Yoshikawa, K. (1984) Biochim. Bio-
504–512. phys. Acta, 767, 192–199.
101. Gange, D.M., Donovan, S., Lopata, 119. Kaplay, M., Kurup, C.K.R., Lam, K.W.,
R.J., and Henegar, K. (1995) Classical and Sanadi, D.R. (1970) Biochemistry, 9,
and Three-dimensional QSAR in Agro- 3599–3604.
chemistry, ACS Symposium Series, Vol. 120. Vinsova, J. and Imramovsky, A. (2004)
606, Chapter 15 (eds C. Hansch and Cesk. Slov. Farm., 53, 294–299.
T. Fujita), American Chemical Society, 121. Ken-Ichi, T., Katsuya, Y., Fumio, F.,
Washington, DC., pp. 199–212. Hiroshi, K., Chieko, I., and Kaoru, K.
102. Labbe-Bois, R., Laruelle, C., and (1993) Patent EP 0566,138.
Godfroid, J.-J. (1975) J. Med. Chem., 122. Heytler, P.G. and Prichard, W.W.
18, 85–90. (1962) Biochem. Biophys. Res. Commun.,
103. Godfroid, J.-J., Deville, C., and Laruelle, 7, 272–275.
C. (1977) Eur. J. Med. Chem., 12, 123. Heytler, P.G. (1963) Biochemistry, 2,
213–217. 357–361.
104. Terada, H., Muraoka, S., and Fujita, T. 124. Goldsby, R.A. and Heytler, P.G. (1963)
(1974) J. Med. Chem., 17, 330–334. Biochemistry, 2, 1142–1147.
105. Guo, Z.-J., Miyoshi, H., Komyoji, T., 125. Danko, P., Flury, T., Hall, R.G., Karrer,
Haga, T., and Fujita, T. (1991) Biochim. F., Kienast, H., Kriz, M., Leuenberger,
Biophys. Acta, 1059, 91–98. K., Pascual, A., Steiger, A., and
106. Brandt, U., Schubert, J., Geck, P., and Trah, S. (2006) Pest Manage. Sci.,
von Jagow, G. (1992) Biochim. Biophys. 62, 229–235.
Acta, 1101, 41–47. 126. Flury, T., Hall, R.G., Karrer, F.,
107. Spycher, S., Escher, B.I., and Gasteiger, Kienast, H., Leuenberger, K., Pascual,
J. (2005) Chem. Res. Toxicol., 18, A., Rindlisbacher, A., Trah, S., and
1858–1867. Widmer, H.-J. (2006) Pest Manage. Sci.,
108. Toyomizu, M., Okamoto, K., Akiba, 62, 236–241.
Y., Nakatsu, T., and Konishi, T. (2002) 127. Parker, V.H. and Summers, L.A. (1970)
Biochim. Biophys. Acta, 1558, 54–62. Biochem. Pharmacol., 19, 315–317.
109. Spycher, S., Smejtek, P., Netzeva, T.I., 128. Konishi, K. and Kuragano, T. (1989)
and Escher, B.I. (2008) Chem. Res. J. Pestic. Sci., 14, 211–221.
Toxicol., 21, 911–927. 129. Okuno, T., Furusawa, I., Matsuura,
110. Spycher, S., Netzeva, T.I., Worth, A.P., K., and Shishiyama, J. (1989) Ann.
and Escher, B.I. (2008) SAR QSAR Phytopath. Soc. Jpn, 55, 281–289.
Environ. Res., 19, 433–463. 130. Okuno, T., Furusawa, I., Matsuura, K.,
111. Truffaut, G. and Pastac, I. (1935) and Shishiyama, J. (1989) Phytopathol-
Patent GB 425,295. ogy, 79, 827–832.
112. Pianka, M. and Polton, D.J. (1966) 131. Matsuura, K., Ishida, Y., Kuragano, T.,
Patent GB 1,019,451. and Konishi, K. (1994) J. Pestic. Sci., 19,
113. Tomlin, C.D.S. (ed.) (2009) The Pes- 325–327.
ticide Manual, 15th edn, British Crop 132. Terada, H., Uda, M., Kametani, F., and
Protection Council, Alton, p. 387, entry Kubota, S. (1978) Biochim. Biophys.
291. Acta, 504, 237–247.
114. Gasztonyi, M. and Lyr, H. (1987) in 133. Kubota, S., Uda, M., Mori, Y.,
Modern Selective Fungicides, Chapter Kametani, F., and Terada, H. (1978)
21 (ed. H. Lyr), Longman, Harlow, pp. J. Med. Chem., 21, 591–594.
309–324. 134. Uda, M., Toyooka, K., Horie, K.,
115. Muraoka, S. and Terada, H. (1972) Shibuya, M., Kubota, S., and Terada,
Biochim. Biophys. Acta, 275, 271–275. H. (1982) J. Med. Chem., 25, 557–560.
135. Nizamani, S.M. and Hollingworth, 149. Helman, D.F., Petracek, P.D., Fugiel,
R.M. (1980) Biochem. Biophys. Res. J.A., and Warrior, P. (2006) Patent US
Commun., 96, 704–710. 6,992,111.
136. Guo, Z.-J., Miyoshi, H., Komyoji, T., 150. Kovacic, P., Pozos, R.S., Somanathan,
Haga, T., and Fujita, T. (1991) Biochim. R., Shangari, N., and O’Brien,
Biophys. Acta, 1056, 89–92. P.J. (2005) Curr. Med. Chem., 12,
137. van Lier, R.B.L. and Cherry, L.D. (1988) 2601–2623.
Fundam. Appl. Toxicol., 11, 664–672. 151. Konishi, K., Kuragano, T., and
138. Addor, R.W., Babcock, T.J., Black, Matsuura, K. (1986) Agric. Biol. Chem.,
B.C., Brown, D.G., Diehl, R.E., Furch, 50, 2427–2428.
J.A., Kameswaran, V., Kamhi, V.M., 152. Konishi, K. and Matsuura, K. (1980)
Kremer, K.A., Kuhn, D.G., Lovell, J.B., Patent EP 0019,450.
Lowen, G.T., Miller, T.P., Peevey, R.M., 153. Akashi, K. and Asogawa, T. (1989)
Siddens, J.K., Treacy, M.F., Trotto, Patent GB 2,210,267.
S.H., and Wright, D.P. Jr (1992) in 154. Tomlin, C.D.S. (ed.) (2009) in The Pes-
Synthesis and Chemistry of Agrochemi- ticide Manual, 15th edn, British Crop
cals III, ACS Symposium Series, Vol. Protection Council, Alton, pp. 497–498,
504, Chapter 25 (eds D.R. Baker, J.G. entry 374.
Fenyes, and J.J. Steffens), American 155. Furuoya, I. (1995) Stud. Surf. Sci.
Chemical Society, Washington, DC, pp. Catal., 92, 315–318.
283–297. 156. Nakayama, M., Matsuura, K., and
Okuno, T. (1996) J. Pestic. Sci., 21,
139. Kuhn, D.G., Kamhi, V.M., Furch, J.A.,
69–72.
Diehl, R.E., Trotto, S.H., Lowen, G.T.,
157. Siegrist, J., Mühlenbeck, S., and
and Babcock, T.J. (1992) in Synthesis
Buchenauer, H. (1998) Physiol. Mol.
and Chemistry of Agrochemicals III, ACS
Plant Pathol., 53, 223–238.
Symposium Series, Vol. 504, Chapter
158. Dreikorn, B.A., Kramer, K.E.,
26 (eds D.R. Baker, J.G. Fenyes, and
Berard, D.F., Harper, R.W., Tao, E.,
J.J. Steffens), American Chemical Soci-
Thompson, L.G., and Mollet, J.A.
ety, Washington, DC, pp. 298–305.
140. Hunt, D.A. (1994) in Advances in the
Chemistry of Insect Control III (ed. G.G.
Series, Vol. 504, Chapter 30 (eds D.R.
Briggs), Royal Society of Chemistry,
Baker, J.G. Fenyes, and J.J. Steffens),
Cambridge, pp. 127–140. American Chemical Society, Washing-
141. Jones, O.T.G. and Watson, W.A. (1967) ton, DC, pp. 336–341.
Biochem. J., 102, 564–573. 159. Kramer, K.E., Dreikorn, B.A.,
142. Schnellmann, R.G. and Manning, R.O. Humphreys, W.H., and Mollet, J.A.
(1990) Biochim. Biophys. Acta, 1016, (1992) in Synthesis and Chemistry of
344–348. Agrochemicals III, ACS Symposium
143. Starkov, A.A. and Wallace, K.B. (2002) Series, Vol. 504, Chapter 31 (eds D.R.
Toxicol. Sci., 66, 244–252. Baker, J.G. Fenyes, and J.J. Steffens),
144. Maruzzella, J.C., Gambocs, J.A., and American Chemical Society, Washing-
Handler, D. (1960) Naturwissenschaften, ton, DC, pp. 342–348.
47, 17. 160. Nakajima, T., Komyoji, T., Akagi, T.,
145. Shaw, P.D. (1967) Antibiotics (USSR), Nagatani, K., Haga, T., Matsuo, N.,
1, 611–612. and Suzuki, K. (1995) Synthesis and
146. Kidd, H. and James, D.R. (eds) (1990) Chemistry of Agrochemicals IV, ACS
European Directory of Agricultural Symposium Series, Vol. 584, Chapter
Products, Royal Society of Chemistry, 38 (eds D.R. Baker, J.G. Fenyes, and
Cambridge, pp. 229–233, entry 415. G.S. Basarab), American Chemical So-
147. Hadar, A. (2005) Patent WO ciety, Washington, DC, pp. 443–448.
2005/022,999. 161. Clarke, E.D., Greenhow, D.T., and
148. Marquet, N. (2007) Phytoma, 600, Adams, D. (1998) Pestic. Sci., 54,
42–44. 385–393.
References 669
162. Akagi, T., Mitani, S., Komyoji, T., and 183. Lemay, A.V., Bailey, J.E., and Shew,
Nagatani, K. (1995) J. Pestic. Sci., 20, B.B. (2002) Plant Dis., 86, 1315–1317.
279–290. 184. Smith, F.D., Phipps, P.M., and Stipes,
163. Akagi, T., Mitani, S., Komyoji, T., and R.J. (1991) Plant Dis., 75, 1138–1143.
Nagatani, K. (1996) J. Pestic. Sci., 21, 185. Tad Smith, F.D., Phipps, P.M., Stipes,
23–29. R.J., and Brenneman, T.B. (1995) Plant
164. Hollingworth, R.M. and Gadelhak, Dis., 79, 517–523.
G.G. (1998) Rev. Toxicol., 2, 253–266. 186. Smith, F.D., Phipps, P.M., and Stipes,
165. Anema, B.P., Bouwman, J.J., Komyoji, R.J. (1992) Peanut Sci., 19, 115–120.
T., and Suzuki, K. (1992) Proc. Brighton 187. Damicone, J.P. and Jackson, K.E.
Crop Prot. Conf. – Pests Dis., 663–668. (1996) Peanut Sci., 23, 81–85.
166. Kapsa, J. (2003) J. Plant Prot. Res., 43, 188. Damicone, J.P. and Jackson, K.E.
191–198. (2001) Peanut Sci., 28, 28–33.
167. Komyoji, T., Sugimoto, K., and Suzuki, 189. Burpee, L.L. (1997) Plant Dis., 81,
K. (1995) Ann. Phytopath. Soc. Jpn, 61, 1259–1263.
145–149. 190. Stewart, T.M. and Long, P.G. (1987)
168. Tucker, R.P., Leaper, D.J., and Laidler, New Zeal. J. Exp. Agric., 15, 97–103.
S.T. (1994) Proc. Brighton Crop Prot. 191. Albert, J.-P., Drouillard, J.-B.,
Conf. – Pests Dis., 295–300. Gueunier, M.M., and Bac, V. (2004)
169. Schepers, H.T.A.M. (1996) Potato Res., Phytoma, 577, 6–8.
39, 541–550. 192. Drouillard, J.-B., Sage, L., Pladeau, V.,
170. Mitani, S., Ohhashi, K., Yamaguchi, T.,
and Dubernet, M. (2003) Phytoma, 565,
and Komyoji, T. (1996) J. Pestic. Sci.,
30–35.
21, 61–63.
193. Drouillard, J.-B., Sage, L., Pladeau,
171. Matheron, M.E. and Porchas, M. (2000)
V., and Kalanquin, D. (2003) Rev.
Plant Dis., 84, 454–458.
Fr. d’CEnol., 202, 10–14.
172. Erlingson, M. (1993) Svenska Vaextsky-
194. Cohen, R., Pivonia, S., Shtienberg,
ddskonferensen, 34, 131–136.
D., Edelstein, M., Raz, D., Gerstl, Z.,
173. Roques, J.-F. and Sylvestre, M. (1994)
and Katan, J. (1999) Plant Dis., 83,
Phytoma, 460, 48–50.
1137–1141.
174. Leonard, R., Dowley, L.J., Rice, B.,
195. Hu, W., Lee, S.J., and Kim, J.-E. (1997)
and Ward, S. (2001) Potato Res., 44,
327–336. Agric. Chem. Biotechnol., 40, 128–133.
175. Dowley, L.J. and O’Sullivan, E. (1995) 196. Yukawa, T., Ohshita, Y., and Watanabe,
Ir. J. Agric. Food Res., 34, 33–37. J. (2003) Nippon Sakumotsu Gakkai Kiji,
176. Cooke, L.R. and Little, G. (1995) Tests 72, 216–218.
Agrochem. Cultivars, 16, 28–29. 197. Porter, I.J., Donald, E.C., and Cross,
177. Platt, H.W., Reddin, R., and Jenkins, S.J. (1998) Tests Agrochem. Cultivars, 19,
S. (1998) Tests Agrochem. Cultivars, 19, 12–13.
22–23. 198. Suzuki, K., Sugimoto, K., Hayashi, H.,
178. Whitehead, R. (ed.) (2006) UK Pesticide and Komyoji, T. (1995) Ann. Phytopath.
Guide 2006, CABI Publishing, Walling- Soc. Jpn, 61, 395–398.
ford, CT, pp. 276–277, entry 232. 199. Cheah, L.-H., Page, B.B.C., and
179. Xu, S., Yao, L., Li, X., and Zhang, X. Koolaard, J.P. (1998) Proc. New Zeal.
(2009) Zhiwu Baohu, 35, 80–85. Plant Prot. Conf., 51, 130–133.
180. Platt, H.W. and Reddin, R. (1996) Tests 200. Donald, E.C., Porter, I.J., and
Agrochem. Cultivars, 17, 16–17. Lancaster, R.A. (2001) Aust. J. Exp.
181. Braithwaite, M., Falloon, R.E., Genet, Agric., 41, 1223–1226.
R.A., Wallace, A.R., Fletcher, J.D., and 201. Mitani, S., Sugimoto, K., Hayashi, H.,
Braam, W.F. (1994) New Zeal. J. Crop Takii, Y., Ohshima, T., and Matsuo, N.
Hort. Sci., 22, 121–128. (2003) Pest Manage. Sci., 59, 287–293.
182. Ryley, M.J., Kyei, N.A., and Tatnell, J.R. 202. Humpherson-Jones, F.M. (1989) Tests
(2000) Aust. J. Agric. Res., 51, 917–924. Agrochem. Cultivars, 10, 36–37.
203. Kurowski, T.P., Majchrzak, B., 216. Syngenta. Shirlan Safety Data Sheet.
Jazwinska, E., and Wysocka, U. (2008) Syngenta Crop Protection, Fulbourn.
Prog. Plant Prot., 48, 212–215. Available at: http://www.syngenta-
204. Kanadani, G., Date, H., and Nasu, N. crop.co.uk/pdfs/products/Shirlan_uk_
(1998) Ann. Phytopath. Soc. Jpn, 64, safety_data.pdf.
139–141. 217. Tomlin, C.D.S. (ed.) (2009) The Pes-
205. Sugimoto, K., Araki, S., and Hayashi, ticide Manual, 15th edn, British Crop
H. (2002) Patent JP 2002/104,907. Protection Council, Alton, pp. 513–514,
206. Sugimoto, K. (2002) Agrochem. Jpn, 80, entry 385.
14–16. 218. Wendt-Rasch, L., Van den Brink,
207. Ziezold, A.M., Hall, R., Reeleder, R.D., P.J., Crum, S.J.H., and Woin, P.
and Proctor, J.T.A. (1998) J. Ginseng (2004) Ecotoxicol. Environ. Saf., 57,
Res., 22, 223–228. 383–398.
208. Ziezold, A.M., Hall, R., Reeleder, R.D., 219. van Wijngaarden, R.P.A., Cuppen,
and Proctor, J.T.A. (1998) J. Ginseng J.G.M., Arts, G.H.P., Crum, S.J.H.,
Res., 22, 237–243. van den Hoorn, M.W., van den
209. Matheron, M.E. and Porchas, M. (2000)
Brink, P.J., and Brock, T.C.M.
Plant Dis., 84, 1038–1043.
(2004) Environ. Toxicol. Chem., 23,
210. Benigni, M., Laville, J., and Bompeix,
1479–1498.
G. (2000) Med. Fac. Landbouww. Univ.
220. Nishiyama, R., Fujikawa, K., Haga, T.,
Gent, 65, 761–769.
Toki, T., Nagatani, K., and Imai, O.
211. Benson, D.M. (1993) Phytopathology, 83,
(1981) Patent EP 0031,257.
1410.
221. Murai, S., Yoshizawa, H., Ohshima,
212. Braithwaite, M., Knight, K.W.L., Saville,
T., Murakami, K., Ando, T., and
D.J., and Alexander, B.J.R. (1996)
Proc. New Zeal. Plant Prot. Conf., 49, Nakamura, T. (2009) Patent WO
157–160. 2009/017,239.
213. O’Neill, T.M., Pye, D., and Locke, T. 222. Murai, S., Yoshizawa, H., Ohshima, T.,
(2002) Ann. Appl. Biol., 140, 207–214. Murakami, K., Ando, T., Nakamura, T.,
214. Anema, B.P., Bouwman, J.J., and Adachi, N., and Isogai, A. (2009) Patent
De Vlugt, J. (1988) Med. Fac. Land- WO 2009/017,241.
bouww. Univ. Gent, 53, 635–641. 223. Murai, S., Yoshizawa, H., Ohshima, T.,
215. Koster, A.T.J. and van der Meer, L.J. Murakami, K., Ando, T., Nakamura, T.,
(1992) Recent Adv. Botrytis Res., Proc. Adachi, N., and Isogai, A. (2009) Patent
Int. Botrytis Symp., 10, 277–281. WO 2009/054,210.
15.5
NADH Inhibitors (Complex I)
Harald Walter
15.5.1
Introduction
Complex I inhibition is an important mode of action of pharmaceuticals and

agrochemicals [1] (for a general introduction, see Chapter 15.1). During the past 20
years, in the area of crop protection, investigations have been mainly focused on
insecticides and acaricides with useful properties. Examples of consequently com-
mercialized insecticide/acaricide compounds include fenazaquin (1), tebufenpyrad
(2), and pyridaben (3) (see Chapter 31.3):
Me
Me Me
Me Me Me
Et Me Me Cl
Cl Me
H S O
O
N
N N Me
N N N
Me O Me Me
N tebufenpyrad 2 pyridaben 3
fenazaquin 1
In general, agrochemical fungicides with useful potency, fungicidal spectrum, and

toxicological properties, and which are of sufficient interest to be considered for
commercialization, are rare. For example, only one compound, diflumetorim (4;
trade name Pyricut) [2] (Figure 15.5.1) has been introduced into the market during
the past 15 years. Diflumetorim (4), which is used mainly in the treatment of
ornamentals (Japan), had estimated sales of only US$ 7 million in 2010 (Cropnosis
database), and has been marketed by SDS Biotech since the start of 2003.4)
Diflumetorim (4) is a member of aminoalkylpyrimidines, which are considered
to be the most interesting class of fungicides with a Complex I mode of action
(Figure 15.5.2). Although all of the major companies in the field pursued this lead
with great intensity, only Ube Industries was successful in developing a commercial
compound. To date, no other broad-spectrum fungicide with a Complex I mode
of action has been identified, despite the discovery of many compounds that have
demonstrated reasonable spectra of activity (e.g., powdery mildew, brown rust, and
leaf spot diseases) and reasonable application rates in the glasshouse.
15.5.2
The Aminoalkylpyrimidines
Fungicides containing pyrimidine moieties with relevant use in agriculture have

been known for over 30 years [3]. The most prominent compounds belong to
the class of anilinopyrimidines, of which mepanipyrim (5), pyrimethanil (6), and
cyprodinil (7) are the most important examples (see Chapter 16.2).
Me
Me
N N N
N N N
H N H N H N
Me Me Me
mepanipyrim 5 pyrimethanil 6 cyprodinil 7
4) In 2003, SDS Biotech acquired the agro-

chemical fungicide business from Ube
Industries, including diflumetorim.
Me Cl Et Figure 15.5.1 Structure of diflumetorim (4).

H
O H
N N F
N H F
Diflumetorim 4
racemic
Lipophilic part
1 2
R A R
(C(R5R6))n X Rlipo
N N
N
R4 Linker
R3
Pharmacophore
(Toxophore)
General aminoalkypyrimidine formula I
R1, R2 = C1-C4-alkyl, C1-C4-haloalkyl, C1-C4-alkoxyalkyl, halogen;

R1 and R2 together may also form an aromatic or nonaromatic 5- to 7-membered ring
system A (e.g., phenyl, thienyl, cyclohexyl, etc.)
R3 = H, Me
R6 = H, C1-C4-alkyl, COC1-C4-alkyl, etc.
R , R6 = H, C1-C4-alkyl
5
Rlipo = phenyl, phenoxyphenyl, benzylphenyl, naphthyl, tetrahydronaphthyl, indanyl,

benzothienyl, benzofuryl, benzoxazinyl, quinolinyl, tetrahydrochinolinyl, cyclopentyl,
heteroatom-containing nonaromtic 5-membered ring systems, cyclohexyl, hetero-
atom containing nonaromatic ring systems
X = direct bond or O
n = 0-3
(Only most important substituents given)
Figure 15.5.2 General structure of the class of aminoalkylpyrimidines.
Although these compounds do not exhibit Complex I inhibition, they have been
reported to inhibit the biosynthesis of methionine. The pioneers of the Complex
I-aminoalkylpyrimidine class fungicides – the research chemists at Ube Indus-
tries – were inspired by a report from Whitehead and Traverso that described
the diuretic properties of certain 4-aminopyrimidine derivatives [4]. Despite
aminoarylalkyl-substituted pyrimidine compounds of the general formula II [5]
(Figure 15.5.3) being first patented in 1988, both insecticidal and fungicidal activ-
ities were claimed for these compounds, notably in the area of fungicides, with
efficacy against rice blast, powdery mildew, and downy mildew. Interestingly, Ube’s
development compound – diflumetorim (4) – had been generically claimed in the
first patent application, but was not described in either the text or respective tables
of the patent.
R1, R2 = C1-C4-alkyl, halogen, etc.

R1 R2
BQ R3 = H, C1-C4-alkyl
A
R4 = H, C1-C4-alkyl, halogen
N N
N H A = lower alkylene
(R4)n
B = direct bond, O, S, lower alkylene or alkyleneoxy
R3
General Ube formula II Q = phenyl, heterocycle, substituted or unsubstituted
alkyl, allyl, geranyl, farnesyl, glycidyl, acetonyl, etc.
Figure 15.5.3 First important Ube subclass.
Further patents from Ube Industries in this area were subsequently obtained
over the years [6–11], with the final one appearing in 2003. The most inter-
esting Ube applications in the area of fungicides were made in 1990 (EP
0370 704) and 1991 (EP 0432 894 - mixture patent), and the development com-
pound diflumetorim (4) {((RS)-5-chloro-N-1-[4-(difluoromethoxy)phenyl]-propyl-6-
methylpyrimidin-4-ylamine} was exemplified for the first time in these patents.
Diflumetorim (Pyricut) was registered as a new fungicide for use in ornamental
plants in Japan in April 1997, and subsequently developed by Ube Industries and
Nissan Chemical Industries, with major fungicidal targets of rose powdery mildew
and chrysanthemum white rust. Diflumetorim has good protective properties,
some curative activity, and instantly arrests fungal growth at any stage, from the
germination of the conidia to formation of the conidiophores [2]. Various properties
relating to the compound’s acute toxicity/ecotoxicity and its environmental fate
(technical material) have been reported by the research team at Ube [2]. Typically, the
oral LD50 -values for diflumetorim of 387 mg kg−1 (male mouse) and 534 mg kg−1
(male rat) were within the more favorable range for this type of compound. As
a consequence of their mode of action, Complex I inhibitors generally possess a
higher degree of acute oral toxicity in mammals than do most modern pesticides
(H. Walter, unpublished results and [12]). As an example, in rats the acute dermal
toxicity (LD50 ) was > 2000 mg kg−1 , while the acute inhalation toxicity (LD50 ) was
0.61 mg l−1 . However, a series of negative in vitro tests to monitor genotoxicity
(Ames test, chromosome aberration test, mouse micronucleus test) appeared
favorable for diflumetorim.
At this point, the ecotoxicological behavior of diflumetorim will be discussed
only briefly. Toxicity towards fish was shown to be high, with 48 h LC50 -values of
0.098 and 0.025 mg l−1 for carp and rainbow trout, respectively, but was moderate
towards Daphnia (3 h LC50 = 0.96 mg l−1 ). In birds, the toxicity was relatively low
(LD50 = 1979 mg kg−1 for mallard duck; 881 mg kg−1 for quail), which indicated
that the risks were likely to be low.
With regards to the soil dissipation behavior of diflumetorim, an aerobic
metabolism study conducted in soil revealed a slow degradation time (DT50 =
4.5 months), with the major route of metabolism being identified as the hydrox-
ylation of diflumetorim in the 2-position of the pyrimidine ring. Soil dissipation
studies performed both in the field and in the laboratory, using various soil types,
revealed DT50 values of 60–140 days, while absorption studies (Koc = 473) indicated
a low potential for the mobility of the compound in the soil.
The chemistry of diflumetorim appears straightforward; a possible technical
synthesis for the compound is shown in Scheme 15.5.1. In this case, the pyrimidine
moiety (4,5-dichloro-6-methylpyrimidine; 11) is synthesized in a two-step sequence
starting with the condensation of 2-chloro-3-oxobutanoic acid methyl ester (8)
and formamidine acetate (9) [13] to give 5-chloro-4-hydroxy-6-methylpyrimidine
(10) in high yield, followed by chlorination of the hydroxy group using standard
synthesis methodology [13–16]. Synthesis of the substituted benzylamine
(14) [17] begins from 4-hydroxypropiophenone (12), which is transformed
into 4-difluoromethoxypropiophenone (13) by reaction of the phenol (12)
in an autoclave with difluorochloromethane in the presence of potassium
carbonate. Reductive amination of the resulting ketone by treatment with liquid
ammonia in methanol in the presence of Raney nickel, again in an autoclave, gives
α-(RS)-ethyl-4-difluoromethoxybenzylamine (14) in 97% yield. The final compound,
5-chloro-6-methyl-N-(α-(RS)-ethyl-4-difluoromethoxybenzyl)-pyrimidine-4-amine
(4) [17] is obtained by reacting 11 with 14 in the presence of triethylamine and a
catalytic amount of dimethylaminopyridine (DMAP).
Structure–activity relationship (SAR) studies, correlating preventive fungicidal
potency against wheat brown rust and barley powdery mildew, have been described
only for special subclasses of N-benzyl-4-pyrimidine-amines [17, 18].
Pyrimidine part:
HN NH2 Me Cl
Me Cl
Cl 9 x MeCOOH POCl3/toluene
H
Me O N OH N Cl
Me 1) NaOMe/MeOH Reflux
N N
O O 3h, rt Yield est.: 80-85%
8 2) Neutralize with H2SO4, 10 11
then 1h, 65°C
(Yield for Et-derivative
according to lit.16: 77%)
Yield: 94%
Aralkylamino part:
CF2ClH
NH3(l), MeOH
O K2CO3/DMF O H
Raney-Ni H2N
OH O H O H
3h, 100°C Autoclave
Et Et F Et F
3h, 120°C
12 Yield: 42% 13 F 14 F
Yield: 97% racemic
Coupling step:
H
H 2N
Me Cl OH Me Cl Et
Et F H
14 F
O H
N Cl N N F
NEt3/toluene F
N N H
cat. DMAP 4
11 8h, reflux racemic
Yield: 70%
Scheme 15.5.1 Synthesis of diflumetorim.

R1 R2 R3 R1 = C1-C4-alkyl
H
O R2 = halogen
N N
R 5
R3 = C1-C4-alkyl
N H R4
R4 = H, F, Cl, OMe, OCHF2, 3,5-Me2
General formula III
R5 = CF2H: subclass IIIa
F F
R6 (R6 = F: subclass IIIb)
F F
R6 = F, CF2H, CF3, Ac, CHO, CN, NO2
Figure 15.5.4 Structures of subclasses IIIa and IIIb.
Recently, a brief SAR analysis of compounds of the general formula III has
been produced by the research group at Ube (Figure 15.5.4) [17]. The preferred
combination of substituents R1 and R2 on the pyrimidine ring seems to be a small
alkyl group (R1 = Me, Et) and a halogen group (R2 = halogen, preferably Cl). The
introduction of a small alkyl group (R3 = Me, Et) at the benzyl position leads to
the most active compounds. Generally, the introduction of further substituents R4
in the benzyl-benzene moiety of the OCF2 H-subclass IIIa does not seem to lead to
any significant improvements of activity (one exception: 2 -F improves the brown
rust activity). For the OC6 F5 -subclass IIIb, the influence of further substituents R4
in the benzyl-benzene moiety was not further investigated. However, a study of
the influence of substituents R6 in the 4 -position demonstrated that replacement
of the 4 -F by substituents such as CHO, Ac, CN, and NO2 leads to compounds
with significantly reduced activity (with the exception of CF2 H!). In summary,
substitution of the 4-position of the benzyl-benzene by electron-withdrawing groups
such as OCF2 H and OC6 F5 leads to the excellent fungicidal aminoalkylpyrimidine
subclasses IIIa and IIIb.
Since diflumetorim (4) was developed for use in ornamentals seemingly confined
to the Japanese market, and not as a cereal fungicide (perhaps due to an unfavorable
price/activity ratio), the sales potential of the compound will remain limited.
15.5.2.1 The Competitors’ Contributions in the Aminoalkylpyrimidine Area

Many companies began synthesis programs based on this novel lead of Complex
I inhibitors, including the legacy companies of Syngenta Crop Protection AG
(Ciba-Geigy AG and Sandoz Ltd), Bayer CropScience (Hoechst AG and Hoechst
Schering AgrEvo), and Du Pont. Eli Lilly, Sumitomo Ind., Shell AG, and BASF also
reported on this lead, albeit on a smaller scale.
It soon became evident that the pyrimidine moiety (toxophore) was already well
optimized for fungicidal activity (to the authors’ best knowledge: 5-halogen, 6-Me,
Et) although, occasionally, substitution patterns of the pyrimidine ring such as R1 =
CH2 OMe and R1 = OMe were proposed. Consequently, synthetic efforts became
concentrated on modifying the linker [C(R5 R6 )n X] and the lipophilic part Rlipo . The
research teams at Ciba-Geigy AG [19–23], Sandoz Ltd [24], and Du Pont [25–27]
R1 R2 R3
H R1 = C1-C4-alkyl
Rlipo R2 = halogen
N N
R3 = C1-C4-alkyl
N H
Rlipo = 2-naphthyl (EP 470 600): subclass IVa
General formula IV
(ex. Ciba-Geigy AG) (WO 94/20490): subclass IVb
N
X R'
R' = C1-C6-alkyl, C2-C6-alkenyl, C3-C7-cycloalkyl

X = O, S
R1 R2 R3
H
Rlipo Rlipo = 2-naphthyl (overlap with Ciba-Geigy): subclass IVa
N N
2-benzothienyl: subclass IVc
N H
General formula IV Subclass IVd

Me
Du Pont (WO 92/08704) Me
Me
R1 R2 R1 R2
X O
N N R N N A R'
N H X N H O
General formula V General formula VI

Hoechst schering AgrEvo Hoechst schering AgrEvo
(WO 93/01950, WO 95/31441 (WO 96/11924)
and WO 97/02264)
R1 = C1-C4-alkyl, CH2OR1; R2 = halogen, C1-C4-alkoxy
R = C1-C8-alkyl, phenyl, subst. phenyl: subclass Va

COO-alkyl: subclass Vb
R' = alkyl, subst. alkyl

A = 5 or 6-membered ring system
X = O, CH2
Figure 15.5.5 Some general structures of competitor subclasses.
each introduced further ring systems, leading to aryl(hetaryl)methylalkylamine

compounds, whereas efforts at Hoechst and Hoechst Schering Agrevo [28–38] led to
the introduction of nonaromatic cyclic amines such as substituted cyclohexylamines
and others (Figure 15.5.5).
(5-Chloro-6-ethylpyrimidin-4-yl)(1-naphthaleno-2-ylethyl)amine (15; Figure 15.5.6),
which was discovered at about the same time by the groups at Ciba-Geigy AG
[19] and Du Pont [25], demonstrated an interesting broad spectrum of activity.
Promising activities against powdery mildew diseases, brown rust, and some
leaf spot diseases of the pyrimidine (15) were also confirmed in field trials;
however, due mainly to the unfavorable cost/activity ratio, development of
the compound was halted at an early stage. Replacement of naphthalene by
Et Cl Et Cl Et Cl
H H H
N N N N S N N
N N N S
N
Me H Me H Me H
15 (racemic) 16 (racemic) 17 (racemic)

(ex. Ciba-Geigy, Du Pont) (Du Pont, ex. Ciba-Geigy) (ex. Ciba-Geigy)
Figure 15.5.6 Structures of the promising competitor compounds 15–17.
benzothiophene [20, 25] also led to active compounds, a representative example

being (1-benzo[b]thiophen-2-ylethyl)(5-chloro-6-ethylpyrimidin-4-yl)amine (16;
Figure 15.5.6). The introduction of the 4,5-dihydro-3H-benzo[b]azepine deriva-
tives led to an improved Oomycetes control of Phytophthora infestans (late blight)
and of Plasmopara viticola (downy mildew) [22]. A representative example is (5-
chloro-6-ethylpyrimidin-4-yl)[1-(2-propylsulfanyl-4,5-dihydro-3H-benzo[b]azepin-7-
yl)ethyl]amine (17; Figure 15.5.6), which showed very high activity against late
blight on tomato and potato in the glasshouse, although its performance under
field conditions was unsatisfactory. The introduction of cyclic nonaromatic amines
did not appear to lead to any fungicides with robust activity under field conditions.
An analysis of the patent literature has suggested that the insecticidal/acaricidal
activity is more pronounced in this subclass.
Subsequently, further applications were made which covered aralkylamino-
quinazolines and other heterocycles (Eli Lilly [39]), new benzyl-substituted
pyrimidines (Sumitomo [40]), aminoalkylspiro-1,3-dioxolanes (Shell [41]), and
hydrazino-substituted pyrimidine compounds (BASF [42]).
15.5.2.2 Summary: Aminoalkylpyrimidines

Aminoalkylpyrimidines represent an interesting class of novel inhibitors of Com-
plex I in the mitochondrial respiration chain, with a broad-spectrum activity having
been identified against many diseases in major crops. However, until now no
commercial products – with the exception of diflumetorim (4) – have reached the
market. This may be due to an unfavorable cost/activity relationship and, in certain
cases, also to issues of high toxicity. Nonetheless, diflumetorim remains the only
Complex I inhibitory fungicide to be introduced to the market, albeit only for use
in horticulture.
15.5.3
Arylacetic Acid Amides Derived from 4-Aminopyridines and 4-Aminoquinolines
Over the years, many companies in the fungicidal arena have undertaken investi-
gations into the N-(4-pyridyl and 4-quinolinyl) acetic acid amide class of the general
formula VII (see Figure 15.5.7), including Bayer CropScience (Hoechst Schering
AgrEvo, Bayer AG), Dow Elanco, Syngenta Crop Protection AG (Ciba-Geigy AG),
and Kumiai Chem. Ind. Co. [43–57]. In 1973, a pharma patent application covering
diphenylalkanoylaminopyridines, and the salts thereof, was published [43] in which
Lipophilic part
R1 A R2 X
CH2 Rlipo
N N
5
R
R3 R4 Linker
Pharmacophore
(Toxophore)
N -(4-pyridyl/4-quinolinyl) aryl acetamides of the general formula VII
R , R2 = C1-C4-alkyl, C1-C4-haloalkyl, C1-C4-alkoxyalkyl, halogen; CN, COO-C1-4-alkyl

1

R3, R4 = H, Me, C1-C4-haloalkyl
X = O, S
Figure 15.5.7 General structure of the class of N-(4-pyridyl/4quinolinyl) aryl acetamides.
O DOS 2325309, 1973

(anti-inflammatory activity claimed)
N N
18
H
Figure 15.5.8 Structure of compound 18.
an example amide derived from 4-aminopyridine, N-(4-pyridyl)biphenyl acetic acid

amide (18; Figure 15.5.8) was described (in this case the pyridine did not bear any
substituents). Strangely, only an anti-inflammatory activity was claimed, but no
mention of fungicidal activity.
Unfortunately, the potential of the pyridine amides in the area of agrochemicals
was not recognized for about 20 years, with details of the first agro-relevant
patent covering N-(4-pyridyl and 4-quinolinyl) arylacetamides not being reported
until 1993, by DowElanco [44]. In this patent, biological data covering fungicidal,
insecticidal, and also nematicidal activity were provided, while a broad-spectrum
fungicidal activity was claimed against Erysiphe graminis, Pyricularia oryzae, Puccinia
recondita, P. viticola, and Septoria nodorum. In the glasshouse, amides such as 19
and 20 (Figure 15.5.9) show 90–100% activity at 100 ppm against powdery mildew,
brown rust, and downy mildew on grapes. The preferred substituents in the
F
F
O O F
Et Cl O Et Cl O
N N 19 N N 20
H H
DowElanco (WO 93/04580)
Figure 15.5.9 Structures of compounds 19 and 20.
pyridine part appear to be a lower alkyl and a halogen, whilst in the quinoline
moiety a fluoro-substituent in the 8-position seems to be preferred.
The next target of the research team at DowElanco was to concentrate on three
strategies aimed at identifying patent gaps:
• To vary the substituents at the pyridine moiety, including quaternization of the

pyridine nitrogen.
• To modify the amido group.
• To vary the lipophilic moiety, Rlipo .
As in the alkylamino-pyrimidine area, it soon became clear to the competitors

that the substitution pattern at the pyridine ring was well optimized for fungal
activity (personal data: R1 = Me, Et and R2 = chloro). In particular, the teams at
Hoechst Schering AgrEvo [45] and Bayer [46] sought alternatives of R1 and R2 and
R1 = CH2 OMe and CH(Me)OR (R = COY, COOY , and CONH2 ) and R2 = cyano
and lower alkylesters have been taken into account as being good alternatives for
lower alkyl and halogen. Another Bayer approach included quaternization of the
pyridine nitrogen, mainly by methyl [47] although, as in some other Bayer patents
covering Complex I inhibitors, the major focus was the insecticidal/acaricidal
activity.
Two possible strategies dominated the modification of the functional group:
(i) the introduction of sulfur instead of oxygen; and (ii) the addition of substituents
to the amido nitrogen. The first approach was used successfully by the research
teams at Syngenta Crop Protection AG [48] and Bayer AG [49]. As representatives
of the thioamide class, compounds 21–23 and 24a,b (see Figure 15.5.10) will be
detailed here. For example, in the glasshouse the thioamides 21 and 22 demonstrate
a broad antifungal spectrum, with activity levels similar to the corresponding
amides. The second approach was used by Bayer AG and Kumiai. The group
at Bayer AG introduced alkoxyalkyl and lower alkyl ester groups [46, 49, 50] as
N-substituents, while the group at Kumai used either alkenyl (e.g., allyl), alkynyl
(e.g., propargyl), or lower alkenyl/alkynyl esters [51]. On the other hand, whilst
the three Bayer applications seemed to focus on the insecticidal/acaricidal activity
(no fungicidal biological data were given!), the Kumai application included both
insecticidal and fungicidal data.
F
O F
+
O N O O F
−
O
Me Cl S F S
N N 21 22
N N
H H
Ciba-Geigy AG (WO 96/14301)
O Cl O R1
Me Me
Et R1 = Cl 24a
O O S Cl S
CN 24b
N N 23 N N
H H
Bayer AG (DE 19695263) - some overlap with Ciba-Geigy WO 96/14301
Figure 15.5.10 Structures of compounds 21–24.
Variation of the lipophilic moiety Rlipo was undertaken by both Syngenta Crop
Protection and Bayer AG. The former group concentrated on using new bicyclic sys-
tems such as benzothienyl, tetrahydro-5H-benzocycloheptenyl, and others [52], and
later focused on the use of benzoxazolyl and benzothiazoly as Rlipo [53], but on this
occasion examined more the insecticidal/acaricidal potential. Some of the amides
developed in glasshouse trials at Syngenta, such as 25 and 26 (Figure 15.5.11),
showed a broad-spectrum fungicidal activity (leaf spots, rusts, and Oomycetes) and
achieved good activity levels. In contrast, the Bayer team focused on the use of new,
para-substituted variants, using initially bisphenyl moieties with para NH-SO2 Me-
and COOallyl substituents [54], with the focus being clearly on insecticidal ac-
tivity. The second approach was to introduced O-alkyl(cycloalkyl)oximes in the
para position of the phenyl group [55] (see compounds 27a–c in Figure 15.5.12),
while the third approach described the use of phenyl moieties substituted in para
position either by pyridine or pyrimidine [56]. Subsequently, when tested in the
glasshouse, compounds such as 28a and 28b (Figure 15.5.12), representing the
Me
Me Cl O S F O
N N 25 N N 26
H H
Ciba-Geigy AG (WO 96/08475)

Me O R'
N
R' = cyclopentyl: 27a
4-CF3benzyl: 27b
Et Cl O tert. butyl: 27c
p -isomers preferred
N N
H Bayer AG (WO 01/72726) - pyridines covered as table examples
O Me
X
N
X = CH: 28a
= N: 28b
Et Cl O
N N
Bayer cropscience (WO 03/059903)
H
pyridinyl/pyrimidinyl phenyl subtype, showed good levels of activity against Po-

dosphaera leucotricha, P. recondita, Sphaerotheca fuliginea, and Venturia inequalis at
application rates of between 100 and 500 g a.i. ha−1 .
15.5.4
Phenylacetic Acid Amides Derived from Aminopyrimidines and Aminoquinazolines
Among the pyrimidine/quinazoline pharmacophore series (see general formula

VIII; Figure 15.5.13), DowElanco, Bayer CropScience (Bayer AG), and Syngenta
Crop Protection AG were the major players [57–60]. Again, the research team from
DowElanco filed their patent application first [58], having used a pyrimidine closely
related to the pyridine (keeping the alkyl substituent in 2-position constant and
substitute C-halogen by a nitrogen in 3-position). The linker and the lipophilic
moieties were identical to those claimed in the pyridine application of 1993 [44].
In this case, a broad fungal spectrum and good activity levels were obtained in
glasshouse trials at rates of 100 ppm for the better representatives of the subclass
of the general formula IX. Compounds 29a and 29b (Figure 15.5.14) are typical
examples.
A few years later the research group at Syngenta used the same approach, but
differed from DowElanco by using new lipophilic parts such as benzoxazolyl and
benzothiazolyl that bore specific substituents in the 2-postion of the lipophilic
heterocycles [59]. Compounds 30a–c (Figure 15.5.14) are discussed here as repre-
sentatives; with all three amides showing again broad-spectrum activities and good
efficacy levels in glasshouse trials.
Lipophilic part
R1 A R2 X
CH2 Rlipo
N N
N
R4
R 3 Linker
Pharmacophore
(Toxophore)
N -(4-pyrimidinyl/4-quinazolinyl) aryl acetamides of the general formula VIII
R1, R2 = C1-C4-alkyl, C1-C4-haloalkyl, C1-C4-alkoxyalkyl, halogen; CN, COO-C1-4-alkyl

R3 = H, Me, C1-C4-haloalkyl
X = O, S
Figure 15.5.13 General structure of the class of

N-(4-pyrimidyl/4quinazolinyl) aryl acetamides.
Bayer AG [60] and Ciba-Geigy (H. Walter, Ciba-Geigy AG, unpublished re-
sults, 1994–1997 and [57]) used pyrimidines that were more closely related
to the alkylaminopyrimidine area (keeping the substituents constant, see, e.g.,
pyrimidine moiety of diflumetorim). Compared to the DowElanco approach, the
substitution pattern of the pyridine was retained, while the nitrogen was added
at another position, thus delivering the new pyrimidine. The Bayer application
[60] also covered compounds derived from hydrazino pyrimidines (see compound
31 in Figure 15.5.14). For example, compound 31 had a restricted spectrum and
was clearly inferior to the better amides covered by the DowElanco application
[58]. The research team at Ciba-Geigy AG was also active in this area, with
6-amino-5-chloro-4-ethylpyrimidine being used mainly as the building block [57],
while the linker was CH2 and halosubstituted phenoxyphenyl moieties were se-
lected for the lipophilic part (Figure 15.5.14). As compounds 32a,b showed only a
marginal biological activity, the inverse amides (33a,b) were also prepared with the
hope of increasing the biological activity, but these failed to show any significant
fungal activity. The disappointing biological results of 32a,b and 33a,b, and also of
some other closer analogs, prevented Ciba-Geigy from filing patent applications
covering these compounds.
Rlipo R1 = H, lower alkyl, Rlipo = subst. phenoxyphenyl, benzoxazolyl

O
N
N N
N H
R1 = Et, Rlipo = O 29a
R1 F
F
O F
General formula IX
29b
O
DowElanco (WO 95/18795)
N
R1 = Et, Rlipo = R'
R' = Me: 30a
O
n-propyl: 30b
CH2-t-butyl: 30c
R2 R3 R1 = H, lower alkyl, R2 = lower alkyl, R3 = halogen,

Rlipo
Rlipo = subst. phenyl, subst. hetaryloxyphenyl, X = direct bond or NH
X
N N O
R1 = H, R2 = Et, R3 = Cl, X = NH (31) and direct bond (32a-c)
N H Et
N
R1 Cl
Rlipo = N
General formula X O 31 Bayer AG (WO 97/28133)
R'
R' = F 32a Ciba-Geigy AG,

Cl 32b unpublished results,
O
CF3 32c 1994-1997
Bayer AG (WO 97/28133)
R2 R3 Rlipo
O
R1 = H, lower alkyl, R2 = lower alkyl, R3 = halogen,
Rlipo = halophenoxyphenyl
N N R'
N H
R' = F 33a
R1 R1 = H, R2 = Et, R3 = Cl, Rlipo = O
Cl 33b
General formula XI
(inverse amides) Ciba-Geigy AG unpublished results + [59]
Figure 15.5.14 Structures of classes IX, X, and XI.
15.5.5
Arylsulfonyl Acid Amides Derived from Heteroarylmethylamines
Among the major agrochemical companies, BASF was the last to enter the
Complex I amide area [61, 64–83]. The first patent covering fungicidal activity
derived from the sulfonamide area (general formula XII; Figure 15.5.15) was
published in 2005 [61]. The novel use of sulfonamides in the Complex I area may
have led to the BASF team being attracted by a Takeda patent [62] of 2000 which
covered the later development candidate, tolnifanide (34; Figure 15.5.16) which,
although not a Complex I inhibitor was shown to interfere with lipid metabolism
[63]. Tolnifanide-type structures are derived from anilines, whereas Complex I
inhibitors of the sulfonamide type are derived from methyl-aminoheterocycles.
It would appear that the BASF approach mainly employed knowledge from the
carboxylic acid amide area. As noted above, the most important aminomethyl
(Ri)n R1 O
A N S Aryl1(Hetaryl1) [Y Aryl2(Hetaryl2)]m
B O
U V
General formula of the BASF type Sulfonamides (formula XII)
A, B, U, V = N or CH (at least one heteroatom in the ring)

Ri = H, halogen, C1-C4-alkyl, C1-C4-haloalkyl, C1-C4-alkoxy, C1-C4-haloalkoxy,
C1-C4-alkylthio, C1-C4-haloalkylthio, C1-C4-alkoxy-C1-C4-alkyl,
C1-C4-haloalkoxy-C1-C4-alkyl, etc.
Aryl1, Aryl2 = phenyl, naphthyl, benzyl, C5-C7-cycloalkyl, partially hydrogenated bicycles

(e.g., tetrahydronaphthalene, etc.)
Hetaryl1, Hetaryl2 = pyridine, pyrimidine, quinoline, etc.
Aryl1, Aryl2, Hetaryl1 and Hetaryl2 may be further subsituted by cyano, halogen, nitro, C1-C8-alkyl,
C1-C6-haloalkyl, C1-C8-alkoxy, C1-C8-haloalkoxy, (C1-C4-alkyl)carbonyl, (C1-C4-alkoxy)carbonyl, etc.
Y = direct bond, O, S or CH2

n = 0-4
m = 0,1
Figure 15.5.15 General structure of the BASF-type sulfonamides.
Cl Figure 15.5.16 Structure of tolnifanide.
O
+
Me S N N O
−
O Et O
Tolnifanide 34 (Takeda)
group-containing heterocycles are pyridine, pyrimidine, and quinoline. In the

phenylacetic acid amides, the CH2 group is in the acid moiety, whereas in the
BASF-type sulfonamides the CH2 group forms part of the aminoheterocycle
moiety (see general formula XII; Figure 15.5.15). The substituents present in the
heterocycle do not differ much from the substituents mentioned in earlier arylacetic
acid amide patents, as filed by the competitors. BASF was – and is still – investing in
the sulfonamide area, and about 20 patent applications have been published to date.
In analogy to the 4-N-aminopyridyl and 4-aminoquinolinyl area, the insecticidal
activity according to the BASF patent data is sometimes also pronounced.
As the area being discussed here is rather new, no clear SAR picture on the
sulfonamides can yet be derived. However, some additional, more fungicide-related,
applications involving compounds derived from aminomethylpyridines [66, 70, 71]
and aminomethylpyrimidines [68, 72] will be discussed in greater detail. Two
types of compound derived from 4-aminomethyl-substituted pyridines have been
identified:
• Type 1 compounds of the general formula XIII (Figure 15.5.17) originate from
2-pyridylsulfonic acids, where the pyridine is further substituted with phenyl [66].
Examples of this class are compounds 35 and 36. In the case of compound 35,
a curative activity against Phakopsora pachyrhizi was mentioned (>95% control
at 250 ppm), while for compound 36 a good control of Alternaria solani and P.
infestans at 250 ppm was reported in glasshouse trials.
• Type 2 compounds of the general formula XIV (Figure 15.5.17) originate from
phenylsulfonic acids, further substituted by pyridyloxy [70, 71]. Examples of type
2 compounds are 37–39. For compound 37, a good activity against Septoria tritici
was detected at 250 ppm, while for compounds 38 and 39 good activities against
P. pachyrhizi were reported.
One major sulfonamide class has been derived from 4-aminomethylpyrimidines

(see general formula XV; Figure 15.5.18). Compounds of the general formula XV
originate from phenylsulfonic acids, the phenyl moiety being further substituted by
either a second phenyl or pyridyloxy. Compounds 40–42 are discussed as examples,
with antifungal data being provided for P. infestans, P. recondita, and P. pachyrhizi.
There are no suggestions from the patent applications that the new type of
Complex I inhibitors could be active at low rates (BASF mentions activity at
a lowest rate of 250 ppm in the glasshouse!). To date, information concerning
BASF’s new sulfonamide class remains incomplete, and no clear judgment can
R1 R2
H O R1, R2 = H, lower alkyl, lower alkoxy, R' = subst. phenyl
N S R'
N O N R1 = R2 = H and R' = 3-fluorophenyl: 35
Me
general formula XIII 36
N O
(type 1 pyridine sulfonamides) Et
BASF AG (WO 07/093599)
R'
R1 = Me, R2 = Et, R' = CF3: 37
1 2
R = OMe, R = H, R' = CF3: 38
R1 R2
H O N BASF AG (WO 071448)
N S O
N O R1 = R2 = Me and R' = H: 39
BASF AG (WO 09/071449)

general formula XIV
(type 2 pyridine sulfonamides)
Figure 15.5.17 Structures of classes XIII and XIV.
H O R1 = H, R' = 4-chlorophenyl: 40
N S R' BASF AG (WO 08/062011)
N O
N Cl
F
1 general formula XV R1 = SCHF2 and R' = O 41
R N F F
1
R = OMe and R' = 4-chlorophenoxy: 42
BASF AG (WO 09/101078)
Figure 15.5.18 Structures of class XV.

yet be made on this class. Neither is any information yet available on development
activities in this area.
15.5.6
Other Leads in the Area of Complex I Inhibitors
Only a few leads in the fungicide area are worthy of mention in this section.
Phenoxan (43) [84], a secondary metabolite isolated from Polyganium sp. strain
PI VO19 with an oxazole-γ -pyrone structure, is discussed as a first example
(Figure 15.5.19).
Phenoxan has been shown to inhibit the growth of agro-relevant fungi such
as Botrytis cinerea and Ustilago maydis, both major diseases in agriculture, in
an agar diffusion test system [84]. Other natural product leads have originated
from fermentations of the Pterula species 82168 (Basidiomycetes), with both
pterulone (44) and pterulinic acid (45; Figure 15.5.19) [85] having been isolated.
Both metabolites have demonstrated antifungal activity against Fusarium species,
Ustilago nuda, and Botrytis cinerea in agar diffusion tests. Present knowledge
indicates that no significant progress has been made in identifying any close
analogs of the natural products 43–45, which have shown useful activity in
glasshouse tests. The aryloxylepidines [86] (Figure 15.5.20), which are closely
O
Me O
Cl
N Me
Me
O O O
O Me
43 (phenoxan) 44 (pterulone)
O O
HO Cl HO
O O Cl
O O
E-45 Z-45
pterulinic acid
Figure 15.5.19 Structures of natural product leads.
R1
X
46: R1 = F, R2 = R3 = Cl, X = O
R2
47: R1 = F, R2 = H, R3 = F, X = CH2O
N
3
R
general formula XVI
Figure 15.5.20 Structures of the general lepidine class XVI;

quinoxyfen (46) and lepidine (47).
References 687
related to the quinazoline compound fenazaquin (1), a commercialized acaricide,

and also to quinoxyfen (21; Figure 15.5.8), a potent powdery mildewicide, represent
another class of leads.
Whereas, fenazaquin (1) is a well-known Complex I inhibitor, the primary
mode of action of quinoxyfen (46) is, as yet, unknown. Although the lepidine
compound 47 (Figure 15.5.20) shows submicromolar activity level (Ustilago maydis,
IC50 0.73 μmol) in the biochemical Complex I assay, the activity level in glasshouse
tests has proved to be highly unsatisfactory.
Clearly, the chances of identifying lepidine derivatives of sufficient biological
activity in agro-relevant disease complexes are rather low [86].
15.5.7
Conclusions and Summary
The identification of Complex I inhibitors with market-relevant fungicidal prop-

erties appears to be much more difficult than finding insecticides/acaricides with
such properties. In the fields of both fungicides and insecticides, only niche
products with restricted market volumes appear to be feasible, yet the reasons
for such a situation are not fully understood. In the case of the fungicidal
aminoalkylpyrimidine class, it has been shown that, on occasion, the cost/field
activity relationship and/or problems with acute oral toxicity might be responsible
and, as a consequence, several promising projects were halted at an early stage.
A similar picture is beginning to emerge with the fungicidal arylacetic amide
class, with the best amides derived from 4-aminopyridines, 4-aminopyrimidines,
and 4-aminoquinolines having shown broad fungicidal spectra at reasonable rates
in glasshouse tests, but none has yet been introduced to the market. The rea-
sons for this might, again, be toxicity issues and/or unfavorable price/field
activity ratios. Today, BASF’s sulfonamide-type fungicides remain the subject
of much investigation, and consequently no statement on their true potential
can be given. For natural products and other leads, however, the potential to
optimize their biological activities and fungicidal spectra appears to be rather
limited.
References
1. Esposti, M.D. (1998) Biochim. Biophys. Rechenberg, Germany, September
Acta, 1364, 222–235. 1999.
2. Fujii, K. and Takamura, S. (1998) 4. Whitehead, C.W. and Traverso, J.J.
Agrochem. Jpn, 72, 14–15. (1958) J. Am. Chem. Soc., 80,
3. Walter, H. (1999) Topic presented 2185–2189.
in part at the 4th Conference 5. Yoshioka, H., Obata, T., Fujii, K.,
of Iminium Salts, Stimpfach- Tsutsumiuchi, K., and Yoshiya, H.
(1988) (Ube Industries). EP 0264, 23. Walter, H. (1995) (Ciba-Geigy AG, now
217. Syngenta Crop Protection AG). WO
6. Fujii, K., Tanaka, T., and Fukuda, Y. 95/01,975.
(1990) (Ube Industries). EP 0370,704. 24. Schaub, F. and Lamberth, C. (1997)
7. Obata, T., Fujii, K., Narita, I., and (Sandoz Ltd., now Syngenta Crop Pro-
Shikita, S. (1991) (Ube Industries). EP tection AG). WO 97/09,316.
0411,634. 25. Drumm, J.E., Lett, R.M., and Stevenson,
8. Iida, S. and Fujii, K. (1991) (Ube Indus- T.M. (1992) (Du Pont). WO 92/08,
tries). EP 0432,894. 704.
9. Obata, T., Fujii, K., Ooka, A., and 26. Lett, R.M. (1994) (Du Pont). WO
Yamanaka, Y. (1995) (Ube Industries). 94/08,976.
27. Drumm, J.E., Lett, R.M., and Stevenson,
EP 0665,225.
T.M. (1995) (Du Pont). WO 95/07,
10. Obata, T., Fujii, K., Tsutsumiuchi, K.,
278.
and Yamanaka, Y. (1996) (Ube Indus-
28. Schaper, W., Salbeck, G., Ehrhardt, H.,
tries). WO 96/06,086.
Braun, P., Knauf, W., Sachse, B.,
11. Fujii, K. (2003) (Ube Industries). WO
Waltersdorfer, A., Kern, M., and
03/022,822. Lümmen, P. (1992) (Hoechst AG,
12. Hollingworth, R.M. (2001) in Handbook now Bayer CropScience). EP 0519,
of Pesticide Toxicology, 2nd edn (ed. R. 211.
Krieger), Academic Press, San Diego, 29. Schaper, W., Freuss, R., Salbeck,
CA, pp. 1169–1261. G., Braun, P., Knauf, W., Sachse,
13. Ataka, K. and Kono, M. (1996) (Ube B., Waltersdorfer, A., Lümmen, P.,
Industries). JP 08,198,858. and Bonin, W. (1993) (Hoechst AG,
14. Yamanka, H., Sakamoto, T., now Bayer CropScience). WO 93/19,
Nishimura, S., and Sagi, M. (1987) 050.
Chem. Pharm. Bull., 35, 3119–3126. 30. Andree, R. and Dehne, H.-W. (1995)
15. Mills, L. and Previdoli, F. (1990) (Lonza (Bayer AG, now Bayer CropScience).
AG). EP 0370,391. WO 95/00496.
16. Kuragano, T. and Tanaka, Y. (2002) 31. Reuschling, D.B., Linkies, A.D.,
(Takeda Chem. Ind.). WO 02/038, Wehner, V., Preuss, R., Schaper, W.,
550. Jakobi, H., Braun, P., Knauf, W.,
17. Yamanaka, Y., Moritomo, M., Fujii, K., Sachse, B., Waltersdorfer, A., Kern, M.,
Tanaka, T., Fukuda, Y., and and Bonin, W. (1995) (Hoechst Schering
Nishimura, K. (1998) Pestic. Sci., 54, AgrEvo, now Bayer CropScience). WO
223–229. 95/07,894.
18. Yamanaka, Y., Moritomo, M., Fujii, K., 32. Schaper, W., Preuss, R., Braun, P.,
Knauf, W., Sachse, B., Waltersdorfer, A.,
Tanaka, T., Fukuda, Y., and
Lümmen, P., and Bonin, W. (1995)
Nishimura, K. (1999) Pestic. Sci., 55,
(Hoechst Schering AgrEvo, now Bayer
896–902.
CropScience). WO 95/31,441.
19. Kristiansen, O., Zondler, H., and
33. Schaper, W., Preuss, R., Braun, P.,
Müller, U. (1992) (Ciba-Geigy AG,
Kern, M., Knauf, W., Sanft, U.,
now Syngenta Crop Protection AG). EP Waltersdorfer, A., Bonin, W., Linkies,
0470,600. A.H., and Reuschling, D.B. (1996)
20. Walter, H. and Havenhand, M. (1993) (Hoechst Schering AgrEvo, now Bayer
(Ciba-Geigy AG, now Syngenta Crop CropScience). WO 96/11,924.
Protection AG). EP 0530,149. 34. Märkl, M., Schaper, W., Knauf, W.,
21. Walter, H. (1994) (Ciba-Geigy AG, now Sanft, U., Kern, M., Reuschling, D.B.,
Syngenta Crop Protection AG). WO Linkies, A.H., and Bonin, W. (1996)
94/19,340. (Hoechst Schering Agrevo, now Bayer
22. Walter, H. (1994) (Ciba-Geigy AG, now CropScience). WO 96/13,487.
Syngenta Crop Protection AG). WO 35. Schaper, W., Krautstrunk, G.,
94/20,490. Knauf, W., Kern, M., Pasenok, S.,
References 689
Reuschling, D.B., Linkies, A.H., and (Hoechst Schering Agrevo, now Bayer
Bonin, W. (1996) (Hoechst Schering CropScience). DE 19,647,317.
AgrEvo, now Bayer CropScience). WO 48. Walter, H. and Zambach, W. (1996)
96/30,345. (Ciba-Geigy AG, now Syngenta Crop
36. Schaper, W., Knauf, W., Sanft, U., Protection AG). WO 96/14,301.
Kern, M., Ehrhardt, H., Linkies, A.H., 49. Bretschneider, T., Heil, M., Kleefeld,
Reuschling, D.B., and Bonin, W. (1997) G., and Erdelen, C. (1998) (Bayer AG,
(Hoechst Schering Agrevo, now Bayer now Bayer CropScience AG). DE 19,625,
CropScience). WO 97/02,264. 263.
37. Jakobi, H., Braun, R., Schaper, W., 50. Bretschneider, T., Heil, M., Alig, B.,
Krautstrunk, G., Märkl, M., Sanft, U., Kleefeld, G., and Erdelen, C. (1999)
Thönessen, M.-T., Kern, M., and (Bayer AG, now Bayer CropScience). DE
Bonin, W. (1998) (Hoechst Schering 19,750,401.
AgrEvo, now Bayer CropScience). WO 51. Takefuji, N., Nakatani, M., Suzuki, J.,
98/22,444. Ozaki, M., and Ueno, R. (1996) (Kumiai
38. Jakobi, H., Ort, O., Schaper, W., Chemical Ind. Co.). WO 96/3,975.
Braun, R., Krautstrunk, G., Märkl, M., 52. Walter, H. (1996) (Ciba-Geigy AG, now
Stark, H., Sanft, U., Kern, M., and Syngenta Crop Protection AG). WO
Bonin, W. (1998) (Hoechst Schering 96/08,475.
AgrEvo, now Bayer CropScience). WO 53. Armstrong, S., Barnes, N.J., Barnett,
98/22,446. S.P., Clarke, E.D., Crowley, P.J., Fraser,
T.E., Hughes, D.J., Mathews, C.J.,
39. Coghlan, M.J., Dreikorn, B.A., Suhr,
Salmon, R., Smith, S.C., Viner, R.,
R.G., and Jourdan, G.P. (1989) (Eli
Whittingham, W.G., Williams, J.,
Lilly). EP 0, 326,328.
Whittle, A.J., Mound, W.R., and Urch,
40. Katoh, T., Takano, H., Fujimoto, H., and
C.J. (2001) (Syngenta Crop Protection
Kisida, H. (1992) (Sumitomo Ltd.). EP 0,
AG). WO 2001/55,135.
467,760.
54. Heil, M., Bretschneider, T., Kleefeld, G.,
41. Pfrengle, W. and Carter, P.A. (1995)
Erdelen, C., Kuck, K.-H., Stenzel, K.,
(Shell Int.). WO 95/11,899.
Turberg, A., and Mencke, N. (1999)
42. Wagner, O., Röhl, F., Lorenz, G.,
(Bayer AG, now Bayer CropScience). DE
Ammermann, E., and Strathmann, S.
19,727,162.
(1998) (BASF AG). DE 19,650,
55. Heil, M., Militzer, H.-C., Gebauer, O.,
378. Haenssler, G., Kuck, K.-H., and
43. Tenconi, F., Tagliabue, R., and Mauler-Machnik, A. (2001) (Bayer
Molteni, L. (1973) (L. Zambeletti S. AG, now Bayer CropScience). WO
p. A, Mailand). DOS 2,325,309. 01/72,726.
44. Hackler, R.E., Jourdan, G.P., Johnson, 56. Maurer, F., Erdelen, C., Kuck,
P.L., Thoreen, B.R., and Samaritoni, K.-H., Mauler Machnik, A.,
J.G. (1993) (Dow Elanco). WO 93/04, Wachendorff-Neumann, U., and
580. Turberg, A. (2003) (Bayer CropScience).
45. Jakobi, H., Knauf, W., Sanft, U., WO 03/059,903.
Kern, M., Reuschling, D.B., and Linkies, 57. Walter, H. (2003) Topic presented in
A.H. (1996) (Hoechst Schering AgrEvo, part at the 6th Conference of Iminium
now Bayer CropScience). DE 4,434, Salts, Stimpfach-Rechenberg, Germany,
637. September, 2003.
46. Bretschneider, T., Heil, M., Alig, B., 58. Johnson, P.L. (1995) (Dow Elanco). WO
Kleefeld, G., and Erdelen, C. (1999) 95/18,975.
(Bayer AG, now Bayer CropScience). DE 59. Armstrong, S., Barnes, N.J., Barnett,
19,750,402. S.P., Clarke, E.D., Crowley, P.J., Fraser,
47. Jakobi, H., Ort, O., Schaper, W., T.E., Hughes, D.J., Mathews, C.J.,
Braun, R., Krautstrunk, G., Märkl, M., Salmon, R., Smith, S.C., Viner, R.,
Stark, H., Sanft, U., Thönessen, Whittingham, W., Williams, J., Whittle,
M.-T., Kern, M., and Bonin, W. (1998) A.J., Mound, R.W., and Urch, C.J.
(2001) (Syngenta AG). WO 01/055, 70. Grammenos, W., Lohmann, J.-K.,

137. Grote, T., Dietz, J., Müller, B., Puhl, M.,
60. Bretschneider, T., Kleefeld, G., Renner, J., Ulmschneider, S.,
Wernthaler, K., Erdelen, C., and Vrettou, M., and Rheinheimer, J. (2009)
Stenzel, K. (1997) (Bayer AG, now (BASF AG). WO 09/071,448.
Bayer CropScience). WO 97/28, 71. Grammenos, W., Lohmann, J.-K.,
133. Grote, T., Dietz, J., Müller, B., Puhl, M.,
61. Grammenos, W., Blettner, C., Renner, J., Ulmschneider, S.,
Mueller, B., Gewehr, M., Vrettou, M., and Rheinheimer, J. (2009)
Tormo, J., Blasco, I., Grote, T., (BASF AG). WO 09/071,449.
Rheinheimer, J., Schäfer, P., 72. Lohmann, J.-K., Glättli, A.,
Schieweck, F., Schwögler, A., Grammenos, W., Montag, J.,
Wagner, O., Götz, N., Strathmann, S., Müller, B., Vrettou, M., Renner, J.,
Schöfl, U., Scherer, M., and Stierl, Ulmschneider, S., Rack, M., and
R. (2005) (BASF AG). WO 05/033, Dietz, J. (2009) (BASF AG). WO
081. 09/101,078.
62. Takanori, Y. and Tetsuhiro, N.M. (2000) 73. Lohmann, J.-K., Glättli, A.,
(Takeda Chem. Ind.). W0 00/065, Grammenos, W., Montag, J.,
913. Müller, B., Vrettou, M., Renner, J.,
63. Nakano, M., Furukawa, M., Tanaka,
Ulmschneider, S., Rack, M., and
C., Miyagawa, H., and Okuno, T.
Dietz, J. (2009) (BASF AG). WO
(2006) Proc. Pestic. Sci. Soc. Jpn, 31,
09/101,079.
63.
74. Dietz, J., Grammenos, W., Müller, B.,
64. Grammenos, W., Rheinheimer, J.,
Lohmann, J.-K., Renner, J.,
Lohmann, J.-K., Grote, T., Puhl, M.,
Ulmschneider, S., Vrettou, M., and
Koradin, C., Baumann, E.,
Puhl, M. (2009) (BASF AG). WO
Von Deyn, W., Langewald, J., Götz, N.,
09/101,082.
Culbertson, D.L., Anspaugh, D.D.,
75. Grammenos, W., Lohmann, J.-K.,
Oloumi-Sadeghi, H., van Tuyl Cotter,
Grote, T., Dietz, J., Müller, B.,
H., and Kuhn, D.G. (2006) (BASF AG).
Puhl, M., Renner, J., Ulmschneider, S.,
WO 06/097,489.
Vrettou, M., and Rheinheimer, J. (2009)
65. Lohmann, J.-K., Grammenos, W.,
Puhl, M., Dietz, J., Müller, B., (BASF AG). WO 09/103,650.
Rheinheimer, J., and Renner, J. (2006) 76. Dietz, J., Grote, T., Grammenos, W.,
(BASF AG). WO 06/093,599. Müller, B., Lohmann, J.-K., Renner, J.,
66. Lohmann, J.-K., Grammenos, W., Ulmschneider, S., Vrettou, M., and
Puhl, M., Dietz, J., Müller, B., Glättli, A. (2009) (BASF AG). WO
Rheinheimer, J., and Renner, J. (2007) 09/112,523.
(BASF AG). WO 07/093,599. 77. Dietz, J., Grote, T., Grammenos, W.,
67. Lohmann, J.-K., Grammenos, W., Müller, B., Lohmann, J.-K., Renner, J.,
Puhl, M., Dietz, J., Müller, B., Ulmschneider, S., Vrettou, M., and
Rheinheimer, J., and Renner, J. (2008) Glättli, A. (2009) (BASF AG). WO
(BASF AG). WO 08/022,937. 09/112,533.
68. Lohmann, J.-K., Grammenos, W., 78. Glättli, A., Grammenos, W., Müller, B.,
Puhl, M., Dietz, J., Müller, B., Lohmann, J.-K., and Vrettou, M. (2009)
Rheinheimer, J., Renner, J., Vrettou, M., (BASF AG). WO 09/124,903.
Ulmschneider, S., and Grote, T. (2008) 79. Grammenos, W., Glättli, A., Lohmann,
(BASF AG). WO 08/06,211. J.-K., Müller, B., and Vrettou, M. (2009)
69. Lohmann, J.-K., Grammenos, W., (BASF AG). WO 09/141,241.
Puhl, M., Dietz, J., Müller, B., 80. Grammenos, W., Glättli, A., Lohmann,
Rheinheimer, J., Renner, J., Vrettou, M., J.-K., Puhl, M., Müller, B., and
Ulmschneider, S., and Grote, T. (2008) Vrettou, M. (2009) (BASF AG). WO
(BASF AG). WO 08/062,012. 09/141,274.
References 691
81. Grammenos, W., Glättli, A., Lohmann, Höfle, G., and Reichenbach, H. (1992) J.
J.-K., Müller, B., and Vrettou, M. (2009) Antibiot., 45, 1549–1552.
(BASF AG). WO 09/141,290. 85. Engler, M., Anke, T., Sterner, O.,
82. Lohmann, J.-K. (2009) (BASF AG). WO
and Brandt, U. (1997) J. Antibiot., 50,
325–329.
09/141,291.
86. Kirby, N.V., Daeuble, J.F., Davis, L.N.,
83. Lohmann, J.-K. (2009) (BASF AG). WO
Hannum, A.C., Hellwig, K., Lawler,
09/141,292. L.K., Parker, M.H., and Pieczko,
84. Kunze, B., Jansen, R., Pridzun, L., M.E. (2001) Pest Manage. Sci., 57,
Jurkiewicz, E., Hunsmann, G., 844–851.
693
16
Fungicides Acting on Amino Acids and Protein Synthesis
16.1
Natural Compounds Used in Agriculture Interfering in Protein Synthesis of Fungi
and Bacteria
16.1.1
Introduction
Whilst only a limited number of antifungal and antibacterial compounds that

interfere in protein synthesis are used in agriculture and horticulture, it is in-
teresting that almost all inhibitors of protein synthesis are natural, rather than
synthetic, compounds. The various processes by which proteins are synthesized
will be summarized in this chapter.
16.1.2
General Mechanisms of Protein Biosynthesis
The information of a gene that is encoded by the sequence of a DNA region

is translated into a particular protein in a process that involves several steps.
During transcription, the DNA region encoding the gene is transcribed into a
complementary messenger RNA (mRNA) [1] which, in eukaryotic cells, is further
modified and, when mature, is exported to the cytoplasm. Here, the mRNA binds
to a ribosome that employs the nucleotide sequence as a template for the synthesis
of a specific polypeptide. In prokaryotic cells, the mRNA is not further modified
and the ribosomes can bind to the nascent mRNA.
Protein synthesis is catalyzed by ribosomes that contain several proteins as well
as ribosomal RNA (rRNA). The ribosomes (cytoplasmic) from both eukaryotes and
prokaryotes exhibit sedimentation coefficients of 80S and 70S, respectively. The
ribosome particles from eukaryotes are composed of 40S and 60S subunits, while
those from prokaryotes comprise 30S and 50S subunits. The rRNA and various
protein units function as structural components of the ribosomes, and play a role
in the process of protein synthesis. In order for the genetic information on the
mRNA to be translated, adaptor molecules are required that are referred to as
transfer RNAs (tRNAs). These each consist of about 80 nucleotides, with each

694 16 Fungicides Acting on Amino Acids and Protein Synthesis
tRNA capable of recognizing a specific codon on the mRNA by a complementary

triplet, called the anticodon. At the 3’-end of each specific tRNA, a particular amino
acid is attached by a specific aminoacyl-tRNA synthetase. In eukaryotic cells, the
small ribosomal subunit first associates with an initiation tRNA (Met-tRNA) and
then binds the mRNA at its 5 cap. After attachment, the complex scans along the
mRNA until reaching the translation start, which is the AUG-codon that binds the
Met-tRNA. Subsequently, during initiation the large ribosomal subunit is added to
the complex and protein synthesis can start in the 5 → 3 direction. Each ribosome
has three binding sites: the first tRNA binding site, the P (peptidyl)-site, contains
the initiation tRNA, while the second A (aminoacyl) site is free to be occupied by
an aminoacyl-tRNA that carries an anticodon complementary to the second codon.
When the A site is occupied, the amino acid of the P site (which is the methionine)
establishes a peptide bond with the amino group of the amino acid at the A site.
The tRNA carrying the dipeptide then moves to the P site, while the unloaded
tRNA will move to the E (exit) site and leave the ribosome. As a result, the A site is
opened for another aminoacyl-tRNA that is complementary to the third codon in
the sequence and, thus, the ribosome moves one codon further downstream. This
process is repeated until a stop codon (UAA, UAG, UGA) is reached, at which point
the newly synthesized polypeptide detaches from tRNA and the ribosome releases
the mRNA.
As a consequence of these reactions, the synthesis of proteins can be subdivided
into four distinct steps: the formation of aminoacyl-tRNA; followed by the initiation,
elongation, and termination of the polypeptide chains.
Whilst the general mechanisms by which proteins are synthesized appear to be
similar in both prokaryotes and eukaryotes, some minor differences have been
identified, particularly with regards to chain initiation and termination, and also in
the designation and chemical characteristics of the various soluble factors.
The mode of action of the antifungal/antibacterial compounds discussed in
this chapter is via an interference of one or more of the major step(s) of protein
synthesis in fungi or bacteria.
16.1.3
Blasticidin S
Blasticidin S (Figure 16.1.1), which represents the first agricultural antibiotic

to be developed in Japan, was first isolated from the culture broth of Strepto-
myces chriseochromogenes [2], after which the chemical structure was elucidated
[3, 4]. Blasticidin S exhibits a broad spectrum of biological actions, including
antibacterial, antiviral, antitumor, and antifungal activities [5, 6]. In particular, the
compound has been developed for the control of rice blast, caused by Pyricularia
oryzae [7].
Blasticidin S and the benzylaminobenzene sulfonate derivative proved to be least
phytotoxic to rice plants, and to display a pronounced antifungal activity against
P. oryzae [8]; hence, this salt has been produced industrially since 1961 for the
control of rice blast. Other derivatives of this antibiotic, blasticidin A and C have
16.1 Natural Compounds Used in Agriculture Interfering in Protein Synthesis of Fungi and Bacteria 695
Me Figure 16.1.1 The structure of

NH2 blasticidin S.
O N
NH NH2 NH
OH
N O
O
H2N N O
Blasticidin S
proved to be inferior to the S derivative in disease control. The amount of blasticidin

S following spray treatments is 10–40 g a.i. ha−1 ; the application at higher levels
led to some phytotoxic effects on the rice plants.
Following spray treatment, using a 14 C-labeled radioactive compound, most of
the blasticidin was found to remain as a residue on the surface of the rice plants,
with only a small proportion being taken up and translocated into the host tissue.
However, the compound was readily absorbed through the wounds of infected
plant parts and translocated to the apices [9].
The antibiotic residues present on the plant surfaces were decomposed by
sunlight, the main degradation product being cysteine. Following the treatment
of paddy field soil with radiolabeled blasticidin S, a significant degradation of
the antibiotic was determined, with several microorganisms (e.g., Pseudomonas
marginalis, P. ovalis, Fusarium oxysporum) that colonized the paddy field being
found to reduce the biological activity of blasticidin S [9]. These findings indicate
that blasticidin S is easily degraded in the environment, and that no environmental
pollution or food contamination should be expected. Residual levels of the antibiotic
in unpolished rice, using biological assays, were shown to be <0.05 ppm [9].
The antifungal effect of blasticidin S against rice blast may be attributed to an
inhibition of the mycelial growth of P. oryzae. Studies on the mode of action of
blasticidin S, using cell-free systems of P. oryzae and Escherichia coli, showed that
it would inhibit the incorporation of 14 C-radiolabeled amino acids into protein very
effectively [10], by binding to the 60S and 50S ribosomal subunits, respectively
[11]. Despite blasticidin S being highly inhibitory towards P. oryzae, it exhibits
no antifungal activity towards Pellicularia sasaki; such differential sensitivity was
found to be caused by species-related differences in the binding affinities of the
compound to the fungal ribosomes [10, 11].
The accidental contact of blasticidin S with the eyes generally causes conjunc-
tivitis; the effect is especially problematic following dust applications, but a lesser
degree of injury has been reported with wettable powder or solution applications.
Direct contact of the mucous membranes or injured skin with blasticidin S may
also result in tissue inflammation. The problems of eye irritation were allevi-
ated by mixing calcium acetate with blasticidin S; this had no adverse effect on
antiblast activity, and was subsequently employed in dusting applications of the

antibiotic.
Several strains of P. oryzae that are resistant to blasticidin S have been selected on
agar media, with relative ease. Subsequently, preparations of cell-free systems from
both blasticidin S-sensitive and -resistant strains proved to be equally inhibited
by the antibiotic. Thus, it was suggested that any reduction in resistance to the
antibiotic was due to a decrease in the ability of the compound to permeate
the plasmalemma [10]. Resistant mutants generated in laboratory experiments
have displayed a decreased pathogenicity [12], but no emergence of resistance to
blasticidin S by P. oryzae has yet been observed in practice. At present, however, it
is unclear whether the lack of resistance under field conditions can be explained by
a reduced fitness of the resistant strains [13].
Both, blasticidin S and blasticidin A were shown to inhibit aflatoxin production
in Aspergillus parasiticus, without causing any reduction in fungal growth. These
results suggested that protein synthesis inhibitors might be useful for controlling
the production of aflatoxin [14].
16.1.4
Kasugamycin
Kasugamycin (Figure 16.1.2), a water-soluble and basic antibiotic, is produced by

Streptomyces kasugaensis [15]. Chemically, the antibiotic is composed of three moi-
eties: d-inositol, kasugamine (2,3,4,5-tetradeoxy-2,4-diaminohexopyranose); and an
imino acetic acid side chain [15–17].
Kasugamycin was developed as a specific and effective antibiotic for the control
of rice blast. The antibiotic controls the disease when applied at 20 ppm in aqueous
solution, and demonstrates both protective and curative activities [18]. Since 1965,
kasugamycin has been used to control rice blast on a large scale, the predominant
application being as a dust containing 0.3% of the active ingredient. In addition,
the pre-treatment of rice seeds with a 2% wettable powder of kasugamycin has
been shown to protect the new rice plants against blast for a period of one month.
Moreover, kasugamycin antibiotic exhibits a high level of crop safety, with no
phytotoxicity having been observed.
Kasugamycin also exhibits activity against plant diseases caused by bacteria; for
example, treatment of the soil in nursery boxes with kasugamycin provided control
over Pseudomonas glumae, which causes severe diseases among rice seedlings [19].
Me HO OH
O
O N O OH
HO NH NH2 HO OH
Kasugamycin Figure 16.1.2 The structure of kasugamycin.

Following foliar applications, kasugamycin has shown both preventive and curative
activities in the control of cucumber angular leaf spot [20]. Although the antibiotic
was not sufficiently effective to control citrus canker evoked by Xanthomonas
campestris pv. citri , its application in combination with copper oxychloride led to an
effective disease control. In this situation, it was suggested that kasugamycin was
displaying a synergistic effect with the copper. In both laboratory and greenhouse
studies, kasugamycin was found to provide an effective control of vegetable soft
rot (Erwinia carotovora), bean halo-blight (P. syringae pv. phaseolicola), cucumber
marginal blight (P. marginalis), and rice sheath brown rot (P. fuscovaginae) [21].
Kasugamycin also exhibits a low toxicity towards animals (e.g., mice, rats,
rabbits, dogs, monkeys). In mice, the oral LD50 was shown to be 2 g kg−1 ,
while a concentration of 1000 μg ml−1 failed to cause any toxic effects in fish.
Moreover, in rat liver the antibiotic had no adverse effect on ribosomal protein
synthesis [22].
Kasugamycin is known to inhibit the mycelial growth of P. oryzae in media with
an acidic pH (pH 5.0), but is barely inhibitory at neutral pH (7.0) [23]. The antibiotic
also interferes with protein synthesis in both P. oryzae and bacteria, having been
shown to react with the 30S ribosomal subunits in cell-free systems of P. fluorescens
and E. coli. In this case, a complex formation between the small ribosomal
subunit and kasugamycin causes an inhibition of the start of protein synthesis, by
destabilizing the initiator aminoacyl-tRNA [24]. However, the antibiotic was shown
not to bind to the 30S subunits of ribosomes derived from resistant strains [25].
In a cell-free system derived from a sensitive strain of P. oryzae, kasugamycin
inhibited the binding of aminoacyl-tRNA to ribosomes, but no such interference
was noted when ribosomes from a resistant strain of P. oryzae were used. Hence,
it was suggested that such resistance was most likely due to a decreased affinity of
the ribosomes for the antibiotic [26]. The results of a genetic analysis of strains that
were resistant to kasugamycin suggested that such resistance could be reduced to
one major gene mutation. Indeed, although three loci for antibiotic resistance have
been detected, only one of these genes was shown also to mediate resistance to
blasticidin S [27].
Strains of P. oryzae that are resistant to kasugamycin can easily be isolated
in vitro, but were found to be only weakly controlled by the antibiotic. Such findings
suggest that kasugamycin selects for the spontaneous emergence of resistant
strains of P. oryzae. Under field conditions, when the antibiotic was used repeatedly
(four to five times per year) and exclusively over three years, a decrease in rice blast
was observed [28–30], but when the treatments were stopped the proportion of
resistant strains was decreased. This disappearance of resistant strains under field
conditions might indicate that the fitness of resistant strains would, in general, be
inferior to that of sensitive strains [31]. When a mixture of spores from sensitive
and resistant strains (in 1 : 1 proportion) was used for inoculation in pot trials in
the absence of the antibiotic, the sensitive strain produced not only more lesions
but also larger lesions than the resistant strain. These results also confirmed
an inferior competitive ability of the resistant strains compared to the wild-type
strains [31]. On an agar medium, however, the resistant and sensitive isolates
showed similar levels of mycelial growth and sporulation [32]. Because of the lower
fitness of resistant strains compared to sensitive strains, the former tended to
disappear under field conditions in the absence of any selection pressure from
kasugamycin. The antibiotic has again been used successfully to control rice blast,
either in combination with fungicides that display different modes of action, or by
employing alternating applications of these materials [13, 21].
16.1.5
Mildiomycin
Mildiomycin (Figure 16.1.3), which has been isolated from the culture filtrate
of Streptoverticillium rimofaciens B98891, is water-soluble and is a member of
the new nucleoside antibiotic group (the pyrimidine base of mildiomycin is
5-hydroxymethylcytosine [33, 34]). Whereas, mildiomycin shows only a weak
activity on agar media against Gram-positive and Gram-negative bacteria, yeast and
some phytopathogenic fungi (e.g., Cochliobolus miyabeanus, Sclerotinia sclerotiorum,
Botrytis cinerea, and Alternaria kikuchiana), it has proven to be highly active against
powdery mildews; in fact, it was for this reason that it was named mildiomycin
[34–36]. Of 13 powdery mildew species tested on 15 plant species, all were
controlled with mildiomycin [36]. In addition, mildiomycin was active against
powdery mildew on green pepper caused by the endoparasitic fungus Leveillula
taurica, and also provided an effective control in benomyl-resistant strains of
cucumber [37]. The compound also demonstrated systemic activity, with root
treatments providing the control of powdery mildew on the cotyledons and leaves
of cucumber plants. A translaminar activity of mildiomycin against powdery mildew
on tobacco and cucumber plants has also been demonstrated [37].
Following the application of mildiomycin, the germination of Sphaerotheca
fuliginea conidia was inhibited at rather low concentrations; moreover, when the
germ tubes were formed they showed either spherical or oval-shaped alterations
NH2
CH2OH
N
O N
O−
O OH
+H3N O
HN OH
HN
HO
NH
H2N
O
Mildiomycin Figure 16.1.3 The structure of mildiomycin.

[37]. The mode of action of mildiomycin is via an interference with protein

synthesis; at low concentration (0.02 mM), mildiomycin inhibited the incorporation
of amino acids into polypeptides in a cell-free system of E. coli, but the synthesis of
polypeptides in a mammalian cell-free system obtained from rabbit reticulocytes
proved less sensitive to the antibiotic [38].
Mildiomycin exhibits a low level of toxicity towards mammals and fishes.
In rats and mice, the LD50 for acute toxicity was 500–1000 mg kg−1 after
intravenous and subcutaneous injection, and 2.5–5.0 g kg−1 after oral adminis-
tration. The compound had no irritant effect on either the cornea or skin of
rabbits [39].
16.1.6
Cycloheximide
Cycloheximide (Figure 16.1.4), β-[2-(3,5-dimethyl-2-oxocyclohexyl)2-hydroxyethyl]

glutarimide, is a member of the glutarimide antibiotics. Cycloheximide was dis-
covered in 1946 in a culture filtrate of Streptomyces griseus, and exhibits a high
efficacy against a wide range of fungi and yeasts. The compound shows inhibitory
activities against several human RNA viruses such as HIV-1, influenza viruses,
coxsackie B virus, enterovirus (EV71), and several DNA viruses [40], but shows
no activity against bacteria. Cycloheximide is not a specific inhibitor of fungi, but
is toxic towards plants and animals [41, 42]. Based on its phytotoxicity, the com-
pound has been used only to a limited degree to control plant diseases caused by
fungi.
Cycloheximide is an effective inhibitor of protein synthesis in eukaryotic organ-
isms, with Kerridge [43] first demonstrating its ability to inhibit protein synthesis
in the yeast, Saccharomyces carlsbergensis. At a cycloheximide concentration of
0.7 μM, which was sufficient to inhibit the growth of intact cells of Saccharomyces
pastorianus, the incorporation of amino acids into proteins in cell-free extracts was
diminished by 50% [44]. Cycloheximide is known to interfere with protein synthesis
by inhibiting the transfer of amino acids from aminoacyl-tRNA into the protein
[26, 42]; however, it did affect neither amino acid activation nor transfer to tRNA.
The ribosomal binding site for cycloheximide is located in the 60S subunit [45, 46].
Cycloheximide-resistant mutants of S. cerevisiae have been isolated with ease in
in vitro experiments [47]. Whilst the incorporation of amino acids into protein in a
O OH N
Me
O
Me
Cycloheximide Figure 16.1.4 The structure of cycloheximide.

cell-free system from wild-type cells was inhibited, the compound failed to prevent
amino acid incorporation into proteins in cell-free extracts from resistant cells [48].
The mutation of a single protein component of the 60S subunit led to a specific
alteration of the binding site [26]. As cycloheximide is used to only a limited extent
for the control of plant diseases, it is not surprising that resistance to cycloheximide
has not been reported.
16.1.7
Streptomycin
Following the successful treatment of bacterial diseases in humans with antibiotics,

investigation into the use of antibiotics to treat the bacterial pathogens of cultivated
plants began in earnest during the 1950s. Whilst many of the antibiotics tested
proved to be highly active in inhibiting the growth of Erwinia amylovora in vitro,
very few of them were suitable for use under practical conditions due to problems
of plant or mammalian toxicity, a lack of systemic activity, or short persistence on
the plant surfaces [49, 50].
Among the antibiotics tested against fire blight caused by E. amylovora, strep-
tomycin – and, to some extent, oxytetracycline and kasugamycin – fulfilled the
requirements for controlling the disease under field conditions. Streptomycin
(Figure 16.1.5), an aminoglycoside antibiotic produced by strains of Streptomyces
griseus, was discovered in 1944 [51] and successfully applied against tuberculosis
NH
NHC NH2
NH
OH
H2NCNH HO Streptidin
HO
O
CHO Streptose
H3C
OH
O
O
HO CH OH
2
H3CHN N-Methyl-L-Glucosamin
OH
Figure 16.1.5 The structure of
Streptomycin streptomycin.
caused by Mycobacterium tuberculosis. Streptomycin has also been tested against dif-
ferent plant-pathogenic bacteria, both in vitro and in vivo, and was found to inhibit
14 species of plant pathogenic bacteria, both Gram-positive and Gram-negative
[52]. In addition, antifungal activity has been reported for species of the Oomycetes
(e.g., Pythium, Phytophthora, and downy mildew) and yeasts [53, 54].
Streptomycin, when applied over a concentration range of between 30 and
240 μg ml−1 , controlled fire blight and caused no phytotoxic effects on the leaves,
nor any fruit russetting [55, 56]. In the USA, streptomycin has been used since
1955, with numerous orchard trials being conducted during the 1950s and 1960s to
determine the efficiency of streptomycin treatments to control fire blight. Because
of the limited systemic activity of streptomycin, any spray treatments should
completely cover all possible infection sites, such as open flowers, shoots, and
leaves. Streptomycin is also used in New Zealand, in some European countries,
and also in Middle Eastern countries to control fire blight [50].
With fire blight having spread across Europe, from country to country, since
the late 1950s, streptomycin is used regularly, applied on an emergency basis, or
is not permitted, depending on the country. The main reason for the banning of
streptomycin in many countries has been the development of resistance to it, not
only in E. amylovora but also in other organisms present on the plant surface or
in the soil and/or water, including potential human and veterinary pathogens [57].
The use of streptomycin is permitted in the USA on tobacco, to control wild fire
(P. syringae pv. tabaci) and blue mold (Peronospora tabacina), the latter organism
being the only fungal pathogen to be controlled by streptomycin [58].
The mode of action of streptomycin has been studied intensively in bacterial
cell-free systems. One molecule of [3 H]-dihydrostreptomycin binds per 30S subunit
[59] and a single ribosomal 30S subunit protein (S12) has been identified as
the binding site of streptomycin [60]. No binding to the 50S subunit has been
determined. Streptomycin also causes misreading of the genetic message in both
whole cells and cell-free systems, which results in miscoded amino acids being
incorporated into proteins [61].
Under in vitro conditions, highly resistant strains of Mycobacterium tuberculosis
were shown to develop shortly after streptomycin had been applied to con-
trol tuberculosis [62]. Streptomycin-resistant strains of Erwinia amylovora were
first detected in 1971 in the pear orchards of California [63, 64]; subsequently,
streptomycin-resistant E. amylovora strains were reported from areas where the
antibiotic had been applied intensively to control fire blight, including several
western states of the USA [65–67] and outside the USA, such as Egypt [68] and
New Zealand [69].
The emergence of streptomycin-resistant strains in the pear orchards of California
in 1971, and in Michigan in 1990, stimulated investigations into the emergence,
development, and mechanisms of streptomycin resistance in E. amylovora. Such
mechanisms include alterations of the ribosomal target site, the production of
streptomycin-modifying enzymes, and a reduced uptake and consequent access to
the target site [70, 71].
Among the E. amylovora populations, two phenotypes – one which is highly

resistant to streptomycin [minimal inhibitory concentration (MIC) of 2000 μg ml−1 ]
and one moderately resistant strain (MIC 500–750 μg ml−1 ) have been detected
[65, 72, 73]. Spontaneous mutants with a high level of resistance to streptomycin
were isolated at a frequency of 4 in 109 bacteria, while mutants with a moderate
resistance level were obtained at a frequency of 0.1 in 109 bacteria.
All streptomycin-resistant E. amylovora strains isolated in the California pear
orchards showed high resistance levels [74]. Resistance in the highly resistant
strains has been attributed to a point mutation in the rps L gene of the ribosomal
S12 protein, by which the streptomycin target site is altered and binding of
streptomycin to the ribosome is prevented [75]. The rps L gene of E. amylovora is
only 375 bp, and mutations in this gene associated with streptomycin resistance in
highly resistant strains of E. amylovora have been identified. The highly resistant
strains contained a mutation in the codon 43. The codon encoding for lysine (AAA)
in the sensitive strains was converted into a codon for arginine (AGA) in most of
the highly resistant strains, or in some other strains for asparagine (AAT or ACC)
or threonine (ACA) [75].
Although the mutation of rps L is the primary mechanism of streptomycin
resistance, resistance in strains isolated from the Michigan apple orchards,
which exhibited a moderate level of resistance to streptomycin, was located
on a 34 kb self-transmissible plasmid, pEa34 [67]. Sequence analysis of the
plasmid in E. amylovora strain CA11 revealed a novel 6.7 kb Tn3-type transpo-
son, Tn5393, which contained two linked strA and strB genes that encoded for
aminoglycoside-3 -phosphotransferase and aminoglycoside-6-phosphotransferase,
respectively [57, 67, 72, 73, 76]. These enzymes mediate resistance by phosphory-
lation of the 3 - or 6-hydroxyl position of the streptomycin molecule, by which the
antibiotic is inactivated. This plasmid-mediated resistance for streptomycin is trans-
ferable by conjugation, and may result in a rapid increase in streptomycin-resistant
strains. Although strains with a moderate degree of resistance have been identified
easily under laboratory conditions, they have seldom been detected in nature,
because of reduced fitness [74]. Although pEa34 is the most common vehicle
for Tn5393 in E. amylovora, some moderately streptomycin-resistant strains carry
Tn5393 on a nontransmissible plasmid, pEA29, which is unrelated to pEa34 [72].
In five streptomycin-resistant strains of E. amylovora, Tn5393 was inserted at five
different positions in pEA29 [58]. Besides pEa34, an 8.7 kb streptomycin-resistant
plasmid, pEA8.7, has been detected in isolates of E. amylovora from apple orchards
in California. This plasmid, which is related to the broad-host-range plasmid
RSF1010, confers resistance to both streptomycin and sulfonamide antibiotics,
encoded by strA-strB and sulII genes, respectively [77].
Furthermore, it may be likely that highly resistant strains with chromo-
somal mutations may also have genes coding for enzymes that modify
streptomycin.
Streptomycin was introduced into the USA during the 1950s to control fire blight.
The development of resistance of E. amylovora to streptomycin in the western states
occurred many years earlier (1971) than in the eastern states (1990), the time
References 703
difference most likely being associated with significant differences in selection

pressure exerted by streptomycin. The number of applications of streptomycin
in western states was between 10 and 14 per season [78, 79], whereas in the
eastern USA it was used initially up to five times per season; since the early 1960s,
however, it was applied only when the environmental conditions were favorable
for infection. The development of risk assessment systems that would ensure an
optimal timing of treatment in relation to the risk of infection led to a marked
reduction in the selection pressure for resistance in the USA [80–84] and also
in European countries, such as England [85, 86], France [87], Belgium [88], and
Germany [89].
Strains of E. amylovora isolated in the western states of USA (California, Wash-
ington, Oregon) expressed high levels of resistance to streptomycin. Although,
following the emergence of resistance, the use of streptomycin was discontinued
[74, 90], these strains were not impaired in terms of their fitness and showed a
long-term survival [91].
The control of fire blight is very difficult in areas where highly resistant strains
to streptomycin prevail. In those regions of the USA where streptomycin-resistant
strains of E. amylavora have become established, growers use oxytetracyline either
alone or in combination with streptomycin [72]. In areas where resistance problems
have not appeared, the management strategies used to prevent disease build-up
have become extremely important, and include the selection of cultivars and
rootstocks with a lower susceptibility to fire blight, improved sanitation methods,
and the use of good forecasting programs to enable the precise timing of each spray
during flowering [92].
References
1. Heldt, H.W. (1996) Pflanzenbio- 8. Asakawa, M., Misato, T., and Fukunaga,
chemie, Spektrum Akademischer T. (1963) Pestic. Tech, 8, 24–29.
Verlag, Heidelberg, Berlin, Oxford, 9. Yamaguchi, I., Takagi, K., and Misato,
pp. 503–511. T. (1972) Agric. Biol. Chem., 36,
2. Takeuchi, S., Hirayama, K., Ueda, K., 1719–1727.
Sasaki, H., and Yonehara, H. (1958) 10. Huang, K.T., Misato, T., and Asuyama,
J. Antibiot. Ser. A, 11, 1–5. H. (1964) J. Antibiot. Ser. A, 17,
3. Otake, N., Takeuchi, S., Endo, T., and 65–70.
Yonehara, H. (1966) Agric. Biol. Chem., 11. Yamaguchi, I. and Tanaka, N. (1966)
30, 132–141. J. Biochem., 60, 632–642.
4. Yonehara, H. and Otake, N. (1966) 12. Nakamura, H. and Sakurai, L. (1962)
Tetrahedron Lett., 1, 3785–3791. Ann. Phytopathol. Soc. Jpn, 27, 84
5. Hirai, T. and Shimomura, T. (1965) Phy- (Abstract).
topathology, 55, 291–295. 13. Dekker, J. (1983) in Pesticide Chemistry:
6. Tanaka, N., Sakagami, Y., Yamaki, H., Human Welfare und the Environment,
and Umezawa, H. (1961) J. Antibiot. Ser. vol. 2 (eds N. Takahashi, H. Yoshioka,
A, 14, 123–126. T. Misato, and S. Matsunaka), Pergamon
7. Misato, T., Ishi, I., Asakawa, M., Press, Oxford, pp. 269–275.
Okomoto, Y., and Fukunaga, K. 14. Tomoya, Y., Yoichi, N., Koji, Y., Hiroshi,
(1959) Ann. Phytopathol. Soc. Jpn, 24, S., Hiromichi, N., and Shohei, S. (2010)
302–306. J. Antibiot., 63, 309–312.
15. Umezawa, H., Okami, G., Hashimoto, 31. Ito, I. and Yamaguchi, T. (1979) Ann.
T., Suhara, Y., Hamada, M., and Phytopathol. Soc. Jpn, 45, 40–46.
Takeuchi, T. (1965) J. Antibiot. Ser. 32. Miura, H., Katagiri, M., Yamaguchi,
A, 18, 101–103. T., Uesugi, Y., and Ito, H. (1976)
16. Suhara, Y., Maeda, K., and Umezawa, Ann. Phytopathol. Soc. Jpn, 42,
H. (1966) Tetrahedron Lett., 12, 117–123.
1239–1244. 33. Harada, S., Mizuta, E., and Kishi,
17. Ikekawa, T., Umezawa, H., and T. (1978) J. Am. Chem. Soc., 100,
Iitaka, Y. (1966) J. Antibiot. Ser. A, 19, 4895–4897.
49–50. 34. Iwasa, T., Suetomi, K., and Kusaka, T.
18. Ishiyama, T., Hashimoto, T., Hamada, (1978) J. Antibiot., 31, 511–518.
M., Okami, Y., Takeuchi, T., and 35. Harada, S. and Kishi, T. (1978) J. An-
Umezawa, H. (1965) J. Antibiot., 18, tibiot., 31, 519–524.
115–119. 36. Suetomi, K. and Kusaka, T. (1979)
19. Tsujimoto, K., Yamamura, H., and Sato, J. Pestic. Sci., 4, 349–353.
K. (1981) Ann. Phytopathol. Soc. Jpn, 47, 37. Iwasa, T. (1983) in Pesticide Chemistry:
402. Human Welfare and Environment, vol.
20. Sato, K., Kanda, M., and Yamamura, H. 2 (eds N. Takahashi, H. Yoshioka, T.
(1976) Ann. Phytopathol. Soc. Jpn, 42, Misato, and S. Matsunaka), Pergamon
61. Press, Oxford, pp. 57–62.
21. Sato, K. (1983) in Pesticide Chemistry: 38. Om, Y., Yamaguchi, I., and Misato, T.
(1984) J. Pestic. Sci., 9, 317–323.
Human Welfare and the Environment, vol.
39. Kusaka, T., Suetomi, S., Iwasa, T., and
2 (eds N. Takahashi, H. Yoshioka, T.
Harada, S. (1979) Br. Crop Prot. Conf. -
Misato, and S. Matsunaka), Pergamon
Pests Dis., 2, 589–595.
Press, Oxford, pp. 293–299.
40. Guo, H., Li, Y., Tao, P., Yi, H.,
22. Tanaka, N., Nishimura, T., Yamaguchi,
Wang, S., He, W., Jiang, J., and Li,
H., Yamamoto, C., Yoshida, Y.,
Z. (2010) Yaoxue Xuebao, 45, 268–273
Sashikata, K., and Umezawa, H. (1965)
(in Chinese).
J. Antibiot., 18, 139–144.
41. Worthing, C.R. (1979) The Pesticide Man-
23. Hamada, M., Hashimoto, T., Takahashi,
ual, 6th edn, British Crop Protection
S., Yoneyama, M., Miyake, T., Takeuchi,
Council, London.
Y., Okami, Y., and Umezawa, H. (1965)
42. Ennis, H.L. and Lubin, M. (1964)
J. Antibiot. Ser. A, 18, 104–106. Science, 146, 1474–1476.
24. Poldermans, B., Goosen, N., and Van 43. Kerridge, D. (1958) J. Gen. Microbiol., 19,
Knippenberg, P.H. (1979) J. Biol. Chem., 497–506.
254, 9085–9089. 44. Siegel, M.R. and Sisler, H.D. (1963)
25. Helser, T.L., Davies, J.E., and Dahlberg, Nature, 200, 675–676.
J.E. (1971) Nature, 233, 12–14. 45. Roa, S. and Grollmann, A.P. (1967)
26. Siegel, M.R. (1977) in Antifungal Com- Biochem. Biophys. Res. Commun., 29,
pounds (eds M.R. Siegel and H.D. 696.
Sisler), Marcel Dekker, New York, 46. Battaner, E. and Vazquez, D. (1971)
pp. 399–438. Biochim. Biophys. Acta, 254, 316.
27. Taga, M., Tsuda, M., and Ueyama, A. 47. Wilkie, D. and Lee, B.K. (1965) Genet.
(1979) Plant Prot., 33, 471–476. Res., 6, 130–138.
28. Miura, H., Kimura, H., and Takahashi, 48. Cooper, D., Banthorpe, D.V., and
S. (1973) Ann. Phytopathol. Soc. Jpn, 39, Wilkie, D. (1967) J. Mol. Biol., 26,
230–240. 347–350.
29. Miura, H., Ito, H., and Takahashi, S. 49. Ark, P.A. (1949) in Streptomycin: Na-
(1975) Ann. Phytopathol. Soc. Jpn, 41, ture and Practical Applications (ed.
415–417. S.A. Waksman), Williams & Wilkins,
30. Goh, N., Yaoita, Y., Aoyagi, K., Ikeda, Baltimore, MD, pp. 607–612.
U., and Sakurai, H. (1977) Proc. Assoc. 50. Psallidas, G. and Tsiantos, J. (2000)
Plant Prot. Hokuriku, 25, 58–60. in Fire Blight – The Disease and its
References 705
Causative Agent (ed. J.L. Vanneste), 70. Davies, J.E. (1986) in Antibiotics in
CABI Publishing, Wallingford, pp. Laboratory Medicine (ed. V. Lorian),
199–234. Williams & Wilkins, Baltimore, MD, pp.
51. Schatz, A., Bugie, E., and Waksman, 790–809.
S.A. (1944) Proc. Soc. Exp. Biol. Med., 55, 71. Amyes, S.G.B. and Gemmell, C.G.
66–69. (1992) J. Med. Microbiol., 36, 4–24.
52. Ark, P.A. (1947) Phytopathology, 37, 842 72. McManus, P.S. and Jones, A.L. (1994)
(abstract). Phytopathology, 84, 627–633.
53. Gottlieb, D. and Shaw, P.D. (1970) 73. Chiou, C.-S. and Jones, A.L. (1995)
Annu. Rev. Phytopathol., 8, 371–402. Phytopathology, 85, 324–328.
54. Tsao, H.P. (1970) Annu. Rev. Phy- 74. Schroth, M.N., Thomson, S.V., and
topathol., 8, 157–186. Moller, W.J. (1979) Phytopathology, 69,
55. Heuberger, J.W. and Poulos, P.L. (1953) 565–568.
Plant Dis. Rep., 37, 81–83. 75. Galili, S., Fromm, H., Aviv, D.,
56. Ark, P.A. and Scott, C.E. (1954) Phy- Edelman, M., and Galem, E. (1989)
topathology, 44, 481 (Abstract). Mol. Gen. Genet., 218, 289–292.
57. Jones, A.L. and Schnabel, E.L. (2000) 76. Shaw, K.J., Rather, P.N., Hore, R.S., and
in Fire Blight – The Disease and its Miller, G.H. (1993) Microbiol. Rev., 57,
Causative Agent (ed. J.L. Vanneste), 138–163.
CABI-Publishing, Wallingford, pp. 77. Palmer, E.L., Teviotdale, B.L., and Jones,
235–251. A.L. (1997) Appl. Environ. Microbiol., 63,
4604–4607.
58. McManus, P.S., Stockwell, V., Sundin,
78. Thomson, S.V., Schroth, M.N., Moller,
G.W., and Jones, A.L. (2002) Annu. Rev.
W.J., and Reil, W. (1975) Phytopathology,
Phytopathol., 40, 443–465.
65, 353–358.
59. Kaji, H. and Tanaka, Y. (1968) J. Mol.
79. Moller, W.J., Schroth, M.N., and
Biol., 32, 221–230.
Thomson, S.V. (1981) Plant Dis., 65,
60. Ozaki, M., Mizushima, S., and Nomura,
563–568.
M. (1969) Nature, 222, 333–341.
80. Luepschen, N.S., Parker, K.G., and
61. Pestka, S. (1971) Annu. Rev. Microbiol.,
Mills, W.D. (1961) Cornell Agric. Exp.
25, 487–562.
Stn Bull., 963, 1–20.
62. Steenken, W. Jr and Wolinsky, M.D.
81. Steiner, P.W. (1990) Acta Hortic., 273,
(1949) in Streptomycin: Nature and Prac-
139–148.
tical Applications (ed. S.A. Waksman), 82. Steiner, P.W. (1990) Acta Hortic., 273,
Williams and Wilkins, Baltimore, MD, 149–158.
pp. 177–196. 83. Smith, T.J. (1993) Acta Hortic., 338,
63. Miller, T.D. and Schroth, M.N. (1972) 153–157.
Phytopathology, 62, 1175–1182. 84. Smith, T.J. (1999) Acta Hortic., 489,
64. Moller, W.J., Beutel, J.A., Reil, W., and 429–436.
Zoller, B.G. (1972) Phytopathology, 62, 85. Billing, E. (1990) Acta Hortic., 273,
779 (Abstract). 163–170.
65. Coyier, D.L. and Covey, R.P. (1975) 86. Billing, E. (1999) Acta Hortic., 489,
Plant Dis. Rep., 59, 849–852. 399–405.
66. Shaffer, W.H. and Goodman, R.N. 87. Jaquart-Romon, C. and Paulin, J.P.
(1985) Phytopathology, 75, 1281 (1991) Agronomie, 11, 511–519.
(Abstract). 88. Timmermans, Y. (1990) Acta Hortic.,
67. Chiou, C.-S. and Jones, A.L. (1991) 273, 121–127.
Phytopathology, 81, 710–714. 89. Berger, F., Zeller, W., Gutsche, V., and
68. EI-Goorani, M.A., EI-Kasheir, H.M., Rosenberg, D. (1996) Acta Hortic., 411,
Shoeib, A.A., and Hassanein, F.M. 155–161.
(1989) J. Phytopathol., 127, 69–74. 90. Loper, J.E., Henkels, M.D., Roberts,
69. Thomson, S.V., Gouk, S.C., Vanneste, R.G., Grove, G.G., Willett, M.J., and
J.L., Hale, C.N., and Clark, R.G. (1993) Smith, T.J. (1991) Plant Dis., 75,
Acta Hortic., 338, 223–230. 287–290.
91. Stockwell, V.O., Sugar, D., Spots, 92. Steiner, P.W. (2000) in Fire Blight –
R., Johnson, K.B., and Loper, J.E. The Disease and its Causative Agent
(1996) Phytopathology, 86, S50 (ed. J.L. Vanneste), CABI Publishing,
(abstract). Wallingford, pp. 339–358.
16.2
Anilinopyrimidines: Methionine Biosynthesis Inhibitors
16.2.1
Introduction
Pyrimidines have long been known as both pharmaceuticals and as crop protection
agents. In terms of plant disease control, the first pyrimidines were introduced more
than 30 years ago while anilinopyrimidines were, to the best of our knowledge, first
prepared in 1901 and later described by ICI as having some potential antimalarial
activity [1]. The anilinopyrimidines were first patented as fungicides in 1981 by VEB
Fahlberg-List, Magdeburg (former German Democratic Republic, DDR) [2] but,
during the late 1980s, were rediscovered independently by Ciba-Geigy, Schering,
and Kumiai/Ihara Chemical Industries. As a result of these research efforts, three
anilinopyrimidines were introduced into the market as novel fungicides between
1992 and 1995: pyrimethanil (1) (reported in 1992) [3], cyprodinil (2) (in 1994) [4],
and mepanipyrim (3) (in 1995) [5] (Figure 16.2.1; Table 16.2.1).
16.2.2
Chemistry of the Anilinopyrimidines
Two general syntheses, here termed methods A and B, have been used to prepare
anilinopyrimidines (Scheme 16.2.1).
Following method A, condensation of phenylguanidine (4) with the correspond-
ing β-di-ketones (5) gives the anilinopyrimidines in a single step [2, 7]. Both
starting materials, phenylguanidines and β-diketones, are easily accessible and
allow the preparation of a wide variety of anilinopyrimidines. As an example,
the β-diketone, 1-cyclopropyl-butane-1,3-dione (5; R1 = CH3 , R2 = cyclopropyl),
intermediate for the preparation of cyprodinil (2), is easily prepared by a Claisen
H H H
N N N N N N
N N N
1 2 3
Figure 16.2.1 Structures of the anilinopyrimidines:

pyrimethanil (1), cyprodinil (2), mepanipyrim (3).
Table 16.2.1 Chemical and physical properties of the anilinopyrimidines [3–6].
Common name Pyrimethanil (1) Cyprodinil (2) Mepanipyrim (3)
Patent no. – EP310550 EP224339/JP

63208581
Melting point (◦ C) 96.3 75.9 132.8
◦
Vapor pressure 2.2 (25 C) Crystal mod. A: 0.51 2.32 × 10−2 (20 ◦ C)
(mPa) Crystal mod. B: 0.47
KOW (log P) (25 ◦ C) 2.84 (pH 6.1) 4.0 (pH 7.0) 3.28 (20 ◦ C)
Solubility (g l−1 ) at Water 0.121 (pH 6.1), Water 0.013 (pH 7.0), Water 0.003.10
25 ◦ C in methanol 176 (pH ethanol 160, acetone (20 ◦ C), acetone 139
6.1), acetone 389 (pH 160, n-hexane 26 (20 ◦ C), methanol
6.1), n-hexane 23.7 15.4 (20 ◦ C),
(pH 6.1) n-hexane 2.06 (20 ◦ C)
Stability Stable: in water Stable: in water Stable: in water
within relevant pH (DT50 >> 1 year, pH (DT50 > 1 year, pH
range; to heat 14 4–9, 25 ◦ C); 4–9); to heat; to light
days at 54 ◦ C photolysis in water in water (DT50
DT50 5–30 days 12.9 days)
pKa 3.52, weak base 4.44, weak base n.d.
(20 ◦ C)
n.d., not determined.
condensation of methyl acetate (9) and methyl cyclopropyl-ketone (10) [8].

Following synthesis method B, 2-anilino-4,6-dimethyl-pyrimidine (1) – that is,
pyrimethanil – was first prepared as early as 1901 [9]. Anilines or, as described
later [10], N-formyl-anilines (7) were reacted with 4,6-disubstituted pyrimidines
(6) carrying a leaving group such as halogen, sulfide, or preferably a sulfonyl
group at position 2. As an example, mepanipyrim (3) was first prepared starting
from 2-methansulfonyl-4-methyl-6-(1-propynyl)pyrimidine (6; L = −SO2 CH3 )
and formanilide (7) in presence of a strong base to give the N-formyl-derivative
(8; R1 = CH3 , R2 = 1-propynyl) which, on hydrolysis, affords mepanipyrim (3) [10].
In analogy to method A, phenylguanidine (4) was condensed with dehydroacetic
acid (11) to give the intermediate 4-(propan-2-one)-6-methyl-2-anilinopyrimidine
(12; Scheme 16.2.2), which was then converted in two steps into mepanipyrim (3):
first, a chlorination to the chloro-allyl compound 13; and second, the elimination
of hydrochloric acid [11].
Zondler and Hubele [12] described the synthesis of N-amino- and N-hydroxy-
2-anilino-pyrimidines in analogy to method B (Scheme 16.2.3). Noteworthy here are
the reaction conditions for the preparation of hydroxylamino compounds 16 and
the hydrazine compounds 17. Substitution with arylhydroxylamines leads, under
acidic reaction conditions, to the desired N-hydroxy-anilinopyrimidines (16); with
arylhydrazines under these conditions, however, the undesired 1-aryl-2-pyrimidinyl
Method A
R1 H 3
H N 2 N R1
N NH2 O 4
+ 1N 5
NH O 6
R2 R2
4 5 1-3
O
O
H3C O
CH3
9 10
Method B H O
R1
N H
N O N N R1
L +
N H N
R2
R2
6 7 8
Scheme 16.2.1 Synthesis of anilinopyrimidines.
O O H
H N N
N NH2
+ N O
NH
O O
4 11 12
H H
N N N
N
N N CI
3 13
Scheme 16.2.2 Synthesis of 4-alkynyl-2-anilinopyrimidines.

OH
CI N R1
H N N R1
N
OH N HCI / Methanol
N
R2
R2
14 6 (L = CI) 16
NH2
N N R1
THF, K+ −O-t-butyl
N
CI N R1
H
N R2
NH2 N 17
+
R2 R1
N
15 6 (L = CI) HCI / Methanol H
N
N N R2
H
18
Scheme 16.2.3 Synthesis of N-hydroxy-anilinopyrimidines

and 1-phenyl-1-pyrimidinyl-hydrazines.
derivatives 18 are produced. The desired 1-aryl-1-pyrimidinyl-derivatives 17 were

obtained under basic reaction conditions only. Under the same conditions, the
N-hydroxy-anilinopyrimidines could not be prepared. These compounds were
described as generally stable crystalline compounds at room temperature, but
decompose to the parent 2-anilino-pyrimidines when exposed to light [13]. By
following these two described methods, numerous anilinopyrimidines displaying
a wide diversity of properties have been prepared.
16.2.3
Biological Activity
The spectrum of fungicidal activity of anilinopyrimidines is restricted to As-

comycetes, including a broad range of pathogens such as: Botryotinia fuckeliana
(Botrytis cinerea) on grapes, fruits, vegetables, and ornamentals; Venturia inaequalis
on apple; and Alternaria and Monilinia spp. causing leaf spot diseases and rot on
a range of vegetables and deciduous fruits [13]. In addition to these pathogens,
cyprodinil also controls a range of cereal diseases caused by Tapesia spp. (Pseudo-
cercosporella herpotrichoides, eyespot), Pyrenophora teres and Rhynchosporium secalis
in barley (net blotch and scald, respectively) and, to a moderate degree, also Lep-
tosphaeria nodorum (Septoria on wheat) and Blumeria (Erysiphe) graminis (powdery
mildew) on cereals. Pyrimethanil has additional activity against Ascochyta spp. in

legumes, Mycosphaerella spp. in banana, pea, and other vegetables and some of
the post-harvest diseases (e.g., Aspergillus and Penicillium spp.) and seed-borne
pathogens (e.g., Pyrenophora graminea, which is controlled also by cyprodinil). To
broaden the spectrum of activity and delay the evolution of resistance, anilinopy-
rimidines are often mixed with sterol biosynthesis inhibitors (SBIs) for their use in
cereals and fruits (e.g., propiconazole, cyproconazole, fluquinconazole, imazalil),
with multisite inhibitors in apple and legumes (e.g., with captan, dithianon,
chlorothalonil), or with fludioxonil in grapes.
Anilinopyrimidines exhibit a strong preventive activity that is based on the inhi-
bition of germ tube elongation during spore germination, of appressoria formation,
and of mycelial growth. The penetration and infection process of the pathogen
into the host tissue is also affected, presumably by inhibiting the secretion of hy-
drolytic enzymes during pathogenesis. Anilinopyrimidines, as systemic fungicides,
are translocated in the apoplast of leaves, which results in the inhibition of later
stages of pathogenesis, such as the formation of haustoria, intercellular growth
of the mycelium, and sporulation. Consequently, anilinopyrimidines can exhibit
a curative activity against V. inaequalis in apples up to three days after infection,
while later stages and spore germination are not affected.
16.2.4
Studies on the structure–activity relationships of the anilinopyrimidines have

clearly demonstrated that the biological activity generally falls sharply with the
introduction of any substituents in positions 2 to 6 of the anilinobenzene ring; the
exception is the 3 or 4 fluoro-substituted compounds, which have demonstrated
some biological activity [13, 14]. Likewise, the introduction of substituents in posi-
tion 5 of the pyrimidine ring greatly reduced the biological activity (Figure 16.2.2).
The photolytic and hydrolytic stabilities of the N-amino-, N-hydroxy- or N-O-alkyl,
or O-acetyl-2-anilinopyrimidine derivatives must be considered when assessing the
biological activity, as these compounds might decompose to the parent compounds
[12]. N-Methyl-anilinopyrimidine showed some activity, but less than that of the
unsubstituted analogs, while higher alkyl substituents at the bridging nitrogen led
to inactivity. No fungicidal activity was observed when the bridging nitrogen was
replaced by sulfur or oxygen. Various substituents such as alkyl, chloro, methoxy,
methylamino-, cyclopropyl-, and alkenyl and alkynyl are tolerated in the 4 and
6 positions of the pyrimidine ring [12, 13]. The highest potency and broadest
R3
6′ 3
X 2 N R1
5′
S 1′ 4
4′ 2′ 1 N 5
6
3′
R2
Figure 16.2.2 General structure of anilinopyrimidines.
spectrum was observed with sterically small and chemically stable combinations,
such as those present in pyrimethanil, cyprodinil, and mepanipyrim.
16.2.5
Mode of Action and Mechanism of Resistance
Anilinopyrimidines are considered to be inhibitors of methionine biosynthesis [15,

16] (Figure 16.2.3). They are single-site inhibitors in the amino acid biosynthesis
pathway, and cross-resistance was observed between cyprodinil, pyrimethanil, and
mepanipyrim [17], which suggested a common mode of action for the entire
class [18].
In addition, the anilinopyrimidines were reported also to affect (potentially
as a consequence) the secretion of hydrolytic enzymes during penetration of
the target pathogens into the plant tissues [19, 20], although the biosynthe-
sis of these enzymes was not affected. In biochemical studies, methionine and
homocysteine – but not cystathionine – reversed the action of the anilinopyrim-
idines [15], while the incorporation of radiolabeled sulfur starting from sulfate
Methionine biosynthesis pathway
Tricar-
bonic acid
cycle
Serine acetyl CoA Aspartate
O-acetylserine Homoserine
CSA HCT
Cysteine O-acetylhomoserine O-succinylhomoserine

CoA
CGL Acetate CGS ‘CGS’ CGS ‘CGS’

Cystathionine
CBL
Pyruvat
Homocysteine
HMT CBL cystathionine-β-lyase
– CH3 CGL cystathionine-γ-lyase
Methionine
CGS cystathionine-γ-synthase
‘CGS’ = HSA = homocysteinesynthase
CSA cysteinesynthase
S-adenosylmethionine
HCT homoserine-O-acetyltransferase
HMT homocytheine-metyltransferase
Figure 16.2.3 Schematic representation of the methionine biosynthesis pathway [18].

suggested an inhibition of methionine biosynthesis [16]. Thus, it was proposed

that either cystathionine-β-lyase (CBL) or cystathionine-γ -synthase (CGS) may
be the target enzymes. However, isolated CBL was not sensitive to cyprodinil
(cell-free assay) and CGS-deficient mutants of Neurospora crassa were insensitive to
anilinopyrimidines [18].
When the sequences of the cbl gene in sensitive and resistant field isolates of
Botrytis cinerea were compared, no mutations were detected that would confer
resistance to anilinopyrimidines [18]. Thus, CBL is unlikely to be the target enzyme
for resistance to anilinopyrimidines, and is probably not the biochemical site of
action. However, two different mutations (S24P and I64V) were found in the cgs
gene of several B. cinerea field isolates that were resistant to anilinopyrimidines [18].
When sensitive and cyprodinil-resistant parents of B. cinerea were crossed and the
8 F1 ascospore progeny was analyzed for resistance (per ascus in two independent
crosses), the segregation of resistance was 1 : 1 [21] (Figure 16.2.4). In both crosses,
the mutations (S24P in cross 1 and I64V in cross 2) cosegregated with the resistant
phenotype and were inherited in a 1 : 1 manner [18] (Figure 16.2.4). Therefore, it can
be assumed that resistance to anilinopyrimidine fungicides in B. cinerea (and proba-
bly also in other target pathogens) is monogenic, but multiallelic. The CGS enzyme
complex is end-product-inhibited and accepts different substrates circumventing
cystathionine for methionine biosynthesis (see Figure 16.2.3). A comparison of the
cgs gene sequences in B. cinerea revealed that the amino acid change in the resistant
Cross 1 Cross 2
Isolate Sensitivity S24P 164V Isolate Sensitivity S24P 164V
CH 9.83 (s) 0.003 wt wt CH 9.83 (s) 0.003 wt wt
Z 103.16 (r) 10 wt mut Z 203.21 (r) 2 mut wt
Ascospore 1 10 wt mut Ascospore 1 s wt wt
Ascospore 2 10 wt mut Ascospore 2 r mut wt
Ascospore 3 10 wt mut Ascospore 3 s wt wt
Ascospore 4 0.007 wt wt Ascospore 4 s wt wt
Ascospore 5 0.016 wt wt Ascospore 5 r mut wt
Ascospore 6 0.012 wt wt Ascospore 6 r mut wt
Ascospore 7 7 wt mut Ascospore 7 r mut wt
Ascospore 8 0.006 wt wt Ascospore 8 s wt wt
wt: wild type, no mutated allele, sensitive to cyprodinil

S24P: exchange of serine by phenylalanine at position 24 in CGS, resistant to APs
I64V: exchange of isoleucine by valine at position 64 in CGS, resistant to APs
Figure 16.2.4 CGS genotype (wt, S24P, I64V) and sensitiv-

ity (s, r, EC50 , mg l−1 ) of parent isolates (CH 9.83, Z 103.16,
Z 203.21) and F1 single ascospore progeny isolates (8 per
ascus in crosses 1 and 2) of Botrytis cinerea to cyprodinil
[18, 21]. s = sensitive; r = resistant; mut = mutant.
References 713
phenotype was located in the regulatory part of the gene [18]. Thus, it was suggested
that the mutated CGS is insensitive to end-product repression, and that the change
in CGS regulation (overexpression) may confer resistance to anilinopyrimidines.
Therefore, the level and extent of resistance evolution is estimated as moderate
[see Fungicide Resistance Action Committee (FRAC) classification]. The mode of
action of the anilinopyrimidine fungicides remains speculative, however.
Although resistance to the anilinopyrimidines was detected several years ago in
some pathogens, including B. cinerea, V. inaequalis, and Tapesia spp. [22–25], it
has not yet evolved to an extent that product performance is affected in practice.
Resistance to anilinopyrimidines was observed at trial sites in France in 1991
for B. cinerea [22], and also in Italy and Switzerland in 1997 for V. inaequalis
after excessive product use (up to 14 applications in orchards) [23]. Resistant
isolates have also been detected repeatedly in Tapesia spp., but their frequency
remained low [24] and product performance was good. As the current sensitivity
assays are normally conducted with bulk samples, there is no solid information
on the proportion of resistance in field populations. Although there is an inherent
risk of resistance evolution to anilinopyrimidines in field populations, the extent
and distribution did not follow the same dynamics as was observed for other
single-gene mechanisms [e.g., in benzimidazoles or quinone outside inhibitors
(QoIs)]. A restriction of the number of applications per season (one to two in
cereals and grapes, three to four in apple, fruits, and vegetables) and the use of
mixtures or alternations, are recommended for the delay of resistance evolution
(see AP-FRAC recommendations) [26].
16.2.6
Degradation and Metabolism
Anilinopyrimidines are decomposed rapidly in water when exposed to ultravi-

olet light [degradation time (DT)50 about two weeks]. In soil, cleavage of the
aniline–pyrimidine linkage represents the major degradation pathway, while other
reactions include hydroxylations, oxidations, and nitrations [27]. Based on their
high Kow values (Table 16.2.1), the anilinopyrimidines show minimal movement
to deeper soil layers. In plants, the major residual components after application
are the active ingredients. The anilinopyrimidines are metabolized mainly via
hydroxylation at the phenyl ring, the pyrimidine, or the methyl moiety [27].
References
1. Curd, F.H.S. and Rose, F.L. (1946) J. 3. Neumann, G.L., Winter, E.H., and Pittis,
Chem. Soc., 343–351. J.E. (1992) Proc. Brighton Crop Prot.
2. Friedrich, F., Klepel, M., Krause, Conf. – Pests Dis., 1, 395.
G., Lehmann, H., and Brämer, B.
4. Heye, U.J., Speich, J., Siegle, H.,
(1981) Deutsche Demokratische
Republik. Patentschrift 151,404 Wohlhauser, R., and Hubele, A. (1994)
(Publ. Date. 21.10.1981), (Inventors), Proc. Brighton Crop Prot. Conf. – Pests
VEB-Fahlberg-List. Dis., 2, 501.
5. Maeno, S., Miura, I., Masuda, K., and 16. Leroux, P., Colas, V., Fritz, R., and
Nagata, T. (1990) Proc. Brighton Crop Lanen, C. (1995) in Modern Fungicides
Prot. Conf. – Pests Dis., 2, 425. and Antifungal Compounds (eds H. Lyr,
6. Tomlin, C.D.S. (ed.) (2004–2005) The P.E. Russell, and H.D. Sisler), Intercept,
e-Pesticide Manual, Version 3.1, 13th Andover, MA, pp. 61–67.
edn, British Crop Protection Council, 17. Leroux, P., Chapland, F., Desbrosses,
Alton. D., and Gredt, M. (1999) Crop Prot., 18,
7. (a) Hubele, A. (1989) EPA 310,550 687–697.
(Publ. Date 05.04.1989), (Inventor), 18. Sierotzki, H., Wullschleger, J., Alt, M.,
Ciba-Geigy AG; (b) Gastner, T., Hölzl, Bruyère, T., Pillonel, C., Parisi, S., and
A., Huber, C., and Mascha, A. (2003) Gisi, U. (2002) in Modern Fungicides and
WO 03/070,708 (Publ. 21.02.2003), (In- Antifungal Compounds III (eds H.W.
ventors), Degussa AG; (c) Ressel, H.-J. Dehne, U. Gisi, K.H. Kuck, P.E. Russell,
and Schlegel, G. (1996) EPA 717,038 and H. Lyr), AgroConcept, Bonn, pp.
(Publ. 19.06.1996), (Inventors), Hoechst 141–148.
Schering AgrEvo GmbH. 19. Miura, I., Kamakura, T., Maeno, S.,
8. (a) Cannon, G. and Whidden, H.L. Hayashi, S., and Yamaguchi, I. (1994)
(1952) J. Org. Chem., 17, 685–692; (b) Pestic. Biochem. Physiol., 48, 222–228.
Muhr, J. (1995) DE 4,404,059 (Publ. 20. Milling, R.J. and Richardson, C.J. (1995)
Date: 10.08.1995), (Inventor), Hüls AG. Pestic. Sci., 45, 43–48.
9. Angerstein, St. (1901) Chem. Ber., 34,
21. Hilber, U.W. and Hilber-Bodmer, M.
3962.
(1998) Plant Dis., 82, 496–500.
10. Ito, S., Masuda, K., Kusano, S., Nagat,
22. Forster, B., Heye, U., Pillonel, C., and
T., Kojima, Y., Sawal, N., and Maeno,
Staub, T. (1995) in Modern Fungicides
S.-I. (1987) EPA 224,339 (Publ. date
and Antifungal Compounds (eds H. Lyr,
03.06.1987), (Inventors), Kumiai Chem-
P.E. Russell, and H.D. Sisler), Intercept,
ical Industries Co., Ltd and Ihara
Andover, MA, pp. 365–376.
Chemical Industry Co., Ltd.
23. Küng, R., Chin, K.M., and Gisi, U.
11. Kimoto, T., Ohi, H., Watanabe, T., and
(1999) in Modern Fungicides and Anti-
Nakayama, T. (1989) EPA 347,866 (Publ.
Date 27.12.1989), (Inventors), Ihara fungal Compounds II (eds H. Lyr, P.E.
Chemical Industry Co., Ltd. Russell, H.W. Dehne, and H.D. Sisler),
12. (a) Zondler, H. and Hubele, A. (1989) Intercept, Andover, MA, pp. 313–322.
EPA 358,609 (Publ. Date 14.03.1989), 24. Leroux, P., Arnold, A., and Gredt, M.
(Inventors), Ciba-Geigy AG; (2002) in Modern Fungicides and Anti-
(b) Zondler, H. (1991) EPA 441,747 fungal Compounds III (eds H.W. Dehne,
(Publ. Date 14.08.1991), (Inventor), U. Gisi, K.H. Kuck, P.E. Russell, and H.
Ciba-Geigy AG. Lyr), AgroConcept, Bonn, pp. 205–212.
13. Müller, U., Hubele, A., Zondler, H., 25. Dux, H., Sierotzki, H., Meier-Runge, F.,
and Herzog, J. (1998) in Synthesis and Gisi, U. (2005) in Modern Fungicides
and Chemistry of Agrochemicals V, and Antifungal Compounds IV (eds H.W.
ACS-Symposium Series, Vol. 686 (eds Dehne, U. Gisi, K.H. Kuck, P.E. Russell,
D.R. Baker, J.G. Fenyes, G.S. Basarab, and H. Lyr), BCPC, Alton, pp. 45–54.
and D.A. Hunt), American Chemical 26. Fungicide Resistance Action Committee
Society, pp. 237–245. (FRAC) (2010) http://www.frac.info.
14. Nagata, T., Masuda, K., Maeno, S., and 27. Leroux, P. (2003) in Encyclopedia of
Miura, I. (2003) Pest Manage. Sci., 60, Agrochemicals, vol. 2 (eds J.R. Plimmer,
399–407. D.W. Gammon, and N.N. Ragsdale),
15. Masner, P., Muster, P., and Schmid, J. Wiley-Interscience, Hoboken, NJ, pp.
(1994) Pestic. Sci., 42, 163–166. 531–536.
715
17
Fungicides Acting on Signal Transduction
17.1
Mode of Action
Andrew Corran
17.1.1
Phenylpyrroles and Dicarboximides
The high-osmolarity glycerol (HOG) pathway in the budding yeast Saccharomyces

cerevisiae is well characterized (for recent reviews, see Refs [1–3]). In this path-
way, the sole histidine kinase Sln1p – as with histidine kinases in most other
eukaryotes – is a hybrid protein in which the kinase domain is fused to the re-
sponse regulator domain. Sln1p is a transmembrane protein that modulates its
cytoplasmic kinase domain activity in response to external stimuli, and conditions
of low osmolarity lead to the autophosphorylation of a histidine residue in the
kinase domain. This phosphate group is then transferred to an aspartate residue
in the Sln1p receiver domain, then to Ypd1p, and finally Ssk1p (Figure 17.1.1).
In this phosphorylated form, Ssk1p is inactive and unable to phosphorylate Ssk2p
or Ssk22p, the two mitogen-activated protein kinase kinase kinases (MAPKKKs)
in the downstream HOG MAP kinase cascade. Conversely, under conditions of
high osmolarity Sln1p, Ypd1p, and Ssk1p are all dephosphorylated. Ssk1p in this
dephosphorylated, active form subsequently phosphorylates and activates the first
kinases of the HOG MAP kinase cascade. This eventually leads to the transcription
of enzymes involved in glycerol production that allow the cell to compensate for
the high external osmotic pressure.
Osmotic-sensitive mutants of Neurospora crassa have been identified, including
os-2, a deletion mutant lacking the MAP kinase orthologous to Hog1p. These mutant
strains grow normally in a low osmotic environment, but cannot adapt to conditions
of high osmolarity and were found to be highly resistant to phenylpyrroles such
as fludioxonil and fenpiclonil, and dicarboximides such as iprodione and vinclo-
zolin [4]. In addition, both phenylpyrroles and dicarboximides stimulate glycerol
production in wild-type strains of Neurospora and cause conidia and hyphal cells
to swell and burst, most likely by the generation of a too-high internal turgor
pressure [4, 5]. Such bursting did not occur in the os-2 mutant, however, which was

716 17 Fungicides Acting on Signal Transduction
Extracellular
hyperosmolarity
Sln1
His-containing
Ypd1 phosphotransfer
protien
Ssk1 Response regulator
Ssk22 Ssk2 MAPKKK
Pbs2 MAPKK
Hog1 MAPK
Gene expression,
adaptation to high osmolarity
and glycerol accumulation
Figure 17.1.1 Signaling events downstream of the histidine

kinase Sln1p following hyperosmotic stress in Saccharomyces
cerevisiae.
found to be unable to synthesize glycerol in response to phenylpyrroles or under

conditions of high osmolarity [6]. This MAP kinase is, therefore, essential for an
adaptation to a high-osmolarity environment and for the expression of fungicidal
activity. Supporting this, in the fungal pathogen Colletotrichum lagenarium, the
orthologous MAP kinase (Osc1p) was found to be rapidly activated and transported
into the nucleus following treatment with either high osmotic stress or fludioxonil
[7]. Kojima and coworkers were also able to demonstrate a similar activation of the
orthologous MAP kinases in both Botrytis cinerea and Cochliobolus heterostrophus
with fludioxonil treatment, indicating an overall similarity between fungal species
in pathways involved in osmotic signaling. The C. lagenarium osc1 null mutant was
fully pathogenic, showed a reduced sensitivity to fludioxonil, and expressed growth
defects under conditions of high osmolarity [7].
In addition to the hyperosmolarity-sensing MAP kinase, other upstream protein
kinases in N. crassa, including the Os-1 histidine kinase, have been similarly
implicated in fungicide resistance and osmotic sensitivity [8, 9]. These data have
now been widely accepted to indicate that phenylpyrroles and dicarboximides act
upstream of the MAP kinase cascade involved in the hyperosmolarity response.
Based on the genomic and biochemical information available to date, filamentous
fungi have similar signaling pathways to S. cerevisiae to detect and respond
to changes in the osmotic environment, in that both histidine kinases and a
downstream MAP kinase cascade have been identified in fungi as well as fungal
homologs of both Ypd1p and Ssk1p [10]. Yet, despite this S. cerevisiae is insensitive to
both phenylpyrroles and diarboximides, which indicates that certain key differences
exist between budding yeasts and filamentous fungi. In addition, whereas budding
yeast has one essential histidine kinase, N. crassa has been reported to have 11
such enzymes, and plant pathogenic fungi were found to contain even more; for
example, B. cinerea is reported to have 20 histidine kinase genes [10, 11]. These
additional kinases most likely reflect the greater need for plant pathogenic fungi to
sense and respond to a complex and changing environment. Another difference in
the histidine kinases between budding yeast and filamentous fungi is that sln1 null
mutants (and ypd1 null mutants) are nonviable in S. cerevisiae, presumably due
to an inappropriate activation of the MAP kinase cascade and glycerol synthesis,
which causes the yeast cells to take in water, swell, and burst. However, the deletion
of histidine kinases in filamentous fungi is not lethal in a low-osmolarity medium,
possibly due to functional redundancy between histidine kinases.
Catlett et al. [10] further clustered histidine kinases into 11 families, some of
which – such as the putative osmotic sensing family (group III) – were found
to be highly conserved between species. In contrast, others appeared to have
few homologs, which suggests that they may have evolved to fulfill a specific
requirement in the lifestyle of a fungus. Histidine kinases that are involved in the
hyperosmotic response have now been cloned and sequenced from a range of fungi,
including B. cinerea [12, 13], N. crassa [14], C. heterostrophus [15], C. lagenarium [7],
Alternaria brassicicola [16], and Pyricularia oryzae [17]. Osmotic-sensing histidine
kinases from filamentous fungi differ from Sln1p in that they contain six N-terminal
tandem amino acid repeats that are predicted to form a coiled-coil structure that is
essential for functioning of the histidine kinase. It has been shown that histidine
kinase null mutants, as well as a range of different mutants with alterations to
the tandem amino acid repeat sequences, can confer high levels of resistance to
dicarboximides and phenylpyrroles [14–17].
In Botrytis, dicarboximide- and phenylpyrrole-resistant laboratory strains [12, 18],
as well as less-sensitive field isolates [13, 19], are usually found to be mutated
in Daf1, the histidine kinase homologous to Os-1. However, field isolates that
were less sensitive to dicarboximide fungicides were reported to retain wild-type
levels of sensitivity to phenylpyrroles [18–20]. When Vignutelli et al. [21] crossed a
field-resistant strain of Botrytis with a sensitive strain, the dicarboximide resistance
was found to be segregated separately from the phenylpyrrole resistance, which
suggested that different genes were regulating the field resistance for these two
fungicides. In addition, when Vignutelli et al. crossed a field-resistant fludioxonil
strain with one generated in the laboratory, they again found the two resistance
genes to be segregated independently, which indicated that fludioxonil resistance
is influenced by at least two different genes. Furthermore, mutations in three

separate genes were reported to result in fludioxonil resistance in strains of Ustilago
maydis generated in the laboratory [22]. Taken together, these data suggest that
a fully functional osmotic-sensing histidine kinase is required for the expression
of phenylpyrrole and dicarboximide fungicidal activity, but that several different
genes can regulate fungal sensitivity to dicarboximides and phenylpyrroles, and
these genes can differ between the two different classes of chemistry.
Recently, high levels of field resistance to both phenylpyrroles and dicarboximides
have been found in A. brassicicola [23]. These Alternaria strains are particularly sig-
nificant because field-resistant fungal strains had not previously shown high levels
of cross-resistance between phenylpyrroles and dicarboximides. One particular
mutant was found that was proposed to lack the first two N-terminal amino acid
repeats, but was thought to retain functional histidine kinase activity [16]. This
mutant was found to be highly resistant to phenylpyrroles and dicarboximides, sug-
gesting that the N-terminal tandem repeat structure itself is required for expression
of fungicide activity.
The orthologous histidine kinase gene (HIK1) from the rice blast disease,
Pyricularia oryzae (telomorph: Magnaporthe grisea), has been isolated by Motoyama
and coworkers [17]. Like N. crassa os-1 and A. brassicicola nik1, gene disruption
of HIK1 in P. oryzae was found not to be a lethal event. Both, appressorium
formation and pathogenicity were unaffected, and the fungus was reported to
be highly resistant to phenylpyrroles and dicarboximides. Interestingly, this null
mutant showed an increased sensitivity to high concentrations of sugars (such as
1 M sorbitol) but, unlike N. crassa os-1 [14], tolerance to high levels of salt was
unaffected; this suggested the existence of differences in perception of osmotic
stress between fungal species. The same group went on to express P. oryzae HIK1
in S. cerevisiae [24], and found that expression of this gene conferred sensitivity to
phenylpyrroles and dicarboximides; this led them to suggest that Hik1p itself could
be the molecular target for these chemistries. The sensitivity of the transformed
yeast toward phenylpyrroles and dicarboximides was also found to depend on the
presence of other genes in the hyperosmotic response pathway; for example, Hik1p
was shown to interact directly with Ypd1p, suggesting that Hik1p transmits the
fungicide signal to Hog1p via Ypd1p.
Clearly, further investigations are required to determine the molecular tar-
get(s) of phenylpyrroles and dicarboximides, and to understand the role that
hyperosmolarity-sensing histidine kinases play in the mode of action of these
fungicides. Still unresolved is the role of the protein kinase PK-III that was isolated
from N. crassa and found to be inhibited by phenylpyrroles, but not by dicarbox-
imides [5]. Another recently reported – and rather surprising – finding was that an
inhibitor of the N. crassa Os-2 MAP kinase was able to antagonize the fungicidal
effect of fenpiclonil, but not that of vinclozolin [25], which again suggested that
these two fungicides had different modes of action.
17.1.2
Quinoxyfen
Quinoxyfen is a protectant fungicide used to control the powdery mildew diseases

of wheat, barley, and grapes, and is a potent inhibitor of appressorium formation
in these fungi (Chapter 17.2) [26–28]. In barley powdery mildew (Blumeria graminis
f.sp. hordei, syn. Erysiphe graminis), the appressorial germ tubes are normally
approximately 20 μm long and have a swollen tip and a hooked apical lobe.
Treatment with quinoxyfen results in the formation of longer germ tubes that
remain unswollen, similar to normal hypha; moreover, no penetration of the host
leaf occurs [28, 29].
Detailed biochemical investigations of the mode of action of quinoxyfen have been
hampered because B. graminis is an obligate plant pathogen, and cannot be cultured
away from its plant host. However, quinoxyfen-resistant strains of barley powdery
mildew have been generated in the laboratory by chemical mutagenesis, and also
have been isolated from quinoxyfen-treated plots [30]. These strains were found to
show no cross-resistance to SBI sterol biosynthesis inhibitors or to ethirimol; in
addition, they require quinoxyfen to be present in the growth medium, and display
a range of unusual phenotypes, including defects in sporulation [30]. As a result
of concerns over the potential for development of resistance to fungicides, the
sensitivity of wheat powdery mildew in Europe to quinoxyfen has been monitored
from 1995 to 2000 [31]. No significant changes were found, which suggests that the
risk of a rapid shift in sensitivity to quinoxyfen is unlikely. When the conidia from
a resistant strain of barley powdery mildew from the quinoxyfen-treated plots were
artificially maintained in the laboratory, they were found to germinate normally
on barley leaves compared to the wild-type strain, and to form appressoria even in
the presence of high concentrations of quinoxyfen. Interestingly, the germination
rates on an artificial membrane were higher in the resistant mutant than in the
wild-type control; this suggests that the perception of the host surface had been
affected in the mutant strain [27, 29].
Wheeler and coworkers went on to use differential display reverse-transcription
polymerase chain reaction (RT-PCR) to identify a gene that is expressed in wild-type
conidia on treatment with quinoxyfen, but which was absent in the resistant
mutant under the same conditions. This gene was putatively identified as a
Ras-type GTPase-activating protein (GAP) [27, 28]. Ras G-proteins have been
reported to play a role in spore germination [32, 33], and the disruption of GAP
expression in quinoxyfen-resistant mutants may therefore cause the G-protein to
be inappropriately activated; this would lead to a disruption of the host recognition
signals and result in unregulated appressorium formation.
More recently, Lee et al. used RT-PCR to demonstrate that the cutinase transcript
levels in a quinoxyfen-resistant field isolate were eightfold higher than in the
wild-type [29]. In subsequent studies, the same group showed that quinoxyfen
would inhibit the serine esterases from wild-type barley powdery mildew, but
not from quinoxyfen-resistant barley powdery mildew, or from barley itself. It was
speculated that quinoxyfen could affect the serine esterase activity, and that resistant
isolates had altered levels of serine esterase expression. Interestingly, a similarity

was also noted in germling morphology between treatment with quinoxyfen and
the protein kinase C (PKC) inhibitor Ro 318220; this suggested that PKC-mediated
signaling might also play a role in the mode of action of quinoxyfen.
It is clear that further studies must be conducted before the mechanism of
action of quinoxyfen can be fully elucidated. Nonetheless, it will be fascinating to
understand the role of these complex signaling pathways in host perception, fungal
infection, and the expression of fungicidal activity.
References
1. Alonso-Monge, R., Navarro-Garcia, F., Ianakiev, P., Bell-Pedersen, D.,

Urena, T., Molina, D., Nombela, C., and Nelson, M.A., Werner-Washburne, M.,
Pla, J. (2003) Rec. Res. Dev. Microbiol., 7, Selitrennikoff, C.P., Kinsey, J.A.,
531–545. Braun, E.L., Zelter, A., Schulte, U.,
2. Westfall, P.J., Ballon, D.R., and Kothe, G.O., Jedd, G., Mewes, W.,
Thorner, J. (2004) Science, 306, Staben, C., Marcotte, E.,
1511–1512. Greenberg, D., Roy, A., Foley, K.,
3. Saito, H. and Tatebayashi, K. (2004) J. Naylor, J., Stange-Thomann, N.,
Biochem., 136, 267–272. Barrett, R., Gnerre, S., Kamal, M.,
4. Fujimura, M., Ochiai, N., Ichiishi, A., Kamvysselis, M., Mauceli, E., Bielke, C.,
Usami, R., Horikoshi, K., and Rudd, S., Frishman, D., Krystofova, S.,
Yamaguchi, I. (2000) J. Pestic. Sci., Rasmussen, C., Metzenberg, R.L.,
25, 31–36. Perkins, D.D., Kroken, S., Cogoni, C.,
5. Pillonel, C. and Meyer, T. (1997) Pestic. Macino, G., Catcheside, D., Li,
Sci., 49, 229–236. W., Pratt, R.J., Osmani, S.A.,
6. Zhang, Y., Lamm, R., Pillonel, C., DeSouza, C.P., Glass, L., Orbach, M.J.,
Lam, S., and Xu, J.-R. (2002) Appl. Berglund, J.A., Voelker, R., Yarden, O.,
Environ. Microbiol., 68, 532–538. Plamann, M., Seiler, S., Dunlap, J.,
7. Kojima, K., Takano, Y., Yoshimi, A., Radford, A., Aramayo, R., Natvig, D.O.,
Tanaka, C., Kikuchi, T., and Okuno, T. Alex, L.A., Mannhaupt, G., Ebbole, D.J.,
(2004) Mol. Microbiol., 53, 1785–1796. Freitag, M., Paulsen, I., Sachs, M.S.,
8. Fujimura, M., Ochiai, N., Ichiishi, A., Lander, E.S., Nusbaum, C., and
Usami, R., Horikoshi, K.I., and Birren, B. (2003) Nature, 422, 859–868.
Yamaguchi, I. (2000) Pestic. Biochem. 12. Cui, W., Beever, R.E., Parkes, S.L.,
Physiol., 67, 125–133. Weeds, P.L., and Templeton, M.D.
9. Fujimura, M., Ochiai, N., Oshima, M., (2002) Fung. Genet. Biol., 36, 187–198.
Motoyama, T., Ichiishi, A., Usami, R., 13. Cui, W., Beever, R.E., Parkes, S.L., and
Horikoshi, K., and Yamaguchi, I. Templeton, M.D. (2004) Phytopathology,
(2003) Biosci. Biotechnol. Biochem., 67, 94, 1129–1135.
186–191. 14. Ochiai, N., Fujimura, M., Motoyama, T.,
10. Catlett, N.L., Yoder, O.C., and Ichiishi, A., Usami, R., Horikoshi, K.,
Turgeon, B.G. (2003) Eukaryot. Cell, and Yamaguchi, I. (2001) Pest Manage.
2, 1151–1161. Sci., 57, 437–442.
11. Galagan, J.E., Calvo, S.E., 15. Yoshimi, A., Tsuda, M., and
Borkovich, K.A., Selker, E.U., Tanaka, C. (2004) Mol. Genet. Genomics,
Read, N.D., Jaffe, D., 271, 228–236.
FitzHugh, W., Ma, L.J., Smirnov, S., 16. Avenot, H., Simoneau, P.,
Purcell, S., Rehman, B., Elkins, T., Iacomi-Vasilescu, B., and
Engels, R., Wang, S., Nielsen, C.B., Bataille-Simoneau, N. (2005) Curr.
Butler, J., Endrizzi, M., Qui, D., Genet., 47, 234–243.
17.2 Chemistry and Biology of Fludioxonil, Fenpiclonil, and Quinoxyfen 721
17. Motoyama, T., Kadokura, K., 26. Longhurst, C., Dixon, K., Mayr, A.,
Ohira, T., Ichiishi, A., Fujimura, M., Bernhard, U., Prince, K., Sellars, J.,
Yamaguchi, I., and Kudo, T. (2005) Prove, P., Richard, C., Arnold, W.,
Fung. Genet. Biol., 42, 200–212. Dreikorn, B., and Carson, C. (1996)
18. Faretra, F. and Pollastro, S. (1993) Brighton Crop Prot. Conf. – Pests Dis., 1,
Mycol. Res., 97, 620–624. 27–32.
19. Leroux, P., Fritz, R., Debieu, D., 27. Wheeler, I., Hollomon, D.W.,
Albertini, C., Lanen, C., Bach, J.,
Longhurst, C., and Green, E. (2000)
Gredt, M., and Chapeland, F. (2002)
Brighton Crop Prot. Conf. – Pests Dis., 3,
Pest Manage. Sci., 58, 876–888.
841–846.
20. Sierotzki, H. and Gisi, U. (2003) Chem-
istry of Crop Protection: Progress and 28. Wheeler, I.E., Hollomon, D.W.,
Prospects in Science and Regulation, Gustafson, G., Mitchell, J.C.,
Wiley-VCH Verlag GmbH, Weinheim, Longhurst, C., Zhang, Z., and Gurr, S.J.
pp. 71–88. (2003) Mol. Plant Pathol., 4, 177–186.
21. Vignutelli, A., Hilber-Bodmer, M., and 29. Lee, S., Gustafson, G., Skamnioti, P.,
Hilber, U.W. (2002) Mycol. Res., 106, Baloch, R., and Gurr, S. (2008) Pest
329–335. Manage. Sci., 64, 544–555.
22. Ziogas, B.N., Markoglou, A.N., and 30. Holloman, D.W., Wheeler, I.E.,
Spyropoulou, V. (2005) Eur. J. Plant Dixon, K., Longhurst, C., and
Pathol., 113, 83–100. Skylakakis, G. (1997) Pestic. Sci., 51,
23. Iacomi-Vasilescu, B., Avenot, H., 347–351.
Bataille-Simoneau, N., Laurent, E., 31. Bernhard, U., Leader, A., Longhurst, C.,
Guenard, M., and Simoneau, P. (2004)
and Felsenstein, F.G. (2002) Pest Man-
Crop Prot., 23, 481–488.
age. Sci., 58, 972–974.
24. Motoyama, T., Ohira, T., Kadokura, K.,
32. Osherov, N. and May, G.S. (2001) FEMS
Ichiishi, A., Fujimura, M.,
Yamaguchi, I., and Kudo, T. (2005) Microbiol. Lett., 199, 153–160.
Curr. Genet., 47, 298–306. 33. Fillinger, S., Chaveroche, M.-K.,
25. Pillonel, C. (2005) Pest Manage. Sci., 61, Shimizu, K., Keller, N., and d’Enfert, C.
1069–1076. (2002) Mol. Microbiol., 44, 1001–1016.
17.2
Chemistry and Biology of Fludioxonil, Fenpiclonil, and Quinoxyfen
17.2.1
Phenylpyrroles: Fenpiclonil and Fludioxonil
17.2.1.1 Chemistry
Fenpiclonil (1) [1] and fludioxonil (2) [2] belong to the class of fungicides known as
phenylpyrroles (Figure 17.2.1); both are nonsystemic fungicides and were developed
by Ciba-Geigy. The discovery of this novel class of agrochemical fungicides is based
on the synthetic optimization of the natural product pyrrolnitrin (3).
17.2.1.1.1 Chemistry Pyrrolnitrin (3) was first isolated in 1964 by Arima, from
the bacterial cells of Pseudomonas pyrocinia [3]. This simple, secondary metabolite
was shown to strongly inhibit the growth of fungi [4], and was developed as
an antimycotic for topical application in human medicine. Owing to its rapid
N N
Cl
4
Cl 3 O Cl
Cl 2 O NO2
N1 F N O N
F
H H H
Fenpiclonil 1 Fludioxonil 2 Pyrrolnitrin 3
Figure 17.2.1 Structures of the phenylpyrroles.
metabolism after oral administration, however, pyrrolnitrin showed only minimal

antimycotic activity [5].
The first use of a pyrrolnitrin analog in plant protection was described in 1975,
in a Japanese patent application [6]. In greenhouse tests, pyrrolnitrin showed an
interesting activity against a range of phytopathogenic fungi but, owing to its
insufficient photostability, this natural compound could not be used under field
conditions. The exposure of several 3-chloro-4-phenylpyrroles to sunlight revealed
that photooxidation of the pyrrole ring led to the production of inactive metabolites
[7]. Consequently, optimization of the lead structure pyrrolnitrin for crop protection
use was focused on the synthesis of analogs with increased photostability.
Attempts were made by several research groups to stabilize 3-chloro-4-phenyl-1H-
pyrroles by varying the substituents on the phenyl ring [8], by acylation of the
pyrrole-nitrogen [9], or by inclusion of 3-chloropyrroles into cyclodextrin [10],
but no commercial crop-protection compound resulted from such efforts. But, it
was shown that the use of biocontrol bacteria, which produce pyrrolnitrin as a
metabolite, might afford protection to plants against infection by soil-borne fungal
pathogens [11].
In 1972, van Leusen described a simple synthesis of 4-phenylpyrroles bearing a
cyano-, a keto-, or an ester-group in the 3-position of the pyrrole ring, using TosMIC
(5) as a key reagent (Scheme 17.2.1) [12]. This approach was quickly copied by sev-
eral groups to prepare new analogs of pyrrolnitrin. Details of the structure–activity
relationship (SAR) resulting from such an optimization have been reported [13],
E
(i)
+ −
+ Tos-CH2-N C Rn
Rn E N
H
4 5 6
(i) NaH in Et2O/DMSO at reflux or E: CN, C(O)R, CO2R

KO-tBu in THF or
K2CO3 in MeOH at reflux
Scheme 17.2.1 Van Leusen pyrrole synthesis.

4 Figure 17.2.2 Structure–activity relationship of the

E phenylpyrroles. E = CN; X = Cl, Br, CF3 , or -O-CF2 -O-;
3 position of X = 2-, 3-, or 2,3-position; n = 1 or 2; R = Hor
Xn 2 a group that can hydrolyze back to the parent 1H-pyrrole.
N
R
7
with the best biological activity being observed for the substituents E, X, and R in
structure 7, as shown in Figure 17.2.2.
The discovery of the 3-cyanopyrroles led to two major problems being solved.
First, these molecules proved to be between 50- and 100-fold more stable towards
exposure to sunlight than their 3-chloro analogs [13], and were readily accessible.
Second, their fungicidal activity was comparable to that of pyrrolnitrin in the
greenhouse, but much more effective in the field.
The first phenylpyrrole fungicide to be developed by Ciba-Geigy for seed treat-
ment, fenpiclonil (1) [14], was followed two years later by fludioxonil (2), which
could be used as either a foliar fungicide [15] or for seed treatment [16].
The production processes described by Ciba-Geigy for fenpiclonil [17] and
fludioxonil [18] both employ TosMIC (5) as a key reagent. Crystalline TosMIC
is thermolabile, has the potential for deflagration and was, therefore, unavailable
in bulk quantities. Subsequently, a production process for TosMIC, and its safe
handling as a solution in organic solvents, was reported [17].
Fenpiclonil (1) can be prepared (Scheme 17.2.2) via a Knoevenagel condensation
of 2,3-dichlorobenzaldehyde (8) with a cyanoacetic acid derivative 9, to provide
an α-cyanocinnamate intermediate 10 that is then reacted with TosMIC in the
presence of a base to produce the desired 1H-pyrrole 1 in high yield.
The research team at Nippon Soda had shown previously [19] that α-cyano-
cinnamates of structure type 10 render much higher yields of 3-cyanopyrroles than
the corresponding phenyl acrylates, such as 4. These findings can be rationalized
by the major differences in the pKa -values of the corresponding intermediates
involved in pyrrole ring formation [20].
An atom-economic route for the preparation of fludioxonil (2) has been patented
[18], whereby known 2,2-difluorobenzodioxole 12 is regioselectively lithiated to
form 13 (Scheme 17.2.3). Then, in a one-pot reaction, intermediate 13 is directly
quenched with 14, followed by conversion of the formed intermediate 15 with
TosMIC into the desired fludioxonil (2). Alternatively, intermediate 13 can be
quenched with dimethylformamide (DMF) to form aldehyde 16 which is then, in
similar fashion to the above-described process, stepwise-reacted with a cyanoacetic
acid derivative to obtain 15; this is followed by ring formation using TosMIC to
deliver fludioxonil (2). The chemical and physical properties of both fenpiclonil (1)
and fludioxonil (2) are listed in Table 17.2.1.
17.2.1.1.2 Safety Profile The toxicological profiles of fenpiclonil and fludioxonil

demonstrate that both compounds can be considered as safe, without restrictions
724
Synthesis of fenpiclonil
N N N
+ −
ToS-CH2-N C
17 Fungicides Acting on Signal Transduction
O + O base Cl
Cl base
O Cl Cl
Cl H H2N Cl H NH2 N
H
8 9 10 1
Scheme 17.2.2 Synthesis of fenpiclonil (1).

Synthesis of fludioxonil
14
EtO N N
i POCI3 (a) COOEt

O Li O
O ii KF or O
O TosMIC / 0 −25 °C O
O HF O F N
F F F H
F F
2
11 12 13
DMF TosMIC
(a) BuLi / TMEDA / −20 °C;
N
CN
O COOEt
O COOR
O
base O H
O H F
F F
F
16 15
Scheme 17.2.3 Synthesis of fludioxonil (2).

17.2 Chemistry and Biology of Fludioxonil, Fenpiclonil, and Quinoxyfen
725
Table 17.2.1 Chemical and physical properties of fenpiclonil and fludioxonil.
N N
Cl O
Cl O
N F N
F
H H
Common name Fenpiclonil Fludioxonil

Melting point (◦ C) 144.9–151.1 199.8
Vapor pressure at 25 ◦ C (Pa) 1.1 × 10−5 3.9 × 10−7
Solubility in water at 25 ◦ C 4.8 1.8
(mg l−1 )
Partition coefficient: Log P = 3.86 (n-octanol water) Log P = 4.12 (n-octanol water)
Light stability T1/2 (h) 48 24.9
Hydrolysis Not hydrolyzed up to 6 h at Not hydrolyzed at 70 ◦ C between
100 ◦ C between pH 3 and 9 pH 5 and 9
From The Pesticide Manual, 11th edn. British Crop Protection Council, Farnham.
for humans, animals, and the environment [21, 22]. Because of its favorable safety
profile, fludioxonil has been classified by the US Environmental Protection Agency
(EPA) as a reduced-risk compound [23].
17.2.1.2 Biology
Fludioxonil, which was introduced in 1990 as a foliar fungicide and for seed
treatment [15, 16], provides broad-spectrum activity across all fungal classes (except
for Oomycetes), especially against species of the genera Aspergillus, Fusarium,
Monilinia, Penicillium, Monographella, and Botrytis. An overview of the in vitro
activity spectrum of fludioxonil is provided in Table 17.2.2.
17.2.1.2.1 Foliar and Post-Harvest Use of Phenylpyrroles Fludioxonil is highly

effective against B. cinerea on grapes [24], fruits [25], vegetables, and ornamentals
[26, 27]. In addition to the very efficient control of Botrytis, fludioxonil also controls
molds such as Penicillium, Aspergillus, and Trichothecium on grapes [28]. Further-
more, it provides protection against Monilinia spp. in stonefruit and Sclerotinia spp.
[29], Rhizoctonia and Alternaria in vegetables, turf, and ornamentals. Its high efficacy
against different molds makes it well suited for use as both pre- and post-harvest
treatments in citrus [30–32], grapes [33], stonefruits [34–36], and top fruits [37–42],
as it is compatible with the different application technologies used for post-harvest
treatments [43, 44].
It has been shown that not only grape products such as wine, juice, and raisins,
but also other crops, may become contaminated with ochratoxin A (OTA), a toxin
produced by Aspergillus and Penicillium species. The fungal species with the highest
Table 17.2.2 In vitro activity spectrum of fludioxonil (2).
Fungal species Growth inhibition (EC50 (mg a.i. l−1 ))
Alternaria solani 0.15

Aphanomyces laevis 10.3
Aspergillus carbonarius 0.18
A. niger 0.02
Botrytis cinerea 0.02
Cercospora arachidicola 0.2
Cladosporium cucumerinum >100
Cochliobolus sativus 0.08
Fusarium culmorum 0.18
F. graminearum 0.02
F. oxysporum 0.08
F. proliferatum 3.3
F. semitectum 0.01
F. sulphureum 0.09
Magnaporthe grisae >100
Monilinia fructicola 0.07
M. fructigena <0.01
M. laxa <0.01
Monographella nivale 0.15
Penicillium digitatum 0.01
Phytopthora infestans >100
Pyrenophora teres 0.05
Pythium ultimum >100
Rhizoctonia cerealis 0.01
R. solani AG1 (rice) 0.02
R. solani AG2 (sugar beet) 0.03
R. solani AG3 (potato) 0.22
R. solani AG4 (cotton) 0.4
Sclerotinia sclerotiorum 0.002
Sclerotium rolfsii 0.22
Venturia inaequalis 6.05
Internal data Syngenta Crop Protection AG.
OTA production potential is A. carbonarius, a secondary fungus on grapes [45, 46].

In both laboratory studies [47] and field trials [48], fludioxonil was shown to control
the production of OTA by restricting growth of the toxin-producing fungi.
The results of biokinetic studies on grape berries have shown fludioxonil to be
nonsystemic (Table 17.2.3), with the major proportion of the applied fludioxonil
being recovered from the plant surface. Although a small proportion (ca. 10%)
of the applied fludioxonil was detected in the wax layer up to 14 days after
application, there was no uptake of the active ingredient into the skin or pulp
[49]. The lack of any systemic properties of fluodioxinil may also be indicated
Table 17.2.3 Biokinetic data for treatment of grape berries with fludioxonil.
Grape berry fraction Time after application

2h 1 day 7 days 14 days
% recovered active ingredient
Surface 96 96 38 48
Wax layer 4 10 3 4
Skin 0 0 0 0
Pulp 0 0 0 0
by a lower efficacy of the fungicide in citrus fruits inoculated by puncturing

[50].
The inherent risk for the development of resistance toward phenylpyrroles is
considered as medium [51]. To date for B. cinerea, no cross-resistance has been
reported between phenylpyrroles and products of other chemical classes, includ-
ing benzimidazoles, dicarboximides, N-phenylcarbamates, and anilinopyrimidines
[52]. Multiple resistance of Alternaria brassicola isolates [53] towards phenylpyrroles
and dicarboximides was observed, though the underlying mechanism(s) require
further clarification. A reduced sensitivity of B. cinerea [54] and P. expansum lab-
oratory mutants [55] and field isolates of B. cinerea [56, 57] and P. digitatum [58]
to fludioxonil has been observed in a very few cases; however, no losses in the
performance of the solo or mixture product were observed under practical com-
mercial conditions. The resistance of laboratory mutants of P. expansum towards
fludioxonil can be associated with lower fitness costs [55]. Reported indications for
a multidrug resistance mechanism in B. cinerea field isolates from grapes cannot
be correlated with reduced performance of fludioxonil [59, 60] in planta.
In order to broaden the spectrum of activity and for resistance management,
fludioxonil is mainly applied in mixtures with the anilinopyrimidine, cyprodinil
[61–63].
17.2.1.2.2 Seed Treatment Use of Phenylpyrroles The first phenylpyrrole fungi-

cide to be introduced by Ciba-Geigy in 1988 as a seed treatment in cereals,
fenpiclonil [14], was quickly followed by the second phenylpyrrole fungicide,
fludioxonil. The latter compound was shown to provide improved biological
properties compared to fenpiclonil, and consequently replaced fenpiclonil for seed
treatment [14–16, 64].
In general, the treatment of seeds with phenylpyrroles shows a limited uptake into
both the seed and seedling, with the major proportion of seed-applied fludioxonil
remaining either on the surface of the seed (9%), or in its immediate vicinity
(87%). As a consequence, a protective layer of fludioxonil becomes built up around
the seed, shielding both it and the seedlings against soil-borne infections, while the
active ingredient adhering directly to the seed will combat diseases located on the
seed surface or just below the seed coat. Approximately 4% of the applied agent has
been shown to penetrate the seed during germination, to provide control against
deep-seated fungal infections. About one-fourth of this (i.e., 1% of the total applied)
reaches the coleoptile, where it controls Fusarium spp., for example, Monographella
nivalis or F. culmorum [65].
One of the strengths of fludioxonil is its broad coverage of many different species
from the Fusarium group, including F. graminearum, F. oxysporum, and F. solani
[66–68]. Besides causing direct effects, including reductions in emergence and
seedling growth, many Fusarium species are producers of potent mycotoxins that
can lead to severe health damage when consumed by livestock or humans [69, 70].
The results of recent studies have indicated that F. culmorum and F. graminearum are
able to infect wheat plants systemically from soil, crop residues, or seeds; however,
treatment of the seeds with fludioxonil can lead to a reduced production of the
mycotoxin desoxnivalenol in the wheat heads, by controlling systemic Fusarium
infections [71].
Fludioxonil is highly active against the causal agent of snow mold in wheat
and rye, Monographella nivale, providing excellent long-term control both in
growth chamber and field trials [72]. Owing to its different mode of action,
no cross-resistance to benzimidazole fungicides occurs [52], and therefore
benzimidazole-insensitive strains of M. nivale are also controlled. The recent
occurrence of quinone outside inhibitor (QoI)-insensitive strains of M. nivale [73]
further underlines the importance of fludioxonil as a seed treatment against snow
mold.
On corn, fludioxonil provides protection against fungi of the genera Fusarium,
Rhizoctonia, and Helminthosporium, and also weakly pathogenic fungi of the genera
Penicillium and Aspergillus [67]. In potato, fludioxonil controls silver scurf caused
by Helminthosporium solani [74], black leg caused by Rhizoctonia solani [75], and
gangrene caused by Phoma exigua and Fusarium dry rots [76, 77]. The first cases
of fludioxonil resistance in F. sambucinum and F. coeruleum have been reported,
however [78].
In oilseed rape, the seedling disease complex caused by Fusarium spp., Rhi-
zoctonia spp., and Alternaria spp. is controlled by fludioxonil [79], whilst in peas
fludioxonil can be used to control the foot rot disease caused by Mycosphaerella
pinodes [80].
For seed-treatment purposes, fludioxonil is generally utilized as an FS (Flowable
for Seed Treatment) formulation. Fludioxonil is very safe for seeds and seedlings,
and has shown no negative effects on either emergence or on the growth and
development of cereals, maize, cotton, and other plant species. Owing to its broad
activity and good crop tolerance, fludioxonil is used worldwide as a seed treatment
in many different crops, against a range of important diseases (Table 17.2.4).
Several combinations of fludioxonil with other fungicides are available to com-
plete the spectrum of activity.
Table 17.2.4 Uses of fludioxonil as a seed treatment.
Crop Diseases Rate (g a.i. per 100 kg seed)
Cereals (wheat, barley, rye) Claviceps purpurea 5

Cochliobolus sativus 5
Fusarium culmorum 5
Monographella nivale 5
Phaeospheria nodorum 5
Pyrenophora graminea 5
Tilletia caries 5
Maize Fusarium graminearum 2.5
Cotton Fusarium spp. 2.5
Rhizoctonia solani 2.5
Peanut Rhizoctonia solani 2.5
Peas Mycosphaerella pinoides 10
Potato Fusarium spp. 2.5
Helminthosporium solani 2.5
Phoma exigua 2.5
Rice Gibberella fujikuroi 5
Soybean Fusarium spp. 2.5
Sclerotinia sclerotiorum 2.5
Internal data Syngenta Crop Protection AG.
17.2.1.2.3 Biological Mode of Action The primary effect of fludioxonil is a highly

efficient inhibition of conidia germination in vitro [81], and this has been confirmed
in experiments with B. cinerea on grape leaves (Table 17.2.5) [82].
The pronounced effect on germination of Botrytis conidia on the leaf surface
explains the excellent efficacy of fludioxonil after preventive application.
Table 17.2.5 Inhibitory effect of fludioxonil on conidia germination for B. cinerea.
Rate (mg a.i. l−1 ) Time after inoculation
24 h 72 h 120 h
% inhibition of conidia germination % control of leaf attack
0.1 9 2 0
1 95 59 95
10 100 100 100
Germination on untreated plants 99%; percentage leaf attack on untreated plants 100%.
17.2.2
Quinoxyfen
17.2.2.1 Chemistry
Quinoxyfen (17; Figure 17.2.3) [83], which was developed by Dow-Elanco, belongs to
a new chemical class of fungicides, the phenoxyquinolines, that possesses specific
activity against powdery mildews.
17.2.2.1.1 Chemistry The excellent control of wheat powdery mildew of

LY-186054 (18; Figure 17.2.3) was discovered by Eli Lilly [84] during routine
screening. After having proved that this 4-phenoxyquinoline showed no
cross-resistance with known commercial mildewicides, an optimization program
was commenced for this new lead. Systematic modifications of ring A through C,
as well as of the linker in 19, led to the SAR shown in Figure 17.2.4.
F F
Cl O O
LY 211795 LY-186054
Cl N Quinoxyfen Cl N
17 18
Figure 17.2.3 Quinoxyfen (LY 211795, 17) and LY-1 186054 (18).
4′
A
Linker 3′
O
5 4 2′
7 C B
Cl N
19
Figure 17.2.4 Structure–activity relationship 2 -NO2 ; 2 -Me-4 -F; linker=O; OCH2 ;

of 7-chloro-4-phenoxyquinoline. Best activity O(CH2 )2 ; CH2 ; OCH(CH3 ). Variation of B:
was seen for: A = 4 -F; 2 ,4 -F2 ; 2 -CF3 ;
R2 R2 R2 R2
O O O O
N
R1 B R1 B R1 B > R1 B
+ N
N N N N
O
C = 5,7-Cl2; 7-Cl; 5,7-(Me)2; 6-F-7-Cl; 5,7-Cl2-6-F; 7-Br; 5-NO2.

Synthesis of quinoxyfen
Cl
Cl O
O O O
+
O Cl N O
Cl NH2 H
O
O O
20 21 22
F
OH Cl X
Cl O
+
Cl N
F
Cl N
23 X = OH
17 25
24 X = Cl
Scheme 17.2.4 Synthesis of quinoxyfen (17).

Interestingly, the 8-chloro analogs of LY-186054 (18) showed a diminished
powdery mildew control, but were active against botrytis and other commercially
interesting species of fungi. This shift in the biological profile is based on their dif-
ferent MOA; 8-haloquinolines are known inhibitors of the enzyme, dihydroorotate
dehydrogenase [85].
Field assessments of several analogs as powdery mildewicides on a variety of
host crops led to the selection of LY-211795, quinoxyfen (17; Figure 17.2.3) as a
development candidate.
The preparation of quinoxyfen [83] involves the synthesis of 4,5,7-
trichloroquinoline (24), and follows known procedures. The addition of aniline 20
to the Michael acceptor 21 is followed by cyclization of 22 to a 4-hydroxyquinoline
23 (Scheme 17.2.4). The subsequent treatment of 23 with phosphoryl chloride
gives 24 which, after heating to about 140 ◦ C together with 4-fluorophenol (25),
provides the desired ether quinoxyfen (17). An improvement in the last step using
4-dialkylaminopyridine as a catalyst has been described [86]. The chemical and
physical properties of quinoxyfen are listed in Table 17.2.6.
17.2.2.1.2 Safety Profile Quinoxyfen can be considered, from a toxicological
standpoint, as a relatively safe compound [87, 88]. With the exception of some
aquatic species, it has a very desirable toxicity profile towards nontarget organisms
encountered in the environment.
17.2.2.2 Biology
Quinoxyfen was introduced in 1996 [88] by Dow-Elanco, and has been in com-
mercial use since 1997 for the control of powdery mildews. It is specifically active
References 733
Table 17.2.6 Chemical and physical properties of quinoxyfen (17).
Cl O
Cl N
Common name Quinoxyfen

Melting point (◦ C) 106–107.5
Vapor pressure at 25 ◦ C (Pa) 1.2 × 10−5
Solubility in water at 20 ◦ C, pH 6.45 (mg l−1 ) 0.116
Partition coefficient Kow log P = 4.66 (pH 6.6, 20 ◦ C)
Hydrolysis In dark at 25 ◦ C, stable to hydrolysis at pH
7 and 9
Light stability Degraded more rapidly in light
From The Pesticide Manual, 13th edn, British Crop Protection Council, Farnham.
against powdery mildews on cereals and horticultural crops [89–92]. Quinoxyfen is

active after protective application but, it does not demonstrate any curative activity.
It does have a long-lasting activity that can persist on cereals for up to 42 days.
Quinoxyfen interferes with conidia germination and appressorium formation
in the life cycle of target fungi [93], but has no effect on either haustorium
formation or any further development of the disease; this explains the compound’s
strong preventive activity and lack of activity in curative situations. Quinoxyfen is
redistributed in and on the treated plant through the xylem, and also by superficial
vapor movement [94].
A high frequency of wheat powdery mildew isolates resistant toward quinoxyfen
has been reported from Northern Germany [95]. Although field isolates of Uncinula
necator with a reduced sensitivity to quinoxyfen were first reported in 2003 [96, 97],
the efficacy of quinoxyfen under practical field conditions has remained unchanged.
Indications for cross-resistance with proquinazid require further clarification.
Both compounds are grouped together in the same Fungicide Resistance Action
Committee code list [98].
References
1. Staub, Th., Dahmen, H., Nyfeler, R., 3. Arima, K., Imanaka, H., Kousaka, M.,
and Williams, R.J. (1987) Patent EP Fukuda, A., and Tamura, G. (1964)
236,272. Agric. Biol. Chem., 28, 575–576.
2. Nyfeler, R. and Ehrenfreund, J. (1986) 4. (a) Arima, K., Imanaka, H.,
Patent EP 206,999. Kousaka, M., Fukuda, A., and
Tamura, G. (1965) J. Antibiot., 18, 19. Genda, Y., Muro, H., Nakayama, K.,
201–204; (b) Arima, K., Tamura, G., Miyazaki, Y., and Sugita, Y. (1987) US
Imanaka, H., Mukaizaka, M., and Patent 4,680,413.
Fukuda, A. (1964) JP 39,025,876. 20. van Leusen, D., van Echten, E., and van
5. Murphy, P.J. and Williams, T.L. (1972) Leusen, A.M. (1992) J. Org. Chem., 57,
J. Med. Chem., 15, 137–139. 2245–2249.
6. Nakanishi, K., Shimizu, K., Ono, K., and 21. Tomlin, C.D.S. (ed.) (1997) A World
Ueda, K. (1975) Patent JP 50,002,011, Compendium. The Pesticide Manual, 11th
1975 (Appl. March 19, 1969) (Chem. edn, British Crop Protection Council,
Abstr., (1975), 83, 38796r). Farnham, pp. 522–524.
7. Knüppel, P.C., Lantzsch, R., and 22. Tomlin, C.D.S. (ed.) (1997) A World
Wollweber, D. (1992) in Synthesis Compendium. The Pesticide Manual, 11th
and Chemistry of Agrochemicals III, edn, British Crop Protection Council,
ACS Symposium Series, Vol. 504 Farnham, pp. 566–568.
(eds D.R. Baker, J.G. Fenyes, and J.J. 23. United States Environmental Protec-
Steffens), American Chemical Society, tion Agency (US EPA) (1998) General
pp. 405–413. Overview: Reduced-risk Pesticide Pro-
8. Hashimoto, S., Nakada, A., Ueda, A., gram. Environment Protection Agency
and Ueki, Ch. (1976) Patent JP Office of Pesticide Programs, Staff
51,088,630. Background, pp. 2–4.
9. Okuma, K., Ueda, A., and Nakata, A. 24. Trespeuch, J. (1994) Proceedings of Con-
(1979) Patent JP 54,070,425. ference International Maladies Plantes,
10. Mikata, S.K.K. (1980) Patent JP Bordeaux, France, December 6–8, pp.
55,149,204. 923–930.
11. Ligon, J.M., Hill, D.S., Hammer, P.E., 25. Paulus, A.O., Vilchez, M., and
Torkewitz, N.R., Hofmann, D., Kempf, Larson, K. (2000) Phytopathology, 90,
H.J., and van Pée, K.H. (2000) Pest 120.
Manage. Sci., 56, 688–695. 26. Lee, M.L., Chen, L.C., and Chen, T.Z.
12. van Leusen, A.M., Siderius, H., (2004) Plant Prot. Bull., 46, 1–13.
Hoogenboom, B.E., and van Leusen, D. 27. Minuto, A., Gullino, M.L., and
(1972) Tetrahedron Lett., 52, 5337–5340. Garibaldi, A. (1996) Med. Fac. Landb.
13. Nyfeler, R. and Ackermann, P. (1992) in Toegen. Biol. Wet. Uni. Gent., 61,
Synthesis and Chemistry of Agrochemicals 453–459.
III, ACS Symposium Series, Vol. 504 28. Albert, J.P., Drouillard, J.B., Gueunier,
(eds D.R. Baker, J.G. Fenyes, and J.J. M.M., and Bac, V. (2004) Phytoma, 577,
Steffens), American Chemical Society, 6–8.
pp. 395–404. 29. Matheron, M.E. and Poches, M. (2004)
14. Nevill, D., Nyfeler, R., and Sozzi, Plant Dis., 88, 665–668.
D. (1988) Proc. Brighton Crop Prot. 30. Zhang, J.X. and Swingle, P.P. (2003)
Conf. –Pests Dis., 1, 65–72. Phytopathology, 93, 94.
15. Gehmann, K., Nyfeler, R., Leadbeater, 31. Agostini, J.P., Peres, N.A., MacKenzie,
A.J., Nevill, D., and Sozzi, D. (1990) S.J., Adaskaveg, J.E., and Timmer, L.W.
Proc. Brighton Crop Prot. Conf. – Pests (2006) Plant Dis., 90, 1419–1424.
Dis., 2, 399–406. 32. Kanetis, L., Förster, H., and Adaskaveg,
16. Leadbeater, A.J., Nevill, D., Steck, B., J.E. (2008) Plant Dis., 92, 261–269.
and Nordmeyer, D. (1990) Proc. 33. Franck, J., Latorre, B.A., Torres, R.,
Brighton Crop Prot. Conf. – Pests Dis., and Zoffoli, J.P. (2005) Postharvest Biol.
2, 825–830. Technol., 37, 20–30.
17. Pfluger, R.W., Indermühle, J., and 34. Northover, J. and Zhou, T. (2002) Can.
Felix, F. (1990) Patent EP 378,046. J. Plant Pathol., 24, 144–153.
18. Ackermann, P., Känel, H.-R., and 35. Förster, H. and Adaskaveg, J.E. (1999)
Schaub, B. (1990) US Patent 5,194,628. Phytopathology, 89, 26.
References 735
36. Föster, H., Driever, G.F., Thompson, 54. Hilber, U.W., Schwinn, F.J., and
D.C., and Adaskaveg, J.E. (2007) Plant Schuepp, H. (1995) J. Phytopathol.,
Dis., 91, 209–215. 143, 423–428.
37. Adaskaveg, J.E. and Förster, H. (2003) 55. Li, H.X. and Xiao, C.L. (2008) Phy-
Phytopathology, 93, 127. topathology, 98, 427–435.
38. Adaskaveg, J.E., Förster, H., Gubler, 56. Baroffio, C., Siegfried, W., and Hilber,
W.D., Teviotdale, L., and Thompson, U.W. (2003) Plant Dis., 87, 662–666.
D.F. (2005) Calif. Agric., 59, 109–114. 57. Zhao, H., Kim, Y.K., Huang, L., and
39. Errampalli, D. and Crnko, N. (2004) Can Xiao, C.L. (2010) Postharvest Biol. Tech-
J. Plant Pathol., 26, 70–75. nol., 56, 12–18.
40. Rosenberger, D.A., Meyer, F.W., Ahlers, 58. Kanetis, L., Förster, H., and Adaskaveg,
C.A., and van Camp, K.L. (2002) Phy- J.E. (2010) Phytopathology, 100,
topathology, 92, 145–146. 738–746.
41. Errampalli, D., Northover, J., Skog, L., 59. Kretschmer, M. and Hahn, M. (2008)
Brubacher, N.R., and Collucci, C.A. J. Plant Dis. Protect., 115, 214–219.
(2005) Pest Manage. Sci., 61, 591–596. 60. Leroch, M., Kretschmer, M., and
42. Adaskaveg, J.E. and Förster, H. (2004) Hahn, M. (2011) J. Phytopathol., 159,
Phytopathology, 94, S149. 63–65.
43. Zhang, J. (2007) Postharvest Biol. Tech- 61. Raum, J. and Hauck, R. (1996) Mit-
nol., 46, 262–270. teilungen aus der Biologischen Bun-
44. Kanetis, L., Förster, H., and Adaskaveg, desanstalt für Lannd-Forstwirtschaft
Berlin-Dahlem, Munster, September
J.E. (2007) Plant Dis., 91, 1502–1511.
23–26, p. 505.
45. Cabaňes, F.J., Accensi, F., Bragulat,
62. Sylvestre, M., Tanne, M.N., Thomas, G.,
M.R., Abarca, M.L., Castellá, G.,
and Litoux, J. (1997) Proceedings of
Minguez, S., and Pons, A. (2002)
Conference International Maladies
J. Food Microbiol., 79, 213–215.
Plantes, Tours, France, December 3–5,
46. Belli, N., Marin, S., Sanchis, V., and
pp. 909–916.
Ramos, A.J. (2006) Food Addit. Contam.,
63. Cloud, G.L., Minto, B., and Bassi, B.
23, 1021–1029.
(2001) Phytopathology, 91, 18.
47. Valero, A., Marin, S., Ramos, A.J., and
64. Koch, E. and Leadbeater, A. (1992) Proc.
Sanchis, V. (2007) Lett. Appl. Microbiol.,
45, 194–199. 1137–1146.
48. Tjamos, S.E., Antoniou, P.P., 65. Mueller, K., Fischer, W.,
Kazantzidou, A., Antonopoulos, D.F., Steinemann, A., Leadbitter, N., and
Papageorgiou, I., and Tjamos, E.C. Mesterházy, Á. (1997) Proceedings of
(2004) J. Phytopathol., 152, 250–255. the Fifth European Fusarium Seminar,
49. Knauf-Beiter, G., Kueng-Faerber, R., Szeged, Hungary.
Pillonel, C., Speich, J., and Broyard, C. 66. Zang, L., Shetty, K., Watrin, C.,
(2000) Proceedings of Conference In- Forster, B., and Biddle, A.J. (2001)
ternational Maladies Plantes, Tours, BCPC Symposium Proceedings Seed Treat-
France, December 6–8, pp. 923–930. ment: Challenges and Opportunities, vol.
50. D’Aquino, S., Schirra, M., Palma, A., 76, British Crop Protection Council,
and Liguori, R. (2010) Acta Hortic., 858, Farnham, pp. 257–262.
357–362. 67. Munkvold, G.P. and O’Mara, J.K. (2002)
51. Fungicide Resistance Action Committee Plant Dis., 86, 143–150.
(FRAC) (2005) Pathogen Risk List. 68. Glynn, N.C., Edwards, S.G., Hare, M.C.,
52. Forster, B. and Staub, T. (1996) Crop Burke, R., and Parry, D.W. (2000) Proc.
Prot., 15, 529–537. Brighton Crop Prot. Conf. – Pests Dis., 1,
53. Iacomo-Vasilescu, B., Avenot, H., 479–482.
Bataille-Simoneau, N., Laurent, E., 69. Logrieco, A., Mulè, G., Morietti, A., and
Guenard, M., and Simoneau, P. (2004) Bottalico, A. (2002) Eur. J. Plant Pathol.,
Crop Prot., 23, 481–488. 108, 597–609.
70. Pascale, M., Visconti, A., and 83. Arnold, W.R., Coghlan, M.J.,
Chelkowski, J. (2002) Eur. J. Plant Krumkalns, E.V., Jourdan, G.P., and
Pathol., 108, 645–651. Suhr, R.G. (1989) Patent EP 326,330.
71. Klix, M., Oostendorp, M., and Zeun, R. 84. Coghlan, M.J., Krumkalns, E.V., Caley,
(2009) BCPC Symp. Proc. Seed Prod. B.A., Hall, H.R., and Arnold, W.R.
Treat., 83, 59–63. (1991) in Synthesis and Chemistry of Agro-
72. West, S., Doppmann, F., Forster, B., chemicals II, ACS Symposium Series,
Zeun, R., and Biddle, A.J. (2001) BCPC Vol. 443 (eds D.R. Baker, J.G. Fenyes,
Symposium Proceedings Seed Treatment: and J.J. Steffens), American Chemical
Challenges and Opportunities, vol. 76, Society, pp. 539–552.
British Crop Protection Council, Farn- 85. Parker, M.H., Durst, G.L., Hannum,
A.C., Henry, M.J., Lawler, L.K., and
ham, pp. 267–272.
Smith, A.J. (2002) in Synthesis and
73. Walker, A.S., Auclair, C., Gredt, M., and
Chemistry of Agrochemicals VI, ACS
Leroux, P. (2009) Pest Manage. Sci., 65,
Symposium Series, Vol. 800 (eds D.R.
906–915.
Baker, J.G. Fenyes, W.K. Moberg,
74. Tsror, L. and Peretz-Alon, I. (2004) Am.
G.P. Lahm, Th.P. Selby, and Th.M.
J. Potato Res., 81, 291–294. Stevenson), American Chemical Society,
75. Bains, P.S., Bennypaul, H.S., Lynch, pp. 303–313.
D.R., Kawchuk, L.M., and Schaupmeyer, 86. Robery, R.L., Alt, C.A., and DeAminis,
C.A. (2002) Am. J. Potato Res., 81, C.V. (1993) Patent EP 569,021.
99–106. 87. Tomlin., C.D.S. (ed.) (1997) A World
76. Bains, P.S., Bennypaul, H., Kawchuk, Compendium. The Pesticide Manual, 11th
L.M., and Holley, J.D. (2001) Can. J. edn, British Crop Protection Council,
Plant Pathol., 23, 184. Farnham, pp. 1083–1084.
77. Filippov, A.V., Kuznetsova, M.A., 88. Longhurst, C., Dixon, K., Mayr, A.,
Barlyuk, T.I., Rogozhin, A.N., and Bernhard, U., Prince, K., Sellars, J.,
Pyushpeki, V.D. (1996) Proc. Brighton Prove, P., Richard, C., Arnold, W.,
Crop Prot. Conf. – Pests Dis., 1, Dreikorn, B., and Carson, C. (1996)
269–274. Proc. Brighton Crop Prot. Conf. – Pests
78. Peters, R.D., Platt, H.W., Drake, K.A., Dis., 1, 27–32.
Coffin, R.H., Moorehead, S., Clark, 89. Henry, H.J. (2003) Encyclopedia of Agro-
M.M., Al-Mughrabi, K.I., and Howard, chemicals, vol. 2 (ed. J.R. Plimmer),
R.J. (2008) Plant Dis., 92, 172. John Wiley & Sons, Inc., Hoboken,
79. Doyle, P., Stypa, M., Schneidersmann, pp. 623–625.
F., Ramachandran, R., and Biddle, A.J. 90. Rougerie, I. and Leroux, P. (1997)
(2001) BCPC Symposium Proceedings Seed Phytoma, 499, 57–58.
91. Green, E.A., Bernhard, U., and
Treatment: Challenges and Opportunities,
Bacci, L. (1998) Proc. Brighton Crop
vol. 76, British Crop Protection Council,
Prot. Conf. – Pests Dis., 3, 857–862.
Farnham, pp. 173–180.
92. Mueller, J.P. and Green, E.A. (2002)
80. Forster, B., Sztor, E., Burke, R.,
Phytopathology, 92, S58.
Follas, G., and Biddle, A.J. (2001)
93. Wheeler, I.E., Hollomon, D.W.,
BCPC Symposium Proceedings Seed Gustafson, G., Mitchell, J.C.,
Treatment: Challenges and Opportunities, Longhurst, C., Zhag, Z., and Gurr, S.J.
vol. 76, British Crop Protection Council, (2003) Mol. Plant Pathol., 4, 177–186.
Farnham, pp. 105–110. 94. Bartlett, D.J., Baloch, R., and
81. Errampalli, D. (2004) Crop Prot., 23, Guegen, F. (1997) Proceedings of Con-
811–817. ference International Maladies Plantes,
82. Pillonel, C., Knauf-Beiter, G., and Tours, France, December 3–5, pp.
Steinemann, A. (2003) in Encyclopedia of 1031–1038.
Agrochemicals, vol. 2 (ed. J.R. Plimmer), 95. Gustafson, G.D., Green, E.A., Bernhard,
John Wiley & Sons, Inc., Hoboken, pp. U.H., and Henry, M.J. (2003) Proceed-
617–623. ings of International Congress of Plant
References 737
Pathology, Christchurch, New Zealand, 97. Green, E.A. and Gustafson, G.D.
February 2–7. (2006) Pest. Manage. Sci., 62,
96. Green, E.A. and Duriatti, A. (2005) Proc. 492–497.
Brighton Crop Prot. Conf. – Pests Dis. 1, 98. Fungicide Resistance Action Committee
163–168. (FRAC) (2010) Code List.
739
18
Fungicides Acting on Mitosis and Cell Division
18.1
Zoxamide: An Antitubulin Fungicide for the Control of Oomycete Pathogens
David H. Young
18.1.1
Introduction
Zoxamide (1) (3,5-dichloro-N-(3-chloro-1-ethyl-1-methyl-2-oxopropyl)-4-methylben-

zamide, RH-7281; Figure 18.1.1) was discovered and commercialized by Rohm and
Haas Company in 2001 for the control of Oomycete pathogens [1]. Until 2008, it was
marketed by Dow AgroSciences LLC, and then acquired by Gowan Co. The primary
markets for zoxamide are late blight on potatoes and downy mildews on vines and
vegetables. Inhibitors of microtubule assembly which have been used as fungicides
include benzimidazole (BZ) and thiophanate fungicides such as carbendazim
and thiophanate-methyl, the N-phenylcarbamate (NPC) diethofencarb, and the
antibiotic griseofulvin. Zoxamide represents the first inhibitor of microtubule
assembly to be used commercially for the control of Oomycetes.
18.1.2
Mechanism of Action
Microtubules are hollow filaments of the cytoskeleton composed primarily of

tubulin, a dimeric protein consisting of α-and β-subunits, each of which is approx-
imately 55 kDa in size. As components of the mitotic spindle, the microtubules
play a critical function in separating the daughter chromosomes during nuclear
division. Since this role requires reversible assembly from free tubulin, nuclear
division can be blocked by agents such as colchicine which inhibit assembly, or
by compounds which stabilize microtubules and prevent their disassembly, such
as paclitaxel [2] and experimental triazolopyrimidine fungicides such as BAS600F
[3, 4].
Zoxamide belongs to a class of benzamides which shows activity toward a
broad range of organisms, including both Oomycete and non-Oomycete fungi
[1], protozoan [5], plant [6], and mammalian cells [7, 8]. The relative potency of
analogs toward different organisms varies widely, depending on their structure

740 18 Fungicides Acting on Mitosis and Cell Division
Figure 18.1.1 Zoxamide.

O
Cl
NH Cl
O
Cl
1
[8]. At the cellular level, these benzamides arrest nuclear division and destroy
the microtubule cytoskeleton [6, 8, 9]; the loss of microtubules results from an
inhibition of microtubule assembly caused by a highly specific covalent binding to
Cys239 on the β-subunit of tubulin [8, 9].
18.1.3
Analysis of the Benzamide Binding Site Using Radioligand Binding Assays
The covalent binding property of benzamides has been exploited in the develop-
ment of cell-based competitive binding assays which measure the ability of other
antitubulin agents to inhibit the binding of radiolabeled benzamides to β-tubulin
in whole cells. The tritiated S-enantiomer of zoxamide has been used to study the
zoxamide binding site in the Oomycete Phytophthora capsici [9]. Tritiated analogs 2
and 3 (RH-4032 and RH-5854; Figure 18.1.2) have been used in similar assays in
plant [6] and mammalian cells [8], respectively.
The experimental benzamide fungicide zarilamide (4) was discovered by ICI
during the 1980s [10], and found to act on microtubules [11]. Later, it was shown
to inhibit the binding of [3 H](S)-zoxamide to β-tubulin in Phytophthora capsici in a
competitive manner [9], indicating a common binding site with zoxamide.
In tobacco cells, the antimitotic herbicides pronamide (5) and the NPC chlor-
propham (6) inhibited the binding of [3 H]RH-4032 to tobacco tubulin, again
suggesting a common binding site with zoxamide-related benzamides [6]. Simi-
larly, colchicine (7) and various other ligands of the colchicine site, including the
BZ nocodazole, podophyllotoxin, tubulozole C, and TN-16, were found to inhibit
the binding of [3 H]RH-5854 to mammalian tubulin [8]. In contrast, the anticancer
drug vinblastine, which is known to bind to a different site from colchicine [2],
enhanced [3 H]RH-5854 binding, presumably through an allosteric effect [8].
O O
Cl O
NH Cl N NH Cl
O O
H 2N
Cl
2 (RH-4032) Cl 3 (RH-5854)
Figure 18.1.2 RH-4032 and RH-5854.

18.1 Zoxamide: An Antitubulin Fungicide for the Control of Oomycete Pathogens 741
Covalent binding to Cys239 in β-tubulin is involved in the mechanism of action

of some other antitubulin compounds, including 2,4-dichlorobenzyl thiocyanate (8)
[12], the experimental anticancer drug T138067 9 [13], and the bifunctional reagent
N,N -ethylenebis(iodoacetamide) [14]. The latter compound forms a crosslink be-
tween Cys239 and Cys354 in a reaction that occurs in free tubulin, but not in intact
microtubules [14]. As in the case of the zoxamide analog RH-5854 [8], the reaction
of Cys239 with these various agents was inhibited by ligands of the colchicine
site [12–14] and, in the case of T138067 and N,N -ethylenebis(iodoacetamide),
was enhanced by vinblastine [13, 14]. Cys239 has also been shown to bind to
chemically reactive analogs of colchicine in which methoxy groups on the A-ring
of colchicine are replaced by chloroacetyl groups [15]. Moreover, modeling stud-
ies of the colchicine binding site place Cys239 in close proximity to the A-ring
[15, 16]. The location of Cys239 in the colchicine binding site correlates well with the
ability of colchicine site ligands to inhibit the binding of radiolabeled benzamides
to tubulin, and implies a common binding region for zoxamide and colchicine.
Representative compounds which are believed to bind at the benzamide site
on the basis of evidence from competitive binding or cross-resistance studies
O
O CN H
Cl Cl N O
N
N O H
H O
Cl
4 Cl 5 6
zarilamide pronamide chlorpropham
O
O
HN
F O O
O Cl
C F S
B CN
S N O
O H
A F F
Cl F
F
O O
7 8 9
colchicine 2,4-dichlorobenzyl thiocyanate T138067
H
N H
H
O N O N
N O
O O
O
11 12
diethofencarb carbendazim
Figure 18.1.3 Representative antitubulin compounds be-

lieved to bind at the benzamide site.
N Figure 18.1.4 Ethaboxam.

H
N N
H S S
O CN
10
(as discussed below) are shown in Figure 18.1.3. Although these compounds differ
from zoxamide in their relative toxicity towards different organisms, they appear
to bind to the same domain on β-tubulin. Selective toxicity may be governed by
structural differences between organisms in this domain.
Ethaboxam (10) (Figure 18.1.4), which was developed for the commercial
anti-Oomycete market in Korea in 1999 by LG Life Sciences [17], has a mode
of action in Phytophthora infestans which involves the disruption of microtubules
[18]. To date, it has not been established whether ethaboxam binds to tubulin
and, if so, whether it binds to the same site as do other benzamides. How-
ever, the structural similarity between ethaboxam and zarilamide, together with
evidence that zarilamide is a competitive inhibitor of zoxamide binding [9],
lend support to the possibility that ethaboxam binds to the zoxamide site on
tubulin.
18.1.4
Cross-Resistance Relationships between Zoxamide, Carbendazim, and Diethofencarb
Although zoxamide is used commercially only to control Oomycete pathogens, it is

also active against other fungi [1]. These include pathogens in which field resistance
to BZs has occurred, such as Botrytis cinerea, Venturia inaequalis, Monilinia fructicola,
Mycosphaerella fijiensis, and Cercospora beticola. Resistance to BZs in the field
occurred shortly after their introduction, due in part to their widespread, exclusive
use [19]. Subsequently, the finding of a negatively correlated cross-resistance
between NPC herbicides and BZs led to the discovery and commercialization
of the NPC diethofencarb (11) to combat BZ-resistant fungi [20]. Unfortunately,
however, the combined use of diethofencarb and the BZ carbendazim (12) resulted
in strains that were resistant to both compounds [21]. Resistance to carbendazim
and diethofencarb in field isolates is determined by allelic mutations at positions
198 and 200 in β-tubulin [22].
The analysis of cross-resistance relationships between zoxamide, carbendazim,
and diethofencarb in such resistant isolates has provided further informa-
tion regarding the zoxamide binding site [23]. As shown in Table 18.1.1,
carbendazim-resistant isolates of Botrytis cinerea, which are sensitive to diethofen-
carb due to a negatively correlated cross-resistance (Rb1 phenotype), show greatly
enhanced sensitivity to zoxamide. Furthermore, strains that are resistant to both
carbendazim and diethofencarb (Rb2 phenotype) are resistant to zoxamide. The
fact that mutations in β-tubulin which alter sensitivity to carbendazim and
Table 18.1.1 Cross-resistance relationships between zoxam-

ide, carbendazim, and diethofencarb in Botrytis cinerea field
isolates.
Phenotype Number of strains EC50 ± S · D · (μg ml−1 )

Zoxamide Carbendazim Diethofencarb
Sba 9 0.97 ± 0.36 0.045 ± 0.009 >50

Rb1b 6 0.073 ± 0.039 >50 0.077 ± 0.028
Rb2c 4 >50 9.3 ± 1.08 >50
a
Sb = sensitive to benzimidazoles.
b
Rb1 = resistant to benzimidazoles and sensitive to diethofencarb.
c
Rb2 = resistant to both benzimidazoles and diethofencarb.
diethofencarb also affect sensitivity to zoxamide suggests a common binding

domain for benzamides, BZs, and NPCs. This conclusion is supported by results
from binding assays, which demonstrated an inhibition of benzamide binding
in mammalian cells by the BZ nocodazole [8], and in plant cells by the NPC
chlorpropham [6]. The conclusion is also consistent with the aforementioned
binding of colchicine to the same region as benzamides, since colchicine and BZs
are believed to bind to the same domain [2].
18.1.5
The α-haloketone-containing benzamide series that led to zoxamide originated in

an herbicide synthesis effort to prepare analogs of pronamide (5) [24]. Although
pronamide is not fungicidal, early α-haloketone analogs such as 13 (Table 18.1.2)
were found to be highly active towards Oomycete fungi, as well as plants. Despite
the fact that such activity was based on the same mode of action [6, 9] it was
found subsequently that the relative potency toward Oomycetes and plants could
be modulated by structural changes.
In attempts to identify analogs with high fungitoxicity and low phytotoxicity, more
than 300 analogs were prepared and tested in parallel in an in vitro fungitoxicity
assay against Pythium ultimum, and in a tobacco root elongation assay. The ratio
of EC50 values obtained provided a measure of the selective toxicity towards
Oomycetes. An appropriate substitution of the phenyl ring was found to be
essential for strong biological activity (Table 18.1.2). Notably, substitution at the 3-
and 5-positions (13) led to a dramatically increased activity over the unsubstituted
ring (14), whereas substitution at the 2-position caused a greatly reduced activity
(15). Both, Me and Et in the R1 and R2 positions (as in 2) were found to be optimal
for fungitoxicity. A key finding here was the discovery that, when R1 = Me and
R2 = Et, certain 4-substituents on the phenyl ring reduced the phytotoxicity with
Table 18.1.2 Optimization of potency and crop safety.
2
O R1 R2
3
NH Cl
O
4
5
Compound 2 3 4 5 R1 R2 Pythium Tobacco Pythium EC50 /

EC50 a EC50 b tobacco EC50
13 H Cl H Cl CH3 CH3 0.024 0.006 4.0

14 H H H H CH3 CH3 12.0 ND –
15 Cl Cl CH3 Cl CH3 C2 H5 3.49 0.030 3.0
2 H Cl H Cl CH3 C2 H5 0.007 0.004 1.7
1 (Zoxamide) H Cl CH3 Cl CH3 C2 H5 0.006 0.017 0.35
EC50 (μg ml−1 ) for inhibition of Pythium ultimum growth.

a
EC50 (μg ml−1 ) for inhibition of tobacco root elongation.

b
ND, not determined.
little or no reduction in fungitoxicity [25]. Thus, the combination of 3,5-dichloro,

4-methyl as favorable substituents on the phenyl ring with Me and Et in the R1 and
R2 positions yielded 1 (zoxamide) as an experimental compound with an improved
fungitoxicity/phytotoxicity ratio that showed outstanding disease control in both
greenhouse and field-tests, and no phytotoxicity in whole plants.
Whilst zoxamide comprises two enantiomers, its biological activity is due almost
entirely to the S-enantiomer [26]. Unfortunately, despite the latter being more
active than the racemate, its manufacture and sale in pure form was found not
to be economically viable; hence, the commercial product is currently sold as the
racemic mixture.
18.1.6
Synthesis of Zoxamide
The key precursors in the preparation of zoxamide are 3,5-dichloro-4-methylbenzoyl

chloride (16) and 3-amino-3-methyl-1-pentyne (17). Compound 16 is prepared from
methyl toluate (Scheme 18.1.1), and 17 from its corresponding acetylenic alcohol
(Scheme 18.1.2).
The precursors 16 and 17 react to yield the alkynyl amide (18), which is
converted to the 5-methylene oxazoline (19) (Scheme 18.1.3). The chlorination of
19 employs trichloroisocyanuric acid (TCIA) as the chlorinating agent to produce
the monochlorinated oxazoline (20), which is converted to zoxamide (1) via an
acid-catalyzed hydrolysis.
CO2CH3 CO2CH3 CO2H COCl
Cl2 /AlCl3 SOCl2
Cl Cl Cl Cl Cl Cl
CH3 CH3 CH3 CH3
16
Scheme 18.1.1 Synthesis of 3,5-dichloro-4-methylbenzoyl chloride.
HCl NH3
OH Cl NH2
17
Scheme 18.1.2 Synthesis of 3-amino-3-methyl-1-pentyne.
COCl O
Cl
N
+ H
Cl Cl NH2
CH3 Cl
16 17 18
N N O
Cl Cl Cl
O O N Cl
Cl
H
O
TCIA HCl
Cl Cl Cl
19 20 1
Scheme 18.1.3 Synthesis of zoxamide.
18.1.7
Resistance Risk
Since its first commercial use in 2001, there have been no reports of any reduced
pathogen sensitivity to zoxamide in the field. In laboratory studies to identify
the potential for resistance development to zoxamide [27] in Phytophthora capsici,
using either chemical mutagenesis or adaptation, and in P. infestans using only
adaptation, no resistant mutants could be isolated. The only known case of
resistance to zoxamide in an Oomycete involved Pythium sylvaticum, for which a
reduction in sensitivity was reported following repeated exposure in the laboratory
[28], although the mechanism responsible was not determined. Laboratory-based
investigations to explore resistance development have also been conducted in
various Oomycetes using zarilamide [29], a benzamide that binds to the same site
as zoxamide on β-tubulin [9]. In the latter studies, attempts to isolate resistant
mutants using chemical mutagenesis, ultraviolet irradiation, or adaptation were
unsuccessful. Taken together, these results suggest that the risk for resistance
development to zoxamide in its commercial target pathogens is relatively low.
Despite the similarity between zoxamide and the BZ fungicides with regards to
their mechanisms of action, the resistance risk for zoxamide in the field contrasts
sharply with the serious resistance problems of the BZ fungicides [19]. One critical
difference between zoxamide and the BZs is the nature of the pathogens against
which these products are used. The Oomycete fungi targeted by zoxamide are
diploid [30], whereas fungi in which BZ-resistance has occurred are haploid. A
likely explanation for the low resistance risk of zoxamide is that a target-site
mutation which affects its binding would likely be recessive and would have little
effect on sensitivity of diploid cells, which were heterozygous with respect to the
mutation [27, 31].
18.1.8
Metabolism and Toxicology
Zoxamide has a low toxicity towards mammals, except for the potential to cause skin
sensitization [32]. Based on the results of laboratory studies, zoxamide poses a very
low risk to most nontarget species [1, 32]. Environmental fate studies have shown
that zoxamide dissipates rapidly in the environment due to hydrolysis, photodegra-
dation in water, and microbial metabolism. It has a half-life in soil of between 2
and 10 days, a low water solubility, and a low soil mobility [1]; consequently, there
is very little potential for zoxamide to leach into the groundwater.
18.1.9
Biology and Use in Agriculture
®
Zoxamide was developed under the trade name Zoxium , but is sold primarily
® ® ®
in mixtures with mancozeb under the trade names Gavel , Electis , Aderio ,
® ® ®
Stimo , Unikat , and Roxam . The application rates are typically within the range
of 125–150 g a·i·ha−1 in formulation with mancozeb at 1.2–1.4 kg a·i· ha−1 . The
spray intervals depend on the crop and disease, but are usually between 7 and 14
days. In addition to the mixtures with mancozeb, zoxamide is also coformulated
®
with cymoxanil and sold under the trade name Harpon .
Zoxamide is highly active towards a broad range of Oomycete fungi, and is used
commercially on potatoes, vines, and vegetables to control late blight and downy
mildew diseases. Activity has also been demonstrated against certain non-Oomycete
fungi, such as Venturia, Sclerotinia, Botrytis, and Monilinia spp. [1]. Zoxamide is
reported to show a synergistic effect with mancozeb in controlling Alternaria species
®
[33], and is registered for this use as Electis in some European countries.
Consistent with the mode of action of zoxamide, the stages in fungal growth that
are susceptible to its inhibitory effects are those that depend on nuclear division.
References 747
Thus, zoxamide inhibits germ tube elongation and mycelial growth [9], and
prevents the correct formation of zoospores by interfering with nuclear division in
the developing sporangia [34]. Zoxamide does not directly affect zoospore motility,
encystment, or germination, but rather arrests germ tube elongation coincident
with the first cycle of nuclear division [9]; it also prevents germ tube penetration
into the host plant. Zoxamide is not a systemic fungicide, but does exhibit curative
activity [1]. The good residual efficacy and excellent rainfastness of zoxamide result
from its high affinity for the plant cuticle [1, 35]. Zoxamide is highly effective in
controlling tuber blight [36, 37], the control mechanism of which does not involve
any direct effect on zoospore motility [34]; moreover, the efficacy of zoxamide
cannot result from any protective action in the tuber or soil, as it is not systemic and
has a short soil half-life. In fact, the mechanism of action of zoxamide in controlling
tuber blight may involve a reduced production of motile zoospores resulting from
an inhibition of nuclear division in the sporangia as they form on the plant
surface [34].
With the possible exception of ethaboxam (see discussion above), zoxamide has
a different mode of action from other products used in the Oomycete market,
and there is no likelihood of cross-resistance in the field to existing products such
as metalaxyl, carboxylic acid amides, cymoxanil, or strobilurins. Consequently,
zoxamide provides a unique tool for resistance management in the Oomycete
fungicide market.
References
1. Egan, A.R., Michelotti, E.L., Young, 8. Young, D.H., Rubio, F.M., and Danis,
D.H., Wilson, W.J., and Mattioda, H. P.O. (2006) J. Biomol. Screen., 11,
(1998) Brighton Crop Prot. Conf. - Pests 82–89.
Dis., 2, 335–342. 9. Young, D.H. and Slawecki, R.A. (2001)
2. Wilson, L. and Jordan, M.A. (1994) Pestic. Biochem. Physiol., 69, 100–111.
in Microtubules (eds J.S. Hyams and 10. Heaney, S.P., Shephard, M.C., Crowley,
C.W. Lloyd), Wiley-Liss, New York, pp. P.J., and Shearing, S.J. (1988) Brighton
59–83. Crop Prot. Conf. - Pests Dis., 2, 551–558.
3. Zhang, N., Ayral-Kaloustian, S., Nguyen, 11. Young, D.H. (1991) Pestic. Biochem.
T., Afragola, J., Hernandez, R., Lucas, J., Physiol., 40, 149–161.
Gibbons, J., and Beyer, C. (2007) J. Med. 12. Bai, R.L., Duanmu, C., and Hamel,
Chem., 50, 319–327. E. (1989) Biochim. Biophys. Acta, 994,
4. Crowley, P.J., Lamberth, C., Mueller, 12–20.
U., Wendeborn, S., Nebel, K., Williams, 13. Shan, B., Medina, J.C., Santha, E.,
J., Sageot, O.-A., Carter, N., Mathie, T., Frankmoelle, W.P., Chou, T.C., Learned,
Kempf, H.-J., Godwin, J., Schneiter, P., R.M., Narbut, M.R., Stott, D., Wu, P.,
and Dobler, M. (2010) Pest Manag. Sci., Jaen, J.C., Rosen, T., Timmermans,
66, 178–185. P.B.M.W., and Beckmann, H.
5. Rohm and Haas Co. (2000) US Patent (1999) Proc. Natl Acad. Sci. USA, 96,
6,107,316. 5686–5691.
6. Young, D.H. and Lewandowski, V.T. 14. Luduena, R.F. and Roach, M.C. (1991)
(2000) Plant Physiol., 124, 115–124. Pharmacol. Ther., 49, 133–152.
7. Rohm and Haas Co. (1998) European 15. Bai, R.L., Covell, D.G., Pei, X.-F., Ewell,
Patent Application 834,311. J.B., Nguyen, N.Y., Brossi, A., and
Hamel, E. (2000) J. Biol. Chem., 275, 27. Young, D.H., Spiewak, S.L., and
40443–40452. Slawecki, R.A. (2001) Pest Manag. Sci.,
16. Ravelli, R.B.G., Gigant, B., Curmi, P.A., 57, 1081–1087.
Jourdain, I., Lachkar, S., Sobel, A., 28. Martinez, C., Levesque, C.A., Belanger,
and Knossow, M. (2004) Nature, 428, R.R., and Tweddell, R.J. (2005) Pest
198–202. Manag. Sci., 61, 767–771.
17. Kim, D.-S., Park, H.-C., Chun, S.-J., Yu, 29. Eacott, C.J.P. (1986) Assessment of the
S.-H., Choi, K.-J., Oh, J.-H., Shin, K.-H., risk of resistance to benzamide fungi-
Koh, Y.-J., Kim, B.S., Hahm, Y.-I., and cides in Phytophthora infestans. Ph.D.
Chung, B.-K. (1999) Plant Pathol. J., 15, Thesis, University of London.
48–52. 30. Shaw, D.S. (1983) in Phytophthora: Its
18. Uchida, M., Roberson, R.W., Chun, S.-J., Biology, Taxonomy, Ecology, and Pathol-
and Kim, D.-S. (2005) Pest Manag. Sci., ogy (eds D.C. Erwin, S. Bartnicki-Garcia,
61, 787–792. and P.H. Tsao), American Phytopatho-
19. Delp, C. (1995) in Modern Selective logical Society, St Paul, MN, pp. 81–94.
Fungicides – Properties, Applications, 31. Cabral, F. and Barlow, S.B. (1991)
Mechanisms of Action (ed. H. Lyr), Gus- Pharmacol. Ther., 52, 159–171.
tav Fischer Verlag, Jena, pp. 291–303. 32. U.S. Environmental Protection
20. Fujimura, M., Hayashi, M., and Hisada, Agency (2001) Internet website:/
Y. (1990) Jpn. Pestic. Inf., 57, 7–11. www.epa.gov/opprd001/factsheets.
21. Leroux, P. (1995) Pestic. Outlook, 6, 33. Edmonds, J. (2005) Congress Proceed-
20–24. ings – BCPC International Congress:
22. Davidse, L.C. and Ishii, H. (1995) in Crop Science and Technology, BCPC,
Modern Selective Fungicides – Properties, Alton, pp. 879–882.
Applications, Mechanisms of Action (eds 34. Young, D.H. and Vjugina, U. (2002)
H. Lyr), Gustav Fischer Verlag, Jena, Phytopathology, 92, S89.
pp. 305–322. 35. Gobert, L., Mattioda, H., Raux, A., Arp,
23. Young, D.H. and Slawecki, R.A. (2005) U., Egan, A.R., Michelotti, E.L., Young,
in Modern Fungicides and Antifungal D.H., and Wilson, W.J. (2000) Meded.
Compounds IV (eds H.W. Dehne, U. Fac. Landbouwkd. Toegep. Biol. Wet.
Gisi, K.H. Kuck, P.E. Russell, and (Univ. Gent), 65 (2b), 799–806.
H. Lyr), The British Crop Production 36. McFadden, A.G., Duttle, A.E., Smith,
Council, Alton, pp. 125–131. R.L., Kemmitt, G.M., Olson, B.D.,
24. Rohm and Haas Co. (1972) US Patent Edmonds, J., and Young, D.H. (2002)
3,661,991. Phytopathology, 92, S53.
25. Rohm and Haas Co. (1994) US Patent 37. Oemichen, B., Olson, B.D., McFadden,
5,304,572. A.G., Kemmitt, G.M., Young, D.H.,
26. Rohm and Haas Co. (2003) US Patent Secor, G.A., and Gudmestad, N.C.
6,566,403. (2003) Phytopathology, 93, S67.
18.2
Pencycuron: A Phenylurea Fungicide for Rhizoctonia solani
Isao Ueyama2 and Yoshio Kurahashi2
18.2.1
Introduction
18.2.1.1 Overview of the Compound

Pencycuron, which was developed by Nitokuno (the Japanese subsidiary of Bayer
CropScience), and for which an application for patent was filed in 1976 [1], is
2
Retired
a fungicide that is specifically active against Rhizoctonia solani (perfect stage:

Thanatephorus cucumeris), the causative agent of several important plant diseases
such as rice sheath blight, potato black scurf, leaf blight in sugar beet, and the
seedling damping-off of various crops.
Pencycuron induces an abnormal branching of the hyphae of the sensitive strains
of R. solani, and its activity is fungistatic [2]. This morphological change implies that
the mode of action of pencycuron would be anti-microtubular (as for carbendazime),
and consequently it is classified as ‘‘B4: Cell Division’’ in the Fungicide Resistance
Action Committee (FRAC) code list. However, whereas carbendazime inhibits
β-tubulin assembly in the mitosis of R. solani, pencycuron does not have such an
action but rather seeks to destroy the cytoskeleton of the microtubules. To date,
a clear-cut explanation of the mode of action of pencycuron is not yet available;
moreover, it is especially interesting as to why pencycuron is effective only against
somewhat limited strains of the Anastomosis Groups of R. solani.
With pencycuron having been introduced to the market as far back as 1985, the
current turnover for the product worldwide is more than US$ 40 million, the major
markets being in Japan, Taiwan, Korea, Netherlands, Germany, France, and the
UK.
18.2.1.2 Pencycuron: Background to the Development

After rice blast, the sheath blight of rice is the second most troublesome disease
encountered among the rice culture, with significant crop damage due to the disease
being reported each year, especially in Northeast Asian countries. During the 1970s,
rice sheath blight in Japan was controlled mainly with organic arsine fungicides
which, although demonstrating a stable efficacy, led to major environmental
concerns with regard to safety and pollution. As a consequence, the sales of
these products were halted. Subsequently, two antibiotics – polyoxin (Kaken) and
OCH(CH3)2 OCH(CH3)2
CO-NH CO-NH
CH3 CF3
Mepronil (Kumiai) Flutolanil (Nihon Nohyaku)
Cl H
N N
Cl CH2
CH3 O
N CO NH
Cl
Pencycuron (Nitokuno, BCS) Diclomezine (Sankyo)
Figure 18.2.1 Representative rice sheath blight control

fungicides developed in Japan since 1980.
validamycin (Takeda) – were developed in place of the arsenic compounds but each
of these proved to be effective over only a very short period of time. The launch of
a new group of fungicides that could control sheath blight while retaining efficacy
and demonstrating good plant compatibility was keenly awaited in the Japanese
market. There followed, in Japan, the development of several compounds: mepronil
(Kumiai) was launched first, in early 1980, while flutolanil (Nihon Nohyaku),
pencycuron, and diclomezine (Sankyo) were each marketed in quick succession.
The chemical structures of these compounds are shown in Figure 18.2.1.
Of these four fungicides, pencycuron was unique in terms of its narrow fungi-
cidal spectra; indeed, its antifungal activity was found to be highly selective.
Subsequently, different isolates that were either sensitive or less-sensitive (inher-
ently resistant) to pencycuron have been identified, within the same Anastomosis
Groups (AGs) of R. solani. However, only a quite narrow range of derivatives of
the urea skeleton showed controlling activity against R. solani. Thus, in retrospect,
it seemed that a miracle was required to identify one active molecule among this
class of chemical structures that would be worthy of development.
18.2.2
Chemistry
18.2.2.1 Developmental History

The perceived primary activity and milestone compounds identified during the
development of pencycuron are summarized in Figure 18.2.2. The compound
was first identified in an ‘‘indication shift’’ that often occurs when chemists
are attempting to synthesize a new compound intended for a particular activity.
When investigating new pesticides, findings would often arise in areas that were
quite different from those originally intended and, indeed, this was the case for
pencycuron, which originated from what was, primarily, a project to create a new
herbicide.
During the late 1940s and early 1950s, many urea compounds with herbicidal
activity had been investigated, and this had led to the development of some
candidates as practical herbicides. Whilst N-3,4-dichlorophenyl-N -dimethylurea
(DCMU) had been successfully developed by Du Pont in 1954, the quest for
more active compounds led to the chemists initially attempting to modify the
urea molecule. The result of these investigations was that most of the new
compounds showed far from improved herbicidal activities, with neither fungicidal
nor insecticidal activities observed during routine screening tests.
In anticipating a new deployment, a substituted benzyl was introduced instead
of a substituted phenyl at the N-position, and a new general formula was proposed
(Figure 18.2.3) based on which many compounds were synthesized but few showed
any biological activity, except for one – NTN 15192 – which showed a weak activity
against R. solani (Table 18.2.1). In agar plate test results, however, NTN 15192
failed to demonstrate any activity against a variety of plant pathogenic fungi,
except for R. solani. Subsequent screening tests conducted under a higher activity
Cl O
CH3 DCMU was the lead compound for
aiming at herbicidal activity.
Cl HN N
CH3
O
NTN 15192 was the first compound, which
Cl CH2 N N showed fungicidal activity on R. solani.
H
C3H7-i
Cl CH2 N N First candidate for development as

H Rhizoctonia fungicide (NTN 16543)
C4H9-s
Cl CH2
Pencycuron, the goal of the project
N CO NH
Figure 18.2.2 Milestone compounds leading to pencycuron,

and the chemical modifications involved.
(low concentration, long interval) again provided unsatisfactory results, and the
project was halted.
After two blank years, however, the fungicidal activity of NTN 15192 was
re-reviewed, the general formula for chemical modification was structured, and
the N-alkyl moiety of the molecule was changed. On the basis of this new scheme,
derivatives modified with N-alkyl and substituents at the benzyl or phenyl ring were
synthesized, from which one compound – NTN 16543 – appeared to be worthy of
promotion to the next stage, as it showed excellent efficacy against sheath blight of
rice under greenhouse conditions (Table 18.2.2). Further studies, which included
field trails, showed NTN 16543 to be very effective against sheath blight of rice
and also damping-off diseases caused by R. solani. Unfortunately, however, the
compound demonstrated a low plant compatibility, the reasons for which were
revealed in a soil metabolism study in which NTN 16543 was shown to decompose
to form a des-benzyl moiety that possessed a slight herbicidal activity. Further
positive efforts at compound derivation were then undertaken in accordance with
the structure–activity relationship (SAR) defined in the study such that, the research
team finally produced an N-cyclopentyl compound (coded NTN 19701) that could
overcome many of the weak points demonstrated by previous compounds. Thus,
O Figure 18.2.3 General formulae of deriva-

Xm R1
tives leading to the development of NTN
CH2 N N 15192.
Yn
Pr-i R2
X, Y: H, lower alkyl, halogen, CN, NO2

R1, R2: H, lower alkyl, phenyl
Cl CH2 N N
H
C3H7-i
NTN 15192
Table 18.2.1 Pot test results of the derivatives modified from urea herbicides.
O
Xm R1
CH2 N N
Yn Effectiveness against R. solani on rice.a
Pr-i R2 Concentration used in screening (ppm)
X,Y R1 R2 First Second Tier
500 250 125
4-Cl CH3 - H 5 – –
4-Cl H 5 – –
4-Cl H 0 2 1
(= NTN 15192)
4-CH3 H 5 – –
H Cl H 5 – –
4-Cl H3C H 4 – –
4-Cl CH3 - 5 – –
2,4-Cl2 H 5 – –
a
0: Excellent, 5: no activity, –: no trial.
®
NTN 19701 became known as pencycuron, with the commercial name of Monceren ,
and was marketed in Japan in 1985.
Based on the results of these experiments, it became clear that unintended small
changes and scant signs observed in the study often proved meaningful to future
progress. Clearly, the careful observation of symptoms in tests, followed by the
minute investigation of the results, are extremely important in order to spot the
signs of primal activity.

With the chemical structures of NTN 15192 and NTN 16543 as starting points, a
variety of N-alkyl compounds was tested (Table 18.2.2), but none of the derivatives
in which alkyls, such as methyl, ethyl, n-propyl, n-butyl, i-butyl, and t-butyl, were
introduced showed any biological activity. In contrast to this, when i-propyl and
s-butyl were substituted the molecules showed a clear activity towards R. solani. A
4-Cl substitution at the benzyl ring was effective, whereas methyl or no substitution
proved ineffective, as was the case for compounds substituted at the phenyl ring.
The next stage was to synthesize derivatives that had been modified in various
regions of their chemical structures, and to evaluate their activity. The repre-
sentative data used to elucidate the relationship between chemical structure and
the R. solani control activity are listed in Table 18.2.3. Based on these data, a
clear-cut, well-defined relationship was gradually unfolded between the chemical
structures and the biological activities. Notably, in the basic skeleton of the chemical
structure (Figure 18.2.4), substituents at the benzyl (Xm) and phenyl rings (Yn)
proved to be strictly limited for the chemicals to be active. In particular, ortho- or
meta-substitutions and multiple substitutions at the benzyl ring led to a loss of
activity. Various alkyls, including cyclo-alkyl combined at the N-atom, were tested,
and although i-propyl and s-butyl were each active, n-propyl and n-, i-, or t-butyl
Table 18.2.2 Pot test results of the urea derivatives aiming for fungicidal activity (I).
O
Xm Yn
CH2 N NH
Effectiveness against R. solani on rice.a
R Concentration used in screening (ppm)
R Xm Yn First Second Tier
500 250 125
CH3 - 4-Cl H 5 – –
C2 H5 - 4-Cl H 5 – –
n-C3 H7 - 4-Cl H 5 – –
i-C3 H7 - (= NTN 15192) 4-Cl H 0 1 2
n-C4 H9 - 4-Cl H 5 – –
s-C4 H9 - (= NTN 16543) 4-Cl H 0 0 0
a
Table 18.2.3 Pot test result of the urea derivatives aiming for fungicidal activity (II).
O
Xm Yn
CH2 N N
Effectiveness against R. solani on rice.a
R1 R2 Concentration in screening (ppm)
Xm R1 R2 Yn First Second Tier
500 250 125
4-F s-C4 H9 - H H 1 3 –
4-Br s-C4 H9 - H H 0 0.5 1
4-Cl s-C4 H9 - H H 0 0 0
(= NTN 16543)
2-Cl s-C4 H9 - H H 5 – –
3-Cl s-C4 H9 - H H 5 – –
2,3-Cl2 s-C4 H9 - H H 5 – –
4-Cl s-C5 H11 - H H 5 – –
4-Cl H H 5 – –
4-CH3 H H 5 – –
4-Cl H H 0 0 0
(= NTN 19701, pencycuron)
4-Br H H 0.5 0.5 1
4-NO2 H H 0.5 1 2
4-Cl H H 4 – –
3,4-Cl2 H H 5 – –
4-Cl H 4-Cl 5 – –
4-Cl CH3 - H 5 – –
4-Cl H 3-OH- 0.5 1 2
4-Cl H 4-OH- 1.5 2 3
a
Z
Xm Yn
CH2 N N′
H
R
Xm: para-position with electron withdrawing substituents

(such as Cl or Br, but not F or I)
R: 3-5 alkyl with branching at the first carbon

(such as i -propyl, s -butyl, cyclopentyl)
N’: One proton is essential.
Ym: No substituents
Z: O is slightly better than S.
Figure 18.2.4 Structure–activity relationships and R. solani

control activity of the various urea skeleton derivatives.
showed no activity at all. Despite branching, this type of structure may be required
at the first carbon atom of the alkyl, although the s-pentyl derivative was not active.
In the case of cycloalkyl groups, which also branch at the first carbon atom, the
N-cyclopentyl compound showed an excellent efficacy, but neither N-cyclohexyl
nor N-cyclopropyl were seen clearly as inferior to the former compound. Hence,
a suitable bulkiness of the alkyl at the N-atom must be necessary to achieve
efficacy.
Based on the results of these screening, a series of rules was proposed regarding
the relationship between chemical structure and R. solani activity (Figure 18.2.4):
• Electron-withdrawing lipophilic substituents such as Cl or Br are necessary at the
para position of benzyl ring. However, the presence of F or I atoms will reduce
the activity, as will the presence of electron-donating groups (such as methyl or
ethyl).
• A C3 − C5 alkyl with branching at the first carbon atom is required (such as
i-propyl, s-butyl, and cyclopentyl group).
• One proton is essential at the N -position.
• Substitutions at the phenyl ring cause the activity to be lost, whereas derivatives
that are substituted at meta- or para-OH cause it to remains. In terms of efficacy,
thiourea derivatives with the same substructure were similar to, but not better
than, the urea derivatives.
18.2.3
Chemical Synthesis and Physico-Chemical Properties
18.2.3.1 Preparation of Pencycuron

The preparation of pencycuron is achieved in a two-step process (Scheme 18.2.1).
Initially, 4-chlorobenzyl chloride (1) was added to a mixture of cyclopentylamine
(2) and NaOH aqueous solution to produce 4-chlorobenzyl-N-cyclopetylamine (3),
which was isolated as a colorless oil (b.p. 109–110 ◦ C/20 Pa).
NH2
(II)
Cl CH2 Cl Cl CH2 NH
(I) (III)
NCO
Cl CH2
(IV) N CO NH
(III)
pencycuron
Scheme 18.2.1 The preparation of pencycuron.
Phenylisocyanate (4) was then added to a solution of (3) in toluene, furnishing

N-4-chlorobenzyl-N-cyclopentyl-N -phenylurea (pencycuron) as a white solid (m.p.
129–134 ◦ C).
18.2.3.2 Physico-Chemical Properties of Pencycuron

The physico-chemical properties of technical pencycuron were first reported in
1986 [2], but have since been updated. The current data indicate melting points of
128 ◦ C for form A and 132 ◦ C for form B, while the vapor pressure is <1.0 × 10−5 Pa
(20 ◦ C), and the water-solubility is 0.3 mg l−1 at 20 ◦ C. The log Pow is 4.68 at 20 ◦ C.
Pencycuron is relatively stable in distilled water in the absence of light; typically,
the half-life is 76 days at pH 5, but no hydrolytic degradation occurs at either
pH 6.6 or 8.8. Under conditions of sunlight (August in Japan, 8 h exposure,
338 W m−2 (300–3000 nm) at 23–27 ◦ C) the half-life in distilled water was only two
days.
18.2.4
Mode of Action and Biology
18.2.4.1 Mode of Action

Following the application of 14 C-labeled pencycuron to four strains of R. solani that
differed in sensitivity to the fungicide [3], none of the metabolites identified in
either the medium or the mycelia showed any greater fungicidal activity than did
the parent compound. This suggested that the active agent is pencycuron itself,
and that the metabolic activation or detoxification of pencycuron appears irrelevant
with regards to any inter-strain differences observed in sensitivity to the fungicide.
Among a series of comparative studies using other anti-Rhizoctonia compounds,
such as validamycin, flutolanil, and polyoxin [4, 5], biochemical experiments
showed that pencycuron had no effect on trehalose biosynthesis, trehalase activity,
or on the biosyntheses of fatty acids, lipids, chitin, protein, and DNA. Thus, it
was concluded that pencycuron’s mode of action differed from that of other rice
sheath blight controlling fungicides. When sensitive strains of R. solani were treated
with pencycuron, a series of morphological changes (seen as abnormal branching)
was observed that was similar to changes effected by benzimidazole fungicides,
such as carbendazime. Following the implication that pencycuron could produce
antimicrotubular effects in fungi [6], Ueyama and Araki used β-tubulin immunoflu-
orescence microscopy to demonstrate that, while carbendazime inhibited β-tubulin
assembly in the mitosis of R. solani, pencycuron instead destroyed the cytoskeleton
of the microtubules [7]. Subsequently, Kim noted that pencycuron had no effect
on the assembly of tubulin extracted from a sensitive strain of R. solani [8]. Rather,
due to its high lipophilicity (log P = 4.82), pencycuron could be accommodated in
the lipid bilayers of fungal cells, and this would result in a change in membrane
fluidity [9]. Despite all of these suggestions, however, a conclusive, clear-cut mode
of action of pencycuron has not yet been discerned; neither has any explanation
been provided as to why this fungicide is effective against several limited strains of
the AGs of R. solani.
18.2.4.2 Biology
The primary infection of sheath blight originates from sclerotia that are floating
in irrigation water in the paddy field, and which come into contact with the tillers
of the growth plants, especially at the tillering stage. Secondary infection occurs
when the hyphae derived from the young lesions extend and proceed laterally to
the neighboring tillers; this occurs between tillering and heading of the rice stage,
and later also to the upper leaf sheath. Disease development is also promoted
under humid and high-temperature conditions, and continues to the heading
stage. Clearly, a long-lasting efficacy is a key factor for controlling rice sheath blight
disease.
As a fungicide, pencycuron has a nonsystemic contact action, is chemically
stable and, when used as a foliar application for rice sheath blight control, exerts a
sufficiently longlasting efficacy during disease outbreak.
In addition to rice sheath blight, pencycuron is also effective against black scurf
of potato, leaf and root rot of beet, and the damping-off diseases of various crop
seedlings caused by R. solani. Black scurf of potato can be well controlled by
dipping the seed potatoes, while leaf and root rot of sugar beet are well controlled
by foliar spraying and/or soil drench application. Although pencycuron is effective
against R. solani-mediated damping-off diseases by treating either the seed or the
soil, it is ineffective against such diseases caused by soil- and seed-borne Pythium
or Fusarium. In this case, a mixed application of pencycuron with other effective
fungicides is recommended for simultaneous control.
18.2.4.3 Sensitivity to Several Anastomosis Groups (AGs) of Rhizoctonia solani

Pencycuron has an extremely specific fungicidal spectrum, and shows no substan-
tial activity against any plant pathogenic fungus, except for R. solani. Moreover,
it proved to be effective against only limited AGs of R. solani. Nonetheless, most
Table 18.2.4 Sensitivity of pencycuron to each Anastomosis

Group (AG) of R. solani and other plant pathogenic fungi.
Fungi and AG of Strain code Isolated plant MIC (μg ml−1 ) Sensitive (S)/tolerant (T)
R. solani
R. solani
AG-1 C-423 Rice 1.6 S
R-1-2-1 Acacia 0.1 S
HS-1 Rice 0.4 S
AG-2-1 C-121 Mat grass 0.4 S
AG-2-2 BV-30 Sugar beet 0.4 S
I Sugar beet 0.4 S
AG-3 C-563 Potato 0.4 S
AG-4 RC Rice seeding 1.6 S
Rh-131 Beet >500 T
AG-5 SH-1 Soil >500 T
SH-19 Soil >500 T
Rhizoctonia orizae RO-23 Rice >500 T
Pyricularia orizae TH 67–22 Rice >500 T
Alternaria mali – Apple >500 T
Corticium rolfsii – Tobaccos >500 T
Pythium sp. – Cucumber >500 T
Sclerotinia – Unknown >500 T
sclerotiorum
MIC, Minimum inhibitory concentration.
economically important Rhizoctonia diseases can be well controlled by the appro-

priate application of pencycuron, including rice sheath blight (AG-1), black scurf
of potato (AG-3), leaf blight and root rot of sugar beet (AG-2-2), stem rot of mat
rash (AG-2-1), and main damping-off diseases of young seedlings (AG-4). Both
sensitive and insensitive strains to pencycuron are categorized in AG-2 and AG-4
(Table 18.2.4). Although, at present, 14 AGs of R. solani have been reported [10]
(most of which incorporate subsets), the sensitivity of pencycuron is not sufficiently
clear for all AGs, or of the subsets.
18.2.5
Toxicology, Ecotoxicology, and Metabolism
18.2.5.1 Toxicology and Ecotoxicology

Toxicology studies with pencycuron in various mammalian species [2] have shown
the toxicity of the compound to be quite low (acute oral LD50 in rats, mice, and dogs
>5 g kg−1 ), while dermal, inhalational, skin function, and chronic toxicities, as well
as teratogenicity, each appear favorable. This tendency towards a ‘‘low’’ toxicity for
pencycuron applies also to other environmental biota such as fish, algae, Daphnia,
and birds. Indeed, the fact that during more than 20 years since pencycuron
was launched, no accidents caused by the fungicide have been reported, clearly
supports the favorable toxicological and ecotoxicological characteristics of this
compound.
18.2.5.2 Metabolism of Pencycuron

The metabolic fate of pencycuron in rice plants has been investigated using
[phenyl-U-14 C]pencycuron [11]. Following the application of [14 C]pencycuron to the
leaves, the radiolabel was seen gradually to penetrate into the leaf tissues, part
showed an acropetal movement to the upper parts of the plant, but most was
retained on the leaf surface. At 40 days after the application of pencycuron, 52%
of the dose remained unchanged, while 7% was metabolized. The metabolites
identified were 1-cyclopentyl-3-phenylurea (5), 1-(4-chlorobenzyl)-3-phenylurea (6),
O O
HN C N Cl CH2 N C N
H H H
(V)
(VI)
O
Cl CH2 N C N
H
Pencycuron
O
Cl CH2 N C N
H
OH
cis (VII) & trans (VIII) hydroxy derivative
b- glucoside conjugates
Scheme 18.2.2 The metabolism of [14 C]pencycuron in rice plants.

1-(4-chlorobenzyl)-1-(cis-3-hydroxycyclopentyl)-3-phenylurea (7) and its trans isomer

(8), and glycoside conjugates (Scheme 18.2.2). When [14 C]pencycuron was sprayed
twice onto rice plants, before heading and at the heading stage, the concentration
of pencycuron in the rice grains was equivalent to 0.56 ppm, while the radiolabel
was located mainly in the bran (85%). Intact pencycuron was detected at a level of
0.018 ppm in hulled rice, and at 0.003 ppm in polished rice, while the radiolabel in
the grains persisted as an unextractable bound residue.
In a metabolism study conducted in rabbits [12], an ion-cluster analysis was
conducted after dosing with proton- (i.e., nonlabeled) and deuterium-labeled pen-
cycuron (each 50% by volume). Extracts of the urine and feces were examined
using gas chromatography/mass spectrometry; a subsequent comparison with over
20 authentic samples revealed the main degradation pathway of pencycuron to be
para-hydroxylation at the phenyl moiety, followed by conjugation with β-glucuronic
acid. Further hydroxylation occurred at the 3-position of the cyclopentyl moi-
ety. In total, 11 metabolites were identified, including five β-glucuronic acid
conjugates.
References
1. Yamada, Y., Saito, J., Tamura, T., and 7. Ueyama, I., Araki, Y., Kurogochi, S.,
Kurahashi, Y. (1976) Japan Patent Ishii, H., and Yamaguchi, I. (1993)
Application No. 51-85,582. Abstracts of the 18th Pesticide Science
2. Yamada, Y. (1986) Jpn. Pestic. Inf., 48, Society Meeting of Japan, Tokyo, Japan,
16–22. March 27–29, p. 73.
3. Ueyama, I., Araki, Y., Kurogochi, S., and 8. Kim, H.T., Kamakura, T., and
Yamaguchi, I. (1993) J. Pestic. Sci., 18, Yamaguchi, I. (1996) J. Pestic. Sci.,
109–117. 21, 159–163.
4. Kuck, K.H., Ueyama, I., Kurogochi, S., 9. Kim, H.T. and Yamaguchi, I. (1996) J.
Yamada, Y., and Schneider-Christians, J. Pestic. Sci., 21, 323–328.
(1988) Abstract of the 5th International
10. Carling, D.E., Kuninaga, R.S., and
Congress of Plant Pathology, Kyoto,
Brainard, K.A. (2002) Phytopathology, 92,
Japan, August 20–27, p. 22.
43–50.
5. Ueyama, I., Araki, Y., Kurogochi, S.,
11. Kurogochi, S., Takase, I., Yamaguchi, I.,
Yoneyama, K., and Yamaguchi, I. (1990)
and Misato, T. (1987) J. Pestic. Sci., 12,
Pestic. Sci., 30, 363–365.
6. Leroux, P., Droughot, V., and Gredt, M. 435–443.
(1990) Abstracts of the 7th Interna- 12. Ueyama, I., Kurogochi, S., Kobori, I.,
tional Congress of Pesticide Chemistry, Hoshino, T., Ishii, Y., and Takase, I.
Hamburg, Germany, August 5–10, (1982) J. Agric. Food Chem., 30,
p. 348. 1061–1067.
761
19
Sterol Biosynthesis Inhibitors
19.1
Sterol Biosynthesis Inhibitor SBI Fungicides in Agriculture
Fungicides that inhibit targets within the fungal sterol biosynthesis have been
the most important group of specific fungicides worldwide for more than the
past three decades. The biochemical basis of this success is the fact that fungi
have specific sterols that differ from those in plants and animals, thus providing
the chance to develop selective inhibitors that cover a broad spectrum of plant
pathogens.
Fungal cell membranes are characterized in most pathogens belonging to the
Ascomycetes and Basidiomycetes by a common dominant sterol component,
ergosterol. The designation ‘‘ergosterol’’ was generated by Tanret in 1889 as a
result of studies with the ergot pathogen, Claviceps purpurea [1].
Despite the general predominance of ergosterol, some exceptions have to be
noted. In the important pathogen group of rust fungi, for example, fungisterol
(ergost-7-enol), stigmast-7-enol, and other sterols were found, but not ergosterol
[2]. Because ergosterol is the major sterol in most true fungi – but not in all – the
group name of sterol biosynthesis inhibitor (SBI) fungicides should be preferred to
the designation ergosterol biosynthesis inhibitor (EBI) fungicides, which has been
partly used in parallel.
Nevertheless, because ergosterol is the typical sterol in the vast majority of all
fungi, the ergosterol content of food and plant material can be used as a quantitative
indicator of fungal contamination or infection in all types of biological material.
Consequently, many studies have been conducted to investigate the ergosterol
content of a wide variety of fungal species from different taxonomic groups
[3, 4].
One important group of plant pathogens, the Oomycetes, lacks taxonomic
affinity with the so-called true fungi (i.e., Ascomycetes and Basidiomycetes). The
Oomycetes, which formerly were regarded as fungi, have been excluded from
the traditional ‘‘true fungi’’ of the kingdom Mycotae and, according to the newer
classification, have been included along with brown algae in the kingdom Chromista
[5]. More recently, Oomycetes have been classified with diatoms, the golden algae

762 19 Sterol Biosynthesis Inhibitors
and the brown algae in a clade referred to as Stramenophiles [6]. Important

differences exist between the Oomycetes and true fungi. For example, Oomycetes
have cell walls composed mainly of β-glucans and cellulose, and have only
minor contents of chitin. Most phytopathogenic Oomycetes are unable to perform
the full de novo synthesis of sterols but can – to different degrees – metabolize
exogenous precursors derived from plants. So, for example, some species of
the order Peronosporales, which are unable to epoxidize squalene – and thus
to synthesize sterols – can metabolize exogenous cycloartenol to lanosterol and,
in some organisms, to fucosterol, ergosterol, and cholesterol [7]. Based on the
most recent results, the Oomycete Aphanomyces euteiches is able to synthezise
fucosterol and cholesterol from the isoprenoid processor [8]. In fact, an in vitro
inhibition of the mycelial growth of A. euteiches by azoles was demonstrated.
Under field conditions, the SBI fungicides do not show any activity against
Oomycetes.
The fact that more than 40 SBI fungicides have reached market stage is proof
of the interesting properties of the inhibitors of this biosynthetic pathway. One
important property is that targets within fungal sterol biosynthesis clearly provide
sufficient chemical room to synthesize a considerable diversity of highly active
fungicides that are, at the same time, safe for treated plants on both the toxicological
and environmental levels. Additionally, SBI fungicides offer a broad spectrum of
activity within the Ascomycetes and Basidiomycetes. SBI fungicides exhibit at
least a locosystemic activity, with the most successful compounds showing a
significant systemicity leading to a longlasting protection. Moreover, most SBIs are
among one of the few fungicide groups that provide a pronounced curative and
eradicative activity. These characteristics of the SBI fungicides – and namely of the
demethylation inhibitor (DMI) group of triazoles – lead to a broad applicability as
seed treatment, and also as foliar and as ear treatments. In addition, beneficial side
effects on the plant health and growth are also used in practical crop protection in
different crops. Finally – and most importantly for fungicides – the resistance risk
of SBI fungicides is generally regarded to be low to medium [9]. If resistance to SBIs
does occur, it usually has a multigenic basis resulting in a stepwise, continuous
selection, and not in a disruptive selection as occurs with benzimidazoles and
strobilurins. As resistance mechanisms, mainly mutations in the target-encoding
CYP51 gene, as well as an enhanced active efflux and the overexpression of the target
encoding CYP51 gene, have been discussed [10–13]. Stepwise selection – which
is also designated as ‘‘shifting’’ – provides good chances for both rational and
effective resistance management. Moreover, gradual losses in activity can often be
overcome by the introduction of new compounds that exhibit a higher intrinsic
activity [14]. The possibility to compensate for efficacy losses is supported by the
fact that – although a general cross-resistance is mostly found within the SBI
classes I and II – a complete cross-resistance between individual members of
each class is, at the same time, quite rare. Although these SBI classes have been
widely used over decades, their performances continue to function on a high
level.
19.1.1
Market Importance of SBI Fungicides
Since 1980, the SBI fungicides have become the most important fungicide class
overall, with total sales of more than ¤35 billion. In 2004, the total fungicide market
was estimated to have a total value of about US$ 7.33 billion [15], of which more
than 30% was accounted for by fungicides interfering with sterol biosynthesis. The
data listed in Table 19.1 show clearly that by far the most important regional market
for SBIs is in Western Europe, followed by Latin America. Taken together, both
regions consume 70% of the overall SBI market.
The regional distribution can be explained by considering the most important
crops for SBI fungicides. The intensive wheat and barley production in Europe
consumes a great part of the worldwide SBI production. Latin America, especially
Brazil, has gained a considerably increased importance for triazole fungicides
because of the epidemic spread of a devastating disease, the Asian soybean rust
(Phakopsora pachyrhizi).
The success of the SBI fungicides is essentially the success of the triazole
fungicides; all other SBI mode of action classes and the nontriazole classes within
the DMI fungicides play only a limited role in terms of sales. It is interesting to note
that, since the introduction of the triazoles, the sales of this group has continued to
increase, despite the introduction of other broad-spectrum fungicide classes such
as the strobilurines (Figure 19.1). The steep increase since 2003/2004 is also driven
by the new Asian soybean rust segment.
19.1.2
Biochemical Targets of SBI Fungicides
During recent years, several detailed reviews have been produced on the fungal
ergosterol biosynthesis pathway, and of fungicides that interfere with it [16, 17].
Consequently, only a brief, simplified overview will be provided at this point.
The main biosynthesis steps, involving 11 enzymes, from squalene to ergosterol
are shown in Figure 19.2, while further information on the enzymes involved
and the targets of agricultural fungicides is provided in Table 19.2. The main
Table 19.1 Regional markets of SBI fungicides in 2009.
Region Market share (%)a
Europe 53
Latin America 22
Asia/Pacific 18
North America 7
a
% of total SBI market, 2010.
Data: Agrowin 2010.
2500
DMI Amines Anilides
2000
1500
m
1000
500
0
19
19
19
19
19
19
19
19
20
20
20
76
81
84
87
90
93
96
99
02
05
08
Figure 19.1 Sales development of the SBI classes in agriculture.
primary biochemical consequences of an impaired ergosterol biosynthesis has

been often a matter of debate. For an explanation, Vanden Bossche [18] cites Sisler
and Ragsdale [19], who noted that a ‘‘Lack of ergosterol impedes the synthesis of
new membranes and leads to deterioration of existing membranes.’’ Associated
with these changes, it was observed that the fatty acid synthesis continues at a
relatively high rate, resulting in a disproportion between fatty acid synthesis and
utilization for phospholipids. Further, detailed studies have shown a multitude of
consequences of ergosterol depletion, on the one hand, and the accumulation of
toxic precursors on the other hand [17].
Since their introduction, distinct plant growth regulatory effects have been noted
with DMI fungicides. Whereas, unspecific signs of phytotoxic activity such as necro-
sis or leaf drop are only occasionally reported with DMI fungicides, pronounced
plant growth-regulatory side effects on plants have more often been noted. Typically,
shorter shoots and internodes designated as ‘‘stunting,’’ smaller dark green leaves,
and an improved stress tolerance of DMI-treated plants are reported. Accordingly,
specific plant growth regulators (PGRs) such as paclobutrazol have been developed
from the triazole group [20]. The beneficial side effects of fungicidal triazoles have
been intensively studied and documented [21], and represent an important part
of the biological profile of DMI fungicides in several crops, such as oilseed rape
(prevention of lodging) and cereal seed treatment (increased frost tolerance).
Investigations on the biochemical mechanisms causing these symptoms in
plants have revealed that the inhibition of the biosynthesis of the plant hormone
gibberellins, and of plant sterols, seem to be the most important targets in plants
[22, 23].
19.1.3
SBI Classes
An overview of the classes of agricultural SBI fungicides, as defined in the

Fungicide Resistance Action Committee (FRAC) classification, is provided in
22 24
(1) 23 25
(2)
9 14 15
3 5 8
4 7
squalene lanosterol HO
O
monooxygenase synthase
squalene 2,3-oxidosqualene lanosterol
sterol C 24
(3) methyltransferase
28
(5) (4) 24
HO sterol C14 HO sterol C14 HO

reductase demethylase
4,4-dimethylfecosterol 4,4-dimethylergosta- 24-methylenedihydrolanosterol
8,14,24(28)-trien-3ß-ol (eburicol)
sterol C 4 methyloxidase
(6a) sterol C 3 dehydrogenase
(7a)
(6b)
sterol C4 methyloxidase
O sterol C3 HO sterol C3 dehydrogenase O
ketoreductase
4a-methylfecosterone 4-methylfecosterol fecosterone
sterol
(7b)
C 3-ketoreductase
(9) (8)
sterol C5
HO sterol Δ8-Δ7-isomerase HO
desaturase HO
Δ5,7,24(28) ergostatrienol episterol fecosterol
(10) sterol C22 desaturase
(11)
sterol Δ24(28)
HO reductase HO
Δ5,7,22,24(28) ergostatetraenol ergosterol
Figure 19.2 Simplified pathway of ergosterol biosynthesis in

most Ascomycetes and Basidiomycetes, indicating the sites
blocked by SBI fungicides. Further information is provided in
Table 19.4 and in the text.
Table 19.2 Enzymes involved in fungal sterol biosynthesis

and targets of agricultural fungicides.
Step Enzyme Gene, other enzyme Agricultural inhibitors

no. designations
1 Squalene mono-oxygenase Erg 1, squalene epoxidase Target of G4 inhibitors such

oxidosqualene synthase as allylamines; side target of
some amines (G2)
2 Lanosterol synthase Erg 7, oxidosqualene cyclase Side target of some amines
(G2)
3 Sterol C24 methyl Erg 6, sterol methyl –
transferase transferase
4 Sterol C14 demethylase Erg 11, CYP51lanosterol Target of the DMI
14α-demethylase fungicides (G1)
5 Sterol C14 reductase Erg 24, sterol 14 reductase Main target of fenpropidin
and spiroxamine (G2)
6 Sterol C4 methyloxidase Erg 25 –
Sterol C3 dehydrogenase Erg 26, sterol C4
decarboxylase
7 Sterol C3 ketoreductase Erg 27 Target of hydroxyanilides
(G3)
8 Sterol 8 −7 isomerase Erg 2, sterol C-8 isomerase Main target of tridemorph
(G2)/secondary target of
other amines (G2)
9 Sterol C-5 desaturase Erg 3, C5 dehydrogenase –
10 Sterol C22 desaturase Erg 5, ergosterol 22 –
desaturase
11 Sterol 24(28) reductase Erg 4, 24-methylene sterol –
(24(28))-reductase
Step numbers are those shown in Figure 19.2.
Table 19.3. As described in Chapter 12 of this book [24], the major purpose of this
classification is to facilitate resistance management at the farmer’s level. The data
in Table 19.3 highlight the need to derive a simple and clear system to distinguish
the mode of action classes that are, at the same time, cross-resistance classes. For
example, the DMI group includes fungicides belonging to five different chemical
classes, although all of these have the same biochemical target in common. A
general (although mostly not complete) cross-resistance within the DMI fungicide
group must be considered, and is indicated by the common FRAC code number.
No cross-resistance has been found between different SBI classes, for example,
between DMIs and amines. Accordingly, in some countries (e.g., in the United
States) the FRAC codes form part of the label information and are the basis of
resistance management programs, as in the UK.
Only the first three SBI classes have practical importance in plant pro-
tection. Squalene epoxidase inhibitors, although used as antimycotics in
Table 19.3 Grouping of SBI fungicides in the FRAC classification.
FRAC codes G: Sterol biosynthesis inhibitors
G1 G2 G3 G4
Group name De-methylation Amines (formerly Hydroxy-anilides Squalene

inhibitors: ‘‘morpholines’’) epoxidase
(DMIs) inhibitors
SBI class I II III IV
Target in sterol Sterol C14 14 Reductase 3-Keto reductase Squalene
biosynthesis demethylase and 7 → 8 epoxidase
isomerase
Chemistry Piperazines Morpholines Hydroxyanilides Thiocarbamates
Pyridines Piperidines Allylamines
Pyrimidines Spiroketalamines
Imidazoles
Triazoles
pharmaceutical applications, have until now not been launched as plant-protection

fungicides.
19.2
SBI Class I: DMI Fungicides
In 1973, while investigating the mode of action of triarimol, Ragsdale and Sisler
[25] first identified fungal sterol biosynthesis in Ustilago maydis as the target of
this pyrimidine derivative. Yet, triarimol – which is chemically closely related to
fenarimol – has never reached the market stage (see Table 19.4). Later, Ragsdale
[26] suggested that C14 demethylase was the most important target site within
sterol biosynthesis to be affected by triarimol. Today, C14 demethylase is the
common target of over 30 agricultural fungicides belonging to diverse chemical
classes grouped together under the designation DMIs.
The DMI fungicides are by far the most important mode of action class, based on
their economic importance within the SBI fungicides. Moreover, within the DMI
fungicides one chemical class – the triazoles – dominates not only by their market
share but also in the number of compounds that have reached market level (see
Figure 19.3). Another aspect becomes clear from Figure 19.3, namely that although
the first DMI fungicides, piperazines, and imidazoles, had been brought to market
level by the late 1960s, the introduction of new DMI compounds continued until the
early 2000s. During the past 15 years, further imidazoles and, primarily, triazoles
have also been presented. This extraordinarily long life-cycle of a fungicidal mode
of action class is unique within the specific fungicides, and provides an indirect
proof of the commercial viability of this fungicide class. Although, since the early
768
Table 19.4 Important piperazine, pyridine, pyrimidine, and imidazole compounds launched before 1990.
Structure Common Chemical class Example for Launch Distributor Patent number
name trade name(s) (year/company) (example)
Triforine Piperazines Saprol® 969 Celaflor DOS 1901421,

OHC Cl
Cl Boehringer/ 1968
N
Celamerck
H Cl
N
19 Sterol Biosynthesis Inhibitors
N
Cl
H
N
Cl
Cl CHO
Pyrifenox Pyridines Podigrol®, 1986 Syngenta EP 49854, 1980

N
Dorado® Hoffmann-
O LaRoche
N
Cl
Cl
Fenarimol Pyrimidines Rubigan®, 1975 Eli Lilly/ Gowan Fr 1.569940,
N N
Rimidin® Dow 1967
OH
Cl Cl
Imazalil Imidazoles Fungazil® 970 Janssen Janssen BP 1244530,
N
1969
N
O
Cl
Cl
Prochloraz Imidazoles Sportak® 1980 Bayer GB 1469772,

N Boots/FBC/ 1973
Cl Cl O N
Schering
N
O
Cl
Triflumizole Imidazoles Trifmine® 1987 Nippon Nippon DOS 2814041,

CF3
N Soda, Uniroyal soda 1978
N N
Cl O
19.2 SBI Class I: DMI Fungicides
769
35
30
25
No. of compounds
s
o le
20 i az
Tr
15
s
o le
10 s
ne az
i id s
id
s
Im ne
ne
im i
5 yr id
zi
yr
ra
P
pe
P
Pi
69 71 73 75 77 79 81 83 85 87 89 91 93 95 97 99 01 03 05
19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 20 20 20
Year
Figure 19.3 Market launch of members of different chemical DMI groups.
1980s, shifts of sensitivity to DMIs have been reported with some pathogens, such
as powdery mildews, an adapted resistance management and the introduction of
new compounds with a higher intrinsic activity has allowed maintenance of the
efficacy of DMI fungicides on an economically highly competitive level for over 30
years [14].
As several reviews on DMI fungicides have been produced [27–29], a short
overview on some of the more important compounds is provided here, with only
the more recent market introductions being treated in more detail.1)
19.2.1
Piperazines, Pyridines, Pyrimidines, and Imidazoles
Fungicides belonging to the piperazines, pyrimidines, and imidazoles were the

first of the DMI fungicides to enter the agricultural market. The first pyridine
compound, pyrifenox, was marketed in 1986. An overview of the most important
compounds launched before 1990 is provided in Table 19.5. One compound,
prochloraz, rapidly gained a dominant position during the 1980s for the control
of the cereal eyespot pathogens (Oculimacula yallundae and O. acuformis = Tapesia
yallundae and T. acuformis = Pseudocercosporella herpotrichoides), whereas the other
1) The pyridine-derived compound SYP-Z048 indication of the mode of action. Based

(3-[5-(4-chlorophenyl)-2,3-dimethyl-1,2- on the structure, however, the most
oxazolidin-3-yl]pyridine; CAS 847749-37-5) likely mode of action is DMI. Details of
from Shenyang Research Institute of separation of the diastereoisomers are
Chemical Industry, has been recently available in DOW EP1035122.
granted an ISO name without any
Table 19.5 Important triazole fungicides launched before 1990.
Structure Common Trade Launch Marketed Patent

name name (s) (year/ by number,
(example) company) (example) priority
Triadimefon Bayleton® 1976 Bayer DE

N N Bayer 02201063,
N 1972
Cl O O
Bitertanol Baycor®, 1979 Bayer DE

N N Sibutol® Bayer 2324010,
N (ST) 1973
O OH
Triadimenol Bayfidan®, 1980 Bayer DE

N N Baytan® Bayer 2324010,
N (ST) 1973
Cl O OH
Propiconazole Tilt® 1980 Janssen, DOS

Cl
Ciba-Geigy Ciba-Geigy 2551560,
N 1974
N
N
Cl O O
®
Penconazole Topas 1983 Syngenta DOS
Cl
N Ciba-Geigy 2735872,
N
N 1976
Cl
®
Flutriafol Impact 1984 Cheminova EP
N
N ICI/Zeneca 0015756,
F N 1979
OH
F
Structure Common Trade Launch Marketed Patent

name name (s) (year/ by number,
(example) company) (example) priority
Flusilazole Punch®, 1986 DuPont EP

N
Nustar® DuPont 0068813,
N N 1981
F Si
F
Hexaconazole Anvil® 1986 Syngenta EP
N ICI/Zeneca 0015756,
1979
N N
Cl HO
Cl
Myclobutanil Systhane® 1986 Dow US
Rohm and 4366165,
N Haas 1977
N
N
Cl
Tebuconazole Folicur®, 1988 Bayer EP 40345,
N
N
Raxil® Bayer 1980
N
OH
Cl
Cyproconazole Alto®, 1988 Syngenta, GB-A-21

N
N Sentinel® Sandoz Bayer 36423,
Cl N 1982
OH
O Cl
N
N
Cl
N
O O
Difenoconazole Score® 1989 Syngenta EP 65485,

Ciba-Geigy 1981
ST, seed treatment.

fungicides shown in Table 19.5 are used predominantly in broadleaved crops and
ornamentals against leaf spot and powdery mildew diseases.
19.2.1.1 Pefurazoate
In 1985, the Japanese companies Ube and Hokko jointly published a patent on
new imidazole derivatives that were said to be particularly useful for disinfecting
plant seeds [30].
From this patent application, a new fungicide with the common name pefurazoate
was developed (see Compound Table 19.1). This new imidazole seed treatment
fungicide exhibited an effective control of major rice seed-borne pathogens, includ-
ing bakanae disease (Fusarium moliniforme), brown spot (Cochliobolus miyabeanus),
and rice blast (Magnaporthe grisea) [31, 32]. Studies on the enantioselective antifun-
gal activity of the two enantiomers of pefurazoate revealed that the (S)-(−) isomer
exhibited much higher activity against Gibberella fujikuroi than the (R)-(+)-isomer
[33, 34].
Pefurazoate is a member of the imidazole class of DMIs. More specifically, unlike
most DMIs – where the nitrogen atom of the heterocycle is attached to an aliphatic
carbon – the nitrogen of the imidazole ring is linked to a carbonyl group, producing
a less flexible and less basic N-imidazolecarbonyl group than the imidazolylmethyl
counterpart in imazalil.
Pefurazoate can be synthesized [35] by the transesterification of methyl
2-bromobutyrate with 4-pentenyl alcohol, substitution of the bromine by
furfurylamine, and imidazolylcarbonylation of the amine by phosgene or
diphosgene and imidazole (Scheme 19.1).
The registered pefurazoate is a mixture of two enantiomers (C2 of the butanoic
acid backbone is chiral), the synthesis of which has been described [28] from chiral
2-aminobutanoic acid. The respective position of the furan and imidazole rings are
very different for each enantiomer, and might account for the observed differences
Compound Table 19.1
Structure/common name Launch (company, year) Melting point

Patent number (year) Water-solubility
trade Name (s) (example) Log POW
Marketed by (example) Vapor pressure
Ube, Hokko (1990) Decomposes at 235 ◦ C

O N
N EP00248086 (1985) 443 mg l –1 (25 ◦ C)
O Healseed®, Healthied® 3.0
N SDS Biotech 0.65 mPa (23 ◦ C)
O O
pefurazoate
O O
Br Br NH2 H
HO N
O O O O K2CO3, DMF
O O
N
O N
ClCOOCCl3 O
N 2
N TEA
N O O
H
Scheme 19.1 Synthesis of pefurazoate.
N
O N
O
N
O O
Scheme 19.2 Biologically most active enan-
(S )-(-) enantiomer of pefurazoate tiomer of pefurazoate.
in biological efficacy, the (S)-enantiomer (Scheme 19.2) being much more active
then the (R)-isomer.
19.2.1.2 Oxpoconazole
Oxpoconazole is a new imidazole derivative launched by Ube and Otsuka in 2000
(see Compound Table 19.2). The compound is used mainly in fruits under the
®
trade name All-Shine , but seems also to be suited to the control of diseases in rice
seedlings, such as Magnaporthe orzyzae and Rhizoctonia solani. The compound has
been developed as its fumarate salt. In addition, unlike most DMIs, oxpoconazole
seems to have a field efficacy against the gray mold pathogen, Botrytis cinerea
[36]. General reports on the synthesis and biological activities of oxpoconazole are
available from Morita and Nishimura [37] and from Li et al. [38]. Typical usage
rates in apples and peaches against Venturia spp., Monilinia spp., and Phomopsis
are around 0.01% active ingredient in the spray.
Oxpoconazole is the most recent example of a DMI that includes an
N-acylimidazole group like its predecessors prochloraz (1980) and pefurazoate
(1990; see above). Owing to the presence of the nonacylated nitrogen of the
imidazole ring, this compound is quite basic, and is sold as a fumarate salt.
The synthesis of the free base has been described from a 5-aryl-2-pentanone
[39], its keto group being transformed into a 1,3-oxazolidine with α, α-dimethyl
Compound Table 19.2

Trade name (s) (example) Log POW
Ube, Otsuka (2000) 123.6–124.5 ◦ C

JP07304774 (1994) 89.5 mg l−1 (25 ◦ C,
N
N pH 4)
O
All-Shine®, 3.69 (pH 7.5;
N Oh-Shine® 25 ◦ C)
O SDS Biotech –
O HO
OH
O
Cl
2
oxpoconazole fumarate
NH2
HO H
N Cl-CO-Cl
Cl O
Cl O
N
N
Cl
N
N 2
N O H
O
N
Cl O Cl
K2CO3 O
DMF
Scheme 19.3 Synthesis of oxpoconazole.
ethanolamine, after which the free NH reacts first with phosgene and second with
imidazole (Scheme 19.3).
The registered compound is a mixture of enantiomers (C2 of the oxazolidine
ring is chiral); however, the synthesis and properties of the enantiomers have not
been described.
19.2.2
Triazoles
19.2.2.1 Triazoles Launched Before 1990

Several of the well-known triazole-based DMI fungicides that were launched before
1990 still have a significant market importance, although recently newer triazoles
have largely taken over the leading position.
The first triazole to be introduced to the market, in 1976, was triadimefon, which
®
became rapidly known under its trade name Bayleton . This first representative
of the triazole group was considered as real progress at the time, because of
its excellent activity against powdery mildew and rust, combined with significant
activity against several leaf spot diseases.
A selection of important further triazoles is listed in Table 19.5. Besides triadime-
fon, Bayer introduced bitertanol and triadimenol during the early 1980s. As a foliar
fungicide, bitertanol offers good control of apple scab (Venturia inaequalis) and of
the Black Sigatoka pathogen in bananas (Mycosphaerella fijiensis); it is also used as
a seed treatment fungicide against snow mold (Microdochium nivale), and common
and dwarf bunt (Tilletia caries, T. controversa). Triadimenol was introduced initially
exclusively as a systemic seed treatment fungicide in cereals, but was later also
developed as a foliar fungicide in monocotyledonous and dicotyledonous plants.
®
Propiconazole, which is also well known under its trade name Tilt , is another
successful triazole offering a broader spectrum of activity and especially good leaf
spot activity in a multitude of crops such as cereals and banana. Owing to a specific
®
strength in the control of barley diseases, flusilazole (Punch ) from Du Pont was for
®
many years a dominant product in this crop. Penconazole (known as Topas ) was
specifically developed for broadleaf crops. Beside the simultaneous control of scab
and powdery mildew in apples, the product is used for the control of powdery mildew
and other diseases in grapes, fruits, and vegetables. Tebuconazole is a very success-
ful triazole that has found worldwide use due to its very broad fungicidal spectrum
of activity. The product was initially introduced in cereals under its trade name
®
Folicur , but it is used nowadays in a multitude of crops such as peanuts, bananas,
®
and soybeans as a foliar fungicide, as well as a cereal seed treatment (Raxil ).
19.2.2.2 Triazole Fungicides Launched Since 1990

Since 1990, a further 11 new triazoles have reached the market stage, and are
presented below in greater detail.
19.2.2.2.1 Tetraconazole Tetraconazole was the first azole fungicide to be

introduced by the Italian company Isagro Ricerca (formerly Montedison); today, it is
distributed by Isagro (see Compound Table 19.3). Information on the comparative
antifungal effects of tetraconazole have been summarized [40], while details of the
compound’s stereoselective interaction with C14 demethylase have been reported
by Gozzo et al. [41]. The vapor pressure of tetraconazole is comparatively high
(0.18 mPa), and this result – together with a pronounced systemicity – in a good
redistribution in plant tissue, although it may lead to evaporation losses at higher
Compound Table 19.3

Montedison (1991) 6 ◦C
F
F N EP0234 242 (1986) 156 mg l−1 (20 ◦ C, pH 7)
F Eminent® 3.56 (20 ◦ C)
F O N N Isagro 0.18 mPa
Cl
Cl
tetraconazole
temperatures. In cereals, application at 125 g a.i. ha−1 of tetraconazole (trade name

®
Eminent ) is used to control powdery mildew and rusts, as well as Stagonospora
nodorum and Septoria tritici [42]. Cercospora leaf spot (Cercospora beticola) in sugar
beets is another indication where tetraconazole shows promising results at rates of
about 100 g a.i. ha−1 [43, 44]. When Bianchi et al. examined the fungitoxicity of the
(R)-(+) and the (S)-(−) enantiomers of tetraconazole with a series of pathogens,
in all cases the (R)-(+)-enantiomer was shown to be more active [45].
The main reason why the structure of this triazole is so different is the presence
of the highly fluorinated side chain. Indeed, the structure of tetraconazole is also
unique because of the unusual position of the oxygen gamma to the triazole, instead
of beta. The synthesis of tetraconazole [46] involves a nucleophilic substitution of
the mesyl group of methyl 2-(2,4-dichlorophenyl)-3-mesylpropanoate by triazole,
followed by reduction of the ester to the alcohol by lithium aluminum hydride and
alkylation of the hydroxyl group so-obtained by tetrafluoroethylene (Scheme 19.4).
The final compound is a mixture of two enantiomers (C2 is chiral) (Scheme 19.5).
In 1991, the use of lipases to separate racemic mixture of a key synthon proved to be
the cornerstone of the synthetic scheme leading to each enantiomer [45]. In 2001,
the two enantiomers of tetraconazole were separated using β-cyclodextrin-mediated
capillary electrophoresis [47].
19.2.2.2.2 Fenbuconazole Fenbuconazole (see Compound Table 19.4) is a triazole

fungicide intended for use as an agricultural and horticultural fungicide spray for
the control of leaf spot, yellow and brown rust, powdery mildew, and net blotch on
wheat and barley and apple scab, pear scab, and powdery mildew on apples and
pears. Fenbuconazole was presented to the public in 1988, by Driant et al. [48].
N
OH
OSO2Me N
N
Cl MeSO2Cl
OMe Cl H
Ether, TEA OMe
Cl O K2CO3 Acetone
Cl O
N N N
N N N
N N N
LiAlH4 F2C = CF2
Cl Cl Cl F
OMe Ether OH NaH DMF O
F
Cl O Cl Cl F F
Scheme 19.4 Synthesis of tetraconazole.
N Scheme 19.5 Biologically most active enantiomer of

N tetraconazole.
N
Cl
OCF2CHF2
Cl
(R )-(+)enantiomer of
tetraconazole
Compound Table 19.4

Rohm and Haas (1991) 124–126 ◦ C

N
N DE03721786 (1986) 3.8 mg l−1 (25 ◦ C)
N
Cl Enable®, Indar® 3.23
N Dow AgroSciences 3.4 × 10−1 mPa (25 ◦ C)
fenbuconazole
®
Under its trade name Indar , fenbuconazole is one of the few fungicides
registered in the USA for the control of Mummy Berry, a blueberry disease caused
by Monilinia vaccinii-corymbosi. Further, the control of soybean rust (Phakopsora
®
pachyrhizi) with the product Enable is currently under investigation in the USA
and in Latin America.
CN Br
Cl Cl
N N
NaH/DMF 50%NaOH
LG CH2Br2
Cl
LG = Leaving group
+ N
Na − N
N N
X N
Cl Cl
N N
N 2
DMSO
X = Cl, Br X = Cl 92%
Scheme 19.6 Synthesis of fenbuconazole.
Like former triazoles developed by Rohm and Haas – fenapanil, and myclobu-
tanil – fenbuconazole is chemically characterized by its nitrile substituent on the
quaternary C2. Its synthesis [49] starts from the phenylethylation of phenylacetoni-
trile by e.g 1-(2-bromoethyl)-4-chlorobenzene (Scheme 19.6). A second alkylation
at the same carbon with dibromomethane then leads to a quaternary carbon still
bearing a reactive bromomethyl group. Fenbuconazole is obtained as a mixture of
enantiomers by nucleophilic substitution of the halogen by triazolyl sodium.
The compound on the market is a mixture of enantiomers (quaternary C2 is
chiral). To date, no reports have been made regarding the synthesis or the biological
properties of each enantiomer.
19.2.2.2.3 Epoxiconazole Epoxiconazole, which was presented to the public in

1990 (see Compound Table 19.5) [50, 51], is a broad-spectrum triazole fungicide
with a pronounced strength against cereal leaf spots and rust fungi, and a prolonged
duration of activity. Owing to the increasing importance of Septoria tritici since
the early 1990s in European wheat production, epoxiconazole has rapidly gained
a prominent role in the cereal fungicide market where it is used alone (trade
®
name: Opus ) at application rates of 90–125 g a.i. ha−1 , or in combination with
strobilurins and several other mixing partners. Detailed investigations on the uptake
and systemic translocation of epoxiconazole are available [52]. The nonfungicidal
effects of epoxiconazole on wheat plants have been studied in detail [53]. Beside
cereal applications, epoxiconazole can be used in other crops such as tea, sugar
beet and coffee, against a broad range of diseases.
Many synthetic methods for epoxiconazole have been described in BASF patents
[54]: six main N-1 intermediates have been reported, which are gathered in
Scheme 19.7 in chronological order, together with the patent application number
and its year of publication:
1) By nucleophilic substitution of the bromine atom of a bromomethyl group

with sodium triazolide.
Compound Table 19.5

Trade name(s) (example) Log POW
BASF (1992) 136 ◦ C

N
N EP196038 (1985) 6.6 mg l−1 (20 ◦ C)
N Opus® 3.33 or 3.44
BASF < 10−3 mPa
Cl O
F
epoxiconazole
2) By epoxidation of the corresponding double bond with maleic acid/hydrogen

peroxide.
3) By cyclization of an erythro-1-bromo-3-chloro-1,2-diaryl-2-propanol with sodium
triazolide.
4) By a Corey–Chaykovsky reaction on a precursor ketone with dimethylsulfo-
nium benzylide.
5) By nucleophilic substitution of the precursor mesylate with sodium triazolide
(ton scale).
6) By deprotection of N-aminotriazolium with sodium nitrite in hydrochloric acid.
Scheme 19.8 highlights a recent example via the mesylate intermedi-

ate (route 5 in the above list) [54g]: epoxidation of the double bond of
3-(2-chlorophenyl)-2-(4-fluorophenyl) acrylaldehyde is conducted with tert-butyl
peroxide under basic conditions. The reduction of the aldehyde to the correspond-
ing alcohol is started before the double bond is completely converted, in order
to minimize side products. The free hydroxyl group is activated by mesylation to
the mesyl derivative which, in turn, undergoes nucleophilic substitution with the
sodium salt of 1,2,4-triazole. The yield is 96.2% of the expected epoxyconazole.
The active ingredient in epoxyconazole is a mixture of the 2R,3S and 2S,3R
enantiomers (C2 and C3 are chiral), corresponding to the Z stereochemistry, which
means that both chlorophenyl and triazolylmethyl groups are on the same side
of the oxirane ring (Scheme 19.9). The preparation and biological efficacy of each
enantiomer has been demonstrated [55].
19.2.2.2.4 Triticonazole Triticonazole was presented in 1991 as a new specific

triazole for the control of cereal seed-borne and foliar diseases by seed treatment
application (see Compound Table 19.6) [56].
N
N
Br OH Cl N
Cl O
EP0431450 EP0515876
1990 1992
F 4 F
3
N N
N N
N OSO2Me
N
2 5
Cl Cl
Cl
O O
WO2002/094817
EP0427061
1990 F F F
H2N +
1 6 N
N
Br EP0196038 WO2004/000835 N
1986
Cl Cl
O O
F F
Scheme 19.7 Access to epoxiconazole.
O O OH
NaOH
H H MeSO2Cl
MeOH
tBuOOH O NaBH4 Cl O
Cl Cl
F PhMe F F
N
OSO2Me − + N
N
Na N
N
N
Cl O DMF
Cl O
F
F
Scheme 19.8 Synthesis of epoxiconazole.
When applied at rates of between 150 g a.i. (cereals) and 600 g a.i. (maize) per
100 kg of seeds, triticonazole was reported [57] to provide a good systemic control of
diseases such as Rhynchosporium secalis in barley or of corn head smut (Sphacelotheca
reiliana). Furthermore, spray applications with triticonazole showed activity against
N N
N N
N N
3 2 3 2
Cl O Cl O
F F
2R,3S 2S,3R
Scheme 19.9 Cis stereoisomers of epoxiconazole.
Compound Table 19.6

Rhone-Poulene (1992) 139–140 ◦ C

N
N
Cl EP 00378953 (1989) 7 mg l−1 (20 ◦ C)
N Charter®, Real® 3.29 (20 ◦ C)
HO Bayer, BASF <1 × 10−5 mPa (50 ◦ C)
triticonazole
several turf grass diseases. Other cereal diseases controlled via seed treatment, such
as rusts, Septoria tritici, powdery mildew, and the W-strains of eyespot (Oculimacula
yallundae), have been described [58].
At lower dose rates of about 5 g a.i. per 100 kg of seeds, triticonazole provides (in
®
products such as Kinto TS , mostly in combination with prochloraz) a good control
of smuts, bunts and other seed, and soil-borne diseases that are usually controlled
®
by DMI fungicides [59]. In products such as Rubin TT , triticonazole is used at
rates of 25 g a.i. ha−1 in combination with two other components. Good systemic
mobility is required for the systemic control of foliar diseases, and the results of
detailed studies on the uptake and distribution of triticonazole in wheat following
seed treatment have been reported [60, 61].
The synthesis of triticonazole is only described in patents as a one-pot
sequence [62]: the Knoevenagel condensation of 4-chlorobenzaldehyde on
2,2-dimethylcyclopentanone gives the α, β-unsaturated ketone, which enters a
Corey–Chaykovsky epoxidation reaction to afford the epoxide which, in turn, is
opened with the potassium salt of 1,2,4-triazole (Scheme 19.10).
O
O
O Cl CHO
Me3S + I −
O Cl
aq. NaOH, EtOH Cl HNa, THF
S
N
N
N N
N K2CO3 HO
N
H 1 E, > 95%
DMF
Cl
Scheme 19.10 Synthesis of triticonazole.
The commercially available triticonazole is a racemic mixture (C1 bearing the

hydroxy group is chiral) with an E double bond. To date, no reports have been made
on the preparation of the enantiomers and their biological properties.
19.2.2.2.5 Bromuconazole In 1990, Rhône-Poulenc presented a new triazole

fungicide, bromuconazole, to the public (see Compound Table 19.7) [63].
In cereals, bromuconazole provides a good control of eyespot (Oculimacula
yallundae), Microdochium nivale, and Fusarium head blight, in addition to those
diseases that are usually combated by triazoles, such as rusts, powdery mildew, and
Septoria tritici. Bromuconazole showed good potential in oilseed rape, vegetables,
and potatoes, where especially Alternaria diseases are well controlled. Various
other crops where the product has shown good control at rates of between 50 and
200 g ha−1 include fruits (Monilinia, Venturia), coffee, turf, and rice. Bromuconazole
as a cereal fungicide is mostly used at 133–200 g a.i. ha−1 [64].
Compound Table 19.7

Rhône-Poulenc (1992) 84 ◦ C
N
Cl N EP00258161 (1986) 50 mg l –1
N
Granit® 3.24
Cl Bayer CropScience 4 × 10−3 mPa (25 ◦ C)
O Br
bromuconazole
N N
N
(1) Mg, Et2O / THF Cl N Cl N
Cl N
H
Br Cl O
(2) K2CO3
Cl DMF
Cl HO Cl HO
Cl
N
Cl N
N
(1) Br2 CHCl3
(2) KOH, MeOH

2
Cl O
4 Br
Scheme 19.11 Synthesis of bromuconazole.
The only synthesis of bromuconazole to have been described [65], from

Rhône-Poulenc Agrochimie, involves three steps. The initial allylmagnesium bro-
mide adds to the carbonyl double bond of 2-chloro-1-(2,4-dichlorophenyl)ethanone
to afford the corresponding alcohol (Scheme 19.11). The aliphatic chlorine is then
substituted by the potassium salt of triazole and the tetrahydrofuran ring is formed
by an attack of the hydroxy group on the brominated double bond.
The compound contains two chiral centers (C2, C4), and exists as a mixture of
roughly equal proportions of two diastereomers (2RS,4RS : 2RS,4SR), each with two
enantiomers [66]. As a consequence, the three substituents of the tetrahydrofuran
are equally distributed up or down versus the heterocyclic ring. Notably, the two
diastereoisomers can be separated by using column chromatography.
19.2.2.2.6 Metconazole The triazole fungicide metconazole was invented by

Kureha, and is codistributed by BASF (see Compound Table 19.8). Metconazole
was first presented in 1992 [67], and to date its main markets are in cereals and
oilseed rape [68, 69].
In cereals, metconazole is one of the few triazoles to show a pronounced effect
against Fusarium head blight caused by a complex of pathogens such as Fusarium
culmorum and F. graminearum. As these Fusarium species can synthesize several
mycotoxins, such as desoxynivalenol (DON), their control has become of increasing
importance. The role of metconazole and of other fungicides such as tebuconazole
and prothioconazole in lowering the mycotoxin contents of cereal grains has,
therefore, been studied intensively [70–73].
Beside Fusarium head blight, metconazole controls the disease complex in
wheat and barley caused by rusts, Septoria, powdery mildew, Rhynchosporium, and
Drechslera species at low rates of between 60 and 90 g a.i. ha−1 . In oilseed rape, the
same rates are needed to combat Sclerotinia sclerotiorum, Phoma, Alternaria, and
other pathogens.
Three-dimensional modelings of C14 demethylase and its interaction with
metconazole have been reported [74, 75], as well as the results of a quantitative
Compound Table 19.8

Kureha (1993) 110–113 ◦ C or 100–108.4 ◦ C

N
Cl EP00329397 (1988) 15 mg l –1
N
N Caramba® 3.85 (20 ◦ C)
HO BASF, Kureha –
metconazole
structure–activity relationship (QSAR) study covering metconazole and ipconazole

[76].
The synthesis scheme is described in two process patents [77]: A
Dieckmann condensation applied to ethyl adipate leads to the salt of 2-ethoxy-
carbonylcyclopentanone (Scheme 19.12). From this compound, methylation
at position 2, followed by a rearrangement under basic medium, brings the
methyl group to position 5. The salt obtained is directly benzylated with 4-
chlorobenzyl chloride. A second methylation then occurs directly at position 5,
while a subsequent decarboxylation allows access to the key cyclopentanone with
all the required substituents present. The final step consists of a one-pot Corey–
Chaykovsky epoxidation reaction in which the cyclopentanone and the trimethyl-
sulfoxonium bromide are added successively to the triazolyl sodium formed
in situ.
Commercially available metconazole is a mixture of cis (1RS,5SR major) and
trans (1RS,5RS minor) isomers (C1 and C5 are chiral); this means that the hydroxy
and benzyl groups are on the same side of the cyclopentane ring (Scheme 19.13).
19.2.2.2.7 Ipconazole Ipconazole, which was launched by Kureha in 1993 (see

Compound Table 19.9), is a systemic fungicide that is suitable for the control of a
wide range of seed diseases in rice and other crops with seed treatment application.
® ® ® ®
Trade names are Tec-Lead , Techlead , Vortex , and Crusoe . Ipconazole is
particularly effective against Bakanae disease caused by Fusarium moniliforme [78],
Helminthosporium leaf spot, and blast (Magnaporthe grisea) on rice.
Information on the QSARs of the enantiomers of ipconazole, as well as
their fungicidal and plant growth-inhibitory activities, has been reported
[79, 80]. The QSARs and three-dimensional shape studies of fungicidal
azoylmethyl-cyclopentanols such as ipconazole and metconazole are also available
[77].
+
Na −
O O O O O
EtO NaOEt MeBr
OEt EtO EtO
Toluene
O DMF
+
Na
−
O O Cl
O
NaOEt Cl Cl
OEt
Toluene
DMF EtOOC
O
NaH MeBr O
Cl AcOH
Toluene Cl
DME H2SO4, H2O
EtOOC
cat. NaI
N
O N
N
Cl
N NaOH NMP 1) HO 1
N Cl
NH Toluene, BaO 2) Me3S(O)+Br − 5
Scheme 19.12 Synthesis of metconazole.
N Scheme 19.13 Metconazole, stereochemistry.

N
N
HO
Cl
(1R,5S ) metconazole
The synthesis scheme for ipconazole [81] (Scheme 19.14) is similar to that
described in Scheme 19.12 for metconazole. Isopropylation takes place at position
2 of the easily available 2-methoxycarbonylcyclopentanone, and a subsequent
rearrangement leads to the less-hindered isomeric 5-isopropyl. The same type of
condensation (without rearrangement) with the 4-chlorobenzyl chloride, followed
by decarboxylation, then gives rise to the key ketone precursor of ipconazole.
Here also, the one-pot final step employs a Corey–Chaykovsky epoxidation with
trimethylsulfoxonium bromide.
Commercially available ipconazole is a mixture of two diastereoisomers: 1RS,
2SR, 5RS and 1RS, 2SR, 5SR, which means that only four enantiomers are present
out of the eight that are theoretically possible. Clues about the ratio of isomers
Compound Table 19.9

Kureha (1993) 88–90 ◦ C

N
EP 00329397 (1988) 6.9 mg l –1 (20 ◦ C)
N
N Tec-Lead®, Vortex® 4.21 (25 ◦ C)
OH Kureha –
chiral
Cl
ipconazole
(1) NaH / DMF

O O
O O Cl Cl O
(1) NaH DMF/ iPrI
OMe Cl
OMe (2) HBr 47% aq.
(2) NaOMe/ MeOH
N
O N
Cl N
N NaOH NMP (1) HO
Cl
N (2) Me3S(O)+Br-
NH Toluene, BaO
Scheme 19.14 Synthesis of ipconazole.
of the three stereo centers (C1, C2, and C5) and their separation through chiral
column can be found in another Kureha patent [82]. To illustrate the position,
up or down, of the different substituents versus the cyclopentane ring, only one
enantiomer of each former pair is shown in Scheme 19.15.
N N N N
N N
HO 1 HO 1
5 5
2 2
Cl Cl
1R, 2S, 5R 1R, 2S, 5S
Scheme 19.15 Stereoisomers of ipconazole.

Compound Table 19.10

FBC/Schering (1993) 191.9–193.0 ◦ C

N
N EP00183458 (1984) 1.1 mg l –1 (pH 6.6)
N N
Castellan®, Galmano® 3.24 (20 ◦ C)
N Bayer, BASF 6.4 × 10 –6 mPa (20 ◦ C)
F
O
Cl Cl
fluquinconazole
19.2.2.2.8 Fluquinconazole Fluquinconazole, a quinazoline-based triazole fungi-

cide, was introduced to the public in 1992 [83] (see Compound Table 19.10). When
used as a foliar fungicide, fluquinconazole is particularly active against pome fruit
diseases such as Venturia inaequalis and Podosphaera leucotricha. Other diseases
controlled include powdery mildews, Monilinia spp., Cercospora spp., and rusts.
As a seed treatment, fluquinconazole protects cereal roots against take-all (Gaeu-
mannomyces graminis) at the rate of 75 g a.i. per 100 kg seeds [84]. Systemic efficacy
against infection by Puccinia and Septoria leaf diseases, as well as the control of
seed-borne diseases such as Tilletia spp. and Ustilago spp., has also been reported
[85, 86].
In contrast to the other triazoles, where there is a certain degree of flexibility
in the vicinity of the triazole heterocycle, fluquinconazole is based on a unique
rigid quinazolone. Furthermore, steric hindrance around the triazole is reinforced
by the presence of the aryl moiety in the ortho position. The synthesis depicted in
Scheme 19.16 is based on the general approach patented in EP0183458 on close
analogs bearing halogens other than fluorine [87]; fluquinconazole preparation
itself is not described per se in any patent. Starting from 5-fluoroanthranilic-amide,
the quinazolone ring is formed by interaction with 2,4-dichloroisocyanatobenzene
(Scheme 19.16). Subsequent treatment with phosphorus oxychloride and reaction
of the newly formed chlorine substituent with the potassium salt of 1,2,4-triazole
gives fluquinconazole.
19.2.2.2.9 Imibenconazole In 1988, Hokko presented to the public HF-6305, a

new triazole fungicide (see Compound Table 19.11) [88] that subsequently became
known by its common name imibenconazole, and which demonstrated a wide range
of activity against diseases in fruit, turf, vegetables, and ornamentals. In apples and
pears, imibenconazole was effective against scab (Venturia spp.), powdery mildew
(Podosphaera leucotricha), and rust. Furthermore, a good performance against grape
O
N
Cl H
(1) N Cl
NH2 N O
AcOEt POCl3
Cl
N N
NH2 F Pyridine F
F
(2) HCl, EtOH O
O O Cl Cl
Cl Cl
N
N N
N N N
N
H
N
K2CO3 CH3CN F
O
Cl Cl
Scheme 19.16 Synthesis of fluquinconazole.
powdery mildew, grape anthracnose (caused by Elsinoe ampelina) as well as the

control of citrus and peach scab, has been reported. With the exception of grape
powdery mildew, these diseases have been difficult to control with most other
triazole fungicides. Studies on the mode of action of imibenconazole have been
reported [89].
The early syntheses of imibenconazole, as reported in 1983 and 1984 [90],
require three steps from 2-chloro-N-(2,4-dichlorophenyl)acetamide (Scheme 19.17).
First, the amide group is transformed into imidoyl chloride with phosphorus
pentachloride, followed by displacement of the two nonaromatic chlorine atoms by
two equivalents of the potassium salt of 1,2,4-triazole. The triazolyl group borne by
the imino bond, being the best leaving group, is replaced when reacted with a good

Hokko (1994) 89.5–90 ◦ C

N
DE03238306 (1981) 1.7 mg l –1 (25 ◦ C)
N
Cl N Manage® 4.94
N Cl Hokko 8.5 × 10 –5 mPa (25 ◦ C)
S
Cl
imibenconazole
N
N
N N
Cl Cl Cl Cl N Cl N
H
N PCl5 N K2CO3, MeCN N
O Cl N N
Cl Cl Cl
N
N
N
N Cl
Cl N N
Cl N
HS
N Cl
N
N N MeCOiBu
Cl S
N Cl
Scheme 19.17 Synthesis of imibenconazole.
nucleophile such as (4-chlorophenyl) methanethiol. The stereochemistry (E or Z)

of the double bond was not reported.
19.2.2.2.10 Simeconazole Simeconazole (see Compound Table 19.12) was devel-

oped by Sankyo and presented to the public in 2000 as a new broad-spectrum
compound for seed treatment in cereals and rice [91]. In studies comparing the
systemic activity of simeconazole with other DMI fungicides, Tsuda et al. [92]
reported a prominent vapor-phase activity, a good translaminar movement, as well
as systemic activity against barley powdery mildew after soil drench application.
Accordingly, seed treatment with simeconazole achieves excellent efficacies against
Ustilago at 4–10 g a.i. per 100 kg seed. At high doses of 50–100 g a.i. per 100 kg
seed, simeconazole was also shown to be effective against Rhizoctonia cerealis,

Sankyo (2000) 118.5–120.5 ◦ C

N
EP00537957 (1991) 57.5 mg l –1 (20 ◦ C)
N
F
N Sanlit®, Mongarit® 3.2
Sankyo 5.4 × 10 –2 mPa (25 ◦ C)
OH
Si
simeconazole
N N N
F F N N
N + N MgCl SiMe3 F
Cl N N
− Na
1
MeCN MgBr2Et2O
Si
O O HO
Scheme 19.18 Synthesis of simeconazole.
Oculimacula yallundae, and Blumeria graminis on barley. In rice, simeconazole is

taken up by plants after submerged application; hence, it can provide a systemic
control of rice sheath blight (Rhizoctonia solani) [93].
The quite straightforward two-step synthesis [94–96] of simeconazole involves
substitution of the chlorine atom of α-chloro-4-fluoro-acetophenone by the sodium
salt of 1,2,4-triazole (Scheme 19.18). The subsequent addition of trimethylsilyl-
methylmagnesium chloride on the carbonyl double bond produces a mixture of the
two enantiomers of simeconazole. Whilst the two steps can be inverted, it appears
that the order depicted in Scheme 19.18 produces the best results.
The preparation of each enantiomer (C1 is chiral) has been described by a
modified route involving the addition of the same Grignard reagent on chiral esters
of phenylglyoxylic acid [97].
19.2.2.2.11 Prothioconazole The latest introduction into the DMI market, proth-
ioconazole, is unique among the triazole fungicides because its toxophore moiety
is a 1,2,4-triazole-3-thione (see Compound Table 19.13) [98, 99].
Introduced into the market in 2004 by Bayer CropScience, prothioconazole
rapidly gained market importance due to its broad spectrum of activity, covering
all important cereal diseases.
In cereal crops, prothioconazole is used at 200 g a.i. ha−1 as a solo product
®
(trade name: Proline ). In mixtures with fungicide partners such as fluoxastrobin
® ® ®
(Fandango ), spiroxamine (Input ), or tebuconazole (Prosaro ), prothioconazole
−1
is used at rates between 125 and 200 g a.i. ha . The disease spectrum controlled by
prothioconazole in wheat covers leaf spot diseases such as Septoria leaf spot (Septoria
tritici) and tan spot (Drechslera tritici-repentis), as well as rust (Puccinia triticina) and
powdery mildew (Blumeria graminis f.sp. tritici). Further, prothioconazole is one
of the rare azoles capable of providing excellent protection against Fusarium ear
blight caused by several Fusarium species [100].
One unique feature of prothioconazole is that it shows equally good
activity against both cereal eyespot species, Oculimacula yallundae (= Tapesia
yallundae = Pseudocercosporella herpotrichoides W-type) and O. acuformis (= Tapesia
acuformis = Pseudocercosporella herpotrichoides R-type), whereas all other triazoles
used against eyespot control only O. yallundae effectively. Surprisingly, in
cross-resistance studies prothioconazole revealed a unique profile, in that no
positive cross-resistance to either triazole-resistant or to prochloraz-resistant
isolates could be detected [101].

Bayer CropScience (2004) 139.1–144.5 ◦ C

H
N DE19528046 (1994) 5 mg l –1 (pH 4, 20 ◦ C)
N 300 mg l–1 (pH 8, 20 ◦ C)
S
Cl N Proline® 3.82 (pH 7)
Bayer CropScience 4 × 10−4 mPa (20 ◦ C)
Cl
HO
prothioconazole
In barley, prothiconazole provides a high level of efficacy against diseases such as

Rhynchosporium secalis and Drechslera teres, as well as good activity against powdery
mildew (Blumeria graminis f.sp. hordei) and barley leaf rust (Puccinia hordei).
In oilseed rape, all economically important pathogens such as Sclerotionia
sclerotiorum, Phoma lingam, and Pyrenopeziza brassicae fungi are controlled by
prothiconazole at 175 g a.i. ha−1 . Further crops where prothioconazole is under
development are peanuts and pulses, including peas, beans, and lentils. Beside
foliar applications, cereal seed treatment products containing prothioconazole at
rates <10 g a.i. per 100 kg seed either alone or in combination with fluoxastrobin,
® ®
tebuconazole, and other agents, are currently marketed (e.g., Redigo , Lamardor ,
® ® ®
Proseed , Scenic , EfA ).
A representative synthesis [102] of prothioconazole starts with an addition of the
Grignard derivative of 2-chlorobenzyl chloride to the carbonyl double bond of chloro
methyl-1-chloro-cyclopropyl ketone (Scheme 19.19). The untouched chlorine atom
of the chloromethyl group is then classically substituted with 1,2,4-triazole. From
this intermediate, one way to obtain the 2,4-dihydro-3H-1,2,4-triazole-3-thione of
prothioconazole is by a direct lithiation of the 1,2,4-triazole at position 5 with n-butyl
lithium, followed by reaction with sulfur. The commercially available compound
is a mixture of two enantiomers (chirality of the quaternary carbon bearing the
hydroxy group).
These enantiomers have been separated [103] using column chromatography with
a chiral auxiliary (N-methacryloyl-l-leucin-3-(2,4-dimethylpentyl)-amide) bound to
silica gel.
H
N
(1) Mg
N
Cl
N N
Cl N S
Cl O Cl Cl N
(2) n BuLi S8
Cl
Cl Cl
N THF
(3) N HO HO
N
H
Scheme 19.19 Synthesis of prothioconazole.
19.3
SBI Class II: Amines
19.3.1
Morpholines and Piperidines
The first SBI fungicides to be introduced as agricultural fungicides were, chemically,

morpholines such as dodemorph and tridemorph, which had already entered the
market during the late 1960s (Table 19.6). Whereas, dodemorph was mainly
developed in ornamentals, tridemorph gained importance in cereal crops, and
bananas [104].
Owing to resistance problems of cereal powdery mildews toward triazole fungi-
cides during the mid-1980s, the morpholine fenpropimorph and the piperidin
fenpropidin gained rapidly in importance as partners for the resistance manage-
ment of triazole fungicides, since amines are not cross-resistant to DMIs. The latest
introduction within the amine group is spiroxamine from Bayer, which is a repre-
sentative of a new chemical class within fungicidal amines, the spiroketalamines.
19.3.2
Biochemical Targets of Amines
Amines are SBI fungicides that inhibit several targets within fungal sterol biosyn-
thesis. Comprehensive reviews on the mechanism of action of cyclic amines
launched until 1995 have been provided [105, 106].
The results of the studies conducted demonstrate that each individual molecule
shows a unique profile with regards to the strength of inhibition at the different
targets sites.
In addition to the compound-dependent inhibitory profile it is found that, in
the different target pathogens, different inhibition profiles can be identified that
are characteristic of the individual species. Accordingly, the dominant site of
inhibition after tridemorph application is the 8 → 7 isomerase, whereas with
fenpropimorph it is predominantly the 14 reductase that is targeted, and the
8 → 7 isomerase is inhibited only at higher concentrations. With fenpropidin,
predominantly 14 reductase besides 8 → 7 isomerase is inhibited [107]. At
794
Table 19.6 The most important morpholine and piperidine compounds launched before 1990.
Common Chemical Trade Launch (com- Marketed by Patent/

name class name(s) pany/year) (example) priority
(example)
Tridemorph Calixin® Morpholines BASF 1969 BASF GB 00988630,

1961
H3C
n
N O
n = 10 to 13
n = 12 : ~70%
Fenpropimorph Corbel® Morpholines BASF 1980 BASF DE 02656747,
N 1976
O
Fenpropidin Patrol® Piperidines Hoffmann- Syngenta DE 02752135,

N
LaRoche, 1976
1985
high concentrations, the accumulation of squalene and 2,3-oxidosqualene indicates

also an inhibitory effect at the earlier steps of sterol biosynthesis.
The complicated and manifold picture of target sites affected by amines was
enlarged by Tiemann et al. [108], using spiroxamine. Although, in general, the
biochemical profile of spiroxamine is similar to that of fenpropidin, Tiemann and
coworkers found that all four isomers of spiroxamine were active on 14 -reductase,
but that other target sites became apparent when the four stereo isomers of
spiroxamine were tested separately. An inhibition of the 8 → 7 isomerase could
be demonstrated for the B/S and B/R isomers. Secondary effects on squalene
mono-oxygenase by the A/S isomer, and on lanosterol synthase by the A/R isomer,
were detected, although these activities were reported to vary in intensity according
to the test fungus.
19.3.3
Spiroxamine: The First of the Spiroketalamines
The first representative of the new chemical class of spiroketalamines within the
amine fungicide group – spiroxamine – was introduced to the public in 1996 see
(Compound Table 19.14) [109]. The chemistry and stereochemistry of spiroxam-
ine have been described by Krämer et al. [110, 111], while the biological spectrum
has been characterized by Dutzmann [112].
Similar to other amine fungicides, spiroxamine is applied in mixtures with
other fungicide partners for the control of powdery mildew in cereals at rates
® ®
of 375 g a.i. ha−1 under the trade names Input and Pronto Plus . Beside a
pronounced preventive, curative, and eradicative activity against powdery mildew,
spiroxamine is also effective against cereal rusts (Puccinia spp.), net blotch (Drech-
slera teres); side effects against Septoria tritici and Stagonospora nodorum are also
reported. In addition to its use in cereals, spiroxamine has other important fields of

Bayer (1997) (Liquid)

O
DE03735555 1987 470 mg l –1 diastereomer A;
O N 340 mg l –1 diastereomer B
(20 ◦ C, pH 7)
spiroxamine
Impulse® 2.79 diastereomer A; 2.98
diastereomeric mixture diastereomer B (20 ◦ C,
cis (dia A): trans (dia B) pH 7)
cis 49-56%, trans 51-44% Bayer CropScience 9.7 × 10−3 Pa (mixture at
20 ◦ C)
HO
H N
HO
5O
Cl O
APTS O O
O Toluene
2 N
Cl
Scheme 19.20 Synthesis of spiroxamine.
O
O N
O
N
O
A form, 2 enantiomers B form, 2 enantiomers
Scheme 19.21 Diastereomers of spiroxamine.
application, in grapes against powdery mildew (Erysiphe necator) at rates between

200 and 400 g a.i. ha−1 , and in bananas against the Black Sigatoka pathogen, My-
cosphaerella fijiensis at 320 g a.i. ha−1 . In grapes, spiroxamine is the only amine
representative with a registration in all major vine-producing countries, due to its
favorable plant selectivity.
The synthesis of spiroxamine involves the formation of a ketal of 4-tert-butyl cyclo-
hexanone with racemic 3-chloro-1,2-propanediol, and substitution of the chlorine
with ethylpropylamine, as described by W. Krämer and coworkers (Scheme 19.20).
The currently available compound is a mixture of two diastereoisomers (chiral
C2 and C5), in the ratio A 49–56%/B 51−44%.
The structures of the diastereoisomers A and B are shown in Scheme 19.21.
Notably, a report from W. Krämer et al. [110] also included the preparation and
description of the biological properties of all four enantiomers.
19.4
SBI Class III: Hydroxyanilides
19.4.1
Fenhexamid: The First of the Hydroxyanilides
During a Bayer synthesis program directed toward photosynthesis (PS) com-

plex II inhibitors as herbicides, the biological activity of synthetic intermediates
as well as of target molecules was tested also against fungi and insects. Sur-
prisingly, the 4-hydroxy-3,5-dichloro-anilides showed a weak, but stable, in vitro
and in vivo activity against Botrytis cinerea in the test systems (Scheme 19.22).
1,4-Hydroxyanilides proved to be of particular interest as the starting point for
chemical research since, depending on the properties of their aromatic sub-
stituents, these molecules can be easily degraded and therefore (at least potentially)
H Scheme 19.22 Hydroxyanilides lead structure

Cl N
O
HO
Cl
Lead structure
z Scheme 19.23 General structure of

1,4-hydroxyanilide fungicides.
N R
X Y2 Y
O
Y1
Particularly active:
X = CO-R1, H
Y1, Y2 = Halogen
Z = H
Y = O
R = tert-Cycloalkyl, tert-Haloalkyl
should have a very favorable toxicological profile and favorable environmental

behavior (Scheme 19.23).
By adapting the aromatic substitution pattern, in particular by the introduction of
chlorine atoms in positions 2 and 3, and by the incorporation of tertiary carboxylic
acids of a certain size as the carboxy part of the molecule (e.g., 1-alkylcycloalkanoyl
or halogen-substituted pivaloyl), highly active botryticides with additional activities
against different other fungi were obtained [113].
The optimum activity was reached in a novel hydroxyanilide with the chemical
name N-(2,3-dichloro-4-hydroxyphenyl)-1-methylcyclohexanecarboxamide; this was
presented by Bayer in 1997 as a new specific botryticide with the common name,
fenhexamid (see Compound Table 19.15) [114, 115].

Patent number (year) Trade Water-solubility Log POW
name(s) (example) Marketed Vapor pressure
by (example)
Bayer (1998) 153 ◦ C

OH
O
EP0339418 (1988) 20 mg l –1 (pH 5–7, 20 ◦ C)
N Cl Teldor®, Elevate® 3.51 at 20 ◦ C (pH 7)
H
Cl Bayer CropScience, Arysta 4 × 10 –7 Pa at 20 ◦ C
LifeScience (extrapolated)
fenhexamid
Cl Cl Cl
Cl Cl Cl
HNO3 Reduction
O2N HOHN
Cl
Cl
Cl OH
Cl OH H 3C O
Toluene, NaOH
O N
H 2N H3 C H
Cl
Fenhexamid
last 3 steps 70%
Scheme 19.24 Synthesis of fenhexamid.
The amide can be easily prepared in high yield and high purity by the reaction of
2,3-dichloro-4-hydroxy-aniline and 1-methylcyclohexane-carboxylic acid chloride,
for example, in toluene with sodium hydroxide as a base (Scheme 19.24). The
starting aniline can be prepared with a Bamberger rearrangement of the interme-
diate hydroxylamine itself obtained by partial reduction of the corresponding nitro
aromatic [116].
19.4.2
Biochemical Target of Fenhexamid
Although it became clear at an early stage during cross-resistance studies that

fenhexamid was the first representative of a new mode of action class, the exact
biochemical target was unknown when the new botryticide was launched in 1998.
In 2001, the research group of Pierre Leroux at the Institut National de la Recherche
Agronomique (INRA) Versailles identified fenhexamid as the first member of a
new class of SBI fungicides [117].
Subsequently, further studies conducted by the same group confirmed the origi-
nal findings [118]. Starting from the observation that the three 3-keto compounds
4α-methylfecosterone, fecosterone, and episterone accumulated after fenhexamid
application, Debieu et al. concluded that fenhexamid is a specific inhibitor of 3-keto
reductase in fungal sterol biosynthesis, an enzyme involved in the C-4 demethy-
lation. Details of the sites of inhibition within the sterol biosynthesis pathway are
indicated in Figure 19.4.
19.4.3
Biology
Fenhexamid is one of the rare SBI fungicides with a quite narrow spectrum of
biological activity. In vitro, it shows excellent activity against Botryotinia fuckeliana
(anamorph: Botrytis cinerea) and most other Botrytis species. Furthermore, the
Cycle I: 4,4-dimethylfecosterol to 4-methylfecosterol
C4-methyloxidase C3-dehydrogenase C3-ketoreductase
34 34 34 34
HO NADPH, O2 HO NADP+ O HO
COOH NADPH
4,4-dimethyl 4-carboxy,4-methyl 4 a-methyl 4 a-methyl

fecosterol fecosterol fecosterone fecosterol
4 a-methyl
episterone
Cycle II: 4-methylfecosterol to fecosterol
C4-methyloxidase C3-dehydrogenase C3-ketoreductase
34 34
34 34
HO NADPH, O2 HO NADP+ O NADPH HO
COOH
4 a-methyl 4-carboxyfecosterol fecosterone fecosterol

fecosterol
episterone
Figure 19.4 Interference of fenhexamid in fungal sterol

biosynthesis according to Debieu et al. The dotted frames
indicate accumulating sterones.
related taxon groups Sclerotinia and Monilinia are affected at low concentrations.
A broad, but moderate, activity against other fungi belonging to the Ascomycetes
and Basidiomycetes becomes visible only at distinctly higher concentrations under
in vitro conditions. As with other SBI fungicides, no activity against Oomycete
pathogens can be detected. In good correlation with the in vitro results is the profile
of use under field conditions. Fenhexamid is used at dosages ranging from 375 to
1000 g a.i. ha−1 in grapes, berries, stone-fruits, citrus, vegetables, and ornamentals
against Botrytis cinerea and the related pathogens Monilinia spp. and Sclerotinia
sclerotiorum [119].
The main target pathogen of fenhexamid, Botrytis cinerea, belongs to the high-risk
pathogens in view of its ability to develop resistance against fungicides. Accordingly,
800
Table 19.7 Squalene epoxidase inhibitors.
Common name Chemical Trade Launch (year) Patent number

class name(s)a company
Terbinafine Allylamines Lamisil® 1991 Novartis, EP 00024587,

Sandoz 1979
Naftifine Allylamines Naftin® Merz Phar- –

maceuticals
N
Pyributicarb Thiocarbamates Toyocarb® 1990 Toyo JP 85067463,

O N N O Soda 1984
S
References 801
intensive studies have been carried out during the pre-market period to clarify
eventual risks. The studies revealed that, even before its market introduction, a
small proportion of the population was able to metabolize fenhexamid under in
vitro conditions [120]. However, as demonstrated by Suty et al., the metabolism took
place only within long periods of undisturbed growth under optimal conditions. As
these requirements are clearly not fulfilled under outdoor conditions, the practical
importance of this resistance mechanism is low. In general, these tendencies have
been confirmed by the group of Pierre Leroux at INRA, Versailles [121]; detailed
studies conducted by the same group also led to a differentiation between three
groups with specific resistances to fenhexamid, namely Hyd R1 to Hyd R3.
19.5
SBI Class IV: Squalene Epoxidase Inhibitors
SBI class IV includes squalene epoxidase inhibitors that are actually not used as
agricultural fungicides. Fungal squalene epoxidases are only very distantly related
to their mammalian and higher plant counterparts in the phylogenetic tree [122],
and are therefore principally suited as targets of selective antimycotics as well as of
herbicides.
Inhibitors of squalene epoxidase belonging to two different chemical classes are
listed in Table 19.7. The allylamines consist of two compounds used as antimycotics
against a wide range of fungi. Terbinafine is used in topical and oral uses, whereas
naftifine is restricted to topical uses.
Pyributicarb is a systemic herbicide that is absorbed by the roots, leaves, and stem
and translocated to active growth sites, where it inhibits elongation of roots and
aerial plant parts. It is mainly used in rice and turf against annual and perennial
grass weeds, such as Echinochloa.
References
1. Windaus, A. (1928) Nobel Lecture, 6. Adl, S.M. et al. (2005) J. Eukaryot.

www.nobelprize.org/chemistry/laureates/ Microbiol., 52, 399–451.
1928/windaus-lecture.pdf. 7. Warner, S.A., Eierman, D.F., Sovocool,
2. Weete, J.D. (1980) Lipid Biochemistry G.W., and Domnas, A.J. (1982) Proc.
of Fungi and other Organisms, Plenum, Natl Acad. Sci. USA, 79, 3769–3772.
New York. 8. Madoui, M.A, Bertrand-Michel, J.,
3. Pasanen, A.L., Yli-Pietilä, K., Pasanen, Gaulin, E., and Dumas, B. (2009) New
P., Kalliokoski, P., and Tarhanen, J. Phytol.,, 183 (2), 291–300.
(1999) Appl. Environ. Microbiol., 65, 9. Fungicide Resistance Action Commit-
138–142. tee (FRAC) (2010) FRAC Code List:
4. Stahl, P.D. and Parkin, T.B. (1996) Soil Fungicides Sorted by Mode of Action.
Biol. Biochem., 28, 847–855. Available at: www.frac.info.
5. Erwin, D.C. and Ribeiro, O.K. (1996) 10. Cools, H.J., Fraaije, B.A., and Lucas
Phytophthora Diseases Worldwide, APS J.A. (2005) Proceedings of the BCPC
Press, St Paul, MN. Congress, Crop Science and Technology,
vol. 1, British Crop Protection Council, 24. Kuck, K.H. and Gisi, U. (2007) FRAC
Alton, pp. 267–274. Mode of Action Classification and
11. Leroux, P., Albertini, C., Gautier, A., Resistance Risk of Fungicides.
Gredt, M., and Walker, A.-S. (2007) 25. Ragsdale, N.N. and Sisler, H.D. (1973)
Pest Manage. Sci., 63 (7), 688–698. Pestic. Biochem. Physiol., 3, 20–29.
12. Cools, H.J., Parker, J.E., Kelly, D.E., 26. Ragsdale, N.N. (1975) Biochim. Biophys.
Lucas, J.A., Fraaije, B.A., and Kelly, Acta, 380, 81–96.
S.L. (2010) Appl. Environ. Microbiol., 76 27. Kuck, K.H., Scheinpflug, H., and
(9), 2866–2872. Pontzen, R. (1995) in Modern Selective
13. Leroux, P. and Walker, A.S. (2011) Pest Fungicides – Properties, Applications,
Manage. Sci., 67, 44–59. Mechanisms of Action (ed. H. Lyr), Gus-
14. Kuck, K.H. (2002) in Modern Fungicides tav Fischer Verlag, Jena, Stuttgart, New
and Antifungal Compounds III (eds York and Wiley-VCH Verlag GmbH,
H.W. Dehne, U. Gisi, K.H. Kuck, P.E. Deerfield Beach, pp. 205–258.
Russell, and H. Lyr), Agroconcept, 28. Green, M.B. and Spilker, D.A. (eds)
Bonn, pp. 21–28. (1986) Fungicide Chemistry, ACS Sym-
15. Phillips McDougall (2005) The posium Series, Vol. 304. American
Global Crop Protection Market; 2004 Chemical Society, Washington, DC.
Overview. ISSN: 0097-6156.
16. Berg, D. and Plempel, M. (eds) (1988) 29. Kuck, K.H. and Scheinpflug, H. (1986)
Sterol Biosynthesis Inhibitors: Phar- in Chemistry of Plant Protection, Sterol
maceutical and Agrochemical Aspects, Biosynthesis Inhibitors and Anti-Feeding
Wiley-VCH Verlag GmbH, Cam- Compounds, vol. 1 (eds G. Haug and
bridge, New York, Basel and Horwood, H. Hoffmann), Springer-Verlag, Berlin,
Chichester. Heidelberg, New York, Tokyo, pp.
17. Mercer, E.I. (1984) Pestic. Sci., 15, 65–96.
33–155. 30. Patent EP 0,248,086 (1985) Ube Indus-
18. Vanden Bossche, H. (1988) in Sterol tries Ltd, Hokko Chemical Industry Co.
Biosynthesis Inhibitors: Pharmaceutical Ltd.
and Agrochemical Aspects (eds D. Berg 31. Takenaka, M. and Yamane, I. (1990)
and M. Plempel), Wiley-VCH Verlag Jpn. Pestic. Inform., 57, 33–35.
GmbH, Cambridge, New York, Basel 32. Wada, T., Kuzuma, S., Takenaka,
and Horwood, Chichester, pp. 79–119. M., and Hirota, Y. (1991) Nippon
19. Sisler, H.D. and Ragsdale, N.N. (1977) Shokubutsu Byori Gakkaiho, 57,
Neth. J. Plant Pathol., 83, 81–91. 153–159.
20. Lürssen, K. (1988) in Sterol Biosynthesis 33. Takenaka, M., Kimura, S., Tanaka, T.,
Inhibitors: Pharmaceutical and Agro- and Wada, K. (1992) J. Pestic. Sci., 17,
chemical Aspects (eds D. Berg and M. 205–211.
Plempel), Wiley-VCH Verlag GmbH, 34. Takenaka, M., Nishimura, T., and
Cambridge, New York, Basel and Hayashi, K. (2001) J. Pestic. Sci., Nippon
Horwood, Chichester, pp. 305–320. Noyaku Gakkaishi, 26, 347–353.
21. Buchenauer, H. (1995) in Modern Se- 35. Patent JP 02,062,871 (1988) and Patent
lective Fungicides (ed. H. Lyr), Gustav JP 2,062,863 (1988), Ube and Hokko.
Fischer Verlag, Jena, Stuttgart, New 36. Hayashi, K. (2003) ABC and MFS
York and Wiley-VCH Verlag GmbH, transporters from Botrytis cinerea in-
Deerfield Beach, pp. 259–290. volved insensitivity to fungicides and
22. Burden, R.S., Cooke, D.T., and natural toxic compounds. University
Carter, G.A. (1989) Phytochemistry, Dissertation, No. 3459,Wageningen.
28, 1791–1804. 37. Morita, T. and Nishimura, T. (2001)
23. Sisler, H.D., Ragsdale, N.N., and Agrochem. Jpn., 79, 10–12.
Waterfield, W.W. (1984) Pestic. Sci., 15, 38. Li, Z., Guan, A., and Liu, C. (2002)
167–176. Xiandai Nongyao, 1 (4), 31–33.
References 803
39. Patent EP 0,412,681 (1989) and Patent Plantes ANPP, December 3–5, Bordeaux,
JP 2000/159,766 (1998), UBE Indus- ANPP, Bordeaux, pp. 941–948.
tries. 57. Mugnier, J., Chazalet, M., Gaulliard,
40. Carzaniga, R., Carelli, A., Farina, G., J.M., Anelich, R.Y., and Gouot, J.M.
Arnoldi, A., and Gozzo, F. (1991) Pestic. (1994) Proc. Brighton Crop Prot.
Biochem. Physiol., 40, 274–283. Conf. – Pests Dis., 325–330.
41. Gozzo, F., Carelli, A., Carzaniga, R., 58. Gauillard, J.M. and Peron, L. (1993)
Farina, G., Arnoldi, A., Lamb, D., and Phytoma, 454, 57–59.
Kelly, S. (1995) Pestic. Biochem. Physiol., 59. Mugnier, J., Gouot, J.M., Hutt, H.,
53, 10–22. Greiner, A., Chazalet, M., Gauillard,
42. Huraux, M. and Prove, P. (1992) Phy- J.M., and Ingram, G. (1993) Meded.
toma, 443, 69–70. Fac. Landbouwkd. Toegep. Biol. Wete.
43. Uchino, H. and Watanabe, H. (1999) (Univ. Gent.), 58, 1411–1419.
Tensai Kenkyu Kaiho, 41, 80–84. 60. Querou, R., Euvrard, M., and Gauvrit,
44. Khan, M.F.R. and Smith, L.J. (2005) C. (1997) Pestic. Sci., 49, 284–290.
Crop Prot., 24, 79–86. 61. Querou, R., Euvrard, M., and Gauvrit,
45. Bianchi, D., Cesti, P., Spezia, S., C. (1998) Pestic. Sci., 53, 324–332.
Caravaglia, C., and Mirenna, L. (1991) 62. Patent FR 2,663,196 (1990),
J. Agric. Food Chem., 39, 197–201. Rhône-Poulenc Agrochimie.
46. Patent EP 0,234,242 (1986), Montedi- 63. Pepin, R., Greiner, A., and Zech,
son S.p.A. B. (1990) Proc. Brighton Crop Prot.
47. Wu, Y.S., Lee, H.K., and Li, S.F.Y. Conf. – Pests Dis., 439–446.
(2001) J. Chromatogr., A, 912, 64. Duroni, O., Gauillard, J.M., and Pepro,
171–179. F. (1992) Phytoma, 440, 43–44.
48. Driant, D., Hede-Hauy, L., Perrot, A., 65. Patent EP 0,258,161 (1986),
Quinn, J.A., and Shaber, S.H. (1988) Rhône-Poulenc Agrochimie.
Proc. Brighton Crop Prot. Conf. – Pests 66. Kenneke, J.F., Mazur, C.S., and
Dis., 33–40. Garrison, W.A. (2005) Abstracts of
49. Patent EP 0,711,775 (1995) and Patent papers, 230th ACS National Meeting,
US 880,990 (1986), Rohm and Haas. Washington, DC. American Chemical
50. Ammermann, E., Loecher, F., Lorenz, Society, Division of Agrochemicals.
G., Janssen, B., Karbach, S., and 67. Sampson, A.J., Cazenave, A.,
Meyer, N. (1990) Proc. Brighton Crop Laffranque, J.P., Jones, R.G.,
Prot. Conf. – Pests Dis., 407–414. Kumazawa, S., and Chida, T. (1992)
51. Sauer, R., Loecher, F., and Schelberger, Proc. Brighton Crop Prot. Conf. – Pests
K. (1990) Proc. Brighton Crop Prot. Dis., 419–426.
Conf. – Pests Dis., 831–836. 68. Laffranque, J.P., Thienpont, E., and
52. Akers, A., Köhle, H.H., and Gold, Garford, B. (1994) Phytoma, 459,
R.E. (1990) Proc. Brighton Crop Prot. 49–51.
Conf. – Pests Dis., 837–845. 69. Gilgenberg-Hartung, A. (1999) Gesunde
53. Siefert, F. and Grossmann, K. (1996) Pflanzen., 51, 55–57.
Gesunde Pflanzen., 48, 224–231. 70. Pirgozliev, S.R., Edwards, S.G., Hare,
54. (a) Patent EP 0,196,038 (1985); (b) M.C., and Jenkinson, P. (2002) Eur. J.
Patent EP 0,427,061 (1989); (c) Patent Plant Pathol., 108, 469–478.
EP 0,431,450 (1989); (d) Patent EP 71. Kang, Z., Huang, L., and Buchenauer,
0,515,876 (1991); (e) Patent WO H. (2001) Z. Pflanzenkr. Pflanz., 108,
2002/094,817 (2001); (f) Patent WO 419–432.
2004/000,835 (2002); (g) Patent WO 72. Paul, P.A., Lipps, P.E., Hershman,
2010/089,353 (2009); all from BASF. D.E., McMullen, M.P., Draper, M.A.,
55. Patent WO 2005/056,548 (2005), and Madden, L.V. (2008) Phytopathol-
Cheminova. ogy, 98, 999–1011.
56. Mugnier, J., Chazalet, M., and 73. Bradley, C.A., Adee, E.A., Ebelhar, S.A.,
Gatineau, F. (1991) Troisième Confèrence Grybauskas, A.P., Hollingsworth, C.R.,
Internationale sur Les Maladies des Kirk, W.W., McMullen, M.P., Milus,
E.A., Osborne, L.E., Ruden, K.R., and 90. Patent DE 3,238,306 (1981) and Patent
Young, B.G. (2009) Proceedings of JP 5,988,473 (1982), Hokko Chemical
the 2009 National Fusarium Head Industries.
Blight Forum, Orlando, Florida, p. 34. 91. Tsuda, M., Itoh, H., Wakabayashi, K.,
Available at: www.scabusa.org. Ohkouchi, T., Kato, S., Masuda, K.,
74. Ito, A., Sudo, K., Kumazawa, S., and Sasaki, M. (2000) Proceedings of the
Kikuchi, M., and Chuman, H. (2005) 2000 Brighton Conference, The British
ACS Symp. Ser., 892, 142–150. Crop Protection Council, Farnham,
75. Chuman, H., Ito, A., Saishoji, T., and UK, pp. 557–562.
Kumezawa, S. (1998) Nippon Noyaku 92. Tsuda, M., Itoh, H., and Kato, S. (2004)
Gakkaishi, 23, 330–335. Pest Manage. Sci., 60 (9), 875–880.
76. Chuman, H., Ito, A., Saishoji, T., and 93. Tsuda, M. and Kato, S. (2003) Kita-
Kumezawa, S. (1995) ACS Symp. Ser., nippon Byogaichu Kenkyukaiho, 54,
606, 171–185. 32–34.
77. Patent EP 1,308,432 (2001) and Patent 94. Itoh, H., Kajino, H., Tsukiyama, T.,
EP 0,655,443 (1993), both from Kureha Tobitsuka, J., Ohta, H., Takahi, Y.,
Kagaku. Tsuda, M., and Takeshiba, H. (2002)
78. Tateishi, H. and Chida, T. (2000) J. Bioorg. Med. Chem., 10, 4029–4034.
Gen. Plant Pathol., 66, 353–359. 95. Itoh, H., Yoneda, R., Tobitsuka, J.,
79. Saishoji, T., Ito, A., Kumazawa, S., and Matsuhisa, T., Kajino, H., Ohta, H.,
Chuman, H. (1998) Nippon Noyaku Hayashi, N., Takahi, Y., Tsuda, M., and
Gakkaishi, 23, 129–136. Takeshiba, H. (2000) Chem. Pharm.
80. Ito, A., Saishoji, T., and Kumazawa, S. Bull., 48, 1148–1153.
(1997) Nippon Noyaku Gakkaishi, 22, 96. Patent EP 0,609,099 (1993) and Patent
119–125. JP 3,007,258 (1993), Sankyo.
81. (a) Ito, A., Saishoji, T., and Kumazawa, 97. Itoh, H., Furukawa, Y., Tsuda, M.,
S. (1997) J. Pestic. Sci., 22, 119–125; and Takeshiba, H. (2004) Bioorg. Med.
(b) Patent EP 0,655,443 (1993), Kureha Chem., 12, 3561–3567.
Chemicals. 98. Mauler-Machnik, A., Rosslenbroich,
82. Patent EP 0,488,395 (1990), Kureha Ka- H.J., Dutzmann, S., Applegate, J., and
gaku. Jautelat, M. (2002) Proc. Brighton Crop
83. Russell, P.E., Percival, A., Coltman, Prot. Conf. – Pests Dis., 389–394.
P.M., and Green, D.E. (1992) Proc. 99. Jautelat, M., Elbe, H.L., Benet-Bucholz,
Brighton Crop Prot. Conf. – Pests Dis., J., and Etzel, W. (2004) Pflanz. Nachr.
411–418. Bayer, 57, 145–162.
84. Löchel, A.M., Wenz, M., Russell, P.E., 100. Dutzmann, S. and Suty-Heinze,
Buschhaus, H., Evans, P.H., Cross, S., A. (2004) Pflanz. Nachr. Bayer, 57,
Puhl, T., and Bardsley, E. (1998) Proc. 249–264.
Brighton Crop Prot. Conf. – Pests Dis., 101. Kuck, K.H. and Mehl, A. (2004) Pflanz.
89–96. Nachr. Bayer, 57, 225–235.
85. Wenz, M., Russell, P.E., Löchel, A.M., 102. Patent WO 99/19,307 (1997), Bayer
Buschhaus, H., Evans, P.H., and AG.
Bardsley, E. (1998) Proc. Brighton Crop 103. Patent DE 19,917,617 (1999), Bayer
Prot. Conf. – Pests Dis., 907–912. AG.
86. Bardsley, E.S., Davies, P.H., and 104. Pommer, E.H. (1995) in Modern Se-
Hopkinson, D. (2000) Proc. Brighton lective Fungicides (ed. H. Lyr), Gustav
Crop Prot. Conf. – Pests Dis., 877–882. Fischer Verlag, Jena, Stuttgart, New
87. Patent EP 0,183,458 (1984), FBC Lim- York and Wiley-VCH Verlag GmbH,
ited. pp. 163–183.
88. Ohyama, H., Wada, T., Ishikawa, H., 105. Kerkenaar, A. (1995) in Modern Se-
and Chiba, K. (1988) Proc. Brighton lective Fungicides (ed. H. Lyr), Gustav
Crop Prot. Conf. – Pests Dis., 519–526. Fischer Verlag, Jena, Stuttgart, New
89. Ogawa, Y. (1995) Agrochem. Jpn., 67, York and Wiley-VCH Verlag GmbH,
20–21. pp. 185–204.
References 805
106. Mercer, E.I. (1988) in Sterol Biosynthesis 115. Rosslenbroich, H.J., Brandes, W.,
Inhibitors: Pharmaceutical and Agro- Krueger, B.W., Kuck, K.H., Pontzen,
chemical Aspects (eds D. Berg and M. R., Stenzel, K., and Suty, A. (1998)
Plempel), Wiley-VCH Verlag GmbH, Proceedings of the 1998 Brighton Confer-
Cambridge, New York, Basel and ence, British Crop Protection Council,
Horwood, Chichester, pp. 120–150. Farnham, pp. 327–335.
107. Schneegurt, M.A. and Henry, M. (1992) 116. Patent EP 0,569,792 (1992) and Patent
Pestic. Biochem. Physiol., 43, 45–62. EP 0,720,980 (1995), Bayer AG.
108. Tiemann, R., Berg, D., Krämer, W., 117. Debieu, D., Bach, J., Hugon, M.,
and Pontzen, R. (1997) Pflanz. Nachr. Malosse, C., and Leroux, P. (2001) Pest
Bayer, 50, 29–48. Manage. Sci., 57, 1060–1067.
109. Dutzmann, S., Berg, D., Clausen, N.E., 118. Leroux, P., Debieu, D., Albertini, C.,
Krämer, W., Kuck, K.H., Pontzen, Arnold, A., Bach, J., Chapeland, F.,
Fournier, E., Fritz, R., Gredt, M.,
R., Tiemann, R., and Weissmüller,
Hugon, M., Lanen, C., Malosse, C., and
J. (1996) Proc. Brighton Crop Prot.
Thiebaud, G. (2002) in Modern Fungi-
Conf. – Pests Dis., 47–52.
cides and Antifungal Compounds III
110. Krämer, W., Weissmüller, J., Gau, W.,
(eds H.W. Dehne, U. Gisi, K.H. Kuck,
Etzel, W., and Stelzer, U. (1997) Pflanz.
P.E. Russell, and H. Lyr), Agroconcept,
Nachr. Bayer, 50, 5–14.
Bonn, pp. 29–40.
111. Patent DE 3,735,555 (1987), Bayer AG. 119. Rosslenbroich, H.J. (1999) Pflanz.
112. Dutzmann, S. (1997) Pflanz. Nachr. Nachr. Bayer, 52, 131–148.
Bayer, 50, 21–28. 120. Suty, A., Pontzen, R., and Stenzel,
113. Krueger, B.-W., Etzel, W., and Goehrt, K. (1999) Pflanz. Nachr. Bayer, 52,
A. (1999) Pflanz. Nachr. Bayer, 52, 149–161.
123–130. 121. Leroux, P., Fritz, R., Albertini, D.C.,
114. Kuck, K.H., Krueger, B.W., Lanen, C., Bach, J., Gredt, M., and
Rosslenbroich, H.J., and Brandes, W. Chapeland, F. (2002) Pest Manage. Sci.,
(1997) Proceedings of ANPP Cinquieme 58, 876–888.
Conference International sur les Maladies 122. Ruckenstuhl, C., Eidenberger, A., Lang,
des Plantes, Tours, ANPP, Paris, pp. S., and Turnowsky, F. (2005) Biochem.
1055–1062. Soc. Trans., 33, 1197–1201.
807
20
Carboxylic Acid Amide (CAA) Fungicides
20.1
Introduction
The chemical group of carboxylic acid amide (CAA) fungicides was officially an-
nounced by the Fungicide Resistance Action Committee (FRAC; www.frac.info) in
2005 as group number 40 in the FRAC code list, including the three subclasses of
cinnamic acid amides (dimethomorph, flumorph), valinamide carbamates (benthia-
valicarb, iprovalicarb, valifenalate), and mandelic acid amides (mandipropamid)
(Figure 20.1). The reason for this classification was a common cross-resistance pat-
tern amongst all members for the vast majority of the tested isolates of Plasmopara
viticola. Other common features are the specific and rather narrow spectrum of
activity, including, within the Oomycetes, pathogens of the Peronosporales (e.g.,
Bremia on lettuce, Peronospora on tobacco, pea, onion, Pseudoperonospora on cucur-
bits, Plasmopara in grape and sunflower, several Phytophthora spp. on many crops
such as potato, tomato, pineapple), except for the entire genus Pythium, which is
insensitive, as are all other pathogens outside the Oomycetes. Dimethomorph (1)
was the first in the class to be introduced in 1988 [1], followed by iprovalicarb (4) in
1998 [2], flumorph (2) in 2000 [3], benthiavalicarb (5) in 2003 [4], mandipropamid
(7) in 2005 [5], and valifenalate (6) in 2008 [6]. Pyrimorph (3) is expected to be
launched in the near future [7]. In addition, several experimental compounds and
compound families have been described within this chemical class, such as the
glyoxylic acid derivatives (54) (related to mandelic acid amides) in 1995 [8], the
mandelic acid amide SX 623509 (35) in 2003 [9], and the aminosulfone XR-539 (31)
in 2005 [10].
The mode of action of CAA fungicides has in the past been associated with an
inhibition of phospholipid biosynthesis, but it has now been confirmed to be an
interference with cell wall deposition and cellulose biosynthesis (see below). The
physico-chemical data of the CAA fungicides are summarized in Table 20.1, while
details of their mammalian toxicology and environmental profile and fate in soil
are listed in Tables 20.2 and 20.3, respectively.

808 20 Carboxylic Acid Amide (CAA) Fungicides
Cinnamic acid amides:
O O
O O
O O O
N N N
O O O
Cl N
Cl F
1 2 3
dimethomorph flumorph pyrimorph
Valinamides:
O O N
H H
N N F
O N O N S
H H O
O
4 5
iprovalicarb benthiavalicarb
Cl
O
H
N
O N
H
O O
6 O
valifenalate
Mandelic acid amides:
O
H
N O
O
Cl O
7
mandipropamid
Figure 20.1 Carboxylic acid amides launched into the market (status 2010).
Table 20.1 Physico-chemical data of launched carboxylic acid amides.
CAA Dimetho- Flumorph Iprovalicarb Benthiava- Mandi-

morph licarb propamid
(1) (2) (4) (5) (7)
Melting point (◦ C) 125–149 105–110 163–165 152.0 96.4–97.3

Solubility in water 42–81 – 11.0 13.1 4.2
(mg l –1 )
Solubility in organic Acetone: 106 – Acetone: 22; – –
solvents (mg l –1 ) DMSO: 42
Vapor pressure (Pa) 9.7 × 10−8 – 7.7 × 10−8 < 3.0 × 10−4 < 9.4 × 10−7
◦
Log POW 2.63 2.20 3.2 (20 C) 2.52 3.2 (25 ◦ C)
Data acquired from The Pesticide Manual (2006), British Crop Protection Council, Alton, UK.
Table 20.2 Mammalian toxicology of launched carboxylic acid amides.
Mammalian Dimetho- Flumorph Iprovalicarb Benthiava- Mandi-

toxicology morph licarb propamid
(1) (2) (4) (5) (7)
Acute oral 3900 >2710 (♂) >5000 >5000 >5000

(LD50 rat) (mg kg –1 ) >3160 (♀)
Acute dermal >5000 >2150 >5000 >2000 >2000
(LD50 rat) (mg kg –1 )
Acute inhalation >4.2 – >4977 mg m –3 >4.6 >5.0
(LC50 rat) (mg l –1 )
Other characteristics a, c, d, f, g d, e, g a, b, c, d, e, f, a, c, d, e, a, e, f, g,
g, h, i f, g, j h, i, j
a, Non-carcinogenic; b, Non-genotoxic; c, Non-irritating to eyes; d, Non-irritating to skin; e,

Non-mutagenic; f, Non-skin-sensitizing; g, Non-teratogenic; h, No reproductive and developmental
toxicity; i, No evidence of neurotoxicity; j, No adverse effect identified on reproduction.
20.2
Chemistry of the Carboxylic Acid Amides
20.2.1
Cinnamic Acid Amides
20.2.1.1 Dimethomorph
Dimethomorph (1) (Figure 20.2) was discovered as a specific Oomycete fungicide
during the early 1980s by the pharmaceutical research group at Celamerck. This
company was subsequently acquired by Shell, whose agrochemical business was
in turn acquired by American Cyanamid, which was then acquired by BASF.
Table 20.3 Environmental profile and fate in soil (half-life)

of launched carboxylic acid amides.
Environmental Dimetho- Flumorph Iprovalicarb Benthiava- Mandi-

profile morph licarb propamid
(1) (2) (4) (5) (7)
Bobwhite quail (LD50 ) >2000 – >2000 >2000 >2250

(mg kg –1 )
Rainbow trout (LC50, 96 h ) 6.2 – >22.7 >10 >2.9
(mg l –1 )
Earthworm (LC50, 14 d ) >1000 – >1000 >1000 >1000
(mg kg –1 soil)
Bee (LD50 , contact ) (μg/bee) >100 >170 200 – >200
Bee (LD50 , oral ) (μg/bee) – – >199 >100 >200
Fate in soil (half-life; days) 14–50 – 1–17 11–19 2–29
O
O
O
N
Cl
1
Figure 20.2 Chemical structure of dimethomorph (1).
Dimethomorph was described in detail by Albert et al. in 1988 [1], and is a mixture
of E and Z isomers. The fungicidal activity resides exclusively in the Z isomer.
However, in sunlight dimethomorph rapidly equilibrates to an E/Z mixture of
about 20 : 80.
A concise synthesis of dimethomorph can be achieved by condensation of
4-chloro-3 ,4 -dimethoxybenzophenone (8) and N-acetylmorpholine (9) with the aid
of potassium hydroxide [11] or sodium tert-amylate (Scheme 20.1) [12].
20.2.1.2 Flumorph
Flumorph (2), a close analog of dimethomorph, was developed by Shenyang
(Figure 20.3) [3]. Herein, the replacement of dimethomorph’s chloro substituent by
a fluorine atom seems to further improve the antisporulant and curative activities.
Flumorph is composed of a mixture of E and Z isomers in an E/Z ratio of
45 : 55.
O O O
KOH or
O O
N NaOt-amylate O
+
O O N
9 O
Cl Cl
8 1
Scheme 20.1 Synthesis of dimethomorph (1).
O
O
O
N
F
2
Figure 20.3 Chemical structure of flumorph (2).
20.2.1.3 Pyrimorph
Pyrimorph (3) seems also to be an Oomycetes-selective fungicide, because it is
described to be active against Phytophthora infestans, Phytophthora capsici, and
Pseudoperonospera cubensis (Figure 20.4). This novel cinnamic acid amide derivative
has been recently announced by China Agricultural University [7].
O
N
Cl N
Figure 20.4 Chemical structure of pyrimorph (3).

20.2.2
Amino Acid Amides
20.2.2.1 Iprovalicarb
Iprovalicarb (4) was the first fungicide introduced into the market from the amino
acid amide carbamate class of compounds with the general formula 10, which was
discovered by Bayer during a synthesis program for new fungicidal lead structures
in 1988 [2]. Even the first representatives of this compound class showed interesting
effects on pathogens of the Oomycetes, such as Plasmopara viticola or Phytophthora
infestans [13]. The structural variability of iprovalicarb permitted optimization
studies to be conducted on the basic chemical structure 10 (Figure 20.5).
Good efficacy is obtained in particular with α-branched alkyl residues or directly
bound aromatic systems in the carbamate section of the underlying structure
(R1 ). The use of valine or isoleucine as the amino acid portion of the molecule
(R2 = isopropyl or sec-butyl) leads to highly active compounds. Finally, the use
of an α-branched arylethylamine as the amine portion of the amino acid amide
carbamate (R3 ) ensures good efficacy. The outcome of a vast selection program was
the development of iprovalicarb (4) (Figure 20.6) [14].
The iprovalicarb molecule contains two chiral centers; the configuration of the
stereocenter in the amino acid function is defined by the use of l-valine as a
natural amino acid component. As the amine portion of the molecule is racemic,
the active substance contains two diastereomers (the S,S- and S,R-diastereomers).
Iprovalicarb (4) is composed of three building blocks: the carbamate compo-
nent isopropyloxycarbonyl; the natural amino acid l-valine (12); and the amine
unit p-methylphenylethylamine (16). In the first step, isopropyl chloroformate
(11) is treated with l-valine (12) in aqueous sodium hydroxide solution to give
isopropyloxycarbonyl-l-valine (13) (Scheme 20.2).
2
O R 4
1
H R
R N
O N
H 3
O R
Figure 20.5 General structure of amino acid amide
10
carbamates.
O
H
N
O N
H
O
4 Figure 20.6 Chemical structure of iprovalicarb (4).
O O
base, water
+ OH OH
O Cl H2N O N
H
O O
11 12 13
Scheme 20.2 Synthesis of iprovalicarb.

CH3COCl,
AlCl 3 H 2, NH3
O H2N
14 15 16
O O O
base
OH + O O
O N O Cl O N
H H
O O O
13 11 17
A short parallel sequence produces the amine component p-methylphenyl-

ethylamine (16) from toluene (14). First, a Friedel–Crafts acylation is used to
selectively convert toluene (14) into p-methylacetophenone (15). In a second step, the
reductive amination of 15 with hydrogen and ammonia in the presence of a Raney
nickel catalyst produces the required p-methylphenylethylamine (16) (Scheme 20.3).
In the last step of the reaction, the carboxylic acid function of
isopropyloxycarbonyl-l-valine (13) is activated by treating it with a second equivalent
of isopropyl chloroformate (11) under basic conditions, using toluene as a solvent.
This produces the reactive mixed anhydride (17) as an intermediate (Scheme 20.4).
Finally, the mixed anhydride 17, which cannot be isolated, is treated with an
auxiliary base and the p-methylphenylethylamine (16), already prepared in toluene
solution, as part of the same reaction step to form the active substance iprovalicarb
(4) with the elimination of carbon dioxide and isopropanol (Scheme 20.5).
The entire reaction sequence can be carried out on a laboratory scale with an
isolated yield of 4 of more than 85%; the reaction may also be employed on the
technical scale [15–17].
Iprovalicarb (4) was first registered in Indonesia in 1998. The product received
®
approval in Germany in 2000 in combination with tolylfluanid (as Melody Multi ),
and has been registered in France and Italy in combination with mancozeb
H 2N
O 16 O
H
O O auxiliary base N
O N O N
H − CO2 H
O O O
− (CH3)2CHOH
17 4
Scheme 20.5 Synthesis of iprovalicarb (final step).

® ® ® ® ®
(as Yorel ), folpet (as Melody Care , Melody Combi , Odena , Sirbel ), and
® ®
with copper (as Melody Compact , Ocarina ). Additionally, iprovalicarb has been
® ® ®
registered in combination with propineb (as Invento , Melody WP , Melody Duo ,
®
Positron ) in many countries. A ternary mixture with mancozeb and fosetyl-Al
®
(Melody Triplo ) was approved in Italy in 2002. These combination products
provide a broad spectrum of activity under various growing conditions in different
target crops and contribute to an effective anti-resistance strategy.
20.2.2.2 Benthiavalicarb
Benthiavalicarb (5, benthiavalicarb-isopropyl, KIF-230; Figure 20.7) was discovered
by Kumiai-Ihara, and has been developed for the control of Oomycete diseases
such as downy mildews (Plasmopara viticola and Pseudoperonospora cubensis) on
grape vine and vegetables and late blight (Phytophthora infestans) on potatoes [4, 18].
The valinamide derivative benthiavalicarb (5) shows a close structural similarity to
iprovalicarb (4).
Application for European Union (EU) approval was submitted in early 2002,
and the dossier was declared complete in 2003. Kumai Chemical received its first
global registrations for benthiavalicarb in Switzerland and Cuba; consequently, the
®
mixture of benthiavalicarb with mancozeb (Valbon ) was launched in Switzerland
®
in 2004 for use against potato late blight. Concurrently, Vincare (benthiavalicarb
®
plus folpet) was launched for use against grape downy mildew. In 2005, Valbon
® ®
was launched in Belgium, the Netherlands, and UK; in 2006, Valbon and Vincare
were introduced in Austria. The benthiavalicarb molecule contains, similarly to
iprovalicarb, two chiral centers. The configuration of the stereocenter in the first
amino acid function is defined by the use of l-valine as a natural amino acid
component. The second portion of the molecule contains the unnatural amino acid
(R)-alanine.
In the first synthesis step, isopropyl chloroformate (11) is treated with l-valine
(12) in aqueous sodium hydroxide solution to give isopropyloxycarbonyl-l-valine
(13), analogous to the iprovalicarb synthesis. In a parallel sequence,
the amine component 2-(1-aminoethyl)-6-fluorobenzothiazole (21) can be
synthesized from 2-amino-5-fluorothiophenol (20) and its reaction with
(R)-alanine ester. Other approaches include the stereoselective reductive
amination of 2-acetyl-6-fluorobenzothiazole (19) or the Grignard reaction of
2-cyano-6-fluorobenzothiazole (18) with methylmagnesium bromide or methyl-
lithium (Scheme 20.6). In the last step of the reaction, the carboxylic acid function
of isopropyloxy-carbonyl-l-valine (13) is activated and, finally, the mixed anhydride
O N
H
N F
O N S
H O
5
Figure 20.7 Chemical structure of benthiavalicarb (5).

N
CN MeMgBr or MeLi
F S
18
N O NH4OAc or RONH2 N NH2
F S F S
19 21
O
NH2
O OH
O N
F SH H 2N H
O O
20 13
O N
H F
N
O N S
H
O
5
Scheme 20.6 Synthesis of benthiavalicarb (5).
17 is treated with an auxiliary base and the 2-(1-aminoethyl)-6-fluorobenzothiazole

(21), already prepared in toluene solution as part of the same reaction step, to
form the active substance benthiavalicarb (5) [19]. An alternative synthesis route
is the amidation reaction of isopropyloxy-carbonyl-l-valine (13) with (R)-alanine,
which is protected at the carbonic acid function with N-hydroxysuccinimide. The
resulting dipeptide 22 can be converted into the target molecule benthiavalicarb
(5) by reaction with the zinc salt of 2-amino-5-fluorothiophenol (23) in
dimethylformamide and water under acidic conditions (Scheme 20.7) [20].
O _
O O F S N
H
N N + Zn2+ _
O N O
H N S F
O O
22 23
O N
H F
N
O N S
H
O
5
Scheme 20.7 Synthesis of benthiavalicarb (5).

20.2.2.3 Valifenalate
® ®
Valifenalate (6), recently launched by Isagro under the trade names Java , Valis ,
®
and Yaba , is a fungicidal dipeptide of the valinamide class of compounds, and is
active against Oomycetes such as Phytophthora sp., Peronospora sp., and Plasmopara
sp.. It is suitable for use in crops such as grapevines, potatoes, and various
vegetables. Detailed information on the biological characteristics of valifenalate has
not yet been reported.
Valifenalate compound can be synthesized in a similar fashion to that described
for iprovalicarb, via the mixed anhydride 17 (Scheme 20.8). A second process is
O O O
base
OH + O O
O N O Cl toluene O N
H H
O O O
13 11 17
Cl
H2 N O
Cl
O
24 H
N
O N
H
O O
6 O
Scheme 20.8 Synthesis of valifenalate (6).
O
O
HN O O N O
+ O Cl
O O
O O
25 11 26
Cl
H 2N O
Cl
O
24 H
N
O N
H
O O
6 O
Scheme 20.9 Synthesis of valifenalate (6).

exemplified by a single preparation of the specified compound, methyl (±)-(RS)-3-

(N-isopropoxycarbonyl-S-valinyl)amino-3-(4-chlorophenyl)propanoate (6). The pro-
cess consists of the addition of N-methylmorpholine to a mixture of isopropyl
chloroformate (11) and 4-isopropyloxazolidine-2,5-dione (25) in ethyl acetate. The
resulting N-alkoxycarbonyloxazolidinedione intermediate 26 is then reacted with
the aminoester 24 to give the desired valifenalate 6 (Scheme 20.9) [21].
20.2.2.4 Aminosulfones (Experimental Compounds)

The experimental fungicide XR-539 (31), discovered by Dow, belongs to the
aminosulfone class of chemistry and shows high activity against Oomycete diseases
such as grape downy mildew and potato late blight [10]. These compounds can
be synthesized as follows. In a first step, the β-amino alcohol 27 is reacted with
a formic acid chloro ester to afford the corresponding carbamate-protected amino
alcohol. The free hydroxy group is then activated with tosyl chloride to give the
‘‘tosylate electrophile’’ 28. In the central step to the desired molecule, this ‘‘tosylate
electrophile’’ is coupled with the ‘‘thiol nucleophile’’ phenethylthiol 29, followed
by oxidation of the resulting sulfide 30 to the sulfone 31 (Scheme 20.10) [22].
XR-539 (31) bears some structural similarity to the amino acid amide
carbamates – that is, iprovalicarb and benthiavalicarb-isopropyl. In radial
growth-inhibition assays against Phytophthora capsici, XR-539 (31) acted similarly
to dimethomorph and iprovalicarb, both in potency and in the shape of
their dose–response curves [10]. Similar to other CAA fungicides, XR-539 is
inactive against Pythium ultimum. Strong evidence was provided that XR-539 is
cross-resistant to other CAAs in Plasmopara viticola, and thus acts by the same
general mechanism as, for example, valinamides and cinnamic acid amides. It
remains to be established whether the various compounds bind to the same target
protein or, alternatively, act on different proteins in the biochemical pathway or
process [10].
X
HS
1. ROCOCl
2. tosyl chloride
O
29
OH R OTos
H2N O N
H
27 28
1. NaH
O O O O
X 2. mCPBA X
R S R S
O N O N
H H
30 31
Scheme 20.10 Synthesis of XR-539 (31).

O O H O O H
S N O S N O
N N N
H H
O O
O O
32 33
Cl
Figure 20.8 Structures of the N-sulfonyl amino acid amides 32 and 33.
20.2.2.5 N-Sulfonyl Amino Acid Amides (Experimental Compounds)

The carbamate moiety of N-carbamoyl amino acid amides can be replaced by a
sulfonamide function, with full preservation of their biological activity. However,
in this case, the α-methylbenzylamine moiety, which is typical for amino acid
amide carbamates, must be exchanged by a special dialkoxy-substituted phenethyl-
amine. As with amino acid amide carbamates, a diverse range of different amino
acids can be transformed into this kind of fungicides [23]. In general, the amino
acid requires a lipophilic backbone for good fungicidal activity. Examples of
suitable amino acids are valine and isoleucine, but nonproteogenic amino acids
such as phenylglycines are also tolerated [23, 24]. The configuration of the chiral
α-carbon atom is also important. The naturally occurring l-forms show, in most
cases, higher activities than their d-enantiomers. The two highly active N-sulfonyl
amino acid amides 32 and 33 demonstrate the similarities and differences be-
tween valinamides and phenylglycinamides (Figure 20.8). Both subclasses have
a common characteristic, in that small sulfonyl groups such as methylsulfonyl,
ethylsulfonyl, and dimethylsulfamoyl achieve the best biological activities [23, 24].
Different structure–activity requirements affect the para-position of the phenethyl-
amine moiety. The valinamide 32 with a chlorophenylpropargyloxy substituent
demonstrates its high efficacy against Phytophthora infestans on tomato, with the
very low EC80 of 0.02 ppm [23]. The much shorter propargyloxy group is the
best substituent for the 4-position of the phenethylamine in phenylglycinamides,
such as 33 [23, 24].
20.2.3
Mandelic Acid Amides
20.2.3.1 Mandipropamid
The antifungal activity of mandelic acid amides (mandelamides) with dialkoxylated
phenethylamine moieties was first discovered for human pathogens by Yu and Van
Scott during the mid-1980s [25]. It was found that 34, which is the acetylated adduct
of mandelic acid and homoveratrylamine, has significant activity against skin
disorders such as mycosis fungoides and psoriasis (Figure 20.9). During the early
1990s this general structure was taken up by chemists at Agrevo (now Bayer), who
O O OH H
H
N O Cl N O
O O
O Cl O
34 35
OH O
H H
N O N O
O O
Br O Cl O
36 7
Figure 20.9 Mandelic acid amides.
found that the mandelamide SX 623509 (35) was active against plant pathogens,
especially Oomycetes [9, 26, 27]. At Novartis (now Syngenta), replacement of the
ethoxy group of 35 by a propargyloxy function resulted in the mandelamide 36, with
an enhanced fungicidal efficacy [28]. The exchange of methoxy and ethoxy groups
by propargyloxy often leads to increased biological activity, as has been reported
previously for compounds with antibacterial [29] and leishmanicidal activity [30].
The introduction of a second propargyl group into the mandelic acid moiety of 36
clearly increased the fungicidal activity further, leading finally to Syngenta’s fungi-
cide mandipropamid (7) – the first derivative of the chemical class of mandelamide
fungicides to be commercialized (Figure 20.9) [5, 24, 31, 32].
Mandipropamid (7) received its first registration in Austria in 2005, and has
since been launched in several different countries, both as a solo product and in
® ®
mixtures under the trade names Revus and Pergado .
An important building block for the amine moiety of mandelic acid amides is
2-(4-hydroxy-3-methoxy)phenethylamine (41), also known as 3-O-methyldopamine
or 3-methoxytyramine. In principle, 41 can be prepared by several different meth-
ods, three of which are highlighted in Scheme 20.11. The most widely applied of
these methods is a reduction of the nitrostyrene 38, which may be obtained by
the Henry reaction of vanillin (37) and nitromethane. This reduction of a nitro
group and an olefin function can be performed in one step, using either catalytic
hydrogenation [33] or lithium aluminum hydride [34]. An alternative, more reliable
approach, which also avoids any undesirable highly exothermic reaction profiles,
involves two steps via the phenylnitroethane 40 [23]. A second strategy is the
catalytic hydrogenation of vanillin cyanohydrin (39) [35]. The third possibility is the
catalytic hydrogenation of the benzyl cyanide 43, which can be obtained directly
from vanillinol (42) through a quinoid transition state [36].
O
O
H
OH
37
NaCN, HCl
MeNO2
OH
O2N O O
N
OH OH
38 39
NaBH4 H2, Pd/C H2, Pd/C
O2 N O H2N O
H2, Pd/C
OH OH
40 41
H2, Pd/C
O O
HO NaCN, Δ
N
OH OH
42 43
Scheme 20.11 Synthesis of 3-O-methyldopamine (41).
The synthesis of mandipropamid (7) is possible via several different routes.

One important approach employs 4-chloromandelic acid (45), which can be pre-
pared from 4-chlorobenzaldehyde (44) via Strecker-type mandelonitriles [37], as
well as with chloroform and sodium hydroxide (Scheme 20.12) [38]. Alternatively,
4-chloroacetophenone (46) can be converted into 45 either in two steps by dichlo-
rination and a subsequent Cannizzaro-type transformation of the intermediate
dichloromethyl ketone [39], or by a ytterbium triflate promoted tandem one-pot
oxidation – the Cannizzaro reaction [40]. 4-Chloromandelic acid can be transformed
with acetone under acidic conditions into the acetonide 47 [41]; this is ring-opened
[42] with 2-(4-hydroxy-3-methoxyphenethyl)amine (41) [23] to the mandelamide 48
[43], which bears both an alcoholic and a phenolic hydroxy function. Both OH
groups may be simultaneously propargylated with propargyl bromide and sodium
hydroxide under phase-transfer conditions to obtain mandipropamid (7).
1. NaHSO3 1. Cl2
O OH O
2. NaCN 2. NaOH
3. HCl, H2O OH 3. HCl
H
O
Cl Cl Cl
44 45 46
CH3COCH3,
H2SO4
O
O
O
Cl OH
H
H2N O 47 N O
O
OH Cl OH
41 48
O
HC CCH2Br, H
N O
NaOH, TBAB
O
Cl O
Scheme 20.12 Synthesis of mandipropamid (7).
In a different approach, the propargylated formamide 50, available in two

steps from 41, can be transformed directly by a Seebach’s modification [44]
of the Passerini reaction [45] into the mono-propargylated mandelamide 51
(Scheme 20.13). The introduction of a second propargyl group into the hydroxy
function of the mandelic acid moiety of 51 leads to mandipropamid (7) [24, 31].
Mandipropamid (7) has been developed as racemic mixture of both enan-
tiomers. The stereoselective synthesis of enantiopure mandelamide fungicides
is also possible via a diastereoselective Passerini reaction with a galacturonic
acid derivative as acid component [46], as well as by an enantioselective hydro-
genation of phenylglyoxylic amides with a homogeneous Rh catalyst system [47].
However, none of these enantiomers, when used alone, offered a biological advan-
tage over the mixture, and therefore mandipropamid was registered as racemate.
Other amides that are closely related to mandelamides can also achieve high
fungicidal activities (Figure 20.10). The glyceric acid amide 52, which bears an
OCH2 spacer between the 4-chlorophenyl ring and the 2-propargyloxyacetamide
function of mandipropamid, has been reported by Syngenta to be highly active
against Phytophthora infestans on tomato (EC80 0.02 ppm) [24, 48]. The hydroferulic
HCO2H, H H C CCH2Br,
H2N O Ac2O H N O
NaOMe
O
OH OH
41 49
Cl3COCO2CCl3, OH
H H
H N O NEt3, 43, TiCl4 N O
O O
O Cl O
50 51
O
H C CCH2Br, H
N O
NaH
O
Cl O
Scheme 20.13 Synthesis of mandipropamid (7).
N
O O
H
O N O O
N
O H
Cl O Cl O
52 53
Figure 20.10 Structures of 52 and 53.
acid amide 53, which is a mandipropamid derivative with an inverted amide

function, possesses powerful anti-Oomycete efficacy and has been patented by
Sumitomo [49].
20.2.3.2 Glyoxylic Acid Derivatives (Experimental Compounds)

The glyoxylic acid derivatives 54 (Figure 20.11) were discovered by Bayer in 1994 as
a new class of Oomycete fungicides by derivatization of advanced research project
compounds with broad fungicidal activities [8].
The glyoxylic acid derivatives 54 exhibit specific activity against the Oomycetes,
including downy mildew on grapes (Plasmopara viticola) and late blight on potatoes
and tomatoes (Phytophthora infestans). In addition, they are active against Oomycetes
in soil, such as Phytophthora in tobacco and citrus. The derivatives exhibit protective,
3 Figure 20.11 General structure of glyoxylic acid deriva-

R
1 tives.
R A 5
N R
2 4
R R
54
curative, eradicative, and antisporulant activities. This class of compounds can be

synthesized via a simple synthetic approach where, in the first step oxalic ester
chloride is added to the substituted phenylic part via a Friedel–Crafts acylation. In
the second step, the keto function of the glyoxylic acid ester 56 is converted into
the oxime ether function by reaction with an alkoxy amine derivative. The resulting
intermediate 57 can be easily transformed into the desired product 59 via an
amidation reaction with a substituted phenethylamine (58) under basic conditions
(Scheme 20.14) [8].
For high fungicidal activity, it is important to have small substituents such as
chlorine, methyl, or ethyl in the meta or para position of the aromatic moiety of the
glyoxylic acid molecule part (R1 , R2 ), or a saturated or aromatic bicyclic system (with
R1 , R2 building a ring together). Ortho substituents lead to a complete inactivity of
the final compound. At the oximether moiety (R) small alkyl substituents such as
methyl are preferred.
During the past decade, several variations of that structural motif have been
developed by various companies, with all such compound families exhibiting
specific activities against the Oomycetes. Some examples of these compounds are
illustrated in Figure 20.12 [50–52].
R
O
O N
1 ClCOCO2C2H5, 1 1
R AlCl3
R O RONH2 R O
2 2 O 2 O
R R R
55 56 57
O
R
H2N O O
N
1
H
58 , NaOC2H5 R N O
2 O
R O
59
Scheme 20.14 Synthesis route to glyoxylic acid amides.

4
R
3 N
R N
1
H
R N O
2 O
R O
60
Bayer
N
1
H
R N O
2 O
R O
61
Bayer
3
R
1
H
R N O
2 O
R O
62
BASF
Figure 20.12 Examples of glyoxylic acid amides.
20.3
Biological Activity of Carboxylic Acid Amides
All of the CAA fungicides are specifically effective against foliar pathogens of the
Oomycetes, including Phytophthora infestans (potato and tomato late blight), Plas-
mopara viticola (grape downy mildew), and Pseudoperonospora cubensis (cucumber
downy mildew). The entire genus Pythium, as well as all pathogens outside the
Oomycetes, are not sensitive to the CAA fungicides. Typically, the CAA fungicides
function by inhibiting the germination of cystospores and sporangia (but not
zoospore release and motility), and also affecting the growth of germ tubes and the
mycelium, thus preventing infection of the host tissue. Following foliar application,
the CAAs are reported to exhibit – besides their strong preventive activity – also
curative activity and some eradicative effects, depending on the quantity of the
fungicide that has been taken up into the leaf, and how it has been distributed
based on translaminar movement.
20.4 Mode of Action and Mechanism of Resistance of the CAA Fungicides 825
Dimethomorph has a good preventive activity, as well as pronounced curative

and antisporulant activities. Following a soil drench or foliar application, iprovali-
carb is reported to be more systemic than both dimethomorph (1) [2, 53, 54]
and mandipropamid (7). As iprovalicarb (4) is a systemic compound [55], it is
distributed in the apoplast (acropetal translocation), and also protects untreated
leaves against infection, especially in grapes. Autoradiography of grape leaves
treated with 14 C-iprovalicarb showed a high level of systemic distribution within
the tissues [56]. Generally, specific ingredients in the formulation have a strong
impact on uptake and fungicidal activity [2], while an elevated temperature and
humidity, together with leaf wetness can also increase the uptake and curative
activity of iprovalicarb [56]. In grape leaves, benthiavalicarb delivers long-lasting
preventive [4, 57] and some loco-systemic activity, but demonstrates a low translam-
inar activity [58]. The results of further studies have shown that benthiavalicarb,
when applied at between one and six days post-inoculation, protected grape
leaves against downy mildew and inhibited the sporulation of P. viticola [58].
Mandipropamid binds rapidly and tightly to the wax layer of the leaf surface,
providing a rain-fast and long-lasting barrier against infections [59]. It also de-
livers strong preventive and translaminar activity, and also provides a robust
control of both Phytophthora infestans and Plasmopara viticola, under severe disease
pressures [5].
20.4
Mode of Action and Mechanism of Resistance of the CAA Fungicides
The results of cytological studies have implied that dimethomorph, iprovalicarb, and
benthiavalicarb inhibit processes involved in cell-wall biosynthesis and assembly
[60–66]. These findings were supported by observations that these agents also
affect the regeneration of protoplasts of P. infestans, alter the staining of cell
walls with fluorochromes, and inhibit the encystment of zoospores of various
Phytophthora species and Plasmopara viticola, or cause their lysis. No inhibition was
observed for zoospore discharge from sporangia, not of zoospore motility. Further
studies have shown that the CAAs have no effect on the transition of zoospores into
cystospores, a process which requires cell-wall synthesis and rearrangement. Taken
together, these findings indicate that cell-wall deposition at this stage is obviously
insensitive [64]. Rather, the most sensitive developmental stage in the life cycle of
the Oomycetes is the germination of cystospores and sporangia. A 1 h incubation
of the cystospores in the presence of CAAs, followed by incubation in water for
another 2 h, was less effective than a continuous exposure. This suggested that the
binding of CAAs to their targets is not completed within 1 h, either because they
did not reach the target, it was not yet ready for binding, or because the binding
was weak [64].
In Oomycetes, the process of cell-wall synthesis is rather complex and remains
poorly investigated. The altered architecture of the cell wall following CAA treat-
ment was associated with effects on cytoskeletal elements or membrane-bound
components (e.g., receptors, enzymes) that were responsible for the transport of
cell-wall precursors, but the enzymes associated with cell-wall biosynthesis were
found not to be inhibited [62–64]. In studies with iprovalicarb, a direct inhibition
of glucan synthase was excluded [63]. Rather, the CAAs were postulated to inhibit
the three-dimensional arrangement and cross-linkage of the complex glucan struc-
ture necessary for germ tube and hyphal growth [65, 66]. Cytological studies with
P. infestans demonstrated a different microtubule organization following treatment
with iprovalicarb than with dimethomorph [62]. Alterations in phospholipid biosyn-
thesis were also proposed, with an inhibition of phosphatidylcholine (lecithin)
biosynthesis as the main target [9]. However, as somewhat high fungicide concen-
trations were used in these studies it remains unclear as to whether the observed
effects were a reaction to general cell death rather than to a specific inhibition
of lecithin biosynthesis. Furthermore, the sequence analysis of two genes coding
for choline phosphotransferase (CPT) in wild-type and laboratory mutants of P.
capsici, selected with pyrimorph on amended agar, failed to show any amino acid
differences in the two potential target genes [67]; this suggested an alternative site
of action (and resistance) for CAA fungicides.
The results of recent studies have contributed to elucidating the mode of action
for CAA fungicides. The in situ immunolocalization of cellulose synthase from
P. infestans with specific antibodies showed an altered distribution and partial
agglomeration of the enzyme in the plasma membrane following treatment with
iprovalicarb [68]. Gene sequencing of artificially generated mutants of P. infestans
that were highly resistant to mandipropamid revealed an amino acid substitution
in the cellulose synthase (CesA3) gene at position 1105 from glycine to serine,
G1105S. In addition, the transformation and expression of a mutated CesA3 allele
in a sensitive P. infestans isolate resulted in a CAA-resistant phenotype [69]. Thus,
cellulose synthase can be postulated as the primary target enzyme for CAA activity
within cell-wall biosynthesis.
Despite dimethomorph having been in use for more than 15 years, resistance
to the CAAs in P. infestans has never been detected in field populations. The
lack of resistant isolates in nature has encouraged several research groups to
produce artificial mutants in vitro [70–76]. For example, mutants resistant to
dimethomorph (1), flumorph (2), or mandipropamid (7) were produced but found
to show reduced growth rates, a reduced frequency of infections on leaves and
tubers, and a lower fitness or survival over several generations compared to wild-type
isolates. Accordingly, based on the lack of practical resistance and stable mutants,
the resistance risk of P. infestans to CAA fungicides has been estimated as low
(FRAC CAA Working Group, CAA FRAC guidelines; www.frac.info). Nonetheless,
in France, resistance to CAAs in P. viticola was reported as far back as 1994,
shortly after the introduction of dimethomorph (1) [75]. As a consequence, when
intensive sensitivity monitoring was carried out across European vineyards by
several companies, resistant isolates were repeatedly detected, mainly in some of
the grape-growing regions in France and Germany. Although cross-resistance was
identified among all CAAs for the vast majority of isolates [77], as might have been
expected no cross-resistance was found between CAAs and other modes of action,
20.4 Mode of Action and Mechanism of Resistance of the CAA Fungicides 827
r
Sensitivity, EC 50 (mg / L) 100
10
s
1
0.1
J.4
J.13
J.17
J.2
J.20
J.23
J.10
J.5
J.24
J.12
J.17
J.25
J.6
J.19
J.3
J.22
J.18
J.26
J.27
J.28
J.7
II.8
II.25
II.26
II.10
II.29
D5
K.18
K.21
K.13
K.21
K.10
K.24
K.7
K.6
K.28
K.15
K.8
K.25
K.27
K.3
K.5
K.1
K.20
K.4
K.9
K.23
K.16
K.19
K.2
K.11
K.12
CH05.2
K.29
G.22
G.2
G.17
G.3
G.30
G.10
G.27
G.4
G.5
G.19
G.11
G.8
G.1
G.12
G.23
G.28
G.15
G.13
G.16
G.26
G.18
G.9
G.20
F2 progeny (n = 69)
Figure 20.13 Sensitivity to mandipropamid (7) of F2

progeny isolates (n = 69) produced from crosses (G, J, K,
white, and hatched columns) between sensitive F1 isolates
(gray columns highlighted by triangles) of Plasmopara viticola
(black columns highlighted by arrows are F0 sensitive and
resistant parents) [76].
such as phenylamides, quinone outside inhibitor (QoI) fungicides, and zoxamide.

Because the group members of the CAA fungicides express different intrinsic
activities, resistance factors (the difference in sensitivity between wild-type and
resistant isolates) can vary significantly.
In order to obtain further information on CAA resistance, the segregation pattern
was investigated in sexual crosses made between sensitive (s) and CAA-resistant (r)
isolates of P. viticola [77]. All F1 progeny isolates were sensitive to CAA fungicides,
reflecting a segregation pattern of s : r = 1 : 0; however, when the F1 progeny
isolates (siblings) were crossed the segregation in the F2 progeny was about 9 : 1
(s : r) (Figure 20.13) [77]. This segregation pattern suggested that the resistance
to CAAs might be controlled by recessive nuclear genes. In the F2 progeny,
the resistance was co-segregated for all of the tested CAAs (mandipropamid,
dimethomorph, iprovalicarb). Sequencing of the cellulose synthase, CesA3 gene
in a CAA-sensitive and -resistant field isolate of P. viticola revealed five single
nucleotide polymorphisms (SNPs) affecting the amino acid structure of the protein
[78]. SNP inheritance in F1 -, F2 -, and F3 -progeny isolates confirmed that resistance
was correlated with one specific SNP located in the CesA3 gene. Only if present in
both alleles, would this SNP lead to the substitution of glycine for serine at position
1105 (G1105S), thus conferring CAA resistance [78]. These results demonstrated
that one recessive mutation in the CesA3 gene would cause an inheritable
resistance to CAAs, and confirmed the involvement of cellulose synthase in CAA
activity. For the entire group of CAA fungicides, the risk and extent of resistance in
P. viticola was classified by the FRAC as ‘‘moderate.’’ As a consequence, resistance
to CAAs in P. viticola should be managed through appropriate usage strategies,
such as the use of mixtures and restriction of the number of applications per
season (see CAA FRAC recommendations; www.frac.info).
References
1. Albert, G., Curtze, J., and Drandarevski, 17. Seitz, T., Benet-Buchholz, J., Etzel, W.,
C.A. (1988) Brighton Crop Prot. Conf., 1, and Schindler, M. (1999) Pflanz.-Nachr.
17–24. Bayer, 52 (1), 5–14 (German Edition).
2. Stenzel, K., Pontzen, R., Seitz, T., 18. Miyake, Y., Sakai, J., Shibata, M.,
Tiemann, R., and Witzenberger, A. Yonekura, N., Miura, I., Kumakura, K.,
(1998) Brighton Crop Prot. Conf., 5A-7, and Nagayama, K. (2005) J. Pestic. Sci.,
367–374. 30 (4), 390–396.
3. Liu, C.L., Liu, W.C., and Li, Z.C. (2000) 19. Isozumi, K. (2000) Patent WO
Brighton Crop Prot. Conf., 549–556. 2001/074,795 (Ihara Chemical Industry
4. Miyake, Y., Sakai, J., Miura, I., and Co., Ltd.).
Nagayama, K. (2003) Brighton Crop Prot. 20. Tanaguchi, S. (2001) Patent JP
Conf., 1, 105–112. 2000/169,458 (Ihara Chemical Industry
5. Huggenberger, F., Lamberth, C., Co., Ltd.).
Iwanzik, W., and Knauf-Beiter, G. (2005) 21. Camaggi, G., Gusmerol, M.,
Proceedings of the BCPC International Mormile, S., and Elmini, A. (2002)
Congress, October 31–November 2, Patent WO 2002/006,304 (Isagro Ricerca
Glasgow, pp. 87–92. S.R.L.).
6. Gonzalez-Rodriguez, R.M., 22. Ricks, M.J., Klittich, C.J.R., Cetusic,
Cancho-Grande, B., and Simal-Gandara, J.R.P., Iamauti, M.T., Morrison, I.M.,
J. (2011) Food Chem., 125, 549–560. Lo, W.C.-L., Sullenberger, M.T., Buysse,
7. Yan, X., Qin, W., Sun, L., Qi, S., A.M., Rieder, B., Mathieson, J.T.,
and Olson, M.B. (2002) Patent WO
Yang, D., Qin, Z., and Yuan, H. (2010)
2002/040,431 (Dow Agrosciences LLC).
J. Agric. Food Chem., 58, 2720–2725.
23. Cederbaum, F., De Mesmaeker, A.,
8. Seitz, T., Hänssler, G., and Stenzel, K.
Jeanguenat, A., Kempf, H.-J.,
(1996) Patent WO 9,623,763 (Bayer AG).
Lamberth, C., Schnyder, A., Zeller, M.,
9. Griffiths, R.G., Dancer, J., O’Neill, E.,
and Zeun, R. (2003) Chimia, 57,
and Harwood, J.L. (2003) New Phytol.,
680–684.
158, 345–353.
24. Lamberth, C., Jeanguenat, A.,
10. Young, D.H., Kemmitt, G.M., and
Cederbaum, F., De Mesmaeker, A.,
Owen, J. (2005) in Modern Fungicides
Zeller, M., Kempf, H.-J., and Zeun, R.
and Antifungal Compounds IV (eds H.W. (2008) Bioorg. Med. Chem., 16,
Dehne, U. Gisi, K.H. Kuck, P.E. Russell, 1531–1545.
and H. Lyr), British Crop Protection 25. Yu, R.J. and Van Scott, E.J. (1986) US
Council, Alton, pp. 145–152. Patent 4,518,789 (Temple Univ.).
11. (a) Curtze, J. (Shell) (1994) Patent WO 26. Ort, O., Döller, U., Reissel, W., Lindell,
94/01,424 (Shell); (b) Curtze, J. (1989) S.D., Hough, T.L., Simpson, D.J.,
Patent EP 294,907 (Shell). and Chung, J.P. (1997) Pestic. Sci.,
12. Curtze, J., Briner, P.H., and Schröder, L. 50, 331–333.
(1990) Patent EP 329,256 (Shell); 27. Döller, U., Braun, P., and Sachse, B.
Curtze, J. and Krummel, G. (1990) (1994) Patent WO 94/29,267 (Agrevo).
Patent DE 3,817,711 (Shell). 28. Zeller, M., Jeanguenat, A., Lamberth, C.,
13. Wollweber, D., Seitz, T., and and Kunz, W. (2000) Patent WO
Brandes, W. (1990) Patent EP 398,072 2000/41,998 (Novartis).
(Bayer AG). 29. Periers, A.-M., Laurin, P., Ferroud, D.,
14. Seitz, T., Wollweber, D., Haesslein, J.-L., Klich, M.,
Brandes, W., and Dehne, H.-W. (1992) Dupuis-Hamelin, C., Mauvais, P.,
Patent EP 472,996 (Bayer AG). Lassaigne, P., Bonnefoy, A., and
15. Stelzer, U., Casser, C., and Seitz, T. Musicki, B. (2000) Bioorg. Med. Chem.
(1996) Patent EP 767,780 (Bayer AG). Lett., 10, 161–165.
16. Rivadeneira, E. (1998) Patent DE 30. de Gomes, D.C.F., Alegrio, L.V.,
19,631,270 (Bayer AG). de Lima, M.E.F., Leon, L.L., and
References 829
Araujo, C.A.C. (2002) Arzneim.-Forsch. 44. (a) Seebach, D., Adam, G.,
Drug Res., 52, 120–124. Gees, T., Schiess, M., and
31. Lamberth, C., Cederbaum, F., Weigand, W. (1988) Chem. Ber.,
Jeanguenat, A., Kempf, H.-J., Zeller, M., 121, 507–517 (b) Schiess, M.
and Zeun, R. (2006) Pest Manage. Sci., and Seebach, D. (1983) Helv. Chim.
62, 446–451. Acta, 66, 1618–1623.
32. Lamberth, C., Zeller, M., Kunz, W., 45. (a) Banfi, L. and Riva, R. (2005) Org.
and Cederbaum, F. (2001) Patent WO React., 65, 1–140; (b) Dömling, A.
2001/87,822 (Syngenta). and Ugi, I. (2000) Angew. Chem., 112,
33. (a) Kohno, M., Sasao, S., and 3300–3344; (c) Angew. Chem. Int. Ed.,
Murahashi, S. (1990) Bull. Chem. Soc. (2000), 39, 3168–3210.
Jpn, 63, 1252–1254; (b) Wagner, D.P., 46. Frey, R., Galbraith, S.G., Guelfi, S.,
Rachlin, A.I., and Teitel, S. (1971) Synth. Lamberth, C., and Zeller, M. (2003)
Commun., 1, 47–50; (c) Brossi, A., Van Synlett, 1536–1538.
Burik, J., and Teitel, S. (1968) Helv. 47. Cederbaum, F., Lamberth, C., Malan, C.,
Chim. Acta, 51, 1965–1979. Naud, F., Spindler, F., Studer, M., and
34. (a) Colombini, M.A. and Burger, A. Blaser, H.-U. (2004) Adv. Synth. Catal.,
(1972) J. Med. Chem., 15, 692–693; 346, 842–848.
(b) Ramirez, F.A. and Burger, A. (1950) 48. Lamberth, C., Kempf, H.-J., and
J. Am. Chem. Soc., 72, 2781–2782. Kriz, M. (2007) Pest Manage. Sci., 63,
35. Buck, J.S. (1933) J. Am. Chem. Soc., 55, 57–62.
49. (a) Sakaguchi, H. (2005)
3388–3390.
Patent WO 2005/000,796
36. (a) Szantay, C., Dörnyei, G., Blasko, G.,
(Sumitomo); (b) Soma, M.
Barczai-Beke, M., and Pechy, P.
(2005) US Patent 2005/282,888 (Sumit-
(1981) Arch. Pharm., 314, 983–991;
omo).
(b) Schwartz, M.A., Zoda, M.,
50. Seitz, T. and Stenzel, K. (1996) Patent
Vishnuvajjala, B., and Mami, I. (1976) J.
WO 9,631,464 (Bayer AG).
Org. Chem., 41, 2502–2503.
51. Seitz, T. and Stenzel, K. (1997) Patent
37. (a) Hallas, G. and Yoon, C. (2001) Dyes
WO 9,714,673 (Bayer AG).
Pigments, 48, 107–119; (b) Corson, B.B.,
52. Grammenos, W., Sauter, H.,
Dodge, R.A., Harris, S.A., and Yeaw,
Cullmann, O., Gewehr, M., Müller, B.,
J.S. (1926) Org. Synth., 6, 58–62; (c) Org.
Tormo i Blasco, J., Goetz, N., Volk, T.,
Synth. Coll., 1, 336–340. Lorenz, G., Ammermann, E., Stierl, R.,
38. Zhou, C.-H., Yuan, D.-Q., and Xie, and Strathmann, S. (2001) Patent WO
R.-G. (1994) Synth. Commun., 24, 43–46; 2001/095,721 (BASF AG).
(d) Merz, A. (1974) Synthesis, 724–725. 53. Gisi, U. (2002) in Advances in
39. (a) Bandyopadhyay, A., Bagchi, U.B., Downy Mildew Research (eds P.T.N.
Podder, G., and Moitra, S.K. (1989) J. Spencer-Phillips, U. Gisi, and A.
Ind. Chem. Soc., 66, 239–240; (b) Aston, Lebeda), Kluwer Academic Publishers,
J.G., Newkirk, J.D., Jenkins, D.M., and Dordrecht, pp. 119–159.
Dorsky, J. (1943) Org. Synth., 23, 48–51; 54. Cohen, Y., Baider, A., and Cohen, B.-H.
(c) Org. Synth. Coll., 3, 538–541. (1995) Phytopathology, 85, 1500–1506.
40. Curini, M., Epifano, F., Genovese, S., 55. Dutzmann, S. (1999) Pflanz.-Nachr.
Marcotullio, M.C., and Rosati, O. (2005) Bayer, 52 (1), 15–32.
Org. Lett., 7, 1331–1333. 56. Stübler, D., Reckmann, U., and
41. Audrieth, L.F. and Sveda, M. (1940) Org. Noga, G. (1999) Pflanz.-Nachr. Bayer,
Synth., 20, 62–64; Org. Synth. Coll., 3, 52 (1), 33–48.
536–538. 57. Hofman, T.W., Boon, S.M., Coster, G.,
42. Khalaj, A. and Nahid, E. (1985) Synthesis, van Oudheusden, Z., Ploss, H., and
115–1155. Nagayama, K. (2003) Brighton Crop Prot.
43. Zeller, M., Faber, D., Vettiger, T., Conf., 1, 413–418.
and Lamberth, C. (2003) Patent WO 58. Reuveni, M. (2003) Eur. J. Plant Pathol.,
2003/042,166 (Syngenta). 109, 243–251.
59. Hermann, D., Bartlett, D.W., University of Düsseldorf, Germany,

Fischer, W., and Kempf, H.-J. (2005) http://docserv.uni-duesseldorf.de/servlets/
Proceedings, British Crop Protection DerivateServlet/Derivate-12680/
Council International Congress, vol. 1, BrittaDelvosDissertation.pdf.
pp. 93–98. 69. Blum, M., Boehler, M., Randall, E.,
60. Albert, G., Thomas, A., and Gühne, M. Young, V., Csukai, M., Kraus, S.,
(1991) Association Nationale de Pro- Moulin, F., Scalliet, G., Avrova, A.O.,
tection des Plantes - 3rd International Whisson, S.C., and Fonné-Pfister, R.
Conference Plant Diseases, Paris, pp. (2010) Mol. Plant Pathol., 11, 227–243.
887–894. 70. Yuan, S.K., Liu, X.L., Si, N.G., Dong, J.,
61. Jende, G., Steiner, U., and Dehne, H.W. Gu, B.G., and Jiang, H. (2006) Plant
(1999) Pflanz.-Nachr. Bayer, 52, 49–60. Pathol., 55, 258–263.
62. Jende, G., Steiner, U., and Dehne, H.W. 71. Rubin, E. and Cohen, Y. (2006) Plant
(2002) in Modern Fungicides and Anti- Dis., 90, 741–749.
fungal Compounds III (eds H.W. Dehne, 72. Stein, J.M. and Kirk, W.W. (2004) Plant
U. Gisi, K.H. Kuck, P.E. Russell, and Dis., 87, 1283–1289.
H. Lyr), AgroConcept, Bonn, pp. 83–90. 73. Bagirova, S.F., Li, A.Z., Dolgova, A.V.,
63. Mehl, A. and Buchenauer, H. (2002) in Elansky, S.N., Shaw, D.S., and Dyakov,
Modern Fungicides and Antifungal Com- Y.T. (2001) J. Russ. Phytopathol., 2,
pounds III (eds H.W. Dehne, U. Gisi, 19–24.
K.H. Kuck, P.E. Russell, and H. Lyr), 74. Chabane, K., Leroux, P., and
AgroConcept, Bonn, pp. 75–82. Bompeix, G. (1993) Pestic. Sci., 39,
64. Cohen, Y. and Gisi, U. (2007) Phy- 325–329.
topathology, 97, 1274–1283. 75. Chabane, K., Leroux, P., Maia, N., and
65. Thomas, A., Albert, G., and Bompeix, G. (1996) in Modern Fungicides
Schlösser, E. (1992) Med. Fac. Land- and Antifungal Compounds (eds H. Lyr,
bouww. Univ. Gent, 57 (2a), 189–197. P.E. Russell, and H.D. Sisler), Intercept,
66. Kuhn, P.J., Pitt, D., Lee, S.A., Andover, pp. 387–391.
Wakley, G., and Sheppard, A.N. (1991) 76. Ziogas, B.N., Markoglou, A.N.,
Mycol. Res., 95 (3), 333–340. Theodosiou, D.I., Anagnostou, A.,
67. Sun, H., Wang, H., Stammler, G., and Boutopoulou, S. (2006) Eur. J. Plant
Ma, J., and Zhou, M. (2010) J. Phy- Pathol., 115 (3), 283–292.
topathol., 158, 244–252. 77. Gisi, U., Waldner, M., Kraus, N.,
68. Delvos, B. (2009) Untersuchungen Dubuis, P.H., and Sierotzki, H. (2007)
der Effekte von Iprovalicarb und Plant Pathol., 56, 199–208.
Dimethomorph auf die Zellwand von 78. Blum, M., Waldner, M., and Gisi, U.
Phytophthora infestans, Dissertation, (2010) Fungal Genet. Biol., 47, 499–510.
831
21
Fluopicolide: A New Anti-Oomycetes Fungicide?
21.1
Introduction
Although the Oomycetes appear to share certain morphological, physiological,

and biochemical features with fungi, they are in fact phylogenetically distant [1].
Oomycetes such as Phythophthora, Pythium, or Plasmopara cause dramatic diseases
in a wide variety of plant species, including potato, vegetables, or grape. However,
because of the differences from other fungi, many effective fungicides – such as
azoles – are not effective against the Oomycetes.
Fluopicolide, which belongs to a new chemical class of fungicides (Figure 21.1),
exhibits a high antifungal activity against a broad spectrum of Oomycetes, such as
Phytophthora infestans, Plasmopora viticola, and various Pythium species. It shows
no cross-resistance to other commercially anti-Oomycete fungicides, and can
inhibit the development of strains that are resistant to phenylamides, strobilurins,
or dimethomorph, and iprovalicarb. These findings lead to the suggestion that
fluopicolide acts with a new mode of action. In addition, fluopicolide affects several
stages of the life cycle of the different Oomycetes studied, such as the release and
the motility of zoospores, the germination of cysts, the growth of the mycelium,
and sporulation.
A detailed biochemical analysis has shown that fluopicolide does not inhibit res-
piration, has no direct effect on membrane composition, and does not significantly
alter tubulin polymerization or the tubulin and actin contents of the cell.
21.2
Chemical and Physical Properties
The physico-chemical properties of fluopicolide (Table 21.1) allow it to be easily

redistributed via the xylem (acropetal systemic activity) and translocated within the
leaf tissues, providing a translaminar activity.

832 21 Fluopicolide: A New Anti-Oomycetes Fungicide?
Cl O Cl Figure 21.1 Fluopicolide.
N
N F
Cl
F
F
Table 21.1 Physico-chemical properties of fluopicolide.
ISO name Fluopicolide

Chemical class Acylpicolides [2]
Chemical name (IUPAC) 2,6-Dichloro-N-{[3-chloro-5-(trifluoromethyl)-2-
pyridinyl]methyl}benzamide
Code number AE C638206
Vapor pressure 3:03 × 10 –7 Pa (20 ◦ C)
Partition coefficient 2200 (log P = 2 : 9) (octanol/water)
Water solubility 2.8 mg l –1 (20 ◦ C, pH 7)
DMSO solubility 183 g l –1 (20 ◦ C)
Photolytic stability Stable
Hydrolytic stability Stable over pH range 4–9
DMSO, dimethyl sulfoxide.
Table 21.2 Mammalian toxicity data for fluopicolide.
Acute oral toxicity (rat) LD50 > 5000 mg kg –1

Acute dermal toxicity (rat) LD50 > 5000 mg kg –1
Eye irritation, rabbit Non-irritant
Skin irritation, rabbit Non- irritant
Acute inhalation in air, 4 h, LC50 (rat) >5160 mg a.i. m –3
Sensitization (guinea pig) Non-sensitizing
Carcinogenicity (mice, rat) No carcinogenic potential
Mutagenicity No genotoxic effects
Chronic toxicity No embryotoxic potential
Teratogenicity, rat, rabbit No teratogenic potential
21.3
Toxicology/Ecotoxicology
Mammalian toxicity data for fluopicolide are listed in Table 21.2, while details
of the compound’s ecotoxicological and environmental properties are listed in
Table 21.3.
21.4 Spectrum of Activity 833
Table 21.3 Ecotoxicological and environmental properties of fluopicolide.
Bird, acute oral, LD50 , quail >2250 mg kg –1

Fish, acute (96 h), LC50 , rainbow trout 0.36 mg l –1
Fish, acute (96 h), LC50 , bluegill sunfish 0.75 mg l –1
Water flea, acute (48 h), EC50 , Daphnia magna >1.8 mg l –1
Algae, growth inhibition (72 h), ErC50 , Selenastrum capricornutum >4.3 mg l –1
Plant, growth inhibition (7 days), EC50 , Lemna gibba >3.2 mg l –1
Earthworm, acute (14 days), LC50 >1000 mg a.i. kg soil –1
Honeybee, contact, LD50 >100 mg per bee
Typhlodromus sp., acute, LR50 0.313 kg ha –1
Aphidius sp., acute, LR50 0.419 kg ha –1
Table 21.4 Crops and pathogens on which fluopicolide has been tested
successfully.
Crop Pathogen
Potato Phytophthora infestans

Tomato Phytophthora infestans
Peppers Phytophthora capsici
Leek Phytophthora porri
Vines Plasmopara viticola
Brassicas Peronospora parasitica
Tobacco Peronospora tabacina
Cucurbits Pseudoperonospora cubensis
Lettuce Bremia lactucae
Roses Peronospora sparsa
21.4
Spectrum of Activity
Fluopicolide has been tested successfully on a range of crops and pathogens

(see Table 21.4). Currently, fluopicolide is undergoing development worldwide, in
combination with other fungicides, for use in a wide variety of crops. The first
commercial launches of fluopicolide are in co-formulations with fosetyl-Al for use
®
in vines (trade name Profiler ), and with propamocarb-HCl for use in potatoes and
®
vegetables (trade name Infinito ).
21.4.1
Effect on Zoospores and Mycelial Growth of P. infestans
Fluopicolide, when applied to the zoospores of P. infestan at a concentration of only

1 μg ml−1 , caused the zoospores to stop swimming within 1 min; the zoospores
subsequently swelled and burst within a few minutes (Figure 21.2).
X 40 X 40
(a) (b)
Figure 21.2 Effect of fluopicolide on P. infestans zoospores.

(a) Control [solvent (dimethyl sulfoxide)-treated] zoospores;
(b) Fluopicolide-treated zoospores (3 ppm) at 10 min after
treatment.
X 100 X 100
(a) (b)
Figure 21.3 Effect of fluopicolide on P. infestans hyphae

(stained with Trypan blue). (a) Control [solvent (dimethyl
sulfoxide-treated)] hyphae; (b) Fluopicolide-treated mycelium
(10 ppm) at 48 h after treatment.
In vitro, fluopicolide strongly inhibited the mycelial growth of P. infestans, with

an 80% inhibition being observed at a concentration as low as 0.1 μg ml−1 over four
to seven days. Distinctive symptoms were also observed on the treated mycelium
(Figure 21.3). Staining of the treated hyphae with Trypan blue indicated leakage of
the cellular contents, and confirmed that fluopicolide had induced mycelial lysis,
notably at the apex of the hyphae.
21.5
Effect of Fluopicolide on Spectrin-Like Protein Distribution
The rapidly induced effects of fluopicolide led to the investigation in greater detail
of proteins known to be associated with the cytoskeleton, since no significant effect
was observed on either tubulin or actin. One candidate here was spectrin, which is
21.5 Effect of Fluopicolide on Spectrin-Like Protein Distribution 835
(a) (b) (c) (d) (e)
Figure 21.4 Kinetics of the effect of fluopicolide on the

distribution of spectrin-like protein(s) in the hyphae of
P. infestans. (a) Control cell; (b–e) Hyphae treated with
10 ppm fluopicolide for 3 min (b), 10 min (c), 2 h (d), and
24 h (e).
known to play a crucial role in membrane stability by anchoring to other cytoskeletal

proteins in animal cells [3]. In order to determine such effect, immunofluorescence
studies using antibodies raised against chicken erythrocyte α/β spectrin (Sigma
S1390) were first conducted on the mycelium of P. infestans. The results showed
that antigen(s) cross-reacting with those antibodies were prominently localized
in the peripheral regions (close to the plasma membrane), along the nontreated
hyphae (Figure 21.4). Upon fluopicolide treatment, a complete loss of plasma
membrane localization of these spectrin-like protein(s) was observed, such that
they became distributed as spherical spots in the cytoplasm of the hyphae cells.
The kinetics of this effect revealed that such spectrin-like protein delocalization
occurred very rapidly, within 3 min of fluopicolide treatment (Figure 21.4). Notably,
such delocalization was maintained with longer treatment times. Clearly, the action
of fluopicolide was to induce a cellular delocalization of spectrin-like protein(s),
from the plasma membrane to the cytoplasm.
Similar effects were observed in zoospores, when they were just ceasing to
move (1 min after being treated with fluopicolide), during swelling (after 5 and
10 min treatment) and before cell lysis (from 15–20 min treatment) (Figure 21.5).
Interestingly, the phenotypic symptoms observed on zoospores correlated very well
with the modification of spectrin-like protein(s) cellular localization.
Subsequently, the actions of anti-Oomycete fungicides and fluopicolide were
compared in terms of their effects of spectrin-like protein(s) localization, with
treatment times ranging from 3 min to 24 h. After a 2 h treatment period, none
of the fungicides tested (iprovalicarb, fenamidone, dimethomorph, metalaxyl, and
zoxamide) had induced any spectrin-like protein redistribution (Figure 21.6).
Whereas, at all treatment times, each of the fungicides produced a peripheral
staining similar to that in untreated (control) cells, no such effect was observed
with fluopicolide.
21.5.1
Characterization of Spectrin-Like Proteins in P. infestans by Bioanalysis
Spectrin was first discovered and described in animal cells in different tissues
and cell types [3]. Interestingly, spectrin-like proteins have also been identified in
(a) (b) (c) (d) (e)
(a) (b) (c) (d) (e)
Figure 21.5 Kinetics of the effect of fluopicolide on the

distribution of spectrin-like protein(s) in zoospores of P.
infestans. Upper row: images under light inducing the fluo-
rescence. Lower row: Images under normal light. (a) Control
cell; (b–e) Zoospores treated with 3 ppm fluopicolide for
1 min (b), 5 min (c), 10 min (d), and 20 min (e).
(a) (b) (c) (d) (e)
(f) (g)
Figure 21.6 Immunofluorescent localization of spectrin-like

protein(s) in hyphae of P. infestans treated with fluopicolide
and known anti-Oomycetes agents. (a) Control cell; (b–g)
After 2 h treatment with (b) 10 ppm fluopicolide, (c) 10 ppm
iprovalicarb, (d) 10 ppm fenamidone, (e) 10 ppm dimetho-
morph, (f) 10 ppm metalaxyl, and (g) 10 ppm zoxamide.
plant and fungi [4–7]; these proteins were characterized by their spatial localization
close to the plasma membrane, and their size determined on Western blots using
an anti-chicken α/β spectrin antibody (Sigma S1390). However, in none of these
organisms has a protein corresponding to spectrin been purified, and the amino
acid sequence identified. A database search to find spectrin-like protein(s) in fungi
(Magnaporthe grisea and Neurospora crassa genome sequences) or in the Oomycetes
[Phytophthora sojae and Phytophthora ramorum genome sequences, partial expressed
21.6 Conclusion 837
sequence tag (EST) sequences of P. infestans] by homology using BLAST (Basic

Local Alignment Search Tool) proved to be unsuccessful. Consequently, a search
by spectrin domain was initiated.
In structural terms, erythrocyte spectrins comprise an anti-parallel heterodimer
of two subunits α (240 kDa) and β (220 kDa), that are characterized by the presence
of specialized domains: (i) a domain formed by a triple-helical repeat of 106–120
amino acids, the so-called spectrin repeat (SpR) (this is present from four to
more than 20 times); (ii) an EF-hand domain; (iii) a calcium-binding domain; and
(iii) a highly conserved N-terminal domain responsible for the binding of actin
filaments.
The SpR domain was used to start a PFAM (protein family database) analysis
[8], a system based on Hidden Markov Models. The SpR domain built only with
mammalian representative domains (PF00435) gave no hit in either fungal or
Oomycete species. To improve this search, a PFAM motif was constructed with a
SMART (a simple modular architecture research tool) alignment [9]. In this system,
the motifs are automatically enriched by new sequences obtained from worldwide
databases, allowing more diversity. The ‘‘seed alignment’’ of the SMART database
(SM00150) was then used to build a new PFAM motif for SpR domain. This
approach provided one hit in P. sojae, to a protein of around 100 kDa (accession
number 137006) that corresponded to a putative protein belonging to the spectrin
family known as α-actinin. To date, far α-actinin has not been found in most
eukaryotic cells, except in plants [10]. This protein contained two SpR domains of
107 and 113 residues, showing a homology to the consensus SpR. A search with
the other domains gave no better results.
Similar results were obtained in fungi; that is, a 113 kDa protein and a 88 kDa
protein can be found in N. crassa (NCU06429.1) and M. grisea (MG06475.4),
respectively; both are closely related to a putative α-actinin.
Gene expression profiling studies have shown that a significant number of the
genes involved in endoplasmic reticulum/Golgi functions were upregulated after
treatment with fluopicolide [11]; these findings suggest that vesicle transport is
affected by fluopicolide. Small GTPases of the Arf family might contribute to the
regulation of both cytoskeleton functions and the assembly of vesicle transport
[12]. At the present time, further investigations are required to reveal the molecular
mechanism involved in spectrin-like delocalization.
21.6
Conclusion
The fluopicolide-induced delocalization of spectrin-like proteins represents a new

mode of action that differs from that of the currently available anti-Oomycete
fungicides. Although, to date, the spectrin-like proteins have been poorly charac-
terized in fungi and the Oomycetes, recent data have indicated the presence of
these proteins in Phytophthora. Further investigations are required to: (i) determine
whether fluopicolide interacts directly with spectrin-like protein(s); and (ii) identify
the role of these cytoskeleton-associated proteins in Oomycete development. The

resolution of these points may lead to the creation of a second generation of active
compounds.
References
1. Tyler, B.M. (2001) Trends Genet., 17, Lassmann, T., Moxon, S., Marshall,
611–614. M., Khanna, A., Durbin, R., Eddy, S.R.,
2. Moloney, B.A., Hardey, D., and Sonnhammer, E.L.L., and Bateman, A.
Saville-Stones, E.A. (1999) Patent WO (2006) Nucleic Acids Res. Database, (34),
09,942,447, (Agrevo UK) Prior. February D247–D251.
19, 1998. 9. Schultz, J., Milpetz, F., Bork, P., and
3. Bennett, V. (1990) Physiol. Rev., 70, Ponting, C.P. (1998) Proc. Natl Acad. Sci.
1029–1065. USA, 95, 5857–5864.
4. Braun, M. (2001) Plant Physiol., 125, 10. Virel, A. and Backman, L. (2007) Mol.
1611–1619.
Biol. Evol., 10, 2254–2265.
5. Degousée, N., Gupta, G.D., Lew, R.R.,
11. Toquin, V., Barja, F., Sirven, C., Gamet,
and Heath, I.B. (2000) Fungal Genet.
S., Mauprivez, L., Perret, P., Latorse,
Biol., 30, 33–44.
M.-P., Zundel, J.-L., Schmitt, F., Lebrun,
6. Slaninova, I., Holubarova, A., and
Svoboda, A. (2003) Can. J. Microbiol., 49, M.-H, and Beffa, R. (2010) in Recent
189–196. Developments in Management of Plant
7. Kaminskyj, S.G. and Heath, I.B. (1995) Diseases (eds U. Gisi, I. Chet, and M.L.
J. Cell Sci., 108, 849–856. Gullino), Springer, pp. 19–36.
8. Finn, R.D., Mistry, J., Schuster-Böckler, 12. Myers, K.R. and Casanova, J.E. (2008)
B., Griffiths-Jones, S., Hollich, V., Trends Cell Biol., 18, 184–192.
839
22
Melanin Synthesis in the Cell Wall
22.1
Biological Occurrence and Function of Melanin in Fungi
In 2002, the melanin biosynthesis inhibitor (MBI) fungicides constituted a minor

segment of the world fungicide market, with a global market share of approximately
10%. The MBI fungicides play an important role in rice production in Asia and in
some Latin American countries. In Japan, among the various specific fungicides
used to treat rice blast (Pyricularia oryzae), the MBIs come a close second position
after the host defense inducers. However, following the report of a MBI-D resistant
strain of Pyricularia oryzae in southern Japan in 2001 (notably, such resistance in
rice blast was reported only in Japan, and not in other countries), the MBIs were
quickly replaced with host defense inducers.
Melanin is a general term used in different biological contexts for various insol-
uble dark or black complex polyphenolic resins, the chemical structures of which
are often only incompletely characterized [1, 2]. Despite its imprecise definition,
melanin is found ubiquitously in the living world, the best known example being
mammalian melanin. The latter is biosynthesized from the amino acids tyro-
sine and 3,4-dihydroxyphenylalanine l-(DOPA) as monomeric building blocks. In
mammals, the melanins undertake a variety of biological functions including, on
occasion, the neuronal system. Mammalian melanin is, however, quite distinct
from the fungal melanin being discussed here.
Fungi such as Pyricularia contain a melanin that is constructed from
1,8-dihydroxynaphthalene (DHN), and which is referred to as DHN melanin [1].
The biosynthetic pathway of DHN melanin is short and simple (Figure 22.1); the
origin of the building blocks is not amino acids, but a polyketide originating from
acetyl coenzyme A and malonyl coenzyme A. Besides DHN, this fungal melanin
may contain other poorly characterized constituents. To date, all the enzymes of
the DHN melanin biosynthesis pathway have been identified, cloned, and are
mostly well described [1, 3].
Knowledge of the enzyme’s sequences allows their presence to be monitored
in different organisms. BLAST (Basic Local Alignment Search Tool) sequence
searches have been used to clearly identify the genes for pathway specific enzymes

840
OH OH 1,3,6,8-tetrahydroxy- OH O
Polyketide naphthalene
synthase reductase
1 × acetyl CoA +
4 × malonyl CoA HO OH HO OH
1,3,6,8-tetrahydroxy-naphthalene (+) scytalone

22 Melanin Synthesis in the Cell Wall
1,3,8-trihydroxy-
Scytalone OH OH O OH Scytalone OH OH
naphthalene
dehydratase dehydratase
reductase
HO HO
1,3,8-trihydroxynaphthalene Vermelone 1,8-dihydroxynaphthalene

(DHN)
OH
O OH
Diphenol oxidase OH Phenol resin
Radical polymerization
(laccase type) (no precise structure,
O O
O OH
crosslinked statistical polymer
HO may contain other building blocks)
DHN melanin
Figure 22.1 Biosynthetic pathway of fungal DHN melanin.

22.1 Biological Occurrence and Function of Melanin in Fungi 841
Oomycota
M Leotiomyceta
Ascomycota
Ustilaginomycetes
Urediniomycetes
Zygomycota Basidiomycota
Glomeromycota
Figure 22.2 Occurrence of DHN melanin biosynthesis en-

zymes in a phylogenetic tree of fungi (unpublished assem-
bly). The red star with the yellow ‘M’ indicates the evolution-
ary point of DHN melanin biosynthesis emergence.
only in Leotiomycetes (Figure 22.2), and not in any other organism. This evolution-
ary appearance of the pathway limits the potential biological spectrum of fungicides
that might inhibit DHN melanin biosynthesis.
Fungal DHN melanin is located in the granular or fibrillar layers in the cell walls
[1] where, as a robust polymer, it mechanically protects the cell walls, while the dark
color shields the cells against the effects of ultraviolet light. In addition, melanin
can adsorb poisonous heavy metals, providing further chemical protection against
any oxygen radicals that are set free by the plant’s defense mechanisms. Melanin
is also resistant to the lytic enzymes (e.g., glucanases, chitinases) of predators and
of prey plants. These defensive characteristics of melanin indicates why fungicides
that inhibit melanin biosynthesis do not kill the fungi directly, nor show any
fungicidal activity towards fungi grown in liquid culture or on agar.
Nevertheless, some phytopathogenic fungi require melanin absolutely for their
pathogenicity (see Refs [4, 5], and references therein). Among these can be included
Pyricularia, which causes rice blast, and Colletotrichum, which causes anthracnoses.
In order to penetrate the plant epidermis, these fungi generate pressure appressoria,
the cell walls of which are heavily fortified by melanin, except for the tip, from
where the penetration peg grows into the plant [1]. Such melanin bracing allows
the build-up of an extremely high turgor pressure, which is used to force the
penetration peg into the plant. Notably, those melanin-containing fungi without
pressure appressoria are not affected by MBI fungicides during their infection
process, further narrowing the biological spectrum of the MBI fungicides.
842 22 Melanin Synthesis in the Cell Wall
22.2
Overview: Fungicides Inhibiting DHN Melanin Biosynthesis
The initial enzyme in the DHN biosynthetic pathway – a specific polyketide syn-
thase – may represent an interesting target for the MBIs. Although polyketide
synthases with diverse functions are found in many different organisms, very few
inhibitors of polyketide synthases have been identified, and even fewer inhibitors
of the polyketide synthase involved in melanin biosynthesis (Table 22.1). Aflastatin
A (1), a complex natural compound, inhibits melanin production in Colletotrichum
lagenarium [6], while both abikoviromycin (2) and a dihydro derivative thereof (3)
also inhibit the polyketide synthase of melanin biosynthesis in C. lagenarium [7].
Unfortunately, no details have been reported of fungicidal assays (i.e., pathogenicity
tests) with either aflastatin A or abikoviromycin. The fungicide KC10017 (4) [8] is
known to inhibit polyketide synthase, while cerulenin (5), which is better known
as a fatty acid synthase (FAS) inhibitor, blocks the polyketide synthase of melanin
biosynthesis [9].
Several of the fungicides that are known to inhibit 1,3,6,8-tetrahydroxy-
naphthalene reductase (MBI-Rs; see Table 22.2) have been employed since the
1970s, including pentachlorobenzyl alcohol (PCBA) (6), tricyclazole (7), pyroquilon
(8), and fthalide (9). However, none of these has encountered any problems of
resistance, and they have retained considerable economical importance, mainly in
Northeast Asia.
Since 1998, several rice fungicides have been introduced into the market that func-
tion by inhibiting the enzyme scytalone dehydratase (SD) (MBI-Ds; see Table 22.3).
These fungicides have been used almost exclusively in Japan as systemic fungicides
for specialized protective applications in rice nursery boxes. The syntheses, as well
as the structural, biochemical and biological aspects, of these melanin biosynthe-
sis inhibitors are detailed in the following subsections. Prominent examples of
these include carpropamid (10), dicyclomet (11), and fenoxanil (12); details of these
agents are shown in Table 22.3, together with those of other inhibitors (the majority
of which have been co-crystallized with their target).
22.3
Biology of Scytalone Dehydratase Inhibitors
The common properties of the MBI-Rs and MBI-Ds include a protective controlling
activity against rice blast, and the inhibition of appressorium pigmentation of
Pyricularia oryzae on agar plates or cellophane membranes [14–16]. The inhibition
of spore liberation from leaf lesions has also frequently been observed in all known
MBI-Rs and MBI-Ds [15, 17–21], and this might be considered an indirect effect of
melanin inhibition in the conidia.
Agronomically, the main advantage of the MBI-Ds to MBI-Rs is their compatible
systemic action, combined with a long-lasting control efficacy. Both, tricyclazole and
pyroquilon are highly water-soluble and show quick mobility, although their action
Table 22.1 Structures of inhibitors of polyketide synthase in DHN melanin biosynthesis.
Compound no. Name and structure Status Biology
1 Aflastatin A Exptl. C
O
N OH OH OH OH
OH
O OH OH OH OH OH OH OH OH OH OH OH OH OH OH O
OH
HO
OH
2 Abikoviromycin Exptl. C
O
H
3 Dihydroabikoviromycin Exptl. C
O
H
22.3 Biology of Scytalone Dehydratase Inhibitors
843
H
N
844
4 KC10017 Exptl. RF
O N
O
O N
22 Melanin Synthesis in the Cell Wall
O
Br
N
5 Cerulenin Exptl. P,C

H O
O O
H
NH2
Abbreviations used in Tables 22.1–22.3: P: inhibition activity in vitro, Pyricularia; C: inhibition activity in vitro, Colletotrichum; RF: Blast control efficacy in rice, foliar spray;
RS: Blast control efficacy in rice, systemic application; MP: commercial product in 2006; Res: Field resistance authorized by Fungicide Resistance Action Committee
(FRAC).
Table 22.2 Structures of inhibitors of 1,3,6,8-tetrahydroxy-

naphthalene reductase in DHN melanin biosynthesis.
6 Pentachlorobenzyl alcohol Withdrawn RF
OH
CI CI
CI CI
CI
7 Tricyclazole Market RF
RS
S MP
N
N
N
8 Pyroquilon Market RS
MP
N O
9 Fthalide Market RF
MP
CI
CI
O
CI
CI O
is hardly long-lasting. Fthalide has an excellent long-lasting efficacy, but no systemic

effect. In clear contrast to those MBI-Rs, carpropamid, diclocymet, and fenoxanil
show systemic effects with moderate levels of water solubility, which enables the
season-long control of leaf blast by a one-shot application at transplanting, and
also provides comparable lasting efficacy to fthalide when sprayed. The lower water
solubility is favorable by itself from an environmental viewpoint of rice cultivation.
In Korea, carpropamid is used also for seed treatment with insecticides for the
long-lasting control of blast and pests in rice. In Japan, the MBI-Ds – together with
Table 22.3 Structures of inhibitors of scytalone dehydratase

(SD) in DHN melanin biosynthesis.
Compound Name and structure Status Biology Comment

no.
10 Carpropamid Market RS Res

RF
CI CI CI CI MP
O O Ki = 19 pMa
N N
H H
CI CI
11 Dicyclomet Market RS Res

RF
N MP
CI
H Ki = 36 pMb
H
N
CI O
12 Fenoxanil Market RS Res

RF
N MP
CI O Ki = 130 pMb
O
N
H
CI
13 OH O Exptl. Ki = 47 pMa –
N
H
Br
F
14 Exptl. Ki = 8 pMd –
N
N
N
N
H

no.
15 Exptl. Ki = 32 pMa –
N N
N
H
F
F
16 CI CI Exptl. Ki = 2300 pMa –

O
SO N
H
Br
17 Exptl. Ki = 26 pMb Not systemic

CO
CI N
CF3 H
Me Br
18 Exptl. Ki = 100 pMb Systemic

CO
CI N
CH3 H
Me Br
19 O Exptl. Ki = 12 pMc Hydrophilic

N variation
N
H
20 Exptl. Ki = 2.2 pMc Hydrophilic

CI CI O
variation
N
H

no.
21 Exptl. Ki = 13 pMc Hydrophilic

N N
variation
N
H
22 HO O Exptl. Ki = 3.8 pMc Hydrophilic

variation
N
H
23 N Exptl. Ki = 3.6 pMc Hydrophilic

N variation
N
H
24 N Exptl. Ki = 15 pMc Hydrophilic

N N variation
N
H
25 O Exptl. Ki = 4.6 pMc Hydrophilic

CI variation
N
H
CI N
26 S Exptl. Ki = 11 pMc Lipophilic

variation
O
N
O
N
H

no.
27 Exptl. Ki = 25 pMc Lipophilic

variation
O
N
O
N
H
28 O Exptl. Ki = 20 pMc Lipophilic

N variation
N
H
Br

N variation
O
N
H
F

N variation
O
N
H
F

N variation
O
N
H
a
From Ref. [10].
b
From Ref. [11].
c
From Ref. [12].
d
From Ref. [13].
chloronicotyinyl insecticides (CNIs) – have contributed significantly to reducing

not only the farmers’ labor costs but also the total amount of rice pesticides
utilized, by the popular use of a protective, one-shot application in the rice nursery.
The overwhelming prevalence of a one-shot application with long-lasting MBI-Ds
led, however, to an enhancement of the relatively early outbreak of field resistance.
This had not been expected for such secondary metabolism inhibitors, based on
the long-term experience of using MBI-Rs without resistance problems.
One other common property of the three commercial MBI-Ds, that is not shared
by the MBI-Rs, is the specific systemic damage to Solanaceae plants, although the
relevance of this specific sensitivity and MBI-D activity has not been investigated.
In practice, however, as MBI-s are used exclusively in an isolated environment for
rice, this is not considered to be a serious problem.
22.4
Biochemical Reaction Mechanism of Scytalone Dehydratase and Structure-Based
Inhibitor Design
The role of SD is to catalyze two analogous steps in DHN melanin biosynthesis

(Figure 22.1), and various biochemical aspects of this catalysis have been inves-
tigated by Douglas Jordan and coworkers, at DuPont [12, 13, 22–25]. Based on
the crystal structure analysis of SD (the details of which, with respect to inhibitor
design, are outlined below), a biochemical reaction mechanism can be proposed
(Figure 22.3).
22.4.1
X-Ray Structures and the Active Site of Scytalone Dehydratase
Although the binding of a target to an enzyme is clearly an essential requirement

for biological activity, other factors such as absorption, distribution, metabolism,
excretion (ADME) are equally important to understand the mechanism of action
involved. For example, the rational design of a new compound will be greatly
enhanced if details are available of the three-dimensional (3-D) structure of the
enzyme–ligand complex.
Based on the results of protein X-ray structures of SD in its apo form, and
cocrystallized with several inhibitors (Table 22.4), the binding niche in the presence
of inhibitors is well known. Unfortunately, no protein X-ray structures have been
determined with the natural substrates of SD, namely scytalone or vermelone, both
of which are considerably smaller than the inhibitors.
The SDs are symmetric trimers built from identical single-domain monomers
belonging to a group of folds referred to as α + β rolls. The active site of each
monomer forms a hydrophobic pocket in the interior of the central β-barrel
formed by a curved, six-stranded β-sheet. Two representations [26, 27] of an SD
monomer cocrystallized with carpropamid (10) – the first member of the novel
class of MBI-Ds – are shown in Figure 22.4 [26, 27].
Only one polar amino acid, Asn131, is available for direct interaction with the
inhibitors. Two conserved water molecules can interact with the inhibitors; these
are fixed by hydrogen bonds to the hydroxyl groups of Tyr30 and Tyr50, and by
the imidazole nitrogens of His85 and His110. Access to the active site might be
possible by a hinge-bending movement of the amphiphilic carboxy-terminal helices
to H2O to H O
H H bound to H H bound2 to
OH O O O OH O O O OH OH
Y30 and Y50 Y30 and Y50
H
HO OH HO O to HO HO HO
H85 and H110 H H
H to
H85 and H110
1,3,8-trihydroxy-
Scytalone Transition state Transition state
naphthalene
to H2O to H O
H H bound to H H bound2 to
OH O O O O OH O O Y30 and Y50 OH OH
Y30 and Y50
H
OH O to
H85 and H110
H H H to
H85 and H110
1,8-dihydroxy-
Vermelone Transition state Transition state naphthalene
Figure 22.3 Biochemical reaction mechanism of scytalone dehydratase in DHN melanin biosynthesis. Note that the molecules flip by 180◦ between
the two steps.
22.4 Biochemical Reaction Mechanism of SD and Structure-Based Inhibitor Design
851
Table 22.4 Publicly available protein X-ray structures of scy-

talone dehydratase (SD) inhibitor complexes.
PDB code Inhibitor class Year Resolution (Å) Reference Inhibitor
1STD Salicylamide 1994 2.9 [28] 13

2STD Cyclopropanecarboxamide 1998 2.1 [29] 10
3STD Cyanocinnoline 1998 1.65 [30] 14
4STD Salicylamide 1999 2.15 [10] 13
5STD Norephedrine 1999 1.95 [10] 15
1IDP Apo Enzyme 2002 1.45 [29] –
Figure 22.4 Cartoons of the 3-D structure of an

SD-monomer with carpropamid in the active site.
H4 and H5, which contribute significantly to the hydrophobic part of the binding
niche. In the apo-structure, the flexibility of the C-terminal renders the amino acids
156–172 invisible.
Comparing the amino acid sequences of the SDs in different organisms –
augmented by the knowledge of 3-D protein structures – enables rationalization of
the reasons for specificity, or for the failure of a fungicide to be active. Especially,
resistance caused by mutations in the fungal target can be explained and, perhaps,
circumvented. The sequences of all SDs known by 2006 are shown in Figure 22.5.
Figure 22.5 Sequence alignment of scytalone dehydratases. Legend: = active site residues; • = further hydrogen-bonding
residues; = further hydrophobic residues; ⇓= V75M mutation. Organisms: Botrci = Botryotinia fuckeliana B05.10 [Botrytis cinerea];
Sclesc = Sclerotinia sclerotiorum 1980; Gibbze = Gibberella zeae PH-1; Cerare = Ceratocystis resinifera SD1; Collla = Colletotrichum lage-
22.4 Biochemical Reaction Mechanism of SD and Structure-Based Inhibitor Design
narium; Glomgr = Glomeralla graminicola M1.001; Vertal = Verticillium albo-atrum VaMs.102; Sordma = Sordaria macrospora S48977;
Ophofl = Ophiostoma floccosum 387N; Bipoor = Bipolaris oryzae; Pyrior = Magnaporthe grisea 70-15 [Pyricularia oryzae] (in green and
853
bold); Neurcr = Neurospora crassa OR74A; Pyrntr = Pyrenophora tritici-repentis Pt-1C-BFP; Aspeni = Aspergillus nidulans FGSC A4;
Aspefu = Aspergillus fumigatus Af293; Aspeor = Aspergillus flavus var. oryzae NRRL3357.
22.4.2
Computational Investigations of the Enzyme Mechanism
Molecular dynamics (MD) calculations used to explore the mobility of the two
water molecules in the SD binding pocket in the presence of the weak inhibitor
N-isopropyl salicylamide [31], have revealed that the water molecule associated with
the inhibitor carbonyl is more labile than that associated with the inhibitor NH.
Although the protein was kept fixed during the simulation and the protonation
states of the two histidines are worthy of discussion, these findings may help to
prioritize drug design efforts.
The same holds true if the detailed enzymatic steps were known. However, as no
complex of the enzyme’s natural substrates – scytalone or vermelone – is available,
the proposed mechanism of SD, the catalysis of a syn β-elimination of water from
scytalone and subsequent aromatization [28], remains to be proven experimentally.
Although much smaller and much less lipophilic, both scytalone and vermelone
share some structural features with the cocrystallized competitive inhibitors. To
investigate the proposed enzyme mechanism, a quantum chemical model system
was built based on the protein structure 4STD [10]. This consisted of the four amino
acids Tyr30, Tyr50, His85, and His110, the catalytic water molecule and vermelone,
the initial position of which was ascertained by superposition on the salicylic ring
of the inhibitor in 4STD. The second conserved water molecule found in the X-ray
structures was omitted, as it was thought to be a product of the enzymatic reaction.
Keeping only the backbone atoms of the model system fixed, a geometry
optimization using density functional theory (DFT; BP86 functional/SVP basis set,
unpublished results), as provided by the TURBOMOLE suite of programs [32],
revealed that only one conformation of vermelone – with the β-hydrogen to be
abstracted in an axial position – avoids being trapped in a local minimum, thus
preventing a subsequent reaction. During optimization, the catalytic water and the
side chains change their positions only marginally, an exception being Tyr50, the
phenoxy ring of which rotates by more than 100◦ to improve its H-bond with
the catalytic water. In contrast, to prepare for the reaction, vermelone moves by
almost two bond lengths from its starting position, shortening the Hβ − Nε85
and OH − Nε110 distances from 3.58 and 5.59 Å to 2.63 and 1.84 Å, respectively.
For the next step – formation of the carbanion intermediate in the E1cb reaction
path – Hβ was attached to Nε85 , keeping His85 either protonated at N-δ, or not. The
calculations indicated that the deprotonation of N-δ is essential for the water-assisted
formation of the enolate and the subsequent abstraction of the hydroxyl-group.
Although the last step in the reaction sequence – abstraction of a proton from
C4, followed by aromatization of the α, β-unsaturated ketone – will occur spon-
taneously without assistance by a protein, it seems reasonable to assume that
product formation is accelerated considerably by an enzymatic mechanism. Again,
DFT calculations suggest that deprotonated His110 could accept a proton from
the previously generated water molecule, which in turn accepts the C4 proton.
Protonation of the ketone could occur by the catalytic water bound by the two
tyrosines.
22.4 Biochemical Reaction Mechanism of SD and Structure-Based Inhibitor Design 855
22.4.3
Comparison of Inhibitor Structures in the SD Binding Niche
With the exception of Asn131 and Asp31, the binding niche of SD is mainly
hydrophobic. Its polar part consists of His85 and His110, and of Tyr30 and Tyr50
coordinating two conserved water molecules that mediate hydrogen bonds to the
inhibitors in the protein–ligand complexes.
All of the five different cocrystallized inhibitor complexes shown in Table 22.4,
and those reported during rational design programs [30, 33–35], demonstrate
a common pattern of H-bonds: the carbonyl oxygens accept an H-bond from
the tyrosine-coordinated water, and the amide NH donates an H-bond to the
histidine-coordinated water. Replacing salicylamide by quinoxaline does not
change this pattern. Superposition of the protein structures shows the varying
flexibility of the binding niche: While the amino acids responsible for recognition
have their side chains nearly unchanged, the hydrophobic part of the binding
pocket exhibits considerable side-chain flexibility. Yet, remarkably the backbone
atoms show almost no structural variation. This is true even for the apo structure,
where only His110 adopts an outward directed side-chain conformation. Clearly,
the inhibitors stabilize the C-terminal helix of SD (Phe156:Lys172).
Some notable backbone differences can be found between the early X-ray
structures 1STD and 2STD obtained at low (pH 4.5, pH 5.1), and those obtained
later at neutral pH (7.5–8) (3STD–7STD), which can be attributed to the pH
differences. Site-directed mutations (see below) support the assumption that these
differences affect the lipophilic inhibitor recognition.
Superposition of the Cα-atoms of 1STD–7STD, and examining the resulting
inhibitor positions, provides an impression of the extensions of the active site
(Figure 22.6) [26].
Once target protein structures are available, several computational techniques can
be applied to support a rational drug design. Such design, based on the protein X-ray
structures, led to several proposals that turned out to bind more effectively than
the original compounds. The catalytic water molecule was successfully replaced by
a part of a ligand designed for this purpose [30]. Modeling techniques can be used
to place other putative or existing inhibitors into the binding site, as is outlined for
the examples of diclocymet (11) and fenoxanil (12) below.
Figure 22.6 Two views of the SD-inhibitors, from backbone

superimposition of the X-ray structures 1STD-7STD.
It is not too difficult to place the two isomers of diclocymet into the known binding
niche as it is supplied as a mixture ((RS)-2-cyano-N-[(R)-1-(2,4-dichlorophenyl)ethyl]-
3,3-dimethylbutyramide), where the specified (R)-configuration mimics that
of carpropamid, but fenoxanil (N-(1-cyano-1,2-dimethylpropyl)-2-(2,4-dichloro-
phenoxy)proprionamide) requires some attention as no information on its
stereochemistry is provided. One of the default approaches would be to start with
poses proposed by a docking program (e.g., FLEXX [36]), which is typically used in
a high-throughput application – the so-called ‘‘virtual screening approach’’ – where
libraries of millions of compounds are docked into the binding niche. In the
fenoxanil case, this approach is inferior to docking by hand, as fenoxanil is not
only larger than the other inhibitors but also has a reversed functionality at the
amide moiety. DFT optimization of the SS (RR) enantiomer reveals that the
conformation fitting best into the binding niche corresponds to a local minimum
in energy, ∼3 kcal mol−1 above the absolute minimum with its internal H-bond.
In the binding conformation, this internal H-bond is replaced by external H-bonds
to water; its proposed orientation within the binding niche of 7STD, superimposed
with carpropamid, is shown in Figure 22.7 [27], together with the Connolly
surfaces of the wild-type and the V75M binding niches.
Highly potent (8 pM < Ki < 48 pM) cyanoacetamide derivatives of norephedrine
were designed [34] using multiple crystal structures, permitting the detection of
variable regions of the active site and optimizing hydrophobic contacts with the
inhibitors. In addition, by combining selected cyclic aliphatic carboxylic acids
with appropriate amides, a combinatorial chemistry was employed to successfully
(a) (b)
Figure 22.7 (a) Connolly surfaces of the binding niches of

native (green) and V75M (red) SD in 7STD; (b) Superpo-
sition of carpropamid and fenoxanil (yellow) in the 7STD
binding niche.
22.5 Chemistry and Stereochemistry of Carpropamid 857
identify new chemical classes [35]. The best representative of these, a cyclobu-
tanecarboxamide with chlorine trans to the trifluoromethyl group 17, showed an
in vitro activity (Ki = 26 pM) comparable to that of carpropamid. From the X-ray
structure [35], it becomes clear why the cis-isomer is less active by almost two
orders of magnitude – the favorable complementary electrostatic interaction of 17
with the side chain of Asn131 is disturbed by the ‘‘wrong’’ spatial arrangement of
the substituents.
The importance of optimizing general physico-chemical properties, in addition
to improving the binding characteristics of the inhibitors, was demonstrated
by a design process where the replacement of CF3 in the exceptionally potent
trifluorosubstituted compound [37] by a methyl group led to a slightly less potent,
but significantly more systemic, compound (18) [11].
22.4.4
Complementary Information by Site-Directed Mutations
The details of other inhibitor complexes have been resolved [34, 35] during the
course of drug design programs, by changing systematically the hydrophobic and
hydrophilic parts of the ligands. Likewise, numerous site-directed mutations [24]
have been conducted, based on the structures served to explore the variability of
the binding site, the aim being to probe the importance of selected amino acids for
binding (see Table 22.3, compounds 19–25 and 26–31).
In the hydrophilic region of the active site, 15 single-point mutants resulted in
binding affinities that ranged from 10-fold enhancements to 1100-fold reductions
for the ligands 19–25, while and five mutations in the hydrophobic part led to
enhancements for ligands 26–31, ranging from three- to 70-fold compared to the
wild-type SD. From the results of these studies it can be concluded that the side
chain of Phe158, the orientation of which differs in the X-ray structures grown at
acidic pH, where it is exposed to the solvent, and at neutral pH, where it points
toward the inhibitors, is important for the hydrophobic interactions in the binding
site. Val75 is critical for resistance effects as it recognizes the chiral methyl groups
of some inhibitors. The hydroxyl groups of Tyr30 and Tyr50 and their H-bonds to
the catalytic water molecule are much less important for inhibitor binding than
the corresponding H-bond network of His85 and His110. Inhibitors with good
acceptor properties interact favorably with the carboxamide of Asn131, whereas its
H-bond to Ser129 does not contribute to the shape of the active site.
22.5
Chemistry and Stereochemistry of Carpropamid
A novel class of fungicides for rice blast control was presented in 1994 at
the Brighton Crop Protection Conference [14], and details of the synthesis of
its most prominent representative, carpropamid (10), are outlined below [38].
The syntheses of the two other marketed MBI-Ds – diclocymet (11) and fenox-
anil (12) – are described elsewhere [39, 40]. Three chiral atoms of carpropamid
give rise to eight stereoisomers, the most active mixture of which is (1RS,
3SR, 1 RR)-2,2-dichloro-N-[1-(4-chlorophenyl)ethyl]-1-ethyl-3-methyl-cyclopropane-
carboxamide [41]. The free energies of the two pairs of enantiomers, having alter-
nating chiralities at the cyclopropyl ring, differ by less than 0.2 kcal mol−1 , which
is well within the computational error (DFT/TZVP/COSMO) [42]. In a multistep
process, the racemic trans-2,2-dichloro-1-ethyl-3-methyl-cyclopropane-acid chloride
(37) is synthesized from commercially available (E)-2-ethyl-crotonaldehyde (32) via
its acid (33) and ethyl ester (34) (Scheme 22.1). This is followed by a stereospecific
addition of dichlorocarbene, saponification of the ester 35 to the acid 36 which is
finally treated with thionyl chloride (Scheme 22.2).
The necessary (R)-(+)-p-chlorophenethylamine is obtained from racemic
p-chlorophenethylamine by racemic cleavage with optically active (S)-phenyl-
carbamate-lactic acid.
In the last reaction step, the racemic trans-cyclopropyl-carbonic acid chloride is
reacted with (R)-(+)-p-chlorophenethylamine in the presence of a base to the two
main products, contaminated with small amounts originating from the (S)-amine
(Scheme 22.3).
Finally, the individual stereoisomers can be analyzed with HPLC, using a chiral
separation phase.
22.6
Resistance Problems and Successful Management in Japan
MBI-D fungicides (MBI-Ds) have been used in Japan since 1998. In 2001,
a reduced performance of MBI-Ds was first reported in a limited area [43],
when Magnaporthe grisea isolates showed a decreased sensitivity to MBI-Ds,
CHO O2 COOH C2H5OH COOC2H5

H3C H3C
C2H5
Ag
C2H5 H2SO4 H3C
C2H5
32 33 34
Scheme 22.1 Carpropamid synthesis, part I.
CI CI CI CI CI
COOC2H5 CHCI3 CI
COOC2H5 NaOH COOH SOCI2 COCI
H3 C NaOH
C 2H 5 H3C C2H5 H3C C2H5 C2H5
H3C
35 36 37
Scheme 22.2 Carpropamid synthesis, part II.

22.6 Resistance Problems and Successful Management in Japan 859
CI Ph
CI
CONH H
H
CH3
CI H3C C2H5
Cl R S
COCI
H CI CH3
CI
H 3C C 2H5 CONH H
H3C NH2 H
Ph
R S H3C C2H5
R S
+
CI
Cl CI
C2H5 CI CI
H3 C C2H5
H3C Ph
H COCI
HS CONH H
(R,S -amine) R CH3
S R CI
CI
C2H5
H 3C CH3
H CONH H
Ph
S R
Scheme 22.3 Carpropamid synthesis, part III.
both in vitro and in vivo [44]. A single-point mutation at Sdh1 (GenBank

Accession Number AB004741), which caused the substitution of one amino
acid in SD (Val75 −→ Met: V75M), was found in those isolates showing a
decreased sensitivity to MBI-Ds, but no change was seen in the metabolism
of the fungicides by the isolates [45]. A practical method for detecting
V75M mutants was established using a primer-introduced restriction analysis
(PIRA)-polymerase chain reaction (PCR) [46]. Subsequently, a correlation between
the incidence of the V75M mutation and the efficacy of MBI-Ds was confirmed
[47], with the V75M variant enzyme retaining a significant level of enzymatic
activity [48].
Based on the 3-D structures of the molecular target, the influence of the mutations
on the properties of the binding niche, or the direct influence on binding, could be
determined. It transpired that the volume of the binding niche was reduced by the
more bulky side chain (Figure 22.7a) and, although leaving the natural substrates
unaffected, the chiral methyl groups of the inhibitors were in particular prevented
from optimally fitting into the niche (Figure 20.7b).
Since 2002, the monitoring of resistant strains has been conducted over a broader
area to establish the resistance management for MBI-Ds, using PIRA-PCR [49].
Another single nucleotide polymorphism (SNP) diagnostic method, PCR-Luminex
[50], is also available for this mutation but has not been widely accepted, for
economic reasons. Nationwide monitoring carried out between 2002 and 2005
showed the V75M mutants to be distributed over wider areas in Japan [51],
while genetic studies of the population suggested that the mutation had occurred
independently in each area [47, 52, 53] with, in some exceptional cases, artificial
transportation by infected seeds being suspected [54, 55].
Based on the monitoring results obtained since 2002, local governments have
vehemently stressed seed sanitation as the first priority. In fact, this countermeasure
was generally successful as, under Japanese conditions, rice blast is essentially a
seedborne disease, with rare exception. If some resistant mutants are found
without problems of reduced control efficacy, then it is the duty of each prefecture
to provide administrative guidance to the farmers’ cooperatives for a complete
renewal of seeds for the following season, with blast-free seeds. If a failure to
control with MBI-D is suspected, and resistant mutants are dominant at several
fields in a village, the replacement of MBI-D is also recommended to the relevant
unit sharing a common seed supplier. Although MBI-Rs or probenazole have
shown no cross-resistance to MBI-Ds [56], when the blast infection rate of the seeds
had reached an extremely high level it became impossible to provide sufficient
control [57]. Since, in such cases, it may take several years to recover the mutants
rate to a controllable level [58, 59], a supply of healthy seeds is always prioritized
in commercial rice production areas [60]. As a consequence of these approaches,
serious problems in the field have seldom been reported since 2004 in major areas.
However, epidemiological investigations with V75M mutants in western Japan
have proved that even some authorized seeds could be seriously contaminated
by blast fungus [51]. In contrast, MBI-Ds were successfully and widely used for
blast control in Japan, mainly before the problem of resistance emerged. Notably,
this was the first successful demonstration of fungicide resistance management
in Japan to be based on an integrated collaboration of molecular biology and
epidemiologic approaches.
A fitness penalty of V75M mutants has not been confirmed; typically, the
temperature tolerance, UV sensitivity and virulence of V75M mutants do not differ
significantly from those of wild-type strains [47, 61]. The mutant rate in Saga
2001 was maintained in 2002, despite no selection pressure from MBI-D [47].
Kimura [61] reported a weaker competitiveness of three samples of V75M mutant
isolate compared to three samples from wild-type strains. The results obtained
from monitoring studies in Shikoku and Kyushu, following the total withdrawal of
MBI-D in those areas, strongly suggested that, in the absence of MBI-D selection
pressure and under field conditions, the resistant mutants were less fit than the
sensitive strains [62, 63].
22.7
Final Remarks
The development of a new MBI-D class of SD inhibitors has involved a truly

interdisciplinary research regimen, ranging from classical biology and chemistry to
molecular biology, biochemistry, protein X-ray crystallography, and computational
chemistry. Based on knowledge of the ligand-binding sites, acquired by determining
References 861
X-ray structures and conducting mutation experiments, numerous variations of

these inhibitors – mainly involving alterations to the lipophilic regions of the
molecule – have been investigated. The ultimate result has been the discovery of
a group of highly potent and novel fungicides capable of providing an efficient
control of rice blast.
Unfortunately, following such discoveries, the fungi responded rapidly by creat-
ing mutations of the molecular target, thus rendering the binding niche smaller.
Subsequently, the reasons behind such resistance were explained convincingly,
and effective resistance management strategies introduced to overcome the prob-
lem. Taken together, these findings emphasize the need to conduct continuous
research into new modes of fungicide action, thus offering new chemical classes
of compounds with access to new and different fungal targets.
References
1. Butler, M.J. and Day, A.W. (1998) Can. 10. Wawrzak, Z., Sandalova, T., Steffens,
J. Microbiol., 44, 1115–1136. J.J., Basarab, G.S., Lundquist, T.,
2. Nosanchuk, J.D. and Casadevall, A. Lindquist, Y., and Jordan, D.B. (1999)
(2003) Cell. Microbiol., 5, 203–223. Proteins: Struct., Funct., Genet., 35,
3. Pihet, M., Vandeputte, P., Tronchin, G., 425–439.
Renier, G., Saulnier, P., Georgeault, S., 11. Jennings, L.D., Rayner, D.R., Jordan,
Mallet, R., Chabasse, D., Symoens, F., D.B., Okonya, J.F., Wawrzak, Z.,
and Bouchara, J.P. (2009) BMC Micro- Amorose, D., Anaclerio, B.M., Lee,
biol., 9, 177. J.K., Schwartz, R.S., and Whitmore, K.A.
4. Sisler, H.D. and Ragsdale, N.N. (1995) (2000) Bioorg. Med. Chem., 8, 897–907.
in Modern Selective Fungicides, 2nd edn 12. Jordan, D.B., Basarab, G.S., Steffens,
(ed. H. Lyr), Gustav Fischer Verlag, J.J., Schwartz, R.S., and Doughty, J.G.
Jena, pp. 543–564. (2000) Biochemistry, 39, 8593–8602.
5. Kurahashi, Y. and Pontzen, R. (1998) 13. Basarab, G.S., Steffens, J.J.,
Pflanz. Nachr. Bayer, 51, 245–256. Wawrzak, Z., Schwartz, R.S.,
6. Okamoto, S., Sakurada, M., Kubo, Y., Lundqvist, T., and Jordan, D.B. (1999)
Tsuji, G., Fujii, I., Ebizuka, Y., Ono, M., Biochemistry, 38, 6012–6024.
Nagasawa, H., and Sakuda, S. (2001) 14. Hattori, T., Kurahashi, K., Kagabu, S.,
Microbiology, 147, 2623–2628. Konze, J., and Kraatz, U. (1994) Brighton
7. Maruyama, H., Okamoto, S., Kubo, Y., Crop Protection Conference – Pests and
Tsuji, G., Fujii, I., Ebizuka, Y., Diseases, vol. 2, British Crop Protection
Furihata, K., Hayakawa, Y., Council, Alton, pp. 517–524.
Nagasawa, H., Nakasako, S.M., 15. Soma, M., Sahara, M., and Oguri, Y.
Motoyama, T., Kurahashi, Y., and (1999) Jpn. J. Phytopathol., 65, 401,
Yamaguchi, I. (1998) Biochemistry, (Abstract).
37, 9931–9939. 16. Sieverding, H., Hirooka, T.,
8. Kim, J.-C., Min, J.-Y., Kim, H.T., Kim, Nishiguchi, Y., Yamamoto, Y.,
B.S., Kim, Y.S., Young, S., Kim, B.T., Spadafora, V.J., and Hasui, H. (1998)
Yu, S.H., Yamaguchi, I., and Cho, K.Y. Brighton Crop Protection Confer-
(1998) Pestic. Biochem. Physiol., 62, ence – Pests and Diseases, British Crop
102–112. Protection Council, pp. 359–366.
9. Kubo, Y., Katoh, M., Furusawa, I., and 17. Kitamura, Y., Yakushiji, K., and
Sishiyama, J. (1986) Exp. Mycol., 10, Wakae, O. (1976) Jpn. J. Phytopathol.,
301–306. 42, 370 (abstract).
18. Okuno, T., Kitamura, Y., and 35. Jennings, L.D., Wawrzak, Z.,
Matsuura, K. (1983) J. Pestic. Sci., 8, Amorose, D., Schwartz, R.S., and
361–362. Jordan, D.B. (1999) Bioorg. Med. Chem.
19. Shiba, Y., Todoriki, J., Hata, M., and Lett., 9, 2509–2514.
Nagata, T. (1983) J. Pestic. Sci., 8, 36. Rarey, M., Kramer, B., Lengauer, T.,
167–171. and Klebe, G.A. (1996) J. Mol. Biol., 261,
20. Sakuma, H. (1999) Annu. Rep. Plant 470–489.
Prot. North Jpn, 50, 32–34. 37. Basarab, G.S., Jordan, D.B., Gehret,
21. Yamamoto, Y., Nishiguchi, T., T.C., Schwartz, R.S., Rand, S., Bonman,
Uchikurobane, T., Hirooka, T., and J.M., and Smith, G.S. (2002) Synthesis
Hino, I. (2000) Jpn. J. Phytopathol., 66, and Chemistry of Agrochemicals VI, ACS
183 (abstract). Symposium Series, Vol. 800, American
22. Basarab, G.S., Jordan, D.B., Gehret, Chemical Society, pp. 278–291.
T.C., and Schwartz, R.S. (2002) Bioorg. 38. Kraatz, U. and Littmann, M. (1998)
Med. Chem., 10, 4143–4154. Pflanz. Nachr. Bayer, 51, 201–206.
23. Zheng, Y.-J., Basarab, G.S., and Jordan, 39. Manabe, A., Maeda, K., Enomoto, M.,
D.B. (2002) Biochemistry, 41, 820–826. Takano, H., Katoh, T., Yamada, Y.,
24. Jordan, D.B., Zheng, Y.-J., Lockett, B.A., and Oguri, Y. (2002) J. Pestic. Sci., 27,
and Basarab, G.S. (2000) Biochemistry, 257–266.
39, 2276–2282. 40. Buck, W. and Raddatz, E. (1988) Euro-
25. Jordan, D.B., Basarab, G.S., Steffens, pean Patent Applications EP 262393.
J.J., Lundqvist, T., Pfrogner, B.R., 41. Kagabu, S. and Kurahashi, Y. (1998)
Schwartz, R.S., and Wawrzak, Z. (1999) J. Pestic. Sci., 23, 145–147.
Pestic. Sci., 55, 277–280. 42. Klamt, A. and Schüürmann, G. (1993)
26. Sybyl 7.1, Tripos Inc., St Louis, MO. J. Chem. Soc., Perkin Trans. 2, 799–805.
27. Wallace, A.C., Laskowski, R.A., and 43. Yamaguchi, J., Kuchiki, F., Hirayae, K.,
Thorton, J.M. (1995) Prot. Eng., 8, and So, K. (2002) Jpn. J. Phytopathol., 68,
127–134. 261 (abstract).
28. Lundquist, T., Rice, J., Hodge, C.N., 44. So, K., Fuji, M., Iwabuchi, H.,
Basarab, G.S., Pierce, J., and Kanayama, M., and Yamaguchi, J. (2002)
Lindquist, Y. (1994) Structure, 2, Jpn. J. Phytopathol., 68, 262 (abstract).
937–944. 45. Takagaki, M., Shimizu, T., Miura, I.,
29. Nakasako, M., Motoyama, T., and Araki, Y., Sawada, H., So, K., and
Yamaguchi, I. (2002) Acta Crystallogr., Nagayama, K. (2002) Jpn. J. Phytopathol.,
Sect. D, 58, 148–150. 68, 262 (abstract).
30. Chen, J.M., Xu, S.L., Wawrzak, Z., 46. Kaku, K., Takagaki, M., Shimizu, T., and
Basarab, G.S., and Jordan, D.B. (1998) Nagayama, K. (2003) Pest Manage. Sci.,
Biochemistry, 37, 17735–17744. 59, 843–846.
31. Jordan, D.B. and Basarab, G.S. (2000) 47. Sawada, H., Sugihara, M., Takagaki, M.,
Bioorg. Med. Chem. Lett., 10, 23–26. and Nagayama, K. (2004) Pest Manage.
32. (a) Ahlrichs, R., Bär, M., Häser, M., Sci., 60, 777–785.
Horn, H., Kölmel, C. (1989) Chem. 48. Yamada, N., Motoyama, T., Nakasako,
Phys. Lett., 162, 165; (b) Treutler, O. and M., Kagabu, S., Kudo, T., and
Ahlrichs, R. (1995) J. Chem. Phys., 102, Yamaguchi, I. (2004) Biosci. Biotechnol.
346. Biochem., 68, 615–621.
33. Jordan, D.B., Lessen, T., Wawrzak, Z., 49. Arai, M. (2004) Plant Prot. (Shokubutsu
Bisaha, J.J., Gehret, T.C., Jansen, S.L., Boeki), 58, 20–23.
Schwartz, R.S., and Basarab, G.S. (1999) 50. Ishii, H., Tanoue, J., Oshima, M.,
Bioorg. Med. Chem. Lett., 9, 1607–1612. Yamaguchi, J., Nemoto, F., and So, K.
34. Basarab, G.S., Jordan, D.B., Gehret, (2005) in Modern Fungicides and Anti-
T.C., Schwartz, R.S., and Wawrzak, Z. fungal Compounds IV (eds H.-W. Dehne
(1999) Bioorg. Med. Chem. Lett., 9, et al.) British Crop Protection Council,
1613–1618. Alton, pp. 31–34.
References 863
51. Arai, M. (2004) Abstracts of the 14th Bug on Rice, January 15, 2004, Takino-
Symposium of Research Committee on gawa Kaikan, Tokyo, Japan, pp. 21–30.
Fungicide Resistance, March 31, 2004, 58. Yasunaga, T., Kusumoto, T., and
Kyushu University, Fukuoka, Japan, pp. Kotani, M. (2004) Abstracts of the 49th
27–36. Conference of Shikoku Plant Protection
52. Sawada, H., Sugihara, M., Takagaki, M., Research Committee, November 16,
Shimizu, T., and Nagayama, K. (2003) 2004, Mielparque Matsuyama, Ehime,
Abstracts of the 3rd Pan Pacific Confer- Japan, p. 10.
ence on Pesticide Science, June 3, 2003, 59. Yamaguchi, J., Inada, M., Furuta, A.,
Hilton Hotel Honolulu, Hawaii, USA, Kuchiki, T., So, K., Arai, M., and
p. 46. Suzuki, F. (2005) Jpn. J. Phytopathol.,
53. Anonymous (2005) National Agricultural 71 (3), 250 (abstract).
Research Centre for Kyushu Okinawa 60. Nemoto, F. (2005) Abstracts, 15th Sym-
Region, Press release, May 24 2005.
posium of Research Committee on
Available at: http://konarc.naro.affrc.go.jp/
Fungicide Resistance, March 28, 2005,
press/20050524/.
Granship Shizuoka, Shizuoka Japan,
54. Anonymous (2004) Official Notice of
pp. 35–44.
Shizuoka Crop Protection Office on
61. Kimura, N. (2005) Jpn. J. Phytopathol.,
October 13, 2004.
71, 202 (abstract).
55. Sasaki, N., Iwadate, Y., Tominaga, T.,
and Katsube, K. (2005) Annu. Rep. Plant 62. Yasunaga, T., Kusumoto, S., and
Prot. North Jpn, 56, 205 (abstract). Kotani, M. (2004) Annual Meeting of
56. So, K. (2003) Abstracts of the 13th the 49th Shikoku Crop Protection Re-
Symposium of Research Committee search Conference, November 16, 2004,
on Fungicide Resistance, March 31, Mielparque Matsuyama, Ehime, Japan,
2003, Meiji University, Tokyo, Japan, p. 4, (congress abstract in Japanese).
pp. 37–47. 63. Suzuki, F., Yamaguchi, J., Koba, A.,
57. Nakajima, T. (2004) Recent Abstracts, Nakajima, T., and Arai, M. (2010) Plant
JCPA Symposium for Blast and Sting Dis., 94 (3), 329–334.
865
23
Fungicides with Unknown Mode of Action
23.1
Introduction
In general, the biochemical mode of action of all major fungicides in current

agricultural use has been well described; indeed, the vast literature on these promi-
nent products has been treated extensively in several chapters of this volume. For
some smaller market products, however, the knowledge is much less complete.
Since, in order to register a product it is sufficient only to fulfill toxicological and
environmental requirements, a number of fungicides are currently available for
which the biochemical mode of action has not been defined. The time and effort
required to determine such information for an active ingredient can hardly be
overestimated, and consequently not only younger but also some older fungicides
still await their mode(s) of action to be elucidated. In this chapter, the details are
provided of six compounds – cymoxanil (1), fosetyl-aluminum (2), flusulfamide (3),
diclomezine (4), triazoxide (5), and tebufloquin (6) – for all of which the mode of
action is, at present, unknown.
23.2
Cymoxanil
Cymoxanil (1) was developed by DuPont and launched in 1977 [1]. It is active
against the Oomycetes, with a weak activity against fungi. Cymoxanil belongs
to the chemical class of cyanohydroxyiminoacetamides and was discovered in
1972 by DuPont [2]. The International Union of Pure and Applied Chemistry
(IUPAC) chemical name of cymoxanil is 2-cyano-N-[(ethylamino)carbonyl]-2-
(methoxyimino)acetamide (CAS-RN.: 57966-95-7) (Figure 23.1). No further
compounds from this class have ever been registered as a commercial product.
Due to its high polarity, the water-solubility of cymoxanil is rather high for a
fungicide. It is stable towards hydrolysis, but unstable under ultraviolet UV light.
Additional physico-chemical properties of cymoxanil are listed in Table 23.1 [3].

866 23 Fungicides with Unknown Mode of Action
O O Figure 23.1 Chemical structure of cymoxanil (1).

H3C N
O N N CH3
H H
N
Table 23.1 Physico-chemical properties of cymoxanil (1).
Melting point (◦ C) 159–160

Hydrolysis (DT50 , 25 ◦ C) 148 days (pH 5), 34 h (pH 7), 31 min (pH 9)
UV stability Sensitive to light
pKa 9.7 ± 0.2
Vapor pressure (mPa, 20 ◦ C) 0.15 (pH 5)
Specific gravity (g cm−3 , 22.5 ◦ C) 1.32
log PO/W 0.59 (pH 5), 0.67 (pH 7)
Solubility (g l−1 , 20 ◦ C) Water (pH 5) = 0.9
Water (pH 7) = 0.8
Acetone = 62.4
Acetonitrile = 57.0
Dichloromethane = 133
Ethyl acetate = 28.0
Hexane = 0.037
Methanol = 22.9
Toluene = 5.29
n-Octanol = 1.43
The synthesis of cymoxanil, according to the literature [4], is rather straight-

forward, starting (as shown in Scheme 23.1) with the nitrosation of 1-(2-cyano-
acetyl)-3-ethylurea (7) with sodium nitrite in water, followed by O-methylation of
the oxime (8) with a methylating reagent such as iodomethane.
Cymoxanil is a protective and curative oomyceticide for foliar application; it is
especially active against Peronospora spp., Phytophthora spp., and Plasmopara spp. in
grapes, hops, tomatoes, and some other vegetables, as well as in potatoes where it is
used for leaf and tuber treatment. Although some activity against fungal pathogens
(e.g., Botrytis cinerea) has been observed in laboratory studies [5], this proved to be
insufficient under practical conditions in the field for a commercial use.
Cymoxanil penetrates rapidly into the plant leaves, and also shows excellent
contact activity via its strong sporulation-inhibiting properties. Due to its high
water solubility, the agent is mobile in the plant, showing favorable curative
and local systemic effects. Cymoxanil degrades very quickly within the plant
[6, 7], leading to a lack of long-lasting activity; consequently, it is used mainly in
combination with other oomyceticides to improve the residual activity [8]. In these
23.2 Cymoxanil 867
O O O O
NaNO2 N
N N CH3 HO N N CH3
H2O CN H H
CN H H
7 8
MeI
CH3CN, NEt3
O O
H3C N
O N N CH3
CN H H
Scheme 23.1 Synthesis of cymoxanil (1).
Table 23.2 Cymoxanil (1): Use against plant diseases of high market significance.
Plant disease Application rates (recommended) Maximum number of

(g a.i. ha−1 ) applications per season
Potato late blight 110–120 (mixtures with mancozeb) 3–4

(Phytophthora infestans) 125 (mixtures with propamocarb) 8
175 (mixtures with famoxadone) 6
Vine downy mildew 50–200 (mixtures with famoxadone) 3

(Plasmopara viticola)
Tomato late blight 90–180 (mixtures with famoxadone) 5
(Phytophthora infestans)
Cucumber downy mildew 75–150 (mixtures with famoxadone) 5
(Pseudoperonospora cubensis)
combinations, the favorable curative properties of cymoxanil are emphasized. Plant

diseases where cymoxanil has a high market significance are listed in Table 23.2.1)
Several companies (e.g., DuPont, BASF, Bayer CropScience, Shanghai Zhongxi,
Sipcam, Staehler, Sundat, and Syngenta) sell cymoxanil in many countries in
Europe, Asia, and the Americas, either as solo products or in mixtures, under
® ® ® ®
numerous trade names (e.g., Curzate , Blizzard , Pulstar , Tanos , Equitation
® ® ® ® ® ®
Pro , Aktuan , Horizon , Wakil , Evolve , Scribe ). Due to its rapid degra-
dation in plants [6, 7], animals [9], and in the environment, the toxicological
and ecotoxicological profile of cymoxanil is favorable (acute oral toxicity rats:
1) Source: German Federal Office of Con-

sumer Protection and Food Safety (BVL).
LD50 = 960 mg kg−1 day−1 ), leading to a classification in World Health Organization

(WHO) toxicity class III (slightly hazardous).
Although, currently, cymoxanil has been used intensively for more than 30 years,
the resistance development of fungal pathogens to this compound is rather limited.
Some studies have indicated a significant decrease in the sensitivity of Plasmopara
viticola to cymoxanil compared to baseline studies performed before its market
introduction [10–12]. Nevertheless, it is still used successfully for the control of this
plant disease. Only a few reports have been made describing a reduced sensitivity of
cymoxanil, against Phytophthora infestans [13, 14], though such reduced sensitivity
occurred only in a few limited regions. In other studies [15–17], no reduction in
sensitivity of this disease could be observed. Consequently, any resistance problems
of cymoxanil against P. infestans will, most probably, not play a major role within
the next years and beyond.
The mode of action of cymoxanil remains unclear according to previous reviews
[5, 12, 18, 19], although some interesting results have been reported. One recent
proposal regarding the mode of action was that cymoxanil might be a prodrug
of as-yet-to-be identified active metabolite, for example, cyanoglycine [20]. Two
reports have described an inhibition of respiration by cymoxanil in yeast at
concentrations of about 10–30 μg ml−1 , which are known to be growth-inhibitory
[21, 22]. Other reports, however, have contradicted any action on respiration.
Cymoxanil, when applied at concentrations up to 100 μg ml−1 , had no effect on the
mycelial respiration and zoospore motility of P. infestans [23], showing that energy
production within this organism is not affected.
In P. infestans [23], cymoxanil applied at concentrations up to 100 μg ml−1 did not
inhibit the uptake of radiolabeled precursors of DNA (thymidine), RNA (uridine), or
proteins (phenylalanine). However, following a less than 2 h treatment, despite the
uptake of thymidine being unaffected, its incorporation was considerably reduced;
at the same time, uridine incorporation was slightly affected and phenylalanine
incorporation proved to be insensitive. In contrast, the application of cymoxanil
at 10 μg ml−1 failed to inhibit both thymidine or uridine incorporation, while the
inhibition of mycelial growth was complete; these findings suggested that the
inhibition of DNA and RNA synthesis were secondary effects. In isolated nuclei,
RNA polymerase activity was unaffected by cymoxanil.
In the mycelium of P. cinnamomi [24], a short-lived treatment (<2 h) with
cymoxanil at up to 100 μg ml−1 led to a moderate inhibition of both, uridine and
phenylalanine (or serine) uptake and incorporation. After longer treatments (4 and
6.5 h), the uptake and incorporation of both uridine and phenylalanine (or serine)
were still inhibited, as was the incorporation of acetate into lipids.
Taken together, these results indicate that DNA, RNA, and protein biosyntheses
are not the primary targets of cymoxanil.
In parallel, some interesting results were obtained in a nontarget organism,
Botrytis cinerea strain B [25], a benzimidazole-resistant strain found to be sensitive
to cymoxanil with an ED50 = 0.7 μg ml−1 on mycelial growth. First, similar to
P. cinnamomi, short-term treatment (<2 h) with cymoxanil at up to 100 μg ml−1
had no effect on respiration, and led to a minimal inhibition of uridine and
phenylalanine uptake and incorporation. A strong inhibition was observed after

long-term treatment (4 and 6.5 h), however, the major effect being on uridine
uptake and incorporation. In contrast to P. cinnamomi, the incorporation of acetate
into lipids was increased, without causing any change in lipid composition [25].
Second, the fungitoxicity of cymoxanil can be partially reversed by the addition of
serine and cysteine to the growth medium, but not of methionine or glutathione
(GSH) [25]. Third, the fact that cymoxanil is rapidly metabolized by the sensitive
strain of B. cinerea, but not by the resistant strain, suggests that it may be a
pro-fungicide [20, 26].
The effect of preventative or curative application of cymoxanil on host–pathogen
interactions during the P. infestans infection of tomato and potato has been studied
using both light and electron microscopy [27]. A cytological analysis showed that, in
the presence of the pathogen and cymoxanil, a hypersensitive-type response (HR)
of the host cells was observed. This response was characterized by granulation,
plasmolysis, and a yellowing of the cytoplasm of the invaded epidermal cells, in
addition to cell-wall thickening and necroses at the infection site.
In conclusion, the available data suggest that cymoxanil has an unknown fungi-
cidal mode of action, and might also induce some host plant defense responses.
23.3
Fosetyl-Aluminum
Another fungicide of high market significance with efficacy mainly against the
Oomycetes is fosetyl-aluminum (2). This was developed by Rhône-Poulenc (now
Bayer CropScience), and first introduced to the market in 1977.
Fosetyl-aluminum belongs to the chemical class of phosphonates, and was
discovered in 1973 by Philagro [28]. The IUPAC chemical name is aluminumethyl-
hydrogenphosphonate (CAS-RN.: 39148-24-8); the chemical structure is shown in
Figure 23.2. Although the official name of this compound is fosetyl, it has been
named elsewhere as phosetyl, fosethyl, or phosethyl.
A related compound – the corresponding sodium salt (fosetyl-sodium) – also
reached an advanced stage of development as an Oomycetes fungicide, but did
not reach the market. Moreover, other salts and the parent compound itself, ethyl
phosphite or fosetyl, are also active against the Oomycetes.
Due to the fact that the compound is a salt, the water-solubility of
fosetyl-aluminum is high. It is also stable towards hydrolysis under neutral
H −
H3C O O Al3+
P
O
3
2 Figure 23.2 Chemical structure of fosetyl-aluminum (2).

Table 23.3 Physico-chemical properties of fosetyl-aluminum (2).
Melting point (◦ C) >200 (dec.)

Hydrolysisa (DT50 , 70 ◦ C) 6 h (pH 1.2), 12 h (pH 12.8)
Vapor pressure (mPa, 25 ◦ C) <0.013
log PO/W ∼2.7 (pH 4)
Solubility (mg l−1 , 20 ◦ C) Water = 120 000
Acetone = 13
Acetonitrile = 5
Ethyl acetate = 5
Hexane = 5
Methanol = 920
Propylene glycol = 80
a
Stable under normal storage conditions.
1. < 20 °C H −
3 PCl3 + 3 EtOH + Al0 + 2 H2O H3C O O Al3+ + 6 HCl
2. white spirit P
90-130 °C
O
85-95 % d.Th. 3
Scheme 23.2 Synthesis of fosetyl-aluminum (2).
conditions, and decomposes only under strong acidic or basic conditions, or

by exposure to strong oxidizing agents. Further physico-chemical properties of
fosetyl-aluminum are listed in Table 23.3 [29].
According to a patent application published in 1996 [30] (Scheme 23.2),
fosetyl-aluminum can be prepared by combining phosphorus trichloride with
a stoichiometric amount of ethanol (85–100%) at temperatures below 20 ◦ C,
followed by dissolving metallic aluminum to produce aluminum chloride. This
mixture then reacts in situ with water to yield diethyl phosphate, containing traces
of ethyl phosphite. The diethyl phosphite reacts with aluminum chloride in an inert
solvent at 90–130 ◦ C to give fosetyl-aluminum in good yields, with HCl generated
as a side product.
Fosetyl-aluminum is a protective and curative fungicide for foliar application. The
compound allows a specific control of Oomycetes (Phytophthora spp., Plasmopara
spp., Bremia spp., etc.) on lettuce, hops, strawberries, pome fruits, citrus fruits,
pineapples, avocados, vines, cucurbits, onions, cocoa, rubber, tobacco, ornamental
plants, and shrubs. It also possesses a wider spectrum of lower-level activity
against other Oomycetes, such as pearl millet downy mildew [31] (Sclerospora
graminicola) and some bacteria. Following a spray application of fosetyl-aluminum,
the compound is rapidly absorbed, predominantly through the leaves but also
through the roots. Within the plant it is translocated both acropetally and basipetally,
leading to a protection of the complete plant, including the roots and fruits [32].
Table 23.4 Fosetyl-aluminum (2): Use against plant diseases of highest market significance.
Plant disease Application rates (recommended) Maximum number of

(g a.i. ha−1 ) applications per year
Downy mildew on grapes 500–2000 (mixtures with 3

(Plasmopara viticola) fenamidone)
Downy mildew on hop 2000–4000 8
(Pseudoperonospora humuli)
Downy mildew on lettuce 2400 3
(Bremia lactucae)
Downy mildew on cucumber 2400–4800 3
(Pseudoperonospora cubensis)
Fosetyl-aluminum acts by inhibiting the germination of spores and by blocking

mycelial development.
Fosetyl-aluminum is mainly used in grapes, vegetables, citrus, and tropical fruits
such as pineapple. Details of its use to control plant diseases of the highest market
significance are listed in Table 23.4.2)
Several companies (e.g., Bayer CropScience, Philagro, Shanghai Zhongxi, Sun-
dat) currently market fosetyl-aluminum either as a solo product or in mixtures
®
under several trade names. These include the most prominent Aliette , as well as
® ® ® ® ® ® ® ®
Mikal , Valiant , Proban , Mikalix , Cap 25 , Odyssee , Aliziman , Rhodax ,
® ® ® ®
Almanach , Alliance , Artimon , and Sillage .
The toxicological profile of fosetyl-aluminum is very favorable (acute oral toxicity
in rats: LD50 = 5800 mg kg−1 day−1 ), leading to a classification in WHO toxicity
class U (unlikely to be hazardous).
Although fosetyl-aluminum has been used intensively for more than 30 years, the
resistance development of fungal pathogens to it has been very limited. However,
some studies have indicated a significant decrease in the sensitivity of P. viticola
to fosetyl-aluminum, with resistance factors of between 5 and 24 [33]. Moreover,
a decreased sensitivity against lettuce downy mildew, Bremia lactucae, has been
observed [34]. Overall, however, the development of resistance to fosetyl-aluminum
appears to have had only a limited impact on the overall product performance.
In some studies, fungal strains that were insensitive to fosetyl-aluminum, as well
as to metalaxyl, were isolated [34, 35]. Although there has a degree of speculation
regarding a possible cross-resistance between these two compounds, it has not
been possible to demonstrate this under field conditions. Rather, it is likely that
the fungal isolates show independent multiple resistance to these fungicides,
2) Source: German Federal Office of Con-

sumer Protection and Food Safety (BVL),
online database.
although these effects might be explained by other mechanisms [36]. In laboratory

experiments, no metalaxyl-tolerant isolates of Phytophthora capsici with insensitivity
against fosetyl-aluminum could be generated by mutagenesis [37]. Nevertheless,
the possibility of obtaining strains of Pythium aphanidermatum that were resistant
to metalaxyl and fosetyl-aluminum, by the exposure of a metalaxyl-resistant field
isolate to a chemical mutagen, has been demonstrated [38]. In summary, the risk of
significant resistance problems with fosetyl-aluminum occurring within the near
future appears to be very low.
The abundant reports on the biochemical mode of action of fosetyl-aluminum,
produced during the 1980s, have been extensively reviewed [5, 39–41], and this
has led to the conclusion that fosetyl-aluminum and phosphonate (its in planta
metabolite) each have both direct and indirect modes of action.
The direct mode of action affects multiple targets in phosphate transport,
phosphate use, and regulatory functions within the fungus. In vitro, the efficacy of
fosetyl-aluminum is generally dependent on the (low) phosphate concentration in
the assay system.
Thus, several enzymes of glucose metabolism [42, 43] as well as inorganic
pyrophosphatase [44] from Phytophthora spp., were found to be inhibited in vitro.
Further to this biochemical activity, physiological responses were measured in
P. palmivora and P. citrophthora. Initially, in treated P. palmivora, decreases in the
levels of NAD and ATP were observed [45]. Longer-term exposures resulted in
changes in phosphorus distribution and lipid composition [46], while the activities
of key enzyme of the pentose phosphate pathway, and also of β-glucan biosynthesis,
were increased several-fold [43]. The former results point to an altered fungal
metabolism, whereas the latter indicate the direct or indirect control of protein
levels [43, 47]. The reported activities of fosetyl-aluminum are in the millimolar
range, consistent with the high concentrations required to inhibit fungal growth.
However, inhibition of the above enzymes is unlikely to account for the specific
anti-Oomycetes effect of phosphonates, as the same enzymes obtained from other
sources (yeast, animals) are also affected.
In contrast, an indirect mode of action involving the promotion of plant defense
responses could easily explain the much higher antifungal activity observed in
planta than in vitro.
In fact, fosetyl-aluminum alone induces both phytoalexin and pathogenesis-
related (PR) protein production in grape vines [48]. In Arabidopsis thaliana, a
broad spectrum of defense-related genes was activated by fosetyl-aluminum that
resembled gene-expression changes induced by microbial defense elicitors [49, 50].
Concurrently, the pathogen Peronospora parasitica was controlled [49]. Likewise, in
a range of other host–pathogen systems – for example, tomato infested by P. capsici
or grapevine infested by Plasmopara viticola – fosetyl-aluminum stimulated the HR
to infection and the production of phytoalexins [51]. A recent study clearly demon-
strated, that fosetyl-aluminum acts rather by priming A. thaliana for pathogen
resistance than by straight defense gene induction [52]. The benefit of priming, in
contrast to direct defense elicitation, represents a substantial resource saving in the
plant [53].
23.4 Flusulfamide 873
In conclusion, fosetyl-aluminum (phosphonate) is a potent primer of plant

defense responses, supported by a limited direct effect on fungal metabolism.
23.4
Flusulfamide
Flusulfamide (3), a niche product with activity against a narrow range of fungal
pathogens, was first registered in Japan in 1992 [54].
Flusulfamide, which belongs to the chemical class of benzenesulfonanilides,
was discovered and developed in 1972 by Mitsui Toatsu [55]. In South Africa, the
compound was codeveloped by Chemserve (formerly Kynoch).
The IUPAC chemical name of flusulfamide is 4-chloro-N-(2-chloro-4-nitro-
phenyl)-3-trifluoromethyl-benzenesulfonamide (CAS-RN.: 106917-52-6). No fur-
ther compounds from this chemical class have ever been placed into development.
The chemical structure of flusulfamide is shown in Figure 23.3.
Flusulfamide is a rather polar compound but, due to its high melting point,
its water solubility is relatively low. The compound is stable in water. Further
physico-chemical properties of flusulfamide are listed in Table 23.5 [56, 57].
Cl
H
O2N N
O
S
O F
F
F
Cl
3 Figure 23.3 Chemical structure of flusulfamide (3).
Table 23.5 Physico-chemical properties of flusulfamide (3).
Melting point (◦ C) 170–172.5

Hydrolysis Stable in acidic media; moderately stable in
alkaline media
Vapor pressure (mPa, 20 ◦ C) 358
Specific gravity (g cm−3 , 23 ◦ C) 1.739
log PO/W 2.4
Solubility (g kg−1 , 25 ◦ C) Water = 0.0029
Acetone = 314
Chloroform = 17
Ethyl acetate = 125
Hexane = 0.05
Methanol = 24
Tetrahydrofuran = 592
Xylene = 14
Cl
Cl H
O
Cl S O2N N
O O
F S
+ O F
O2N NH2 F
F F
Cl F
Cl
9 10 3
Scheme 23.3 Synthesis scheme of flusulfamide (3).
Table 23.6 Flusulfamide (3): Use against plant diseases of the highest market significance.
Plant disease Application rates (recommended) (g a.i. ha−1 )
Cabbage/cauliflower clubroot 600 (soil treatment)

(Plasmodiophora brassicae)
Potato powdery scab 1800 (soil-borne, soil treatment)
(Spongospora subterranea) 1 (seed-borne, seed tuber dip)a
a
Application rate (recommended) (g a.i. l).
Flusulfamide can be prepared by the reaction of 2-chloro-4-nitro-phenylaniline (9)

with 4-chloro-3-trifluoromethyl-benzenesulfonyl chloride (10) [57] (Scheme 23.3).
Flusulfamide has been developed to control diseases caused by soil-borne
pathogens from the order Plasmodiophorales (the only order of the Plasmodio-
phorid group). In the past, this isolated group of obligate plant pathogens has been
classified in either the protoctists kingdom or in the fungi kingdom. Flusulfamide
is used mainly as a soil treatment for the control of the causal agent of clubroot
disease of Brassicae (crucifer) crops, Plasmodiophora brassicae. Furthermore, it
has demonstrated efficacy against another member of the Plasmodiophorales in
reducing the incidence of powdery scab on potatoes (Spongospora subterranea var.
subterranea) in field trials, following soil treatment prior to planting or after spray
treatment of the seed tubers (tuber dip). As potato powdery scab serves as a vector
of potato mop top virus, an indirect efficacy could also be demonstrated against this
disease [56]. Although flusulfamide also shows antifungal activity on a cell test level
against fungal plant pathogens, such as B. cinerea, Pythium aphanidermatum, and
others [57], it has never been registered for use against these diseases. Details of
the use of flusulfamide to control plant diseases of the highest market significance
are listed in Table 23.6.
Mitsui & Co. Ltd. and Certis Europe (the agroscience company of Mitsui & Co. in
Europe) market flusulfamide in products for soil treatment under the trade names
® ® ®
Nebijin , Scablok , and Hoganna . Flusulfamide is also distributed by Elliott.
Flusulfamide demonstrates an acute toxicity towards mammals (LD50 :
180 mg kg−1 for male rats [58]; 132 mg kg−1 for female rats [58]; and
23.5 Diclomezine 875
245–254 mg kg−1 for mice), leading to a WHO toxicity classification of II

(moderately hazardous). However, it has not demonstrated show mutagenic,
teratogenic, reproductive, or oncogenic effects. Studies on the translocation of
flusulfamide from the soil to cabbage and turnip plants showed there to be almost
no movement. Consequently, as the compound accumulates neither in the soil
nor in mammals, potential toxicological effects to the consumers and to the
environment are very low [57].
Flusulfamide inhibits the germination of resting spores, and thus prevents
root-hair infection; spore integrity and viability is not affected, however [59].
To date, no reports have been made on the biochemical mode of action of
flusulfamide. However, its chemical structure with the acidic sulfonamide proton
suggests that an uncoupling activity on respiration might exist.
23.5
Diclomezine
Diclomezine (4) is used against several sclerotial diseases of rice plants. The
compound was discovered in 1972 by Sankyo [60], and first registered in Japan in
1987.
Diclomezine belongs to the chemical class of pyridazinones. The IUPAC
chemical name is 6-(3,5-dichloro-4-methylphenyl)-3(2H)-pyridazinone (CAS-RN.:
62865-36-5); the structure is shown in Figure 23.4. No other compounds from this
class have ever been placed into development.
The melting point of diclomezine is high, its water-solubility is very low, it is
stable in water and it decomposes slowly under UV light. Further physico-chemical
data relating to diclomezine are listed in Table 23.7 [61, 62].
Cl
H3C O
N N
Cl H
Figure 23.4 Chemical structure of diclomezine (4).
Table 23.7 Physico-chemical properties of diclomezine (4).

Hydrolysis Stable under acidic, neutral, and alkaline conditions
UV stability Slow decomposition
Vapor pressure (mPa, 60 ◦ C) ≤0.015
Solubility (g l−1 , 23 ◦ C) Water = 0.74a
Acetone = 3.4
Methanol = 2.0
a
Solubility (mg l−1 , 25 ◦ C).
O
Cl O Cl COOH
OH
CH3 MeS− Na+
H
H3C H3C
O AcOH O H2O
Cl Cl
11 12
Cl COOH Cl S CH3
H2NNH2 . H2O
H3C S CH3 H3C O
EtOH
O N N
H
Cl Cl
13 14
Cl
HCl / H2O
H3C O
N N
H
Cl
4
Scheme 23.4 Synthesis of diclomezine (4).
The synthesis of diclomezine, starting from 1-(3,5-dichloro-4-methylphenyl)-

ethanone (11), according to the literature [62], is shown in Scheme 23.4. The
procedure commences with an aldol condensation of the acetophenone
(11) with glyoxylic acid, yielding the corresponding benzoylacrylic acid (12).
The addition of sodium methanethiolate in water affords 4-(3,5-dichloro-4-
methylphenyl)-2-methylsulfanyl-4-oxo-butyric acid (13). The latter can be
cyclized with hydrazine hydrate in ethanol to afford 6-(3,5-dichloro-4-
methylphenyl)-4-methylsulfanyl-4,5-dihydro-(2H)-pyridazin-3-one (14), which can
be aromatized under acidic conditions by the elimination of methyl mercaptan to
generate diclomezine (4).
Diclomezine is a highly effective fungicide against rice sheath blight caused
by Rhizoctonia solani. When applied by foliar application at panicle initiation
to heading stage at rates of 160–480 g ha−1 , it exhibits a high protective and
curative activity against this and other sclerotial diseases, including Rhizoctonia
oryzae, Sclerotium fumigatum, and Sclerotium oryzae-sativae. Diclomezine adheres
to the sheath surfaces of rice plants and persists for a long period, thus showing
long-lasting efficacy. Furthermore, it shows efficacy against white mold (Sclerotium
rolfsii) and twig rot (R. solani) of peanuts, as well as against Rhizoctonia diseases
of turf grass. Due to the narrow range of pathogens controlled by diclomezine, its
market importance has remained limited. Currently, it is marketed under the trade
®
name Monguard by Sankyo. No information about the resistance development of
diclomezine is available.
23.6 Triazoxide 877
The toxicological and ecotoxicological profile [61] of diclomezine is favorable. In

rats, it shows no acute oral toxicity (LD50 ≥ 12 000 mg kg−1 day−1 ), and it is neither
mutagenic nor teratogenic. In soil, it is readily adsorbed onto the soil particles.
An inhibition of septum formation and leakage of cytoplasm were observed in the
mycelium of R. solani after 2–3 h treatment with diclomezine at a level of 1 ppm [61].
The primary biochemical mode of action of diclomezine remains unknown,
however.
23.6
Triazoxide
Triazoxide (5), which was discovered by Bayer in 1978 [63], fills a classical gap of
other fungicides in the control of Pyrenophora-mediated seedborne diseases.
Triazoxide belongs to the chemical class of 1,2,4-benzotriazines. The IU-
PAC chemical name is 7-chloro-3-(1H-imidazol-1-yl)-1,2,4-benzotriazine-1-oxide
(CAS-RN.: 72459-58-6). Although other 1,2,4-benzotriazine-1-oxides have demon-
strated fungicidal activity, triazoxide is the only such compound to have been
commercialized. The chemical structure of triazoxide is shown in Figure 23.5.
The aqueous stability of triazoxide depends largely on the pH. Moreover, its
water-solubility is rather low and it is fairly unstable under UV light. Further
physico-chemical properties of triazoxide are listed in Table 23.8 [64, 65].
−
O
+
Cl N
N
N N
N
5 Figure 23.5 Chemical structure of triazoxide (5).
Table 23.8 Physico-chemical properties of triazoxide (5).

Hydrolysis (DT50 , 22 ◦ C, extrapol) 1 yr (pH 4), 3.6 yr (pH 7), 22.6 days (pH 9)
UV stability May be decomposed by light
Vapor pressure (Pa) 1.5 × 10−12 (20◦ C)
5.2 × 10−12 (25◦ C)
2.4 × 10−4 (120◦ C)
Specific gravidity (g cm−3 , 22.5 ◦ C) 1.577
log PO/W (23 ◦ C) 2.0
Solubility (g l−1 , 20 ◦ C) Water = 0.03
Dichloromethane = 50–100
Hexane = <1
Isopropanol = 2–5
Toluene = 20–50
Cl NO2 Cl NO2 Cl NO2

COCl2 NH3
toluene toluene
NH2 N=
=C=
=O NH
15 16 17 O NH2
−
O −
+ 1. HCl / H2O O
NaOH / H2O +
Cl N 2. SOCl2
N Cl N
toluene N
toluene
N ONa
N Cl
18 19
−
O
N +
HN Cl N
N
NaHCO3
toluene N N
N
Scheme 23.5 Synthesis of triazoxide (5).
The most appropriate technical synthesis of triazoxide [66], as described in

Scheme 23.5, starts with phosgenation of 2-nitro-4-chloroaniline (15), followed
by ammonolysis of the isocyanate (16). Cyclization of the formed arylurea (17)
occurs by treatment with aqueous sodium hydroxide to obtain the sodium salt of
3-hydroxy-7-chloro-1,2,4-benzotriazine-1-oxide (18). After acidification, the hydroxy
group can be converted into the corresponding imidazolyl-containing triazoxide
after being transformed into the chloro precursor (19) with thionyl chloride.
Triazoxide has been developed for the control of soilborne Pyrenophora diseases.
It is used only as a seed dressing primarily for the control of Pyrenophora graminea,
the cause of leaf stripe on barley [64]. This is one of the most important seedborne
diseases of barley, and is difficult to control using most seed-treatment compounds
such as triazoles. As the spectrum of fungicidal activity is very narrow, triazoxide
is sold only in mixtures with other seed-dressing compounds, such as triazoles.
As it is not translocated within barley plants, the fungicidal activity of triazoxide
is limited to seedborne Pyrenophora diseases. Therefore, Pyrenophora teres, the
net blotch pathogen of barley – the spores of which can also be dispersed by
wind – cannot be fully eliminated. However, the control of seedborne P. teres
is desirable as it may delay the onset of this disease [64]. The recommended
application rates of triazoxide used in seed treatments are listed in Table 23.9.
Bayer CropScience sells triazoxide as a seed dressing in mixtures with other
®
fungicides such as prothioconazol and triadimenol (trade name Baytan ), tebu-
®
conazole (trade name Raxil S ), as a quaternary mixture with fluoxastrobin,
®
prothioconazole, and tebuconazole (trade name EfA ), or with tebuconazole and
®
the insecticide imidacloprid (trade name Gaucho Orge ).
Table 23.9 Triazoxide (5): Use in seed treatment against

plant diseases of market significance.
Plant disease Application rates (recommended) (g a.i. per 100 kg seed)
Leaf stripe on barley 1.6–3.6

(Pyrenophora graminea) (mixture with fluoxastrobin, prothioconazol, tebuconazol)
Loose smut on barley 1.6–3.8

(Ustilago nuda) (mixture with fluoxastrobin, prothioconazol, tebuconazol)
Of note is the acute oral toxicity of triazoxide in rats (LD50 = 100–200 mg kg−1
day−1 [64]), leading to a classification in WHO toxicity class II (moderately haz-
ardous). After oral intake, triazoxide is excreted rapidly in the urine and the feces.
No mutagenic or teratogenic effects have been observed with triazoxide. As the
dose required for a complete control of the named pathogens is very low, and as
the amount of triazoxide taken up by plants after seed treatment is negligible, no
studies investigating the metabolism of triazoxide in plants have been reported.
Although, within the soil triazoxide degrades steadily, no leaching risk of the parent
compound could be observed as it is almost totally immobile in the soil.
Currently, no information available on the biochemical mode of action of
triazoxide. The FRAC Code List reports no resistance development to date.
23.7
Tebufloquin
As a rapid resistance development against fungal diseases remains a severe problem

in several chemical classes, there will always be a need for compounds with new
modes of action. This is the case especially in the most important rice disease, rice
blast.
In order to overcome this problem, tebufloquin (6) has been developed as
a compound that is active against this and some other diseases. Tebufloquin
is currently in the process of being registered in Japan [67]; the plan is to
®
commercialize it under the trade name Try .
Tebufloquin belongs to the chemical class of quinolines, and was dis-
covered in 2000 by Meiji Seika Kaisha [68]. The IUPAC chemical name is
6-tert-butyl-8-fluoro-2,3-dimethylquinolin-4-yl acetate (CAS-RN.: 376645-78-2). The
chemical structure of tebufloquin is shown in Figure 23.6. Following its launch,
tebufloquin will be the second commercial product in this chemical class after
quinoxyfen (see Chapter 17.2). However, as both the substitution pattern and the
spectrum of activity of tebufloquin are very different from those of quinoxyfen, it
should be regarded in its own chemical subclass.
O Figure 23.6 Chemical structure of tebufloquin (6).
CH3 O CH3
CH3
H3C
H3C
N CH3
F
Table 23.10 Physico-chemical properties of tebufloquin (6).
Melting point (◦ C) 92.3

log PO/W (23 ◦ C) 4.02
Solubility in water (g l−1 ) 0.0202
Tebufloquin is a rather unpolar compound that demonstrates a surprisingly high

water solubility with respect to its lipophilicity. The physico-chemical properties of
tebufloquin that are publicly available are listed in Table 23.10 [67].
A straightforward synthesis of tebufloquin, according to the literature [69], is
described in Scheme 23.6. It starts with an N-acylation of 4-tert-butyl-aniline
H3C CH
H3C CH 3 H3C CH
3 F 3
1. Ac2O-Py H3C 6M HCl F
H3C H3C
2. Selectfluor NH
NH2 NH2
O CH3
20 21 22
O O
+
toluene
H3C O
reflux
CH3
CH3
H3C O
CH3
H3C
H3C O
O N CH3 CH3
H
F
H3C CH O CH3 H3C CH O
3 3 23
CH3 CH3
H3C Ac2O-Py H3C Ph2O, 250 °C
N CH3 N CH3
H
F F
6 24
Scheme 23.6 Synthesis of tebufloquin (6).

(20), followed by an ortho-fluorination of the N-acyl amino compound by using

®
Selectfluor . After removal of the acyl group by hydrolysis with a 6 M solution
of hydrochloric acid, the amino group of 22 is transformed into an enamine (23)
by reacting with ethyl 2-methyl-3-oxobutanoate in refluxing toluene. This enamine
(23) is then cyclized into 6-tert-butyl-8-fluoro-2,3-dimethylquinolin-4(1H)-one (24)
by heating it to 250 ◦ C in diphenylether. Finally, tebufloquin can be obtained by
O-acylation of 24 with a mixture of acetic anhydride and pyridine.
Tebufloquin has been developed mainly for the control of rice blast (Magnaporthe
grisea). It can be used to control this disease under both protective and curative
conditions, at application rates of 600–800 g a.i. ha−1 (in the case of a dust
formulation). However, it will most likely be recommended also for use against
diseases in soybeans, tomatoes, and other cultures, as it was tested in official
trials in Japan [67]. The estimated application rates of tebufloquin are listed in
Table 23.11.
Table 23.11 Tebufloquin (6): Use in plant diseases tested in official trials in Japan.
Plant disease Application ratesa

(estimated, g a.i. ha−1 )
Rice blast 600–800b

(Magnaporthe grisea) 200–300c
Rice dirty panicle 800b

(Cochliobolus miyabeanus)
Purple stain on soybean 200–400
(Cercospora kikuchii)
Powdery mildew on tomato 200–400
(Erysiphe cichoracearum)
Leaf spot on Chinese cabbage 200–400
(Alternaria brassicae)
Rust on Welsh onion 200–400
(Puccinia allii)
Leaf spot on Welsh onion 200–400
(Alternaria porri)
Gray blight on Tea 200–400
(Pestalotiopsis longiseta)
Anthracnose on Tea 400
(Colletotrichum theae-sinensis)
a
Application rates are converted into g a.i. ha−1 based on the official test
results in Japan (2005–2010).
b
Dust formulation.
c
WG (water-dispersible granule) formulation.
To date, no reports have been made regarding the toxicological effects of

tebufloquin, nor on its behavior in the environment.
To date, the mode of action of tebufloquin is unknown. As no cross-resistance
was detected towards methyl benzimidazole carbamate (MBC), phenylamide (PA),
quinone outside inhibitor (QoI), or demethylation inhibitor (DMI) fungicides,
and tebufloquin has also been shown as effective against rice blast strains that
are resistant against phosphorothiolate, kasugamycin, or melanin biosynthesis
inhibitor (MBI)-D, it is likely that the mode of action of tebufloquin is via a novel
process.
References
1. Serres, M. and Garraro, G.A. (1976) cymoxanil in the rat. Pestic. Sci., 12,
DPX-3217, a new fungicide for the 27–36.
control of grape downy mildew, potato 10. Gullino, M.L., Mescalchin, E., and
late blight and other Peronosporales. Mezzalama, M. (1997) Sensitivity to
Med. Fac. Landbouww. Univ. Gent, 41, cymoxanil in populations of Plasmopara
645–650. viticola in northern Italy. Plant Pathol.,
2. Davidson, S.H. (1976) 2-Cyano-2-hydrox 46, 729–736.
iminoacetamides as plant disease control 11. Klinkenberg, H.J., Stierl, R., and
agents. US Patent 3,954,992, published Dehne, H.W. (1998) Investigations
May 4, 1976 and priority March 15, on fungicide resistance in Oomycetes.
1972. (Continuation-in-part of patent Med. Fac. Landbouww. Univ. Gent, 63
application US Patent 1972/234,997). (3b), 1009–1015.
3. United States Environmental Protection 12. Gisi, U. and Sierotzki, H. (2008) Fungi-
Agency (EPA) (1998) Office of Preven- cide modes of action and resistance in
tion, Pesticides and Toxic Substances. downy mildews. Eur. J. Plant Pathol.,
Fact Sheet Cymoxanil, April 21 1998. 122, 157–167.
4. Martin, H. (1980) Oxime derivatives for 13. Grunwald, N.J., Sturbaum, A.K.,
the protection of cultivated crops. Br. Montes, G.R., Serrano, E.G.,
UK Patent Application GB 2,029,223, Lozoya-Saldana, H., and Fry, W.E.
published March 19, 1980 and priority (2006) Selection for fungicide resis-
September 1, 1978. tance within a growing season in field
5. Schwinn, F. and Staub, T. (1995) in populations of Phytophthora infestans at
Modern Selective Fungicides, 2nd edn the center of origin. Phytopathology, 96,
(ed. H. Lyr), Gustav Fischer Verlag, pp. 1397–1403.
326–339. 14. Perez, W., Lara, J., and Forbes, G.A.
6. Cohen, Y. and Gisi, U. (1993) Up- (2009) Resistance to metalaxyl-M and cy-
take, translocation and degradation of moxanil in a dominant clonal lineage of
[14 C]cymoxanil in tomato plants. Crop Phytophthora infestans in Huanuco, Peru,
Prot., 12, 284–292. an area of continuous potato production.
7. Belasco, I.J., Han, J.C.Y., Chrzanowski, Eur. J. Plant Pathol., 125 (1), 87–95.
R.L., and Baude, F.J. (1981) Metabolism 15. Hamlen, R.A. and Power, R.J. (1998)
of [14 C]cymoxanil in grapes, potatoes, Distribution of sensitivity responses to
and tomatoes. Pestic. Sci., 12, 355–364. cymoxanil within global populations of
8. Rana, S. and Beer, A. (eds) (2004) Phytophthora infestans. Pestic. Sci., 53,
Agrow: New Developments in Fungicides, 101–103.
2004 edn, PJB Publication Ltd, London. 16. Barchietto, T. and Genet, J.L. (2002)
9. Belasco, I.J. and Baude, F.J. (1981) Sensitivity of European isolates of Phy-
The metabolism of carbon-14-labeled tophthora infestans to famoxadone and
References 883
cymoxanil. Proc. Brighton Crop Protect. cinerea. Pestic. Biochem. Physiol., 78,
Conf. – Pests Dis., 2, 835–840. 151–160.
17. Reis, A., Ribeiro, F.H.S., Maffia, L.A., 27. Howard, R.J., Ferrari, M., Shillingford,
and Mizubuti, E.S.G. (2005) Sensitivity C., Stidham, M., Power, R., and
of Brazilian isolates of Phytophthora in- Hamlen, R. (1996) Biology of cymox-
festans to commonly used fungicides in anil action against Phytophthora infestans
tomato and potato crops. Plant Dis., 89, infection of tomato and potato. Proc.
1279–1284. Brighton Crop Prot. Conf. – Pests Dis.,
18. Corbett, J.R., Wright, K., and Baillie, 933–936.
A.C. (1984) The Biochemical Mode of 28. Ducret, J., Lacroix, G., and Gaulliard,
Action of Pesticides, 2nd edn, Academic J.M. (1975) Fungicide containing alkyl
Press, London, pp. 312–313. phosphite. Ger. Offen. DE 2,456,627,
19. Griffith, J.M., Davis, A.J., Grant, B.R., published June 19, 1975 and priority
and Köller, W. (1992) in Target Sites of December 14, 1973.
Fungicide Action (ed. W. Köller), CRC 29. United States Environmental Protection
Press, Boca Raton, FL, p. 81. Agency (EPA) (1991) Office of Pesti-
20. Tellier, F., Fritz, R., Kerhoas, L., Ducrot, cides and Toxic Substances. Fact Sheet
P.H., Carlin-Sinclair, A., Einhorn, J., Fosetyl-Al (Aliette), January 1991.
and Leroux, P. (2009) Metabolism of 30. Kun, A.Z., Maniu, V., Mihaly, L.,
fungicidal cyanooximes, cymoxanil and Fenesan, I., Crucin, V., Fagarasan,
analogues in various strains of Botrytis G., Gherasim, I., and Hantz, A.A. (1996)
Aluminum tris(O-ethyl phosphite)
cinerea. Pest Manage. Sci., 65, 129–136.
preparation process. Rom. RO 111,271,
21. Ribeiro, I.C., Verissimo, I., Moniz, L.,
published August 30, 1996 and priority
Cardoso, H., Sousa, M.J., Soares,
April 25, 1994.
A.M.V.M., and Leao, C. (2000) Yeasts
31. Deepak, S.A., Chaluvaraju, G.,
as a model for assessing the toxicity of
Basavaraju, P., Amuthesh, K.N.,
the fungicides penconazol, cymoxanil
Shekar Shetty, H., and Oros, G. (2005)
and dichlofluanid. Chemosphere, 41,
Response of pearl millet downy mildew
1637–1642.
(Sclerospora graminicola) to diverse
22. Esteve, K., Poupot, C., Dabert, P.,
fungicides. Int. J. Pest Manage., 51,
Mietton-Peuchot, M., and Milisic, V.
7–16.
(2009) A Saccharomyces cerevisiae-based
32. Williams, D.J., Beach, B.G.W., Horriere,
bioassay for assessing pesticide toxi- D., and Marechal, G. (1977) LS 74-783,
city. J. Ind. Microbiol. Biotechnol., 36, a new systemic fungicide with activity
1529–1534. against phycomycete diseases. Proc.
23. Ziogas, B.N. and Davidse, L.C. (1987) Brighton Crop Prot. Conf. – Pests Dis.,
Studies on the mechanism of action 565–573.
of cymoxanil in Phytophthora infestans. 33. Khilare, V.C., Deokate, A.S., and
Pestic. Biochem. Physiol., 29, 89–96. Gangawane, L.V. (2003) Occurrence of
24. Fritz, R., Despreaux, D., and Leroux, P. aluminum phosethyl (Allitte) resistance
(1984) Studies on the mode of action in Plasmopara viticola causing downy
of cymoxanil. Tag. – Akad. Landwirtsch. mildew of grapevine in Maharashtra. J.
Dtsch. Demokrat. Rep., 222, 65–69. Phytol. Res., 16, 239–241.
(Systemic Fungicides and Antifungal 34. Brown, S., Koike, S.T., Ochoa, O.E.,
Compound). Laemmlen, F., and Michelmore, R.W.
25. Despreaux, D., Fritz, R., and Leroux, P. (2004) Insensitivity to the fungicide
(1981) Mode d’action biochimique du fosetyl-aluminum in California isolates
cymoxanil. Phytiatr.- Phytopharm., 30, of the lettuce downy mildew pathogen,
245–255. Bremia lactucae. Plant Dis., 88, 502–508.
26. Tellier, F., Carlin-Sinclair, A., Fritz, R., 35. Cohen, Y. and Samoucha, Y. (1984)
Cherton, J.C., and Leroux, P. (2004) Cross-resistance to four systemic
Activity and metabolism of cyano-oxime fungicides in metalaxyl-resistant
derivatives in various strains of Botrytis strains of Phytophthora infestans and
Pseudoperonospora cubensis. Plant Dis., (phosphite). J. Gen. Microbiol., 136,

68, 137–139. 1285–1291.
36. Clerjeau, M., Moreau, C., Piganeau, B., 46. Niere, J.O., Griffith, J.M., and Grant,
Bompeix, G., and Malfatti, P. (1984) Ef- B.R. (1990) 31 P NMR studies on the
fectiveness of fosetyl-AL against strains effect of phosphite on Phytophthora
of Plasmopara viticola and Phytophthora palmivora. J. Gen. Microbiol., 136,
infestans that have developed resistance 147–156.
to anilide fungicides. Proc. Brighton Crop 47. Singh, V.K., Wood, S.M., Knowles, V.L.,
Prot. Conf. – Pests Dis., 497–502. and Plaxton, W.C. (2003) Phosphite
37. Bower, L.A. and Coffey, M.D. (1985) accelerates programmed cell death in
Development of laboratory tolerance phosphate-starved oilseed rape (Brassica
to phosphorous acid, fosetyl-Al, and napus) suspension cell cultures. Planta,
metalaxyl in Phytophthora capsici. Can. J. 218, 233–239.
Plant Pathol., 7, 1–6. 48. Bonomelli, A., Franchel, J., Mercier,
38. Sanders, P.L., Coffey, M.D., Greer, L., Mauro, M.C., and Boulay, M. (2002)
G.D., and Soika, M.D. (1990) Action du fosétyl d’aluminium sur la
Laboratory-induced resistance to stimulation des défenses naturelles de
fosetyl-Al in a metalaxyl-resistant field la vigne. Journées Jean Chevaugeon,
isolate of Pythium aphanidermatum. IVe Rencontres de Phytopatholo-
Plant Dis. London, 74, 690–692. gie/Mycologie, Aussois, France, March
39. Corbett, J.R., Wright, K., and Baillie, 13– 17, Poster No. 1.
A.C. (1984) The Biochemical Mode of 49. Molina, A., Hunt, M.D., and Ryals, J.A.
Action of Pesticides, 2nd edn, Academic (1998) Impaired fungicide activity in
Press, pp. 311–312. plants blocked in disease resistance
40. Bompeix, G. (1989) Fungicides and signal transduction. The Plant Cell, 10,
host-parasite interactions: the case of 1903–1914.
phosphonates. C.R. Acad. Agric. Fr., 75, 50. Chuang, H.W., Hsieh, T.F., Duval,
183–189. M., and Thomas, T.L. (2003) Genomics
41. Griffith, J.M., Davis, A.J., Grant, B.R., of Plants and Fungi, Mycology Series,
and Köller, W. (1992) in Target Sites of Vol. 18, Marcel Dekker, New York, pp.
Fungicide Action (ed. W. Köller), CRC 237–253.
Press, Boca Raton, FL, pp. 81–84. 51. Bompeix, G., Fettouche, F., and
42. Stehmann, C. and Grant, B.R. (2000) Saindrenan, P. (1981) Mode d’Action du
Inhibition of enzymes of the glycolytic Phosetyl Al. Phytiatr.- Phytopharm., 30,
pathway and hexose monophosphate 257–272, and references cited therein.
bypass by phosphonate. Pestic. Biochem. 52. Latorse, M.P., Mauprivez, L., Sirven, C.,
Physiol., 67, 13–24. Gautier, P., and Beffa, R. Comparison
43. Barchietto, T., Saindrenan, P., and of fosetyl-Al and another phosphonate
Bompeix, G. (1992) Physiological re- on plant downy mildew protection and
sponses of Phytophthora citrophthora to on Arabidopsis thaliana gene expression.
a subinhibitory concentration of phos- Proceedings of the 6th Workshop of
phonate. Pestic. Biochem. Physiol., 42, Grapevine Downy and Powdery Mildew,
151–166. Bordeaux, France, July 4–9, 2010 (eds
44. Martin, H., Grant, B.R., and Stehmann, A. Callonec, F. Delmotte, B. Emmett, D.
C. (1998) Inhibition of inorganic py- Gadoury, C. Gessler, D. Gubler, K.H.
rophosphatase by phosphonate – a site Kassemeyer, P. Magarey, M. Raynal, and
of action in Phytophthora spp? Pestic. R. Seem), p. 158.
Biochem. Physiol., 61, 65–77. 53. Beckers, G.J.M. and Conrath, U. (2007)
45. Griffith, J.M., Smillie, R.H., and Grant, Priming for stress resistance: from the
B.R. (1990) Alterations in nucleotide and lab to the field. Curr. Opin. Plant Biol.,
pyrophosphate levels in Phytophthora 10, 425–431.
palmivora following exposure to the an- 54. Yoshimoto, T. and Fujita, T. (1997)
tifungal agent potassium phosphonate Control of plant pathogens in soil
References 885
with flusulfamide, new fungicide. Phy- 62. Takeshiba, H., Kinoto, T., and Jojima,
toparasitica, 25, 360 (The Japan-Israel T. (1983) Antifungal pyridazinone
Workshop on Novel Approaches for derivatives. Eur. Pat. Appl. EP 89,650,
Controlling Insect Pests and Plant published September 28, 1983 and
Diseases). priority March 19, 1982.
55. Yoshimoto, T., Umemoto, M., 63. Sasse, K., Gauss, W., Frohberger, P.E.,
Igarashi, K., Kubota, Y., Yamazaki, Kraus, P., and Paul, V. (1979) 3-Azolyl
H., Enomoto, Y., and Yanagida, H. benzo triazines and their 1-oxides useful
(1986) N-(2-chloro-4-nitrophenyl)-3- in combating plant diseases. Ger. Offen.
trifluoromethylbenzenesulfonamides as DE 2,802,488, published July 26, 1979
fungicides. Jpn. Kokai Tokkyo Koho JP and priority January 20, 1978.
61,197,553, published September 1, 1986 64. Dutzmann, S. (1994) Triazoxide – a seed
and priority February 27, 1985. treatment fungicide for the control of
56. Dixon, G.R., Craig, M.A., Burgess, P.J., seed borne Pyrenophora species. BCPC
and Thomas, J. (1994) MTF 651: a new Monogr., 57, 85–89.
soil-applied fungicide for the control of
65. Meister, R.T. and Sine, C. (eds) (2005)
plasmodial fungi. Proc. Brighton Crop
Crop Protection Handbook, Meister Media
Worldwide, Willoughby, OH.
57. Yoshinari, M., Kochi, S., Kubota, Y.,
66. Heinrich, J. and Doering, F.
Inami, S., and Fujita, T. (1997) Develop-
(1998) Preparation of 3-(imidazol-
ment of a new fungicide, flusulfamide.
1-yl)-7-chlorobenzo-1,2,4-triazine-1-oxide.
J. Pestic. Sci., 22, 176–184.
Ger. Offen. DE 19,641,925, published
58. Fujita, T. (1994) Nebijin (flusulfamide,
MTF-651), a new soil fungicide. April 16, 1998 and priority October 11,
Agrochem. Jpn, 65 (65), 17–19. 1996.
59. Tanaka, S., Kochi, S., Kunita, H., Ito, 67. Matsumura, M. (2010) 27th Meeting,
S., and Kameya-Iwaki, M. (1999) Bio- Bioactivity of Pesticides Committee,
logical mode of action of the fungicide, Tokyo, April 23, 2010, Pesticide Science
flusulfamide, against Plasmodiophora Society of Japan.
brassicae (clubroot). Eur. J. Plant Pathol., 68. Yamamoto, K., Teraoka, T., Kurihara,
105, 577–584. H., Matsumura, M., and Yoshitake, K.
60. Jojima, T. and Takahi, Y. (1977) Pyrida- (2001) Rice blast control agents. WO
zone derivative-containing agricultural Patent 2001/092,231.
fungicides. Ger. Offen. DE 2,640,806, 69. Nishizuka, T., Kurihara,H.,
published March 24, 1977 and priority Yamamoto, K., and Yoshitake,
September 11, 1975. K. (2004) Process for producing
61. Takahi, Y. (1988) Diclomezine 2,3,6-trialkyl-8-fluoro-4-quinoline deriva-
(Monguard). Jpn Pestic. Inf., 52, 31–35. tive, WO Patent 2004/007,460.
887
24
Recently Introduced Powdery Mildew Fungicides
Jochen Dietz
24.1
Introduction
Powdery mildew pathogens continue to infest a large number of field and specialty
crops. During recent years, the widespread appearance of strains with increased
tolerance to existing powdery mildew fungicides such as benzimidazoles, demethy-
lation inhibitors (DMIs), or strobilurines has resulted in a significant drop in
the efficacy of these active ingredients. Consequently, there has been an urgent
need for new mildewicides with novel modes of action in order to ensure a
highly effective control of powdery mildew, and to allow for a smart resistance
management. On this basis, several companies have initiated R&D programs
to identify innovative solutions designed specifically to combat powdery mildew
pathogens.
Ultimately, cyflufenamid (Nippon Soda), metrafenone (BASF SE), pyriofenone
(Ishihara Sangyo Kaisha), proquinazid (Du Pont de Nemours), and flutianil
(Otsuka) have been developed as new benchmark mildewicides for cereals and
specialty crops.
24.2
Cyflufenamid
Cyflufenamid (1), a benzamidoxime fungicide, was developed under the code

NF-149 by Nippon Soda Co., Ltd. (Nisso), and has been introduced into the
market as a powdery mildewicide for fruits, vegetables, and cereals (Table 24.1)
[1–4].
24.2.1
Discovery
Due to the changing market environment in the 1990s for powdery mildew fungi-
cides, Nippon Soda began to re-evaluate the potential of benzamidoximes for this

888 24 Recently Introduced Powdery Mildew Fungicides
Table 24.1 Structure, physical, toxicological, and ecotoxico-

logical properties of cyflufenamid [1, 5].
O
Structure F N O
F
N
H
CF3 1
Melting point 61.5–62.5 ◦ C

Vapor pressure 3.54 × 10−5 Pa˜(20 ◦ C)
Water solubility 0.52 mg l−1 (20 ◦ C, pH 6.5)
Log Pow 4.70 (25 ◦ C, pH 6.75)
Acute toxicity (oral, rat) LD50 > 5000 mg kg−1
Acute toxicity (dermal, rat) LD50 > 2000 mg kg−1
Acute toxicity (inhalation, rat) LD50 = 4760 mg m−3
Eye irritation (rabbit) Slightly irritating
Skin irritation (rabbit) Non-irritating
Skin sensitization (guinea pig) Non-sensitizing
Mutagenicity Non-mutagenic
Acute toxicity to fish LC50 > 1.14 mg l−1 (carp, 96 h)
Acute toxicity to algae EC50 > 0.83 mg l−1 (72 h)
target [2]. According to the company, the core structure of the benzamidoximes was
a combination of structural features derived from several older pesticides such as
the miticide benzoximate (2), the herbicides alloxydim-sodium (3) and sethoxydim
(4), and the fungicide metalaxyl (5) (Figure 24.1). As a result of the re-evaluation,
compounds 6 and 7 were found to show a high efficacy against powdery mildew
pathogens, combining excellent curative and residual activities. However, systemic-
ity – which is crucial for preventing powdery mildew infestations on newly grown
leaves – was not observed with these compounds. Further optimization then led
to the discovery of the highly potent derivative 8. Although this compound had
a higher activity than its precursors, it still did not exhibit satisfactory in-plant
mobility. To circumvent this obstacle, a different strategy was chosen for the
distribution of the active ingredient, which was then proved to be successful. The
introduction of one or more additional fluorine atoms into the molecule eventually
led to novel analogs that exhibited a sufficiently high vapor pressure to allow
for distribution to newly grown leaves via the vapor phase [1, 2]. Ultimately, the
2,3-difluoro-6-trifluoromethyl derivative 1, cyflufenamid, was selected as the devel-
opment candidate, outbalancing field performance, physico-chemical parameters,
synthesis simplicity, production costs, and safety [2–5].
24.2 Cyflufenamid 889
O O
OMe N O ONa N
Cl
O
OMe O 3
2 CO2Me
O
OH N CO2Me
N
CH2OMe
4 O 5
S O
R1
O R2 O OMe
Cl N O F N O
N N
H H
Cl CF3 8
6: R1 = Me, R2 = OMe
7: R1 = Cyclopropyl, R2 = H
Figure 24.1 Structural precursors (2–5) of cyflufenamid,

lead compounds (6, 7), and initial candidate 8.
24.2.2
Cross-Resistance and Mode of Action
The cross-resistance of cyflufenamid to DMIs, morpholines, quinone outside in-

hibitors (QoIs), benzimidazoles, cyprodinil, and quinoxyfen has not been observed.
Morphologically, cyflufenamid inhibits the infection process by preventing haus-
torium formation and development, the growth of secondary hypha, germ tube
elongation, and conidiospore formation. In contrast, spore germination and ap-
pressorium formation are not inhibited [1, 2, 5, 6]. Although the exact biochemical
target that cyflufenamid interacts with has not yet been identified, several possibili-
ties could be excluded. In a test system using Monilinia fructicola, no influence was
observed on cell division, sterol biosynthesis, lipid biosynthesis, cell membrane
functions, or respiration [2]. Meanwhile, cyflufenamid has been characterized as a
powdery mildewicide with a low resistance risk. Owing to the inherently linked high
pathogen resistance risk, recommendations on resistance management strategies
have, however, been developed [7].
Cl 1. n-BuLi
Cl Cl
Cl 2. HCO2Me or CO2 Cl X Cl CN
CF3 CF3 CF3

X = CHO or CO2H
O
F F N O
KF F CN F
N
H
CF3 CF3 1
Figure 24.2 Access to 2,3-difluoro-6-trifluoromethyl-

benzonitrile, a synthetic key intermediate for the synthesis
of cyflufenamid.
24.2.3
Manufacturing Process
The arrangement of the four consecutive substituents on the benzene moiety

represented the key challenge for cycflufenamid’s synthesis [2]. Although several
routes were scouted, it was decided ultimately that an ortho-lithiation strategy
would be the best method to introduce this substitution pattern (Figure 24.2).
Toward this end, 3,4-dichlorobenzotrifluoride – a raw material commercially avail-
able in bulk quantities – was chosen as starting point and converted into the
carbaldehyde or, alternatively, the carboxylic acid, by a metalation reaction.
The latter functional groups were then converted into a carbonitrile function,
and subsequent chlorine–fluorine exchange with potassium fluoride afforded
2,3-difluoro-6-trifluoromethylbenzonitrile, a suitable precursor for cyflufenamid
equipped with all necessary functional groups in the desired positions.
24.2.4
Fungicidal Profile
Cyflufenamid shows both preventive and curative activities on powdery mildew

pathogens in cereals and specialty crops. In addition, it is highly active against
brown rot on stone fruits. Cyflufenamid exhibits a good residual activity, a remark-
able vapor phase activity, and a good translaminar mobility; however, only poor
translocation within the host plant was observed [1–3, 6, 8].
24.2.5
Registration, Products, Formulations, and Crops
Cyflufenamid was first registered in 2002 for the treatment of fruits and veg-
etables in Japan, and launched by Certis Europe for cereals in the UK in 2005.
24.3 Metrafenone 891
Since that time, additional registrations have been granted in other European
countries.
In order to avoid resistance development, cyflufenamid’s main formulation
for fruits and vegetables is a water-dispersible granule (WG) formulation with
®
triflumizole, with the trade name Pancho TF.
−1 ®
For cereals, a 50 g l emulsion oil-in-water (EW) formulation named Cyflamid
was introduced [8]; however, as this product is a solo formulation a tank mix with a
broad-spectrum fungicide is recommended to avoid resistance development. Very
recently, a patent application covering an emulsifiable concentrate (EC) formulation
of cyflufenamid and the announcement of the introduction of a 10% suspension
®
concentrate (SC) formulation (Miltrex ) for specialty crops in the US, have widened
the spectrum of potential formulation options [9, 10].
24.3
Metrafenone
Metrafenone (9), the first fungicide to be launched from the benzophenone

family, was originally discovered by American Cyanamid as a result of structural
considerations of previously investigated fungicides (Table 24.2) [11, 12]. It was
transferred during the development phase to BASF SE, following the merger
of the two companies in 2000. In 2004, Metrafenone was introduced in the
European market as a potent powdery mildewicide for use in cereals and specialty
crops [13].

logical properties of metrafenone [14, 15].
OMe MeO
O OMe
Structure
OMe
Br
9
Melting point 99.2–100.8 ◦ C

Vapor pressure 2.56 × 10−4 Pa˜(25 ◦ C)
Water solubility 0.49 mg l−1 (20 ◦ C, pH 7)
Log Pow 4.3 (pH 4)
Eye irritation (rabbit) Non-irritating
Acute toxicity to fish LC50 > 94 mg l−1 (96 h, Oncorhynchus mykiss)
Acute toxicity to algae EC50 = 2.9 mg l−1 ˜(72 h)
24.3.1
No cross-resistance of metrafenone with other products has been observed to date.

Although the mode of action is not yet fully understood, it has been proven to differ
from that of other major fungicides, and extensive investigations to clarify the
biochemical target(s) are ongoing [16–19]. The results of initial studies with barley
powdery mildew have indicated that metrafenone disturbs the organization or
polarization of the actin cytoskeleton [16]. In addition, morphological investigations
have shown that metrafenone interacts at the early stages of the development of
wheat powdery mildew [20], by inhibiting mycelia growth and penetration into
the leaf surface. Furthermore, the development of appressoria, the formation of
haustoria, and sporulation are also each reduced.
Comprehensive studies on the current sensitivity situation of wheat powdery
mildew have revealed the appearance of strains that were not fully controlled at
registered dose rates. For a sustainable use of metrafenone, resistance management
strategies and continued sensitivity monitoring are, therefore, essential [21].
24.3.2
Processes for the synthesis of metrafenone have been described in two BASF patents
[22, 23]. The benzophenone can be obtained by an iron(III) chloride-catalyzed
Friedel–Crafts acylation of 3,4,5-trimethoxytoluene. The appropriate benzoyl chlo-
ride is easily accessible by bromination of 2-methoxy-6-methyl benzoic acid and
subsequent conversion to the acid chloride (Figure 24.3).
24.3.3
Fungicidal Profile
Metrafenone provides excellent preventative, curative, and residual activities against

powdery mildew pathogens in cereals, grapes, and vegetables. In addition, it is
effective against eye spot on wheat and barley. Metrafenone shows significant
translaminar action, good acropetal translocation and, similar to cyflufenamid,
distribution by vapor-phase diffusion.
OMe
OMe
OMe OMe OMe MeO
O OMe
CO2H COCl OMe
cat. FeCl3 OMe
Br Br
9
Figure 24.3 Synthesis of metrafenone.

24.3.4
Registration, Products, Formulation, and Crops
Metrafenone was first launched in the UK in 2004 to control powdery mildew

in wheat and barley. Subsequently, registrations for use in field crops have been
granted in the Netherlands and in Germany, as well as for powdery mildew
control in grapes in Germany [24–26]. BASF SE has since gained further national
registrations for both sectors in other countries.
® ®
Metrafenone is sold as Flexity (300 g l−1 SC) for use in cereals, and as Vivando
−1
(500 g l SC) for powdery mildew control in grapevines. The recommended use
rate is 0.25–0.5 l ha−1 for powdery mildew control, and 0.5 l ha−1 to reduce eye
spot. Since cereal powdery mildew is a high-risk pathogen with regards to resistance
development, a spray program with azole fungicides for broad-spectrum purposes
is recommended [27]. In grapes, alternate applications with non-metrafenone
fungicides (e.g., strobilurine-containing products) are advised [25].
24.4
Pyriofenone
In January 2010, ‘‘pyriofenone’’ was provisionally approved as the ISO name for
Ishihara Sangyo Kaisha, Ltd’s newest powdery mildewicide from the benzophenone
class, which was developed under the code name IKF-309 [28]. Thus, it is the most
recent of the new-generation powdery mildew fungicides discussed herein and,
consequently, publicly available information on this compound remains scarce.
Recent information from regulatory authorities has allowed for the conclusion that
this compound is currently in the process of registration [29].
Pyriofenone (10) is structurally very closely related to metrafenone, and it can be
assumed that the mode of action of these two fungicides is the same (Figure 24.4).
Due to the conspicuous structural similarity, it can also be speculated that the
large-scale synthesis would be conducted in similar fashion to that of metrafenone.
24.5
Proquinazid
Proquinazid (11), the first fungicide belonging to the quinazolinone class, was
developed under the code DPX-KQ926 by Du Pont de Nemours (Table 24.3), and
OMe MeO
O OMe
N
OMe
Cl
10 Figure 24.4 Structure of pyriofenone.

logical properties of proquinazid [31, 34].
O
Structure I
N
N O
11
Melting point 48–49 ◦ C

Acute toxicity (dermal, rat) LD50 > 5000 mg kg−1
Eye irritation Irritating
Acute toxicity to fish LC50 = 2.3 mg l−1 (96 h, Oncorhynchus mykiss)
Acute toxicity to algae EC50 = 3.3 mg l−1 (72 h)
has been introduced as a potent preventative powdery mildewicide for the cereal
and the grapevine markets [30–33].
24.5.1
Discovery
According to the inventors, proquinazid was derived from the initial lead compound
12, derived from Du Pont’s random screening program for novel fungicides
(Figure 24.5) [30]. Although pyridopyrimidone 12 showed an interesting, but weak,
activity at a high dose rate against wheat powdery mildew, the lead optimization
process led to a variety of novel analogs of 12 with the same core structure, as well
as to the synthesis of novel core-modified derivates 13 [31, 35, 36].
Whilst many of these analogs were highly active in glasshouse screening, only
the quinazolinone derivatives 13 also showed good activity in the field. Eventually,
proquinazid was chosen as a development candidate as it showed outstanding
pathogen control in field trials in cereals, grapes, and other crops at low dose rates;
notably, it also demonstrated an excellent residual activity [30].
O O
X R1
N N
N OMe N R2
Y
Br
12 13
Figure 24.5 Initial lead compound 12 for the optimization

program of proquinazid and structural development.
I CO2H I CO2R Figure 24.6 Synthesis of pro-

quinazid.
NH2 NH2
nPrNCS 1. Cl2C=S
2. H2NnPr
O
I
N
N S
H
1. MeI
2. NaOnPr
O
I
N
N O
11
24.5.2
Proquinazid can be prepared starting from 5-iodoanthranilic acid (Figure 24.6)

[31, 37, 38]. Cyclization with n-propyl isothiocyanate affords 2,3-dihydro-6-iodo-
3-propyl-2-thioxo-4(1H)-quinazolinone. Alternatively, this key intermediate can
be obtained by the reaction of an appropriate anthranilate with thiophosgene
and n-propylamine. The subsequent introduction of a propoxy substituent via a
methylation displacement sequence concludes the synthesis.
24.5.3
Morphologically, the active ingredient inhibits spore germination and appres-

sorium formation [33]. In July 2010, proquinazid was moved by the Fungicide
Resistance Action Committee (FRAC) from ‘‘Unknown Mode of Action’’ to the E1
class (‘‘signal transduction’’) as a result of recent findings in Erysiphe necator that
this new mildewicide showed characteristics similar to those of quinoxyfen. The
exact molecular target, however, remains unknown and is considered to be distinct
from that of quinoxyfen [39–41].
24.5.4
Fungicidal Profile
Proquinazid acts in a preventive manner, and does not show any significant
curative activity against powdery mildew pathogens. It is locally systemic and
allows – similar to cyflufenamid and metrafenone – for the protection of untreated
leaves or neighboring plants by distribution of the active ingredient via the vapor
phase [33].
24.5.5
Registration, Products, Formulation, and Crops
®
Proquinazid obtained its first registration and sales as the cereal fungicide Talius
in 2005. In addition, proquinazid could successfully be registered as the grapevine
®
fungicide Talendo in Hungary and Austria in 2005. Du Pont has since gained
more registrations in key countries.
®
Talius (200 g l−1 EC) controls powdery mildew infections on cereals at ap-
proximately 40 g a.i. ha−1 , and provides an excellent residual activity of up to six
weeks from a single application. The product can be applied twice in a season,
but for resistance management reasons it should be mixed with a broad-spectrum
fungicide or a powdery mildewicide with a different mode of action.
®
Talendo (200 g l−1 EC) is recommended for the control of Uncinula necator in
grapes, at a rate of approximately 50 g a.i. ha−1 .
24.6
Flutianila
‘‘Flutianil’’ (17) was announced as the provisional ISO name of Otsuka’s newest
mildewicide OK-5203 in July 2008 (Figure 24.7) [42]. It is structurally distinct
from all other new powdery mildewicides discussed herein, and publicly available
information remains scarce.
According to published reports, flutianil can be synthetically accessed by a mul-
tistep procedure, starting from an appropriately substituted aniline. Diazotization
and conversion of the diazonium salt into a dithiocarbonate affords intermediate
14 that is subsequently deprotected and cyanomethylated to obtain the nitrile 15.
Reaction with 2-methoxyphenylisothiocyanate leads to precursor 16, that ultimately
is cyclized with 1,2-dibromoethane to afford flutianil [43].
The commercial product named Pinpoint™ is being developed by Valent, under
the code V-10118. With regards to the mode of action, it has been reported that
flutianil is not cross-resistant to DMIs and strobilurines, although the exact mode
of action has not yet been fully elucidated. In morphological studies, however, an
inhibitory effect on mycelia growth was observed [44].
S
H2N
F 1. HCl/NaNO2 EtO S 1. LiAlH4
F
2. EtOCS2K 2. ClCH2CN
F
F F
F
F
F
14
NCS O
H SH
OMe N
S
NC F S
NC F
F
F F
F
F
F
15 16
O
S
N
Br
Br
S
NC F
F
F
17 F
Figure 24.7 Synthesis of flutianil.
24.7
Conclusions
Within the past decade, five new powdery mildew fungicides have been launched
for the treatment of both field and specialty crops. The active ingredients were
designed to combat powdery mildew infestations in cereals, fruits, vegetables,
and grapevines and, in combination with other fungicides, these innovations have
proved to be particularly useful solutions for the broad-spectrum control of fungi
in a variety of crops. All of these compounds have a new mode of action, and
do not show cross-resistance to any existing market product. Consequently, smart
resistance management should avoid the possible emergence of resistant strains
and allow for an effective and sustainable powdery mildew control in cereals and
specialty crops in the future.
Acknowledgments
The author thanks his colleagues Drs T. Grote, R. Riggs, and J. Montag for
stimulating discussions, valuable input, and careful revision of the manuscript.
References
1. Yokota, C. (2004) Agrochem. Jpn, 84, 12. Curtze, J., Morschhäuser, G., Stumm,
12–14. K.-O., Albert, G., Reichert, G., Simon,
2. Kasahara, I. (2005) Fain Kemikaru (Fine W., Waldeck, A., Cotter, H.V.T., and
Chem.), 34, 29–37. Rehnig, A.E.E. (1999) European Patent
3. Ma, Y.-S. and Liu, C.-L. (2005) Nongyao Applications EP 897,904 A1, American
(Chin. J. Pest.), 44, 128–129. Cyanamid Company, USA, 29 pp. (b)
4. (a) Kasahara, I., Ooka, H., Sano, S., Curtze, J., Morschhäuser, G., Stumm,
Hosokawa, H., and Yamanaka, H. (1996) K.-O., Albert, G., Reichert, G., Simon,
PCT International Application WO W., Waldeck, A., Cotter, H.V.T., and
9,619,442 A1, Nippon Soda Co., Ltd., Rehnig, A.E.E. (1999) Chem. Abstr., 130,
Japan, 89 pp. (b) Kasahara, I., Ooka, H., 209501.
Sano, S., Hosokawa, H., and Yamanaka, 13. Michel, P. (2003) Phytoma, 566, 33–35.
H. (1996) Chem. Abstr., 125, 167579. 14. Bundesamt für Verbraucher-
5. Sano, S., Kasahara, I., and Yamanaka, schutz und Lebensmittelsicherheit,
H. (2007) J. Pestic. Sci., 32, 137–138. Pflanzenschutzmittel-Verzeichnis –
6. Haramoto, M., Yamanaka, H., Metrafenone, www.bvl.bund.de (accessed
Hosokawa, H., Sano, H., Sano, S., 2011).
and Otani, H. (2006) J. Pestic. Sci., 31, 15. BASF Aktiengesellschaft (2005)
116–122. ®
Flexity Material Safety Data Sheet,
7. Haramoto, M., Hamamura, H., Sano, S.,
http://www.agrar.basf.de/ (accessed
Felsenstein, F.G., and Otani, H. (2006)
2011).
J. Pestic. Sci., 31, 397–404.
16. Opalski, K. (2005) Cell polarity in plant
8. Agrow World Crop Protection News
defense and fungal pathogenesis in
(2005) Article ID A00884184 (June 2,
the interaction of barley with powdery
2005), A00901062 (November 3, 2005).
mildew fungi. Ph.D. Thesis. University
Available at: www.agrow.co.uk (accessed
of Giessen, Germany.
2006).
9. (a) Tanaka, T., Dairiki, K., and Ono, 17. Köhle, H., Opalski, K., and
K. (2010) Jpn. Kokai Tokkyo Koho JP Hückelhoven, R. (2004) 54. Deutsche
2010/001,268 A, Nippon Soda Co., Ltd., Pflanzenschutztagung, Hamburg, Ger-
Japan, 12 pp. (b) Tanaka, T., Dairiki, K., many, September 20–23, Section 45-7,
and Ono, K. (2010) Chem. Abstr., 152, http://pub.jki.bund.de/index.php/MittBBA
113150. /article/viewFile/721/656 (accessed
10. See, for example, http://ir4.rutgers.edu/ 2011).
FoodUse/FUWorkshop/Industry%20Talks/ 18. Opalski, K., Hückelhoven, R., Tresch,
Pathology09/NissoCyflufenamidTalk2009. S., Kogel, K.-H., Grossmann, K., and
pdf (accessed 2011). Köhle, H. (2006) Pest Manage. Sci., 62,
11. (a) Curtze, J., Rudolph, C.H.G., 393–401.
Schröder, L., Albert, G., Rehnig, A.E.E., 19. Schmitt, M.R., Carzaniga, R., Cotter,
and Sieverding, E.G. (1996) European H.V.T., O’Connell, R., and Hollomon,
Patent Applications EP 727,141 A2, D. (2006) Pest Manage. Sci., 62,
American Cyanamid Company, USA, 383–392.
63 pp. (b) Curtze, J., Rudolph, C.H.G., 20. Agrow World Crop Protection News
Schröder, L., Albert, G., Rehnig, A.E.E., (2004) Article ID A00831684 (February
and Sieverding, E.G. (1996) Chem. 6, 2004). Available at: www.agrow.co.uk
Abstr., 126, 7819. (accessed 2006).
References 899
21. Felsenstein, F., Semar, M., and http://www2.dupont.com/Crop_Protection/

Stammler, G. (2010) Gesunde Pflanz., de_DE/assets/downloads/pdfs/TALIUS.pdf
62, 29–33. (accessed 2011).
22. Kameswaran, V. (2001) PCT Interna- 35. (a) Selby, T. (1993) PCT International
tional Application WO 2001/051,440 Application WO 9,323,398 A1, E. I. Du
A1, BASF Aktiengesellschaft, Germany, Pont de Nemours & Co., USA, 65 pp.
21 pp. (b) Kameswaran, V. (2001) (b) Selby, T. (1993) Chem. Abstr., 120,
Chem. Abstr., 135, 107143. 217721.
23. (a) Maywald, V., Hoffmann, N., Keil, 36. Selby, T.P., Sternberg, C.G., Bereznak,
M., Vogelbacher, U.J., and Wevers, J.H. J.F., Coats, R.A., and Marshall, E.A.
(2004) PCT International Application (2007) The discovery of proquinazid: a
WO 2004/054,953 A1, BASF Aktienge-
new and potent powdery mildew con-
sellschaft, Germany, 19 pp. (b) Maywald,
trol agent, in: Synthesis and Chemistry
V., Hoffmann, N., Keil, M., Vogelbacher,
of Agrochemicals VII, ACS Symposium
U.J., and Wevers, J.H. (2004) Chem.
Series, Vol. 948, American Chemical
Abstr., 141, 71349.
Society, pp. 209–222.
24. Agrow World Crop Protection News
(2005) Article ID A00895966 (September 37. Bereznak, J.F., Marshall, E.A., Sternberg,
16, 2005). Available at: www.agrow.co.uk C.G., Sternberg, J.A., and Sun, K.-M.
(accessed 2006). (1997) PCT International Applica-
25. BASF Aktiengesellschaft, Prod- tion WO 9,748,684 A1, E. I. Du Pont
uct information. Available at: de Nemours & Co., USA, 65 pp.
http://www.agrar.basf.de/ (accessed (b)Bereznak, J.F., Marshall, E.A.,
2011). Sternberg, C.G., Sternberg, J.A., and
26. Agrow World Crop Protection News Sun, K.-M. (1997) Chem. Abstr., 128,
(2005) Article ID A00903288 (November 88927.
24, 2005). Available at: www.agrow.co.uk 38. (a) Bereznak, J.F., Chang, Z.-Y., Selby,
(accessed 2006). T.P., and Sternberg, C.G. (1999) US
®
27. BASF Aktiengesellschaft Flexity Prod- Patent Application US 5,945,423 A, E. I.
uct Brochure, http://www.agrar.basf.de/ Du Pont de Nemours & Co., USA, p. 23;
(accessed 2011). (b) Bereznak, J.F., Chang, Z.-Y., Selby,
28. http://www.alanwood.net/pesticides/ T.P., and Sternberg, C.G. (1999) Chem.
pyriofenone.html (accessed 2011). Abstr., 131, 170360.
29. see e.g. http://edocket.access.gpo.gov/2010/ 39. Fungicide Resistance Action Commit-
2010-22331.htm (accessed 2011). tee. Available at: www.frac.info (accessed
30. Selby, T.P., Sternberg, C.G., Bereznak, 2011).
J.F., Coats, R.A., and Marshall, E.A. 40. Genet, J.-L. and Jaworska, G. (2009) Pest
(2004) Abstract of Papers, 228th ACS Manage. Sci., 65, 878–884.
National Meeting, Philadelphia, PA, 41. Gilbert, S.R., Cools, H.J., Fraaije, B.A.,
August 22–26, 2004, AGRO-001.
Bailey, A.M., and Lucas, J.A. (2009)
31. (a) Bereznak, J.F., Chang, Z.-Y., Selby,
T.P., and Sternberg, C.G. (1994) PCT
42. http://www.alanwood.net/pesticides/
International Application WO 9,426,722
flutianil.html (accessed 2011).
A1, E. I. Du Pont de Nemours & Co.,
43. (a) Hayashi, M., Endo, Y., and Komura,
USA, 73 pp. (b) Bereznak, J.F., Chang,
Z.-Y., Selby, T.P., and Sternberg, C.G. T. (2000) Jpn. Kokai Tokkyo Koho JP
(1994) Chem. Abstr., 123, 169646. 2000/319,270 A, Otsuka Chemical Co.,
32. Michel, P. (2004) Phytoma, 577, 47–48. Ltd., Japan; Otsuka Chemical Holdings
33. Agrow World Crop Protection News Co., Ltd., 40 pp. (b) Hayashi, M., Endo,
(2005) Article ID A00905436, December Y., and Komura, T. (2000) Chem. Abstr.,
15, 2005. Available at: www.agrow.co.uk 133, 350209.
(accessed 2006). 44. http://ir4.rutgers.edu/FoodUse/
® FUWorkshop/Industry%20Talks/Disease/
34. Du Pont de Nemours, Talius
200EC Sicherheitsdatenblatt (2009) Valent.ppt (accessed 2011).
901
25
Newest Aspects of Nucleic Acid Synthesis Inhibitors:
Metalaxyl-M
25.1
Introduction
The control of diseases caused by plant pathogens of the Oomycetes has been a
major target since the beginning of modern chemical crop protection. Today, a
wide range of fungicides is available, and new products are being introduced to
the market at regular intervals [1–3]. The phenylamide fungicides include com-
pounds such as metalaxyl (3), metalaxyl-M (1) (Figure 25.1), furalaxyl, benalaxyl,
ofurace, and oxadixyl [4]. When metalaxyl – the first of this class of fungicides – was
introduced to the market in 1977, it marked a breakthrough in chemical disease
control, and metalaxyl soon became the most important compound of the class in
this market segment. The unique properties of the phenylamide fungicides, such
as the control of all members of the Peronosporales and Pythiales, the longlasting
preventive and curative activities, the high systemicity, and the excellent safety
profile have been reviewed [4]. In 1996, Ciba-Geigy announced the introduction
of the active enantiomer of metalaxyl [5, 6]. By introducing a pure enantiomer,
and thus replacing the racemic metalaxyl –a process sometimes referred to as a
‘‘chiral switch’’ – a new chapter was opened in both the control of Oomycetes
by phenylamide fungicides and the use of chiral crop protection agents in
general.
To date, metalaxyl-M (which is also known as mefenoxam in the USA) [7], is
an indispensable product in the control of plant pathogenic Peronosporales and
Pythiales.
25.2
Chemistry of Metalaxyl-M/Mefenoxam
Both enantiomers of metalaxyl were first prepared by the classical procedure

of fractional crystallization of the salts of d, l-N-(2,6-xylyl)-alanine with (+)
and (−)-α-phenethylamine, followed by Fischer esterification and acylation with
methoxyacetyl chloride in ethyl acetate in the presence of triethylamine as base,

902 25 Newest Aspects of Nucleic Acid Synthesis Inhibitors: Metalaxyl-M
O O O O O O
O CH3 O CH3 O CH3
O H O CH3 O
H3C N CH3 H3C N H H3C N CH3
H3C CH3 H3C CH3 H3C CH3
1 2 3
Figure 25.1 Metalaxyl-M (USA: mefenoxam): methyl-N-

(methoxyacetyl)-N-(2,6-xylyl)-D-(−)-alaninate (1); methyl-N-
(methoxyacetyl)-N-(2,6-xylyl)-L-(+)-alaninate (2); metalaxyl:
methyl-N-(methoxyacetyl)-N-(2,6-xylyl)-rac alaninate (3).
or in toluene using sodium carbonate as base, at room temperature. Optical

purity was determined using nuclear magnetic resonance (NMR) spectroscopy
using chiral shift reagents, and the absolute configuration was assigned by
connecting the enantiomers to l-lactic acid of the chiral pool. The reaction of
2,6-dimethylaniline with the p-nitrobenzene sulfonate of l-methyl lactate gave
methyl d-N-(2,6-dimethylphenyl)-alaninate through inversion at the chiral center
[8]. When this process was further developed, the key step was the preparation
of d-N-(2,6-dimethylphenyl)-alanine esters, which, elaborated in a general synthe-
sis of N-substituted α-amino acids, starts with (S)-2-(trifluoromethylsulfonyloxy)-
carboxylic acid esters that are then reacted with various amines and anilines
(Scheme 25.1) [9, 10]. The sulfonylation of methyl (S)-lactate 4 gave pure sul-
fonylated ester 5 in yields up to 96%. Surprisingly, the sterically hindered
2,6-dimethylaniline 6 also reacted easily to afford the corresponding methyl
(R)-N-(2,6-dimethylphenyl)-propionate 7 in good yields (90%) and high optical
O O
O O O O NH2 H
H H + HN CH3
OH O
H3C H3C
SO2R′
4 5 6
7
O O
O
O
O H
O
Cl N CH3
8
Scheme 25.1 Synthesis of metalaxyl-M (1) starting from

(S)-lactic acid methyl ester (chiral pool).
25.2 Chemistry of Metalaxyl-M/Mefenoxam 903
Table 25.1 Chemical and physical properties of metalaxyl-M [7].
Common name Metalaxyl-M (BSI, E-ISO, F-ISO) mefenoxam

(only in USA)
Patent no. WO96/01559
Specific rotation (−); [α]24 D = –57 ± 1 (ca. 1.807%, H3 CCOCH3 ) [9]
(S)-Isomer (2) (+); [α]24 D = +57 ± 1 (ca. 2.883%, H3 CCOCH3 )
[9]
Form Pale yellow to light brown, viscous liquid
Vapor pressure (mPa) (25 ◦ C) 3.3
KOW (log P) (25 ◦ C) 1.71
Solubility (g l –1 ) Water = 26 (25 ◦ C); n-hexane = 59. Miscible with
acetone, ethyl acetate, methanol, toluene,
n-octanol
Stability Hydrolytic stability pH ≤ 7, DT50 > 200 days; pH
9 (25 ◦ C), DT50 = 116 days
purity. Later, this process was generalized by reacting the aniline 6 with alkyl and,
especially, methyl (S)-2-(methylsulfonyloxy)-propionate, claiming yields of 90%
(methylsulfonate) and a R/S ratio of 97.5 : 2.5 of 7. Acylation of the intermedi-
ate 7 with methoxy-acetyl chloride 8 yielded metalaxyl-M (1; properties listed in
Table 25.1) in 94% chemical yield without any racemization (R/S ratio 97.5 : 2.5)
(Scheme 25.1) [11, 12].
Clearly, the classical separation of enantiomers by fractional crystallization, as
described above, would hardly be economic for the large-scale production of a pes-
ticide. Thus, a selective enzymatic hydrolysis of esters of d,l-N-(2,6-xylyl)-alanine
to the corresponding (R)-N-(2,6-xylyl)-alanine and subsequent synthesis of
metalaxyl-M, was recently developed [13]. Following the pioneering breakthrough
in the large-scale production of the herbicide aR/aS,1’S-metolachlor, and employ-
ing an enantioselective catalytic hydrogenation process for the preparation of a key
intermediate [14], Spindler and Blaser elaborated the basic principles governing
the use of such catalytic processes [15]. Prerequisite for the successful development
of such processes for the large-scale production of pharmaceuticals and agrochem-
icals is a broad experience in catalysis, and the capacity to optimize both the chiral
catalyst and the reaction conditions. This has been well demonstrated in the de-
velopment of catalytic processes for the production of metalaxyl-M (Scheme 25.2),
O O O O
O O
[Rh(nbd)2]BF4/(R,R)-Me-duphos H
O O
N CH2 N CH3
10
9 1
Scheme 25.2 Enantioselective catalysis in the preparation of metalaxyl-M (1).

where the easily prepared enamide 9 was subjected to catalyst screening. Of the 34
chiral Rh diphosphine catalysts tested, twelve produced an enantiomeric excess (ee)
of >90% of metalaxyl-M, but most showed an insufficient fungicidal activity to be
considered for large-scale production. Further optimization led to the discovery of
the catalyst [Rh(nbd)2 ]BF4 /(R,R)-Me-duphos (10), and to a hydrogenation process
with hydrogen at 10 bar, 60 ◦ C and a substrate-to-catalyst ratio of 5 × 104 , which
resulted in 95.6% ee and a turnover frequency of 5.2 × 103 h−1 [16].
25.3
Biological Activity
Like metalaxyl, metalaxyl-M controls all pathogens of the Oomycetes in the or-
ders Peronosporales, Sclerosporales, and Pythiales, such as the downy mildews of
the genera Albugo, Bremia, Peronospora, Peronosclerospora, Plasmopara, Pseudoper-
onospora, Sclerophtora, and Sclerospora, as well as Pythium and Phytophtora species.
In all applications, whether foliar, soil, or seed treatment, the outstanding level of
control by metalaxyl-M is achieved at up to half the rate of its predecessor metalaxyl
3 [5, 6]. As shown in greenhouse trials, the (R)-(−)-enantiomer 1 is the active iso-
mer, and the (S)-(+)-enantiomer 2 the almost inactive isomer. This also indicates
that there is no racemization; that is, formation of the (S)-(+)-enantiomer 2 on or
within the plant or pathogen after treatment, which is an important prerequisite
for the introduction of pure enantiomers as products. Metalaxyl-M is mostly used
in mixture with multisite fungicides to protect a wide range of crops, and is a major
pillar product in the control of diseases caused by pathogens of the Oomycetes
® ®
[17]. Major brand names are Ridomil Gold and Apron XL . Both, metalaxyl and
metalaxyl-M are extremely safe to crop plants. For example, in a study conducted
by Singh, Mersie, and Brlansky on the control of foot rot and root rot (Phytophtora
spp.) in citrus, the slight herbicidal effect of metalaxyl was shown to be associated
with the (S)-(+)-enantiomer 2, while no such effects could be detected for the
(R)-(−)-enantiomer (metalaxyl-M) [18].
25.4
Mode of Action and Mechanism of Resistance
The phenylamide fungicides – including metalaxyl and metalaxyl-M – inhibit ri-

bosomal RNA synthesis, specifically RNA polymerization (polymerases). In the
mycelium of Phytophthora megasperma, metalaxyl primarily affected the polymerase
I complex of rRNA synthesis, which is considered the primary site of action [19].
The endogenous RNA polymerase activity of isolated nuclei of P. megasperma and
P. infestans was highly sensitive to metalaxyl, unless the isolates were from resistant
strains. This led to the hypothesis that a mutation in the target site was responsible
for the resistance [20], a proposal that was further supported by the observation
that [3 H]metalaxyl binds to cell-free mycelial extracts of metalaxyl-sensitive but
25.4 Mode of Action and Mechanism of Resistance 905
not of metalaxyl-resistant isolates [20]. Although metalaxyl, metalaxyl-M, oxadixyl,

benalaxyl, and ofurace exhibit different levels of intrinsic activity and rRNA poly-
merase inhibition [20], cross-resistance was observed between all phenylamide
fungicides [21].
In the Oomycetes, the phenylamide fungicides affect especially hyphal growth
and the formation of haustoria and spores [22]. As spores typically will contain many
ribosomes to support the early growth stages of the fungus, RNA synthesis is fully
operational only after spore germination; hence, the later developmental stages are
the most sensitive [19]. As a consequence of RNA inhibition, the precursors of RNA
synthesis (i.e., nucleoside triphosphates) are accumulated. These in turn activate
the β-1,3-glucan synthetases that are involved in cell wall formation [19], with the
result that metalaxyl-treated hyphae often produce thicker cell walls than untreated
hyphae.
Shortly after the commercial introduction of metalaxyl, resistant isolates were
detected in Pseudoperonospora cubensis, Phytophthora infestans, Peronospora tabacina,
and Plasmopara viticola [23]. In most cases, this effect was coupled to a decline in
disease control. As a consequence, strict recommendations for the use of phenyl-
amides were designed and enforced by the PhenylAmide Fungicide Resistance Ac-
tion Committee (PA-FRAC) to prevent and further delay resistance evolution [24].
These recommendations involved the preventive use of prepacked mixtures with
well-defined amounts of nonphenylamide fungicides, a limited number of applica-
tions per crop and per season, and no soil use for the control of airborne pathogens.
These recommendations have been successfully implemented such that today,
products containing phenylamides remain important fungicides, offering specific
advantages for the control of diseases caused by Oomycetes, although resistant
isolates can be found worldwide, and on many crops.
Phenylamide resistance has been described as monogenic. The majority of
the F1 progeny produced from metalaxyl-resistant (r) and metalaxyl-sensitive (s)
parental isolates of P. infestans had an intermediate sensitivity to metalaxyl. Crosses
between two F1 isolates with intermediate sensitivity yielded a 1s : 2i : 1r ratio of
progeny in the F2 generation [25]. This Mendelian segregation pattern reflects
a single-gene (monogenic) resistance [26] based on an incompletely dominant
gene [27]. Resistance to metalaxyl was also reported to be controlled by a sin-
gle incompletely dominant gene in Phytophthora capsici [28], P. sojae [29], and
B. lactucae [30]. However, a continuous sensitivity segregation pattern was ob-
served in the F1 generation received from r × s crosses of European and Mexican
P. infestans parents. This suggested that one semidominant locus, together with
several minor loci, may be involved in resistance [31]. Resistance in these isolates
was associated with two loci, MEX1 and MEX2, with the second locus mapping
to the same linkage group as MEX1 but to a distinct site [32]. Although many
investigations on the mode of action and mechanism of resistance to phenyl-
amide fungicides have been undertaken over the past 25 years, the responsible
resistance gene(s) and the site of mutation(s) in the genome have not yet been
elucidated.
Although phenylamides are considered to bear a high intrinsic resistance risk

[33], they have failed to fully eliminate the sensitive subpopulations from nature,
even after 25 years of intensive use [17]. The proportion of resistant isolates
in P. infestans and P. viticola fluctuates from year to year, and also within the
season. The proportion increases within a season, more rapidly in fields treated
with phenylamides than in untreated fields, begins to decline at the end of the
season, and is significantly lower at the start of the next season when compared
to the proportion at the end of the previous year [23]. The appearance of new
P. infestans genotypes in recent populations of many European countries (e.g.,
‘‘blue13’’ isolates, which are mostly resistant to phenylamides and A2 mating type
[34]) and the USA (US-22 isolates, which are mostly sensitive to phenylamides and
A2 mating type [35]) may be a result of migration (the import of infected tubers
and plant material) rather than selection through fungicide use.
25.5
Degradation and Metabolism of the Two Enantiomers
One of the goals of making the ‘‘chiral switch’’ – that is, to develop the pure
enantiomer of metalaxyl – was to reduce the amount of chemicals dispersed in the
environment. Clearly, the application of half the amounts compared with metalaxyl
will result in a reduction of the active ingredient in the environment. However,
just as they have different biological activities, the (R) and (S) enantiomers may be
expected to behave differently in the environment, as their degradation processes
are mostly of an enzymatic nature. The metabolic pathways of metalaxyl-M in
soil, plants, and animals are very similar to those of metalaxyl [4]. The main
metabolite of metalaxyl-M in soil is, as for metalaxyl, the corresponding acid
of metalaxyl-M. Although, because of the extremely wide variation of soil and
climatic conditions, a uniform degradation process cannot be expected for the two
enantiomers under natural conditions, some general trends have emerged from
the extensive studies carried out in this field. Racemization was neither observed
in plants nor in the different soil types investigated [36–38]. Indicators of soil
processes, such as changes in ammonium and nitrate concentrations, as well as
the activity of soil enzymes (e.g., phosphatases, β-glucosidases, or dehydrogenases)
can be affected, but no uniform correlation to the use of metalaxyl-M could be
found. The changes of these soil processes depend very much on the number
of treatments, the amount of the active ingredient applied, and on whether
the soil investigated had been previously treated with the fungicide (enhanced
degradation) [39].
In conclusion, the use of metalaxyl-M is safe to the environment, with no specific
effects having been found that could be attributed to the (R)-(−)-optical isomer and
which would impair the fungicide’s safety profile. Moreover, the ability to reduce
the application rate by almost half (compared to metalaxyl) while maintaining the
spectrum of activity and level of potency, clearly demonstrates an innovative step
in the development of metalaxyl-M.
References 907
References
1. Henningsen, M. (2003) Chem. Unserer 15. Spindler, F. and Blaser, H.-U. (1999)
Zeit, 37, 98–111. Enantiomer, 4, 557–568.
2. Knauf-Beiter, G. and Hermann, D. 16. Spindler, F., Pugin, B., Buser, H.-P.,
(2005) BCPC Int. Congr. Crop Sci. Tech- Jalett, H.-P., Pittelkow, U., and
nol., 1, 99–104. Blaser, H.-U. (1998) Pestic. Sci., 54
3. Tafforeau, S., Wegmann, T., Latorse, (3), 302–304.
M.P., Gouot, J.M., Duvert, P., and 17. Gisi, U. (2002) in Advances in
Bardsley, E. (2005) BCPC Int. Congr. Downy Mildew Research (eds P.T.N.
Crop Sci. Technol., 1, 79–86. Spencer-Phillips, U. Gisi, and A.
4. Gisi, U. and Ziegler, H. (2003) in En- Lebeda), Kluwer Academic Publishers,
cyclopedia of Agrochemicals (eds J.R. Dordrecht, New York, pp. 119–159.
Plimmer, D.W. Gammon, and N.N. 18. Singh, M., Mersie, W., and Brlansky,
Ragsdale), John Wiley & Sons, Inc., New R.H. (2003) Plant Dis., 87 (9),
York, pp. 609–616. 1144–1147.
5. Nunninger, C., Watson, G., 19. Davidse, L.C. (1995) in Modern Selective
Leadbitter, N., and Ellgenhausen, H. Fungicides, 2nd edn (ed. H. Lyr), Gustav
(1996) Proc. Brighton Crop. Prot. Conf., 1, Fischer, Jena, pp. 347–354.
41–46. 20. Davidse, L.C. (1988) in Fungicide Resis-
6. Nunninger, C., Goggin, J.E.N., and tance in North America (ed. C.J. Delp),
Sozzi, D. (1996) Patent WO 96/01,559 APS Press, St Paul, MN, pp. 63–65.
(Publ. date 25.01.1996), (Inventors), 21. Diriwächter, G., Sozzi, D., Ney, C., and
Staub, T. (1987) Crop Prot., 6, 250–255.
Ciba-Geigy AG.
22. Schwinn, F.J. and Staub, T. (1995) in
7. Tomlin, C.D.S. (2004–2005) The
Modern Selective Fungicides, 2nd edn
e-Pesticide Manual, Version 3.1, 13th
(ed. H. Lyr), Gustav Fischer, Jena,
edn, British Crop Protection Council.
pp. 323–346.
8. Staub, T. and Hubele, A. (1981)
23. Gisi, U. and Cohen, Y. (1996) Annu.
in Chemie der Pflanzenschutz-und
Rev. Phytopathol., 43, 549–572.
Schädlingsbekämpfungsmittel (ed. R.
24. Urech, P.A. and Staub, T. (1985) EPPO
Wegler), Springer-Verlag, Berlin, pp.
Bull., 15, 539–543.
389–422, ISBN: 3-540-10307.
25. Shattock, R.C. (1986) Proc. Brighton Crop
9. Effenberger, F., Burkard, U., and
Willfahrt, J. (1986) Liebigs Ann. Chem., 26. Shattock, R.C. (1988) Plant Pathol., 37,
1986 (2), 314–333. 4–11.
10. Drauz, K., Burckard, U., and 27. Shaw, D.S. and Shattock, R.C. (1991)
Effenberger, F. (1985) Patent DE in Phytophthora (eds J.A. Lucas, R.C.
3,328,986 (Publ. date 21.02.1985), Shattock, D.S. Shaw, and L.R. Cooke)
(Inventors), Degussa A.G. Cambridge University Press, Cambridge,
11. Zanardi, G. and Confalonieri, G. (2000) pp. 218–230.
Patent WO 00/76,960 (Publ. date 28. Lucas, J.A., Greer, G., Oudemans, P.V.,
21.12.2000), (Inventors), Isagro S.P.A. and Coffey, M.D. (1990) Physiol. Mol.
12. Stutz, W. and Brünisholz, J. (2000) Plant Pathol., 36, 175–187.
Patent CH 690,367 (Publ. date 29. Bhat, R.G., McBlain, B.A., and
15.08.2000), (Inventors), Novartis AG. Schmitthenner, A.F. (1993) Mycol.
13. Park, O.-J., Lee, S.-H., Park, T.-Y., Lee, Res., 97, 865–870.
S.-W., and Cho, K.-H. (2005) Tetrahe- 30. Crute, I.R. and Harrison, J.M. (1988)
dron: Asymmetry, 16, 1221–1225. Plant Pathol., 37, 231–250.
14. Blaser, H.-U., Buser, H.-P., Coers, K., 31. Fabritius, A.L., Shattock, R.C., and
Hanreich, R., Jalett, H.-P., Jelsch, E., Judelson, H.S. (1997) Phytopathology, 87,
Pugin, B., Schneider, H.-D., Spindler, 1034–1040.
F., and Wegmann, A. (1999) Chimia, 53, 32. Judelson, H.S. and Roberts, S. (1999)
275–280. Phytopathology, 89, 754–760.
33. Gisi, U. and Staehle-Csech, U. (1988) 36. Zadra, C., Marucchini, C., and
Proc. Brighton Crop Prot. Conf. – Pests Zazzerini, A. (2002) J. Agric. Food
Dis., 1, 359–366. Chem., 50, 5373–5377.
34. Gisi, U., Walder, F., Resheat Eini, 37. Buerge, I.J., Poiger, T., Müller, M.D.,
and Buser, H.-R. (2003) Environ. Sci.
Z., Edel, D., and Sierotzki, H. (2011)
Technol., 37, 2668–2674.
J. Phytopathol., 159, 223–232.
38. Monkiedje, A. and Spiteller, M. (2005)
35. Ristaino, J.B. (2010) Contribution 5-S Int. J. Environ. Res. Public Health, 2 (2),
(Special Session), APS 2010 Annual 272–285.
Meeting, August 2010, Charlotte, NC, 39. Droby, S. and Coffey, M.D. (1991) Ann.
USA. Appl. Biol., 118, 543–553.
909
26
Host Defense Inducers
26.1
Introduction
In the natural environment, the defense reactions of plants are induced by a variety
of factors that include microorganisms and ectoparasites, as well as abiotic stresses
such as wind or heat. Today, numerous species of bacteria and fungi, as well
as pathogen-derived molecules, cell-wall components of fungi, peptides or plant
extracts have been commercialized as either biological or natural agents for the
control of crop diseases. Subsequently, some of these have been proven to induce
systemic acquired resistance in plants, in similar fashion to synthetic chemical
inducers [1, 2].
In this chapter, attention will be focused on those synthetic chemicals that
have been defined as host defense inducers by the Fungicide Resistance Action
Committee (FRAC) as: code P1, such as acibenzolar-S-methyl (2; ASM); code P2,
such as probenazole (1; PBZ); and code P3, such as tiadinil (3) and isotianil (4)
(Figure 26.1) [3].
On a practical basis, these compounds have become very well established as
major countermeasures for specific diseases, notably rice blast in northeast Asia.
Yet, despite the practical use of biological control agents increasing worldwide the
economical impact of these individual products – when compared to chemicals – is,
as yet, rather limited.
Since the 1970s, numerous investigations into chemical inducers of host de-
fense systems have been conducted against a background of functional problems
associated with modern fungicides inhibiting a single target site. Examples of this
include the rapid emergence of critical resistance, and/or a lack of efficacy against
certain bacterial diseases.
During the 1980s – in parallel with the commercial success of PBZ (1) in the
rice blast fungicide market of Japan – intensive research was conducted worldwide
into inducers of host defense systems. Unfortunately, at that time the quantitative
evaluation of defense induction activity was not possible by using in vitro screen-
ing; neither could it be assessed by the classical in vivo screening of fungicidal
compounds in the greenhouse.

910 26 Host Defense Inducers
O S
O CH3
O
S S
N N
O
1 2
probenazole (PBZ) acibenzolar-S-methyl (ASM)
CH3 Cl Cl NC
N H Cl H
N N
N N
S CH3 S
O O
3 4
tiadinil isotianil
Figure 26.1 Synthetic chemicals defined as host defense in-

ducers: probenazole (1, PBZ) acibenzolar-S-methyl (2, ASM),
tiadinil (3), and isotianil (4).
During the 1990s, however, the rapid development of biochemical and molecular
biological assays based on the genomic, proteomic, and metabolomic analyses of
defense mechanisms in model plants (mainly tobacco and Arabidopsis) permitted
a more efficient discovery of new lead structures, and also allowed the quantitative
profiling of compounds from known classes of defense inducers [4, 5]. Yet, despite
many reports of – and patent applications relating to – chemical host defense
inducers and genetically modified (GM) crops with an enhanced expression of
defense genes, extremely limited numbers of compounds achieved commercial
status. And, even today, disease-resistant GM crops have still not established a
significant share in the market of crop-protection materials.
26.2
General Mechanism of Induced Resistance
26.2.1
Induced Resistance
It is assumed that all plants possess an intrinsic capacity to defend themselves

against attacks by pathogens. Such enhanced defenses can be triggered by a variety
of stimuli that includes biotic and abiotic stress, beneficial microorganisms, or
chemicals. ‘‘Induced resistance’’ is typically a systemic response with longlasting
effects that confers a broad spectrum of resistance which can be regulated by a
network of signaling pathways that involve endogenous phytohormones such as the
signal molecules salicylic acid (SA), jasmonic acid (JA) (Figure 26.2), and ethylene.
26.2 General Mechanism of Induced Resistance 911
COOH O
OH CH3
COOH
salicylic acid (SA) jasmonic acid (JA)
Figure 26.2 Phytohormones salicylic acid (SA) and jasmonic acid (JA).
According to the inducing agent involved and the subsequent molecular mecha-
nism, two major types of induced resistance have been identified: systemic acquired
resistance (SAR), which depends on SA; and induced systemic resistance (ISR),
which requires JA and ethylene, but not SA [6]. Typically, SAR is induced by a
limited primary infection caused by a necrotizing pathogen, whereas ISR is induced
by nonpathogenic organisms that colonize the roots.
Both, SAR and ISR confer an enhanced defense capacity. Upon pathogen attack,
the defense response occurs at an earlier stage and is stronger, leading to a broader
resistance spectrum [7]. It is well known that SAR is most efficient against biotrophic
and hemibiotrophic pathogens, and leads to the expression of pathogenesis-related
(PR) genes [8]; in contrast, necrotrophic pathogens are generally controlled by ISR.
As noted above, chemicals can also induce resistance; however, as the compounds
reviewed here are inducers of SAR, attention will be focused on the mechanisms
of such resistance.
26.2.2
Systemic Signals in Systemic Acquired Resistance
Despite the fact that SAR has been studied extensively in a wide variety of species,
the identity of the systemic signal that triggers the defense response remains
unknown. In dicotyledonous plants, such as Arabidopsis and tobacco, the onset of
SAR is associated with an accumulation of SA [9]. Indeed, the exogenous application
of SA (or of its functional analogs) has been shown to induce SAR. Yet, the results
of studies involving transgenic plants with a reduced level of SA have suggested
that the latter is not the mobile signal, but rather is required in distant tissues for
the establishment of SAR [10]. Other putative candidates that have been proposed
for this role include methyl salicylate (MeSA), lipid-derived signals (including JA)
and, more recently, azelaic acid [11–13].
However, in contrast to dicotyledonous and other monocotyledonous crops, in
the case of rice the role of SA in SAR remains unclear. Rice is known to contain
high levels of SA, and no further accumulation is observed with disease resistance
[14]. In addition, the depletion of endogenous SA in transgenic rice does not
measurably affect defense gene expression [15]; nevertheless, it has been suggested
that the role of SA might be age-dependent [16]. The results of other studies have
shown that, in some rice cultivars, a tight correlation can be observed between
the level of blast fungus resistance and the endogenous SA level, which in turn
suggests that SA might contribute to rice defenses [15].
26.2.3
Signal Perception and Transduction
The analysis of an Arabidopsis mutant that was nonresponsive to SA led to the

discovery of a key regulator of SAR, so-called NPR1 genes (non-expressor of
PR genes). NPR1 is essential for transducing the SA signal and to activate PR
gene expression [17, 18]. Upon cellular redox changes induced by SA, NPR1
migrates from the cytoplasm to the nucleus, where it interacts with a bZIP (basic
region/leucine zipper motif) transcription factor and activates the defense genes
[19]. In addition, both SA and pathogen infection lead to an enhanced expression
of the NPR1 gene [20, 21]. Recently, a global transcriptional approach was used to
identify genes involved in the very early responses to SA [22]. In this case, it was
found that most of the induced genes were NPR1-dependent and that, among such
genes, some coded for proteins involved in signaling pathways such as kinases,
disease-resistance proteins such as AtMLO2 (Arabidopsis thaliana mildew resistance
locus o2), and RPS2 (resistance to Pseudomonas syringae 2) and the superfamily of
WRKY transcription factors. Together, these are important for the onset of plant
immune responses and the SAR-mediated regulation of PR genes.
Rice was used as monocotyledonous plant model to investigate the molecular
mechanisms of induced resistance. In this context, an SA NPR1-dependent signal-
ing pathway has also been characterized [23, 24], and suggests that at least some
defense signaling components are conserved between dicotyledonous and mono-
cotyledonous plants. Furthermore, overexpression of the Arabidopsis NPR1 gene in
rice [25] results in an enhanced resistance against bacterial and fungal pathogens,
as it does in Arabidopsis [26], tomato [27], and wheat [28]. In rice, the transcrip-
tion factor Oryza sativa WRKY45 (OsWRKY45) plays an essential role in induced
resistance [29, 30]. Whilst this pathway is independent of NPR1, it participates
actively in blast resistance, as demonstrated in knockdown experiments. To date,
no counterpart of OsWRKY45 genes has been identified in Arabidopsis, although its
overexpression in Arabidopsis increases the resistance to bacterial pathogens [31].
Thus, both NPR1 and WRKY represent key node proteins in SAR.
26.2.4
Biochemical Changes in Systemic Acquired Resistance
Transcription factors differentially regulate a number of genes involved in defense,

leading ultimately to the production of defense proteins. The first proteins to be
discovered that were associated with SAR were the PR proteins. These proteins,
which have been classified in 17 families, are mainly extracellular and possess
antimicrobial activities, thus supporting the concept of a ‘‘direct’’ role in defense
[8]. The induction of PR genes serves as part of the defense response. Typically, the
activation of additional defenses is necessary to halt the development of pathogens.
In this case, a complex array of antimicrobial processes is activated that includes a
strengthening of the plant cell wall by callose and hydroxyproline-rich glycoproteins
depositions, and also an accumulation of peroxidases [7].
26.2.5
Priming in Systemic Acquired Resistance
In SAR-expressing plants, the defense response occurs more rapidly and/or more
efficiently on pathogen challenge. This phenomenon – known as priming – exists
not only for SAR but also for other forms of induced resistance [32]. An investigation
into the priming effect in parsley cells has shown that chemicals or elicitors present
at low doses do not directly induce expression of the phenylalanine ammonia-lyase
(PAL) defense-related gene, although in these cells the PAL gene is known to be
more robustly expressed following a challenge [33]. Similarly, in intact plants, a
low level of SA will prime the plant for a potentiated expression of the PR1 and
PAL genes, which becomes detectable only after pathogen attack. It has also been
shown that NPR1 is essential for priming in Arabidopsis [34].
The exact molecular mechanisms of priming are, nevertheless, not clearly under-
stood. It is possible that an enhancement of the signaling pathway, together with
an accumulation of post-translational modifications and of crucial transcription
factors may be important for the priming effect [35]. Indeed, an accumulation of
two mitogen-activated protein kinases (MAPK3 and MAPK6), both of which are
involved in signaling, was recently reported in primed Arabidopsis plants [36].
26.3
Market Products
26.3.1
Probenazole
Probenazole (1) was introduced to the market in 1975 by Meiji Seika, to control
fungal blast disease and bacterial blight in rice. In Japan, PBZ has been the
best-selling rice blast control agent since 1980s. Because of the unstable efficacy of
PBZ after foliar spraying, it was applied for the first 20 years of use by spreading the
granular formulation on the water surface of the paddy field (2400–3200 g a.i. ha−1 ).
The same formulation has various label recommendations, including those for
bacterial diseases in brassica, cucurbits, and onions. However, the prevalence of
PBZ in vegetables has been limited, most likely due to its high application rates
(4800–7200 g a.i. ha−1 ). The recommended timing for the application of PBZ was
about 10 days before the first outbreak of leaf blast, though this could not be always
forecast accurately.
During the late 1990s, a new granular formulation of PBZ was developed that
could be applied to the nursery box at transplanting time, with sufficient crop
tolerance. At the same time, an additional formulation for mixing into fertilizer
was introduced, but this had to be injected mechanically in the rhizospheres of
the transplanted seedlings. Together, these new application technologies secured
an easier application of PBZ and a more stable control of rice blast, which was
independent from the timing of the first infection.
Currently, PBZ is mainly used as combination granule formulations with differ-

ent insecticides (e.g., fipronil, dinotefuran) for nursery box application in Japan,
providing longlasting control against a variety of pests in rice. In 2010, a new
granular formulation of PBZ in combination with insecticides was launched, that
is safe for nursery box application at sowing time.
26.3.1.1 Synthesis of Probenazole

PBZ can easily be prepared by the reaction of 3-chloro-1,2-benzisoxazole-1,1-dioxide
(5) with allylalcohol (6) (Scheme 26.1) [37].
26.3.1.2 Systemic Acquired Resistance Induction by Probenazole

On treating rice with PBZ, the induction of PR genes (e.g., PBZ1 and OsPR1a)
has been reported [38]. A PBZ-inducible (PBZ1) gene (PR10-related gene), which
was first discovered by screening genes responding to PBZ [39], represents a
useful marker for SAR in rice. In addition to PR genes, other genes are known
to be involved in signal transduction pathways, such as kinase and the rice
PBZ-responsive (RPR1) gene; the latter gene codes for a disease-resistant-like
protein that is also activated upon infection. In addition, an enhanced activity in the
phenylpropanoid metabolism is also observed in rice plants treated with PBZ [40].
In dicotyledonous plants, it is assumed that PBZ induces defense responses
through the SA signaling pathway [41]. It has been shown that the level of
total SA [free SA + salicylic acid-glucoside (SAG)] is significantly increased
by treatment with PBZ. Moreover, PBZ and its active metabolite 1,2-
benzisothiazol-3(2H)-one-1,1-dioxide (BIT) (Scheme 26.1) require SA accumula-
tion to induce SAR in Arabidopsis and tobacco, as well as a functional NPR1 gene
[41, 42]. Indeed, both compounds are ineffective in nahG transgenic plants that
are unable to accumulate SAR. In addition, these compounds do not involve JA
and ethylene pathways. Thus, PBZ is assumed to act upstream of SA, unlike ASM
(2) and tiadinil (3) (see Sections 26.3.2.2 and 26.3.3.2).
In contrast, in rice SAR is independent of the accumulation of SA, the exact
role of which remains unclear. Nevertheless, SA accumulation has been reported
to occur in adult rice plants following treatment with PBZ [38]. Umemura and
coworkers [43] have reported that salicylic acid glucosyltransferase (OsSGT1),
which catalyzes the conversion of free SA into the SA-O-β-glycoside (SAG), might
contribute actively to PBZ-induced resistance [43]. These authors found that PBZ
HO
O O O
O O O
S S S
6
N N NH
reflux
Cl O O
5 1 BIT
Scheme 26.1 Synthesis of probenazole (1, PBZ) and its

metabolite 1,2-benzisothiazole-3(2H)-one-1,1-dioxide (BIT).
would induce an accumulation of SAG that correlated with activation of the OsSGT1
gene. Furthermore, an OsSGT1 knockdown mutant showed an impaired capacity
to develop PBZ-induced resistance against blast disease.
26.3.2
Acibenzolar-S-Methyl
Acibenzolar-S-methyl (ASM; 2) was first described by Ciba-Geigy in 1987 [44], and

launched in 1996 for the treatment of cereals in Germany. Between 1998 and
2006, ASM maintained its pesticide registration in Japan, as a granule combination
with insecticides for nursery box application in rice. In the USA, formulations of
ASM are applied as a foliar spray and as seed treatment in various dicotyledonous
crops, although neither of these formulations has played a major role in the
agrochemical market. Despite its limited economical impact, ASM is the most
widely investigated molecule as a positive marker of SAR in various species of
plants, diversified study approaches, and also in many countries. Typically, ASM
induces a broad resistance spectrum on an extended diversity of dicotyledonous
and monocotyledonous plants.
26.3.2.1 Synthesis of Acibenzolar-S-Methyl

Benzo[1,2,3]thiadiazole-7-carboxylic acid (8) is the key intermediate for the syn-
thesis of ASM (2). For the preparation of thiadiazoles, two main methodolo-
gies have been reported [44]: (i) by a cyclization reaction of the diazonium
salt of 2-alkylthio-3-aminobenzoic acids 7 (Scheme 26.2; Method A) or by the
O OH O OH
SR1 S
Diazotization
Method A
N
NH2 N
7 8
Aromatization,
hydrolysis
O OH
O OH
R2 SOCl2 S
NH (Hurd Mori) N
N Method B N
H
9
10
R2 = -tosyl, -COOR, -CONR2
Scheme 26.2 Synthesis of benzo[1,2,3]thiadiazole-7-carboxylic acid (8) by methods A

and B.
cyclization of N-acyl or N-tosylhydrazones 9 bearing an adjacent α-methylene

group (Scheme 26.2; Method B). The latter is possible by treatment with thionyl
chloride (Hurd Mori reaction), followed by spontaneous aromatization of the
intermediate 10 with subsequent transformation to 8.
An elaboration of these syntheses, leading to ASM, is shown in Scheme 26.3.
Esterification of 2-chloro-3-nitrobenzoic acid (11) resulted in the formation of its
methylester 12 which, following the substitution of chlorine by benzylmercaptan,
gave compound 13. Following reduction of the nitro group in 13, the methyl
3-amino-2-benzylthiobenzoate (14) was formed; this can be cyclized by diazotation
into methyl benzo[1,2,3]thiadiazole-7-carboxylate (15) according to Method A in
Scheme 26.2. Subsequent hydrolysis of 15 produced 8. Starting with this key
intermediate, ASM was prepared via the corresponding acid chloride 16. It should
be noted that other synthesis concepts have also been reported [45, 46].
26.3.2.2 Systemic Acquired Resistance Induction by Acibenzolar-S-Methyl

It was reported recently that the transformation of ASM (2) by methyl salicylate
esterase (SABP2) is required to induce a full SAR response [47]. At the molecular
O OH O O O O
CH3 CH3
Cl Cl HS S
CH3OH/H2SO4
(99%)
NO2 NO2 NO2
11 12 13
H2/RaNi
O OH O O O O
CH3 CH3
S NaOH,H2O
S
S NaNO2 / H+
N N
diazotation
N N NH2
8 15 14
SOCl2
O Cl O S
CH3
S S
N N
N N
16 2
Scheme 26.3 Elaborated synthetic pathway of acibenzolar-S-methyl (2, ASM).

level, ASM induces the same characteristic set of SAR genes as does biological
or SA induction [48]. ASM directly activates the PR-1 gene, primes the plants
for stronger PAL gene induction, and improves callose deposition [49]. Various
studies conducted with rice have shown that ASM induces diverse components of
the defense signaling cascade such as OsBIMK2 coding for an MAP kinase [50],
OsBIANK1 encoding a plasma membrane-anchored protein [51], and OsBISERK1
encoding a protein belonging to somatic embryogenesis receptor kinase (SERK)
[52]. In dicotyledonous plants, ASM induces SAR without any accumulation of
SA [48]; moreover, its action does not require SA accumulation as ASM induces
SAR in transgenic nahG plants. However, ASM necessitates a functional NPR1
gene, as does SA [53]. This situation has been further confirmed by transcriptional
profiling, which showed almost all ASM-responsive genes to be NPR1-dependent
[54]. Therefore, ASM may be considered as a functional analog of SA, which
suggests that it might act at the same site.
In rice, an induced resistance by ASM requires the two transcriptional regula-
tors – NPR1 and the rice-specific OsWRY45 [55]. In contrast to Arabidopsis, it was
shown that only a subset of ASM-responsive genes is OsNPR1-dependent in rice
[56]. While all of the ASM upregulated genes, categorized under defense response,
are NPR1-dependent in Arabidopsis, only 5.9% of them are OsNPR1-dependent
in rice. The expression of PR1a and PR1b is controlled by OsNPR1, whereas
glutathione-S-transferase (GST) and cytochrome P450 (CYP450) genes are reg-
ulated by OsWRK45. Thus, ASM-induced resistance in rice would depend on
two complementary pathways – NPR1 and OsWRKY45 – with the latter being cru-
cial for blast resistance, induced not only by ASM but also by PBZ and tiadinil
(3) [55].
26.3.3
Tiadinil
Tiadinil (3) was first reported in 1995 by Nihon Nohyaku, and registered in Japan
in 2003. To date, it has been developed only in Japan and Korea for rice blast and
bacterial diseases. The primary method of application for tiadinil is by granule
spreading with insecticide (e.g., imidacloprid, spinosad) combinations in nursery
boxes, just before transplanting. A second from of granule formulation of tiadinil,
in combination with insecticides, was later introduced that could be applied to the
nursery box at sowing time. Finally, ready-made mixture granule formulations with
herbicides (e.g., bensulfuron-methyl, clomeprop), to be spread on the paddy water
surface in classical fashion, have been developed as a new labor-saving approach to
the use of tiadinil.
26.3.3.1 Synthesis of Tiadinil

The β-ketoester 17 can be treated with an N-acylated hydrazine to form the
hydrazone 18. Following its cyclization in thionyl chloride, the ethyl 4-methyl-
1,2,3-thiadiazole-5-carboxylate (19) was prepared, and then hydrolyzed to obtain
O
O CH3 N CH3
H2NNHCOR R N
OC2H5 H
OC2H5
O O
17 18
SOCl2
CH3 CH3
N Hydrolysis N
OH N OC2H5
N S
S
O O
20 (SV-03) 19
Halogenation
CH3 CH3
H2N Cl N H
N Et3N Cl
Cl N
N + THF
N
S CH3 S
O O CH3
21 22 3
Scheme 26.4 Synthesis of tiadinil (3) and its metabolite

4-methyl-1,2,3-thiadiazole-5-carboxylic acid (20, SV-03).
the 4-methyl-1,2,3-thiadiazole-5-carboxylic acid (20). Subsequent conversion to the

acid chloride 21, and its reaction with the 3-chloro-4-methyl-aniline (22), led to
tiadinil (3) (Scheme 26.4).
26.3.3.2 Systemic Acquired Resistance by Tiadinil

Tiadinil treatment in rice induces the expression of PR genes such as PBZ1, and
leads to callose deposition upon infection [57]. The mode of action of tiadinil
and its active metabolite 4-methyl-1,2,3-thiadiazole-5-carboxylic acid (20; SV-03)
has been mostly studied in tobacco and Arabidopsis. Both compounds induce
resistance to a broad spectrum of pathogens as well as the expression of SAR
marker genes [58]. Like ASM, tiadinil and its metabolite SV-03 are able to induce
resistance in nahG plants, but require a functional NPR1 [58]. By using JA- and
ethylene-insensitive mutants, it was possible to show that these compounds acted
independently of the JA and ethylene pathways. Therefore, tiadinil and its active
metabolite SV-03 induce plant defense responses via the SA signaling pathway, at
a site between SA and NPR1.
26.3.4
Isotianil
Isotianil (4), a newly developed plant defense inducer that controls leaf blast and
bacterial leaf blight in rice, was discovered by Bayer in 1997 [59], developed jointly
with Sumitomo [60, 61], and launched in 2010 in Japan and Korea.
26.3.4.1 Synthesis of Isotianil

The 3,4-dichloro-isothiazole-5-carboxylic acid (24) is the key intermediate in the
synthesis of isotianil (4) [62–64]. Starting from sodium cyanide, carbon disulfide,
and gaseous chlorine the 5-cyano-3,4-dichloro-isothiazole (23) is synthesized. The
carboxylic acid 24 can be produced by hydrolysis of 23 in 2 N sodium hydroxide
solution. Following preparation of the heterocyclic acid chloride 25 and its coupling
with 2-cyano-aniline (26), isotianil was obtained (Scheme 26.5).
26.3.4.2 Systemic Acquired Resistance by Isotianil

Isotianil, by itself, does not exhibit any activity against pathogens but rather
protects plants against infection when applied at an early developmental stage.
Hence, isotianil is readily accepted in Japan and Korea where prophylactic rice blast
control methods – for example, nursery box application, water surface application,
and seed treatment – are well established by the use of melanin biosynthesis
inhibitors and PBZ. Although the practical use of isotianil has been limited in
rice so far, it demonstrates a broader control spectrum as a host defense inducer
in various plants (Table 26.1). The disease control spectrum is basically similar to
those of PBZ, ASM, and tiadinil, which covers typical biotrophic pathogens such as
Pyricularia, powdery mildews, Xanthomonas, and some leaf spot diseases; however,
isotianil is generally negative for necrotrophic pathogens.
Isotianil is distinctive for its lower application dosages in systemic applications,
and applicability for seed treatment among commercial host defense inducers for
Cl Cl Cl Cl
2N NaOH
NaCN + CS2 + Cl2(g) N
N CN reflux COOH
S S
23 24
CN SOCl2
H2N
Cl Cl NC Cl Cl
H
N 26
N N
S S COCl
O
4 25
Scheme 26.5 Synthesis of isotianil (4).

Table 26.1 Disease control spectrum of isotianil (4).
Crop Disease Pathogen Efficacy
Rice Blast Pyricularia grisea +++

Sheath blight Rhizoctonia solani –
Bacterial leaf blight Xanthomonas. oryzae pv. oryzae +++
Leaf spot Cochliobolus miyabeanus –
Seedling rot Burkholdelia glumae +
Damping off Rhizopus chinensis –
Wheat Powdery mildew Blumeria graminis f.sp tritici +++
Yellow spot Pyrenophora tritici-repentis –
Potato Late blight Phytophthora infestans –
Cucumber Downy mildew Pseudoperonospora cubensis –
Anthracnose Colletotrichum orbiculare +++
Bacterial leaf spot Pseudomonas syringae pv. lachlymans +++
Pumpkin Powdery mildew Sphaerotheca fuliginea ++
Strawberry Anthracnose Glomerella cingulata ++
Powdery mildew Sphaerotheca aphanis +
Eggplant Powdery mildew Sphaerotheca fuliginea +
Tomato Powdery mildew Oidiopsis sicula +
Late blight Phytophthora infestans +
Soybean Purple stain Cercospora kikuchii –
Chinese cabbage Leaf spot Alternaria brassicae ++
Soft rot Erwinia carotovora subsp. carotovora –
Citrus Melanose Diaporthe citri –
Peach Bacterial shot hole Xanthomonas pruni ++
Banana Black Sigatoka Mycosphaerella fijiensis ++
Tea Shoot blight Pestalotia longiseta ++
Tobacco – TSWV (tomato spotted wilt virus) +
Efficacy by spray or drench application to pot-planted or field crops at 100–250 ppm are summarized.
+++: Equal or higher than 80% control.
++: Equal or higher than 70% control.
+: Equal or higher than 50% control (for virus disease, 20%).
–: Lower than 50% control.
Adapted from Ref. [60].
rice (Table 26.2). By water surface application, isotianil shows a greater than 90%
control efficacy at 5 g a.i. ha−1 under greenhouse conditions, and it has a stable
efficacy at 300 g a.i. ha−1 in the field (Figure 26.3).
26.3.4.3 Gene Expression Profiling Experiments

In attempting to identify the mode of action of isotianil, gene expression profil-
ing experiments were conducted using an Affymetrix array of the whole genome
sequence of rice under two conditions – with and without pathogen challenge.
Without pathogen pressure, three-week-old rice seedlings were drenched in an iso-
tianil solution (20 mg l−1 , at a dose range corresponding to agronomical relevance),
Table 26.2 Practical application rates of commercial host defense inducers in rice.
Type of application Application rates (g a.i. ha –1 )

Probenazole (1) Tiadinil (3) Isotianil (4)
Seed treatment Not applicable Not applicable 100a

Water surface application 2000–2400 1800 300
Nursery box application 1000–2400 1200 200–300
a
On sowing density at 300 nursery boxes or 50 kg seed ha.−1
and the gene expression profile was then studied at two time points (Figure 26.4).
Mock-treated plants were used as controls. A Student’s t-test analysis using twofold
threshold increase or decrease in gene expression (P < 0.001), identified five
isotianil-responsive genes at one day post-treatment, and 15 more genes after four
days of treatment.
A first analysis indicated that only very few genes were directly responsive
to isotianil itself. All of these genes were upregulated, and most were involved
in defense responses. The first group of interesting genes was shown to code
for transcription factors involved in the SA pathway. Thus, an upregulation of
a NPR1 homolog gene, called NPR3 (or NH3), was observed that was induced
sixfold. Likewise, OsWRKY45 – another key regulator of induced resistance – was
induced 2.9-fold (P < 0.0017). Other transcription factors implicated in the SA
pathway were also upregulated, namely OsWRKY62 and OsWRKY76, with fold
changes of 18 and 6.5, respectively. The second group of interesting genes was
involved in SA catabolism, such as OsSGT1 coding for an SA glucosyltransferase
(sevenfold induction) and OsBMST1 coding for a salicylate methyltransferase
(13.5-fold induction).
To investigate whether isotianil would affect gene expression upon infection,
both isotianil- and mock-treated three-week-old rice seedlings were challenged by
fungal infection with P. oryzae at four days after treatment. The gene expression
was then analyzed (Figure 26.4) at one and two days post-inoculation (dpi). Using
the same statistical test as described above to select differentially regulated genes,
the number of differentially expressed genes in infected mock-treated plants, that
were susceptible, was found to be very low at these time points (Figure 26.5).
In contrast, in infected isotianil-primed plants, that were resistant to fungal
blast disease, variation in gene expression was observed as early as 1 dpi and then
strongly increased at 2 dpi. As the greatest difference in gene expression between
susceptible and resistant rice was observed at 2 dpi, the analysis was first focused
on this time point. At 2 dpi, in isotianil-primed plants, with a cut-off set above 2, a
total of 481 genes was differentially expressed in response to infection, with most
being upregulated. Some 13% of these genes were also induced in noninfected
isotianil-primed plants at the same time point (data not shown). Hence, 87% of
the differentially expressed genes were due specifically to isotianil treatment upon
922
Isotianil : dose optimization for water surface application

(summary of 11 field trials in 2002-2004)
100
26 Host Defense Inducers
40
90 Abnormal value > 3x
35
30
80
25
20 Abnormal value >1.5
% Control
70
15 Maximum
Q3 : 75 percentile
10
Mean
60
5 Median
Q1 : 25 percentile
(g a.i. ha-1) 0 Minimum
50 1 Untreated
100 200 300 400 500 1000 2000 2400
Probenazole
Isotianil
Figure 26.3 Leaf blast control efficacy of isotianil (4) by water surface application.
Figure 26.4 Experimental procedure of gene At the same time points, samples from sim-
expression analysis. Three-week-old seedlings ple infection (e) as well as controls (d) and
were treated with isotianil (4) by drenching (f) were also investigated. All experiments
(step 1) (20 mg l−1 in 1% acetone, 0.1% were performed in triplicate. In parallel, dis-
Tween 20 solution). Samples (a, b) were ease resistance against fungal blast was
harvested for microarray analysis at 1 and evaluated 7 dpi, by measuring disease sever-
4 dpt. Mock-treated plants were used as ity (chlorotic regions) in comparison with
controls. After four days of treatment, rice mock-treated plants. Isotianil (4) showed an
seedlings were inoculated with Pyricularia efficiency of up to 85% in these experimental
grisea. Samples (c) were harvested at 1 and conditions.
2 dpi and subjected to microarray analysis.
600
Genes down regulated by atleast a factor 2
Genes up regulated by atleast a factor 2
Number of genes
400
200
0
Control Isotianil Control Isotianil
1dpi 2dpi
Figure 26.5 Numbers of differentially expressed genes dur-

ing the time course of fungal infection in isotianil-primed
and mock-treated three-week-old rice seedlings.
infection. These differentially expressed genes were implicated in diverse cellular

functions, and most were related to responses to stress and defense. Indeed,
typical markers of SAR have been identified. In total, a set of 17 PR genes was
upregulated, including PR1a, PBZ, and PR genes involved in antifungal activities
such as chitinase and β-glucanase (Table 26.3).
The expression of these PR genes was increased in comparison to nontreated
plants, with some genes displaying a stronger induction and others being induced
only upon priming. Defense-associated cellular processes were also studied, such as
the expression level of PAL-encoding genes, a key enzyme in the phenylpropanoid
pathway leading to defense-related secondary metabolites [65] (e.g., phytoalexins,
lignin-like polymers); and of chalcone synthase and chalcone isomerase, two genes
involved in the phytoalexins biosynthetic pathway. These three genes were shown to
be activated in isotianil-primed plants, but no activation was observed in nontreated
plants (Table 26.3).
In summary, these results together indicated that isotianil-treated plants were
potentiated for defense responses. As transcription factors such as those belonging
Table 26.3 Differential expression of genes in isotianil-primed and mock-treated plants.a
Locus ID Annotation Fold change in Fold change in

isotianil-primed mock-treated
plants plants
Defense response : pathogenesis-related proteins

LOC_Os07g03730.1 PR1a-like 5.4 2.6
LOC_Os01g51570.1 PR2/β-glucanase 3.1 1.4
LOC_Os01g47070.1 PR3/acidic chitinase 16.9 6.4
LOC_Os03g04060.1 PR3/basic chitinase 4.2 1.5
LOC_Os12g36880.1 PBZ/PR10a 19.9 4.0
Secondary metabolism: phenylpropanoids, flavonoids
LOC_Os02g41670.1 Phenylalanine ammonia-lyase 5.44 1.55
(PAL1)
LOC_Os11g32650.1 Chalcone synthase (CHS) 3.68 1.41
LOC_Os11g02440.2 Chalcone isomerase 5.78 1.53
Transcription factors
LOC_Os03g46440.1 NPR1 homolog (NPR3) 6.64 1.04
LOC_Os05g25770.1 OsWRKY45 (class III) 9.4 1.5
LOC_Os09g25070.1 OsWRKY62 (class II) 18.5 1.06
LOC_Os05g39720.1 OsWRKY70 (class I) 3.7 1.77
SA catabolism
LOC_Os09g34230.1 UDP-glucosyl transferase 65.66 1.4
family protein (OsSGT1)
LOC_Os09g34250.1 UDP-glucosyl transferase 12.4 0.84
family protein (OsSGT1)
a
The list includes part of genes upregulated by atleast a factor 2 (P < 0.001).
to WRKY classes and NRP1 are critical for the expression of defense-related genes,
their expression was also studied at 2 dpi. Similar to treatment with isotianil in
the absence of infection, the NPR1 homolog gene (NPR3) and OsWRK45 were
upregulated as well as OsWRK62 (Table 26.3). Additional WRKY genes were also
upregulated, including OsWRK70 ; these genes encoding transcription factors are
not activated in nontreated plants. Finally, OsSGT1 (Os09g34230), which is induced
in the absence of a pathogen, and its closed homolog Os09g34250, which has been
shown to play an important role in PBZ-induced resistance [43], were induced by
factors of 65 and 12, respectively.
These initial results show that isotianil, when applied at the dose range used
under agronomical conditions and without pathogen pressure, induces very few
changes in gene expression. Nevertheless, upon pathogen challenge, isotianil will
prime the rice plants for an increased defense genes activation compared to simple
infection. Thus, isotianil represents a novel type of defense inducer that functions
by priming the plants intrinsic disease-resistance mechanisms. Based on these
results, the mode of action of isotianil would involve the SA pathway. Indeed,
OsWRKY45, OsWRKY62, OsWRKY70, and OsWRKY76, all of which are activated
by SA and by incompatible interaction between P. oryzae and rice [66, 67], are also
strongly induced in isotianil-primed plants. Another putative key component of the
SA pathway in rice, the NPR1 homolog gene (NPR3), is also induced by isotianil.
The results of a recent study highlighted a possible direct involvement of NPR3
in defense [68]. In addition, isotianil-induced resistance would directly involve SA
catabolism by strongly activating OsSGT1. Such activation would, most likely, lead
to an increased SAG production that is believed to be more than a reservoir for
free SA in rice. The observed activation of OsBMST1 would lead to MeSA that
in tobacco, has been reported to be the systemic signal of SAR [69], and would
also be important for induced resistance. Isotianil-primed plants also display a
potentiated SAR response upon infection, including an enhanced activation of PR
genes and of genes involved in secondary metabolism, such as the phenylpropanoid
and flavonoid pathways. Finally, isotianil induces an upregulation of key players
involved in signal transduction that have been suggested to form part of the priming
phenomenon that also occurs in induced resistance.
26.4
Summary and Outlook
The excellent historic reputation of host defense inducers in Japan can be attributed
to:
• Established protective applications on schedule, supported by a labor-saving
background.
• A high risk of fungicide resistance, due to a lower genetic diversity of rice and
blast fungus.
• An utter dominance of blast-susceptible rice varieties, to ensure a stable efficacy
of defense inducers.
Consideration of these factors indicates a clear demand for host defense inducers
in other areas, especially if the cost of a curative spray application exceeds that of
a protective control, such as seed treatment. The situation is most acute in the rice
fields, where curative spraying without large-scale tractors during the irrigation
period is not only labor-intensive but the workers are also exposed to other dangers,
such as snakes. This situation may be replaced by a timely protective application of
host defense inducers, which would also reduce the high risk of resistance outbreak
by the curative use of single-site inhibitors. Although, by their very nature, host
defense inducers cannot provide perfect control (even at very high application
rates), they offer – in terms of their biodiversity – a more favorable approach to
resistance management than the complete eradication of sensitive pathogens by the
direct application of fungicides. Undoubtedly, host defense inducers – particularly
when applied at low rates, such as by seed treatment – can provide a sustainable
disease control with low environmental impact.
References
1. Verhagen, B.W.M., Glazebrook, J., 11. Park, S.-W., Kaimoyo, E., Kumar, D.,
Zhu, T., Chang, H.-S., van Loon, Mosher, S., and Klessig, D.F. (2007)
L.C., and Pieterse, C.M.J. (2004) Mol. Science, 318, 113–116.
Plant-Microbe. Interact., 17, 895–908. 12. Katsir, L., Chung, H.S., Koo, A.J.K., and
2. Takahashi, H., Ishihara, T., Hase, S., Howe, G.A. (2008) Curr. Opin. Plant
Chiba, A., Nakaho, K., Arie, T., Teraoka, Biol., 11, 428–435.
T., Iwata, M., Tugane, T., Shibata, D., 13. Jung, H.W., Tschaplinski, T.J., Wang,
and Takenaka, S. (2006) Phytopathology, L., Glazebrook, J., and Greenberg, J.T.
96, 908–916. (2009) Science, 324, 89–91.
3. Fungicide Resistance Action 14. Silverman, P., Seskar, M., Kanter, D.,
Committee (FRAC) (2010) Schweizer, P., Métraux, J.P., and Raskin,
http://www.frac.info/frac/publication/ I. (1995) Plant Physiol., 108, 633–639.
anhang/FRAC_Code_List_2010.pdf 15. Yang, Y., Qi, M., and Mei, C. (2004)
(accessed 13 December 2010). Plant J., 40, 909–919.
4. Ohshima, M., Itoh, H., Matsuoka, M., 16. Iwai, T., Seo, S., Mitsuhara, I., and
Murakami, T., and Ohashi, Y. (1990) Ohashi, Y. (2007) Plant Cell Physiol., 48,
Plant Cell, 2, 95–106. 915–924.
5. Uknes, S., Dincher, S., Friedrich, 17. Dong, X. (2004) Curr. Opin. Plant Biol.,
L., Negrotto, D., Williams, S., 7, 547–552.
Thompson-Taylor, H., Potter, S., Ward, 18. Grant, M. and Lamb, C. (2006) Curr.
E., and Ryals, J. (1993) Plant Cell, 5, Opin. Plant Biol., 9, 414–420.
159–169. 19. Mou, Z., Fan, W., and Dong, X. (2003)
6. Pieterse, C.M.J., Leon-Reyes, A., van der Cell, 113, 935–944.
Ent, S., and van Wees, S.C.M. (2009) 20. Cao, H., Glazebrook, J., Clarke, J.D.,
Nat. Chem. Biol., 5, 308–316. Volko, S., and Dong, X. (1997) Cell, 88,
7. Hammerschmidt, R. (2009) Adv. Bot. 57–63.
Res., 51, 173–222. 21. Ryals, J., Weymann, K., Lawton, K.,
8. Loon, L.C., Rep, M., and Pieterse, C.M.J. Friedrich, L., Ellis, D., Steiner, H.Y.,
(2006) Annu. Rev. Phytopathol., 44, Johnson, J., Delaney, T.P., Jesse, T., Vos,
135–162. P., and Uknes, S. (1997) Plant Cell, 9,
9. Garcion, C. and Métraux, J.P. (2006) 425–439.
Annu. Plant Rev., 24, 229–255. 22. Blanco, F., Salinas, P., Cecchini, N.M.,
10. Parker, L.E. (2009) Sci. Signal., 2, 1–3. Jordana, X., Hummelen, P.V., Alvarez,
References 927
M.E., and Holuigue, L. (2009) Plant Mol. and Conrath, U. (2009) Plant Cell, 21,
Biol., 70, 79–102. 944–953.
23. Chern, M.S., Fitzgerald, H.A., Yadav, 37. Seki, S., Sekizawa, Y., Nishibata, K.,
R.C., Canlas, P.E., Dong, X., and Watanabe, T., Kikuchi, T., and Igarashi,
Ronald, P.C. (2001) Plant J., 27, H. (1971) Meiji Seika Kaisha Ltd, US
101–113. Patent 3,629,428.
24. Chern, M., Fitzgerald, H.A., Canlas, 38. Iwai, T., Seo, S., Mitsuhara, I., and
P.E., Navarre, D.A., and Ronald, P.C. Ohashi, Y. (2007) Plant Cell Physiol., 48,
(2005) Mol. Plant-Microbe. Interact., 18, 915–924.
511–520. 39. Nakashita, H., Yoshioka, K., Takayama,
25. Quilis, J., Penas, G., Messeguer, J., M., Kuga, R., Midoh, N., Usami, R.,
Brugidou, C., and Segundo, B.S. Horikoshi, K., Yoneyama, K., and
(2008) Mol. Plant-Microbe. Interact., Yamaguchi, I. (2001) Biosci. Biotechnol.
21, 1215–1231. Biochem., 65, 205–208.
26. Cao, H., Li, X., and Dong, X. (1998) 40. Iwata, M. (2001) Pestic. Outlook, 12,
Proc. Natl Acad. Sci. USA, 95, 28–31.
6531–6536. 41. Yoshioka, K., Nakashita, H., Klessig,
27. Lin, W.C., Lu, C.-F., Wu, J.-W., Cheng, D.F., and Yamaguchi, I. (2001) Plant J.,
M.-L., Lin, Y.-M., Yang, N.-S., Black, 25, 149–157.
L., Green, S.K., Wang, J.-F., and 42. Nakashita, H., Yoshioka, K., Yasuda,
Cheng, C.-P. (2004) Transgenic Res., M., Nitta, T., Arai, Y., Yoshida, S., and
Yamaguchi, I. (2002) Physiol. Mol. Plant
13, 567–581.
Pathol., 61, 197–203.
28. Makandar, R., Essig, J.S., Schapaugh,
43. Umemura, K., Satou, J., Iwata, M.,
M.A., Trick, H.N., and Shah, J. (2006)
Uozumi, N., Koga, J., Kawano, T.,
Mol. Plant-Microbe. Interact., 19,
Koshiba, T., Anzai, H., and Mitomi, M.
123–129.
(2009) Plant J., 57, 463–472.
29. Shimono, M., Sugano, S., Nakayama,
44. Schurter, R., Kunz, W., and Nyfeler, R.
A., Jiang, C.-J., Ono, K., Toki, S., and
(1989) Ciba-Geigy A.-G., EP 0,313,512.
Takatsuji, H. (2007) Plant Cell, 19,
45. Kunz, W., Schurter, R., and Maetzke, T.
2064–2076.
(1997) Pestic. Sci., 50, 275–282.
30. Takatsuji, H., Jiang, C.-J., and
46. Kunz, W. and Jau, B. (1997) Novartis
Sugano, S. (2010) Jpn. Agric. Res. Q.,
A.-G., EP 780,372.
44, 217–2230. 47. Tripathi, D., Jiang, Y.-L., and Kumar, D.
31. Qiu, Y. and Yu, D. (2009) Environ. Exp. (2010) FEBS Lett., 584, 3458–3463.
Bot., 65, 35–47. 48. Friedrich, L., Lawton, K., Ruess, W.,
32. Conrath, U., Beckers, G.J.M., Flors, V., Masner, P., Specker, N., Rella, M.G.,
Garcı́a-Agustı́n, P., Jakab, G., Mauch, Meier, B., Dincher, S., Staub, T., Uknes,
F., Newman, M.-A., Pieterse, C.M.J., S., Métraux, J.-P., Kessmann, H., and
Poinssot, B., Pozo, M.J., Pugin, A., Ryals, J. (1996) Plant J., 10, 61–70.
Schaffrath, U., Ton, J., Wendehenne, 49. Kohler, A., Schwindling, S., and
D., Zimmerli, L., and Mauch-Mani, B. Conrath, U. (2002) Plant Physiol., 128,
(2006) Mol. Plant-Microbe. Interact., 19, 1046–1056.
1062–1071. 50. Song, D., Li, G., Song, F., and Zheng,
33. Conrath, U., Pieterse, C.M.J., and Z. (2008) Mol. Biol. Rep., 35, 275–283.
Mauch-Mani, B. (2002) Trends Plant 51. Zhang, X., Li, D., Zhang, H., Wang,
Sci., 7, 210–216. X., Zheng, Z., and Song, F. (2010) Mol.
34. Kohler, A., Schwindling, S., and Biol. Rep., 37, 653–660.
Conrath, U. (2002) Plant Physiol., 128, 52. Song, D., Li, G., Song, F., and Zheng,
1046–1056. Z. (2008) Mol. Biol. Rep., 35, 275–283.
35. Goellner, K. and Conrath, U. (2008) 53. Lawton, K.A., Friedrich, L., Hunt, M.,
Eur. J. Plant Pathol., 121, 233–242. Weymann, K., Delaney, T., Kessmann,
36. Beckers, G.J.M., Jaskiewicz, M., Liu, Y., H., Staub, T., and Ryals, J. (2006) Plant
Underwood, W.R., He, S.Y., Zhang, S., J., 10, 71–82.
54. Wang, D., Amornsiripanitch, N., and 61. Ogawa, M., Sakuma, H., Sawada, H.,
Dong, X. (2006) PLoS Pathog., 2, e123. and Ishikawa, R. (2008) Jpn. J. Phy-
55. Takatsuji, H., Jiang, C.-J., and Sugano, topathol., 74, 267.
S. (2010) Jpn. Agric. Res. Q., 44, 62. Mailey, E.A. (1967) Pennsalt Chemicals
217–223. Corp. US Patent 3,341,547.
56. Sugano, S., Jiang, C.-J., Miyazawa, S.-I., 63. Mailey, E.A. (1968) Pennsalt Chemicals
Masumoto, C., Yazawa, K., Hayashi, N., Corp. US Patent 3,393,992, 1968.
Shimono, M., Nakayama, A., Miyao, M., 64. Ishikawa, K., Tanigawa, H., Kawashima,
and Takatsuji, H. (2010) Plant Mol. Biol., H., Kanemoto, Y., Shimotori, H.,
74, 549–562. Yanase, J., Sekino, T., Tomura, N.,
and Kuwazuka, T. (1993) Mitsui Toatsu
57. Hirooka, T. and Umetani, K. (2004)
Chemicals, JP 05, 059,024.
Agrochem. Jpn, 85, 12–15.
65. Dixon, R.A., Achnine, L., Kota, P., Liu,
58. Yasuda, M. (2007) J. Pestic. Sci., 32,
C.-J., Reddy, M.S.S., and Wang, L.
281–282.
(2002) Mol. Plant Pathol., 3, 371–390.
59. Assmann, L., Kuhnt, D., Elbe, H.-L.,
66. Song, Y., Ai, C.-R., Jing, S.-J., and Yu,
Erdelen, Ch., Dutzmann, S., Hänsler,
D.-Q. (2010) Rice Sci., 17, 60–72.
G., Stenzel, K., Mauler-Machnik, A., 67. Ryu, H.-S., Han, M., Lee, S.-K., Cho,
Kitagawa, Y., Moka, T., Sawada, H., J.-I., Ryoo, N., Heu, S., Lee, Y.-H., Bhoo,
Yuki, I., Sakuma, H., Haruhiko, O., and S.H., Wang, G.-L., Hahn, T.-R., and
Oyama, T. (1999) Bayer A.G., DE 19, Jeon, J.-S. (2006) Plant Cell Rep., 25,
750,012. 836–847.
60. Sakuma, H., Araki, Y., Tanaka, K., 68. Bai, W., Chern, M., Ruan, D., Canlas,
Kinbara, T., Imanishi, K., Shigyo, T., P.E., Sze-to, W.H., and Ronald, P.C.
Kuchii, Y., Ogawa, M., Ishikawa, R., and (2010) Plant Biotechnol. J., 9, 1–11.
Sawada, H. (2008) Jpn. J. Phytopathol., 69. Shah, J. (2009) Curr. Opin. Plant Biol.,
74, 267. 12, 459–464.
929
Part III
Insecticides

931
Overview
Peter Jeschke
The use of insecticides belongs to the oldest plant protection measurements,

starting with the application of natural insecticides such as nicotine, rotenone,
or pyrethrum. With the discovery of organophosphorus insecticides on the one
hand, and the insecticidal chlorohydrocarbons on the other hand, the era of the
industrially produced insecticides began during the 1940s and 1950s. The most
important innovation in the field of insecticides was the discovery and introduction
of the synthetic pyrethroids during the 1970s and 1980s. Since that time, however,
the research and development of new insecticides has changed dramatically. Today,
aspects of high selectivity, minimal of risks of acute and long-term toxicity, reduced
chemical stability (persistence in the environment), and the protection of nontarget
organisms have become the center of attention for the agrochemical industry,
the regulatory authorities, the farmers, food companies, and the general public.
Today, the use of beneficial insects, combined with insecticides or acaricides or
new formulation concepts and versatile application methods (such as using soil
and seed treatments against insects such as aphids as important vectors of plant
virus diseases) has intensified with the development of compounds with greater
target site-selectivity. This, in turn, has led to the development of effective active
ingredients with novel modes of action (MoAs) but narrower insecticidal spectra,
perhaps against only sucking or certain chewing pests. Each of these aspects is
reflected in the contributions to this volume, on Insecticides.
Initially, in Chapter 27, a broad overview is provided of the different insecticide
classes, based on the Insecticide Resistance and Mode of Action Classification of
Insecticides of the Insecticide Resistance Action Committee (IRAC). The classi-
fication scheme (v. 7.0, August 2010) presented in this chapter enumerates the
main groups of insecticides and also specifies the active ingredients within those
groups, based on available evidence of their MoAs. A survey is also provided of
the ‘‘older’’ insecticides, as described previously in the Chemistry of Pesticides (John
Wiley and Sons). The objectives of the IRAC, the mission, and the structure and or-
ganization of its activities, clearly indicate the cooperation of regulatory authorities,
the agrochemical industry, university research groups and growers in preventing
the development of resistance against insecticides. This involves the practice of

932 Overview
alternations, sequences, rotations, or applying mixtures of compounds from the

different MoA groups as a sustainable and effective approach towards Integrated
Resistance Management (IRM).
In contrast to Volumes I and II, on Herbicides and Fungicides, respectively, the
sequence of contributions in this Volume begins with the physiology of insects,
describing insecticides that act on insect molting and metamorphosis (Chapter 28)
and chitin biosynthesis (Chapter 29), in relation to highly specific insecticidal
targets that are not present in mammals or other non-arthropod organisms.
Bacillus thuringiensis (Bt) toxins, which act on the midgut of insects, form
the basis of the transgenic crops protected by Bt genes against damage caused
by chewing insects. With the introduction of transgenic crops such as cotton,
maize, and potato that express Bt toxins active against lepidopteran and specific
coleopteran pest species, the insecticide market has changed dramatically, and will
continue to change. During the past few years, significant progress has been made
in understanding the special role of Cry proteins for the MoA, and identifying its
receptor in different insect species. In Chapter 30, an update is provided of the
current state of these insecticides, together with details of modern methods used
to combat insects. Formulations of Bt spore-crystal mixtures as natural insecticides
have now been used for over 45 years, and have shown Bt to be a highly specific,
effective, and safe bioinsecticide based on its crystal proteins that act selectively on
the midgut of insect pests.
Since the 1970s, a major topic of new insecticide discovery has related to inhibitors
of metabolic processes. The main challenge having been to identify compounds and
compound classes that are highly selective towards their insect targets. In Chapter
31, the details are provided of uncouplers of oxidative phosphorylation, inhibitors
of mitochondrial electron transport and, especially, inhibitors of lipid synthesis. To-
gether, these illustrate very well the major efforts and successes of the agrochemical
industry in this area during the past 25 years. The development of inhibitors that act
on metabolic pathways has been achieved using the ‘‘prodrug’’ concept, whereby
acute toxic effects towards non-target organisms (including mammals) following
in vivo uptake can be minimized, based on the differing metabolism of the parent
compound and its derivative(s). Hence, specific ‘‘prodrugs’’ have been designed
with modified physico-chemical properties that result in improved stability, solu-
bility, lipophilicity, and distribution that can be metabolized by insects, but not by
mammals. The production of an active ingredient in this way, via insect-mediated
metabolism, allows the differentiation between insect and nontarget organisms.
Such differentiation can also be achieved if a metabolic process is biochemically
diverse, as this may lead to strong interactions between the receptors of nontarget
organisms or insects and the corresponding inhibitors.
Insecticides that act on the central nervous system are very effective in preventing
crop damage within a short time, either by killing the insects or stopping them
from sucking or chewing (sometimes within seconds). This rapid efficacy forms the
basis of the farmers’ preference for such insecticides. Whereas, during the 1980s,
organophosphorus insecticides and, subsequently, the pyrethroids dominated the
insecticide market, the introduction during the 1990s of neonicotinoids with their
Overview 933
systemic properties (mainly against sucking insects) and of spinosyns (mainly

against chewing insects) led to a dramatic change in the market that will clearly
continue (see Chapters 32.1, 32.2, and 32.4). This situation is also described in
Chapter 32.3, for a new member of the neonicotinoid class that is effective on
a wide range of sap-feeding insects. Much progress has also been made with
insecticides that function by controlling the activity of sodium channels, notably via
a compound that achieves selectivity through the prodrug concept (Chapter 32.5.1),
by a new insecticide of the semicarbazone class (Chapter 32.5.2), and by high
selectivity towards insect chloride channels (Chapter 32.7). The fiproniloids, which
were developed as a compound class in 1985 as a spin-off of herbicidal research,
represent insecticide structures that act as antagonists on gamma-aminobutyric
acid (GABA)-gated chloride channels. Unfortunately, however, these were so highly
competitive (due to their systemic properties) that they could not be used as seed
treatment compounds (Chapter 32.6).
Selective homopteran feeding blockers, synthetic inhibitors, and neuroactive
miticides or nematicides of so far unknown or uncertain MoAs are described in
Chapters 33.1–33.4.
The details of highly specific ryanodine receptor (RyR) modulators, which
demonstrate similar symptoms to those seen in insects treated with the toxic plant
alkaloid ryanodine (see Chapters 34), have led to the discovery of new class of
insecticides. An understanding of the MoA of these compounds, together with
details of their specific binding sites and diversity of the functional regulation of
the RyR, has led to a new era of insecticides. Based on their broad-spectrum activity
against insect pests, their remarkable target specificity, and their low toxicity, this
innovative class of insecticides should be well suited to both IRM and Integrated
Pest Management (IPM) programs. They are sure to have a major influence on the
crop-protection market of the future.
935
27
IRAC: Insecticide Resistance, and Mode of Action
Classification of Insecticides
27.1
Introduction
The effective management of pest insect populations in most of the world’s

agriculture and horticulture is dependent on a variety of inputs, including a ready
supply of safe, highly efficacious chemical insecticides. Likewise, the effective
control of insect vectors of diseases is also highly dependent on the availability of
insecticidal products. With their abundant numbers and short life-cycles, popula-
tions of pest insects can readily develop resistance to the insecticides used against
them, with the result that once-effective insecticides are no longer able to control
the pests for which they were intended [1]. Accordingly, resistance may be usefully
defined as ‘‘. . . a heritable change in the sensitivity of a pest population that is
reflected in the repeated failure of a product to achieve the expected level of control
when used according to the label recommendation for that pest species.’’ Although
this definition differs slightly from others described elsewhere, it is believed to rep-
resent the most accurate, practical definition of relevance to farmers and growers.
The agrochemical industry views resistance as an extremely serious threat and an
issue that needs a proactive approach. Effective insecticide resistance management
(IRM) is essential, and the Insecticide Resistance Action Committee (IRAC) has
been dedicated to making this a reality for the past two decades [2–4].
27.2
Objectives of the IRAC
The IRAC was formed in 1984 to provide a coordinated crop protection industry
response to either prevent or delay the development of resistance in insect and mite
pests [3]. The mission of the IRAC is to facilitate communication and education on
insecticide resistance, and to promote the development of resistance management
strategies in crop protection and vector control, so as to maintain efficacy and
support sustainable agriculture and improved public health.

936 27 IRAC: Insecticide Resistance, and Mode of Action Classification of Insecticides
The IRAC is an inter-company organization acting as a Specialist Technical

Group of croplife. IRAC International not only supports resistance management
project teams but also provides a central coordination role to regional, country,
and technical groups around the world. IRAC International’s main focus is on
education, communication, and the regulation of IRM.
27.3
Structure and Organization of the IRAC
The organization is implementing comprehensive strategies to confront resistance

through a range of activities. Along with the other Resistance Action Commit-
tee (RACs) – for example, the Fungicide Resistance Action Committee (FRAC) –
the IRAC operates under the umbrella of Croplife International and, as such,
is recognized by the Food and Agriculture Organization (FAO) and the World
Health Organization (WHO) of the United Nations (UN) as an advisory body.
The group’s activities are coordinated via the IRAC Executive Committee, IRAC
International, and Country or Regional Committees, with the information dissem-
inated through meetings, workshops, educational materials, and the IRAC web site
(www.irac-online.org). Groups are comprised primarily of key technical personnel
from the agrochemical companies affiliated with croplife through membership in
the relevant National Associations (ECPA, croplife America, etc.). Current member
companies are: BASF, Bayer CropScience, Belchim Crop Protection, Cheminova,
Chemtura, Dow Agrosciences, DuPont, FMC, Makhteshim Agan Industries, Mon-
santo, Nihon Nohyaku, Nufarm, Sumitomo Chemical, Syngenta, and Vestergaard
Frandsen.
27.3.1
Project and Functional Teams
Project Teams are set up with specific resistance management objectives and time
lines dealing with a local, national, or international insect resistance issues. Once
the Project Team (Working Group, WG) has completed its objectives, the work
and outcome of the project is reported and the team then disbanded. WGs are set
up and disbanded on an as-needed basis; for example, Codling Moth, Lepidoptera,
Oilseed Rape Pests, or Diamide WG (currently 13 different IRAC WGs are active;
www.irac-online.org).
There are also several functional teams that currently exist within the IRAC
International Group; these include The Communication and Education Team, the
Public Health Team, and the Biotech Team.
27.3.2
Country Groups
IRAC International encourages the formation of Country Groups to interact with

local experts and research institutions, and to form Project Teams to address local
27.4 Activities 937
resistance issues. Currently, Country Groups exist in the USA, Brazil, South Africa,
Spain, India, Southeast Asia, and Australia, but clearly there are still many resistance
problems that need tackling by new Country or Regional Groups coming together.
In some European countries, so-called Resistance Action Group (RAGs) have been
formed, such as IRAG UK or NORBARAG (Nordics and Baltics Resistance Action
Group), and working closely together with IRAC. Other countries, such as Germany
(including participants from Austria), Benelux, Poland, and Italy, have very recently
established similar groups.
27.4
Activities
The IRAC groups are actively involved, and on certain occasions provide funding for
a variety of resistance management projects around the world. These are generally
driven or coordinated by the local Country Group, and in some cases a Specific
Project Group is set up to lead and ultimately report results and findings into the
public domain. Examples of these have been the long-term monitoring of mosquito
resistance in Mexico, and the monitoring of pyrethroid resistance of Helicoverpa
armigera in West African cotton. Recently, a new Project Group was set up within
IRAC International to investigate codling moth resistance in a number of countries
worldwide. Also, a Neonicotinoid Project Group has recently been established in
order to define and implement guidelines for the use of this valuable chemical
class of insecticides (this group later merged with the IRAC Sucking Pest Team).
Other activities focus on issues relating to education, communication, and
regulatory approvals, as well as providing expert technical support. These more
general activities are wide-ranging but can be grouped under the following headings.
27.4.1
Resistance-Monitoring Methods
Reliable data on resistance rather than anecdotal reports or assumptions, is

the cornerstone of successful resistance management, and key to this is the
availability of sound baseline data on the susceptibility of the target pest to the
insecticide/acaricide. Baseline data can be defined as . . .data obtained from a strain
(or several strains) with no history of selection with the toxicant or related toxicants
showing cross-resistance. Currently, a wide range of bioassay and biochemical
tests are employed to characterize the susceptibility of target pests to insecticides
and acaricides. Unfortunately, the results from varied test methods may not
be comparable as they may measure different parameters, and this can lead to
difficulties over the interpretation of monitoring data. The IRAC, in fulfilling its
aim of providing expert advice to Croplife International on all technical matters
relating to insecticide and acaricide resistance, has addressed this issue with the
aim of recommending a range of bioassay techniques to monitor insecticide and
acaricide susceptibility for selected pest species of economic importance. The
IRAC has evaluated and validated a wide range of testing methods which have been
published and are also freely available on the IRAC web site.
27.4.2
IRAC and the Regulatory Requirements of Resistance Management
IRAC – along with the Herbicide Resistance Action Committee (HRAC) and
FRAC – have taken a leading role as an expert group providing industry responses
to proposals from regulatory bodies. For example, there is now a regulatory re-
quirement in the European Union (EU) under Directive 91/414/EEC for companies
to provide an assessment of the potential risk of resistance being developed by
target organisms, and for management strategies to be introduced to address such
risks [2, 5]. The RACs have been instrumental in developing workable guidelines
for companies, and this has resulted in the publication of an official Guidance
Document. Similarly, the US Environmental Protection Agency and the Pest Man-
agement Regulatory Agency of Canada have been developing a voluntary pesticide
resistance management labeling scheme based on mode or target site of action on
the pest. The RACs have been heavily involved in classifying pesticides into specific
groups and families to enable the scheme to work. Development has been carried
out under the auspices of the North American Free Trade Association, and has
resulted in the issue of a Pesticide Registration (PR) Notice in the US.
In addition, some of the IRAC’s other resources are being used by Regula-
tory Authorities such as the Mode of action (MoA) Classification Scheme, the
IRAC Monitoring Methods, and the Michigan State University (MSU) Arthropod
Pesticide Resistance Database (APRD) (see below).
27.4.3
Education and Communication
For the IRAC, education and communication play a key role in the global man-
agement of resistance, and many steps have been taken over the years to provide
resources to academia, research groups, industry, and growers.
The IRAC education material has been put together to provide a basic un-
derstanding of insecticide resistance, and to explain how resistance can be best
managed. In addition to IRAC’s centrally developed resources, most of the IRAC
Country Groups have their own educational programs in place, tailored to meet
local needs. The IRAC US, for example, publishes articles on a regular basis in
grower magazines, while IRAC Brazil holds training workshops in different loca-
tions. Other IRAC Groups such as Australia, South Africa, Southeast Asia, Spain,
and India have similar initiatives ongoing.
Workshops, seminars, conferences, and exhibitions serving as important plat-
forms for communication and education are often organized, attended, or spon-
sored by the IRAC. As an output from these meetings, a large number of reports
have been published by members on behalf of the IRAC. A full bibliographic listing
27.4 Activities 939
of more than 100 key papers provides an interesting overview on recent findings in
the different areas of IRM/acaricide resistance management (www.irac-online.org).
The biannual Resistant Pest Management Newsletter of the Centre for Integrated
Plant Systems (CIPS), in cooperation with the IRAC and the Western Regional Co-
ordinating Committee (WRCC-60), updates the most recent findings on insect/mite
resistance.
The existing IRAC web site (http://www.irac-online.org) has now been on-line
since 2001, and has become the main home for IRAC information. Data available
includes the monitoring methods, the MoA classification scheme, Project and
Country Group updates, meeting minutes, member contact details, useful links,
details of published articles, and copies of new posters recently produced. A
special section for growers and other relevant associations has been established
to facilitate practical advice for resistance management. The econnection (IRAC’s
quarterly published newsletter) has been developed in conjunction with the new
IRAC web site to update users about new information appearing on the web site
and advise of ongoing IRAC activities supporting IRM.
27.4.4
Resistance Database Managed by Michigan State University and Supported by IRAC
For many years, the IRAC maintained the Resistance Survey which was built
up from information received from sources around the world. In recent years,
however, this has become out of date and the decision was taken in collaboration
with croplife to sponsor MSU to extend their APRD to include and extend the
information that was contained in the original survey. The database includes reports
of resistance cases from 1914 to the present day. The introductory text explains that
pesticide resistance is a dynamic, evolutionary phenomenon, and a record in the
database may or may not be indicative of a specific area. Similarly, the absence of
a record in this database does not indicate the absence of resistance. This project
has been – and continues to be – a major effort for the IRAC, and has become a
valuable resource for the management of insecticide/acaricide resistance.
27.4.5
The Mode of Action Classification Scheme
The IRAC MoA Classification provides farmers, growers, advisors, extension staff,
consultants, and crop-protection professionals with a guide to the selection of
insecticides or acaricides for use in a rotation-based approach that can provide
effective and sustainable IRM or acaricide resistance management strategy. In
addition to presenting the MoA classification for a wide range of insecticides
and acaricides, this document outlines the background to – and the purposes
of – the classification list, and also provides guidance on how it is used for IRM
purposes.
27.5
Principles of Resistance
As mentioned earlier, resistance to insecticides may be defined as:
A heritable change in the sensitivity of a pest population that is reflected in

the repeated failure of a product to achieve the expected level of control when
used according to the label recommendation for that pest species (IRAC).
Although this definition differs slightly from others in the literature, the IRAC
believes that it represents the most accurate, practical definition of relevance to
farmers and growers. Resistance most commonly arises through the over-use or
mis-use of an insecticide or acaricide against a pest species, and results in the
selection of resistant forms of the pest and the consequent evolution of populations
that are resistant to that insecticide or acaricide.
27.5.1
Mode of Action, Target-Site Resistance, and Cross-Resistance
In the majority of cases, not only does resistance render the selecting compound
ineffective but it often also confers cross-resistance to other chemically related
compounds. This is because compounds within a specific chemical group usually
share a common target site within the pest, and thus share a common MoA. A com-
mon mechanism of resistance involves a genetic modification to the insecticide’s
target site [6]. When this occurs, the interaction of the selecting compound with its
target site is impaired and the compound loses its pesticidal efficacy. Because all
compounds within the chemical subgroup share a common MoA, there is a high
risk that the resistance that has developed will automatically confer cross-resistance
to all compounds in the same subgroup. It is this concept of cross-resistance within
chemically related insecticides or acaricides that is the basis of the IRAC MoA
Classification.
27.5.2
Non-Target-Site Resistance Mechanisms
It is fully recognized that the resistance of insects and mites to insecticides

and acaricides can – and frequently does – result from enhanced metabolism by
enzymes overexpressed due to insecticide selection pressure [6]. Such metabolic
resistance mechanisms are not linked to any specific site of action classification,
and therefore they may confer cross-resistance to insecticides in more than one
IRAC MoA group. Where such metabolic resistance has been characterized and
the cross-resistance spectrum is known, it is possible that certain alternations,
sequences, or rotations of MoA groups cannot be used. Similarly, mechanisms of
reduced penetration of the pesticide into the pest, or behavioral changes of the pest,
may also confer resistance to multiple MoA groups [6, 7]. Where such mechanisms
27.6 The Mode of Action (MoA) Classification Scheme v7.0, August 2010 941
are known to give cross-resistance between MoA groups, the use of insecticides
should be modified appropriately.
Where the resistance mechanism(s) is unknown, the intelligent use of alterna-
tions, sequences, or rotations of compounds from different MoA classes remains
an entirely viable resistance management technique, since such a practice will
minimize selection pressures [8].
27.6
The Mode of Action (MoA) Classification Scheme v7.0, August 2010
The following classification scheme, developed and endorsed by the IRAC, is based
on the best available evidence of the MoA of available insecticides (Table 27.1).
Details of the listing have been agreed by IRAC companies and approved
by internationally recognized industrial and academic insect toxicologists and
biochemists.
27.6.1
Rules for Inclusion of a Compound in the MoA List
• Chemical nomenclature is based on that appearing in The Pesticide Manual,

15th edition, November 2009 (ed. C.D.S. Tomlin), published by The British Crop
Protection Council. ISBN 9781901396188.
• To be included in the active list, compounds must have – or be very close to
having – a minimum of one registered use in at least one country.
• In any one MoA classification subgroup, where more than one active ingredient
in that chemical subgroup is registered for use, the chemical subgroup name is
used.
• In any one MoA classification subgroup, where only one active ingredient is
registered for use, the name of that exemplifying active ingredient is used.
• Where more than one chemical subgroup or exemplifying active ingredient
appears in a single MoA group, each is named according to the above rules;
chemical subgroups have precedence over single active ingredients.
It is the aim of the IRAC to ensure that insecticide and acaricide users are
aware of MoA groups, and that they have a sound basis on which to implement
season-long, sustainable resistance management through the effective use of
alternations, sequences, rotations, or even mixtures of insecticides with different
MoA. In order to help delay resistance development, it is strongly recommended
that growers also integrate other control methods (e.g., biological, cultural, etc.)
into insect or mite control programs (further advice is provided in Table 27.1).
The organophosphates and carbamates, as inhibitors of acetylcholinesterase
(IRAC MoA Groups 1A and 1B), still form the largest group of insecticides,
followed by the pyrethroids (IRAC MoA Group 3A), which are known to act on
voltage-gated sodium channels (these classes are briefly described below in terms
Table 27.1 IRAC mode of action classification v. 7.0, August 2010a.
Main group and primary site Chemical subgroup Active ingredients

of action or exemplifying
active ingredient
1b 1A
Acetylcholinesterase (AChE) Carbamates Alanycarb, aldicarb, bendiocarb,
inhibitors benfuracarb, butocarboxim,
Nerve action (strong butoxycarboxim, carbaryl, carbofuran,
evidence that action at this carbosulfan, ethiofencarb, fenobucarb,
protein is responsible for formetanate, furathiocarb, isoprocarb,
insecticidal effects) methiocarb, methomyl, metolcarb, oxamyl,
pirimicarb, propoxur, thiodicarb,
thiofanox, triazamate, trimethacarb, XMC,
xylylcarb
1B
Organophosphates Acephate, azamethiphos, azinphos-ethyl,
azinphos-methyl, cadusafos,
chlorethoxyfos, chlorfenvinphos,
chlormephos, chlorpyrifos,
chlorpyrifos-methyl, coumaphos,
cyanophos, demeton-S-methyl, diazinon,
dichlorvos/DDVP, dicrotophos,
dimethoate, dimethylvinphos, disulfoton,
EPN, ethion, ethoprophos, famphur,
fenamiphos, fenitrothion, fenthion,
fosthiazate, heptenophos, imicyafos,
isofenphos, isopropyl
O-(methoxyaminothio-phosphoryl)
salicylate, isoxathion, malathion,
mecarbam, methamidophos,
methidathion, mevinphos,
monocrotophos, naled, omethoate,
oxydemeton-methyl, parathion,
parathion-methyl, phenthoate, phorate,
phosalone, phosmet, phosphamidon,
phoxim, pirimiphos-methyl, profenofos,
propetamphos, prothiofos, pyraclofos,
pyridaphenthion, quinalphos, sulfotep,
tebupirimfos, temephos, terbufos,
tetrachlorvinphos, thiometon, triazophos,
trichlorfon, vamidothion
2 2A
GABA-gated chloride Cyclodiene Chlordane, endosulfan
channel antagonists organochlorines
Nerve action (strong
evidence that action at this
protein is responsible for
insecticidal effects)

active ingredient
2B
Phenylpyrazoles Ethiprole, fipronil
(fiproles)
3b 3A
Sodium channel modulators Pyrethroids Acrinathrin, allethrin, d-cis-trans allethrin,
Nerve action (strong Pyrethrins d-trans allethrin, bifenthrin, bioallethrin,
evidence that action at this bioallethrin S-cyclopentenyl isomer ,
protein is responsible for bioresmethrin, cycloprothrin, cyfluthrin,
insecticidal effects) beta-Cyfluthrin, cyhalothrin,
lambda-cyhalothrin, gamma-cyhalothrin,
cypermethrin, alpha-cypermethrin,
beta-cypermethrin, theta-cypermethrin,
zeta-cypermethrin, cyphenothrin
[(1R)-trans-isomers], deltamethrin,
empenthrin [(EZ)- (1R)-isomers],
esfenvalerate, etofenprox, fenpropathrin,
fenvalerate, flucythrinate, flumethrin,
tau-Fluvalinate, halfenprox, imiprothrin,
kadethrin, permethrin, phenothrin
[(1R)-trans-isomer], prallethrin, pyrethrins
(pyrethrum), resmethrin, silafluofen,
tefluthrin, tetramethrin, tetramethrin
[(1R)-isomers], tralomethrin, and
transfluthrin
3B
DDT methoxychlor DDT methoxychlor
4 4A
Nicotinic acetylcholine Neonicotinoids Acetamiprid, clothianidin, dinotefuran,
receptor (nAChR) agonists imidacloprid, nitenpyram, thiacloprid, and
Nerve action (strong thiamethoxam
evidence that action at one 4B Nicotine
or more of this class of Nicotine
5
Nicotinic acetylcholine Spinosyns Spinetoram spinosad
receptor (nAChR) allosteric – –
activators
Nerve action (strong
evidence that action at one
or more of this class of

active ingredient
6
Chloride channel activators Avermectins, Abamectin, emamectin benzoate,
Nerve and muscle action milbemycins lepimectin, milbemectin
(strong evidence that action – –
at one or more of this class
of protein is responsible for
7 7A
Juvenile hormone mimics Juvenile hormone Hydroprene, kinoprene, methoprene
Growth regulation (target analogs
protein responsible for 7B
biological activity is Fenoxycarb Fenoxycarb
unknown, or 7C
uncharacterized) Pyriproxyfen Pyriproxyfen
8 8A
Miscellaneous nonspecific Alkyl halides Methyl bromide and other alkyl halides
(multisite) inhibitors 8B
Chloropicrin Chloropicrin
8C
Sulfuryl fluoride Sulfuryl fluoride
8D
Borax Borax
8E
Tartar emetic Tartar emetic
9 9B
Selective homopteran Pymetrozine Pymetrozine
feeding blockers 9C
Nerve action (target protein Flonicamid Flonicamid
responsible for biological
activity is unknown, or
uncharacterized)
10 10Ab
Mite growth inhibitors Clofentezine Clofentezine, hexythiazox, diflovidazin
Growth regulation (target Hexythiazox –
protein responsible for Diflovidazin –
biological activity is 10B
unknown, or Etoxazole Etoxazole
uncharacterized)

active ingredient
11
Microbial disruptors of Bacillus Bacillus thuringiensis subsp. Israelensis
insect midgut membranes thuringiensis or Bacillus sphaericus
(includes transgenic crops Bacillus sphaericus Bacillus thuringiensis subsp. Aizawai
expressing Bacillus and the insecticidal Bacillus thuringiensis subsp. Kurstaki
thuringiensis toxins; proteins they Bacillus thuringiensis subsp. Tenebrionis
however, specific guidance produce Bt crop proteins: Cry1Ab, Cry1Ac, Cry1Fa,
for resistance management Cry2Ab, mcry3a, Cry3Ab, Cry3Bb,
of transgenic crops is not Cry34/35Ab1
based on rotation of modes
of action)
12 12A
Inhibitors of mitochondrial Diafenthiuron Diafenthiuron
ATP synthase 12B
Energy metabolism Organotin Azocyclotin, cyhexatin, fenbutatin oxide
(compounds affect the miticides
function of this protein, but 12C
it is not clear that this leads Propargite Propargite
to biological activity) 12D
Tetradifon Tetradifon
13
Uncouplers of oxidative Chlorfenapyr Chlorfenapyr
phosphorylation via DNOC DNOC
disruption of the proton Sulfluramid Sulfluramid
gradient
Energy metabolism
14
Nicotinic acetylcholine Nereistoxin analogs Bensultap, cartap hydrochloride,
receptor (nAChR) channel thiocyclam, thiosultap-sodium
blockers
Nerve action (compounds
affect the function of this
protein, but it is not clear
that this leads to biological
activity)
15
Inhibitors of chitin Benzoylureas Bistrifluron, chlorfluazuron,
biosynthesis, type 0 diflubenzuron, flucycloxuron,
Growth regulation (target flufenoxuron, hexaflumuron, lufenuron,
protein responsible for novaluron, noviflumuron, teflubenzuron,
biological activity is and triflumuron
unknown, or
uncharacterized)

active ingredient
16
Inhibitors of chitin Buprofezin Buprofezin
biosynthesis, type 1
Growth regulation (target
protein responsible for
biological activity is
unknown, or
uncharacterized)
17
Molting disruptor, dipteran Cyromazine Cyromazine
Growth regulation (target
unknown, or
uncharacterized)
18
Ecdysone receptor agonists Diacylhydrazines Chromafenozide, halofenozide,
Growth regulation (strong methoxyfenozide, tebufenozide
19
Octopamine receptor Amitraz Amitraz
agonists
Nerve action (good evidence
that action at one or more of
this class of protein is
responsible for insecticidal
effects)
20 20A
Mitochondrial Complex III Hydramethylnon Hydramethylnon
electron transport inhibitors 20B
Energy metabolism (good Acequinocyl Acequinocyl
evidence that action at this 20C
protein complex is Fluacrypyrim Fluacrypyrim
effects)
21 21A
Mitochondrial Complex I METI acaricides Fenazaquin, fenpyroximate, pyrimidifen,
electron transport inhibitors and insecticides pyridaben, tebufenpyrad, tolfenpyrad
Energy metabolism (good 21B
evidence that action at this Rotenone Rotenone (Derris)
protein complex is
effects)

active ingredient
22b 22A
Voltage-dependent sodium Indoxacarb Indoxacarb
channel blockers 22B Metaflumizone
Nerve action (good evidence Metaflumizone
that action at this protein
complex is responsible for
23
Inhibitors of acetyl CoA Tetronic and Spirodiclofen, spiromesifen, spirotetramat
carboxylase. tetramic acid
Lipid synthesis, growth derivatives
regulation (good evidence
that action at this protein is
effects)
24 24A
Mitochondrial Complex IV Phosphine Aluminum phosphide, calcium phosphide,
electron transport inhibitors phosphine, and zinc phosphide
Energy metabolism (good
24B
Cyanide Cyanidea
protein complex is
effects)
25
Mitochondrial Complex II Cyenopyrafen Cyenopyrafen
electron transport inhibitors
Energy metabolism (good

protein complex is
effects)
28
Ryanodine receptor Diamides Chlorantraniliprole, flubendiamide
modulators
Nerve and muscle action
(good evidence that action at
this protein complex is
effects)

active ingredient
Un compounds of unknown Azadirachtin Azadirachtin

or uncertain MoAc (target Benzoximate Benzoximate
unknown, or
uncharacterized)
Un compounds of unknown Azadirachtin Azadirachtin
or uncertain MoAc (target Benzoximate Benzoximate
protein responsible for Bifenazate Bifenazate
biological activity is Bromopropylate Bromopropylate
unknown, or Chinomethionat Chinomethionat
uncharacterized) Cryolite Cryolite
Cyflumetofen Cyflumetofen
Dicofol Dicofol
Pyridalyl Pyridalyl
a
Inclusion of a compound in the list above does not necessarily signify regulatory approval.
b
Please see footnotes for further information on the use of compounds between subgroups.
c
A compound with an unknown or controversial MoA or an unknown mode of toxicity will be held in
category ‘‘un’’ until evidence becomes available to enable that compound to be assigned to a more
appropriate MoA class.
Notes to be read in association with the above classification scheme:
MoA assignments will usually involve identification of the target protein responsible for the
biological effect, although groupings can be made where compounds share distinctive physiological
effects and have related chemical structures.
Criteria for descriptors of the quality of MoA information:
1. (Strong evidence that action at this Potent effects on the function of the target
protein (or protein complex) is responsible protein and either resistance due to
for insecticidal effects) mutation/overexpression/removal of this
protein or correlation of potency between
effects on the protein and biological activity
for a set of analogs.
2. (Good evidence that action at this Highly potent effects on the function of the
protein (or protein complex) is responsible protein, combined with clearly consistent
for insecticidal effects) physiological effects.
3. (Compounds affect the function of this Compounds (or their metabolites) have
protein, but it is not clear that this leads to moderate or low potency on the function of
biological activity) the protein, and there is little or no evidence
associating this effect with biological activity.
Compounds may be grouped because of
similarity of structure and distinctive
physiological effect.

active ingredient
4. (Target protein responsible for biological Compounds may be grouped because of

activity is unknown, or uncharacterized) similarity of structure and distinctive
physiological effect.
Notes regarding subgroups:

Subgroups represent distinct structural classes believed to have the same MoA. In principle, they
provide a useful level of differentiation between compounds that may bind at the same target site but
are, nevertheless, structurally different enough that the risk of metabolic cross-resistance is lower
than for close chemical analogs. Subgroups are likely to be metabolized by different enzymes, and
may bind differently enough within the target site that the chance of selection for either metabolic or
target-site resistance is reduced compared to close analogs. In the absence of other alternatives, it may
be possible to rotate compounds between subgroups if it is clear that cross-resistance mechanisms do
not exist in the target populations. By definition, subgroups are established to represent distinct
chemical classes with a common MoA. Whether they should be rotated or not will depend on
knowledge and experience of cross-resistance patterns, resistance mechanisms and, furthermore, on
the pest, crop, and region considered.
Subgroup number Notes
1A and B If there are no other alternatives, compounds from groups 1A and 1B

may be rotated in situations where cross-resistance mechanisms are
known to be absent in the insect populations to be treated.
3A and B If there are no other alternatives, compounds from groups 3A and 3B
may be rotated in situations where cross-resistance mechanisms (e.g.,
kdr) are known to be absent in the insect populations to be treated.
Because DDT is no longer used in agriculture, this is only applicable for
the control of human disease vectors such as mosquitoes, because of a
lack of alternatives.
10A Hexythiazox is grouped with clofentezine because they commonly
exhibit cross-resistance, even though they are structurally distinct, and
the target site for neither compound is known. Diflovidazin has been
added to this group because it is a close analog of clofentezine and is
expected to have the same mode of action.
22A and B Although these compounds are believed to have the same target site,
they have been subgrouped because they are chemically distinct, and
current evidence indicates that the risk of metabolic cross-resistance is
low.
Table 27.2 Recommendations for successful resistance management.
Consult a local agricultural advisor or extension services in the area for up-to-date
recommendations and advice on IPM and IRM programs
Consider options for minimizing insecticide use by selecting early-maturing or
pest-tolerant varieties of crop plants
Include effective cultural and biological control practices that work in harmony
with effective IRM programs. Where possible, adopt all nonchemical techniques
known to control or suppress pest populations, including biological sprays such as
Bt’s, resistant varieties, within-field refugia (untreated areas), and crop rotation
Where possible, select insecticides and other pest management tools which
preserve beneficial insects
Use products at their full, recommended doses. In many cases, reduced
(sublethal) doses quickly select populations with average levels of tolerance, whilst
doses that are too high may impose excessive selection pressures
Appropriate, well-maintained equipment should be used to apply insecticides.
Recommended water volumes, spray pressures, and optimal temperatures should
be used to obtain optimal coverage
Where larval stages are being controlled, target younger larval instars where
possible because these are usually much more susceptible and therefore much
more effectively controlled by insecticides than older stages
Use appropriate local economic thresholds and spray intervals
Follow label recommendations or local expert advice for use of alternations or
sequences of different classes of insecticide with differing modes of action as part
of an IRM strategy
Where there are multiple applications per year or growing season, alternate
products of different MoA classes
In the event of a control failure, do not reapply the same insecticide but change the
class of insecticides to one having a different mode of action and to which there is
no (locally) known cross-resistance
Mixtures may offer a short-term solution to resistance problems, but it is essential
to ensure that each component of a mixture belongs to a different insecticide MoA
class, that each component is used at its full rate, and that there is no existing
resistance to either component in the target insect pest(s)
Consideration should be given to monitoring for the incidence of resistance in the
most commercially important situations, and gage the levels of control obtained
Withholding the use of a product to which resistance has developed until
susceptibility returns may be a valid tactic if sufficient alternative chemical classes
remain to provide effective control
of their MoA and biological value). The details of other significant chemical classes
are provided elsewhere in this book.
27.6.2
Organophosphates and Carbamates
Organophosphates (OPs) were first introduced to the agrochemical market in

1944, and are economically still the most successful and diverse chemical class of
insecticides ever invented [9]. More than 100 different active ingredients belonging
to this class are known, with the best selling OP of all being chlorpyrifos.
All OPs act as neuroactive compounds that bind irreversibly to the enzyme
acetylcholinesterase (AChE), thus preventing hydrolysis of the neurotransmitter
acetylcholine in the central nervous system. This leads to prolonged periods of nerve
excitation, and results in paralysis and subsequent death of the treated pest insects
[10, 11]. The OPs are used to control almost all pest species from a wide variety
of insect orders, including Lepidoptera, Coleoptera, Diptera, Hemiptera (including
aphids), and many more [12]. Additionally, the OPs can be used to control
phytopathogenic nematodes and phytophageous mites. The major disadvantage
of most OPs is their toxicity to vertebrates, including humans. Some OPs have
LD50 -values (rat oral) of less than 5 mg kg−1 (e.g., Disulfoton), although the
majority show LD50 -values of between 5 and 50 mg kg−1 (e.g., Methamidophos). A
few examples, such as acephate (rat oral LD50 > 600 mg kg−1 ) and malathion (rat
oral LD50 > 800 mg kg−1 ) are known to exhibit a lower toxicity. The broad toxicity
of the OPs tend to minimize their selectivity among insect pests. Likewise, the
often strong efficacy toward beneficial insects (e.g., those of biocontrol importance)
has driven the agrochemical industry to seek alternative compounds to replace the
OPs in the long term.
Another class of AChE inhibitors which is of economic importance but usually
less toxic to nontarget organisms are the carbamates, which were introduced to the
insecticide market during the late 1950s [11]. One of the most important aphicides
among this group, pirimicarb, was launched during the early 1970s, exhibits
appreciable toxicity to vertebrates (as shown by its oral LD50 of 140 mg kg−1 in rats).
However, it is more than 100-fold less toxic than other widely used carbamates,
such as Aldicarb (oral LD50 of 0.9 mg kg−1 in rats) which is used primarily as a soil
insecticide.
27.6.3
Pyrethroids
One of the most important chemical classes of insecticides are the pyrethroids,
which act as ligands of voltage-gated sodium channels in nerve axons [13, 14].
During the mid-nineteenth century, an insecticidal powder derived from the dried
flower heads of the genus Pyrethrum (Chrysanthemum) was introduced from Africa
to central Europe. The insecticidal components of this material were identified as
pyrethrins. Due to several asymmetric centers, these compounds have many enan-
tiomeric forms, and only a few of them are insecticidally active [14–16]. The natural
pyrethrins are unstable, sensitive to photodegradation, and relatively expensive, but
have been used as templates to generate synthetic analogs – the so-called ‘‘synthetic
pyrethroids’’ [15–18]. Modern synthetic pyrethroids are well-optimized compounds
with respect to their potency, residual activity, and photostability. When considering
the symptomatology of poisoning induced by these contact insecticides, pyrethroids
can be separated into two classes [19]: (i) type I pyrethroids (e.g., permethrin), which
cause hyperactivity and incoordination; and (ii) type II pyrethroids, which contain
an α-cyano substituent that induces nerve depolarization and subsequent paralysis

of the insect.
The recommended application rates of the pyrethroids are often in the range
of 5–25 g a.i. ha−1 . In contrast, fenvalerate – as one of the most intensively used
pyrethroids (especially in cotton) – is applied at 100–200 g a.i. ha−1 ; the high dose
rate reflects the low vertebrate toxicity of fenvalerate (oral LD50 of 450 mg kg−1 in
rats) compared to deltamethrin (120 mg kg−1 ).
Although the pyrethroids are highly active against lepidopteran pest species, their
speed of action – which leads to a rapid pest knockdown – renders them useful
in many cropping systems against numerous pests of different insect orders,
including Lepidopteran, coleopteran, and many sucking pests (including aphids).
27.7
Effective IRM Strategies and Approved Principles
The objective of successful IRM is to prevent or delay the evolution of resistance

to insecticides, or to help regain susceptibility in insect pest populations in which
resistance has already arisen. Effective IRM is, therefore, an important element in
maintaining the efficacy of valuable insecticides. It is important to recognize that
it is usually easier to proactively prevent resistance from occurring than it is to
reactively regain susceptibility. As such, the IRAC MoA Classification will always
provide valuable guidance to the design of effective IRM strategies.
Experience has shown that all effective management strategies seek to minimize
the selection for resistance from any one type of insecticide or acaricide. In practice,
alternations, sequences, rotations, or even mixtures of compounds from different
MoA groups provide an effective – and, in some cases, a sustainable – approach
to IRM [8]. This type of an approach ensures that selection from compounds in
any one MoA group is minimized. The IRAC classification in this document is
provided as an aid to insecticide selection for these types of IRM strategy.
Applications are often arranged into MoA spray windows or blocks that are
defined by the stage of crop development and the biology of the pest(s) of concern
[8]. Local expert advice should always be followed with regard to spray windows
and timings. Although several sprays of a compound may be possible within each
spray window, it is generally essential to ensure that successive generations of the
pest are not treated with compounds from the same MoA group.
The principles endorsed by the IRAC as basic tools for successful resistance
management are listed in Table 27.1.
To assist users in the selection of insecticides for use in IRM strategies employing
sequences, rotations, or alternations of MoA groups, the IRAC is encouraging
producers to clearly indicate the IRAC MoA group number and description on the
product label, and to accompany this with appropriate advice of the type indicated
below. Thus, in addition to the detailed product information, handling, and safety
information required by local regulations, a typical title label should clearly indicate
the IRAC MoA Group number, description, and brief advice in IRM.
27.8 Future Market Trends 953
27.8
Future Market Trends
The major insecticidal classes, and their market share in 2008, are shown in
Figure 27.1.
With the expansion in numbers of insect-resistant crops, the insecticide market
may decline slightly over the next few years [20]. The incorporation of Bacillus
thuringiensis (Bt) genes into plants, to express intrinsic insect resistance, has already
had an impact on insecticide sales, particularly in the cotton and maize sectors.
Whilst this impact has been modest compared to the effect of herbicide-tolerant
crops on herbicide sales, it is expected that insect-resistant crops will have a
greater negative influence as the technology improves. Although, at present, Bt
crops control only a limited pest range (primarily the cotton bollworm and corn
borer), this situation is slowly being expanded to cover other pests through stacking
technology and the discovery of advanced toxins; an example of this is Cry1F in
Herculex maize and the forthcoming Vip gene. With the commercial launch of
corn rootworm-resistant maize, another important insecticide market is now also
under pressure.
Other key factors include increasing regulatory pressures and generic competi-
tion. Regulatory restrictions in Western Europe and North America are affecting
many old, but still commercially important, insecticides. To a certain extent, substi-
tution with alternative products will minimize this impact, although some negative
effect on sales is inevitable. Generic manufacture is also affecting the sales of
several chemistry groups, this being particularly true in Far East markets such as
China [20].
The increasing demand for high-quality vegetables will have a positive effect on
insecticide use, whereas in cotton and maize – and eventually also in rice – negative
effects due to Bt-technology are to be expected. Yet, the long-term use of Bt crops
Natural products 7%
Organochlorines 1%
5,6
2A
Others 24%
Benzoylureas 2% Organophosphates 15%
15 1B
Neonicotinoids 24% Carbamates 10%

4A Pyrethroids 17% 1A
3
Total Global Insecticide Sales 2008 = $10,658 million

[ Source Cropnosis 2009 ]
[ Natural products includes avermectins and spinosyns ]
Numbers in italics = IRAC MoA classification number
Figure 27.1 Major chemical classes of insecticides and their market share.
will create opportunities for emerging sucking pests such as aphids, whiteflies,
bugs, spider mites, and Lepidoptera that are not controlled by this technology.
27.9
Conclusions
Given the ever-increasing costs and difficulties associated with the discovery of
new active ingredients with novel MoA that not only circumvent existing resistance
problems but also pass increasingly stringent regulatory hurdles, the IRAC believes
that it is absolutely vital to ensure the sustained efficacy of the broad range of the
current and future modern, safe, and effective insecticides that the agrochemical
industry produces. The concept that susceptibility is a highly valued commodity is
clearly central to this approach. Indeed, such a resource should not be squandered
indiscriminately through either the misuse or over-use of insecticides. Effective
IRM is, therefore, not an option – it is essential, and one of the most challenging
issues in modern applied entomology. As a responsibility to both the industry and
to its customers, and in the interests of protecting the industry’s products, the IRAC
is undertaking a broad range of activities to help make effective IRM possible.
References
1. Whalon, M.E., Mota-Sanchez, D., and Hollingworth), CABI, Wallingford,

Hollingworth, R.M. (2008) in Global Pes- pp. 40–89.
ticide Resistance in Arthropods (eds M.E. 7. Sparks, T.C., Lockwood, J.A., Byford,
Whalon, D. Mota-Sanchez, and R.M. R.L., Graves, J.B., and Leonard, B.R.
Hollingworth), CABI, Wallingford, pp. (1989) The role of behaviour in insect
5–31. resistance. Pestic. Sci., 26, 383–399.
2. Mccaffery, A. and Nauen, R. (2006) 8. Roush, R.T. (1989) Designing resistance
The insecticide resistance action com- management programs: how can you
mittee (IRAC): public responsibility choose. Pestic. Sci., 26, 423–441.
and enlightened industrial self-interest. 9. Eto, M. (1974) Organophosphorus Pesti-
Outlooks Pest Manage., 2, 11–14. cides: Organic and Biological Chemistry,
3. Thompson, G.D. and Leonard, P.K. CRC Press, Boca Raton, FL.
(1996) in Molecular Genetics and Evo- 10. Lund, A.E. (1985) in Comprehensive
lution of Pesticide Resistance (ed. T.M. Insect Physiology, Biochemistry and Phar-
Brown), American Chemical Society, macology, vol. 12 (eds G.A. Kerkut and
Washington, DC, pp. 187–195. L.I. Gilbert), Pergamon Press, Oxford,
4. Leonard, P.K. (1997) There has never pp. 9–56.
been a better time or a greater need for 11. Eldefrawi, A.T. (1985) in Comprehensive
resistance management. Pestic. Sci., 51, Insect Physiology, Biochemistry and Phar-
387–390. macology, vol. 12 (eds G.A. Kerkut and
5. OEPP/EPPO (1999) – EPPO Standard L.I. Gilbert), Pergamon Press, Oxford,
PP 1/213(1) Resistance risk analysis, pp. 115–130.
OEPP/EPPO Bull. 29, 325–347. 12. Siegfried, B.D. and Scharf, M.E. (2001)
6. Hollingworth, R.M. and Dong, K. in Biochemical Sites Important in In-
(2008) in Global Pesticide Resistance secticide Action and Resistance (ed.
in Arthropods (eds M.E. Whalon, I. Ishaaya), Springer-Verlag, Berlin,
D. Mota-Sanchez, and R.M. Heidelberg, pp. 269–291.
References 955
13. Narahashi, T. (1992) Nerve membrane 7, Chemie der Pflanzenschutz-und

Na+ channels as targets of insecticides. Schädlingsbekämpfungsmittel,
Trends Pharmacol. Sci., 13, 236–241. Springer-Verlag, Berlin.
14. Zlotkin, E. (2001) in Biochemical Sites 18. Ruigt, G.S.F. (1985) in Comprehensive
Important in Insecticide Action and Resis- Insect Physiology, Biochemistry and Phar-
tance (ed. I. Ishaaya), Springer-Verlag, macology, vol. 12 (eds G.A. Kerkut and
Berlin, Heidelberg, pp. 43–76. L.I. Gilbert), Pergamon Press, Oxford,
15. Elliot, M., Farnham, A.W., Janes, N.F., pp. 183–262.
Needham, P.H., and Pulman, D.A.
19. Gammon, D.W., Brown, M.A., and
(1974) Synthetic insecticides with a new
Casida, J.E. (1981) Two classes of
order of activity. Nature, 248, 710–711.
pyrethroid action in the cockroach.
16. Elliot, M., Janes, N.F., and Porter, C.
(1978) The future of pyrethroids in in- Pestic. Biochem. Physiol., 15, 181–191.
sect control. Annu. Rev. Entomol., 23, 20. Cheung, K. and Sirur, G. (2003) Agro-
443–469. chemical Service, Update of the Products
17. Naumann, K. (1981) Chemie der syn- Session Insecticides, Cropnosis Limited,
thetischen Pyrethroidinsektizide, vol. pp. 1–36.
957
28
Insect Molting and Metamorphosis
28.1
Bisacylhydrazines: Novel Chemistry for Insect Control
28.1.1
Introduction
The discovery of insecticidal bisacylhydrazine (BAH) compounds reinforced the

original concept of Carrol Williams [1] for the discovery and development of insect
hormone mimics as a third generation of novel and reduced-risk insecticides.
Although this concept was proposed several decades ago, it was not until the
mid-1970s that the first insecticidal compounds that mimicked insect juvenile
hormone (JH) were discovered (for reviews, see Refs [2, 3]); this was followed
by the discovery of BAH nonsteroidal agonists of the insect molting hormone
20-hydroxyecdysone (20E; Figure 28.1.1; 1) during the late 1980s (for reviews, see
Refs [4–6]). Initial attempts to synthesize insecticides with 20E activity had failed
because compounds based on the cholesterol backbone and structural similarities
to ecdysteroids are chemically and metabolically unstable [7, 8]. It was not until
the late 1980s that the first nonsteroidal ecdysone agonist was identified at Rohm
and Haas Company by Hsu and colleagues [9], and the first prototype, RH-5849
(Figure 28.1.1; 3), was characterized by its insecticidal spectrum of activity and
ability to compete with ecdysteroids for binding to ecdysone receptor in insect
cell preparations [10, 11]. Since then, four members of the BAH chemistry – three
discovered at Rohm and Hass Company and one at Nippon Kayaku/Sankyo
Companies – have been developed commercially as insecticides (see Table 28.1.1).
In this chapter, the structures and biology (physiological, biochemical, and
molecular basis of mode of action, insect activity spectrum, and ecotoxicological
safety) of the commercialized BAH nonsteroidal ecdysone agonist insecticides
are reviewed. Further details are available in various reviews on this topic
[4–6, 12].
28.1.1.1 Physiological and Molecular Basis of Insect Molting Hormone Action

Arthropods achieve growth and development by molting several times as immature
nymphal or larval instars. Molting is required due to the absence of an endoskeleton,

958 28 Insect Molting and Metamorphosis
OH R2
R1 Cl O
21 25 26 O
22 24
18 20 23 OH N N
12 17 27 N N
19 11 C 13 D 16 H H
14 15
O O
HO 1 9 Cl
2 10 8
A B OH
3 5 7
4 6
HO
H O
1 2 3
Figure 28.1.1 Chemical structures of its ecdysone effects at the cellular and
20-hydroxyecdysone (20E; 1 R1 = OH), the whole-insect level, as well as binding to
first bisacylhydrazine found to have an Drosophila melanogaster cell extracts con-
ecdysone agonist effect in insect assays (2), taining ecdysteroid receptor complexes. The
and the first bisacylhydrazine (RH-5849; numbers on the 20E structure represent the
3), which has been well characterized for carbon-numbering system.
which is accomplished through a sequence of steps that include: cessation of

feeding; separation of the old cuticle from the underlying epidermal cells (apolysis);
synthesis of a new cuticle; degradation of the old cuticle by secreted chitinolytic
enzymes; and a hormone-dependent eclosion behavior that allows the molting
insect stage to emerge from the old cuticle. The presence of a new cuticle before it
become sclerotized allows the molted life-stage to expand and resume growth by
continuing to feed.
Growth and development in insects is regulated by two primary hormones: the
steroidal insect molting hormone, 20E (Figure 28.1.1; 1); and the sesquiterpene,
JH, of which there are four main types (see Refs [13, 14]). Initiation of the molting
process is characterized by rising 20E titers in the insect larval hemolymph, and
the cessation of feeding by the larva. As the 20E titers continue to rise, the old
cuticle separates from the underlying epidermal cells (apolysis); this allows inactive
chitinolytic enzymes to fill the ecdysial space and signals the epidermal cells
to secrete proteins that would form the new cuticle. As the 20E titers decline,
the chitinolytic enzymes are activated to digest away the old endocuticle, and the
secreted proteins for the new cuticle are layered systematically. By the time that the
20E titers have declined to a basal level, only remnants of the old exocuticle remain,
the new cuticle is fully formed, and the molting larva is ready to ecdyse from its
old cuticle into a new one. At this time, a neuropeptide – eclosion hormone – is
released to cause secretion of the ecdysis trigger hormone, which in turn enables
the ecdysis behavior and allows the larva to depart from its old cuticle shell. It is
important that the 20E titers have declined to a basal level; otherwise, the eclosion
hormone is not released, and the ecdysis or eclosion behavior will not occur [15–17].
Following the completion of larval ecdysis, the newly molted larva resumes feeding,
while deposition of the endocuticle continues during the intermolt period.
The manifestation of 20E effects during molting or other developmental stages
(e.g., reproduction) are brought about by the interaction of 20E with the ecdysone
receptor complex. The latter is a heterodimer of the ecdysone receptor (EcR) protein
Table 28.1.1 Commercialized bisacylhydrazine ecdysone agonist insecticides: structures, common and coded names, and pest spectrum.
O O
O O
N
N N
N H N N
H N N
O H H
O
O O O Cl O
Common Tebufenozide Methoxyfenozide Chromafenozide Halofenozide

name
Coded AS RH-5992 RH-2485 ANS-118; CM-001 RH-0345
®
Registered MIMIC™ INTREPID™ MATRIC MACH2™
®
names CONFIRM™ RUNNER™ KILLAT
ROMDAN™ PRODIGY™
FALCON™
Melting point 191 204–205 186.4 153–155
( ◦ C)
Log it P 4.25 3.7 2.7 3.42
Pest spectrum Lepidoptera Lepidoptera Lepidoptera Coleoptera
Lepidoptera
a a b a
Industry
a
Trademarks of Dow AgroSciences LLC.
b
Nipon Kayaku Company, Saitame, Japan and Sankyo Company, Ibaraki, Japan.
28.1 Bisacylhydrazines: Novel Chemistry for Insect Control
959
and the ultraspiracle protein (USP), a homolog of the vertebrate retinoid X receptor
(RXR) protein (see Ref. [18]). Both, EcR and USP are members of the steroid
receptor superfamily of ligand-dependent transcription factors. Characteristically,
these family members have both DNA-binding protein domains (DBDs) and
ligand-binding domains (LBDs) in between the transactivation domains at the N
and C termini. The binding of ecdysteroids to the EcR takes place only when both
EcR and USP exist as heterodimers. However, a recent report has suggested a
low-affinity binding of tritiated ponasterone A (a phytoecdysteroid) to EcR proteins
from the rice stemborer, Chilo suppressalis [19] and the Colorado potato beetle,
Leptinotarsa decemlineata [20]. cDNAs encoding EcRs and USPs (or RXR-like USPs)
from several arthropods have been cloned, and the proteins expressed for ligand
and/or DNA binding (see Refs [5, 18]), or crystal structural studies [21, 22].
28.1.2
Discovery and Structures of Commercialized Bisacylhydrazine Insecticides
Several years after the first insecticidal BAH (Figure 28.1.1; 2) had been discovered
serendipitously at the Rohm and Haas Company [9], the first prototype of a bona fide
nonsteroidal ecdysone agonist BAH, RH-5849 (Figure 28.1.1; 3), was discovered
[23]. Characterization of the spectrum of activity of RH-5849, in addition to its ability
to bind to an EcR-containing preparation from Drosophila Kc cells [10, 11], led to an
intense chemical synthesis program at Rohm and Haas Company. Subsequently,
the use of this compound in both in vitro and in vivo studies furthered an under-
standing of the developmental and reproductive physiology of susceptible insects.
Continued investigations into the structure–activity relationship of RH-5849, which
had broad-spectrum activity against several lepidopteran, coleopteran, and dipteran
insects, led to the discovery and commercialization of three BAH compounds
as insect-selective insecticides (Table 28.1.1), namely tebufenozide (RH-5992),
methoxyfenozide (RH-2485), and halofenozide (RH-0345).
Halofenozide, which is predominantly active on coleopteran and lepidopteran
larvae, has been commercialized for the turf market in the USA, while both
tebufenozide and methoxyfenozide are predominantly active on lepidopteran lar-
val pests of vegetable crops, fruits, nuts and vines, corn, and cotton. Typically,
methoxyfenozide is more potent than tebufenozide, and also has a broader spec-
trum of activity for lepidopteran larval pests.
Finally, a fourth BAH – chromafenozide (Table 28.1.1) – was discovered and
jointly commercialized by Nippon Kayaku Company and Sankyo Company for
the control of lepidopteran larval pests of vegetables, fruits, vines, tea, rice, and
ornamentals in Japan [24, 25].
28.1.3
Synthesis of Commercial Bisacylhydrazines
Halofenozide (8) is prepared via a regioselective acylation of t-butyl hydrazine

hydrochloride (5) (Scheme 28.1.1). For this, 4-chloro-benzoyl chloride (4) in butyl
.HCl
N O n-butylacetate
N O
COCl K2CO3/water O
5 N
N N
NaOH/K2CO3 N
Cl n-butylacetate Cl 6
water COOH Cl 8
4 SOCl2
Scheme 28.1.1 Synthesis of halofenozide.
.HCl
N O n-butylacetate
COCl N N K2CO3/water O
5 N O
NaOH/K2CO3 N
10 N
9 n-butylacetate
water COOH SOCl2 12
11
Scheme 28.1.2 Synthesis of tebufenozide.
acetate is added to an aqueous solution of t-butyl hydrazine and sodium hy-

droxide, and potassium carbonate. The Schotten–Baumann conditions afford the
4-chlorobenzoic acid N -t-butyl-hydrazide (6) in high purity and regioselectivity.
This material is additionally acylated with benzoyl chloride (prepared from benzoic
acid 7) to yield halofenozide in excellent yield and purity [26].
Tebufenozide (12) is prepared by the same process (Scheme 28.1.2), acylating first
with 4-ethylbenzoyl chloride (9) to produce 4-ethylbenzoic acid N -t-butyl-hydrazide
(10). A second acylation with 3,5-dimethylbenzoyl chloride produced from the
corresponding benzoic acid 11, affords tebufenozide [26].
Methoxyfenozide (17) is produced as shown in Scheme 28.1.3. The inter-
mediate 3-methoxy-2-methylbenzoic acid (15) is prepared in two steps from
2,6-dichlorotoluene (13). Nucleophilic substitution of 13 with sodium methox-
ide in dimethylsulfoxide (DMSO) at 140–160 ◦ C yields 1-chloro-3-methoxy-2-
methylbenzene (14) in high yields and purity. This compound is converted
into 15 by Grignard formation and subsequent quenching with carbon dioxide
[27]. Preparation of the corresponding benzoyl chloride, followed by regiospe-
cific Schotten–Baumann acylation of t-butylhydrazine hydrochloride (5), gives
3-methoxy-2-methylbenzoic acid N -t-butyl-hydrazide (16). Finally, 16 is again
acylated with 3,5-dimethylbenzoyl chloride in methylene chloride with aqueous
sodium hydroxide to afford methoxyfenozide [28].
Chromafenozide (24) is the most synthetically complex compound in the BAH
class of chemistry; the published procedure for its production is outlined in
O
Cl Cl NaOCH O Cl Mg/THF O COOH (1) SOCl2 O N
3
DMSO (2) N N
CO2/HCl
N
.HCl 5
16
13 14 15 NaOH/water COOH
DCM SOCl2
DCM
NaOH/water 11
O
O
O N
N
17
Scheme 28.1.3 Synthesis of methoxyfenozide.
Scheme 28.1.4 [29]. In this case, 2-t-butyl-5-methylphenol is O-alkylated with

propargyl bromide to give 1-t-butyl-4-methyl-2-prop-2-ynyloxybenzene (18). This
material can be efficiently cyclized to the 8-t-butyl-5-methyl-2H-chromene (19)
in refluxing N,N-diethylaniline. Catalytic hydrogenation with 5% palladium on
carbon affords 8-t-butyl-5-methylchroman (20). A low-temperature (0 ◦ C) Friedel–
Crafts acylation of 20 with acetyl chloride and anhydrous aluminum chloride
gives the 1-(8-t-butyl-5-methyl-chroman-6-yl)-ethanone (21). Bromination of
21 in 1,4-dioxane, followed by hydrolysis and subsequent de-t-butylation with
aluminum chloride, gives 5-methylchroman-6-carboxylic acid (22), which is
converted into the acid chloride by typical methods and then used to mono-acylate
t-butyl hydrazine hydrochloride (5). The resulting 5-methylchroman-6-carboxylic
acid N -t-butyl-hydrazide (23) is again acylated with the acid chloride of
3,5-dimethylbenzoic acid to afford chromafenozide (24).
28.1.4
Structure–Activity Relationship of Ecdysteroids and Bisacylhydrazines
The structure–activity relationship (SAR) of the BAHs, both during and after the
discovery of the commercial compounds, has been extensively studied. This was
partly driven by the novelty of the chemistry, the mode of action (ecdysone agonists
via interaction with the EcR), and the availability of suitable assays (tissue, cell,
and target-site-based). Numerous reports have been made on this subject, and the
reader is referred to an excellent review by Dinan and Hormann [6] as a starting
point. The collective findings of the various research groups are highlighted and
summarized in the following subsections.
When considering the SARs of BAHs for the discovery of new and novel ecdysone
agonists, it is essential first to understand the SAR of ecdysteroids. This helps in
defining the three-dimensional (3-D) space of ecdysteroids in the EcR LBD, and
O (1) Br2 O
N,N '-diethylaniline H2 AcCl NaOH-H2O
OH
O Heat Pd-C AlCl3
O O
(2) AlCl3
O O
22
18 19 20 21
N
SOCl2 N
.HCl
O 5
O O
DCM
N
N N
N
O
O
COOH SOCl2
24 23
11
Scheme 28.1.4 Synthesis of chromafenozide.

28.1 Bisacylhydrazines: Novel Chemistry for Insect Control
963
also allows for overlaps and comparison with BAH or other ecdysone agonist
chemotypes (as described below).
28.1.4.1 Structure–Activity Relationship (SAR) of the Ecdysteroids

The results of earlier studies on the SAR of ecdysteroids, based on several simple
different insect bioassays [30], were much later substantiated for the most part
using very comprehensive SARs based on sets of ecdysteroids and their quantitative
effects in cell- and tissue-based ecdysone-responsive assays. These were conducted
in addition to comparative molecular field analysis (CoMFA) and four-dimensional
qualitative structure–activity relationship (4-D-QSAR) approaches to analyze the
data [31–33] (see also Ref. [6]). In general, the results of the two approaches
are similar in that all of the hydroxyl groups – except for 14-OH and 25-OH,
and the steroid side chain (Figure 28.1.1; 1) – play important roles in hydrogen
bonding and the activity of ecdysteroids, respectively. The general picture to
emerge from the CoMFA and 4-D-QSAR analyses is that the ecdysteroid side chain
lies in a sterically restricted hydrophobic cavity in the ligand-binding pocket of
the EcR, in which position both 20-OH and 22-OH provide hydrogen-bonding
functions. Many of these conclusions have been supported by homology modeling
of insect EcR LBD based on low levels (24–27%) of sequence homology to published
sequences, and crystal structures of the vertebrate steroid receptor LBD coupled with
ecdysteroid docking studies [34, 35]. The presence of 25-OH on 20E significantly
diminishes the activity of 20E as compared to that of ponasterone A, which lacks
25-OH.
The publication of the crystal structure of the Heliothis viresence ecdysone receptor
(HvEcR) LBD, heterodimerized with the LBD of the H. viresence ultraspiracle
protein (HvUSP) with and without ponasterone A (phytoecdysteroid) or a BAH
ecdysone agonist, provided a more realistic conformation of the HvEcR LBD and
the ligands in the LBD [21]. Here, the most surprising result was that ponasterone
A cocrystallized in the HvEcR LBD in an orientation opposite to that predicted
by the CoMFA, 4-D-QSAR, and homology modeling studies. In this case, the
steroid nucleus was furthest away from helix 12, while the steroid side chain
was closest to it. Subsequent crystal structure studies revealed that HvEcR LBD
amino acid residues R383, E309, T343/T346, Y408, and A398 interacted with 2-OH,
3-OH, 14-OH, 20-OH, and the C-6 carbonyl, respectively, for hydrogen bonding
(Figure 28.1.2; see also Figure 28.1.4). Interestingly, all of these amino residues
were seen to be conserved amongst the EcR LBDs of insects from different orders
(see sequence review in Ref. [18]), which was consistent with the observation
that the insect EcRs are similarly responsive to the binding of active or potent
ecdysteroids to produce biological effects.
28.1.4.2 Structure–Activity Relationship (SAR) of the Bisacylhydrazines

Following the initial discovery of the first few analogs of BAHs and their charac-
terization as ecdysone agonists not only in both in vivo insect assays but also in in
vitro cell-based and target-site-based assays [10, 11], this class of chemistry contin-
ued to enjoy extensive SAR analysis (for reviews, see Refs [6, 36–38]). From over
C-region
t-butyl (unoptimized
A- and B-regions);
neopentyl, 1-t-butyl-
ethyl, or 1,1-dimethyl-
benzyl (A- and B-
regions optimized)
B-ring
A-ring (monosubstituted,
(monosubstituted, C 5 A = phenyl)
B = phenyl)
O 4
2 B
2-Cl, 2-CH3, 3-Cl, 3- N 2-ethyl, 2-I, 2-Br, 2-NO2,
3 N 3
OCH3, 4-Et, 4-Br, 4-l, 2-Cl, 3-Br, 3-ethyl, 3-methyl,
4-CH3, 4CF3, 4- A H 2
4 3-Cl, 4-Cl, 4-F>H>2-OCH3,
OCH3 > H > 2-Br, 2-NO2, D O 2-CH3, 2-NH2, 3-CF3,
2-CF3, 2-NH2, 2- OCH3, 5
3-OCH3, 3-NO2, 3-NH2,
3-CH3, 3-Et, 3-NO2, 4-NO2, 4-CH3, 4-OCH3
4-Cl, 4-iPr, 4-t-Bu
D-region
H or hydrolyzable
group
Backbone: C=O>C=S, S=O; NH-N(tbu)>CH2-N(tbu), NH-CH(tbu)
Figure 28.1.2 Chemical structure of the first nonsteroidal

ecdysone agonist compound (RH-5849) with insecticidal
activity. Different substitutions on this molecule led to the
discovery of the four bisacylhydrazine commercial insecti-
cides. The letters and numbers refer to substitutions shown
in the boxes around the structure.
4000 analogs synthesized, three compounds – tebufenozide, methoxyfenozide, and

halofenozide – were subsequently commercialized by Rohm and Haas Company,
and are currently owned by Dow AgroSciences LLC. Before elucidation of the
crystal structure of HvEcR LBD in the absence and presence of ponasterone A, or
a BAH (BY-106830) in the ligand-binding pocket, most SARs using CoMFA and
4-D-QSAR approaches were conducted based on various BAHs and internally gen-
erated data sets (extensively reviewed in Refs [6, 37, 38]). For this, the investigators
employed target-site ligand binding, cell-based, tissue-based, and whole-insect as-
says to predict the descriptors (substitutions) for BAH responsible for lepidopteran
and coleopteran activity.
A summary of the SAR of BAH, as proposed by Rohm and Haas Company
investigators, based on the activity of these compounds in southern armyworm
larvae, is shown in Figure 28.1.2.
These SAR studies led to the discovery and commercialization of two
Lepidoptera-specific (tebufenozide and methoxyfenozide) BAHs, and one
Coleoptera- and Lepidoptera-specific (halofenozide) BAH. Subsequently, Nipon
Kayaku and Sankyo Co. announced the discovery of another commercial

insecticidal BAH, chromafenozide [25, 39, 40], for which a qualitative SAR limited
to structures related to this compound [41–43] were reported. Sawada et al.
[41–43] showed that heterocycles fused to the 3,4-positions of the A-ring were
insecticidal to the common cutworm, Spodoptera litura. The main departure in the
structure of chromafenozide from tebufenozide is in the A-ring, where the most
active compounds have oxygen- and carbon-containing, five- or six-membered
fused rings that are devoid of bulky or hydrogen bond-donating substitutions,
and two methyl groups on the B-ring (as for tebufenozide and methoxyfenozide)
(Table 28.1.1).
Dinan and Hormann [6] made the following observations for a BAH pharma-
cophore with toxicity-related features common to species from sensitive insect
orders (mainly Lepidoptera and Coleoptera):
1) Two hydrogen acceptor or polar negative atoms that are ∼3.5–4.0 Å apart.
2) A bulky, conformational-determining lipophilic group located asymmetrically
between the two negative centers.
3) Moderately sized (about six carbons) groups on either side of the negative
centers. Aryl groups are favored for both lepidopteran and coleopteran activity.
a. Lepidopteran activity is enhanced with the following substitutions on the
A-ring; 4-position with one to two carbon lipophilic groups or, alternatively,
with a 2,3- or a 2,[3,4]-ring patterns. Substitutions on the B-ring are less
specific, though substitutions at the 2-, 2,5-, 3,5-, or 3,4,5-positions can be
favorable.
b. Coleopteran activity, in contrast, is favored by one or two small group
substitutions on the aryl rings, as exemplified by halofenozide, which has a
4-Cl substitution on the A-ring.
4) A hydrogen bond-donating group located near the alternate negative center.
28.1.5
Mode of Action of BAH Insecticides
The discovery that RH-5849 acts as an ecdysone agonist in Drosophila Kc cells

[10] and in whole insects [11] stimulated extensive research in insects and other
arthropods at the cellular, tissue, and whole-insect levels (see Refs [4, 5, 12]). For
example, Wing [10] showed that in Drosophila Kc cells, RH-5849 could elicit cell
aggregation and growth inhibition in a similar manner as active ecdysteroids. In
the same study, the competitive displacement of tritiated ponasterone A bound to
Kc cell nuclear extracts containing EcR complexes by excess amounts of RH-5849
indicated that the latter would manifest its ecdysone agonist effects via an interaction
with the same macromolecule (EcR) as did 20E and ponasterone A. Subsequently,
morphological cellular effects (cellular aggregation, clumping, and inhibition of
cell growth) of tebufenozide, methoxyfenozide, and halofenozide similar to those
induced by 20E were demonstrated for several cell lines derived from tissues or
embryos of different insects [13, 44–49]. Once again, several groups showed that
Table 28.1.2 Comparison of relative binding affinities

of ecdysteroids, tebufenozide, methoxyfenozide, and
halofenozide to either cellular extracts or in vitro-expressed
EcR and USP proteins from representatives of different insect
orders.
Insect (Order) Kd (nM)
20E Pon Aa Tebufenozide Methoxyfenozide Halofenozide
Drosophila 145 0.9 336 – –

melanogaster
(Diptera)
Aedes aegypti 28 2.8 30 – –
(Diptera)
Chironomus – 0.35 – – –
tentans (Diptera)
Spodoptera 158 – 86 24.3 –
littoralis
(Lepidoptera)
Spodoptera 166 8.9 1.5 3.5 –
frugiperda-Sf9
cells
(Lepidoptera)
Anthonomus 247 6.1 >12 000 – –
grandis
(Coleoptera)
Leptinotarsa 425 – 1316 – –
decemlineata
(Coleoptera)
Tenebrio molitor – 6 >10 000 >10 000 >10 000
(Coleoptera)
Locusta 1000 1.8 >10 000 >10 000 >10 000
migratoria
(Orthoptera)
Bemesia – 8 >10 000 >10 000 >10 000
argentfoli
a
Pon A = ponasterone A.
RH-5849 and the commercial BAH insecticides would bind to the EcRs in imaginal
wing discs, cellular extracts or in vitro-expressed EcR and USPs from different orders
of insect (see Refs [5, 19, 20, 50]). The relative binding affinities of ecdysteroids and
BAHs to the EcR complexes from different orders of insect are listed in Table 28.1.2.
Clearly, while ponasterone A binds to EcR complexes from different orders of insect
with similar affinities (Kd = 0.9 to 9 nM), tebufenozide binds only to lepidopteran
EcRs with an affinity similar to that of ponasterone A. The binding affinity of
tebufenozide to EcRs from dipteran and coleopteran receptors was two to four
orders of magnitude lower than was its binding to lepidopteran EcRs. However,
the binding of tebufenozide to homopteran and orthopteran EcRs could not be
detected, even at concentrations up to 10 μM. These binding data correlated very
well with the insect-selective toxicity of tebufenozide and methoxyfenozide, which
are known to be predominantly active against lepidopteran insects. For at least
three of the four BAH insecticides that are predominantly lepidopteran-specific,
the affinity of the BAH ecdysone agonists for the lepidopteran EcR was seen to
correlate directly with the toxicity manifested in that order of insects [19, 50] (see
also Ref. [5]). However, a similar correlation was not identified for halofenozide,
which despite a very weak binding affinity to EcRs from both Coleoptera and
Lepidoptera was toxic in select members of both orders of insect [4, 5, 20].
Although predominantly selective for lepidopteran larval pests, both tebufenozide
and methoxyfenozide do demonstrate limited toxicity towards some dipterans,
such as the midge, Chironomus tentans [46, 51], and a few mosquito species
[52, 53]. Interestingly, tebufenozide shows very disparate binding affinities to
EcRs from three different dipteran species, Drosophila melanogaster (Dm), Aedes
aegypti (Aa), and C. tentans (Ct). In fact, tebufenozide binds to bacterially produced
glutathione-S-transferase (GST)-fusions of DmEcR/DmUSP, AaEcR/AaUSP, and
CtEcR/CtUSP with determined Kd -values of approximately 300, 30, and 3 nM,
respectively, which were directly proportional to susceptibilities in that order
(C. tentans > A. aegypti > D. melanogaster; see Refs [4, 5]). The determined Kd -value
for tebufenozide binding to CtEcR/CtUSP was equal that of tebufenozide binding
to EcR/USP heterodimer from the spruce budworm, Choristoneura fumiferana. In
both cases, tebufenozide exhibited biological potency.
In the discovery of new compounds based on target-site assays, it is important to
bear in mind that the mere binding of a compound to its target site does not automat-
ically translate into an in vivo biological or toxic function. For example, even though
tebufenozide binds to DmEcR/DmUSP with submicromolar affinity (Kd ), which is
about twofold the Kd for 20E for the same receptor (Table 28.1.2), tebufenozide is
not toxic towards Drosophila larvae. Dhadialla and colleagues [5] (also unpublished
results) further investigated the significance of binding of tebufenozide to EcRs
from D. melanogaster, A. aegypti and the spruce budworm, Choristoneura fumiferana
(Cf), employing a limited proteolysis of different radiolabeled EcRs in EcR/USP
heterodimers following equilibrium binding with either muristerone A (a potent
ecdysteroid) or tebufenozide. The results showed that the binding of muristerone
A and tebufenozide to CfEcR/CfUSP and AeEcR/AeUSP would induce similar
conformational changes in the EcR (as indicated by protease-resistant EcR peptide
fragments of the same molecular size). In contrast, the binding of tebufenozide to
DmEcR/DmUSP did not afford any protease resistance (indicative of a lack of ligand
induced conformation), whereas muristerone A did. These results re-enforced the
concept that ligand–receptor interaction alone is insufficient for biological activity;
rather, such an interaction must result in a conformational change in the receptor
that leads to subsequent steps that are important for the biological manifestation
of the ligand.
Elucidation of the crystal structure of HvEcR/HvUSP heterodimeric LBDs in

the absence or presence of steroidal or nonsteroidal ligands [21] demonstrated
conclusively the binding of BAH in the EcR LBD. These results also allowed
verification of the pharmacophore structural requirements for both an ecdysteroid
(ponasterone A) and a nonsteroidal ecdysone agonist BAH (BY106830) interaction
with residues specifically in a lepidopteran EcR LBD (Figure 28.1.3a and b,
respectively). This also allowed verification of the CoMFA, 4-D-QSAR, and in-silico
docking of BAH or ecdysteroid molecules in homology models of EcR LBD that
were developed from the crystal structures of vertebrate steroid receptor LBDs
that had a <30% sequence homology to insect EcR LBDs. Based on the results
H5
V384
R383
H1
M380 R387
OH E309(O)
Y408 Q310
H6 OH
L396 2
3 P311
H7
H F397
M413 HO 14
6 β-sheet
HO O
20 A398(N)
OH
V416 22
T346 M342
L420
T343 I339
H11 N504
H3
W526
(a) H12
H5
M380 M381
S377 V384
Y408
M413 H3C CH3
H6
H7 O
V416 H
D419 Y403
N
L420 O N O
H10 L500 O CH3

H3C CH
Q503 N504 3 T343 T340
H11 M507 H3
W526 I339
(b) L511 H12
Figure 28.1.3 Hydrogen bond interac- the relative locations of HvEcR LBD helices.
tions of ponasterone A (a) and BAH, The hydrogen bonds formed by the two lig-
BY106830 (b) with amino acid residues in ands and the amino acid residues are shown
the ligand-binding cavity of H. viresence EcR by arrows. Adapted from Ref. [21].
are shown schematically. H1. . .H12 represent
(a)
(b)
Figure 28.1.4 (a) Relative positions and largely different spaces in the binding cavity
conformations of ponasterone A (yellow) and [more clearly shown in panel (b)] with over-
BY106830 (green) in the ligand-binding cavity lapping space for the side chain of ponas-
of HvEcR. The purple ribbons represent the terone A and the t-butyl group of BY106830.
helices surrounding the binding cavity. The Adapted from 1R1K.pdb and 1R20.pdb files
steroidal and nonsteroidal molecules occupy deposited by Ref. [21].
of crystal structure studies conducted by Billas et al. [21], it became apparent that
the two ligands would occupy distinctly different – but overlapping – cavities in
the EcR ligand-binding pocket (Figure 28.1.4). While ponasterone A localizes in a
long and slender conformation and a deeply buried cavity located distal to helix
12 in the EcR LBD, the BAH assumes a much more globular conformation in
a bulky V-shaped cavity, with an open cleft between H7 and H10 proximal to
H12 in the EcR LBD. The two ligands overlap over the t-butyl group of BAH
and the side chain of ponasterone A. The crystal structure results of EcR/USP
LBD heterodimer, when liganded with ponasterone A, showed that the steroid
binds with the fused A/B rings furthest away from helix 12 and the side chain
closest to helix 12. However, earlier homology modeling and ligand-docking studies
assumed the reverse orientation of the steroid molecule in the EcR ligand-binding
cavity [35, 55]. Additionally, while precrystal structure studies failed to predict an
important role for the 14-OH on the steroid molecule, the crystal structure showed
the interaction of 14-OH with not only one amino acid residue but two threonine
residues, T343 and T346 (Figure 28.1.3a). The strong interaction of the hydroxyl
groups, 2-OH and 3-OH, with residues R383 and E309, respectively, of the H1–H2
loop, H5 and the β-sheet brings stability to the conformation of H2, while the
interaction of 20-OH with Y408 on H6 is important (as previously predicted) for
boosting the in vivo activity of 20E by about 100-fold over that of ecdysone (E), which
lacks the 20-OH moiety. The three hydroxyl groups have long been known to be
important for ecdysteroid activity. The C-6 carbonyl on ponasterone A interacts with
A398.
The complexing of the BAH, BY106830, with the EcR LB cavity resulted in
slight differences in the conformation of EcR LBD, which were distinct from
those induced by the steroidal ponasterone A. BY106830, which lodges in a
different but slightly overlapping (with the side chain of ponasterone A) space
in the EcR ligand-binding cavity, destabilizes the helical conformation of H2 and
disrupts the interactions between the second and third β-sheet strands [21]. As
indicated in Figure 28.1.3b (derived from1R1K.pdb and 1R20.pdb files deposited
[21]), hydrogen bond interactions of this BAH are made on the A-ring carbonyl
with N504, B-ring carbonyl with T343, and NH group with Y408. The t-butyl
group lies in a hydrophobic cavity formed by residues from H3, H11, the H6/H7,
and the H11/H12 loops. Billas et al. [21] proposed that the basis of lepidopteran
specificity of BAH, specifically BY106830, would lie in the V384 residue in H5,
which is conserved in lepidopteran insect EcR LBDs but replaced by methionine
in all other insects. However, this does not explain the activity of some of the
lepidopteran-specific BAHs such as tebufenozide that bind strongly with EcRs
from a few dipterans (e.g., the midge, C. tentans and the mosquito, A. aegypti
[42, 49]; T.S. Dhadialla, unpublished results). Moreover, halofenozide, which has
only a 4-Cl substitution on the A-ring, binds both lepidopteran and coleopteran
EcRs, albeit less tightly than tebufenozide and methoxyfenozide bind to the
lepidopteran EcRs [4, 5, 19, 50].
Although tebufenozide and methoxyfenozide show a very high affinity for EcRs
from Lepidopteran and a few dipteran insects, 20E binds to EcRs from all insects to
manifest its effects. Kumar et al. [35] have shown that the binding of 20E to CfEcR
LBD could be eliminated by the mutation of a single residue, A110 (where alanine
is the 110th residue if numbering of residues is started from first residue in helix
one of LBD, which otherwise is A393 in the full-length CfEcR), to proline. Tests
of the binding and responsiveness of these single-point A110P-mutated CfEcR
LBD in ligand-binding and cell-based transactivation assays, respectively, indicated
that both ponasterone A and 20E were ineffective. However, although there was a
30% decrease in transactivation assays for methoxyfenozide, its ability to bind the
mutated EcR LBD was not affected. Interestingly, Kumar et al. [35] predicted the
amino acid residues for mutational analysis, based on homology models derived
from the crystal structures of vertebrate steroid receptor LBDs (<30% homology to
insect EcR LBDs) and ligand docking, before the crystal structure of HvEcR LBD
[21] became available. This was achieved despite the ecdysteroid being docked in
an orientation opposite to that revealed by crystal structure data.
The crystal structure of the LBD of the EcR from the whitefly, (Bt), has been
reported [22]. A comparison of the crystal structures of the BtEcR and HvEcR
LBDs revealed that the regions of the ligand-binding pocket surface in contact
with 20E were remarkably well conserved, not only in shape but also in their
overall hydrophobic and polar characteristics. BtEcR, when heterodimerized with a
heterologous USP, does not bind any of the commercial BAHs (despite the same
complex binding 20E or ponasterone A; T.S. Dhadialla, unpublished results). In
the absence of a BAH capable of binding the BtEcR/BtUSP complex, Carmichael
et al. [22] compared the crystal structures of BtEcR and HvEcR using 1.2 and 1.4 Å
probes. The results indicate flexibility in the wall of the HvEcR ligand binding
pocket close to where the side chain of bound 20E docks. How an active BAH,
as a nonsteroidal agonist of 20E, interacts with an EcR ligand-binding pocket
of an insect EcR depends on which insect EcR is the target. On binding of a

BAH to a ligand-binding pocket of HvEcR, the flexible wall extends outside. On
the other hand, the same extension of the ligand-binding pocket wall does not
take place for interaction of BAH and the BtEcR ligand-binding pocket. This
might perhaps explain the lack of binding of commercialized BAHs to the BtEcR
LBD.
A more recent review of insecticides that affect insect growth and disrupt their
development is available in Ref. [54].
28.1.5.1 Whole-Organism Effects

Commercialized BAH insecticides manifest their toxicity towards susceptible
insects almost exclusively via their ingestion; their contact-mediated toxicity is very
low, and only becomes apparent when the BAH is applied at 10-fold higher doses
than would induce oral toxicity. The effects of BAHs have been studied in several
susceptible insects of the Lepidoptera, Coleoptera, and Diptera [20, 25, 40, 55–63]
(for reviews, see Refs [4, 5]). In insects, the high metabolic stability of BAHs
compared to that of ecdysteroids, when coupled to their mode of action, creates the
condition of ‘‘hyperecdysonism’’ in susceptible insects following their intoxication
with BAHs [1]. This causes the susceptible larvae to stop feeding within 3 to 14 h
of their ingesting the BAH [56, 58, 64], although they do not actually die until two
to four days later; the cessation of feeding prevents further damage to the host
plant, however. After several hours of feeding inhibition, the intoxicated larva slips
its head capsule (this part of the molting process is prematurely induced by BAH
insecticides); this, in turn, prevents the intoxicated larva from completing the molt,
which would normally culminate in the larva eclosing from its old, digested cuticle
into a new one. Instead, the intoxicated larva remains moribund and ultimately
dies as a result of starvation, hemolymph loss due to hemorrhage, or predation.
Typically, when applied to susceptible insects, the BAH insecticides are most active
during the larval stages.
The effects of lethal doses of commercial BAH insecticides have been investigated
at the ultrastructural, biochemical, and molecular level in tissues of intoxicated
larvae. Ultrastructural studies have been conducted in the larvae of the beet
armyworm, Spodoptera exigua [58], spruce budworm, Choristoneura fumiferana [64],
and the Colorado potato beetle, Leptinotarsa decemlineata [5]. The results have shown
that, following the ingestion of tebufenozide or halofenozide by lepidopteran or
coleopteran larvae, respectively, synthesis of the new cuticle begins as soon as 3 h
after insecticide ingestion, and this is followed by apolysis of the new cuticle from
the old one. As the old cuticle is digested, the new cuticle is malformed, as indicated
by a disordered lamellate layering of the epicuticule proteins and vacuolation of the
epidermal cells secreting the new cuticle proteins. A comparison of the integument,
which consists of epidermal cells and the endo- and exo-cuticle, showed the
cuticle of the intoxicated larva to be dramatically thinner than that of a control
larva [5].
Differences at the biochemical and molecular levels between control and BAH
insecticide-treated larvae may further help to understand the mode of action of these
insecticides. During a normal molt, the rise and decline of 20E levels modulate
the expression and repression of certain genes, while the decline to basal levels of
20E results in a release of the eclosion hormone, allowing the eclosion behavior
to occur. Following treatment with tebufenozide, a rapid rise of the insecticide
levels in the larval hemolymph results in the expression of 20E-dependent genes.
However, owing to its much greater metabolic stability and potency than 20E, the
continued presence of tebufenozide in the hemolymph and the target tissues does
not allow for the regulation of those genes that normally depend on declining titers
of 20E. In addition, owing to the continued presence of tebufenozide (the same
most likely applies to other BAH insecticides) the eclosion hormone is not released,
so that the intoxicated larva is left midway through the molt process; that is, with a
malformed new cuticle, a slipped head capsule, and an inability to eclose from the
old cuticle. Retnakarn et al. [65] have reported that, in intoxicated spruce budworm
larvae, dopa-decarboxylase – an enzyme which is important for sclerotization and
tanning of the new cuticle – is not expressed. The expression of this enzyme is
normally suppressed by the presence of 20E, and in this case by tebufenozide,
which mimics 20E.
28.1.5.2 Basis for Selective Insect Toxicity of Bisacylhydrazine Insecticides

Of the four commercially available BAH insecticides, tebufenozide,
methoxyfenozide, and chromafenozide are selectively toxic to lepidopteran
larvae. Tebufenozide and methoxyfenozide have also been shown to have
insecticidal activity towards mosquito species [53, 56, 66] and the midge, C.
tentans [51]. Although the mode of action of BAH insecticides is manifested via
an interaction with the EcR, the reasons for their selective insecticidal activity
were initially confusing, as all insects have EcRs and almost all employ the same
ecdysteroid, 20E, as the molting hormone. As noted above, 20E manifests its
action via an interaction with the EcR LBD.
Three major causes have been investigated as the basis for the selective toxicity
of BAH insecticides, namely their metabolism, pharmacokinetics, and differences
in their target sites. By using 14 C-labeled RH-5992 (tebufenozide), both Smagghe
and Degheele [67] and T.S. Dhadialla and C.S. Thompson (unpublished results)
showed there to be no differences in the pharmacokinetics and metabolism of in-
gested RH-5992 in insect species that were either susceptible (e.g., the armyworms,
S. exigua, S. exempta) or nonsusceptible (Colorado potato beetle, L. decemlineata
and Mexican bean beetle, Epilachna verivesta) to this insecticidal BAH. Conse-
quently, attention was focused on the relative binding affinities of tebufenozide,
methoxyfenozide, and halofenozide to either EcRs in extracts from cell lines from
different insect Orders [4, 5], or proteins expressed from cloned cDNAs encod-
ing EcRs and USPs from insect species representing different Orders of insects.
The results obtained with both tebufenozide and methoxyfenozide were revealing.
While these compounds had an extremely high affinity for EcR proteins from
lepidopteran insects, the binding affinities for the two compounds fell by between
one and three orders of magnitude for EcR/USP heterodimers from insects that
were weakly or not at all susceptible to either tebufenozide or methoxyfenozide
(Table 28.1.2). Notably, the very high affinity of the insecticides for lepidopteran
EcRs correlated directly with their selective toxicity towards members of this insect
order. In cases where tebufenozide was active against a non-lepidopteran insect
(e.g., larvae of the midge, C. tentans), the same correlation was seen to hold. In
insects such as mosquitoes (e.g., Aedes aegypti), where tebufenozide is not very
active, its affinity for the EcR was intermediate between that for susceptible and
nonsusceptible insects (Table 28.1.2).
When Sundaram et al. [68] investigated other possible reasons for the selective
insect toxicity of tebufenozide, they found that both lepidopteran (C. fumiferana)
and dipteran (D. melanogaster) cell lines responded equally to 20E or ponasterone
A to generate the ecdysone-inducible genes, Hormone Receptor 3 (HR3) from C.
fumiferana or D. melanogaster, respectively. However, the two cell lines responded
differently to RH-5992. Other than the more than 100-fold higher binding affinity
of RH-5992 to CfEcR compared to DmEcR, the lepidopteran cells retained much
higher levels of RH-5992 than did the D. melanogaster cells. The results of this study
confirmed that the differential retention of RH-5992 in the two cell lines was due to
an active efflux mechanism in dipteran cells, which was temperature-dependent and
could be blocked with 10−5 M oubain (an inhibitor of Na+ , K+ -dependent ATPase).
It would be of interest to determine if similar Na+ , K+ -dependent ATPases were
also present in the cells of other dipterans such as C. tentans and A. aegypti, both
of which are significantly more susceptible to tebufenozide than D. melanogaster.
The presently available data indicate, however, that lepidopteran EcR affinities for
methoxyfenozide and chromafenozide are most likely the primary drivers for their
selective toxicity in lepidopteran larvae.
Whilst the very high affinities of tebufenozide, methoxyfenozide, and chro-
mafenozide for lepidopteran EcRs may help to explain the basis for their selective
lepidopteran toxicities, the same does not apply to the fourth BAH insecti-
cide, halofenozide, which is toxic to both lepidopteran and coleopteran larvae.
Halofenozide has a significantly reduced affinity for EcRs from the two insect
orders; rather, it appears that the relatively weak affinity of halofenozide to the EcR
of the target susceptible insect may be compensated by its increased metabolic
stability in the same insect.
28.1.6
Spectrum of Activity of Commercial Bisacylhydrazine Insecticides
Whilst a brief, but important, description of the spectrum of activity of the four BAH
insecticides is provided in the following subsections, more in-depth information,
together with specific bibliographies on these insecticides, are available in excellent
reviews [4, 5].1)
1) MIMIC™, CONFIRM™, ROMDAN™, FALCON™, and MACH 2™ are registered

RUNNER™, INTREPID™, PRODIGY™, Trademarks of Dow AgroSciences LLC.
28.1.6.1 Tebufenozide (MIMIC™; CONFIRM™; ROMDAN™; RH-5992),

Methoxyfenozide (RUNNER™; INTREPID™; PRODIGY™; FALCON™; RH-2485), and
Chromafenozide (MATRIC® ; KILLAT® ; ANS-118; CM-001)
All three of these insecticides are predominantly toxic towards lepidopteran lar-
vae. However, while tebufenozide is toxic to most lepidopteran species, it lacks
substantive commercial activity on Heliothis and Ostrinia species due to its lower
potency and poor systematic activity. Both, methoxyfenozide and chromafenozide,
in contrast, are active on major pests from the two genera. Chromafenozide is
registered for use against lepidopteran pests on vegetables, fruits, vines, tea, rice,
arboriculture, ornamentals, and other crops in Japan. Both, tebufenozide and
methoxyfenozide have been registered worldwide for lepidopteran pests on the
same crops, and also to treat lepidopteran pests in forests, and fruit and nut
trees. The typical usage rates for CONFIRM™ (tebufenozide) and RUNNER™
(methoxyfenozide) are in the range of 60 to 500 g a.i. ha−1 ; methoxyfenozide is
approximately twice as potent as tebufenozide.
These insecticides manifest their toxic effects primarily by ingestion, and are
only weakly active when applied topically. The feeding inhibition and lethal
effects of these insecticides are manifested during the insects’ larval stages.
Nonetheless, examples of sublethal reproductive or ovicidal effects of tebufenozide
and methoxyfenozide have been reported for some lepidopteran adults, or when
their eggs are deposited on surfaces treated with tebufenozide [59, 69–72].
28.1.6.2 Halofenozide (MACH 2™; RH-0345)

Unlike the three above-described BAH insecticides, halofenozide is more
soil-systemic and is active not only against lepidopteran but also coleopteran
larvae. It has been developed primarily for the control of beetle grub and
lepidopteran larval pests of turf in lawns and on golf courses. Halofenozide is
highly efficacious against the soil-dwelling larval stages of scarbaeid beetles such
as the Japanese beetle, Popillia japonica, the oriental beetle, Exomala orientalis,
and Phyllophaga, Cyclocephala, and Hyperodes spp., as well as various soil- or
sod-dwelling caterpillars such as cutworms and webworms [73–75]. Halofenozide
was not active on the Asiatic garden beetle, Maladera castanaea (Arrow), even at
high doses. The recommended rates for control of lepidopteran larvae and beetle
grubs for halofenozide (MACH 2™) are in the range of 0.9 kg a.i. acre−1 .
28.1.7
Ecotoxicological and Mammalian Reduced-Risk Profiles
The mammalian and ecotoxicological data for the four commercial BAH ecdysone
agonist insecticides are listed in Table 28.1.3. In 1998, methoxyfenozide was only
one of the four pesticide products to be awarded the ‘‘Presidential Green Chemistry
Award’’ by the US Government in recognition of outstanding chemical processes
and products that reduce negative impact on human health and the environment.
Both, tebufenozide and methoxyfenozide were registered by Environmental Pro-
tection Agency (EPA) under its Reduced Risk Pesticide Program. Both of these
Table 28.1.3 Mammalian and ecotoxicological reduced-risk

profiles of registered bisacylhydrazine insecticides.
Tebufenozide Methoxyfenozide Chromofenozide Halofenozide
Mammalian
Acute oral LD50 >5000 >5000 >5000 2850
(rat, mouse)
(mg kg−1 )
Acute dermal LD50 >5000 >2000 >2000 >2000
(mg kg−1 )
Eye irritation (rabbit) Non-irritating Non-irritating Slightly irritating Moderately
irritating,
positive for
contact
Dermal sensitization Non-sensitizer Negative Mildly sensitizing Allergy
(guinea pig)
Ames test Negative Negative Negative Negative
Acute inhalation >4.3 >4.3 – >2.7
(mg l−1 )
Reproduction (rat) No effect No effect No effect No effect
Ecotoxicological
Avian: mallard duck, >5000 >5620 – >5000
LC50 (8 day dietary)
(mg kg−1 )
Bobwhite quail, LC50 >5000 >5620 >5000 (Japanese 4522
(8 day dietary) quail, 14 day)
(mg g−1 )
Aquatic: bluegill 3.0 >4.3 – >8.4
sunfish, acute acute
LC50 (96 h) (mg l−1 )
Daphnia magna, 3.8 3.7 >189 (3 h) 3.6
acute EC50 (48 h)
(mg l−1 )
Honeybee (oral and 234 100 >100
contact) acute LC50 >100 (contact)
(μg per bee) >133 (oral)
Earthworm, LC50 1000 1213 >1000 980
(14 day) (mg kg−1 )
pesticides (as shown in Table 28.1.3) have low acute and chronic mammalian
toxicity, and are safe towards most beneficial arthropods. In fact, considering their
mode of action (ecdysone agonists), their highly selective toxicity to lepidopteran
larvae is amazing. When tested on 150 insect species from different insect Or-
ders (Lepidoptera, Hymenoptera, Coleoptera, Hemiptera, Diptera, Homoptera, and
Neuroptera), both tebufenozide and methoxyfenozide were devoid of any toxicity
to members of these insect orders, except for toxicity on a few of Dipteran species
such as the midge, C. tentans, and mosquito species (G. Carlson, unpublished
results). In separate studies, these insecticidal compounds were found to have very
little or no toxicity in several non-lepidopteran (Coleoptera, Homoptera, mites,
and nematodes) Orders [49, 76]. Both, tebufenozide and methoxyfenozide are also
nontoxic to bees.
28.1.8
Resistance Mechanisms and Resistance Potential
The history of insecticides has shown that, depending upon how an insecticide
is used, target insect species will inevitably develop some resistance to a given
insecticide at one time/place or another.
Since the initial use of tebufenozide during the mid-1990s, the first documented
case of codling moth (C. pomonella) resistance to tebufenozide was reported in
southern France by Sauphanor and Bouvier [77] and Sauphanor et al. [78], and
subsequently in the greenheaded leaf roller, Planotortrix octo, in New Zealand by
Wearing [79]. The resistance reported by Sauphanor and Bovier [77] seemed almost
too rapid from the initial time of launch of a product with a new insecticidal
mode of action. A major contributing factor for this resistance may have been
an existing multiresistant codling moth population following the extensive use
of other insecticides. Attempts to select laboratory colonies of beet armyworm
(Spodoptera exigua) that had been continuously exposed to sublethal amounts of
tebufenozide in the diet led to a selected strain crash after 12 generations of selective
pressure [80]. Both, in this study and in studies conducted at the author’s laboratory
with susceptible beet armyworm larvae, the oxidative metabolism of tebufenozide
was found to be the main route for detoxification [80] (also T.S. Dhadialla,
unpublished observations). Interestingly, the first few oxidative metabolites of
tebufenozide (mono-alcohols at the ethyl and methyl substitutions on the two
phenyl rings) continue to retain affinity to the ecdysone receptor, albeit much
lower than the parent (T.S. Dhadialla, unpublished observations). The oxidative
metabolism of tebufenozide in beet armyworm selected over six generations with
tebufenozide could be dramatically reduced with the use of piperonyl butoxide, an
inhibitor of cytochrome P450 mono-oxygenases, but not with DEF (S,S,S-tributyl
phosphorothioate), an esterase inhibitor [80]. These results supported the oxidative
metabolism of tebufenozide as being the main mechanism for detoxification and
resistance development.
Following reports of a decrease in the field efficacy of MIMIC™ insecticide
for the control of beet armyworm on vegetables outside Bangkok, Thailand,
Moulton et al. [81] amplified the resistance level in generations of field-collected

beet armyworm larvae to levels that reached 150-fold over the susceptible strain of
the same insect. The selected strain was about 120-fold lower in its susceptibility
to methoxyfenozide, which suggested a common mechanism of resistance. These
regions in Thailand, as in the south of France, have witnessed a rapid development
of resistance to insecticides with new and old modes of action, due to the insufficient
implementation of resistance management strategies.
While the available data have suggested oxidative metabolism as being the main
route for detoxification of at least two of the four BAH insecticides (tebufenozide
and methoxyfenozide), no evidence has been presented any of target-site
resistance.
28.1.9
Fufenozide: A New Bisacylhydrazine in Development
In addition to the four commercially available BAHs described above, a new

BAH analog – JH114 (fufenozide; 29), invented by the Jiangsu Pesticide Research
Institute Company Limited – has been described to have lepidopteran-specific
activity [82].
The synthesis of fufenozide was first reported in late 2001 [83]. As shown
in Scheme 28.1.5, the key intermediate in the sequence is the B-ring heterocycle-
2,7-dimethyl-2,3-dihydro-1-benzofuran-6-carboxylic acid (27). This can be efficiently
prepared from 3-hydroxy-2-methylbenzoate (25) via an O-alkylation with allyl chlo-
ride, followed by a thermal Claisen rearrangement and acid-catalyzed cyclization
and hydrolysis. The carboxylic acid is treated with thionyl chloride, followed by
t-butyl hydrazine hydrochloride (5), and the resulting carbohydrazide (28) is again
O O O
O Cl O (1) 200 °C
O O
O O
K2CO3 (2) H+
cyclohexane 27
25 26
(1) SOCl2 (2) N N
.HCl
5
O
O
O O N
N
O N
N SOCl2
28
29
COOH
11
Scheme 28.1.5 Synthesis of fufenozide.

regioselectively acylated with 3,5-dimethylbenzoyl chloride (from 11) to provide

technical-grade fufenozide (29).
In laboratory bioassays, fufenozide was shown to be active against larvae of
the diamond-back moth (Plutella xylostella), corn borer (Ostrinia furnaclis), and
the rice stem borer (Chilo suppressalis). In leaf dip feeding assays, the activities
of fufenozide and RH5992 (tebufenozide) were similar for control of the third
instar larvae of diamond-back moth (LC50 = 69 mg kg−1 ). However, in the case
of the third instar larvae of corn borer, fufenozide was significantly more effec-
tive than tebufenozide (LC50 = 48.69 and 74.1 mg kg−1 , respectively). In parallel
tests, albeit with topical application, tebufenozide and fufenozide showed similar
activities (LC50 = 0.02 μg per insect) against the fourth instar larvae of rice stem
borer.
In limited field trials, fufenozide was shown to provide control for the beet
armyworm (Spodoptera exigua) and diamond-back moth larvae.
In toxicology tests in rats, the acute oral LD50 and acute dermal LD50 of fufenozide
were both > 5000 mg kg−1 . Fufenozide did not show any mutagenic effect in the
Ames test, and was non-irritant in rabbit eye and skin irritancy tests. Currently,
no information is available regarding the commercialization of this insecticidal
BAH.
28.1.10
Other Chemistries and Potential for New Ecdysone Agonist Insecticides
At least two other chemotypes – tetrahydroquinolines (Figure 28.1.5, 30) [84, 85]
and amidoketones (Figure 28.1.5, 31) [86, 87] (also reviewed in Ref. [6]) – have
been shown to interact either directly (via ligand-binding assays) or indirectly
(via cell-based reporter gene transactivation assays) with the EcR. These new
EcR-binding chemistries may lead to new products capable of controlling insect
pests that cannot be controlled with currently available BAH insecticides.
Y O R1 R2
C
O
HN N
A H
X A B B
X
N
Y
O D Z
25 26
Figure 28.1.5 Generalized structures of rings in the two chemotypes. R1 and R2 can
two additional chemotypes, tetrahydroquino- be 4- or 5-attached carbons, either as two
lines (25) and amidoketones (26), that bind acyclic substituents or, preferably, as a five-
EcRs from several insects. X, Y, and Z rep- or six-membered ring (reviewed in Ref. [6]).
resent different substitutions on the phenyl
28.1.11
Conclusions and Future Prospects of Ecdysone Agonist Chemistries
The BAHs are well-understood insecticides in terms of their mode of action

at physiological, biochemical, and molecular (including crystal structures of
ligand–receptor) levels. The availability of the crystal structures of EcR LBDs
in the absence and presence of ligands (both steroidal and nonsteroidal), and the
availability of at least four different chemotypes (20E, BAH, tetrahydroquinolines,
and amidoketones), offers great potential for the rational design and discovery
of new nonsteroidal ecdysone agonist insecticides with activities against different
spectra of pests. Owing to the novel mode of action of BAH insecticides, their
insect selectivity and reduced-risk ecotoxicity and mammalian toxicity profiles,
these insecticides are ideally suited for use in integrated insect resistance and
management programs.
An additional potential use of the registered BAH insecticides (not discussed in
this chapter) is to apply these agents to the area of gene switching, to regulate genes
or traits in mammalian or plant systems. Details of the use of tebufenozide and
methoxyfenozide for ligand-dependent gene expression in plant and mammalian
systems in which the EcR-based gene switch was reconstituted are available in two
excellent reviews [5, 88].
Acknowledgments
The authors thank David Demeter for creating Figure 28.1.4 from the 1R1.pdf and
1R20.pdf files deposited by Billas et al. [21], Mark Hertlein and Steve Evans for their
critical reading of the manuscript, and W. Kleschick for his support and approval
for the writing of this chapter.
References
1. Williams, C.M. (1967) Biol. Bull., 121, Gilbert, K. Iatrou, and S.S. Gill), Elsevier
572–585. Pergamon, New York, pp. 55–100.
2. Retnakaran, A., Grannet, J., and 6. Dinan, L. and Hormann, R.E. (2005) in
Ennis, T. (1985) in Comprehensive Insect Comprehensive Insect Molecular Science,
Physiology, Biochemistry and Pharmacol- vol. 3 (eds L.I. Gilbert, K. Iatrou, and
ogy, vol. 12 (eds G.A. Kerkut and L.I. S.S. Gill), Elsevier Pergamon, New York,
pp. 198–236.
Gilbert), Pergamon Press, Oxford, pp.
7. Watkinson, L.A. and Clarke, B.S.
529–601.
(1973) Proc. Natl Acad. Sci. USA, 19
3. Staal, G.B. (1975) Annu. Rev. Entomol.,
(4), 488–509.
20, 407–460.
8. Dinan, L. (1989) in Ecdysone: From
4. Dhadialla, T.S., Carlson, G.R., and Le, Chemistry to Mode of Action (ed. J.
D.P. (1998) Annu. Rev. Entomol., 43, Koolman), Thieme Verlag, Stuttgart,
545–569. pp. 345–354.
5. Dhadialla, T.S., Retnakaran, A., and 9. Hsu, A.C.-T. (1991) in Synthesis and
Smagghe, G. (2005) in Comprehensive Chemistry of Agrochemicals, II, ACS Sym-
Insect Molecular Science, vol. 6 (eds L.I. posium Series, 443 (eds D.R. Baker, J.G.
References 981
Fenyes, and W.K. Moberg), American 25. Reiji, I., Shinya, N., Takashi, O.,
Chemical Society, Washington, DC, pp. Keiji, T. et al. (2000) Annu. Rep. Sankyo
478–490. Res. Lab., 52, 59–62.
10. Wing, K.D. (1988) Science, 241, 26. Rohm and Haas Company (1995) Euro-
467–469. pean Patent 0,639,559(A1).
11. Wing, K.D., Slawecki, R., and 27. Rohm and Haas Company (2000) US
Carlson, G.R. (1988) Science, 241, Patent 6,124,500.
470–472. 28. Rohm and Hass Company (1996) US
12. Oberlander, H., Silhacek, D.L., and Patent 5,530,028.
Porcheron, P. (1995) Arch. Insect 29. Swada, Y., Yanai, T., Nakagawa, H.,
Biochem. Physiol., 28, 209–223. Tsukamoto, Y., Yokoi, S. et al. (2002)
13. Riddiford, L.M. (1996) Arch. Insect Pest Manage. Sci., 59, 36–48.
Biochem. Physiol., 32, 271–286. 30. Horn, D.H.S. and Begamasco, R. (1985)
14. Goodman, W.G. and Granger, N.A. in Comprehensive Insect Physiology Bio-
(2005) in Comprehensive Insect Molecular chemistry and Pharmacology, vol. 7 (eds
Science, vol. 3 (eds L.I. Gilbert, K. Iatrou, G.A. Kerkut and L.I. Gilbert), Pergamon
and S.S. Gill), Elsevier Pergamon, New Press, Oxford, pp. 185–248.
York, pp. 319–408. 31. Dinan, L., Savchenko, T., Whiting, P.,
15. Zitnan, D. and Adams, M.E. (2005) in and Sarkar, S.D. (1999) Pestic. Sci., 55,
Comprehensive Insect Molecular Science, 331–335.
vol. 3 (eds L.I. Gilbert, K. Iatrou, and 32. Nakagawa, Y., Hattori, K.,
S.S. Gill), Elsevier Pergamon, New York,
Minukuchi, C., Kugimiya, S., and Ueno,
pp. 1–60.
T. (2000) Steroids, 65, 117–123.
16. Truman, J.W., Rountree, D.B.,
33. Saez, E., Nelson, M.C., Eshelman, B.,
Reiss, S.E., and Schwartz, L.M. (1983) J.
Banayo, E., Koder, A. et al. (2000) Proc.
Insect Physiol., 29, 895–900.
Natl Acad. Sci. USA, 97, 14512–14517.
17. Zitnanova, I., Adams, M.E., and
34. Wurtz, J.-M., Guillot, B., Fagart, J.,
Zitnan, D. (2001) J. Exp. Biol., 204,
Moras, D., Tietjen, K. et al. (2000)
3483–3495.
Protein Sci., 9, 1073–1084.
18. Henrich, V.C. (2005) in Comprehensive
35. Kumar, M.B., Fujimoto, T.,
Insect Molecular Science, vol. 3 (eds L.I.
Potter, D.W., Deng, Q., and Palli, S.R.
Gilbert, K. Iatrou, and S.S. Gill), Elsevier
(2002) Proc. Natl Acad. Sci. USA, 99,
Pergamon, New York, pp. 243–286.
19. Minakuchi, C., Nakagawa, Y., 14710–14715.
Kamimura, M., and Miyagawa, H. 36. Toya, T., Yamaguchi, K., and Endo,
(2003) Eur. J. Biochem., 270 (20), Y. (2002) Bioorg. Med. Chem. Lett., 10,
4095–4104. 953–961.
20. Ogura, T., Minakuchi, C., Nakagawa, Y., 37. Nakagawa, Y., Takahashi, K.,
Smagghe, G., and Miyagawa, H. (2005) Kishikawa, H., Ogura, T., Minakuchi, C.,
FEBS J., 272, 4114–4128. and Miyagawa, H. (2005) Bioorg. Med.
21. Billas, I.M.L., Twema, T., Garnier, J.-M., Chem. Lett., 13, 1333–1340.
Mitschler, A., Rochel, N. et al. (2003) 38. Wheelock, C.E., Nakagawa, Y.,
Nature, 426, 91–96. Harada, T., Oikawa, N., Akamatsu, M.,
22. Carmichael, J.A., Lawrence, M.C., Smagghe, G. et al. (2006) Bioorg. Med.
Graham, L.D., Pilling, P.A., Epa, Chem. Lett., 14, 1143–1159.
V.C. et al. (2005) J. Biol. Chem., 280, 39. Keiji, T., Yoshihisa, T., Yoshihiro, S.,
22258–22269. Atsushi, K., Hiroki, H., Reiji, I. et al.
23. Aller, H.E. and Ramsay, J.R. (1988) (2001) Annu. Rep. Sankyo Res. Lab., 53,
Brighton Crop Prot. Conf. – Pests Dis., 2, 1–49.
511–518. 40. Mikio, Y. (2000) Agrochem. Jpn, 76,
24. Yanagi, M., Watanabe, T., Masui, A., 16–18.
Yokoi, S., Tsukamoto, Y. et al. (2000) 41. Sawada, Y., Yanai, T., Nakagawa, H.,
Proc. Brighton Crop Prot. Conf. – Pests Tsukamoto, Y., Yokoi, S. et al. (2003)
Dis., 2, 27–32. Pest Manage. Sci., 59, 25–35.
42. Sawada, Y., Yanai, T., Nakagawa, H., Brownright, A.J. et al. (1997) J. Insect
Tsukamoto, Y., Yokoi, S. et al. (2003) Physiol., 43, 55–68.
Pest Manage. Sci., 59, 36–48. 60. Retnakaran, A., Krell, P., Feng, Q., and
43. Sawada, Y., Yanai, T., Nakagawa, H., Arif, B. (2003) Arch. Insect Biochem.
Tsukamoto, Y., Tamagawa, Y. et al. Physiol., 54, 187–199.
(2003) Pest Manage. Sci., 59, 49–57. 61. Carton, B., Heirman, A., Smagghe, G.,
44. Clement, C.Y., Bradbrook, D.A., and Tirry, L. (2000) Med. Fac. Land-
Lafont, R., and Dinan, L. (1993) Insect bouww. Univ. Gent., 65, 311–322.
Biochem. Mol. Biol., 23, 187–193. 62. Borchert, D.M., Walgenbach, J.F.,
45. Smagghe, G., Braeckman, B.P., Kennedy, G.G., and Long, J.W. (2004) J.
Huys, N., and Raes, H. (2003) J. Appl. Econ. Entomol., 97, 1342–1352.
Entomol., 127, 167–173. 63. Seth, R.K., Kaur, J.J., Rao, D.K., and
46. Spindler-Barth, M., Turberg, A., and Reynolds, S.E. (2004) J. Insect Physiol.,
Spindler, K.-D. (1991) Arch. Insect 50, 505–517.
Biochem. Physiol., 16, 11–18. 64. Retnakaran, A., Macdonald, A.,
47. Sohi, S.S., Palli, S.R., and Tomkins, W.L., Davis, C.N.,
Retnakaran, A. (1995) J. Insect Physiol., Brownright, A.J. et al. (1997) J. Insect
41, 457–464. Physiol., 43, 55–68.
48. Dhadialla, T.S. and Tzertzinis, G. 65. Retnakaran, A., Hiruma, K., Palli, S.R.,
(1997) Arch. Insect Biochem. Physiol., and Riddiford, L.M. (1995) Insect
35, 45–57. Biochem. Mol. Biol., 25, 109–117.
49. Trisyono, A., Goodman, C.L.,
66. Darvas, B., Pap, L., Kelemen, M., and
Grasela, J.J., McIntosh, A.H., and
Laszlo, P. (1998) J. Econ. Entomol., 91,
Chipendale, G.M. (2000) In Vitro Cell.
1260–1264.
Dev. Biol. Anim., 36, 400–404.
67. Smagghe, G. and Degheele, D. (1994)
50. Minakuchi, C. (2005) J. Pest. Sci. (Tokyo,
Japan), 30, 233–238.
68. Sundaram, M., Palli, S.R., Krell, P.J.,
51. Smagghe, G., Dhadialla, T.S., and Lezzi,
Sohi, S.S., and Dhadialla, T.S. (1998)
M. (2002) Insect Biochem. Mol. Biol., 32,
Insect Biochem. Mol. Biol., 28, 693–704.
187–192.
69. Sun, X., Song, Q., and Barrett, B.
52. Boudjelida, H., Bouaziz, A., Soin, T.,
(2003) Arch. Insect Biochem. Physiol.,
Smagghe, G., and Soltani, N. (2005)
Pestic. Biochem. Physiol., 83, 115–123. 52, 115–129.
53. Beckage, N.E., Marion, K.M., 70. Sun, X., Barrett, B., and Song, Q. (2004)
Walton, W.E., Wirth, M.C., and Tan, J. Econ. Entomol., 39, 417–425.
F.F. (2004) Arch. Insect Biochem. Physiol., 71. Knight, A.L. (2000) J. Econ. Entomol., 93,
57, 111–122. 1760–1767.
54. Dhadialla, T.S. (2010) in Insect Control 72. RohMid LLC (1996) Technical Informa-
(eds L.I. Gilbert and S.S. Gill), Academic tion Bulletin 9.
Press, San Francisco, CA, pp. 182–184. 73. Cowles, R.S. and Villani, M.G. (1996) J.
55. Kasuya, A., Sawada, Y., Tsukamoto, Y., Econ. Entomol., 89, 1556–1565.
Tanaka, K., Toya, T., and Yanagi, M. 74. Cowles, R.S., Alm, S.R., and
(2003) J. Mol. Model., 9, 58–65. Villani, M.G. (1999) J. Econ. Entomol.,
56. Slama, K. (1995) Eur. J. Entomol., 92, 92, 427–434.
317–323. 75. Carlson, G.R., Dhadialla, T.S.,
57. Smagghe, G., Vinuela, E., Budia, F., Hunter, R., Jansson, R.K., Jany, C.S.
and Degheele, D. (1996) Arch. Insect et al. (2001) Pest. Manage. Sci., 57,
Biochem. Physiol., 32, 121–134. 115–119.
58. Smagghe, G., Eelen, H., Verschelde, E., 76. Medina, P., Budia, F., Tirry, L.,
Richter, K., and Degheele, D. (1996) Smagghe, G., and Vinuela, E. (2001)
Insect Biochem. Mol. Biol., 26, 687–695. Biocontrol Sci. Technol., 11, 597–610.
59. Retmakaran, A., Macdonald, 77. Sauphanor, B. and Bouvier, J.C. (1995)
A., Tomkins, W.L., Davis, C.N., Pestic. Sci., 45, 369–375.
78. Sauphanor, B., Bouvier, J.C., and Chiu, G. et al. (2000) A new class
Brosse, V. (1998) J. Econ. Entomol., of potent ecdysone agonists:
91, 1225–1231. 4-phenylamino-1,2,3,4-tetrahydro-
79. Wearing, C.H. (1998) Pestic. Sci., 54, quinolines. Presentation at the Mid-
203–211. dle Atlantic Regional ACS Meeting,
80. Smagghe, G., Dhadialla, T.S., University of Delaware, May 15–16,
Derycke, S., Tirry, L., and Degheele, 2000.
D. (1998) Pestic. Sci., 54, 27–34. 85. Tice, C.M., Hormann, R.E., Thompson,
81. Moulton, J.K., Pepper, D.A., C.S., Friz, J.L., Cavanough, C.K. et al.
Jansson, R.K., and Dennehy, T.J. (2002) (2003) Bioorg. Med. Chem. Lett., 13,
J. Econ. Entomol., 95, 414–424. 475–447.
82. Zhang, X., Li, Y., Ni, Y., Zhu, L., Hu, J., 86. Tice, C.M., Hormann, R.E.,
and Jiang, M. (2003) Pesticides, 42 (12), Thompson, C.S., Friz, J.L., Cavanough,
18–20. C.K. et al. (2003) Bioorg. Med. Chem.
83. Zhang, X., Li, Y., Zhu, L., Liu, L., Lett., 13, 1883–1886.
Sha, X., Xu, H., Ma, H., Wang, F., 87. Palli, S.R., Hormann, R.E.,
Ni, Y., and Guo, L. (2001) Preparation of Schlattner, U., and Lezzi, M. (2005)
diacylhydrazines insecticides and their Vitam. Horm., 73, 59–100.
intermediates. Faming Zhuanli Shenqing 88. Smith, H.C., Cavanough, C.K., Friz, J.L.,
Gongkai Shuomingshu, 11. Thompson, C.S., Saggers, J.A. et al.
84. Dixson, J.A., Elshenawy, Z.M., (2003) Bioorg. Med. Chem. Lett., 13,
Eldridge, J.R., Dungan, L.B., 1943–1946.
28.2
Pyriproxyfen: A New Juvenoid
Makoto Hatakoshi
28.2.1
Introduction
Pyriproxyfen is an insect growth regulator (IGR) which disrupts insect development

at specific stages, and is classified among the IGR group as a ‘‘juvenoid.’’ During
their growth, insects undergo molting and metamorphosis, both of which are
controlled by the endocrine system. The activity of the endocrine system has
been studied in detail in many insect species, especially lepidopterans [1]; indeed,
it has been shown that, in insects, many phenomena – including reproduction,
egg development, phase polymorphism, diapause, and pheromone synthesis – are
controlled by the endocrine system.
As juvenoids are known to affect the insect-specific endocrine system, they would
likewise be expected to demonstrate insect-specific insecticidal actions [2]. Certain
naturally occurring compounds have been shown to possess juvenoid activity;
examples include farnesol (1) and farnesal (2) (Figure 28.2.1), both of which have
been isolated from the feces of the mealworm, Tenebrio molitor [3].
Subsequently, it was confirmed [4] that farnesol and its related compounds
demonstrated juvenoid activity against the bloodsucking bug, Rhodnius prolixus,
while paper products created from the balsam fir, Abies balsamea, showed juvenoid
activity against the hemipteran bug, Pyrrhocoris apterus [5]. This juvenoid compound
Farnesol 1
CH2OH
Farnesal 2
CHO
Juvabione 3 O
(paper factor)
COOCH3
Figure 28.2.1 Chemical structures of naturally occurring juvenoids.
was subsequently named the ‘‘paper factor,’’ and it chemical structure confirmed
as the methyl ester of todomatuic acid, juvabione (3) [6].
Although many compounds with juvenoid activity have been identified in plants,
they tend to be impractical as insecticides due to their chemical instability and
complexity of synthesis. Nonetheless, a wide variety of juvenoid compounds have
been synthesized and their activities monitored against insects in attempts to create
more active and stable compounds. The details of juvenoids reported to date are
provided in the following sections.
28.2.2
History of Juvenoid Research
Following the discovery by Bowers [7] that some compounds used as insecticide
synergists (e.g., piperonyl butoxide) possessed juvenoid activity, a group of synergist
analogs was synthesized that included aromatic terpenoid ether compounds. When
the morphogenetic activity of these analogs was subsequently examined against
T. molitor and the milkweed bug, Oncopeltus fasciatus, the first synthetic compound
(4) with high activity was identified (Figure 28.2.2) [8].
As a consequence of this finding, a variety of compounds in which different
substituents were introduced into the phenyl ring and/or the side chain was altered
were produced; among these compounds, the 4-ethylphenyl ether (5) was found to
have a high juvenoid activity [9].
In contrast, the research group at Zoecon Corporation identified a high juvenoid
activity in the alkyl (2E,4E)-3,7,11-trimethyl-2,4-dodecadienoates, some of which
were subsequently commercialized as methoprene (6) (ZR-515) [10], kinoprene (7)
(ZR-777) [11], and hydroprene (8) (ZR-512) [10]. Unfortunately, as these compounds
possessed double bonds and an ester bond in the molecule, they could not be applied
in the open field, where an essential requirement for their use is stability against
sunlight.
This problem was overcome, however, by exchanging the unstable terpenoid
structure with a 4-phenoxyphenyl group such that fenoxycarb (9), a carbamate with
a 4-phenoxyphenyl group, was the first juvenoid to be developed for full agricultural
use [12]. Later, the ether compound pyriproxyfen (10) was developed by Sumitomo
Chemical [13].
Aromatic terpenoid ether 4 O O

O
4-Ethylphenyl ether 5 O
O
O
O
Methoprene 6 O
O
Kinoprene 7 O
O
Hydroprene 8 O
O
O
N O
Fenoxycarb 9 H
O
O
O N
Pyriproxyfen 10
O
Figure 28.2.2 Chemical structures of some synthetic juvenoids.
The major insect targets of the juvenoids are listed in Table 28.2.1. In the
case of methoprene, these include mosquitoes, sciarid flies, storage pests, filter
flies, and midges. As methoprene has an asymmetric carbon atom at C-7, optical
isomers will be present in these juvenoids; indeed, it appears that the (S)-form
[(S)-methoprene] has a higher level of insecticidal activity [14] and is today used in all
such products. In the case of kinoprene-containing products, the active ingredient
is also the (S)-form, and this is used to control aphids, thrips, whiteflies, scales,
and mealybugs. For hydroprene, (S)-hydroprene is used in products to control
cockroaches, fruit and drain flies, bedbugs, and storage pests. Fenoxycarb is used
to control lepidopteran insects on fruits and grapes, while pyriproxyfen is applied
for the control mainly of whiteflies on vegetables and cotton, and scales on citrus
fruits.
28.2.3
Process of Pyriproxyfen Research
Until 1981, when investigations into the juvenoids were first started at Sumit-
omo Chemical, studies of insecticidal action had been largely focused on the
Table 28.2.1 Commercialized juvenoids and their target insects.
Common name Trade name Target insect(s)
(S)-Methoprene Altosid Mosquitoes

Apex Sciarid flies (mushrooms)
Diacon II Storage pests (e.g., L. serricorne)
Strike Filter flies, midges
(S)-Kinoprene Enstar II Aphids, thrips, whiteflies
(greenhouse)
(S)-Hydroprene Gentrol Cockroaches
Fenoxycarb Insegar Lepidoptera (fruits, grapes)
use pyrethroids, which not only acted quickly on the insects but also had a
wide insecticidal spectrum. A major problem that arose with the pyrethroids,
however, was that the insects were becoming increasingly resistant, due mainly
to the frequent use of these compounds. Clearly, from the viewpoint of inte-
grated pest management (IPM), there was a need to develop highly selective
insecticides.
Following synthesis of the thiolcarbamate (11; Figure 28.2.3) in 1981, the primary
screening studies failed to demonstrate any insecticidal activity, other than against
spider mites. However, exposure of the tobacco cutworm, Spodoptera litura, to the
thiolcarbamate caused the worm’s body color to be changed to red. This led to
the presumption that the compound was a juvenoid, and possessed a completely
different mechanism of action compared to conventional insecticides. This action
was subsequently confirmed using the Galleria wax test [15].
Following the screening of many analogs, and the creation of an evaluation
system using the larvae of the common mosquito (Culex pipiens pallens) and
housefly (Musca domestica) – which at the time were the main target insects of
juvenoids – compound 11 was selected as the most active, although it failed to
demonstrate a sufficient efficacy against mosquito larvae in the field. When, as a
result of these investigations, the oxime ether compound 12 (Figure 28.2.3) – which
has the same juvenoid action – was quickly identified, it was clear that this chemical
group had a remarkably high inhibitory activity against adult emergence, as
O
O
S N
Thiolcarbamate 11
O
O N
O
Oxime ether 12
O
Figure 28.2.3 Chemical structures of 4-phenoxyphenyl ju-

venoids discovered in the early stage of research at Sumit-
omo Chemical.
demonstrated in a series of laboratory tests [16, 17]. Nonetheless, the residual

efficacy against mosquito larvae in field tests remained unsatisfactory, and further
investigations were conducted to identify more stable compounds [17]. Based on
previous results, it was deduced that the thiolcarbamate and oxime ether groups
controlled the stability of the compound. Consequently, in an effort to improve
the chemical stability, the synthesis of heterocyclic compounds that would cyclize
these groups was initiated in 1982; in the following year, pyriproxyfen was selected
as a candidate compound with a high IGR activity and a remarkably improved
stability [18]. Subsequently, a variety of laboratory tests were conducted using
both household [19–21] and agricultural insect pests [22–27]. This, in turn, led to
the quest for further promising target insects, with which many field trials were
conducted.
28.2.4
Activity of Optical Isomers
The existence of an asymmetric carbon atom in the pyriproxyfen molecule led

to a series of investigations of the insecticidal activities of the optical isomers,
using larvae of the housefly, M. domestica. Typically, the activity ratio of the (R)-
and (S)-forms was about 1 : 9 (R : S), with the (S)-form showing the higher level of
activity (Table 28.2.2).
28.2.5
Mechanism of Action
Pyriproxyfen acts only at specific growth stages to control insects by juvenoid

activity [28], namely the early egg stage, the last instar larva, the pupa, and the adult.
Thus, the action of pyriproxyfen involves: (i) an inhibition of egg hatching (ovicidal
activity); (ii) an inhibition of metamorphosis; (iii) an inhibition of adult emergence;
and (iv) an inhibition of reproduction (decreased number of eggs oviposited and/or
decreased hatchability) [29]. It should be noted that the mechanism of action of
pyriproxyfen is dependent on the insect species involved, and that there is no
Table 28.2.2 Inhibition of adult emergence of optical

isomers of pyriproxyfen against larvae of housefly, Musca
domestica.
Compound e.e. (%) R/S IC50 (ppm)a
Pyriproxyfen – 50/50 0.017

(R)-Pyriproxyfen 99.4 99.7/0.3 0.068
(S)-Pyriproxyfen 96.3 1.85/98.15 0.0090
a
Half-inhibitory concentration of adult emergence.
e.e., enantiomeric excess.
general tendency of mechanism; rather, the mechanism should be identified for

each insect species.
As the molecular mode of action of juvenoids is unknown, the endocrine
mechanism of supernumerary larval molt [30] is employed in the following
subsection as an example of the mode of action of pyriproxyfen.
The application of various amounts of pyriproxyfen to day 0 last instar larvae of
the tobacco cutworm, S. litura, caused the larvae to molt into pupae, seventh instar
larvae (super larva), or larval–pupal intermediates, depending on the dose applied
(Table 28.2.3). Notably, the last instar larval period was also affected (Table 28.2.3).
Although the untreated last instar larval period was 5.6 ± 0.7 days (mean ± SE),
an increase in the pyriproxyfen dose (to 100 μg per insect) led to a prolongation
(to 9.1 ± 1.1 days) of this period in individuals that molted into pupae and
larval–pupal intermediates. In contrast, a supernumerary larval molt was ob-
served at a dose level of more than 3 μg, and all treated larvae molted into seventh
instar supernumerary larvae at a dose of 100 μg. The larval period of individuals
that molted into super larvae was about four days, irrespective of the dose level.
Notably, the super larvae obtained continued to feed, such that their average body
weight reached about 1.9 g (typically, the average maximum body weight of an
untreated last instar larvae is ca. 0.8 g).
When changes in the ecdysteroid titer of the hemolymph of untreated and
pyriproxyfen-treated (100 μg) last instar larvae were investigated [13], a peak level
of 100 ng ml−1 was seen in pyriproxyfen-treated larvae on day 3, whereas a peak of
300 ng ml−1 was noted on day 4 in untreated larvae. Clearly, as ecdysone secretion
Table 28.2.3 Effects of pyriproxyfen on the development of

last instar larvae of Spodoptera litura.a
Dose N Molted into (%) Sixth instar larval periodb

(μg/larva)
Seventh L/Pc Pupa Seventh L/P + Pupa
100 14 100 0 0 4.4 ± 0.6 –

15 60 20 20 4.0 ± 0.5 9.0 ± 1.1
30
14 7 50 43 4.0 7.9 ± 0.9
10
15 7 53 40 4.0 7.1 ± 0.7
3
15 0 80 20 – 6.4 ± 0.6
1
15 0 67 33 – 5.7 ± 0.5
0.3
Untreated 20 0 0 100 – 5.4 ± 0.5
control
a
Day 0 last instar larvae were treated with pyriproxyfen.
b
Mean ± SE (day).
c
Larval–pupal intermediate.
by the prothoracic gland of pyriproxyfen-treated larvae was only one-third that of

untreated larvae one day earlier, pyriproxyfen had affected the secretory activity of
the prothoracic glands, either directly or indirectly.
As the timing of ecdysone release was shown to be affected by pyriproxyfen,
ligation between the head and thorax was conducted in untreated and 100 μg
pyriproxyfen-treated larvae to investigate the release schedule of prothoracicotropic
hormone (PTTH), which triggers ecdysone release. As a consequence, PTTH was
released at 13:40 on day 3 in untreated larvae, and at 10:00 on day 2 in treated larvae
(almost 28 h earlier); in other words, pyriproxyfen has a stimulatory effect on the
brain to cause an accelerated release of PTTH.
The location of brain cells containing PTTH was confirmed by staining the
neurosecretory substance with paraldehyde-fuchsin; this led to two pairs of large
neurosecretory cells (ca. 20 μm wide) and two pairs of small neurosecretory cells
(ca. 10 μm wide) being identified in the pars intercerebrum of the brain. Moreover,
staining of the large cells was seen to change with insect development. Although
staining of the large cells was barely changed between days 0 and 2 in untreated
larvae, it was decreased rapidly on day 3. In contrast, while staining of the large
cells in pyriproxyfen-treated larval brain was similar on days 0 and 1 to that in
untreated larvae, it decreased rapidly on day 2. Based on these results, PTTH
was considered to be released from two pairs of large neurosecretory cells in the
pars intercerebrum of the brain. The presence of PTTH was also reported in
one pair of lateral neurosecretory cells of the tobacco hornworm (Manduca sexta)
[31], in five pairs of the dorsolateral part of the protocerebrum of the waxmoth
(Galleria mellonella) [32], and in four pairs of dorsomedial neurosecretory cells of
the silkworm (Bombyx mori) [33].
100 1000
14
Pyriproxyfen (ng/ml)
Ecdysteroid (ng/ml)
Staining of NSC
100
50
9
10
Pyriproxyfen
0 1
0 1 2 3
Day of last instar
Figure 28.2.4 Effects of topically applied pyriproxyfen (trian-

gles) on the staining of neurosecretory cells (NSC; squares),
PTTH release (arrows) and ecdysteroid titer (circles) in the
last instar larvae of Spodoptera litura. Filled symbols indicate
untreated larvae; open symbols indicate 100 μg pyriproxyfen
treatment on day 0.
When pyriproxyfen titers in the hemolymph were monitored using gas

chromatography-mass spectrometry (GC-MS), a titer of 144.7 ng ml−1 pyriprox-
yfen was seen to occur just after treatment, to reach a peak of 992.3 ng ml−1
on day 1, and to fall to 319.3 and 21.5 ng ml−1 on days 2 and 3, respectively
(see Figure 28.2.4). It was considered that the last instar larvae treated with
pyriproxyfen had a high concentration of pyriproxyfen in the hemolymph on day
2 when PTTH was released. Based on these results, if a quantity of pyriproxyfen
were to be administered to day 0 last instar larvae of the tobacco cutworm and to
persist in the hemolymph for some time, the brain would release PTTH about one
day earlier. The prothoracic glands would secrete ecdysone in the same pattern as
seen in fifth instar larvae in the presence of pyriproxyfen, in order to induce a
supernumerary larval molt.
28.2.6
Biological Activity
As pyriproxyfen is used mainly to control agricultural pests, details of the laboratory

and field evaluations of these insects are described in the following subsections.
The biological activity of pyriproxyfen against household insect pests has been
reviewed elsewhere [30].
28.2.6.1 Laboratory Evaluations

The activity of pyriproxyfen against various agricultural insects has been evaluated
in Japan and other countries. When tests on the adult cotton whitefly, Bemisia
tabaci (which had become an important pest) were carried out in the Middle and
Near East in 1984 [34], the results were summarized as follows:
• There was almost no effect on adults (no lethality towards adults).
• Although eggs were laid, most of them failed to hatch; when the adults were
released just after spraying, none of the eggs hatched.
• This tendency showed minimal concentration dependency.
• Few eggs were oviposited just after spraying.
• Over time, the number of eggs oviposited was increased, and a development to
nymphs and pupae was observed.
Taken together, these results indicated that pyriproxyfen should be applied
preventively, due to its sterilizing effect.
Bearing these results in mind, the characteristics of pyriproxyfen were in-
vestigated in greater detail in Japan, with the activity against each stage being
monitored in the laboratory using the diamond-back moth, Plutella xylostella, and
the greenhouse whitefly, Trialeurodes vaporariorum. Consequently, ovicidal activity
was identified at very low pyriproxyfen concentrations (Table 28.2.4), together with
an inhibitory activity against adult emergence when applied to larvae (Table 28.2.5)
[34]. Further evaluations of the greenhouse whitefly, aphids, and lepidopterans
were carried out in the greenhouse or in the field, with spray concentrations, spray
timing, numbers of sprays, and spray intervals being closely examined. In addition,
Table 28.2.4 Ovicidal activity of pyriproxyfen against eggs of

whitefly, Trialeurodes vaporariorum.
Egg stagea LC50 (ppm)
0–1 0.46
1–2 0.34
2–3 0.21
3–4 >30
4–5 >30
a
Days after oviposition.
Table 28.2.5 Effects of pyriproxyfen on the development of

whitefly, Trialeurodes vaporariorum.
Treated stage N Adult emergence (%)
Hatching 46 0
First instar nymph 165 0
Second instar nymph 54 0
Pupa 44 93.2
Untreated control 68 76.5
as sterilizing and ovicidal activities of pyriproxyfen had been demonstrated with

various insects, there remained some doubt that the response was specific to the
test insects. Consequently, when the responses of all developmental stages to
pyriproxyfen were examined using only P. xylostella, an ovicidal activity, together
with an inhibition of pupation (when applied to larvae), of adult emergence, of
adult emergence when applied to pupae, and of sterilizing activity when applied to
adults, were confirmed [29].
The modes of action of pyriproxyfen in a range of insects are summarized in
Table 28.2.6.
28.2.6.2 Field Evaluations

In order to control insects by spraying with pyriproxyfen, it is important to
reconsider: (i) the target insect; (ii) the target stage; and (iii) whether pyriproxyfen
can reach the target insect, based on the above-described information. Processes
to investigate the ultimate application method are detailed in the following
subsections.
Based on information acquired from the sterilizing effect on cotton whitefly
(B. tabaci), pyriproxyfen was sprayed three times at intervals of about three to four
weeks on a 100 m2 experimental cotton field in Sudan (an untreated plot served
as a control). Initially, the number of adults in the treated and control plots was
Table 28.2.6 Effects of pyriproxyfen on each stage of insects.
Insect Treated stage
Egg Larva/nymph Pupa Adult
Bemisia tabaci Most active Treated to first −→ No adults; eggs do not

[24, 27] within 24 h after to second hatch
oviposition
Trialeurodes Active within Treated to first −→ No adults
vaporariorum [25] three days old to third
Aonidiella aurantii – High activity – No offspring when
[23] against first males or females treated
stage
Myzus persicae [25, – – – No offspring
26]
Cydia pomonella [35] Active within 24 h – – Oviposited eggs were
after oviposition unhatched when males
or females treated
Spodoptera litura [36] – Matured larva Just after Low adult emergence;
pupation decreased number of
−→ eggs and ability to hatch
Thrips palmi [37] – Reared on −→ Inhibition of adult
treated emergence
leaf = dermal
uptake from
treated leaf
effectively the same, as was the number of eggs; however, the number of first instar
nymphs was not increased (Figure 28.2.5). The increased adult number, despite
pyriproxyfen treatment, was considered due to the narrow width of the experimental
field and an immigration of adults from the control plot. This hypothesis was
confirmed by a report from Turkey where, after spraying pyriproxyfen twice
onto a 1 ha cotton field, the number of adults was not increased immediately after
application, but showed a clear increase when the residual efficacy of the compound
seemed to be lost (see Figure 28.2.6). Thus, it appeared that the adults had migrated
to the treated field from untreated areas. Based on the results obtained in these two
countries, it was clear that: (i) the application dose of pyriproxyfen should be less
than 75 g ha−1 ; and (ii) the inter-application period should be two weeks.
The next point to consider was the spray timing. For this, the number of adults
per leaf on cotton was incorporated into the spray timing index; changes in nymph
numbers are shown in Figure 28.2.7. The results obtained suggested that spraying
should, preferably, be started when 50 or fewer adults per 100 leaves were counted
(i.e., one adult per two leaves). However, based on the consideration that the above
number was still low, an index of four to five adults per leaf was instituted. The spray
No. of nymphs/15 leaves 10
0
5 10
Weeks after first application
Figure 28.2.5 Efficacy of pyriproxyfen on the nymphs of

whitefly, Bemisia tabaci on cotton. Arrows indicate the appli-
cation timing of pyriproxyfen (100 g a.i. ha−1 ): (•) treated,
() untreated.
1000
No. of adults/30 leaves
500
0 10 20 30
Days after first application
Figure 28.2.6 Efficacy of pyriproxyfen on adult white-

fly, Bemisia tabaci on cotton. Arrows indicate the applica-
tion timing of pyriproxyfen (100 g a.i. ha−1 ): (•) treated,
() untreated.
frequency was limited to one application, bearing in mind the assumption that resis-
tance to this compound may develop on repeated use (as observed in many examples
of resistance development). Consequently, a rotational system was adopted.
The effects of pyriproxyfen were investigated for not only whitefly but also other
insects. An example of the results obtained with California red scale, Aonidiella
aurantii, in an orchard is shown in Table 28.2.7 [34]. In this investigation, the char-
acteristics of inhibiting reproduction and metamorphosis were well demonstrated
and, as a result of such treatment, high-quality fruits without any infestation of the
scales could be harvested (Table 28.2.8) [34].
In Japan, the control of greenhouse whitefly and cotton whitefly that infest
vegetables and ornamentals has been attempted using a yellow plastic tape formu-
lation containing pyriproxyfen. Extensive use of this tape has shown that it can
2000
1500
No. of nymphs/60 leaves
1000
500
0 20 40 60 80 100 120
Days after first application
Figure 28.2.7 Effects of the application of number of adults indicated. Arrows indi-
timing of pyriproxyfen on the nymphs of cate the application timing of pyriproxyfen
whitefly, Bemisia tabaci, on cotton. Pyriprox- (200 g a.i. ha−1 ): () 50 adults per 100
yfen was applied successively three times. leaves; (•) 200 adults per 100 leaves; ()
The first application was made at the time 250 adults per 100 leaves; () untreated.
Table 28.2.7 Efficacy of pyriproxyfen on California red scale,

Aonidiella aurantii on apple trees.
Concentration (ppm) N Mortality (%)a

Young stage Females Reproducing
females
200 200 91.8 69.5 66.0

Untreated control 200 27.3 37.0 4.0
a
Mortality was observed after 71 days.
Table 28.2.8 Effect of pyriproxyfen on fruit damage

by California red scale, Aonidiella aurantii, on mature
apple trees.
Concentration (ppm) Clean fruits (%)
200 98
Untreated control 45
cause a reduction in the whitefly population for several months after installation,
but have a minimal influence on any natural enemies and pollinators. The system
functions by the adult flies being attracted to the yellow tape; on touching the tape,
body contact is made with pyriproxyfen, the ovicidal activity of which causes the
hatching of any oviposited eggs to be greatly inhibited [38, 39].
28.2.6.3 Resistance
Resistance to pyriproxyfen has been observed only in whitefly, and reported in
Israel [40], the United States [41], and Spain [42]. In Israel, where pyriproxyfen
was introduced in 1991, the compound was sprayed once each season to control
whitefly (B. tabaci). However, by 1996 the whitefly had developed a high resistance
to pyriproxyfen in some regions, and its use was stopped in 1996 and 1997
[43]. In the laboratory, the susceptibility of field-collected B. tabaci population to
pyriproxyfen was seen to recover partially when pyriproxyfen exposure was avoided
for 13 generations [44]. Although the mechanism of resistance is unknown, it
has been reported that piperonyl butoxide (an oxidase inhibitor) does not have any
synergistic effect [45]. The resistance has been identified as incompletely or partially
dominant [46], and the susceptibility of whitefly was recovered when pyriproxyfen
use was halted [43, 47].
28.2.7
Synthesis
The synthetic route to pyriproxyfen is shown in Figure 28.2.8. The optical isomer
of pyriproxyfen is synthesized using optically active lactic acid as a starter material
[48, 49], or by employing an enantioselective hydrolysis with enzymes [50, 51].
28.2.8
Physico-Chemical Properties and Formulation
28.2.8.1 Physico-Chemical Properties

Some of the physico-chemical properties of pyriproxyfen are listed in Table 28.2.9.
The active ingredient of pyriproxyfen is an odorless white crystal with a melting
point of 48.1 ◦ C. The vapor pressure of pyriproxyfen is below 1.0 × 10−7 mmHg
(22.8 ◦ C), and its solubility in water at 25 ◦ C is 0.367 ppm.
O O
OH O N
O O
Cl N
Figure 28.2.8 The synthetic route to pyriproxyfen.

Table 28.2.9 Physico-chemical properties of

pyriproxyfen.
Property Value

Vapor pressure <1.0 × 10−7 mmHg (22.8 ◦ C)
Log P 5.37 (25 ◦ C)
Solubility
Water 0.367 mg l−1 (25 ◦ C)
n-Hexane 42 g l−1 (20 ◦ C)
Methanol 44 g l−1 (20 ◦ C)
Acetone >500 g l−1 (20 ◦ C)
28.2.8.2 Stability
Pyriproxyfen is stable and hardly decomposes, even if stored at 50 ◦ C for six
months. However, it is easily decomposed if maintained at a higher pH and a
higher temperature. Although pyriproxyfen is promptly decomposed by ultraviolet
rays, its decomposition by the action of sunlight (>290 nm) is only slight.
28.2.8.3 Formulation
Pyriproxyfen is available commercially for spray applications as an emulsifiable
concentrate and as an emulsion in water; microcapsular and granular formulations
are also available. A tape formulation for hanging applications is also available in
Japan. The physico-chemical properties of the formulations are very good, as are the
storage stabilities. Effective mixtures of pyriproxyfen with a variety of insecticides
have also been developed for commercial use.
28.2.9
Toxicology
The very favorable mammalian toxicity of pyriproxyfen, together with details of its
animal and plant metabolism, environmental toxicity and residues, and its effects
on nontarget organisms, have been described in a technical report [34].
28.2.10
Conclusions
The sale of pyriproxyfen was started under temporary registration in the Middle
and Near East in 1988, and this registration was approved in 1991. In the United
States, temporary registration was approved in 1996 and an emulsifiable concentrate
®
named Knack , containing 10% pyriproxyfen, is available that shows a high efficacy
against whitefly. The number of countries in which pyriproxyfen was registered
was expanded by setting whiteflies and scales on various crops as the main targets;
References 997
consequently, pyriproxyfen is now sold to control whitefly on cotton and vegetables,

thrips (Thrips palmi) on vegetables, scales on citrus fruits, psylla on pears, and
leafroller and scales on fruit trees. In 2008, an emulsion-in-water formulation
containing 10% pyriproxyfen was registered in Columbia as Epingle®. In Japan,
the tape formulation, containing pyriproxyfen at 1 g m2 , for whitefly control in
greenhouses was registered in 1995 as Lano® tape. Pluto® MC, a microcapsule
suspension containing 9% pyriproxyfen, for the control of scale insects on tea, was
also registered in Japan in 2007 [52].
Pyriproxyfen is also sold as Sumilarv® granules (0.5% pyriproxyfen) to control
mosquito and housefly larvae for household use; this use was later expanded to
control midges in Japan. In other countries, pyriproxyfen has also been developed
to control mosquitoes and houseflies, and sold as Sumilarv® 0.5% granules. In
addition, pyriproxyfen has been developed for home and pest control operator
(PCO) uses, and registered by the Environmental Protection Agency (EPA) in
1995.
Thus, although initially attention was focused on the development of pyriproxyfen
for household use, the breakthrough to agricultural use was made by investigating
the insect-sterilizing activity of the compound. Notably, this sterilizing effect was
linked to the development of Lano® tape, which controls whiteflies without a need
for spraying.
References
1. Riddiford, L.M. (1985) in Comprehensive 11. Nassar, S.G., Staal, G.B., and
Insect Physiology, Biochemistry and Phar- Armanious, N.I. (1973) J. Econ. Entomol.,
macology, vol. 8 (eds G.A. Kerkut and 66, 847–850.
L.I. Gilbert) Pergamon Press, New York, 12. Dorn, S., Frischknecht, M.L., Martinez,
pp. 37–84. V., Zurfluh, R., and Fischer, U. (1981)
2. Williams, C.M. (1967) Sci. Am., 217, Z. Pflanzenkrankh. Pflanzenschutz, 88,
13–17. 269–275.
3. Schmialek, P. (1961) Z. Naturforsch., 13. Hatakoshi, M., Agui, N., and Nakayama,
166, 461–464. I. (1986) Appl. Ent. Zool., 21, 351–353.
4. Wigglesworth, V.B. (1963) J. Insect Phys- 14. Henrick, C.A. (1982) in Insecticide Mode
iol., 9, 105–119. of Action (ed. J.R. Coats), Academic
5. Slama, K. and Williams, C.M. (1966) Press, New York, pp. 315–402.
Biol. Bull., 130, 235–246. 15. Hatakoshi, M., Kisida, H., Fujimoto, I.,
6. Bowers, W.S., Fales, H.M., Thompson, Itaya, N., and Nakayama, I. (1984) Appl.
M.J., and Uebel, E.C. (1966) Science, 154, Entomol. Zool., 19, 523–526.
1020–1021. 16. Hatakoshi, M., Osumi, T., Kisida, H.,
7. Bowers, W.S. (1968) Science, 161, Itaya, N., and Nakayama, I. (1985) Jpn. J.
895–897. Sanit. Zool., 36, 327–331.
8. Bowers, W.S. (1969) Science, 164, 17. Hatakoshi, M., Osumi, T., Kisida, H.,
323–325. Itaya, N., and Nakayama, I. (1986) Jpn. J.
9. Pallos, F.M., Menn, J.J., Letchworth, Sanit. Zool., 37, 99–104.
P.E., and Miaullis, J.B. (1971) Nature, 18. Hatakoshi, M., Nishida, S., Kisida, H.,
232, 486–487. and Oouchi, H. (2003) Nippon Nogeika-
10. Henrick, C.A., Staal, G.B., and Siddal, gaku Kaishi, 77, 730–735.
J.B. (1973) J. Agric. Food Chem., 21, 19. Kawada, H., Dohara, K., and Shinjo, G.
354–359. (1987) Jpn. J. Sanit. Zool., 38, 317–322.
20. Hatakoshi, M., Kawada, H., Nishida, S., 36. Hatakoshi, M. (1992) J. Insect Physiol.,
Kisida, H., and Nakayama, I. (1987) Jpn. 38, 793–801.
J. Sanit. Zool., 38, 271–274. 37. Nagai, K. (1990) Appl. Entomol. Zool., 25,
21. Kawada, H., Kojima, I., and Shinjo, G. 199–204.
(1989) Jpn. J. Sanit. Zool., 40, 195–201. 38. Nakamura, S., Inoue, M., Fujimoto, H.,
22. Cooper, R.M. and Oetting, R.D. (1985) and Kasamatsu, K. (1994) Appl. Entomol.
J. Entomol. Sci., 20, 429–434. Zool., 29, 454–456.
23. Peleg, B.A. (1988) J. Econ. Entomol., 81, 39. Oouchi, H. and Langley, P. (2005) J. Pes-
88–92. tic. Sci., 30, 50–52.
24. Ascher, K.R.S. and Eliyahu, M. (1988) 40. Horowitz, A.R. and Ishaaya, I. (1994)
Phytoparasitica, 16, 15–21. J. Econ. Entomol., 87, 866–871.
25. Yamamoto, H. and Kasamatsu, K. 41. Li, A.Y., Dennehy, T.J., and Nichols,
(1990) in Advances in Invertebrate Re- R.L. (2003) J. Econ. Entomol., 96,
production, vol. 5 (eds M. Hoshi and O. 1307–1314.
Yamashita), Elsevier Science Publisher 42. Fernandez, E., Gravalos, C., Haro, P.J.,
B. V., Amsterdam, pp. 393–398. Cifuentes, D., and Bielza, P. (2009) Pest
26. Hatakoshi, M., Shono, Y., Yamamoto, Manage. Sci., 65, 885–891.
H., and Hirano, M. (1991) Appl. Ento- 43. Horowitz, A.R., Kontsedalov, S.,
mol. Zool., 26, 412–414. Denholm, I., and Ishaaya, I. (2002)
27. Ishaaya, I. and Horowitz, A.R. (1992) Pest Manage. Sci., 58, 1096–1100.
J. Econ. Entomol., 85, 2113–2117. 44. Wilson, M., Moshitzky, P., Laor, E.,
28. Retnakaran, A., Granett, J., and Ennis, Ghanim, M., Horowitz, A.R., and
T. (1985) in Comprehensive Insect Phys- Morin, S. (2007) Pest Manage. Sci.,
iology, Biochemistry and Pharmacology, 63, 761–768.
vol. 12 (eds G.A. Kerkut and L.I. 45. Devine, G.J., Ishaaya, I., Horowitz, A.R.,
Gilbert), Pergamon Press, New York, and Denholm, I. (1999) Pestic. Sci., 55,
pp. 529–601. 405–411.
29. Oouchi, H. (2005) Appl. Entomol. Zool., 46. Horowitz, A.R., Gorman, K., Ross, G.,
40, 145–149. and Denholm, I. (2003) Arch. Insect
30. Hirano, M., Hatakoshi, M., Kawada, H., Biochem. Physiol., 54, 177–186.
and Takimoto, Y. (1998) Rev. Toxicol., 2, 47. Horowitz, A.R., Kontsedalov, S.,
357–394. Khasdan, V., and Ishaaya, I. (2005) Arch.
31. Agui, N., Granger, N., Gilbert, L.I., and Insect Biochem. Physiol., 58, 216–225.
Bollenbacher, W.E. (1979) Proc. Natl 48. Ghirardelli, R.G. (1973) J. Am. Chem.
Acad. Sci. USA, 76, 5694–5698. Soc., 95, 4987–4990.
32. Muszynska-Pytel, M. (1987) Arch. Insect 49. Mori, K. and Kisida, H. (1986) Tetrahe-
Biochem. Physiol., 5, 211–224. dron, 42, 5281–5290.
33. Mizoguchi, A., Hatta, M., Sato, S., 50. Sugiura, M., Iwai, M., Fukumoto, J., and
Nagasawa, H., Suzuki, A., and Ishizaki, Okamoto, Y. (1977) Biochim. Biophys.
H. (1990) J. Insect Physiol., 36, 655–664. Acta, 488, 353–358.
34. Hatakoshi, M., Kisida, H., Kawada, H., 51. Mitsuda, S., Umemura, T., and
Oouchi, H., Isobe, N., and Hagino, Hirohara, H. (1988) Appl. Microbiol.
S. (1997) Pyriproxyfen: A new insect Biotechnol., 29, 310–315.
growth regulator. Technical Report, 52. Isayama, S. and Tsuda, N. (2008) Devel-
Sumitomo Chemical Co. Ltd, vol. I, pp. opment of a novel formulation: Pluto®
4–20. MC to control mulberry scale on tea.
35. Yokoyama, V.Y. and Miller, G.T. (1991) Technical Report, Sumitomo Chemical
J. Econ. Entomol., 84, 942–947. Co., Ltd, vol. II, pp. 4–12.
999
29
Chitin Synthesis
29.1
Chitin Synthesis and Inhibitors
Joel J. Sheets
29.1.1
Introduction
Next to cellulose, chitin is the second most abundant biopolymer found in nature
[1]. Similar to cellulose, chitin is also a carbohydrate, consisting of long un-
branched chains of polymerized N-acetyl-glucosamine (poly-N-acetyl-glucosamine)
monomers linked where the adjacent sugars have opposing orientations
(Figure 29.1.1). The only chemical difference between chitin and cellulose is the
presence of aminoacetyl side groups found in chitin for the C-2 hydroxyl groups
found in cellulose. Chitin is present in the cuticle of insects, and is also found in
other organisms including the shells of all crustaceans, in protozoa, fungi, algae,
and nematodes [2–4]. Notably, as chitin is completely absent from vertebrates and
higher plants, its biosynthetic pathway represents an attractive target site for the
action of insect-specific insecticides [1, 5].
The cuticular exoskeleton of insects is composed of both chitin and proteins,
to provide a rigid support structure for muscle attachment, locomotion, and also
to protect the insect from environmental contaminants and desiccation [2]. Chitin
is also synthesized and secreted by endodermal cells of the midgut of insects,
combining with proteins and glycoproteins to form the peritrophic matrix. Chitin
is a major component of the peritrophic matrix that lines the interior of the insect
gut and separates its contents from the intestinal epithelium. The peritrophic
matrix helps provide protection to the gut from mechanical damage, functions as
a semi-permeable membrane to regulate passage of molecules between different
midgut compartments, and also serves as a barrier to protect insects from microbial
and parasitic attacks [6–13].

1000 29 Chitin Synthesis
CH2OH CH2OH CH2OH CH2OH H NHCOCH3

H OH HO H OH O
H H O H
H H H H OH H
OH H H OH 2
OH H OH H H
OH O OH OH OH H H O
O
H OH OH H H OH H NHCOCH3 CH2OH
n
Trehalose B-D-Glucose Chitin
Figure 29.1.1 Abbreviated biosynthetic pathway of the

monomeric N-acetyl-glucosamine structure of chitin, starting
from trehalose.
29.1.2
Chitin Structure and Biosynthesis
Insects employ three different types of chitin (α, β, and γ ), that differ in the relative
orientations of the adjacent chitin polymer chains. α-Chitin, which is the most
abundant form found in the insect cuticle, is composed of polymeric chains of
N-acetyl-glucosamine arranged in an anti-parallel orientation. This arrangement
allows for the formation of microfibrils consisting of closely packed crystalline
arrays of individual chitin chains that utilize extensive hydrogen bonding between
the amine and carbonyl groups to help provide mechanical strength [4, 14]. β-Chitin
is found in the insect gut, along with α-chitin, as a component of the peritrophic
matrix. The adjacent chitin polymers in β-chitin are parallel, as opposed to the
anti-parallel orientation of α-chitin [15]. This results in the adjacent chitin chains
forming fewer hydrogen-bond linkages, and allows the structure to be less rigid
and more hydrated [16]. The third, and less predominant form of chitin – γ -chitin –
exists primarily in cocoons, and has a structure that consists of two parallel strands
of chitin polymers positioned next to a single chain of chitin running in the opposite
direction.
29.1.2.1 Chitin Synthase

In insects, the biosynthetic pathway for chitin starts with the disaccharide
trehalose; this is synthesized in the fat body, and is the most predomi-
nant sugar found in insects [17–19]. Trehalose is cleaved into two glucose
molecules that are phosphorylated, isomerized, and acetylated, affording the
precursor UDP-N-acetylglucosamine (Figure 29.1.1). Chitin synthase (CHS; EC
2.4.2.26) is the final enzyme of the pathway that utilizes the activated sugar
UDP-N-acetylglucosamine, polymerizing it to form chitin. The activity and
structure of this enzyme have been described in several excellent reviews [18–22].
CHS is a 180 kDa protein belonging to the family of β-glycosyltransferases, and
is considered to be the key enzyme involved in chitin synthesis. The enzyme
has been shown to require divalent cations such as Mg2+ , Mn2+ , or Ca2+ for
activity [23]. Investigations to localize CHS in the midgut of Manduca sexta by
immunohistochemistry, using fluorescently labeled anti-CHS antibodies, have
shown the midgut brush border membranes to be heavily labeled, with the
immunofluorescence labeling localized at the apical areas of the microvilli. In
addition, CHS is localized in the apical membranes of salivary glands and tracheal
cells [24]. Immunochemistry performed on the epiprot of the American cockroach
Periplaneta americana show CHS also to be located in the apical region of the
epidermis [4].
Based on data acquired from insects investigated to date, two different forms of
CHS have been found encoded by two different genes (CHS-A and CHS-B). These
genes are differentially regulated and expressed in different tissues, including the
integument, midgut, and trachea [25–31]. CHS-A is expressed in the epidermis
for the formation of chitin in embryonic and pupal cuticles, whereas CHS-B is
associated with the expression of chitin associated with the peritrophic matrix of
the midgut [25, 31]. Tellam and coworkers were the first to determine the cDNA
sequence of an insect CHS from the Australian sheep blowfly, Lucilia cuprina. The
amino acid sequence of the protein showed minimal similarities to yeast CHSs, and
contained between 15 and 18 putative transmembrane regions, which indicated
that the enzyme is an integral membrane protein [29].
There is evidence that fungal CHS is initially synthesized as a zymogen, and
requires proteolytic activation for full expression of its biological activity [32, 33].
Based on results from tissue assays, CHS has also been proposed to be synthesized
as a zymogen in arthropods [18], though evidence to support this proposal has
only recently been obtained from in vitro CHS preparations from the midgut of
Manduca sexta [30]. In these studies, the activation of enzymatic activity is observed
when crude cell extracts from midgut tissues are treated with trypsin. However,
when the 12 000 × g membrane fraction from this preparation was treated with
trypsin, no activation of CHS activity was observed even though this fraction should
be enriched with CHS. The addition of the soluble fraction back to the membrane
fraction restored the ability of trypsin to activate CHS activity, which suggested that
trypsin acts on a soluble protein or some other factor(s) that, in some way, activates
CHS located in the membrane fraction [30]. Purification of an active CHS from
the midgut of the tobacco hornworm showed the protein to exist in an oligomeric
form [34].
Assays designed for measuring chitin synthesis rely on several unique physical
properties of chitin, including its insolubility in most solvents, combined with
its ability to be deacetylated in boiling alkali to chitosan, which is also insoluble.
The treatment of chitin with concentrated hot acids results in deacetylation and
hydrolysis, yielding soluble glucosamine. Chitin is also sensitive to degradation by
chitinases [35]. Cultured insect imaginal wing disks provide a convenient in vitro
preparation for measuring chitin synthesis. This tissue preparation responds to
the addition of ecdysteroids by stimulating the rate of incorporation of [14 C]GlcAc
into base-insoluble material that is readily digested with chitinase [36–39]. Hajjar
and Casida have described an in vitro assay to the measure rate of incorporation of
[14 C]glucose into [14 C]chitin, using the abdomen of newly emerged adult milkweed
bugs Oncopeltus fasciatus [40]. The study results showed a good correlation between
the activity of 24 different benzoylphenyl urea analogs to inhibit chitin synthesis
and their toxicity toward O. fasciatus nymphs, demonstrating the convenience of
such an in vitro assay system to measure the activity of new chitin synthesis
inhibitors. A nonradioactive, high-throughput assay for measuring CHS activity in

yeast has also been described that could be adapted for measuring this activity in
insect cell lines [41].
29.1.3
Inhibitors of Chitin Synthesis
Polyoxin D and nikkomycin Z are both Streptomyces-derived peptidyl nucleoside

antibiotics that serve as competitive inhibitors of CHS in both fungal and insect in
vitro systems [42–47]. The fact that polyoxin D and nikkomycin Z each have struc-
tural similarities to the substrate UDP-N-acetylglucosamine most likely accounts
for the competitive nature of their ability to inhibit CHS (Figure 29.1.2). Polyoxin
D is a moderate inhibitor of chitin synthesis in isolated integument tissues from
various different insects, whereas nikkomycin has a greater inhibitory potency [43].
Using [14 C]glucose as a substrate, polyoxin D was shown to inhibit the formation
of [14 C]chitin by 50% at 12 μM in an in vitro system using Oncopeltus fasciatus
[40]. Diflubenzuron, however, in the same system was about 22-fold more potent.
Nikkomycin reversibly inhibited chitin synthesis in a dose-dependent manner
when injected into fifth instar larvae of the tobacco hornworm, Manduca sexta
[48]. When nikkomycin is applied either topically or as an oral dose, much higher
concentrations are required to inhibit chitin synthesis than if the compound is
injected directly into the insect. The poor inhibitory activity of these nucleosides,
O
HOOC
NH
N O
O
O
HO
O OH
HO N
H OH
H2N O NH2
O
Polyoxin D
NH
N O
O
O
HO
O OH
H3C N
H OH
HO NH2
N OH
Nikkomycin Z
Figure 29.1.2 Structure of peptidyl nucleoside antibiotic inhibitors of chitin synthesis.

when applied to insects, is most likely due their poor pharmacokinetic properties,
which in turn greatly limits their agricultural use as insecticides. Instead, they have
found limited use as fungicides, and as valuable tools in the study of CHS [5, 23].
29.1.3.1 Benzoylphenyl Ureas

Diflubenzuron, as an early representative of the benzoylphenyl ureas, was discov-
ered by Philips-Duphar B.V. as an inhibitor of chitin synthesis, and commercialized
in 1977 for the control of lepidopteran and coleopteran pests in fruit, cotton, soy-
beans, and vegetable crops [49–54] (Figure 29.1.3). Triflumuron represents an
additional early benzoylphenyl urea registered during the early 1980s by Bayer AG
for the control of insect pests on fruits and vegetables [55]. These older representa-
tives have found additional roles in the urban pest management arena, due to their
activity against noncrop insects such as mosquitoes, termites, and cockroaches
[56–58].
Chlorfluazuron was introduced during the late 1980s in Australia, Hungary,
Japan, Philippines, and Vietnam by Ishihara Sangyo Kaisha, for the control of
lepidopteran pests on cotton, tea, vegetables, and fruits, especially in situations
requiring the management of insecticide resistance. It was never registered in the
USA. Chlorfluazuron possesses ovicidal activity against adult German cockroach
(Blattella germanica), and the common cutworm (Spodoptera litura) [57, 59], and has
also been evaluated for uses in noncrop applications such as fly control [60] and
for use as part of a termite bait matrix [61, 62] that may extend the use of this now
‘‘mature’’ compound.
R4
R3 R5
R1 O O
N N R6
H H
R7
R2
Chemical R1 R2 R3 R4 R5 R6 R7
Diflubenzuron F F H H Cl H H
Chlorfluazuron F F H Cl 3-Cl-5-CF3-pyrindin-2-yloxy Cl H
Teflubenzuron F F F Cl F Cl H
Triflumuron H Cl H H -OCF3 H H
Flufenoxuron F F F H 2-Cl-4-CF3-phenoxy H H
Hexaflumuron F F H Cl -OCF2CF3 Cl H
Noviflumuron F F F Cl -OCF2CHFCF3 Cl H
Novaluron F F H Cl -OCF2CHFOCF3 H H
Lufenuron F F H Cl -OCF2CHFCF3 H Cl
Bistrifluron F F Cl CF3 H CF3 H
Figure 29.1.3 Structure of representative benzoylphenyl ureas.

Teflubenzuron was introduced in 1986 by American Cyanamid Corporation

(now BASF) into parts of Europe and Africa for the control of lepidopteran pests in
fruits, citrus, vegetables, and cotton. It also has mosquito larvicidal activity and is
currently being investigated for use as a feed treatment for sea lice on fish grown
in captivity [63].
Flufenoxuron (Cascade) was introduced by American Cyanimide Corporation
(now BASF) in 1989 for the foliar control of insects and mites on fruit, citrus
vegetables, and cotton. During 2008, the sales of flufenoxuron reached US$ 45
million. When formulated as a wettable powder, flufenoxuron has also shown
potential for the control of urban pests such as the German cockroach [64].
Most recently, it has been formulated as a bait (FLUROX™) for the control of
subterranean termite infestations.
Hexaflumuron, which originally was developed by Dow as an insecticide for the
control of Lepidoptera, Coleoptera, Homoptera, and Diptera pests on cotton, top
fruit, and potatoes, was first introduced into Latin America in 1987. It was later
registered in the USA in 1995 as the first reduced-risk pesticide for use as part of a
®
termite bait matrix system called Sentricon [62, 65–69]. Hexaflumuron has since
been replaced with another benzoylphenyl urea, noviflumuron, which is more
potent, faster-acting [70], and also demonstrates activity against other household
pests such as German cockroach (Blattella germanica) when applied either as a
spray or as part of a bait matrix [71–73].
Novaluron, as the most recently introduced benzoylphenyl urea, was originally
developed by Isagro and subsequently sold to Makhteshim. This molecule is
registered in Europe and in the USA for use on ornamentals, cotton, fruits, and
vegetables. It is very potent on the cotton leafworm (S. littoralis) and the Colorado
potato beetle (Leptinotarsa decemlineata), showing some contact and good residual
activity [74, 75]. At present, novaluron is also undergoing investigations for use in
vector control programs against Aedes aegypti [76].
Lufeneron (Ciba-Geigy), which is the current market leader of the benzoylphenyl
ureas [77], has long been in use in Europe and Japan for the treatment of cotton
and vegetables, and its markets are today increasing in both Latin America and
South Korea. In the past, the major application of lufeneron has been in the animal
health market, for the control of fleas on domestic dogs, cats, and other animals
[78–81]. It is also currently being developed as a bait for termite control [77].
Bistrifluron (DBI-3204), a benzoylphenyl urea recently introduced by Dongbu
Hannong of Korea, is active against whiteflies (Trialeurodes vaporariorum and
Bemisia tabaci) and lepidopteran pests (e.g., Spodoptera exigua, and Plutella xylostella)
[82]. The compound has also shown promise for use as a bait for the control of ants
and cockroaches in domestic environments [83].
29.1.3.2 Other Chitin Synthesis Inhibitors
Buprofezin (2-tert-butylimino-3-isopropyl-5-phenyl-1,3,5-thiadiazinan-4-one; Appl-
®
aud , NNI-750; Nihon Nohyaku) is an insect growth regulator based on the
thiadiazine class of chemistry. Buprofezin was introduced in 1984 as a nonsystemic
contact insecticide for the Japanese rice market, having particularly good activity as
a foliar spray against the brown planthopper Nilaparvata lugens. It inhibits molting,
H
S H2N N N
N N
N N N
O
NH2
Buprofezin Cyromazine
Figure 29.1.4 Structures of other chitin synthesis inhibitors.
leading to the suppression of ecdysis, presumably through an inhibition of chitin

synthesis, but has little effect on egg viability [84, 85] (Figure 29.1.4).
Cyromazine (N-cyclopropyl-1,3,5-triazine-2,4,6-triaminel; Trigard, CGA 72662;
Ciba-Geigy now Syngenta AG) is another member of the thiadiazine class of
chemistry. Cyromazine was introduced in 1985 as a foliar spray with systemic and
translaminar activities against leaf miners in vegetables, potatoes, and ornamentals;
it is also selective against Dipterous species and is used to control flies in commercial
poultry operations. Cyromazine interferes with molting, presumably due to an
inhibition of chitin synthesis, though the ultrastructural changes in the cuticle that
result from its action appear to be distinct from those caused by diflubenzuron
[86]. The exact mode of action of cyromazine remains unclear, however.
29.1.4
The Future of Chitin Synthesis Inhibitors for Crop Protection
Chitin synthesis inhibitors possess the valuable property of having a non-neurotoxic

mode of action at a target site that is absent in vertebrates, which means in turn
that they are generally safe to mammals if ingested. The availability of a target site
that is distinct from that of the more commonly employed neurotoxic insecticides
also allows these molecules to be used as important tools in insecticide resistance
management (IRM) programs [75, 87]. In addition, the low contact activity of
benzophenyl ureas reduces their impact on predatory insects, which in turn
promotes their use in IRM programs.
Resistance to the benzoylphenyl ureas has been measured both in the field and
in the laboratory, but is usually correlated with insects acquiring an increased
capacity to metabolize and eliminate the compound [88, 89], as opposed to the
generation of target-site resistance. The benzoylphenyl ureas may be toxic towards
chitin-synthesizing invertebrates such as Daphnia, and crustaceans, that exist lower
down the food chain, and this may result in significant environmental problems if
such compounds are inadvertently allowed to enter natural water sources such as
streams and/or lakes.
Typically, the onset of action of the benzoylphenyl ureas is slow, with several
days being required before any reduction is observed in larval numbers, although
cessation of larval feeding often occurs much earlier. This slow activity has resulted
in a need to educate farmers and growers, who are accustomed to the rapid
knockdown activity of neurotoxic pesticides, regarding the correct application
timing of these compounds, and the time required to observe their full activity. The
lack or limited contact activity of the benzoylphenyl ureas has also limited their use
against insects that are hidden feeders. Interestingly, the activity and onset of action
of biological control agents such as Spodoptera litura (F) nucleopolyhedrovirus
(SINPV) can be increased by their co-administration with benzophenyl ureas, due
to an ability of the chemical to disrupt the peritrophic matrix of insects and allow
an easier entry of the virus through the gut [90]. Indeed, this combined approach
may open new avenues for the use of benzylphenyl ureas with nonchemical agents.
Overall, during the five-year period up to 2009, the global sales of benzoylphenyl
ureas have increased to about US$ 413 million, with an annual growth rate of
about 7% [77]. This increase has in part been due to the use of these agents in IRM
programs, as well as their use outside of crop protection as part of a bait matrix
for the long-term control of urban pests, or as veterinarian medicinal products for
the control of fleas and ticks in companion animals and livestock. These niche
markets represent significant opportunities to extract further value from these
chemistries beyond their use in crops, and typically represent significantly higher
gross margins than are generally obtained in commodity agricultural markets. The
continual development of transgenic crops such as cotton and corn expressing
genes that produce intrinsic insect resistance will, most likely, exert pressure to
erode the use of these chemicals for crop protection, unless widespread resistance
breaks out – either towards the transgenic crops or towards other insecticides with
neurotoxic activity.
Today, the inhibition of chitin synthesis remains an important and underutilized
target site for the control of agricultural insect pests. Given the high turnover and
important role of chitin synthesis in the maintenance of the peritrophic matrix
of insects, this mode of action represents an opportunistic target in the gut of
insects to inhibit through transgenic means. Recent advancements using RNA
interference (RNAi) technology to control insects have identified CHS as a valid
target to downregulate its expression, resulting in toxicity to insects [91, 92]. Such
modern molecular approaches will most likely become increasingly important in
the future, and chitin synthesis offers an exciting target site to be exploited in this
respect. The discovery of proteins that are capable of inhibiting chitin synthesis in
the gut of insects, and the subsequent expression of such proteins in transgenic
plants, might also represent an attractive strategy for controlling pests in the future.
Moreover, such an approach will be made easier as the exact mechanism used by
benzylphenyl ureas to inhibit chitin synthesis in insects is revealed [93]. Research
investigations towards this goal should present more precise target sites that could
be used to screen for new contact-active chemicals or gut-active proteins with the
ability to inhibit this critical biological activity in insects, and lead to new methods
of insect control.
Acknowledgments
The author wishes to thank Dr Thomas C. Sparks for his careful review
of this manuscript, and Gene F. Tisdell for providing his insights into the
References 1007
2,6-diphenyl-heterocyclic chemistries. These studies were supported by Dow

AgroSciences.
References
1. Neville, A.C. (1975) Biology of the Arthro- membrane (PM): identification of po-
pod Cuticle, Springer-Verlag, New York. tential PM target sites for insect control.
2. Andersen, S.O. (1979) Biochemistry of Arch. Insect Biochem. Physiol., 47 (2),
insect cuticle. Annu. Rev. Entomol., 24, 110–118.
29–61. 12. Zimmermann, U., Mehlan, D., and
3. Fuhrman, J.A. and Piessens, W.F. (1985) Peters, W. (1973) Investigations on
Chitin synthesis and sheath morphogen- the transport function and structure of
esis in Brugia malayi microfilariae. Mol. peritrophic membranes. 3. Periodic in-
Biochem. Parasitol., 17, 93–104. corporation of glucose, methionine and
4. Merzendorfer, H. and Zimoch, L. (2003) cysteine into the peritrophic membranes
Chitin metabolism in insects: structure, of the blowfly Calliphora erythrocephala
function and regulation of chitin syn- Mg. in vivo and in vitro. Comp. Biochem.
thases and chitinases. J. Exp. Biol., 206, Physiol., B, 45, 683–693.
4393–4412. 13. Richards, A.G. and Richards, P.A. (1977)
5. Cohen, E. (1993) Chitin synthesis and The peritrophic membranes of insects.
degradation as targets for pesticide ac- Annu. Rev. Entomol., 22, 219–240.
tion. Arch. Insect Biochem. Physiol., 22, 14. Carlstrom, D. (1957) The crys-
245–261. tal structure of alpha-chitin
6. Edwards, M.J. and Jacobs-Lorena, M. (poly-N-acetyl-D-glucosamine). J. Cell
(2000) Permeability and disruption Biol., 3, 669–683.
of the peritrophic matrix and caecal 15. Pervaiz, S.M. and Abdul, H.M. (1975)
membrane from Aedes aegypti and Studies on the structure of beta-chitin, I.
Anopheles gambiae mosquito larvae. Z. Naturforsch., C, 30, 571–574.
J. Insect Physiol., 46, 1313–1320. 16. Gardner, K.H. and Blackwell, J. (1975)
7. Elvin, C.M., Vuocolo, T., Pearson, R.D., Refinement of the structure of β-chitin.
East, I.J., Riding, G.A., Eisemann, C.H., Biopolymers, 14, 1581–1595.
and Tellam, R.L. (1996) Characteriza- 17. Becker, A., Schloder, P., Steele, J.E.,
tion of a major peritrophic membrane and Wegener, G. (1996) The regula-
protein, peritrophin-44, from the larvae tion of trehalose metabolism in insects.
of Lucilia cuprina: cDNA and reduced Experientia, 52, 433–439.
amino acid sequences. J. Biol. Chem., 18. Cohen, E. (1987) Chitin biochemistry:
271 (15), 8925–8935. synthesis and inhibition. Annu. Rev.
8. Lehane, M.J. (1997) Peritrophic ma- Entomol., 32, 71–93.
trix structure and function. Annu. Rev. 19. Cohen, E. (2001) Chitin synthesis and
Entomol., 42, 525–550. inhibition: a revisit. Pest Manage. Sci.,
9. Ramos, A., Mahowald, A., and 57, 946–950.
Jacobs-Lorena, M. (1994) Peritrophic 20. Merz, R.A., Horsch, M., Nyhlen, L.E.,
matrix of the black fly Simulium vitta- and Rast, D.M. (1999) Biochemistry of
tum: formation, structure, and analysis chitin synthase. EXS, 87, 9–37.
of its protein components. J. Exp. Zool., 21. Merzendorfer, H. (2006) Insect chitin
268 (4), 269–281. synthases: a review. J. Comp. Physiol. B,
10. Tellam, R.L., Wijffels, G., and 176, 1–15.
Willadsen, P. (1999) Peritrophic ma- 22. Kramer, K.J. and Muthukrishnan, S.
trix proteins. Insect Biochem. Mol. Biol., (2005) Chitin metabolism in insects.
29 (2), 87–101. Comp. Mol. Insect Sci., 4, 111–144.
11. Wang, P. and Granados, R.R. (2001) 23. Cohen, E. and Casida, J.E. (1982)
Molecular structure of the peritrophic Properties and inhibition of insect
integumental chitin synthase. Pestic. Muthukrishnan, S. (2005) Chitin

Biochem. Physiol., 17 (3), 301–306. synthase genes in Manduca sexta: char-
24. Zimoch, L. and Merzendorfer, H. (2002) acterization of a gut-specific transcript
Immunolocalization of chitin synthase and differential tissue expression of
in the tobacco hornworm. Cell Tissue alternately spliced mRNAs during devel-
Res., 308, 287–297. opment. Insect Biochem. Mol. Biol., 35,
25. Arakane, Y., Hogenkamp, D.G., Zhu, 529–540.
Y.C., Kramer, K.J., Specht, C.A., 32. Machida, S. and Saito, M. (1993)
Beeman, R.W., Kanost, M.R., and Purification and characterization of
Muthukrishnan, S. (2004) Characteri- membrane-bound chitin synthase.
zation of two chitin synthase genes of J. Biol. Chem., 268, 1702–1707.
the red flour beetle, Tribolium casta- 33. Palli, S.R. and Retnakaran, A. (1999)
neum, and alternate exon usage in one Molecular and biochemical aspects of
of the genes during development. Insect chitin synthesis inhibition. EXS, 87,
Biochem. Mol. Biol., 34, 291–304. 85–98.
26. Arakane, Y., Muthukrishnan, S., 34. Maue, L., Meissner, D., and
Kramer, K.J., Specht, C.A., Merzendorfer, H. (2009) Purification
Tomoyasu, Y., Lorenzen, M.D., of an active, oligomeric chitin synthase
Kanost, M., and Beeman, R.W. (2005) complex from the midgut of the tobacco
The Tribolium chitin synthase genes hornworm. Insect Biochem. Mol. Biol.,
TcCHS1 and TcCHS2 are specialized 39, 654–659.
for synthesis of epidermal cuticle and
35. Arakane, Y., Zhu, Q., Matsumiya, M.,
midgut peritrophic matrix. Insect Mol.
Muthukrishnan, S., and Kramer, K.J.
Biol., 14, 453–463.
(2003) Properties of catalytic, linker
27. Bolognesi, R., Arakane, Y.,
and chitin-binding domains of insect
Muthukrishnan, S., Kramer, K.J.,
chitinase. Insect Biochem. Mol. Biol., 33,
Terra, W.R., and Ferreira, C. (2005)
631–648.
Sequences of cDNAs and expression
36. Mikolajczyk, P., Zimowska, G.,
of genes encoding chitin synthase and
Oberlander, H., and Silhacek, D.L.
chitinase in the midgut of Spodoptera
(1995) Chitin synthesis in Spodoptera
frugiperda. Insect Biochem. Mol. Biol., 35,
frugiperda wing imaginal disks. III: role
1249–1259.
of the peripodial membrane. Arch. Insect
28. Gagou, M.E., Kapsetaki, M., Turberg, A.,
and Kafetzopoulos, D. (2002) Biochem. Physiol., 28 (2), 173–187.
Stage-specific expression of the chitin 37. Oberlander, H. (1976) Hormonal control
synthase DmeChSA and DmeChSB of growth and differentiation of insect
genes during the onset of Drosophila tissues cultured in vitro. In Vitro, 12,
metamorphosis. Insect Biochem. Mol. 225–235.
Biol., 32, 141–146. 38. Mikolajczyk, P., Oberlander, H.,
29. Tellam, R.L., Vuocolo, T., Johnson, S.E., Silhacek, D.L., Ishaaya, I., and
Jarmey, J., and Pearson, R.D. (2000) Shaaya, E. (1994) Chitin synthe-
Insect chitin synthase cDNA sequence, sis in Spodoptera frugiperda wing
gene organization and expression. Eur. imaginal disks: I. Chlorfluazuron,
J. Biochem., 267, 6025–6043. diflubenzuron, and teflubenzuron in-
30. Zimoch, L., Hogenkamp, D.G., hibit incorporation but not uptake of
Kramer, K.J., Muthukrishnan, S., and [14 C]N-acetyl-D-glucosamine. Arch. Insect
Merzendorfer, H. (2005) Regulation of Biochem. Physiol., 25 (3), 245–258.
chitin synthesis in the larval midgut 39. Nakagawa, Y., Matsutani, M.,
of Manduca sexta. Insect Biochem. Mol. Kurihara, N., Nishimura, K., and
Biol., 35, 515–527. Fujita, T. (1992) Inhibition of
31. Hogenkampa, D.G., Arakanea, Y., N-acetylglucosamine incorporation into
Zimochc, L., Merzendorferc, H., the cultured Integument of Chile sup-
Kramera, K.J., Beemanb, R.W., pressalis by diflubenzuron. Pest. Biochem.
Kanosta, M.R., Spechtd, C.A., and Physiol., 42, 242–247.
References 1009
40. Hajjar, N.P. and Casida, J.E. (1978) armigera (Hub.) (Lepidoptera: Noctu-
Insecticidal benzoylphenyl ureas: idae) in the field. J. Entomol. Res., 24 (2),
structure-activity relationships as chitin 177–184.
synthesis inhibitors. Science, 200 (4349), 51. Post, L.C. and Vincent, W.R. (1973) A
1499–1500. new insecticide inhibits chitin synthesis.
41. Lucero, H.A., Kuranda, M.J., and Bulik, Naturwissenschaften, 60, 431–432.
D.A. (2002) A Nonradioactive, high 52. Post, L.C., De Jong, B.J., and Vincent,
throughput assay for chitin synthase ac- W.R. (1974) 1-(2,6-Disubstituted
tivity. Anal. Biochem., 305 (1), 97–105. benzoyl)-3-phenylurea insecticides:
42. Cabib, E. (1991) Differential inhibi- inhibitors of chitin synthesis. Pestic.
tion of chitin synthases 1 and 2 from Biochem. Physiol., 4 (4), 473–483.
Saccharomyces cerevisiae by polyoxin D
53. Van Laecke, K. and Degheele, D. (1991)
and nikkomycins. Antimicrob. Agents
Detoxification of diflubenzuron and
Chemother., 35, 170–173.
teflubenzuron in the larvae of the beet
43. Cohen, E. and Casida, J.E. (1980)
armyworm (Spodoptera exigua) (Lepi-
Inhibition of Tribolium gut chitin syn-
doptera: Noctuidae). Pestic. Biochem.
thase. Pestic. Biochem. Physiol., 13 (2),
129–136. Physiol., 40 (2), 181–190.
44. Endo, A. and Misato, T. (1969) Poly- 54. Verloop, A. and Ferrell, C.D. (1977) in
oxin D, a competitive inhibitor of Pesticide Chemistry in the 20th Century,
UDP-N-acetylglucosamine: chitin Symposium, 1976, American Chemi-
N-acetylglucosaminyltransferase in cal Society Symposium Series, vol. 37
Neurospora crassa. Biochem. Biophys. Res. (ed. J.R. Plimmer), American Chemical
Commun., 37, 718–722. Society, New York, pp. 237–270.
45. Gaughran, J.P., Lai, M.H., Kirsch, D.R., 55. Ammar, I.M.A., Darwish, E.T.E., Farag,
and Silverman, S.J. (1994) Nikkomycin A.I., and Eisa, A.A. (1986) Detrimental
Z is a specific inhibitor of Saccharomyces effects of five molt-inhibiting insect
cerevisiae chitin synthase isozyme growth regulators on the development
Chs3 in vitro and in vivo. J. Bacteriol., and reproduction of the cabbage aphid,
176, 5857–5860. Brevicoryne brassicae (L.). J. Appl. Ento-
46. Hector, R.F. and Pappagianis, D. (1983) mol., 102 (4), 417–422.
Inhibition of chitin synthesis in the cell 56. Batra, C.P., Mittal, P.K., Adak, T., and
wall of Coccidioides immitis by polyoxin Ansari, M.A. (2005) Efficacy of IGR
D. J. Bacteriol., 154, 488–498. compound Starycide 480 SC (Triflu-
47. Turnbull, I.F. and Howells, A.J. (1983) muron) against mosquito larvae in clear
Integumental chitin synthase activity in and polluted water. J. Vector Borne Dis.,
cell-free extracts of larvae of the Aus- 42, 109–116.
tralian sheep blowfly, Lucilia cuprina,
57. Demark, J.J. and Bennett, G.W. (1990)
and two other species of diptera. Aust. J.
Ovicidal activity of chitin synthesis
Biol. Sci., 36, 251–262.
inhibitors when fed to adult German
48. Schluter, U. and Seifert, G. (1989)
cockroaches (Dictyoptera: Blattellidae).
Chitin synthesis inhibition by
J. Med. Entomol., 27, 551–555.
Nikkomycin in the integument of
Manduca sexta: an ultrastructural 58. Ho, C.M., Wu, S.H., and Wu, C.C.
and fluorescence microscopic study. (1990) Evaluation of the control of
J. Invertebr. Pathol., 53, 387–391. mosquitoes with insect growth regula-
49. Ascher, K.R.S. and Nemny, N.E. tors. Gaoxiong Yi Xue Ke Xue Za Zhi, 6,
(1976) Toxicity of the chitin synthe- 366–374.
sis inhibitors, diflubenzuron and its 59. Perveen, F. and Miyata, T. (2000) Effects
dichloro-analog, to Spodoptera littoralis of sublethal dose of chlorfluazuron on
larvae. Pestic. Sci., 7 (1), 1–9. ovarian development and oogenesis in
50. Chakraborti, S. and Chatterjee, M.L. the common cutworm Spodoptera litura
(2000) Effect of four benzophenylureas (Lepidoptera: Noctuidae). Ann. Entomol.
on the chickpea pod borer, Heliothis Soc. Am., 93 (5), 1131–1137.
60. Quesada, B.L. and Montoya-Lerma, J. for controlling the subterranean ter-
(1994) Laboratory evaluation of chlorflu- mite Coptotermes curvignathus (Isoptera:
azuron against larval phlebotomine sand Rhinotermitidae) in Malaysia. J. Econ.
flies (Diptera: Psychodidae). J. Econ. Entomol., 93, 429–433.
Entomol., 87, 1129–1132. 69. Su, N.Y., Ban, P.M., and Scheffrahn,
61. Peters, B.C. and Fitzgerald, C.J. (2003) R.H. (2000) Control of Coptotermes
Field evaluation of the bait toxicant havilandi (Isoptera: Rhinotermitidae)
chlorfluazuron in eliminating Coptoter- with hexaflumuron baits and a sensor
mes acinaciformis (Froggatt) (Isoptera: incorporated into a monitoring and
Rhinotermitidae). J. Econ. Entomol., 96, baiting program. J. Econ. Entomol., 93,
1828–1831. 415–421.
62. Rojas, M.G. and Morales-Ramos, J.A. 70. Karr, L.L., Sheets, J.J., King, J.E., and
(2004) Disruption of reproductive activ- Dripps, J.E. (2004) Laboratory perfor-
ity of Coptotermes formosanus (Isoptera: mance and pharmacokinetics of the
Rhinotermitidae) primary reproductives benzoylphenylurea noviflumuron in
by three chitin synthesis inhibitors. eastern subterranean termites (Isoptera:
J. Econ. Entomol., 97, 2015–2020. Rhinotermitidae). J. Econ. Entomol., 97,
63. Scottish Environmental Protection 593–600.
Agency (1999) Calicide (Tefluben- 71. Ameen, A., Wang, C., Kaakeh, W.,
zuron) – Authorisation for Use as an Bennett, G.W., King, J.E., Karr, L.L.,
Infeed Sea Lice Treatment in Marine and Xie, J. (2005) Residual activity and
Cage Salmon Farms. Risk Assess- population effects of noviflumuron for
ment, EQS Recommendations No. 29, German cockroach (Dictyoptera: Blat-
pp. 1–15. tellidae) control. J. Econ. Entomol., 98,
64. Reid, B.L., Appel, A.G., Demark, J.J., 899–905.
and Bennett, G.W. (1992) Oral tox- 72. King, J.E. (2005) Ovicidal activity of nov-
icity, formulation effects, and field iflumuron when fed to adult German
performance of flufenoxuron against cockroaches (Dictyoptera: Blattellidae).
the German cockroach (Dictyoptera: J. Econ. Entomol., 98, 930–932.
Blattellidae). J. Econ. Entomol., 85 (4), 73. Wang, C. and Bennett, G.W. (2006) Effi-
1194–1200. cacy of noviflumuron gel bait for control
65. Getty, G.M., Haverty, M.I., Copren, of the German cockroach, Blattella
K.A., and Lewis, V.R. (2000) Response of germanica (Dictyoptera: Blattellidae) -
Reticulitermes spp. (Isoptera: Rhinotermi- laboratory studies. Pest Manage. Sci., 62,
tidae) in northern California to baiting 434–439.
with hexaflumuron with Sentricon ter- 74. Cutler, G.C., Scott-Dupree, C.D.,
mite colony elimination system. J. Econ. Tolman, J.H., and Harris, C.R. (2005)
Entomol., 93, 1498–1507. Acute and sublethal toxicity of noval-
66. Haagsma, K.A. and Rust, M.K. (2005) uron, a novel chitin synthesis inhibitor,
Effect of hexaflumuron on mortality to Leptinotarsa decemlineata (Coleoptera:
of the Western subterranean termite Chrysomelidae). Pest Manage. Sci., 61,
(Isoptera: Rhinotermitidae) during and 1060–1068.
following exposure and movement of 75. Ishaaya, I., Kontsedalov, S., and
hexaflumuron in termite groups. Pest Horowitz, A.R. (2003) Novaluron
Manage. Sci., 61, 517–531. (Rimon), a novel IGR: potency and
67. Rojas, M.G. and Morales-Ramos, J.A. cross-resistance. Arch. Insect Biochem.
(2001) Bait matrix for delivery of Physiol., 54, 157–164.
chitin synthesis inhibitors to the For- 76. Mulla, M.S., Thavara, U., Tawatsin, A.,
mosan subterranean termite (Isoptera: Chompoosri, J., Zaim, M., and Su, T.
Rhinotermitidae). J. Econ. Entomol., 94, (2003) Laboratory and field evaluation of
506–510. novaluron, a new acylurea insect growth
68. Sajap, A.S., Amit, S., and Welker, J. regulator, against Aedes aegypti (Diptera:
(2000) Evaluation of hexaflumuron Culicidae). J. Vector Ecol., 28, 241–254.
References 1011
77. Phillips McDougall Crop Protec- immatures of Chrysoperla rufilabris (Neu-

tion & Agricultural Biotechnology roptera: Chrysopidae). J. Econ. Entomol.,
Consultants: Benzoylurea (2010) 93, 234–239.
AgriService – Products Section – 2009 86. Binnington, K.C. (1985) Ultrastructural
Market, pp. 181–186. changes in the cuticle of the sheep
78. Blagburn, B.L., Vaughan, J.L., Lindsay, blowfly, Lucilia, induced by certain
D.S., and Tebbitt, G.L. (1994) Efficacy insecticides and biological inhibitors.
dosage titration of lufenuron against Tissue Cell, 17, 131–140.
developmental stages of fleas (Cteno- 87. Perng, F.S. and Sun, C.N. (1987) Sus-
cephalides felis) in cats. Am. J. Vet. Res., ceptibility of diamondback moths
(Lepidoptera: Plutellidae) resistant to
55, 98–101.
conventional insecticides to chitin syn-
79. Blagburn, B.L., Hendrix, C.M., Vaughan,
thesis inhibitors. J. Econ. Entomol., 80
J.L., Lindsay, D.S., and Barnett, S.H.
(1), 29–31.
(1995) Efficacy of lufenuron against
88. Kotze, A.C., Sales, N., and Barchia,
developmental stages of fleas (Cteno-
I.M. (1997) Diflubenzuron tolerance
cephalides felis felis) in dogs housed in
associated with monooxygenase activity
simulated home environments. Am. J. in field strain larvae of the Australian
Vet. Res., 56, 464–467. sheep blowfly (Diptera: Calliphoridae).
80. Dean, S.R., Meola, R.W., Meola, S.M., J. Econ. Entomol., 90, 15–20.
Sittertz-Bhatkar, H., and Schenker, R. 89. Perng, F.S., Yao, M.C., Hung, C.F.,
(1998) Mode of action of lufenuron and Sun, C.N. (1988) Teflubenzuron
on larval cat fleas (Siphonaptera: resistance in diamondback moth (Lepi-
Pulicidae). J. Med. Entomol., 35, doptera: Plutellidae). J. Econ. Entomol.,
720–724. 81 (5), 1277–1282.
81. Dean, S.R., Meola, R.W., Meola, S.M., 90. Guo, H.F., Fang, J.C., Liu, B.S., Wang,
Sittertz-Bhatkar, H., and Schenker, R. J.P., Zhong, W.F., and Wan, F.H. (2007)
(1999) Mode of action of lufenuron in Enhancement of the biological activity of
adult Ctenocephalides felis (Siphonaptera: nucleopolyhedrovirus through disruption
Pulicidae). J. Med. Entomol., 36, of the peritrophic matrix of insect larvae
486–492. by chlorfluazuron. Pest Manage. Sci., 63,
82. Kim, K.S., Chung, B.J., and Kim, H.K. 68–74.
(2000) DBI-3204: a new benzoylphenyl 91. Chen, X., Tian, H., Zou, L., Tang, B.,
urea insecticide with a particular activity Hu, J., and Zhang, W. (2008) Disruption
against whitefly. Brighton Crop. Prot. of Spodoptera exigua larval development
Conf. – Pests Dis., 1, 41–46. by silencing chitin synthase gene A with
RNA interference. Bull. Entomol. Res.,
83. Aki, M. (2003) Insecticidal Baits
98, 613–619.
Containing Bistrifluron and Method
92. Zhang, J., Liu, X., Zhang, J., Li, D.,
for Control of Ants and Cock-
Sun, Y., Guo, Y., Maa, E., and Zhu,
roaches. Japanese Patent 2003-
K.Y. (2010) Silencing of two alternative
6,767[2004/217,569], (Shinto Fine
splicing-derived mRNA variants of chitin
Co., Ltd., Japan) January 15, 2003,
synthase 1 gene by RNAi is lethal to the
p. 5. oriental migratory locust, Locusta migra-
84. Izawa, Y., Uchida, M., and Yasui, M. toria manilensis (Meyen). Insect Biochem.
(1986) Mode of action of buprofezin Mol. Biol., 40, 824–833.
on the twenty-eight-spotted ladybird, 93. Matsumura, F. (2010) Studies on the
Henosepilachna vigintioctopunctata action mechanism of benzoylurea insec-
Fabricius. Agric. Biol. Chem., 50 (5), ticides to inhibit the process of chitin
1369–1371. synthesis in insects: a review on the
85. Liu, T.X. and Chen, T.Y. (2000) Effects status of research activities in the past,
of the chitin synthesis inhibitor bupro- the present and the future prospects.
fezin on survival and development of Pest. Biochem. Physiol., 97, 133–139.
29.2
Mite Growth Inhibitors: Clofentezine, Hexythiazox, Etoxazole
29.2.1
Introduction
Phytophagous mites are important pests in many cropping systems worldwide,

including those of fruits, vegetables, grapes, and ornamentals. Two major problems
in the control of these mites are their high reproductive potential and extremely
short life cycle, both of which facilitate a rapid resistance development to many
acaricides, often after only a few applications. Consequently, the history of spider
mite control has become a head-to-head race between: (i) resistance development
in frequently exposed populations; (ii) suitable measures for resistance control by,
for example, spray programs for commercial products; and (iii) the development of
acaricides with a new mode of action that are not affected by established resistance
mechanisms [1].
During recent decades, many different biochemical targets in mites have been
addressed in the quest for new acaricides [2, 3]. One such group of acari-
cides – the mite growth inhibitors – have been classified as Group 10 of the
Insecticide Resistance Action Committee (IRAC) mode of action classification
scheme (www.irac-online.org). In this chapter, the details are presented of mite
growth regulators such as clofentezine (1), diflovidazin (2), hexythiazox (3), and
etoxazole (4), on the basis that these compounds share some common characteris-
tics (Figure 29.2.1).
These compounds interfere with mite development, and demonstrate activity
against all developmental stages of mites (eggs, larvae, nymphs), except the adult
stage. As cross-resistance occurs between some of these compounds – such as
Cl F
Cl Cl
N N
N N
N N
N N
F
Clofentezine 1 Diflovidazin 2
O
Me
N N F
H N
S O
OEt
O
Cl F
Hexythiazox 3 Etoxazole 4
Figure 29.2.1 Structures of the mite growth inhibitors.

clofentezine and hexythiazox – they have been allocated together in IRAC Group
10, as mentioned above [4].
In this chapter, details are provided of the discovery, synthesis, structure–activity
relationships (SARs), biology, and biochemistry of these compounds.
29.2.2
Tetrazines (Clofentezine, Diflovidazin = Flutenzine)
The mite ovicidal activity of ortho-halogen phenyl-substituted tetrazines was first

discovered at Chesterford Park Research Station in 1976. In contrast to the inactive
unsubstituted bis-phenyl derivative (entry 1, Table 29.2.1), introduction of the
2-chloro-phenyl residue on one side of the tetrazine led to an interesting ovicidal
mite activity (entry 2, Table 29.2.1) that served as a lead for further research [5].
Some representative structure–activity data for these compounds are listed in
Table 29.2.1.
While retaining the 2-chloro-phenyl substitution at position 3 of the tetrazine,
many different substituents at position 6 were screened, with highly active com-
pounds being identified with 2-chloro-phenyl (entry 5) and a cyclohexyl (entry
9) derivatives. In field trials, the bis-2-chloro derivative showed clear advantages,
especially with regards to its longlasting activity, and this was finally selected for
development by Schering AG (which later was incorporated into AgrEvo and Bayer
CropScience AG) under the common name, clofentezine (Table 29.2.2) [6].
Clofentezine was presented by Schering at the 1981 British Crop Protection
Council Conference [7], launched in 1983 under the main trade name Apollo,
and subsequently proved to be a quite successful product in the top fruit sector.
Table 29.2.1 Selected structure–activity data for the diaryl-tetrazines.

N N
R1 R2
N N
Entry Compound number R1 R2 Relative ovicidal

mite activitya
1 Ph Ph 0
2 Lead compound 2-Cl-Ph Ph 2
3 2-Cl-Ph 4-Cl-Ph 0
4 2-Cl-Ph 3-Cl-Ph 0
5 Clofentezine 2-Cl-Ph 2-Cl-Ph 4
6 2-Cl-Ph 2-Br-Ph 3
7 2-Cl-Ph 2-I-Ph 3
8 2-Cl-Ph CH2 -Ph 2
9 2-Cl-Ph Cyclohexyl 4
10 2-Cl-Ph CH2 -cyclohexyl 3
a
0 = weak to 4 = excellent.
Table 29.2.2 Data for clofentezine.

Cl Cl
N N
N N
Common name Clofentezine

IUPAC name 3,6-bis(2-Chlorophenyl)-1,2,4,5-tetrazine
Development code NC 21314
Patent EP5912 (priority 1978-05-02)
Launch 1983
log P 4.1
Water solubility < 1 mg l−1
Toxicity (LD50 ; rat, oral) > 5200 mg kg−1
In 2001, Aventis CropScience sold clofentezine to the Israeli pesticide producer

Makhteshim-Agan Industries (MAI). More recently, clofentizine has also been
®
marketed by Arysta as Acaristop .
During the early 1990s, the Hungarian company Chinoin (now Agro-Chemie)
began chemical research in the area of the miticidal tetrazines, with the goal
of finding compounds with improved properties [8, 9]. Subsequently, a series of
unsymmetrical substituted tetrazines was synthesized, whereby the introduction
of a fluorine atom into the ortho position of the phenyl ring resulted in acaricides
with improved translaminar and transovarian properties, and with higher vapor
activities [10]. Of those derivatives, the 2,6-difluoro compound 2 was selected for
further development and presented under the code name SZI 121 at the 1994
British Crop Protection Council Conference [11]. The initially proposed common
name flufenzine was later changed to diflovidazin (provisionally approved by ISO
in 2004). The product was launched in Hungary in 1997 under the trade name
®
Flumite 200 (Table 29.2.3). Recently, it was reported that some Chinese companies
(e.g., Baoye Chemical and Zhejiang Heben) are interested in flufenzine for their
domestic market, and will institute its production when government approval
is acquired [12]. Very recently, diflovidazin was added to IRAC mode of action
classification subgroup 10A (www.irac-online.org, list Version 7.0).
The synthesis routes leading to clofentezine and diflovidazin are shown in
Scheme 29.2.1. The synthesis of clofentezine begins with a twofold acylation
of hydrazine hydrate with 2-chlorobenzoyl chloride, followed by a phosphorus
pentachloride-mediated activation; a second reaction with hydrazine hydrate then
produces the 1,2-dihydrotetrazine derivative, which is finally oxidized to clofen-
tezine using sodium nitrite [5, 13, 14].
29.2.2.1 Biology and Biochemistry

Clofentezine and diflovidazin have been developed for the control of a wide range
of spider mite species, such as Tetranychus spp., Panonychus spp., and Eriophyid
Table 29.2.3 Data for diflovidazin.

Cl F
N N
N N
F
Common name Diflovidazin

IUPAC name 3-(2-Chlorophenyl)-6-(2,6-difluorphenyl)-1,2,4,5-tetrazine
Development code SZI 121
Patent EP635499 (priority 1993-07-21)
Launch 1997
log P 4.1
Water solubility < 1 mg l−1
mites, especially on top fruits and vines. Clofentezine is a specific contact acaricide
that acts primarily as an ovicide but with some effect on young motile stages,
and has a long residual activity; typically, when sprayed onto grape leaves prior
to the hatching of winter eggs of P. ulmi, its activity lasts for more than 60 days
[15]. Clofentezine has no activity against adult mites; rather, it interferes with cell
growth and differentiation during the final phases of embryonic and early larval
development. Clofentezine is particularly effective against mite eggs, including
winter eggs of the European red mite, P. ulmi. The compound is marketed in
different formulations and combinations with other acaricides and insecticides to
®
broaden the spectrum also against adults (e.g., Viktor CL is effective against all
®
stages of P. ulmi, T. urticae, and Eotetranychus carpini), additional insects (Torant ),
or to prevent the rapid development of resistance (Table 29.2.4).
Nevertheless, resistance to clofentezine was identified in different populations
of mites and areas, such as P. ulmi from orchards in Ontario after about five
years of use [16], or in populations of T. urticae from Australia [17, 18]. In these
cases, the resistance factors were between 770-fold and >2000-fold at LC50 - and
LC90 -values, respectively. Observations on such field populations indicated that
resistance persisted for at least two seasons [16]. Cross-resistance to hexythiazox
was also observed [17]. An enhanced detoxification by an increased activity of
mono-oxygenases and esterases is at least partially responsible for the observed
resistance and cross-resistance [19].
Both, clofentezine and diflovidazin have very favorable ecotoxicological proper-
ties. In particular, they are safe towards beneficial arthropods, including predatory
mites (of the genus Amblyseius, Phytoseiulus, Typhlodromus, and Zetzelia), predatory
insects (of the genus Anthocoris, Chrysoperla, Orius, and Stethorus), pollinating bees,
and parasitic wasps. This property leads to preferred uses of these agents under
integrated pest management conditions. Typically, the usage rates in the different
crops vary between 7.5 and 40 g a.i. hl−1 , depending on the water volume used for
Cl Cl
H2N-NH2
O
COCl
N NH2
H
F
O
H2N-NH2
Cl
F
Cl Cl Cl F
O O O O
N N N N
H H H H F
PCl5 PCl5
Cl Cl Cl F
Cl Cl Cl Cl
N N N N
F
H2N-NH2
H2N-NH2
EtOH
EtOH
Cl H H Cl Cl H H F
N N N N
N N N N
F
NaNO2 NaNO2
AcOH AcOH
Cl F
Cl Cl N N
N N
N N
N N F
Clofentezine 1 Diflovidazin 2
Scheme 29.2.1 Synthesis of the tetrazine acaricides clofentezine (1) and diflovidazin (2).
spraying in top fruits, soft fruits, vegetables (basic dose rate: 100–200 g a.i. ha−1 ),
and 150–250 g a.i. ha−1 in cotton (50–100 g a.i. hl−1 at 150–300 l ha−1 ).
Diflovidazin was introduced in 1997 to Eastern European and Asian markets
(Table 29.2.5),and has also been submitted for registration in the EU. In field trials,
diflovidazin (SZI-121) provides longlasting control at application rates as low as
80 g a.i. ha−1 against Panonychus, Tetranychus, Aculus, and Calipitrimerus spp. in
apple and vine [11]. It has both translaminar and transovarial activity, and is also
Table 29.2.4 Different formulations and mixtures of clofentezine.
Trade name Use Crops Content
Apollo 50 SC Control of a wide range Topfruit, citrus, vines, 500 g l−1

(Apollo 50 SC; of mite species cotton, vegetables, clofentezine
Acaristop 50 SC; ornamentals, soft
Sa-lan, Cara) and fruit, tea
20 SC
Apollo Plus (6SE) Control of a wide range Topfruit, citrus, vines, 60 g l−1 clofentezine
of mite species, cotton, vegetables, plus 540 g l−1
especially under ornamentals, soft mineral oil (SE)
circumstances of fruit, tea
developing tolerance to
clofentezine
Victor Tetranychus spp., Vegetables, vines, SE containing
Panonychus ulmi, others 200 g l−1
Eriophyid mites, esp. clofentezine
Calepitrimerus vitis +100 g l−1
Leafhoppers on vines fenpropathrin
Torant CL, Percut Tetranychus spp., P. Vines, vegetables; SC containing
ulmi, Eriophyid mites, strawberries, 200 g l−1
esp. Calepitrimerus vitis; ornamentals and clofentezine
grape berry moth roses (Percut) +40 g l−1 bifenthrin
Orion CL Tetranychus spp. P. ulmi, Topfruit and vines Twinpack with
Eriophyid mites suitable for IPM 500 ml clofentezine
situations 20 SC + 1000 ml
propargite 57 EW
Torero Control of mites Vegetables, vines, Twinpack with
others 500 ml clofentezine
20 SC + 300 ml
tau-fluvalinate 24 SC
Apollo/Kelthane Control of mites, esp. P. Citrus Twinpack with
citri and T. cinnabarinus 300 ml clofentezine
20 SC + 1000 ml
dicofol 48 LE
Apollo/Sanmite Control of mites with Citrus Twinpack with
side effect on whitefly 500 ml clofentezine
20 SC + 500 ml
pyridaben 20 SC
effective via the vapor phase. Like clofentezine, diflovidazin acts primarily as an
ovicide but in laboratory trials was also investigated for its activity against the
chrysalis stage of T. urticae. Interestingly, the LC50 of 0.39 ppm indicated a much
higher activity than for clofentezine (LC50 > 20 ppm) [9]. In soil, diflovidazin is
degraded more rapidly than clofentezine.
Table 29.2.5 Diflovidazin registrations (status 2004).
Country Trade name Crops Status Date
Hungary Flumite Vines, apples, peaches, plums Launched 1997

Georgia Flumite Apples, grapes, citrus Approved 1998
Kazakhstan Flumite Apples, cotton Approved 1998
UAE Flumite Vegetables Approved 1998
Uzbekistan Flumite Cotton Approved 1998
Yugoslavia Flumite Apples Launched 1998
29.2.3
Thiazolidinones (Hexythiazox)
The discovery of the thiazolidinone acaricides in the laboratories of Nippon Soda

began during studies on fungicidal thiazolo[2,3-b]triazine derivatives, when a weak
miticidal activity was observed with derivative 5.
O O O
Me Me Me
N N N N N N
H H
S N O S N S O
Me
5 6 7
Subsequently, several triazine ring-opened derivatives were screened, among

which the trans-configured derivatives 6 – and, especially, the thiazolidinone (7) –
not only showed very interesting activities against T. urticae but also served as leads
for a broad chemical optimization program. Selected structure–activity data for
these compounds are listed in Table 29.2.6 [20].
These examples illustrate the importance of a small alkyl group as R1 (methyl is
the optimum), the cyclohexyl group on the amide R2 (even cyclopentyl is less active),
and an electron-withdrawing group in the para position of the phenyl ring (X).
With regards to these constraints after field trials with different candidates, Nippon
Soda selected the derivative with the internal code NA-73 (entry 10 in Table 29.2.6)
for development under the common name hexythiazox (Table 29.2.7) [21]. Only the
trans diastereomer showed any acaricidal activity; separation of the enantiomers
showed that the activity was related to the (4R,5R) enantiomer, whereas the (4S,5S)
enantiomer was inactive [20].
Commercial hexythiazox is a racemic mixture of the two trans enantiomers
(the main synthetic pathways are shown in Scheme 29.2.2 [13, 20, 22]. Starting
from 4-chloropropiophenone, the key intermediate erythro amino-alcohol may be
obtained by a stereoselective catalytic reduction of the corresponding hydroxy
iminoketone, or by sodium borohydride reduction of the aminoketones obtained
via a Gabriel synthesis. Different routes lead from this amino-alcohol to the
Table 29.2.6 Selected structure–activity data for the thiazolidinones.
O
R1 R2
N N
H
S O
X
Entry R1 R2 X Relative to Tetranychus

activitya
1 H Cyclohexyl H 0
2 Lead compound Me Cyclohexyl H 2
3 Me Cyclohexyl H 1
4 Me n-Hexyl H 0
5 Me i-Pr H 0
6 Me Ph H 0
7 Me Cyclohexyl 2-Cl 1
8 Me Cyclohexyl 3-Cl 2
9 Me Cyclohexyl 3,4-Cl2 1
10 Hexythiazox Me Cyclohexyl 4-Cl 3
11 Et Cyclohexyl 4-Cl 2
12 n-Pr Cyclohexyl 4-Cl 0
13 i-Pr Cyclohexyl 4-Cl 0
14 Me Cyclohexyl 4-CF3 3
15 Me Cyclohexyl 4-Me 2
16 Me Cyclohexyl 4-OMe 2
a
Score: EC50: 0 = >125 ppm; 1 = 125−10 ppm; 2 = 10−1 ppm; 3 = <1 ppm.
trans-thiazolidinone system; the basis of all routes is activation of the hydroxy

group, for example, in the form of the sulfonate and a ring-forming reaction with
carbon disulfide or carbonyl sulfide. Final acylation of the NH group with cyclohexyl
isocyanate leads to hexythiazox. In this case, process optimization studies were
conducted by a group from the East China University of Science and Technology
[23].
Hexythiazox was presented by Nippon Soda during the very early 1980s [24], and
® ® ®
launched in 1985 as Nissorun (Nippon Soda), Cesar (AgrEvo), and Ordoval
(BASF).

Hexythiazox shows a broad acaricidal spectrum against different mite species such
as Tetranychus spp. and Panonychus spp., with LC50 -values between 0.2 and 1.1 ppm
[24]. Hexythiazox has both ovicidal and larvicidal activity but, similar to tetrazines,
it shows poor activity against adult mites (LC50 > 500 ppm).
Table 29.2.7 Data for hexythiazox.

O
Me
N N
H
S O
Cl
(4R,5R)-(+) Enantiomer of Hexythiazox
Common name Hexythiazox

IUPAC name trans-5-(4-Chlorophenyl)-N-cyclohexyl-4-methyl-2-
oxo-1,3-thiazolidine-3-carboxamide
Development code NA-73
Patent DE 03037105 (priority 1979-10-03), US4442116
Launch 1985
log P 2.5
Water solubility 0.5 mg l−1
Hexathiazox is used as an acaricidal treatment in apples, citrus, vine, vegetables,

and cotton; typical application rates are listed in Table 29.2.8 [24].
Hexythiazox has a very longlasting activity; for example, against Brevipalpus
phoenicis it has been shown to be efficient for up to 127 days after application [25].
Hexythiazox has a highly favorable ecotoxicological profile, being safe against
predatory mites and beneficial insects. Consequently, it is especially useful in
integrated pest management (IPM) situations [26], or where predatory mites have
been used together with an acaricide to suppress T. urticae populations [27].
Resistance to hexythiazox has developed in different strains of mites, such as
T. urticae in Australia [17, 18], P. ulmi [17], and P. citri [28]. Although such
resistance has recently been described as polygenic in T. urticae [29], other studies
have suggested the presence of a single major gene mutation [28].
29.2.4
Oxazolines (Etoxazole)
During its oxazoline chemistry program, Yashima identified the high acarici-
dal activities of the 2,4-diphenyl-1,3-oxazolines of the structure type shown in
Table 29.2.9 [30]. Various structure–activity data of selected compounds synthe-
sized during the optimization program are also provided in Table 29.2.9 [31].
Starting from the unsubstituted lead compound (entry 1, Table 29.2.9), it
was found that ortho substitution X would in particular enhance the activity;
the 2,6-difluoro pattern was kept constant in further optimizations due to its
good aphicidal activity (entry 10, Table 29.2.9). With regards to the substituents
Y in the second phenyl ring, it was recognized that alkyl substituents in the
Me
O
1. Chlorination (e.g. Cl2)
t -BuONO
Cl 2. Gabriel synthesis
HCl
OH Me
Me NH2
N
O
O
Cl
Cl
H2
Pd-C
NaBH4
Me Me S Ph
NH2 CS2
N S
Ph-CH2Br H
OH OH
Cl erythro Cl
H2SO4 HCl
Me Me
NH2 NH2
CS2
Ph
OSO3H S S
NaOH
O
Cl Cl
Me H COS
N NaOH
S S NaOH
Cl Me H
H2O2
N
trans
NaOH S O
Cl
NCO
O
Me
N N
H
S O
Cl Hexathiazox
Scheme 29.2.2 Synthetic pathways to hexythiazox.

Table 29.2.8 Field applications of hexythiazox.
Crop Mite Dosage (g a.i. hl−1 )
Apple European red mite (P. ulmi) 3.0–5.0

Two-spotted spider mite (Tetranychus urticae)
Citrus Citrus red mite (P. citri) 2.5–5.0
Vine European red mite (P. ulmi) 2.5–5.0
Two-spotted spider mite (T. urticae)
Yellow grape mite (Eotetranychus carpini f. vitis)
Other fruits Two-spotted spider mite (T. urticae) 3.0–5.0
European red mite (P. ulmi)
Vegetables Two-spotted spider mite (T. urticae) 3.0–5.0 + adulticide
Carmin spider mite (T. cinnarabinus)
Desert spider mite (T. desertorum)
para position showed especially favorable acaricidal and aphicidal activities (e.g.,
entries 14 and 17 in Table 29.2.9). An additional ortho substituent in this ring can
increase the activity, possibly via suppression of the oxidative detoxification of the
oxazoline to an oxazole heterocycle. An optimal combination was found with the
2-ethoxy, 4-t-butyl pattern, which showed high activity against Tetranychus, Plutella,
and Myzus (entry 20, Table 29.2.9), and was therefore chosen for development
under the internal code YI-5301 (common name etoxazole). Recently, both two-
and three-dimensional quantitative structure–activity relationship (QSAR) studies
were reported for ovicidal activity of the 2,4-diphenyl-1,3-oxazoline analogs against
the two-spotted spider mite, T. urticae [32].
The synthesis of etoxazole is shown in Scheme 29.2.3 [22, 31]. Starting from
2-ethoxy-4-t-butyl acetophenone, standard procedures lead to an oxime interme-
diate, which is reduced to the corresponding amino-alcohol. Acylation of this
amino-alcohol with 2,6-difluorobenzoyl chloride, followed by base-catalyzed cy-
clization after activation of the hydroxy group, leads to etoxazole (4). An alternative
route starts with the amino acid ester; this is first acylated using 2,6-difluorobenzoyl
chloride and then reduced with sodium borohydride to the same final inter-
mediate. In 2008, further studies on the synthesis of etoxazole, starting from
erythro-2-amino-1-(chloro-phenyl)-1-propanol via esterification, carbon disulfide
cyclocondensation, hydrogen peroxide oxidation, and condensation with cyclohexyl
isocyanate, was reported by the East China University of Science and Technology
[23].
Etoxazole was first launched in 1998 by Yashima in Japan under the trade name
®
Baroque , and is further marketed together with Sumitomo (Table 29.2.10). In
2005, etoxazole was added to Annex I of the EU agrochemical registration Directive,
®
and in 2006 Sumitomo Chemical launched its acaricide Borneo in Italy through
Isagro Italia for use on stone fruit, pome fruit, and grapevines. In 2009, the Chinese
Table 29.2.9 Selected structure–activity data of the diphenyl-oxazolines.

Y
X N
Compound number X Y Relative to Relative to Relative to

acaricidal insecticidal aphicidal
TETRUR PLUTMA MYZUPE
activity, eggs a activity b activity b
1 Lead compound H H 1 3 0
2 2-Cl H 2 2 0
3 3-Cl H 0 3 0
4 4-Cl H 0 3 0
5 2-Me H 0 3 3
6 2-OMe H 1 1 0
7 2-F H 1 2 0
8 2,6-Cl2 H 2 1 1
9 2-Cl,6-F H 4 0 1
10 2,6-F2 H 2 0 5
11 2,6-F2 2-Cl 3 2 5
12 2,6-F2 3-Cl 2 0 0
13 2,6-F2 4-Cl 6 0 0
14 2,6-F2 4-Me 3 0 5
15 2,6-F2 2-OMe 1 1 0
16 2,6-F2 2-OEt 1 1 0
17 2,6-F2 4-t-Bu 6 0 5
18 2,6-F2 2,4-C2 4 3 5
19 2,6-F2 2,4-Me2 1 2 5
20 Etoxazole (4) 2,6-F2 2-OEt, 4-t-Bu 6 5 5
a
b
company Guangdong Tianhe Agricultural and Sumitomo signed an agreement to

jointly promote etoxazole [33].
In contrast to clofentezine, diflovidazin, and hexythiazox, which belong to the
IRAC subgroup 10A, etoxazole is grouped in the separate subgroup 10B.

Like hexythiazox, etoxazole has excellent activity on the eggs, larvae, protonymphs,
and deutonymphs of susceptible spider mites, but is ineffective against adults.
However, in contrast to other insecticides that interfere with the molting process,
etoxazole shows good effects on the juvenile stages of aphids; in the laboratory, the
molting process was shown to be incomplete after treatment with etoxazole,
with LC50 -values of 0.5 and 2.2 ppm for A. gossypii and M. persicae nymphs,
O
Me OEt
1. Br2
2. KOAc
O
OEt
AcO
NH2OH
H2N
HO OEt
N O OEt
OEt
AcO F
Cl
LiAIH4
THF O
F
Amino-alcohol
route F H
H2N Amino-acid
N
OEt route
HO OEt
O
O OEt
F
F
Cl
NaBH4
O MeOH
F H
F N
OEt
O OH
F
1. SOCl2 or MsCl
2. NaOH
F
N
OEt
O
F
Etoxazole 4
Scheme 29.2.3 Synthetic pathways leading to etoxazole.

Table 29.2.10 Data for etoxazole.
F
N
OEt
O
F
Common name Etoxazole

IUPAC name 5-tert-Butyl-[2-(2,6-difluorophenyl)-4,5-dihydro-
1,3-oxazol-4-yl]-phenetole
Development code YI-5301 (Yashima), S-1283 (Sumitomo)
Patent WO9322297 (priority 1992-04-28) [25]
Launch 1998
log P 5.59
Water solubility 0.075 mg l−1
respectively [34] (etoxazole showed no activity against adult aphids). The activity
of etoxazole (YI-5301) on the eggs of four major spider mite species (T. urticae,
T. kanzawai, P. citri, P. ulmi) was described to be 100-fold higher than for
hexythiazox, which was also demonstrated by treatment of the larvae, protonymphs,
and deutonymphs of these mite species. Etoxazole was equally active against
the eggs of hexythiazox-resistant P. citri and T. kanzawai (hexythiazox LC50 >
1000 ppm), there being no cross-resistance [34]. The biochemical pathway on
which etoxazole has an effect in mites and aphids was suggested to be very similar
to that affected by the benzoylureas [31]. This proposal was supported by the
findings of Nauen and Smagghe, who described chitin biosynthesis inhibition by
etoxazole in Spodoptera frugiperda with similar symptomatology to poisoning with
triflumuron [35]. These authors also showed chitin biosynthesis to be inhibited in
both whole larvae and isolated integuments.
Under field conditions, etoxazole can be used in many crops (apples, cherries,
citrus, cotton, cucumbers, egg plants (aubergines), fruit, ground covers, Japanese
medlar, melons, ornamental plants, ornamental trees, peas, shrubs, strawberries,
tea, tomatoes, watermelons, vegetables, vines), against all important mites (Ambl-
yseius fallacis, E. carpini, E. lewisi, Oligonychus illicis, O. ununguis, P. citri, P. ulmi,
Stethorus punctum, T. cinnabarinus, T. pacificus, T. urticae). The field application
rates range from 5 to 10 g hl−1 and from 100 to 150 g ha−1 , depending on the
crop and water volume used. The lowest rate necessary for control is in cotton, at
32–50 g a.i. ha−1 .
Currently, etoxazole been launched in several countries and will shortly be
launched elsewhere (Table 29.2.11).
Table 29.2.11 Marketing status of etoxazole.
Country Trade name Crops Status Date
Japan Baroque Citrus, melons, cherries, Launched 1998

watermelons, apples,
peas, peaches, tea,
strawberries,
cucumbers, egg plants,
Japanese medlar
South Korea Zoom Citrus Launched 1998
Turkey Zoom Apples, citrus, tomatoes, Launched 1998
cotton
Taiwan Zoom – Launched 2000
US Zeal Cotton Approved 2003
Tetrasan Outdoor uses: shrubs, Launched 2004
ornamental trees, Launched 2003
flowering crops, foliage
crops, ground covers
France Bornéo Vines, apples, peaches, Launched 2002
pears, nectarines,
apricots
Australia Paramite Apples, cotton Approved 2004
South Africa Smite (Philagro) Roses, apples, tomatoes, Approved –
Rose Spider Mite pears
(Ball Straathof)
EU – Fruit, vegetables, cotton Included in 2005
Annex I
Italy (Isagro Italia) Bornéo Vines, fruit trees Launched 2006
China (Guangdong) – – To be launched 2009
Resistance against etoxazole has been described in T. urticae, and shown to be

under monogenic control [29, 36].
The ecobiological properties of etoxazole are less favorable than those described
for clofentezine and hexythiazox. In experiments on the comparative toxicity of
acaricides to the predatory mite Phytoseiulus persimilis and the two-spotted spider
mite, T. urticae, etoxazole had no serious effect on the survival and reproduction
of adult female predatory mites, but caused high mortality rates in the eggs and
larvae of P. persimilis [37].
References
1. van Leeuwen, T., Vontas, J., 2. Dekeyser, M.A. (1994) Pestic. Sci., 40,
Tsagkarakou, A., Dermauw, W., and 85–101.
Tirry, L. (2010) Insect Biochem. Mol. 3. Dekeyser, M.A. (2005) Pest Manage. Sci.,
Biol., 40 (8), 563–572. 61, 103–110.
References 1027
4. Insecticide Resistance Action Committee 21. Iwataki, I., Kaeriyama, M., Matsui, N.,
(IRAC). Available at: http://www.irac- and Yamada, T. (1979) Nippon Soda
online.org. DE 03,037,105, (Prio: 03. 10. 1979),
5. Brooker, P.J., Parsons, J.H., Reid, J., corresponds to US Patent 4,442,116.
and West, P.J. (1987) Pestic. Sci., 18, 22. Becker Associates, Paris (2003) Pes-
179–190. ticide Production Database. Available
6. Parsons, J.H. (1978) FBC Ltd, Patent EP at: http://www.beckerdata.com/new/web/
5,912. index.php?p=ppd.
7. Bryan, K.M.G., Geering, Q.A., and 23. Lou, J., Liao, D., Wu, Z., Wang, M., and
Reid, J. (1981) Proc. Brighton Crop Prot. Li, C. (2008) Nongyao, 47 (5), 328–329,
Conf. – Pests Dis., 11 (1), 67–74. 332.
8. Hajimichael, J., Horvat, G., Pap, L., 24. Soda, N. (1984) Jpn. Pestic. Inf., 44,
Botar, S., and Szekely, I. (1996) J. Envi- 21–24.
ron. Sci. Health, B31 (3), 515–519. 25. Sato, M.E., Raga, A., Ceravolo, L.C.,
9. Pap, L., Hajimichael, J., and Bleicher, E. Cezario, A.C., and Rossi, A.C. (1995)
(1996) J. Environ. Sci. Health, B31 (3), Sci. Agric. (Piracicaba, Brazil), 52 (2),
521–526. 282–286.
10. Hajimichael, J.B., Bleicher, E., Pap, L., 26. Sterk, G. and Peregrine, D.J. (1989) Belg.
Szekely, I., Marmarosi Keller, K., and Meded. Fac. Landbouw. Univ. Gent., 54
Ori, J. (1993) Chinoin EP 635,499, (Prio: (3b), 969–973.
21. 07. 1993). 27. Cote, K.W., Schultz, P.B., and Lewis,
11. Pap, L., Hajimichael, J., Bleicher, E., E.E. (2004) J. Entomol. Sci., 39 (2),
Botar, S., and Szekely, I. (1994) Proc. 267–227.
Brighton Crop Prot. Conf. – Pests Dis., 1, 28. Yamamoto, A., Yoneda, H., Hatano, R.,
75–82. and Asada, M. (1995) Nippon Noyaku
12. Anonymous (2010) Crop Prot. Gakkaishi, 20 (4), 513–519.
China News, 4 (13), 7. Available at: 29. Asahara, M., Uesugi, R., and Osakabe,
http://www.cnchemicals.com. M.H. (2008) J. Econ. Entomol., 101 (5),
13. Unger, T.A. (1996) Pesticides Synthesis 1704–1710.
Handbook, Noyes Publications, Brack- 30. Suzuki, J., Kikuchi, Y., Toda, K.,
nell. Itoh, Y., Ishida, T., Ikeda, T., and
14. Lu, D., Hu, J., and Zhang, Y. (2005) Tsukidate, Y. (1992) Yashima WO
Xiandai Nongyao, 4 (1), 10–11. 93/22,297 (Prio: 28. 04. 1992).
15. Rauch, F., Lagouarde, P., and Batalla, 31. Suzuki, J., Ishida, T., Kikushi, Y., Ito, Y.,
J.C. (1988) Def. Vegetaux, 249–250, Morikawa, C. et al. (2002) J. Pestic. Sci.,
28–32. 27, 1–8.
16. Pree, D.J., Bittner, L.A., and Whitty, 32. Roy, K. and Paul, S. (2009) QSAR Comb.
K.J. (2002) Exp. Appl. Acarol., 27 (3), Sci., 28 (4), 406–425.
181–193. 33. Anonymous (2010) Insecticides
17. Nauen, R., Stumpf, N., Elbert, A., China News, 3 (1), 11. Available at:
Zebitz, C.P.W., and Kraus, W. (2001) http://www.cnchemicals.com.
Pest Manage. Sci., 57 (3), 253–261. 34. Ishida, T., Suzuki, J., Tsukidate, Y.,
18. Herron, G., Edge, V., and Rophail, J. and Mori, Y. (1994) Br. Crop Prot.
(1993) Exp. Appl. Acarol., 17 (6), Conf. – Pests Dis., 1, 37–44.
433–440. 35. Nauen, R. and Smagghe, G. (2006) Pest
19. van Leeuwen, T., van Pottelberge, S., Manage. Sci., 62, 379–382.
and Tirry, L. (2005) Pest Manage. Sci., 61 36. Uesugi, R., Goka, K., and Osakabe, M.
(5), 499–507. (2002) J. Econ. Entomol., 95 (6),
20. Kasahara, I., Matsui, N., Yamada, T., 1267–1274.
Kaeriyama, M., and Ishimitsu, K. (1991) 37. Kim, S. and Yoo, S. (2002) BioControl,
ACS Symp. Ser., 443, 340–351. 47 (5), 563–573.
1029
30
Midgut–Transgenic Crops Expressing Bacillus thuringiensis Cry
Proteins
30.1
Introduction
Worldwide, preharvest crop losses have been estimated as being 13.8% due to
insects and other arthropods, 11.6% due to disease (fungi, bacteria, and viruses), and
9.5% due to weeds [1]. In Africa and Asia – the continents with the largest annual
human population increases – total crop losses typically reach almost 50% [2]. In
order to control insects efficiently and in a sustainable way, synthetic insecticides
must be integrated with alternative pest control methods. One such method
involves the use of resistant plant varieties obtained through ‘‘classical breeding,’’
while another is to use biological insecticides such as sprayable formulations based
on Bacillus thuringiensis (Bt). Today, however, due to their limited field stability,
a low capacity to reach cryptic insects and a narrow spectrum of activity, Bt
sprays represent only a minor fraction of the insecticide market. Transgenic plants
expressing Bt insecticidal crystal protein (Cry) proteins can be used to overcome the
first two of these drawbacks. The potential benefits of Bt crops include increased
crop yields, a reduction in broad-spectrum insecticide use and the associated
application costs and energy input, a reduced need for scouting, an improvement
of the health conditions of farm workers, and savings in time. However, these
benefits should be balanced against putative safety and environmental risks, as
compared to the benefits and risks of insect control in conventional agriculture.
30.2
Plant Engineering
Since the first successful transformations of plants, significant progress has been
made such that the capacity to introduce and express foreign genes in plants now
extends to over 120 species, including some previously classified as recalcitrant [3].
• Agrobacterium-mediated transformation has proven an efficient and reliable
method to engineer different traits in a wide range of crops, both dicotyledonous
and monocotyledonous plants [4]. The main advantages of this DNA transfer

1030 30 Midgut–Transgenic Crops Expressing Bacillus thuringiensis Cry Proteins
method are the low level of rearrangements in the transforming DNA, and the
high number of plants with a single insertion of the transgene.
• In contrast, direct gene delivery systems – such as particle bombardment or
protoplast electroporation – frequently result in a higher frequency of complex
patterns of transgene integration.
• Equally important was the development of tissue culture techniques that allow
the production of highly regenerable tissues from immature undifferentiated
tissue, and the development of tools to control the expression of a transgene in a
plant.
• Today, plant transformation remains a random process with respect to the
integration site of the transgene into the plant genome, as it sometimes results in
suboptimal transgene expression or may have a negative impact on the expression
of endogenous plant genes.
• Somaclonal variation is another aspect that can potentially lead to transgenic
plants with suboptimal characteristics.
• Gene silencing – most likely due to the presence of multiple copies of foreign
gene sequences – has also been observed in transformed plants.
Together, these phenomena necessitate the generation of a large number of
transgenic plant lines (events) from which those plants with the best performance
(elite events) must be selected through several rounds of laboratory and field
evaluations – a process sometimes referred to as elite event selection.
30.3
Insecticidal Crystal Proteins from B. thuringiensis
Formulations of Bt spore–crystal mixtures have been used for more than 40 years,
and have shown Bt to be a highly specific, effective, and safe bioinsecticide. The
insecticidal activity of Bt is due mainly to the presence of the insecticidal crystal
proteins (Cry and Cyt proteins) and vegetative insecticidal proteins (Vips) [5].
Of the Vips, which are mostly produced during the vegetative stage of growth
of the bacterium, Vip1 and Vip2 binary toxins are specific for coleopteran insects,
whereas Vip3 proteins are specific for Lepidoptera [6]. Knowledge of the mode of
action of these toxins is rather limited [7–10].
In the case of the Cry proteins, which are produced during sporulation, much
information is available on their mode of action, based mainly on studies of Cry1A
proteins. The following model for the pathway of toxic action of these proteins has
been proposed [11–13]. When ingested by susceptible insects, the crystals dissolve
in the insect gut such that the protoxins are liberated and activated proteolytically to
a toxic fragment. This fragment passes through the peritrophic membrane, binds
to a specific cadherin on the brush border membrane of gut epithelial cells, and
then oligomerizes into a tetramer. The oligomer binds to a secondary receptor
such as aminopeptidase N or alkaline phosphatase, which is located in lipid rafts,
and (partially) inserts into the membrane, thus generating pores. The change in
membrane permeability leads to a colloid osmotic lysis of the gut epithelial cells and,
ultimately, to death of the insect. Also in dipteran and coleopteran insect species,
30.3 Insecticidal Crystal Proteins from B. thuringiensis 1031
the binding of Cry proteins appears to involve several different proteins [14–16].
The functional role of these receptors in toxicity has been assessed by using various
approaches, including RNA interference (RNAi) and ectopic expression in insect
cells [17]. The significance of cadherin-like proteins as Cry receptors is further
corroborated by the presence of mutated cadherin genes in resistant Heliothis
virescens [18, 19], Helicoverpa armigera [20], and Pectinophora gossypiella [21, 22]
insect strains. Likewise, a deletion mutation in an aminopeptidase N gene is likely
to confer resistance to Cry1Ac in a H. armigera strain [23]. In addition, glycolipids
and glycosylated proteins have been implicated in Cry binding [17, 24, 25]. Recently,
an alternative model has been proposed in which cell death is caused by activation of
a Mg+2 -dependent signal cascade pathway following the interaction of monomeric
Cry toxin with a cadherin receptor [26]. The observation that some engineered Cry
proteins are toxic to insect species with mutated or reduced cadherin receptors [27],
and that dominant-negative mutant Cry proteins can block the oligomerization and
toxicity of wild-type toxin [28], seem to argue against a predominant role of this
alternative toxicity pathway.
Today, more than 300 Cry sequences are known and have been classified solely
on the basis of sequence homology of the full-length proteins into 68 Cry classes
[29, 30]. Although there is no simple correlation between sequence and insecticidal
spectrum, some generalizations can be made. For example, Cry1 and Cry9 proteins
are active on lepidopteran larvae, whereas Cry3, Cry7, and Cry8 proteins are active
on coleopteran larvae. However, within a certain class, Cry proteins may have
widely differing activity spectra. This specificity remains one of the most intriguing
aspects of Cry proteins, as any step in the mode of action can influence the activity
spectrum.
Many Cry proteins, such as Cry1 and Cry9, are protoxins of about 120–140 kDa
that are proteolytically processed to an active toxic fragment of about 60–70 kDa.
Characterization of the proteolytic fragment and fragments generated by the
expression of truncated cry genes has indicated that, while only few amino acids
can be removed from the N terminus without interfering with biological activity,
about half of the protoxin can be removed at the C terminus. Other Cry proteins,
such as Cry2, are proteins of about 70 kDa and appear to require no C-terminal
activation for toxicity. Upon alignment of the Cry amino acid sequences, sequence
variation is clearly not distributed in a random fashion. Five conserved sequence
blocks can be distinguished in the activated fragment of most Cry proteins.
Today, the crystal structures of eight activated Cry proteins has been resolved
[31–38]. These proteins – Cry1Aa, Cry1Ac, Cry2Aa, Cry3Aa, Cry3Bb, Cry4Aa,
Cry4Ba, and Cry8Ea – have a very similar architecture and are composed of three
structural domains. The N-terminal domain [residues 58–290 (in Cry3A)] contains
seven α-helices, with the central more hydrophobic helix (α5) encircled by six outer
amphipathic helices. The second domain (residues 291–500) consists of three
β-sheets, packed as three sides of a prism. The third, C-terminal, domain (residues
501–644) is a β-sandwich with the outer sheet facing the solvent and the inner
sheet facing the other two domains. The level of amino acid sequence homology
between some of the eight Cry proteins is very low, yet their global structure is
quite similar. This suggests that most other Cry proteins possess a similar global
architecture. Based on these structures, and on the characterization of Cry mutants
and hybrids, the following hypotheses have been proposed regarding the function
of the three structural domains of Cry proteins: (i) the long amphipathic helices
of domain I would be responsible for pore formation; (ii) domain II would play a
major role in receptor binding; and (iii) domain III also plays a role in receptor
binding and perhaps modulates pore formation [39, 40].
30.4
Bt Plants
The first experiments to create plants expressing B. thuringiensis cry genes (Bt
plants) used T-DNA vectors in Agrobacterium carrying the coding sequence for
Cry1A protoxins. Only very low levels of Cry proteins and no significant insecticidal
activity related to Bt was observed [41–43]. The first successes were obtained
by expressing gene fragments encoding the toxic part of the Cry protein only.
The expression of truncated cry1Aa [42] and cry1Ab [43] (Figure 30.1) genes in
tobacco resulted in significant levels of protein and a high insecticidal activity to
Manduca sexta larvae feeding from the leaves. Tomato plants engineered with a
truncated cry1Ac gene [44] were also shown to be protected from feeding damage
by M. sexta, and this resulted in either mortality or growth inhibition of H. virescens
and Helicoverpa zea larvae. Tubers from different potato varieties engineered with a
truncated cry1Ab gene and infested with potato tuber moth (Phthorimea operculella)
larvae did not show tunneling or feeding damage following a two-month storage
period [45].
When tested under agronomic conditions in the field, transgenic tomato [46]
and tobacco plants [47] expressing truncated cry genes showed substantial levels of
insecticidal activity against their primary pest insects. Yet, it became apparent that,
for certain crops or insect pests, higher Cry protein expression levels were needed
Figure 30.1 Tobacco plants expressing a truncated cry1Ab

gene resulting in significant levels of Cry1Ab protein and
high insecticidal activity to Manduca sexta larvae feeding
from the leaves.
30.4 Bt Plants 1033
to achieve complete insect control in the field. Typically, expression levels of native
truncated cry genes in plants (usually about 0.001% of total soluble protein) were
lower than levels obtained with other transgenes. Plant genes generally have a
high G+C content, whereas bacterial cry genes typically have a high A+T content.
A+T-rich regions in native cry genes contain cryptic intron splice sites [48] and
potential polyadenylation sites [38], that results in aberrant splicing or premature
polyadenylation, both of which lead to nonfunctional mRNA. Furthermore, the
codon usage in cry and plant genes is significantly different. The presence of
rare plant codons in native cry genes could result in ribosomal pausing [49], and
perhaps also in an accelerated degradation of the cry gene messenger. However,
experimental evidence has suggested that the presence of rare codons per se does
not dramatically interfere with mRNA accumulation [50, 51]. On the other hand,
a comparison of mRNA and protein levels in plants transformed with truncated
cry1Ab genes with different degrees of modification led Perlak et al. [52] to suggest
that the presence of predominantly plant-preferred codons improved the overall cry
gene translational efficiency.
Several groups have shown that modifications in a specific region of the cry
coding sequence could result in significant improvements in expression. For
example, Perlak et al. [52] reported that changes in the 5 half of the cry1Ab coding
sequence were more efficient in achieving increased expression levels than changes
in the 3 half. Cornelissen et al. [53] identified a region between nucleotides 785
and 1285 in cry1Ab where transcript elongation was retarded. Tobacco plants
transformed with a cry1Ab gene with 63 translationally neutral substitutions in
this region showed up to 20-fold higher levels of the cry1Ab transcript [54]. It was
also shown that modifications which removed cryptic splice sites caused further
increases in transcript levels. Although modifications in a specific region may
result in significant improvements in expression, translationally neutral nucleotide
changes throughout the cry coding region are mostly used to obtain the highest
levels of expression of cry genes integrated in the nuclear genome. A truncated cry1A
gene was rendered more ‘‘plant-like’’ by modifications in the codons used, which
included the removal of potential polyadenylation signals and ATTTA sequences
by changing 62 of the 1743 nucleotides [52]. Transformation of tobacco and tomato
with constructs containing this modified gene were reported to result in a higher
number of insecticidal plants and higher expression levels (0.02% of total soluble
protein) than constructs containing the wild-type genes. Another modified cry1A
gene, which contained additional changes to increase the overall G+C content
and to introduce more plant-preferred codons, was reported to increase expression
levels up to about 0.2% of the total soluble proteins. Similar results were obtained
for a modified truncated cry1Ac gene. Cotton engineered with these modified
genes was reported to show protection against feeding damage caused by the main
lepidopteran pests [55, 56]. More recently, cry genes have been optimized for plant
expression by taking into account the above parameters, and constructed using
total gene synthesis [57].
During the early 1990s, the two main monocotyledonous crops – maize and
rice – were successfully transformed to express Cry proteins. Maize transformed
with a truncated modified cry1Ab gene [58], driven by either a constitutive

cauliflower mosaic virus (CaMV) 35S promoter or the combination of a green
tissue and pollen-specific promoter, was reported to result in plants with high
levels of expression (up to 4 μg mg−1 total plant protein). Field trials confirmed that
the tunneling of corn stalks by the European corn borer (Ostrinia nubilalis) was
dramatically reduced in such plants. Armstrong et al. [59] transformed maize with
a modified cry1Ab gene driven by the constitutive CaMV 35S promoter, and found
excellent European corn borer control. Similarly, maize lines transformed with a
modified cry9C or cry1Ab gene, driven by the CaMV 35S promoter and including
5 -untranslated leader sequences, showed a complete inhibition of stalk tunneling
by O. nubilalis in both greenhouse and field trials, as well as reduced feeding
damage by Agrotis ipsilon [60] (Figure 30.2). Certain modifications outside the
coding region may also contribute to Cry protein expression in plants. With rice (a
monocotyledonous plant), introns are frequently introduced to increase Cry protein
expression levels giving rice stem borer control [61–64]. In maize, Armstrong et al.
[59] also introduced an intron to increase Cry protein expression levels.
Modified or ‘‘synthetic’’ cry1A genes have now been used for the transformation
of several additional plant species, including peanut, Chinese flowering cabbage,
canola, broccoli and soybean [57], and coffee [65], as well as ‘‘exotic’’ crops such as
persimmon [66] and walnut [67]. Similarly, a cry1C gene redesigned for high-level
expression in plants provided protection to Spodoptera littoralis and Spodoptera
exigua in transgenic tobacco and alfalfa [68], and protection from Plutella xylostella
in transgenic broccoli [69]. Chickpea expressing a sequence-modified cry2Aa gene
demonstrated up to 98% mortality in bioassays on the H. armigera pod borer [70].
Using plastid transformation, high levels of expression can be obtained without
the use of modified or synthetic genes. It has been observed that bacterial genes
are well expressed in plant plastids, without any optimization of the codon usage.
In this way, high levels of Cry1Ac [71], Cry2Aa2 [72], and Cry1Ia5 [73] in tobacco,
and high levels of Cry1Ab in soybean [74], were obtained.
Figure 30.2 Cry1Ab corn event (right) gives excellent

European corn borer control, compared to the nontransgenic
control B73 (left). Split corn stalks and ears are shown fol-
lowing artificial infestation of corn plants with more than
1000 neonate Ostrinia nubilalis larvae per plant.
30.4 Bt Plants 1035
A next important step was the first registration in 1995 by the U.S. Environmental
Protection Agency (EPA) of Bt maize, Bt cotton, and Bt potato. Now, 15 years later,
about 133 million hectares of transgenic crops are grown worldwide [75], with
the USA, Brazil and Argentina growing most transgenic crops, followed by India,
Canada, China, Paraguay, and South Africa [75].
The most dominant Bt crop is Bt maize; in 2009, this was grown commercially
in the USA, Argentina, Brazil, Canada and South Africa and, on a lesser acreage, in
Uruguay, the Philippines, Spain, Chile, Honduras, the Czech Republic, Portugal,
Romania, Poland, Egypt, and Slovakia. In total, 42 million ha of mainly Bt maize
and Bt/herbicide-tolerant maize, as well as some herbicide-tolerant maize, were
grown [75].
The different Bt maize events target two maize pest complexes: lepidopteran
corn borers; and coleopteran corn rootworms:
• Maize events Mon810 and Bt11 both constitutively express the Cry1Ab protein,
and have an excellent control of corn borers [76]; both are sold under the trade
name ‘‘Yieldgard.’’
• In 2003, the maize event DAS-TC1507-1, containing a different Cry protein,
Cry1F, was introduced in North America as ‘‘Herculex I.’’ In addition to excellent
corn borer control, this was reported to provide control of armyworms and
cutworms.
• In the same year, the first event to control corn rootworm (Diabrotica spp.) – the
most destructive pest of maize in North America – was registered for commercial
sale in the USA [77]. The Mon863 event, sold as ‘‘Yieldgard Rootworm,’’ contains
a modified cry3Bb1 gene, optimized for expression in monocotyledons and
driven by a root-enhanced promoter. Two year later, a second Cry3Bb1 corn
event, MON88017, was registered. Additional to the corn rootworm resistance,
this event was also glyphosate-tolerant.
• Also in the USA, the event DAS-59122-7, ‘‘Herculex rootworm,’’ was registered in
2005. In this case, a binary delta-endotoxin Cry34/35Ab1 from the B. thuringiensis
strain PS149B1 was introduced into the maize and was reported to give excellent
corn rootworm control [78].
• The most recent corn rootworm resistant event was commercially registered
in 2006: MIR604, expressing a modified Cry3A protein. Like the unmodified
Cry3Bb1 protein, the unmodified Cry3A protein shows a low corn rootworm
toxicity. However, an engineered chymotrypsin/cathepsin G site in domain I
renders the Cry3A protein active against western corn rootworm larvae [79].
Recently, the industry has moved towards the pyramiding or stacking of multi-
genes in corn, with the intent of having two modes of action for lepidopteran pests,
and/or two modes of action for coleopteran pests [80]. For example, Syngenta’s
‘‘Agrisure Viptera™ 3111GT’’ is a breeding stack of the corn rootworm-resistant
MIR604 event and two lepidopteran resistant corn events: Bt11 and the new
Viptera™ MIR162 event which expresses a different type of insecticidal protein
from Bt, Vip3A [6, 81]. It has no sequence or structural homology with the crystal
proteins of B. thuringiensis, and has been reported to have a different mode of
action [7, 8]. In 2009, Monsanto and Dow AgroSciences received EPA approval for
the ‘‘Smartstax™ ’’ hybrid corn product with six different insect resistance genes,
besides different herbicide tolerance genes [82]. This is a breeding stack of four
corn transgenic events: the events Mon88017 and DAS-59122-7, which provide
two modes of action against corn rootworm; and the events DAS-01507-1 and
MON89034, which provide different modes of action against lepidopteran pests.
The new MON89034 event is a molecular stack of the cry2Ab gene and the cry1A105
gene, expressed in one plant. The Cry1A105 toxin is a hybrid Cry1A (domains I
and II)–Cry1F (domain III) protein.
Even 15 years after the introduction of the first commercial Bt crop, no Bt
soybean event has been commercially released. One of the main reasons for this
is that lepidopteran pests are not primary pests in the northern soybean growing
areas of the USA. However, now that soybean acreage is expanding in Argentina
and Brazil it is expected that Bt soybean events will become commercialized during
the coming years in South America. The first candidates for commercialization in
South America are soybean events expressing the Bt gene, tic107, a synthetic cry1A
gene very similar to cry1Ac [83]. Extensive testing has shown virtually complete
control of Anticarsia gemmatalis, Epinotia aporema, Rachiplusia ni, and Spilosoma
virginica in Argentina [84], and of A. gemmatalis, Pseudoplusia includens, and Hypena
scabra in the USA [84, 85].
In 2009, Bt cotton, Bt/herbicide-tolerant cotton, and some herbicide-tolerant
cottons were grown on 16.1 million ha, mainly in the USA, India, and China,
and also in Brazil, South Africa, Australia, and Argentina [75]. Of special mention
here is India, with an increase from 50 000 ha in 2002 to 8.4 million ha of Bt
cotton in 2009, planted by 5.6 million, mostly resource-poor, farmers. The event
Mon531 – containing the cry1Ac gene driven by a constitutive promoter and sold
as ‘‘Bollgard’’ in the US or as ‘‘Ingard’’ in Australia – has been the most important
Bt cotton event in the first 10 years. This provides near-100% control of square and
boll damage against the tobacco budworm (H. virescens) [86] and pink bollworm
(P. gossypiella). However, against the cotton bollworm (H. armigera in the Old Word,
H. zea in the New World), control was not always complete [87] and extra foliar
insecticide applications were required [88]. Since the introduction of ‘‘Bollgard,’’
sprays to control lepidopteran pests have been reduced by about half, however. In
China, besides the Mon531 event, cotton varieties GK using a modified Bt fusion
gene cry1Ab/cry1Ac were developed by public research institutes led by the Chinese
Academy of Agricultural science (CAAS), and have been grown since 1997 [89]. A
newer, genetically engineered variety, SGK321, produced by the CAAS was also
approved [90]. In this case, two pesticidal genes – the Bt fusion gene cry1Ab/cry1Ac
and the cowpea trypsin inhibitor – were inserted into cotton; the CAAS expects
that the bollworm will require more generations to develop insect resistance when
using these two genes. Singh et al. [91] have described the production of cotton
lines to control the cotton pest insect Spodoptera litura. Here, a hybrid Cry protein
was created by replacing amino acid residues 530–587 in a Cry1Ea protein with
the corresponding 70 amino-acid region of Cry1Ca, and expressed in cotton.
The event T-24 provided complete control of S. litura. Although the T-24 event
30.4 Bt Plants 1037
exhibited growth inhibition of the main lepidopteran cotton pest, H. armigera, it

will presumably have to be stacked with another event, such as a Cry1Ac event, in
order to be a commercially viable product.
In 2003, a second generation of Bt cotton, which expresses two insecticidal Cry
proteins, was introduced in the US. The ‘‘Bollgard II’’ event Mon15985 was created
by inserting the cry2Ab gene into the ‘‘Bollgard’’ event Mon531. ‘‘Bollgard II’’
expresses more toxin, and the levels of insect control are higher than in ‘‘Bollgard’’
[92]. Field studies showed that ‘‘Bollgard II’’ provided excellent cotton bollworm
control and an increased efficacy against armyworms and loopers [93]. Another
two-insecticidal protein product, ‘‘Widestrike,’’ was produced by crossbreeding
two insect-resistant cotton events: DAS-24236-5, producing constitutively Cry1F
protein; and DAS-21023-5, producing constitutively Cry1Ac protein. Introduced
into the US market in 2005, this was reported to provide excellent control of bud-
and bollworm [94, 95], while the presence of the Cry1F protein in the transgenic
variety was reported to increase the control of fall armyworm (Spodoptera frugiperda),
soybean looper (P. includens) and, to some extent, beet armyworm (S. exigua)
[96, 97]. Under extremely high insect pressure – that is, 100% of white flowers
artificially infested with cotton bollworm larvae – significant yield reductions could
be observed on both ‘‘Bollgard II’’ and ‘‘Widestrike’’ cotton [98]. This was confirmed
in naturally infested efficacy field trials, where cotton bollworm caused feeding
injury to 100% of bolls in non-Bt cotton, and where injury levels reached 13% in
‘‘Bollgard II’’ varieties and about 40% in ‘‘Bollgard’’ and ‘‘Widestrike’’ varieties
[99]. Supplemental insecticide applications may be needed.
The ‘‘Vip3A’’ Bt cotton event Cot102 expresses a different type of insecticidal
protein from Bt, Vip3A. Extensive field evaluation was reported to indicate an
efficacious control of bud- and bollworms, beet armyworm, and soybean looper
[100]. Bacheler and Mott [101] compared all of the above-mentioned Bt cotton
events in adjacent fields under the same cotton bollworm pressure and agronomic
conditions. In this study, with high bollworm insect pressure ‘‘Bollgard II’’ provided
excellent control, with 6% peak boll damage at the end of the season. The
‘‘Widestrike’’ and the ‘‘Vip3A’’ Cot102 event gave 15% and 14% peak boll damage,
respectively – much less than the adjacent ‘‘Bollgard’’. Field trials in Australia
confirmed that the Cot102 plants were highly efficacious against H. armigera [102].
Other ‘‘Vip3A’’ events, Cot202 and Cot203 – where the vip3A gene is driven by
a stronger constitutive promoter – are being field tested under high bollworm
pressure [103]. These showed about 1% boll damage, which was much better than
Cot102 and the nontransgenic control, which caused 10% and 73.2% boll damage,
respectively. Currently, the ‘‘Vip3A’’ Cot102 event is being pyramided with the
Cot67B cotton event, which produces the full-length version of the Cry1Ab crystal
protein [104]. This breeding stack will most likely be launched in the near future as
‘‘VipCot™ ’’ cotton; such a combination of the two insecticidal proteins expressed in
the ‘‘VipCot™ ’’ cotton has been shown to improve heliothine efficacy levels above
that of the single protein in the ‘‘Vip3A’’ Cot102 cotton [105].
In India, Bt brinjal or Bt eggplant would have been the first genetically engineered
food crop, had it not been caught in a ‘‘political hurricane’’ [106, 107], and
at the end of 2010 it was still unclear when approval for commercial growing
might be expected. In this case, a cry1Ac gene, which had already been used
in the ‘‘Bollgard’’ cotton, is expressed in brinjal and provides excellent control
against Leucinodes orbonalis [108]. This insect, a fruit and shoot borer, is very
difficult to control with insecticides: up to 40 insecticide sprays are necessary per
season.
The Bt rice variety developed by the Agricultural Biotechnology Research Institute
at Karaj was officially released in Iran in 2004 on 2000 ha. Full commercialization
was expected in 2006 in Iran, when it was expected that 10 000–20 000 ha would
be planted. The aromatic variety Tarom Molaii was transformed with a synthetic
cry1Ab gene under the control of the phosphoenolpyruvate carboxylase (PEPC)
promoter [109]. Alinia et al. [110] showed, in greenhouse tests, that stem borer
and leaf folder control was good in the early plant stages, but declined at the
flowering stage. However, as this Bt rice variety had not been commercialized
by 2010 the early prediction that China would be the first country to introduce
Bt rice to the market for the control of economically important lepidopteran rice
pests, such as striped stem borer (Chilo suppressalis), yellow stem borer (Scirpophaga
incertulas), and rice leaf folder (Cnaphalocrocis medinalis), may still hold true. In
the fall of 2009, the Chinese Ministry of Agriculture issued biosafety certificates
for the commercial production of two Bt rice lines – the parent line Huahui No.1
and the derived hybrid, Bt Shanyou 63 [75, 111]. The indica hybrid rice Bt Shanyou
63 expresses a cry1Ab/cry1Ac fusion gene, driven by the rice actin I promoter
[112], and has been extensively field tested [113]. The same transgene, driven by
the rice actin I promoter has been introduced in an elite indica rice IR72 [114]. In
both cases, a high level of protection was found against rice stem borers and rice
leaf folders. The KMD1 and KMD2 lines, generated by transforming Xuishi 11 (a
commercial Chinese japonica rice) with a synthetic cry1Ab gene under control of
a maize ubiquitin promoter [115] has been extensively field-tested in China. Shu
et al. [116] reported, in 2000, that the KMD1 line was resistant to eight different
lepidopteran pests. Many years of field testing [117, 118] have confirmed a high level
of stable resistance against stem borers and leaf folders. Chen et al. [119, 120], at
the Huazhong University, China, is developing Cry2A rice: in this case, a modified
cry2A gene under the control of the maize ubiquitin promoter was introduced
into the Minghui 63 line, and provided excellent stem borer control. The same
group has also transformed a cry1C gene under the maize ubiquitin promoter
into the Minghui 63 line. Subsequently, hybrids were evaluated in the field that
showed excellent stem borer and leaf folder control, and resulted in a significantly
increased yield [121]. Although the KMD lines, Cry1C rice and Cry2A rice, are
currently undergoing preproduction testing, they have not yet received a biosafety
certificate [111]. In japonica rice, Cry1C transgenic rice events have been generated
driven by the CaMV 35S promoter [122] and the rice rubisco small subunit rbcS
promoter [123]. The R5J line, expressing a cry1C gene under the control of the rice
rbcS promoter, possessed a normal agronomic performance and a high resistance
to leaf folders and stem borers, although when compared to other Bt rice events
it had very low Cry protein expression levels in the endosperm. A Korean group
30.5 Insect Resistance to Bt 1039
[124, 125] has also used the rbcS promoter to drive a cry1Ac gene in rice; in this case,
the expression was targeted to the chloroplast, which led to an increase in Cry1Ac
protein expression. Currently, basmati Bt rice is being field-tested and developed
in Pakistan: these involve rice events expressing a synthetic cry1Ab gene under the
control of different promoters, and also other events expressing a synthetic cry2A
gene under control of the CaMV 35S promoter [126, 127].
In contrast to most groups that use constitutive or tissue-specific promoters to
drive the expression of cry genes, such as cry1Ab, cry1Ac, cry1C, cry1Ab/cry1Ac,
and cry2A, Breitler et al. [128] used the −689/+197 region of the maize protease
inhibitor gene to direct the wound-induced expression of a cry1B gene in the elite
japonica cultivar Ariete. As a consequence, satisfactory levels of striped stem borer
(C. suppressalis) control were identified, with a low level of stem penetration, but
with more external symptoms compared to the rice lines where the expression is
constitutively driven by the maize ubiquitin promoter. It was suggested by these
authors that such a difference might be due to the time lag that occurs before the
plant is protected by the Cry1B protein.
As with corn and cotton, gene pyramiding or gene stacking is currently under-
going active investigation in rice, including basmati rice expressing Cry1Ac and
Cry2A in Pakistan [129], and different combinations of Cry1Ab/Cry1Ac, Cry1C,
and Cry2A rice in China [130].
30.5
Insect Resistance to Bt
The evolution of resistance in insect populations represents a serious threat to

the continued success of Bt crops. The first report of resistance to Bt was made
in 1985, when a 250-fold level of resistance to Bt was observed in a Plodia
interpunctella population from grain bins that were regularly treated with Bt. Since
then, a substantial number of strains of different insect species with various
levels of resistance to Cry proteins have been obtained via laboratory selection
experiments, or have been established from field collections [131]. In the case of
laboratory selection experiments, it is impossible to predict whether resistance will
develop in the field, or which resistance mechanisms will be selected; however,
the results may indicate the repertoire of resistance mechanisms available within
a certain population. Insects might, in principle, become resistant to Cry proteins
due to mutations in genes encoding the proteins that are involved in any of the
different steps in the mode of action. Several mechanisms have been observed
in laboratory-selected insect strains [131], such as an altered binding to midgut
receptors, altered protoxin activation, toxin degradation, more efficient repair
(or replacement) of the damaged midgut cells, esterase sequestration [132], and
elevated immune status [133]. In contrast, only one major mechanism – altered
binding – has yet been detected in field-selected resistant insects. In most such
cases, the pattern of cross-resistance parallels the pattern of binding specificity
of the Cry proteins in the particular insect species. For example, a P. xylostella
strain collected from fields in Hawaii, which had been treated with a sprayable Bt
product (DiPel) and further selected in the laboratory, had high levels of (cross-)
resistance to Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, and Cry1Ja, but no significant
(cross-) resistance to Cry1Ba, Cry1Bb, Cry1Ca, Cry1Da, Cry1Ia, or Cry2Aa [134].
The binding of Cry1Ab and Cry1Ac, but not Cry1Ca, was strongly reduced in this
resistant strain. A very similar pattern of resistance and binding characteristics was
observed in a P. xylostella strain from Pennsylvania [135], the Philippines [136],
and Florida [137], as well as in two P. gossypiella resistant strains [138]. Indeed,
while there was a complete lack of Cry1Ab binding in the P. xylostella strain from
Florida, binding of Cry1B and Cry1C was unaltered [137]. These cross-resistance
and binding data in P. xylostella can be understood in view of the model for the
Cry binding sites in this species. According to this model, one site (site 1) is
recognized only by Cry1Aa, another (site 2) is shared among Cry1Aa, Cry1Ab,
Cry1Ac, Cry1F, and Cry1J, and two additional sites bind Cry1Ba (site 3) and
Cry1Ca (site 4) [131]. Site 1 appears to be a nonfunctional binding site. Resistant
P. xylostella strains, selected with Cry1A containing Bt products, appear to have
an altered site 2 (which explains their cross-resistance to Cry1F and Cry1J), while
their sites 3 and 4 have remained unaltered (which explains their full susceptibility
toward the two Cry proteins). The lack of such cross-resistance can be exploited
in resistance management strategies. Resistance – at least in field-evolved resistant
insect strains – appears to be autosomally inherited and, in many cases, involves a
single major locus (as assessed from backcross data) and reverts upon withdrawal
of selection; this suggests fitness costs associated with resistance genes or closely
linked loci. On a molecular genetics level, the mutant alleles of a cadherin gene,
encoding a putative functional Cry1A receptor, have been linked to resistance in H.
virescens [18, 19], H. armigera [20], and P. gossypiella [21, 22]. The level of dominance
has been tested in five resistant insect strains using transgenic plants expressing
high levels of Cry proteins; in all of these cases, resistance to the expressed Cry
protein was demonstrated to be functionally recessive [21,139–142]. Interestingly,
resistance to Cry proteins in diet or leaf dip bioassays does not necessarily enable
resistant larvae to survive on Bt plants [143].
Recently, evidence for field-developed insect resistance to Bt crops – defined as
a genetically based decrease in the susceptibility of a population to a toxin by
exposure of the population to the toxin in the field – has been reported in some
populations of five lepidopteran pest species, namely Busseola fusca in South Africa,
S. frugiperda in Puerto Rico, P. gossypiella in western India, H. zea in southeastern
USA, and H. armigera in Australia [144, 145], although some of this evidence has
been disputed [146, 147].
30.6
Resistance Management with Bt Plants
In insects, resistance is a preadaptive phenomenon that develops by the selection

of rare individuals of a population that can survive a certain insecticide treatment.
30.6 Resistance Management with Bt Plants 1041
Resistance management strategies attempt to prevent or diminish the selection of

the rare individuals carrying resistance genes, and hence to keep the frequency of
resistance genes below levels that would result in inefficient insect control.
Several different strategies have been developed that should – at least in the-
ory – slow down the development of insect resistance in Bt plants [148, 149]:
• Tissue-specific Bt protein expression (protecting only the critical tissues).
• Wound-induced Bt protein expression (if expression were only to be induced at
a threshold level of insect damage).
• Chemical-induced Bt protein expression.
• Moderate Bt expression.
• Ultrahigh Bt protein expression (making the plant a nonhost for the pest).
• Rotation of crops expressing different types of Bt protein.
• Pyramiding (expression of different Bt protein types in the same crop plant).
When Bt crops were first grown commercially in 1996, there was a consensus
among population geneticists and insect resistance experts that the best insect
resistance management (IRM) tactic for Bt crops was the high-dose strategy
combined with a refuge. The principle of the high-dose strategy is that the
plant tissues express a Bt protein dose sufficiently high to kill all of the most
common carriers of resistance – that is, the heterozygote resistant insects. By
using modified Bt genes, a high-dose (defined as 25-fold the dose needed to kill
all homozygous susceptible larvae) should be achieved in plants. A refuge is an
area free of toxin-expressing plants that allows homozygote susceptible insects to
survive. Provided that the initial allele frequency for resistance is low, any rare
homozygous resistant insect – emerging from the Bt crop area – will more likely
mate with susceptible insects from the refuge than with other resistant insects.
Such crosses will result in a heterozygous resistant progeny, which will be killed
by the transgenic crop plants, and hence cause a dilution of resistance. Refugia
that are temporally and spatially contiguous with the transgenic crop should
ensure random mating between homozygote resistant and susceptible adults, and
should produce at least 500 susceptible moths for each homozygous resistant moth
emerging from the Bt crop. Another prerequisite for the high-dose/refuge strategy
is that the resistance is recessive, or at least is partially recessive. As noted above,
resistance to Cry proteins, as tested on high-dose transgenic Bt plants, is indeed
functionally recessive. Although the high-dose/refuge strategy may be difficult to
realize for sprayable insecticides, it seems likely to be efficacious for Bt plants.
Whereas, the validity of the high-dose/refuge strategy was originally based only
on projections from computer models simulating insect population growth under
various conditions, more recent studies have provided experimental support for this
strategy. Indeed, the selection of P. xylostella under laboratory conditions resulted
in resistance in colonies without refuge more rapidly than in those provided with
a refuge [150]. Furthermore, controlled greenhouse trials [151] and field trials [152]
involving Cry1Ac-expressing broccoli plants and artificial P. xylostella populations,
with known Cry1A resistance allele frequencies, have demonstrated that resistance
could be delayed by increasing the percentage of refuge plants. These experiments
also indicated that, under those conditions, a 20% refuge, separated from the Bt
plants, was more effective in maintaining the population of susceptible insects than
a 20% mixed refuge, created by planting a mixture of seeds of Bt and non-Bt plants.
Based on the results of these and other studies, Shelton et al. [76] concluded that
separate refuges are superior to seed mixtures for delaying resistance for insects
that can move between plants in the larval stage.
Unprecedented in the field of insect control, the US EPA required a compulsory
IRM plan, based on the high-dose/refuge strategy, in 1996 with the introduction of
Bt crops. Based on the experience with Bt crops grown under different agronomic
conditions, the plan is further optimized on a regular basis. An overview of the
current refuge requirements set by the US-EPA for Bt crops is described by
MacIntosh [153]. For example, the IRM plan for Lepidoptera controlling maize
expressing a single Cry protein requires a structured refuge of at least 20%
non-Bt maize, but 50% in cotton growing areas due to the extra potential selection
pressure on H. zea from Cry1A-expressing cotton. The refuge corn can be treated
with insecticides only when the level of pest pressure meets or exceeds the economic
threshold, and sprayable Bt insecticides must not be applied to the refuge. The
refuge must be placed within 1 km [0.5 mile, but 0.5 km (0.25 mile) preferred] of
the Bt maize field, and may be a separate field, a block within the maize field,
the field perimeters, or an alternation of four or more consecutive rows of refuge
maize with Bt maize. For coleopteran-controlling maize expressing a single Cry
protein, on each farm at least 20% non-Bt should be planted. The refuge maize
can be treated for corn rootworm larvae and other soil pests, and must be planted
within or adjacent to the Bt maize fields. Alternatively, the refuge may be planted
as in-field or perimeters strips, which must be at least six consecutive rows wide.
Whereas, structured refuges are required for cotton varieties expressing a single Cry
protein, and for cotton varieties expressing two Cry proteins in Arizona, California,
New Mexico, and west Texas, no such refuges are required for these latter cotton
varieties in southeast USA. Indeed, the US EPA determined that the amount of
natural refuges for H. virescens and H. zea – the two cotton key pests species in
this region – would be effective for the cultivation of these cotton varieties [154].
In such Bt plants, the high-dose/refuge strategy is combined with the strategy of
pyramiding or stacking two or more Bt toxins, with a different mode of action, into
one variety [155]. In this strategy, all insects – except for the extremely rare double
homozygous resistant individuals (with complete resistance) – will be killed, and
the development of resistance to stacked toxins is expected to be much slower than
to single toxin plants [149]. Computer models [149] have shown that the refuge
size can be reduced from 30–40% when using single Bt plants sequentially to
5–10% for stacked or ‘‘dual’’ Bt plants. Zhao et al. [156] showed, experimentally in
the greenhouse, that the use of transgenic plants expressing two Bt toxins binding
to a different site in the target insect delayed the development of resistance :
a population of P. xylostella containing genes for resistance against Cry1Ac and
Cry1C developed resistance more slowly to the stacked Cry1Ac/Cry1C broccoli with
20% refuge than to the Cry1Ac broccoli with 20% refuge. Also, compared to single
Bt plants deployed in mosaics (with 20% refuge), the resistance development was
delayed.
Other requirements of the IRM plan are annual resistance monitoring, grower
education, compliance assurance, research, and reporting. The research can be
on the mode of action, pest biology, and resistant insect colonies. There is also a
requirement for a remedial action plan should insect resistance develop in the field
[153].
In those species for which field-evolved resistance has been reported in some
populations, it appears that some of the criteria for the high dose–refuge tactic
have not been met. For example, resistance development in S. frugiperda to Bt
corn in Puerto Rico is likely associated with a lack of high dose and sufficiently
abundant refuges [157]. Resistance development in B. fusca in South Africa to Bt
corn is suspected to be due, at least in part, to a lack of grower compliance with the
refuge strategy [158].
IRM plans need to take into account the specific agricultural practices found in
local and regional environments. In China, cotton is typically cultivated alongside
a number of crops that are hosts for H. armigera and can function as natural
refuge crops; structured refuges have, therefore, not been required for Bt cotton
production [159]. For Bt rice, Cohen et al. [160] have proposed the creation of
refuges by limiting the number of Bt rice cultivars that can be released in a
certain growing area, and to focus the implementation of a refuge system on large
rice-growing estates, collectives, and well-organized farmer organizations. Cohen
suggested that the stacked Bt rice be released as fast as possible, such that fewer
refuge fields will be needed.
Clearly, Bt plants cannot be considered as a stand-alone product, but rather as an
additional insect control tool that should be integrated with other pest management
tools, such as crop rotation, the manipulation of insect predators and parasites,
spray-on insecticides, and the destruction of larval overwintering sites.
30.7
Safety of Bt Plants
The premarket regulatory review of genetically modified crops – including Bt

crops – assesses the food derived from, and the environmental safety of, such
crops. Based on the concept of substantial equivalence, this safety assessment
focuses on the proteins encoded by the genes that have been introduced into
the novel variety. The principal components of food safety assessments include an
evaluation of potential toxicity and allergenicity, in the context of anticipated human
dietary exposure [161]. Animal toxicity studies conducted with the Cry proteins
present in currently commercial Bt crops have indicated the absence of any acute
or chronic effects [162, 163]. The evaluation of potential allergenicity is mainly
based on in vitro digestibility assays using simulated gastric fluid, and amino acid
sequence comparisons with known allergens. The Cry proteins in current Bt crops
are all degraded rapidly by gastric fluid, and do not show any sequence similarity
with known allergens [162, 163]. In conclusion, none of these Cry proteins shows
any characteristics of toxins or food allergens [76,161–163].
In many locations, protection from the European corn borer by Bt corn has
resulted in a significant reductions in the production of fumonisins by certain
Fusarium species [164–166]. However, such reductions in fumonisin levels have
not been observed under conditions where H. zea, rather than O. nubilalis, is the
predominant pest insect on Cry1Ab-expressing corn [167, 168]. This situation is
most likely explained by the significantly lower susceptibility of H. zea to Cry1Ab,
as compared with O. nubilalis. Fumonisins are acutely toxic to a variety of animals,
they are carcinogenic in rats, and they have been associated with the presence of
esophageal cancer in humans [169]. Corn borers such as O. nubilalis larvae may
serve as a vector of Fusarium spores from the plant surface to damaged kernels, or
to the interior of stalks, or they may provide entry wounds for fungi. Thus, Bt corn
has the potential to reduce the levels of fumonisin mycotoxins in field-harvested
grain, and hence their dietary intake, especially in regions of the world with a high
incidence of Fusarium and high levels of corn consumption [164].
The adoption of Bt crops (especially Bt cotton) has resulted in significant
reductions in chemical pesticide applications [76,170–173]. For example, in 2001 in
China, Pray et al. [170] estimated a reduction in pesticide use of 78 000 tons of
formulated pesticide. Between 1996 and 2008, savings in insecticides in Bt cotton
of 128 × 106 kg have been reported [173]. Such reduced pesticide exposure may
benefit the farmers’ health, especially in countries where pesticides are applied
under conditions that are not always optimal with respect to worker protection.
Such positive effects on farmers’ health have been reported for both Bt rice and
Bt cotton in China [174, 175]. The regional suppression of pests by Bt crops has
been reported for P. gossypiella, H. virescens, H. armigera, and O. nubilalis [176–179].
A reduction in pest populations on non-Bt crops grown close to Bt crops – a
situation referred to as the ‘‘halo-effect’’ – has been observed for Bt cotton and Bt
corn, and this may lead to further reductions in insecticide use. In some regions,
large reductions in insecticide use for target lepidopteran pests in Bt cotton have
increased the importance of particular pest species, such as mirid plant bugs, which
require specific treatments for their control. Nevertheless, total insecticide use in
Bt cotton has continued to decline over the past decade [173, 180].
Bt sprayable biopesticides are generally regarded as safe for use as biological
control agents, and are promoted in both organic and integrated pest management
(IPM) systems [181]. In Bt crops, the Cry proteins are often present as noncrys-
talline proteins, are often truncated, and present throughout the growing season.
Therefore, their impact on nontarget insects may be different from that of Bt
sprayable insecticides, and this must be monitored individually as part of a risk
assessment, where risk is defined on the basis of both potential hazard and expo-
sure. Such assessment has involved studies of different levels of complexity, from
laboratory tests conducted under very high exposure to studies where the responses
of organisms are analyzed under more realistic conditions and, ultimately, to field
studies.
In view of the specificity of Cry proteins, the effect of Bt crops on most nontarget
insects can be expected to be minimal, especially when compared to the effects of
broad-spectrum insecticides. In the case of Bt cotton and Bt corn plants, both of
which express lepidopteran-specific Cry proteins, some nontarget Lepidoptera may
be negatively affected when challenged with tissues of such Bt plants. Losey et al.
[182] found that, under laboratory conditions, when Bt corn pollen was dusted over
milkweed plants it led to a decrease in survival of the larvae of the monarch butterfly,
Danaus plexippus, an effect that, in their view, might potentially have profound
implications for the conservation of monarch butterflies. Although criticized on
methodological grounds, this report was seen by many as an example of agricultural
biotechnology (specifically pollen from Bt corn) disrupting Nature [183]. However,
the critical question was not whether some Lepidoptera were susceptible to the
Cry protein expressed in tissue of Bt plants, but whether – or to what extent – the
larvae were exposed to the protein under field conditions.
When, in a subsequent experiment, Jesse and Obrycki [184] placed potted
milkweed plants in corn fields at different distances from the field edge, disks from
field-sampled milkweed plants dusted with Bt corn pollen caused a significantly
higher mortality than disks from plants dusted with non-Bt corn pollen. Bioassays
using pollen extracted from the tassels of Bt corn and non-Bt corn, and applied to
milkweed leaf disks, also indicated an increased mortality from the Bt corn pollen.
Based on these results, it was predicted that transgenic Bt corn would have a negative
impact on D. plexippus larvae in, and adjacent to, Bt corn fields. However, the pollen
samples collected from tassels were shown to contain substantial amounts of plant
debris as contaminants, and this may have caused significant mortality [185].
A more recent series of studies examined the impact of Bt corn pollen more
rigorously, in order to quantify the potential risk to monarch butterflies associated
with the large-scale growing of Bt corn [185–190]. Whilst the hazard posed by
Cry1Ab was confirmed in laboratory bioassays, the exposure was shown to depend
significantly on the expression level of Cry1Ab in pollen in different Bt corn events
[185, 188]. The exposure of larvae to Bt-expressing pollen also varied significantly,
depending on the distance from the corn field, the position of the leaf of milkweed
plants (i.e., upper leaves versus middle leaves), the site of pollen deposition on
the leaf (i.e., along the midrib or areas flanking the midrib), and the occurrence
of rainfall [187]. Both, spatial and temporal overlap between the presence of
susceptible life stages of D. plexippus and the corn pollen shed represent another
important determinant of exposure [186, 188]. In essence, the results of these
studies have indicated that the currently registered corn events have little or no
impact on monarch butterfly populations. Most notably, the only Bt corn event
producing pollen with substantial toxicity towards the monarch butterfly larvae – Bt
corn event 176 – has been phased out. Gatehouse et al. [191] have stated that:
Contrary to media hype, the primary threat to the monarch population is

loss of crucial winter habitats in southern California and central Mexico,
rather than commercial growing of Bt-maize.
Likewise, Pimentel and Raven [192] mentioned that:
Although Bt corn pollen under certain conditions has the potential of

adversely affecting the population levels of monarch butterflies and other
non-target Lepidoptera, we consider these impacts to be minimal when
compared with habitat loss and the widespread use of pesticides throughout
the ecosystem.
Another study that attracted much media attention was conducted by Hilbeck
et al. [193], in which an increased mortality was noted of the larvae of the predatory
green lacewing (Chrysoperla carnea) when offered O. nubilalis or S. littoralis larvae
fed on Cry1Ab-expressing Bt corn. In an effort to differentiate between a direct
effect of the toxin, and an indirect effect due to reduced nutritional quality of the
Bt-fed prey, Hilbeck et al. [194] compared the survival of C. carnea larvae developing
on Ephestia kuehniella eggs or on an artificial diet, with or without Cry1Ab. The use
of an artificial diet increased the mortality to 30%, compared to 8% when using
eggs as the diet. The inclusion of Cry1Ab at 100 μg ml−1 in the diet increased
mortality to 57%. However, Dutton et al. [195] found that C. carnea larvae were
not affected by feeding on Bt corn-reared Tetranychus urticae spider mites, while
the prey insects contained higher levels of Cry1Ab than Bt corn-reared S. littoralis
larvae. These observations were in line with the fact that lepidopteran larvae
represent a low-quality prey for C. carnea larvae, as compared to other prey such as
aphids, spider mites, or lepidopteran eggs. Recently, Romeis et al. [196] developed
an improved bioassay for C. carnea using a sucrose-based artificial diet, and showed
that Cry1Ab at a concentration of 1 mg ml−1 had no direct toxic effect on C. carnea.
The amount of Cry1Ab consumed by C. carnea was calculated to be 10 000-fold
more than that ingested through Bt corn-reared S. littoralis larvae. These results
were corroborated by data which showed the absence of either direct or indirect,
prey-mediated toxic effects of Cry proteins, as well as an absence of Cry1Ac binding,
in C. carnea [197]. Likewise, C. carnea larvae were not negatively affected when
fed Cry1Ac-resistant H. armigera larvae that had fed Bt cotton expressing Cry1Ac
[198]. In conclusion, the above-described data confirmed that: (i) it is crucially
important to use a high-quality artificial diet when assessing direct toxic effects; (ii)
predators should not be forced to feed exclusively on prey species that constitute
only a relatively minor portion of their natural diet in the field; and (iii) the earlier
reported negative effects of Bt corn were due to the low nutritional quality of the
prey, rather than to any direct toxic effects. The question then remains as to what
these data mean with respect to the risk of Bt corn to C. carnea? Since the larvae
of this predator species are known to prefer aphids to lepidopteran larvae in the
field, and aphids are not harmed by Bt corn, then the risk of Bt corn to C. carnea
is considered negligible [196]. This suggestion has been confirmed by results from
field studies comparing the densities of beneficial insects, including C. carnea, on Bt
and non-Bt corn [199–203]. The above-described data on the monarch butterfly and
green lacewing illustrate that considerable care should be taken when extrapolating
laboratory findings to natural field conditions. In particular, factors such as the
significance of the crop as a food source, and the degree of specialization of the
predator or parasite species, are likely to be important in estimating the impact
under field conditions. Clearly, in evaluating the risk to nontarget organisms, both
toxicity and exposure must be taken into account. Likewise, any impact of Bt crops
should be judged alongside conventional insect control methods. Whereas, early
field studies were usually performed on a rather limited scale, a series of recent
investigations on the effects of Bt crops on nontarget insects have been reported,
most of which were conducted on a medium- to large-scale, during several years
and at multiple locations [204–214]. Collectively, the results of these studies have
shown only minor changes in the abundance of a few nontarget taxa, and almost
all of these effects could be explained by expected changes in the size of the target
pest populations. Importantly, a five-year field trial of Bt cotton indicated essentially
no effects of Bt cotton on natural enemy function, and also showed that minor
reductions in the density of several nontarget taxa in Bt cotton may have little
ecological meaning with regards to the natural biological control of key cotton
pests [210]. Most likely, reductions in the abundance and associated function of
any one species (especially predators of the natural enemy complex of cotton) are
offset by other members of the community. The study results also showed that
the use of broad-spectrum insecticides, as an alternative insect control measure,
had a significantly greater impact on nontarget arthropods [211]. According to
O’Callaghan et al. [215], the extensive testing on nontarget plant-feeding species,
and on beneficial species that has accompanied the long-term and wide-scale use
of Bt plants, has not detected any significant adverse effects. When Romeis et al.
[216] evaluated all peer-reviewed studies published until 2006 on the effect of Bt
crops on predators and parasites, they concluded that:
• Laboratory and greenhouse studies have revealed effects on natural enemies only
when Bt-susceptible, sublethally affected herbivores were used as the prey or
host, with no indication of direct effects.
• Field studies have only revealed minor, transient, or inconsistent effects of
Bt crops on parasitoids and predators as compared to non-Bt crops, with the
exception of specialist natural enemies.
• The applications of conventional insecticides have usually resulted in severe
negative impacts on biological control organisms.
• Since Bt transgenic varieties can lead to substantial reductions in insecticide use
in some crops, such Bt crops can contribute to a better IPM system with a strong
biological control component [216].
Several reviews have essentially confirmed the conclusions of O’Callaghan and

Romeis [173, 198, 217–221]. With respect to reductions in the abundance of special-
ist natural enemies that depend on the target pest, any control method (including
biological control and conventional host–plant resistance) resulting in a drastic
reduction in the target pest density will have similar effects. As Marvier et al. [217]
have noted, whereas the lack of a difference in abundance of nontarget inverte-
brates is generally considered a signal of environmental safety, it is more difficult
to interpret whether statistically significant differences in abundance translate into

ecologically important changes.
Various studies have been conducted to examine the potential effects of Bt crops,
through root exudates and post-harvest plant residues, on soil ecosystems. Cry
proteins have been detected in root exudates from Bt corn, potato, and rice, but not
in Bt canola, cotton, and tobacco [222–224]. The degradation of Cry proteins in the
soil most likely depends on various parameters, including the plant and soil type,
the composition of the soil microbial communities, agricultural practices, and envi-
ronmental conditions. This may explain, at least in part, the wide variation observed
when assessing the presence over time of Cry proteins in soils from root exudates
and/or post-harvest plant material. The highest potential for the persistence of Cry
proteins is in soils with a high content of clay and/or organic matter, as the proteins
can bind to such soil constituents and thus be protected against microbial degrada-
tion and also retain their insecticidal activity [225, 226]. The results of some studies
have indicated that Cry proteins degrade rather slowly in soil environments [227],
whereas others have shown a rapid decay [228–231]. Moreover, a study conducted
over several years failed to detect Cry1Ac (using either ELISA or insect bioassays) in
soil samples taken from fields where Cry1Ac-expressing cotton cultivars had been
grown for three to six consecutive years [232]. The impact of Bt corn on the cul-
turable bacterial species of soil communities [233–235], as well as on actinomyces,
fungi, protozoa, nematodes, earthworms [235], springtails (Collembola) [205, 236],
and various members of the community of soil-dwelling invertebrates [200, 204],
was determined as being minimal or not significant. Cultivation-independent, more
sensitive molecular techniques indicated either no adverse effects on bacterial com-
munity structure [237], or only small shifts in bacterial communities between the
Bt and non-Bt plant varieties [233, 238, 239]. The environmental relevance of the
latter observations is unclear, however. Interestingly, in this context, a field study
conducted at two sites during three consecutive years of Cry1Ab-expressing corn in-
dicated that environmental factors such as the field site and plant age caused greater
differences in rhizosphere communities than did the expression of Cry1Ab [240].
Perhaps more important than changes in the soil microbacterial community
per se are potential changes in soil microbial activity by Bt crops. A two-year
field study using Cry3Bb-expressing corn found no effects on microbial activity
measures such as N mineralization potential, short-term nitrification rate, and soil
respiration rate [237]. Similarly, no (or only small) differences were identified in
the decomposition rate between Cry1Ab-expressing and nontransgenic maize by
Hopkins and Gregorich [230] and Castaldini [241], respectively. In contrast, Flores
et al. [234] and Stotzky [242] each reported different decomposition rates between
Bt and non-Bt varieties in corn and other crop species from laboratory incubation
studies, using soil amended with ground biomass. An additional prerequisite for the
correct interpretation of observations is the use of appropriate controls (e.g., plant
lines from the same cultivar that have been transformed and regenerated but do
not express the transgene), as well as unrelated plant cultivars, since differences
between the transgenic and nontransgenic plant may be smaller than those between
different cultivars. In conclusion, while there appears to be variation in the soil
decay rate of Cry proteins from Bt crops, no major impact of Cry protein residues
on soil (micro)biota has been observed [243].
The movement of genes – from both conventional and transgenic plants – to
wild relatives of the crop might result in the evolution of increasingly weedy
and/or invasive plants. Pollen-mediated gene flow depends on the geographic
distributions of donor crops and the recipient wild plant, the distance of pollen
movement, the rate of outcrossing, the synchrony of flowering between donor
and recipient plant, and the sexual compatibility between both. The fertility of the
hybrids and their offspring is an important factor in determining the likelihood
of transgene introgression. Seed-mediated gene flow depends on seed persistence
and dispersal. The consequences of gene flow will depend on the nature of the
(trans)gene and its expression level in the hybrid, and the biology and ecology
of the recipient plants [244]. While hybridizations between crops and their wild
relatives may be relatively widespread, the likelihood for such hybridizations in
Bt corn and Bt cotton in, for example, the USA and Europe, seem essentially
nonexistent as either no wild relatives of these crops occur in such regions or
they are incompatible with cultivated varieties [162]. Gene flow can occur not only
from crops to wild plants but also between crops; for example, between transgenic
cultivars and landraces. It has been suggested that transgenic DNA, including a cry
gene, had introgressed into maize landraces in Mexico, despite there being a ban of
transgenic corn in this country [245]; the report was later retracted, as introgression
per se was not shown [246]. A subsequent report failed to find evidence for the
presence of such transgenes in maize landraces from the same area [247]. The
latter authors considered it unlikely that the presence of a few transgenes would
reduce the genetic diversity of the landraces to a greater extent than would gene
flow from conventional modern cultivars. Resistance traits such as insect resistance
due to Cry expression might potentially confer an increased fitness on recipient
plants by reducing lepidopteran damage and increasing seed production. Thus, the
ecological effect of a Bt gene introgressed into landraces or wild plants is likely
to depend to a large extent on the importance of lepidopteran herbivores in these
populations, as well as to the degree that the transgene is linked to domestication
genes or any gene that would be selected against [247–249]. Studies of the effects
on the fitness of the presence of a cry gene in a wild relative of either sunflower or
oilseed rape have yielded different results, depending on the plant species and the
location of the field test site [250, 251]. In this context, whilst gene flow has been
introducing pest-resistance genes from conventional crops to wild relatives for
generations, there are no known examples of increased invasiveness owing to the
introgression of those alleles [252]. Furthermore, fitness-related measures do not
necessarily predict invasiveness [249, 253]. In summary, the potential for – and the
risk of – gene flow to wild relatives from commercially available Bt crop varieties
in those areas where they are currently grown seems very limited. The risk of Bt
transgenes being introgressed into landraces or modern crop cultivars depends
on many factors, should be addressed on a case-by-case basis, and can likely – at
least to some extent – be minimized by (physical) containment measures, such as
increased isolation distances and border rows.
30.8
Conclusions
Bt crops have provided farmers with a valuable additional tool to control insect pests
on corn and cotton, and Bt rice may be commercialized in the near future. At least
in the USA, when growing such crops, farmers must agree to implement certain
IRM tactics, aimed at preventing or delaying the development of resistance in
insect populations. Yet, these tactics are being refined as knowledge is acquired on
insect pest biology and insect/crop plant interactions. To date, the field evaluations
conducted have failed to identify any negative impacts of Bt crops on nontarget and
beneficial insects, except for the expected reduction in specialized natural enemies.
Especially in cotton, significant reductions in synthetic insecticide sprays have
been realized upon the adoption of Bt crops. The judicious use of this novel insect
control tool should result in sustainable benefits to farmers and the environment
and, as a consequence, also to the consumer.
References
1. Chrispeels, M.J. and Sadava, D.E. (eds) 10. Singh, G., Sachdev, B., Sharma,
(1994) Plants, genes and agriculture. N., Seth, R., and Bhatnagar, R.K.
Jones and Bartlett Publishers, Boston. (2010) Appl. Environ. Microbiol., 76,
2. Oerke, E.C., Weber, A., Dehne, H.W., 7202–7209.
and Schonbeck, F. (1994) in Crop 11. Bravo, A., Gill, S.S., and Soberon, M.
Production and Crop Protection: Esti- (2005) in Comprehensive Molecular In-
mated Losses in Major Food and Cash sect Science (eds L.I. Gilbert, I. Kostas,
Crops (eds E.C. Oerke, A. Weber, H.W. and S.S. Gill), Elsevier, Oxford, pp.
Dehne, and F. Schonbeck), Elsevier, 175–205.
Amsterdam, pp. 742–770. 12. Soberon, M., Pardo, L., Munoz-Garay,
3. Birch, R.G. (1997) Annu. Rev. Plant C., Sanchez, J., Gomez, I., Porta, H.,
Physiol. Plant Mol. Biol., 48, 297–326. and Bravo, A. (2010) Adv. Exp. Med.
4. Hansen, G. and Wright, M.S. (1999)
Biol., 677, 127–142.
Trends Plant Sci., 4, 226–231.
13. Groulx, N., Juteau, M., and Blunck, R.
5. Raymond, B., Johnson, P.R.,
(2010) J. Gen. Physiol., 136, 497–513.
Nielsen-LeRoux, C., Lereclus, D., and
14. Likitvivatanavong, S., Chen, J., Bravo,
Crickmore, N. (2010) Trends Microbiol.,
A., Soberon, M., and Gill, S.S. (2011)
18, 189–194.
Appl. Environ. Microbiol., 77, 24–31.
6. Estruch, J.J., Warren, G.W.,
Mullins, M.A., Nye, G.J., Craig, A., 15. Dechklar, M., Tiewsiri, K.,
and Koziel, G. (1996) Proc. Natl Acad. Angsuthanasombat, C., and Pootanakit,
Sci. USA, 93, 5389–5394. K. (2011) Insect Biochem. Mol. Biol.,
7. Lee, M.K., Miles, P., and Chen, J.S. 407, 708–713.
(2006) Biochem. Biophys. Res. Commun., 16. Fabrick, J., Oppert, C., Lorenzen,
339, 1043–1047. M.D., Oppert, B., and Jurat-Fuentes,
8. Lee, M.K., Walters, F.S., Hart, H., J.L. (2009) J. Biol. Chem., 284,
Palekar, N., and Chen, J.S. (2003) Appl. 18401–18410.
Environ. Microbiol., 69, 4648–4657. 17. Pigott, C.R. and Ellar, D.J. (2007)
9. Leuber, M., Orlik, F., Schiffler, B., Microbiol. Mol. Biol. Rev., 71, 255–281.
Sickmann, A., and Benz, R. (2006) 18. Gahan, L.J., Gould, F., and Heckel,
Biochemistry, 45, 283–288. D.G. (2001) Science, 293, 857–860.
References 1051
19. Jurat-Fuentes, J.L., Gahan, L.J., Gould, 32. Galitsky, N., Cody, V., Wojtczak, A.,
F.L., Heckel, D.G., and Adang, M.J. Ghosh, D., Luft, J.R., Pangporn, W.,
(2004) Biochemistry, 43, 14299–14305. and English, L. (2001) Acta Crystallogr.
20. Zhao, J., Jin, L., Yang, Y., and Wu, Y. Sect. D, 57, 1101–1109.
(2010) Insect Biochem. Mol. Biol., 40, 33. Grochulski, P., Masson, L., Borisova,
113–118. S., Pusztai-Carey, M., Schwartz, J.L.,
21. Morin, S., Biggs, R.W., Sisterson, M.S., Brousseau, R., and Cygler, M. (1995)
Shriver, L., Ellers-Kirk, C., Higginson, J. Mol. Biol., 254, 447–464.
D., Holley, D., Gahan, J.J., Heckel, 34. Li, J., Carroll, J., and Ellar, D.J. (1991)
D.G., Carrière, Y., Dennehy, T.J., Nature, 353, 815–821.
Brown, J.K., and Tabashnik, B.E. 35. Li, J., Derbyshire, D.J., Promdonkoy,
(2003) Proc. Natl Acad. Sci. USA, 100, B., and Ellar, D.J. (2001) Biochem. Soc.
5004–5009. Trans., 29, 571–577.
22. Tabashnik, B.E., Liu, Y.B., Unnithan, 36. Morse, R.J., Yamamoto, T., and Stroud,
D.C., Carrière, Y., Dennehy, T.J., and R.M. (2001) Structure, 9, 409–417.
Morin, S. (2004) J. Econ. Entomol., 97, 37. Boonserm, P., Mo, M.,
721–726. Angsuthanasombat, C., and Lescar,
23. Zhang, S., Cheng, H., Gao, Y., Wang, J. (2006) J. Bacteriol., 188, 3391–3401.
G., Liang, G., and Wu, K. (2010) Insect 38. Guo, S., Ye, S., Liu, Y., Wei, L., Wu,
Biochem. Mol. Biol., 39, 421–429. H., Song, P., Zhang, J., Wu, X.,
24. Griffitts, J.S., Haslam, S.M., Yang, T., Huang, D., and Rao, Z. (2009) J. Struct.
Garczynski, S.F., Mulloy, B., Morris, Biol., 168, 259–266.
H., Cremer, P.S., Dell, A., Adang, M.J., 39. de Maagd, R., Bravo, A., Berry, C.,
and Aroian, R.V. (2005) Science, 307, Crickmore, N., and Schnepf, H.E.
922–925. (2003) Annu. Rev. Genet., 37, 409–433.
25. Jurat-Fuentes, J.L. and Adang, 40. Schnepf, E., Crickmore, N., Van Rie, J.,
M.J. (2004) Eur. J. Biochem., 271, Lereclus, D., Baum, J., Feitelson, J.,
3127–3135. Zeigler, D.R., and Dean, D.H. (1998)
26. Ibrahim, M.A., Griko, N., Junker, M., Microbiol. Mol. Biol. Rev., 62, 775–806.
and Bulla, M.A. (2010) Bioeng. Bugs, 1, 41. Adang, M.J., Firoozabady, E., Klein, J.,
31–50. DeBoer, D., Sekar, V., Kemp, J.D.,
27. Soberon, M., Pardo-Lopez, L., Lopez, I., Murray, E.E., Rocheleau, T.A.,
Gomez, I., Tabashnik, B., and Bravo, Rashka, K., Staffeld, G., Stock, C.,
A. (2007) Science, 318, 1640–1642. Sutton, D., and Merlo, D.J. (1987) in
28. Rodriguez-Almazan, C., Zavala, L.E., Molecular Strategies for Crop Protection,
Munoz-Garay, C., Jimènez-Juarez, N., (eds C.J. Arntzen and C. Ryan), Alan R.
Pacheco, S., Masson, L., Soberon, M., Liss, Inc., New York, pp. 345–353.
and Bravo, A. (2009) PLos ONE, 4, 42. Barton, K., Whiteley, H., and Yang,
e5545. N.-S. (1987) Plant Physiol., 85,
29. Crickmore, N., Zeigler, D.R., Feitelson, 1103–1109.
J., Schnepf, E., Van Rie, J., Lereclus, 43. Vaeck, M., Reynaerts, A., Höfte,
D., Baum, J., and Dean, D.H. (1998) H., Jansens, S., De Beuckeleer, M.,
Microbiol. Mol. Biol. Rev., 62, 807–813. Dean, C., Zabeau, M., Van Montagu,
30. Crickmore, N., Zeigler, D.R., Schnepf, M., and Leemans, J. (1987) Nature, 327,
E., Van Rie, J., Lereclus, D., Baum, 33–37.
J., Bravo, A., and Dean, D.H. (2005) 44. Fischhoff, D.A., Bowdish, K.S., Perlak,
Revision of the Nomenclature for F.J., Marrone, P.G., McCormick,
the Bacillus thuringiensis Pestici- S.M., Niedermeyer, J.G., Dean, D.A.,
dal Crystal Proteins. Available at: Kusano-Kretzmer, K., Mayer, E.J.,
http://www.lifesci.sussex.ac.uk/home/ Rochester, D.E., Rogers, S.G., and
Neil_Crickmore/Bt/intro.html. Fraley, R.T. (1987) Bio/Technology, 5,
31. Boonserm, P., Davis, P., Ellar, D.J., 807–813.
and Li, J. (2005) J. Mol. Biol., 348, 45. Peferoen, M., Jansens, S.,
363–382. Reynaerts, A., and Leemans, J. (1990)
in Molecular and Cellular Biology of the Hill, M., Kadwell, S., Launis, K.,
Potato (eds M.E. Vayda and W.C. Park), Lewis, K., Maddox, D., McPherson, K.,
C.A.B. International, Wallingford, pp. Meghji, M.R., Merlin, E., Rhodes, R.,
193–204. Warren, G.W., Wright, M., and
46. Delannay, X., LaVallee, B.J., Proksch, Evola, S.V. (1993) Bio/Technology,
R.K., Fuchs, R.L., Sims, S.R., 11, 194–200.
Greenplate, J.T., Marrone, P.G., 59. Armstrong, C.L., Parker, G.B.,
Dodson, R.B., Augustine, J.J., Pershing, J.C., Brown, S.M., Sanders,
Layton, J.G., and Fischoff, A. (1989) P.R., Duncan, D.R., Stone, T., Dean,
Bio/Technology, 7, 1265–1269. D.A., DeBoer, D.L., and Hart, J. (1995)
47. Warren, G.W., Carozzi, N.B., Desai, Crop Sci., 35, 550–557.
N., and Koziel, M.G. (1992) J. Econ. 60. Jansens, S., van Vliet, A., Dickburt, C.,
Entomol., 5, 1651–1659. Buysse, L., Piens, C., Saey, B.,
48. Brown, J.W.S. and Simpson, C.G. De Wulf, A., Gosselè, V., Goebel,
(1998) Annu. Rev. Plant Physiol. Plant E., and Peferoen, M. (1997) Crop Sci.,
Mol. Biol., 49, 77–95. 37, 1616–1624.
49. Gallie, D.R. (1993) Annu. Rev. Plant 61. Alam, M.F., Datta, K., Abrigo, E.,
Physiol. Plant Mol. Biol., 44, 77–105. Oliva, N., Tu, J., Virmani, S.S., and
50. De Rocher, E.J., Vargo-Gogola, T.C., Datta, S.K. (1999) Plant Cell Rep., 18,
Diehn, S.H., and Green, P. (1998) 572–575.
Plant Physiol., 117, 1445–1461. 62. Fujimoto, H., Itoh, K., Yamamoto, M.,
51. Van Hoof, A. and Green, P.J. (1997)
Kyozuka, J., and Shimamoto, K. (1993)
Plant Mol. Biol., 35, 383–387.
Bio/Technology, 11, 1151–1155.
52. Perlak, F.J., Fuchs, R.L., Dean, D.A.,
63. Nayak, P., Basu, D., Das, S., Basu,
McPherson, S.L., and Fischhoff, D.A.
A., Ghosh, D., Ramakrishnan, A.,
Ghosh, M., and Sen, S.K. (1997) Proc.
3324–3328.
Natl Acad. Sci. USA, 94, 2111–2116.
53. Cornelissen, M., Soetaert, P., Stam,
64. Wünn, J., Klöti, A., Burkhardt,
M., and Dockx, J. (1991) International
P.K., Biswas, G.C.G., Launis, K.,
application published under the patent
Iglesias, V.A., and Potrykus, I. (1996)
cooperation treaty (PCT). Patent WO
Bio/Technology, 14, 171–176.
93/09,218.
65. Leroy, T., Henry, A.-M., Royer, M.,
54. van Aarssen, R., Soetaert, P., Stam, M.,
Dockx, J., Gosselè, V., Seurinck, J., Altosaar, I., Frutos, R., Duris, D., and
Reynaerts, A., and Cornelissen, M. Philippe, D. (2000) Plant Cell Rep., 19,
(1995) Plant Mol. Biol., 28, 513–524. 382–389.
55. Perlak, F.J., Deaton, R.W., Armstrong, 66. Tao, R., Dandekar, A., Uratsu, S.,
T.A., Fuchs, R.L., Sims, S.S., Vail, P., and Tebbets, J. (1997) J. Am.
Greenplate, J.T., and Fischhoff, D.A. Soc. Hortic. Sci., 122, 764–771.
(1990) Bio/Technology, 8, 939–943. 67. Dandekar, A.M., McGranahan, G.H.,
56. Wilson, F.D., Flint, H.M., Deaton, Vail, P.V., Uratsu, S.L., Leslie, C.A.,
W.R., Fischoff, D.A., Perlak, F.J., and Tebbets, J.S. (1998) J. Plant Cell
Armstrong, T.A., Fuchs, R.L., 131, 181–193.
Berberich, S.A., Parks, N.J., and 68. Strizhov, N., Keller, M., Mathur, J.,
Stapp, B.R. (1992) J. Econ. Entomol., Koncz-Kalman, Z., Bosch, D.,
85, 1516–1521. Prudovsky, E., Schell, J., Sneh, B.,
57. Mazier, M., Pannetier, C., Tourneur, J., Koncz, C., and Zilberstein, A.
Jouanin, L., and Giband, M. (1997) in (1996) Proc. Natl Acad. Sci. USA,
Biotechnology Annual Review, vol. 3 (ed. 93, 15012–15017.
M.R. El-Gewely), Elsevier, Amsterdam, 69. Cao, J., Tang, J.D., Strizhov, N.,
pp. 313–347. Shelton, A.M., and Earle, E.D. (1999)
58. Koziel, M.G., Beland, G.L., Mol. Breed., 5, 131–141.
Bowman, C., Carozzi, N.B., Crenshaw, 70. Acharjee, S., Sarmah, B.K., Kumar,
R., Crossland, L., Dawson, J., Desai, N., P.A., Olsen, K., Mahon, R., Moar, W.J.,
References 1053
Moore, A., and Higgens, T.J.V. (2010) 81. Yu, C.G., Mullins, M.A., Warren, G.W.,
Plant Sci., 178, 333–339. Koziel, M.G., and Estruch, J.K.J. (1997)
71. Mcbride, K.E., Svab, Z., Schaaf, D.J., Appl. Environ. Microbiol., 63, 532–536.
Hogan, P.S., Stalker, D.M., and Maliga, 82. Environmental Protection Agency. Cur-
P. (1995) Bio/Technology, 13, 362–365. rent & Previously Registered Section
72. Kota, M., Daniell, H., Varma, S., 3 PIP Registrations. Available at:
Garczynski, S.F., Gould, F., and www.epa.gov/oppbppd1/biopesticides/
William, M.J. (1999) Proc. Natl Acad. pips/pip_list.htm.
Sci. USA, 96, 1840–1845. 83. Miklos, J.A., Alibhai, M.F., Bledig,
73. Reddy, V.S., Leelavathi, S., S.A., Connor-Ward, D.C., Gao, A.G.,
Selvapandiyan, A., Raman, R., Holmes, B.A., Kolacz, K.H., Kabuye,
Giovanni, F., Shukla, V., and V.T., MacRae, T.C., Paradise, M.S.,
Bhatnagar, R.K. (2002) Mol. Breed., Toedebusch, A.S., and Harrison, L.A.
9, 259–269. (2007) Crop Sci., 47, 148–157.
74. Dufourmantel, N., Tissot, G., 84. McPherson, R.M. and MacRae,
Goutorbe, F., Garçon, F., Muhr, C., T.C. (2009) J. Econ. Entomol., 102,
Jansens, S., Pelissier, B., Peltier, G., 1640–1648.
and Dubald, M. (2005) Plant Mol. Biol., 85. MacRae, T.C., Baur, M.E., Boethel,
58, 659–668. D.J., Fitzpatrick, B.J., Gao, A.G.,
75. James, C. (2009) Global Status of Gamundi, J.C., Harrison, L.A., Kabuye,
Commercial Biotech/GM Crops: V.T., McPherson, R.M., Miklos, J.A.,
Paradise, M.S., Toederbusch, A.S., and
2009, ISAAA Briefs No. 41, Ithaca,
Viegas, A. (2005) J. Econ. Entomol., 98,
NY. Available at: http://www.isaaa.
577–587.
org/resources/publications/briefs/41/
86. Williams, M.R. (2000) in Beltwide
default.asp.
Cotton Conference Proceedings (eds P.
76. Shelton, A.M., Zhao, J.-Z., and Roush,
Dugger and D. Richter), National Cot-
R.T. (2002) Annu. Rev. Entomol., 47,
ton Council, Memphis, pp. 894–913.
845–881.
87. Bacheler, J.S. and Mott, D.W. (1997)
77. Vaughn, T., Cavato, T., Brar, G.,
in Beltwide Cotton Conference Proceed-
Coombe, T., DeGooyer, T., Ford,
ings (eds P. Dugger and D. Richter),
S., Groth, M., Howe, A., Johnson,
National Cotton Council, Memphis, pp.
S., Kolacz, K., Pilcher, C., Purcell,
858–861.
J., Romano, C., English, L., and 88. Burd, T., Bradley, J.R. Jr, and Van
Pershing, J. (2005) Crop Sci., 45, Duyn, J.W. (1999) in Beltwide Cotton
931–938. Conference Proceedings (eds P. Dugger
78. Moellenbeck, D.J., Peters, M.L., Bing, and D. Richter), National Cotton Coun-
J.W., Rouse, J.R., Higgins, L.S., cil, Memphis, pp 931–934.
Sims, L., Nevshemal, T., Marshall, 89. Pray, C.E., Huang, J., Ma, D., and
L., Ellis, R.T., Bystrak, P.G., Lang, Qiao, F. (2001) World Dev., 29,
B.A., Stewart, J.L., Kouba, K., Sondag, 813–825.
V., Gustafson, V., Nour, K., Xu, D., 90. Guo, S.D., Cui, H., Xia, L., Wu, D., Ni,
Swenson, J., Zhang, J., Czapla, T., W.C., Zhang, Z., Zhang, B., and Xu, Y.
Schwab, G., Jayne, S., Stockhoff, B.A., (1999) Sci. Agric. Sin., 32, 1–7.
Narva, K., Schnepf, H.E., Stelman, S.J., 91. Singh, P.K., Kumar, M., Chaturvedi,
Poutre, C., Koziel, M., and Duck, N. C.P., Yadav, D., and Tuli, R. (2004)
(2001) Nat. Biotechnol., 19, 668–672. Transgenic Res., 13, 397–410.
79. Walters, F.S., Stacy, C.M., Lee, M.K., 92. Greenplate, J.T., Penn, S.R., Shapply,
Palekar, N., and Chen, J.S. (2008) Appl. Z., Oppenhuizen, M., Mann, J.,
Environ. Microbiol., 74, 367–374. Reich, B., and Osborn, J. (2000) in
80. Que, Q., Chilton, M.-D.M., de Fontes, Beltwide Cotton Conference Proceedings
C.M., He, C., Nucccio, M., Zhu, T., (eds P. Dugger and D. Richter), Na-
Wu, Y., Chen, J.S., and Shi, L. (2010) tional Cotton Council, Memphis, pp.
GM Crops, 1, 1–10. 1041–1043.
93. Adamczyk, J.J. Jr, Adams, L.C., and 107. Jayaraman, K. (2010) Nat. Biotechnol.,
Hardee, D.D. (2001) J. Econ. Entomol., 28, 296.
91, 539–545. 108. Choudhary, B. and Gaur, K. (2009)
94. Huckaba, R.M., Lassiter, R.B., The Development and Regulation of Bt
Huang, X., Blanco, C.A., Langston, Brinjal in India, ISAAA Briefs No. 38,
V.B., Braxton, L.B., Haile, F.J., Ithaca, NY.
Richardson, J.M., and Pellow, J. (2003) 109. Ghareyazie, B., Alinia, F., Menguito,
in Beltwide Cotton Conference Proceed- C.A., Rubia, G., De Palma, J.M.,
ings (eds P. Dugger and D. Richter), Liwanag, E.A., Cohen, M.B., Khush,
National Cotton Council, Memphis, pp. G.S., and Bennett, J. (1997) Mol. Breed.,
1293–1298. 3, 401–414.
95. Siebert, M.W., Nolting, S., Leonard, 110. Alinia, F., Ghareyazie, B., Rubia, L.,
B.R., Braxton, L.B., All, J.N., Van Duyn, Bennett, J., and Cohen, M. (2000) J.
J.W., Bradley, J.R., Bacheler, J., and Econ. Entomol., 93, 484–493.
Huckaba, R.M. (2008) J. Econ. Entomol., 111. Chen, M., Shelton, A., and Ye, G.
101, 1950–1959. (2011) Annu. Rev. Entomol., 56,
96. Adamczyk, J.J. Jr and Gore, J. (2004) 81–1101.
Florida Entomol., 87, 424–432. 112. Tu, J., Datta, K., Alam, M.F., Khush,
97. Tindall, K.V., Siebert, M.W., Leonard, G.S., and Datta, S.K. (1998) Plant
B.R., All, J., and Haile, F.J. (2009) J. Biotechnol., 15, 183–191.
Econ. Entomol., 102, 1497–1505. 113. Tu, J., Zhang, G., Datta, K., Xu, C.,
98. Greene, J.K. and Robinson, D. (2010) He, Y., Zhang, Q., Khush, G.S., and
in Beltwide Cotton Conference Proceed- Datta, S.K. (2000) Nat. Biotechnol., 18,
ings (eds P. Dugger and D. Richter), 1101–1104.
National Cotton Council, Memphis, pp. 114. Ye, G.Y., Tu, J., Hu, C., Datta, K., and
1297–1302. Datta, S.K. (2001) Plant Biotechnol., 18,
99. Gore, J., Adamczyk, J.J. Jr, Catchot, A., 125–133.
and Jackson, R. (2008) J. Econ. Ento- 115. Cheng, X., Sardana, R., Kaplan, H.,
mol., 101, 1594–1599. and Altosaar, I. (1998) Proc. Natl Acad.
100. Mascarenhas, V.J., Shotkoski, J.F., and Sci. USA, 95, 2767–2772.
Boykin, R. (2003) in Beltwide Cotton 116. Shu, Q.U., Ye, G.Y., Cui, H.R., Cheng,
Conference Proceedings (eds P. Dugger X.Y., Xiang, Y.B., Wu, D.X., Gao,
and D. Richter), National Cotton Coun- M.W., Xia, Y.W., Hu, C., Sardana, R.,
cil, Memphis, pp. 1316–1322. and Altossar, I. (2000) Mol. Breed., 6,
101. Bacheler, J.S. and Mott, D.W. (2004) 433–439.
in Beltwide Cotton Conference Proceed- 117. Ye, G.Y., Shu, Q.Y., Yao, H.W., Cui,
ings (eds P. Dugger and D. Richter), H.R., Cheng, X., Hu, C., Xia, Y.W.,
National Cotton Council, Memphis, pp. Gao, M., and Altosaar, I. (2001) J. Econ.
1365–1368. Entomol., 94, 271–276.
102. Llewellyn, D.J., Mares, C.L., and Fitt, 118. Ye, G.Y., Yao, H.W., Shu, Q.Y.,
G.P. (2007) Agric. Forest Entomol., 9, Cheng, X., Hu, C., Xia, Y.W., Gao,
93–101. M., and Altosaar, I. (2003) Crop Prot.,
103. Bradley, J.R., Van Duyn, J.W., and 22, 171–178.
Jackson, R. (2004) in Beltwide Cotton 119. Chen, H., Tang, W., Xu, C., Li, X., Lin,
Conference Proceedings (eds P. Dugger Y., and Zhang, Q. (2005) Theor. Appl.
and D. Richter), National Cotton Coun- Genet., 111, 1330–1337.
cil, Memphis, pp. 1362–1364. 120. Chen, H., Zhang, G., Zhang, Q., and
104. Kurtz, R.W., McCaffery, A., and Lin, Y. (2008) J. Econ. Entomol., 101,
O’Reilly, D. (2007) J. Invertebr. Pathol., 182–189.
95, 227–230. 121. Tang, W., Chen, H., Xu, C., Li, X., Lin,
105. Bommireddy, P.L. and Leonard, B.R. Y., and Zhang, Q. (2006) Mol. Breed.,
(2008) J. Entomol. Sci., 43, 349–361. 18, 1–10.
106. Shelton, A.M. (2010) Crop Prot., 29, 122. Zaidi, M.A., Ye, G., Yao, H., You,
412–414. T.H., Dean, D.H., Riazuddin, S., and
References 1055
Altosaar, I. (2009) Mol. Breed., 43, 137. Tang, J.D., Shelton, A.M., Van Rie, J.,
232–242. De Roeck, S., Moar, W.J., Roush, R.T.,
123. Ye, R., Huang, H., Yang, Z., Chen, and Peferoen, M. (1996) Appl. Environ.
T., Liu, L., Li, X., Chen, H., and Microbiol., 62, 564–569.
Lin, Y. (2008) Pest Manage. Sci., 65, 138. Tabashnik, B.E., Liu, Y.B., de Maagd,
1015–1020. R.A., and Dennehy, T.J. (2000) Appl.
124. Lee, K.R., Shin, K.S., Suh, S.C., Kim, Environ. Microbiol., 66, 4582–4584.
K.Y., Jeon, Y.H., Park, B.S., Kim, J.K., 139. Bird, L.J. and Akhurst, R.J. (2005) J.
Kweon, S.J., and Lee, Y.H. (2009) Plant Econ. Entomol., 97, 1699–1709.
Biotechnol. Rep., 3, 317–324. 140. Liu, Y.B., Tabashnik, B.E., Dennehy,
125. Kim, E.H., Suh, S.C., Park, B.S., Shin, T.J., Patin, A.L., and Bartlett, A.C.
K.S., Kweon, S.J., Han, E.J., Park, S.H.,
(1999) Nature, 400, 519.
Kim, Y.S., and Kim, J.K. (2009) Planta,
141. Metz, T.D., Roush, R.T., Tang, J.D.,
230, 397–405.
Shelton, A.M., and Earle, E.D. (1995)
126. Bashir, K., Husnain, T., Fatima, T.,
Mol. Breed., 1, 309–317.
Latif, Z., Mehdi, S.A., and Riazuddin,
142. Zhao, J.Z., Collins, H.L., Tang, J.D.,
S. (2004) Mol. Breed., 13, 301–312.
127. Hushnain, T., Asad, J., Maqbool, S.B., Cao, J., Earle, E.D., Roush, R.T.,
Datta, S.K., and Riazuddin, S. (2002) Herrero, S., Escriche, B., Ferrè, J.,
Euphytica, 128, 121–128. and Shelton, A.M. (2000) Appl. Environ.
128. Breitler, J.-C., Cordero, M.-J., Royer, Microbiol., 66, 3784–3789.
M., Meynard, D., San Segundo, B., and 143. Tabashnik, B.E., Carrière, Y., Dennehy,
Guiderdoni, E. (2001) Mol. Breed., 7, T.J., Morin, S., Sisterson, M.S.,
259–274. Roush, R.T., Shelton, A.M., and
129. Rahman, M., Rashid, H., Shahid, A.A., Zhao, J.-Z. (2003) J. Econ. Entomol.,
Bashir, K., Husnain, T., and Riazuddin, 96, 1031–1038.
S. (2007) Electron. J. Biotechnol., 10, 144. Tabashnik, B.E., Van Rensburg, J.B.J.,
240–251. and Carriere, Y. (2009) J. Econ. Ento-
130. Chen, H., Lin, Y.J., and Zhang, Q. mol., 102, 2011–2025.
(2009) Chin. Sci. Bull., 54, 4049–4068. 145. Carriere, Y., Crowder, D.W., and
131. Ferrè, J. and Van Rie, J. (2002) Annu. Tabashnik, B.E. (2010) Evol. Appl., 3,
Rev. Entomol., 47, 501–533. 561–573.
132. Gunning, R.V., Dang, H.T., Kemp, 146. Moar, W., Roush, R., Shelton, A.,
F.C., Nicholson, I.C., and Moores, G.D. Ferrè, J., MacIntosh, S., Leonard, B.R.,
(2005) Appl. Environ. Microbiol., 71, and Abel, C. (2008) Nat. Biotechnol., 26,
2558–2563. 1072–1074.
133. Ma, G., Roberts, H., Sarjan, M., 147. Bagla, P. (2010) Science, 327, 1439.
Featherstone, N., Lahnstein, J.,
148. Bates, S.L., Zhao, J.Z., Roush, R.T.,
Akhurst, R., and Schmidt, O. (2005)
and Shelton, A.M. (2005) Nat. Biotech-
nol., 23, 57–62.
134. Tabashnik, B.E., Malvar, T., Liu, Y.-B.,
149. Roush, R.T. (1997) Pest. Sci., 51,
Finson, M., Borthakur, D., Shin, B.-S.,
328–334.
Park, S.-H., Masson, L., de Maagd,
R.A., and Bosch, D. (1996) Appl. Envi- 150. Liu, Y.-B. and Tabashnik, B.E. (1997)
ron. Microbiol., 62, 2839–2844. Proc. R. Soc. London B, 264, 605–610.
135. Tabashnik, B.E., Liu, Y.-B., Malvar, T., 151. Tang, J.D., Collins, H.L., Metz, T.D.,
Heckel, D.G., Masson, L., Balleste, V., Earle, E.D., Zhao, J.Z., Roush, R.T.,
Granero, F., Mènsua, J.L., and Ferrè, and Shelton, A.M. (2001) J. Econ.
J. (1997) Proc. Natl Acad. Sci. USA, 94, Entomol., 94, 240–247.
12780–12785. 152. Shelton, A.M., Tang, J.D., Roush, R.T.,
136. Ferrè, J., Real, D.M., Van Rie, Metz, T.D., and Earle, E.D. (2000) Nat.
J., Jansens, S., and Peferoen, M. Biotechnol., 18, 339–342.
(1991) Proc. Natl Acad. Sci. USA, 153. MacIntosh, S. (2009) Pest Manage. Sci.,
88, 5119–5123. 65, 100–106.
154. Head, G. and Dennehy, T. (2010) 171. Wu, K.M. and Guo, Y.Y. (2005) Annu.
in Cotton, Biotechnology in Agricul- Rev. Entomol., 50, 31–52.
ture and Forestry (ed. U.B. Zehr), 172. Brookes, G. and Barfoot, P. (2010)
Springer-Verlag, Berlin, pp. 113–125. AgBioForum, 13, 76–94.
155. Roush, R.T. (1998) Philos. Trans. R. 173. Naranjo, S.E.J. (2011) Agric. Food
Soc. B, 353, 1777–1786. Chem., 59, 5842–5851.
156. Zhao, J.-Z., Cao, J., Collins, H.L., 174. Huang, J., Rozelle, S., Pray, C.,
Bates, S.L., Roush, R.T., Earle, E.D., and Wang, Q. (2002) Science, 295,
and Shelton, A.M. (2005) Proc. Natl 674–677.
Acad. Sci. USA, 102, 8426–8430. 175. Huang, J., Hu, R., Rozelle, S., and
157. Storer, N.P., Babcock, J.M., Schlenz, Pray, C. (2005) Science, 308, 688–690.
M., Meade, T., Thompson, G.D., Bing, 176. Carriere, Y., Ellers-Kirk, C., Sisterson,
J.W., and Huckaba, R.M. (2010) J. M., Antilla, L., Whitlow, M., Dennehy,
Econ. Entomol., 103, 1031–1038. T.J., and Tabashnik, B. (2003) Proc.
158. Kruger, M., Van Rensburg, J.B.J., and Natl Acad. Sci. USA, 100, 1519–1523.
Van den Berg, J. (2009) Crop Prot., 28, 177. Adamczyk, J.J. and Hubbard, D. (2006)
684–689. J. Cotton Sci., 10, 155–160.
159. Qiao, F., Huang, J., Rozelle, S., and 178. Wu, K., Lu, Y., Feng, H., Jiang, Y.,
Wilen, J. (2010) J. Sci. Chin. Life Sci., and Zhao, J. (2008) Science, 321,
10, 1227–1238. 1676–1678.
160. Cohen, M.B., Gould, F., and Bentur, 179. Hutchison, W.D., Burkness, E.C.,
Mitchell, P.D., Moon, R.D., Leslie,
J.S. (2000) Int. Rice Res. Notes, 25,
T.W., Fleischer, S.J., Abrahamson, M.,
4–10.
Hamilton, K.L., Steffey, K.L., Gray,
161. Chassy, B.M. (2002) J. Am. Coll. Nutr.,
M.E., Hellmich, R.L., Kaster, L.V.,
21, 166S–173S.
Hunt, T.E., Wright, R.J., Pecinovsky,
162. Betz, F.S., Hammond, B.G., and
K., Rabaey, T.L., Flood, B.R., and Raun,
Fuchs, R.L. (2000) Reg. Toxicol. Phar-
E.S. (2010) Science, 330, 222–225.
macol., 32, 156–173.
180. Lu, Y., Wu, K., Jiang, Y., Xia, B., Li, P.,
163. Mendelsohn, M., Kough, J., Vaituzis,
Feng, H., Wuckhuys, K.A.G., and Guo,
Z., and Matthews, K. (2003) Nat.
Y. (2010) Science, 328, 1151–1154.
Biotechnol., 9, 1003–1009.
181. Glare, T.R. and O’Callaghan M. (eds)
164. Hammond, B.G., Campbell, K.W.,
(2000) in Bacillus thuringiensis, Biology,
Pilcher, C.D., Degooyer, T.A., Ecology and Safety, John Wiley & Sons,
Robinson, A.E., McMillen, B.L., Ltd, Chichester.
Spangler, S.M., Riordan, S.G., Rice, 182. Losey, J.E., Rayor, L.S., and Carter,
L.G., and Richard, J.L. (2004) J. Agric. M.E. (1999) Nature, 399, 214.
Food Chem., 52, 1390–1397. 183. Shelton, A.M. and Sears, M.K. (2001)
165. Munkvold, G.P. (2003) Annu. Rev. Phy- Plant J., 27, 483–488.
topathol., 41, 99–116. 184. Jesse, L.C.H. and Obrycki, J.J. (2000)
166. Papst, C., Utz, H.F., Melchinger, A.E., Oecologia, 125, 241–248.
Eder, J., Magg, T., Klein, D., and Bohn, 185. Hellmich, R.L., Siegfried, B.D., Sears,
M. (2005) Agron. J., 97, 219–224. M.K., Stanley-Horn, D.E., Daniels, M.J.,
167. Clements, M.J., Campbell, K.W., Mattila, H.R., Spencer, T., Bidne, K.G.,
Maragos, C.M., Pilcher, C., Headrick, and Lewis, L.C. (2001) Proc. Natl Acad.
J.M., Pataky, J.K., and White, D.G. Sci. USA, 98, 11925–11930.
(2003) Crop Sci., 43, 1283–1293. 186. Oberhauser, K.S., Prysby, M.D.,
168. Dodd, P.F. (2001) J. Econ. Entomol., 94, Mattila, H.R., Stanley-Horn, D.E.,
1067–1074. Sears, M.K., Diveley, G., Olson, E.,
169. Bennett, J.W. and Klich, M. (2003) Pleasants, J.M., Lam, W.-K.F., and
Clin. Microbiol. Rev., 16, 497–516. Hellmich, R.L. (2001) Proc. Natl Acad.
170. Pray, C.E., Huang, J., Hu, R., and Sci. USA, 98, 11913–11918.
Rozelle, S. (2002) Plant J., 31, 187. Pleasants, J.M., Hellmich, R.L., Dively,
423–430. G.P., Sears, M.K., Stanley-Horn, D.E.,
References 1057
Mattila, H.R., Foster, J.E., Clark, T.L., 202. Pilcher, C.D., Obrycki, J.J., Rice, M.E.,
and Jones, G.D. (2001) Proc. Natl Acad. and Lewis, L.C. (1997) Environ. Ento-
Sci. USA, 98, 11919–11924. mol., 26, 446–454.
188. Sears, M.K., Hellmich, R.L., 203. Wold, S.J., Burkness, E.C., Hutchison,
Stanley-Horn, D.E., Oberhauser, K.S., W.D., and Venette, R.C. (2001) J.
Pleasants, J.M., Mattila, H.R., Siegfried, Entomol. Sci., 36, 177–187.
B.D., and Dively, G.P. (2001) Proc. Natl 204. Bhatti, M.A., Duan, J., Head, G., Jiang,
Acad. Sci. USA, 98, 11937–11942. C., McKee, M.J., Nickson, T.E., Pilcher,
189. Stanley-Horn, D.E., Dively, G.P., C.L., and Pilcher, C.D. (2005) Environ.
Hellmich, R.L., Mattila, H.R., Sears, Entomol., 34, 1325–1335.
M.K., Rose, R., Jesse, L.C.H., Losey, 205. Bitzer, R.J., Rice, M.E., Pilcher, C.D.,
J.E., Obrycki, J.J., and Lewis, L. Pilcher, C.L., and Lam, W.-K.F. (2005)
(2001) Proc. Natl Acad. Sci. USA, Environ. Entomol., 34, 1346–1376.
98, 11931–11936. 206. Daly, T. and Buntin, G.D. (2005)
190. Zangerl, A.R., McKenna, D., Wraight, Environ. Entomol., 34, 1292–1301.
C.L., Carroll, M., Ficarello, P., Warner, 207. Dively, G.P. (2005) Environ. Entomol.,
R., and Berenbaum, M.R. (2001) Proc. 34, 1267–1291.
Natl Acad. Sci. USA, 98, 11908–11912. 208. Head, G., Moar, W., Eubanks, M.,
191. Gatehouse, A.M.R., Ferry, N., and Freeman, B., Ruberson, J., Hagerty,
Raemaekers, R.J.M. (2002) Trends A., and Turnipseed, S. (2005) Environ.
Genet., 18, 249–251. Entomol., 34, 1257–1266.
209. Lopez, M.D., Prasifka, J.R., Bruck,
192. Pimentel, D.S. and Raven, P.H.
D.J., and Lewis, L.C. (2005) Environ.
(2000) Proc. Natl Acad. Sci. USA,
Entomol., 34, 1317–1324.
97, 8198–8199.
210. Naranjo, S.E. (2005) Environ. Entomol.,
193. Hilbeck, A., Baumgartner, M., Fried,
34, 1211–1223.
P.M., and Bigler, F. (1998) Environ.
211. Naranjo, S.E. (2005) Environ. Entomol.,
Entomol., 27, 480–487.
34, 1193–1210.
194. Hilbeck, A., Moar, W.J., Pusztai-Carey,
212. Pilcher, C.D., Rice, M.E., and Obrycki,
M., Filippini, A., and Bigler, F. (1998)
J.J. (2005) Environ. Entomol., 34,
Environ. Entomol., 27, 1255–1263.
1302–1316.
195. Dutton, A., Klein, H., Romeis, J., and
213. Torres, J.B. and Ruberson, J.R. (2005)
Bigler, F. (2002) Ecol. Entomol., 27,
Environ. Entomol., 34, 1242–1256.
441–447. 214. Whitehouse, M.E.A., Wilson, L.J., and
196. Romeis, J., Dutton, A., and Bigler, F. Fitt, G.P. (2005) Environ. Entomol., 34,
(2004) J. Insect Physiol., 50, 175–183. 1224–1241.
197. Rodrigo-Simon, A., De Maagd, R.A., 215. O’Callaghan, M., Glare, T.R., Burgess,
Avilla, C., Bakker, P.L., Molthoff, J., E.P.J., and Malone, L.A. (2005) Annu.
Gonzalez-Zamora, J.E., and Ferrè, J. Rev. Entomol., 50, 271–292.
(2006) Appl. Environ. Microbiol., 72, 216. Romeis, J., Meissle, M., and Bigler, F.
1595–1603. (2006) Nat. Biotechnol., 24, 63–71.
198. Lawo, N.C., Wackers, F.L., and 217. Marvier, M., McCreedy, C., Regetz,
Romeis, J. (2010) J. Insect Physiol., J., and Kareiva, P. (2008) Science, 316,
56, 1702–1710. 1475–1477.
199. Bourguet, D., Chaufaux, J., Micoud, 218. Duan, J.J., Marvier, M., Huesing, J.,
A., Delos, M., Naibo, B., Bombarde, F., Dively, G., and Huang, Z.Y. (2008)
Marque, G., Eychenne, N., and Pagliari, PLos ONE, 3, e1415.
C. (2002) Environ. Biosafety Res., 1, 219. Chen, M., Zhao, J., Collins, H.L., Earle,
49–60. E.D., Cao, J., and Shelton, A.M. (2008)
200. Candolfi, M.P., Brown, K., Grimm, PLos ONE, 3, e2284.
C., Reber, B., and Schmidli, H. (2004) 220. Wolfenbarger, L.L., Naranjo, S.,
Biocontrol Sci. Technol., 14, 129–170. Lundgren, J.G., Bitzer, R.J., and
201. Orr, D.B. and Landis, D.A. (1997) J. Watrud, L.S. (2008) PLos ONE, 3,
Econ. Entomol., 90, 905–909. e2118.
221. Naranjo, S.E. (2009) CAB Rev.: Perspect. 238. Blackwood, C.B. and Buyer, J.S. (2004)
Agric. Vet. Sci. Nutr. Nat. Resour., 4 J. Environ. Qual., 33, 832–836.
(11). doi: 10.1079/PAVSNNR20094011. 239. Donegan, K.K., Palm, C.J., Fieland,
Available at: http://ddr.nal.usda.gov/ V.J., Porteous, L.A., Ganio, L.M.,
bitstream/10113/48264/1/IND44494096. Schaller, D.L., Bucao, L.Q., and Seidler,
pdf. R.J. (1995) Appl. Soil Ecol., 2, 111–124.
222. Saxena, D., Flores, S., and Stotzky, G. 240. Baumgarte, S. and Tebbe, C. (2005)
(1999) Nature, 402, 480.
Mol. Ecol., 14, 2539–2551.
223. Saxena, D., Flores, S., and Stotzky, G.
241. Castaldini, M., Turrini, A., Sbrana,
(2002) Soil Biol. Biochem., 34, 133–137.
C., Benedetti, A., Marchionni, M.,
224. Saxena, D., Steward, C.N., Altosaar, I.,
Shu, Q., and Stotzky, G. (2004) Plant Mocali, S., Fabiani, A., Landi, S.,
Physiol. Biochem., 42, 383–387. Santomassimo, F., Pietrangeli, B., Nuti,
225. Crecchio, C. and Stotzky, G. (1998) Soil M.P., Miclaus, N., and Giovannetti,
Biol. Biochem., 4, 463–470. M. (2005) Appl. Environ. Microbiol., 71,
226. Stotzky, G. (2000) J. Environ. Qual., 29, 6719–6729.
691–705. 242. Stotzky, G. (2004) Plant Soil, 266,
227. Zwahlen, C., Hilbeck, A., Gugerli, P., 77–89.
and Nentwig, W. (2003) Mol. Ecol., 12, 243. Icoz, I. and Stotzky, G. (2008) Soil Biol.
765–775. Biochem., 40, 559–586.
228. Herman, R.A., Evans, S.L., Shanahan, 244. Dale, P.J., Clarke, B., and Fontes,
D.M., Mihalial, C.A., Bormett, G.A., E.M.G. (2002) Nat. Biotechnol., 20,
Young, D.L., and Buehrer, J. (2001) J. 567–574.
Physiol. Chem. Ecol., 30, 642–644. 245. Quist, D. and Chapela, I.H. (2001)
229. Herman, R.A., Scherer, P.N., and Wolt, Nature, 414, 541–543.
J.D. (2002) J. Physiol. Chem. Ecol., 31, 246. Metz, M. and Fütterer, J. (2002) Nature,
208–214.
416, 600–601.
230. Hopkins, D.W. and Gregorich, E.G.
247. Ortiz-Garcia, S., Ezcurra, E., Schoel,
(2003) Eur. J. Soil Sci., 54, 793–800.
B., Acevedo, F., Soberon, J., and Snow,
231. Sims, S. and Holden, L. (1996) Physiol.
Chem. Ecol., 25, 659–664. A.A. (2005) Proc. Natl Acad. Sci. USA,
232. Head, G., Surber, J.B., Watson, J.A., 102, 12338–12343.
Martin, J.W., and Duan, J.J. (2002) 248. Pilson, D. and Prendeville, H.R.
Commun. Ecosyst. Ecol., 31, 30–36. (2004) Annu. Rev. Ecol. Evol. Syst.,
233. Brusetti, L., Francia, P., Bertolini, C., 35, 149–174.
Borin, S., Sorlini, C., Abruzzese, A., 249. Stewart, C.N., Halfhill, M.D., and
Sacchi, G., Viti, C., Giovannetti, L., Warwick, S.I. (2003) Nat. Rev., 4,
Giuntini, E., Bazzicalupo, M., and 806–817.
Daffonchio, D. (2004) Plant Soil, 266, 250. Halfhill, M.D., Sutherland, J.P., Moon,
11–21. H.S., Poppy, G.M., Warwick, S.I.,
234. Flores, S., Saxena, D., and Stotzky, Weissinger, A.K., Rufty, T.W., Raymer,
G. (2005) Soil Biol. Biochem., 37, P.L., and Stewart, C.N. (2005) Mol.
1073–1082. Ecol., 14, 3177–3189.
235. Saxena, D. and Stotzky, G. (2001) Soil 251. Snow, A.A., Pilson, D., Rieseberg, L.H.,
Biol. Biochem., 33, 1225–1230.
Paulsen, M.J., Plescak, N., Reagon,
236. Heckmann, L.H., Griffiths, B.S., Caul,
M.R., Wolf, D.E., and Selbo, S.M.
S., Thompson, J., Pusztai-Carey, M.,
(2003) Ecol. Appl., 13, 279–286.
Moar, W.J., Andersen, M.N., and
252. Traynor, P.L. and Westwood, J.H.
Krogh, P.H. (2006) Environ. Pollut.,
142, 212–216. (1999) Information Systems for Biotech-
237. Devare, M.H., Jones, C.M., and nology, Blacksburg, p. 129.
Thies, J.E. (2004) J. Environ. Qual., 253. Bergelson, J. (1994) Ecology, 75,
33, 837–843. 249–252.
1059
31
Metabolic Processes
31.1
Inhibitors of Oxidative Phosphorylation
Josef Ehrenfreund
31.1.1
Introduction
Mitochondria produce most of the energy in cells by the process of oxidative

phosphorylation, which combines two distinct but tightly coupled parts: (i) electron
transport; and (ii) the phosphorylation of ADP to ATP (for details, see Chapter 15.1).
Most modern insecticides and acaricides that disrupt mitochondrial ATP synthesis
[1] interfere with the electron transport (mainly at Complex I, less frequently at
Complex III) (see Chapter 31.3).
In this chapter, attention is focused on compounds that disrupt oxidative phos-
phorylation by direct inhibition of the mitochondrial ATP synthase (Complex V),
with the main emphasis being on diafenthiuron, the only modern representative
of that class.
31.1.2
Mitochondrial ATP Synthase as a Target for Insecticides and Acaricides
Within the process of oxidative phosphorylation, the mitochondrial ATP synthase

(also referred to synonymously as Complex V, F1 F0 -ATPase, or F1 F0 -ATP synthase)
must fulfill two main tasks:
1) To discharge the electrochemical potential gradient that has been generated
by the expulsion of protons across the inner mitochondrial membrane. It does
so by actively channeling protons across the inner mitochondrial membrane,
from the cytoplasmic side back to the matrix side.
2) To catalyze the phosphorylation of ADP and to release the so-formed ATP into
the cell.
ATP synthase is an enzyme of enormous complexity and efficiency (for a detailed
discussion of its structure and reaction mechanism, see Chapter 15.1). Importantly,
the mechanism of the transmembrane proton conduction is highly conserved and

1060 31 Metabolic Processes
may be common to an entire class of membrane channels [2]. It involves an

essential free carboxyl group of subunit c (Asp61 in Escherichia coli, Glu in all other
organisms) inside the phospholipids bilayer which transfers a proton to a nearby
basic Arg of the stator. The lipophilic carbodiimide N,N-dicyclohexylcarbodiimide
(DCCD) binds irreversibly to this essential carboxylic group, and therefore acts as
general inhibitor of ATP synthase.
Despite the large and complex machinery of ATP synthase, its essential role
in cellular bioenergetics, and the existence of inhibitors of structural diversity
(e.g., see Refs [3–9]), very few agrochemicals – mostly specific acaricides – have
been reported as inhibitors of the enzyme. These include chlorfenson, tetradifon,
chloropropylate, bromopropylate, flubenzimine, oxythioquinox, and propargite
[10]. However, for most of these compounds there is no clear evidence that their
biological activity in vivo is due to ATP synthase inhibition. Of these compounds,
only tetradifon and propargite are still commercially available [11].
In the case of the acaricide fenazaquin [4-(4-tert-butylphenethoxy)quinazoline],
which is a potent inhibitor of electron transport at Complex I (see Section 31.1.3),
an additional low-affinity binding site has been recently identified in the stalk region
of ATP synthase. However, the relevance of this newly discovered site is unknown,
as the enzymatic activity of ATP synthase is not impaired by fenazaquin [12].
Some obsolete members of the class of organotin acaricides cause a significant
inhibition of ATP synthesis at the ATP synthase level. Indeed, enzyme prepara-
tions from nonmammalian sources are reportedly more sensitive to organotins
(I50 -values in the range 0.1 to 100 nM) than are typical mammalian enzymes
(I50 -values ranging from 1000 to 10 000 nM), which may explain their apparent
selective toxicity towards invertebrates [1]. It has been suggested that some of
these chemicals bind to the F0 component of mitochondrial ATP synthase at a site
different to the known inhibitors oligomycin or dicyclohexylcarbodiimide (DCCD),
in such a way that the rapid rotary motion required to maintain efficiency of ATP
synthesis is inhibited [13]. However, organotins exhibit a range of additional biolog-
ical effects that may well be as relevant as ATP synthase inhibition for their effects
in vivo; examples of these are the mitochondrial uncoupling caused by the hydroxide
ion shuttle across the inner membrane, or the inhibition of Ca2+ ATP-ases [1].
ATP synthase from DDT (1,1-bis-(p-chlorophenyl)-2,2,2-trichlorethane) suscep-
tible insects is inhibited by DDT, albeit at relatively high concentrations. Where as
ATP synthase from DDT-resistant insects is not inhibited at all. Recently, it has
been suggested that this inhibition is associated with the presence of a specific
protein in the F0 component at a site different from the binding sites of oligomycin
and DCCD. As this specific protein is present only in insect strains susceptible to
DDT, however, it is thought to be the target for DDT, the primary mode of action
of which is via the inhibition of ATP synthase [14].
To date, convincing evidence that an inhibition of mitochondrial ATP synthase
is responsible for biological activity in vivo, and therefore represents the mode of
action, is available for only one recently introduced pesticide, diafenthiuron (1)
(Figure 31.1.1). This compound, which was introduced by Ciba-Geigy AG (now
Syngenta Crop Protection AG) in 1988 [15] and launched in 1991, is currently
Figure 31.1.1 Diafenthiuron (1). Structure and

H acute toxicity data.
N
O N
H
S
1; diafenthiuron (PoloTM; PegasusTM)

LD50: 2068 mg kg−1 rat, oral, acute
marketed as an insecticide and acaricide under the main trade names Pegasus®
and Polo®.
The biochemical mode of action of diafenthiuron, together with some aspects of
its chemistry and the main biological features that make it a unique and highly
valuable crop-protection tool for the farmer, are described in the following sections.
31.1.3
Diafenthiuron: Mode of Action
The results of all available studies support the conclusion that diafenthiuron is a
proinsecticide [16] that is activated by oxidative desulfurization to the insecticidal
carbodiimide 2 (Figure 31.1.2). The evidence for this can be summarized [17] as
follows:
1) Diafenthiuron (1) itself had no effect in biochemical and neurophysiological

assay systems, including mitochondria, cuticle formation, axonal sodium
channel, and main neuronal receptors. However, it proved to be a substrate for
cytochrome P-450 [18].
2) The conversion of 1 into 2 occurs readily; photochemically on glass plates
[19] or in water [20], on cotton [19] or Chinese cabbage [21] in the field, in
microsomes, as well as in whole organisms of insects and mammals [18, 22].
The activation of diafenthiuron to 2 and its precursor diafenthiuron S-monoxide
competes with metabolic deactivation, mainly to hydroxylated derivatives and
diafenthiuron-ureas. Quantitatively, the proportion of activation to deactivation
varies significantly in different animals and organs. Rat liver microsomes do
not accumulate significant amounts of 2, but hydroxylate diafenthiuron at the
O N C N
Figure 31.1.2 Structure of diafenthiuron

2; diafenthiuron carbodiimide (CGA140408) carbodiimide (CGA140408) (2).
4 -position; this efficient deactivation may explain the low acute oral toxicity of
diafenthiuron in rats [23].
3) The carbodiimide 2, in contrast to 1, displays biological effects in vitro that may
be responsible for the activity in vivo. In particular, 2 is a potent inhibitor of
mitochondrial ATP synthesis at the ATP synthase level both in vitro [10, 18]
and in vivo [22, 24, 25]. Radiolabeling experiments with [14 C]-2 have confirmed
that it binds covalently to the 8 kDa proteolipid of F0 of the mitochondrial
ATP synthase in mitochondria isolated from insect flight muscle and rat liver.
Because binding is competitively blocked by DCCD, and partly inhibited by
venturicidin, it has been concluded that 2 and the classical and well-studied
inhibitor DCCD [26] share the same binding site on the F0 proteolipid [27].
Additionally, both the carbodiimide 2 and DCCD bind to porin, a 30 kDa
voltage-dependent anion channel located in the outer mitochondrial mem-
brane. Again, a common binding site involving covalent interaction with an
essential carboxylate is postulated. However, in contrast to DCCD, 2 binds
to porins from insects specifically [24, 27]. The binding of 2 to porin does
not dramatically impair the channel, but does induce changes of its voltage
dependency [28]. Porin has important biological functions that are still being
explored [29]. DCCD binding reportedly affects some of these functions [30];
however, the effect of 2 on these functions has not been explored. Carbodi-
imide 2 reportedly also stimulates the octopamine-sensitive adenylate cyclase
of the bulb mite Rhizoglyphus echinopus [31], adults of the diamond-back
moth Plutella xylostella [32], and the lantern of the firefly Photinus pyralis,
an organ known to be a rich source of octopamine-sensitive adenylate cy-
clase [33]. As poisoning symptoms of diamond-back moth adults treated with
diafenthiuron or 2 resembled closely those of the known octopaminergic ago-
nist N -(4-chloro-o-tolyl)-N-methylformamidine [34], and differed strongly from
those of DCCD, it was suggested that 2 acts in vivo by affecting octopaminergic
transmission [35]. However, these results could not be extended to other insects
and mites [23, 36].
4) The following evidence supports the causality between oxidative activation of
diafenthiuron to its carbodiimide 2, ATP synthase inhibition by 2 and biological
activity in vivo. Both, diafenthiuron and 2 inhibit the respiration of locusts in
vivo at low rates; however, only 2 is active in vitro [22, 27]. In vivo, the onset of
poisoning symptoms is more rapid for 2 than for 1 [18, 32, 35]. Photosensitizers
such as Bengal Red, which promote [37] the photochemical conversion of
diafenthiuron into 2, accelerate the acaricidal effects of diafenthiuron in vivo
[38]. Complementarily, the toxicity of diafenthiuron 1 in insects is antagonized
by piperonyl butoxide (PBO) [18], a commonly used synergist and potent
inhibitor of cytochrome P-450-dependent mono-oxygenases. However, this
antagonistic effect of PBO was not apparent in mice [22].
In insects, ATP synthesis in certain organs is strongly and progressively af-

fected following the application of 2. When locusts were topically treated with
2, mitochondrial ATP synthase activity in the abdominal ganglia was decreased
by 46% at the onset of the first symptoms, and by 63% when the animals were
paralyzed. In addition, a severe block of ATP synthase in the gut (78%) and in
the jumping leg muscle (83%) was noted. In all of these organs, the inhibition of
ATP synthesis also caused a significant decrease in the actual ATP levels. On the
one hand, mitochondrial ATP synthase activity of flight muscles and heads of the
flies Calliphora erythrocephala and Phormia regina (both tissues that contain a high
number of mitochondria) was not significantly reduced by either diafenthiuron 1 or
by its carbodiimide 2 at the onset of paralysis. In addition, total energy metabolism
in fly thoraces was not affected by lethal doses of diafenthiuron or 2 [24].
Evidently, therefore, the mitochondria of different organs are not equally sensitive
to 2. A supportive indication that mitochondria from different tissues can be affected
differently by pesticide action was observed with fenpyroximate, a Complex I
inhibitor of the respiration chain (see Chapter 31.3), which causes morphological
changes of the mitochondria in peripheral nerve cells, but not in muscular cells [39].
A complementary line of evidence demonstrates that the acute toxicity of diafen-
thiuron in mice may also be attributed to its conversion into 2 and the inhibition
of mitochondrial ATP synthase in different target organs [22, 40, 41].
In summary, most of the available evidence leads to the conclusion that di-
afenthiuron, due to its conversion to the carbodiimide 2, acts in strikingly similar
fashion to the well-known carbodiimide DCCD in its reaction toward potential
binding proteins [22, 23]. DCCD reportedly binds to many channels that conduct
protons [42, 43], as well as to the calcium channel of the sarcoplastic reticulum [44].
More generally, it interacts (or may be expected to interact) with the manifold of
proteins that catalyze ATP-triggered reactions and that contain Walker sequence
motifs at their active site [13]. As a theoretical consequence, it may be expected that
the carbodiimide 2 – similar to DCCD – may also affect other essential proteins.
Consequently, further studies may be required to clarify the extent to which other
binding sites contribute to the mode of action of diafenthiuron [23].
31.1.4
Diafenthiuron: Discovery, Structure–Activity Relationships, and Production
Process Chemistry
The chemistry-driven optimization of chemical leads that originally have been iden-
tified by competitor companies represents a classical approach to crop-protection
discovery [45]. Inspired by patent applications that had been filed by Bayer in
1976/1977 and claimed N-aryl-N -alkyl (or cycloalkyl)-thioureas and isothioureas as
insecticides and acaricides [46], it was soon established that the introduction of a
(substituted) phenoxy substituent into the 4-position of the original ‘‘Bayer lead’’
(Figure 31.1.3) [47] would result in a profound shift of the biological spectrum
in the greenhouse. Whereas the systemic activity of the lead against the brown
planthopper (an important pest of rice) was decreased, a strong activity against
phytophagous mites and some Lepidoptera became apparent [48].
Unexpected field results with diafenthiuron (1), which was selected as the most
promising compound for further development, triggered a series of chemodynamic
R1 R1
H H
(R3)n N R4 (R3)n N R4
Optimization
N O N
H H
S S
R2 R2
Ciba Geigy 1981

Bayer 1978/1979
R1, R2, : saturated or
R1, R2, R3: saturated or
unsaturated alkyl, cycloalkyl unsaturated alkyl, cycloalkyl
n : 0−2 R3: Halogen, alkoxy, nitro, CF3
R4: secondary/ tertiary alkyls or n : 0−3
R4: saturated or unsaturated
cycloalkyl
alkyl or cycloalkyl
Figure 31.1.3 Thiourea insecticides: first optimization at Ciba-Geigy.
R1
R3 A R4
Y
R2
A = NHCSNH; N=C(SAlkyl)-N; N=C=N

R1;R2 = Alkyl, Cycloalkyl
R3 = X - Aryl; X - Heteroaryl;
X = O,S, CH2;CH(CH3);C(CH3)2;NH; N(Alkyl); NCHO
Y = CH, N
R4 = secondary or tertiary alkyl
Figure 31.1.4 Diafenthiuron optimization: scope of Ciba-Geigy patent applications.
studies. When sprayed onto cotton leaves in the field, 1 was quickly converted into
2 by sunlight, with a half-life of 3 h [19, 48, 49]. The newly formed 2 was – in
comparison with 1 – much more light-stable and biologically more active against
mites, but less stable against hydrolysis in acid media and, unfortunately, quite
phytotoxic [48]. In accordance with this result, the extensive optimization of this
chemical class at Ciba-Geigy was extended to include, besides thioureas and
isothioureas, also the corresponding carbodiimides (Figure 31.1.4).
Although only limited detailed information has been reported, some general
structure–activity relationship (SAR) principles have been formulated [48]. For
excellent potency:
• Both R1 and R2 should be alkyl; isopropyl often leads to maximum activity.

• R4 must be a sterically demanding alkyl or cycloalkyl, t-butyl is often best.
• Isothioureas excel against lepidopterous larvae; however, at least for one example
a high mammalian toxicity has been observed.
• Carbodiimides generally are the most potent acaricides; however, they are often
toxic to fish and some show only limited crop tolerance.
S S
O N N O N N
N N O
O
3 4
UV-light
O N C N
Figure 31.1.5 1,4-Dihydro-5H-tetrazol-5-thione (3) and

5-thiono-1,2,4-oxazolidinone (4) as precursors of insecticidal
carbodiimide (5).
These rules were confirmed by combined quantitative multivariate SAR

and chemodynamic studies in the subclass of N-(pyrid-3-yl)thioureas and
-carbodiimides (Figure 31.1.4: Y equals N). These studies support the SAR concept
that, for thiourea analogs of diafenthiuron, the rate and efficiency of carbodiimide
formation and its photolytic and chemical stability govern the overall biological
potency [50, 51].
This concept has been extended in two further examples. Based on a re-
port that 1,4-dihydro-1-phenyl-5H-tetrazol-5-thiones photochemically form car-
bodiimides by the extrusion of nitrogen and sulfur [52], and the expectation that
5-thiono-1,2,4-oxazolidinones may similarly lose carbon dioxide and sulfur to form
carbodiimides, 3 and 4 were prepared as potential proinsecticides. Both com-
pounds were efficiently converted into the carbodiimide (5) by ultraviolet light,
and displayed the expected strong insecticidal and acaricidal properties in vivo
(Figure 31.1.5) [53–55].
The large-scale production process for diafenthiuron (1), starting from the
industrial chemical 2,6-diisopropylaniline, is outlined in Scheme 31.1.1 [56, 57].
Although the technical route involves only classical well-known reactions, im-
portant improvements were necessary to realize a viable industrial process; for
example, a conventional bromination of 2,6-diisopropylaniline in acetic acid gives
the desired 4 -4 -brominated product in only about 90% yield and purity. In con-
trast, the bromination of 2,6-diisopropylaniline hydrochloride in nonpolar solvents
yields 6 in nearly quantitative yield and excellent purity. As 4-bromoanilines are no-
toriously thermolabile and, therefore, cannot be safely distilled on a large scale, this
improvement was of critical importance for the successful implementation of the
overall production process. Additionally, the use of thiophosgene for the conversion
(a) (b)
NH2 × HCl Br NH2 × HBr O NH2
99.9% 80%
7
6
96% (c)
(e) (d)
1 O N C S O NHCSNH2
93%
9 8
(a) Br2/Cyclohexane/70 °C
(b) C2H5OK/CuCO3/DMF/140 °C
(c) NaSCN/HCI + H2O/o-Xylene/90 °C
(d) o -Xylene/150 °C
(e) t-C4H9NH2
Scheme 31.1.1 Diafenthiuron (1): structure and acute toxicity data.
of 7 into 9 [47] was not acceptable for an industrial, large-scale process; rather, an
optimized version of a known [58] two-step reaction sequence had to be developed.
31.1.5
Diafenthiuron: Mammalian Toxicology and Ecotoxicology
The key toxicological and ecotoxicological data of diafenthiuron have been sum-
marized [59]. As the compound is very readily degraded in the environment, the
ecotoxicological hazard has been rated as low.
31.1.6
Diafenthiuron: Biological Activity and Significance for Crop Protection
Originally, the development of diafenthiuron at Ciba-Geigy (now Syngenta Crop

Protection AG) was based on its potent activity against spider mites in cotton,
citrus, and deciduous fruit. Its high potential for the control of the sweet potato
whitefly Bemisi tabaci was first unexpectedly observed during acaricide trials in
cotton [60], but was decisive for the positioning of the compound in the market.
Since its commercial introduction in 1991, diafenthiuron has established itself as an
important tool for crop protection, especially for multiple-spray programs in cotton.
Diafenthiuron has a useful spectrum of activity that cannot be found in insec-
ticides of other chemical classes: at the recommended rate of 300–400 g a.i. ha−1
[60] it controls not only the important sucking insect complex of cotton, especially
the cotton whitefly, cotton aphid, cotton leafhoppers, but also tetranychid and
tarsonemid mites and young larvae of noctuids [61].
In addition, although it is not systemic it displays a translaminar activity: pests
located on the underside of the leaf are controlled even if they are not directly hit by
the spray. This property is especially valuable in crops that produce dense canopies
such as cotton, and may be the result of vapor-phase activity [15, 19] and/or an
efficient uptake into the leaf cuticle [21].
The efficacy of diafenthiuron against different stages of whiteflies on cotton
seedlings has been characterized in the laboratory [61]: the progeny of female
adults was highly suppressed, even at the low rate of 5 mg l−1 . Larvae were the most
susceptible stage (LC50 6.5 mg l−1 ; LC90 49.2 mg l−1 for second instar), followed
by adults (LC50 23 mg l−1 ; LC90 102.4 mg l−1 ) and pupae (LC50 45 mg l−1 ), but the
reduction of egg hatching was much less pronounced.
A similar stage sensitivity pattern has been observed in leaf dip tests with the mite
Tetranychus urticae: at a rate of 200 mg a.i. l−1 the larvae and adult females were
more susceptible than nymphs, while egg hatch was insufficiently controlled [15].
More recently, the sensitivity of different developmental stages of the carmine mite
Tetranychus cinnabarinus toward diafenthiuron has been determined and compared
with dimethoate and propargite [62].
The strong translaminar activity of diafenthiuron, which is an important ben-
efit for its overall field performance, was also confirmed in the laboratory with
Tetranychus cinnarabinus. Careful treatment of the upper surface only of cotton
leaves with diafenthiuron (300 mg a.i. l−1 ) gave – in contrast to the commercial
standard propargite applied at the same rate – a good overall control of the mobile
stages [21]. However, in the laboratory neither the translaminar activity nor the
strong gas-phase activity became apparent against cotton whitefly larvae. However,
as this effect is clearly observable under field conditions, it was proposed that the
vapor-phase activity might be stronger in the field because of the larger amount of
vapor produced under field spray conditions [61].
Besides its main application against the sucking pest complex in Asian, Aus-
tralian, and Latin American cotton [15, 63–65], diafenthiuron has important specific
additional uses against lepidopterous pests in brassicas in southeast Asia and
the Far East. Specifically, good activity against susceptible and resistant strains of
diamond-back moth, the lesser armyworm, the small white butterfly, and Spodoptera
litura at rates ranging from 30 to 60 g a.i.100 l−1 have been recorded [15, 66–69].
Under field conditions, diafenthiuron is harmless toward the main beneficial
arthropods, especially those of cotton. It is, therefore, highly compatible with
Integrated Pest Management (IPM) spray programs. Neither mite stimulation nor
aphid and whitefly resurgence phenomena – which sometimes are observed with
less-selective insecticides – have ever been reported with diafenthiuron.
Although some toxicity against nymphs and adults of the predatory bugs has
been observed in the laboratory [70, 71], the effects are not significant under
field conditions [72, 73]. Because diafenthiuron has a unique mode of action,
no cross-resistance with any other insecticide or acaricide has been reported.
Most importantly, the white flies Bemisia tabaci and Trialeurodes vaporiorum,
and the aphid Aphis gossypii, which have rapidly developed strains with tol-
erance/resistance against all major insecticides (including organophosphates,
pyrethroids, growth regulators, and neonicotinoids) remained fully susceptible
to diafenthiuron [64, 65, 74, 75]. A similar lack of cross-resistance has been
reported for strains of the diamond-back moth, which had acquired multiple re-
sistance against organophosphates, acylureas, pyrethroids, and abamectin [66, 76].
In addition, resistance development to diafenthiuron may be slow: during field
cage selection pressure studies, carried out in Malaysia and Thailand, the tested
populations of diamond-back moth developed no observable resistance after 25
generations in Malaysia, or even after 55 generations in Thailand [77]. Similarly,
resistance-monitoring trials in Taiwan confirmed that, while Plutella xylostella
strains in heavily treated areas have become considerably less susceptible to mod-
ern insecticides such as abamectin, emamectin benzoate, fipronil, chlorfenapyr,
and spinosad, the susceptibility to diafenthiuron remained unchanged [78].
In summary, diafenthiuron remains a singular active ingredient in
crop-protection chemistry because of its unique chemical class and biochemical
mode of action. Owing to its unparalleled biological spectrum, translaminar,
and gas-phase activity, selectivity toward beneficial arthropods and the lack of
cross-resistance with all other established insecticide classes, it continues to be an
important component of rotational spray regimes, mainly in cotton and vegetable
crops.
References
1. Hollingworth, R.M. (2001) Handbook of 10. Kadir, H.A. and Knowles, C.O. (1991) J.
Pesticide Toxicology, Chapter 57, vol. 2, Econ. Entomol., 84, 801–805.
Academic Press, San Diego, CA. 11. Meister, R.T. (ed.) (2005) Crop Protection
2. Boyer, P.D. (1997) Annu. Rev. Biochem., Handbook, Meistermedia, Willougby,
66, 717–749. OH, p. 91.
3. Salomon, A.R., Voehringer, D.W., 12. Wood, E., Latli, B., and Casida, J.E.
Herzenberg, L.A., and Khosla, C. (2001) (1996) Pestic. Biochem. Physiol., 54,
Chem. Biol., 8, 71–80. 135–145.
4. Sekya, J., Ito, F., and Kitani, S. 13. Matsuno-Yagi, A. and Hatefi, Y. (1993)
(1997) Japan Kokai Tokkyo Koho JP J. Biol. Chem., 268, 1539–1545 and
09,040,695. 6168–6173.
5. Glick, G.D. (2004) US Patent Applica- 14. Younis, H.M., Abo-El-Saad, M.M.,
tion Publication US 2004/241,781. Abdel-Razik, R.K., and Abo-Seda, S.A.
6. Martin-Galiano, A.J., Gorgojo, B., (2002) Biotechnol. Appl. Biochem., 35,
Calvin, M., and De la Campa, A.G. 9–17.
(2002) Antimicrob. Agents Chemother., 46, 15. Streibert, H.P., Drabek, J., and
1680–1687. Rindlisbacher, A. (1988) Proc. Brighton
7. Zheng, J. and Ramirez, V.D. (2000) Br. Crop Prot. Conf. – Pests Dis., 1, 25–32.
J. Pharmacol., 130, 1115–1123. 16. Ujvary, I. (2003) in Encyclopedia of Agro-
8. Zheng, J. and Ramirez, V.D. (1999) Eur. chemicals, vol. 2 (eds J.R. Plimmer,
J. Pharmacol., 368, 95–102. D.W. Gammon, and N.N. Ragsdale),
9. McEnery, M.W. and Pedersen, P.L. Wiley-Interscience, John Wiley & Sons,
(1986) J. Biol. Chem., 261, 1745–1762. Inc., Hoboken, NJ, pp. 1263–1276.
References 1069
17. Ruder, F.J., Benson, J.A., and Kayser, H. 37. Alder, A. (1989) European Patent Appli-
(1992) Insecticides: Mechanism of Action cation EP 307,361.
and Resistance, Intercept Ltd, Andover, 38. Alder, A., Rindlisbacher, A.,
pp. 263–276. Streibert, H.P., and Baenninger, R.
18. Ruder, F.J., Guyer, W., Benson, J.A., (1990) European Patent Application EP
and Kayser, H. (1991) Pestic. Biochem. 390,743.
Physiol., 41, 207–219. 39. Motoba, K., Suzuki, T., and Uchida, M.
19. Steinemann, A., Stamm, E., and Frei, B. (1992) Pestic. Biochem. Physiol., 43,
(1990) Pestic. Outlook, 1, 3–7. 37–44.
20. Keum, Y.-S., Kim, J.-H., Kim, Y.-W., 40. Chander, A. (1992) Biochim. Biophys.
Kim, K., and Li, Q.X. (2002) Pest Man- Acta, 1123, 198–206.
age. Sci., 58, 496–502. 41. Ruder, F.J., Kaeding, D., Kayser, H., and
21. Keum, Y.-S., Liu, K.H., Seo, J.S., Kobel, W. (1992) Proceedings of the XIX
Kim, J.-H., Kim, K., Kim, Y.-H., and International Congress of Entomology,
Kim, P.J. (2002) Bull. Environ. Contam. Beijing.
Toxicol., 68, 845–851. 42. Solioz, M. (1984) Trends Biochem. Sci., 9,
22. Petroske, E. and Casida, J.E. (1995) 309–312.
Pestic. Biochem. Physiol., 53, 60–74. 43. Hassinen, I.E., Vuckila, E., and Petti, T.
23. Kayser, H. and Eilinger, P. (2001) Pest (1993) Biochim. Biophys. Acta Bioenerg.,
Manage. Sci., 57, 975–980. 1144, 107–124.
24. Ruder, F.J. and Kayser, H. (1993) Pestic. 44. Feng, W. and Shoshan-Barmatz, V.
Biochem. Physiol., 46, 96–106. (1996) Mol. Membr. Biol., 13, 85–93.
25. Ruder, F.J. and Kayser, H. (1994) 45. Stetter, J. and Lieb, F. (2000) Angew.
Biochem. Soc. Trans., 22, 241–244. Chem. Int. Ed., 39, 1725–1744.
26. Beechey, R.B., Roberton, A.M., 46. (a) Enders, E., Stendel, W., and
Holloway, C.T., and Knight, I.G. (1967) Hammann, I. (1979) Ger. Offen
Biochemistry, 6, 3867–3879. DE 2,727,416; (b) Enders, E.,
27. Ruder, F.J. and Kayser, H. (1992) Pestic. Hammann, I., and Stendel, W. (1979)
Biochem. Physiol., 42, 248–261. Ger. Offen DE 2,727,529; (c) Enders,
28. Wiesner, P., Popp, B., Schmid, A., E., Stendel, W., and Hammann, I.
Benz, R., and Kayser, H. (1996) Biochim. (1978) Ger. Offen DE 2,639,748; (d)
Biophys. Acta, 1282, 216–224. Enders, E., Stendel, W., Hammann, I.,
29. Rostovsteva, T. and Colombini, M. and Behrenz, W. (1979) Ger. Offen DE
(1997) Biophys. J., 72, 1954–1962. 2,730,620.
30. Nakashima, R.A., Mangan, P.S., 47. (a) Drabek, J. and Boeger, M. (1981) Bel-
Colombini, M., and Pedersen, P.L. gium Patent BE 888,179; (b) Drabek,
(1986) Biochemistry, 25, 1015–1021. J. and Boeger, M. (1981) Ger. Offen DE
31. Kadir, H.A. and Knowles, C.O. (1991) 3,034,905.
Pestic. Biochem. Physiol., 39, 261–269. 48. Drabek, J., Boeger, M., Ehrenfreund, J.,
32. Kadir, H.A. and Knowles, C.O. Stamm, E., Steinemann, A., Alder, A.,
(1992) Comp. Biochem. Physiol., 103C, and Burckhardt, U. (1990) Recent Ad-
303–307. vances Chemistry Insect Control, vol. 2,
33. (a) Nathanson, J.A. (1985) Proc. Natl Special Publication-Royal Society of
Acad. Sci. USA, 82, 599–603; (b) Chemistry, Cambridge, pp. 170–183.
Hashemzadeh, H., Hollingworth, R.M., 49. Steinemann, A., Stamm, E., and Frei, B.
and Voliva, A. (1985) Life Sci., 37 (5), (1989) Aspects Appl. Biol., 21, 203–213.
443–440. 50. Pascual, A. and Rindlisbacher, A. (1994)
34. Hollingworth, R.M. and Murdock, L.L. Pestic. Sci., 42, 253–263.
(1980) Science, 208, 74–77. 51. Pascual, A., Rindlisbacher, A., Schmidli,
35. Kadir, H.A. and Knowles, C.O. (1991) J. H., and Stamm, E. (1995) Pestic. Sci., 44,
Econ. Entomol., 84, 780–784. 369–379.
36. Hollingworth, R.M. and Gadelhak, G.G. 52. Quast, H. and Nahr, U. (1985) Chem.
(1998) Rev. Toxicol., 2, 253–266. Ber., 118, 526–540.
53. Ehrenfreund, J. and Stamm, E. (1991) 67. Liu, A., Li, S., Wu, Y., Yu, G., and Lio, J.
European Patent Application EP (2003) Xiandai Nongyao, 2, 37–38 (Chem.
406,163. Abstr., 140, 212500).
54. Krenzer, J. (1970) Patent US 3,505,454. 68. Acharya, N.G. (2000) Int. Pest Control,
55. Ehrenfreund, J., Stamm, E., and 42, 134–137.
Alder, A. (1994) 8th IUPAC Interna- 69. Kao, C.H., Chiu, C.S., and Cheng, E.Y.
tional Congress of Pesticide Chemistry, (1990) Zhonghua Nongye Yanjiu, 39,
August 1994, Washington, Book of 221–227 (Chem. Abstr., 115, 129968).
Abstracts 2, p. 84. 70. Delbeke, F., Vercruysse, P., Tirry, L.,
56. Haessig, R. (1989) European Patent De Clerq, P., and Deghele, D. (1997)
Application EP 347,380. Entomophaga, 42, 349–358.
57. Haessig, R. (1991) Patent US 4,997,967. 71. De Cock, A., de Clerq, P., Tirry, L., and
58. Baxter, J.N., Cymerman-Craig, J., Deghele, D. (1996) Environ. Entomol., 25,
Moyle, M., and White, R.A. (1954) 476–480.
Chem. Ind., 27, 785. 72. Gerling, D. (1992) Phytoparasitica, 20,
59. Tomlin, C.D.S. (ed.) (1997) The Pesti- 70.
cide Manual, 11th edn, British Crop 73. Silva-Torres, J.B., Silva, M.R., and
Protection Council.
Ferreira, J.F. (2002) Neotrop. Entomol.,
60. Drabek, J. (1987) European Patent Appli-
31, 311–317.
cation EP 210,487.
74. Ishaaya, I. and Horowitz, A.R. (1995)
61. Ishaaya, I., Mendelson, Z., and
Pestic. Sci., 43, 227–232.
Horowitz, A.R. (1993) Phytoparasitica,
75. Gorman, K., Wren, J., Devine, G., and
21, 199–204.
Denholm, I. (2003) Proceedings – BPCP
62. Deep, K. and Dhooria, M.S. (2004) J.
International Congress: Crop Sci-
Res. (Punjab Agric. Univ.), 41, 74–80.
ence & Technology, Glasgow, UK,
63. Horowitz, A.R. (1993) Phytoparasitica,
21, 281–291. pp. 783–788.
64. Otoidobiga, L.C., Vincent, C., and 76. Wu, Q., Zhang, W., Zhang, Y., Xu, B.,
Stewart, R.K. (2003) J. Environ. Sci. and Zhu, G. (2002) Zhiwu Baohu Xue-
Health, Part B, B38, 757–769. bao, 29, 239–243 (Chem. Abstr., 139,
65. Denholm, I., Rolett, A.J., Cahill, M.R., 161037).
and Ernst, G.H. (1995) Proceedings 77. Uk, S. (1995) 13th International Plant
Beltwide Cotton Conference, vol. 2, pp. Protection Congress, The Hague, NL.
991–994. 78. Kao, C.-H. and Cheng, E.Y.
66. Streibert, H.P. and Kaeding, D. (1994) (2001) Zhongua Nongye Yanjiu,
Proc. Brighton Crop Prot. Conf. - Pests 50 (4), 80–89 (Chem. Abstr., 137,
Dis., 2, 743–748. 228073).
31.2
Inhibitors of Oxidative Phosphorylation via Disruption of the Proton Gradient
31.2.1
Introduction
Molecules targeting mitochondrial functions represent viable alternatives to clas-

sical neurotoxicants. Several naturally occurring compounds that target respi-
ration processes within the cell have been identified, including the annon-
ins [1], the anacardic acids [2], and sesquiterpenes [3]. As part of a program
to identify novel microbial metabolites having insecticidal activity, Carter and
31.2 Inhibitors of Oxidative Phosphorylation via Disruption of the Proton Gradient 1071
coworkers – using bioassay-guided fractionation – isolated dioxapyrrolomycin, a

member of the pyrrolomycin family of pyrrole antibiotics (1, Figure 31.2.1) from
the fermentation of Streptomyces fumanus (Sveshnikova) [4]. Although this com-
pound displayed a broad yet moderate insecticidal and acaricidal activity [5], its
relatively high oral toxicity in mice precluded it from being developed.
However, the simplicity of the structure did warrant the use of 1 as a starting
point for a synthesis program to optimize the insecticidal activity, while attempting
to reduce mammalian toxicity. The result of this program was chlorfenapyr (2)
(Scheme 31.2.1), an intermediate of which, with the common name tralopyril
[2-(p-chlorophenyl)-3-cyano-4-bromo-5-trifluoromethyl pyrrole], has been commer-
cialized by Janssen PMP as a marine antifouling agent for incorporation into
paints to control invertebrate fouling organisms such as barnacles, tubeworms,
bivalve mollusks, and sponges [6]. To date, tralopyril has not been developed for
crop-protection purposes.
Cl
Cl NO2
H
Cl N Cl
H O O
Species LC50 (ppm)
Southern armyworm (Spodoptera eridania) 40

Tobacco budworm (Heliothis virescens) 32
Two-spotted spider mite (Tetranychus urticae) 10
Western potato leafhopper (Empoasca abrupta) < 100
Figure 31.2.1 Structure and insecticidal activity of dioxapyrrolomycin (1).
Cl
Cl NO2 Br CN
H
Cl Cl F3C N
N
H O O Cl
O
dioxapyrrolomycin, 1 chlorfenapyr, 2
m.p. = 100–101 °C
vP< 1.2 × 10−2 mPa (20 °C)
Scheme 31.2.1 Synthetic evolution from dioxapyrrolomycin to chlorafenapyr.

31.2.2
Biochemical Mode of Action
Based on the structure of dioxapyrrolomycin and related compounds, it was postu-

lated that the insecticidal activity of these compounds was due to the uncoupling of
oxidative phosphorylation. This was subsequently confirmed through mouse-liver
mitochondrial assays [6].
Uncoupling activity is dependent on two physico-chemical parameters: (i) the
lipophilicity (log P) that allows the molecule to move across the mitochondrial
membrane [7, 8]; and (ii) the acidity (pKa ) that allows the molecule to disrupt the
proton gradient necessary for the conversion of ADP into ATP [7–9]. The results
of various studies have shown that a log P of 6.0 ± 1 and a pKa range of 7.0–7.9 are
necessary for optimal insecticidal activity [10].
An examination of the structure of chlorfenapyr (2) revealed that, whilst it is
a lipophilic molecule, it lacks the acidic proton necessary for potent uncoupling
activity. Although, in in vitro studies using intact Sf9 insect cells, chlorfenapyr failed
to demonstrate any significant inhibition of respiration [11], the N-dealkylated
compound 3 proved to serve as a potent uncoupler of oxidative phosphorylation
in insect mitochondria. Taken together, the results of these studies suggested that
chlorfenapyr would act as a proinsecticide and that 3, when liberated by metabolic
dealkylation of the parent – partially under the action of mixed function oxidases
(MFOs) – was the active compound in insects (Scheme 31.2.2). The fact that both
the parent molecule and the N-dealkylated compound were equipotent in vivo
added credence to the proinsecticide concept.
Further support for the proinsecticide concept was derived using Colorado potato
beetles that had been pretreated with piperonyl butoxide (PBO), an inhibitor of
the MFOs. In this study, at a dose rate of 10 ppm, chlorfenapyr provided complete
control of the untreated insects. Subsequent treatment of the insects with PBO
reduced the level of control to <10% [12].
Br CN Br CN
MFO
F3C N F3C
N -dealkylation N
Cl Cl
H
O
Chlorfenapyr, 2 3
Lipophilic Lipophilic
No Acidic proton Acidic proton present
LC50 (TBW): 5.9 ppm LC50 (TBW): 3.3 ppm
U50 : > 1000 nM U50 : 2.4 nM
Scheme 31.2.2 U50 = concentration required to produce 50% uncoupling.

The N-ethoxymethyl group provided the best balance between metabolic activa-
tion, while avoiding phytotoxic effects seen for the parent, 3.
31.2.3
Chemistry
Although, at the outset of the analog program, several interesting synthetic chal-
lenges were identified, the methods available to prepare densely functionalized
pyrroles were limited. Moreover, the preparation of trifluoromethyl-substituted
pyrroles in a regiospecific manner had not yet been developed. Nonetheless,
both of these issues were addressed by the development of a new cycloaddi-
tion reaction [13, 14], whereby thermal cycloaddition of the oxazolinone (4) with
2-chloroacrylonitritrile in the presence of a base gave the trisubstituted pyrrole (5)
in good yields (Scheme 31.2.3) [15].
The synthesis of chlorfenapyr was completed by the introduction of bromine
onto 5, using standard conditions to give 3, followed by alkylation on the pyrrole
nitrogen. The results are summarized in Scheme 31.2.4.
With the discovery of the activity of chlorfenapyr, alternate routes for preparing
the trisubstituted intermediate pyrrole (5) were investigated. The cycloaddition
routes shown in Scheme 31.2.5allowed for the regiospecific preparation of 5, while
avoiding the preparation of the oxazolinone (4) [16, 17].
Subsequently, the use of benzoyl chloride (or benzoic acid) led to manufacturing
opportunities that would have been limited by the previous routes. Moreover, new
intermediates such as imidoyl chlorides or amides containing fluoroalkyl groups
were made available for biological screening.
The need to prepare densely functionalized pyrroles has led to the development
of cycloaddition reactions that proceed in high yield, and in a regiospecific fashion.
These routes have helped to facilitate structure–activity relationship studies to
optimize the biological activity for this class of chemistry.
31.2.4
Pest Species and Markets
Chlorfenapyr is active against larvae and adults of many pest species, including in-
sects and crop mites of the orders Lepidoptera, Coleoptera, Thysanoptera, Isoptera,
Orthoptera, Hymenoptera, and Acarina. Chlorfenapyr’s broad spectrum of activity
Cl
CN
CN
Cl
N 1 eq
F3C N
O Et3N / 1 eq H Cl
F3C O
4 5
Scheme 31.2.3 Cycloaddition to the 2,3,5-trisubstituted pyrrole nucleus.

1074
31 Metabolic Processes
CN
Br CN Br CN
Br2 ClCH2OCH2CH3
F 3C N Acetic acid F3C N (CH3)3CO−K+ F3C
H Cl N
H Cl THF Cl
O
5 3 Chlorfenapyr, 2
Scheme 31.2.4 Conversion of the 2,3,5-trisubstituted pyrrole to chlorafenapyr.

O Cl
N CF3 PCl5 / Δ N CF3

H H
Cl Cl
CN
Na2CO3
Cl DMF/rt
CN
F3C N
H Cl
5
CN
Et3N / toluene
Cl
Cl
O
N CF3
N CF3 PCl5 / Δ
H
Cl
Cl
Scheme 31.2.5 Alternate cycloaddition routes to the 2,3,5-trisubstituted pyrrole nucleus.
has provided commercial opportunities for its use in control of pests in a wide
range of crops, including vegetables, tree fruits, vines, cotton, and ornamentals.
The first crop registrations for chlorfenapyr were achieved in Israel in January
1995; this was followed by South Africa, Japan, Honduras, Taiwan, and Chile, with
registrations for major crop uses against Lepidoptera, Coleoptera, and Acari in
1996. The US Environmental Protection Agency (EPA) registration for greenhouse
use on fruiting vegetables and noncrop registration for termite control was granted
in 2001. In addition to the noncrop registration in the US, chlorfenapyr is also
registered for noncrop (professional) use in Australia, Costa Rica, Indonesia, South
Korea, Mexico, Saudi Arabia, and Thailand. Chlorfenapyr is approved for noncrop
use in France, and is also currently undergoing European evaluation as a biocide
(Biocide Products Directive 98/8/EC). Chlorfenapyr is currently registered for use
in over 30 countries worldwide.
The uptake of chlorfenapyr is mainly by ingestion and, secondarily, by direct
contact. Owing to its unique mode of action, chlorfenapyr is able to control pests
that are resistant to other insecticide chemical classes, and no instances of target
site cross-resistance have been observed. Unfortunately, although chlorfenapyr
exhibits a good translaminar movement in plants, it has a very limited systemicity

and/or ovicidal activity.
As chlorfenapyr is nonrepellant, it has found particular use in noncrop pest
control. For example, when applied as a barrier treatment around buildings,
termites fail to detect its presence in the soil and hence move through the
treated zone, contacting a lethal dose. In fact, termite-control professionals have
found chlorfenapyr to be an important tool for eliminating termites from houses,
and providing effective residual protection from further termite attack [19]. Also
due to its nonrepellant nature, chlorfenapyr is highly effective as a spray and in
baits for the control of cockroaches, ants, and other household pests that tend to
avoid irritant-type insecticides.
More recently, chlorfenapyr has been used to control pyrethroid- and
DDT-resistant mosquitoes, and is currently being evaluated for malaria control via
residual spraying and treated net applications [24]. Because of its pro-insecticide
properties whereby, in the insect body it must first be converted to the active
insecticidal form by the action of MFOs, chlorfenapyr has proven to be relatively
benign towards natural enemies. For example, field studies conducted in Australia
and the USA have shown chlorfenapyr to have significantly less impact on
predatory bugs and various parasitic wasps and spiders than other broad-spectrum
insecticides used in the treatment of cotton [AmCy commissioned (unpublished)
studies: S. Simpson, R. Lloyd, and D. Murray, QDPI Australia, and M. Sullivan,
Clemson University, USA].
31.2.5
Chlorfenapyr Resistance
Since the first commercial use of chlorfenapyr some 15 years ago, very few
cases of resistance have been documented, and none of those reported has been
associated with target-site resistance. Rather, reported cases have been associated
with metabolic cross-resistance to pyrethroids via esterases in Australian Helicoverpa
armigera bollworms [20], and increased esterase and/or glutathione-S-transferase
activity in Tetranychus urticae mites in Australia and Japan [21]. Because of the
bioactivation of chlorfenapyr by MFOs, some pyrethroid-resistant species may
develop a negative cross-resistance to chlorfenapyr, as has been hypothesized for
Haematobia irritans hornflies in the USA [22, 23]. Because of its unique mode of
action (IRAC Group 13), however, chlorfenapyr remains an important resistance
management tool for use in rotation, or in mixtures with insecticides having other
modes of action.
31.2.6
Conclusions
The discovery and development of chlorfenapyr – a potent uncoupler of oxidative

phosphorylation – as an insect control agent have been summarized in this chapter.
Typically, chlorfenapyr demonstrates activity against a broad spectrum of crop and
References 1077
urban pests, while having minimal impact on beneficial insects. Moreover, the
manipulation and improvement of the biological activity of the natural product lead,
dioxapyrrolomycin, has confirmed that the use of naturally occurring compounds
as scaffolds for synthetic programs remains a viable avenue for the discovery of
new insecticides.
References
1. Londershausen, M., Leicht, W., Lieb, F., Chemical Society, Washington, DC, pp.
and Moeschler, H. (1991) Pestic. Sci., 33, 199–212.
427–438. 12. Black, B., Hollingworth, R.,
2. Toyomizu, M., Okamoto, K., Ahammadsahib, K., Kukel, C., and
Ishibashi, T., Chen, Z., and Nakatsu, Donovan, S. (1994) Pestic. Biochem.
T. (1999) Life Sci., 66, 229–234. Physiol., 50, 115–128.
3. Castelli, M., Lodeyro, A., Malherios, A., 13. Kuhn, D. (1997) in Phytochemicals
Zacchino, S., and Roveri, O. (2005) for Pest Control, ACS Symposium
Biochem. Pharmacol., 70, 82–89. Series, vol. 606 (eds P. Hedin, R.
4. Carter, G., Nietsche, J., Goodman, J., Hollingworth, E. Masler, J. Miyamoto,
Torray, M., Dunne, T., Borders, D., and D. Thompson), American Chemical
and Testa, R. (1987) J. Antibiot., 40, Society, Washington, DC, pp. 195–205.
233–236. 14. Kuhn, D., Kamhi, V., Furch, J.,
5. Addor, R., Babcock, T., Black, B., Diehl, R., Trotto, S., Lowen, G., and
Brown, D., Diehl, R., Furch, J., Babcock, T. (1992) in Synthesis and
Kameswaran, V., Kamhi, V., Kremer, K., Chemistry of Agrochemicals III, ACS
Kuhn, D., Lovell, J., Lowen, G., Miller, Symposium Series, vol. 504 (eds D.
T., Peevey, R., Siddens, J., Treacy, M.,
Baker, J. Fenyes, and J. Steffens), Ameri-
Trotto, S., and Wright, D. (1992) in Syn-
thesis and Chemistry of Agrochemicals III,
pp. 298–305.
ACS Symposium Series, vol. 504 (eds D.
15. For a discussion of the use of
Baker, J. Fenyes, and J. Steffens), Ameri-
alkyl-substituted oxazolinones in pyr-
role synthesis, see: (a) Benages, I. and
pp. 283–297.
Albonico, S. (1978) J. Org. Chem.,
6. van der Flaas, M.A.J. and Crawley, L.S.
43, 4273–4276; (b) Cardozo, M.,
(1996) European Patent Application
Pizzorno, M., and Albonico, S. (1986)
746,979.
7. Treacy, M., Miller, T., Black, B., Gard, I., Tetrahedron, 42, 5857–5862.
Hunt, D., and Hollingworth, R. (1994) 16. Addor, R., Furch, J., and Kuhn, D.
Biochem. Soc. Trans., 22, 244–247. (1991) US Patent 5,030,735, 1991.
8. Hansch, C., Kiehs, K., and Lawrence, G. 17. Kameswaran, V. (1992) US Patent
(1965) J. Am. Chem. Soc., 87, 5,145,986, 1992.
5770–5773. 18. Kameswaran, V. (1999) US Patent
9. Tollenaere, J. (1973) J. Med. Chem., 16, 5,965,773, 1999.
791–796. 19. Anonymous (2001) Int. Pest Control, 43,
10. Corbett, J., Wright, K., and Baille, A. (July/August).
(eds) (1984) The Biochemical Mode of 20. Gunning, R.V. and Moores, G.D. (2002)
Action of Pesticides, 7th edn, Academic Brighton Crop Protect. Conf. - Pests Dis.,
Press, New York, pp. 1–49. 2, 793–798.
11. Gange, D., Donovan, S., Lopata, R., 21. van Leeuwen, T., van Pottelberge, S.,
and Henegar, K. (1995) in Classical and and Tirry, L. (2006) Pest Manage. Sci., 62
Three-Dimensional QSAR in Agrochem- (5), 425–433.
istry, ACS Symposium Series, vol. 606 22. Sheppard, D.C. and Joyce, J.A. (1998) J.
(eds C. Hansch and T. Fujita), American Econ. Entomol., 91 (2), 398–400.
23. Barros, A.T.M., Andress, E., Doscher, 24. N’Guessan, R., Boko, P., Odjo, A.,
M.E., and Foil, L.D. (1999) Southwest. Akogbeto, M., Yates, A., and Rowland,
Entomol., 24 (4), 331–338. M. (2007) Acta Trop., 102 (1), 69–78.
31.3
Inhibitors of Mitochondrial Electron Transport: Acaricides and Insecticides
31.3.1
Introduction
Insect control agents – including acaricides – act through three broad mechanisms:
(i) disruption of the nervous system; (ii) insect development (insect growth regula-
tors); and/or (iii) respiration [1]. The insect nervous system has long been the target
for most insect control agents, past and present, as exemplified by the organophos-
phorus, carbamate, pyrethroid, cyclodiene, and DDT-related families of insecticides
[1–3]. Until the early 1990s, examples of insect-control agents acting through a
disruption of insect respiration had been limited to the dinitrophenols, organotins,
and a few natural products such as rotenone and piericidin A [3]. However, during
the past 20 years several new insecticidal and acaricidal molecules have emerged
that exert their effects through the disruption of respiratory processes, including
those of mitochondrial electron transport (MET) and oxidative phosphorylation [1,
4, 5]. The MET chain consists of a series of sequentially acting electron carriers
(metalloproteins) bound to the inner membrane of the mitochondrion [6–8]. These
carriers move electrons from NADH though a sequence of four metalloprotein
Complexes (I–IV) to, ultimately, molecular oxygen [7, 8]. Potential sites for inhibi-
tion exist throughout the MET chain, and several of these (Complexes I–III) have
been exploited as sites of action for insecticides and acaricides [4, 8, 9].
In this chapter, attention is focused on insecticides and acaricides that act by
inhibiting MET at these three sites. Further details on this subject are provided in
Chapters 15.1 [6] and 15.5 [10], respectively. Likewise, Ehrenfreund [11] and Kuhn
[12], in Chapters 31.1 and 31.2, respectively, provide information on insect-control
agents that interfere with aspects of oxidative phosphorylation.
31.3.2
Complex I Inhibitors
Rotenone (Figure 31.3.1) is a well-known insecticidal constituent from plant

species belonging to the genera Derris and Lonchocarpus [13]. The mode of action
of rotenone is inhibition of the MET at Complex I [8, 13, 14], and for many
years rotenone and its related analogs (rotenoids) were the primary insecticidal
compounds employing this mode of action. Thus, during the early to mid-1980s,
research investigations conducted at different companies in the US and Japan, all
of which involved unrelated chemistry, led to the discovery of acaricidal molecules.
31.3 Inhibitors of Mitochondrial Electron Transport: Acaricides and Insecticides 1079
O Figure 31.3.1 Structure of rotenone.
HO
O O
Rotenone
O O
Each if these was unique and distinct in its chemistry, yet all possessed an
identical and, until that time, an underexploited mode of action, namely the in-
hibition of site 1 in the MET system. Since the development of this first group
of MET-I (Complex I) inhibitors, other compounds and analogs that act at this
target site have been investigated, leading in some cases to the creation of other
new acaricidal and insecticidal products. An overview of the properties, toxicol-
ogy, and pest-control spectra for these different MET compounds is provided in
Table 31.3.1.
As a group, the MET-I inhibitor-based acaricides are broadly active against
a diverse array of mite species, especially spider mites. In many cases, rust
mites are also controlled, as are other mite species. While the initial MET-I
inhibitors were primarily active on mites, more recent compounds (e.g., tolfenpyrad,
and perhaps others) also provide control of an expanded spectrum of insect
species. Compared to many of the ‘‘older’’ acaricides, the MET-I acaricides tend
to be active on all mite stages (Table 31.3.1), thus enhancing their utility to the
grower. Additionally, in contrast to some new acaricides that function via growth
regulation or the inhibition of fatty acid synthesis [4, 15], the MET inhibitor
chemistry tends to be fast-acting with a good knockdown as well as a good
residual effect [16]. Unfortunately, the MET-I acaricides and insecticides tend to
be much more active against aquatic species (Table 31.3.1; fish and daphnia LC50
0.001–0.1 mg l−1 ), and this may represent a potential problem to be considered
in some cropping systems. All of the MET-I inhibitors possess rat/mouse oral
toxicities (LD50 ) that are generally in the range of 100 to 997 mg kg−1 for technical
materials (formulated materials typically display improved mammalian selectivity),
with dermal and avian values typically >2000 mg kg−1 (Table 31.3.1). Notably,
mammalian selectivity in the form of acute rat oral toxicity for the MET-II and
MET-III inhibitors is, in general, better than that observed for the MET-I inhibitors
(Table 31.3.1).
31.3.2.1 Fenpyroximate
Interestingly, each of the four of the initial companies that developed MET-I
acaricides employed rather different methodologies in their discovery efforts. At
Nihon Nohyaku, the discovery of fenpyroximate (NNI-850; Table 31.3.1) was the
result of an effort focused on discovering a new acaricide, using a chemistry-based
approach. Here, the effort was initiated using chloroformylpyrazole as a template
because it was easily synthesized and possessed several sites for substitution [17].
About 2000 analogs were synthesized and tested for acaricidal activity, including
some pyrazoloxime ethers that exhibited high levels of mite activity. Further
Table 31.3.1 Commercial and experimental insecticidal and acaricidal MET inhibitors.
1080
Common Name, Trade Manufacture Physical Properties use Toxicologyb Spectrum [references]
Names, Code Yr. Introduced propertiesa rates (gr/ha)
Complex inhibitors
Rotenone 1848 – primarily foliar 60-1500 ro broadly active:

short residual >2000 rbd caterpillars, aphids, mites,
2600 md beetles, thrips, hoppers, etc.

0.031 tr also as a piscicide [3]
Fenpyroximate Nihon Nohyaku mp 101-102 primarily foliar 245-480 ro Mite eggs & motile forms
Danitron®, FujiMite® 1991 log Kow 5.01 quick knockdown >2000 rd tetranychids, tarsonemids, tenupalpids
Kendo®, Acaban®, good residual 25-50 >2000 md eriophyids, grape leafhoppers
Ortus®, 0.0061 cp pear physlla, grape mealy bug [57]
Akari® NNI-850
Pryidaben Nissan mp 111-112 rapid knockdown 358-435 ro Mite motile forms
Sanmite®, Pyramite®, 1991 log Kow 6.37 good residual >2000 rd broad spectrum acaricide
Nexter® 100-250 >2250 rd whitefly, thrips, leafhoppers
NC-129, NCI-129 0.0083 cp mealybugs, plant bugs [57]
Fenazaquin Dow mp 78-80 primarily foliar>134 ro Mite eggs & motile forms
Magister®, Matador® AgroSciences log kow 5.51 contact active 1480 mo spider mites [57]
EL-436, XDE-436 (now Gowan) 56-560 >5000 rbd
1993 >2000 md
0.004-0.034 cp
Tebufenpyrad Mitsubishi mp 61-62 rapid knockdown 595-997 ro Mite eggs & motile forms
Masai®, Pyranica® (now Nihon log Kow 5.04 good residual >2000 rd broad spectrum acaricide
MK-239 Nohyaku) translaminar >2000 md [57, 135]
1993 100-250 0.073 cp
Tolfenpyrad Mitsubishi mp 87-89 ovicidal effects 75-86 ro Broad spectrum insecticide
Hachihachi® (now Nihon – anti-feedant 107-114 mo aphids, thrips, whiteflies,
OMI-88 Nohyaku) effects >2000 rd lepidopteran leaf miners, some mites
2002 75-200 0.0029 cp Diamondback moth [136]
Pyrimidifen Sankyo/Ube mp 69–91 25-75 110-148 ro spider & rust mites, all stages
Miteclean® 1995 log Kow 4.59 >2000 rd other pests, diamondback moth
SU 8801, SU 9118 445 md [57]
0.093 cp
Flufenerimc Ube – – – broader spectrum development of
S-1560 (now – – – pyrimidifen, incl. some lepidopterans
Makhteshim-Agan) [16, 56]
In development
XR-100 Dow – – >10– <50 mo Mites, eggs & motile forms,
LY 247356 AgroSciences lepidopteran eggs [67]
discontinued
LY 809460 Dow – – – Broad spectrum insecticide
AgroSciences – – – esp. lepidopterans [67]
Discontinued
LY 823089 Dow – – <0.0001 tr Broad spectrum insecticide
AgroSciences esp. lepidopterans [68]
Discontinued
SAN 548A Sandoz – – – ticks and possibly other
Discontinued insect / mite pests [30]
Hoe 11077 Hoechst – – – cockroaches and possibly
Discontinued other insect / mite pests [50, 70]
AE F117223 Aventis – – – broad spectrum acaricides
31.3 Inhibitors of Mitochondrial Electron Transport: Acaricides and Insecticides
Discontinued and insecticides [137]
1081
1082
Common Name, Trade Manufacture Physical Properties Use Toxicologyb Spectrum [references]
Names, Code Yr. Introduced propertiesa rates (gr/ha)
Complex II Inhibitors
Cyenopyrafen Nissan mp 106.7-108,2 – >5000 rd spider mites

Starmite® 2008 log Kow 5.6 0.018 rt all stages [83]
NC-510
Cyfelmetofenc Otsuka mp 77.9-81.7 100–800 >2000 ro spider mites

Danisaraba® 2007 log Kow 4.3 >5000 rd all stages [83]
OK 5101
IKA-2002c ISK BioSciences – – – mites (all stages) and some
Experimental insect pests [83]
Thiapronil Schering mp 182-183 113–500 >5000 ro broad spectrum insecticide

SN 72129 (now China) >2000 rbd [85]
1983
Complex III Inhibitors
Acequinocyl DuPont mp 59-60 contact-oral >5000 ro broad spectrum acaricide

Kanemite®, Shuttle® (now Agro- log Kow >6.2 activity >2000 rd esp. motile stages
DPX-3792, AKD-2023 Kanesho) good residual >2000 md [57]
1999 130 97 cp
Fluacrypyrim Nippon Soda mp 107-109 – >5000 ro Spider mites
Titaron® 2002 log Kow 4.51 >5000 rd [57]
NA-83
Hydramethylnon AmCy (now mp 189-191 oral active >1131-1300 ro bait for ants and cockroaches [138]
Amdro® BASF) bait >2000 rhd
AC 217,300 1980 >2510 md
0.16 tr
Bifenazatec Uniroyal mp 123-125 non-systemic >5000 ro phytophagous mites [138]
Acramite® (now Chemtura) logKow 3.4 log residual >5000 rd
Floramite® 2000 150–750 726 md
D2341 0.76 tr
SYP-4903c Shenyang Res. – – – lepidopterans and mites [93]

Inst. of Chem.
Ind. (SYRICI)
In development
HNPC-A3066c Hunan Res. mp 79.6–80.1 – – acaricide

Inst. of larvicidal & ovicidal activity
of Chem. Ind. [93, 94]
(HRICI)
In development
a
1 – mp = melting point co.
b
2 – ro = acute rat oral LD50 (mg/kg), mo = acute mouse oral LD50 (mg/kg), rd = acute rat dermal LD50 (mg/kg), rbd = acute rabbit dermal LD50 (mg/kg),
md = acute avian toxicity, mallard duck LD50 (mg/kg), cp = acute aquatic toxicity, carp LC50 (mg/L), tr = acute aquatic toxicity, trout LC50 (mg/L); data
adapted from 3, 8, 14, 21, 29, 47, 112–119.
c
3 – mode of action classification appears likely, but is not yet proven.
31.3 Inhibitors of Mitochondrial Electron Transport: Acaricides and Insecticides
1083
activity-directed optimization led to the identification of methyl groups in the 1,3

positions of the pyrazole, along with a phenoxy group in the 5-position, as being
highly active. When coupled with a 4-tert-butyl ester moiety on the benzyl ring,
the resulting compound appeared optimal and was selected for development as
fenpyroximate [17]. Subsequent studies demonstrated that fenpyroximate inhibits
MET at Complex I [18–21]. Interestingly, fenpyroximate can assume the same
nonlinear molecular shape as the other MET-I inhibitors, including pyridaben and
tebufenpyrad [22]. Fenpyroximate is active against a wide variety of mite species
(Table 31.3.1), and is registered for use in a wide variety of crops, including citrus,
pome fruit, vegetables, beans, vines, strawberries, melons, hops, and ornamentals
[23].
The synthesis of fenpyroximate is shown in Scheme 31.3.1. The pyrazolone ring,
formed by the condensation of ethyl acetoacetate with methylhydrazine, is then
subjected to the Vilsmeier–Haack chloroformylation using dimethylformamide
(DMF) and POCl3 to give the 5-chloro-4-formylpyrazole [24, 25]. The chloride in
the 5-position of the pyrazole is substituted with phenol through a nucleophilic
displacement reaction [26]. Fenpyroximate is then generated by condensation of the
4-formyl-5-phenoxypyrazole with hydroxylamine, followed by a Williamson ether
H3C HCON(CH3)2 H3C

OEt CHO
CH3NHNH2 POCl3
N N
O O 20 °C 100 °C
N O N Cl
H3C H3C
KOH
H3C H3C
OH CHO NaOH
OH
NH2OH.HCl N
N N
DMSO, 80 –100 °C MeOH, 60 °C
N O N O
H3C H3C
O
KOH
O O
H3C
Br O O
N
N
DMSO
N O Fenpyroximate
H3C
AIBN
O Br2 O 1. SOCl2 O
CHCl3 2. t-BuOH
OH Br OH Br O
pyridine
Scheme 31.3.1 Synthesis of fenpyroximate.

synthesis with the side chain t-butyl (4-bromomethyl)benzoate [17, 24, 27, 28]. The
side chain is prepared by free-radical bromination of p-toluic acid, followed by
formation of the acid chloride and condensation with t-butanol [29, 30].
31.3.2.2 Pyridaben
In contrast to fenpyroximate, the discovery of pyridaben (NCI-129; Table 31.3.1)
by Nissan Chemical Industries exemplifies where activity in one product area can
lead to product level activity in other product areas. This also demonstrates the
value of broad screening chemistries as a means to unearth new activity. In this
case, research into the herbicidal activity of pyridazinones led to the discovery that
one of the analogs possessed acaricidal activity [31]. A subsequent exploration of
the structure–activity relationships (SARs) around this chemistry, now focusing
on acaricidal activity, led to the identification of pyridaben [31]. Like others in this
group, pyridaben inhibits insect respiration and has been shown specifically to
impede Complex I oxidations [20]. More recent studies using a pyridaben derivative
as a photoaffinity label have demonstrated that pyridaben, as well as other MET-I
inhibitors, interacts with the PSST subunit of Complex I [32–34]. Pyridaben is a
broad-spectrum, contact acaricide (Table 31.3.1) registered for use on various crops,
including tree fruit (apples, almonds, cherries, plums, pears), citrus, vegetables,
grapes, strawberries, and ornamentals [23].
The synthesis of pyridaben is shown in Scheme 31.3.2. The pyridazinone ring is
formed though a two-stage process involving the condensation of mucochloric acid
with t-butylhydrazine at low temperature to give a t-butylhydrazone intermediate
that is then cyclized at elevated temperature with catalytic acid [35, 36]. The
chlorine in the 5-position of the pyridazinone is then substituted with sodium
thiolate to give a mercaptan that is reacted with p-tert-butylbenzyl chloride to
NHNH2 . HCl Cl Cl cat. O

Cl Cl acetic acid Cl
NaOH N
MeOH, 10–15 °C NHN HO O toluene, 35 – 45 °C N
HO O O Cl
O
O Cl
Cl N
Cl Na2CO3
NaSH N N
S
H2O N DMF
SH
Pyridaben
Cl2
Radical initiator
Cl
Scheme 31.3.2 Synthesis of pyridaben.

produce pyridaben [37]. The p-tert-butylbenzyl chloride is prepared in high yield by

free-radical chlorination of p-tert-butyltoluene [38].
31.3.2.3 Fenazaquin
The discovery of fenazaquin (EL-436, XDE-436; Table 31.3.1) resulted from yet
another approach. During the early 1980s, as part of Elanco’s random screening
program, a quinazoline (LY-176771) was found to exhibit some fungicidal activity
against grape downy mildew [39]. Based on the observed activity, coupled with
a review of available information, a series of quinazoline ethers was investigated
[40] with the goal of improving fungicidal activity. However, a broad screening
of these new compounds identified a series of analogs that exhibited activity
against lepidopteran insects. Further refinement of the SAR focused on acaricidal
activity, ultimately leading to fenazaquin [40]. Both, internal and external studies
demonstrated that fenazaquin inhibits MET at Complex I [20, 41–43]. Fenazaquin
is particularly effective against tetranychid mite species, including two-spotted
spider mite (TSSM) and red mites (Table 31.3.1), and is registered for use on
various tree fruit (apples, citrus, pears, plums), vines, vegetables, and ornamentals
[23].
The synthesis of fenazaquin is shown in Scheme 31.3.3. The quinazoline ring is
formed by the condensation of anthranilic acid with formamide, and subsequent
halogenation at the 4-position is accomplished using the Vilsmeier reagent [44].
The 4-chloroquinazoline is coupled to the side chain 4-t-butylphenylethanol with
the aid of anhydrous HCl [45]; this yields fenazaquin as the HCl salt, which is
liberated as the free base with aqueous ammonia.
31.3.2.4 Tebufenpyrad
The approach that Mitsubishi Chemical Corporation took in its efforts to develop
a new acaricide lies somewhere between the strict chemistry-based approach and
a simple broad screening approach, specifically targeting chemistry that possesses
biological activity in one area of interest and exploring it for activity in other product
arenas. It was noted that N-phenylpyrazole carboxamides exhibited both fungicidal
O OH SOCl2
Cl
HCONH2 Me3NHCl
OH N N
Δ DMF, 50 –100 °C
NH2 N N
anhyd. HCl
OH O
NH4OH N Fenazaquin
chlorobenzene, 35 –40 °C
N
Scheme 31.3.3 Synthesis of fenazaquin.

and herbicidal activity, and because of their interesting chemical structure and
known activity in other areas, a targeted synthesis effort around pyrazolecarbox-
amides, focusing on acaricidal activity, was initiated [46]. The outcome was the
identification of pyrazole-5-carboxamides with acaricidal activity, ultimately lead-
ing to the discovery of tebufenpyrad (MK-239; Table 31.3.1) [46]. As with the
above-described compounds, subsequent studies showed that tebufenpyrad func-
tions as an inhibitor of MET Complex I [34, 41]. Tebufenpyrad is a broad-spectrum
acaricide, possessing translaminar activity (Table 31.3.1), and is registered for use
on a variety of crops that includes pome and stone fruit, ornamentals, strawberries,
hops, melons, citrus, and tomato [23].
The preparations of tebufenpyrad and tolfenpyrad (see below) are outlined in
Scheme 31.3.4. The pyrazole ring is prepared from a Claisen condensation of
2-butanone with diethyl oxalate; the resulting acylpyruvate is then treated with hy-
drazine [47, 48]. The pyrazole ring is then alkylated with dimethyl sulfate at 50–60 ◦ C
without base to give, selectively, the 1-methylpyrazole-5-carboxylate. The pyrazole is
then chlorinated in the 4 position and saponified to produce the pyrazolecarboxylic
acid. The acid chloride of the pyrazolecarboxylic acid is formed and coupled with
4-t-butylbenzylamine or 4-(p-tolyloxy)benzylamine to yield tebufenpyrad and tolfen-
pyrad, respectively. The side chain compound 4-t-butylbenzylamine is prepared by
the reductive amination of 4-t-butylbenzaldehyde with aqueous ammonia. The
compound 4-(p-tolyloxy)benzylamine is prepared by coupling 4-fluorobenzonitrile
with sodium p-cresol, followed by reduction with Raney nickel in aqueous ammonia
[49, 50].
31.3.2.5 Tolfenpyrad
Mitsubishi Chemical Corporation’s interest in the pyrazolecarboxamides did not
end with the discovery of tebufenpyrad. Rather, further syntheses were undertaken
to improve and expand on the acaricidal activity, leading to the identification of
a weak activity against some insect species for some analogs of tebufenpyrad [47,
51–53]. The results of further studies indicated that replacement of the t-butyl tail
with electron-withdrawing groups would greatly improve the insecticidal activity
against hemipterans and some lepidopterans, although the mammalian toxicity
of the product was also increased [47, 53]. Further investigations [47] led to the
identification of a N-tolyloxybenzyl derivative, tolfenpyrad (OMI-88; Table 31.3.1),
which possessed good insecticidal activity coupled with an acceptable mammalian
selectivity. Tolfenpyrad is a broad-spectrum miticide/insecticide (Table 31.3.1) that
is currently registered for use on vegetables and orchards in Japan [23].
31.3.2.6 Pyrimidifen
Pyrimidifen (SU 8801; Table 31.3.1) is an acaricide jointly patented and de-
veloped by Sankyo Company and Ube Industries. This acaricide chemistry
appeared to be the result of a long line of research, starting with N-(substituted
phenoxyalkyl)-4-quinazolinamines exhibiting fungicidal activity, morphing into
N-benzyl-4-pyrimidinamines that displayed moderate lepidopteran and mite activ-
ity [54–58], and further evolving into analogs from which pyrimidifen emerged
1088
O
O
O O
NH2NH2 . H2O
O
O N O
O NaOEt
O O N
H O
Cl
(CH3)2SO4 Cl2
NaOH
N O
50 – 60 °C N SO2Cl2 N O
N H2O
CH3 O O
CH3
Cl NH2 Cl
Cl R
R
SOCl2 H
N OH N
N Cl N N
N Et3N, toluene N
CH3 O
CH3 O CH3 O
tebufenpyrad, R = t -butyl
tolfenpyrad, R = p -tolyloxy
H2, Ni NH2
CHO
NH3, H2O
ONa
H 2 , Ni NH2
F CN O CN O
DMSO NH 3 , H 2 O
Scheme 31.3.4 Synthesis schemes of tebufenpyrad and tolfenpyrad.

[57]. Based on similarity to other close pyrimidine compounds and specific studies,
pyrimidifen appears to act at Complex I [4, 59, 60]. Pyrimidifen is effective on
various mite species, including spider and rust mites and certain other insect pests,
and has been registered for use in tree fruit (e.g., apples, pears) citrus, vegetables,
and ornamentals [23].
The synthesis of pyrimidifen is shown in Scheme 31.3.5. The pyrimidine ring
in pyrimidifen is prepared via a condensation of methyl 2-chloro-oxovalerate
with formamidine acetate in the presence of a base [61, 62]. The resulting
4-hydroxypyrimidine is then chlorinated with phosphorous oxychloride to produce
4,5-dichloro-6-ethylpyrimidine. The chlorine in the 4-position is then substituted
with the side chain 2-[4-(2-ethoxyethyl)-2,3-dimethylphenoxy]ethylamine through
nucleophilic displacement [56, 63]. The side chain is prepared from the reaction of
2,3-xylenol with chloroacetaldehyde dimethylacetal [61]. The resulting acetal is then
brominated in the 4-position, converted into the Grignard reagent, and reacted
HN NH2 . CH 3 CO2 H OH Cl
O O
CH3ONa H2SO4 Cl POCl3 Cl
N N
OMe H2O, 65 oC
MeOH
Cl N N
O O
NH2 O
HN
Et3N Cl
N O
toluene Pyrimidifen
N
OMe CH3ONa OMe

OH + Cl O
OMe OMe
Br2 OMe OMe

Br O O HO O
dichloroethane Mg
OMe OMe
THF
nBu4NCl
20% aq. NaOH
NH2OH .
Et2SO4 H2SO4
OMe NOH
O O O O
toluene MeOH,
OMe 70 °C
H2, Raney Ni NH2

O O
MeOH
Scheme 31.3.5 Synthesis of pyrimidifen.

with ethylene oxide to yield the phenethyl alcohol that is then converted into the
ethyl ether with diethyl sulfate under phase-transfer conditions. The resulting
substituted phenoxyacetaldehyde dimethylacetal is converted into the oxime with
hydroxylamine under acidic conditions, and the oxime reduced with hydrogen in
the presence of Raney nickel to produce the substituted phenoxylethylamine side
chain [64].
31.3.2.7 Flufenerim
Flufenerim (S-1560; Table 31.3.1; see Scheme 31.3.6) is an acaricide/insecticide that
has been under development for some time, and which appears to be a more recent
derivative of pyrimidifen [23] but possesses less-labile substitutions on the head
and tail regions (Figure 31.3.2). Available information suggests that flufenerim
has acaricidal activity as well as activity against sap-feeding and lepidopteran
insect pests [23, 65–67]. The added spectrum of activity may be one result of
the incorporation of less-labile substituents compared to the earlier member of
this chemistry, pyrimidifen. Data included in this recent study suggested that
Cl
Cl Cl KF
Cl
Cl Cl2 Cl Bu4NBr N
N N
N N
N N N
O F
Cl 110–115 °C
OCF3
OCF3
H2N HN
Et3N Cl
N
toluene Flufenerim
N
F
CH3(CH2)15 NMe3Br
SOCl2 ZnCl2
paraformaldehyde NaCN
CF3O CF3O CF3O
H3PO4 60 C o
Cl CN
H2
Raney Ni
CF3O
NH2
Scheme 31.3.6 Synthesis of flufenerim.

O O CF3
N N N N
F F N
O CF3 N
H
XR-100 LY 809460
N S O N N
O
N
N CF3 H
H
Cl
LY 823089 SAN 548a
N+ N
N N O S S O
O
N −O
H S OEt
O
Hoe 11077 FMC4 O
S
O Cl
Cl CF3 O Cl O S
N
N
N N H
H CN CN
AE F117233 IKA-2002 Thiapronil
O CF3 O
N N O O O O
N
O F3C O
S
SPY-4903 HNPC-A3066
Figure 31.3.2 Structures of experimental and other MET acaricides and insecticides.
flufenerim can reduce acetylcholinesterase (AChE) activity; however, it was also

suggested that the effect on AChE might be the result of flufenerim acting on other
systems [67]. Although the MET activity of flufenerim has not been proven, its
chemical structure – which is close to that of pyrimidifen and other proven MET
Complex I inhibitors (Figure 31.3.1) – makes this likely.
The synthesis of flufenerim is shown in Scheme 31.3.6. The pyrimidine ring
is formed from a Claisen condensation of methyl 2-fluoropropionate with methyl
acetate [68], followed by reaction of the fluoroacetopropionate with ammonia
in phosphomolybdic acid [69] and cyclization with formamide in the presence

of a base [70]. This gives the 4-pyrimidone ring, which is converted into the
4,5-dichloro-6-(1-fluoroethyl)pyrimidine by the action of sulfuryl chloride in the
presence of DMF [71]. The chlorine on the 4-position of the pyrimidine ring is
substituted with the side chain 2-[4-(trifluoromethyl)phenyl]ethylamine through a
nucleophilic substitution reaction [65]. The latter side chain is prepared by the
chloromethylation of trifluoromethoxybenzene, followed by displacement of the
chlorine atom with cyanide and reduction of the nitrile with Raney nickel [72].
31.3.2.8 Experimental MET Complex I Compounds

In addition to the above-described compounds, which are either already in use or
under development, other compounds which perhaps are of a more experimental
nature, or which failed to progress during development, have also been examined.
Following the discovery of fenazaquin, the SAR around this chemistry was explored,
including fused pyrimidine derivatives of fenazaquin [40]. Pyridine and pyrimidine
amide analogs were also investigated [73], as were quinoline/quinazoline derivatives
of fenazaquin with modifications to the tail (t-butyl) region [74]. These investigations
led to the identification of analogs such as XR-100 (Figure 31.3.2) and O-haloalkyl
pyridyl derivatives such as LY 809460 (Figure 31.3.2; compound 11 in Hackler
et al. 1998 [74]) that were more effective than fenazaquin at inhibiting MET
activity, and which exhibited an expanded spectrum and proved to be active against
lepidopterans [74]. A series of isothiazolyl phenylacetamides was also examined [75]
that displayed a good broad-spectrum insect activity. For both series of compounds,
however, optimization of their insecticidal activity led to increases in mammalian
and/or fish toxicities [74, 76] that could not be adequately remedied, thereby limiting
their utility.
In addition to the above-mentioned efforts of Mitsubishi and Dow AgroSciences,
during the early 1990s other companies also investigated chemistry acting as
MET-I inhibitors, including SAN 548A [41], AE F117233 [58], and Hoe 11077
(Figure 31.3.2; Table 31.3.1), and related compounds [51, 59, 60, 77]. Recently,
a new group of MET-I inhibitors emerged from efforts to develop novel chitin
synthesis inhibitors (CSIs) from cyanine dyes [78]. Interestingly, the resulting
lepidopteran-active compounds (e.g., FMC4) were found not to be CSIs, but rather
inhibitors of MET Complex I (Figure 31.3.2; Table 31.3.1) [78].
31.3.3
Complex II Inhibitors
31.3.3.1 Cyenopyrafen
Following a recent expansion of MET inhibitor chemistries, other acarici-
dal/insecticidal chemistries have been identified that act through an inhibition of
the MET Complex II. Cyenopyrafen (NC-512; Table 31.3.1), a recently discovered
acaricide reported to be an inhibitor of Complex II [79, 80], is a pro-acaricide
that is bioactivated through cleavage of the ester; the hydroxyl analog produced
subsequently inhibits MET Complex II [79].
N
O
N CN Cl CN
EtO N N N
CN O N
N
O Xylene, NEt 3 O
NaOEt O
heptane
O O cyenopyrafen
1.
O
OEt MeNHNH2 H2N OEt toluene N
Br N EtO N
O toluene O 2. K2CO3, EtOH,
relux
Scheme 31.3.7 Synthesis of cyenopyrafen.
The synthesis of cyenopyrafen is shown in Scheme 31.3.7. Compound

4-t-butylphenylacetonitrile is condensed with ethyl 1,3,4-trimethylpyrazol-5-
carboxylate [81] to give 3-oxo-2-(4-t-butylphenyl)-3-(1,3,4-trimethylpyrazol-5-yl)prop-
ionitrile. The latter is then reacted with pivaloyl chloride in xylene to yield
cyenopyrafen [82].
31.3.3.2 Cyflumetofen
In addition to cyenopyrafen, thiapronil appears to be a Complex II inhibitor (see
below), and two other closely related chemistries may act at Complex II. Although
no mode of action studies have been reported for either cyflumetofen (OK-5101)
or IKA-2002 (Table 31.3.1), by analogy to cyenopyrafen these two compounds may
also be MET Complex II inhibitors. In the case of cyflumetofen, an acaricide
developed by Otsuka Chemical Co. [83–85], it has been proposed that in the TSSM
a metabolite of cyflumetofen blocks the MET Complex II, though further studies
are required to confirm this mode of action [84].
The synthesis of cyflumetofen is shown in Scheme 31.3.8. Here, compound
4-t-butylphenylacetonitrile is condensed with dimethylcarbonate to yield the phenyl-
cyanoacetic methyl ester. Transesterification with 2-methoxyethanol, followed by
condensation with 2-trifluoromethylbenzoyl chloride under phase-transfer condi-
tions, yields cyflumetofen [86].
31.3.3.3 Experimental and Other MET Complex II Compounds

Based on structural analogy, another potential Complex II inhibitor is IKA 2002
(Figure 31.3.2). This experimental acaricide has been in Japanese Official trials for
several years [87]. Based on chemical analogy, thiapronil (SN 72129) (Figure 31.3.2)
would also appear to be a MET Complex II inhibitor and recent data supports this
assumption [88]. Thiapronil is active on coleopteran, lepidopteran, hemipteran,
and dipteran species [89] and thus appears to be broader spectrum than the other
molecules in the category. In contrast to the other Complex II inhibitors, thiapronil
1094
O CN CN
OMe O
CN MeO OMe O O
OH
O O
NaOMe, 90 °C
100 °C
CF3
CF3
Cl
O NC O
O
Bu4NBr O
K2CO3
toluene/water
O
cyflumetofen
Scheme 31.3.8 Synthesis of cyflumetofen.

is likely not a proinsecticide since there is no derivatization at the keto (hydroxyl)

moiety as is observed for the other molecules.
31.3.4
Complex III Inhibitors
Complex III has been very successfully exploited as a target site for fungicides such
as the strobilurins, famoxadone, and fenamidone [90]. However, it has only been
in the last few years that insecticidal or acaricidal products have used inhibition at
Complex III as a mode of action.
31.3.4.1 Acequinocyl
Originally discovered by DuPont during the 1970s and referred to as DPX-3792,
acequinocyl (Table 31.3.1) was subsequently licensed and brought to market by
Agro-Kanesho (ADK-2023) during the early 1990s [23]. Unlike the MET-I inhibitors,
acequinocyl is a pro-insecticide that is bioactivated via deacylation to its deacetyl
metabolite, 2-hydroxy-3-n-dodecyl-1,4-naphthoquinone (DHN). Studies conducted
by Koura et al. [91] have demonstrated that acequinocyl, via DHN, acts at the
ubiquinol oxidation site (Q0 ) of Complex III. Acequinocyl is a broad-spectrum
acaricide (Table 31.3.1) that is registered for use in pome and stone fruit, citrus,
melons, fruiting vegetables, and ornamentals [23].
The synthesis of acequinocyl is shown in Scheme 31.3.9. In this case,
1,4-naphthaquinone is epoxidized and acidified to produce 2-hydroxy-1,4-
1. NaOCl/H2O
O Bu 4NBr CH3(CH2)10 CHO
O O
2. NaOH OH BuNH2 OH
MeOH
toluene (CH2)10CH3
3. H2SO4
O O O NHBu
O O
H2
H2SO4 OH OH
Pd/C air
110 °C
(CH2)9CH3 (CH2)11CH3
O O
O
pTsOH
Ac2O OAc
acequinocyl
toluene, 110 °C (CH2)11 CH3
O
Scheme 31.3.9 Synthesis of acequinocyl.

naphthaquinone [92]. The dodecyl group is introduced through a condensation of

2-hydroxy-1,4-naphthaquinone with dodecanal in the presence of n-butylamine
to yield 3-(1-butylaminododecyl)-2-hydroxy-1,4-naphthaquinone. The latter com-
pound is then thermally eliminated to produce the dodecenyl derivative [93],
which is in turn hydrogenated and air-oxidized to provide 2-dodecyl-3-hydroxy-1,4-
naphthaqiunone; the latter is acetylated to produce acequinocyl [93].
31.3.4.2 Fluacrypyrim and β-Methoxyacrylate/Strobilurin-Based Compounds

The β-methoxyacrylates and strobilurins are well-known fungicides that act at
Complex III of the MET chain [90, 94]. In addition to their fungicidal activity, many
companies have patents that claim both insecticidal and fungicidal activities for
their strobilurin-related chemistry. Fluacrypyrim (NA-83, Table 31.3.1), discovered
by BASF and licensed to Nippon Soda [23], is the first methoxyacrylate-derived
compound to be marketed other than as a fungicide, in this case as an acaricide.
Fluacrypyrim appears to be a MET-III inhibitor [4], in part based on analogy
with the strobilurins. Fluacrypyrim is targeted for mite control, especially spider
mites (Table 31.3.1), on vegetables and tree fruit such as citrus, apples, and
pears [23]. In addition to fluacrypyrim, the β-methoxyacrylate/strobilurin-based
chemistry continues to be of interest. Further research concerning the strobilurins
as potential acaricides was recently reported [95, 96], and other new products may
eventually emerge from this area of chemistry. Two molecules reported to be in
development are SYP-4903 [97] and HNPC-A3066 [97, 98] (Figure 31.3.2).
The synthesis of fluacrypyrim is shown in Scheme 31.3.10. The pyrimidine ring
is formed through the condensation of trifluoroacetoacetate with o-isopropylisourea
hydrochloride in the presence of a base [99]. The product is then O-alkylated with
methyl 2-(chloromethyl)phenylacetate in the presence of a copper salt to produce the
pyrimidyloxy derivative [100, 101]. The acrylic acid group is introduced in a two-step
Cl
MeO2C
CO2Me
Oi Pr
OH P(Oi Pr)3 O
O O
H2N NH HCl
N Cu2O N
CF3 OEt
CF3 N Oi Pr CF3 N Oi Pr
Bz(Bu)3NCl
Et3N OH MeO2C OMe
MeO2C KOH
TiCl4 (CH3)2SO4
HC(OMe)3
O O
N N
Fluacrypyrim
CF3 N Oi Pr CF3 N Oi Pr
Scheme 31.3.10 Synthesis of fluacrypyrim.

process, wherein the pyrimidyloxy derivative is reacted with trimethylorthoformate

in the presence of a Lewis acid and base to give the 3-hydroxyacrylic acid ester
derivative [102]; the latter is alkylated with dimethyl sulfate under phase-transfer
conditions to provide fluacrypyrim [103].
31.3.4.3 Hydramethylnon
Hydramethylnon is a well-known chemistry that is used widely in insect baits for
the control of ants and cockroaches [104]. Hydramethylnon has been reported to
be an inhibitor of Complex III [105], though minimal additional information is
available on its action at the target site.
The synthesis of hydramethylnon is shown in Scheme 31.3.11. Condensation
of 4-trifluoromethylbenzaldehyde with acetone under phase-transfer conditions
yields 1,5-bis[4-(trifluoromethyl)phenyl]-1,4-pentadien-3-one. The latter is then con-
densed with 2-hydrazino-5,5-dimethyl-1,4,5,6-tetrahydropyrimidine hydrochloride
[106] under phase-transfer conditions to yield hydramethylnon [107].
31.3.4.4 Bifenazate
Early reports on the mode of action of bifenazate suggested that a neuroactive
mechanism was involved [108]. The results of recent studies have indicated that
bifenazate is a pro-acaricide [109] that acts on MET Complex III [80, 110–112].
The available data also suggest that acequinocyl and bifenazate may be
cross-resistant through mutations in Complex III [111, 112] (see also Ref. [107]).
31.3.5
Metabolism
31.3.5.1 MET-I Inhibitors

The MET-I inhibitors are each composed of three sections, namely the head, linker,
and tail. In addition, most commercial MET-I acaricides possess a t-butyl, t-butyl
ester, or other long-chain moiety in the 4-position of the tail (Figure 31.3.3). The
t-butyl ester of fenpyroximate is rapidly cleaved via a mono-oxygenase-mediated
hydroxylation and ensuing transesterification (Figure 31.3.3) [113, 114]. Other
fenpyroximate-based metabolites also arise via mono-oxygenase activity, including
oxidation of the 3-pyrazolo-methyl group, N-demethylation, isomerization, and
cleavage of the oxime ether bond (Figure 31.3.3) [16, 114].
Metabolism studies conducted in rats have also demonstrated the importance of
mono-oxygenase activity in the case of pyridaben. Here, the metabolic pathways
include hydroxylation of either of the two t-butyl moieties, and cleavage of the
thioether linkage (Figure 31.3.3) [31].
As observed with fenpyroximate and pyridaben, a primary metabolic pathway
for fenazaquin in mammalian systems involves oxidation of the t-butyl moiety
(Figure 31.3.3) [115]. Other mono-oxygenase-mediated metabolic reactions include
oxidation of the quinazoline ring and cleavage of the ether linkage (Figure 31.3.3)
[115]. Fenazaquin is also rapidly metabolized in lepidopterous insects [116], with
oxidation of the t-butyl moiety being the predominant route of metabolism [74].
1098
O
HN NH
HN NH
NaOH HCl
CHO O PTC catalyst N
NHNH2
+ N
heptane/ NaOH
F3C water isopropanol/water
F3C CF3 40 ° C
NH F3C CF3
+ HCl N2H4 H2O
125 ° C
hydramethylnon
NH2 NH2 H2N NH2 HN NH
HCl
NHNH2
Scheme 31.3.11 Synthesis of hydramethylnon.

Oxidation O
O
O
N N N N
Ester cleavage
N
O O
Isomerization /
LINKER cleavage
HEAD TAIL
N-demethylation Fenpyroximate
Oxidation Oxidation
Oxidation N N
S O
N
N
Cl Thioether
Ether cleavage
O cleavage
Ring hydroxylation
Pyridaben Fenazaquin
Oxidation
Oxidation
N N N N O
H H
N N
Cl O Cl O Tolfenpyrad
Amide cleavage
Oxidation Tebufenpyrad Oxidation
O
O OH
N N
N N
CN CN
Cyenopyrafen OH- Cyenopyrafen – Active form
O OH
O O
O O
Acequinocyl Acequinocyl – Active form
Figure 31.3.3 Sites of metabolism for MET acaricides and insecticides.

Tebufenpyrad, as other MET-I acaricides, is subject to oxidative metabolism

at the t-butyl moiety (Figure 31.3.3) [117]. The ethyl substituent of the pyrazole
head is also subject to oxidation [91], which leads to the formation of a 1-hydroxy
derivative. Unlike the above-mentioned acaricides, tebufenpyrad contains an amide
moiety in the linker between the pyrazole head and the phenyl tail, such that other
significant metabolic routes involve amide cleavage (Figure 31.3.3) [117]. The close
structural similarity between tebufenpyrad and tolfenpyrad suggests that some of
the metabolic pathways identified for tebufenpyrad (Figure 31.3.3) are likely also
to apply to tolfenpyrad. Indeed, this appears to be the case, with the primary route
of metabolism seeming to be an oxidation of the 4-methyl group on the tolyl-tail,
coupled with hydroxylation of the ethyl moiety on the pyrazole head [118]. To
date, information on the metabolism of pyrimidifen and flufenerim appears to be
lacking. However, by analogy with the other members of this class it is reasonable
to assume that mono-oxygenase-mediated metabolic pathways would predominate
for both of these compounds.
Based on the results of studies with XR-100 [74], it appears likely that the
metabolites and rate of metabolism of flufenerim may be altered by the presence
of a trifluoromethyl group on the tail and a 1-fluoroethyl moiety attached to the
pyrimidinyl head. Clearly, studies specifically targeting the metabolism of these
compounds are required to assess the above-described hypotheses.
31.3.5.2 MET-II Inhibitors

Unlike the MET-I acaricides and insecticides, cyenopyrafen – a MET Complex II
inhibitor – requires bioactivation for activity. Cyenopyrafen is hydrolyzed to its
hydroxyl-form (Figure 31.3.3) that is active on Complex II [79]. In light of the
required bioactivation for cyenopyrafen, a similar process might also be involved in
the activity of cyflumetofen and IKA-2002, as has been suggested for cyflumetofen
[84], though further studies are required to test this hypothesis.
31.3.5.3 MET-III Inhibitors

As observed for cyenopyrafen, the MET Complex III inhibitor acequinocyl is
also a pro-insecticide that requires biological activation for activity. Within the
mitochondrion, acequinocyl is hydrolyzed to the corresponding deacyl derivative,
DHN (Figure 31.3.3) [8, 91]. This process is consistent with the observation of a
time lag in MET inhibition with acequinocyl [89], and the finding that acequinocyl
itself does not have any direct inhibitory effect on MET activity [119].
31.3.6
Resistance and Resistance Mechanisms
As noted above, the MET inhibitor-based acaricides are, as a group, broadly active
against a wide variety of mite species, including the TSSM, Tetranychus urticae.
The TSSM is among the most prevalent pest mite species, with a long history of
developing resistance to available acaricides, and is presently resistant to more
insecticides/acaricides than any other mite or insect species [120]. One of the
initial advantages of the MET-I acaricides was their efficacy against resistant mite
species. However, it was quickly realized that as several of these new acaricides
all possessed the same mode of action and were brought to the market place at
about the same time, resistance might become an issue. Previously, in Europe,
the Insecticide Resistance Action Committee (IRAC) had put in place an acaricide
resistance management program [121] of which the MET-I acaricides had become
part as they entered the market place [122].
During the 20 years that the MET-I inhibitors have been in the market place,
their high levels of efficacy on all mite stages, their long residual and limited
mobility, coupled with the rapid reproductive rate of mites, have all contributed
to the likelihood of resistance developing. Thus, despite great efforts having been
made to minimize the chances of resistance [121, 122], it is not surprising that
resistance to the MET-I inhibitors has developed in several mite species, including
the TSSM. In fact, high levels of resistance (in some cases, beyond 1000-fold) have
been observed towards pyridaben and fenpyroximate in field strains of TSSMs that
have been exposed to extensive MET-I acaricide use [123–128]. Lower levels of
resistance have been observed for tebufenpyrad (6.7- to 97-fold) and fenazaquin (8-
to 168-fold) [123, 126–130]. Although cross-resistance between the MET-I inhibitors
and older acaricides is less common, that within the MET-I acaricide family is more
frequent. For example, TSSMs resistant to fenpyroximate (252-fold) also exhibited
a degree of cross-resistance to pyridaben (38-fold) and tebufenpyrad (24-fold), but
less so to fenazaquin (7.2-fold) [125]. Likewise, TSSMs resistant to tebufenpyrad
(63-fold) were also cross-resistant to pyridaben (>210-fold) and fenpyroximate
(24.6-fold) [126].
Consistent with the above-mentioned trend of minimal MET-I cross-resistance
from mites resistant to other types of acaricide, the MET-I resistant strains
are, likewise, less likely to confer cross-resistance to other classes of acari-
cides and insecticides. MET-I acaricide-resistant TSSM strains exhibited little
cross-resistance to dicofol, amitraz, or chlorfenapyr [123, 128]. In the reverse
case, a methidathion-resistant strain of the predatory mite Amblyseius womer-
sleyi showed no cross-resistance to pyridaben [131]; neither did clofentezine
(or clofentazine–dicofol) -resistant strains of red mites and TSSM show any
cross-resistance to MET-I acaricides such as pyridaben, tebufenpyrad, or ace-
quinocyl [132, 133]. Exceptions to these observations have been the TSSM strains
resistant to dimethoate and methamidophos that also exhibit cross-resistance to
pyridaben [134].
As the MET-I acaricides are primarily metabolized by the mono-oxygenases [111,
112], metabolically based cross-resistance among the MET-I insecticides/acaricides
is not necessarily surprising. Despite the rather different chemistries involved,
the MET-I compounds share similar molecular features and can assume a similar
molecular shape [22], while the substituents on the ‘‘tail’’ region typically include
a t-butyl or alkyl moiety. As noted in Section 31.3.4, since the t-butyl tail is also
a common site for metabolism, any strain developing an enhanced metabolism
to one of these compounds might reasonably be expected to exhibit some level of
enhanced metabolism to the other members of the group and, in general, this has
been observed [111, 112]. In some cases, however, the resistance mechanism(s)
to some of the MET-I acaricides may not be the same as that for other MET-I
inhibitors [111]. In fact, the results of some recent studies have provided evidence
to support the presence of a target-site mechanism [112].
31.3.7
The Future for MET Acaricides and Insecticides
Included in the key considerations for mite control are: (i) a high degree of
efficacy at several growth stages; and (ii) a lack of cross-resistance with acaricides
possessing other modes of action. Among others benefits, the MET-I acaricides
bring these very valuable attributes to the marketplace. The diverse chemistries
of the MET-I acaricides and insecticides identified to date have suggested that
other novel chemistries may yet be discovered that are capable of exploiting these
target sites. Although the first molecules of this type were introduced over 20
years ago, new compounds such as the MET-II acaricides are still being discovered
and developed. Indeed, the ubiquitous nature of all three MET target sites holds
great potential for the development of true broad-spectrum (sucking and chewing)
insect-control agents. To date, however, attempts to capitalize on the potential
for broad-spectrum insect-control agents have met with limited success. Among
the MET-I compounds, there is a very similar, three-dimensional, whole-molecule
shape [22], and the presence of the same substituents on many MET-I acaricides
will likely contribute to their similar metabolic profiles. Moreover, as many of the
major lepidopteran pests possess potent metabolic systems, it is not surprising
that the acaricidal MET-I inhibitors are relatively inactive towards lepidopteran
pests [74]. Yet, the elimination of these metabolically labile sites (e.g., t-butyl or
n-alkyl moieties) by the substitution of aromatics and/or halo-alkyl substituents
may lead to compounds with a broader spectrum and efficacy, though a higher
mammalian toxicity [74] as mammals employ similar metabolic pathways. In the
case of tolfenpyrad, an expanded pest insect spectrum was available compared
to tebufenpyrad and the other MET-I acaricides, while the mammalian selectivity
appeared to be reduced (Table 31.3.1). Likewise, the altered spectrum of flufenerim
may reflect the compound’s reduced susceptibility to metabolism.
Attempts to improve upon fenazaquin have also led to improved activities against
sucking and chewing insects, albeit with an associated increase in mammalian tox-
icity [74]. A pro-insecticidal approach also met with only limited success [76]. Thus,
current chemistries have been unable to strike an optimal balance between the
compounds’ spectra and efficacies, and mammalian and environmental selectivi-
ties. This has resulted in the development of a true broad-spectrum insect-control
agent with a toxicological profile comparable to that of some of the other newer
chemistries, such as indoxacarb, spinetoram, and chlorantraniliprole.
Inhibitors acting on the MET- II and MET-III sites present an interesting contrast
to the MET-I inhibitors. Based on a very limited number of molecules and available
data, compounds in the MET-III group appear to exhibit – at least in terms of
acute rat oral toxicity – a far more favorable toxicological profile than do the MET-I
References 1103
inhibitors discovered to date. Whereas, Complex III and the strobilurin motif have
been widely exploited for the control of fungicide pests, to date only fluacrypyrim
has been developed that is capable of exploiting this motif for the control of mites
or insects.
Clearly, the need for new insect control agents utilizing novel or underutilized
modes of action is ever-present. As such, the MET chain remains an attractive
target site.
Acknowledgments
The authors thank Drs Joel Sheets, Mark Hertlein, Frank Burroughs, Kirk Brewster,
and Cliff Gerwick for their valuable suggestions and discussions, and Mike
Delporte and Carol Foreman for assistance in obtaining the diverse reference
materials used in the writing of this chapter. These studies were supported by Dow
AgroSciences.
References
1. Sparks, T.C. (2004) in The Manage- 9. Holllingworth, R.M. (2001) in Agro-

ment of Diamondback Moth and Other chemical Discovery: Insect Weed and
Crucifer Pests: Proceedings of the 4th Fungal Control (eds D.R. Baker and
International Workshop, Melbourne, N.K. Umetsu), American Chemical So-
Australia (eds N.M. Endersby and P.M. ciety, Washington, DC, pp. 238–255.
Ridland), The Regional Institute Ltd, 10. Walter, H. (2007) in Modern Crop Pro-
Gosford, pp. 37–44. tection Compounds (eds W. Kramer
2. Casida, J.E. and Quistad, G.B. (1998) and U. Schirmer), Wiley-VCH Verlag
Annu. Rev. Entomol., 43, 1–16. GmbH, Weinheim, pp. 528–538.
3. Ware, G.W. and Whiteacre, D.M. 11. Ehrenfreund, J. (2007) in Modern Crop
(2004) The Pesticide Book, 6th edn, Protection Compounds (eds W. Kramer
MeisterPro Information Resources, and U. Schirmer), Wiley-VCH Verlag
Willoughby, OH, p. 487.
GmbH, Weinheim, pp. 867–878.
4. Dekeyser, M.A. (2005) Pest. Manage.
12. Kuhn, D. (2007) in Modern Crop Pro-
Sci., 61, 103–110.
tection Compounds (eds W. Kramer
5. Yu, S.J. (2008) The Toxicology and
and U., Schirmer), Wiley-VCH Verlag
Biochemistry of Insecticides, Academic
GmbH, Weinheim, pp. 879–885.
Press, New York.
13. Fukami, J.-I. (1985) in Comprehensive
6. Earley, F. (2007) in Modern Crop Protec-
tion Compounds (eds W. Kramer and U. Insect Physiology, Biochemistry and Phar-
Schirmer), Wiley-VCH Verlag GmbH, macology, Insect Control, vol. 12 (eds
Weinheim, pp. 433–457. G. Kerkut and L.I. Gilbert), Pergamon,
7. Nelson, D.L. and Cox, M.M. (2005) New York, pp. 291–311.
Lehninger: Principles of Biochemistry, 4th 14. Fukami, J.-I. (1985) in Approaches to
edn, W. H. Freedman and Co., New New Leads for Insecticides (eds H.C.
York. von Keyserlingk, A., Jager, and C. von
8. Lummen, P. (2007) in Insecticides Szczepanski), Springer-Verlag, New
Design Using Advanced Technologies York, pp. 47–69.
(eds I. Ishaaya, R., Nauen, and A.R. 15. Bertschneider, T., Fischer, R., and
Horowitz), Springer-Verlag, Berlin, pp. Nauen, R. (2007) in Modern Crop Pro-
197–215. tection Compounds (eds W. Kramer
and U. Schirmer), Wiley-VCH Verlag and Casida, J.E. (1999) Proc. Natl Acad.
GmbH, Weinheim, pp. 909–925. Sci. USA, 96, 4149–4153.
16. Wachendorff, U., Bruck, E., Elbert, A., 34. Schuler, F. and Casida, J.E. (2001)
Fischer, R., Nauen, R., Stumpf, N., Biochim. Biophys. Acta, 1506, 79–87.
Tiemann, R. (2000) Brighton Crop Prot. 35. Suzuki, H., Kawamura, Y., and Ogura,
Conf. - Pests Dis., 1, 53–59. T. (1986) Patent EP 169,372 A2.
17. Hamaguchi, H., Kajihara, O., and 36. Hirata, K., Kudo, M., Miyake, T.,
Katoh, M. (1995) J. Pestic. Sci., 20 (2), Kawamura, Y., and Ogura, T. (1990)
173–175. Brighton Crop Prot. Conf. - Pests Dis., 1,
18. Motoba, K., Suzuki, T., and Uchida, M. 41–48.
(1992) Pestic. Biochem. Physiol., 43, 37. Taniguchi, M., Hirose, M., Baba, M.,
37–44. Hirata, K., and Ochiai, Y. (1989) US
19. Jewess, P.J. (1994) Biochem. Soc. Trans., Patent 4,877,787.
22, 247–251. 38. Hata, H., Hatta, M., and Iida, Y. (1990)
20. Hollingworth, R.M., Ahammadsahib, Patent JP 02,015,037 A2.
K.I., Gadelhak, G., and McLaughlin, 39. Dreikorn, B.A., Jourdan, G.P., Davis,
J.L. (1994) Biochem. Soc. Trans., 22, L.N., Suhr, R.G., Hall, H.R., and
230–233. Arnold, W.R. (1991) in Synthesis and
21. Lummen, P. (1999) Biochem. Soc. Chemistry of Agrochemicals II (eds
Trans., 27, 602–606. D.R. Baker, J.G. Fenyes, and W.K.
22. Akagi, T., Takahashi, Y., and Sasaki, Moberg), American Chemical Society,
S. (1996) Quant. Struct.–Act. Relat., 15, Washington, DC, pp. 553–565.
290–295. 40. Hackler, R.E., Suhr, R.G., Sheets, J.J.,
23. Davies, M. (ed.) (2005) AGRO Projects– Hatton, C.J., Johnson, P.L., Davis, L.N.,
PEST Projects, PJB Publications, Lon- Edie, R.G., Kaster, S.V., Jourdan, G.P.,
don. Jackson, J.L., and Krumkalns, E.V.
24. Hamaguchi, H., Takaishi, H., (1994) in Advances in the Chemistry of
Ohshima, T., Konno, T., Miyagi, Y., Insect Control III (ed. G.G. Briggs),
Shiraiwa, Y., and Akita, T. (1987) Royal Society of Chemistry, Cambridge,
Patent EP 234,045 A2. pp. 70–84.
25. Kishida, M., Hamaguchi, H., and 41. Hollingworth, R.M. and
Akita, T. (1988) Patent JP 63,267,762 Ahammadsahib, K.I. (1995) in Re-
A2. views in Pesticide Toxicology, vol. 3 (eds
26. Takaishi, H., Hamaguchi, H., R.M. Roe and R.J. Kuhr), Toxicology
Nishimura, A., and Yanaka, K. (1987) Communications Inc., Raleigh, NC, pp.
Patent JP 62,053,969 A2. 277–302.
27. Takaishi, H., Hamaguchi, H., 42. Sparks, T.C., Dreikorn, B.A., Davis,
Nishimura, A., and Yanakaq, K. (1987) N.L., Thompson, G.D., Scott, B.A.,
Patent JP 62,053,970 A2. and Hatton, C. (1993) AGRO 018,
28. Oshima, T., Hamaguchi, H., Shiraiwa, 206th National ACS Meeting, Chicago,
Y., Miyagi, Y., and Akita, T. (1989) August 1993.
Patent JP 01,013,086 A2. 43. Wood, E., Latli, B., and Casida, J.E.
29. Kishida, M., Hamaguchi, H., and (1996) Pestic. Biochem. Physiol., 54,
Akita, T. (1988) Patent JP 63,267,745. 153–145.
30. Fujihira, A., Kodaira, T., and Yabutani, 44. Dreikorn, B.A., Suhr, R.G., Jourdan,
K. (1989) Patent JP 01,313,453 A2. G.P., and Wright, I.G. (1989) Patent EP
31. Hirata, K., Kawamura, Y., Kudo, M., 326,329 A2.
and Igarashi, H. (1995) J. Pestic. Sci., 45. Tai, J.J., Ringer, J.W., Krumel, K.L.,
20 (2), 177–179, 213–222. and Krauss, R.C. (1992) Patent EP
32. Schuler, F. and Casida, J.E. (2001) Pest. 537,600 A1.
Manage. Sci., 57, 932–940. 46. Okada, I., Okui, S., Takahashi, T., and
33. Schluer, F., Yano, T., Di Benardo, S., Fukuchi, T. (1991) J. Pestic. Sci., 16,
Yagi, T., Yankivskaya, V., Singer, T.P., 623–629.
References 1105
47. Okada, I., Okui, S., Wada, M., Fukuchi, 64. Ataka, K. and Imaoka, K. (1990) Patent
T., Yoshiya, K., and Takahashi, Y. EP 357,310 A1.
(1998) in Synthesis and Chemistry of 65. Obata, T., Fujii, K., Ooka, A., and
Agrochemicals V (eds D.R. Baker, Yamanaka, Y. (1995) European Patent
J.G. Fenyes, G.S. Basarab, and D.A. Application EP 665,225, 1995/0,802.
Hunt), American Chemical Society, 66. Fugii, K. and Nakamoto, Y. (1999)
Washington, DC, pp. 168–177. Patent JP 11,012,253 A2.
48. Okada, I. and Fukuchi, T. (2000) J. Pes- 67. Ghanim, M., Lebedev, G.,
tic. Sci., 25 (3), 310–320. Konstesdalov, S., and Ishaaya, I. (2011)
49. Okada, I., Okui, S., Fukuchi, T., and J. Agric. Food Chem., 59, 2839–2844.
Yoshiya, K. (1999) J. Pestic. Sci., 24 (4), 68. Yoshida, H., Omori, K., Fuse, K.,
393–396. Morita, K., Onduka, Y., and Yokota, N.
50. Okada, I., Okui, S., Takahashi, Y., (1999) Patent WO 99/31,044 A1.
and Fukuchi, T. (1990) US Patent 69. Yoshida, H., Omori, K., Fuse, K.,
4,950,668. Morita, K., Onzuka, Y., and Yokota, N.
51. Okada, I., Okui, S., Sekine, M., (2000) Patent JP 2000/119,234 A2.
Takahashi, Y., and Fukuchi, T. (1992) J. 70. Omori, K., Onzuka, Y., Fuse, K.,
Pestic. Sci., 17, 69–73. Morita, K., and Yokota, N. (2000)
52. Okada, I., Okui, S., Tanaka, T., Patent JP 2000/178,259 A2.
Hosakawa, A., Kyomura, N., Fukuchi, 71. Omori, K., Onzuka, Y., Fuse, K.,
T., and Takahashi, Y. (1994) J. Pestic. Morita, K., and Yokota, N. (2000)
Sci., 19, 317–320. Patent JP 2000/159,752 A2.
53. Okada, I., Okui, S., Wada, K., and 72. Fujii, K., Shikita, S., and Nakamoto, Y.
Takahashi, Y. (1996) J. Pestic. Sci., 21, (2002) Patent WO 2002/064,538 A1.
305–310. 73. Johnson, P.L., Hackler, R.E., Sheets,
54. Nakagami, K., Yokoi, S., Nishimura, K., J.J., Worden, T., and Gifford, J. (1998)
Nagai, S., Honda, T., Oda, K., Fujii, K., in Synthesis and Chemistry of Agrochem-
Kobayashi, R., and Kojima, M. (1978) icals V (eds D.R. Baker, J.G. Fenyes,
Patent DE 78-2,824,768 19,781,214. G.S. Basarab, and D.A. Hunt), Ameri-
55. Tsuji, H., Yamamoto, S., Nakagami, K., can Chemical Society, Washington, DC,
Honda, T., Fujii, K., Obata, T., Kokima, pp. 136–146.
M., Akiyoshi, Y., and Kobayashi, T. 74. Hackler, R.E., Hatton, C.J., Hertlein,
(1982) Eur. Patent Appl. EP 57,440 A1 M.B., Johnson, P.L., Owen, J.M.,
19,820,811. Renga, J.M., Sheets, J.J., Sparks, T.C.,
56. Matsumoto, K., Yokoi, S., Fujii, K., and and Suhr, R.G. (1998) in Synthesis
Akiyoshi, Y. (1986) Patent EP 196,524 and Chemistry of Agrochemicals V (eds
A2. D.R. Baker, J.G. Fenyes, G.S. Basarab,
57. Ataka, K. and Asada, H. (1991) Patent and D.A. Hunt), American Chem-
JP 03,005,466 A2. ical Society, Washington, DC, pp.
58. Obata, T., Fujii, K., Yoshiya, H., 147–156.
Tsutsumiuchi, K., and Yoshioka, H. 75. Samaritoni, J.G., Arndt, L., Bruce,
(1992) Pestic. Sci., 34, 133–138. T.J., Dripps, J.E., Gifford, J., Hatton,
59. Lummen, P. (1998) Biochim. Biophys. C.J., Hendrix, W.H., Schoonover,
Acta, 1364, 287–296. J.R., Johnson, G.W., Hegde, V.B., and
60. Okun, J.G., Lummen, P., and Thornburgh, S. (1997) J. Agric. Food.
Brandt, U. (1999) J. Biol. Chem., 274, Chem., 45, 1920–1930.
2625–2630. 76. Sheets, J.J., Schmidt, A., Samaritoni,
61. Ataka, K. and Kohno, M. (1989) Patent J.G., and Gifford, J.M. (1997) J. Agric.
EP 357,348 A2. Food. Chem., 45, 4826–4832.
62. Ataka, K. and Kohno, M. (1996) Patent 77. Lummen, P., Preuß, R., and Schaper,
JP 08,198,858 A2. W. (1994) in Eighth IUPAC Interna-
63. Obata, T., Fujii, K., Yoshiya, H., and tional Congress of Pesticide Chemistry,
Tsutsumiuchi, K. (1991) Patent JP American Chemical Society, Washing-
03,161,485. ton, DC, p. 235 (Abstract 207).
78. Henrie, R.N. II, Cullen, T., Dugan, B., 88. Lisse, D., Huang, L.S., Gasteiger, T.,
Zhang, L., Deng, Y., Simpson, S.F., Berry, E.A., and Lummen, P. (2010)
Black, B., Schuler, F., and Yu, S.J. Biochim. Biophys. Acta – Bioenerg., 1797
(2007) in Synthesis and Chemistry of (Suppl. 1), 18–19.
Agrochemicals VII (eds J.W. Lyga and 89. Joppien, H., Puttner, R., and Winter,
G. Theodoridis), American Chemical E. (1983) in 10th International Congress
Society, Washington, DC, pp. 83–92. of Plant Protection 1983, vol. 1, British
79. Nakahira, K., Takii, S., and Murakami, Crop Protection Council, London, pp.
H. (2006) Mode of action of a novel 392–399.
miticide cyenopyrafen (ISO proposed, 90. Bartlett, D.W., Clough, J.M., Godwin,
NC-512) – inhibitory characteristics of J.R., Hall, A.A., Hamer, M., and
complex II of the respiratory chain. Parr-Dobrzanski, B. (2002) Pest. Man-
11th IUPAC International Congress of age. Sci., 58, 649–662.
Pesticide Chemistry, Book of Abstracts 91. Koura, Y., Kinoshita, S., Takasuka, K.,
(2), August Kobe, Japan, Abstract Koura, S., Osaki, N., Matsumoto, S.,
II-1-i-23B. and Miyoshi, H. (1998) J. Pestic. Sci.,
80. Casida, J.E. (2009) Chem. Res. Toxicol., 23, 18–21.
22, 609–619. 92. Sonobe, A., Tanaka, T., and Suganuma,
81. Suzuki, H. (2008) Patent JP H. (1999) Patent JP 11,035,517 A2.
2008/208,047; A 2008/0,911. 93. Suganuma, H. and Fujimura, H.
82. Fukuda, K., Kondo, Y., Tanaka, N., (1989) Patent EP 330,186 A2.
Suzuki, H., Ohnari, M., and Nishio, K. 94. Beautement, K., Clough, J.M., de
(2004) PCT International Application Fraine, P.J., and Godfrey, C.R.A. (1991)
WO 2004/087,674 A1 2004/1,014. Pestic. Sci., 31, 499–519.
83. Sasama, Y., Takahashi, N., Tanji, I., 95. Chai, B.S., Liu, C.L., Li, H.C., He,
Kobayashi, M., Ishii, N., Hayashi, N., X.-M., Luo, Y.M., Huang, G., Zhang,
Miyata, T., Imai, T., Andoh, A., and H., and Chang, J.B. (2010) Pest Man-
Umetsu, N. (2006) Biological proper- age. Sci., 66, 1208–1214.
ties and field efficacy of cyflumetofen, 96. Li, M., Liu, C.L., Li, L., Yang, H., Li,
11th IUPAC International Congress of Z.N., Zhang, H., and Li, Z.M. (2010)
Pesticide Chemistry, Book of Abstracts Pest. Manage. Sci., 66, 107–112.
(2), August Kobe, Japan, Abstract 97. Agranova. Available at:
II-1-i-22A. http://www.agranova.co.uk (accessed
84. Sasama, Y., Takahashi, N., Ishii, N., 11 December 2010).
Hayashi, N., Miyata, T., Imai, T., and 98. Liu, A., Wang, X., Chen, C., Pei, H.,
Andoh, A. (2007) XVI International Mao, C., Wang, Y., He, H., Huang, L.,
Plant Protection Congress Proceedings, Liu, X., Hu, Z., Ou, X., Huang, M.,
vol. 1, The British Crop Protection and Yao, J. (2009) Pest Manage. Sci., 65,
Council, pp. 46–51. 229–234.
85. Takahashi, N., Gotoda, S., Ishii, N., 99. Kirstgen, R., Oberdorf, K., Schuetz, F.,
Sasama, Y., Imai, T., and Umetsu, N. Theobald, H., and Harries, V. (1996)
(2006) Discovery, synthesis and acari- Patent WO 96/16,047 A1.
cidal activity of cyflumetofen and its 100. Takase, M., Miyazawa, Y., and
derivatives. 11th IUPAC International Tsubokura, S. (1999) Patent WO
Congress of Pesticide Chemistry, Book 99/44,969 A1.
of Abstracts (2), August Kobe, Japan, 101. Sagae, T. (2001) Patent JP
Abstract I-1-i-21C. 2001/220,382 A2.
86. Nakagawa, H. and Hayashi, M. (2004) 102. Miyazawa, Y., Sagae, T., Ishii, H.,
PCT International Application WO Yazaki, H., Funabora, M., Takase,
2004/007,433 A1 2004/0,122. M., Iiyoshi, Y., Yamazaki, S., and
87. Agrow Intelligence. Available at: Kawahara, N. (2000) Patent WO
www/agrow.com/AgrowIntelligence/ 2000/040,537 A1.
IKA2002-267079 (accessed 18 Novem- 103. Yahihara, T., Suzuki, T., and Ozaki, A.
ber 2010). (2001) Patent JP 2001/181,234 A2.
References 1107
104. Milio, J.F., Koehler, P.G., and 118. Anonymous (2004) Evaluation Report –
Patterson, R.S. (1986) J. Econ. Entomol., Tolfenpyrad. Pesticides Expert Commit-
79, 1280–1286. tee, Food Safety Commission, Japan,
105. Hollingshaus, J.G. (1987) Pestic. p. 37.
Biochem. Physiol., 27, 61–70. 119. Kinoshita, S., Koura, Y., Kariya, H.,
106. Lees, R.G. and Tomcufcik, A.S. (1981) Ohsaki, N., and Watanabe, T. (1999)
US Patent 4,262,122. Pestic. Sci., 55, 659–660.
107. Cortese, N.A. Jr and Gastrock, W.H. 120. Whalon, M.E., Mota-Sanchez, D., and
(1985) US Patent 4,521,629. Hollingworth, R.M. (2008) in Global
108. Dekeyser, M.A. (2007) in Modern Pesticide Resistance in Arthropods (eds
Crop Protection Compound, vol. 3 M.E. Whalon, D. Mota-Sanchez, and
(eds W. Kramer and U. Schirmer), R.M. Hollingworth), CABI, Cambridge,
Wiley-VCH Verlag GmbH, Weinheim, MA, pp. 5–21.
pp. 1103–1110. 121. Sterk, G. (1992) in Insecticides: Mech-
109. Van Leeuwen, T., Tirry, L., and Nauen, anism of Action and Resistance (eds
R. (2006) Insect Biochem. Molec. Biol., D. Otto and B. Weber), Intercept Ltd,
36, 869–877. Andover, pp. 427–432.
110. Van Nieuwenhuyse, P., Van Leeuwen, 122. Leonard, P.K. (1997) Pestic. Sci., 51,
T., Khajehali, J., Vanholme, B., and 387–390.
Tirry, L. (2009) Pest Manage. Sci., 65, 123. Nauen, R., Stumpf, N., Elbert, A.,
404–412. Zebitz, C.P.W., and Kraus, W. (2001)
Pest. Manage. Sci., 57, 253–261.
111. Van Leeuwen, T., Vontas, J.,
124. Stumpf, N. and Nauen, R. (2001) J.
Tsagkarakou, A., and Tirry, L. (2009) in
Econ. Entomol., 94, 1577–1583.
Biorational Control of Arthropod Pests:
125. Kim, Y.-J., Lee, S.-H., Lee, S.-W., and
Application and Resistance Management
Ahn, Y.-J. (2004) Pest. Manage. Sci., 60,
(eds A.R. Horowitz and Ishaaya I.),
1001–1006.
Springer, New York, pp. 347–393.
126. Herron, G.A. and Rophail, J. (1998)
112. Van Leeuwen, T., Vontas, J.,
Exp. Appl. Acarol., 22, 633–641.
Tsagkarakou, A., Dermauw, W., and
127. Goka, K. (1998) Exp. Appl. Acarol., 22,
Tirry, L. (2010) Insect Biochem. Mol.
699–708.
Biol., 40, 563–572.
128. Devine, G.J., Barber, M., and Denholm,
113. Motoba, K., Nishizawa, H., Suzuki, I. (2001) Pest Manage. Sci., 57,
T., Hamaguchi, H., and Uchida, M. 443–448.
(1992) Biosci. Biotechnol. Biochem., 56, 129. Gorman, K., Devine, G.J., and
366–367. Denholm, I. (2000) Brighton Crop
114. Nishizawa, H., Motoba, K., Suzuki, Prot. Conf. - Pests Dis., 1, 459–464.
T., Ohshima, T., and Hamaguchi, H. 130. Gorman, K., Hewitt, F., Denholm, I.,
(1993) J. Pestic. Sci., 18, 59–66. and Devine, G.J. (2001) Pest. Manage.
115. Roberts, T. and Hutson, D. (eds) (1999) Sci., 58, 123–130.
Metabolic Pathways of Agrochemicals, 131. Sato, M.E., Miyata, T., Kawai, A., and
Part 2: Insecticides and Fungicides, The Nakano, O. (2000) Appl. Entomol. Zool.,
Royal Society of Chemistry, Cambridge, 33, 393–399.
pp. 747–751. 132. Pree, D.J., Bittner, L.A., and Whitty,
116. Sparks, T.C., Sheets, J.J., Skomp, J.R., K. (2002) Exp. Appl. Acarol., 27,
Worden, T.V., Larson, L.L., Bellows, 181–193.
D., Thibault, S., and Wally, L. (1997) 133. Van Leeuwen, T., Van Pottelberge, S.,
Proceedings of the 1997 Beltwide Cot- and Tirry, L. (2005) Pest. Manage. Sci.,
ton Production Conference, National 61, 499–507.
Cotton Council, Memphis, TN, pp. 134. Richter, P. and Otto, D. (1992) in In-
1259–1264. secticides: Mechanism of Action and
117. Ogawa, K. and Ihashi, Y. (1994) J. Resistance (eds D. Otto and B. Weber),
Pestic. Sci., 19, 169–179. Intercept Ltd, Andover, pp. 433–441.
135. Kyomura, N., Kukuchi, T., Kohyama, and Waltersdorfer, A. (2002) Insectici-
Y., and Motojime, S. (1990) Brighton dal aminopyrimidines: broad spectrum
Crop Prot. Conf. - Pests Dis., 1, 55–62. foliar insecticides and acaricides. 10th
136. Nonaka, N. (2003) Agrochem. Jpn, 83, IUPAC International Congress on the
17–19. Chemistry of Crop Protection, Basel,
137. Schaper, W., Braun, R., Abstract 3a27.
Jakobi, H., Klein, R., Knauf, W., 138. Tomlim, C.D.S. (2010) The Pesti-
Lummen, P., Markl, M., cide Manual, 15th Ed. British Crop
Preuss, Rwarning., Stark, H., Protection Council, Alton, UK
31.4
Inhibitors of Lipid Synthesis: Acetyl-CoA-Carboxylase Inhibitors
31.4.1
Introduction
Acetyl-CoA-carboxylase (ACCase), a biotinylated enzyme, plays a fundamental role

in fatty acid metabolism by catalyzing the carboxylation of acetyl-CoA.
In eukaryotes and prokaryotes, ACCase is a key enzyme in fatty acid biosynthesis
[1]. The reaction product, malonyl-CoA, is both an intermediate in the de novo
synthesis of primary fatty acids and also a substrate in the formation of long-chain
fatty acids and flavonoids in plants [2, 3]. The aryloxyphenoxypropionates (APPs)
and cyclohexandiones (CHDs) are two chemical classes of molecules that selectively
inhibit homomeric, chloroplastic ACCase from grasses [4, 5]; consequently, these
materials have become used worldwide as postemergent herbicides to control
grassy weeds.
31.4.2
The Cyclic Ketoenols, Spirodiclofen, and Spiromesifen: A New Generation
of ACCase Inhibitors
During a synthesis program in the field of protoporphyrinogen-IX-oxidase (PPO)

inhibitors, a group of hydantoins of type 1 was synthesized which was found to
demonstrate a strong activity against broad-leaved weeds (Figure 31.4.1). However,
owing to strong competitor activities in this field, with overlapping patent applica-
tions, the decision was taken to substitute the central nitrogen atom by a carbon
atom, and this led to similar (but not yet claimed) C-aryl compounds. The result of
these syntheses were the tetramic acids 2 and 3 (Figure 31.4.1).
Interestingly, the herbicidal activity of the 2,4-dichloro derivative 3 switched
from the original activity of compound 1 to a weak activity against grassy weeds
where, physiologically, the symptoms were similar to those occurring with APPs
and CHDs. Subsequently, following intensive biochemical investigations based
on a previous report [6], it became clear that these new compounds represented
a new class of herbicides which acted as ACCase inhibitors [7]. After various
O F OR
Xn
N Y
N N
O X O
1 X = OAlk, S-Alk 2 R = H; Xn = 4-Cl
Y = Me, CN 3 R = H; Xn = 2,4-Cl2
4a R = CO-CMe3; Xn = 2,4-Cl2
OR Me OR Me
Me
Me
Me Me
N HN
O Me O Me
4i R = CO-t Bu 5a R = H
5b R = CO2-CH(Me)Et
OR Me OR
Me Cl
O O
O Me O Cl
6a R = H 7a R = H
6b R = CO-t Bu
Et Me Me
Me
O Me
Me O
O
O Me
Cl
O Me
O
O Cl
O Me
7f (Spirodiclofen) 8a (Spiromesifen)
3-(2,4-dichlorophenyl)- 3-mesityl-2-oxo-1-
2-oxo-1-oxaspiro[4.5]- oxaspiro[4,4]non-3-en-
dec-3-en-4-yl- 4-yl-3,3-dimethylbutanoate
2,2-dimethylbutyrate
logPow 5.83 logPow 4.55

melt. point 95°C melt. point 98°C
water sol. 0.05 mg ml−1 water sol. 0.05 mg ml−1
LD50 (rat oral) >2500mg/kg LD50 (rat oral) >2500mg/kg
Figure 31.4.1 Discovery of spirodiclofen (7f) and spiromesifen (8a).

attempts at chemical optimization, it proved even more surprising that acylated

derivatives (e.g., compound 4a; Figure 31.4.1) showed a weak acaricidal efficacy
against the spider mite Tetranychus urticae. Consequently, in an effort to improve
this activity many different acylated compounds and aromatic substitution patterns
were screened (Table 31.4.1).
Although the 2,4,6-trimethylphenyl (mesityl) compound 4i [8] showed an im-
proved acaricidal potential against T. urticae, this proved unsatisfactory under
field conditions with regards to another important mite species, Panonychus ulmi.
Hence, in order to increase the efficacy against P. ulmi, a broad synthesis and
screening of substituents on positions 1 and 5 of the lead structure was carried out.
The monocyclic 5,5-dimethyl tetramic acid derivatives 5a and 5b [9] (Figure 31.4.1)
showed good and broad activities against many important mite species under field
conditions, but unfortunately caused some severe phytotoxic effects in certain crops.
In an attempt to overcome this problem, the core structure of the molecules was
modified by switching to other cyclic ketoenol subclasses, such as tetronic acids,
pyrazolidin-3,5-diones, 4-hydroxy-pyrones, and 6-hydroxy-thiazinones.
Only the synthesis of spirocyclic tetronic acid analogs 6a and especially acylated
compounds such as the pivaloyl derivative 6b [10] revealed excellent acaricidal
performance with improved plant compatibility. In some sensitive crops, however,
Table 31.4.1 Acaricidal efficacy of some pivaloyl-substituted

bicyclic tetramic acids (4), depending on the phenyl
substitution pattern.
Me O
Me
O
Me
N Xn
4 O
Entry Xn Efficacy against T. urticaea
4a 2,4-Cl2 +
4b 2-Cl, 6-F ++
4c 2,6-Cl2 +
4d 2,4,6-Cl3 +
4e 2-Cl,4-CF3 ,6-F +
4f 2,6-Cl2 ,4-CF3 ++
4g 2,4-Me2 ++
4h 2-Me,4-t-Bu +
4i 2,4,6-Me3 +
4j 2,4,5-Me3 +
4k 2,3,4,6-Me4 ++
a
+: weak; ++: moderate; +++: good; ++++: very good.
such as stone fruits or grapes, phytotoxic effects were still observed under special
conditions. Consequently, in a ‘‘back to the roots’’ approach, the mesityl substitution
pattern was changed back to the 2,4-dichloro type examined earlier in the program.
This led to the tetronic acid 7 as a template that combined good acaricidal activity
against many important mite species, as well as good plant compatibility in all rel-
evant crops. These properties were ‘‘fine-tuned’’ by scanning a large set of different
acylating reagents (Table 31.4.2); subsequently, the optimum was achieved with
the 2,2-dimethylbutyric acid derivative, which was selected for development under
®
the common name spirodiclofen (7f) (BAJ2740, trade name: Envidor ) [11, 12].
Somewhat surprisingly, during the acaricidal optimization process a good efficacy
was observed against the white fly species Bemisa tabaci with 6b in some field trials.
However, during the optimization process it transpired that, in particular, acylated
3-mesityl tetronic acids with spiro-cyclopentyl or spiro-cyclohexyl rings in position 5
were highly active against spider mites, while showing simultaneously an excellent
performance against B. tabaci.
The fine-tuning process regarding activity, pest spectrum, toxicology, and plant
compatibility finally led to the 3,3-dimethyl-1-butyric acid derivative spiromesifen
®
(8a) (BSN2060, trade name Oberon ) [13] (Table 31.4.3). The physico-chemical
properties of the new products 7f and 8a are listed in Figure 31.4.1.
31.4.3
Synthesis of Spirodiclofen and Spiromesifen
The first central intermediate of the spirodiclofen (7f) synthesis is ethyl

1-hydroxycyclohexanecarboxylate (9), which is synthesized from cyclohexanone
by hydrocyanic acid addition to the cyanohydrin, followed by saponification
and esterification. The second central intermediate is 2,4-dichloro-phenylacetyl
chloride (10), which is synthesized from 2,4-dichlorobenzyl chloride by cyanide
exchange, saponification, and acid chloride preparation.
Table 31.4.2 Structure–activity relationships of different

acylated compounds 7b–7h of tetronic acid (7a) against T.
urticae and P. ulmi.
Entry R Efficacy against T. urticaea Efficacy against P. ulmia
7a (enol) H ++ +++
7b CO-Me +++ +++
7c CO-n-Pr ++ ++
7d CO-i-Pr + +
7e CO-t-Bu ++++ Not tested
7f (Spirodiclofen) CO-CMe2 -Et +++++ +++++
7g CO-CMe2 -n-Pr ++++ ++++
7h CO-CMe2 -i-Pr +++ +++
a
: +: weak; ++: moderate; +++: good; ++++: very good; +++++: excellent.
Table 31.4.3 Structure–activity relationships of acylated

five- and six-membered spirocyclic mesityl tetronic acids
against B. tabaci.
Entry Ring size R Efficacy against B. tabacia
8a 5 t-Bu-CH2 ++++(+)
8b 5 i-Bu ++(+)
8c 5 t-Bu +++
8d 5 i-Pr +++(+)
6b 6 t-Bu ++++
6c 6 i-Pr +++
6d 6 t-Bu-CH2 +++(+)
a
: +: weak; ++: moderate; +++: good; ++++: very good; +++++: excellent.
The combination of these two building blocks leads in a convergent manner to

the ‘‘diester’’ 11, which is treated with a base (e.g., potassium-tert-butylate; KOtBu)
to form the tetronic acid 6. The final O-acylation with 2,2-dimethyl-butyryl chloride
leads to 7f (Scheme 31.4.1).
Several possibilities for a large-scale synthesis of mesitylacetic acid (12), a
central building block in the synthesis of spiromesifen (8a), were examined
(Scheme 31.4.2). By using the classical standard route, mesitylene (13) is trans-
ferred into mesityl acetonitrile (14) via chloromethylation and cyanide exchange,
which is then saponified to the aryl acetic acid 12. Another route examined was the
(1) NaCN (1) NaCN

(2) HCI
CO2Et CI (2) H2SO4/H2O
(3) EtOH, p-TsOH CI (3) SOCI2 CI
+ CI
O OH O CI
9 CI
10
CO2Et
CI
O
11 CI
O
Et
KOtBu Me
O
Et Me
OH Me
O O
Me
CI CI
O CI
Et3N O
O CI
6 7f O CI
Scheme 31.4.1 Synthesis of spirodiclofen (7f ).

Me
Me
13
Me
BUO2C OSO2Me
(1) HCHO, HCI
(2) NaCN CI CI AICI3
AICI3
Me Me Me
CI Me Me
Me
MeO
NC
Me 15 Me
Me 14 O 16
(1) O3, MeOH

(1) NaOH (2) AcOH/H2O2
NaOH
(2) HCI
Me
Me
HO
O Me 12
Scheme 31.4.2 Synthesis of mesityl acetic acid (12).
Friedel–Crafts alkylation of 13 with 1,3-dichloro-propene to the adduct 15, which is

ozonolyzed to the corresponding aldehyde in the form of its dimethyl acetal and then
further oxidized with hydrogen peroxide under acidic conditions to mesityl acetic
acid (12) [14]. A straightforward route is the AlCl3 mediated Friedel–Crafts alkyla-
tion of mesitylene (13) with the C2-building block butyl[(methylsulfonyl)-oxy]acetate
yielding 16, which is then saponified to the free acid 12 [15].
The further route to spiromesifen (8a) is similar to the above-described synthesis
of spirodiclofen (7f) (Scheme 31.4.3) [16]. Acylation of the cyclopentyl hydroxyester
17 (synthesized from cyclopentanone via the classical cyanohydrin route in three
steps) with mesitylacetyl chloride (18) leads to the intermediate 19, which is cyclized
to the tetronic acid 20 using, for example, KOtBu in N,N-dimethylformamide
(DMF).
Several syntheses for the 3,3-dimethylbutyric acid (23) (Scheme 31.4.3), used as
an acyl side chain in spiromesifen (8a), were investigated. One interesting route
starts from trimethylpyruvic acid (21), which is transferred in a Wolff–Kishner
reaction to the corresponding hydrazone 22 using hydrazine hydrate (optionally
in a solvent, e.g., triethylene glycol), followed by a reductive cleavage with a base
(e.g., KOH) at elevated temperatures. The final acylation of the enol 20 with
3,3-dimethylbutyryl chloride leads to spiromesifen (8a). The main process may also
be conveniently carried out in a single-step/one-pot procedure starting from the
intermediates 9 and 10 for spirodiclofen (7f), and from 17 and 18 for spiromesifen
(8a) [17].
O Me
Me
(1) NaCN Me
(2) HCI (see Scheme 28.4.2)
(3) EtOH, p-TsOH
Me
CO2Et
Me
CI
17 OH
O Me 18
Me
CO2Et
Me
O
19 O Me
Me Me
KOt Bu
Me Me
Me
Me O
O
OH Me
O Me
CI
Me
O Me
O
20 O Me SOCI2 8a
O Me
Me Me Me Me Me Me
Me Me Me
N2H4 O KOH
O O
O N
OH OH NH2 OH
21 22 23
Scheme 31.4.3 Synthesis of spiromesifen (8a).
31.4.4
Biology and Mode of Action
Whiteflies (e.g., B. tabaci) and spider mites (e.g., T. urticae), which are among
the most serious sucking pests in many cropping systems, have developed a
high degree of resistance to many chemical classes of the currently commercially
available insecticides and acaricides (see Refs [18–20] and references cited therein).
Consequently, it is imperative that new active ingredients with novel modes of

action should be developed to be included in resistance management programs for
the control of these pests, in an efficient manner.
The symptomatology of poisoning observed with the new tetronic acid derivatives
indicated a new biochemical mode of action not yet observed with any commercially
available acaricide or insecticide. Although the compounds showed no signs of
neurotoxic activity, they acted on the development of both mites and whitefly. In the
case of spirodiclofen (7f), the compound exhibited activity against all developmental
stages of the mites, including the eggs, but did not kill the adult mites. Spirodiclofen
also reduced the fecundity of the female adults, with the result that the number
of eggs laid was greatly reduced. Moreover, the eggs of female mites exposed to
sublethal doses of spirodiclofen were infertile. Notably, the lipid content in treated
female adults of T. urticae was seen to be significantly decreased, which suggested
that the compound had interfered with lipid biosynthesis (Figure 31.4.2). These
findings were in line with the slightly delayed onset of activity of these compounds.
In contrast, both compounds demonstrated an excellent longlasting effect and
good plant compatibility under field conditions. The biological profile of spirod-
iclofen (7f) has recently been reviewed [21, 22]. Both, spirodiclofen (7f) and
spiromesifen (8a) were tested extensively on several strains of T. urticae, collected
worldwide, that showed a high level of resistance to established commercial acari-
cides. Both compounds were shown to perform with outstanding activity [13, 18, 23].
Similar to spirodiclofen (7f), the second compound in this class – spiromesifen
(8a) – is also particularly active against insect juvenile stages. However, it also has a
strong effect on the fecundity of mite and whitefly adults in a dose-dependent man-
ner by transovarial effects. Typically, spiromesifen demonstrates ovicidal effects in
mites, whereas egg hatch in whiteflies was markedly reduced through transovarial
effects upon the pre-exposure of female adults to the compound. Spiromesifen
was also shown to be extremely effective against Tetranychus strains that were
resistant to abamectin, pyridaben, fenpyroximate, hexythiazox, and clofentezine
(Table 31.4.4), and against whiteflies resistant to pyrethroids, organophosphates,
carbamates, cyclodienes, and neonicotinoids [13, 24].
18 Untreated
16
Lipid, μg/10 Spider mites
Treated
14
12
10
8
6
4
2
0
2h 2d 5d
Figure 31.4.2 Lipid decrease in spirodiclofen (7f)-treated spider mites.

Table 31.4.4Resistance factors of several Tetranychus strains

against commercial acaricides and spiromesifen (8a).
Compound Strain
NL-00 Akita UK-99 AU
Abamectin 54 3 – 2
Pyridaben 22 2000 860 13
Fenpyroximate – 1400 74 5
Hexythiazox – 4 – 1100
Clofentezine – 4 – >770
Spiromesifen 4 1 1 3
Field simulation studies have also revealed that spiromesifen is a valuable tool in
the control of pyriproxyfen-resistant whiteflies (Figure 31.4.3). In particular, when
combined with neonicotinoid (chloronicotinyl) insecticides such as imidacloprid,
spiromesifen was considered a new valuable component in resistance management
strategies for whitefly control [14].
31.4.5
Development, Registration, and Integrated Pest Management Suitability
of EnvidorTM and OberonTM
®
Envidor , with the active ingredient spirodiclofen (7f), is a new nonsystemic foliar
acaricide that provides excellent longlasting activity and is effective in early to
late season applications. Currently, spirodiclofen is undergoing development for
worldwide use in pome fruit, stone fruit, citrus, grapes, almonds, and nuts, and
has demonstrated good to excellent efficacy against all economically important
mite species in these crops. The performance of spirodiclofen is at least equal or
superior to acaricidal standards of different chemical classes, such as abamectin,
pyradaben, and hexythiazox. The recommended application rates are in the range
of 0.0048 to 0.0144% a.i. l−1 spray solution, depending on the crop and pest species.
Spirodiclofen may also be used to control some insect pests, including Psylla piri
®
and Lepidosaphes ulmi. In addition to the worldwide trade name Envidor , other
® ®
trade names such as Ecomite (Japan, pome fruit), Daniemon (Japan, citrus),
® ®
and Sinawi (Korea) have been employed for spirodiclofen. Envidor was first
launched in Korea 2002, and has subsequently been registered in several important
countries including Brazil, USA, Japan, China, Spain, Germany, and Turkey.
®
Oberon , which contains the active ingredient spiromesifen (8a), is a new
foliar contact insecticide–acaricide that has been developed worldwide for use on
vegetables, fruits, cotton, corn, beans, tea, and some ornamentals. Spiromesifen
provides good to excellent control of whiteflies (Bemisia spp., Trialeurodes spp.), and
is also highly effective against mites, including spider mites such as Tetranychus
spp., Tarsonemid mites (e.g., broad mite) and Eriophyd mites (e.g., tomato russet
Imidacloprid .v. spiromesifen

ESP99 (imidacloprid resistant)
5000
Imidacloprid full rate
Spiromesifen full rate
4000
Total whiteflies
3000
2000
1000
0
−2 8 18 28 38 48 58
Imidacloprid Spiromesifen
treatment (foliar)
(a) (drench)
Pyriproxyfen .v. spiromesifen

hot carmel (pyriproxyfen resistant)
5000
Pyriproxyfen full rate
Spiromesifen full rate
4000
Total whiteflies
3000
2000
1000
0
−2 8 18 28 38 48 58
(b) Foliar treatment
Figure 31.4.3 Efficacy of spiromesifen 8a against whitefly

strains resistant to (a) imidacloprid and (b) pyriproxyfen.
Reproduced from [25].
®
mite). In recent field trials conducted in the USA and Central America, Oberon also
proved to be highly effective against tomato and pepper psyllids. For spiromesifen,
the recommended application rates are in the range of 50 to 280 g a.i. ha−1 or
0.0072–0.018% a.i.l−1 , depending on the crop and the pest species being treated.
The new mode of action and lack of cross-resistance to commercial products
supports the use of spiromesifen as a valuable tool for mite and whitefly resistance
®
management [26]. Following its initial registration in Indonesia in 2003, Oberon
was subsequently registered in other important countries such as USA, Brazil, and
Mexico.
With many studies having been conducted with both spirodiclofen and spirome-
sifen against beneficial insects, predatory mites, and spiders, it was concluded that
these compounds should be considered as very safe towards beneficial insects, on
the basis of results obtained from both laboratory and field tests. No permanent
damaging effects were observed on beneficial bugs, lacewings, and parasitoids.
Such excellent selectivity offers the possibility of combining these compounds with
beneficial; as a consequence, both can be recommended for use in integrated pest
management (IPM) programs [27].
31.4.6
Discovery of Spirotetramat
Parallel to the discovery of acaricidally active tetronic acid derivatives, attempts were
also made to improve both the acaricidal and herbicidal efficacies in the subclass
of tetramic acid derivatives. Starting with 1-amino-4-methyl-cyclohexanecarboxylic
acid methyl ester, prepared by the Bucherer–Bergs reaction [27], the tetramic acid
24a and its O-acetyl derivative 24b were synthesized (Figure 31.4.4); subsequently,
a significant improvement was found in herbicidal efficacy compared to that of
unsubstituted spirocyclic analogs. An excellent acaricidal performance was also
registered in the case of 24b and, surprisingly, a moderate to good efficacy against
the peach–potato aphid Myzus persicae that, previously, had never been noted.
Further evaluation in this area led to a series of alkoxy-substituted spirocyclic
tetramic acid derivatives. For this, an alternative synthesis route was utilized,
starting from 4-methoxy-1-aminocyclohexanecarbonitrile (25), which was prepared
by a Strecker synthesis [27], in order to reduce the number of synthesis steps [28]
in the preparation of compounds 28a and 28b (Scheme 31.4.4).
The shortage of 28b for biological testing led to larger amounts being required.
During the work-up of these compounds, the minor isomer (which was identified
as the cis-compound 29; Scheme 31.4.4) demonstrated a very good control of M.
persicae, the efficacy of which was very close to that of the best aphicidal standard,
imidacloprid. Unfortunately, however, these early favorable results were marred by
an elevated herbicidal efficacy against the crops.
R
Me O Me
Me
N
H
O Me
24 a R = H Figure 31.4.4 Structure of the tetramic acid (24a)

24 b R = CO-Me and its O-acetyl derivative (24b).
Me O Me
NH2
Et3N N Me
MeO + Me COCI
MeO H
CN
CN Me
Me
25 18 26
O Me MeO HO Me
1. KOt-Bu
1. H2SO4
N Me
MeO H 2. HCI Me
2. MeOH N
CO2Me Me H
27 28a O Me
Me
Me O Me
Me O H
COCI N
MeO O Me Me
Me CC MeO
Me Silicagel O Me
N Me
Et3N
H
28b O Me 29 O
Me
Scheme 31.4.4 Discovery of substituted spirocyclic tetramic acid derivatives.
Inspired by these results, however, attention was redirected to the

Bucherer–Bergs reaction for the synthesis of substituted 1-aminocyclohexanecar-
boxylic acids, which provided higher yields of the desired cis-isomer. Thus,
an optimization process was commenced to retain the aphicidal efficacy
while improving crop compatibility. Following a three-year period of intensive
studies, which included computer-aided calculations, it appeared impossible to
separate the excellent aphicidal efficacy from the severe phytotoxic symptoms.
However, an unconventional approach of combining the herbicidally most potent
4-methoxy-spirocyclic fragment with herbicidally weak phenyl moieties, thus
creating compound 30 (Figure 31.4.5), proved successful [29]. The derivative
30 demonstrated a good performance against the economically most important
species M. persicae and Aphis gossypii. In comparison to 29, a significant
improvement in crop compatibility towards vegetables was observed, in addition
to good whitefly control. In the case of the enol 31 (Figure 31.4.5), an improvement
in aphicidal activity was identified, while the favorable plant compatibility was
preserved [30]. On the completion of a four-year fine-tuning process, compound
32 (Figure 31.4.5) [31] was selected as a development candidate on the basis of
its physico-chemical parameters, efficacy, pest spectrum, plant compatibility, tox-
icology, environmental fate behavior, and economy of production. Consequently,
the worldwide registration of compound 32, spirotetramat, is planned for the
future.
O Me
O Me H
H N
N Me
Me MeO
MeO
O Me
O Me Me
Me
29 30 O
O Me
Me
O Me
O Me H
H N
N
Me MeO
MeO
O Me
HO Me Me
EtO
31 O
32 (Spirotetramat)
cis-4-(ethoxycarbonyloxy)-
8-methoxy-3-(2,5-xylyl)-
1-azaspiro[4.5]dec-3-en-2-one
logPow 2.5
melt. point 142°C
water sol. 30 mg ml−1
LD50 (rat oral) >2000mg/kg
Figure 31.4.5 The discovery of spirotetramat (32).
31.4.7
Synthesis of Spirotetramat
Spirotetramat (32) can be synthesized via a 12-step convergent synthesis in which

the first key intermediate, cis-4-methoxy-1-aminocyclohexanecarboxylic acid methyl
ester hydrochloride (37), is synthesized in a five-step sequence (Scheme 31.4.5a)
[32]. The first reaction step involves the hydrogenation of 4-hydroxy-anisol (33)
to 4-methoxycyclohexanone (34), followed by a Bucherer–Bergs reaction to form
the hydantoin, 35a. After separation of the cis-isomer 35b, the hydantoin is
hydrolyzed to the amino acid 36, which is then esterified with thionyl chlo-
ride/methanol to the ester hydrochloride, 37 [33]. The second key intermediate,
2,5-dimethyl-phenylacetyl chloride (42), can be synthesized in a straightforward,
four-step route (Scheme 31.4.5b). This starts with para-xylene (38), which is acylated
in an AlCl3 -mediated Friedel–Crafts reaction to the chloroacetophenone 39, fol-
lowed by ketalization with neopentylglycol to form the ketal, 40. Following sodium
acetate-catalyzed 1,2-aryl shifting and saponification of the intermediate, the resul-
tant 2,5-dimethyl-phenylacetic acid (41) is transformed into the acid chloride 42
[34].
Acylation of 37 with 42 leads to the phenylacetylaminoester, 43. The tetramic
acid 44 is then formed in a Dieckmann-cyclization with KOtBu, and finally acylated
with ethyl chloroformate to generate spirotetramat (32) (Scheme 31.4.5c).
O O
H H
N N
OH O H2N CO2H H2N CO2Me
(NH4)2CO3 HN HN
O aq. NH3 O
H2/Pd NaCN NaOH SOCI2
a x HCI
H 2O Separation MeOH
33 OMe 34 OMe 35a OMe 35b OMe 36 OMe 37 OMe

Me Me
Me Me
Me Me O Me Me Me
O O
CICH2COCI 1. NaOAc
OH OH CO2H COCI
b SOCI2
AICI3 CI CI 2. NaOH
38 Me 39 Me pTsOH 40 Me 41 Me 42 Me
Me O Me H
H2N CO2Me H N O
N Me
Et3N KOt-Bu MeO
COCI MeO
c + CO2Me
DMF
HO
42 Me 37 OMe x HCI 43 Me 44
Me
H
N O
CICO2Et MeO Me
Et3N
EtO O
O
31.4 Inhibitors of Lipid Synthesis: Acetyl-CoA-Carboxylase Inhibitors
32 Spirotetramat Me
1121
Scheme 31.4.5 Synthesis of spirotetramat (32).

31.4.8
Biology and Mode of Action of Spirotetramat
Spirotetramat (32) has the same mode of action as both spirodiclofen (7f) and
spiromesifen (8a), namely the inhibition of lipid biosynthesis [35]. Spirotetramat
has been shown to greatly reduce the lipid content of aphids feeding on leaves
treated with the compound (R. Nauen, unpublished results), as shown previously
for spirodiclofen (7f) in spider mites (Figure 31.4.2). More detailed investigations
using [14 C]acetate as a radiolabeled precursor of fatty acids have revealed a full
inhibition of the de novo synthesis of lipids in aphids [35]. Typically, spirotetramat
has demonstrated an excellent efficacy against insecticide-resistant pests (including
neonicotinoid resistant whiteflies), and has been classified in group 23 –together
with spirodiclofen (7f) and spiromesifen (8a) [36] – of the Insecticide Resistance
Action Committee (IRAC) mode of action classification scheme. Clearly, spirote-
tramat will in future prove to be an invaluable new tool for the management of
insecticide resistance in many crops and pests worldwide.
The physico-chemical properties of spirotetramat differ notably from those of
7f and 8a; moreover, spirotetramat has an effective action on a much broader
spectrum of pests, having demonstrated an excellent efficacy against a vari-
ety of aphid species including M. persicae, A. gossypii, and Phorodon humuli
(Table 31.4.5).
Based on its mode of action as an inhibitor of lipid biosynthesis, the juve-
nile stages of aphids are particularly affected by spirotetramat, whereas adults
are greatly affected in terms of their fecundity (R. Nauen, unpublished re-
sults). In practice, under field conditions, this would lead to a drastic reduc-
tion in population development. However, whilst spirotetramat – when applied
in foliar fashion – exhibited an excellent systemic efficacy against aphids and
whiteflies, its contact efficacy against these pests proved to be rather limited
(Figure 31.4.6).
Table 31.4.5 Physico-chemical and biological properties

of spirotetramat (32) compared with spiromesifen (8a) and
spirodiclofen (7f).
Spirotetramat Spiromesifen Spirodiclofen
Log POW 2.5 4.6 5.8

Water solubility (mg l –1 ) 30 0.13 0.05
Melting point (◦ C) 142 98 95
Spider mites + + +
Whiteflies + + –
Aphids + – –
+, active; –, not active.

100
80
Leaf-dip
Mortality, %
Aphid-dip
60
40
20
0
0.1 1 10 100 1000
Concentration [ppm]
Figure 31.4.6 Efficacy of spirotetramat transferred to the leaves. Aphid-dip: Aphids

against three- to four-day-old nymphs of were dipped (5 s) in serial dilutions of the
Myzus persicae (72 h). Leaf-dip: the leaves compound and then transferred to untreated
were dipped (for 5 s) in serial dilutions of leaves.
the compound and, after drying, aphids were
Whilst the enol compound 44 also exhibited activity against aphids, following its
penetration into the plant it needs no further conversion in planta, as does spirote-
tramat which, in leaves, is readily transformed to its enol form 44. Spirotetramat
can be considered as a pro-insecticide, and its above-mentioned systemic properties
were shown to be significantly improved by the coapplication of [14 C]spirotetramate
with an adjuvant, such as rape oil methyl-ester. Once taken up by the leaf, the
radiolabel was distributed through the entire plant, protecting in particular the
younger leaves (Figure 31.4.7).
The spirotetramat-enol 44 shows a notable water solubility and is a weak acid
(pKa 4.9), thus rendering the compound mobile within the symplast (phloem) of
the plant, according to the ‘‘weak acid hypothesis’’ [37]. Hence it can move both
acropetally and basipetally, and may even protects the plant roots when applied in
foliar fashion.
31.4.9
Development and IPM Suitability of MoventoTM
®
Movento is the trade name for the active ingredient spirotetramat (32), the first
broad-acting phloem mobile insecticide to be developed. Currently, spirotetramat
is undergoing development for worldwide use in pome fruits, stone fruits, citrus,
grapes, almonds, nuts, hops, tea, vegetables, cotton, and tropical fruits, and also
performs well to excellent against a broad spectrum of sucking pests, including
Aphididae (Aphis spp., Myzus spp., Dysaphis spp., Toxoptera spp., P. humuli),
Pemphigidae (Eriosoma spp., Pemphigus spp.), root aphids (Phylloxera spp.), Psyllids
(Psylla spp., Paratrioza cockerelli), scales (Ceroplastes spp., Pulvinaria spp., Aonidiella
spp., Quadraspidiotus spp., Orthezia praelonga), mealy bugs (Pseudococcus spp.,
Planococcus spp.), and whiteflies (Bemisia spp., Trialeurodes vaporarium) [38–40].
No additive + 0.1 % RME
(a) Picture 1 (b) Picture 2
Figure 31.4.7 Uptake and translocation of added in combination with rape oil
[14 C]spirotetramate (32) applied as a sus- methyl-ester (RME(; 0.1% RME). Two
pension concentrate (SC) 240 formulation to droplets (each 5 ml; 0.4 mg a.i.) of
cabbage plants. (a) [14 C]spirotetramate added [14 C]spirotetramate were applied to the first
alone (no additive); (b) [14 C]spirotetramate true leaf at two days before the analysis.
Spirotetramat has a slow initial, but very good and longlasting, efficacy with
excellent larvicidal activity. Moreover, new shoots and roots of the plant are also
protected. In addition, spirotetramat has a very favorable ecotoxicological profile,
®
which makes it of great interest for use in IPM programs. Movento was first
launched in Tunisia in 2007, but has since been registered in several important
countries, including USA, China, Mexico, Australia, and Turkey.
31.4.10
Conclusions
The discovery process of the new chemical class of cyclic ketoenols began with
a herbicidal spectrum shift from broadleaved weeds to grassy weeds, associated
with a change in the mode of action (in the present case from PPO to ACCase),
followed by an indication shift from herbicidal to acaricidal activity. The careful
follow-up of an initially weak acaricidal efficacy observed in the first ‘‘hits’’ in
chemistry, combined with an enthusiastic biological research team, and knowledge
of the physiology and mode of action of the compound, each played fundamental
roles during the initial optimization and development process of both spirodi-
clofen (7f) and spiromesifen (8a). Subsequently, another spectrum shift – from
acaricidal to aphicidal activity – proved to be the trigger for the discovery of the
aphicidal ketoenols. During the predevelopment and development stages of this
process, chemistry was identified as an important partner to determine the most
economic routes for the commercial large-scale production of these products. As
References 1125
a consequence, many different synthesis routes for the key intermediates were
examined, as shown in case of mesitylacetic acid (12).
Subsequent investigations in areas of physiology, biology, and biochemistry re-
vealed an inhibition of ACCase to be the mode of action for the cyclic ketoenols.
This was seen as a novel target in acaricidal/insecticidal chemistry and, as a con-
sequence, these compounds demonstrated high activities against pest populations
that had been shown resistant to conventional chemistry. This particular attribute
of the new ketoenols, when (in the case of spirotetramat) combined with their
excellent longlasting efficacy, favorable environmental profile and full systemic
properties, makes them a most valued tool for farmers worldwide.
References
1. Harwood, J.L. (1988) Annu. Rev. Plant Proc. Brighton Crop Prot. Conf., 1 (2A-6),
Physiol., 31, 101–138. 53.
2. Focke, M., Gieringer, E., Schwan, S., 12. Fischer, R. and Benet-Buchholz, J.
Jänsch, L., Binder, S., and Braun, H.-P. (2002) Pflanz.-Nachr. Bayer (Engl. Ed.),
(2003) Plant Physiol., 133, 875–884. 55, 137–148.
3. Nikolau, B.J., Ohlrogge, J.B., and 13. Nauen, R., Bretschneider, T.,
Wurtele, E.S. (2003) Arch. Biochem. Brück, E., Elbert, A., Reckmann, U.,
Biophys., 414, 211–222. Wachendorff-Neumann, U., and
4. Rendina, A.R., Craig-Kennard, A.C., Tiemann, R. (2002) Proc. Brighton Crop
Beaudoin, J.D., and Breen, M.K. (1990) Prot. Conf., 1 (2A-3), 39.
J. Agric. Food Chem., 38, 1282–1287. 14. Lantzsch, R. and Fuchs R. (1995) (Bayer
5. Burton, J.D., Gronwald, J.W., AG), Patent EP 676,388, 1995.
Keith, R.A., Somers, D.A., Gegenbach, 15. Stelzer, U. (1995) (Bayer AG), Patent EP
B.G., and Wyse, D.L. (1991) Pestic. 665,212, 1995.
Biochem. Physiol., 39, 100–109. 16. Bretschneider, T., Fischer, R., and
6. Kobek, K., Focke, M., and Lichtenthaler, Benet-Buchholz, J. (2005) Pflanz.-Nachr.
H.-K. (1988) Z. Naturforsch., 43c, 45–54. Bayer (Engl. Ed.), 58, 307–318.
7. Babczinski, P. and Fischer, R. (1991) 17. Stelzer, U. (1998) (Bayer AG), Patent EP
Pestic. Sci., 33, 455–466. 884,299, 1998.
8. Becker, B., Fischer, R., Hagemann, H., 18. Nauen, R., Stumpf, N., and Elbert, A.
Krebs, A., Lürssen, K., Marhold, A., (2000) Proc. Brighton Crop Prot. Conf., 1
Santel, H.-J., Schaller, K., Schmidt, (4D-9), 453.
R.-R., and Stendel, W. (1990) (Bayer 19. Nauen, R., Stumpf, N., Elbert, A.,
AG), Patent EP 355,599, 1990. Zebitz, C.P.W., and Kraus, W. (2001)
9. Erdelen, C., Fischer, R., Krauskopf, B., Pest Manage. Sci., 57, 253.
Lürssen, K., Santel, H.-J., Schmidt, 20. Denholm, I., Devine, G., Foster, S.,
R.-R., and Wachendorff-Neumann, U. Gorman, K., and Nauen, R. (2002) Proc.
(1991) (Bayer AG), Patent EP 456,064, Brighton Crop Prot. Conf., 1, 161.
1991. 21. Wachendorff-Neumann, U., Nauen, R.,
10. Bachmann, J., Bretschneider, T., Schnorbach, H.-J., Rauch, N., and
Erdelen, C., Fischer, R., Krüger, B.-W., Elbert, A. (2002) Pflanz.-Nachr. Bayer,
Lürssen, K., Santel, H.-J., Schmidt, 55, 149–176.
R.-R., and Wachendorff-Neumann, U. 22. Wachendorff- Neumann, U., Nauen, R.,
(1993) (Bayer AG) Patent EP 528,156, Schnorbach, H.-J., Stumpf, N., and
1993. Elbert, A. (2002) Pflanz.-Nachr. Bayer
11. Wachendorff-Neumann, U., Brück, E., (Engl. Ed.), 55, 149–176.
Elbert, A., Fischer, R., Nauen, R., 23. Rauch, N. and Nauen, R. (2003) Pestic.
Stumpf, N., and Tiemann, R. (2000) Biochem. Physiol., 74, 91.
24. Nauen, R., Schnorbach, H.-J., and Schneider, U., Turberg, A., and
Elbert, A. (2005) Pflanz.-Nachr. Bayer Wachendorff-Neumann, U. (1998)
(Engl. Ed.), 58, 417–440. (Bayer AG), Patent WO 98/05,638.
25. Guthrie, F., Denholm, I., Devine, G.J., 32. Fischer, R. and Weiß, H.-C. (2008)
and Nauen, R., (2003) Biological evalu- Pflanz.-Nachr. Bayer (Engl. Ed.), 61,
ation of spiromesifen against bemisia 127–140.
tabaci and an assessment of resis- 33. Fischer, R., Gallenkamp, B.,
tance risks. Proceedings of the BCPC Himmler, T., Knops, H.-J., and Mulder,
conference, vol. 2, 795–800. L. (2002) (Bayer AG), Patent WO
26. Elbert, A., Brück, E., Melgarejo, J., 02/002,532.
Schnorbach, H.-J., and Sone, S. (2005) 34. Himmler, T. (2005) (Bayer CropScience
Pflanz.-Nachr. Bayer (Engl. Ed.), 58, AG), Patent WO 05/075,401.
441–468. 35. Nauen, R. (2005) J. Pestic. Sci., 30, 272.
27. Munday, L. (1961) J. Chem. Soc., 4372ff. 36. Nauen, R., Reckmann, U., Thomzik, J.,
28. Beck, G. and Fischer, R. (1994) (Bayer
and Thielert, W. (2008) Pflanz.-Nachr.
AG), Patent EP 595,130, 1994.
Bayer (Engl. Ed.), 61, 245–278.
29. Bretschneider, T., Dahmen,
37. Crisp, C.E. (1972) in Pesticide Chemistry:
P., Dollinger, M., Erdelen, C.,
Proceedings 2nd IUPAC Congress (ed.
Fischer, R., Hagemenn, H.,
A.S. Tahori), Gordon and Breach, New
Lieb, F., Ruther, M., Santel, H.-J.,
York, pp. 211–264.
Wachendorff-Neumann, U., and
Widdig, A. (1997) (Bayer AG), Patent 38. Kühnhold, J., Klueken, A.M., de
WO 97/01,535. Maeyer, L., van Waetermeulen, X.,
30. Bretschneider, T., Erdelen, C., Brück, E., and Elbert, A. (2008)
Fischer, R., Graff, A., Hagemann, H., Pflanz.-Nachr. Bayer (Engl. Ed.), 61,
Lieb, F., Ruther, M., Schneider, U., 279–306.
Wachendorff-Neumann, U., and 39. Bell, J., Krueger, S., and Steffens, R.
Widdig, A. (1997) (Bayer AG), Patent (2008) Pflanz.-Nachr. Bayer (Engl. Ed.),
WO 97/36,868. 61, 307–328.
31. Andersch, W., Bretschneider, 40. Lozano, F., Kemper, K., and Tundisi, H.
T., Erdelen, C., Fischer, R., (2008) Pflanz.-Nachr. Bayer (Engl. Ed.),
Graff, A., Lieb, F., Ruther, M., 61, 329–454.
1127
32
Nervous System
32.1
Nicotinic Acetylcholine Receptor Agonists: Target and Selectivity Aspects
32.1.1
Introduction
One of the insecticide molecular target sites of growing importance (2009 mar-
ket share, 26.8%) is the nicotinic acetylcholine receptor (nAChR), which plays
a central role in the mediation of fast excitatory synaptic transmission in the
insect central nervous system (CNS). Despite long-term use of the alkaloid
(S)-(−)-nicotine (1) as a natural insecticide (aqueous tobacco extract), the nAChR
has been an underexploited biochemical target for modern insecticides, with an
estimated total insecticide world market share of about 1.5% in 1987. Because
of its high mammalian toxicity and relatively low level of insecticidal activity,
no major class could be established through taking 1 as lead structure [1].
However, more recently the nAChR has become an important target in crop
protection with the discovery and commercialization of three classes of insecticide
(Table 32.1.1):
• The very small group of so-called nereistoxin (2) analogs (N,N-dimethylamino-1,2-

dithiolane-4-amines), such as the bis(thiocarbamate) proinsecticide cartap (3) [2],
bensultap (4) [3–5], and thiocyclam (5) [6, 7].
• From the lead structure 2-nitromethylene-tetrahydro-1,3-thiazine (6, nithiazine)
[8, 9], resulting neonicotinoids [10] such as the open-chain compounds [e.g.,
nitenpyram (8), acetamiprid (9), clothianidin (12), dinotefuran (13); see
Chapter 32.2.1 the five-membered ring systems [e.g., imidacloprid (7),
thiacloprid (11); see Chapter 32.2.2], and the six-membered ring systems [e.g.,
thiamethoxam (10), AKD 1022 (14); see Chapter 32.2.3].
• The spinosyns, as a family of fermentation-derived insecticidal macrocyclic
lactones, including: the bioinsecticide spinosad (15) [11, 12], a natu-
rally occurring mixture of two active components, spinosyn A (primary
component) and spinosyn D; and the semi-synthetic spinetoram (16) [13], a

1128 32 Nervous System
Table 32.1.1 Evolution of nAChR agonists 1–17 used as insecticides.
Compounda Common name Manufacturer Agonist classes Remarks

(year introduced)
(S)-(–)-nicotine 1814 Nicotinoid Natural product,

N extracts of tobacco
H
Me
N
1
b c Nereistoxin Marine annelid
Me Me
N
S S
2
Me Me Cartap SumiTaked (1964) Nereistoxin analog Prodrug of 2
N hydrochloride
HCI
S S
H2N NH2
O O
3
Me Me Bensultap SumiTaked (1968) Nereistoxin analog Prodrug of 2
N
O S S O
Ph S S Ph
O O
4
Me Me Thiocyclam Sandoz (1979) Nereistoxin analog Prodrug of 2
N
(COOH)2
S S
S
5
Nithiazine Shell (1978) Neonicotinoid First lead structure
for CNIsb
HN S
CH-NO2
6
32.1 Nicotinic Acetylcholine Receptor Agonists: Target and Selectivity Aspects 1129
Compounda Common name Manufacturer Agonist classes Remarks

(year introduced)
CI Imidacloprid Bayer CropScience Neonicotinoid First

(1991) commercial
N CNI with
N NH highest
turnover
N-NO2
7
CI Nitenpyram SumiTaked (1995) Neonicotinoid Open-chain
Et H
nitromethylene
N N N
Me
CH-NO2
8
CI Acetamiprid Nippon Soda (1995) Neonicotinoid Open-chain
Me N-cyano-
N N Me acetamidine
N-CN
9
O Thiamethoxam Syngenta (1998) Neonicotinoid Six-membered
N
heterocyclic
CI N N nitroguanidine
S Me
N-NO2
10
CI Thiacloprid Bayer CropScience Neonicotinoid Five-membered
(2000) N-cyano
amidine
N
N S
N-CN
11
N H H Clothianidin SumiTake/d Bayer Neonicotinoid Open chain
CI CropScience (2000) N-nitroguanidine
N N
S Me
N-NO2
12
H H Dinotefuran Mitsui Toatsu (2002) Neonicotinoid Racemic
O open-chain
N N
Me neonicotinoid
N-NO2
13
Compounda Common name Manufacturer Agonist Remarks

(year introduced) classes
Me AKD-1022 Agro Kanesho Neonicotinoid Prodrug of

N 12 not com-
N
mercialized
CI N N
S Me
N-NO2
14
O Spinosad Dow Spinosyns Natural
O (Principal AgroSciences and product
N
components (1997) spinosoids (different
O O
O O
spinosyn A target site,
(R = H) and D IRAC group)
O
(R = CH3 ))
O
O
O R
15
O Spinetoram Dow Spinosyns Semi-
(Principal AgroSciences and synthetic
N O
components spinosoids spinosyn
O O
O spinosyn J derivative
O
(R = H; (different
O
C5 -C6 = single target site,
O
bond) and L IRAC group)
5 6
(R = CH3 ;
O
O R C5 -C6 = double
16 bond))
Me Sulfoxaflore Dow Sulfoximine Mixture of
O AgroSciences two diastere-
Me
S omeric
N-CN pairs
F3C N
17
a
Further details for each compound are given in subsequent sections of this chapter.
b
No common name.
c
Never commercialized for agricultural use.
d
Sumitomo Chemical Takeda Agro Company Ltd.
e
ISO-proposed common name.
CNI = chloronicotinyl insecticide.
mixture of 3 -O-ethyl-5,6-dihydro-spinosyn J (primary component) and

3 -O-ethyl-5,6-dihydro-spinosyn L (Chapter 32.3) [different binding sites, different
IRAC mode of action (MoA) classification number (5); see Chapter 27].
The N,N-dimethylamino-1,2-dithiolane-4-amines are based on the neurotoxic

and insecticidally active natural occurring insect-paralyzing factor 2, isolated from
the salivary glands of the nereid annelid worm Lumbriconercis heteropoda Marenz
[14, 15]. This natural product is active on cholinergic synapses [16]. Compounds
3–5 may be proinsecticides that are converted metabolically in the insect body
into 2, which then competes with acetylcholine (ACh) to block the nACh-mediated
signal [17, 18].
While nicotinoids are structurally similar to neonicotinoids, they primarily differ
by containing an ionizable basic amine or imine substituent. Today, the seven
commercial neonicotinoids 7–13 are the fastest-growing and major group of insec-
ticides [followed by pyrethroids, organophosphates (OPs), and methylcarbamates],
with widespread use against a broad spectrum of sucking and chewing pest insects
by several modes of application [19, 20] in most countries, and in many agronomic
cropping systems. These compounds act selectively on insect nAChRs, and are
used worldwide in insect pest management (IPM) programs [21, 22].
As the source for the family of novel tetracyclic macrolide polyketides, the
spinosyns (15, spinosad; 16, spinetoram) [23, 24] were found to be secondary
metabolites of the soil bacterium Actinomycete Saccharopolyspora spinosa [25, 26].
The spinosyn biosynthetic gene cluster has been cloned from S. spinosa and
sequenced, and the results have been used to formulate a proposed biosynthetic
pathway [27]. Spinetoram (16) is produced by a two-step post-fermentation synthetic
modification of a mixture of spinosyns J and L [28] (Chapter 32.3).
Recently, methyl[1-(2-trifluoromethylpyridin-5-yl)ethyl]-N-cyano-sulfoximine 17
(sulfoxaflor; common name ISO-proposed) was described as member of the
a new class of sulfoximines, containing a N-cyano-sulfoximine [-S(O)=N-CN]
pharmacophore variant [29] (see Chapter 32.3).
As is known, only minor structural variations of ligands can confer selectivity
among the mammalian nAChR subtypes and between insects and mammals. The
deployment of multidisciplinary approaches has led to an enhanced understanding
of the molecular basis of this selectivity, though electrophysiology has been
particularly instructive in identifying a rich diversity of actions of effectors on
nAChRs [30]. These diverse actions depend not only on the chemical structure of
the active ingredient but also on the subunit composition of the nAChRs tested.
In contrast, important physico-chemical parameters such as electrostatic inter-
action, H-bonding, π,π-stacking interaction, dipole–dipole interaction, and van
der Waals contacts all act closely together with the insecticide action. Therefore,
knowledge of the functional architecture and molecular aspects of insect versus
mammalian nAChRs and their ligand-binding site serves as the basis for the contin-
ued design of receptor subtype-selective active ingredients with high effectiveness
and maximal safety [31]. Especially, the neonicotinoids have created a renaissance
in the investigation of insect nAChRs [32], with the past decade having witnessed
rapid progress in studies of the nAChRs. This reflects the importance of these
receptors as a continuous source for the rational design of novel insecticides [33]
as well as medicinal drugs.
32.1.2
Structure of the Nicotinic Acetylcholine Receptor
The vertebrate nAChRs are agonist-gated ion channels responsible for rapid
excitatory neurotransmission. The nAChRs are well-characterized, large pentameric
transmembrane allosteric proteins (molecular weight ∼290 kDa), and are involved
in the rapid gating of ions elicited by ACh at the vertebrate neuromuscular junction,
and also in all animal central and peripheral nervous systems [34, 35].
Muscular nAChR is assembled from a ring of five homologous subunits (α, γ ,
α, β, δ), each of which is divided into three domains arranged around a central
ion channel: (i) a large extracellular N-terminal ligand-binding domain (LBD);
(ii) a membrane-spanning pore; and (iii) a smaller intracellular domain [36]. The
cation-selective nAChRs belongs to the ‘‘Cys-loop’’ superfamily of ligand-gated ion
channels (LGICs) [37] that also includes ionotropic glutamate, the anion-selective
glycine receptors [38], γ -aminobutyric acid type A and C (GABAA and GABAC )
receptors [39] and 5-hydroxytryptamine type 3 (5-HT3 ) receptors [40, 41], and has
facilitated an impressive number of physiological, pharmacological, and structural
investigations [42, 43]. Based on studies of invertebrate genetic models, such
as Drosophila melanogaster (fruit fly) and Caenorhabditis elegans (nematode), addi-
tional LGICs have been discovered, including GABA-gated cation channels [44],
5-HT3 -gated chloride channels [45], glutamate-gated chloride channels [46], and
histamine-gated chloride channels [47]. The nAChRs play important roles in both
neuronal and neuromuscular functions [48].
The nAChRs are homo- or heteromeric pentamers of structurally related subunits
that encompass an extracellular N-terminal domain which has six distinct regions
(loops A–F) involved in ligand binding, as well as the Cys–Cys loop, four C-terminal
transmembrane-spanning domains (TM1–TM4) that form the cation-permeable
channel [49], and an intracellular region extending from TM3 to TM4 [50]. Each
subunit comprises approximately 500 amino acids, is encoded by a separate gene,
and possesses the four transmembrane domains (TM1–TM4). The subunits are
orientated around a central pore [51, 52], such that the resulting transmembrane
ion channel is formed by a pentameric rearrangement of the TM2 helical segments
contributed by each of the five proteins [53]. Consequently, nAChRs possess
the structural elements necessary to convert a chemical signal into an electric
signal mediated by the opening of the cation channel. The nAChRs exist in four
conformational states, each with distinctive sensitivities to the nicotinic ligands that
dictate channel gating and function: basal or resting (closed, but rapidly activatable);
activated (open); and two desensitized (closed) states [54]. The latter are refractory to
activation on a time scale of milliseconds or minutes, depending on the desensitized
state, but have high affinity (picomolar to nanomolar) [55]. Ligand binding triggers
conformational changes that are transmitted to the transmembrane-spanning

region, leading to gating and changes in the membrane potential.
Among vertebrate species, 17 receptor subunits have been identified, including
ten α (α1 −α10 ), four β (β1 −β4 ), one δ, and one γ (this is replaced by ε in the
later stages of development) [56, 57]. The skeletal muscle or electric ray (Torpedo)
subtype is composed of two α1 subunits and one each of β1, γ , and δ (or ε in adult
muscle) subunits. The human nAChR gene family consists of 16 subunits (α1−α7,
α9, α10, β1−β4, δ, ε, and γ ) [58], whilst chicken possesses an additional subunit
(α8). An analysis of the genome of the pufferfish, Fugu rubripes, has revealed the
largest known set of vertebrate nAChR genes (16α and 12 non-α subunits), the
genesis of which is most likely through genome duplication [59]. In contrast, the
animal nematode model C. elegans possesses the most-diverse nAChR gene family
currently known, consisting of at least 27 subunits (20α and 7 non-α) [60, 61].
To date, the smallest nAChR gene family is that of D. melanogaster, consisting
of seven α and three non-α-subunits [62, 63]. Seven of these subunits, Dα1
(otherwise known as alpha-like subunit; ALS), Dα2 or second alpha-like subunit
Drosophila (SAD), and Dα3−Dα7 are α subunits, whilst Dβ1 or acetylcholine
receptor Drosophila (ARD), Dβ2 or second beta-like subunit Drosophila (SBD), and
Dβ3 are non-α subunits (Figure 32.1.1).
As described, the Dα5−Dα7 subunits are most closely related to vertebrate
α7, sharing 45% peptide sequence identity. Dα1−Dα4 and Dβ1−Dβ2 are most
closely related to each other (30–50% identity), reflecting 25–40% identity with
vertebrates. Dα3 is the most distantly related, showing only 20% sequence identity
with other nAChR subunits of both invertebrates and vertebrates. Dα2 shares the
closest sequence identity with other insect α subunits. The additional diversity
of Drosophila nAChRs arises from alternative splicing (four of the 10 subunits),
while RNA editing (first described for nAChRs in the fly) [64] serves to dramatically
increase the number of possible subunit isoforms (in theory, over 30 000 Dα6
isoforms are possible).
The assembly of five subunits can form channels of a wide variety of multiple
homo- or heteromeric neuronal nAChR subtypes [65] which allow a diversity of
physiological and pharmacological properties [66].
The nAChR is composed of two ligand-binding (α) and three non-α subunits
(γ , δ, or ε) or five α subunits [67]. The most common subunit stoichiometry
has been determined as (αX)2 (βY)3 (X = 2–4; Y = 2–4) for heteromeric subtypes,
and (αZ)5 (Z = 7–10) for homomeric subtypes [68], although other more complex
combinations have also been reported [69]. nAChR subtypes are found in different
locations of the central and peripheral nervous systems, and have been assigned
different pharmacological functions (cf. α7, α3β2, α3β4, α4β2, and α4β4) [70, 71].
The nAChRs contain multiple binding domains that can accommodate different
classes of endogenous and exogenous ligands. The nAChR LBD consists of
seven loops (A–G) spaced on the protein chains of the α and non-α subunits
[34, 37]. Sequences for each nAChR subunit predict hydrophilic extracellular
domains containing a binding site for cholinergic ligands and four transmembrane
hydrophobic segments (TM1 to TM4). The TM2 domain of the five subunits is
vert GABA-Aα1 (outgroup)
Dβ3
vert α9
vert α10
1134
Dα6
Dα5
Dα7 α7-like subunits
vert α7
vert α8
32 Nervous System
vert α1
vert α2
vert α4
vert α3 vertebrate subunits
vert α6
vert α5
vert β3
Dβ1
Dβ2
Dα3
Drosophila subunits
Dα4
Dα1
Dα2
vert β1
vert δ
vert ε vertebrate
non-α subunits
vert β2
vert β4
Figure 32.1.1 Tree showing relationships of Drosophila nAChR subunits and vertebrate nAChR subunits. The tree was
constructed using protein sequences aligned by the ClustalX program, and displayed using the TreeView application.
considered to form the lumen of the cation-channel [72], while a large intracellular
loop extends from TM3 to TM4.
The agonistic ACh-binding site is located at the interface of two adjacent subunits
(α and non-α), and is formed by six distinct regions (loops A–F) in the extracellular
N-terminal LBD, with each of the adjacent subunits contributing three loops.
Subunits that have two adjacent Cys residues in loop C, which are essential for
ACh binding [73], are referred to as α subunits, whereas subunits lacking this Cys
doublet are referred to as non-α or β, δ, ε, or γ subunits.
The ends of the internal lumen of nAChR are highly polar and negatively charged.
This domain can be viewed as a cation selector in which non-competitive inhibitors
bearing a positive charge (e.g., amine moiety) are trapped and directed down the
channel by an electrostatic gradient [67, 74].
Investigations utilizing electron microscopy, on helical tubes grown from Tor-
pedo marmorata postsynaptic membranes [75, 76], using a rapid spray-freezing
technique to mimic the synaptic release of ACh and trap the open-channel form,
have provided insight into the structural mechanism of gating, showing that the
binding of ACh imitates two interconnected events in the LBD.
The recently resolved crystal structures of integral membrane proteins of the
prokaryotic Erwinia chrysanthemi ELIC (at 3.3 Å resolution) [77] and the cyanobac-
terium Gloeobacter violaceus GLIC (at 3.1 Å resolution) [78], which both display
an architecture very close to that of the pentameric LGIC of Torpedo nAChR,
have constituted an important model system for the study of mechanisms of ion
permeation and gating within the receptor family.
32.1.2.1 Agonist-Binding Sites

Several investigations have allowed the identification of key interactions that
lead to the binding of small molecules such as ACh, (S)-(−)-nicotine (1), and
(–)-epibatidine (18) at the agonist-binding site of nAChRs [79].
Agonist ligands acting at vertebrate neurotransmitter-gated ion channels are
characteristically cationic in nature, and the binding induces a conformational
change in the receptor, causing opening of the ionic pore and a cation influx across
the cell membrane. Early biochemical studies of the embryogenic muscle nAChR
identified two agonist-binding sites localized to the α/δ and α/γ interfaces [82–84].
Upon sustained agonist exposure, a progressive loss of response is observed; this
is referred to as desensitization.
Unfortunately, as with many other integral membrane proteins, it has not
been possible to obtain crystals of any nAChR of sufficient quality to conduct
high-resolution X-ray crystallography studies.
An important breakthrough in understanding the Cys-loop receptor (CLR) struc-
ture, in particular with respect to the ligand-binding mechanisms, derived from the
characterization and structural determination of various soluble homopentameric
acetylcholine-binding proteins (AChBPs). The AChBP subtypes are secreted from
glia cells in the CNS of mollusks, such as the fresh-water snail Lymnaea stagnalis
(L-AChBP) [85, 86], Bulinus truncatus (B-AChBP) [87], the pearl oyster Pinctada fu-
cata (P-AChBP) [88], and the saltwater mollusk Aplysia californica (A-AChBP) [89].
These subtypes share only a 20–24% sequence identity with nAChR, but display
a striking structural resemblance with the T. tamorata α –(γ ) nAChR subunit or
mouse α1 nAChR protomer [90]. In addition, A-AChBP shares only 33% amino
acid identity with L-AChBP, but possesses all of the functional residues identified
in L-AChBP [91]. Recently, a homolog of the molluscan AChBPs in the marine
polychaete Capitella teleta (C-AChBP) from the annelid phylum was identified,
thus demonstrating a broader distribution of AChBPs in the animal kingdom. The
amino acid sequence of C-AChBP is 12–30% identical with those of the known
molluscan AChBPs [92].
Nicotinic ligand binding to the A- and L-AChBPs was assayed through different
methods, such as radioligand [125 I]α-BgTx (α-BgTx is a toxin from the elapid snake
Bungarus multicinctus) displacement or surface plasma resonance [93]. Conse-
quently, all of the AChBPs were found to display a pharmacological profile close to
that of the homo-pentameric α7 nAChR. Essential differences were also indentified
in the sensitivities of these AChBPs to various α-neurotoxins and α-conotoxins,
representing functional differences between the nAChR subtypes [94].
Furthermore, the resolved X-ray structure of the monomeric mouse α1 nAChR
subunit in complex with α-BgTx (at 1.94 Å resolution) demonstrated that AChBP
constitutes an excellent model for understanding nAChRs [95]. Whereas, A-AChBP
had a similar high sensitivity for both electronegative neonicotinoids and cationic
nicotinoids, the L-AChBP demonstrated lower neonicotinoid and higher nicotinoid
sensitivities. Thus, these two AChBPs have a distinct pharmacology suggestive of
the nAChRs from species divergent as mammals and insects (see Section 32.1.6)
[96].
In addition, a refined 4 Å resolution electron microscopy structure of the het-
eropentameric T. marmorata muscle type, (α1)2 βγ δ nAChR showed considerable
structural similarity to the L-AChBP with the nAChR LBD [36]. Because of this
similarity, L-AChBP is now considered a structural and functional surrogate of the
extracellular N-terminal LBD of insect nAChRs [97].
Therefore, the ACh binding site of the crystalline structures of AChBPs can be
used as an example for the N-terminal domain of an α-subunit of insect nAChRs
as a template for in-silico docking simulations.
Since 2002, several three-dimensional (3-D) models of LBDs of nAChR subtypes
with ACh, 1, and (-)-epibatidine (18) docked to the binding site, have been described
[98, 99]. A cationic center is contained in almost all nAChR agonists such as ACh,
1, and (-)-epibatidine (18). As a recognition strategy of cations by biological
molecules, the cation–π interaction, stabilizing the interaction between a cation
and the electron-rich aromatic ring, has been reported for several years [100–102].
Investigations of the muscle-type nAChR, using unnatural amino acid mutagenesis,
showed that a key tryptophan (Trp α149) reveals this potent cation–π interaction
with ACh to occur at the agonist-binding site [103]. On the other hand, the L-AChBP
confirmed a H-bonding interaction from the + N–H of 1 to the backbone carbonyl
in the same region of the agonist-binding site [104]. In contrast, (-)-epibatidine
(18) achieves its high potency by taking advantage of both the cation–π interaction
and the backbone H-bond. However, an important limitation of such modeling
studies is the absence of the membrane-spanning helices and intracellular domain

of nAChR, which might play an important role in receptor dynamics. In addition,
the L-AChBP is not a neuroreceptor (<25% sequence identity to its closest relative
in the nAChR family, α7), and its crystal structure most likely represents the
desensitizing state of the receptor. No conformational changes were observed
in the L-AChBP that could explain receptor gating; consequently, the functional
significance of structural insights into nAChRs resulting from L-AChBP remains
to be determined [105]. However, together with acquired cryoelectron microscopy
data of the membrane domains of Torpedo nAChRs [106], a model of the α7 nAChR
was built to explore its gating mechanism [107].
The crystal structure of A-AChBP in the apo form reveals a more open loop C
and distinctive positions for other surface loops, compared to previous structures.
An analysis of Aplysia AChBP complexes with nicotinic ligands showed that loop
C, which does not significantly change conformation on the binding of antagonists
(e.g., methyllycaconitine), further opens to accommodate peptidic antagonists (e.g.,
α-conotoxin), but wraps around agonists such as 18 [108]. The structures also
revealed extended and non-overlapping interaction surfaces for the antagonists,
outside the binding loci for agonists.
Some years ago, a description was provided of the principal pathway that links
agonist binding to channel gating, by using Unwins atomic scale model of T.
mamorata in nAChRs at 4 Å resolution [109]. The primary coupling pathway
integrates contributions from several structural domains, with its distal limb most
likely representing the point at which the binding domain triggers opening of the
channel.
32.1.3
Insect nAChRs
In insects, genes have been identified that encode multiple nAChR subunits, which
suggests the existence of diverse insect receptor subtypes across species [110, 111].
As an agonist-gated ion channel complex for rapid excitatory neurotransmission,
the nAChR is widely distributed in the insect CNS, and constitutes a major
target for insect action. However, the functional architecture, diversity, and 3-D
structure of the native insect nAChRs are poorly understood compared to their
vertebrate counterparts [112]. Today, it remains difficult to express functional
insect nAChRs not only in Xenopus laevis oocytes but also in several insect cell
lines (see Section 32.1.7). In general, insect nAChRs are diverse in structure, as
are those from vertebrates. In insects, as in vertebrates, the nAChRs mediate a
rapid synaptic transmission via an excitatory neurotransmitter–receptor complex
distributed widely in the synaptic neutrophil regions of the insect brain [58, 113].
Insect nAChR gene families are among the smallest nAChR families known
[114]. Genes encoding the ligand-binding α and structural β nAChR subunits have
been cloned in several insect species.
To date, various complete insect nAChR gene families have been described: the
genetic model organism D. melanogaster possesses only 10 nAChR subunits (seven
Dα1–7 and three Dβ1–3) [115], as is the case for the malarial mosquito vector
Anopheles gambiae (nine Agamα1−α9 and one Agamβ1). Eleven subunits have
been described in the honey bee Apis mellifera, which include nine Amelα (α1−α9)
and two Amelβ (β1−β2) [116]. The nAChR gene families of these three insects
have core groups of subunits that are highly conserved between different species.
Subunit equivalents of Dα1–7 and Dβ1 and Dβ2 are evident in A. gambiae and A.
mellifera, respectively. In addition, D. melanogaster, A. gambiae, and A. mellifera each
possess at least one divergent nAChR subunit that shows low sequence homology
(less than 20% identity) to all other known subunits, representing species-specific
receptor subtypes. Genome sequencing has revealed a similar level of nAChR
subunit diversity in other insect species, such as the silkworm Bombyx mori (nine
α-type subunits and three β-type subunits) [117] and the flour beetle Tribolium
castaneum [118].
Interestingly, all insect orthologs of Dβ2 characterized to date have been α
subunits (e.g., Agamα8) [119]. The loop C sequences for Dα2 and Agamα8 are very
similar, with most changes occurring within the vicinal Cys that define α subunits;
this has led to the suggestion that this represents a recent evolutionary transition
between an α and a non-α subunit. It appears that insects have several types of
nAChR subunit that could associate to form channels of disparate pharmacology,
and this might explain some of the complex binding and electrophysiology seen
with the insect cholinergic system. Seven nAChR subunits (four α-type, genomically
nine α-types and three β-type, which exist only in D. melanogaster) have been cloned
from fruit fly D. melanogaster.
Although, three further putative nAChR α subunits (Dα5−Dα7) with sequence
similarity to the vertebrate α7 subunit have been identified from Drosophila genome
sequence data, there have to date not been any reports of their characterization by
heterologous expression [120]. The remaining insect subunits do not have such
close sequence relationships with those of vertebrates.
Generally, insect nAChRs clearly vary with specificity of their interaction with
neonicotinoid insecticides; however, the appropriate subunit is, as yet, unclear.
However, an investigation has been discussed that supports the hypothesis of there
being a conserved neonicotinoid special sensitive subtype of the nAChR binding
site in different insects such as Musca domestica, D. melanogaster, Aphis craccivora,
and Myzus persicae [121].
When it has not been possible to obtain crystals of any nAChR of suffi-
cient quality to conduct high-resolution X-ray crystallography studies, both the
crystal structure of a soluble homopentameric AChBP and the refined model
of the membrane-associated Torpedo AChR [36], based on the crystal structure
of AChBP, can be used to provide considerable support to the understanding of
ligand–receptor interactions.
32.1.3.1 Consideration of AChBP versus nAChR α-Subunit

The AChBP has the same overall architecture as the extracellular portion of
the nAChR [122], and the presence of the vicinal Cys pair characteristic of
ligand-binding receptor subunits. Most of the key residues that have been shown
to contribute to the agonist-binding domain of the nAChRs were also conserved in
AChBP. The latter is not an ion channel, but does show numerous nAChR proper-
ties, including the binding of known nAChR agonists and competitive antagonists
(e.g., ACh, 1, dTC, and α-Bgtx). Therefore, the ACh binding-site on the crystal
structure of the AChBP can be used as an example of the N-terminal domain of
an α-subunit of nAChRs as a template for docking simulations of competitive ACh
ligands such as agonists and antagonists [123] by modeling methods [124].
Recently, 3-D models of the N-terminal part of nAChR were constructed and
docked in the putative ligand-binding pocket. Ligand binding is driven by enthalpy,
and accompanied by conformational changes in the LBS. These hypothetical
docking models offer a structural basis for the rational design of drugs, such as
neonicotinoids, which bind differentially to resting and active (or desensitized)
conformations of the nAChR site.
32.1.3.2 Interaction of Loop D of the α7 nAChR with Neonicotinoids

It is well known that AChBP can form a homo-pentamer like the α7 subunit. The
binding site for ACh and other agonists such as neonicotinoids is formed by six
loops (A–F) in the extracellular N-terminal domain. Of these loops, A–C have been
confirmed to α subunit, whereas loops D–F are present either in non-α subunits of
α/non-α heteromers or in the α subunits of homomers and α/α heteromers (e.g.,
α9/α10) [125]. In its crystal structure, Y164 in loop F (corresponding to G189 of the
α7 subunit) faces the agonist-binding site. Subsequently, it was found that Q55,
which corresponds to Q79 in loop D of the α7 nAChR, is close to Y164 in loop F of
the AChBP. Furthermore, the agonist responses of the α7 nAChR to 7 and 8 were
markedly reduced by the Q79E mutation, whereas the responses were increased by
mutations Q79K and Q79R. On the other hand, the agonist actions of des-N-nitro-7
were increased by the Q79E mutation, whilst responses were reduced by the Q79K
and Q79R mutations. Thus, it was postulated that a glutamine (Gln) residue Q79 in
loop D (Table 32.1.2) and glycine (Gly) G189 in the loop F of the chicken α7 subunit
can interact with the nitro group of neonicotinoids such as 8 (=CH–NO2 ), 7, 10,
and 12–14 (each with =N–NO2 ). This ‘‘induced-fit’’ mechanism can also account
for the selective actions of other neonicotinoids such as 9 and 11, containing the
N-cyano group. Finally, the crystal structures of L-AChBP in complex with 7 and 12
showed that Gln in loop D, corresponding to the basic residues of insect nAChRs,
can participate in H-bonding with the N-nitro group and the neonicotinoid-unique
stacking and CH-π bonds at the LBD [126].
In most insect non-α subunits, amino acids residues corresponding to Q79
of the α7 subunit are lysine (Lys) or arginine (Arg) moieties (Table 32.1.2).
These basic residues may interact with the nitro group of neonicotinoids through
electrostatic forces and H-bonding, thus strengthening the nACh–insecticide
interaction. Moreover, its substitution can result in a reduction of the insecticide
sensitivity of nAChRs, but not in any reduction of affinity.
Table 32.1.2 Alignment of amino acid sequence in loop D

of the ACh binding site of vertebrate and insect nAChRs.
Subunits Amino acid number of chicken β subunita
73 74 75 76 77 78 79 80 81 82
Vertebrates
Chicken α7 N I W L Q M Y W T D
Chicken β2 N V W L T Q E W E D
Chicken β4 N V W L N Q E W I D
Human β2 N V W L T Q E W E D
Human β4 N V W L K Q E W T D
Human δ N V W I E H G W T D
Human ε S V W I G I D W Q D
Rat β1 K V Y L D L E W T D
Rat β2 N V W L T Q E W E D
Rat β4 S I W L K Q E W T D
Insects
Fruit fly Dβ1 N V W L R L V W Y D
Fruit fly Dβ2 N L W V K Q R W F D
Fruit fly Dβ3 H C W L N L R W R D
Locusta N V W L R L V W N D
migratoria β
Myzus N V W L R L V W R D
persicae β1
Heliothis N V W L R L V W M D
virescens β1
a
Residue numbering is from the start methionine.
Data taken from Ref. [125].
32.1.4
Nicotinic Pharmacophore Models
Before X-ray crystallographic studies of the AChBP had been conducted, the
structure of the nAChR binding site(s), and the rational design of potent and
selective nAChR ligands, was facilitated by the identification of a specific 3-D
arrangement of essential chemical groups common to nAChR ligands, the so-called
‘‘nicotinic pharmacophore.’’ Designation of the nicotinic pharmacophore is the
first essential step towards understanding the interaction between nAChR and
the class of neonicotinoids (including commercial products). Although several
early ‘‘nicotinic pharmacophores’’ were described, these either did not take into
consideration any specific binding data, or they were derived on the basis of
pharmacological data from peripheral nAChR assays [127].
For example, in 1970 a useful nicotinic pharmacophore model was described by

Beers and Reich and subsequently improved by Sheridan et al. [128]. Starting from
different models, a distance from the onium group to a point on the van der Waals
surface of the H-bond acceptor of 5.9 Å was common to several ligands. To date,
three binding models of neonicotinoids have been proposed (cf. Figure 32.1.2) [129].
Based on the results of structure–activity relationship (SAR) studies, the first
two models I and II (Figure 32.1.2b) suggested a primary role for the nitrogen at
the 1-position of the neonicotinoid, equating it to N-methyl-pyrrolidine nitrogen
of (S)-(−)-nicotine (1). Yamamoto et al. [80] proposed that the nitrogen atom of
the 6-chloro-pyrid-3-yl moiety and the nitrogen atom at the 1-position of the
imidazolidine ring in 7 (X = N; E = NH; R1 -R2 = CH2 CH2 ) would interact with the
H-donating and electron-rich sites of nAChR, respectively, because the distance
between these two nitrogen atoms was similar to that between the two nitrogen
atoms on 1 (Figure 32.1.2a). In an earlier study, the sp2 pyridine nitrogen of 1
neutral form had been identified as the only H-bond acceptor site [130]. However,
R2 R1 H
1
E N Cl Model I
δ+ N
H
X
O2 N
−
O +
2 N O H
R E
X Model II
Me Me R1 N1
Me δ+ X = CH, N
+ R′
N
Me O O ACh
X C N H
R2
R1
E
(S)- Nicotine (1) N1 −
O
N X
+ H N+ Model III
N
Me O
H Cl N
δ+
H H
- -
5.9 Å 5.9 Å
nAChR nAChR
(a) (b)
Figure 32.1.2 Interaction of (a) ACh, (S)-(−)-nicotine (1)

and (b) neonicotinoids (7–14) with the H-donating and
electron-rich sites of nAChR, exemplified by three models
(I – III). Adapted from Refs [80–81].
it was found later that, in solution, both nitrogens of 1 are involved in the
H-bond interactions, with 90% of the H-bonded complexes being formed on the
pyridine nitrogen [131]. This result was in accordance with the binding of 1 and
carbamoylcholine observed in AChBP [132].
Recently, the high-resolution crystal structures of A-AChBP–neonicotinoid com-
plexes with 7 and 11 have shown that a water or solvent molecule is located close
to the pyridine nitrogen, bridging to loop B or loop E amino acids, or both.
In contrast, Kagabu proposed that the nitrogen atom at the 1-position of the
imidazolidine ring of 7, and one of the oxygen atoms of the nitro group within the
[=N–NO2 ]-pharmacophore (at the van der Waals surface), played important roles
in the interaction with the binding sites on nAChR (Figure 32.1.2b, cf. Model II).
Thus, the π-conjugated system composed of an N-nitro-imino- or N-cyano-imino
group and the conjugated nitrogen in 1-position are considered essential moieties
for the binding of neonicotinoids to the putative cationic subsite in insect nAChR.
The third, most recent model III (Figure 32.1.2b), involves a crucial role for the
N-nitro group within the [=X–NO2 ]-pharmacophore, an important contribution
from the 6-chloro-pyrid-3-yl ring nitrogen, and also a supplemental role for the
nitrogen in 1-position. A first confirmation of this model III is exemplified by
the interaction of 7 with the α7 nAChR based on the AChBP [133], and later by
the high-resolution crystal structures of L-AChBP–neonicotinoid complexes with
7 and 12, both with N-nitroimine moieties and affinities of 1600 and 7300 nM,
respectively.
In order to estimate how the physico-chemical parameters at the pharmacophore
are related to the insecticidal potency of 1, a quantitative structure–activity rela-
tionship (QSAR) study was conducted that involved its injection into American
cockroaches (Periplaneta americana L.). Subsequently, the biological activity of 1
(and thiacloprid, 9; see Section 32.2.2.3) was shown to be determined not only by
the pharmacodynamic factors associated with the interaction involving the pharma-
cophores, but also by the pharmacokinetic factors associated with the movement
and distribution of the neonicotinoids in the insect fluid [134].
32.1.5
Mode of Action in Insects
During recent years, the great effort applied to the creation of insecticides with a
high affinity to the nAChR has resulted in the development of nereistoxin analogs
(e.g., 3–5), neonicotinoids (e.g., 7–13), and spinosyns (e.g., 15 and 16).
32.1.5.1 Nereistoxin and Analogs

In contrast to nAChR agonists causing excitatory effects, 2 induces inhibitory
neurotoxicity. In fact, multiple actions of 2 have been reported with relatively
high concentrations in vertebrate nAChRs, including: (i) a potent blockade of
the mechanosensory-giant interneuron cholinergic synapses in the cockroach (P.
americana) terminal abdominal (TA) ganglion, and also of frog and rat muscle
endplates [135]; (ii) a partial agonist activity [136]; and (iii) a non-competitive or
open-channel blocker (NCB) for the open status of the nAChR/channel from the
Torpedo electric organ. Dual actions have also been proposed at the honeybee (A.
mellifera) nAChR, since 2 binds to both the NCB and ACh sites with high and
low affinities [137]. At low concentrations (0.1 μM), a voltage-dependent inhibitory
effect of 2 on nAChRs was observed [138]. In addition, 2 was shown to inhibit
the specific binding of [125 I]α-BgTx, although the high concentration (0.17 mM)
required to inhibit such binding by 50% suggested that the site of blocking action
by 2 might be distinct from the αBtx binding site.
Cartap [1,3-bis(carbamoylthio)-2-(N,N-dimethylamino)propane] (3), which was
the first commercial insecticide to be derived from 2, functions in a similar manner
at the cercal afferent-interneuron synapses [139]. Indeed, the coapplication of ACh
and 3 (10 μM) induced an opening of the nAChR channel, albeit for a shorter time,
thus generating the appearance of a ‘‘burst’’ [140].
Recent studies on recombinant chicken α7 and α4β2 receptors, and also on
Drosophila/chicken hybrid receptors Dα2β2 and Dα1β2, have shown that 2 is an
effective blocker of both native and expressed vertebrate nAChRs, acting as a NCB
of the nAChR [7]. However, 2 proved to be slightly more potent on recombinant
Drosophila/chicken hybrid receptors than on chicken nAChRs [141].
32.1.5.2 Neonicotinoids
The biochemical MoA of neonicotinoids has been investigated and characterized
during the past 15 years. In insects, all neonicotinoids act selectively at the
postsynaptic nAChRs at the nanomolar level (except for 1, at micromolar level) and
bind to the ACh binding site located on the hydrophilic extracellular domain of the α
subunits. The neonicotinoid binding site in insects is essentially similar or is closely
coupled to that of ACh, 1, and α-BgTx; however, unlike 1 – which is hydrolyzed
by acetylcholine esterases (AChEs) – nicotinic agonists and antagonists lacking the
ester linkage can generate, respectively, a sustained activation or blockade of the
nAChRs.
The ability of the neonicotinoids to displace tritiated imidacloprid (7) ([3 H]-7)
from its binding site correlates well with their insecticidal efficacy [142, 143].
Indeed, the insecticidal activity of neonicotinoids is due to their action as insect
nAChR agonists, causing channel opening. This effect was first demonstrated
using both electrophysiological and [125 I]α-BgTx-binding studies with 6 and the
cockroach nerve cord [144, 145]. This finding was supported by other studies
that demonstrated a high correlation between nerve activity induced in cockroach
preparations and their potential to control numerous target pests species [146–148].
These results were subsequently verified using either [3 H]-7 or [125 I]α-BgTx in bind-
ing studies with insect brain membranes [149, 150]. Two α-BgTx-sensitive nAChR
subtypes in cockroach neurons have been identified as desensitizing (nAChRD),
selectively inhibitable with 100 mM 7, and as non-desensitizing (nAChRN), selec-
tively inhabitable with 100 pM methyllycaconitine. Although the desensitizing rate
of nAChRD receptors is highly variable, their pharmacology is largely independent
and can be measured specifically by using binding assays with radiolabeled 7 [151].
More definitive confirmations were obtained with [3 H]-7 by structure–activity

correlations for the displacement of binding potency with knockdown activity [152,
153] and electrophysiological responses. An agonistic action on nAChRs causes
first hyperexcitation followed by paralysis, as shown with 7 on different insect
species [154–156]. In contrast, antagonistic ligands of mammalian nAChR were
generally less active as insecticides [157, 158].
Since the discovery of 7, a diversity of imidacloprid-related insecticides referred
to as neonicotinoids have been synthesized. Similar to 7, all of the commercial
neonicotinoids 8–13 bind with high affinity (I50 ∼ 1 mM) to [3 H]-7 binding sites
on insect nAChRs.
One notable omission here is the six-membered thiamethoxam (10) (see
Chapter 32.2.3), for which binding affinities of up to 10 000-fold less than other
neonicotinoids have been demonstrated, using housefly head membrane prepa-
rations. This low affinity may be attributed to the compound’s proneonicotinoid
structure, as it was shown to be activated to the open-chain clothianidin (12)
(Chapter 32.2.1) in both plants and insects [159]. The latter exhibits high activity as
an agonist on isolated neurons at concentrations as low as 30 mM.
Recently, it was shown that cholinergic neurons express nAChRs that are highly
sensitive to 7, while a role was demonstrated for voltage-gated calcium channels in
amplifying the 7-induced increase in intracellular calcium levels [160].
Details of the MoA of neonicotinoid insecticides have been provided in several
excellent reviews [161–163]. These describe the current knowledge of the struc-
ture and function of insect nAChRs, as characterized by receptor-binding studies,
together with phylogenetic considerations with regards to receptor homologies
between orthologs from different animal species, and electrophysiological investi-
gations.
32.1.5.3 Spinosyns and Semi-Synthetic Analogs (Spinosoids)

Spinosyns cause hyperexcitation and, ultimately, disruption of the insect CNS
[164] by allosterically activating nAChRs and prolonging the responses of those
receptors to agonists, such as 1 and ACh [165]. Initially, the spinosyns cause
spontaneous involuntary muscle contractions, prostration with tremors by exciting
motor neurons in the CNS [166], paralysis and, finally, death. Spinosyn A can
directly excite the CNS when applied to isolated insect ganglia; this indicates that,
in vivo, the neuronal excitatory effect is due directly to 15 or 16 (Table 32.1.1), and
not to a bioactivated metabolite. These effects are consistent with the activation
of both nAChRs and also the GABA receptor functions of neurons, which may
increase their activity [167]. Insecticidal spinosyns and spinosoids have been
shown to disrupt GABA receptor function in small neurons from the CNS of
P. americana (L), whereas spinosyns lacking insecticidal activity have no such
effect [168]. However, both spinosyns and spinosoids appear not to affect the
binding site of either nicotinic or GABA receptor radioligands such as avermectins,
fiproles, or cyclodienes; this suggests that the macrolactones do not interact directly
with known binding sites for other nicotinic or GABAergic insecticides [169].
Rather, they appeared to suppress the amplitude of GABA responses and to
activate a picrotoxin-sensitive chloride current in small neurons from the CNS of

P. americana [170].
Evidence derived from electrophysiological studies indicates that spinosyns can
alter nicotinic currents in neuronal cell bodies from the CNS of P. americana (L),
an effect which is correlated with toxicity towards neonate Heliothis virescens larvae.
It might also be suggested that the spinosyns affect nAChR and GABA receptors
through a so-called ‘‘undetermined’’ mechanism, in difference to neonicotinoids
[171]. Finally, the action of spinosyn A was shown to differ on both the desensitizing
and non-desensitizing subtypes of α-Btx-sensitive nAChRs. Whereas, spinosyn A
is a highly effective activator of non-desensitizing subtypes, it showed a blocking
effect on the desensitizing subtypes of nAChRs.
Recently, the Dα6 subunit of the nAChR has been described as a molecular
target site for the spinosyns in Drosophila [172, 173] (further details are provided in
Chapter 32.3).
32.1.6
Selectivity for Insect versus Vertebrate nAChRs
Although, neonicotinoid insecticides are more than 100-fold selective for insect
nAChRs than for vertebrate nAChRs, very little is known regarding the mechanism
of such selectivity [174]. This opposing selectivity profile is based largely on the
differential sensitivity of the insect and vertebrate nAChR subtypes, which is in
turn attributable to their unique chemical features. Previously, several groups have
reported evidence related to the submolecular basis of this selectivity, based on
the nAChR subunit composition and properties, as well as on the steric charge
distribution characteristics of neonicotinoids [67, 175–177]. For example, Yao
et al. [178] have shown that amino acids located within insect-specific loops D,
E, and F play key roles in the selective interactions of heteromeric nAChRs with
neonicotinoids.
An opposing selectivity profile was also recently described in two AChBP subtypes
(see Section 32.1.2.1). Aplysia AChBP is highly sensitive to neonicotinoids (e.g., 7,
9, and 11) and nicotinoids (e.g., 1 and 18), whereas the Lymnaea AChBP subtype has
a lower activity for neonicotinoids than for nicotinoids. Thus, the A-AChBP might
serve as a plausible structural surrogate for the interactions of both neonicotinoids
with the insect nAChR, and nicotinoids with the vertebrate nAChR. Furthermore,
the L-AChBP may represent a homolog for the vertebrate nAChR [179, 180]. The
prolonged activation, modulation, or inhibition of LGICs such as nAChRs can result
in toxic effects; however, selective toxicity involving low hazards for vertebrates
but high potency towards insect pests represents an essential requirement for the
future identification of safe and effective insecticides.
32.1.6.1 Neonicotinoids
Debnath and coworkers [181] have demonstrated, in a QSAR study performed
using electrotopological state atom indices, that compounds with a [=N–NO2 ]
(e.g., 7, 10, 12, 13, and 14), [=CH–NO2 ] (e.g., 8) or [=N–CN]-pharmacophore (e.g.,
9 and 11) are more active, selectively, to Drosophila nAChR and safe for humans,
whereas N-unsubstituted imines having affinity to mammalian receptor.
It has been shown that two important enzymes in metabolism of neonicotinoids,
the liver microsomal CYP3A4 (mainly oxidation of imidazolidine moiety) [182],
and cytosol aldehyde oxidase (AOX, reduction at the [=N–NO2 ]-group) [183] result
in either an increase or decrease of agonist potency, depending on the compound
and specificity of the nAChR [184]. Some neonicotinoids such as 10 and 14 act
as pro-insecticides with oxidative activation by CYP450s to more potent agonists
such as 12 [185] (Chapter 32.2.1). On the other hand, neonicotinoid resistance or
cross-resistance is more commonly due to CYP450 detoxification than to nAChR
mutants or variants as described for the Colorado potato beetle (Leptinotarsa
decemlineata) [186]. With the vertebrate α4β2 nAChR, AOX enhances potency of
7 but CYP3A4 does not. The AOX system coupled with the Drosophila receptor
strongly inactivates the neonicotinoids like 7, 10, 12, or 13; with nitromethylenes 6
and 8 some inactivation was found.
In contrast to S-(−)-nicotine (1; rat oral LD50 = 50–60 mg a.i. kg−1 , high mam-
malian oral and dermal toxicity), neonicotinoid insecticides display excellent
selectivity profiles that are largely attributable to specificity for insect versus
vertebrate nAChRs. This is exemplified by the fact that the radioligand [3 H]-7 serves
as an excellent probe for insect, but not vertebrate, nAChRs. On the other hand,
both [3 H]epibatidine (18) ([3 H]-18) [187] and [125 I]- or [3 H]α-BgTx [188] proved to be
important probes for characterizing the vertebrate α4β2 and α7 nAChR subtypes,
respectively. In native insect nAChRs, the [3 H]-7 binding site in Drosophila is
distinct from that of [3 H]α-BgTx [189]. Specific [3 H]-18 binding has been identified
in some insects such as P. americana, but not in others such as the housefly
M. domestica.
Neonicotinoids have little or no effect on the vertebrate peripheral nAChR α1
γ α1δβ1 subtype [190–193], or on some neuronal subtypes (α3β2 (and/or β4)
α5, α4β2, and α7) [194–198]. Minor structural modifications of neonicotinoids
confer differential subtype selectivities in vertebrate nAChRs [199]; in general,
nitromethylene analogs with strong insecticidal efficacy show comparable or higher
affinity than that of 1 to the α3β2β4α5 or α7 subtype (Table 32.1.3).
The results of comparative binding studies have indicated that imidacloprid (7)
and related neonicotinoids have little or no affinity for several mammalian nAChRs.
Numerous electrophysiological measurements have revealed that nAChRs are
widely expressed in the insect CNS on both postsynaptic and presynaptic nerve
terminals, on the cell bodies of interneurons, motorneurons, and sensory neurons
[200–204].
Likewise, the results of both electrophysiological and biochemical binding stud-
ies have shown the primary target of the neonicotinoids to be the nAChRs [150,
205–211]. Typically, electrophysiological studies have indicated that 7 acts as
an agonist on two distinct nAChR subtypes on cultured cockroach dorsal un-
paired motoneurons (DUM)s [212], an α-BgTx-sensitive nAChR with ‘‘mixed’’
nicotinic/muscarinic pharmacology, and an α-BgTx-intensive nAChR. These in-
vestigations were supported by binding studies conducted with [3 H]-7 in membrane
Table 32.1.3 Specificity of commercial neonicotinoids

(7–13) for insect and vertebrate α4β2 nAChRs.
Neonicotinoid Insecta IC50 value (in nM) Selectivity ratio

vertebrate α4β2a,b
Imidacloprid (7) 4.6 2 600 565

Nitenpyram (8) 14 49 000 3500
Acetamiprid (9) 8.3 700 84
Thiamethoxam (10) 5000 >100 000 >20
Thiacloprid (11) 2.7 860 319
Clothianidin (12) 2.2 3 500 1591
(±)-Dinotefuran (13) 900 >100 000 >111
a
IC50 -values for displacing [3 H]-7 binding to the house fly (M. domestica) 9, aphid (M. persicae) 10, and
fruit fly (other neonicotinoids) receptor, and [3 H]-1 binding to the vertebrate α4β2 nAChR.
b
IC50 -values (μM) for the vertebrate α7 nAChR subtype (assayed by [125 I]α-BgTx binding) are 7, 270;
8, >300; 9, 290; 10, >300; 11, 100; 12, 190; 13, >1000.
According to Ref. 2005 [199].
preparations from M. persicae; such ligand competition studies revealed the pres-
ence of high- and low-affinity nAChR binding sites for 7 in this insect [213]. The
identification of multiple putative nAChR subunits by molecular cloning, however,
was consistent with a substantial diversity of insect nAChRs [214].
32.1.6.2 Spinosyns
Spinosad (15) and the semi-synthetic spinosyn spinetoram (16), as reduced-risk
insecticides with wider margins of safety for non-target organisms, also demon-
strate the different sensitivities of insect compared to mammalian nAChRs (see
Chapter 32.3).
32.1.7
Insect Selectivity in Recombinant nAChRs
Pharmacological profiles of the recombinant hybrid insect α/vertebrate β nAChRs

are poorly defined, and the binding sites have not been established for identified
subunits versus native receptors. The functional expression of insect nAChRs of
known subunit compositions facilitates an understanding of the mechanism under-
lying these molecular interactions. However, it is difficult to express heterologously
functionally robust nAChRs, not only in Xenopus oocytes but also in Drosophila
S2 cells [215]. To date, only a few functional receptors have been obtained after
the expression of different subunit combinations in Xenopus oocytes or cell lines.
Nevertheless, Drosophila nAChR α subunits can form homo-oligomeric functional
nAChRs when coexpressed with a vertebrate β2 (non-α) subunit in Xenopus oocytes.
As yet, this has been demonstrated for:
• Four locust nAChR subunits (three α-subunits: Lα1 from Schistocerca gregaria
[216, 217], and Locα2 and Locα3 from Locusta migratoria; and one non-α-subunit:
Locβ1 from L. migratoria) [218].
• Six D. melanogaster nAChR subunits (four α-subunits: Dα1, Dα2 [219], Dα3 [220],
and Dα4 [221]; and two non-α-subunits: ARD and SBD [222]).
• One Manduca sexta nAChR subunit [223].
• Five M. persicae nAChR subunits (Mpα1–4 and Mpβ1) [224, 225].
• Two Nilaparvata lugens nAChR subunits (Nlα3 and Nlα8) [226].
The expression of these subunits was not very effective (low amplitude-current,
5–50 nA), however, following the application of 1 or ACh. In addition, complemen-
tary DNAs (cDNAs) of Locα1 and Locα4 from L. migratoria and Mpα5 from M.
persicae have been cloned partially.
Alternatively, all three Drosophila α subunits (ALS, SAD, and Dα2) can form
functional receptors in Xenopus oocytes when coexpressed with a chicken neuronal
α4β2 subunit [227, 228]; this suggests that additional insect nAChR subunits remain
to be cloned. Of the hybrid receptors, the Drosophila SAD-chicken β2 hybrid nAChR
has been found to be highly sensitive at much lower concentrations to the agonist
actions of 7 and related neonicotinoids (more neonicotinoid-sensitive than the
α4β2 receptor), which suggests that the Drosophila α subunits Dα1 and Dα2 have
structural features favorable for selective interactions with neonicotinoids.
However, other studies using recombinant nAChRs stably expressed in cell lines
have shown that the binding of 7 and methylcarbamoylcholine to the Dα3/β2
receptor could be abolished by replacing the β2 with a vertebrate β4 subunit. This
suggests that non-α subunits may also be important in determining sensitivity to
neonicotinoids and other agonists.
The results of radioligand-binding studies using several M. persicae α subunits
coexpressed with a rat β2 subunit in the Drosophila S2 cell line have also reflected
pharmacological diversity in M. persicae.
Recently, investigations were conducted into the [3 H]-7, [3 H]-18, and [3 H]α-BgTx
binding sites in hybrid nAChR consisting of D. melanogaster or M. persicae α2
coassembled with rat β2 subunits (Dα2/Rβ2 and Mpα2/Rβ2) in comparison with
native insect and vertebrate α4β2 nAChRs [229]. The study findings supported
the conclusion that the nAChR agonist-binding site for neonicotinoids is located
at the interface region between subunits in insects α/vertebrate β hybrids as well
as in native insect receptors. Such results have demonstrated that imidacloprid
(7)-selective targets were formed by Mpα2 and Mpα3 subunits, but not Mpα1
subunits.
On the other hand, a so-called ‘‘cleavage’’ of the imidazolidine 5-ring of 7 led to
the efficacy of the open-chain 12, which was higher than that of ACh or 7 on the
SADβ2 hybrid nAChR expressed in Xenopus oocytes. The superagonist action of
12 and related ligands may account for the more potent action of 12 than that of 7
over a wide range of insect pests [230].
By expressing hybrid nAChRs containing N. lugens (a susceptible, laboratory-
developed strain of brown planthopper) α- and rat β2 subunits, evidence was
obtained to demonstrate that mutation Y151S was responsible for a substantial

reduction in specific [3 H]-7 binding [231]. It seemed plausible that this mutation
might cause an induced conformational change within the nAChR binding site
region.
Recently, a nAChR point mutation (Y151S) was identified as being associated
with neonicotinoid resistance in N. lugens selected under laboratory conditions
with imidacloprid. Subsequently, a Nlα3Y151S mutation was shown to have only a
minimal effect on the agonist potency of ACh, but a dramatic effect on neonicoti-
noid insecticides (reducing Imax values and increasing EC50 values). The apparent
affinity of the neonicotinoids was higher, and the effect of the Y151S muta-
tion on neonicotinoid agonist potency was more profound in Nlα3-containing,
rather than Nlα1-containing nAChRs. In addition, evidence has been obtained
for the coassembly of Nlα1 and Nlα2 subunits into ‘‘triplet’’ nAChRs of subunit
composition Nlα1/Nlα2/β2. Evidence has also been obtained showing that the
resistance-associated Y151S mutation has a significantly reduced effect on neoni-
cotinoid agonist activity when Nlα1is coassembled with Nlα2 rather than expressed
as the sole α subunit in a heteromeric nAChR [232]. However it has also been
shown that fitness costs, such as a reduced fecundity, are linked to the mutation.
These examples confirm the often difficult-to-understand complexity of insect
nAChRs. The considerable diversity of potential subunit combinations most likely
accounts for the multiplicity of distinctive pharmacological profiles in insect
nAChRs. In that context, electrophysiology will play an essential role in determining
the significance of certain subunit combinations in the MoAs of neonicotinoid and
further insecticidally active ligands.
32.1.8
Whole-Cell Voltage–Clamp of Native Neuron Preparations
The use of isolated neurons from the insect CNS for electrophysiological studies
represents a valid tool for investigation of the MoAs of new insecticidal compounds
that act on a range of neuronal target sites. Likewise, primary neuronal cell cultures
from H. virescens larvae – one of the most important lepidopteran pest species – is
a suitable tool for this purpose.
The neurons of H. virescens respond to the application of ACh, with a fast inward
current of up to 5 nA at a holding potential of −70 mV. The current is reversed at
a holding potential close to 0 mV, indicating the activation of non-specific cation
channels – that is, nAChRs.
The whole-cell currents elicited by the application of 1 μM nithiazine (6) and
the commercial open-chain neonicotinoids nitenpyram (8), acetamiprid (9), and
clothianidin (12), are shown in Figure 32.1.3. In the H. virescens preparation, each
of these neonicotinoids acts as an agonist on the nAChR, though the potency
and agonistic efficacy of each were quite different. Typically, the five-membered
imidacloprid (7) and the open-chain clothianidin (12) were the most potent, with
an EC50 of 0.3 μM (Table 32.1.4).
1 μM: Nithiazine (6) Acetamiprid (9) Nitenpyram (8) Clothianidine (12)
400 pA
1s
Heliothis neurons
1.0
Rel. response [1000 μM ACh = 1]
0.8
0.6
0.4 EC50
[μM] nH
0.2 Nithiazine 12.3 1.1
Acetamiprid 1.5 1.0
Nitenpyram 2.1 1.2
0.0
Clothianidine 0.3 0.96
0.0001 0.001 0.01 0.1 1 10 100 1000

Agonist concentration [μM]
Figure 32.1.3 Whole-cell current responses the relative amplitude elicited by 1000 μM
of a neuron isolated from the central ner- ACh. EC50 -values given correspond to the
vous system of Heliothis virescens, after the half-maximal activation of nAChR by each ag-
application of different neonicotinoids. The onist. The Hill coefficient (nH ) of all tested
dose–response curve was fitted by the Hill compounds was close to 1. Upper inset:
equation. All currents were first normal- Corresponding responses for the neonicoti-
ized to mean amplitudes elicited by 10 μM noids at 1 μM (holding potential −70 mV).
ACh before and after each test concentra- All currents were obtained from the very
tion was applied, and then normalized to same neuron [146].
In the case of 7 there was a good agreement with electrophysiological measure-

ments recorded from isolated cockroach neurons, where the EC50 was 0.36 μM
[233]. Neonicotinoids such as 8, 9, and the natural toxin (±)-epibatidine (18) ex-
hibited EC50 -values of between 1 and 2 μM; similar values were observed for the
cockroach preparation (EC50 between 0.5 and 0.7 μM for 18 and 9, respectively),
while nithiazine (6) showed the lowest potency (EC50 = 10 μM). Both of the com-
mercial open-chain neonicotinoids (8 and 12) were full agonists, whereas 7, 9, and
18 were partial agonists. With isolated cockroach and locust neurons [234] , 7 was
found to act as a partial agonist on insect nAChRs; such partial agonistic action
was also identified with chicken α4β2 nAChRs, and on a hybrid nAChR formed
by the coexpression of a Drosophila α-subunit (SAD) with chicken β2-subunit in
Xenopus oocytes. Imidacloprid (7) was shown to activate very small inward currents
in clonal rat phaeochromocytoma 12 (PC 12) cells, thus also indicating partial
agonistic actions [235]. Single-cell analysis revealed that 7 activates predominantly
Table 32.1.4 Comparison between electrophysiological and

[3 H]-7 displacement potencies for different neonicotinoids
6–9, 12, and (±)-epibatidin (18) on insect nAChRs.
Compound n EC50 (μM ± SD) Relative efficacy [3 H]IMI pI50

(1 mM ACh = 1)
6 4 9.60 ± 3.20 0.79 ± 0.06 6.8

7 4 0.31 ± 0.15 0.14 ± 0.02 9.3
8 3 1.66 ± 0.38 0.98 ± 0.07 8.6
9 3 1.07 ± 0.37 0.56 ± 0.05 8.7
12 3 0.33 ± 0.03 0.99 ± 0.08 9.2
(±)-18 3 1.69 ± 0.79 0.20 ± 0.05 6.2
Electrophysiological data (EC50 and relative (agonist) efficacy) were obtained from neuron cell bodies
isolated from the CNS of H. virescens. EC50 and relative efficacy values represent the mean of separate
experiments on different neurons. The inhibition of [3 H]-7 binding to nAChR in housefly head
membrane preparations by the compounds is expressed as pI50 value (pI50 values (= − log M)
correspond to the concentration of cold ligand displacing 50% of bound [3 H]-7 from housefly head
membranes.
IMI, imidacloprid.
Reproduced with permission from Ref. [146].
a subconductance of approximately 10 pS, whereas in the main ACh activated a

high-conductance state with 25 pS. Multiple conductance states were also observed
in an insect nAChR reconstituted into planar lipid bilayers [236], and on locust
neurons [237].
32.1.8.1 Correlation between Electrophysiology and Radioligand-Binding Studies

A good correlation was identified between electrophysiological measurements,
using isolated Heliothis neurons, and radioligand-binding studies on housefly head
membranes with regards to the affinity of the ligand to nAChRs (Figure 32.1.4).
This correlation for the commercial neonicotinoids 8, 9, and 12 may indicate that
houseflies (binding data) and tobacco budworms (electrophysiology) have similar
binding sites for 7 and related compounds.
The high-affinity [3 H]-7 binding site was shown to be conserved in terms of
neonicotinoid sensitivity and specificity across a broad range of insects. The results
of biochemical investigations, using the displacement of [3 H]-7 as a radioligand
in many different insect membrane preparations [including M. persicae, B. tabaci,
N. cincticeps (Homoptera), Manduca sexta, H. virescens (Lepidoptera), Lucilia seri-
cata, D. melanogaster (Diptera) P. americana (Orthoptera), and Ctenocephalides felis
(Siphonaptera)] have indicated that many – if not all – insects have high numbers
of specific imidacloprid-binding sites, with Kd values of between 1 and 10 mM.
As with 7, all neonicotinoids bind with high affinity (I50 s ∼1 mM) to [3 H]-7
binding sites on nAChRs (Table 32.1.5). Whereas, nithiazine (6), the open-chain
compounds (8, 9, and 12), and the five-membered compounds (7 and 11) have
low I50 -values (in the range 0.5 to 2 mM), N-methylimidacloprid (I50 = 1600 mM)
Electrophysiology [pEC50]
7
Imidacloprid (7)
Anatoxin
6 Epibat.
Acetamiprid (9)
Nicotine
Nitenpyram (8)
Cytisine
5
ACh Nithiazine (6)
5 6 7 8 9 10 11
[3H]imidacloprid replacement [pI50]
Figure 32.1.4 Comparison between elec- to the half-maximal activation of nAChR

trophysiological and binding potencies of by each agonist. Binding data: pI50 -values
different neonicotinoids (6–9) and nicoti- (= –log M) correspond to the concentra-
noids. Electrophysiological data were ob- tion of cold ligand displacing 50% of bound
tained from neuron cell bodies isolated from [3 H]imidacloprid from housefly head mem-
the central nervous system of H. virescens. branes [146].
The pEC50 -values (= –log M) correspond
and the six-membered compound thiamethoxam (10; I50 = 5000 mM) showed only
a weak activity in displacing [3 H]-7 from its nAChR binding site in housefly
head membrane preparations. In other words, 10 is up to 10 000-fold less active
than other neonicotinoids, such as 11 (I50 = 0.5 mM). In attempting to clarify
this difference, various pharmacokinetic studies were conducted in a range of
insect species. Recently, the results of further investigations showed that the
six-membered compound 10 could be rapidly metabolized as a prodrug [238–241]
to open-chain clothianidin (12) (see Chapter 32.2.1), which itself demonstrates a
high affinity to nAChRs in both binding assays and whole-cell voltage clamp studies.
In addition, Kayser et al. [242, 243] have presented an alternative explanation for
the obvious lack of 7 competition with all known tritiated nAChR ligands.
Only the homopteran species appeared to have an additional very high-affinity
binding site, however. In general, the pI50 -values obtained by displacement of
specifically bound [3 H]-7 from housefly head membrane were two to four orders
of magnitude higher than the electrophysiologically determined pEC50 -values
obtained from isolated Heliothis neurons (cf. different vertebrate nAChRs) [65].
One possible explanation for this might be that each nAChR can exist in multiple
stages – that is, a resting state, an active (open) state, and one or more desensitized
state(s) – each of which has different affinities for the ligands. The active state has a
Table 32.1.5 Displacement of [3 H]-7 by different neonicoti-

noids 7–12 from nAChR preparations from housefly head
membranes, expressed as I50 (in nM; this represents the con-
centration needed to displace half of the radioligand from its
binding site).
Neonicotinoid I50 (nM)
Imidacloprid (7) 0.79

Nitenpyram (8) 2.00
Acetamiprid (9) 1.26
Thiamethoxam (10) 5000.00
Thiacloprid (11) 0.50
Clothianidin (12) 0.60
N-Methylimidacloprid 1600.00
low affinity for ACh (Kd from about 10 to 1000 μM), whereas the desensitized state(s)
has a higher affinity (Kd from about 10 mM to 1 μM) for nicotinic ligands [244].
The kinetics of the transitions between these states have been resolved for Tor-
pedo nAChRs in vitro. The rate of isomerization between the resting and active
state lies in the microsecond to millisecond timescale, and within the desensitized
state over a timeframe of seconds to minutes. Because binding studies are con-
ducted over a timescale of minutes to hours, the results may reflect an interaction
with the desensitized state(s), whereas electrophysiological studies measure the
interaction of ligands with the active state. Taking this into consideration, there
is – surprisingly – a direct correlation between electrophysiological and biochemi-
cal binding studies for natural alkaloids such as 1, cytosine, (–)-epibatidine (18),
and anatoxin (Figure 32.1.4). For these compounds, the pI50 - and pEC50 -values
were in the same range, with a good correlation. Generally, natural alkaloids
such as 1 and 18 exhibit an agonistic potency in electrophysiological assays on
isolated cockroach neurons and locust neurons, which is comparable to that of
highly insecticidal neonicotinoids such as 7. In 1998, Matsuda et al. [228], using a
hybrid receptor formed by coexpression of the Drosophila α subunit SAD with the
chicken β2, observed a comparable agonistic potency for both 1 and 7; however, the
agonistic potency of 18 was about two orders of magnitude greater. In contrast, all
binding studies using [3 H]-7 on housefly head membranes, whitefly preparations,
and Myzus preparations have indicated that imidacloprid (7) has a considerably
higher potency in replacing specifically bound [3 H]-7 than (S)-(−)-nicotine (1).
32.1.8.2 nAChR Agonists versus Antagonists

The main advantage of electrophysiological measurements over biochemical bind-
ing assays is their ability to distinguish between agonists and antagonists of the
nAChRs. This functional difference in the MoA of ligands with high specificity
for the nAChR is extremely important for insecticidal potency. Electrophysiolog-
ical measurements in isolated housefly neurons showed that compounds which
acted agonistically on nAChR were, in general, insecticidal; this was verified by

compounds 7–13, which already have been introduced to the market.
The agonist actions of commercial neonicotinoids were also investigated using
single-electrode voltage–clamp electrophysiology on nAChRs expressed by neurons
isolated from the thoracic ganglia of P. americana (L) [245]. Based on the maximal
inward currents, neonicotinoids could be allocated to two subgroups (as defined
above): (i) the five-membered ring systems 7 and 11, as well as 1, were relatively
weak partial agonists, causing only 20–25% of the maximal ACh current; and
(ii) open-chain compounds (e.g., 12), which were much more effective agonists,
producing 60–100% of the maximal ACh current. Thiamethoxam (10), even at
100 μM, failed to cause an inward current and showed no competitive interaction
with other neonicotinoids on nAChRs; this indicated that it is not a direct-acting
agonist, nor antagonist [246].
In such context, all neonicotinoids form part of a single MoA group 4A, as defined
by the Insecticide Resistance Action Committee (IRAC) for resistance management
purposes. Subsequently, in studies with insect nAChRs, both nitenpyram (8) and
clothianidin (12) were found to act as full agonists, whereas acetamiprid (9) and
imidacloprid (7) acted as partial agonists.
In contrast, the fully antagonistic neonicotinoids were shown to have a very
limited insecticidal efficacy. Such a general observation was supported by other
investigations highlighting the positive correlation between nerve activity induced
in cockroach preparations, and the insecticidal activity of neonicotinoids against
green rice leafhopper (N. cincticeps).
32.1.9
Molecular Features of a nAChR Agonists
The selectivity of neonicotinoids for insect over vertebrate nAChRs is likely to

result from a selective recognition by insect nAChRs of its structural features, and
vice versa. In an attempt to elucidate the mechanism of selectivity, the structural
features of neonicotinoids and insect nAChRs contributing to this selectivity have
been examined.
Although both neonicotinoids 7–14 and nicotinoids (e.g., 1) have common
structural features, they have different protonation states at physiological pH. As
all insect-selective neonicotinoids 7–14 are neither acids nor bases at pH 4–10,
the electronegative pharmacophore (bioisosteric electron-withdrawing groups 7,
10, 12–14 [=NNO2 ]; 6, 8 [=CHNO2 ]; 9, 11 [=NCN]) plays a crucial role in
the high affinity and selectivity for insect nAChRs. In contrast, the nitrogen
in the N-methyl-pyrrolidine ring of 1 is mostly protonated, having a positive
charge. Based on the fact that both nitrogens in the imidazoline ring of 7 are
conjugated with the N-nitro group, the 2-N-nitro-imino-imidazolidine moiety of 7
has a planar structure, as demonstrated with X-ray crystallography. Subsequently,
semi-empirical molecular-orbital calculations (method PM3 combined with the
AMSOL program) have shown that the nitrogens in the imidazolidine ring of 7 are
changed to positive as soon as the N-nitro group oxygens have formed a H-bond
insects mammals
loop D loop D
HN HN
OH loop A OH loop A
Cl Cl
S NH OH S NH OH
N N
N N
S O S
N N N H
H (+)
OH O OH
H
H
loop C N (+)
NH loop C NH
H
loop B loop B
Figure 32.1.5 Binding subsite specificity shown as hypothetical schematic models for the chloronicotinyl insecticide (CNI) imida-
cloprid (7) binding in the insect nAChR and nicotinoid N-des-nitro-7 binding in the mammalian nAChR, each at the ACh agonist site.
32.1 Nicotinic Acetylcholine Receptor Agonists: Target and Selectivity Aspects
1155
with the positively charged ammonium cation. These results suggest that Lys or
Arg in most insect non-α subunits of the nAChRs could play an important role
in determining their neonicotinoid sensitivity. The basic residues may interact
with the N-nitroimino group of neonicotinoids (e.g., 7, 10, and 12–14) through
electrostatic forces and H-bonding in a cationic subsite of the insect nAChRs [247,
248], suggesting a topological divergence of the agonist-binding subsites in insect
and vertebrate nAChRs (see also Chapter 32.1.8.2; Figure 32.1.5).
Recently, electrophysiological studies on native nAChRs and on wild-type and mu-
tagenized recombinant nAChRs, have shown that the basic residues (particularly in
loop D of insect nAChRs) are likely to interact electrostatically with the N-nitroimino
group of the neonicotinoid insecticides [249]. The coplanar system between the
electronegative pharmacophore and guanidine–amidine moiety extends the con-
jugation, and facilitates a negative charge flow towards the electron-withdrawing
group; this will enhance any interaction with the proposed cationic subsite, such as
Lys and Arg in insect nAChRs. In addition, the high-resolution crystal structures
of L-AChBP with 7 and 12 have shown that the guanidine moiety in both cases
will stack with tyrosine (Tyr) 185, whereas the N-nitroimino group of 7 (but not
of 12) will form an H-bond with Gln55. The H-bond of NH at position 1 with the
backbone carbonyl group of Trp143, offers – in the case of 12 – an explanation for
the diverse actions of neonicotinoids in insect nAChRs. Furthermore, a water or
solvent molecule is captured close to the pyridine or thiazole nitrogen bridging to
relevant amino acids in the active ingredient binding pocket [250].
Characteristic for several agonists (such as 7 and 11), loop C largely envelops
the ligand, positioning the aromatic side chains to interact with conjugated and
hydrophobic regions of the neonicotinoids; this suggestion is consistent with the
results of solution-based photoaffinity labeling studies [251].
In contrast, cation–π interaction is a prominent feature in agonist recognition
by the neurotransmitter-gated vertebrate nAChR [81]. The nicotinoids – including
1, 18, and the N-unsubstituted imine (des-N-nitro or des-N-cyano) analogs of
neonicotinoids (cationic compounds) – demonstrate a cationic character and can
undergo this specific cation–π interaction with Trp at mammalian neuronal
nAChR subsite at the α4β2 interface. Thus, binding subsite specificity will play a
major role in the selective toxicity of neonicotinoids in insects, and of protonated
nicotinoids in mammals.
32.1.10
Conclusions
To date, it remains impossible to create a perfect model of the insect native nAChR
in terms of its structure and diversity. Moreover, whilst the identification and char-
acterization of insect nAChR subtypes remains an important area of research, the
provision of such data may lead to a new era of subtype-selective insecticides with
specific biological profiles and maximal safety margins. In the past, the neonicoti-
noids have been identified as highly effective probes for the structural investigation
of insect nAChRs; indeed, over the past 10 years a wide variety of high-resolution
References 1157
crystal structures of soluble homopentameric AChBPs from different snail species

such as L. stagnalis and A. californica (which are homologs to the extracellu-
lar N-terminal LBD of nAChRs) have been obtained. Both, Lymnea-AChBP and
Aplysia-AChBP, demonstrate a distinct pharmacology that most likely reflects the
differences that might be expected between the nAChRs of mammals and insects.
The crystal structures of the AChBPs have demonstrated a conserved architectural
fold, and provided the theoretical foundation for the construction of homology
models for the LBDs of insect nAChRs [252]. This, in turn, would serve as a
useful basis for in-silico docking simulations, or aid in the virtual screening of
chemical libraries to identify new insecticidally active nAChR ligands acting on
these practically relevant channels. Although the models of nAChR extracellular
domains represent so-called frozen ‘‘snapshots’’ of a particular state constrained
by the crystal structure of AChBP [253], the ligand–receptor interactions of these
AChBP subtypes have been established by the application of photoaffinity labeling
and high-resolution crystal structures of AChBP-neonicotinoid complexes with im-
idacloprid (7), thiacloprid (11), and clothianidin (12), to demonstrate their binding
conformations. The first insights into the 3-D structure of the ion channel, in its
closed and open conformations, led to a refined model of the membrane-associated
Torpedo nAChR at 4 Å resolution, which in turn confirmed that all channels of the
Cys-loop LGIC-superfamily were constructed around the same global principle.
Whilst, in the past, the AChBP has been employed to resolve the various models
of (S)-(−)-nicotine (1), ACh and neonicotinoid binding, it may also provide novel
explanations for important issues on both the nAChR and the ligand site. This may
be achieved by addressing topics such as charge compensation and inter-nitrogen
distances in the nicotinoid or neonicotinoid pharmacophore.
References
1. Jeschke, P., Nauen, R., Schindler, M., 7. Lee, S.J., Tomizawa, T., and Casida,
and Elbert, A. (2011) J. Agric. Food J.E. (2003) J. Agric. Food Chem., 51,
Chem., 59, 2897–2908. 2646–2652.
2. Konishi, K. (1970) Agric. Biol. Chem., 8. Soloway, S.B. Henry, A.C.
34, 935–940. Kollmeyer, W.D. Padgett, W.M.
3. Sakai, M. (1969) Rev. Plant Prot. Res., 2, Powell, J.E. Roman, S.A. Tiemann,
17–28. C.H. Corey, R.A., and Horne, C.A.
(1978) Pesticide and Venom Neu-
4. Konishi, K. (1968) Agric. Biol. Chem.,
rotoxicology (eds D.L. Shankland,
32, 678–679.
R.M. Hollingworth, and T. Smyth
5. Richter, R., Otto, D., and Mengs, H.J.
Jr), Plenum Press, New York, pp.
(1989) in Chemistry of Plant Protection
153–158.
2: Degradation of Pesticides, Desiccation 9. Kollmeyer, W.D., Flattum, R.F., Foster,
and Defoliation, ACh-Receptors as Tar- J.P., Powel, J.E., Schroeder, M.E., and
gets (eds G. Haug and H. Hoffmann), Soloway, S. (1999) in Neonicotinoid
Springer-Verlag, Berlin, Heidelberg, pp. Insecticides and the Nicotinic Acetyl-
157–195. choline Receptor (eds I. Yamamoto and
6. Berg, W. and Knutti, H.J. (1975) Proc. J.E. Casida), Springer Press, Berlin,
Br. Insectic. Fungic. Conf., 2, 683–691. Heidelberg, New York, pp. 71–89.
10. Yamamoto, I. and Casida J.E. (eds) Worden, T.V., and Thibault, S.T. (1998)
(1999) Nicotinoid Insecticides and J. Econ. Entomol., 91, 1277–1283.
the Nicotinic Acetylcholine Receptor, 25. Mertz, F.P. and Yao, R.C. (1990) Int. J.
Springer-Verlag, Tokyo, p. 300. Syst. Bacteriol., 40, 34–39.
11. Thompson, G.D., Dutton, R., and 26. Bret, B.L., Larson, L.L., Schoonover,
Sparks, T.C. (2000) Pest Manage. Sci., J.R., Sparks, T.C., and Thompson, G.D.
56, 696–702. (1997) Down to Earth, 52 (1), 6–13.
12. Copping, L.G. and Menn, J.J. (2000) 27. Waldron, C., Matsuhima, P., Rosteck,
Pest Manage. Sci., 56, 651–676. P.R., Broughton, M.C., Turner, J.,
13. Crouse, G.D., Dripps, J.E., Orr, N.T., Madduri, K., Crawford, K.P., Merlo,
Sparks, C., and Waldron, C. (2007) in D.J., and Baltz, R.H. (2001) Chem.
Modern Crop Protection Compounds, vol. Biol., 8, 487–499.
3 (eds W. Krämer and U. Schirmer), 28. Salgado, V.L. and Sparks, T.C. (2010)
John Wiley & Sons, Inc., New York, in Insect Control, Biological and Syn-
pp. 1013–1031. thetic Agents (eds G.I. Lawrence
14. Nitta, S. (1934) Yakugaku Zasshi, 54, and S.S. Gill), Academic Press, pp.
648–652. 207–246.
15. Okaichi, T. and Hashimoto, Y. (1962) 29. Loso, M.R., Nugent, B.M., Huang,
Agric. Biol. Chem., 26, 224–227. J.X., Rogers, R.B., Zhu, Y., Renga,
16. Narahashi, T. (1973) in Marine Pharma- J.M., Hegde, V.B., and Demark, J.J.
cognosy. Actions of Marine Biotoxins at (2007) Patent WO 095,229 A2 (Dow
the Cellular Level (eds D.F. Martin and Agrosciences LLC).
G.M. Padilla), Academic Press, New 30. Matsuda, K., Shimomura, M., Ihara,
York, pp. 107–126. M., Akamatsu, M., and Sattelle, D.B.
17. Sakai, M. (1967) Botyu-Kagaku, 32, (2005) Biosci. Biotechnol. Biochem., 69,
21–33. 1442–1452.
18. Sattelle, D.B., Harrow, I.D., David, J.A., 31. Tomizawa, M. and Casida, J.E. (2009)
Pelhate, M., Callec, J.J., Gepner, J.I., Acc. Chem. Res., 42, 260–269.
and Hall, L.M. (1985) J. Exp. Biol., 118, 32. Honda, H., Tomizawa, M., and Casida,
37–52. J.E. (2006) J. Agric. Food Chem., 54,
19. Elbert, A., Nauen, R., and Leicht, W. 3365–3371.
(1998) in Insecticides with Novel Mode of 33. Jeschke, P. (2007) in Insecticide Design
Action: Mechanism and Application (eds Using Advanced Technologies (eds I.
I. Ishaaya and D. Degheele), Springer Ishaaya, R. Nauen, and A.R. Horowitz),
Press, pp. 50–74. Springer, pp. 150–195.
20. Jeschke, P., Schindler, M., and 34. Changeux, J.P. and Edelstein, S.J.
Beck, M. (2002) Br. Crop Protect. Conf. - (1998) Neuron, 21, 959–980.
Pests Dis., 1, 137–144. 35. Karlin, A. (2002) Nat. Rev. Neurosci., 3,
21. Denholm, I., Devine, G., Foster, S., 102–114.
Gorman, K., and Nauen, R. (2002) 36. Unwin, N. (2005) J. Mol. Biol., 346,
Br. Crop Protect. Conf. - Pests Dis., 1, 967–989.
161–168. 37. Le Novère, N. and Changeux, J.P.
22. Nauen, R. and Denholm, I. (2005) (1999) Nucleic Acids Res., 27, 340–342.
Arch. Insect Biochem. Physiol., 58, 38. Legendre, P. (2001) Cell Mol. Life Sci.,
200–215. 58, 760–793.
23. Kirst, H.A., Michel, K.H., Martin, 39. Bormann, J. (2000) Trends Pharmacol.
J.W., Creemer, L.C., Chio, E.H., Yao, Sci., 21, 16–19.
R.C., Nakatsukasa, W.M., Boeck, L., 40. Lukas, R.J., Changeux, J.P.,
Occolowitz, J.L., Paschal, J.W., Deeter, Le Novere, N., Albuquerque, E.X.,
D.B., Jones, N.D., and Thompson, Balfour, D.J., Berg, D.K., Bertrand,
G.D. (1991) Tetrahedron Lett., 32, D., Chiappinelli, V.A., Clarke, P.B.,
4839–4842. Collins, A.C., Dani, J.A., Grady, S.R.,
24. Sparks, T.C., Thompson, G.D., Kirst, Kellar, K.J., Lindstrom, J.M., Marks,
H.A., Hertlein, M.B., Larson, L.L., M.J., Quik, M., Taylor, P.W., and
References 1159
Wonnacott, S. (1999) Pharmacol. Rev., 56. Changeux, J.-P. and Edelstein, S.J.
51, 397–401. (2005) in Nicotinic Acetylcholine Re-
41. Lester, H.A., Dibas, M.I., Dahan, D.S., ceptors: From Molecular Biology to
Leite, J.F., and Dougherty, D.A. (2004) Cognition (ed. O. Jacob), Johns Hopkins
Trends Neurosci., 27, 329–336. University Press, New York, p. 284.
42. Corringer, P.J., Le Novère, N., and 57. Millar, N.S. and Gotti, C. (2009) Neu-
Changeux, J.P. (2000) Annu. Rev. ropharmacology, 56, 237–246.
Toxicol., 40, 431–458. 58. Millar, N.S. (2003) Biochem. Soc. Trans.,
43. Jensen, A.A., Frølund, B., Liljefors, T., 31, 869–874.
and Krogsgaard-Larsen, P. (2005) J. 59. Jones, A.K., Elgar, G., and Sattelle,
Med. Chem., 48, 4705–4745. D.B. (2003) Genomics, 82, 441–451.
44. Beg, A.A. and Jorgensen, E.M. (2003) 60. Jones, A.K. and Sattelle, D.B. (2004)
Nat. Neurosci., 6, 1145–1152. BioEssays, 26, 39–49.
45. Ranganathan, R., Cannon, S.C., and 61. Jones, A.K., Grauso, M., and Sattelle,
Horwitz, H.R. (2000) Nature, 408, D.B. (2005) Genomics, 85, 176–187.
470–475. 62. Littleton, J.T. and Ganetzky, B. (2000)
46. Vassilatis, D., Elliston, K.O., Paress, Neuron, 26, 35–43.
P.S., Hamelin, M., Arena, J.P., 63. Sattelle, D.B., Jones, A.K., Sattelle,
Schaeffer, J.M., van der Ploeg, L.H.T., B.M., Matsuda, K., Reenan, R., and
and Cully, D.F. (1997) J. Mol. Evol., 44, Biggin, P.C. (2005) BioEssays, 27,
501–508. 366–376.
64. Grauso, M., Reenan, R.A., Culetto, E.,
47. Engel, A.G., Ohno, K., and Sine, S.M.
and Sattelle, D.B. (2002) Genetics, 160,
(2003) Muscle Nerve, 27, 4–25.
1519–1533.
48. Sattelle, D.B., Jones, A.K., Sattelle,
65. Holladay, M.W., Dart, M.J., and
B.M., Matsuda, K., Reenan, R., and
Lynch, J.K. (1997) J. Med. Chem.,
Biggin, P.C. (2005) BioEssays, 27,
40, 4169–4194.
366–376.
66. D’hoedt, D. and Bertrand, C. (2009) Ex-
49. Miyazawa, A., Fujiyoshi, Y.,
pert Opin. Ther. Targets, 13, 395–411.
Stowell, M., and Unwin, N. (1999)
67. Arias, H.R. (1997) Brain Res. Rev., 25,
J. Mol. Biol., 288, 765–786.
133–191.
50. Sine, S.M. and Engel, A.G. (2006)
68. Le Novere, N., Corringer, P.J., and
Nature, 440, 448–455.
Changeux, J.P. (2002) J. Neurobiol., 53,
51. Albuquerque, E.X., Alkondon, 447–456.
M., Pereira, E.F.R., Castro, N.G., 69. Lloyd, K.G. and Williams, M. (2000) J.
Schrattenholz, A., Barbosa, C.T., Pharmacol. Exp. Ther., 292, 461–467.
Bonfante-Cabarcas, R., Aracava, Y., 70. Jozwiak, K., Ravichandran, S., Collins,
Eisenberg, H.M., and Maelicke, A. J.R., and Wainer, I.W. (2004) J. Med.
(1997) J. Pharmacol. Exp. Ther., 280, Chem., 47, 4008–4021.
1117–1136. 71. Lloyd, K.L. and Williams, M. (2000) J.
52. Hucho, F., Tsetlin, V.I., and Machold, Pharmacol. Exp. Ther., 292, 461–467.
J. (1996) Eur. J. Biochem., 239, 72. Tomizawa, M. and Casida, J.E. (2001)
539–555. Pest Manage. Sci., 57, 914–922.
53. Changeux, J.-P., Galzi, J.L., 73. Kao, P.N. and Karlin, A. (1986) J. Biol.
Devillers-Thiery, A., and Bertrand, D. Chem., 261, 8085–8088.
(1992) Q. Rev. Biophys., 25, 395–432. 74. Krauss, M., Korr, D., Herrmann, A.,
54. Hansen, S.B., Sulzbacher, G., and Hucho, F. (2000) J. Biol. Chem.,
Huxford, T., Marchot, P., Taylor, P., 275, 30196–30201.
and Bourne, Y. (2005) EMBO J., 24, 75. Brisson, A. and Unwin, N. (1984) J.
3635–3646. Cell Biol., 99, 1202–1211.
55. Cassels, B.K., Bermúdez, I., Dajas, F., 76. Toyoshima, C. and Unwin, N. (1990) J.
Abin-Carriquiry, J.A., and Wonnacott, Cell Biol., 111, 2623–2635.
S. (2005) Drug Discov. Today, 10, 77. Hilf, R.J. and Dutzler, R. (2008) Nature,
1657–1665. 452, 375–379.
78. Hilf, R.J. and Dutzler, R. (2009) Nature, Yakel, J.L. (2010) Biochemistry, 49,
457, 115–118. 2279–2287.
79. Schmitt, J.D. (2000) Curr. Med. Chem., 93. Dutertre, S., Ulens, C., Buttner, R.,
7, 749–800. Fish, A., Van Elk, R., Kendel, Y.,
80. Yamamoto, I., Yabuta, G., Hopping, G., Alewood, P.F., Schröder,
Tomizawa, M., Saito, T., Miyamoto, C., Nicke, A., Smit, A.B., Sixma, K.T.,
T., and Kagabu, S. (1995) J. Pestic. Sci., and Lewis, R.J. (2007) EMBO J., 26,
20, 33–40. 3858–3867.
81. Tomizawa, M., Zhang, N., 94. Tsetlin, V., Utkin, U., and Kasheverow,
Durkin, K.A., Olmstead, M.M., and I. (2009) Biochem. Pharmacol., 78,
Casida, J.E. (2003) Biochemistry, 42, 720–731.
7819–7827. 95. Dellisanti, C.D., Yao, Y., Stroud, J.C.,
82. Grutter, T. and Changeux, J.P. (2001) Wang, Z.Z., and Chen, L. (2007) Nat.
Trends Biochem. Sci., 26, 459–463. Neurosci., 10, 953–962.
83. Karlin, A. (2000) Nat. Rev. Neurosci., 3, 96. Tomizawa, M., Maltby, D., Talley,
102–114. T.T., Durkin, K.A., Medzihradszky,
84. Corringer, P.-J., Le Novère, N., and K.F., Burlingame, A.L., Taylor, P., and
Changeux, J.-P. (2000) Annu. Rev. Casida, J.E. (2008) Proc. Natl Acad. Sci.
Pharmacol. Toxicol., 40, 431–458. USA, 105, 1728–1732.
85. Brejc, K., van Dijk, W.J., Klaassen, 97. Talley, T.T., Harel, M., Hibbs, R.E.,
R.V., Schuurmans, M., van der Oost, Radic, Z., Tomizawa, M., Casida, J.E.,
J., Smit, A.B., and Sixma, T.K. (2001)
and Taylor, P. (2008) Proc. Natl Acad.
Nature, 411, 269–276.
Sci. USA, 105, 7606–7611.
86. Smit, A.B., Syed, N.I., Schaap, D.,
98. Le Novere, N., Grutter, T., and
van Minnen, J., Klumperman, J., Kits,
Changeux, J.-P. (2002) Proc. Natl Acad.
K.S., Lodder, H., van der Schors, R.C.,
Sci. USA, 99, 3210–3215.
van Elk, R., Sorgedrager, B., Brejc, K.,
99. Henchman, R.H., Wang, H.-I., Sine,
Sixma, T.K., and Geraerts, W.P. (2001)
S.M., Taylor, P., and McCammon, J.A.
Nature, 411, 261–268.
(2003) Biophys. J., 85, 3007–3018.
87. Celie, P.H., Klaasen, R.V., van
100. Dougherty, D.A. (1996) Science, 271,
Rossum-Fikkert, S.E., van Elk, R.,
163–168.
van Nierop, P., Smit, A.B., and
101. Ma, J.C. and Dougherty, D.A. (1997)
Sixma, T.K. (2005) J. Biol. Chem.,
280, 26457–26466. Chem. Rev., 97, 1303–1324.
88. Ma, Z., Huang, J., Sun, J., Wang, G., 102. Zacharias, N. and Dougherty, D.A.
Li, C., Xie, L., and Zhang, R.J. (2007) J. (2002) Trends Pharmacol. Sci., 23,
Biol. Chem., 282, 23253–23263. 281–287.
89. Hansen, S.B., Talley, T.T., Radic, Z., 103. Zhong, W., Gallivan, J.P., Zhang, Y.,
and Taylor, P. (2004) J. Biol. Chem., Li, L., Lester, H.A., and Dougherty,
279, 24197–24202. D.A. (1998) Proc. Natl Acad. Sci. USA,
90. Rucktooa, P., Smit, Ab., and Sixma, 95, 12088–12093.
T.K. (2009) Biochem. Pharmacol., 78, 104. Schapira, M., Abagyan, R., and
777–787. Totrov, M. (2002) BMC Struct. Biol.,
91. Celie, P.H., Kasheverow, I.E., 2, 1–8.
Mordvintsev, D.Y., Hogg, R.C., 105. Cashin, A.L., Petersson, E.J., Lester,
van Nierop, P., van Elk, R., van H.A., and Dougherty, D.A. (2005) J.
Rossum-Fikkert, S.E., Zhamak, M.N., Am. Chem. Soc., 127, 350–356.
Bertrand, D., Tsetlin, V., Sixma, T.K., 106. Miyazawa, A., Fujiyoshi, Y., and
and Smit, A.B. (2005) Nat. Struct. Mol. Unwin, N. (2003) Nature, 423,
Biol., 12, 1–7. 949–955.
92. McCormack, T., Petrovic, R.M., 107. Taly, A., Delarue, M., Grutter, T.,
Mercier, K.A., DeRose, E.F., Nilges, M., Le Novere, N., Corringer,
Cuneo, M.J., Williams, J., Johnson, P.-J., and Changeux, J.-P. (2005) Bio-
K.L., Lamb, P.W., London, R.E., and phys. J., 88, 3954–3965.
References 1161
108. Hansen, S.B., Sulzenbacher, G., 126. Ihara, M., Okajma, T., Yamashita, A.,
Huxford, T., Marchot, P., Taylor, P., Oda, T., Hirata, K., Nishiwaki,
and Bourne, Y. (2005) EMBO J., 25, H., Morimoto, T., Akamatsu, M.,
3635–3646. Ashikawa, Y., Kuroda, S., Mega, R.,
109. Lee, W.L. and Sine, S.M. (2005) Nature, Kuramitsu, S., Sattelle, D.B., and
438, 243–247. Matsuda, K. (2008) Invert. Neurosci., 8,
110. Thany, S.H., Lenears, G., 71–81.
Raymond-Delpech, V., Sattelle, D.B., 127. Glennon, R.A., Dukat, M., and Liao,
and Lapid, B. (2006) Trends Pharmacol. L. (2004) Curr. Top. Med. Chem., 4,
Sci., 28, 14–22. 631–644.
111. Millar, N.S. and Denholm, I. (2007) In- 128. Sheridan, R.P., Nilakantan, R.,
vert. Neurosci., 7, 53–66. Dixon, J.S., and Venkataraghavan,
112. Gundelfinger, E.D. and Schulze, R. R. (1986) J. Med. Chem., 29, 899–906.
(2000) Insect nicotinic acetylcholine 129. Zhang, N., Tomizawa, M., and Casida,
receptors: genes, structure, physic- J.E. (2004) J. Org. Chem., 69, 876–881.
ochemical and pharmacological 130. de Taey, J. and Zeegers-Huyskens, T.
properties, in Handbook of Experi- (1987) Bull. Soc. Chim., 96, 1–6.
mental Pharmacology: Neuronal Nicotinic 131. Graton, J., Berthelot, M., Gal, J.F.,
Receptors, vol. 44 (eds F. Clementi, Laurence, C., Lebreton, J., Le Questel,
D. Fornasari, and C. Gotti), Springer J.Y., Maria, P.C., and Robbins, R.
Press, Berlin, pp. 497–521. (2003) J. Org. Chem., 68, 8208–8221.
113. Tang, Z. (2002) Xiandai Nongyao, 1,
132. Celie, P.H.N., van Rossum-Fikkert,
1–6.
S.E., van Dijk, W.J., Brejc, K., Smit,
114. Jones, A.K., Brown, L.A., and Sattelle,
A.B., and Sixma, T.K. (2004) Neuron,
D.B. (2007) Invert. Neurosci., 7, 67–73.
41, 907–914.
115. Breer, H. and Sattelle, D.B. (1987) J.
133. Shimomura, M., Yokata, M.,
Insect Physiol., 33, 771–790.
Amura, M., Matsuda, K., Akamatsu,
116. Jones, A.K., Raymond-Delpech, V.,
M., Sattelle, D.B., and Komai, K. (2003)
Thany, S.H., Gauthier, M., and
Brain Res., 991, 71–77.
Sattelle, D.B. (2006) Genome Res.,
134. Kagabu, S. (2011) J. Agric. Food Chem.,
16, 1422–1430.
59, 2887–2896.
117. Shao, Y.M., Dong, K., and Zhang, C.X.
(2007) BMC Genomics, 8, 324. 135. Eldefrawi, A.T., Bakry, N.M., Eldefrawi,
118. Jones, A.K. and Sattelle, D.B. (2007) M.E., and Tsai, M.-C. (1980) Mol. Phar-
BMC Genomics, 8, 327–332. macol., 17, 172–179.
119. Holt, R.A. et al. (2002) Science, 298, 136. Sherby, S.M., Eldefrawi, A.T., David,
96–97. J.A., Sattelle, D.B., and Eldefrawi, M.E.
120. Lansdell, S.J. and Millar, N.S. (2004) J. (1986) Arch. Insect Biochem. Physiol., 3,
Neurochem., 90, 479–489. 431–445.
121. Zhang, A., Kayser, H., Maienfisch, P., 137. Tomizawa, M., Otsuka, H.,
and Casida, J.E. (2000) J. Neurochem., Miyamoto, T., Eldefrawi, M.E., and
75, 1294–1303. Yamamoto, I. (1995) J. Pestic. Sci., 20,
122. Unwin, N., Miyazawa, A., Li, J., and 57–64.
Fujiyoshi, Y. (2002) J. Mol. Biol., 319, 138. Raymond-Delpech, V., Matsuda, K.,
1165–1176. Sattelle, B.M., Rauh, J.J., and Sattelle,
123. Sine, S.M. (2002) J. Neurobiol., 53, D.B. (2005) Invert. Neurosci., 5,
431–446. 119–133.
124. Dutertre, S. and Lewis, R.J. (2004) Eur. 139. Bettini, S., D’Ajello, V., and Maroli, M.
J. Biochem., 271, 2327–2334. (1973) Pestic. Biochem. Physiol., 3,
125. Shimomura, M., Yokata, M., Ihara, M., 100–205.
Akamatsu, M., Sattelle, D.B., and 140. Nagata, K., Iwanaga, Y., Shono, T., and
Matsuda, K. (2006) Mol. Pharmacol., 70, Narahashi, T. (1997) Pestic. Biochem.
1255–1263. Physiol., 59, 119–128.
141. Raymond-Delpech, V., Ihara, M., (eds I. Yamamoto and J.E. Casida),
Coddou, C., Matsuda, K., and Sattelle, Springer, New York, p. 109–125.
D.B. (2003) Invert. Neurosci., 5, 29–35. 158. Nauen, R., Ebbinghaus, U., and
142. Liu, M.-Y. and Casida, J.E. (1993) Tietjen, K. (1999) Pestic. Sci., 55,
Pestic. Biochem. Physiol., 46, 40–46. 608–610.
143. Lui, M.-Y., Lanford, J., and Casida, 159. Nauen, R., Ebbinghaus-Kintscher, U.,
J.E. (1993) Pestic. Biochem. Physiol., 46, Salgado, V.L., and Kaussmann, M.
200–206. (2003) Pest. Biochem. Physiol., 76,
144. Schröder, M.E. and Flattum, R.F. 55–69.
(1984) Pestic. Biochem. Physiol., 22, 160. Jepson, J.E.C., Brown, L.A., and
148–160. Sattelle, D.B. (2006) Invert. Neurosci., 6,
145. Sattelle, D.B., Buckingham, S.D., 33–40.
Wafford, K.A., Sherby, S.M., Bakry, 161. Kagabu, S. (1997) Rev. Toxicol., 1,
N.M., Eldefrawi, A.T., Eldefrawi, M.E., 75–129.
and May, T.E. (1989) Proc. R. Soc. Lond. 162. Matsuda, K., Buckingham, S.D.,
Ser. B: Biol. Sci., 237, 501–514. Kleier, D., Rauh, J.J., Grause, M.
146. Nauen, R., Ebbinghaus-Kintscher, A., et al. (2001) Trends Pharmacol. Sci., 22,
Elbert, A., Jeschke, P., and Tietjen, K. 573–578.
(2001) in Biochemical Sites of Insecticide 163. Tomizawa, M. and Casida, J.E. (2003)
Action and Resistance (ed. I. Ishaaya), Annu. Rev. Entomol., 48, 339–364.
Springer Press, pp. 77–105. 164. Salgado, V.L., Sheets, J.J.,
Watson, G.B., and Schmidt, A.L. (1998)
147. Nishimura, K., Kanda, Y., Okazawa, A.,
and Ueno, T. (1994) Pestic. Biochem.
165. Salgado, V.L., Watson, G.B., and
Physiol., 50, 51–59.
Sheets, J.J. (1997) Proceedings of the
148. Nishimura, K., Tanaka, M., Iwaya, K.,
Beltwide Cotton Conference, vol. 2, Na-
and Kagabu, S. (1998) Pestic. Biochem.
tional Cotton Council, Memphis, pp.
Physiol., 62, 172–178.
1082–1084.
149. Abbink, K. (1991) Pflanz.-Nachr. Bayer
166. Salgado, V.L. (1998) Pestic. Biochem.
(Ger. Ed.), 44, 183–195.
Physiol., 60, 91–102.
150. Bai, D., Lummis, S.C.R., Leicht, W.,
167. Thompson, G. and Hutchins, S. (1999)
Breer, H., and Sattelle, D.B. (1991)
Pestic. Outlook, 10, 78–81.
Pestic. Sci., 33, 197–204.
168. Sparks, T.C., Crouse, G.D., and
151. Salgado, V.L. and Saar, R. (2004) J. Durst, G. (2001) Pest Manage. Sci.,
Insect Physiol., 50, 867–879. 57, 896–905.
152. Liu, M.-Y., Lanford, J., and Casida, 169. Thompson, G.D. and Sparks, T.C.
J.E. (1993) Pestic. Biochem. Physiol., 46, (2002) in Advancing Sustainability
200–206. through Green Chemistry and Engineer-
153. Tomizawa, M., Latli, B., and Casida, ing, ACS Symposium Series, vol. 823
J.E. (1999) in Nicotinoid Insecticides and (eds R.L. Lankey and P.T. Anastas),
the Nicotinic Acetylcholine Receptor (eds American Chemical Society, Washing-
I. Yamamoto and J.E. Casida), Springer ton, DC, pp. 61–73.
Press, Tokyo, pp. 271–192. 170. Watson, G.B. (2001) Pestic. Biochem.
154. Sone, S., Nagata, K., Tsuboi, S., and Physiol., 71, 20–28.
Shono, T. (1994) J. Pestic. Sci., 19, 171. Dunbar, S.J., Goodchild, J.A., and
69–72. Cutler, P.M. (1998) 9th International
155. Nauen, R. (1995) Pestic. Sci., 44, Congress of Pesticide Chemistry, Au-
145–153. gust 2–7, London, Book of Abstracts,
156. Mehlhorn, H., Mencke, N., and vol. 1, pp. 4B–040.
Hansen, O. (1999) Parasitol. Res., 172. Perry, T., McKenzie, J.A., and
85, 625–637. Batterham, P. (2007) Insect Biochem.
157. Wollweber, D. and Tietjen, K. (1999) Mol. Biol., 37, 184–188.
in Neonicotinoid Insecticides and 173. Watson, G.B., Chouinard, S.W.,
the Nicotinic Acetylcholine Receptor Cook, K.R., Geng, C., Gifford, J.M.,
References 1163
Gustafson, G.D., Hasler, J.M., 191. Methfessel, C. (1992) Pflanz.-Nachr.

Larrinua, I.M., Letherer, T.J., Mitchell, Bayer (Ger. Ed.), 45, 369–380.
J.C., Pak, W.L., Salgado, V.L., and 192. Zwart, R., Oortgiesen, M., and
Sparks, T.C. (2010) Insect Biochem. Mol. Vijverberg, H.P.M. (1994) Pestic.
Biol., 40, 376–384. Biochem. Physiol., 48, 202–213.
174. Matsuda, K. and Sattelle, D.B. (2005) 193. Tomizawa, M., Otsuka, H., Miyamoto,
New Discoveries in Agrochemicals, ACS T., and Yamamoto, I. (1995) J. Pestic.
Symposium Series, vol. 892, American Sci., 20, 49–56.
Chemical Society, Washington, DC, pp. 194. Yamamoto, I., Tomizawa, M., Saito,
172–182. T., Miyamoto, T., Walcott, E.C., and
175. Huang, Y., Williamson, M.S., Sumikawa, K. (1998) Arch. Insect.
Devonshire, A.L., Windass, J.D., Biochem. Physiol., 37, 24–32.
Lansdell, S.J., and Millar, N.S. (1999) J. 195. Chao, S.L. and Casida, J.E. (1997)
Neurochem., 73, 380–389. Pestic. Biochem. Physiol., 58, 77–88.
176. Landsdell, S.J. and Millar, N.S. (2000) 196. Nagata, K., Aistrup, G.L., Song, J.H.,
Neuropharmacology, 39, 671–679. and Narahashi, T. (1996) Neuroreport, 7,
177. Wiesner, P. and Kayser, H. (2000) J. 1025–1028.
Biochem. Mol. Toxicol., 14, 221–230. 197. D’Amour, K.A. and Casida, J.E. (1999)
178. Yao, X., Song, F., Chen, F., Zhang, Y., Pestic. Biochem. Physiol., 64, 55–61.
Gu, J., Liu, S., and Liu, Z. (2008) Insect 198. Ihara, M., Matsuda, K., Otake, M.,
Biochem. Mol. Biol., 38, 384–840. Kuwamura, M., Shimomura, M.,
179. Tomizawa, M., Talley, T.T., Maltby, D., Komai, K., Akamatsu, M., Raymond,
Durkin, K.A., Medzihradsky, K.F., V., and Sattelle, D.B. (2003) Neurophar-
Burlingame, A.L., Taylor, P., and macology, 45, 133–144.
Casida, J.E. (2007) Proc. Natl Acad. Sci. 199. Tomizawa, M. and Casida, J.E. (2005)
USA, 104, 9075–9080. Annu. Rev. Pharmacol. Toxicol., 45,
180. Tomizawa, M. and Casida, J.E. (2011) J. 247–268.
Agric. Food Chem., 59, 2825–2828. 200. Goodman, C.S. and Spitzer, N.C.
181. Depnath, B., Gayen, S., Naskar, S.K., (1980) Receptors for Neurotransmitters,
Roy, K., and Jha, T. (2003) Drug Des. Hormones and Pheromones in Insects,
Discov., 18, 81–89. Elsevier, Amsterdam, pp. 195–307.
182. Schulz-Jander, D.A. and Casida, J.E. 201. Harrow, L.D. and Sattelle, D.B. (1983)
(2002) Toxicol. Lett., 132, 65–70. J. Exp. Biol., 105, 339–350.
183. Dick, R.A., Kanne, D.B., and Casida, 202. Sattelle, D.B., Harrow, I.D., Hue, B.,
J.E. (2005) Chem. Res. Toxicol., 18, Pelhate, M., Gepner, J.I. et al. (1983) J.
317–323. Exp. Biol., 107, 473–489.
184. Honda, H., Tomizawa, M., and Casida, 203. Breer, H. (1988) Neurotox 88: Molecu-
J.E. (2006) Toxicol. Lett., 161, 108–114. lar Basis of Drug and Pesticide Action,
185. Casida, J.E. (2011) J. Agric. Food Chem., Excerpta Medica, Amsterdam, pp.
59, 2923–2931. 301–309.
186. Alyokhin, A., Baker, M., Mota-Sanchez, 204. Restifo, L.L. and White, K. (1990) Adv.
D., Dively, G., and Grafius, E. (2008) Insect. Physiol., 22, 115–219.
Am. J. Potato Res., 85, 395–413. 205. Benson J.A. (1989) in Progress and
187. Houghtling, R.A., Dávilia-Garcia, M.I., Prospects in Insect Control, BCPC Mono-
and Kellar, K.J. (1995) Mol. Pharmacol., graph, vol. 43 (eds N.R. McFarlane
48, 280–287. and A.W. Farnham), British Crop
188. Anand, R., Peng, X., Ballesta, J.J., and Protection Council, pp. 59–70.
Lindstrom, J. (1993) Mol. Pharmacol., 206. Leech, C.A., Jewess, P., Marshall, J.,
44, 1046–1050. and Sattelle, D.B. (1991) FEBS Lett.,
189. Zhang, N., Tomizawa, M., and Casida, 290, 90–94.
J.E. (2004) Neurosci. Lett., 271, 56–59. 207. Cheung, H., Clarke, B.S., and Beadle,
190. Tomizawa, M., Lee, D.L., and Casida, D.J. (1992) Pestic. Sci., 34, 187–193.
J.E. (2000) J. Agric. Food Chem., 48, 208. Tomizawa, M. and Yamamoto, I. (1992)
6016–6024. J. Pestic. Sci., 17, 231–236.
209. Tomizawa, M. and Yamamoto, I. (1993) Lansdell, S.J., and Millar, N.S. (2000)
J. Pestic. Sci., 18, 91–98. Neurosci. Lett., 284, 116–120.
210. Zwart, R., Oortgiesen, M., and 225. Sgard, F., Fraser, S.P., Katkowska, M.J.,
Vijverberg, H.P. (1992) J. Pharmacol., Djamgoz, M.B.A., Dunbar, S.J., and
228, 165–169. Windass, J.D. (1998) J. Neurochem., 71,
211. Tomizawa, M., Latli, B., and Casida, 903–912.
J.E. (1996) J. Neurochem., 67, 226. Yixi, Z., Liu, Z., Han, Z., Song, F., Yao,
1669–1676. X., Shao, Y., Li, J., and Millar, N.S.
212. Buckingham, S.D., Lapied, B., (2009) J. Neurochem., 110, 1855–1862.
Le Corronc, H., Grolleau, F., and 227. Bertrand, D., Ballivet, M., Gomez,
Sattelle, D.B. (1997) J. Exp. Biol., 200, M., Bertrand, S., Phannavong, B.,
2685–2692. and Gundelfinger, E.D. (1994) Eur. J.
213. Lind, R.J., Clough, M.S., Reynolds, Neurosci., 6, 869–875.
S.E., and Early, E.G.P. (1998) Pestic. 228. Matsuda, K., Buckingham, S.D.,
Biochem. Physiol., 62, 3–14. Freeman, J.C., Squire, M.D., Baylis,
214. Gundelfinger, E.D. (1992) Trends H.A., and Sattelle, D.B. (1998) Br. J.
Neurosci., 15, 206–211. Pharmacol., 123, 518–524.
215. Landsdell, S.J., Schmitt, B., Betz, H., 229. Tomizawa, M., Millar, N.S., and
Sattelle, D.B., and Millar, N.S. (1997) J. Casida, J.E. (2005) Insect Biochem.
Neurochem., 68, 1812–1819. Mol. Biol., 35, 1347–1355.
216. Marshall, J., Buckingham, S.D., 230. Ihara, M., Matsuda, K., Shimomura,
Shingai, R., Lunt, G.G., Goosey, M.W.,
M., Sattelle, D.B., and Komai, K.
Darlison, M.W., Sattelle, D.B., and
(2004) Biosci. Biotechnol. Biochem., 68,
Barnard, E.A. (1990) EMBO J., 9,
761–763.
4391–4398.
231. Liu, Z., Williamson, M.S., Lansdell,
217. Amar, M., Thomas, P., Wonnacott, S.,
S.J., Denholm, I., Han, Z., and Millar,
and Lunt, G.G. (1995) Neurosci. Lett.,
N.S. (2005) Proc. Natl Acad. Sci. USA,
199, 107–110.
102, 8420–8425.
218. Hermsen, B., Stetzer, E., Thees, R.,
232. Liu, Z., Han, Z., Zhang, Y., Song, F.,
Heiermann, R., Schrattenholz, A.,
Yao, X., Liu, S., Gu, J., and Millar, N.S.
Ebbinghaus, U., Kretschmer, A.,
(2009) J. Neurochem., 108, 498–506.
Methfessel, Ch., Reinhardt, S., and
233. Orr, N., Shaffner, J., and Watson, G.B.
Maelicke, A. (1998) J. Biol. Chem., 273,
18394–18404. (1997) Pestic. Biochem. Physiol., 58,
219. Sawruk, E., Schloss, P., Betz, H., 183–192.
and Schmitt, B. (1990) EMBO J., 9, 234. Nauen, R., Reckmann, U., Armborst,
2671–2677. S., Stupp, H.P., and Elbert, A. (1999)
220. Schulz, R., Sawruk, E., Mülhardt, Pestic. Sci., 55, 265–271.
C., Bertrand, S., Baumann, A., 235. Nagata, K., Aistrup, G.L., Song, J.-H.,
Phannavong, B., Betz, H., Bertrand, and Narahashi, T. (1996) Neuroreport, 7,
D., Gundelfinger, E.D., and Schmitt, B. 1025–1028.
(1998) J. Neurochem., 71, 853–862. 236. Hancke, W. and Breer, H. (1986)
221. Lansdell, S.J. and Millar, N.S. (2000) Nature, 321, 171–175.
Neuropharmacology, 39, 2604–2614. 237. van den Breukel, I., van Kleef,
222. Sawruk, E., Udri, C., Betz, H., and R.G.D.M., Zwart, R., and Oortgiesen,
Schmitt, B. (1990) FEBS Lett., 273, M. (1998) Brain Res., 789, 263–273.
177–181. 238. Jeschke, P. and Nauen, R. (2004) 228th
223. Eastham, H.M., Lind, R.J., Eastlake, Jl., ACS National Meeting, Philadelphia,
Clarke, B.S., Towner, P., Reynolds, PA, August 22–26, Abstract of Papers
S.E., Wolstenholme, A.J., and AGRO-003 (Chem. Abstr., 655197).
Wonnacott, S. (1998) Eur. J. Neurosci., 239. Nauen, R., Ebbinghaus-Kintscher, U.,
10, 879–889. and Jeschke, P. (2005) 230th ACS
224. Huang, Y., Williamson, M.S., National Meeting, Washington, DC,
Devonshire, A.L., Windass, J.D., August 28–September 1, Abstract
of Papers AGRO-026 (Chem. Abstr., 245. Ihara, M., Brown, L.A., Ishida, C.,
735867). Okuda, H., Sattelle, D.B., and Matsuda,
240. Jeschke, P. and Nauen, R. (2007) Syn- K. (2006) J. Pestic. Sci., 31, 35–40.
thesis and Chemistry of Agrochemicals 246. Tan, J., Galligan, J.J., and
VII, ACS Symposium Series, vol. 948 Hollingworth, R.M. (2007) Neuro-
(eds J.W. Lyga and G. Theodoridis), toxicology, 28, 829–842.
American Chemical Society, Washing- 247. Casida, J.E. and Quistad, G.B. (2004) J.
ton, DC, pp. 51–65. Pestic. Sci., 29, 81–86.
241. Benzidane, Y., Touinsi, S., Motte, E., 248. Wang, Y., Cheng, J., Qian, X., and
Jadas-Hécart, A., Communal, P.-Y., Zhong, L. (2007) Bioorg. Med. Chem.,
Leduc, L., and Thany, S.H. (2010) Pest 15, 2624–2630.
249. Matsuda, K., Kanaoka, S.,
Manage. Sci., 66, 1351–1359.
Akamatsu, M., and Sattelle, D.B. (2009)
242. Kayser, H., Lee, C., Decock, A.,
Mol. Pharmacol., 76, 1–10.
Baur, M., Haettenschwiler, J., and
250. Ohno, I., Tomizawa, M., Durkin, K.A.,
Maienfisch, P. (2004) Pest Manage. Sci.,
Casida, J.E., and Kagabu, S. (2009) J.
60, 945–958.
Agric. Food Chem., 57, 2436–2440.
243. Kayser, H., Wellmann, H., Lee, C., 251. Tomizawa, M., Talley, T.T., Park, J.F.,
Decock, A., Gomes, M., Cheek, B., Maltby, D., Medzihradszky, K.F.,
Lind, R., Bauer, M., Hattenschwiler, J., Durkin, K.A., Cornejo-Bravo, J.M.,
and Maienfisch, P. (2007) in Synthesis Burlingame, A.L., Casida, J.E., and
and Chemistry of Agrochemicals VII, Taylor, P. (2009) J. Med. Chem., 52,
ACS Symposium Series, vol. 948 (eds 3735–3741.
J.W. Lyga and G. Theodoridis), Ameri- 252. Sine, S.M., Wang, H.L., and Gao, F.
can Chemical Society, Washington, DC, (2004) Curr. Med. Chem., 11, 559–567.
pp. 67–81. 253. Grutter, T., Le Novère, N., and
244. Lèna, C. and Changeux, J.-P. (1993) Changeux, J.P. (2004) Curr. Top. Med.
Trends Neurosci., 16, 181–186. Chem., 4, 645–651.
32.2
Chemical Structural Features of Commercialized Neonicotinoids
Peter Jeschke
Introduction
In general, all commercialized neonicotinoids can be divided into open-chain

compounds (see Chapter 32.2.1) and neonicotinoids having ring systems such as
five-membered (see Chapter 32.2.2) and six-membered compounds (see Chapter
32.2.3) that differ in their molecular characteristics. The structural requirements
for both neonicotinoids having open-chain structures and ring-system contain-
ing neonicotinoids consist of different segments, as listed below (Figure 32.2.1;
Tables 32.2.1 and 32.2.2) [1–3].
1) For open-chain compounds, the separate substituents (R1 , R2 ) and for five-
and six-membered ring systems the bridging fragment (R1 -R2 , R1 -Z-R2 ; Z = O,
NMe).
2) The heterocyclic or heteroalicyclic group A with a bridging chain [-CHR-; R = H;
e.g., A-CH2 -: 6-chloro-pyrid-3-ylmethyl (CPM), 2-chloro-1,3-thiazol-5-ylmethyl
(CTM), and (±)-6-tetrahydro-fur-3-ylmethyl (TFM; see Table 32.2.2)].
1166 32 Nicotinic Acetylcholine Receptor Agonists: Target and Selectivity Aspects
(i) Figure 32.2.1 Structural segments for neonicotinoids.
R1 R2
(ii) A N E
R X
Y
(iii)
3) The functional group [= X-Y] as part of the different pharmacophore types

[-N-C(E) = X-Y].
In all seven neonicotinoids launched to date, the methylene group (–CH2 –) is

normally used as the bridging chain. Their pharmacophore (iii in Figure 32.2.1)
can be represented by the group [−N−C(E) = X−Y], where [= X−Y] is an
electron-withdrawing group, while E is NH, NMe, sulfur, or methyl. Due to
the fact that the pharmacophore type can influence the insecticidal activity of
neonicotinoids, the commercialized neonicotinoids have ring systems that differ
in their pharmacophore types [4]:
• Open-chain compounds: [−N−C(E) = CH−NO2 ; E = NHMe] nitroenamines

(nitromethylenes); [−N−C(E) = N−NO2 ; E = NHMe] N-nitroguanidines;
[−N−C(E) = N−CN; E = Me] N-cyanoamidines (see Chapter 32.2.1).
• Five- and six-membered compounds: [−N−C(E) = N−NO2 ; E = NH, NMe]
N-nitroguanidines; [−N−C(E) = N−CN; E = S] N-cyanoamidines (see
Chapters 32.2.2 and 32.2.3, respectively).
Besides its influence on biological activity, the pharmacophore is also responsible

for some specific physico-chemical properties such as photolytic stability, water
solubility, lipophilicity, systemicity, degradation behavior in soil, metabolism in
plants and insects, as well as toxicity to bees and different animals [5–13]. Recently,
the influence of the pharmacophore on neonicotinoid binding conformations
conferring selective nicotinic acetylcholine receptor (nAChR) interactions were
investigated by comparison of two of acetylcholine-binding proteins (AChBPs) (see
Chapter 32.1) [14].
The Term Neonicotinoid
The term ‘‘neonicotinoid’’ [15] was originally proposed for imidacloprid (see Chapter
32.2.2) and related insecticidal compounds with a structural similarity to the
insecticidal alkaloid (S)-(−)-nicotine, which has a similar mode of action (see
Chapter 32.1) [8, 16]. Until now, various terms have been used to subdivide these
important commercial products, based on their structural fragments, including:
Table 32.2.1 Structure types of neonicotinoid insecticides,

including commercialized products (1, 3–10).
Pharmacophores Five- and six-membered Open-chain compounds

[-N-C(E)=X-Y] compounds (R1 -R2 , (R1 , R2 )
R1 -Z-R2 )
Nitromethylenes/ Et H
nitroenamines
HN S N N
[-N-C(E)=CH-NO2 ], CPM Me
E = S, NH CH-NO2
CH-NO2
Nithiazine (3, CH2CH2CH2)a Nitenpyram (4, Et, Me)
N-Nitroimines/
H H
N-nitroguanidine N NH
[-N-C(E)=N-NO2 ], CPM N N
CTM Me
E = NH, NMe N-NO2 N-NO2
Imidacloprid (1, CH2CH2) Clothianidin (7, H, Me)
O
H H
N N N N
CTM Me TFM Me
N-NO2 N-NO2
Thiamethoxam (5, CH2-O-CH2) Dinotefuran (8, H, Me)
Me
N
N N
CTM Me
N-NO2
AKD 1022 (6, CH2-NMe-CH2)b
N-Cyanoimines/ Me
N-cyanoamidines N S
CPM N Me
[-N-C(E)=N-CN], CPM
E = S, Me N-CN N-CN
Thiacloprid Acetamiprid
(9, CH2CH2) (10, Me, Me)
a
Launched in USA for use in poultry (1997–1998, Quick Strike®, Wellmark). Fly abatement strip
Quick Strike® uses nithiazine plus triple-action attractant to achieve rapid kill of nuisance flies and
house flies.
b
Not commercially used.
1168 32 Nicotinic Acetylcholine Receptor Agonists: Target and Selectivity Aspects
Table 32.2.2 Structural segments of commercialized neonicotinoids.
Structure of A-CHR- Chemical name of this moiety Abbreviation
CI CH2− 6-Chloro-pyrid-3-ylmethyl CPM

N
N
CH2− 2-Chloro-1,3-thiazol-5-ylmethyl CTM
CI S
O CH2− (±)-6-Tetrahydro-fur-3-ylmethyl TFM

*
∗
Mixture of (R)- and (S)-enantiomers
1) Heterocyclic- or heteroalicyclic group A with bridging chain/A-CH2 -: chloro-

nicotinyls (CNIs)/CPM [17], thianicotinyls/CTM [18], and furanicotinyls/TFM
[19] or first generation (CPM), second generation (CTM versus TFM) [20], and
third generation (TFM).
2) Functional group [= X − Y] as part of the pharmacophore types [−N−C(E) =
X−Y]: N-nitroimines [= N−NO2 ] or N-nitroguanidines [−N−C(N) = N−NO2 ],
nitromethylenes [= CH−NO2 ] or nitroenamines [−N−C(N) = CH−NO2 ]
and N-cyanoimines [= N−CN] or N-cyanoamidines [−N−C(E) = N−CN;
E = S, Me].
The subdivision of commercialized neonicotinoids into different generations im-
plies their ranking regarding novelty – a view not based on chemical or biochemical
classification. Since the introduction of imidacloprid (see Chapter 32.2.2) in 1991,
all neonicotinoids have been classified in the same mode of action class (nAChR
agonists, group 4A) by the Insecticide Resistance Action Committee (IRAC).
References
1. Nauen, R., Ebbinghaus-Kintscher, U., Agents (eds L.I. Gilbert and S.S. Gill),
Elbert, A., Jeschke, P., and Tietjen, K. Elsevier, Academic Press, pp. 61–113.
(2001) in Biochemical Sites of Insecticide 4. Jeschke, P. and Nauen, R. (2008) Pest
Action and Resistance (ed. I. Ishaaya), Manage. Sci., 64, 1084–1098.
5. Kagabu, S., Moriya, K., Shibuya, K.,
Springer, New York, pp. 77–105.
Hattori, Y., Tsuboi, S., and Shiokawa, K.
2. Jeschke, P. and Nauen, R. (2005) in
(1992) Biosci. Biotechnol. Biochem., 56,
Comprehensive Molecular Insect Science, 362–363.
vol. 5 (eds L.I. Gilbert, L. Latrou, and 6. Moriya, K., Shibuya, K., Hattori, Y.,
S.S. Gill) Elsevier, Oxford, pp. 53–105. Tsuboi, S., Shiokawa, K., and Kagabu,
3. Jeschke, P. and Nauen, R. (2010) in S. (1992) Biosci. Biotechnol. Biochem., 56,
Insect Control – Biological and Synthetic 364–365.
7. Minamida, I., Iwanaga, K., Tabuchi, T., Sumikawa, K. (1998) Arch. Insect.
Aoki, I., Fusaka, T., Ishizuka, H., and Biochem. Physiol., 17, 24–32.
Okauchi, T. (1993) J. Pestic. Sci., 18, 16. Tomizawa, M. and Casida, J.E. (2003)
41–48. Annu. Rev. Entomol., 48, 339–364.
8. Tomizawa, M. and Yamamoto, I. (1993) 17. Leicht, W. (1993) Pestic. Outlook, 4,
J. Pestic. Sci., 18, 91–98.
17–24.
9. Shikawa, K., Tsuboi, S., Iwaya, K., and
18. Maienfisch, P., Brandl, F., Kobel, W.,
Moriya, K. (1994) J. Pestic. Sci., 19,
209–217, 329–332. Rindlisbacher, A., and Senn, R. (1999)
10. Tabuchi, T., Fusaka, T., and Iwanaga, K. in Neonicotinoid Insecticides and the
(1994) J. Pestic. Sci., 19, 119–125. Nicotinic Acetylcholine Receptor (eds I.
11. Iwasa, T., Motoyama, N., Ambrose, J.T., Yamamoto and J.E. Casida), Springer,
and Roe, R.M. (2004) Crop Prot., 23, New York, pp. 177–209.
371–378. 19. Wakita, T., Konoshita, K., Yamada,
12. Tomizawa, M. and Casida, J.E. (2005) E., Yasui, N., Kawahara, N., Naoi, A.,
Annu. Rev. Pharm. Toxicol., 45, Nakaya, M., Ebihara, K., Matsuno, H.,
247–268. and Kodaka, K. (2003) Pest Manage. Sci.,
13. Jeschke, P., Nauen, R., Schindler, M., 59, 1016–1022.
and Albert, A. (2011) J. Agric. Food
20. Maienfisch, P., Huerlimann, H.,
Chem., 59, 2897–2908.
Rindlisbacher, A., Gsell, L., Dettwilwer,
14. Tomizawa, M. and Casida, J.E. (2011) J.
Agric. Food Chem., 59, 2825–2828. H., Haettenschwiler, J., Sieger, E., and
15. Yamamoto, I., Tomizawa, M., Saito, Walti, M. (2001) Pest Manage. Sci., 57,
T., Miyamoto, T., Walcott, E.C., and 165–176.
32.2.1
Open-Chain Compounds
Peter Jeschke
To date, four open-chain-type neonicotinoids have been commercialized, mainly by
Japanese companies. These open-chain type neonicotinoids can have, as separate
substituents: (R1 , R2 ), for example, R1 = hydrogen or alkyl such as ethyl (1,
nitenpyram) and methyl (2, acetamiprid, E − R2 = Me); or, in the case of E = NH
for the substituent R2 , an alkyl group such as methyl (3, clothianidin and 4,
dinotefuran) (see Chapter 32.2; Table 32.2.1.1 and Figure 32.2.1.1).
32.2.1.2 Nitenpyram
Starting from the cyclic nithiazine (5) [1–4], nitenpyram (1, 1995, Takeda Chemical
Industries Ltd., now Sumitomo Chemical Takeda Agro Company Ltd) [5] was
Table 32.2.1.1 Chemical classification of nitenpyram (1).
Common name Nitenpyram

® ®
Trade names Bestguard , Capstar
Development codes TI-304, CGA 246916
discovered during the optimization of substituents of an open-chain nitroethene [6].

®
It was introduced to the Japanese market in 1995 under the trade name Bestguard .
In 1999, Novartis Animal Health introduced nitenpyram as a systemic, fast-acting,
adult flea control product in cats and dogs in veterinary medicine under the trade
®
name Capstar (oral tablet formulation, adulticide) [7–9].
HN S
CH-NO2
5
32.2.1.2.1 Chemical Classification and Physico-Chemical Properties The ni-

tromethylene (1) is characterized by its extremely high water solubility (840 g l−1 ),
low partition coefficient (−0.64) (Tables 32.2.1.1 and 32.2.1.2) [10], and a similar
poor photostability as 5. This latter property is responsible for the compound’s
rapid decomposition in water under light and soil under aerobic conditions.
The outstanding characteristic of nitenpyram is its insecticidal activity, by
translaminar action and translocation. Because of its high water solubility, niten-
pyram shows good systemic activity and no phytotoxic effects; it can be used to
control pests by special soil treatment methods such as planting hole application,
plant foot treatment before and after transplanting, and soil incorporation (e.g.,
application combined with fertilizer using irrigation systems). With a half-life
(degradation time; DT50 ) of 1–15 days, the polar nitenpyram has a relatively short
persistence in soil, which probably offsets the relatively weak sorption that might
otherwise lead to mobility through the soil [11].
32.2.1.2.2 Chemistry Nitenpyram (1) can be prepared via two synthetic pathways
(Scheme 32.2.1.1). According to pathway A, 1,1-dichloro-2-nitroethene (6) [12] is
transformed into 1,1-bis(methylthio)-2-nitro-ethene (7) [13], which reacts in the first
step with N-methylamine to give 1-methylthio-1-(N-methyl)-amino-2-nitroethene
(8), and in the second step with N-(6-chloro-pyrid-3-ylmethyl)-N-ethyl-amine (9b,
CPM-NHEt) [14, 15] to yield 1. By pathway B, 6 is treated with 9b to give
1-chloro-1-[N-(6-chloro-pyrid-3-ylmethyl)-N-ethyl]-amino-2-nitroethene (10) in situ,
which reacts with N-methylamine to form 1 [16].
Table 32.2.1.2 Physico-chemical properties of nitenpyram (1).

Partition coefficient (log POW at 25 ◦ C) –0.64
Vapor pressure (mPa at 20 ◦ C) 1.1 × 10−6
Solubility in water (g l−1 at 20 ◦ C, pH 7.0) 840
Solubility in organic solvents (g l−1 at 20 ◦ C) Methanol: 670, acetonitrile: 430,
ethanol: 89, xylene: 4.5
Dissociation constant pKa (at 20 ◦ C) 3.1 and 11.5
H
S S H2N-Me
S N CPM-NH-Et (9b)
Me Me Me Me
CH-NO2 CH-NO2 pathway A
Et
H
7 8 N N
CPM Me
CH-NO2
Et 1
Cl Cl CPM-NH-Et (9b) N Cl H2N-Me
CPM
pathway B
CH-NO2 CH-NO2
6 10
Scheme 32.2.1.1 Synthetic pathways for preparation of nitenpyram (1).
32.2.1.2.3 Efficacy on Target Pests and Application Rates Nitenpyram controls

homopterous insect pests, such as leaf hoppers (e.g., Empoasca spp.), plant hoppers
(e.g., Nilaparvata lugens) on rice, whiteflies (e.g., Bemisia argentifolii) and aphids on
vegetables, and is also effective against thysanopterous insect pests on vegetables,
fruit trees, tea, and glasshouse crops. It is more active by ingestion than by contact.
At low concentrations, the product inhibits the pest’s feeding. When nitenpyram
is used as a foliar spray it shows translaminar activity; in addition, it has ovicidal
activity and shows no crop phytotoxicity. As directions for use in Japan, the product
is available as [17]:
• Water-soluble granules (WSG)s Bestguard® 10% soluble powder (SP) (1 = 10%)
for foliar spray (15–75 g a.i. ha−1 ) in rice and field crops.
• Granule Bestguard® 1% G (1 = 1.0%) for rice.
• Bestguard® 0.25% Dust (1 = 0.25%, 75–100 g a.i. ha−1 ) (Table 32.2.1.3).
®
Nitenpyram, when applied at the rate of 75 g a.i. ha−1 as Bestguard 0.25% Dust
formulation, strongly suppressed brown planthopper density for over three weeks.
However, with higher application rates such as 100 g a.i. ha−1 a controlling of
stink bugs (mixed population of Nezara viridula, Leptocorisa chinensis, and Cletus
punctiger) in rice fields has been described [18]. The results of trials in Japan have
indicated that the soil application of granular 1 formulations has good potential
for the control of Liriomyza trifolii in tomatoes and chrysanthemums grown under
greenhouse conditions.
Combination products contain mixtures of nitenpyram and the insecticide
cartap-hydrochloride, developed for the simultaneous control of these Hemipterous
pests, and the fungicide validamycin, respectively.
32.2.1.3 Acetamiprid
The N-cyano-acetamidine acetamiprid (2, 1995, Nippon Soda Co., Ltd) [19–21] con-
tains, in analogy to nitenpyram and the five-membered ring systems imidacloprid
(11) and thiacloprid (12), the CPM moiety (see Chapter 32.2.2). Acetamiprid was
discovered by Nippon Soda as part of its nitromethylene research program during

optimization studies of special 2-N-cyanoimino compounds with an imidazoline
five-membered ring obtained from Nihon Bayer Agrochem K. K. [22]. Methyl and
ethyl were identified as the favorable substituent R1 on the secondary amino group,
and both compounds led to potent activity against the cotton aphid.
Cl Cl
N N
N NH N S
N-NO2 N-CN
11 12
Table 32.2.1.3 Direction for use of nitenpyram (1) formula-

®
tions in Japan: Bestguard 1% G and 0.25 Dust.
Crop Insect pest Dose
®
Bestguard 1% G Rice Planthoppers 30–40 kg ha−1
Green rice leafhopper 30–40 kg ha−1
Cucumber Aphids 1–2 g per plant
Melon thrips 2 g per plant
Eggplant Aphids 1–2 g per plant
Melon thrips 1–2 g per plant
Tomato Aphids 2 g per plant
Silver leaf whitefly 2 g per plant
(Water)melon Aphids 2 g per plant
®
Bestguard 0.25% Dust Rice Planthoppers 30–40 kg ha−1
Green rice leafhopper 30–40 kg ha−1
Table 32.2.1.4 Chemical classification of acetamiprid (2).
Common name Acetamiprid

® ® ® ® ® ®
Trade names Adjust , Assail , Calex , Chipco Tristar , Conquest , Epik ,
® ® ® ® ® ®
Gazel , Gazelle , InSyst , Intruder , Manik , Mospilan ,
® ® ® ® ®
Molan , Mortal , Mothpiran , Mothpyran , Pristine ,
® ® ® ® ®
Profile , Rescate , Saurus , Supreme , Tata Manik ,
® ®
Vapcomere , Volley
Development codes NI-25
32.2.1.3.1 Chemical Classification and Physico-Chemical Properties Because of

its high water solubility (4.25 g l−1 at 25 ◦ C), acetamiprid shows a systemic and
translaminar insecticidal efficacy (Tables 32.2.1.4 and 32.2.1.5).
Acetamiprid is stable in water at pH 4, 5, and 7 at all temperatures, and
at pH 9 at 22 ◦ C, but is hydrolyzed at pH 9 at 35 ◦ C and 45 ◦ C, forming two
major hydrolytic transformation products. Furthermore, acetamiprid undergoes
phototransformation at pH 7, with a half-life of 34 days.
In laboratory studies the systemic properties and the translaminar aphicidal
activity of acetamiprid was studied in comparison with imidacloprid (11) [23].
The translaminar residual activity of 11 on cabbage leaves was superior to that of
acetamiprid, whereas its translaminar efficacy against Aphis gossypii on cotton was
inferior. However, the efficacy of both compounds against Myzus persicae and A.
gossypii in oral ingestion bioassays, using an artificial double-membrane feeding
system, revealed no significant differences in their intrinsic activities.
Investigations have shown that, in most soils, acetamiprid is a moderately mobile
(according to Koc values) and rapidly biodegradable chloronicotinyl insecticide (CNI)
[24]. The DT50 values of acetamiprid in clay loam or light clay soils were in range of
one to two days; however, in soil from the field, or in that used in container studies,
the degradation of acetamiprid varied widely (typical half-life 12 days).
Table 32.2.1.5 Physico-chemical properties of acetamiprid (2).

Partition coefficient (log POW at 25 ◦ C) 0.80
Vapor pressure (mPa at 25 ◦ C) <1.0 × 10−6
Solubility in water (g l−1 at 25 ◦ C) 4.25 (distilled water)
2.95 (pH 7 puffer system)
Solubility in organic solvents (g l−1 at Soluble in ethanol: >200, dichloromethane:
20 ◦ C) >200, hexane: 0.00654
Dissociation constant pKa (at 25 ◦ C) 0.7 weak base
O Me CPM-NH-Me (9a)
Me
N-CN
Me
16 N Me
CPM
CPM-NH2 (14)
N-CN
H 2
H2N Me CPM-NH2 (14) N Me (MeO)2SO2
CPM
N-CN N-CN
13 15
Scheme 32.2.1.2 Synthetic pathways for preparation of acetamiprid (2).

Table 32.2.1.6 Spectrum of insecticidal activity (LC50 in ppm) of acetamiprid (2).
Species Developmental stage LC50 (ppm)
Hemiptera Aphis craccivora Larvae and adults 0.91

Aphis gossypii First instar nymph 0.056
Aphis spiraccola First instar nymph 0.17
Myzus persicae First instar nymph 0.21
Brevicoryne brassicae First instar nymph 0.039
Rhopalosiphum padi Larvae and adults 0.032
Bemisia tabaci Eggs 4.8
Planococcus citri Larvae and adults 1.8
Lepidoptera Carposina niponensis Eggs 2.8

Grapholita molesta Eggs 3.1
Mamestra brassicae Second instar larvae 13
Plutella xylostella First instar larvae 4.4
Spodoptera litura First instar larvae 9.6
Thysanoptera Thrips palmi Adults 3.4
Isoptera Reticulitermes speratus Adults 0.16
32.2.1.3.2 Chemistry The synthetic routes to acetamiprid (2) are outlined in

Scheme 32.2.1.2. The reaction of the key intermediate methyl N-cyano-acetimidate
13 [25] with N-(6-chloro-pyridin-3-ylmethyl)amine (14, CPM-NH2 ) [26–28] in
aqueous methanol leads to 15 (or by treatment of methyl N-cyano-acetimidate
(16) [29] with 14), which is further N-methylated by dimethyl sulfate and
inorganic base, giving acetamiprid in an excellent yield. Alternatively,
acetamiprid can be synthesized in one step by the reaction of 16 with
N-(6-chloro-pyridin-3-ylmethyl)-N-methyl-amine (9a, CPM-NHMe) [30, 31].
32.2.1.3.3 Efficacy on Target Pests and Application Rates Acetamiprid is a broad-

spectrum insecticide that is ovicidal and larvicidal against a wide range of sucking
insects such as Hemiptera (especially aphids) or the adults of Thysanoptera,
Isoptera, and Coleoptera pests. It has contact, stomach and systemic longlasting
actions, and is moderately activity against Lepidoptera such as the peach fruit
moth (Carposina niponensis), the oriental fruit moth (Grapholita molesta), and the
diamond-back moth (Plutella xylostella). Acetamiprid also has ovicidal effects on
these species (Table 32.2.1.6) [32].
However, the penetration of acetamiprid through the cuticle is low. Other
hemipterous pests, such as sweet potato whitefly, Bemisia tabaci, and the cit-
rus mealybug, Planococcus citri, or melon thrips, Thrips palmi, and the termite,
Reticulitermes speratus, are also very susceptible to acetamiprid.
Acetamiprid is suitable for use on a wide range of crops, including cereals,

®
citrus, and sugar beet (Mospilan ), fruit, vegetables, vines, and horticultural crops
® ® ® ® ®
(Assail ), ornamentals (Chipco Tristar ), cotton (Assail , Manik , Intruder ,
® ® ® ®
Supreme ), tobacco (Epik , Profil ), and tea (Mospilan ). It can be applied to the
soil, and also to the foliage or as a seed treatment.
The translaminar efficacy of acetamiprid against the green peach aphid (M.
persicae) was assessed using a root-dipping method at low concentration [LC50 (in
ppm) = 0.031 (eggplant); 0.023 (radish)]; activity against Plutella xylostella was about
10-fold lower in this test. In the case of the diamond-back moth, acetamiprid was
shown to be active at different development stages. Typically, the LC50 against the
first instar larva was much lower than for the third- and fourth instar larvae; this is
an important point for timing the compound application. Furthermore, acetamiprid
exhibits a high activity towards insects that are either organophosphate (OP) and/or
pyrethroid-resistant, as well as towards susceptible strains. Because of its strong
repellent and toxic activities on termites such as Riticulitermes, acetamiprid is also
used for the control of termites and household pests [33].
It should be noted that, as acetamiprid is toxic to honeybees, it must not be
applied when bees are present in the area being treated.
32.2.1.4 Clothianidin
During optimization of the open-chain neonicotinoids, the research group at
Takeda was able to show that compounds containing the N-nitroguanidine moiety,
coupled with the chlorothiazolylmethyl (CTM) residue, demonstrate an increased
activity against some lepidopteran pests [34, 35]. Subsequently, clothianidin (3,
2002, Takeda Chemical Industries Ltd, now Sumitomo Chemical Takeda Agro
Company Ltd, and Bayer CropScience) [36, 37] emerged as the most promising
derivative from this program. In this open-chain structure, the N-nitro-guanidine
pharmacophore is similar to the five-membered neonicotinoids imidacloprid (11)
(see Chapter 32.2.2), but the CPM group has been replaced by the CTM moiety [as
also described in Chapter 32.2.3 for thiamethoxam (17) and AKD 1022 (18)].
32.2.1.4.1 Chemical Classification and Physico-Chemical Properties Because cloth-

ianidin demonstrates no acidic or alkaline properties at the relevant pH [38], the
pH of the aqueous system has no influence on the compound’s physico-chemical
properties. Although clothianidin is stable to hydrolysis in the pH range 4 to 9,
its photolysis contributes significantly to its degradation in the environment, and
this results in an elevated mineralization rate. The degradation of clothianidin
in water/sediment systems was observed to be significantly faster (by a factor
of 2–3) under anaerobic conditions than aerobic conditions. The water solubil-
ity (0.327 g l−1 at 20 ◦ C), vapor pressure (1.3 × 10−10 Pa at 25 ◦ C) and volatility
of clothianidin are relatively low compared to other neonicotinoids that have a
N-nitroguanidine pharmacophore (Tables 32.2.1.7 and 32.2.1.8).
This point is also reflected by the octanol–water partition coefficient of clothi-
anidin, which indicates a favorable absorption to soil (log POW = 0.7 at 25 ◦ C) at
25 ◦ C). The plant uptake of clothianidin occurs via the cotyledons and roots of the
emerging seedlings, and through the roots of established plants (see Chapter 29).
The results of translocation studies have shown that clothianidin moves in both
an acropetal and a basipetal manner. Typically, the active ingredient taken up by
the roots is transported rapidly to the leaves, while translaminar activity results in
an efficient transport of the compound across the leaf tissues, from one surface to
the other.
On the basis of these physico-chemical properties, clothianidin should not
bioaccumulate. Likewise, as the compound is nonvolatile, no significant amounts
should be expected in the atmosphere.
32.2.1.4.2 Chemistry In the laboratory, the active ingredient clothianidin (3)

can be obtained, for example, by the treatment of S-methyl- or O-methyl-
N-nitro-N -phthaloyl-isothiourea (20a or 20b) [39, 40], synthesized from S-methyl-
or O-methyl-N-nitro-isothiourea (19a or 19b) [41] and phthaloyl chloride, with
2-chloro-5-(aminomethyl)-1,3-thiazole (15, CTM-NH2 ) [42–48].
According to other synthesis methods, clothianidin can be prepared by coupling
of the N1,5 -dialkylated 2-(N-nitroimino)-hexahydro-1,3,5-triazine (21, R = alkyl, ary-
lalkyl) with 2-chloro-5-(chloro-methyl)-1,3-thiazole (22, CCMT; Table 32.2.1.9) at the
3-position, followed by ring-cleavage reaction of the resulting bis-aminal structure
within the six-membered system of 23 (Scheme 32.2.1.3) [49–51].
Alternatively, 19a or 19b may be treated in the first step with 15 to give the N-
alkylated N -nitro-guanidine (24), which is transformed into the N3,5 -dialkylated
2-(N-nitroimino)-hexahydro-1,3,5-triazine (25). In the second step, N1 -alkyla-
tion with methyl iodide leads to the N1,3,5 -trialkylated 2-(N-nitroimino)-hexahydro-1,
3,5-triazine (23a), which is cleaved under acid conditions to form clothianidin (3).
Table 32.2.1.7 Chemical classification of clothianidin (3).
Common name Clothianidin

® ® ® ® ® ®
Trade names Arena , Belay , Celero , Clutch , Deter , Dantotsu ,
® ® ® ® ®
Dantop , Focus , Fullswing , Poncho , Prosper
Development codes TI-435
Table 32.2.1.8 Physico-chemical properties of clothianidin (3).

Vapor pressure (Pa at 25 ◦ C) 1.3 × 10−10 (extrapolated)
Solubility in water (g l−1 at 20 ◦ C) 0.327
Solubility in organic solvents (g l−1 at Methanol: 6.26, dichloromethane: 1.32, 1-octanol:
25 ◦ C) 0.938, xylene: 0.0128, n-heptane: <0.00104
Dissociation constant pKa (at 25 ◦ C) 11.09
Table 32.2.1.9 Selection of synthetic pathways for prepara-

tion of the key intermediate CCMT (22).
Direct precursor Reaction conditions Reference
Cl − 1. H2 O, KI, I2 [52]
NH-CS-S Na +
2. CH2 Cl2 , SO2 Cl2
CH2
Cl
N=C=S SO2 , Cl2 , 50 ◦ C [53]
CH2 Cl2 , MeCN, 10–15 ◦ C
H 2C NCS, AlBN CH2 Cl2 , Cl2 , [55]

N=C=S −15 to −10 ◦ C, (1 h) [56]
ClCH2 SO2 Cl2 , 50–60 ◦ C, (3 h) [57]

N=C=S
Cl
NH-CHO SOCl2 , SCl2 , reflux, (24 h) [57]
CH2
Cl
NC CCl4 , SCl2 , 40 ◦ C, (4 h) [58]
CH2
CH2
CHCl3 , Cl2 , −10 ◦ C [59]
S
S N
H
[60]
CH2Cl CH2 Cl2 , (2 h) [59]
S
BnS N
CH2OH SOCl2 , CH2 Cl2 , (3 h) [61]

S
BnS N OH
CHO 1. H2 , PtO2 , FeCl2 ·4 H2 O, [61]

S AcOEt, (2 h)2. SOCl2 ,
CH2 Cl2 , (3 h)
BnS N
CH3
S NCS, DBPO, CCl4 , reflux [52]
[62]
Cl N
Bn = benzyl; NCS = N-chloro-succinimide; AIBN = 2,2 azobis[isobutyronitrile]; DBPO = dibenzoyl
peroxide.
Me Me
N N
H
N NH2 Me-NH2, HCHO N NH MeI
CTM CTM N N
CTM Me
N-NO2 N-NO2 N-NO2
24 25
23a
CTM-NH2(15)
COCl O HCl, EtOH
H2N X COCl N X CTM-NH2 (15)

Me Me
N-NO2 O N-NO2
X = S (19a) X = S (20a) H H
X = O (20b) N N
X = O (19b) CTM Me
N-NO2
R R
N N 3
CCMT (22)
HN N N N - R-NH2
Me CTM Me
N-NO2 N-NO2
21 R = alkyl, arylalkyl 23
Scheme 32.2.1.3 Synthetic pathways for preparation of clothianidin (3).
Numerous synthetic pathways for CCMT (22) have been investigated by Bayer
CropScience or Sumitomo Chemical Takeda Agro Company, as well as other
companies, to develop practical and economic processes for this intermediate [63].
Consequently, several patent applications and reports have been made with regards
to its technical synthesis [64]. As outlined in Table 32.2.1.9, various attractive
synthetic routes are known, based on commercially available heterocyclic and
open-chain starting materials.
The results of molecular modeling calculations (e.g., force-field methods,
MMFF94s) at room temperature, in addition to those of nuclear magnetic reso-
nance (NMR) studies, have shown the preferred orientation of the functional group
[=N−NO2 ] in clothianidin to be in the trans-position; the energy of the (Z)-isomer
(2.6 kcal mol−1 ) was somewhat higher than that of the optimal (E)-isomer [65].
Both, calculations and X-ray structure analyses have shown that the three C=N
bonds involving the C5 atom have some double-bond characteristics. The N-methyl
group of clothianidin can flip easily from the anti- to the syn-position. The en-
ergies of the respective conformers, relative to the optional structure, are below
1.5 kcal mol−1 .
Table 32.2.1.10 Spectrum of activity of clothianidin (3) seed

treatment for corn root worm and secondary corn pests.
Coleoptera Corn root worm Diabrotica spp.

Wireworm Melanotus spp.
Flea beetle Chaetocnema pulicania
Grape colaspis Colaspis brunnen
White grub Lachnosterna implicata
Lepidoptera Black cutworm Agrotis ypsilon
Diptera Seed corn maggot Hylemyia platura
Homoptera Corn leaf aphid Rhopalosiphum maidis
Hemiptera Chinch bug Blissus leucopterus
Stink bug Nezara viridula
Hymenoptera Imported fire ant Solenopsis spp.
32.2.1.4.3 Efficacy on Target Pests and Application Rates Clothianidin, which has a
broad spectrum of activity, acts as an acute contact and stomach poison, combining
highly systemic properties with a relatively low application rate. Together, these
properties make clothianidin suitable for application to the soil, foliage, and/or
seeds [66]. Clothianidin is particularly active against sucking insects such as aphids,
leaf hoppers, whiteflies, and thrips, while various species of beetles (e.g., Atomaria
spp., Agriotes lineatus, Diabrotica spp.) and some species of flies (e.g., Oscinella
frit and Pegomyia spp.) and cutworm (e.g., Agrotis spp.) can also be controlled
very effectively. Because of its excellent root systemicity, clothianidin is very
active against a broad spectrum of root-, stem-, and leaf-feeding pests, as well as
soil-inhabiting pests that dwell in the halo around the seed. This spectrum includes
Coleoptera, Lepidoptera, and Diptera, which together include most of the early-
and mid-season corn pests in the USA (Table 32.2.1.10) [67, 68].
®
Clothianidin is marketed as insecticide: (i) for foliar application as Dantop or
®
Fullswing (Sumitomo Chemical Takeda Agro Company; the latter was launched as
a water-dispersible granule on turf, providing control of beetle larvae and bluegrass
®
worms); (ii) for soil application as a WSG (cf. Dantotsu ); and (iii) as flowable
concentrate for seed treatment (FS; flowable concentrate for seed treatment) as 600
®
FS Poncho (Bayer CropScience).
®
In order to control insects in different crops by using Poncho , usage rates
of the active ingredient clothianidin are recommended as follows for: cereals
(20–50 g a.i. 100 kg−1 ); corn (0.25–1.25 mg a.i. per seed); sugar beet (10–60 g a.i.
per 100 000 seeds) [69]; oilseed rape (4–10 g a.i. kg−1 seeds); or sunflower
®
(20–37.5 g a.i. per 150 000 seeds). The seed treatment (Poncho 250) is applied at
a low rate of 0.25 mg a.i. per kernel in maize, and provides early season seed and
seedling protection against cutworms (Agrotis spp.), wireworms (Melanotus spp.),
seed corn maggots (Hylemyia plature), white grubs (Phyllophaga spp.), chinch bugs
Z
in vivo H H
N N N N
CTM Me CTM Me
N-NO2 N-NO2
Thiamethoxam (17) (Z = O) 3
AKD-1022 (18) (Z = NMe)
Scheme 32.2.1.4 In vivo transformation of six-membered

neonicotinoids into clothianidin (3).
(Blissus leucopterus), flea beetles (Chaetocnema pulicaria), and numerous other

pests.
For rice, different formulations of clothianidin have been developed, including
®
Dantotsu 1 kg granule (1% a.i.), in addition to various combination products such
®
as: (i) Dantotsupadan (clothianidin + cartap), which provides additional control
®
of stem borers and leaf rollers; (ii) nursery box formulations such as Dantotsu
®
Nursery Box Granule (1.5% a.i.), Windantotsu (clothianidin + carpropamid), and
®
Delausdantotsu (clothianidin + diclocymet), to provide additional control against
®
rice blast; and (iii) dust formulations for use in rice, such as Dantotsu Dust
®
DL (0.15% a.i.), Dantotsupadanvalida (clothianidin + cartap + validamycin) to
®
provide control of skippers, green rice caterpillars, and sheath blight, and Hustler
(clothianidin + cartap + validamycin + ferimzone + phthalide) for the control of
rice blast, panicle blight, and Curbularia spp.
Further different combination products with insecticides and fungicides
®
are known for seed treatment; an example is Poncho Beta (clothianidin +
®
beta-cyfluthrin), for the treatment of sugar beet. Likewise, Prosper 200 contains
®
a mixture of clothianidin and the systemic and contact fungicides Vitavax
(cabothiin), Thiram, and Metalaxyl for the control of flea beetles, from emergence
®
up to second-leaf stage, while Prosper 400 (which contains a higher dosage of
clothianidin) can be used to extend control up to the fourth leaf stage [70].
Similar to imidacloprid (11; see Chapter 32.2.2.3), clothianidin can be used to
control important vectors of virus diseases in sugar beet [71].
Both, AKD 1022 (18) and thiamethoxam (17) (see Chapter 32.2.3) can undergo
transformation to the open-chain neonicotinoid, clothianidin (3), by ring cleavage,
either by hydrolysis (see above) or by metabolism in vivo (Scheme 32.2.1.4).
The use of thiamethoxam has been demonstrated for important and relevant
insects, including wireworm larvae Agrotis segetum, corn root worm Diabrotica
balteata, and the Colorado potato beetle, Leptinotarsa decemlineata, in an in vivo
assay (see Scheme 32.2.1.4) [72] (the latter finding points to thiamethoxam having a
proinsecticidal mode of action; MoA) [73, 74]. Differences in the binding site of thi-
amethoxam have also been suggested, in contrast to the findings of Wellmann and
Kayser [75–77]. Further pharmacokinetic investigations with thiamethoxam in the
Colorado potato beetle (one of the most relevant species targeted by neonicotinoid
insecticides [78]) have revealed a rapid conversion of thiamethoxam to clothianidin
when applied either topically or orally. Recently, thiamethoxam was shown to be

transformed to clothianidin inside tomato fruits [79], while the inhibitory effect of
thiamethoxam locomotor activity in cockroaches also appeared to be associated with
clothianidin [80]. Additional evidence for clothianidin being the active principle of
thiamethoxam was provided by the consideration of neonicotinoid cross-resistance
data. All neonicotinoids are classified within group 4A of the Insecticide Resistance
Action Committee (IRAC) MoA classification.
32.2.1.5 Dinotefuran
The discovery of the N-nitroguanidine dinotefuran (4, 2002, Mitsui Chemicals)
[81–84] resulted from the idea of incorporating an N-nitroimino group into
the acetylcholine structure as a lead compound [85]. Following the synthesis of
neonicotinoids containing a N-(3-methoxy-propyl) moiety (hydrogen acceptor site),
the investigation of cyclic ether groups led to the discovery of a novel tetra-
hydrofuryl (THF) moiety, 6-tetrahydro-fur-3-ylmethyl (TFM) which showed a more
than 10-fold increase in insecticidal activity.
In contrast to other commercial neonicotinoids, dinotefuran has an alicyclic and
racemic (R,S)-(±)-TFM moiety instead of the halogenated heteroaromatic CPM
and CTM moieties (see Chapter 32.2). The nonaromatic oxygen atom of the TFM
residue is situated in the position corresponding to that of the aromatic nitrogen
atom of the other heterocyclic moieties of neonicotinoids; consequently, the TFM
structure can be taken as an isostere of the CPM and CTM moieties [86].
Recently, a new topical spot-on ectoparasiticide containing a combination of
dinotefuran, permethrin, and pyriproxyfen was introduced into veterinary medicine
under the trade name Vectra 3DTM , to combat Amblyomma americanum and the
Gulf Coast tick A. maculatum in dogs, [87].
32.2.1.5.1 Chemical Classification and Physico-Chemical Properties Dinotefuran

(4), which is characterized by its high water solubility (54.3 g l−1 ) but low partition
coefficient (−0.644) (Tables 32.2.1.11 and 32.2.1.12), is stable in water at pH 4, 7,
and 9 at 20 ◦ C. In aerobic soil metabolism studies conducted with loamy sand soil,
the DT50 -value was 51.7 days, whilst in a 320-day aquatic water-sediment system
the DT50 -value was 23–49 days in the aerobic water phase, and 45–128 days in the
largely anaerobic sediment layer. In addition, dinotefuran has excellent systemic
and translaminar actions in many plants.
Table 32.2.1.11 Chemical classification of dinotefuran (4).
Common name Dinotefuran

® ® ® ® ®
Trade names Alubarin , Alburin , Daepo , Oshin , Phantom ,
® ® ® ® ®
Safari , Shuriken , Starkle , Starkul , Venom
Development codes MTI-446
Table 32.2.1.12 Physico-chemical properties of dinotefuran (4).

Partition coefficient (log POW at 25 ◦ C, pH 7) –0.644
Vapor pressure (mPa at 25 ◦ C) n.d.
Solubility in water (g l−1 at 20 ◦ C) 54.3 ± 1.3 (purified water)
Solubility in organic solvents (g l−1 at 20 ◦ C) n.d.
Dissociation constant pKa (at 25 ◦ C) No dissociation in range pH value of 1.4–12.3
n.d. = not described.
32.2.1.5.2 Chemistry Generally, dinotefuran (4) can be synthesized by the treat-

ment of N-nitro-N -[((R,S)-(±)-tetrahydrofur-3-yl)methylamino]-guanidine (27) [88],
synthesized from S-methyl-N-nitro-isothiourea (19), and (R,S)-(±)-tetrahydrofur-
3-ylmethylamine (26, TFM-NH2 ) [89], with methylamine and formaldehyde.
Dinotefuran is obtained following N -alkylation of the resulting N-alkylated
2-(N-nitroimino)-hexahydro-1,3,5-triazine (28) with methyl iodide, and subsequent
ring cleavage reaction of the resulting bis-aminal structure 31 within the
six-membered system (Scheme 32.2.1.5).
Alternatively, 31 can be prepared from N-methyl 2-(N-nitroimino)-hexahydro-1,3,
5-triazine (29) by N-alkylation with the O-triflate (30) (TFM-OTf, Tf = SO2 CF3 ) [90].
Subsequent ring cleavage is possible under either basic (method A) or acidic
(method B) conditions (Scheme 32.2.1.5).
Structural modifications of dinotefuran with regards to the substitution pattern
of the TFM moiety, modification of the nitrogen substituents, or variation of
Me
N
TFM-NH2 (26) H MeNH2, HCHO N N
19 N NH2 H
TFM TFM
N-NO2 N-NO2
27 28
MeI
Me Me
N N
TFM-OTf (30) method A or B H H
HN N N N N N
Me TFM Me - R-NH2 TFM Me
N-NO2 N-NO2 N-NO2
29 31 4
Ring cleavage: method A = NaH, DMF, 50 °C, [2 h]; (via 31 in-situ)

method B = 1N HCl, EtOH, 40 °C, [1 h]
Scheme 32.2.1.5 Synthetic pathways for preparation of dinotefuran (4).

Table 32.2.1.13 Biological activities of imidacloprid (11),

clothianidin (3), (R,S)-(±)-dinotefuran (4), and the separated
enantiomers (R)-(−)-(4) and (S)-(+)-(4) against houseflies.
Entry Insecticidal log(1/EC50 )(M) Binding log(1/IC50 )(M)

(observed) (radioligand [3 H]-11)
11 5.93 (± 0.12) (3) 7.71 (± 0.14) (2)

3 5.32 (± 0.17) (2) 8.28 (± 0.07) (2)
(R,S)-(±)-4 5.02 (± 0.19) (2) 6.67 (± 0.09) (2)
(S)-(+)-4 5.14 (± 0.03) (2) 6.82 (± 0.01) (2)
(R)-(−)-4 3.93 (± 0.34) (2) 5.73 (± 0.30) (2)
the pharmacophore, may each result in drastic changes of insecticidal potency

[91]. Moreover, the incorporation of structural fragments known from previous
neonicotinoid insecticides does not necessarily lead to compounds retaining a
higher activity.
Similar to the other open-chain neonicotinoids, dinotefuran has an agonistic
action on nicotinic acetylcholine receptors (nAChRs). On examining the neu-
ral activities of racemic (R,S)-(±)-dinotefuran, the separated enantiomers, and
a competitive nAChR antagonist ([125 I]α-BGTX) in inhibiting the binding of
[3 H]epibatidine (32) to the nerve cord membranes of the American cockroach,
Periplaneta americana (L), the (R)-(−)-enantiomer was found to be about twofold
less effective. In contrast, the (S)-(+)-enantiomer of dinotefuran was approximately
50-fold more insecticidally active than the (R)-(−)-enantiomer [92].
The insecticidal activity of (R,S)-(±)-4, its enantiomers and the neonicotinoids
clothianidin (3) and imidacloprid (11) against the housefly, Musca domestica (L), and
their binding activity using housefly head membrane preparations, were monitored
by using [3 H]-11 as a radioligand (Table 32.2.1.13) [93]. Subsequently, dinotefuran
was shown to be less active than clothianidin and imidacloprid by a factor of 10 in
molar concentrations. Finally, the enantiomer (S)-(+)-4 was more potent than its
counterpart (R)-(−)-4, though the (S)-(+)-4 and (R,S)-(±)-4 enantiomers showed
almost equal efficacy in target-site and insecticidal potency evaluations.
32.2.1.5.3 Efficacy on Target Pests and Application Rates Dinotefuran exhibits

activity against numerous insects such as Hemiptera, Lepidoptera, Coleoptera,
Diptera, Dictyoptera, and Thysanoptera, and also against some other important
pests (e.g., stink bugs, fruit moths, flea beetles, leaf miners) in various crops
(e.g., sugar beet, fruit, vegetables, turf, cotton, and ornamentals) at rates of
100–200 g a.i. ha−1 via ingestion and contact, and at 150–600 g a.i. ha−1 by soil
application, including root-systemic activity. Dinotefuran can be applied by either a
® ®
foliar route (112–224 g in 380 l, Safari ) or as a soil drench (680 g in 380 l, Safari )
application (Table 32.2.1.14).
Table 32.2.1.14 Spectrum of activity (LC50 ; range) of dinote-

furan (4) after foliar and leaf dipping application under labo-
ratory conditions.
Insect LC50 (ppm a.i.) Application method
Sogatella furcifera 1−0.1 Foliar

Nilapavarta lugens 1−0.1 Foliar
Laodelphax striatellus 10−1 Foliar
Nephotettix cincticeps 1−0.1 Foliar
Myzus persicae 10−1 Foliar
Aphis gossypii 10−1 Foliar
Trialeurodes vaporariorum 10−1 Foliar
Bemisia tabaci 10−1 Foliar
Thrips palmi 10−1 Foliar
Phyllotreta striolata 100−10 Foliar
Plutella xylostella 100−10 Leaf dipping
Pieris rapae 100−10 Leaf dipping
Spodoptera litura 100−10 Leaf dipping
Liriomyza trifolii 100−10 Foliar
The toxicological and environmental profile of dinotefuran is favorable, and

includes low mammalian, avian, and aquatic toxicities. The product is available
in different formulations: (i) as a 2% granule for use in paddy rice nursery boxes
® ® ®
(Oshin ); (ii) as a 0.5% dust for foliar rice applications (Starkle , Phantom ); (iii)
®
as a 1% granule for soil incorporation in vegetables (Alubarin ); and (iv) as a 20%
®
soluble granule for foliar applications to fruit and vegetables (Safari ).
32.2.1.6 Open-Chain Compounds versus Ring Systems

Today, numerous examples exist of isosterism between open-chain and ring sys-
tems among bioactive molecules [94]. In comparison to the corresponding five-
and six-membered ring systems (see Chapters 32.2.2 and 32.2.3, respectively), the
open-chain compounds exhibit a similar broad-spectrum insecticidal activity, form-
ing a so-called ‘‘quasi-cyclic’’ conformation when binding to the insect nAChR [95].
Generally, the open-chain neonicotinoids are less lipophilic than the correspond-
ing neonicotinoids with a ring structure (see Chapters 32.2.2 and 32.2.3). Recently,
a binding model for imidacloprid (11) has been described, based on comparative
molecular field analysis (CoMFA) results, which suggested that the nitrogen of the
CPM moiety would interact with a hydrogen-donating site of the nAChR, and that
the nitrogen atom at the 1-position of the imidazolidine ring would interact with
the negatively charged domain [96–101]. Thus, the binding activities of open-chain
structures (e.g., 1, 2, and related compounds) to the nAChR of houseflies were
monitored, and the results analyzed using CoMFA.
Generally, the nitrogen-containing hetarylmethyl group as N-substituent, such
as CPM (11, 1, 2, and 12) and CTM (3, 17, 18), has a remarkably strong influence
(a) (b)
Figure 32.2.1.1 Superposition of van der Waals volumes of

neonicotinoid insecticides in the minimum-energy confor-
mations. (a) Open-chain neonicotinoids (1–4); (b) The ring
system (7, 11, 17).
on the insecticidal activity of not only open-chain neonicotinoids but also ring
systems. In comparison with both groups, replacement by the isosteric TFM group
(e.g., dinotefuran) resulted in a markedly weaker H-bond acceptor at the target
site. Atom-based alignments of open-chain neonicotinoids (e.g., 2–4) as well as the
ring system (11) showed that the (R,S)-(±)-tetrahydrofur-3-yl ring of dinotefuran
is more or less perpendicular to the heteroaromatic ring systems of the other
neonicotinoids [102, 103].
The superposition of van der Waals volumes of open-chain neonicotinoids
such as 1, 2, 3, and 4, as well as the ring systems 7, 11, and 17, are shown in
Figure 32.2.1.1.
The acquisition of high-resolution crystal structures of the acetylcholine-binding
protein (A-AChBP) with thiacloprid (12) and imidacloprid (11), as well as of acetyl-
choline receptor-binding protein (L-AChRBP) with imidacloprid and clothianidin
(3), has provided a deeper insight into the molecular determinants of the neonicoti-
noid agonists, and how they interact with their respective binding sites on insect
nAChRs. A water or solvent molecule is captured close to the CPM or CTM nitrogen
bridging to relevant amino acids in the neonicotinoid binding pocket [104, 105]
(see Chapter 32.1). One characteristic of several agonists, such as thiacloprid and
imidacloprid, is that loop C largely envelops the ligand, such that the aromatic side
chains are positioned to interact optimally with the conjugated and hydrophobic
regions of the neonicotinoid insecticides.
In contrast, the cocrystallization of L-AChBP with clothianidin and imidacloprid
has suggested that, in both cases, the guanidine moiety stacks with Tyr185, whereas
the N-nitro group of imidacloprid – but not of clothianidin – forms an H-bond
with Gln55. The H-bond of NH at position 1 with the backbone carbonyl group of
Trp143 offers, in the case of clothianidin, an explanation for the diverse actions of
neonicotinoids on insect nAChRs [106].
References
1. Soloway, S.B., Henry, A.C., Kollmeyer, 10. Kashiwada, Y. (1996) Agrochem. Jpn, 68,
W.B., Padgett, W.M., Powell, J.E., 18–19.
Roman, S.A., Thiemann, C.H., Corey, 11. Tsumura, Y., Nakamura, Y., Tonagai,
R.A., and Horne, C.A. (1978) in Ad- Y., Kamimoto, Y., Tanaka, Y. et al.
vances in Pesticide Science, Part 2 (eds (1998) Shokuhin Eiseigaku Zasshi, 39,
H. Geissbühler, G.T. Brooks, and 127–134.
C. Kearney), Pergamon Press, pp. 12. Aoki, I., Tabuchi, T., and Minamida, I.
206–227. (1990) Patent EP 381,130 A2 (Takeda
2. Soloway, S.B., Henry, A.C., Kollmeyer, Chem. Ind., Ltd).
W.D., Padgett, W.M., Powell, J.E., 13. Gompper, R. and Schaefer, H. (1967)
Roman, S.A., Tieman, C.H., Corey, Chem. Ber., 100, 591–604.
R.A., and Horne, C.A. (1979) Ple- 14. Minamida, I., Iwanago, K., and
nary Lecture Symposium Paper, 4th Okauchi, T. (1989) Patent EP 302,389
International Congress on Pesti- A2 (Takeda Chem. Ind., Ltd).
cide Chemistry, Adv. Pestic. Sci., 2, 15. Diehr, H.-J. (1993) Patent EP 556,684
206–217 A1 (Bayer AG).
3. Schröder, M.E. and Flattum, R.F. 16. Minamida, I., Iwanaga, K., Tabuchi,
(1984) Pestic. Biochem. Physiol., 22, T., Aoki, I., Fusaka, T. et al. (1993)
J. Pestic. Sci., 18, 41–48.
148–160.
17. Kashiwada, Y. (1996) New Pestic., 68,
4. Kollmeyer, W.D., Flattum, R.F., Foster,
18–19.
J.P., Powel, J.E., Schroeder, M.E., and
18. Elbert, A., Haas, M., Springer, B.,
Soloway, S.B. (1999) in Nicotinoid
Thielert, W., and Nauen, R. (2008) Pest
Insecticides and the Nicotinic Acetyl-
Manage. Sci., 64, 1099–1105.
choline Receptor (eds I. Yamamoto
19. Takahashi, H., Mitsui, J., Takakusa, N.,
and J.E. Casida), Springer, Tokyo,
Matsuda, M., Yoneda, H., Suzuki, J.,
pp. 71–89.
Ishimitsu, K., and Kishimoto, T. (1992)
5. Minamida, I., Iwanaga, K., Tabuchi,
Brighton Crop Protect. Conf. – Pests Dis.,
T., Aoki, I., Fusaka, T., Ishizuka, H.,
1, 89–96.
and Okauchi, T. (1993) Nihon Noy- 20. Matsuda, M. and Takahashi, H. (1996)
aku Gakkaishi (J. Pestic. Sci.), 18, Agrochem. Jpn, 68, 12–20.
41–48. 21. Kiriyama, K., Itazu, Y., Kagabu, S., and
6. Minamida, I., Iwanaga, K., Tabuchi, Nishimura, K. (2003) J. Pestic. Sci., 28,
T., Uneme, H., Dantsuji, H., and 8–17.
Okauchi, T. (1993) J. Pestic. Sci., 18, 22. Yamada, T., Takahashi, H., and
31–40. Hatano, R. (1999) in Neonicotinoid
7. Rust, M.K., Waggoner, M.M., Hinkle, Insecticides and Nicotinic Acetylcholine
N.C., Stansfield, D., and Barnett, S. Receptor (eds I. Yamamoto and J.E.
(2003) J. Med. Entomol., 40, 678–681. Casida), Springer, New York, pp.
8. Baynes, R.E. (2009) in Veterinary Phar- 149–176.
macology and Therapeutics, 9th edn (eds 23. Buchholz, A. and Nauen, R. (2001) Pest
J.E. Riviere and M.G. Papich), Black- Manage. Sci., 58, 10–16.
well Publishing Professional, Ames, IA, 24. Tokieda, M., Ozawa, M., Kobayashi,
pp. 1181–1201. S., Gomyo, T., and Takeda, M. (1999)
9. Vo, D.T., Hsu, W.H., Abu-Basha, J. Pestic. Sci., 24, 115–122.
E.A., and Martin, R.J. (2010) J. Vet. 25. Kishimoto, T., Suzuki, J., Ishimitsu,
Pharmacol., 33, 315–322. K., Mitsui, J., Iwasa, T., Yamamoto, A.,
References 1187
and Takakusa, N. (1993) Abstracts of 45. Hamada, Y. (2000) Patent WO

the 18th Annual Meeting of Pesticide 2000/021,943 A1 (Ihara Chem. Ind.
Science Society of Japan, p. 39. Co., Ltd).
26. Gesing, E., (1989) Patent DE 3,727,126 46. Stelzer, U., Lantzsch, R., and
A1 (Bayer AG). Hupperts, A. (1998) Patent DE
27. Maurer, F. (1989) Patent DE 3,726,993 19,653,586 A1 (Bayer AG).
A1 (Bayer AG). 47. Uneme, H. and Minamida, I. (1993)
28. Diehr, H.-J. (1990) Patent EP 391,205 Patent JP 05,286,936 A2 (Takeda
A1 Bayer AG. Chem. Ind., Ltd).
29. Arnold, B. and Regitz, M. (1980) Tetra- 48. Kaku, S., Ichihara, R., Seshimo, A.,
hedron Lett., 21, 909–912. and Yamazaki, M. (1992) Patent JP
30. Ieno, K. (1993) Patent JP 05,294,932 A2 04,022,174 A2 (Nippon Soda Co., Ltd).
(Koei Chem. Co.). 49. Van Laak, K. and Wollweber, D. (1999)
31. Ieno, K. and Kawanami, Y. (1994) Patent DE 19,806,469 (Bayer A.-G.).
Patent EP 609,811 A1 (Koei Chem. 50. Wollweber, D., Krämer, W., and
Co.). Rivadeneira, E. (1998) Patent WO
32. Matsuda, M. and Takahashi, H. (1996) 98/42,690 (Bayer A.-G.).
Plant Prot., 50, 248. 51. Maienfisch, P., Huerlimann, H., and
33. Doi, S. and Nishioka, H. (2003) Patent Haettenschwiler, J. (2000) Tetrahedron
JP 2003/063,902 A (Koshii Preserving Lett., 41, 7187–7191.
Co., Ltd). 52. Ciba-Geigy AG (1997) Patent WO
34. Uneme, H., Iwanaga, K., Higuchi, N., 97/23,469.
Kando, Y., Okauchi, T., Akayama, A., 53. Syngenta AG (2002) Patent WO
and Minamida, I. (1999) Pestic. Sci., 55, 2000/2,016,335 A2.
202–205. 54. Bayer AG (2000) Patent EP 1,031,566
35. Uneme, H., Konobe, M., Akayama, A1.
A., Yokota, T., and Mitzuta, K. (2006) 55. DSM Fine Chemicals (2001) Patent
Sumitomo Kagaku, 2, 20–33. WO 2001/090,089.
36. Ohkawara, Y., Akayama, A., Matsuda, 56. Bayer AG (1988) Patent DE 3,631,538
A., and Andersch, W. (2002) Brighton A1.
Crop Protect. Conf. – Pests Dis., 57. Kuraray Co. Ltd (1997) Patent EP
1, 51–58. 794,180 A1.
37. Uneme, H. (2011) J. Agric. Food Chem., 58. Ciba-Geigy AG (1997) Patent WO
59, 2932–2937. 97/10,226.
38. Stupp, H.-P. and Fahl, U. (2003) 59. Bayer AG (1997) Patent EP 780,384
Pflanz.-Nachr. Bayer (Ger. Ed.), 56, A2.
59–74. 60. Novartis AG (1997) Patent WO
39. Kando, Y., Uneme, H., and Minamida, 97/23,469 A1.
I. (1991) Patent EP 452,782 A1 (Takeda 61. Ciba-Geigy AG (1998) Patent WO
Chem. Ind., Ltd). 98/27,075 A1.
40. Uneme, H., Konobe, M., Ishizuka, 62. Kureha Chem. Ind. Co., Ltd (1997)
H., and Kamiya, Y. (1997) Patent Patent EP 775,700 A1.
WO 97/00,867 A1 (Takeda Chemical 63. Göbel, T., Gsell, L., Hüter, O.F.,
Industries, Ltd). Maienfisch, P., Naef, R., O’Sullivan,
41. Hafner, J.S. and Evans, R. (1959) A.C., Pitterna, T., Rapold, T., Seifert,
J. Org. Chem., 24, 1157–1159. G., Senn, M., Szczepanski, H., and
42. Takano, N., Seko, S., and Tanaka, K. Wadsworth, D. (1999) Pestic. Sci., 55,
(2005) Patent WO 2005/123,704 A1 343–389.
(Sumitomo Chem. Comp., Ltd). 64. Jeschke, P., Schindler, M., and Beck,
43. Rauchschwalbe, G. (2002) Patent US M.E. (2002) Brighton Crop Protect. Conf.
6,403,803 B1 (Bayer AG). – Pests Dis., 1, 137–144.
44. Konobe, M., Yamada, J., and Miyazaki, 65. Jeschke, P., Uneme, H.,
K. (2000) Patent JP 2000/143,648 A2 Benet-Buchholz, J., Stölting, J., Sirges,
(Takeda Chem. Ind., Ltd). W., Beck, M.E., and Etzel, W. (2003)
Pflanz.-Nachr. Bayer (Ger. Ed.), 56, and Chemistry of Agrochemicals VII,

5–24. ACS Symposium Series, vol. 948 (eds
66. Altmann, R. (2003) Pflanz.-Nachr. Bayer J.W. Lyga and G. Theodoridis), Ameri-
(Ger. Ed.), 56, 102–110. can Chemical Society, Washington, DC,
67. Schwarz, M., Christie, D., Andersch, pp. 67–81.
W., Kemper, K., Fellmann, K., and 79. Nauen, R., Ebbinghaus-Kintscher,
Altmann, R. (2002) Brighton Crop U., and Jeschke, P. (2005) 230th ACS
Protect. Conf. – Pests Dis., 1, 59–64. National Meeting Washington, DC,
68. Andersch, W. and Schwarz, M. (2003) August 28–September 1, 2005, Abstract
Pflanz.-Nachr. Bayer (Ger. Ed.), 56, of Papers AGRO-026. (Chem. Abstr.,
147–172. (2005), 735867).
69. Meredith, R.H. and Morris, D.B. (2003) 80. Karmakar, R. and Kulshrestha,
Pflanz.-Nachr. Bayer (Ger. Ed.), 56, G. (2009) Pest Manage. Sci., 65,
111–126. 931–937.
70. Hopkins, A., Graham, C., Schwarz, M., 81. Kodaka, K., Kinoshita, K., Wakita, T.,
and Kleyla, C. (2006) Proceedings of Yamada, E., Kawahara, N., and Yasui,
the Beltwide Cotton Conference, Jan- N. (1998) Brighton Crop Protect. Conf.
uary 3-6, San Antonio, Texas, USA, pp. – Pests Dis., 1, 21–26.
1560.1561 / 1–1560.1561/2. 82. Wakita, T., Yasui, N., Yamada, E.,
71. Dewar, A.M., Haylock, L.A., Garner, and Kishi, D. (2005) J. Pestic. Sci., 30,
B.H., Baker, P., Sands, R.J.N., 122–123.
Foster, S.P., Cox, D., Mason, N., and
83. Wakita, T. (2008) Yuki Gosei Kagaku
Denholm, I. (2003) Pflanz.-Nachr. Bayer
Kyokaishi, 66, 716–720.
(Ger. Ed.), 56, 127–146.
84. Wakita, T. (2011) J. Agric. Food Chem.,
72. Nauen, R., Ebbinghaus-Kintscher,
59, 2938–2942.
U., Salgado, V.L., and Kaussmann,
85. Wakita, T., Kinoshita, K., Yamada,
M. (2003) Pest. Biochem. Physiol., 76,
E., Yasui, N., Kawahara, N., Naoi, A.,
55–69.
Nakaya, M., Ebihara, K., Matsuno, H.,
73. Jeschke, P. and Nauen, R. (2004) 228th
and Kodaka, K. (2003) Pest Manage.
ACS National Meeting, Philadelphia,
Sci., 59, 1016–1022.
PA, August 22–26, 2004, Abstract
86. Kagabu, S., Matsuda, K., and Komai, K.
of Papers AGRO-003 (Chem. Abstr.,
(2002) Nippon Noyaku Gakkaishi (J. Pes-
(2004), 655197).
74. Jeschke, P. and Nauen, R. (2007) in tic. Sci.), 27, 374–377.
Synthesis and Chemistry of Agrochemicals 87. Coyne, M.J. (2009) Vet. Ther. Res. Appl.
VII, ACS Symposium Series, vol. 948 Vet. Med., 10, 17–23.
(eds J.W. Lyga and G. Theodoridis), 88. Wakita, T., Kinoshita, K., Yasui, N.,
American Chemical Society, Washing- Yamada, E., Kawahara, N., and Kodaka,
ton, DC, pp. 51–65. K. (2004) J. Pestic. Sci. (Tokyo), 29,
75. Benzidane, Y., Touinsi, S., Motte, E., 348–355.
Jadas-Hécart, A., Communal, P.-Y., 89. Kinoshita, K., Matsunaga, H., Odaka,
Leduc, L., and Thany, S.H. (2010) Pest K., Kawahara, N., and Shiraishi, S.
Manage. Sci., 66, 1351–1359. (1996) Patent JP 08,176,132 A2 (Mitsui
76. Wellmann, H., Gomes, M., Lee, C., Toatsu Chemicals, Japan).
and Kayser, H. (2004) Pest Manage. 90. Kodaka, K., Kinoshita, K., Wakita, T.,
Sci., 60, 959–970. Shiraishi, S., Ohnuma, K., Yamada,
77. Kayser, H., Lee, C., Decock, A., E., Yasui, N., Nakaya, M., Matsuno,
Baur, M., Haettenschwiler, J., and H. et al. (1995) Patent EP 649,845 A1
Maienfisch, P. (2004) Pest Manage. Sci., (Mitsui Toatsu Chemicals, Inc.).
60, 945–958. 91. Kiriyama, K. and Nishimura, K. (2002)
78. Kayser, H., Wellmann, H., Lee, C., Pest Manage. Sci., 58, 669–676.
Decock, A., Gomes, M., Cheek, B., 92. Mori, K., Okumoto, T., Kawahara, N.,
Lind, R., Bauer, M., Hattenschwiler, J., and Ozoe, Y. (2002) Pest Manage. Sci.,
and Maienfisch, P. (2007) in Synthesis 58, 190–196.
93. Kiriyama, K., Nishiwaki, H., Nakagawa, A.R. Horowitz), Springer Press, pp.
Y., and Nishimura, K. (2003) Pest Man- 151–195.
age. Sci., 59, 1093–1100. 101. Jeschke, P., Nauen, R., Schindler, M.,
94. Koyanagi, T. and Haga, T. (1995) in and Albert, A. (2011) J. Agric. Food
Synthesis and Chemistry of Agrochemi- Chem., 59, 2897–2908.
cals IV, Chapter 2 (eds R. Baker, J.G. 102. Jeschke, P. and Nauen, R. (2005) in
Fenyes, and G.S. Basarab), American Comprehensive Molecular Insect Science,
Chemical Society, Washington, DC, pp. vol. 5 (eds L. Gilbert, K. Latrou, and S.
15–24. Gill), Elsevier, Oxford, pp. 53–105.
95. Kagabu, S. (2003) in Chemistry of Crop 103. Jeschke, P. and Nauen, R. (2010) in
Protection: Progress and Prospects in Sci- Insect Control – Biological and Syn-
ence and Regulation (eds G. Voss and thetic Agents (eds L.I. Gilbert and S.S.
G. Ramos), Wiley-VCH Verlag GmbH, Gill), Elsevier, Academic Press, pp.
New York, pp. 193–212. 114–119.
96. Okazawa, A., Akamatsu, M., Ohaka, A.,
104. Gao, F., Mer, G., Tonelli, M., Hansen,
Nishiwaki, H., Cho, W.-J., Nakagawa,
S.B., Burghard, T.P., and Taylor,
Y., Nishimura, K., and Ueno, T. (1998)
P. et al. (2006) Mol. Pharmacol., 70,
Pestic. Sci., 54, 134–144.
1230–1135.
97. Nakayama, A. and Sukekawa, M. (1998)
105. Talley, T.T., Harel, M., Hibbs, R.E.,
Pestic. Sci., 52, 104–110.
98. Okazawa, A., Akamatsu, M., Nishiwaki, Radic, Z., Tomizawa, M., Casida, J.E.,
H., Nakagawa, Y., Miyagawa, H., and Taylor, P. (2008) Proc. Natl Acad.
Nishimura, K., and Ueno, T. (2000) Sci. USA, 105, 7606–7611.
Pest Manage. Sci., 56, 509–515. 106. Ihara, M., Okajima, T., Yamashita,
99. Jeschke, P. and Nauen, R. (2008) Pest A., Oda, T., Hirata, K., Nishiwaki, H.,
Manage. Sci., 64, 1084–1098. Morimoto, T., Akamatsu, M., Ashiwaki,
100. Jeschke, P. (2007) in Insecticides Y., Kuroda, S., Mega, R., Kuramitsu, S.,
Design Using Advanced Technolo- Sattelle, D.B., and Matsuda, K. (2008)
gies (eds I. Ishaaya, R. Nauen, and Intervertebr. Neurosci., 8, 71–81.
32.2.2
Five-Membered Compounds: Imidacloprid and Thiacloprid
Two commercial neonicotinoids containing five-membered ring systems, namely
imidacloprid (1) and thiacloprid (9), belong to this group.
32.2.2.2 Imidacloprid
In 1984, the discovery of the five-membered neonicotinoid imidacloprid (1,
1991, Bayer CropScience; see Scheme 32.2.2.1) [1–8] was the result of a search
for improved activity by altering the structure of the originally announced
six-membered nithiazine (2) [9–12]. Because of the photolabile 2-nitromethylene
group, nithiazine was never commercialized for broad agricultural use.
However, during the early 1980s chemists at the subsidiary company of
Bayer in Japan (Nihon Tokushu Noyaku Seizo K. K., now Bayer CropScience)
recommenced their investigations into a synthesis based on the lead structure
of nithiazine. In this case, by introducing an N-containing heteroaryl-methyl
H2N NH2
CPM-NH-CH2CH2-NH2 (4)
N- NO2
H2N-CH2CH2-NH2
HN NH CCMP (6) N NH
CPM
Base
N- NO2 N- NO2
5 1
Scheme 32.2.2.1 Synthetic pathways for preparation of imidacloprid (1).
group (e.g., N-(6-chloro-pyrid-3-ylmethyl)-N-ethyl-amine; CPM) as a substituent

of the 2-nitromethylene-imidazolidine five-membered ring system (NTN32692,
X–Y = CH–NO2 ; see Figure 32.2.1 in Chapter 32.2) [13], the insecticidal activity
could be enhanced remarkably. Following the preparation of about 2000 com-
pounds, imidacloprid emerged from this program and was selected for commercial
use based on its insecticidal potential, photostability, and longlasting effect under
greenhouse and field conditions, as well as its good systemic properties [14].
HN S
CH-NO2
2
As the first member of the chloronicotinyl insecticide (CNI) family, imidacloprid

has become the most successful, highly effective, and largest-selling insecticide
worldwide for agricultural use (it has been registered in more than 120 coun-
tries worldwide and applied to over 140 crops) [15–18], and for applications in
® ®
nonagricultural areas such as termite control (Hachikusan , Japan; Premise , cf.
® ®
USA) [19, 20], as a garden professional care product (Merit , Provado ) [21], or
® ®
in veterinary medicine as an ectoparasiticide (Advantage , AdvantageMulti , K9
® ®
advantix , Advantix ) [22, 23].
32.2.2.2.1 Chemical Classification and Physico-Chemical Properties The water sol-
ubility and low partition coefficient in octanol–water of imidacloprid are not
influenced by pH values between 4 and 9, at 20 ◦ C (Tables 32.2.2.1 and 32.2.2.2) [24].
The low partition coefficient of imidacloprid indicates that it has no potential
to accumulate in biological tissues nor, consequently, in the food chain. The
rapid uptake and translaminar transport of imidacloprid is excellent, as observed
Table 32.2.2.1 Chemical classification of imidacloprid (1).
Common name Imidacloprid

® ® ® ® ® ®
Trade names Admire , Akteur , Alias , Amigo , Blue Sky , Confidor ,
® ® ® ® ®
Conidor , Connect , Commando , El Hombre , Encore ,
® ® ® ® ® ®
Escocet , Evidence , Faibel , Gaucho , Genesis , Ghoulish ,
® ® ® ® ®
Hachikusan , Imex , Imicide , Impower , Intercept ,
® ® ® ® ® ®
Kohinor , Legend , Lizetan , Marathon , Merit , Muralla ,
® ® ® ® ® ®
Pasada , Plural , Pre-Empt , Premise , Prescribe , Provado ,
® ® ® ® ®
Quick Bayt , Rapid , Seed-one , Tatamida , Termex ,
® ® ® ® ®
Trimax , Trust , Vaplex , Warrant , Winner , Yi Sha
® ® ®
Guang , Yunta , Zorro FS 236,3
Development codes NTN 33893
Table 32.2.2.2 Physico-chemical properties of imidacloprid (1).
Vapor pressure (hPa at 20 ◦ C) 4 × 10−12
Solubility in water (g l−1 at 20 ◦ C) 0.61, no influence of pH-value

−1 ◦
Solubility in organic solvents (g l at 20 C) Dichloromethane: 67, acetone: 50,
methanol: 10, 2-propanol: 2.3, toluene:
0.68, n-hexane: <0.1
Dissociation constant pKa (at 20 ◦ C) Not determined
in cabbage leaves [25] and in rice and cucumber [26]. Whilst imidacloprid has
a considerable acropetal mobility in the xylem of plants, its penetration and
translocation in cotton leaves is less pronounced, as proven using phosphor-imager
autoradiography [27]. Such xylem mobility makes imidacloprid especially useful
for seed treatments and soil applications, though it is equally active when applied
via a foliar route. Owing to its lack of any acidic hydrogen, the pKa of imidacloprid
is >14 and, therefore, its transport within the phloem is unlikely [28, 29]. The
systemic properties of imidacloprid have been investigated using the 14 C-labeled
compound.
The metabolism of imidacloprid is greatly influenced by the method of application
[30–32]. Based on the results of soil metabolism studies, imidacloprid was shown
to be degraded completely to carbon dioxide, and not to persist in the soil. Under
standard laboratory conditions, the aerobic degradation of imidacloprid has a
half-life (degradation time; DT50 ) of 156 days.
32.2.2.2.2 Chemistry The first laboratory synthesis of imidacloprid (1) was car-
ried out by the cyclocondensation of N-nitro-guanidine (3) [33] and N-(6-chloro-
pyrid-3-ylmethyl)-ethylenediamine (4, PEDA) [34–36] in water at 80 ◦ C to give
imidacloprid in good yield [37].
Alternatively, imidacloprid can be prepared by N-alkylation of the 2-N-nitro-
imidazolidine system (5) [38], obtained by the cyclocondensation of 3 and ethylene-
diamine, with 6-chloro-3-chloromethyl-pyridine (CCMP, 6) (Scheme 32.2.2.1).
Numerous syntheses have been investigated to develop practical and economical
processes for the key intermediate 6, based on different commercially available
starting materials, as outlined in Table 32.2.2.3.
The crystallographic analysis of imidacloprid revealed a coplanar relationship of
the five-membered imidazolidine ring to the N-nitroimino group at the 2-position
[48, 49]. An intramolecular H-bond between 1 NH and O2 N–N=C2 was confirmed
using nuclear magnetic resonance (NMR) techniques and infrared (IR) spec-
troscopy (highly chelated 1 NH absorption) [50]. Investigations were also carried out
using comparative molecular field analysis (CoMFA) [51, 52]. The deduced electron
deficiency of the nitrogen atom of imidacloprid was proved explicitly by using
Table 32.2.2.3 Selection of synthetic pathways for prepara-

tion of the key intermediate CCMP (6).
Precursor R1 , R2 Reaction conditions Reference
R2
R1 N
Cl, CH3 (a) AIBN, Cl2 [39]

(b) tert-BuOCl, hν
(c) CCl4 , K2 CO3
SO2 –Ph, CH3 AIBN, SOCl2 [40]
Cl, CH2 NH2 (a) NOCl, HCl [41]

(b) aqueous NaNO2 , HCl
Cl, CH2 OH (a) PCl3 , POCl3 [42]
(b) SOCl2
OCH3 , CH2 OCH3 (a) POCl3 :PCl5 (1 : 2) [43]
Cl, COOH – [44]
Cl, Cl3 (a) Zn powder or [45]

(b) Sn powder, aqueous HCl
OH, CHO – [46]
O-alkyl, CHO – [47]
AIBN, azo-bis-(isobutyronitrile).
15
N NMR spectroscopic measurements [53]. By employing the MNDO method,
combined with the PM3 method (a semi-empirical molecular orbital technique for
calculating electronic structure), Tomizawa et al. [54] calculated that the N-nitro
group of imidacloprid is much more important for binding at the receptor than the
bridgehead nitrogen, which was only marginally positive. The important contribu-
tion of this N-nitro group and its H-bondable property for the insecticidal activity
had already been predicted by Kagabu [55].
32.2.2.2.3 Efficacy on Target Pests and Application Rates The neonicotinoid im-
idacloprid is characterized by its extremely high intrinsic insecticidal potency,
and excellent systemic properties. Uptake of the active ingredient via the roots is
an important prerequisite for soil-directed application, for example, via irrigation
systems (drip or drench), in-furrow-application, granular application, or seed treat-
®
ment [56, 57]. Therefore, imidacloprid can be used as a seed dressing (Gaucho )
[58] as well as a foliar spray, as a soil treatment (e.g., by irrigation), as granules
®
(Admire ) in seedling box applications in rice [59], in floating box systems for
® ®
tobacco seedlings (e.g., Confidor S ), or as plant rodlets (Provado ) or compacts
® ®
(e.g., Confidor , Admire ). Furthermore, imidacloprid can be applied to plants or
®
plant parts (e.g., the stem) as a spray, as a wettable powder (Admire ), by pelleting,
implantation, dipping, injection (e.g., bud), by application to the base of the trunk,
and/or painting. These methods have led to a more economic and environmentally
friendly use of imidacloprid.
Imidacloprid has a broad spectrum of activity, a good longlasting effect, and plant
®
compatibility. The main pests to be controlled with imidacloprid (as Confidor ) are
a wide range of sucking insects, such as aphids, whiteflies, plant- and leafhoppers
(jassids), thrips (except for certain Frankliniella spp.), scales, mealybugs, plant bugs,
and psyllids, including those that are already resistant to conventional insecticides
(Tables 32.2.2.4 and 32.2.2.5) [60, 61].
Many of the sucking insects treated with imidacloprid are known to be vectors
of plant viruses; examples include aphids [62], whiteflies, thrips, and leafhoppers.
Alternatively, they may transmit bacterial diseases (phytoplasma); examples include
leafhoppers and psyllids. Due to their plant systemic properties, neonicotinoids
such as imidacloprid, thiamethoxam (7) (see Chapter 32.2.3), and clothianidin
(8) (see Chapter 32.2.1) can control important vectors of virus diseases, thereby
suppressing the secondary spread of viruses in various crops. In the case of imida-
cloprid, this control was discovered during its early development, and was supported
by the anti-feedant effects of sublethal doses of imidacloprid in whiteflies [63].
An outstanding crop protection was achieved with either seed treatment or
foliar applications of imidacloprid to cereals for controlling the persistent barley
yellow dwarf virus (BYDV) vectors transmitted by Rhopalosiphum padi and Sitobion
avenae [64–66], in tobacco against thrips and tomato spotted wild virus (TSWV)
[67], in tomato against whiteflies and tomato yellow leaf curl virus (TYLCV), and
in citrus against glassywinged sharpshooters as vector of the bacterium Xylella
fastdiosa [18]. Sugar beet seed pelleted (90 g a.i. per unit) with imidacloprid [64] also
Table 32.2.2.4 Insecticidal efficacy of imidacloprid (1) after

foliar application against Homoptera pest insects under labo-
ratory conditions.
Pest species Developmental stage LC95 , rounded (ppm)
Homoptera
Aphis fabae Mixed 8
Aphis gossypii Mixed 1.6
Aphis pomi Mixed 8
Brevicoryne brassicae Mixed 40
Myzus persicae Mixed 1.6
Myzus persicae (tobacco) Mixed 8
Phorodon humuli Mixed 0.32
Laodelphax striatellus Third instar 1.6
Nephotettix cinctiteps Third instar 0.32
Nilapavarta lugens Third instar 1.6
Sogatella furcifera Third instar 1.6
Pseudococcus comstocki Larvae 1.6
Bemisia tabaci Second instar 8
Trialeurodes vaporariorum Adult 40
Hecinothrips femoralis Mixed 1.6
LC, lethal concentration.

provided protection especially against infections of beet mild yellow virus (BMYV)
transmitted by the peach potato aphid (M. persicae) [65].
O
N
Cl N N
S Me
N-NO2
7
N H H
Cl N N
S Me
N-NO2
8
Imidacloprid provides an additional control of Coleopteran species (e.g., rice water

and tobacco weevil, Colorado potato beetle, rice leaf beetle, wireworms, grubs, and
other soil beetles) and Dipteran species (e.g., fruit fly, beet fly, bean, and onion
fly), and of selected micro-lepidopteran species (e.g., citrus, apple, and potato leaf
Table 32.2.2.5 Insecticidal efficacy of imidacloprid (1) after

foliar application against Coleoptera and Lepidoptera pest
insects under laboratory conditions.
Pest species Developmental stage LC95 , rounded (ppm)
Coleoptera
Leptinotarsa decemlineata Second instar 40
Lema oryzae Adult 8
Lissorhoptrus oryzophilus Adult 40
Phaedon cochleariae Second instar 40
Lepidoptera
Chilo suppressalis First instar 8
Helicoverpa armigera Second instar 200
Plutella xylostella Second instar 200
Heliothis virescens Eggs 40
Spodoptera frugiperda Second instar 200
miner), ants (Hymenoptera), termites (Isoptera), cockroaches, grasshoppers, and

crickets (Orthoptera) [66].
Several combination products of imidacloprid with other insecticides and fungi-
cides have been developed over the years for the foliar and soil treatments of
®
a wide range of crops. Examples include: Confidor Supra (100 EC, imidaclo-
® ®
prid combined with cyfluthrin), Confidor Energy (with deltamethrin), Leverage
® ® ®
(324 SC, with cyfluthrin), Imprimo or Montur (with tefluthrin), Connect ,
® ® ® ®
Solomon , Thunder or Chinook (with beta-cyfluthrin), Favilla (23% WP, with
® ® ®
methiocarb), Aeris or CropStar (with thiodicarb), Monceren Star (50 WP, with
®
pencycuron), Nemacur Multi (246 SC, with fenamiphos; mainly for greenhouse
® ®
application at planting), WinAdmire (6 GR, with carpropamid), Camena (4 GR,
® ®
with carpropamid), BeamAdmire (6 GR, with tricyclazole), Gaucho Blé (with
®
bitertanol + anthraquinone), or Gaucho Orange (with tebuconazole + triazoxide).
The novel combinations of imidacloprid with pyrethroids have been developed with
the aim of broadening the spectrum, and to substitute World Health Organization
(WHO) Class I products from older chemical classes [68].
Apart from a direct insecticidal activity, imidacloprid also possesses several
sublethal side effects. Such effects, which sometimes are dose-dependent, include
repellency, a reduction or cessation of feeding, a reduction or cessation of repro-
ductive activities, an overall reduction of movement or activity, and an increased
susceptibility to biological control. The excellent anti-feeding effects of imidacloprid
result in less time for transmission and a reduced period of xylem contact, so that
the number of infections per time units is reduced considerably.
In addition, the results of field studies have indicated that multiple foliar spray
applications of imidacloprid led to improved health and an increased plant growth,
even in situations without insect infestation. Treatment with imidacloprid led

to a considerable reduction in yield losses due to drought stress (stress shield
technology) compared to other neonicotinoids [69]. Water-deficit field studies
®
confirmed the potential of Trimax (an optimized formulation of imidacloprid)
to moderate water stress in plants, with an average lint yield increase in cotton
of 10% [70, 71]. All results have indicated that, in addition to its insecticidal
activity, a ‘‘stress shield MoA (mode of action)’’ of imidacloprid supports plants
in moderating the effects of abiotic and biotic stress. As a trigger of such an
effect, details of the 6-chloro-nicotinic acid metabolite of imidacloprid [69], and/or
the further bioactivated 6-chloro-2-hydroxy-nicotinic acid – which is known to be
a potent inducer of the pathogenesis-related protein 1 (PR1) and an inhibitor of
salicylic acid-sensitive enzymes have been discussed [72].
An additional important aspect with regards to the use of imidacloprid is its
excellent fit into integrated pest management (IPM) systems since, depending on
the application method and timing, nontarget organisms are not affected. Indeed,
the safety of beneficial organisms and pollinators has especially been optimized by
selectivity in space [73]. The application of imidacloprid into the soil via different
methods allows it to be transported to the pest within the plant, without harming
any beneficial organisms [74].
The metabolism of imidacloprid is greatly influenced by its method of application
[75]. Depending on the time and the plant species involved, imidacloprid is
effectively degraded completely, as has been revealed in comparative studies
conducted in many field crops [76].
32.2.2.3 Thiacloprid
In connection with the excellent biological performance and market acceptance
of imidacloprid, a further, extensive research and development program led to
the discovery and development of the five-membered neonicotinoid thiacloprid (9)
(2000, Bayer CropScience) [77, 78], the second member of the CNI family. Similar
to imidacloprid, this neonicotinoid also contains the CPM residue attached to the
cyclic 2-(N-cyanoimino)-thiazolidine (CIT, 11) [79] moiety.
32.2.2.3.1 Chemical Classification and Physico-Chemical Properties Once applied

to the leaves, thiacloprid is stable toward hydrolysis, even under conditions of
heavy rain and sunlight, providing sufficient plant-uptake of the substance by a
continuous penetration of the active ingredient into the leaf. The half-life in water
at pH 5, 7, and 9 is in excess of 500 h (Tables 32.2.2.6 and 32.2.2.7).
Table 32.2.2.6 Chemical classification of thiacloprid (9).
Common name Thiacloprid

® ® ® ®
Trade names Alanto , Bariard , Biscaya , CaLypso
Development codes YRC 2894
Table 32.2.2.7 Physico-chemical properties of thiacloprid (9).

◦
Vapor pressure (hPa at 20 C) 3 × 10−12
Solubility in water (g l−1 at 20 ◦ C) 0.185 (not influenced by pH in the range pH 4–9)

−1
Solubility in organic solvents (g l at Dichloromethane: 160, dimethyl sulfoxide: 150,
20 ◦ C) acetone: 64, acetonitrile: 52, ethyl acetate: 9.4,
propan-2-ol: 3.0, 1-octanol: 1.4, xylene: 0.3,
n-heptane: <0.1
Dissociation constant pKa (at 20 ◦ C) No acidic or basic properties in aqueous solutions;

not possible to specify dissociation constants for
water
The photolysis of thiacloprid in water (buffered at pH 7) shows a half-life of more

than 100 days. Thiacloprid is also stable under sunlight irradiation on oil surfaces.
Because of the single peak maximum at 242 nm in the ultraviolet (UV) spectrum,
the photostability of thiacloprid is superior to that of other neonicotinoids.
The penetration and translocation behavior of [14 C]thiacloprid [80] in cabbage was
comparable to that reported for imidacloprid. The translaminar and acropetal aphi-
cidal efficacy of thiacloprid showed clearly that it can be translocated systemically;
in fact, the translocation pattern of [14 C]thiacloprid revealed xylem-mobility (i.e.,
translocation in an upward direction) only one day after its application to cabbage
leaves.
The metabolic pathway of systemic thiacloprid, in both quantitative and quali-
tative terms, is similar in all of the crops investigated (e.g., fruiting crops, cotton)
[81]. In soil, the degradation of thiacloprid under aerobic conditions occurs rapidly,
with a mean half-life (degradation time; DT50 ) of approximately 16 days (range: 9 to
27 days, depending on soil type). The anaerobic aquatic metabolism of thiacloprid
occurs in very similar fashion, though slightly more slowly than the aerobic route.
In a water-sediment system, thiacloprid was degraded completely to carbon dioxide
[82]. In general, the main degradation steps of thiacloprid are hydrolysis, oxidation,
and conjugation [83].
32.2.2.3.2 Chemistry Thiacloprid can be synthesized by a simple, convergent

one-step technical process, starting from two key intermediates, CIT (11) and
CCMP (6) [84, 85] according to Scheme 32.2.2.2.
The first building block, commercially available CIT, is synthesized by a
base-catalyzed cyclization reaction of N-cyano-S,S-dimethyldithioimidocarbonate
(CIDT, 10a) [86, 87] or N-cyano-O,O-dimethyl-imidocarbonate (DCC, 10b) [88]
with cysteamine hydrochloride. From the technical process for thiacloprid, sev-
eral attractive synthetic routes have been described for the preparation of CCMP
X X + HN S N S
Me CI − H3N-CH2CH2-SH CCMP (6) CPM
Me
N- CN N-CN N-CN
X = S CIDT (10a) CIT (11) 9

X = O DCC (10b)
Scheme 32.2.2.2 Synthetic pathways for preparation of thiacloprid (9).
(Table 32.2.2.3). Finally, the technical process is readily available by N-alkylation of

CIT with CCMP.
Following its synthesis, thiacloprid crystallizes in two different modifications,
depending on the solvent. The neonicotinoid crystallizes from dichloromethane as
form I (m.p. = 136 ◦ C), and from iso-propanol as form II (m.p. = 128.3 ◦ C). Based on
the physico-chemical data, the technically active ingredient is form I. With regards
to the configuration of the pharmacophore [–N–C(S)=N–CN], thiacloprid exists
only in the (Z)-configuration in both forms, I and II. In conjunction with X-ray
analysis, thiacloprid forms in solution exclusively the stable (Z)-configuration.
Molecular modeling studies (e.g., force-field methods, MMFF94s) have shown
that the (Z)-configured thiacloprid is about 4 kcal mol−1 lower in energy than the
(E)-isomer. The preference for the (Z)-configuration stems mainly from steric
reasons. Quantum chemical calculations have shown that the strong delocalization
of the C=N double bond does not reduce the so-called ‘‘double bond character’’ to
any significant degree.
32.2.2.3.3 Efficacy on Target Pests and Application Rates Thiacloprid has been
developed especially for foliar treatments, and is applied at rates ranging from 48
to 180 g a.i. ha−1 , on up to three occasions per season, depending on the target
® ®
crop. Typically, a suspension concentrate (480 SC CaLypso or Alanto ) is used
as a standard formulation, although water-dispersible granules (WG 30 and 70
® ®
Bariard ) and a new formulation oil dispersion (OD) technology (Biscaya O-TEQ
240) have been developed that also provide a stable spray solution. Thiacloprid
is known to possess not only very good systemic characteristics but also to have
excellent stomach-related and contact properties. Moreover, these benefits are
combined with a relatively low rate of application, a superior plant compatibility
in different crops (e.g., canola, cereals, cotton, fruits, potatoes, rice, ornamentals,
oilseed rape, and vegetables), and a favorable ecotoxicological profile [89].
The spectrum of activity of thiacloprid covers three groups of target pest [90]:
• The ‘‘traditional’’ insects of the CNI spectrum, including aphids, whiteflies, and
some thrips and beetles, such as the rice water weevil from rice (Lissoropterus
oryzophilus), the apple weevil (Anthonomus pomorum), and micro-lepidopterans
such as Phyllocnistis citrella in citrus.
• The ‘‘traditional’’ insects from the CNI spectrum; however, at comparatively lower
dosages thiacloprid can be used to control a variety of beetle species, including
Colorado potato beetle (Leptinotarsa decemlineata) and some leafminers, such as

Lithocolletis blancardella and Lyonetia clerkella.
• A completely new spectrum of control for lepidopteran pests, tortricides such as
Cydia pomonella and Cydia molesta in pome and stone fruit, as well as coleopterans
such as Anthonomus grandis in cotton and Meligethes aenus in rape. Furthermore,
thiacloprid is highly effective in controlling dipteran species such as Rhagolethis
cerasi, Dacus oleae, and Ceratitis capitata in fruit crops such as peaches and olives.
The activities of thiacloprid against important agricultural pests following leaf-dip

application are summarized in Table 32.2.2.8 [91].
Thiacloprid demonstrates an excellent performance against the first and second
instar larvae of the codling moth (C. pomonella), which are the stages most likely
to be exposed to spray coverage in a real field situation. The speed of action
of thiacloprid against these most susceptible larval instars was remarkable, with
the lowest concentration (8 ppm) affecting 100% of the larvae after only 4 h of
exposure (e.g., a short period of hyperexcitation and total paralysis of the larva).
More strikingly, the higher (practical) concentrations of 40 and 200 ppm affected
®
100% of the larvae after only 60 and 120 min, respectively. Thiacloprid (CaLypso )
can also be used to control freshly laid insect eggs, the optimal time for spray
application being between the start and peak of egg-laying. The ovicidal efficacy of
sprayed thiacloprid in C. pomonella was described as excellent (Table 32.2.2.9).
Thus, in comparison to insect growth regulators (IGRs), thiacloprid not only
has a wider window of application but also allows a more flexible timing of
application – both of which are highly beneficial in fruit farming.
One other major advantage of thiacloprid is that it has no effect on pollinating
insects, such as honey bees, bumble bees and/or parasitic wasps; this, in turn,
allows its application before, during, and after the flowering period of fruit crops [92,
93]. Moreover, as thiacloprid does not cause any disturbance to the predator–prey
Table 32.2.2.8 Activity of thiacloprid (9) against important

agricultural pests after leaf-dip application.
Pest species LC50 (mg a.i. l−1 ) 95% confidence limit
Myzus persicae, mpa 1.5 1.4–1.7

Aphis fabae, mp 0.8 0.7–0.9
Aphis fabae, mp ≤0.6 –
Aphis gossypii, mp 0.8 0.7–0.9
Bemisia tabaci, mp 1.1 0.3–2.4
Nephotettix cincticeps, L2 0.6 0.5–0.7
Cydia pomonella, L2,3 1.1 0.8–1.4
Phaedon cochleariae, L2 18.5 15.9–21.6
Lissorhoptrus oryzophilus, ad 1.8 1.2–2.7
a
Soil application, LD95 .
mp = mixed population; L = larval stage; ad = adult.
Table 32.2.2.9 Ovicidal activity of thiacloprid (9) after spray

application against eggs of codling moth (C. pomonella).
Concentration (ppm) No. of eggs at day 2 No. of hatched larvae
40 41 0
200 80 0
Control 32 32
equilibrium, it is also ideal for use in IPM programs (see also imidacloprid in
Section 32.2.2.2.3) [94].
To date, several combination products of thiacloprid have been developed for
®
foliar treatments, including the suspo-emulsion marketed as Monarca (SE 112.5,
®
thiacloprid + beta-cyfluthrin) and Proteus (thiacloprid + deltamethrin). The latter
®
product is based on the new O-TEQ formulation, which facilitates leaf penetration
of the active ingredients, especially under suboptimal conditions for foliar uptake
[39–47, 95–97]. Once on the leaf, thiacloprid demonstrates a smooth spreading
of the oil following evaporation of the spray water, and this results in an optimal
coverage and distribution.
In addition to formulations for foliar treatment, granules for rice seedling box
applications have also been developed; these are marketed under the trade names
® ®
1.5 GR and 4.5 GR w , and as a combination product WinBariard (5.5 GR,
thiacloprid + carpropamid).
References
1. Elbert, A., Overbeck, H., Iwaya, K., and D.W. Gammon, and N.N. Rangsdale),
Tsuboi, S. (1990) Proc. Brighton Crop. John Wiley & Sons, Inc., pp. 933–944.
Prot. Conf. – Pests Dis., 1, 21–28. 8. Jeschke, P. and Nauen, R. (2008) Pest
2. Diehr, H.-J., Gallenkamp, B., Jelich, K., Manage. Sci., 64, 1084–1098.
Lantzsch, R., and Shiokawa, K. (1991) 9. Soloway, S.B., Henry, A.C., Kollmeyer,
Pflanz. Nachr. Bayer (Ger. Ed.), 44, W.D., Padgett, W.M., Powell, J.E.,
113–136. Roman, S.A., Thiemann, C.H., Corey,
3. Leicht, W. (1993) Pestic. Outlook, 4, R.A., and Horne, C.A. (1978) in Pesti-
17–21. cide and Venom Neurotoxicity, (eds D.L.
4. Elbert, A., Nauen, R., and Leicht,
Shankland, R.M. Hollingworth, and T.
W. (1998) in Insecticides with Novel
Smyth Jr), Plenum Press, New York, pp.
Mode of Action: Mechanisms and
153–158.
Applications (eds I. Ishaaya and
10. Soloway, S.B., Henry, A.C., Kollmeyer,
D. Degheele), Springer-Verlag,
Berlin, Heidelberg, New York, pp. W.D., Padgett, W.M., Powell, J.E.,
50–73. Roman, S.A., Tieman, C.H., Corey, R.A.,
5. Leicht, W. (1996) Pflanz. Nachr. Bayer and Horne, C.A. (1979) Advances in Pes-
(Ger. Ed.), 49, 71–84. ticide Science, Part 2. Pergamon Press,
6. Shiokawa, K., Tsuboi, S., Iwaya, K., pp. 206–227.
and Moriya, K. (1994) J. Pestic. Sci., 19, 11. Schröder, M.E. and Flattum, R.F. (1984)
7. Kagabu, S. (2003) in Encyclopedia of 12. Kollmeyer, W.D., Flattum, R.F., Foster,
Agrochemicals, vol. 2 (eds J.R. Plimmer, J.P., Powel, J.E., Schroeder, M.E., and
References 1201
Soloway, S.B. (1999) in Nicotinoid In- Pflanz. Nachr. Bayer (Ger. Ed.), 45,
secticides and the Nicotinic Acetylcholine 327–368.
Receptor (eds I. Yamamoto and J.E. 29. Tröltzsch, C.M., Führ, F., Wieneke,
Casida), Springer, Tokyo, pp. 71–89. J., and Elbert, A. (1994) Pflanz. Nachr.
13. Kagabu, S., Moriya, K., Shibuya, K., Bayer (Ger. Ed.), 47, 249–303.
Hattori, Y., Tsuboi, S., and Shiokawa, K. 30. Nauen, R., Reckmann, U., Armborst, S.,
(1992) Biosci. Biotechnol. Biochem., 56, Stupp, H.P., and Elbert, A. (1999) Pestic.
362–363. Sci., 55, 265–271.
14. Moriya, K., Shibuya, K., Hattori, Y., 31. Roberts, T.R. and Hutson, D.H. (1999)
Tsuboi, S., Shiokawa, K., and Kagabu, Metabolic Pathways in Agrochemicals, Part
S. (1992) Biosci. Biotechnol. Biochem., 56, 2, Insecticides and Fungicides, Cambridge
364–365. University Press, Cambridge.
15. Moriya, K., Shibuya, K., Hattori, Y., 32. Koester, J. (1992) Proc. Brighton Crop.
Tsuboi, S., Shiokawa, K., and Kagabu, Prot. Conf. – Pests Dis., 2, 901–906.
S. (1993) Biosci. Biotechnol. Biochem., 57, 33. Knobloch, J. and Mueller,
127–128. K. (1987) International
16. Kagabu, S. (1997) Rev. Toxicol., 1, Jahrestagung–Fraunhofer-Institut
75–129. fuer Treib- und Explosivstoffe, 18th
17. Kagabu, S. (2003) in Chemistry of Crop (Technology Energy Matter), pp. 1–5,
Protection: Progress and Prospects in Sci- 19.
ence and Regulation (eds G. Voss and G. 34. Shiokawa, K., Tsuboi, S., Kagabu, S.,
Ramos), Wiley-VCH Verlag GmbH, New and Moriya, K. (1985) EP 163,855 A1,
York, pp. 193–212. (Nihon Tokushu Noyaku Seizo K. K.,
18. Elbert, A., Haas, M., Springer, B., Japan).
Thielert, W., and Nauen, R. (2008) 35. Diehr, H.J. (1992) EP 474,057 A1,
Pest Manage. Sci., 64, 1099–1105. (Bayer AG).
19. Jacobs, D.E., Hutchinson, M.J., Fox, 36. Lantzsch, R. (1993) EP 542,086 A1,
M.T., and Krieger, K.J. (1997) Am. J. Vet. (Bayer AG).
Res., 58, 1260–1262. 37. Kagabu, S. (2011) J. Agric. Food Chem.,
20. Dryden, M.W., Perez, H.R., and 59, 2887–2896.
Ulitchny, D.M. (1999) J. Am. Vet. Med. 38. McKay, A.F. and Wright, G.F. (1948) J.
Assoc., 215, 36–39. Am. Chem. Soc., 70, 430–431.
21. Armbrust, K.I. and Peeler, H.B. (2002) 39. (a) Lantzsch, R. and Stelzer, U. (1998)
Pest Manage. Sci., 58, 702–706. Patent WO 98/41,504, Bayer AG; Wen,
22. Mencke, N. and Jeschke, P. (2002) Curr. J., Mao, G., and Qiao, P. (2004) Patent
Top. Med. Chem., 2, 701–715. CN 1,517,338 A, Tianjin University;
23. Wenzel, U., Heine, J., Mengel, H., Guenther, A. (1991) Patent DE 4,016,175
Erdmann, F., Schaper, R., Heine, S., A1, Bayer AG; (b) Hu, L. et al. (1998)
and Daugschiess, A. (2008) Parasitol. Huaxue Shijie, 20, 313–316; (c) (1988)
Res., 103, 231–234. Patent DE 3,630,046 A1, Bayer AG.
24. Krohn, J. and Hellpointer, E. (2002) 40. Terashima, S., Kato, T., and Kajiyashiki,
Pflanz. Nachr. Bayer (Germ. Ed.), 55, T. (1996) Patent WO 96/26,188 A1,
3–26 (Special Edition). Sagami Chemical Research Center.
25. Elbert, A., Becker, B., Hartwig, J., and 41. (a) Kraus, H., Lindel, H., and Diehr,
Erdelen, C. (1991) Pflanz. Nachr. Bayer H.-J. (1995) Patent EP 632,025 A1,
(Ger. Ed.), 44, 113–136. Bayer AG; (b) Nahata, T. and Meisho,
26. Ishii, Y., Kobori, I., Araki, Y., Kurogochi, S. (1993) Patent JP 05,178,835 A2, Koei
S., Iwaya, K., and Kagabu, S. (1994) J. Chem. Co.
Agric. Food Chem., 42, 2917–2921. 42. (a) Werbitzky, O. and Studer, P. (1993)
27. Buchholz, A. and Nauen, R. (2001) Mitt. Patent EP 569,947 A1, Lonza AG; (b)
Dtsch. Ges. Allg. Angew. Entomol., 13, Latli, B. and Casida, J.E. (1992) J. Lab.
227–232. Comp. Radiopharm., 31, 609–613.
28. Stein-Dönecke, U., Führ, F., Wienecke, 43. Jelich, K. (1990) Patent EP 393,453 A2,
J., Hartwig, J., and Leicht, W. (1992) Bayer AG.
44. Cabanal-Duvillard, I. and Berrien, J.-F. 62. Epperlein, K., Fuchs, E., Grüntzig, M.,
(1999) Heterocycl. Commun., 5, 257–262. and Kunze, L. (1995) Arch. Phytopathol.
45. (a) Ieno, K. (1993) Patent JP 05,320,132 Pflanzenschutz, 29, 401–415.
A2, Koei Chem. Co.; (b) Awazu, T., 63. Nauen, R., Koob, B., and Elbert, A.
Okada, H., and Matsumoto, M. (1992) (1998) Entomol. Exp. Appl., 88, 287–293.
Patent EP 512,436 A1, Ishihara Sangyo 64. Dewar, A., Read, L., Prince, J., and
Kaisha, Ltd. Ecclestone, P. (1993) Beet Rev., 61, 5–8.
46. Jelich, K. (1990) Patent EP 373,463 A2, 65. Dewar, A.M. and Read, L.A. (1990) Proc.
Bayer AG. Brighton Crop. Prot. Conf. – Pests Dis., 2,
47. Jelich, K. (1990) EP 373,464 A2, Bayer 721–726.
AG. 66. Mullins, J.W. (1993) Pest Control with
Enhanced Environmental Safety, ACS
48. Born, L. (1991) Pflanz. Nachr. Bayer
Symposium Series No. 524, vol. 203,
(Ger. Ed.), 44, 137–144.
American Chemical Society, Washing-
49. Kagabu, S. and Matsuno, H. (1997) J.
ton, DC, pp. 183–198.
Agric. Food Chem., 45, 276–281.
67. Rudolph, R.D. and Rogers, W.D. (2001)
50. Kagabu, S., Yokoyama, K., Iwaya, K.,
Pflanz. Nachr. Bayer (Engl. Ed.), 54,
and Tanaka, M. (1998) Biosci. Biotechnol. 311–336.
Biochem., 62, 1216–1224. 68. Jeschke, P., Nauen, R., Schindler, M.,
51. Cramer, R.D., Paterson, D.E., and and Elbert, A. (2011) J. Agric. Food
Bunce, J.D. (1988) J. Am. Chem. Soc., Chem., 59, 2897–2908.
110, 5959–5967. 69. Thielert, W. (2006) Pflanz. Nachr. Bayer
52. Okazawa, A., Akamatsu, M., Ohaka, A., (Engl. Ed.), 59, 73–86.
Nishiwaki, H., Cho, W.-J., Nakagawa, Y., 70. Oosterhuis, D.M. and Brown, R.S.
Nishimura, K., and Ueno, T. (1998) Pest. (2003) Proceedings, Beltwide Cotton
Sci., 54, 134–144. Conferences, January 7–10, Nashville,
53. Yamamoto, I., Yabuta, G., Tomizawa, Tennessee. National Cotton Council,
M., Saito, T., Miyamoto, T., and Kagabu, Memphis, TN, pp. 1881–1884.
S. (1995) J. Pestic. Sci., 20, 33–40. 71. Brown, R.S., Oosterhuis, D.M., and
54. Tomizawa, M., Lee, D.L., and Casida, Gonias, E. (2004) Proceedings, Beltwide
J.E. (2000) J. Agric. Food Chem., 48, Cotton Conferences, January 5–9, San
6016–6024. Antonio, Texas. National Cotton Council,
55. Kagabu, S. (1996) J. Pestic. Sci., 21, Memphis, TN, pp. 2231–2237.
237–239. 72. Ford, K.A., Casida, J.E., Chandran, D.,
56. Küthe, K. (1995) Gesunde Pflanzen, 47, Gulevich, A.G., Okrent, R.A., Durkin,
139–150. K.A., Sarpong, R., Bunnelle, E.M., and
57. Elbert, A. and Nauen, R. (2004) in Wildermuth, M.C. (2010) Proc. Natl
Acad. Sci. USA, 107, 17527–17532.
Insect Pest Management – Field and Pro-
73. Epperlein, K. and Schmidt, H.W. (2001)
tected Crops (eds A.R. Horowitz and
Pflanz. Nachr. Bayer (Ger. Ed.), 54,
I. Ishaaya), Springer Press, Berlin,
369–398.
Heidelberg, New York, pp. 29–44.
74. Hernandez, D., Mansanét, V., and
58. Altmann, R. (1991) Pflanz. Nachr. Bayer
Puiggrós, J.M. (1999) Pflanz. Nachr.
(Ger. Ed.), 44, 159–174. Bayer (Ger. Ed.), 52, 374–385.
59. Avery, M.L., Decker, D.G., and Fischer, 75. Nauen, R., Hungenberg, H., Tollo, B.,
D.L. (1994) Crop Prot., 13, 535–540. Tietjen, K., and Elbert, A. (1998) Pestic.
60. Nauen, R., Strobel, J., Tietjen, K., Otsu, Sci., 53, 133–140.
Y., Erdelen, C., and Elbert, A. (1996) 76. Placke, F.-J. and Weber, E. (1993)
Bull. Entomol. Res., 86, 165–171. Pflanz. Nachr. Bayer (Ger. Ed.), 46,
61. Jeschke, P. and Nauen, R. (2005) in 109–182.
Comprehensive Molecular Insect Science, 77. Elbert, A., Erdelen, C., Kühnhold, J.,
vol. 5 (eds L.I. Gilbert, L. Latrou, and Nauen, R., and Schmidt, H.W. (2000)
S.S. Gill), Elsevier Ltd., Oxford, pp. Proc. Brighton Crop. Prot. Conf. – Pests
53–105. Dis., 1, 21–26.
78. Yaguchi, Y. and Sato, T. (2001) 88. Robinson, G.E. (1980) EP 14,064, (Impe-
Agrochem. Jpn, 79, 14–16. rial Chem. Ind. Ltd).
79. Neidlein, R. and Reuter, H. (1997) Arch. 89. Schmuck, R. (2001) Pflanz. Nachr. Bayer
Pharm., 305, 731–737. (Ger. Ed.), 54, 161–184.
80. Buchholz, A. and Nauen, R. (2002) Pest 90. Erdelen, C. (2001) Pflanz. Nachr. Bayer
Manage. Sci., 58, 10–16. (Ger. Ed.), 54, 291–306.
81. Klein, O. (2001) Pflanz. Nachr. Bayer 91. Elbert, A., Buchholz, A.,
(Ger. Ed.), 54, 209–240. Ebbinghaus-Kintscher, U., Erdelen,
82. Krohn, J. (2001) Pflanz. Nachr. Bayer C., Nauen, R., and Schnorbach, H.-J.
(Ger. Ed.), 54, 281–290. (2001) Pflanz. Nachr. Bayer (Ger. Ed.),
83. Placke, F.-J. and Schöning, R. (2001) 54, 185–208.
Pflanz. Nachr. Bayer (Ger. Ed.), 54, 92. Schmuck, R., Stadler, T., and Schmidt,
241–260. H.-W. (2003) Pest Manage. Sci., 59,
84. Wollweber, D. and Tietjen, K. (1999)
279–286.
in Neonicotinoid Insecticides and the
93. Decourtye, A. and Devillers, J. (2010)
Nicotinic Acetylcholine Receptor (eds I.
Adv. Exp. Med. Biol., 683, 85–95.
Yamamoto and J.E. Casida), Springer
94. Schuld, M. and Schmuck, R. (2000) Eco-
Press, Tokyo, pp. 109–125.
toxicology, 9, 197–205.
85. Jeschke, P., Moriya, K., Lantzsch, R.,
Seifert, H., Lindner, W., Jelich, K., 95. Vermeer, R. and Baur, P. (2007) Pflanz.
Göhrt, A., Beck, M.E., and Etzel, W. Nachr. Bayer (Engl. Ed.), 60, 7–26.
(2001) Pflanz. Nachr. Bayer (Ger. Ed.), 96. Baur, P., Arnold, R., Giessler, S.,
54, 147–160. Mansour, P., and Vermeer, R. (2007)
86. Timmons, R.J. and Wittenbrook, L.S. Pflanz. Nachr. Bayer (Ger. Ed.), 60,
(1967) J. Org. Chem., 32, 1566–1572. 27–42.
87. Jensen, A.A. and Henriksen, L. (1968) 97. Haas, M. and Kühnhold, J. (2007)
Acta Chem. Scand. (1947–1973), 22, Pflanz. Nachr. Bayer (Ger. Ed.), 60,
1107–1128. 59–70.
32.2.3
Six-Membered Heterocycles: Thiamethoxam and AKD 1022
Peter Maienfisch
The neonicotinoids represent one of the more recent highlights in the area
of insect control. In this chapter the discovery, chemistry, and properties of
six-membered neonicotinoids are reviewed. The most prominent representatives
of this subclass are nithiazine, AKD-1022, and thiamethoxam. Nithiazine has
served as lead structure for the discovery of the neonicotinoid sales products, while
thiamethoxam is the only six-membered neonicotinoid yet to have entered the
marketplace.
32.2.3.2 History of Six-Membered Neonicotinoids

This novel class of neonicotinoid insecticides (see Chapters 32.1 and 32.2), which
was discovered in 1973 by the research group at Shell, included six-membered
saturated nitromethylene heterocycles [1]. The patent application submitted by
the group depicted the synthesis and insecticidal activity of 2-nitromethylene-
Table 32.2.3.1 Neonicotinoid insecticides developed by Shell during the early 1970s.
Compound type General structure Patent application Publication year Reference
1 (n = 1) NO2 DE 2321523 1973 [1]

2 (n = 0) DE 2321522 1973 [2]
R
N
n = 0, 1
[ ]n
3 NO2 DE 2445421 1975 [3]
R
N NH
n = 0,1 and 2
[ ]n
4 NO2 FR 2270251 1975 [4]
R
N S
5 NO2 US 4297496 1981 [5]

N
R
N NH
Development compound
6 NO2 FR 2270251 1975 [4]

Nithiazine
HN S
piperidines (1) (Table 32.2.3.1). Compounds of type 1 were claimed to pos-

sess good activity against houseflies, pea aphids, corn earworm, mosquito larva,
and cabbage loopers. A second patent application [2], filed on the same date,
revealed the insecticidal activity of the corresponding five-ring analogs, the
2-nitromethylene pyrrolidines (2), and indicated that five- and six-membered
nitromethylenes possessed similar insecticidal activities. Further inventions by
Shell covered 2-nitromethylene-1,3-diazacycloalkanes such as the imidazolidine,
pyrimidine, and diazepine nitromethylenes of type 3 [3], thiazines 4 [4] and, most
importantly, also nitroguanidines of the general structure 5 [5].
Shell’s pioneering work [6–8] during the 1970s on insecticidal nitromethylene
heterocycles led to the invention of the nitroenamine and nitroguanidine pharma-
cophores, and cumulated in the discovery of nithiazine (6), a compound which
Table 32.2.3.2Inventions (six-membered heterocycles) made

by Nihon Tokushi Noyaku/BayerAG (selection).
Compound type General structure Patent application Publication year Reference
7 NO2 EP 163855 1986 [9]

X EP 192060 1986 [10]
R
Het N N n = 0, 1, 2
X = CH, N
[ ]n
Examples
NO2 NO2
X X
N NH S N NH
Cl
Cl N [ ]n N [ ]n
X = CH, N X = CH, N
n = 0,1 and 2 n = 0,1 and 2
Sales product
8 Imidacloprid NO2 EP 192060 1986 [10]

N
N NH
Cl N
has never been commercialized as a crop-protection agent. This was due mainly
to the compound’s rapid degradation under both hydrolytic and photochemical
conditions [7], as well as its limited potency.
Nonetheless, these compounds (1–4 and 6) have served as neonicotinoid lead
structures, such that 13 years later Nihon Tokushu Nohyaku (a subsidiary of Bayer
AG in Japan), with the synthesis of nitromethylene and nitroguanidine derivatives
of imidazolidines, perhydro-pyrimidines, and diazepines of type 7 (Table 32.2.3.2),
achieved an important breakthrough in this area of chemistry [9, 10].
The extremely high insecticidal activity of neonicotinoids of the imidacloprid-type
7 (Chapter 32.2.2) – triggered extensive research activities within several other
companies, including Ciba-Geigy (later Novartis, now Syngenta), Takeda, Nippon
Soda, Agro Kanesho, Mitsui Chemicals, all of which immediately entered this
promising area of research [11–13]. Although each of these companies began to
investigate novel structural modifications, at that time little was known regarding
the influence of the nitroimino-heterocycle on biological activity. Consequently,
compounds possessing an additional heteroatom in the nitroimino-heterocycle
were designed and synthesized, and many patent applications were filed [14–21]
(Table 32.2.3.3).
Table 32.2.3.3 Novel six-membered nitroimino-heterocycle with an additional heteroatom.
Compound General Company Patent Publication Reference

type structure application year
Triazinane (hexahydro-1,3,5-triazines)
9 NO2 Nihon Tokushu EP 386565 1990 [14]

N
Noyaku/Bayer
R1 Nippon Soda WO 9101978 1991 [15]
Het N N
Agro-Kanesho EP 428941 1991 [16]
N Ciba-Geigy EP 483055 1992 [17]
R2 Ciba-Geigy EP 483062 1992 [18]
Oxadiazinanes (hexahydro-1,3,5-oxadiazines)
10 NO2 Ciba-Geigy EP 580553 1994 [19]

N
Nihon Tokushu JP 07224062 1995 [20]
R1 Noyaku
Het N N
Mitsui Toatsu JP 08291171 1996 [21]
O Chemicals
Thiadiazinanes (hexahydro-1,3,5-thiadiazines)
11 NO2 Nihon Tokushu JP 07224062 1995 [20]

N
Noyaku
R1
Het N N
Development compound and sales product
12 AKD-1022 Agro-Kanesho EP 428941 1991 [16]

NO2
N
S CH3
N N
Cl
N N
CH3
13 Ciba-Geigy EP 580553 1994 [19]

NO2
Thiamethoxam N
S CH3
N N
Cl
N O
The investigations into the six-membered neonicotinoids described above yielded

three development compounds, namely nithiazine (6), AKD-1022 (12), and thi-
amethoxam (13), the latter being the only one to enter the marketplace.
32.2.3.3 Biological Activity and Structure–Activity Relationship

Compounds of types 9–11 provide a good control of a broad range of commercially
important pests, such as aphids, whiteflies, thrips, rice hoppers, Colorado potato
beetle, flea beetles, wireworms, and leaf miners, as well as some lepidopterous
species. When the influence of the nitroimino-heterocycle on the biological activity
was investigated in the greenhouse with compounds 14–16 [22], the bioassays
revealed that among these compounds, the 4-nitroimino-1,3,5-oxadiazinane 15
exhibited a clearly better insecticidal activity than the 2-nitroimino-1,3,5-triazinane
14 and the 4-nitroimino-1,3,5-thiadiazinane 16, and that its potency was close to
that of imidacloprid (8) and its six-ring analog 17 (Table 32.2.3.4).
32.2.3.3.1 Structure–Activity Relationship The general structure–activity profile
for six-membered neonicotinoids [22–25] is shown in Table 32.2.3.5, the most
favorable structural features being:
• The nitroguanidine pharmacophore (X–Y = NNO2 ) leads to very high insecticidal

activity, followed by the cyanoguanidine pharmacophore (X–Y = NCN).
• As a pharmacophore ‘‘backbone,’’ a 1,3,5-oxadiazinane ring (Z = O) is more
favorable than other heterocyclic ring systems.
Table 32.2.3.4 Insecticidal activity of six-membered neon-

icotinoids 14–16 in comparison to imidacloprid 8 and its
six-ring analog 17.
Compound Structure type LC80 (mg AI l−1 )
NO2
N Aphis Myzus Diabrotica
craccivora persicae balteata
N NH m.p.Pea, m.p.Pea, L2Filter paper,
foliar spray into water spray
Cl N X
14 Triazinane >200 3 200

X = NCH3
15 Oxadiazinane 50 0.05 3
X=O
16 Thiadiazinane 200 0.8 12
X=S
17 Hexahydro-pyrimidine 12 0.2 3
X = CH2
8 Imidacloprid Imidazolidine 12 0.05 0.8

X = bond
Table 32.2.3.5 Structure–activity profile for six-membered neonicotinoids.
Y
R2 X
R1
Het N3 5
N
Structural Structure–activity relationship

feature
Pharmacophore X–Y N–NO2 > N–CN >> O, S, NH

Pharmacophore Z O > N–CH3 , S, CH2
backbone
Pharmacophore R1 CH3 > H > C2 H5 , n-Pr, allyl, propargyl >> larger substituents
substituent CH3 > COR , COOR CH2 OR
Bridge R2 H > CH3 > larger groups
substituent
Heterocyclic Het
group
S N
Cl > , , Cl >
N Cl N O
Cl N N
, >> others
O Cl
Chlorothiazoyl Het
as heterocyclic S
group R R: Cl > Br, H >> SR1, OR1
N
• The 2-chloro-5-thiazolyl moiety provides a better overall insecticidal activity than

the 6-chloro-3-pyridyl, which is present in the first-generation neonicotinoids,
and is superior to that of all other heterocyclic groups investigated.
• In contrast to the structure–activity relationships in the imidacloprid series, the
introduction of a methyl group at N(5) (R1 = CH3 ) led to a powerful increase in
insecticidal activity.
32.2.3.4 Synthesis
General synthetic methodologies involving Mannich-type cyclization reactions
as the key step have been developed for the synthesis of the 2-nitroimino-
1,3,5-triazinanes (9) [14–18, 22,26–29] and the 4-nitroimino-1,3,5-oxadiazinanes

(10) [19–23, 29] (Schemes 32.2.3.1 and 32.2.3.2). These methodologies allow an
introduction of the heterocyclylmethyl group from either a heterocyclylmethyl
amine or a heterocyclylmethyl chloride, respectively. Thus, treatment of the readily
available S-methyl-N-nitroisothiourea with amines affords the monosubstituted
nitroguanidines in excellent yields; these can then be converted to the monosubsti-
tuted nitroimino-triazanes or oxadiazinanes, respectively. Alkylation leads then, in
good to excellent yields, to the target compounds 9 and 10, respectively.
NO2
N
NO2 R2
NO2 N Het N1 3N
N HetCH2NH2 R2NH2, HCHO
5
EtOH, 80°C
Het N NH2 N
NH2 H EtOH, 50−80°C
CH3S
R1
R1Hal, K2CO3
R1NH2
DMF, 40−80°C
EtOH, 50°C
NO2 NO2
NO2 N N
N R2NH2, HCHO R1 HetCH2Cl, R1
HN N Het N N
H2N NH EtOH, 50−80°C K2CO3, DMF, 50°C
N N
R1
R2 R2
9
Scheme 32.2.3.1 Synthesis of 2-nitroimino-hexahydro-1,3,5-triazines (9).
NO2
NO2 NO2 N
N Het/Aryl-CH2NH2 N HCHO, HCOOH
N NH
H2N SCH3 EtOH, 80°C Het/Aryl N NH2 80−90°C Het/Aryl
H
O
R1NH2 R1-Hal, K2CO3

EtOH, 80°C DMF, 40−80°C
NO2 NO2
NO2 N N
N HCHO, HCOOH R1 Het/Aryl-CH2Hal R1
R1 HN N Het/Aryl N N
H2N N 80−90°C K2CO3, DMF
H O 50−80°C O
10
Scheme 32.2.3.2 Synthesis of 4-nitroimino-1,3,5-oxadiazinanes (10).

32.2.3.5 Hydrolytic Stability of the Six-Membered Nitroimino-Heterocycle

Six-membered nitroimino-heterocycles of type 9–11 are cyclic Mannich adducts
containing a bis-aminal structure (Scheme 32.2.3.3). It is generally known that such
Mannich adducts can cleave into their noncyclic compounds, depending on the
reaction conditions. When the hydrolysis (ring cleavage) of compounds 9–11 was
studied in detail [12, 17, 24, 27, 28, 30–34], it was found that triazinanes 9 and
thiadiazinanes 11 decayed in physiological salt solution (pH 7.6) at 25 ◦ C to the
corresponding acyclic compounds 18, whereas the oxadiazinanes 10 were stable
under these conditions [34]. In contrast to compounds of type 10 (X = O), the
cleavage of compounds 9 (X = NR2 ) was also observed under both acidic and basic
aqueous conditions [24, 33]. Based on these findings, it was concluded that the
triazinanes 9 could be regarded as hydrolytic proinsecticides of the corresponding
acyclic nitroguanidine 18, but that this prodrug concept would not apply to the
oxadiazinanes (10) [34].
32.2.3.6 AKD-1022
During the late 1980s, Agro Kanesho announced the development of their
own neonicotinoid insecticide, AKD-1022 (12); this was a representative of the
2-nitro-1,3,5-triazinane subclass, and contained a 2-chloro-5- thiazolyl moiety as the
heterocyclic group [35]. Unfortunately, this compound never achieved commercial
status, possibly due to the crowded patent situation at the time (see Section 32.2.3.2)
or the lack of hydrolytic stability (see Section 32.2.3.5). Subsequently, AKD-1022
(12) has been described [36] as a possible proinsecticide of the acyclic nitroguandine
clothianidin (19) (Scheme 32.2.3.4; see also Section 32.2.1.4).
NO2
N
NO2
R1 N
Het N N − 2 H2O
H H R1
18 Het N N
H H + 2 H 2O
O O X
H Mannich type
X H
cyclisation reaction 9 X = NR2
H H
10 X = O
11 X = S
Scheme 32.2.3.3 Hydrolytic degradation of nitroimino-heterocycle (9–11).
NO2 NO2
N N
S CH3 S
N N N NH
Cl Cl H
N CH3
N N
CH3
AKD-1022 Clothianidin
13 19
Scheme 32.2.3.4 Structures of AKD-1022 (12) and clothianidin (19).

Initially, the synthesis of this compound was described by Agro Kanesho [16]
(further preparations are discussed in Section 32.2.3.4). As with all neonicotinoids,
AKD-1022 (12) interacts with nicotinic acetylcholine receptors (nAChRs), but is
much less potent than either imidacloprid (8) or other commercially available neon-
icotinoids. In particular, this has been demonstrated with Myzus and Drosophila
membranes [26], as well as on American cockroaches [34]. It has been speculated,
that AKD-1022 (12), as a basic molecule, is ionized in the fluids of insects and
therefore reaches the synapse only very slowly, through the lipophilic cuticles
and ion barriers. During retarded movement, the compound is prone to decom-
pose, most likely due to a partial hydrolysis that is mediated enzymatically and/or
nonenzymatically [34]. Consequently, acyclic nitroguanidines such as 19 may also
contribute to the insecticidal activity observed in glasshouse and field studies.
32.2.3.7 Thiamethoxam (CGA 293’343)

In 1985, Ciba Geigy (since 1996, Novartis; now Syngenta) commenced a research
program on neonicotinoids that resulted in the discovery of thiamethoxam (13) [13,
22], a second-generation neonicotinoid belonging to the thianicotinyl subclass [13].
The combination of an oxadiazine ring with an N-methyl group as pharmacophore
substituent proved to be unique, and appeared to shape the biological properties of
thiamethoxam (13, CGA 293’343).
Thiamethoxam was first synthesized in 1991, and has been marketed since 1998
under the trade names Actara® (for foliar and soil treatment) and Cruiser® (for seed
treatment). In all of its varying usages, thiamethoxam provides excellent control
of a broad range of commercially important pests, including aphids, whiteflies,
thrips, rice hoppers, Colorado potato beetle, flea beetles, wireworms, and leaf
miners, as well as some lepidopteran species [13, 25, 37]. Moreover, the low usage
rates, flexible application methods, excellent efficacy, and favorable safety profile
of thiamethoxam mean that it is extremely well suited to modern integrated pest
management (IPM) programs, in many cropping systems.
32.2.3.7.1 Discovery Ciba Geigy’s studies on the influence of the nitro-

imino-heterocycle on biological activity led initially to the 4-nitroimino-1,3,5-
oxadiazinane derivatives (10) [13, 22]. However, in a second optimization cycle
the oxadiazinane lead structure 15 was further improved, and this resulted in
thiamethoxam (13) being identified as the optimum representative of this chemical
subclass (Scheme 32.2.3.5).
32.2.3.7.2 Synthesis Thiamethoxam was first synthesized in 1991 [19], at

a time when no practical methods were available for the preparation of
4-nitroimino-1,3,5-oxadiazinanes [22]. After much experimentation, however,
optimized procedures [22, 24, 38–44] were developed (Scheme 32.2.3.6).
The key step here is the conversion of N-methyl-nitroguanidine (20) to the
oxadiazinane (21) by treatment with formaldehyde in the presence of formic
acid [22, 24]. Subsequent alkylation with the thiazole (26) [38, 44–49] in
dimethylformamide and potassium carbonate as a base afforded thiamethoxam in
NO2
N
CH3
N N
Cl N O
Improved activity against

NO2 sucking insects NO2
N (Aphis, Myzus, Nilaparvata) N
S CH3
N NH N N
Cl
Cl N O N O
NO2
N
15 Thiamethoxam (13)
S N NH Improved activity against
Cl
N chewing and sucking insects
O
(Spodoptera, Diabrotica,
Aphis, Myzus, Nilaparvata)
Improved activity against
chewing insects
(Spodoptera, Diabrotica)
Scheme 32.2.3.5 Optimization of 4-nitroimino-1,3,5-oxadiazinane lead structure 15.
NO2
NO2 N
N HCHO, HCOOH, 90°C
CH3
CH3 HN N
H 2N N 70−91%
H
O
20 21
S Cl
RS
N 23
K2CO3, DMF S K2CO3, DMF
Cl
50°C Cl
50°C; 70−80%
N
22
NO2 NO2
N N
S CH3 Cl2 S CH3

N N N N
RS Cl
N O N O
24 Thiamethoxam (13)
Scheme 32.2.3.6 Syntheses of thiamethoxam (13).

good to excellent yields. Alternatively, the oxadiazinane (21) can be alkylated with
a 2-mercapto-thiazol-5-ylmethyl chloride (23) to afford compound 24, which can
then be converted to thiamethoxam (13) by chlorination [50–52].
32.2.3.7.3 Chemical and Physical Properties The physico-chemical properties of

thiamethoxam are listed in Table 32.2.3.6. Such properties favor a rapid and
efficient uptake in plants, as well as xylem transport [25, 53]. Indeed, it is this
systemic activity of thiamethoxam that leads to all plant parts situated acropetally
from the thiamethoxam application site being efficiently protected.
Thiamethoxam is hydrolytically very stable at pH 5 (half-life more than one year at
room temperature), and stable at pH 7 (estimated half-life at room temperature ca.
200–300 days). The compound is, however, more labile at pH 9 (half-life of only a few
days). Two major degradation pathways have been observed in the pH range from
5 to 9 [24, 33]]; the first of these led to the corresponding 1,3,5-oxadiazinan-4-one
(25), while the second pathway led to the ring-opened N-nitro-urea (26) and hence
to the 2-chloro-5-aminomethyl-thiazole (27) (Scheme 32.2.3.7). Hydrolytic cleavage
of the 1,3,5-oxadiazinane ring to the corresponding acyclic nitroguanidine (19;
clothianidin) was not observed [24, 33].
Photolytically, thiamethoxam is rapidly degraded (half-life ca. 1 h as a droplet
deposit on Teflon). Although no decomposition was observed after storage of the
active ingredient or formulations at 54 ◦ C for two months, at temperatures above
150 ◦ C an exothermic decomposition was seen to occur [13, 25].
In laboratory soils, thiamethoxam degrades at moderate to slow rates. Typically,
the half-life will range from 34 to 75 days under favorable conditions, but may be
increased by a factor of three under unfavorable conditions. Under field conditions
the degradation is generally faster, mainly because field soils usually have a higher
Table 32.2.3.6 Chemical and physical properties of thiamethoxam (13).
Feature Property

Vapor pressure at 25 ◦ C 6.6 × 10−9 Pa
Water solubility at 25 ◦ C 4.1 mg l−1
pH value 6.84 (saturated solution in water)
Partition coefficient (n-octanol/water at –0.13
25 ◦ C (log Pow ))
Dissociation constant CGA 293 343 has no dissociation within the
range of pH 2–12
Hydrolysis: (estimated half-life at 25 ◦ C) pH 5: 6990 days
pH 7: 152 days
pH 9: 6.1 days
Photodecomposition (half-life as droplet 1h
deposit on Teflon)
NO2 NO2
N N
S CH3 S CH3
N N N N
Cl Cl
H H
N O N
pathway 1
13 pathway 2 19
O NO2
HN
S CH3 S
N N S NH2
Cl NH O Cl
N Cl
O N
N
25 26 27
Scheme 32.2.3.7 Hydrolytic degradation pathways of thiamethoxam (13).
microbial activity. Exposure to light represents another important degradation

pathway for thiamethoxam [13].
32.2.3.7.4 Mode of Action

Target Sites in the Nervous System Typically, the neonicotinoids target the nAChRs,
as has mainly been demonstrated by studies with imidacloprid (8). However,
compared to imidacloprid and the other neonicotinoid sales products, it appears
that, in aphids, thiamethoxam binds in a different manner, possibly to a different
site of the receptor [54–57].
The results of saturation binding studies have revealed the following data
for the binding affinity (Kd ) and binding capacity (Bmax ) for thiamethoxam and
imidacloprid, using fresh membranes from M. persicae assayed at 2 ◦ C:
• Thiamethoxam: Kd = 11.4 nM; Bmax = 700 fmol mg−1 protein.

• Imidacloprid: Kd = 2.5 nM; Bmax = 1400 fmol mg−1 protein.
Nonspecific binding was fairly low with both radioligands, typically about 10%
with [3 H]thiamethoxam and about 5% with [3 H]imidacloprid.
Taken together, these data confirmed that thiamethoxam, is similar fashion to
imidacloprid and the other neonicotinoids, binds with high affinity to the nAChRs
[56]. There were, however, clear differences between thiamethoxam and the other
commercial neonicotinoids, as documented by a ‘‘kinetic’’ analysis of competition
experiments [55]. Whereas, [3 H]thiamethoxam was shown to bind to the receptors
with nanomolar affinity, micromolar concentrations were required to displace
[3 H]imidacloprid. Furthermore, the interaction between the two compounds was
seen to be ‘‘noncompetitive’’; in other words, the binding of thiamethoxam would
reduce the binding capacity of the receptor preparation for imidacloprid, but not
its affinity. Notably, thiamethoxam shares this unusual mode of inhibition with
other neonicotinoids (not commercialized) that contain an N-methyl group as the
pharmacophore substituent [55, 57].
Thiamethoxam has also been found to be highly potent for [3 H]epibatidine

binding [58] and to act, at least in part, directly on the Homolodisca receptor [59].
Kagabu identified a high neuroblocking activity of thiamethoxam in American
cockroach [36]. Taken together, the stability, insecticidal, and neuroblocking tests
performed revealed (in accordance with Syngenta results [57]) that the bioactivation
of thiamethoxam is not necessary. Further evidence that thiamethoxam is an
outright nAChR agonist was derived from two separate studies conducted at the
laboratory of Neil Millar. First, thiamethoxam was shown to stimulate a cationic
current in Xenopus oocytes expressing either Nlα1 with ratβ2 or Nlα3/Nlα8 with
ratβ2, with similar potency to the other commercial neonicotinoids [60, 61]. These
findings do not support the conclusions [62–64], that thiamethoxam is likely to be
a neonicotinoid precursor for clothianidin (19), and is not active by itself.
In summary, varied and minor structural differences in the neonicotinoid
molecules may confer diversity in their binding modes, depending on insect species,
and may also explain the unique receptor-binding behavior of thiamethoxam
[55–57], as well as that of dinotefuran [65].
Biological Mode of Action Thiamethoxam shows a very rapid action in target insects,
with initial symptoms apparent at 15–30 min after uptake in aphids and Colorado
potato beetle, and after 1 h in whiteflies. When feeding ceases the sucking insects
withdraw their stylets, stretch their legs, and move the antennae forwards. Even if
the insects die only 24 h later, the effects are comparable to those of knock-down
compounds, as the feeding stop is irreversible and affected insects do not try to
penetrate again [66].
32.2.3.7.5 Biological Activity and Use Recommendation In laboratory tests and

under field conditions, after foliar, drench, and seed treatment application at
very low concentrations, thiamethoxam shows good to excellent activity against
homopteran, coleopteran, and some lepidopteran pests. Thysanopteran pests are
best controlled after drench or seed treatment application [13, 22, 25].
Under field conditions, thiamethoxam is active at very low rates against many key
pests in many crops. The currently recommended uses after foliar spray and soil
application of Actara®, formulated mainly as WG 25, SC 240, or GR 1, are listed in
Table 32.2.3.7. The soil applications have been optimized for each use, and include
soil surface, soil drench, soil drench surface, soil drench soil granule, trunk spray
application, seedling box, into water, and post-transplanting drench application.
Additionally, many other crops and noncrop uses are currently under evaluation.
This includes ornamentals, grapes, cocoa, pineapple, tea, hazelnuts, date palms,
papaya, durian, pecan, and cereals, as well as noncrop uses such as leafcutting
ants, termites in pastures, and ants. The recommended rates of active ingredient
(a.i.) per ha vary among crops and pests but, in most cases, an application rate of
between 10 and 100 g a.i. ha−1 is sufficient to fully control the target pests [13, 25,
37, 67].
As a seed treatment, thiamethoxam is highly active against a broad range
of soil-dwelling insects. It also offers an effective control of a wide range
Table 32.2.3.7 Recommended foliar and soil applications.
Crops Target Foliar Soil Remarks

pests spray application
Citrus Leafminers X X Soil application as nursery

only
Aphids X X
Citrus whitefly X –
Mealybugs X –
Longicorn beetle X –
Soft scales X –
Citrus psylla X –
Brazilian X –
sharpshooter
Coffee Coffee leafminer – X
Cicades – X
Cotton Aphids X –
Jassids X –
Thrips X –
Whiteflies X –
Lygus bugs X –
Flea hoppers, X –
Mirids
Lettuce Aphids X X
Mango Mango hoppers X –
Pepper/ Aphids X X
eggplant
Whiteflies X X
Jassids X X
Pepper weevil X –
Tomato bug X –
Colorado potato X X
beetle
Thrips – X
Pome Aphids X – Soil application: trunk spray
fruits or soil drench application
Leafhoppers X –
Plum curculio X –
Apple sawfly X –
Apple blossom X –
weevil
Pear psylla X –
Comstock X –
mealybug
Wooly apple X –
aphid
Crops Target Foliar Soil Remarks

pests spray application
Potato Aphids X X
Colorado potato X X
beetle
Leafhoppers X X
Diabrotica X X
Wireworms – X
Potato psyllid X X
Rice Hoppers X X Soil application: seedling
box or into water application
Rice stink bugs X –
Rice leaf beetle – X
Rice water weevil – X
Soybean Stinkbugs X –
and beans
Whiteflies X –
Sugarcane Termites – X
Sugarcane – X
froghoppers
Tobacco Aphids X X Soil application: post
planting drench application
Brown tobacco X X
leaf beetle
Tobacco flea X –
beetle
Wireworms – X
Thrips – X
Tomatoes Aphids X X
Whiteflies X X
Jassids X X
of early-season, leaf-feeding (Coleoptera and Lepidoptera) and sucking insects

(Homoptera and Thysanoptera). Due to its rapid action on sucking insects, thi-
amethoxam also limits the transmission of plant pathogenic viruses (e.g., in cereals
and sugar beets). The current seed treatment recommendations for Cruiser in key
crops are listed in Table 32.2.3.8. Typically, the application rate per 100 kg seed
ranges between 30 and 400 g a.i., although in potato rates of 5–7.5 g a.i. per 100 kg
seed are sufficient [13, 25, 37, 68–71].
32.2.3.7.6 Resistance Despite the extensive use of neonicotinoid insecticides to

control a wide diversity of agricultural pests, relatively few cases of significant
resistance development have been identified during the 20-year period that this
class of insecticide chemistry has been registered for agricultural use. Although the
Table 32.2.3.8 Recommended seed treatment applications.
Crop Target pests
Beans White flies Leaf hoppers

Stem borer Bean leaf beetle
Root worm Seed corn maggot
Canola/oilseed rape Aphids Saw fly
Flea beetle –
Cereals Aphids Soft scale
Cereal ground beetle Coleoptera
Fruit fly Bugs
Wireworms –
Corn/maize Aphids False wireworm
Fruit flies White grubs
Bugs Root maggot
Leaf hopper Grasshoppers
Black maize beetle Corn flea beetle
Cornstalk borer Maize weevil
Wireworms –
Cotton Aphids Thrips
Cotton leaf worm Wireworms
Jassids Cotton boll weevil
Peanuts Thrips Leaf hopper
Peas Aphids Thrips
Pea weevils Seed corn maggot
Potato Aphids Wireworms
Colorado potato beetle Cucumber beetles
Potato leafhopper Thrips
Rice Stem borer Green leaf hoppers
Grasshoppers Termites
Rice grain beetle Cane borer
Thrips –
Sorghum Aphids Green bug aphids
Chinch bug Wireworm
Seed corn maggot –
Soybean Soybean weevil Bean leaf beetle
Termites Thrips
Whiteflies Corn stalk borer
Sugar beet Aphids Wireworm
Mangold pygmy beetle Weevils
Flea beetle Leaf hopper
Maggot fly –
Sunflower Aphids Coleoptera (white grubs)
Jassida Wireworm
Ground weevil –
Stored pest Rice weevil Saw-tooth grain beetle
Indian meal moth –
development of neonicotinoid resistance in the tobacco whitefly (Bemisia tabaci)

[72, 73], glasshouse whitefly (Trialeurodes vaporariorum) [74], brown plant hopper
(Nilaparvata lugens) [75, 76], and Colorado potato beetle (Leptinotarsa decemlineata)
[77], has impaired the control of these pests with this chemistry, other insecticide
chemistries have been found to fill the needs of growers. The use of pymetrozine
(see Chapter 33.11) to control neonicotinoid-resistant brown plant hoppers [76] is
one example of how novel chemistries can provide control of resistant populations,
but can also be used to prevent resistance development from occurring if applied
as part of a resistance management strategy.
32.2.3.7.7 Safety Profile

Mammalian Toxicology Thiamethoxam is rapidly and completely absorbed and
readily eliminated, predominantly as the unchanged compound, via the urine.
When administered to rats either orally (LD50 = 1563 mg kg−1 ), dermally (LD50
>2000 mg kg−1 ), or by inhalation (LC50 (4 h) > 3720 mg m−3 ), thiamethoxam
had a low acute mammalian toxicity, placing it in WHO hazard class III.
Thiamethoxam was also found to be nonirritant to the skin and eyes, and devoid
of any skin-sensitizing potential (Table 32.2.3.9) [13, 25, 37].
In repeated dose studies in rodents and dogs, the liver and kidneys (rat only) were
the main target organs. In lifetime rodent studies, only mice showed increased
incidences of liver tumors, which were found to be specific to this species [78–80].
The tumors were regarded to be mediated by a non-genotoxic threshold mechanism,
and of no relevance to humans at normal levels of application. Thiamethoxam has
no mutagenic potential; neither have reproductive toxicity studies demonstrated
any evidence of developmental impairment or teratogenic potential [13, 25]. Both,
applicator and consumer safety are very favorable for the label-recommended uses.
Table 32.2.3.9 Acute toxicity.
Acute toxicity test Result EPA toxicity

category
LD50 (rat acute oral) 1563 mg kg−1 III

LD50 (rat acute dermal) > 2000 mg kg−1 III
LC50 (rat inhalation, 4 h) > 3720 mg m−3 III
Skin irritation (rabbit) Nonirritant IV
Eye irritation (rabbit) Nonirritant IV
Skin sensitization (guinea pig) Nonsensitizing IV
Genotoxicity Nongenotoxic –
Nonmutagenic
Reproduction No –
developmental,
nor teratogenic
potential
Ecotoxicology Thiamethoxam has a favorable ecological profile, as it is practically

nontoxic or only slightly harmful to water vertebrates and invertebrates, avians
and soil invertebrates, and beneficial arthropods (with the exception of honey
bees and bumble bees, to which it must be considered toxic). However, following
drip irrigation according to label recommendations, thiamethoxam showed no
adverse effects towards bumble bees in tomatoes. Thiamethoxam showed no
bioaccumulation potential, is moderately mobile in soil and, under field conditions,
is degraded at rapid to moderate rates (Table 32.2.3.10) [13, 25].
Effects on Beneficial Arthropods Thiamethoxam is classified as slightly to moderately

harmful to most beneficial insects, but safe to predatory mites in the field. This
rating is similar to that of other neonicotinoid compounds (see Table 32.2.3.10) [13].
32.2.3.7.8 The Thiamethoxam Vigor Effect Crops grown in soil from seeds treated
with thiamethoxam show increased levels of plant vigor and health beyond a
standard plant response to early season insect control [81]. This effect is also
observed after foliar or into soil applications of thiamethoxam at early growth
stages. This ‘‘vigor effect’’ was first disclosed by Syngenta in 2001 [82], and has
been observed in many countries and on multiple crops. The vigor effect is seen
particularly under conditions of abiotic stresses, such as drought (Figure 32.2.3.1),
heat, cold, low pH, and soil salinity. In particular, under these stress conditions
thiamethoxam-treated plants grow more vigorously than either untreated plants
Table 32.2.3.10 Ecological toxicology characteristics of thiamethoxam (13).
Acute toxicity test Species LD50 /LC50 EPA toxicity category
Avian oral Bobwhite quail 1552 mg kg−1 Slightly toxic

LD50 Mallard duck 576 mg kg−1 Slightly toxic
Avian dietary Bobwhite quail >5200 ppm Practically nontoxic
LC50 Mallard duck >5200 ppm Practically nontoxic
Freshwater fish Rainbow trout > 125 mg l−1 Practically nontoxic
LC50 (96 h) Bluegill > 114 mg l−1 Practically nontoxic
Marine fish LC50 (96 h) Sheepshead minnow > 111 mg l−1 Practically nontoxic
Freshwater invertebrate Daphnia magna > 100 mg l−1 Practically nontoxic
EC50 (48 h)
Marine invertebrate Mysid shrimp 6.9 mg l−1 Moderately toxic
EC50 (96 h) Eastern oyster > 119 mg l−1 Practically nontoxic
Algae EC50 (72 h) Green algae > 81.8 mg l−1 None
Earthworm EC50 (14 day) Eisenia foetida > 1000 mg kg−1 None
soil
Bee contact LD50 Honey bee 0.024 μg/bee Highly toxic
Other seed treatment Cruiser treated seeds
Figure 32.2.3.1 The thiamethoxam vigor effect. The Cruiser

(thiamethoxam)-treated maize (grown in China, in 2009)
shows a greater tolerance towards drought conditions.
or those treated with other insecticides, and are more tolerant towards difficult
growing conditions [81, 83, 84].
The thiamethoxam vigor effect can be expressed in many different ways, depend-
ing on the crop species and variety, as well as on environmental factors:
• A faster germination and earlier emergence (Figure 32.2.3.2).

• A quicker canopy closure and greener, more vigorous plants (Figure 32.2.3.3).
• A greater number of, and more healthy, roots that can make use of water and
nutrient resources in a more efficient way (Figure 32.2.3.4).
• Higher yields, even in situations where there is no visible insect attack.
In direct comparisons, thiamethoxam consistently shows higher yield effects

than other seed treatments.
Cruiser treated seeds Other seeds Other seed treatment Cruiser treated seeds
(a) (b)
Figure 32.2.3.2 The thiamethoxam vigor seeds exposed to other seed treatments;
effect. (a) Faster germination: Cruiser (b) Earlier field emergence: Cruiser-treated
(thiamethoxam)-treated maize seeds, sub- sunflower seeds showed an earlier visible
sequently grown on agar, showed a greater above-ground growth than those exposed to
development of roots and leaves than maize other seed treatments.
Cruiser treated seeds Other seed treatment Other seed treatment Cruiser treated seeds
(a) (b)
Figure 32.2.3.3 The thiamethoxam vigor effect. The Cruiser

treatment of seeds results in a quicker canopy closure and
greener, more vigorous plants; this, in turn, impacts on the
biologically active biomass, leading to a higher yield and
the suppression of weed growth. (a) Peas, grown in the UK,
2007; (b) soybeans grown in Brazil, 2006.
Cruiser treated seeds Other seed treatment

(a) (b)
Figure 32.2.3.4 The thiamethoxam vigor effect. (a) Maize

seeds treated with thiamethoxam (Cruiser) and grown in the
USA (in 2008) develop a greater number of fine roots, with
more forking and a greater total root mass than seeds ex-
posed to other treatments (b).
References
1. Roman, S.A. (1973) Ger. Offen DE 4. Powell, J.E. and Roman, S.A. (1975) Fr.
2,321,523, (Shell). Demande FR 2,270,251, (Shell).
2. Roman, S.A. (1973) Ger. Offen DE 5. Davis, R.H. and Davies, J.H. (1981) US
2,321,522, (Shell). Patent US 4,297,496, (Shell).
3. Tieman, C.H., Kollmeyer, W.D., and 6. Soloway, S.B., Henry, A.C., Kollmeyer,
Roman, S.A. (1975) Ger. Offen DE W.D., Padgett, W.M., Powell, J.E.,
2,445,421, (Shell). Roman, S.A., Tieman, C.H., Corey, R.A.,
References 1223
and Horne, C.A. (1978) Nitromethylene Application EP 428,941, (Agro-Kanesho

heterocycles as insecticides. Pesticide Co., Ltd).
and Venom Neurotoxicity, (Selected 17. Maienfisch, P., Kristiansen, O., and
Paper from the 15th International Gsell, L. (1992) European Patent Appli-
Congress of Entomology), pp. 153–158. cation EP 483,062, (Ciba-Geigy AG).
7. Soloway, S.B., Henry, A.C., Kollmeyer, 18. Maienfisch, P., Kristiansen, O., and
W.D., Padgett, W.M., Powell, J.E., Gsell, L. (1992) European Patent Appli-
Roman, S.A., Tieman, C.H., Corey, cation EP 483,055, (Ciba-Geigy AG).
R.A., and Horne, C.A. (1979) in Ad- 19. Maienfisch, P. and Gsell, L. (1994) Eu-
vances in Pesticide Science, Part 2 (eds ropean Patent Application EP 580,553,
H. Geissbühler, G.T. Brooks, and (Ciba-Geigy AG).
P.C. Kearney), Pergamon Press, pp. 20. Moriie, K., Ootsu, J., Hatsutori, Y.,
206–217. Watanabe, A., and Ito, A. (1995) Japan
8. Kollmeyer, W.D., Flattum, R.F., Foster, Patent Application JP 07,224,062, (Nihon
J.P., Powell, J.E., Schroeder, M.E., and Tokushu Noyaku Seizo KK).
Soloway, S.B. (1999) in Nicotinoid In- 21. Matsuo, S., Wakita, T., Odaka, K., and
secticides and the Nicotinic Acetylcholine Shiraishi, S. (1995) Japan Patent Ap-
Receptor (eds I. Yamamoto and J.E. plication JP 08,291,171, (Mitsui Toatsu
Casida), Springer-Verlag, Tokyo, pp. Chemicals).
71–89. 22. Maienfisch, P., Huerlimann, H.,
9. Shiokawa, K., Tsuboi, S., Kagabu, S., Rindlisbacher, A., Gsell, L., Dettwiler,
and Moriya, K. (1985) European Patent
H., Haettenschwiler, J., Sieger, E., and
Application EP 163,855, (Nihon Tokushu
Walti, M. (2001) Pest Manage. Sci., 57,
Noyaku Seizo K. K.).
165–176.
10. Shiokawa, K., Tsuboi, S., Kagabu, S.,
23. Maienfisch, P., Rindlisbacher, A.,
and Moriya, K. (1986) European Patent
Huerlimann, H., Haettenschwiler, J.,
Application EP 192,060, (Nihon Tokushu
Desai, A.K., Ekkundi, V.S., and Gangan,
Noyaku Seizo K. K.).
V.D. (2002) Synthesis and Chemistry
11. Wollweber, D. and Tietjen, K. (1999) in
of Agrochemicals VI, ACS Symposium
Nicotinoid Insecticides and the Nicotinic
Series, vol. 800, American Chemical
Acetylcholine Receptor (eds I. Yamamoto
and J.E. Casida), Springer-Verlag, Tokyo,
pp. 109–125. 24. Maienfisch, P. (2006) Z. Naturforsch. B:
12. Jeschke, P. and Nauen, R. (2005) Compr. Chem. Sci., 61, 353–359.
Mol. Insect Sci., 5, 53. 25. Maienfisch, P., Angst, M., Brandl, F.,
13. Maienfisch, P., Brandl, F., Kobel, W., Fischer, W., Hofer, D., Kayser, H.,
Rindlisbacher, A., and Senn, R. (1999) Kobel, W., Rindlisbacher, A., Senn, R.,
in Nicotinoid Insecticides and the Nicotinic Steinemann, A., and Widmer, H. (2001)
Acetylcholine Receptor (eds I. Yamamoto Pest Manage. Sci., 57, 906–913.
and J.E. Casida), Springer-Verlag, Tokyo, 26. Zhang, A., Kayser, H., Maienfisch, P.,
pp. 177–209. and Casida, J.E. (2000) J. Neurochem.,
14. Shiokawa, K., Tsuboi, S., Moriya, K., 75, 1294–1303.
Hattori, Y., Honda, I., and Shibuya, K. 27. Maienfisch, P., Huerlimann, H., and
(1990) European Patent Applicaation EP Haettenschwiler, J. (2000) Tetrahedron
386,565, (Nihon Tokushu Noyaku Seizo Lett., 41, 7187–7191.
K.K.). 28. Kagabu, S., Maienfisch, P., Zhang, A.,
15. Ishimitsu, K.S.J., Kishimoto, T., and Granda-Minones, J., Haettenschwiler, J.,
Ohishi, H. (1991) PCT International Ap- Kayser, H., Maetzke, T., and Casida, J.E.
plication WO 9,101,978, (Nippon Soda (2000) J. Med. Chem., 43, 5003–5009.
Co., Ltd). 29. Maienfisch, P., Haettenschwiler,
16. Wu, F., Kariya, A., Katsuyama, N., Tsuji, J., Rindlisbacher, A., Decock, A.,
A., Takasuka, K., Segami, S., Nanjo, Wellmann, H., and Kayser, H. (2003)
K., and Sato, J. (1991) European Patent Chimia, 57, 710–714.
30. Wu, F., Katsurayama, T., Segami, S., 45. Beck, G. and Heitzer, H. (1988) Ger. Of-
and Takasuka, S. (1991) Japan Patent fen DE 3,631,538.
Application JP 03,291,267. 46. Uneme, H., Higuchi, N., and Minamida,
31. Maienfisch, P. and Widmer, H. (1998) I. (1991) European Patent Application
PCT International Application WO EP 446,913.
9,856,764. 47. Pitterna, T. et al. (1997) PCT Interna-
32. Ebihara, K., Ura, D., Miyamoto, M., and tional Application WO 97/10,226.
Kaiho, T. (1998) European Patent Appli- 48. Jackson, A., Heyes, G., Grayson, J.I.,
cation EP 869,120. and Clarke, R. (1997) European Patent
33. Widmer, H., Steinemann, A., and Application EP 763,531.
Maienfisch, P. (1999) Chemical and 49. Wakasugi, T., Miyakawa, T., and
physical properties of thiamethoxam Tanonaka, T. (1997) European Patent
(CGA 293’343). Book of Abstracts, 218th Application EP 775,700.
ACS National Meeting, August 22–26, 50. O’Sullivan, A.C., Gsell, L., Naef, R.,
New Orleans, AGRO-134. Senn, M., Pitterna, T., and Wadsworth,
34. Kagabu, S., Murata, N., Hibino, R., D.J. (1997) PCT International Applica-
Hanzawa, M., and Nishimura, K. (2005) tion WO 97/23,469.
J. Pestic. Sci., 30, 111–115. 51. Szczepanski, H., Gobel, T., Huter, O.F.,
35. Yamamoto, I. and Casida, J.E. (eds) O’Sullivan, A.C., Senn, M., Rapold, T.,
(1999) Nicotinoid Insecticides and
Maienfisch, P., and Pitterna, T. (1997)
the Nicotinic Acetylcholine Receptor,
PCT International Application WO
Springer-Verlag, Tokyo, pp. 1–300.
97/20,829.
36. Bryant, R. (2001) Agro Food Ind. Hi-Tech,
52. Pitterna, T., Szczepanski, H.,
12, 58–63.
Maienfisch, P., Huter, O.F., Rapold,
37. Senn, R., Hofer, D., Hoppe, T., Angst,
T., Senn, M., Gobel, T., O’Sullivan, A.C.,
M., Wyss, P., Brandl, F., Maienfisch,
and Seifert, G. (1998) PCT International
P., Zang, L., and White, S. (1998) Proc.
Application WO 98/27,074.
53. Fischer, W. and Widmer, H. (2001)
27–36.
Chemodynamic behaviour of the new
38. Gobel, T., Gsell, L., Huter, O.,
insecticide thiamethoxam as seed treat-
Maienfisch, P., Naef, R., O’Sullivan,
ment. BCPC Symposium Proceeding,
A.C., Pitterna, T., Rapold, T., Seifert,
G., Senn, M., Szczepanski, H., and Seed Treatment, vol. 76, pp. 203–208.
Wadsworth, D.J. (1999) Pestic. Sci., 55, 54. Wiesner, P., Kayser, H. (2000) J.
355–357. Biochem. Mol. Toxicol., 14, 221–230.
39. Seifert, G., Rapold, T., and Gisin, V. 55. Kayser, H., Lee, C., Decock, A., Baur, B.,
(2001) PCT International Application Haettenschwiler, J., and Maienfisch, P.
WO 2001/000,623. (2004) Pest Manage. Sci., 60, 945–958.
40. Kern Faber, N. (2001) Ger. Offen DE 56. Wellmann, H., Gomes, M., Lee, C., and
1994/7,332. Kayser, H. (2004) Pest Manage. Sci., 60,
41. Faber, D., Desponds, O., Rapold, T., and 959–970.
Passafaro, M. (2002) PCT International 57. Kayser, H., Wellmann, H., Lee, C.,
Application WO 2002/016,334. Decock, A., Gomes, M., Cheek, B.,
42. Desponds, O., Faber, D., Gressly, R., Lind, R., Baur, M., Hattenschwiler, J.,
Rapold, T., and Passafaro, M. (2002) and Maienfisch, P. (2007) Synthesis and
PCT International Application WO Chemistry of Agrochemicals VIII, ACS
2002/016,335. Symposium Series, vol. 948, pp. 67–81.
43. Rapold, T., Seifert, G., and Senn, M. 58. Mori, K., Okumoto, T., Kawahara, N.,
(2002) PCT International Application and Ozoe, Y. (2002) Pest Manage. Sci.,
WO 2002/034,734. 58, 190–196.
44. Kovganko, N.V. and Kashkan, 59. Honda, H., Tomizawa, M., and Casida,
Z.N. (2004) Russ. J. Org. Chem., 40, J.E. (2006) J. Agric. Food Chem., 54,
1709–1726. 3365–3371.
References 1225
60. Liu, Z., Williamson, M.S., Lansdell, S.J., 71. Hofer, D., Brandl, F., Druebbisch, B.,
Han, Z., Denholm, I., and Millar, N.S. Doppmann, F., and Zang, L. (2001)
(2006) J. Neurochem., 99, 1273–1281. Thiamethoxam (CGA 293’343) – a
61. Zhang, Y., Liu, Z., Han, Z., Song, F., novel insecticide for seed-delivered
Yao, X., Shao, Y., Li, J., and Millar, N.S. insect control. BCPC Symposium Pro-
(2009) J. Neurochem., 110, 1855–1862. ceedings: Seed Treatment, vol. 76,
62. Nauen, R., Ebbinghaus-Kintscher, U., pp. 41–46.
Salgado, V.L., and Kaussmann, M. 72. Elbert, A. and Nauen, R. (2000) Pest
(2003) Pestic. Biochem. Physiol., 76, Manage. Sci., 56, 60–64.
55–69. 73. Wang, Z., Yan, H., Yang, Y., and Wu, Y.
63. Jeschke, P. and Nauen, R. (2004) Thi- (2010) Pest Manage. Sci., 66, 1360–1366.
amethoxan: A neonicotinoid precursor 74. Karatolos, K., Denholm, I., Williamson,
converted to clothianidin in insects and M., Nauen, R., and Gorman, K. (2010)
plants. Abstracts of Papers, 228th ACS Pest Manage. Sci., 66, 1304–1307.
National Meeting, August 22–26, 2004, 75. Matsumura, M., Takeuchi, H., Satoh,
Philadelphia, PA. AGRO-003. M., Sanada-Morimura, S., Otuka, A.,
64. Nauen, R., Ebbinghaus-Kintscher, U., Watanabe, T., and Van Thanh, D. (2008)
and Jeschke, P. (2005) Pharmacoki- Pest Manage. Sci., 64, 1115–1121.
netics, toxicodynamics and activation 76. Lind, R., Slater, R., Pedroni, D., and
to clothianidin of thiamethoxam in Senn, R. (2008) International Congress
Colorado potato beetles. Abstracts of of Entomology, Proceedings, Sympo-
Papers, 230th ACS National Meet- sium Number 3.02, July 6–12, 2008,
ing, August 28–September 1, 2005, Durban. Abstract number 1375.
Washington, DC. AGRO-026. 77. Alyokhin, A., Dively, G., Patterson, M.,
65. Miyagi, S., Komaki, I., and Ozoe, Y. Castaldo, C., Rogers, D., Mahoney, M.,
(2006) Pest Manage. Sci., 62, 293–298. and Wollam, J. (2007) Pest Manage. Sci.,
66. Harrewijn, P., De Kogel, W.J., and 63, 32–41.
Piron, P.G.M. (1998) Proc. Brighton Crop 78. Green, T., Toghill, A., Lee, R., Waechter,
Prot. Conf. – Pests Dis., 3, 813–818. F., Weber, E., and Noakes, J. (2005) Tox-
67. Lawson, D.S., Dunbar, D.M., White, icol. Sci., 86, 36–47.
S.M., and Ngo, N. (1999) Actara 79. Green, T., Toghill, A., Lee, R., Waechter,
25 WG: control of cotton pests F., Weber, E., Peffer, R., Noakes, J., and
with a new neonicotinoid insecti- Robinson, M. (2005) Toxicol. Sci., 86,
cide, thiamethoxam.Proceedings of 48–55.
Beltwide Cotton Conferences, vol. 2, 80. Pastoor, T., Rose, P., Lloyd, S., Peffer,
pp. 1106–1109. R., and Green, T. (2005) Toxicol. Sci., 86,
68. Hofer, D. and Brandl, F. (1999) 56–60.
Cruiser/Adage performance features 81. Schade, M., Maienfisch, P., Grimm,
of thiamethoxam as a seed treatment C., Nina, M., Hofer, D., and Skillman,
in worldwide cotton. Proceedings of S. (2010) 12th IUPAC International
Beltwide Cotton Conferences, vol. 2, pp. Congress of Pesticide Chemistry, Mel-
1101–1104. bourne, Australia, July 4–8, 2010. Poster
69. Zang, L., Ngo, N., and Minto, B. (1999) Abstract: Abstract no. 647.
Actara 25 WG: control of cotton pests 82. Senn, R., Hofer, D., Thieme, T., and
with a new neonicotinoid insecti- Zang, L. (2001) PCT International
cide, thiamethoxam. Proceedings of Application WO 2001/026,468.
Beltwide Cotton Conferences, vol. 2, pp. 83. Schade, M., Klaveano, R., Cassidy, E.,
1104–1106. and Grimm, C. (2007) Proceedings
70. Hofer, D., Brandl, F., and Fischer, of the 12th International Rapeseed
W. (2000) Cruiser/Adage uptake dy- Congress, March 26–30, 2007, Wuhan,
namic and biological performance China.
under different environmental condi- 84. Castro, P.R.C., Pitelli, A.M.C.M., Peres,
tions. Proceedings of Beltwide Cotton L.E.P., and Aramaki, P.H. (2008) Rev.
Conferences, vol. 2, pp. 1024–1027. Agric., 83, 208–213.
32.3
Sulfoxaflor
Thomas C. Sparks, Michael R. Loso, Gerald B. Watson, Jonathan M. Babcock, Vincent J.
Kramer, Yuanming Zhu, Benjamin M. Nugent, and James D. Thomas
32.3.1
Introduction
Sulfoxaflor (Figure 32.3.1) is the first molecule in development from the new
sulfoximine class of insect control agents. Effective on a wide range of sap-feeding
insects, sulfoxaflor is notable for its efficacy against sap-feeding insect pests that
are resistant to currently available insecticides [1].
Sap-feeding insects constitute a major pest complex that includes a range of
aphids, whiteflies, hoppers, and others. Included within this pest complex are
several well-known pests such as green-peach aphid (Myzus persicae), cotton aphid
(Aphis gossypii), and sweetpotato whitefly (Bemisia tabaci). These sap-feeding pests
have developed resistance to a broad range of insect control agents, including
organophosphates, carbamates, pyrethroids and, most recently, the neonicotinoids
[2]. Although initially slow to develop, resistance to neonicotinoids is becoming
an expanding problem for some insect pest management (IPM) systems [2, 3]. In
light of the growing resistance of key sap-feeding pests to neonicotinoid and other
insecticides, and of regulatory actions that limit the use of older insecticides (e.g.,
organophosphates and carbamates), there is a clear continuing need for new insect
control agents that are both effective and active on these resistant insect pests.
The discovery of new insect control agents may emerge from a wide vari-
ety of sources including natural products, the screening of diverse chemistries,
literature-inspired efforts, and biorational approaches [4–7]. Privileged structures
or scaffolds can also serve as a starting point for new biologically active chem-
istry [8, 9]. The discovery of sulfoxaflor represents just such an approach, where
explorations involving a chemical moiety that was novel to agrochemicals – a
sulfoximine – initially resulted in molecules with weak fungicidal activities. Yet,
further explorations of the substituents attached to the sulfoximine core resulted in
the identification of a new class of insect-active sulfoximines [10–12]. Subsequent
optimization efforts resulted in the discovery of sulfoxaflor, the first member of
this new class of insect control agents which, is currently, undergoing development
by Dow AgroSciences [11, 12].
32.3.2
Chemistry, Structure–Activity Relationships, and Toxicology
32.3.2.1 Synthesis
The synthesis of sulfoxaflor and its closely related analogs is outlined in
Schemes 32.3.1 and 32.3.2. Precursor sulfides, utilized for the synthesis of
sulfoximines, were accessed by the methods depicted in Scheme 32.3.1. Acyclic
sulfides of type I obtained by the reaction of substituted chloromethyl pyridines
CH3 Figure 32.3.1 Structure of sulfoxaflor.

CH3
S
O N CN
F 3C N
NaSMe
Cl S
R1 N R1 N
I
thiourea
Cl
NH
i) NaOH Base
S NH2 S
ii) Br Cl
S
R1 N R1 N
R1 N
II
Scheme 32.3.1 Synthesis of key sulfide intermediates.
Route A
NaN3 Ac2O; HNO2
Het S Het S Het S
mCPBA O O NH O N NO2
Het S
A B C
I
or TFAA;
BrCN
K2CO3
H2NCN, PhI(OAc)2
Het S
mCPBA Het =
II
Het S Het S
Route B R1 N
N CN O N CN
E D
Scheme 32.3.2 Synthetic routes to sulfoximines.
with sodium thiomethoxide. Cyclic sulfides of type II were prepared in a three-step

sequence that included the nucleophilic addition of thiourea to a 6-substituted
chloromethyl pyridine, a subsequent one-pot hydrolysis and alkylation with
1-bromo-3-chloropropane, followed by cyclization under basic conditions [10, 11].
Two routes, as shown in Scheme 32.3.2, were utilized to convert sulfides into
either N-nitro or N-cyano sulfoximine derivatives. In Route A, precursor sulfides
of type I or II are first oxidized to sulfoxides A with meta-chloroperoxybenzoic acid
(mCPBA), and then treated with sodium azide to obtain unsubstituted sulfoximines
B [10, 13]. Subsequent nitration or cyanation [14] provided targeted N-substituted
R3
1) Base
S S
O N R2 2) R3-X O N R2
R1 N R1 N
I III
Sulfoxaflor: R1 = CF3, R2 = CN, R3 = Me
Scheme 32.3.3 Derivatization of acyclic sulfoximines.
sulfoximines C or D, respectively [10]. A more efficient, scalable route utilizes the

mild oxidant iodobenzene diacetate [15] to prepare N-cyano sulfilimines E via an
oxidative addition of cyanamide to sulfides I and II (Route B) [10]. Subsequent
oxidation of E gave targeted N-cyano sulfoximines D. Access back to unsubstituted
sulfoximine B was accomplished via the decyanation of D with trifluoroacetic
anhydride, followed by basic hydrolysis [16]. This reaction, taken together with the
previous two steps of Route B, provided a scalable three-step route to the versatile
sulfoximine intermediate B.
Acyclic sulfoximines derived from sulfides of type I can be further derivatized
at the carbon linking the sulfoximine moiety to the pyridyl ring, under standard
alkylation conditions, to provide substituted sulfoximines III (Scheme 32.3.3).
Sulfoxaflor, which features a methyl substituent at R3, was obtained via alkylation
with methyl iodide [11].

Investigations into a diverse set of linkers and heterocycles revealed that a
6-substituted pyridine, attached at the pyridyl 3-position to a sulfoximine moi-
ety via a single carbon linker, was among the most efficacious of combinations [10,
17]. From there, analogs were synthesized with diversity in three primary regions:
substitutions at the 6-position of the pyridine (R1); on the carbon linker (R3); and
on the sulfoximine nitrogen (R2). For R1, a halogen or short-chain haloalkyl substi-
tution was found to be most effective, with CF3 being the most active (Table 32.3.1).
For R3, substituents of limited size were found to be favorable, with methyl being
the most active. Investigations into R2 revealed two efficacious moieties, a nitro
group and a cyano group. In all cases, nitro substitution was found to be less potent
against green-peach aphid than cyano substitution. Cyclic sulfoximines 8–10 were
found to be less active than open-chain analogs with a methyl substitution at R3.
32.3.2.3 Physico-Chemical Properties of Sulfoxaflor

Following extensive studies into the structure–activity relationship (SAR) of the
sulfoximines against a variety of sap-feeding insect pests, sulfoxaflor was selected
for further development. The physico-chemical properties of sulfoxaflor are listed
in Table 32.3.2. Sulfoxaflor has appreciable water solubility, and is also soluble in
a variety of organic solvents, especially polar solvents such as methanol, acetone,
and ethyl acetate.
Table 32.3.1 Structure–activity relationships (SAR):

sulfoximine analogs and green-peach aphid (Myzus persicae)
efficacy.
R3
S Green-peach
O N R2
R1 N aphid efficacy
R1 R2 R3 LC50 (ppm.)
Cl NO2 H 1 2.0
Cl CN H 2 2.9
Cl CN Me 3 0.3
CF3 NO2 H 4 31.9
CF3 CN H 5 1.2
CF3 CN Me 6a 0.1
Me CN H 7 72.7
S Green-peach
O N R2 aphid efficacy
R1 N
R1 R2 LC50 (ppm.)
Cl CN 8 2.2
CF3 CN 9 2.8
Cl NO2 10 55.6
a
Sulfoxaflor.
Following its application to plant foliage, sulfoxaflor is rapidly absorbed into

the tissues, with the compound’s water solubility and log Kow values at biologically
relevant pH levels indicating a likely movement within the xylem (Table 32.3.2).
The high pKa value suggests that active movement within the phloem is unlikely,
however, as only pest-control molecules with ionizable groups have shown this
tendency. The application of sulfoxaflor to plant roots has confirmed its rapid
movement in an acropetal direction. Phosphor images of 14 C-labeled sulfoxaflor,
when applied at distinct points midway across a leaf, showed a clear movement of
the compound to the leaf margins, which is consistent with xylem movement. The
results of both laboratory-based and field studies have shown that sulfoxaflor, when
applied as a spray to whole plants, confers a broad systemic protection across all
tissues, including those that were not completely covered by the spray application.
Notably, sulfoxaflor is quite stable within plant tissues, such that newly developing
foliage may be protected against pests for up to 14 days after its application at
typical usage rates. Although the application of sulfoxaflor to the roots and to seeds
Table 32.3.2 Physical and chemical properties of sulfoxaflor.
Molecular weight 277.27 g mol−1

Appearance Off-white powder (solid)
Octanol/water partition coefficient pH 7 buffer solution: Low Kow = 0.802
(log Kow ) at 19 ◦ C
Dissociation constant (pKa ) >10 (does not fully dissociate within environmentally
relevant pH ranges)
Hydrolytic stability (DT50 ) Stable
Aqueous photostability (DT50 ) Expected to be stable in sterile conditions
Soil photolysis (DT50 ) Expected to be stable in sterile conditions (DT50 < 1
day in aerobic soil)
Solubility in water ( mg l−1 , 20 ◦ C)
Purified water 670

Buffered water, pH 5 1380
Organic solvent solubility (g l−1 , 20 ◦ C)
Methanol 93.1
Acetone 217
Xylene 0.743
1,2-Dicloroethane 39.6
Ethyl acetate 95.2
Heptane 0.000242
Octanol 1.66
DT = dissipation time.
results in high levels of insect efficacy, the parent compound is rapidly degraded in
the soil (via microbial action) to inactive metabolites.
32.3.3
Mammalian and Environmental Toxicology
32.3.3.1 Mammalian Toxicology

Sulfoxaflor exhibits a low acute mammalian toxicity; neither were any adverse
effects identified in any genotoxicity studies. The results of subchronic toxicity
studies revealed the liver to be the primary target organ, albeit with effects of
limited concern or of no relevance to humans. A summary of the mammalian
toxicology data is provided in Table 32.3.3.
32.3.3.2 Environmental Toxicology: Bees

The effects of sulfoxaflor on the honey bee (Apis mellifera) and bumble bee (Bom-
bus terrestris) have been studied both in the laboratory and in tunnel tests to
Table 32.3.3 Mammalian toxicology data for sulfoxaflor.
Study Animal or test system Result
Acute oral LD50 Rat 1000 mg kg−1

Acute dermal LD50 Rat > 5000 mg kg−1
Acute inhalation LC50 Rat > 2.09 mg l−1
Dermal irritation Rabbit Minimal
Eye irritation Rabbit Slight
Skin sensitization Mouse None
Four-week dietary exposure Rat NOAEL = 24 mg kg−1
Four-week dermal exposure Rat NOAEL = 1000 mg kg−1
Genotoxicity Ames test Negative
Chromosomal aberration Negative
Mouse micronucleus (in vivo) Negative
Acute neurotoxicity Rat NOAEL = 25 mg kg−1 (top dose)
NOAEL = No observed adverse effect level.
simulate field conditions. Under laboratory conditions, sulfoxaflor was acutely

toxic to honey bees after oral (48 h LD50 = 0.146 μg a.i. per bee) or contact (48 h
LD50 = 0.379 μg a.i. per bee) administration, with sulfoxaflor technical and formu-
lated products demonstrating similar levels of toxicity. Based on data for technical
material, as reported in the US Environmental Protection Agency (EPA) Pesticide
Ecological Effects Database (http://www.ipmcenters.org/ecotox/), the contact toxicity
of sulfoxaflor was central among values reported for honey bees with regards to
insecticides used to control sap-feeding insects. When honey bees were exposed to
field-aged dry residues of sulfoxaflor on alfalfa foliage, low mortality rates (<20%)
were observed. In tunnel tests in which honey bees from small colonies were
allowed to forage among flowering plants (Phacelia tanacetifiolia) treated previously
with sulfoxaflor or with a neonicotinoid or an organophosphate (OP) insecticide,
the mortality of bees was similar in sulfoxaflor-treated and control (nontreated)
plots at one to seven days after application. The foraging activity of bees was also
similar in sulfoxaflor-treated and control plots, but was essentially halted for several
days in the OP- and neonicotinoid-treated plots. No long-term effects of sulfoxaflor
have been observed on honey bee brood development.
32.3.3.3 Environmental Toxicology: Other Nontarget Organisms

Acute environmental toxicology studies with sulfoxaflor have been conducted to
fulfill the requirements of the US EPA and other regulatory agencies. Sulfoxaflor
exhibits a very low acute toxicity to fish, freshwater crustaceans (Daphnia magna),
oysters, algae, and aquatic vascular plants. Midge larvae (Chironomus dilutus) and
the mysid shrimp (Americamysis bahia, a saltwater free-swimming crustacean) may
be sensitive to sulfoxaflor. Data generated from these studies are summarized in
Table 32.3.4.
Table 32.3.4 Environmental toxicology data for sulfoxaflor.
Acute toxicity to birds Oral LD50 = 676 mg kg−1 body weight (bobwhite quail)
Dietary toxicity to birds 5-day dietary LC50 >a 5620 mg kg−1 diet (bobwhite quail,
mallard duck)
Acute toxicity to fish 96 h LC50 > 387 mg l−1 (rainbow trout)
96 h LC50 > 363 mg l−1 (bluegill sunfish)
96 h LC50 > 402 mg l−1 (common carp)
96 h LC50 = 266 mg l−1 (sheepshead minnow)
Acute toxicity to invertebrates Daphnia magna – 48 h EC50 > 399 mg l−1
Chironomus dilutus – 96 h LC50 = 0.622 mg l−1
Chironomus dilutus – 10 day LC50 = 0.161 mg kg−1
sediment
Mysid shrimp – 96 h LC50 = 0.643 mg l−1
Eastern oyster – 96 h EC50 (shell deposition) = 86.5 mg l−1
Acute toxicity to aquatic plants 7 day EC50 > 99 mg l−1 (Lemna gibba, duckweed)
a
Symbol indicates that the endpoint was greater than the highest concentration tested.
32.3.4
Mode of Action
Green-peach aphids treated with sulfoxaflor exhibit excitatory symptoms of intox-

ication such as tremors, followed by paralysis. Similar symptoms are exhibited by
other insects such as fruit flies (Drosophila melanogaster) and American cockroach
(Periplaneta americana), after treatment with sulfoxaflor. The initial excitation in-
duced by sulfoxaflor indicated an interaction with excitatory neural pathways, and
sulfoxaflor was ultimately shown to interact with nicotinic acetylcholine receptors
(nAChRs) in the insect nervous system.
Fruit fly α-type nAChR subunits can be expressed in frog oocytes when coex-
pressed with a vertebrate β-type nicotinic receptor subunit (e.g., Ref. [18]). Thus,
expressed Drosophila α2 subunits, coexpressed with chicken β2 subunits, were
used to study the action of sulfoxaflor with the two-electrode voltage–clamp elec-
trophysiology technique. The results of these studies revealed that sulfoxaflor is
a nAChR agonist that induces very large amplitude currents when compared to
many of the neonicotinoid insecticides (e.g., Figure 32.3.2).
Tritium-labeled imidacloprid ([3 H]IMI) defines an insecticidally relevant binding
site at the nAChR [19]. Although, in light of its potent insecticidal activity, sulfoxaflor
might be expected to displace [3 H]IMI from nAChRs with high potency, it displaced
[3 H]IMI from green-peach aphid nAChRs only very weakly when compared to
imidacloprid [12].
The ability of sulfoxaflor to induce very large-amplitude currents, coupled to its
apparent weak affinity for the nAChR (as defined by [3 H]IMI binding) suggests a
complex interaction of sulfoxaflor with insect nAChRs. Further, this combination
400
300
(% Ach Current)
Response
200
Sulfoxaflor
100
Imidacloprid
0
−9 −8 −7 −6 −5 −4 −3
log [compound] (M)
Figure 32.3.2 Dose–response of sulfoxaflor and imida-

cloprid on inducing currents in Dα2/chick β2 nAChRs
expressed in oocytes.
of effects appears unique to sulfoxaflor when compared to other nAChR-acting

insecticides, such as the neonicotinoids. Consequently, studies aimed at character-
izing the interaction of sulfoxaflor with insect nAChRs are ongoing.
32.3.5
Biology and Field Use Patterns
32.3.5.1 Biology
Sulfoxaflor has been characterized under laboratory conditions for the control of
a wide range of insects. In general, the greatest potency is expressed against a
broad range of aphids and of other Heteroptera, such as whiteflies, plant bugs,
scales, mealybugs as well as plant, and leafhoppers (Table 32.3.5). The potency
of sulfoxaflor against other pests groups (Coleoptera, Diptera, Lepidoptera, and
Thysanoptera) is typically much less.
32.3.5.2 Field Use Patterns

Field trials have demonstrated the control of many important sap-feeding pests
with sulfoxaflor, with quick knockdown and good residual activity (see Table 32.3.5
for a partial list). Significant yield responses have been observed in cotton, rice,
and cereals. Field trials have also shown reductions in insect-vectored viral dis-
eases in vegetables and cereals. Field use rates range from approximately 12 to
150 g a.i. ha−1 , depending on the crop, pest, and population level.
32.3.6
Efficacy on Resistant Pests
Since the early 1990s, imidacloprid and the other neonicotinoid insecticides have
been at the forefront for the control of a wide variety of sap-feeding insect pests
Table 32.3.5 Partial list of pests controlled by sulfoxaflor

and crops in which field efficacy trials have been conducted.
Scientific name Common name Crop(s)
Myzus persicae Green-peach aphid Leafy, cole, and fruiting vegetables;

potato; stone fruits
Aphis gossypii Cotton/melon aphid Cotton, cucurbits
Lygus spp. Tarnished plant bugs Cotton, fruiting vegetables
Nilaparvata lugens Brown planthopper Rice
Nephotettix cincticeps Green leafhopper
Sogatella furcifera White-backed planthopper
Laodelphax striatella Small brown planthopper
Bemisia spp. Whitefly Leafy, cole, cucurbit, and fruiting
vegetables; cotton
Trialeurodes spp. Whitefly Fruiting vegetables
Eriosoma lanigerum Wooly apple aphid Pome fruit
Aphis pomi Apple aphid
Dysaphis plantaginea Rosy apple aphid
Aonidiella aurantii California red scale Citrus
Planococcus citri Citrus mealybug
Nezara viridula Southern green stink bug Soybean
Aphis glycines Soybean aphid
[20, 21]. Although initially gradual, resistance to the neonicotinoids has recently
become an increasing issue in the control of many such pests [2, 3, 22, 23]. One key
attribute of sulfoxaflor is its lack of cross-resistance in sap-feeding insect strains
that are resistant to the neonicotinoids and other insecticides ([1, 12], Table 32.3.6).
Assays on a variety of green-peach aphid, whitefly, and brown planthopper strains
resistant to imidacloprid and other neonicotinoids showed no cross-resistance to
sulfoxaflor ([1, 12], Table 32.3.6). In addition, cotton aphid populations that have
shown a reduced susceptibility to thiamethoxam respond similarly to sulfoxaflor
as those that are fully susceptible. For example, whilst a reduced control with
thiamethoxam was observed in field trials with cotton aphid populations in the
mid-South of the US, the efficacy of sulfoxaflor was excellent and consistent with
expectations [23].
Resistance to neonicotinoid insecticides develops almost entirely through
metabolic mechanisms. Whilst the single example of target site-based resistance
in neonicotinoid-resistant sap-feeding insects was found in a laboratory-selected
strain of brown planthopper [24], this mutation has yet to be found in a
field-collected strain [25]. In all other studies of neonicotinoid resistance
mechanisms in sap-feeding insect pests, resistance has been associated with an
enhanced metabolism, specifically via mono-oxygenases [3, 12]. For example,
field-collected strains of imidacloprid-resistant brown planthoppers from China
and India were shown to possess enhanced levels of mono-oxygenase activity
Table 32.3.6 Sulfoxaflor cross-resistance in resistant sap-feeding insect pests.
Species – strains Compound Resistance ratio Reference
Green-peach aphid (Myzus persicae)

R-4013A Imidacloprid 17.1 [12]
Sulfoxaflor 0.4 –
Sweetpotato whitefly (Bemisia tabaci)
PBI Imidacloprid 870 [1]
R-4991BT1 Imidacloprid >230 [12]
R-2971BT9 Imidacloprid 1022 [12]
R-CHLORAKA Imidacloprid >833 [12]
Brown planthopper (Nilaparvata lugens)
Ogori-R Imidacloprid 438 [1]
OH
N N
Cl N NH NH
N Cl N N
NO2 NO2
imidacloprid N
Cl N NH
N
NO2
CYP6G1
S
No reaction
O N CN
F3 C N
sulfoxaflor
Figure 32.3.3 Metabolism of imidacloprid compared to sul-

foxaflor in a cell line expressing CYP6G1.
[25, 26]. Likewise, an enhanced mono-oxygenase activity was also associated with
neonicotinoid resistance in strains of green-peach aphids [27] and whiteflies [28].
The lack of cross-resistance to sulfoxaflor in neonicotinoid-resistant sap-feeding
insects suggests that the unique chemical structure of sulfoxaflor may not be
susceptible to the mono-oxygenases involved and, indeed, the results of recent
studies have provided support for this hypothesis. One strain of Drosophila that was
resistant to imidacloprid and other insecticides demonstrated enhanced levels of a

mono-oxygenase, CYP6G1 [29, 30], that has been shown to metabolize imidacloprid
[31]. However, when CYP6G1 was expressed in a Drosophila cell line, imidacloprid
and acetamiprid – but not sulfoxaflor – were found to be extensively metabolized
[12] (Figure 32.3.3), with two metabolites of imidacloprid being identified (G.
DeBoer, unpublished results). The lack of metabolism of sulfoxaflor – and hence
of cross-resistance – suggests that this compound might serve as a poor substrate
for CYP6G1, most likely due to its unique chemical structure.
32.3.7
Conclusions
Sulfoxaflor, the first member of the new sulfoximine class of chemistry to be

developed (registration is anticipated in 2012), is a broad-spectrum insecticide that
is active against sap-feeding insects and has the potential to fit into many IPM pro-
grams. Notably, sulfoxaflor possesses an excellent mammalian and environmental
toxicological profile.
Sulfoxaflor is distinct from all other insect-control agents in that it acts at nAChRs
as a high efficiency agonist that only weakly displaces imidacloprid. This suggests
the existence of either a complex or unique interaction with insect nAChRs
relative to the neonicotinoids or other nicotinic-acting insecticides, such as the
spinosyns or nereistoxin analogs. Sulfoxaflor may be further differentiated from
the neonicotinoids by its lack of cross-resistance in strains that are highly resistant
to imidacloprid and other neonicotinoids. The latter property which appears, at
least in part, to be related to the compound’s novel chemical structure, which
prevents its metabolism by certain mono-oxygenases (e.g., CYP6G1) associated
with resistance to imidacloprid. Of equal importance, Drosophila larvae possessing
target-site resistance to neonicotinoids (e.g., imidacloprid) show no cross-resistance
to sulfoxaflor (T. Perry and T.C. Sparks, unpublished results). Thus, when compared
to other insect-control agents currently available for the control of sap-feeding insect
pests, sulfoxaflor is chemically and biochemically unique.
Acknowledgments
The authors thank James Hasler for generating the Dα2 and CYP6G1 constructs,
and Gerrit DeBoer for the metabolism data.
References
1. Babcock, J.M., Gerwick, B.C., Huang, (eds M.E. Whalon, D. Mota-Sanchez,

J.X., Loso, M.R., Nakamura, G.E., and R.M. Hollingworth), CAB Interna-
Nolting, S., Rogers, R.B., Sparks, T.C., tional, Wallingford, pp. 5–31.
Thomas, J., Watson, G.B., and Zhu, Y. 3. Jeschke, P. and Nauen, R. (2010) in
(2011) Pest Manage. Sci., 67, 328–334. Insect Control (eds L.I. Gilbert and S.S.
2. Whalon, M.E., Mota-Sanchez, D., and Gill), Academic Press, New York, pp.
Hollingworth, R.M. (2008) in Arthropods 114–119.
References 1237
4. Eto, M. (1992) in Rational Approaches and Daeuble, J.F. (2010) US Patent 7,

to Structure, Activity, and Ecotoxicology 705,156 B2.
of Agrochemicals (eds W. Draber and T. 18. Ihara, M., Matsuda, K., Otake, M.,
Fujita), CRC Press, Boca Raton, FL, pp. Kuwamura, M., Shimomura, M., Komai,
147–161. K., Akamatsu, M., Raymond, V., and
5. Addor, R.W. (1995) in Agrochemi- Sattelle, D.B. (2003) Neuropharmacology,
cals from Natural Products (ed C.R.A. 45, 133–144.
Godfrey), Marcel Dekker, New York, pp. 19. Liu, M. and Casida, J.E. (1993) Pest.
1–62. Biochem. Physiol., 46, 40–46.
6. Clark, J.M. and Ohkawa, H. (eds) (2005) 20. Elbert, A., Haas, M., Springer, B.,
New Discoveries in Agrochemicals, Amer- Thielert, W., and Nauen, R. (2008)
ican Chemical Society, Washington, Pest Manage. Sci., 64, 1099–1105.
DC. 21. Jeschke, P. and Nauen, R. (2008) Pest
7. Ishaaya, I., Nauen, R., and Horowitz, Manage. Sci., 64, 1084–1098.
A.R. (eds) (2007) Insecticides Design using 22. Gorman, K., Liu, Z., Denholm, I.,
Advanced Technologies, Springer-Verlag, Bruggen, K.U., and Nauen, R. (2008)
Berlin. Pest. Manage. Sci., 64, 1122–1125.
8. Kubinyi, H. (2006) in Analogue-based 23. Gore, J., Cook, D., Catchot, A., Leonard,
Drug Discovery (eds J. Fischer and C.R. R., Lorenz, G., and Stewart, S. (2010)
Ganellin), Wiley-VCH Verlag GmbH, Proceedings of the Beltwide Cotton Produc-
Weinheim, pp. 53–68. tion Conference, 2010, January 4–7, New
Orleans, LA. National Cotton Council,
9. Welsch, M.E., Snyder, S.A., and
pp. 1207–2010.
Stockwell, B.R. (2010) Curr. Opin. Chem.
24. Liu, Z., Williamson, M.S., Lansdell, S.J.,
Biol., 14, 1–15.
Denholm, I., Han, Z., and Millar, N.S.
10. Zhu, Y., Rogers, R.B., and Huang,
J.X. (2010) Insecticidal N-substituted
8420–8425.
sulfoximines. US Patent 7, 678,920.
25. Puinean, A.M., Denholm, I., Millar,
11. Loso, M.R., Nugent, B.M., Huang,
N.S., Nauen, R., and Williamson, M.S.
J.X., Rogers, R.B., Zhu, Y., Renga,
(2010) Pestic. Biochem. Physiol., 97,
J.M., Hegde, V.B., and Demark, J.J.
129–132.
(2010) Insecticidal N-substituted
26. Wen, Y., Liu, Z., Bao, H., and Han,
(6-haloalkylpyridin-3-yl)alkyl sulfox-
Z. (2009) Pestic. Biochem. Physiol., 94,
imines. US Patent 7, 687,634. 36–42.
12. Zhu, Y., Loso, M.R., Watson, G.B., 27. Philippou, D., Field, L., and Moores, G.
Sparks, T.C., Rogers, R.B., Huang, J.X., (2009) Pest Manage. Sci., 66, 390–395.
Gerwick, B.C., Babcock, J.M., Kelley, 28. Karunker, I., Bneting, J., Lueke, B.,
D., Hegde, V.B., Nugent, B.M., Renga, Ponge, T., Nauen, R., Roditakis, E.,
J.M., Denholm, I., Gorman, K., DeBoer, Vontas, J., Gorman, K., Denholm, I.,
G.J., Hasler, J., Meade, T., and Thomas, and Morin, S. (2008) Insect Biochem.
J.D. (2011) J. Agric. Food Chem., 59, Mol. Biol., 38, 634–644.
2950–2957. 29. Daborn, P.J., Boundy, S., Yen, J.,
13. Johnson, C.R., Haake, M., and Schroeck, Pittendrigh, B., and ffrench-Constant,
C.W. (1970) J. Am. Chem. Soc., 92, R. (2001) Mol. Genet. Genomics, 266,
6594–6598. 556–563.
14. Mutti, R. and Winternitz, P. (1986) Syn- 30. Daborn, P.J., Yen, J.L., Bogwitz, M.R.,
thesis, 5, 426–427. Le Goff, G., Feil, E., Jeffers, S., Tijet,
15. Ou, W. and Chen, Z.C. (1999) Synth. N., Perry, T., Heckel, D., Batterham,
Commun., 29, 4443–4449. P., Feyereisen, R., Wilson, T.G., and
16. Okamura, H. and Bolm, C. (2004) Org. ffrench-Constant, R.H. (2002) Science,
Lett., 6, 1305–1307. 297, 2253–2256.
17. Loso, M.R., Nugent, B.M., Zhu, Y., 31. Joussen, N., Heckel, D.G., Haas, M.,
Rogers, R.B., Huang, J.X., Renga, Schuphan, I., and Schmidt, B. (2008)
J.M., Whiteker, G.T., Breaux, N.T., Pest Manage. Sci., 64, 65–73.
32.4
Spinosad and Spinetoram, a New Semi-Synthetic Spinosyn
and Clive Waldron
32.4.1
Introduction
Spinosyns (Figure 32.4.1) are a class of fermentation-derived macrocyclic lactone

bioinsecticides, produced by the Actinomycete Saccharopolyspora spinosa [1]. They
are active against a broad range of insect pests, and exhibit activity through both oral
(ingestion) and contact routes of exposure. Their high level of efficacy, combined
with a unique mode of action (Section 32.4.2) and favorable environmental and
toxicological profiles [2], have led to their rapid adoption in numerous insect pest
management settings.
Spinosad, the first active ingredient to be developed from this family of sec-
ondary metabolites, was first registered in 1997 and received a Presidential Green
Chemistry Challenge Award in 1999. Spinetoram (see Section 32.4.5) is the second
member of this class of insecticides, and was first registered in 2007. Spinetoram
was awarded a Presidential Green Chemistry Challenge Award in 2008 in recogni-
tion of its expanded range of insect control relative to spinosad, while maintaining
favorable toxicological and environmental profiles.
The unique chemistry and biological profile of the spinosyns has spurred a
considerable amount of research since their commercial introduction in 1997.
During the three-year period between 2007 and 2009, over 600 studies on the
chemistry, biology, and biochemistry of spinosyns have been reported or presented
[3–7]. Some of the newer aspects of the science and utility of this class of insect
control agents will be described in this chapter.
O O
N O O N O O
O O
O O
O O
O O O
O
56
O O
O O
(a) R (b) R
Figure 32.4.1 (a) Structure of spinosad (a mixture of

spinosyn A (1, R = H) and spinosyn D (2, R = CH3 ); (b)
Spinetoram (major component: 3, single bond at C5–C6,
R = H; minor component: 4, double bond at C5–C6,
R = CH3 ).
32.4.2
Biological Activity and Primary Uses of Spinosad
Spinosad is currently registered for use in over 80 countries, and its labels include
uses on over 250 crops. It is marketed in several formulations for different
application conditions. Spinosad has been widely used for the control of insect
pests of vegetables and tree crops, row crops, ornamental plants, turfgrass and tree
farms, and home and garden pests. Products containing spinosad are certified for
use in organic agriculture in a number of countries.
®
An organic-certified bait formulation of spinosad, GF-120NF , is used to control
numerous destructive tephritid fruit fly species in tree fruits, nuts, vines, vegetables,
and ornamental crops [8–11]. Spinosad is also used as the toxicant in SPLAT-MAT™
Spinosad ME, a baiting technology that controls oriental fruit fly (Bactrocera
dorsalis) populations by attracting and killing the male fruit flies, thus preventing
reproduction [12].
Although the physical characteristics of the spinosyns are not conducive to most
systemic applications in plants, they have been reported to control pests through
root uptake under conditions where soil binding is minimal [13], and to control
insect pests of cabbage and cauliflower as a seed treatment [14].
The evaluation of spinosad as a protectant for stored grains has also been
reviewed [15]. An excellent control of Indian meal moth (Plodia interpunctata), and
of lesser grain borer (Rhyzopertha dominica) and other grain beetle pests, has been
demonstrated [16–18]. Spinosad also controls stored tobacco pests such as cigarette
beetle, Lasioderma serricorne, and the tobacco moth, Ephestia elutella [19].
Spinosad is also used to control insect vectors [20, 21]. A recent review sum-
marizes the effectiveness of spinosad for larval mosquito control [22]. Spinosad
has also been reported to control tsetse fly [23]. Recent studies have demonstrated
the utility of spinosad for use in control of parasitic pests in humans and other
mammals. Spinosad is currently used for the control of blowfly and lice in sheep
in Australia [24], and also has been reported to control both ticks and fleas in cattle
[25] and in companion animals [26]. Recently, the results of field studies were
reported showing the safety and efficacy of an orally administered spinosad tablet
for controlling fleas on dogs [27]. The compound is also currently being developed
for control of human head lice [28].
Spinosad is well known to present a low risk to nontarget insects. Extensive field
experience has indicated that the overall impact of spinosad on beneficial insects is
generally limited and transitory, and it fits well into Integrated Pest Management
(IPM) programs. Spinosad demonstrates large margins of safety to predacious
insects such as lady beetles (Coccinelliedae), lacewings (Neuroptera), big-eyed
bugs (Geocoris spp.), minute pirate bugs (Orius spp.), and others. Field studies
conducted on various crops using typical spinosad usage rates have demonstrated
that spinosad has a low risk to adult honey bees, and has little or no effect on hive
activity and brood development [29].
32.4.3
Mode of Action of Spinosyns
Susceptible insects treated with spinosyn A exhibit a number of excitatory

symptoms, including involuntary muscle contractions and tremors [30]. It was
subsequently shown that spinosyns produce neuronal excitation caused by an
interaction with a target site within the insect central nervous system [30, 31].
Spinosyn A-induced symptoms were unique from those produced by other insecti-
cides, and from this it was hypothesized that the mode of action was likely unique
among the known insecticides.
A number of insecticidal target sites exist, the disruption of which could
produce the excitatory symptoms of the spinosyns. Early studies on cockroach
(Periplaneta americana) neurons demonstrated that spinosyn A induced an
α-bungarotoxin-sensitive current and also prolonged acetylcholine (ACh)-induced
currents [32]. This finding indicated that spinosyns interact with insect nicotinic
acetylcholine receptors (nAChRs). Further studies demonstrated that spinosyn A
interacts with a nondesensitizing subtype of nAChR in cockroach neurons, and it
is likely that spinosyns generate inward currents through this receptor subtype
[33]. Studies using established radioligand-binding assays failed to demonstrate
a meaningful interaction with known insecticidal target sites, including those
for the neonicotinoid-selective nAChRs, γ -aminobutyric acid (GABA) receptors,
and avermectins [34]. Thus, it was hypothesized that the insecticidal actions of
the spinosyns must occur through a novel target site, or a novel target site of a
known insect neurotransmitter receptor, and that this receptor was likely a novel
nAChR.
By using chemical mutagenesis it was shown subsequently that, for the fruit
fly (Drosophila melanogaster), a null mutation of the Dα6 nAChR subunit leads
to high levels of resistance to spinosyns, spinosad, and spinetoram [35]. In a
similar study, a Dα6 knockout strain of D. melanogaster was also demonstrated
to be highly resistant to spinosad [36]. Both studies supported a role for the Dα6
nAChR subunit in the toxicity of the spinosyns in D. melanogaster, and suggested
that an interaction by spinosyns with Dα6-like subunits might underlie their
insecticidal actions. Interestingly, for the diamond-back moth (Plutella xylostella),
field-generated spinosad resistance appears to be due to mutation of a Dα6
homolog, Pxα6, that results in the expression of truncated – and presumably
unusable – nAChR protein subunits [37]. These data suggest that Dα6 nAChR
homologs could represent important insecticidal target sites for the spinosyns in
several insect species.
In Xenopus laevis oocytes, coexpression of the Dα6 subunit with another nAChR
subunit, Dα5, resulted in a nAChR with pharmacological properties consistent
with those from native nAChRs [35]. The Dα6/Dα5 nAChR expressed in oocytes
was activated by ACh, and also by spinosyn A and spinetoram, but was insen-
sitive to the neonicotinoid insecticide imidacloprid. These data suggest that the
Dα6/Dα5 heteromeric nAChR is distinguishable from the nAChR activated by
the neonicotinoid insecticides. Further, spinosyn A does not directly activate the
neonicotinoid-sensitive D. melanogaster Dα2 nAChR or the chicken α7 nAChR

(G. Watson, unpublished observations); this, in turn, indicates that the spinosyns
are highly selective for specific nAChR subtypes. Further, it is possible that the
spinosyns are selective for subtypes of insect nAChRs only, and this could, in part,
underlie the species selectivity of the spinosyn insecticide class.
There is evidence that there may be other target sites for spinosyns in some
susceptible species. For example, although spinosad resistance in housefly (Musca
domestica), appears to be target-site based, existing evidence suggests that it does
not involve homologs of Dα6, nor any other nAChR [38]. Findings such as this,
together with a previously described interaction of insecticidal spinosyns with
certain types of insect γ -aminobutyric acid (GABA) receptors [39], provide evidence
that other – perhaps secondary – target sites for the spinosyns exist in some insect
species.
32.4.4
Spinosyn Analogs
32.4.4.1 Core-Modified Analogs (Aglycone)

As with most complex natural products, the development of a systematic
structure–activity relationship (SAR) for spinosyns is impractical. Potential
modifications are limited to existing reactive sites, and specific modifications
of the spinosyns are limited by their susceptibility to acidic hydrolysis (loss of
forosamine), basic hydrolysis (loss of forosamine and/or dehydration at C17),
photolysis [40], and oxidation (formation of N-oxides and N-demethylation).
Despite these limitations, a considerable amount of structural modification has
been accomplished [3, 4]. As with many other complex natural products, most of
the modification efforts have resulted in a decrease or complete elimination of
biological activity. For example, the loss of either or both sugar units, or disruption
of the enone unit, results in analogs that retain <10% of the activity of the original
natural product [41]. Similarly, the removal of one or more methyl groups from the
rhamnose sugar is highly detrimental to biological activity [3]. As a general rule,
biological activity is closely related to the lipophilicity of the analog; modifications
that increase the overall lipophilicity tend to improve biological activity, and the
reverse holds for polar modifications.
A readily exploitable synthetic handle in spinosyn A is the isolated double
bond at C5=C6. Electrophilic attack on the isolated double bond of spinosyn
A proceeds with high π-diastereofacial selectivity (Scheme 32.4.1) [42]. Despite
greater steric hindrance to electrophilic attack from the concave β face, epoxida-
tion was found to favor the β epoxide (5) by a 5 : 1 ratio, an effect attributed to
torsional steering. Similarly, oxymercuration/reduction resulted in a 5α-hydroxy
derivative (6) with >30 : 1 selectivity. Hydrogenation using homogeneous cata-
lysts (e.g., Wilkinson’s catalyst) results in selective reduction of the 5,6-double
bond. Although heterogeneous catalysts such as Pd/C do show selectivity towards
mono-reduction of the 5,6-double bond (7), careful monitoring of hydrogen uptake
is required to avoid also an over-reduction of the C13–C14 bond. Analogs with a
MCPBA, CH2Cl2
O
O 5
N O O
O 1. Hg(CF3CO2)2
O
O 2. NaBH4
O O
HO
6
O
O H2, Cat
1
Scheme 32.4.1 Modifications to the C5–C6 double bond of spinosyn A.
O
N O R3′
O
O
O
O O
R21 O
O
Figure 32.4.2 C21-modified spinosyn analogs.
reduced 5,6-double bond show marginally improved biological as well as residual

activity [4].
Among the spinosyn factors isolated from S. spinosa, only two different substitu-
tions at C21 have been found. With the exception of spinosyn E (8; Figure 32.4.2),
which has a methyl group at C21, all other factors are substituted with an ethyl
group. This minor structural variation is critical to the biological activity, since
spinosyn E retains only about 10% of the activity of the corresponding ethyl
derivative [3]. The lack of an appropriate synthetic handle had prevented fur-
ther exploration of larger alkyl groups at C21. However, the recent discovery
of a new spinosyn-producing organism, Saccharopolyspora pogona, now allows
more extensive exploration of this and other parts of the molecule [43–45]. The
most prevalent side chain produced by S. pogona is a trans-2-butenyl group (9;
Table 32.4.1), although other unsaturated and hydroxylated side chains (10–13) are
also produced.
An olefin metathesis route was used to further expand the C-21 substituent set
[46]. Despite being significantly larger – and, in some cases, more polar – their
biological activity is similar to that of the corresponding natural derivatives,
demonstrating the flexibility of this portion of the molecule in accepting a broad
range of substitutions with little loss of potency (Table 32.4.1).
Table 32.4.1 Spinosyns modified at C-21.
Compound R21 R3 Source H. virescens H. virescens

LC50 a topicalb
1 –C2 H5 CH3 S. spinosa 0.31 0.03

8 –CH3 CH3 S. spinosa 4.6 –
9 trans–C2 H5 CH=CH– CH3 S. pogona 0.29 –
10 trans–CH3 CH=CH– CH3 S. pogona – –
11 trans–H2 C=CH–CH=CH– CH3 S. pogona – –
12 trans–CH3 CH(OH)CH=CH– CH3 S. pogona – –
13 trans–CH2 =CHCH=CH CH3 S. pogona – –
14 trans–CH3 CH(OH)CH=CH– OH S. pogona – –
15 CH2 =CH– C2 H5 S. pogonac 0.2 –
16 trans–C4 H9 CH=CH– C2 H5 S. pogonac – –
17 trans–C6 H13 CH=CH– C2 H5 S. pogonac – –
18 trans–C6 H5 –CH=CH– C2 H5 S. pogonac – –
19 trans–MeO2 C–CH=CH– C2 H5 S. pogonac 0.2 –
20 –n–C3 H7 CH3 S. spinosad 0.16 0.02
21 –i–C3 H7 CH3 S. spinosad – 0.025
22 CH3 CH(OH)– CH3 Streptomyces – –
(from
spinosyn A)
a
LC50 of neonate H. virescens larvae (ppm).
b
LC50 of neonate H. virescens topical assay (μg per insect).
c
Semi-synthetic analog generated from 14 via olefin metathesis and ethylation of C3 − OH.
d
Analogs were generated from a bioengineered strain of S. spinosa (for details, see text).
See Figure 32.4.2.
Other novel spinosyns have been created by the genetic engineering of spinosyn
biosynthetic genes. The loading module from the avermectin polyketide synthase
(PKS) gene cluster was introduced into S. spinosa to form a hybrid PKS gene
cluster that initiated polyketides with branched-chain carboxylic acids, yielding
spinosyns with a broad range of alkyl groups at C21 [47]. This engineered PKS
could also initiate chains from fed cyclic carboxylic acids to produce C21-cyclobutyl
and C21-cyclopropyl spinosyns.
The creation of a synthetic handle on the C21 side chain through microbial
oxidation has been reported [48]. The transformation of spinosyn A or its aglycone
using a Streptomyces strain results in selective oxidation at C22, to generate 22
(Table 32.4.1). This functional group could then be used, in principle, to prepare
various modifications, although no further analogs derived from this compound
have been described.
Other novel core-modified factors have also been isolated from S. pogona fer-
mentation broths, including analogs containing an expanded 14-membered lactone
ring (23) and a hydroxyl group at C8 (24) (Figure 32.4.3).
O O
N
O O N O O
O O
O O
O O
O O O
O
O O OH
O O
23 24
Figure 32.4.3 Novel core-modified spinosyn analogs iso-

lated from Saccharopolyspora pogona.
N
N
1 Me2N S Me2N O
O O
1 N H2SO4
80 °C
Ag(OTf)2, CH2Cl2 a-D-forosamine

O b-D-forosamine (spinosyn A)
HO O (a:b ratio 3:2)
O
O O
O O O
CCl3CH2O Me2N
N
O
O O
O
a-L-ossamine
25 1. PPTS b-L-ossamine (spinosyn G)
2. Zn, AcOH (a:b ratio 1:2)
3. MeI
HO
HO O
O
Mutational biosynthesis
using bioengineered s. erythrea a-L mycarosyl
HO
HO O
O
a-L olivosyl
Scheme 32.4.2 Synthesis of spinosyns containing modified C-17 sugars.
32.4.4.2 Modifications Involving the C17 Sugar

Most natural spinosyns have an amino sugar (generally, β-d-forosamine) attached
at C17. Since forosamine is a 2-deoxy sugar, hydrolytic removal to form 24 can be
accomplished selectively under mild conditions [49] (Scheme 32.4.2). Glycosylation
to regenerate a β-linked sugar, however, is more problematic. The reattachment of
O
R O
O
O
O O
O
O
MeO OH
O
O HO O
O
O O
HO
26, R = 4"-O -methyl-b-D-oleandroside 27, R = 3"-O -methyl-b-D-glucopyranoside
OH
O
HO O
O O
N
28, R = -b-D-amicetoside 29, R = 2"-hydroxy-b-D-forosaminoside
Figure 32.4.4 Novel C17-saccharides isolated from Saccharopolyspora pogona.
forosamine, using a 2-mercaptopyrimidinyl activating group, is accomplished in

17% yield and favors the α sugar by a 3 : 2 ratio [50]. The N-protected dihydropyran
used in the synthesis of spinosyn G, on the other hand, was attached in 36% yield
(Scheme 32.4.2) [51]. In this case, the desired β anomer was favored by a 2 : 1
margin.
Novel sugar residues have also been incorporated at C17 through mutational
biosynthesis. When 16 was fed to a strain of Saccharopolyspora erythrea engineered
with the spnP gene, the product was found to have incorporated l-mycarose [52].
Spinosyns bearing novel sugars at C17 were also isolated from the
butenyl-producing S. pogona (Figure 32.4.4) [43, 45]. Besides d-forosamine,
other sugars include the neutral sugars 4 -O-methyl-β-d-oleandroside (26),
β-d-amicetoside (27), 3 -O-methyl-β-d-glucopyranoside (28), and 3 -hydroxy-β-d-
forosaminoside (29). The bioactivity and pest spectrum of these neutral sugar
analogs is similar to the bioactivity and spectrum of the amino spinosyns [45].
32.4.4.3 Modifications Involving the C9 Sugar: Rhamnose Derivatives

In addition to a traditional analysis of the SARs for spinosyns and synthetic analogs,
an application of neural network analysis to some of the SARs led to a prediction that
the modification of certain sites on the spinosyn scaffold could lead to analogs with
increased potency [53]. One such site was the 3 position on the per-O-methylated
rhamnose sugar. The availability of spinosyns lacking one or more of the O-methyl
groups from rhamnose (Figure 32.4.5) has allowed for a wide variety of synthetic
variation at this portion of the macrolide, and has also led to some of the most active
analogs. Spinosyns are generally quite base-labile, such that standard alkylating
Me2N R4′ Figure 32.4.5 Modified rhamnose

O R3′ analogs.
O
O
R2′
O O
O
O
conditions (Williamson ether synthesis using NaH or K2 CO3 ) led to an extensive

loss of forosamine. A nonaqueous, phase-transfer alkylation protocol involving
powdered KOH and a quaternary salt such as (n-Bu)4 NI in a halocarbon solvent
avoids these undesired reactions, and leads to high yields of the corresponding
alkylated derivatives 39–41 (Table 32.4.2) [4]. The corresponding acetate esters or
ketones (33–38) show generally weaker activity than the corresponding ethers [54].
Deoxygenation of the free OH groups of spinosyns H, J, or K also resulted in active
analogs; the 2 -deoxy analog 42 was more active than the parent spinosyn A [54].
Table 32.4.2 Structure and insecticidal activity of rhamnose-modified spinosyn analogs.
Compound Position Substituenta Precursor Reagent(s) H.

virescens
no. LC50 b
1 – – Natural factor 0.31

30 R2 –OH Natural factor 3.2
31 R3 –OH Natural factor >64
32 R4 –OH Natural factor 3.5
⎫
33 R2 =O ⎪
30⎬ NCS, (CH3 )2 S, Py 3.5
34 R3 =O 31 10
⎪
35 R4 =O 32⎭ –
⎫
36 R2 –OCOCH3 30⎪
⎬ Ac2 O, Py 1.2
37 R3 –OCOCH3 31 33
⎪
38 R4 –OCOCH3 32⎭ 1.3
⎫
39 R2 –OC2 H5 30⎪
⎬ C2 H5 I, KOH (powdered), Bu4 NI 0.11
40 R3 –OC2 H5 31 0.035
⎪
41 R4 –OC2 H5 32⎭ 0.24
⎫
42 R2 H 30⎪
⎬ 1. NaH, CS2 , MeI; 0.23
43 R3 H 31 2. Bu4 SnH, AIBN 0.36
⎪
44 R4 H 32⎭ 4.1
a
The two remaining rhamnose substituents groups are –OCH3 .
b
LC50 of neonate H. virescens larvae.
See Figure 32.4.5.
AIBN, azobis(isobutyryonitrile); NCS, N-chlorosuccinimide.
In general, the SAR of rhamnose analogs follows two rules: (i) more lipophilic
substituents are more active than their less lipophilic counterparts; and (ii) modi-
fications to the 3 position are more impactful than are modifications to either the
2 - or 4 -positions [4]. Whereas, the difference in activity between analogs bearing
the most polar (OH) and least polar substituents is approximately 10- to 30-fold for
2 and 4 -positions, the difference is almost 2000-fold at the 3 -position.
32.4.5
Spinetoram
Efforts to improve the efficacy of natural spinosyns through chemical modification

have led to the development of spinetoram, a new semi-synthetic analog based on
the improved activity of analogs modified at the 3 -position on the rhamnose sugar
(Table 32.4.2). Spinetoram (Figure 32.4.1) is derived from spinosyns J and L which,
like spinosad, differ by the presence of a hydrogen or methyl group at C6. These
factors are both O-ethylated at the 3 -position of the rhamnose, and the major factor
(spinosyn J) is further reduced to the 5,6-dihydro derivative.
One effect of these chemical modifications is that spinetoram is intrinsically
more active against pest insect species than spinosad. For example, in a laboratory
bioassay of topical toxicity to beet armyworm (Spodoptera exigua) larvae, spinetoram
was 48-fold more active than spinosad (Table 32.4.3). Similar results were observed
in a bioassay of ingestion activity, where spinetoram was 58-fold more active on
beet armyworm than spinosad. Improved potency of spinetoram has also been
observed against other lepidopterous insects (Table 32.4.3).
Another result of the chemical modifications is an improved residual control of
spinetoram compared to spinosad. In a simulated field comparison, spinetoram
showed significant enhancements in both residual control and potency against
codling moth, Cydia pomonella, larvae (Figure 32.4.6) [4]. Improved residual control
and potency were also evident in a field trial against fall armyworm, Spodoptera
frugiperda, infesting maize in Brazil (Figure 32.4.7).
The nontarget organism toxicological profile and environmental fate profile of
spinetoram are similar to those of spinosad, and are very favorable. Interestingly, al-
though spinetoram is more active against pest insect species, under field conditions
Table 32.4.3 Activity of spinosad and spinetoram against

lepidopterous larvae in laboratory bioassays.
Species Topical activity LD50 Ingestion activity LC50

(μg per larva) (mg l−1 on diet)
Spinetoram Spinosad Spinetoram Spinosad
Tobacco budworm (Heliothis virescens) 0.02 0.03 0.87 1.7

Beet armyworm (Spodoptera exigua) 0.013 0.63 0.1 5.8
Cabbage looper (Trichoplusia ni) 0.025 0.03 0.13 0.44
400
Spinosad SC*
LC50 (mg l−1)

300
Spinetoram SC*
200
100
0
Immediately after 14 days after
application application
Figure 32.4.6 Activity of spinetoram and spinosad against

codling moth (Cydia pomonella) larvae on treated apples in a
simulated field exposure study. *, indicates suspension con-
centrate (SC) formulation.
100
80
% Control
60
40
20
0
2 5 9 12
Days after application
Spinetoram 24 g Ha−1 Spinetoram 48 g Ha−1

−1
Spinosad 24 g Ha Spinosad 48 g Ha−1
Figure 32.4.7 Control of fall armyworm (Spodoptera

frugiperda) in maize with spinetoram and spinosad, each ap-
plied at 24 and 48 g a.i. ha−1 .
spinetoram has a minimal impact on most beneficial arthropod populations. This

has been demonstrated in a field trial in Mississippi, USA, in which the populations
of all beneficial insects and spiders in eggplant were measured (Figure 32.4.8).
Following application, changes in total numbers of beneficial arthropods in the
spinetoram-treated plots (and the spinosad-treated plots) paralleled the population
changes observed in the untreated plots. In contrast, the total number of beneficial
arthropods in plots treated with the pyrethroid insecticide λ-cyhalothrin decreased
immediately after application and slowly recovered over the next 14 days.
In field trials around the world, spinetoram has demonstrated excellent,
broad-spectrum control of significant pests attacking tree fruit, tree nut, vine, and
vegetable crops. It is particularly effective against lepidopterous larvae (including
Spodoptera spp., codling moth (C. pomonella), oriental fruit moth (Grapholita
molesta), and tortricid leafrollers), dipterous leafminers (Agromyzidae), thrips
(Thysanoptera), and psyllids (Psyllidae).
80%
Spinosad
60% Spinetoram
Percent change in total number of
Lambda-cyhalothrin
40% Untreated
beneficial insects
20%
0%
−20%
−40%
−60%
−80%
0 2 4 6 8 10 12 14
Figure 32.4.8 Effect of spinetoram SC (70 g a.i. ha−1 ),

spinosad SC (70 g a.i. ha−1 ), and λ-cyhalothrin emulsifiable
concentrate (EC) formulation (28 g a.i. ha−1 ) applications
on the total number of beneficial arthropods in eggplant.
The percentage change is measured relative to the popula-
tion levels before application (Day 0).
As of late 2010, spinetoram has been registered in 27 countries including the

USA, Canada, Mexico, Australia, Indonesia, and Korea; many additional regis-
trations are being pursued worldwide. The major insecticide products containing
® ®
spinetoram are Delegate WG (250 g a.i. kg−1 water-dispersible granule), Radiant
SC (120 g a.i. l−1 suspension concentrate; SC), and Exalt™ SC (60 g a.i. l−1 SC).
Spinetoram use is currently focused on the control of crop pests, and other pest
management uses are likely to be developed in the future.
32.4.6
Biosynthesis and Genetics of the Spinosyns
Synthesis of the spinosyns, like other macrolides, is initiated by stepwise condensa-

tion of acylated carboxylic acids to form a linear polyketide. Each step involves the
addition, by an acyltransferase (AT), of a malonyl or methylmalonyl residue to an
acyl carrier protein (ACP). The acyl group is then transferred to the growing polyke-
tide in a decarboxylative condensation catalyzed by a ketoacyl-ACP synthetase (KS).
Following these obligatory steps, there may be additional reactions that modify the
preceding residue. The keto group may be reduced to a hydroxyl by a ketoacyl-ACP
reductase (KR), changed by a KR and dehydratase (DH) to leave a double bond, or
completely reduced to a backbone of saturated carbons by a KR, a DH, and an enoyl
reductase (ER). The full-length polyketide is cyclized by the action of a thioesterase

(TE) to form a macrocyclic lactone. Each of these reactions is catalyzed by a unique
domain in a large complex of multifunctional polypeptides called a Type I polyketide
synthase. The carboxylic acid origin of spinosyns has been confirmed by incorpo-
ration studies with 13 C-labeled acetate and propionate [55]. The steps of polyketide
synthesis were deduced from DNA sequence homologies to characterized PKS
genes [56], and supported by the novel pentaketide structure produced from a
truncated PKS expressed heterologously [57]. Unlike most macrolides, spinosyn
polyketides are internally crosslinked by a process resembling an intramolecular
cycloaddition. The resulting tetracyclic aglycone is the first detectable biosynthetic
intermediate. The next intermediate is the pseudoaglycone (PSA), which contains
a tri-O-methylated rhamnose at C9. It is not known if the rhamnose is normally
methylated before or after its addition to the aglycone, but S. spinosa cells can
complete the methylation of fed PSAs lacking some rhamnose-O-methyl groups.
The final step in spinosyn biosynthesis is the addition of forosamine at the C17
position. This glycosylation only occurs after rhamnose addition; it does not oc-
cur on the C17 of the aglycone. The forosamine must be di-N-methylated before
incorporation because precursors lacking one or both of these methyl groups are
not modified when fed to S. spinosa cultures. Unlike the C-methyl groups on the
polyketide (which come from the carboxylic acid precursors), the sugar methyl
groups are all derived from S-adenosyl-methionine [55].
The spn genes that encode the spinosyn biosynthetic enzymes have been cloned
and sequenced. Most of these are clustered together in an 80 kb region of the
S. spinosa chromosome. One half of the cluster consists of the five large PKS
genes that code for the five polypeptides containing the 11 modules (one for each
carboxylic acid) which produce the macrocyclic lactone. Together, these constitute
a 2 MDa complex that catalyzes 50 different enzymatic reactions. The other half of
the cluster contains 14 genes encoding the enzymes involved in modification of
the polyketide, and in the synthesis and addition of the deoxysugars. As polyketide
crosslinking is rare, it was difficult to identify the genes whose products contribute to
this process. However, four candidates were tentatively assigned roles in aligning
the polyketide chain and in C–C bridge formation, based on limited sequence
homology to genes of related function. A more detailed model of the crosslinking
emerged from structural analysis of the novel monocyclic intermediate produced
by a truncated PKS [57]. The role of the spnJ product as a flavin-dependent C15
oxidase was confirmed using the purified protein expressed heterologously [58].
The five genes encoding the enzymes required to convert the common deoxysugar
precursor nucleotidyl diphosphate (NDP)-4-keto-6-deoxy-d-glucose into forosamine
were much easier to identify, because their sequences were strikingly similar to
the corresponding genes in other organisms. The role of three of these genes
in the forosamine pathway was confirmed by gene disruption leading to an
accumulation of PSA. The catalytic functions of all five gene products have since
been demonstrated biochemically using heterologously expressed pure proteins
[59]. Three O-methyltransferase genes and two glycosyltransferase genes were also
easy to recognize by sequence homology. Disruption of these genes had pleiotropic
effects that made it difficult to determine the specificity of the genes responsible
for rhamnose methylation. However, the heterologous expression and purification
of gene products elucidated the precise functions of the O-methyltransferases [60].
Precursor feeding provided clear evidence that rhamnose addition is encoded by
spnG, and forosamine addition by spnP. The four genes coding for the enzymes
that generate NDP-4-keto-6-deoxy-d-glucose and then convert it into rhamnose are
not present in the spn cluster. Rhamnose is an essential component of the cell
wall as well as spinosyn; hence, its unique biosynthetic genes cannot be located in
the region of chromosome containing the other spn genes because it is prone to
deletion [61, 62].
S. spinosa produces a family of closely related spinosyns that differ in the
methylation patterns at C6, C16, or C21 of the polyketide nucleus, or on the
sugars. These are either biosynthetic intermediates, or shunt products derived
from them that are generated by incomplete processing. They are generally present
at low levels in the wild-type strain. Spinosyn D is an exception, in that it is
produced to about 20% of the level of the major product, spinosyn A. Spinosyn D
is generated when a methylmalonyl-CoA is incorporated instead of malonyl-CoA
by the AT domain of PKS module 8, resulting in a methyl group at C6. Some
minor factors, such as spinosyn H (19), are accumulated to much higher levels in
mutant strains generated by treatment of cells with N-nitrosoguanidine. In these
strains, a biosynthetic function has been lost completely due to a point mutation
in one of the spn genes. Other strains derived in the same way accumulate
similar intermediates, such as spinosyn K (22), that were undetectable in the
wild-type [63]. The presence of a butenyl group in analogs isolated from S. pogona
is due to an extra module in the PKS that incorporates an additional carboxylic
acid into the polyketide. Another variant, the 14-membered ring lactone (14),
is presumably due to an altered cyclization pathway of the longer polyketide.
Other minor factors carry neutral sugars such as amicetose, O-methyl glucose,
or O-methyl oleandrose instead of forosamine at C17 (see Figure 32.4.4). These
probably reflect the presence of a glycosyltransferase with a broad specificity, and
sugar biosynthetic pathways that are not functional in S. spinosa. The unique
hydroxylated spinosyns 10 and 15, produced by S. pogona, could result from the
incorporation of hydroxylated precursors by the PKS or by the action of unique
cytochrome P450 mono-oxygenases [64].
Another strain of S. spinosa was engineered to replace the AT domain of module
3 with AT domains that preferentially incorporate ethyl malonyl-CoA. As this
precursor is not normally synthesized by S. spinosa, a crotonyl-CoA reductase gene
from Streptomyces cinnamonensis was introduced at the same time. The engineered
strains produced C21-n-propyl and C6-ethyl spinosyns (rather than the targeted
C16-ethyl spinosyn), presumably due to an incorporation of ethyl malonyl-CoA
by the native loading module or module 8, respectively [47] [65, 66]. Spinosyns
containing a novel sugar residue (l-mycarose or l-olivose) at C17 (Scheme 32.4.2)
were generated by a strain of Saccharopolyspora erythraea engineered with the
spnP gene and fed PSA. Clearly, the glycosyltransferase product of this gene has
the ability to incorporate sugars other than forosamine [52, 67]. Similar substrate
promiscuity was demonstrated for the purified rhamnosyltransferase product of

the spnG gene in vitro [68].
32.4.7
Metabolism and Penetration of the Spinosyns
Spinosyn A, as a representative of the spinosyns in general, has several sites that

could be targets for metabolism. A few potential metabolic degradation routes
are the N-demethylation of the forosamine, O-demethylation of one or more of
the methoxy groups on the rhamnose, epoxidation of the 5,6- or 13,14-double
bonds, opening of the macrocyclic lactone, hydroxylation of the tetracycle, or some
combinations thereof. Indeed, studies of spinosyn A and D metabolism in rats
showed that the parent molecules (spinosyn A and D) accounted for only a small
fraction of the material present at 24 h post treatment in fecal extracts [69]. Thus,
a substantial amount of metabolism had taken place, for both spinosyns A and
D, with loss of a methyl group from the forosamine nitrogen (N-demethylation)
and loss of a methyl group from the rhamnose (O-demethylation) representing the
primary pathways [3, 5]. These observations suggest that, in rats, the spinosyns are
readily metabolized. Likewise, studies of spinosyn metabolism in lactating goats
detected the presence of eight metabolites for spinosyn A and five for spinosyn
D. As observed in the rat metabolism studies, N-demethylation was an important
metabolic route, although hydroxylation of the macrolide ring was also noted
as important in this respect [70]. Like spinosad, the components of spinetoram
(Figure 32.4.1) also undergo metabolism in animal systems, leading to loss of a
methyl group from the forosamine nitrogen and O-dealkylation on rhamnose [5].
As noted for the mammalian studies, spinosyn metabolism studies in avian
systems (poultry) also identified N-demethylation of the forosamine nitrogen and
O-demethylation of the rhamnose as the primary metabolic pathways [71]. In these
studies, O-demethylation of the rhamnose at the 2 - and 4 -positions – yielding
spinosyns H and K, respectively – was favored over the 3 -position (spinosyn J
formation). An additional secondary metabolic pathway involves a loss of the
forosamine sugar to form the C17-PSA [71]. As this study and the above-described
mammalian studies clearly show, the spinosyns A and D are readily metabolized,
with N-demethylation of the forsamine nitrogen as a predominant route in all three
species [3, 5]. N-Demethylation, coupled with O-demethylation of the rhamnose
and macrolide ring hydroxylation, are consistent with oxidative metabolism via
mono-oxygenases and/or the action of glutathione-S-transferases [3].
In contrast to mammalian and avian studies, available information from studies
with insects has suggested that the metabolism of spinosyns is limited in these
species. In studies of spinosyn A metabolism in the tobacco budworm (Heliothis
virescens), the only component to be detected (up to 24 h post treatment) in
homogenates of topically treated larvae was the parent, spinosyn A [72, 73]. In
contrast, the same larvae readily metabolized the acaricide fenazaquin [3, 72],
showing a clear capacity to metabolize xenobiotics. Studies with H. virescens larvae
that were highly resistant to spinosad [74] also failed to identify any evidence
for the metabolism of spinosyn A [75]. Metabolism studies with beet armyworm
(Spodoptera exigua) larvae showed only modest levels of metabolism, with no
differences between spinosad and spinetoram. This observation suggests that the
improved activity of spinetoram compared to spinosad against S. exigua larvae is
possibly associated with an enhanced activity at the target site. In a like manner,
the 3 -O-ethyl analog of spinosyn J was found to be more potent at the nicotinic
receptor than spinosyn A [3].
The apparent lack of spinosyn A sensitivity to metabolic processes in insect pests
was further supported by studies that highlighted a general lack of cross-resistance
to spinosad in various insecticide-resistant strains, many of which involved an
enhanced metabolism [3]. Likewise, synergist studies with house flies, using the
mono-oxygenase inhibitor piperonyl butoxide (PBO), demonstrated the ability of
PBO to synergize the activity of permethrin (a pyrethroid insecticide), but not
that of spinosyn A [76]. Thus, within the limits of the available data, insect pests
appear to have a limited capacity to metabolize spinosyns such as spinosyn A.
The lack of spinosyn metabolism in insects may be a reflection, in part, of the
substrate specificity of enzyme systems, as well as the very high molecular weight
and unusually complex structure of the spinosyns.
The spinosyns are more active than most organophosphate and carbamate
insecticides, and as active as many pyrethroids [3, 77, 78]. Although, compared to
many other insecticides, the spinosyns are relatively slow to penetrate the insect
cuticle [3, 72, 73], this apparent drawback is partly offset by their limited metabolism
by insects. Indeed, the balance between low penetration and lack of metabolism
might be a key factor in the high efficacy of the spinosyns, as any compound that
does penetrate will remain intact to exert a long-term pesticidal effect.
32.4.8
Summary
The spinosyns, as a class of fermentation-derived compounds, are unique in the

field of natural product chemistry. Following the discovery, more than two decades
ago, of Saccharopolyspora spinosa and the secondary metabolites that it produces,
subsequent investigations have led to the development of two extremely useful and
effective insecticidal compounds, spinosad and spinetoram, both of which have
received the Presidential Green Chemistry Challenge Award in recognition of the
environmental and human health benefits that each provides. Indeed, spinosad
continues to have a major impact on insect pest management, being used to control
insect pests on more than 250 crops in more than 80 countries. In addition, it is
currently used (or is being developed) to promote animal health, human health,
vector control, and stored grain protection. The chemical novelty of the spinosyns is
reflected in their unique insecticidal mode of action, as agonists of the α6 subunit
of insect nAChRs.
Following the discovery of spinosad, an extensive discovery program led to the
development of an array of synthetic modifications to the spinosyn chemistry
(as outlined in this chapter), the major result of which was spinetoram. This
compound demonstrated a greater intrinsic activity and greater photostability than

spinosad, and consequently displayed not only a broader range of insect pest
management uses than spinosad but also a favorable environmental fate and
similar nontarget organism effects. More recently, the continuing exploration of
the spinosyns has incorporated genetic techniques that provide access to areas of
the spinosyn structure (e.g., C21 position, some sugars) which, previously, were
difficult to address by strictly synthetic means. Clearly, given the unique chemical
and biological attributes of the spinosyns, these novel natural products will continue
to be an area for scientific investigation.
References
1. Mertz, F.P. and Yao, R.C. (1990) Int. J. 10. Stark, J.D., Vargas, R., and Miller,
Syst. Bacteriol., 40, 34–39. N. (2004) J. Econ. Entomol., 97 (3),
2. Cleveland, C.B., Mayes, M.A., and Cryer, 911–915.
S.A. (2002) Pest Manage. Sci., 58 (1), 11. Prokopy, R.J., Miller, N.W., Pinero, J.C.,
70–84. Barry, J.D., Tran, L.C., Oride, L., and
3. Salgado, V.L. and Sparks, T.C. (2005) Vargas, R.I. (2003) J. Econ. Entomol., 96
in Comprehensive Insect Molecular Sci- (5), 1485–1493.
ence, Control, vol. 6 (eds L.I. Gilbert, K. 12. Vargas, R.I., Pinero, J.C., Mau, R.F.L.,
Stark, J.D., Hertlein, M., Mafra-Neto,
Iatrou, and S. Gill), Elsevier, Oxford, pp.
A., Coler, R., and Getchell, A. (2009)
137–173.
Entomol. Exp. Appl., 131 (3), 286–293.
4. Crouse, G.D., Sparks, T.C., Schoonover,
13. Van Leeuwan, T., van de Veire, M., and
J., Gifford, J., Dripps, J., Bruce, T., Tirry, L. (2005) Exp. Appl. Acarol., 37
Larson, L.L., Garlich, J., Hatton, C., Hill, (1-2), 93–105.
R.L., Worden, T.V., and Martynow, J.G. 14. Ester, A., de Putter, H., and van Bilsen,
(2001) Pest Manage. Sci., 57, 177–185. J.G.P.M. (2002) Crop Prot., 22, 761–768.
5. Dripps, J.E., Boucher, R.E., Chloridis, 15. Sarfraz, M., Dosoll, L.M., and Keddie,
A., Cleveland, C.B., DeAmicis, C.V., B.A. (2005) Outlook Pest Manage., 16 (2),
Gomez, L.E., Paroonagian, D.L., 78–84.
Pavan, L.A., Sparks, T.C., and Watson, 16. Fang, L., Subramanyam, B.H., and
G.B. (2011) in Green Trends in In- Arthur, F.H. (2002) J. Econ. Entomol., 95,
sect Control (eds Ó. López and J.G. 640–650.
Fernández-Bolaños), RSC Publishing, 17. Fang, L., Subramanyam, B.H., and
pp. 162–212. Dolder, S. (2002) J. Econ. Entomol., 95,
6. Huang, K.-X., Xia, L., Zhang, Y., Ding, 1102–1109.
X., and Zahn, J.A. (2009) Appl. Microbiol. 18. Daglish, G.J. and Nayak, M.K. (2005)
Biotechnol., 82, 13–23. Int. Pest Control, 47 (3), 130–132.
19. Blanc, M.P., Panighini, C., Gadani, F.,
7. Kirst, H.A. (2010) J. Antibiot., 63,
and Rossi, L. (2004) Pest Manage. Sci.,
101–111.
60 (11), 1091–1098.
8. Moreno, D.S. and Mangan, R.L. (2002)
20. Darriet, F., Duchon, S., Hougard, J.M.,
in Invasive Arthropods in Agriculture (eds and Montpellier, F. (2005) J. Am. Mosq.
G. Hallman and C.P. Schwalbe), Sci- Control Assoc., 21 (4), 495–496.
ence Publishers Inc., Enfield, NH, pp. 21. Huseyin, C., Yanikoglu, A., and Cilek,
333–362. J.E. (2005) J. Vector Ecol., 30 (1),
9. Vargas, R.I., Peck, S.L., McQuate, 151–154.
G.T., Jackson, C.G., Stark, J.D., and 22. Hertlien, M.B., Mavrotas, C.,
Armstrong, J.W. (2001) J. Econ. Ento- Jousseaume, C., Lysandrou, M.,
mol., 94 (4), 817–825. Thompson, G.D., Jany, W., and Ritchie,
References 1255
S.A. (2010) J. Am. Mosq. Control Assoc., 37. Baxter, S.W., Chen, M., Dawson,
26 (1), 67–87. A., Zhao, J.Z., Vogel, H., Shelton,
23. de Deken, R., Speybroeck, N., Gillain, A.M., Heckel, D.G., and Jiggins, C.D.
G., Sigue, H., Batawi, K., and van den (2010) PLoS Genet., 6, e1000802. doi:
Bossche, P. (2004) J. Med. Entomol., 41 10.1371/journal.pgen.1000802.
(5), 814–818. 38. Scott, J.G. (2008) J. Pestic. Sci., 33,
24. Kirst, H.A., Creemer, L.C., Naylor, S.A., 221–227.
Pugh, P.T., Snyder, D.E., Winkle, J.R., 39. Watson, G.B. (2001) Pestic. Biochem.
Lowe, L.B., Rothwell, J.T., Sparks, T.C., Physiol., 71, 20–28.
and Worden, T.V. (2002) Curr. Top. Med. 40. Berard, D.F. and Graper, L.K. (1996)
Chem., 2 (7), 675–699. Book of Abstracts, 211th ACS National
25. Mertens, C., Dohrmann, H., and Rshaid, Meeting, New Orleans, LA, American
G.A.M. (2005) PCT International Appli- Chemical Society, Washington, DC.
cation WO 2005/041,950. 41. Crouse, G.D. and Sparks, T.C. (1998)
26. Snyder, D.E. (2001) PCT International Rev. Toxicol., 2, 133–146.
Application WO 2001/011,962. 42. Graupner, P.R., DeAmicis, C.V.,
27. Robertson-Plouch, C., Baker, K.A., Erickson, J.A., Paschal, N.W., Kirst,
Hozak, R., Zimmerman, A.G., Parks, H.A., Creemer, L.C., and Fanwick, P.E.
S.C., Herr, C., Hart, L.M., Jay, J., (2001) J. Org. Chem., 66, 8431–8435.
Hutchens, D.E., and Snyder, D.E. (2008) 43. Lewer, P., Hahn, D.R., Karr, L.L.,
Vet. Ther., 9 (1), 26–36.
Graupner, P.R., Gilbert, J.R., Worden,
28. Janssen, H., Ho, K., Nystrand, G.,
T.V., Yao, R.C., and Norton, D.W. (2003)
Williams, D., and Lamb, S.C. (2002) Eu-
PCT International Application WO
ropean Patent Application EP 1,252,820.
2001/019,840.
29. Mayes, M.A., Thompson, G.D.,
44. Hahn, D.R., Gustafson, G., Waldron, C.,
Husband, B., and Miles, M.M. (2003)
Bullard, B., Jackson, J.D., and Mitchell,
Rev. Environ. Contam. Toxicol., 179,
J. (2006) J. Ind. Microbiol. Biotechnol., 33
37–71.
(2), 94–104.
30. Salgado, V.L. (1998) Pestic. Biochem.
45. Lewer, P., Hahn, D.R., Karr, L.L.,
Physiol., 60 (2), 91–102.
Dubelbeis, D.O., Gilbert, J.R., Crouse,
31. Salgado, V.L., Sheets, J.J., Watson, G.B.,
G.D., Worden, T., Sparks, T.C.,
and Schmidt, A.L. (1998) Pestic. Biochem.
Physiol., 60 (2), 103–110. McKamey, P., Edwards, R., and
32. Salgado, V.L., Watson, G.B., and Sheets, Graupner, P.R. (2009) Bioorg. Med.
J.J. (1997) Proceedings of the 1997 Chem., 17, 4185–4196.
Beltwide Cotton Production Conference, 46. Daeuble, J., Sparks, T.C., Johnson, P.,
National Cotton Council, Memphis, TN, and Graupner, P.R. (2009) Bioorg. Med.
pp. 1082–1086. Chem., 17, 4197–4205.
33. Salgado, V.L. and Saar, R. (2004) J. 47. Sheehan, L.S., Lill, R.E., Wilkinson,
Insect Physiol., 50, 867–879. B., Sheridan, R.M., Vousden, W.A.,
34. Orr, N., Shaffner, A.J., Richey, K., and Kaja, A.L., Crouse, G.D., Gifford, J.,
Crouse, G.D. (2009) Pestic. Biochem. Graupner, P.R., Karr, L., Lewer, P.,
Physiol., 95, 1–5. Sparks, T.C., Leadlay, P.F., Waldron, C.,
35. Watson, G.B., Chouinard, S.W., Cook, and Martin, C.J. (2006) J. Nat. Prod., 69,
K.R., Geng, C., Gifford, J.M., Gustafson, 1702–1710.
G.D., Hasler, J.M., Larrinua, I.M., 48. Eberz, G., Mohrle, V., Frode, R., Velten,
Letherer, T.J., Mitchell, J.C., Pak, W.L., R., and Jeschke, P. (2003) Ger. Offen.
Salgado, V.L., Sparks, T.C., and Stillwell, Application DE 10,135,550.
G.E. (2010) Insect Biochem. Mol. Biol., 49. Creemer, L.C., Kirst, H.A., and Paschal,
40, 376–384. J.W. (1998) J. Antibiot., 51 (8), 795–800.
36. Perry, T., McKenzie, J.A., and 50. Paquette, L.A., Collado, I., and Purdie,
Batterham, P. (2007) Insect Biochem. M. (1998) J. Am. Chem. Soc., 120 (11),
Mol. Biol., 37, 184–188. 2553–2562.
51. Graupner, P.R., Martynow, J., and 64. Hahn, D.R., Gustafson, G., Waldron, C.,
Anzeveno, P.B. (2004) J. Org. Chem., 70 Bullard, B., Jackson, J.D., and Mitchell,
(6), 2154–2160. J. (2006) J. Ind. Microbiol. Biotechnol., 33,
52. Gaisser, S., Martin, C.J., Wilkinson, B., 94–104.
Sheridan, R.M., Lill, R.E., Weston, A.J., 65. Burns, L.S., Graupner, P.R., Lewer, P.,
Ready, S.J., Waldron, C., Crouse, G.D., Martin, C.J., Vousden, W.A., Waldron,
Leadlay, P.F., and Staunton, J. (2002) C., and Wilkinsson, B. (2003) PCT Inter-
Chem. Commun., 618–619. national Application WO 2003/070,908.
53. Sparks, T.C., Crouse, G.D., Dripps, J.E., 66. Martin, C. (2003) Proceedings of the
Anzeveno, P., Martynow, J., DeAmicis, 13th International Symposium on the
C.V., and Gifford, J. (2008) J. Comput. Biology of Actinomycetes, Melbourne,
Aided Mol. Des., 22 (6-7), 393–401. Australia.
54. Creemer, L.C., Kirst, H.A., Paschal, J.W., 67. Gaisser, S., Carletti, I., Schell, U.,
and Worden, T.V. (2000) J. Antibiot., 53, Graupner, P.R., Sparks, T.C., Martin,
171–178. C.J., and Wilkinson, B. (2009) Org.
55. Kirst, H.A., Michel, K.H., Mynderse, Biomol. Chem., 7, 1705–1708.
J.S., Chio, E.H., Yao, R.C., Nakatsuka, 68. Chen, Y.L., Chen, Y.H., Lin, Y.C., Tsai,
W.M., Boeck, L., Occolowitz, J.L., and K.C., and Chiu, H.T. (2009) J. Biol.
Pascal, J.W. (1992) in Synthesis of Agro- Chem., 284, 7352–7363.
chemicals III (eds D.R. Baker, J.G. 69. Domoradzki, J.Y., Stewart, H.S.,
Feynes, and J.J. Steffens), American Mendrala, A.L., Gilbert, J.R., and
Markham, D.A. (1996) Society of Toxi-
Chemical Society, Washington, DC, pp.
cology Meeting, Anaheim, CA, March
214–255.
11, 1996.
56. Waldron, C., Matsushima, P., Rosteck,
70. Rainey, D.P., O’Neill, J.D., and Castetter,
P.R. Jr, Broughton, M.C., Turner, J.,
S.A. (1996) Book of Abstracts, American
Madduri, K., Crawford, K.P., Merlo, D.J.,
Chemical Society Meeting, New Orleans,
and Baltz, R.H. (2001) Chem. Biol., 8,
LA, March 24–28, 1996.
487–499.
71. Magnussen, J.D., Castetter, S.A., and
57. Martin, C.J., Timoney, M.C., Sheridan,
Rainey, D.P. (1996) American Chemi-
R.M., Kendrew, S.G., Wilkinson, B.,
cal Society Meeting, New Orleans, LA,
Staunton, J., and Leadlay, P.F. (2003)
March 24-28, 1996.
Org. Biomol. Chem., 1, 4414–4417.
72. Sparks, T.C., Sheets, J.J., Skomp, J.R.,
58. Kim, H.J., Pongdee, R., Wu, Q.Q., Worden, T.V., Hertlein, M.B., Larson,
Hong, L., and Liu, H.W. (2007) J. Am. L.L., Bellows, D., Thibault, S., and
Chem. Soc., 129, 14582–14584. Wally, L. (1997) Proceedings of the 1997
59. Hong, L., Zhao, Z.B., Melancon, C.E., Beltwide Cotton Production Conference,
Zhang, H., and Liu, W.H. (2008) J. Am. National Cotton Council, Memphis, TN,
Chem. Soc., 130, 4954–4967. pp. 1259–1265.
60. Kim, H.J., White-Phillip, J.A., 73. Sparks, T.C., Crouse, G.D., and
Ogasawara, Y., Shin, N., Isiorho, E.A., Durst, G. (2001) Pest Manage. Sci.,
and Liu, H.W. (2010) J. Am. Chem. Soc., 57, 896–905.
132, 2901–2903. 74. Bailey, W.D., Young, H.P., and Roe,
61. Matsushima, P., Broughton, M.C., R.M. (1999) Proceedings of the 1999
Turner, J.R., and Baltz, R.H. (1994) Beltwide Cotton Production Conference,
Gene, 146, 39–45. National Cotton Council, Memphis, TN,
62. Madduri, K., Waldron, C., and Merlo, pp. 1221–1224.
D.J. (2001) J. Bacteriol., 183, 5632–5638. 75. Roe, R.M., Young, H.P., Iwasa, T.,
63. Sparks, T.C., Thompson, G.D., Kirst, Wyss, C.F., Stumpf, C.F., Sparks,
H.A., Hertlein, M.B., Mynderse, J.S., T.C., Watson, G.B., Sheets, J.J., and
Turner, J.R., and Worden, T.V. (1999) in Thompson, G.D. (2010) Pestic. Biochem.
Biopesticides: Use and Delivery (eds F.R. Physiol., 96, 8–13.
Hall and J.J. Menn), Humana Press, 76. Sparks, T.C., Thompson, G.D., Larson,
Totowa, NJ, pp. 171–188. L.L., Kirst, H.A., Jantz, O.K., Worden,
T.V., Hertlein, M.B., and Busacca, J.D. J.W., Nakatsukasa, W.M., Paschal, J.W.,
(1995) Proceedings of the 1995 Beltwide and Worden, T.V. (1996) Proceedings
Cotton Production Conference, National of the 1996 Beltwide Cotton Production
Cotton Council, Memphis, TN, pp. Conference, National Cotton Council,
903–907.
Memphis, TN, pp. 692–696.
77. Sparks, T.C., Kirst, H.A., Mynderse, J.S.,
78. Sparks, T.C., Thompson, G.D., Kirst,
Thompson, G.D., Turner, J.R., Jantz,
O.K., Hertlein, M.B., Larson, L.L., Baker, H.A., Hertlein, M.B., Larson, L.L.,
P.J., Broughton, M.C., Busacca, J.D., Worden, T.V., and Thibault, S.T. (1998)
Creemer, L.C., Huber, M.L., Martin, J. Econ. Entomol., 91, 1277–1283.
32.5
Sodium Channel-Blocking Insecticides
32.5.1
Sodium Channel-Blocking Insecticides: Indoxacarb
Stephen F. McCann, Daniel Cordova, John T. Andaloro, and George P. Lahm
32.5.1.1 History and Discovery of the Sodium Channel Blockers

The pioneering studies of pyrazoline insecticides conducted by Kobus Wellinga and
Rudolph Mulder provided the first leads toward the sodium channel-blocking in-
secticides [1]. Details of these early compounds are summarized in Figure 32.5.1.1.
Pyrazoline PH 60-41 was found to have moderate activity on Lepidoptera,
Coleoptera, and Diptera, while the optimization of PH 60-41 produced 5-phenyl
derivatives with improved insecticidal activity across all three insect orders, with
PH I-9 perhaps the most active of the group [2].
Further modification led to the discovery of the 4-aryl derivatives, such as
PH 60-42 (Figure 32.5.1.2), with excellent activity on a broad range of insects;
these compounds demonstrated increased activity over the 5-aryl analogs by
a factor of between 3 and 100 [3]. Modification of the substituent groups on
the pyrazoline, 3-aryl, 4-aryl, and carbamoyl rings provided further information
CI
O N O
N
N N
NH CI NH CI
Cl CF3
PH 60-41 PH I-9
Phillips-Duphar Phillips-Duphar
1972 1972
Figure 32.5.1.1 Pyrazolines: the first insecticides in the field of sodium channel blockers.
Me
CO2Me
O O
N N
N N
NH CI NH CI
CI F3C
PH 60-42 RH 3421
Phillips-Duphar Rohm & Haas
1974 1984
Figure 32.5.1.2 Optimized versions of early pyrazoline sodium channel blockers.
pertaining to structure–activity relationships [4]. Whilst compounds containing

a 4-trifluoromethoxyaniline substituent (which is perhaps the most active to be
identified) were first reported by the research group at Bayer [5], several significant
issues associated with the early pyrazolines most likely contributed to their lack of
commercialization. These problems included a perceived lack of photostability [6],
environmental persistence [7], and difficulties associated with long-term toxicity
and bioaccumulation [8]. Subsequently, the 4-methyl-4-carbomethoxypyrazolines
discovered at Rohm and Haas provided good progress towards a solution to these
problems through the prevention of photoaromatization and inducing a significant
reduction in soil half-life [9].
Modifications to the pyrazoline nucleus were first discovered at DuPont, and
provided information relevant to the orientation and spatial relationships of sodium
channel blockers (Figure 32.5.1.3). Carboxamides such as 1 provided new insight
into the arrangement of atoms in the pyrazoline ring [10]. For the first time, these
‘‘inverse’’ pyrazolines demonstrated that the orientation of substituent groups
around the ring was in fact the critical component for activity, and was not specific
to the known N-carboxamide-3-aryl pyrazolines. Conformationally constrained
pyrazolines, including indazoles such as 2 and 3, provided structural insights
into the spatial relationships of the aryl rings, carboxamide group, and pyrazoline
nucleus, and suggested that a planar arrangement of aryl rings was important [11].
CF3 CI CI
CI Me CO2Me Ph CO2Me
CF3 CF3
N
N N N N N
N N N
O H O H O H
1 2 3
Carboxamides Phenyl indazoles Carbomethoxy indazoles
Figure 32.5.1.3 Discovery of 3-carboxamide pyrazolines and indazole insecticides.

CI CO2Me
CI
Me OCF3
H OCF3
H
H N N
N N
N N
O O H
H
4 5
Phenyl semicarbazones Carbomethoxy semicarbazones
Figure 32.5.1.4 Semicarbazone sodium channel-blocking insecticides.
CI
CO2Me
CF3
N N
N
O H CO2Me CF3
CI
3
N N
N
CI CO2Me H
O
OCF3
H 6
N N
Pyridazines
N
O H
Figure 32.5.1.5 Discovery of pyridazine insecticides.
Indazoles such as 2 provided the basis for the design of semicarbazones [12] such
as 4 and 5, owing to the observation that the indanone-derived semicarbazones pro-
vided a good spatial match with the indazoles (Figure 32.5.1.4). These compounds
showed good insecticidal properties although, on balance, they were somewhat less
than the better pyrazolines. Interestingly, they departed significantly from the de-
veloped structure–activity profile with 2-aryl compounds. Compounds containing
a 2-phenyl group (e.g., 4) showed the best activity, while 2-carbomethoxy analogs
(e.g., 5) showed only weak activity.
Pyridazines [13] such as 6 were discovered from a combination of structural fea-
tures derived from indazoles and semicarbazones such as 3 and 5 (Figure 32.5.1.5).
The pyridazines were some of the most potent analogs evaluated at DuPont, with
activity in the laboratory being observed below 1 ppm, which was significantly
better than insecticide standards; compound 6 was, in fact, the most potent com-
pound evaluated, with excellent field performance on Lepidoptera. Although the
pyridazines lacked acceptable soil residual properties, this problem was solved by
the introduction of the oxygen found in oxadiazines, such as 7 (Figure 32.5.1.6).
OCF3 CO2Me OCF3

CO2Me
O
CI CI
N N N N
N N
H O H
O
6 7
Pyridazines Oxadiazines
Figure 32.5.1.6 Discovery of oxadiazine insecticides.
This newest class of sodium channel blockers led to indoxacarb [14], following an
extensive optimization of chemical and biological attributes within the class.
32.5.1.2 Discovery of Indoxacarb

The problems associated with a slow breakdown in soils that had been identified
for 6 were due, in part, to the chemical robustness of the tricyclic pyridazine ring
system. In an attempt to resolve this problem, it was envisaged that heteroatom
substitutions into the core pyridazine would lead to more potential sites for
chemical or metabolic breakdown. One possible target, the oxadiazine 7 [15], is a
cyclic O,N acetal that could potentially ring-open under acidic conditions, exposing
a product that could undergo further breakdown.
Subsequently, it was found that compound 7 would degrade rapidly in soil test
systems, displaying a soil half-life that ranged from approximately one to four
weeks. This observation was made along with the finding that a high insecticidal
activity was maintained in the new, environmentally labile oxadiazine ring-system.
Moreover, the oxadiazine ring was also easier to prepare than the analogous
pyridazine. The synthetic routes for pyridazines 6 and oxadiazines 7 are compared
in Schemes 32.5.1.1 and 32.5.1.2. Of particular note is the low-yielding step that
installs the ethylene bridge atoms of 10. The reaction of salts of 2-carbomethoxy
indanone anion 8 with 1,2-dibromoethane provided an undesirable 75 : 25 ratio
of O-alkylated versus C-alkylated products, 9 and 10, respectively. The separation
of 9 and 10 required tedious chromatography, and the low yield (<25%) of the
slower-eluting 10 was problematic. Pyridazine 6 was prepared by subsequent
treatment with hydrazine and aryl isocyanate.
The analogous ring-forming steps in the synthesis of 7 involves the oxidation [16]
of 8 to the hydroxy indanone 12, followed by formation of the hydrazone 13, capping
with an aryl isocyanate to form the semicarbazone [17] 14. Finally, ring-closure
with formaldehyde [18] or a formaldehyde equivalent provides the O,N-acetal 7.1)
All steps are high-yielding and do not require chromatography.
The structure activity relationships (SARs) for oxadiazines were found to be
consistent with those for pyrazolines, indazoles, and pyridazines. The wealth
1) Attempts to carry-out the analogous cy-

clization of hydrazone DP-23 were not
successful.
Br
CO2Me CO2Me CO2Me

CI CI
NaH CI
+
O O
Br Br O
Br
8 9 10
Undesired Desired
75% 25%
CO2Me OCF3
CO2Me
1. NH2NH2 CI CI
ArNCO
10
N N N N
2. Base N
H H
O
11 6
Scheme 32.5.1.1 Synthesis of pyridazine.
CO2Me CO2Me
MCPBA NH2NH2 Cl OH
Cl OH
8
O HOAc N NH
2
12 13
CO2Me CO2Me OCF3

Cl OH OCF3 O
ArNCO (CH2O)n Cl
13 N H
N
H+ (cat.)
N N
N N
CH3CN
O O H
H
14 7
Scheme 32.5.1.2 Synthesis of oxadiazine.
of data for pyrazoline-type insecticides allowed for the rapid preparation and
identification of highly active analogs. The selection of candidate DPX-JW062 was
based on a combination of observed high insecticidal efficacy, safety to nontarget
organisms (including predatory insects as well as fish, birds, and mammals), and
a rapid dissipation in the environment [19].
Separation of the DPX-JW062 enantiomers using chiral high-pressure
liquid chromatography and subsequent bioassay showed the (+)-enantiomer,
DPX-KN128, to be approximately twice as active as the racemic material, while

the (−)-enantiomer was inactive as an insecticide. This finding, which was
consistent with that reported for analogous pyrazolines [20], prompted an
investigation of chiral synthesis methods for the selective preparation of the
(+)-enantiomer DPX-KN128. A wide variety of reagents and conditions were
screened [14a,b] for the asymmetric hydroxylation of 8. Use of the alkaloid
cinchonine as a chiral basic catalyst, in combination with t-butyl-hydroperoxide as
the stoichiometric oxidant [21], provided optimum results, yielding 15 as a 75 : 25
mixture of enantiomers (50% e.e.). Compound 15 was then converted into the
enantiomerically enriched oxadiazine, DPX-MP062, consisting of a 75 : 25 mixture
of (+)- and (−)-enantiomers (Scheme 32.5.1.3). The analysis of a derivative by
X-ray diffraction showed the active (+)-enantiomer, DPX-KN128, to have the
(S)-absolute configuration. DPX-MP062 is currently sold as a 75 : 25 mixture of
enantiomers under the generic name, indoxacarb. The commercial synthesis of
indoxacarb is also shown in Scheme 32.5.1.3 [21].
Subsequently, a chiral zirconium-based catalyst was found for the conversion
of 8 to 15 in greater than 95% e.e.. This material can then be converted to the
O
CO2Me
NH2
CO2Me Ph O N CI OH
H
Cinchonine H
Cl OH N N
8
t -butyl- O O
hydroperoxide
O
15 Ph
75% (S )-enantiomer 16
25% (R )-enantiomer
CI O
N O
CO2Me O
F3CO CO2Me
(EtO)2CH2 Cl O
18 Cl O
N N
acid cat.
O H2, Pd/C N N
Ph base H
O
17 Unstable
generated in situ
CO2Me OCF3
O DPX-MP062 - indoxacarb
Cl 75:25 mixture of S:R enantiomers
(the (S )-isomer, DPX-KN128, is shown)
N N
N
O OMe
O
Scheme 32.5.1.3 Synthesis of indoxacarb.

(S)-enantiomer of KN128 in greater than 95% e.e., using the same synthesis as
shown in Scheme 32.5.1.3.
32.5.1.3 Insecticidal Activity and Properties of Indoxacarb

Indoxacarb is a very effective crop-protection product, with low toxicity to nontarget
organisms [22], and a short persistence in the environment. It was designated by the
U.S. Environmental Protection Agency (EPA) as a ‘‘reduced-risk’’ pesticide, which is
defined as having both health and environmental advantages over existing products.
The physico-chemical, toxicological, and environmental properties of indoxacarb
are listed in Table 32.5.1.1 [23]. Details of DuPont’s sodium channel-blocking
products, registered formulations, and the contents of the insecticidally active
enantiomers, are listed in Table 32.5.1.2.
Although indoxacarb is a broad-spectrum lepidopteran insecticide, it is also active
against other pests from several insect orders [24]. Insects controlled by indoxacarb
include most of the globally important lepidopteran pests, such as species of
Heliothis (bollworms), Spodoptera (armyworms), Trichoplusia (loopers), Plutella
(diamond-back moth), Ostrinia (borers), Lobesia (berry moths), Cnaphalocrocis
(leaf folders), Pandemis (leafrollers), Tuta (pinworms), and Agrotis (cutworms). In
addition, indoxacarb controls selected sucking insect pests, including leafhoppers,
fleahoppers, and plant bugs, as well as beetles, sawflies, leafminers, and apple
Table 32.5.1.1 Physico-chemical properties of indoxacarb.
Physical state (99% DPX-KN128) Solid, powder

Formula weight 527.84
Melting point (DPX-KN128) 88.1 ◦ C
Solubility (DPX-MP062) Water: 0.20 ppm
n-Heptane: 1.72 g l−1
1-Octanol: 14.5 g l−1
Methanol: 103 g l−1
Xylene: 117 g l−1
Acetonitrile: 139 g l−1
Ethyl acetate: 160 g l−1
Dichloromethane: > 250 g l−1
Acetone: > 250 g l−1
Dimethylformamide: > 250 g l−1
Partition coefficient Log KOW (DPX-KN128) 4.65
Vapor pressure (DPX-KN128) 9.8 × 10−9 Pa(20 ◦ C)
2.5 × 10−8 Pa(25 ◦ C)
Acute toxicity (DPX-MP062) Oral LC50 : 1730 mg kg−1 (rat male)
Oral LC50 : 268 mg kg−1 (rat female)
Dermal LD50 : > 5000 mg kg−1 (rat)
Inhalation LC50 (4 h): > 5.5 mg l−1
Dermal irritation: nonirritant
Eye irritation: moderate eye irritant
Ames test: negative
Table 32.5.1.2 Indoxacarb product summary.
Code KN128 (%) Formulation: products
®
JW062 50 SC: Tornado
® ® ® ® ®
MP062 75 SC, WDG: Avaunt , Steward , Rumo , Avatar , Ammate
® ®
KN128 100 EC: Steward , Avaunt
SC, suspension concentrate; WDG, water-dispersible granule.
maggot flies. Indoxacarb is also one of the most effective fire ant products for
home, golf-course, and public-property uses [25]. It is used in a bait matrix to
control numerous cockroach and ant species, with commercial activity observed on
termites, fleas, mosquitoes, flies, and silverfish.
The primary route of entry of indoxacarb into the target insects is through
ingestion, although it may also be absorbed through the cuticle. Although the
larval life stage is the major focus of control, indoxacarb is also a very effective
ovilarvicide (i.e., it kills developing larvae in the egg and prevents hatching) as
well as an adulticide. Indoxacarb causes very strong feeding inhibition, even at
sublethal rates; typically, insects exposed to a sublethal dose of indoxacarb eat much
less, develop more slowly, and pupate and emerge later than untreated larvae. The
inhibition of insect feeding occurs very rapidly, which results in a rapid crop
protection, although live insects may be observed for up to 24 h after application.
Typically, these insects are partially paralyzed, smaller, desiccated, and shrunken
with no defense against environmental perils [26]. Other affected behaviors include
reduced egg-laying, mating disruption, inability to molt, difficulty to emerge from
pupal case, inability to excavate soil for pupation, repellency, and uncoordinated F1
progeny. Unlike synthetic pyrethroids, high temperatures are positively correlated
with indoxacarb control, thus hastening the decline of pest populations.
Indoxacarb typically provides excellent crop protection for between 5 and 14
days, depending on the application rate and the crop. It is highly lipophilic and is
absorbed into the waxy cuticle of leaves; this contributes to indoxacarb’s residual
control, aids in translaminar activity, and helps to provide an excellent rainfastness.
The oil-based formulations can penetrate the leaf, resulting in the control of
various sucking insects. The dry formulation is also translaminar, but tank-mixing
an oil-based surfactant can increase the activity. The chemical stability of a pesticide
in a spray tank is primarily dependent on the temperature and pH of the spray mix.
Indoxacarb formulations exhibit an excellent tank stability under a wide pH range
(5–9). In addition, the spray tank temperatures have no effect on indoxacarb stability
over the range 12.5 to 32 ◦ C (35–115 ◦ F). Indoxacarb formulations have proven to be
compatible with tank mix partners when added to the tank in the correct sequence.
The application of indoxacarb, although typically by air and ground equipment,
can also be made through center-pivot or fixed-sprinkler irrigation systems. All
indoxacarb formulations are rainfast with excellent ultraviolet stability.
32.5.1.4 Indoxacarb: Mode of Action
32.5.1.4.1 Overview of Insect Voltage-Gated Sodium Channels Indoxacarb is the

most recent commercialized insecticide to target voltage-gated sodium channels
(VGSCs). These channels play a critical role in the intercellular transmission of
electrical impulses throughout the nervous system of vertebrates and invertebrates
alike. As with vertebrate channels, insect VGSCs exists in three basic states: (i) a
resting (closed) state where the channel is nonconductive; (ii) an activated (open)
state, in which an inward flow of Na+ occurs through the channel, depolarizing
the cell, and ultimately generating an action potential; and (iii) an inactivated state
(closed), in which the channel becomes nonconductive and is unable to achieve
activation (Figure 32.5.1.7). The return of inactivated channels to the resting state
is a voltage-dependent process, where cells remain in a refractory state until the
cell membrane becomes repolarized.
Various organisms such as spiders, scorpions, and carnivorous marine mollusks
have developed highly selective neurotoxins that paralyze their prey through action
on VGSCs (for reviews, see Refs [27–30]). Nine distinct VGSC binding sites have
been identified through the use of neurotoxins, synthetic insecticides, and local
anesthetics (Table 32.5.1.3). Pyrethroids and DDT bind to site 7, altering channel
activation through a shift in voltage dependence [31]; N-alkylamides, by contrast,
bind to site 2 (batrachatoxin- and veratridine-binding site), where they stimulate
persistent channel activation [32].
32.5.1.4.2 Proinsecticide Action of Indoxacarb Indoxacarb is a proinsecticide,

requiring bioactivation to confer potent insecticidal activity. In metabolism
studies where pest insects were treated with 14 C-DPX-JW062 (a 50 : 50 mixture
of the active and inactive enantiomers of indoxacarb), rapid conversion into the
N-decarbomethoxylated metabolite, DCJW (7) was demonstrated (Figure 32.5.1.8)
[33]. This bioactivation was attributed to hydrolytic esterase and amidase
metabolism in the midgut and fat bodies, with cytochrome P450 inhibitors having
Activated state
(open)
Resting state Inactivated state

(closed) (closed)
Figure 32.5.1.7 Various voltage-gated sodium channel

(VGSC) states, and whether the channel is conductive
(open) or nonconductive (closed).
Table 32.5.1.3 Known neurotoxin and insecticide binding sites on the VGSC.
Site Neurotoxin/insecticide Physiological effect
1 Tetrodotoxin, saxitoxin, μ-conotoxin Inhibition of ion transport

2 Batrachatoxin, veratridine, aconitine, Persistent activation
grayanotoxin, N-alkylamides
3 α-Scorpion toxins, sea anemone II toxin Enhancement of persistent activation
4 β-Scorpion toxins Shift voltage dependence of activation
5 Brevetoxins, ciguatoxins Shift voltage dependence of activation
6 δ-Conotoxins (δ-TxVIA) Inhibition of activation
7 DDT-type chemistry, pyrethroids Inhibition of activation
8 Goniopora coral toxin, Conus striatus toxin Inhibition of activation
9 Local anesthetics, anticonvulsants, Inhibition of ion transport
dihydropyrazoles
OCF3 CO2Me OCF3

CO2Me
O metabolic O
CI
CI decarboxylation
N N N N
N N
CO2Me O H
O
7
Indoxacarb DCJW
Figure 32.5.1.8 Indoxacarb, as a proinsecticide, is metabolized to DCJW.
minimal impact on bioactivation. When using a preparation from the central

nervous system of the Lepidoptera, Manduca sexta, DCJW exhibited a greater than
25-fold higher potency than DPX-JW062 in its ability to block nerve conduction
[34]. Moreover, the insecticidal activity was attributed to the (S)-enantiomer, given
the twofold greater potency over the racemic mixture.
32.5.1.4.3 Blockade of VGSCs by Indoxacarb and Dihydropyrazoles Lepidopteran

larvae treated with indoxacarb produced neurotoxic symptoms, beginning with
ataxia and feeding cessation and followed by tremors, mild convulsions, and
progressing to a flaccid paralysis [34]. Such symptoms mirror those observed with
the insecticidal dihydropyrazole, RH-3421 [35]. The extracellular recording of nerve
activity in the cockroach P. americana or lepidopteran larvae M. sexta poisoned
with RH-3421, or indoxacarb, demonstrated a complete block in the spontaneous
activity from sensory and central nervous system (Figure 32.5.1.9).
The results of voltage–clamp experiments using identified neurons [dorsal
unpaired median (DUM) neurons from the terminal abdominal ganglion of
P. americana], showed that DCJW inhibited the peak Na+ current with an inhibitory
(a)
100 μV
200 msec
(b)
Figure 32.5.1.9 Extracellular recordings of spontaneous

central nervous system activity from nerve cords of fifth
instar M. sexta injected with (a) dimethyl sulfoxide or (b)
10 mg g−1 DCJW. Adapted with permission from Ref. [34];
© 1998, Wiley-Liss, Inc.
concentration (IC)50 of 28 nM [36]. DCJW (7) (100 nM) induced a hyperpolarization

of DUM neurons, associated with a block of background Na+ channels involved in
maintenance of the resting potential. Whilst the peak Na+ current was inhibited,
DCJW had no effect on either activation or inactivation kinetics (Figure 32.5.1.10).
Similarly, Zhao et al. (2005) observed no effect on activation or inactivation kinetics
[37], However in their study two distinct types of VGSC were identified (termed type
I and type II), with DCJW and indoxacarb (1 μM) exhibiting differential activities
on these currents. Although, both compounds inhibited type I Na+ channels at a
membrane potential of −100 mV, less-negative potentials (−60 to −40 mV) were
required to observe a block in type II Na+ channels. This difference was attributed
to distinct inactivation kinetics between the two types of Na+ current, where the
type I Na+ channels exhibited significant inactivation at −100 mV but type II Na+
channels inactivated at less-negative potentials. Furthermore, the inhibition of type
I Na+ channels with DCJW was found to be irreversible, whereas the inhibition by
indoxacarb was fully reversed on compound washout (Figure 32.5.1.10). Thus, it
was postulated that the higher insecticidal potency of DCJW, relative to indoxacarb,
could be attributed to its irreversible nature. However, given the higher potency
(>250-fold) of DCJW for blocking compound action potential generation in M. sexta
relative to indoxacarb [34], the reversibility may have been less of a factor than
inherent potency at the VGSC.
Oxadiazine and Dihydropyrazole Binding Site Identification Although details of

radioligand-binding studies with indoxacarb and other oxadiazine species have
not been reported, studies conducted with dihydropyrazoles have shown that
these molecules bind to a site distinct from DDT and pyrethroids. Rather,
dihydropyrazoles most likely bind to site 9 with allosteric interaction at site 2
of the VGSC. RH-3421 was found to inhibit the binding of [3 H]batrachatoxin-B
Potential (mV)
−100 −80 −60 −40 −20 0 20 40 60
Current amplitude (nA)

0
−2
−4
100 nM DCJW −6
Control 4 nA −8 100 nM DCJW

Control
5 ms −10
(a) (b)
Normalized sodium conductance
1.0
Fast
1.0 Control
Slow
I/lmax
0.5 0.5
100 nM DCJW Fast 100 nm

0.0 DCJW
Control Slow
0.0
−60 −40 −20 0 20 40 −140 −120 −100 −80 −60 −40 −20 0 20
(c) Potential (mV) (d) Potential (mV)
Figure 32.5.1.10 Effects of DCJW (7) on potential before and after application of
the DUM neuron voltage-dependent inward 100 nm DCJW; (c) Voltage dependence of
sodium current. (a) Sodium inward cur- the normalized sodium conductance of the
rent traces obtained by a 30 ms depolarizing inward current in normal saline before and
pulse to −10 mV from a holding potential after the application of 100 nm DCJW; (d)
of −90 mV, in the absence and presence of Superimposed voltage dependence of 100 nm
100 nm DCJW; (b) Effect of DCJW on the DCJW. All data are mean ± SEM. Reprinted
current–voltage relationship of the inward with permission from Ref. [36]; © 2001,
sodium current. The maximum peak current MacMillan Publishers Ltd.
amplitude was plotted versus membrane
([3 H]BTX-B) to mouse brain synaptosomes in a noncompetitive manner [38].

Typically, RH-3421 decreased the number of available [3 H]BTX-B binding sites,
without impacting binding affinity. Given the similar mode of action between
dihydropyrazoles and local anesthetics, which bind to site 9, Payne et al. (1998)
investigated the combined effects of RH-3421 and dibucaine on [3 H]BTX-B binding
[39]. RH-3421 was shown to decrease the potency of dibucaine as an inhibitor
of [3 H]BTX-B, consistent with binding to site 9. Evidence that DCJW similarly
binds to this site was supported by the ability of the local anesthetic, lidocaine, to
reduce DCJW’s suppression of the VGSC current in P. americana DUM neurons.

Surprisingly, it was recently shown that, for a single isoform of the rat VGSC
(Nav 1.4) expressed in Xenopus laevis oocytes, the current-blocking efficacy of DCJW
was reduced in the presence of indoxacarb, whereas the RH-3421-induced current
suppression was unaffected [40]. This finding suggested that the binding site for
indoxacarb overlaps that of DCJW, but is separate from the RH-3421 binding site.
32.5.1.4.4 Action of Indoxacarb on Mammalian VGSCs To date, few studies have

been conducted on the effect of indoxacarb and DCJW on mammalian VGSCs.
Zhao et al. (2003) have investigated the ability of these oxadiazines to inhibit
tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium currents
in rat dorsal root ganglion (DRG) neurons [41]. As observed with insect Na+
channels, indoxacarb and DCJW each inhibited sodium currents upon binding
to the inactivated state, although both oxadiazines exhibited a much weaker
potency against rat sodium channels compared to those of insects. Indoxacarb
and DCJW (1 μM) irreversibly inhibited TTX-S sodium currents by 30% and 80%,
respectively (Figure 32.5.1.11b). Likewise, in another study, indoxacarb was found
to be less potent (10-fold) than DCJW against TTX-R sodium currents in DRG
neurons (Figure 32.5.1.11a) [42]. However, in this case the indoxacarb-induced
sodium current suppression was partially reversible following compound washout,
whereas the DCJW-induced suppression was irreversible.
More recently, the effects of indoxacarb, DCJW, and the dihydropyrazole,
RH-3421, were investigated on a single rat sodium channel isoform (Nav 1.4)
120 120
1 μM Insecticides 1 μM Insecticides
100 100
Relative current (%)
Relative current (%)
80 80
60 60
40 40
20 Indoxacarb (n = 5) 20 Indoxacarb (n = 5)
DCJW (n = 6) DCJW (n = 5)
0 0
0 5 10 15 20 0 5 10 15 20
(a) Time (min) (b) Time (min)
Figure 32.5.1.11 Effect of indoxacarb (•) and DCJW (◦) on

(a) TTX-R and (b) TTX-S sodium currents of rat dorsal root
ganglion neurons. Sodium currents were evoked by a 10 ms
step depolarization to 0 mV from a holding potential of
−80 mV. Adapted with permission from Ref. [41]; © 2003,
Elsevier.
expressed in Xenopus laevis oocytes [40]. As observed with DRG neurons, both
DCJW and RH-3421 (10 μM) irreversibly inhibited the Nav 1.4 currents in a
voltage-dependent manner, but indoxacarb (10 μM) had no such inhibitory effect.
Despite having activity against mammalian VGSCs, indoxacarb demonstrates
excellent mammalian safety. Differential sodium channel affinity represents a
major factor contributing to the safety of indoxacarb. In insects, DCJW is highly
potent, with an IC50 value below 30 nM, as compared to the low micromolar
IC50 value for rat VGSCs. Furthermore, indoxacarb – which has 10-fold lower
potency than DCJW against rat VGSCs – is the predominant oxadiazine in mam-
mals [23]. Minimal conversion of indoxacarb into DCJW occurs in mammals,
whereas indoxacarb is rapidly metabolized into DCJW in insects [34] (as noted
above).
32.5.1.4.5 Indoxacarb Resistance Indoxacarb has proven to be an effective

crop-protection product in regions where insects have developed resistance to
organophosphates, carbamates, synthetic pyrethroids, benzyl urea insect growth
regulators, and organochlorines. Because indoxacarb is bioactivated via esterase
and amidase enzymes, the overproduction of esterases in insects that are resistant
to organophosphates or pyrethroids might lead to a more rapid liberation of
the active toxin than in nonresistant insects. This suggests that certain resistant
insects may, in fact, develop a negative cross-resistance to indoxacarb, as has
been observed in laboratory strains [43] and field populations of Helicoverpa
armigera [44]. Nonetheless, after multiple years of repeated use on the island of
Oahu, Hawaii, where growers had access to very few insecticides, a population of
diamond-back moth (Plutella xylostella) developed resistance to indoxacarb [45].
The mechanism of resistance was believed to be enzymatic, though this has
yet to be verified. Certain geographic populations of oblique-banded leaf roller,
Choristoneura rosaceana, a pest of apples, are also known to be highly tolerant to
Indoxacarb WG (wettable granule). Ahmad has suggested that such resistance
might involve an enhanced oxidative degradation, through a mechanism originally
selected by field exposure, to azinphosmethyl [46]. Failure to activate the compound
does not appear to be involved as a resistance mechanism.
Indoxacarb is characterized by attributes that offer a total plant protection package
for cotton, vegetables, tree fruit, vines, and other agricultural crops. It represents
a new chemical class and a novel mode of action that is well-suited for rotation
in resistance management programs. Since indoxacarb is extremely potent on
its biochemical target, this results in low field use rates, and excellent safety for
both workers and consumers. Combined with a general safety to predacious and
parasitic arthropods, indoxacarb is an ideal fit in grower pest-control programs,
and a good choice to alternate, replace, or complement existing insecticides. While,
currently, the field of competitive sodium channel-blocking insecticides is narrow,
it is anticipated that this will grow with time as new products such as metaflumizone
move to market.
References 1271
References
1. (a) Mulder, R., Wellinga, K., and Van T.P. Selby, and T.M. Stevenson), Ameri-
Daalen, J.J. (1975) Naturwissenschaften, can Chemical Society, Washington, DC,
62, 531–532; (b) Wellinga, K. and pp. 121–132.
Mulder, R. (1976) US Patent 3,991,073. 11. Lahm, G.P., Harrison, C.R., Daub, J.P.,
2. Wellinga, K., Grosscurt, A.C., and Van Shapiro, R., Long, J.K., Allen, D.E.,
Hes, R. (1977) J. Agric. Food Chem., 25, March, W.A., Griswold, S.M., March,
987–992. R.W., and Reeves, B.M. (2002) in Syn-
3. Van Hes, R., Wellinga, K., and thesis and Chemistry of Agrochemicals
Grosscurt, A.C. (1978) J. Agric. Food VI (eds D.R. Baker, J.G. Fenyes, G.P.
Chem., 26, 915–918. Lahm, T.P. Selby, and T.M. Stevenson),
4. (a) Mulder, R. and Van Daalen, J.J. American Chemical Society, Washing-
(1979) US Patent 4,174,393; (b) Ozawa, ton, DC, pp. 110–120.
K., Nakajima, Y., Tsugeno, M., Ishii, S., 12. Lahm, G.P., Lett, R.M., Long, J.K.,
Hatanaka, M., Hirose, M., and Kudo, Lowder, P.D., Stevenson, T.M., Currie,
M. (1982) Eur. Patent 058,424; (c) Giles, M.J., Folgar, M.P., Griswold, S.M.,
D.P. and Willis, R.J. (1983) Eur. Patent Lucas, M.A., March, R.W., and March,
113,213. W.A. (2002) in Synthesis and Chemistry
5. Sirrenberg, W., Klauke, E., Hammann, of Agrochemicals VI (eds D.R. Baker, J.G.
I., and Stendel, W. (1978) Ger. Offen. Fenyes, G.P. Lahm, T.P. Selby, and T.M.
DE 270,0289. Stevenson), American Chemical Society,
6. (a) Scheele, B. (1980) Chemosphere, 9,
13. Amoo, V.E., Harrison, C.R., Lahm, G.P.,
483–494; (b) Meier, G.A., Silverman,
Lowder, P.D., Stevenson, T.M., Long,
R., Ray, P.S., Cullen, T.G., Ali, S.F.,
J.K., Shapiro, R., March, R.W., Allen,
Marek, F.L., and Webster, C.A. (1992) in
D.E., Richmond, M.D., March, W.A.,
Synthesis and Chemistry of Agrochemicals
Chun, G., Folgar, M.P., and Griswold,
III (eds D.R. Baker, J.G. Fenyes, and J.J.
S.M. (2002) in Synthesis and Chemistry of
Steffens), American Chemical Society,
Agrochemicals VI (eds D.R. Baker, J.G.
Fenyes, G.P. Lahm, T.P. Selby, and T.M.
7. Fuehr, F., Mittelstaedt, W., and
Stevenson), American Chemical Society,
Wieneke, J. (1980) Chemosphere, 9 (7–8),
469–482. 14. (a) McCann, S.F., Annis, G.D., Shapiro,
8. Meier, G.A., Silverman, R., Ray, P.S., R., Piotrowski, D.W., Lahm, G.P., Long,
Cullen, T.G., Ali, S.F., Marek, F.L., J.K., Lee, K.C., Hughes, M.M., Myers,
and Webster, C.A. (1992) in Synthe- B.J., Griswold, S.M., Reeves, B.M.,
sis and Chemistry of Agrochemicals III March, R.W., Sharpe, P.L., Lowder, P.,
(eds D.R. Baker, J.G. Fenyes, and J.J. Barnette, W.E., and Wing, K.D. (2001)
Steffens), American Chemical Society, Pest Manage. Sci., 57 (2), 153–164; (b)
Washington, DC, pp. 313–326. McCann, S.F., Annis, G.D., Shapiro,
9. (a) Jacobson, R.M. (1987) US Patent R., Piotrowski, D.W., Lahm, G.P., Long,
4,663,341; (b) Jacobson, R.M. (1989) J.K., Lee, K.C., Hughes, M.M., Myers,
in Recent Advances in the Chemistry of B.J., Griswold, S.M., Reeves, B.M.,
Insect Control (ed. L.E. Crombie), The March, R.W., Sharpe, P.L., Lowder, P.,
Royal Society of Chemistry, London, pp. Tseng, P., Barnette, W.E., Wing, and
206–211. Keith, D. (2002) in Synthesis and Chem-
10. Stevenson, T.M., Harrison, C.R., istry of Agrochemicals VI (eds D.R. Baker
Lowder, P.D., Crouse, B.A., March, J.G. Fenyes, G.P. Lahm, T.P. Selby, and
Robert, W., Currie, M.J., Folgar, M.P., T.M. Stevenson), American Chemical
and Chan, D.M.T. (2002) in Synthesis Society, Washington, DC, pp. 166–177;
and Chemistry of Agrochemicals VI (eds (c) Lahm, G.P., McCann, S.F., Harrison,
D.R. Baker, J.G. Fenyes, G.P. Lahm, C.R., Stevenson, T.M., and Shapiro, R.
(2001) Agrochemical Discovery, ACS Sym- Larvae, in Proceedings, Beltwide Cotton

posium Series, vol. 774 (eds D.R. Baker Conference, January 9–13, Anaheim,
and N.K. Umetsu), American Chemical CA. National Cotton Council, Memphis,
Society, Washington, DC, pp. 20–34. TN.
15. Annis, G.D., Barnette, W.E., McCann, 27. Wang, S. and Wang, G.K. (2003) Cell.
S.F., and Wing, K.D. (1992) PCT Int. Signal., 15, 151–159.
Appl. WO 92/11,249. 28. Blumenthal, K.M. and Seibert, A.L.
16. Heathcock, C.H., Mahaim, C., Schlecht, (2003) Cell Biochem. Biophys., 38,
M.F., and Utawanit, T. (1984) J. Org. 215–238.
Chem., 49, 3264–3274. 29. Anger, T., Madge, D.J., Mulla, M., and
17. Daub, J.P., Lahm, G.P., and Martin, B.S. Riddall, D. (2001) J. Med. Chem., 44,
(1990) Eur. Patent 377,304. 115–137.
18. Katritzky, A.R., Jones, R.A.Y., and 30. Zlotkin, E. (1999) Annu. Rev. Entomol.,
Trepanier, D.L. (1971) J. Chem. Soc. B, 44, 429–455.
1300–1302. 31. Bloomquist, J.R. (1993) Rev. Pestic. Toxi-
19. Harder, H.H., Riley, S.L., McCann, col., 2, 185–230.
S.F., and Irving, S.N. (1996) Proc. 32. Catterall, W.A. (1980) Annu. Rev. Phar-
Brighton Crop Prot. Conf. – Pests Dis., macol. Toxicol., 20, 15–43.
2, 449–454. 33. Wing, K.D., Sacher, M., Kagaya, Y.,
20. Bosum-Dybus, A. and Neh, H. (1991) Tsurubuchi, Y., Mulderig, L., Connair,
Liebigs Ann. Chem., 823–825. M., and Schnee, M. (2005) Crop Prot.,
21. Shapiro, R., Annis, G.D., Blaisdell, 19, 537–545.
C.T., Dumas, D.J., Fuchs, J., Griswold, 34. Wing, K.D., Schnee, M.E., Sacher, M.,
S.M., Higley, G.W. Jr, Hollinsed, W.C., and Connair, M. (1998) Arch. Insect
Mrowca, J.A., Sternberg, J.A., and Biochem. Physiol., 37, 91–103.
Wojtkowski, P. (2002) in Synthesis and 35. Salgado, V. (1990) Pestic. Sci., 28,
Chemistry of Agrochemicals VI (eds D.R. 389–411.
Baker, J.G. Fenyes, G.P. Lahm, T.P. 36. Lapied, B., Grolleau, F., and Sattelle,
Selby, and T.M. Stevenson), American D.B. (2001) Br. J. Pharmacol., 132,
Chemical Society, Washington, DC, pp. 587–595.
178–185. 37. Zhao, X., Ikeda, T., Salgado, V.L., Yeh,
22. Michaud, J.P. and Grant, A.K. (2003) J. J.Z., and Narahashi, T. (2005) Neurotoxi-
Insect Sci., 3, 18. cology, 26, 455–465.
23. Environmental Protection Agency 38. Deecher, D.C., Payne, G.T., and
(2000) Fact Sheet on Indoxacarb, Is- Soderlund, D.M. (1998) Pest. Biochem.
sued October 30, 2000, Washington, DC, Physiol., 41, 265–273.
http://www.epa.gov/opprd001/factsheets/ 39. Payne, G.T., Deecher, D.C., and
indoxacarb.pdf (accessed January 2006). Soderlund, D.M. (1998) Pest. Biochem.
24. Andaloro, J.T., Williams, R., and Physiol., 60, 177–185.
Sherrod, D.W. (2002) Indoxacarb in- 40. Silver, K. and Soderlund, D.M. (2005)
secticide: review of efficacy, behavior Neurotoxicology, 26, 397–406.
and insect spectrum. Entomological 41. Zhao, X., Ikeda, T., Yeh, J.Z., and
Society of America Meeting, November Narahashi, T. (2003) Neurotoxicology, 24,
17–20, Fort Lauderdale, FL, p. 185. 83–96.
25. Barr, C.L. (2004) How Fast is Fast?: 42. Tsurubuchi, Y. and Kono, Y. (2003) Pest
Indoxacarb Broadcast Bait. Proceed- Manage. Sci., 59, 999–1006.
ings of the 2004 Annual Red Imported 43. Gunning, R.V. and Devonshire, A.L.
Fire Ant Conference, Baton Rouge, LA, (2003) Negative Cross resistance be-
pp. 46–49. tween indoxacarb and pyrethroids
26. Andaloro, J.T., Edmund, R.M., Castner, in the cotton bollworm, Helicoverpa
E.P., Williams, C.S., and Sherrod, D.W. armigera, in Australia: a tool for resis-
(2001) Steward® SC Field Performance tance management. BCPC International
Against Heliothines: Speed of Action, Congress – Crop Science Technology,
Symptomology, and Behavior of Treated Glasgow, UK, pp. 789–794.
44. Ramasubramanian, T. and Regupath, A. Shelton, A.M. (2006) J. Econ. Entomol.,

(2004) J. Entomol., 1, 21–23. 99, 176–181.
45. Zhao, J.Z., Collins, H.L., Li, Y.X., Mau, 46. Ahmad, M., Hollingworth, R.M., and
R.F.L., Thompson, G.D., Hertlein, Wise, J.C. (2002) Pest Manage. Sci., 8,
M., Andaloro, J.T., Boykin, R., and 834–838.
32.5.2
Semicarbazone Insecticides: Metaflumizone
Metaflumizone, a new insecticide belonging to the semicarbazone class of chem-
istry, was discovered by Nihon Nohyaku Co., Ltd (NNC) and developed globally
in cooperation with BASF SE as a broad-spectrum insecticide with activity on
Lepidoptera and Coleoptera, certain Hemiptera, and noncrop insects.
32.5.2.2 Discovery
PH-6042 (2) [1], followed by several other pyrazoline compounds (3) and (4) [2–5],
first attracted the attention of NNC over 30 years ago (Figure 32.5.2.1). The structure
of these molecules, as specified in the patent literature [6–8], was very different
from other commercial lepidopteran insecticides, such as the organophosphates,
carbamates, pyrethroids, and benzoyl ureas. Following the synthesis one of these
OCF3 Cl
O
O
N N
F3C N N Cl N N
H H H
NC
1 2
OCF3
Cl O
O
N N
N N N
N F2HCO H
Cl H
Me
CO2Me
3 4
Figure 32.5.2.1 Chemical structures of metaflumizone and insecticidal pyrazolines.

compounds, and the examination of its potency, spectrum of activity, and long
residual activity, the chemical area became of much more interest. In addition, the
compound was characterized as employing a completely different mode of action
([9], see discussion below) from that of the already-known insecticides.
In order to identify and develop novel molecules with significant insecticidal
activities, NNC embarked upon a new synthesis program, the initial synthetic
methodology of which is shown schematically in Figure 32.5.2.2. Subsequently,
one analog derived from this program (5) demonstrated insecticidal activity that
was comparable to that of the original pyrazoline derivatives. During the course of
this extensive program, a research group at NNC suggested that a pyrazoline with
three phenyl rings as substituents should be carefully examined from the point of
view of its bioaccumulation, as this might be associated with the compound having
a high log P value, and this would consequently affect its potential for registration.
Furthermore, the stability of these compounds in the soil was determined to be too
high. Unfortunately, despite extensive synthetic efforts being carried out, which
included the introduction of a variety of water-soluble substituents, the log P value
of the resultant compounds was not improved to any dramatic extent.
In parallel, a synthesis of the N-phenylpyrazoline isomer was achieved utiliz-
ing 1,3-dipolar cycloaddition chemistry with styrene derivatives [10], as shown in
Figure 32.5.2.3. Interestingly, compounds in this series showed the same insecti-
cidal activity, but less residual activity, compared to the original pyrazolines. This
lack of residual activity was shown to be due to the facile oxidation to a pyrazole
under photolytic conditions (Figure 32.5.2.4). Confirmation was also provided that
this pyrazole was not active against Lepidoptera.
On the basis of this knowledge, all subsequent efforts were devoted to find-
ing new derivatives that showed good insecticidal activity, but less potential to
bioaccumulate. Some such attempts are depicted schematically in Figure 32.5.2.5.
Following several rounds of synthesis, it was found that cleavage at both bonds a
and b (see Figure 35.2.5), produced a compound (6) that showed weak activity, while
the open-chain compound still retained the semicarbazone substructure found in
the original pyrazoline.
These results encouraged NNC to examine the substituent effects on the three
phenyl rings (phenyl A, phenyl B, and phenyl C), the aim being to maximize
the activity; the results of this structure–activity optimization program (which are
summarized in Tables 32.5.2.1–32.5.2.3) led to the discovery of metaflumizone (1).
Subsequently, further extensions of this knowledge were applied to the isomeric
hydrazone series, and this in turn led to the creation of compounds with good insec-
ticidal activity. Details of the structure–activity relationship (SAR) are summarized
in Table 32.5.2.4.
Typically, a substituent on the para position in ring B of the open-chain com-
pounds shows the highest activity, while a substituent on the meta position of ring
D shows the highest activity. In both series, two phenyl rings are seen to be con-
nected by five atoms which, despite being arranged slightly differently, produces a
resonance system between the two phenyl rings in both open-chain compounds.
This system is also recognized in two types of pyrazoline compound. The matrix of
R
S S
X N Z Z
NH Z N N
X X
N N N N
SCN H H
R-Hal
Y
Y Y
Me
OCF3
Me S
N N
N
Cl
Figure 32.5.2.2 Initial synthetic methodology by NNC.

32.5 Sodium Channel-Blocking Insecticides
1275
1,3-dipolar cycloaddition
O
Cl X N
OR
N N
X Y
NH OR
O
O
Z
X N
N
N H
Figure 32.5.2.3 Synthesis of pyrazoline isomers.
O Sun light O
Z Z
X N X N
N N
N H N H
Y Y Inactive
Figure 32.5.2.4 Facile oxidation of pyrazoline isomers.
activity and structures is shown in Figure 32.5.2.5. The activities of both open-chain
compounds were almost identical both laboratory-based and field tests although,
on considerations of its ease of synthesis, metaflumizone was selected for further
investigation. As expected, this semicarbazone was easily isomerized under light
irradiation conditions, and also readily hydrolyzed.
O O O
Z Z Z
X N X N X N
N a N N N
N a H N H a H
Me
b b
Y b Y Y
O
Z
X N
N
N H
Me
a+b a+b
Inactive
O O
Z Z
X N X N N
N N
N H H H
Optimization
Y Y
Active
6,Active
OCF3
O
N
F3C N N
H H
32.5 Sodium Channel-Blocking Insecticides
NC Metaflumizone, 1
1277
Figure 32.5.2.5 Some of the attempts to identify new pyrazoline derivatives.

Table 32.5.2.1 Structure–activity relationship on substituent X.
OCF3
O
X
N
N N
H H
X Y S. litura LC50 (ppm)
2-Cl H 300
3-Cl H 30–100
4-Cl H >1000
3-F CN 1–3
3-Cl CN 0.3–1
3-Br CN 0.3–1
3-CH3 CN 1
3-CF3 CN 0.3–1
(Metaflumizone, 1)
4-CF3 4-CN ––
––: not tested (<50% at 500 ppm, first screening).
Table 32.5.2.2 Structure–activity relationship on substituent Y.
OCF3
O
N
X N N
H H
Y
X Y S. litura LC50 (ppm)
H 2-Cl > 1000

H 3-Cl 30–100
H 4-Cl 3
H 4-CH3 30–100
H 4-CN 1
H 4-NO2 0.3–1
H 4-OCH3 10–30
CF3 H >1000
CF3 4-CN 0.3–1
(Metaflumizone, 1)
Table 32.5.2.3 Structure–activity relationship on substituent Z.
O
Z
N
X N N
H H
NC
X Z S. litura LC50 (ppm)
H H ––
H 2-Cl ––
H 3-Cl ––
H 4-Cl 30–100
H 4-CF3 3–10
H 4-OCF3 1
H 4-NO2 ––
CF3 4-OCF3 0.3–1
(Metaflumizone, 1)
––: not tested (<50% at 500 ppm, first screening).
Table 32.5.2.4 Structure–activity relationship on isomeric hydrazone series.
O
X Z
N
N N
H
Y
X Y Z S. litura LC50 (ppm)
H Cl 4-Cl >500
H Cl 4-OCF3 10–100
3-Cl Cl 4-OCF3 10–100
3-Cl Cl 4-CH3 >500
3-Cl Cl 3-Cl >500
3-Cl Cl 2-Cl >500
3-Cl CN 4-OCF3 0.3–1
3-CF3 CN 4-OCF3 0.3–1
3-CF3 SOCHF2 4-OCF3 1
32.5.2.3 Insecticidal Activity

The first crop registrations for metaflumizone were achieved in Colombia in
2006, followed by Germany, Austria, Romania, and Malaysia in 2007. The major
registrations are in potatoes against Colorado beetle (Leptinotarsa decemlineata),
and in fruiting vegetables against chewing insect pests (Heliothis sp.). The US
Environmental Protection Agency (EPA) registration for noncrop ant baits was
granted in 2007. Currently, metaflumizone is under evaluation under Council
Directive 91/414/EEC in Europe as a ‘‘new’’ active substance. In addition to those
listed above, metaflumizone is also registered in a further 21 countries.
The biological profile of metaflumizone includes good to excellent activity
against the caterpillars of most economically important lepidopteran species, and
certain other pests of economic importance in the orders Coleoptera, Hemiptera,
Hymenoptera, Diptera, Isoptera, and Siphonaptera, in both crop and noncrop
situations. Due to the fact that it is nonrepellent, metaflumizone has been de-
veloped for applications in baits for pests such as ants, flies, and cockroaches.
Control is achieved primarily through the ingestion of residues, and secondarily
by contact activity. Subsequently, the insects exhibit paralysis that begins with the
appendages and culminating with the cessation of feeding. These effects normally
begin between 15 min and 12 h after treatment, depending on the species being
treated.

Metaflumizone, as a voltage-dependent sodium channel blocker insecticide (SCBI)
[6], causes blockade of the sodium channel. This insecticidal mode of action was
first demonstrated for the pyrazoline insecticides [11–13].
Metaflumizone, when injected into fifth instar larvae of the southern armyworm,
Spodoptera frugiperda, at a dose of 15 μg g−1 , produced 100% prostration within 4 h
and paralysis within 24 h. A gross electrophysiological analysis of the paralyzed
insects at 24 h after treatment showed that all nerve activity had been blocked.
Such blockade of the action potential generation, which results from blocking of
voltage-dependent sodium channels, is also seen in insects treated with toxic doses
of pyrazolines and indoxacarb [11, 14].
Recordings were made from the abdominal stretch receptor organs of Spodoptera
eridania, to confirm and quantify the direct blocking effect on action potential
generation. These tonic mechanoreceptors normally generate action potentials
constantly at a low rate, and are very sensitive to blockage by SCBIs [1, 2].
As shown in Table 32.5.2.1, metaflumizone blocked Spodoptera stretch receptor
activity with a threshold concentration of 1 μM, but had no effect at 3 × 10−7 M
within 1 h.
By contrast, DCJW – the bioactive N-decarbomethoxyllated metabolite of indox-
acarb (see Section 32.5.1.4.2 and Chapter 32.5.1) – is almost 100-fold more active
than metaflumizone on the Spodoptera eridania stretch receptor organ, causing an
average of 40% block within 1 h at 1 × 10−8 M, and 100% block within 20–40 min
at 3 × 10−8 M [14].
In order to directly assess the effect of metaflumizone, Na+ channels were

studied in primary cultured neurons isolated from the central nervous system of
fifth instar Manduca sexta larvae, using the whole-cell voltage–clamp method. In
the presence of 30 mM tetraethylammonium (TEA) and 20 mM 4-aminopyridine
(4-AP) to block potassium channels, and 500 μM Cd2+ to block voltage-dependent
calcium channels, depolarization activated the voltage-gated sodium channels
transiently, so that they could conduct an inward sodium current. Although,
under a maintained depolarization, about 50% of the channels were inactivated, a
current was maintained throughout the duration of the 150 ms depolarizing pulse.
Following the application of 5 μM metaflumizone, the sodium current gradually
decreased and had completely disappeared by 20 min. These results confirmed
that metaflumizone has a direct blockade effect on insect Na+ channels.
It has long been known that the Na+ channel-blocking action of the SCBIs is
enhanced under depolarizing conditions, because these compounds can selectively
block the channels in an inactivated state [4]. This state-dependent situation is
thought to arise because the conformational changes in the para-polypeptide
associated with channel opening leads to formation of the SCBI binding site, which
does not exist in the closed channel. Specifically, the outward tilting of the four
pore-forming S6 segments is thought to make the pore conductive, and to create
the site where the state-dependent blockers can bind. Several families of bioactive
compounds are known to bind to this site, including class I anti-arrhythmics, local
anesthetics, pyrazoline insecticides, and DCJW [14, 15].
In practical terms, binding of the SCBIs is usually measured to the
slow-inactivated state, because the open state is only transiently available and the
compounds bind only very slowly. The SCBI binding site is thought to exist in all
open and inactivated states, but not in the resting state [14].
Metaflumizone, when applied for 10 min at −80 mV had very little effect on
the sodium current, up to a concentration of 10 μM. In contrast, even 0.1 μM
metaflumizone caused a significant depression of the sodium current under
depolarizing conditions, thus confirming the voltage-dependent nature of its
action [16].
32.5.2.5 Cross-Resistance Potential

While indoxacarb and metaflumizone are believed to act at the same target site
it is likely that, because of their very different structures, they recognize different
amino acid side chains within the binding site. Therefore, it seems possible that if
target site resistance to one or the other compound should develop, it would not
necessarily confer cross-resistance to the other compound.
Although metaflumizone has been tested in the laboratory on various insect
species and populations that have exhibited tolerance or resistance to commercial
insecticides, including Plutella xylostella and Heliothis virescens, none of the bioassays
has demonstrated any cross-resistance in any of the strains tested [17].
Due to the lack of any known cross-resistance between indoxacarb and metaflumi-
zone, under the Insecticide Resistance Action Committee (IRAC) mode of action
classification system these compounds are designated to separate subgroups,

namely groups 22A and 22B, respectively.
Metaflumizone, as the second member of the voltage-dependent sodium channel
group of insecticides (IRAC group 22), is structurally different from the first
member indoxacarb, such that any potential for cross-resistance would be expected
to be low. The key target markets for metaflumizone include leafy, fruiting
and root vegetables, potatoes and tree crops for the control of Lepidoptera and
Coleoptera, in addition to insecticide baits in the noncrop markets for pests
such as ants and flies. With a favorable environmental and toxicological profile,
including a low mammalian toxicity, safety to beneficial insects and a unique
mode of action, metaflumizone is clearly an important candidate in insect-control
programs.
References
1. Mulder, R., Wellinga, K., and van 12. For a recent review of the mode of
Daalen, J.J. (1975) Naturwissenschafen, action for sodium channel-blocking
62, 531–532. insecticides, see Silver, K.S., Song, W.,
2. Wellinga, K., Grosscurt, A.C., and van Nomura, Y., Salgado, V.L., and Dong,
Hes, R. (1977) J. Agric. Food Chem., 25, K. (2010) Pestic. Biochem. Physiol., 97,
987–992. 87–92.
3. van Hes, R., Wellinga, K., and 13. Takagi, K., Hamaguchi, H., Nishmatsu,
Grosscurt, A.C. (1978) J. Agric. Food T., and Konno, T. (2007) Vet. Parasitol.,
Chem., 26, 915–918. 150, 177–181.
4. Grosscurt, A.C., van Hes, R., and 14. Wing, K.D., Andaloro, J.T., McCann,
Wellinga, K. (1979) J. Agric. Food Chem., S.F., and Salgado, V.L. (2005) Com-
27, 406–409. prehensive Molecular Insect Science, in
5. Jacobson, R.M. (1989) in Recent Insect Control, vol. 6 (eds L.I. Gilbert,
Advances in the Chemistry of In- K. Iatrou, and S. Gill), Elsevier B.V., pp.
sect Control (ed. L.E. Crombie), The 30–53.
Royal Society of Chemistry, London, 15. A discussion of the role of the S6
pp. 206–211. transmembrane segment of the
6. van Daalen, J.J. and Mulder,R. (1976) sodium channel in the action of
US Patent 3, 991,073, 1976. SCBIs can be found in: Silver, K.S.,
7. Jacobson, R.M. (1987) US Patent Nomura, Y., Salgado, V.L., and
4,663,341, 1987. Dong, K. (2009) Neurotoxicology, 30,
8. Ozawa, K., Nakajima, Y., Tsugeno, M., 613–621.
Ishii, S., Hatanaka, M., and Hirose, M. 16. Salgado, V.L. and Hayashi, J.H. (2007)
(1982) Patent EP 58,424, 1982. Vet. Parasitol., 150, 182–189.
9. Salgado, V.L. (1990) Pestic. Sci., 28, 17. Jose, L., Armes, N.J., Farlow, R., and
389–411. Aldridge, K. (2007) Metaflumizone, a
10. Ruccia, M., Vivano, N., Cusmano, G., new broad-spectrum insecticide for crop
Marino, M.L., and Piozzi, F. (1973) protection, in Proceedings, XVI Interna-
Tetrahedron, 29, 3159. tional Plant Protection Congress, vol. 1,
11. Salgado, V.L. (1992) Mol. Pharmacol., 41, British Crop Production Council, pp.
120–126. 74–81.
32.6
Ligand-Gated Chloride Channel Antagonists (Fiproles)
32.6.1
Discovery and Development of Fipronil and Other Fiproles
Fiprole insecticides belong to the chemical class of insecticidal phenylpyrazoles

(arylpyrazoles) [1], discovered independently by Bayer AG [2] and by May & Baker, a
subsidiary of Rhône-Poulenc [3–5], while investigating herbicidal phenylpyrazoles
[protoporphyrinogen oxidase (PPO)-inhibiting herbicides, e.g., nipyraclofen] [6, 7].
Rhône-Poulenc Agrochimie (later Aventis CropScience) launched fipronil (1) as
a broad-spectrum crop insecticide in 1993 [8]. Within the context of the acquisition
of Aventis CropScience by Bayer CropScience AG in 2002, the fipronil business
was sold to BASF Aktiengesellschaft in early 2003.
Fipronil is highly effective against a significant range of economically important
insect pests, and has become a cornerstone in control programs for both crop and
noncrop insect pests in many areas of the world. Fipronil is currently registered in
over 70 countries for the control of insect pests in more than 100 crops, ranging
from row crops such as rice, corn, potatoes and small grains, to specialty crops
such as ornamentals, mangoes and chili peppers. Fipronil is used in a variety of
formulations, either as a foliar spray, soil application, or seed treatment, depending
on the crop/pest situation. Since fipronil it is not repellent at commercially
effective rates, it is extremely effective in bait applications. Notably, it not only
protects crops from insects, but in some cases can also actively increase yields
through incompletely understood plant health effects.
Within the noncrop area, fipronil has grown rapidly to become the leading
insecticide, such that today, it is the world’s leading termiticide and is also a key
component in urban pest control programs against cockroaches and ants. It is also
used for the control of mole crickets and fire ants in turf, and is one of the leading
veterinary ectoparasiticides [9, 10].
Fipronil is highly effective against insects that are resistant to other insecticides,
in part because of its unique mode of action. Whilst it was recognized even
before its launch that fipronil could block chloride channels gated by the inhibitory
neurotransmitter gamma-aminobutyric acid (GABA) [11–13], it has been shown
only recently that fipronil and/or its predominant sulfone metabolite also potently
block two types of glutamate-gated chloride channel (GluCl) in the insect central
nervous system (CNS) [14]. Thus, fipronil acts at three target sites, with high
affinity.
Ethiprole (2) was launched as a crop insecticide by Bayer CropScience in 2005.
Compared to fipronil, ethiprole has improved plant systemicity and controls a
broader spectrum of sucking pests, but has much less activity on lepidopteran
insects. Pyriprole (6) was recently introduced by Novartis Animal Health as an
ectoparasiticide for fleas and ticks on dogs. Three other phenylpyrazoles have
reached development status as crop insecticides (Figure 32.6.1).
O O
NC S CF3 NC S CF3
NC S C2H5
N N OMe
NH2 N N N
N N NH2
Cl Cl Cl Cl OH
Cl Cl
CF3 CF3
CF3
Fipronil 1 Ethiprole 2 Vaniliprole 3
O O NC S CH2F NC S CHF2
H3C S CH3
N N N N
N N N N N
N NH2
Cl Cl N Cl Cl
Cl Cl
CF3 CF3
CF3
Acetoprole 4 Pyrafluprole 5 Pyriprole 6
Figure 32.6.1 Fiproles on the market (1, 2, 6), or under development (3–5).
32.6.2
Mode of Action
Fipronil and its predominant sulfone metabolite are unique among insecticides
in that they have three known high-affinity target sites – the three ligand-gated
chloride channels that mediate most inhibitory transmission in the insect nervous
system: GABA receptors, and the two subtypes of GluCl that have been described
in insects [14, 15]. This multiplicity of highly sensitive target sites reduces the
potential for target-site resistance. Furthermore, fipronil and its sulfone are much
more potent against insect than against mammalian GABA receptors. Typically,
GABA receptors and GluCls mediate most rapid inhibitory transmission in the
insect nervous system. Inhibitory synapses are widespread in the CNS, and are
thought to be involved in the fine-tuning of all types of behavior [16]. Whilst a certain
level of inhibition is always present in the nervous system, its disruption leads
to hyperexcitation and convulsions; for this reason, GABA-gated chloride channel
blockers are also referred to as convulsants. Although it is assumed that GluCls
play a similar role to GABA receptors in inhibitory neurotransmission, this has not
been investigated. GABA receptors also mediate rapid inhibitory transmission at
insect nerve–muscle junctions. Whilst it has been well established that CNS effects
are important in the convulsant actions of insecticides, the role of muscle effects is
not at all clear.
32.6.2.1 Discovery of the GABA Receptor as an Insecticide Target Site

Between the mid-1940s and the late 1970s, more than 1.3 billion kilograms of
polychlorocycloalkane (PCCA) insecticides were used worldwide [17], and resistance
was becoming widespread; indeed, the PCCAs represented 60% of all known
cases of insecticide resistance [18]. Cross-resistance between the three classes of
PCCAs – lindane, toxaphene, and the cyclodienes (Figure 32.6.2) – suggested at an
early stage that these compounds all had a common target site. Moreover, the
observation [19] that several cyclodiene-resistant insect strains were cross-resistant
to the botanical convulsant picrotoxin, which had long been used in ointments to
control lice [20] and was known to be a noncompetitive antagonist (NCA) of GABA
receptors [21], pointed towards the GABA receptor as the most likely PCCA target
site. Subsequently, the action of PCCAs on GABA receptors was confirmed by
their ability to inhibit GABA-induced chloride flux into cockroach muscle, and also
the binding of [3 H]dihydropicrotoxinin to the NCA site in rat brain synaptosome
GABA receptors [19]. Furthermore, the mammalian toxicity of PCCAs was closely
correlated with displacement of [35 S]TBPS, another ligand for the NCA site, from rat
brain GABA receptors [22]. The blockade of GABA responses in cockroach neurons
by lindane and the cyclodiene endrin was confirmed electrophysiologically [23].
O +
O S O *S
O
P
O O
CI CI O
CI CI
CI CI
CI CI CI CI
CI CI
Dieldrin Alpha-endosulfan [35S]-TBPS
N CI
* CI CI
CI
N CI N
* CF3
CF3 CI Lindane
[3H]-BIDN
CI O O
CI CI O
O O O
CI O OH O
CI
CI
CI
CI
Toxaphene, most * * * *
toxic component [3H]-DHPTX [3H]-EBOB
Figure 32.6.2 Structures of the fiproles and other GABA

antagonists. Dieldrin and α-endosulfan are examples of cy-
clodienes. The location of the radiolabels in the radioligands
are indicated by asterisks.
Although [3 H]dihydropicrotoxinin was the first successful radioligand for the

NCA site, newer ligands have improved properties [17]. For example, [35 S]TBPS is
used extensively for the GABA receptor NCA site in mammalian brain, but does
not identify a toxicologically relevant site in insects; this is consistent with the high
mammalian toxicity and poor insecticidal activity of the bicyclophosphorous esters.
However, the structurally related bicycloorthocarboxylate esters, which are highly
toxic to both insects and mammals, yielded [3 H]EBOB, now the ligand of choice
for the insect GABA receptor [17]. [3 H]BIDN has also been used as a ligand for the
NCA site [24].
The mode of action of fipronil as an NCA of GABA-gated chloride channels was
reported in 1992, soon after the announcement of its development [8]. Fipronil
and other insecticidal phenylpyrazoles had been observed to cause symptoms in
house flies and mice that were similar to those caused by the known GABA
antagonists, dieldrin and EBOB [11]. A dieldrin-resistant house fly strain was
shown to be 20-fold cross-resistant to fipronil, while fipronil inhibited the specific
binding of [3 H]EBOB to GABA receptors in house fly head membranes, with a
50% inhibitory concentration (IC50 ) of 2.3 nM. Furthermore, the potency among
several phenylpyrazoles in inhibiting [3 H]EBOB binding correlated with insecticidal
activity [11], while fipronil was shown to antagonize homomeric Rdl GABA receptors
heterologously expressed in Xenopus oocytes [25–27].
32.6.2.2 Cloning the Insect GABA Receptor and Resistance Mutations

Ffrench-Constant and coworkers isolated and cloned the Rdl gene [28] from a
strain of Drosophila melanogaster that exhibited high levels of resistance [29] and
nerve-insensitivity [30] to cyclodienes and picrotoxinin [28]. This single gene gave
4000-fold resistance to dieldrin and 22 000-fold resistance to picrotoxinin [31], and
had a high homology to vertebrate GABAA receptor subunits [28].
Rdl orthologs have since been cloned from many other insect species and,
when heterologously expressed in the Xenopus oocyte expression system or in cell
lines, produce membrane receptors that behave like insect GABA receptors. The
Rdl receptors are activated by GABA but not by glutamate, and are blocked by
picrotoxinin, dieldrin, and fipronil, but not by bicucculine. Rdl is widely expressed
in adult Drosophila brain and thoracic ganglia, and occurs in regions that also stain
for GABA and glutamic acid decarboxylase (GAD), the enzyme that catalyzes the
synthesis of GABA from glutamate. Rdl is not expressed in muscle, although insect
muscle is known to have GABA receptors [32, 33].
A single base-pair mutation leading to an alanine to serine (A→S) substitution
near the cytoplasmic end of transmembrane domain II (designated the M2 seg-
ment) of Rdl at position 302, designated A302S, was found to be invariably present
in many dieldrin-resistant strains of D. melanogaster, but not in susceptible strains.
A mutation at this site was also invariably correlated with dieldrin resistance in D.
simulans, but in this case the mutation was also sometimes to glycine (A302G) [34].
Elegant confirmation that this single base-pair mutation in Rdl confers resistance
was provided by using a susceptible Rdl allele to transform resistant individuals to
susceptibility [28, 35].
A mutation in Rdl corresponding to A302S is associated with dieldrin resistance

in many other species. Owing to variation in sequence length, a relative numbering
system starting from the cytoplasmic end of the M2 segment is convenient
when comparing different subunits. Thus, A302S becomes A2 S. Dieldrin-resistant
aphids, Nasonovia ribis-nigri with the A2 S mutation [36], and Myzus persicae with
A2 G [37] have been described, as well as the mosquitoes Aedes aegypti, Anopheles
stephensi [38], Anopheles gambiae, and Anopheles arabiensis [39] with A2 S.
The functional consequence of the A2 S and A2 G mutations to the NCA site was
established first with receptor binding studies. Both mutations reduced the affinity
and density of the [3 H]EBOB binding sites, and also reduced the potency of eight
NCAs in inhibiting [3 H]EBOB binding. In D. simulans, A2 G was less effective than
A2 S in protecting the chloride channel from the blockers, but equally effective
in protecting the flies from their lethal effects [40]. In that study, D. melanogaster
flies with the A2 S mutation were 73-fold resistant to fipronil, whereas D. simulans
with A2 S and A2 G were 23- and 41-fold resistant, respectively. Similar levels
of cross-resistance to fipronil in dieldrin-resistant house flies were seen [12]. In
contrast, fipronil resistance in dieldrin-resistant German cockroaches was only
eightfold, even though resistance to some fipronil analogs was much higher [41,
42]. Resistance levels are often highly structure-dependent when a mutation affects
binding directly. The organochlorine-resistant (OCR) house fly strain has the A2 S
mutation, and is 1800-fold resistant to dieldrin and 32-fold resistant to fipronil.
However, while the affinity of the GABA receptors for dieldrin was decreased
45-fold in the resistant insects, it was decreased only twofold for fipronil [43], which
does not easily explain the 32-fold level of resistance.
Recently, a voltage–clamp study of GABA receptors in Rdl insects was conducted
in neurons isolated from susceptible and dieldrin-resistant German cockroaches.
Typically, dieldrin blocked GABA currents with an IC50 of 3 nM in wild-type
cockroaches, and 383 nM in resistant insects, yielding a resistance ratio of 128.
Fipronil sulfone blocked GABA currents with an IC50 of 0.8 nM in susceptible
insects, and 12.1 nM (i.e., 15-fold higher) in resistant insects [44]
Rdl homomultimers containing the A2 S mutation, when heterologously-
expressed in Xenopus oocytes, were highly resistant to dieldrin and picrotoxinin
[45]. Careful measurement of the effect of fipronil on wild-type D. simulans Rdl
homomultimers showed that the IC50 for blockade of the peak current decreased
from 31 nM after 15 min of incubation to 3.6 nM after 30 min, in line with the
binding data cited above. Because the equilibration of fipronil with mutant
receptors is almost complete after 15 min, the resistance ratio was increased from
only 3 at 15 min to 23 at 30 min [46]. Thus, the level of resistance to channel block
by fipronil corresponds very well to the levels measured in bioassays (as discussed
above).
A second mutation in Rdl, T350M, was isolated from a D. melanogaster strain
selected in the laboratory for high levels of resistance to fipronil. This mutation
made the peak GABA activated current fivefold resistant to fipronil. Despite the
A302S/T350M double-mutant receptor being 50-fold resistant to fipronil, even
both of these mutations together could not account for the 20 000-fold resistance
to fipronil in this strain. However, other factors, such as metabolism, were not
excluded [46].
32.6.2.3 Ligand-Gated Chloride Channel Structure and Classification

The molecular biology and classification of GABA-receptors and GluCls of insects
has recently been reviewed [16, 47–49]. Both GABA receptors and GluCls are
members of the cys-loop family of ionotropic neurotransmitter receptors, which
includes nicotinic acetylcholine receptors (nAChRs), 5-HT3 (serotonin type 3)
receptors, and strychnine-sensitive glycine receptors of vertebrates, as well as
5-HT- and histamine-gated chloride channels of invertebrates. Cys-loop recep-
tors are pentameric transmembrane proteins composed of five subunits arranged
symmetrically around an integral ion-conducting pore. Each subunit has four
transmembrane regions, M1–M4, with a large intracellular loop containing phos-
phorylation sites between M3 and M4, and a long N-terminal extracellular region
that is involved in ligand binding. The pore is formed largely from the M2 helices,
with the large N-terminal regions forming two neurotransmitter binding sites per
receptor.
The 2 position in the M2 segment is now well established as a key residue in the
NCA binding site for all ligand-gated chloride channels. Homomers of subunits
containing 2 A are sensitive to NCAs, whereas those with S, T, M, G, or N in
this position show a reduced sensitivity. The NCA sensitivity of heteropentamers
depends on their complement of 2 A-containing subunits (see Section 32.6.2.5).
32.6.2.3.1 GABA Receptors Vertebrate ionotropic GABA receptors are subdivided

into GABAA and GABAC receptors, based on their pharmacology and kinetics. The
GABAA receptors are complex allosteric proteins that desensitize, are antagonized
by the alkaloid bicucculine, and contain distinct binding sites for barbiturates,
benzodiazepines, pregnane steroids, furosemide, loreclazole, picrotoxinin, zinc,
lanthanum, volatile anesthetics, and the anesthetic propofol. The GABAC receptors
are generally nondesensitizing, and are insensitive to bicucculine and many of the
modulators of GABAA receptors.
Nineteen different ionotropic GABA receptor subunits, named α1 – 6 , β1 – 3 , γ1 – 3 , δ,
ε, π and θ , and ρ1 – 3 , have been cloned from vertebrates. The results of experiments
using recombinant expression and immunoprecipitation have indicated that native
vertebrate GABAA receptors contain at least α, β, and γ subunits, sometimes also
with δ, ε, π, or θ subunits, whereas GABAC receptors are believed to be composed
only of ρ subunits.
The GABAC receptors, which are found in the retina, cerebellum, and spinal cord
of mammals, are composed of three types of rho subunit, ρ1 to ρ3 . While the ρ2 and
ρ3 subunits contain S in position 2 and would be expected to be insensitive to many
NCAs, the 2 P residue of ρ1 is unique. The ρ1 homomers bind lindane and EBOB
quite well, but are highly resistant to fipronil [50]. Thus, while GABAC receptors
may play a significant role in the mammalian toxicology of some insecticides, they
are probably not significantly affected by fipronil.
Insect GABA-receptors do not fit into the vertebrate classification, because

most are simultaneously insensitive to the GABAA -diagnostic antagonist bicuc-
culine but sensitive to the GABAA -specific benzodiazepines and barbiturates.
Bicucculine-sensitive receptors that are sensitive to allosteric modulators of GABAA
receptors have also been observed in insects [26].
Two distinct types of ionotropic GABA receptor subunits have been cloned
from insects. Rdl has been cloned from many species, and ligand-gated chloride
channel homolog 3 (LCCH3) has been cloned from Drosophila. A third subunit,
the glycine-receptor-like subunit of Drosophila (GRD), has 40–44% identity to
vertebrate GABAA receptor α subunits and 40–41% identity to glycine receptor α
subunits, but shows more similarity to ligand-gated cation channels in a region
that determines ion selectivity. Accordingly, it coexpresses with LCCH3 to form a
GABA-gated cation channel [51]. While this receptor is very sensitive to blockade by
picrotoxin (PTX), it is insensitive to dieldrin and lindane; its sensitivity to fipronil
is not known.
The Rdl polypeptide of insects has about 30–38% identity to vertebrate GABA
receptor subunits (about the same as exists among the different types of vertebrate
subunits), and is no more similar to the GABAA than the GABAC types. In
fact, it resembles vertebrate glycine receptors more than it does GABA receptors,
but Rdl homomers do not respond to glycine. Rdl is expressed throughout the
adult and embryonic insect nervous system, but not in muscle; moreover, its
pharmacology resembles that of insect CNS GABA receptors and is distinct from
that of muscle GABA receptors. Homomeric Rdl receptors differ significantly from
native GABA receptors of cockroach dorsal unpaired median (DUM) neurons with
respect to sensitivity towards benzodiazepines [26], and single-channel studies have
demonstrated clear differences in conductance and gating between homomeric Rdl
and native Drosophila GABA receptors in isolated neurons [52]. On the other hand,
other native GABA receptor subtypes may exist that are not measured in the somata
of isolated neurons.
LCCH3 has 47% sequence identity to vertebrate GABA receptor β subunits, but
is unlikely to be a subunit of native insect GABA receptors. Although it can form
functional heteromultimers with Rdl subunits, these are unlike any known native
insect GABA receptors in that they are bicucculine-sensitive, PTX-insensitive, and
undergo a slow desensitization [26, 52]. Furthermore, the distributions of LCCH3
and Rdl in the Drosophila nervous system do not overlap [53].
32.6.2.3.2 Glutamate-Gated Chloride Channels A single insect GluCl gene,

DmGluClα, has been cloned from Drosophila, the expression of which yields
homomeric receptors with a pharmacology that is distinct from that of native
Drosophila GluCls and indicates that, like Rdl, it assembles with other subunits
in native receptors [54–56]. One population of receptors containing DmGluClα
but not Rdl, exists in the Drosophila nervous system and binds avermectin but
not nodulisporic acid, both of which are allosteric activators of GluCls. Another
population, assembled from both DmGluClα and Rdl subunits and binding both
avermectin and nodulisporic acid, also exists in the Drosophila nervous system.
However, the coexpression of DmGluClα and Rdl in the Xenopus oocyte expression
system does not yield functional heteromultimers, indicating that other subunits
may be needed [57].
Whilst the effect of NCAs on homomeric DmGluClα was not tested, the presence
of S at position 2 suggests that these channels would have a low sensitivity to most
NCAs. The Caenorhabditis elegans chloride channel subunit GluClα, with 2 T, was
almost insensitive to PTX, with an IC50 of 59 μM. GluClβ, but with 2 A it was very
sensitive, with an IC50 of 77 nM; mutation to 2 S conferred more than 10 000-fold
resistance [58]. GLC-3, a 2 T-containing GluCl from C. elegans, was insensitive to
PTX but was blocked by BIDN with an IC50 of 0.2 μM, and weakly by fipronil, with
an IC50 of 11.5 μM [59].
Isolated American cockroach neuronal somata contain two GluCl sub-
types – desensitizing (GluClD) and nondesensitizing (GluClN) [15] – which were
blocked by fipronil with IC50 -values of 800 and 10 nM, respectively. Fipronil
sulfone, a major bioactive metabolite of fipronil [60], blocked these two receptors
even better than did fipronil, with IC50 -values of 25 and 9 nM, respectively.
By comparison, fipronil and its sulfone blocked GABA receptors in American
cockroach neurons with IC50 -values of 27 and 20 nM, respectively [14].
GluClD channels in dieldrin-resistant German cockroaches containing the A2 S
mutation were less sensitive to glutamate and desensitized more slowly, compared
to GluClD channels from wild-type insects, which suggested that they might
contain the Rdl subunit. Fipronil sulfone blocked GluClD from both strains equally
well, with an IC50 of 38–40 nM. The nondesensitizing GluCls were rare in isolated
German cockroach neurons, and could not be studied [44].
32.6.2.3.3 Histamine-, Proton-, and Glycine-Gated Chloride Channels Two

histamine-gated chloride channel subunits (HisCl1 and HisCl2; Figure 32.6.3),
expressed in the Drosophila eye, both contain T at position 2 and are insensitive
to PTX and fipronil [61]. In addition, three novel proton-gated chloride channel
subunits that are splice variants from a single gene (pHClA, B, and C in
Figure 32.6.3), of unknown function, each contain M at position 2 and are also
insensitive to PTX and fipronil [62].
32.6.2.4 Mechanism of Blockade

The action of channel blockers can depend on the state of the channel. For example,
ligand-gated ion channels are predominantly in the resting state in the absence of
agonist, and can transition to activated, or open, states, and to desensitized states,
when an agonist is applied. The activation of GABA and GluCl receptors enhances
the blocking action of fipronil and its sulfone [14, 63, 64]. Furthermore, recovery
of the desensitizing GluCl from block by either fipronil or its sulfone requires
activation, whereas the recovery of nondesensitizing GluCls does not [14, 64]. PTX
is also an open channel blocker of GluCl-N, and prevents the access of fipronil to
its binding site in the channel [64].
Figure 32.6.3 Alignment of the cytoplasmic halves of the

M2 segments of various ligand-gated chloride channel
subunits. Species are given before the subunit name as RN
(rat), DM (Drosophila melanogaster), HV (Heliothis virescens),
and CE (Caenorhabditis elegans).
Single-channel measurements have shown that BIDN and fipronil reduce

the mean open time and increase the mean closed time, consistent with an
open-channel blocking mechanism [27, 65].
32.6.2.5 Structure of the Binding Site

While some earlier studies proposed multiple, partially overlapping NCA sites in
the GABA receptor [66], it is now thought that all NCAs bind at a single site within
the GABA receptor pore [67].
Because the A2 S mutation confers widely varying levels of resistance to various
NCAs, it was recognized as directly affecting their binding [68], thus locating
the NCA site within the M2 domain. The systematic mutation of suspected
channel-lining residues to cysteine, and showing that they were accessible to
irreversible modification by charged sulfhydryl reagents in functional receptors,
has been used to identify pore-lining residues [69]. This method has confirmed
that A2 lies within the pore, near its cytoplasmic mouth. T6 , located just one turn
deeper into the pore along the M2 helix, was also shown by this method to line the
pore.
A different approach [67] led to the same conclusion, and also showed that
residues A − 1 , A2 , T6 , and L9 , which are consecutively aligned on one face of
the M2 helix, line the pore. Furthermore, mutation of residues A2 , T6 , and L9
dramatically reduced binding of the ligands [3 H]EBOB and [3 H]BIDN, indicating
that these three residues contribute to binding interactions at the NCA site. By using
a homology model of the GABAA receptor [67], various NCAs could be manually
docked into the proposed binding site of a homopentameric mammalian GABAA -β3
receptor. Figure 32.6.4 shows the interactions of fipronil with, from the cytoplasmic
end outward, A2 , T6 , and L9 , when docked in a similar model. The detailed study
L
L
T T
T
A
A
A
Figure 32.6.4 Possible binding interactions Schrödinger Suite 2006 Induced Fit Dock-
of fipronil with side chains of residues A2 , ing protocol; Glide version 4.0, Schrödinger,
T6 , and L9 in the NCA site. Fipronil was LLC, New York, NY, 2005; Prime version
docked into a model of the NCA binding 1.5, Schrödinger, LLC, New York, NY, 2005.
site of a homopentameric mammalian Figure courtesy of Carsten Beyer, BASF AG.
GABAA -β3 receptor (after Ref. [68]), using the
of homology models may prove useful for a better understanding of the quantitative
structure–activity relationship (QSAR) of NCAs and the mechanism of resistance.
32.6.2.6 Mechanism of Insect Selectivity of Fipronil

Among the GABA receptor NCA insecticides, fiproles are the most insect-selective,
due to selectivity at the receptor level that is, in turn, dependent on subunit
composition, and which is seldom known [68]. Among most native ligand-gated
chloride channels, the NCA sites differ materially only with respect to the 2
residues of their component subunits (Figure 32.6.3). Native vertebrate GABAA
receptors require a β subunit for functionality, and there are a variety of receptor
subtypes in mammalian brain, all of which contain at least one β subunit. Casida’s
group has recently focused on the mammalian β3 subunit, the only mammalian
GABAA receptor subunit that forms functional homomers. Like all of the β
subunits, β3 contains 2 A, and β3 homomers are accordingly highly sensitive to
the NCA insecticides lindane, α-endosulfan, and fipronil. However, additional
subunits make the receptors more selective, especially with respect to fipronil.
Accordingly, rat β3 homomers were approximately as sensitive as house fly head
GABA receptors to fipronil, while α1 β3 γ2 multimers and native rat brain receptors
(the subunit compositions of which are unknown) were 10- and 800-fold less
sensitive, respectively [68–70].
32.6.3
Chemistry
32.6.3.1 Chemistry and Synthesis of Fiproles and Intermediates

The physico-chemical properties of fipronil and ethiprole are summarized in
Table 32.6.1.
A remarkable ring-closure reaction was elaborated for the synthesis of 3-
cyano-1-(phenyl)pyrazoles [5]. Initially, 2,6-dichloro-4-trifluoromethylaniline (7)
Table 32.6.1 Chemical and physical properties of fipronil and ethiprole [71, 72].
Property BSI common name
Fipronil (1) Ethiprole (2)
Melting point 201 ◦ C 160–165 ◦ C

Vapor pressure 3.7 × 10−7 Pa 9.1 × 10−8 Pa
Water solubility (20 ◦ C) 1.9–2.4 mg l−1 9 mg l−1
Log Pow (20 ◦ C) 4.00 2.9
Solubility in acetone 545.9 g l−1 90.7 g l−1
Patent EP 295117 DE 19653417
Company Rhône-Poulenc; Bayer Rhône-Poulenc; Bayer
CropScience, BASF CropScience, BASF
BSI, British Standards Institute.

was diazotized and then coupled with 2,3-dicyanopropionate to obtain phenyl-

diazoester (8); this was then cyclized and decarboxylated to 5-amino-3-cyano-
1-(2,6-dichloro-4-trichloromethyphenyl)pyrazole (9) [73] (Scheme 32.6.1).
The 4-trifluoromethylsulfinyl group can be introduced directly by treatment
of 9 with trifluoromethylsulfinyl chloride. Alternatively, the pyrazole-sulfide (10)
can be synthesized by sulfenylation of 9 with trifluoromethylsulfenyl chloride, or
with disulfur-dichloride to yield the dipyrazole-disulfide. The latter is then trifluo-
romethylated with bromotrifluoromethane in a radical-anion mediated reaction [5,
74, 75]. Oxidation of the pyrazole-sulfide (10) with peroxy agents leads to fipronil
(1) (Scheme 32.6.2).
O
CN NC
NH2 EtO CN
CI CI N N
N N NH2
CI CI CI CI
CF3
CF3 CF3
7 8 9
Scheme 32.6.1 Synthesis of 5-amino-3-cyano-pyrazole (9).
NC
CF3 N NH2
N
CI CI
CI CI
N
N NH2 S2CI2 CF3 9
F3CS(O)-CI
NC S F3CS-CI
O
NC S NC S CF3 NC S CF3
N 2F3C-Br N N
NH2 NH2 NH2
N Na2S2O4 N N
CI CI CI CI CI CI
H2O2
CF3 CF3 CF3

Dipyrazole-disulfide 10 1
Scheme 32.6.2 Three routes for sulfenylation and trifluoromethylation of

aminopyrazole (9).

A broad program of chemical structure variation has been carried out by several
agrochemical, pharmaceutical, and veterinary companies, as well as univer-
sity groups. The 2,6-dichloro-4-(trifluoromethyl)phenyl group always gave the
best insecticidal activity, and became known as the ‘‘Parnellophore’’ or ‘‘Magic
Aryl’’ group. Active phenyl variations are the 2-pyridyl analogs 12 [76, 77], the
2,6-dichloro-4-(pentafluorosulfenyl)phenyl group 13 [78], oxyfluormethylenes [79],
biphenyls [80], and other CF3 -substitutes [81]. All oxidation states of sulfur in
the 4-(halo)alkylthio group are suitable for a good intrinsic activity, but sulfides,
sulfoxides, and sulfones (11) deliver different in vivo activity levels due to their
polarity-related properties (Figure 32.6.5).
The 2,6-dichloro-4-(trifluoromethyl)phenyl-heterocycle-4-(halo)alkylthio scaffold
has been considered the essential toxophore. The heterocyclic unit and the sub-
stituents at the 3- and 5-positions on the heterocycle permit more variability, and
may serve as sites for prodrug attachment [82–85].
The heterocycles investigated include pyrazoles, triazoles [86–90], condensed
pyrazoles [91], pyridones, pyrimidones [92], pyrroles [93, 94], imidazoles [95, 96],
and indoles (14–21; Figure 32.6.6), but pyrazoles are preferred [97].
Structural extensions to the essential toxophore concept include replacement of
the 4-(halo)alkylthio group by imidazole [98] and other five-membered-heterocycles
[99], by phenyl [100], by cyclopropyl, and other cycloalkyls [101, 102], by alkyl,
haloalkyl, alkenyl, and alkinyl groups [81, 103] (22–25; Figure 32.6.7).
The aim of these structural variation studies was to improve the pest spectrum,
target receptor specificity, nontarget safety, environmental behavior, degradation,
and photostability. However, despite the tremendous effort of so many research
teams, none of the numerous variations has yet resulted in further development
products.
Although fipronil has a chiral, asymmetric sulfoxide group, various studies
have shown that there are no significant differences in activity between the
two enantiomers on cotton stainer (Dysdercus cingulatus), grain weevil (Sitophilus
granarius), or house fly (Musca domestica) [104].
R S(O)n (halo)alkyl R S(O)0,1,2 (halo)alkyl
N N
N NH2 NH2
N
CI CI CI
A
CF3 R4
11 n = 0 sulfide 12 A = N, R4 = CF3 : Pyridyl analogs

n = 1 sulfoxide
n = 2 sulfone 13 A = C-CI, R4 = SF5 : ICI-Fiproles
Figure 32.6.5 Sulfur oxidation states and phenyl variations.

SR R1 X
R1 R2 W Y
N N
N N
R N N R2 N N
N N
N R
CI CI CI CI CI CI
CI CI
CF3 CF3 CF3

CF3
Triazoles: 14 15 16 Annelated pyrazole 17
F3C CF3 R4 SR SR
Hal N N
Hal
N O N O R5 R2 R2 N R5
N
CI CI CI CI CI CI CI CI
CF3 CF3 CF3 CF3
Pyridones 18 Pyrimidones 19 Pyrroles 20 Imidazoles 21
Figure 32.6.6 Heterocycle variations.
R
R R4
R R Cyclopropyl R R4
N
R N N
N R N N
R N R
N
N CI CI
R CI CI CI CI
N
CI CI
CF3
CF3 CF3
R4 = Phenyl
CF3 R4 = Alkyl
Heteroaryl
Alkenyl
Alkinyl
22 23 24 25
4-Imidazolylpyrazole 4-(Het)arylpyrazole 4-Cyclopropylpyrazole 4-Alkyl/Alkenyl/Alkinyl-
pyrazole
Figure 32.6.7 Variations at the fourth-position of the heterocycle.
32.6.4
Biological Properties
32.6.4.1 General
Although fipronil has contact activity, it is particularly effective by ingestion.
Because the target receptors are in the insect CNS, mortality may appear to be
somewhat slow, although feeding cessation and other symptoms may be noted
soon after treatment.
Fipronil is not highly systemic, and the transport of fipronil and its significant
metabolites following uptake from soil is primarily in the xylem, with limited
movement in the phloem. When applied to the soil or seed, only small amounts are
taken up into plants, although highly susceptible leaf- and stem-feeding insects can
be controlled through seed treatment or in-furrow application in some cropping
systems at early growth stages [105].
32.6.4.2 Biological Spectrum

Fipronil has a high activity over a significant range of insects, and is registered for
the control of over 140 species on more than 100 crops worldwide:
• Insects in the orders Orthoptera (cockroaches, locusts), Isoptera (termites), and
Diptera (flies) are generally highly sensitive to fipronil, and can be controlled at
field use rates ranging between <1 and 25 g a.i. ha−1 .
• Insects in the orders Coleoptera (beetles and weevils), Thysanoptera (thrips),
Hemiptera (true bugs), as well as some families within the Lepidoptera (Plutelli-
dae, Pyralidae), show a high susceptibility to fipronil, and are generally controlled
with field use rates of 25–75 g a.i. ha−1 .
• The Homoptera (aphids and whiteflies) and Lepidoptera, Noctuidae (bollworms,
armyworms) show a low susceptibility to fipronil, requiring use rates of more
than 200 g a.i. ha−1 [8, 105].
The potential biological activity of fipronil is described in following subsections.
It should be borne in mind however that, as for any active substance, prod-
ucts containing fipronil must always be used according to applicable laws and
regulations.
32.6.4.3 Soil Applications

As a soil treatment, fipronil provides excellent control of a wide range of insect
pests in numerous crops, at rates of 50–200 g a.i. ha−1 .
One of the major uses of fipronil is the in-furrow, at-planting control of termites
(e.g., Heterotermes tenuis, Odontotermes takensis) in sugar cane, oil palm, and other
plantation crops. At higher usage rates (200–300 g a.i. ha−1 ), a single treatment
provides very good control in these long-cycle crops. In maize, good control of the
corn rootworm (D. virgifera virgifera), the first-generation European corn borer (Os-
trinia nubilalis), and wireworm (Agriotes spp.) is obtained with 100–150 g a.i. ha−1 ,
applied either as a granule or in-furrow spray at planting [105].
In paddy rice, the granular application of fipronil at 25–75 g a.i. ha−1 provides
control of virtually all major insect pests, including stem borers (Chilo spp.,
Tryporyza spp., Rupela spp., Ostrinia spp.), brown plant hopper (Nilaparvata lugens),
rice water weevil (Lissorhoptrus spp.), and thrips (Frankliniella spp., Stenchaetothrips
spp., Thrips spp., etc.). Fipronil can also be applied to rice seedbeds as a seedling
box treatment, with excellent results against a broad range of rice pests, including
rice water weevil, stem borers, and rice leaf folder (Cnaphalocrocis medinalis) [102].
Fipronil is also used for the control of insect pests in specialty crops. In bananas,
a good control of banana weevil (Cosmopolites sordidus) and of some thrips species
can be achieved with fipronil granules applied to the soil at the base of the mat
at 0.1–0.2 g a.i. ha−1 [105]. In vegetable crops, the in-furrow application of both
liquid and granular formulations of fipronil provides good control of root maggots
and thrips [106, 107].
32.6.4.4 Seed Treatment

Fipronil may be applied to seeds using several application methods, from
small-scale equipment to industrial-scale seed treatment stations.
Very good control of wireworm and white grubs in corn, and of wireworm in sun-
flower and sugar beet, has been demonstrated at the equivalent of 50–200 g a.i. ha−1
[71, 105]. A fipronil-based soybean seed treatment provides excellent control of white
grubs and soybean stem weevil (Sternechus subsignanthus), and has become a stan-
dard treatment in this key crop in Brazil [105]. At 10–50 g a.i. ha−1 , a good control
of several species of thrips (Thysanoptera) is obtained from a seed treatment in
cotton [108].
Fipronil’s high intrinsic activity against Dipterans allows its successful use as a
seed treatment in several crops for the control of root maggots. In cereals, fipronil
provides excellent control of wireworm and wheat bulb fly (Delia coarctata) at rates
of 50 g per 100 kg of seed [8]. In leeks, seeds that have been film-coated with fipronil
provided an excellent control of onion fly (Delia antiqua), as well as of thrips and
onion moth [109, 110].
32.6.4.5 Use in Crop Baiting Systems

Fipronil-based baiting systems for agricultural and fruit fly pests have either been
recently developed, or are currently in development. If suitable attractants and/or
feeding matrices are available, the nonrepellency and high activity of fipronil should
allow for the development of more of these insect control systems in the future.
Fipronil-based baits can be used to control ant pests in crops at rates as low as a
few grams, or even milligrams per hectare [105]. A bait matrix containing 0.003%
fipronil provided an excellent control of leafcutting ants (Atta spp.) in citrus and
eucalyptus [105, 111], while sucrose-based liquid baits permitted excellent control
of Argentine ants (Linepithema humile) and, indeed, may prove to be a valuable
tool for controlling these attendant ants in Californian grapes [112]. A low-assay
bait produced an excellent control of two pest species of Iridomyrmex in Australian
citrus [113].
Tephritid fruit flies are extraordinarily susceptible to fipronil which, when
combined with appropriate attractants, makes highly active bait formulations for
use in control and eradication programs. ‘‘Attract and Kill’’ stations have been
used successfully in eradication programs in South Pacific islands such as Nauru
and the Cook Islands [114]. Such stations, when using Cue-lure as an attractant,
have been shown to attract and control melon fly (Bactrocera cucurbitae) for up to
77 days, and oriental fruit fly (Bactrocera dorsalis) for up to 21 days [115]. Fipronil
has good activity in baits that target the blueberry maggot (Rhagoletis mendax)
when combined with olfactory and visual attractants [116, 117]. Several different
fipronil-based systems have been commercialized or are currently in development
for fruit fly control.
32.6.4.6 Urban Pest Control Applications

Outstanding activity against several urban insect pests, formulation flexibility (gel,
liquid, granule, bait, etc.), and horizontal transfer in termite, ant, and cockroach
populations, has quickly led to fipronil becoming one of the most successful urban
pest control agents.
As a liquid termiticide, fipronil provides long-term (>10 years) control of many
urban termite species when applied as registered [118]. The compound is not
detected in the soil by termites [119, 120], and its relatively slow action against both
subterranean termites (Reticulitermes flavus) and Formosan termites (Coptotermes
formosanus) [121] allows the transfer, through several routes, to other members
of the colony, leading to colony elimination [122]. The combination of excellent
toxicity to termites with an ability to be transferred to nestmates, has made fipronil
one of the best-selling liquid termiticides worldwide.
Fipronil is extraordinarily active against cockroaches, and shows no
cross-resistance with currently available chemistry [123, 124]. Gel bait formulations
assist transfer of the active ingredient from exposed individuals to unexposed
adults and nymphs, and this has been shown to be significant through several
different routes of exposure [125–127]. Fipronil-based gel bait formulations are
highly palatable to cockroaches, which appears to enhance their efficacy [128]. The
high intrinsic activity, and an excellent, palatable formulation have made gel baits
containing fipronil a key component in cockroach control.
Fipronil is also highly active against many nuisance ants. Exterior perimeter, or
surface barrier, treatments have provided excellent results against most key species,
including Pharaoh ants (Monomorium pharaonis) [129], Argentine ants (Linepithema
humile) [130] and a mixed population of eight different species [131]. Fipronil was
shown to be readily transferable among Argentine ants after crossing a treated sand
barrier [130], and this may partly explain the success of this treatment method.
The use of fipronil-based exterior perimeter sprays has become a key strategy for
the long-term control of ants infesting the interiors of structures. Fipronil provides
long-term control of red imported fire ant (Solenopsis invicta) in baits at rates as
low as 25.5–51 mg a.i. ha−1 , or with broadcast granular treatments [132].
32.6.4.7 Turf and Ornamental Applications

In turf and ornamentals, fipronil is extremely effective as a granular formulation
against the larvae of black vine weevils (Otiorhynchus sulcatus) in containerized
ornamentals [133], and also against many orthopteran insects, including mole
crickets (Scapteriscus spp.) [134] in turfgrass. Fipronil provides excellent control of
the Japanese beetle (Popillia japonica), when applied as a soil drench or injection to
field-grown ornamental trees.
32.6.4.8 Animal and Human Health Uses

Fipronil has high intrinsic activity against a wide range of animal health pests,
and has become the standard treatment for flea and tick control on domesticated
animals. Cat fleas (Ctenocephalides felis) are extremely sensitive to fipronil [135]
when applied as either a spray [136] or a ‘‘spot-on’’ [137] formulation to companion
animals. Fipronil is also highly active towards ticks [138, 139], and has been shown
to reduce the transmission of the tick-borne causative agents of canine diseases
[140, 141]. Fipronil has also been shown to control biting lice on both dogs and cats
[142, 143]; in this case, it can be applied as both a spray and a ‘‘spot-on’’ treatment,
as well as a combination product with S-methoprene.
A system for managing tick vectors of human Lyme disease has been developed
in the United States. The system comprises a plastic box that allows white-footed
mice – which are hosts to the nymphal deer tick vector (Ixodes scapularis) – to
enter and receive a swipe of a liquid fipronil formulation on their backs. This
system significantly reduces the number of adult and larval ticks on the mice, the
infection rate of the spirochete (Borrelia burgdorferi) in the mice, and the number of
host-seeking nymphs on treated properties [144]. The system effectively interrupts
the natural disease cycle, and, when used correctly, can lead to reduced numbers
of human cases of Lyme disease.
32.6.4.9 Resistance and Its Management

Resistance to fipronil was first recognized in Southeast Asia as early as 1996,
in the diamond-back moth (Plutella xylostella), within three years of fipronil’s
introduction. The high intrinsic activity of fipronil, coupled to a lack of alternatives
during the mid-1990s, led growers in countries such as Thailand to use it up to 40
times per year on cruciferous crops. Consequently, by early 1997 many populations
were resistant and field failures were widespread. At the same time, use of the
product against diamond-back moth began to decrease as new, novel chemistries
(indoxacarb, chlorfenapyr, spinosad) entered the Asian market.
By late 1998, a resistance monitoring program that had been put in place in 1995
began to show a dramatic recovery of sensitivity to fipronil in P. xylostella populations
from several locations in Thailand. In fact, in some areas the LC50 had returned
almost to baseline levels. The results of subsequent laboratory studies indicated that
resistant populations collected from Thailand demonstrated a significant loss of
fitness (K.A. Holmes, unpublished results). Studies on cockroaches demonstrated a
similar trend, with fipronil-resistant insects having a lower fitness than susceptible
insects [145]. Fipronil resistance in Plutella is incompletely recessive and controlled
by a single locus [146] which, in combination with the high fitness penalty, makes
rotation with other insecticides a very effective means of resistance management.
As the resistance monitoring program showed in Thailand, when selection pressure
was removed from the local populations of P. xylostella, the LC50 values returned
almost to baseline levels.
The Insecticide Resistance Action Committee (IRAC) mode of action (MoA)
classification (fiproles are classified as group 2B) is one of the most important tools
available for resistance management [1]. IRAC recommends rotation of insecticides
References 1301
so that successive generations of a population are not treated with insecticides from
the same MoA group.
References
1. Insecticide Resistance Action Commit- 15. Zhao, X., Salgado, V.L., Yeh, J.Z., and
tee (IRAC) (2011) Mode of Action Clas- Narahashi, T. (2004) Neurotoxicology,
sification, http//www.irac-online.org. 25, 967–980.
2. Jensen-Korte, U., Gehring, R., 16. Buckingham, S.D. and Sattelle, D.B.
Schallner, O., Stetter, J., Wroblowsky, (2004) in Comprehensive Molecular In-
H.-J., Becker, B., Homeyer, B., and sect Science, Insect Control, vol. 6 (eds
Behrenz, W. (1985) Patent EP 201,852, L.I. Gilbert, K. Iatrou, and S.S. Gill),
Bayer AG. Elsevier B.V., Oxford, p. 107.
3. Hatton, L.R., Hawkins, D.W., Parnell, 17. Casida, J.E. (1993) Arch. Insect Biochem.
E.W., Pearson, C.J., and Roberts, D.A. Physiol., 22, 13–23.
(1985) Patent WO 8,703,781, May & 18. Georghiou, G.P. (1986) Pesticide Re-
Baker. sistance, Strategies and Tactics for
4. Hatton, L.R., Hawkins, D.W., Parnell, Management, National Academy Press,
E.W., Pearson, C.J., and Roberts, D.A. Washington, DC, p. 14.
(1985) Patent EP 579,280, May & 19. Matsumura, F. and Ghiasuddin, S.M.
Baker. (1983) J. Environ. Sci. Health B, 18,
5. Buntain, I.G., Hatton, L.R., 1–14.
Hawkins, D.W., Pearson, C.J., and 20. Bentley, R. and Trimen, H. (1875)
Medicinal Plants, Being Descriptions with
Roberts, D.A. (1987) Patent EP 295,117,
Original Figures of the Principal Plants
May & Baker.
Employed in Medicine and an Account of
6. Schallner, O., Gehring, R., Klauke, E.,
Their Properties and Uses, Chapter Part
Stetter, J., Wroblowsky, H.-J., Schmidt,
2, Item 14, J&A Churchill, London,
R.R., and Santel, H.-J. (1984) Patent
p. 5.
DE 03,402,308, Bayer AG.
21. Takeuchi, A. and Takeuchi, N. (1969) J.
7. Hatton, L.R., Parnell, E.W., and
Physiol., 205, 377–391.
Roberts, D.A. (1982) patent WO
22. Lawrence, L.J. and Casida, J.E. (1984)
8,300,331, May&Baker.
Life Sci., 35, 171–178.
8. Colliot, F., Kukoroski, K.A., Hawkins,
23. Wafford, K.A., Sattelle, D.B., Gant,
D.W., and Roberts, D.A. (1992) D.B., Eldefrawi, A.T., and Eldefrawi,
Brighton Crop Prot. Conf. - Pests Dis., 1, M.E. (1989) Pestic. Biochem. Physiol., 33,
29–34. 213–219.
9. Meinke, P.T. (2001) J. Med. Chem., 44, 24. Rauh, J.J., Benner, E., Schnee, M.E.,
641–659. Cordova, D., Holyoke, C.W., Howard,
10. Londershausen, M. (1996) Pestic. Sci., M.H., Bai, D., Buckingham, S.D.,
48 (4), 269–292. Hutton, M.L., Hamon, A., Roush,
11. Cole, L.M., Nicholson, R.A., and R.T., and Sattelle, D.B. (1997) Br. J.
Casida, J.E. (1993) Pestic. Biochem. Pharmacol., 121, 1496–1505.
Physiol., 46, 47–54. 25. Buckingham, S.D., Hosie, A.M.,
12. Bloomquist, J.R. (1993) Comp. Biochem. Roush, R.L., and Sattelle, D.B. (1994)
Physiol. C, 106, 301–314. Neurosci. Lett., 181, 137–140.
13. Gant, D.B., Chalmers, A.E., Wolff, 26. Hosie, A.M., Aronstein, K.,
M.A., Hoffman, H.B., and Bushey, D.F. Sattelle, D.B., and Ffrench-Constant,
(1998) Rev. Toxicol., 2, 147–156. R.H. (1997) Trends Neurosci., 20,
14. Zhao, X., Yeh, J.Z., Salgado, V.L., and 578–583.
Narahashi, T. (2005) J. Pharmacol. Exp. 27. Grolleau, F. and Sattelle, D.B. (2000)
Ther., 314, 363–373. Br. J. Pharmacol., 130, 1833–1842.
28. Ffrench-Constant, R.H., Mortlock, D.P., 45. Ffrench-Constant, R.H., Rocheleau,

Shaffer, C.D., MacIntyre, R.J., and T.A., Steichen, J.C., and Chalmers, A.E.
Roush, R.T. (1991) Proc. Natl Acad. Sci. (1993) Nature, 363, 449–451.
USA, 88, 7209–7213. 46. Le Goff, G., Hamon, A., Berge, J.B.,
29. Ffrench-Constant, R.H., Roush, R.T., and Amichot, M. (2005) J. Neurochem.,
Mortlock, D., and Dively, G.P. (1990) J. 92, 1295–1305.
Econ. Entomol., 83, 1733–1737. 47. Raymond, V. and Sattelle, D.B. (2002)
30. Bloomquist, J.R., Ffrench-Constant, Nat. Rev. Drug Discov., 1, 427–436.
R.H., and Roush, R.T. (1991) Pestic. 48. Raymond-Delpech, V., Matsuda, K.,
Sci., 32, 463–469. Sattelle, B., Rauh, J., and Sattelle, D.
31. Ffrench-Constant, R.H., and Roush, (2005) Invertebr. Neurosci., 5, 119–133.
R.T. (1991) Genet. Res., 57, 17–21. 49. Knipple, D.C. and Soderlund, D.M.
32. Harrison, J.B., Chen, H.H., Sattelle, (2010) Pestic. Biochem. Physiol., 97,
E., Barker, P.J., Huskisson, N.S., Rauh, 140–148.
J.J., Bai, D., and Sattelle, D.B. (1996) 50. Ratra, G.S., Erkkila, B.E., Weiss, D.S.,
Cell Tissue Res., 284, 269–278. and Casida, J.E. (2002) Toxicol. Lett.,
33. Aronstein, K. and Ffrench-Constant, R. 129, 47–53.
(1995) Invertebr. Neurosci., 1, 25–31. 51. Gisselmann, G., Plonka, J., Pusch, H.,
34. Ffrench-Constant, R.H., Steichen, J.C., and Hatt, H. (2004) Br. J. Pharmacol.,
Rocheleau, T.A., Aronstein, K., and 142, 409–413.
Roush, R.T. (1993) Proc. Natl Acad. Sci. 52. Zhang, H.G., Lee, H.J., Rocheleau, T.,
Ffrench-Constant, R.H., and Jackson,
USA, 90, 1957–1961.
M.B. (1995) Mol. Pharmacol., 48,
35. Ffrench-Constant, R.H. (1993) Comp.
835–840.
Mol. Neurol., 63, 210–223.
53. Aronstein, K., Auld, V., and
36. Rufingier, C., Pasteur, N., Lagnel, J.,
Ffrench-Constant, R. (1996) Invertebr.
Martin, C., and Navajas, M. (1999)
Neurosci., 2, 115–120.
54. Smith, M.M., Warren, V.A., Thomas,
37. Anthony, N., Unruh, T., Ganser, D.,
B.S., Brochu, R.M., Ertel, E.A., Rohrer,
and Ffrench-Constant, R.H. (1998) Mol.
S., Schaeffer, J., Schmatz, D., Petuch,
Gen. Genet., 260, 165–175.
B.R., Tang, Y.S., Meinke, P.T.,
38. Ffrench-Constant, R.H., Anthony,
Kaczorowski, G.J., and Cohen, C.J.
N., Aronstein, K., Rocheleau, T., and
(2000) Biochemistry, 39, 5543–5554.
Stilwell, G. (2000) Annu. Rev. Entomol., 55. Kane, N.S., Hirschberg, B., Qian, S.,
45, 449–466. Hunt, D., Thomas, B., Brochu, R.,
39. Du, W., Awolola, T.S., Howell, P., Ludmerer, S.W., Zheng, Y., Smith, M.,
Koekemoer, L.L., Brooke, B.D., Arena, J.P., Cohen, C.J., Schmatz,
Benedict, M.Q., Coetzee, M., and D., Warmke, J., and Cully, D.F.
Zheng, L. (2005) Insect Mol. Biol., 14, (2000) Proc. Natl Acad. Sci. USA,
179–183. 97, 13949–13954.
40. Cole, L.M., Roush, R.T., and Casida, 56. Cully, D.F., Paress, P.S., Liu, K.K.,
J.E. (1995) Life Sci., 56, 757–765. Schaeffer, J.M., and Arena, J.P. (1996)
41. Scott, J.G. and Wen, Z.M. (1997) J. J. Biol. Chem., 271, 20187–20191.
Econ. Entomol., 90, 1152–1156. 57. Ludmerer, S.W., Warren, V.A.,
42. Bloomquist, J.R. (1994) Arch. Insect Williams, B.S., Zheng, Y., Hunt, D.C.,
Biochem. Physiol., 26, 69–79. Ayer, M.B., Wallace, M.A., Chaudhary,
43. Ozoe, Y., Ishikawa, S., Tomiyama, S., A.G., Egan, M.A., Meinke, P.T., Dean,
Ozoe, F., Kozaki, T., and Scott, J.G. D.C., Garcia, M.L., Cully, D.F., and
(2007) in Synthesis and Chemistry of Smith, M.M. (2002) Biochemistry, 41,
Agrochemicals VII (eds J.W. Lyga and 6548–6560.
G. Theodoridis), American Chemical 58. Etter, A., Cully, D.F., Liu, K.K., Reiss,
Society, Washington, DC, p. 39. B., Vassilatis, D.K., Schaeffer, J.M., and
44. Zhao, X. and Salgado, V.L. (2010) Arena, J.P. (1999) J. Neurochem., 72,
Pestic. Biochem. Physiol., 97, 153–160. 318–326.
References 1303
59. Horoszok, L., Raymond, V., Sattelle, 75. Clavel, J.-L., Pelta, I., Le Bars, S.,
D.B., and Wolstenholme, A.J. (2001) and Charreau, P. (1999) Patent WO
Br. J. Pharmacol., 132, 1247–1254. 01/30,760, Aventis CropScience.
60. Hainzl, D., Cole, L.M., and Casida, 76. Stetter, J., Alig, B., Marhold, A.,
J.E. (1998) Chem. Res. Toxicol., 11, Mencke, N., Mrusek, K., and Turberg,
1529–1535. A. (1994) Patent EP 679,650, Bayer
61. Zheng, Y., Hirschberg, B., Yuan, J., AG.
Wang, A.P., Hunt, D.C., Ludmerer, 77. Phillips, J.L., Timmons, P.R., Powell,
S.W., Schmatz, D.M., and Cully, D.F. G.S., Pilato, M.T., Chou, D.T.-W., and
(2002) J. Biol. Chem., 277, 2000–2005. Huang, J. (1991) Patent EP 500,209,
62. Schnizler, K., Saeger, B., Pfeffer, C., Rhône-Poulenc.
78. Salmon, R. (1991) Patent WO
Gerbaulet, A., Ebbinghaus-Kintscher,
93/06,089, ICI.
U., Methfessel, C., Franken, E.M.,
79. Uekawa, T. and Tomoika, H. (1997)
Raming, K., Wetzel, C.H., Saras, A.,
Patent EP 913,398, Sumitomo.
Pusch, H., Hatt, H., and Gisselmann,
80. Herman, N.D., Huber, S.K., Huang, J.,
G. (2005) J. Biol. Chem., 280,
and Timmons, P. (1996) Patent EP
16254–16262. 839,810, Rhône-Poulenc.
63. Zhao, X., Salgado, V.L., Yeh, J.Z., and 81. Casida, J.E., Sammelson, R.E., Caboni,
Narahashi, T. (2003) J. Pharmacol. Exp. P., and Durkin, K.A. (2004) Bioorg.
Ther., 306, 914–924. Med. Chem., 12, 3345–3355.
64. Zhao, X., Yeh, J.Z., Salgado, V.L., and 82. Okui, S., Kyomura, N., Fukuchi, T.,
Narahashi, T. (2004) J. Pharmacol. Exp. and Tanaka, K. (1997) Patent WO
Ther., 310, 192–201. 98/45,274, Mitsubishi Chem.
65. Ikeda, T., Nagata, K., Kono, Y., Yeh, 83. Manning, D., Pilato, M., Wu, T.-T.,
J.Z., and Narahashi, T. (2004) Pest and Hawkins, D.W. (1996) Patent WO
Manage. Sci., 60, 487–492. 98/28,279, Rhône-Poulenc.
66. Deng, Y.L., Palmer, C.J., and Casida, 84. Kando, Y. and Kiji, T. (2000) Patent
J.E. (1993) Pestic. Biochem. Physiol., 47, WO 01/40,203, Takeda Chem.
98–112. 85. Huang, J., Lowder, P.D., Ray, N.C.,
67. Chen, L., Durkin, K.A., and Casida, J.E. and Hawkins, D.W. (1996) Patent EO
(2006) Proc. Natl Acad. Sci. USA, 103, 780,378, Rhône-Poulenc.
5185–5190. 86. Tomioka, H., Furukawa, T., Takada, Y.,
68. Ratra, G.S., Kamita, S.G., and Casida, and Takano, H. (1996) Patent EP
J.E. (2001) Toxicol. Appl. Pharmacol., 780,381, Sumitomo.
172 (3), 233–240. 87. Willis, R.J. and Marlow, I.D. (1989)
69. Caboni, P., Samuelson, R.E., and Patent EP 400,842, Schering Agro-
chemicals.
Casida, J.E. (2003) J. Agric. Food Chem.,
88. O’Mahony, M.J., Boddy, I.K., Briggs,
51 (24), 7055–7061.
G.G., Harrison, R.P., Jones, T.H.,
70. Ratra, G.S. and Casida, J.E. (2001) Tox-
Marlow, I.D., Roberts, B.G., Willis, R.J.,
icol. Lett., 122 (3), 215–222.
Bardsley, R., and Reid, J. (1996) Pestic.
71. Wilde, G., Roozeboom, K., Claassen,
Sci., 48, 189–196.
M., Janssen, K., and Witt, M. (2004) J. 89. Ozoe, Y., Alam, M.S., Kajiki, R.,
Agric. Urban Entomol., 21 (2), 75–85. Hanatani, H., Kong, X., Ozoe, F.,
72. Anonymous. Ethiprole Technical Matsui, Y., and Matsumura, F. (2006)
Bulletin, Bayer CropScience. J. Agric. Food Chem., 54, 1361–1372.
73. Hawkins, D.W., Roberts, D.A., 90. O’Mahony, M.J. and Willis, R.J. (1988)
Wilkinson, J.H., and Clavel, J.-L. (1997) Patent EP 350,237, Schering Agro-
Patent WO 97/32,843, Rhône-Poulenc. chemicals.
74. Wakselman, C., Clavel, J.-L., Langlois, 91. Dhanoa, D., Meegalla, S., Soll, R.M.,
B., Nantermet, R., and Tordeux, M. Doller, D., Sha, D., Liu, R., and Silver,
(1992) J. Chem. Soc. Perkin Trans. 1, G. (1999) Patent WO 01/25,241, 3-DP,
3371–3375. Heska Corporation.
92. Whittle, A.J. (Zeneca) (1994) in Ad- 109. Ester, A., De Vogel, R., and Bouma, E.
vances in the Chemistry of Insect Control (1997) Crop Prot., 16 (7), 673–677.
III (ed. G.G. Briggs), Royal Society of 110. Ester, A. and Huiting, H.F. (2001)
Chemistry, Cambridge, pp. 156–170. Brighton Crop Protection Conference,
93. Timmons, P., Outcalt, R., Cramp, S., Symposium Proceedings, vol. 76 (Seed
Kwiatkowski, P., Lopes, A., Sinodis, D., Treatment), pp. 159–166.
and Cain, P. (1988) Patent EP 372,982, 111. Grosman, D.M., Upton, W.W.,
Rhône-Poulenc. McCook, F.A., and Billings, R.F.
94. Timmons, P., Outcalt, R., Kwiatkowski, (2002) Southwest. Entomol., 27 (3/4),
P., Lopes, A., Sinodis, D., Cain, P., 251–256–.
Hall, L.S., and Vors, J.-P.A. (1990) 112. Hooper-Bui, L.M. and Rust, M.K.
Patent EP 460,940, Rhône-Poulenc. (2000) J. Econ. Entomol., 93 (3),
95. Powell, G.S., Sinodis, D.N., Timmons, 858–869.
P.R., and Wu, T.T. (1989) Patent EP 113. Stevens, M.M., James, D.G., and
396,427, Rhône-Poulenc. Schiller, L.J. (2002) J. Appl. Entomol.,
96. Powell, G.S., Sinodis, D.N., Timmons, 126 (9), 490–496.
P.R., Wu, T.T., Chou, D.T.-W., 114. Allwood, A.J., Vueti, E.T., Leblanc, L.,
Newsome, P.W., and Hall, L.S. (1990) and Bull, R. (2002) in Proceedings of the
Patent EP 484,165, Rhône-Poulenc. International Conference on Eradication
97. Huang, J., Huber, S.K., Smith, P.H.G., of Island Invasives (eds C.R. Veitch
and Wilkinson, J.H. (1996) Patent EP and M.N. Clout), IUCN Publications
738,713, Rhône-Poulenc. Services Unit, Cambridge, UK, pp.
98. Willis, R.J., O’Mahony, M.J., and 19–25.
Roberts, B.G. (1989) Patent EP 412,849, 115. Vargas, R.I., Stark, J.D., Mackey, B.,
Schering Agrochemicals. and Bull, R. (2005) J. Econ. Entomol., 98
99. Banks, B.J. (1996) Patent EP 846,686, (5), 1551–1159.
Pfizer. 116. Barry, J.D., Polavarapu, S., and
100. Meegalla, S.K., Doller, D., Sha, D., Soll, Teixeira, A.F.L. (2004) J. Econ. Entomol.,
R., Wisnewski, N., Silver, G.M., and 97 (6), 2006–2014.
Dhanoa, D. (2004) Bioorg. Med. Chem. 117. Barry, J.D. and Polavarapu, S. (2005)
Lett., 14, 4949–4953. Fla Entomol., 88 (3), 268–277.
101. Banks, B.J. (1996) Patent WO 118. Wagner, T.L., Peterson, C.J.,
98/24,767, Pfizer. Mulrooney, J.E., and Shelton, T.G.
102. Gladwell, I., Matthews, J.G., and (2005) Pest Control, 43–50.
Pettman, A.J. (2003) Patent WO 119. Remmen, L.N. and Su, N. (2005) J.
2005/023,773, Pfizer. Econ. Entomol., 98 (3), 906–910.
103. Banks, B.J. (1995) Patent WO 120. Ping, H.X. (2005) J. Econ. Entomol., 98
97/07,102, Pfizer. (2), 509–517.
104. Teicher, H.B., Kofoed-Hansen, B., and 121. Remmen, L.N. and Su, N. (2005) J.
Jacobsen, N. (2003) Pest Manage. Sci., Econ. Entomol., 98 (3), 911–915.
59, 1273–1275. 122. Shelton, T.G. and Grace, J.K. (2003) J.
105. Holmes, K. (2006) Fipronil Worldwide Econ. Entomol., 96 (2), 456–460.
Technical Bulletin, BASF Corporation. 123. Kaakeh, W., Reid, B.L., and Bennett,
106. Hoepting, C.A., Scott-Dupree, C.D., G.W. (1997) Entomol. Exp. Appl., 84 (3),
Harris, C.R., Ritcey, G., and McDonald, 229–237.
M.R. (2000) Brighton Crop Prot. Conf. 124. Tilak, R., Tilak, V.W., Yadav, J.D., and
– Pests Dis., 1, 279–284. Gupta, K.K. (2002) J. Commun. Dis., 34
107. Jukes, A.A., Collier, R.H., and Finch, S. (1), 659.
(2001) Meded.–Fac. Landbouwkd. 125. Durier, V. and Rivault, C. (2000) J.
Toegep. Biol. Wet. (Univ. Gent), 66 Econ. Entomol., 93 (2), 434–440.
(2a), 395–402. 126. Le Patourel, G. (2000) Pest Manage.
108. Hamon, N., Shaw, R., and Yang, H. Sci., 56 (9), 732–736.
(1996) Proc. Beltwide Cotton Conf., 2, 127. Buczkowski, G. and Schal, C. (2001) J.
759–764. Econ. Entomol., 94 (3), 680–685.
32.7 Chloride Channel Activators/New Natural Products: Avermectins and Milbemycins 1305
128. Durier, V. and Rivault, C. (2000) Med. 138. Davey, R.B., George, J.E., Hunter, J.S.
Vet. Entomol., 14 (4), 410–418. III, and Jeannin, P. (1999) Exp. Appl.
129. Buczkowski, G., Scharf, M.E., Ratliff, Acarol., 23 (4), 351–364.
C.R., and Bennett, G.W. (2005) J. Econ. 139. Burridge, M.J., Simmons, L., and
Entomol., 98 (2), 485–492. Allan, S.A. (2004) J. Agric. Urban
130. Soeprono, A.M. and Rust, M.K. (2004) Entomol., 20 (4), 207–219.
J. Econ. Entomol., 97 (6), 2021–2028. 140. Davoust, B., Marie, J.L., Mercier, S.,
131. Scharf, M.E., Ratliff, C.R., and Bennett, Boni, M., Vandeweghe, A., Parzy, D.,
G.W. (2004) J. Econ. Entomol., 97 (2), and Beugnet, F. (2003) Vet. Parasitol.,
601–605. 112 (1-2), 91–100.
132. Loftin, K., Hopkins, J., Gavin, J., and 141. Jacobson, R., McCall, J., Hunter, J.
III, Alva, R., Irwin, J., Eschner, A.,
Shanklin, D. (2004) J. Agric. Urban
Jeannin, P., and Boeckh, A. (2004) J.
Entomol., 20 (3), 151–156.
Appl. Res. Vet. Med., 2 (1), 39–45.
133. Parsons, R.G., Pearce, M.A., Hingley,
142. Pollmeier, M., Pengo, G., Jeannin, P.,
P.J., Lankford, W.T., and James, D.A.
and Soll, M. (2002) Parasitology, 107
(1998) Brighton Crop Prot. Conf. – Pests
(1-2), 127–136.
Dis., 3, 819–822.
143. Pollmeier, M., Pengo, G., Longo, M.,
134. Brandenburg, R.L., Xia, Y., and and Jeannin, P. (2004) Vet. Parasitol.,
Watson, B. (2005) J. Entomol. Sci., 121 (1-2), 157–165.
40 (2), 115–125. 144. Dolan, M.C., Maupin, G.O., Schneider,
135. Moyses, E.W. and Gfeller, F.J. (2001) J. B.S., Denatale, C., Hamon, N.,
Med. Entomol., 38 (2), 193–196. Cole, C., Zeidner, N.S., and Stafford,
136. Payne, P.A., Dryden, M.W., Smith, V., K.C. III (2004) J. Med. Entomol., 41 (6),
and Ridley, R.K. (2001) Vet. Parasitol., 1043–1054.
102 (4), 331–340. 145. Wang, C., Scharf, M.E., and Bennett,
137. Medleau, L., Clekis, T., McArthur, T.R., G.W. (2004) J. Econ. Entomol., 97 (6),
Alva, R., Barrick, R.A., Jeannin, P., and 2067–2072.
Irwin, J. (2003) J. Small Anim. Pract., 146. Sayyed, A.H. and Wright, D.J. (2004) J.
44 (2), 71–75. Econ. Entomol., 97 (6), 2043–2050.
32.7
Chloride Channel Activators/New Natural Products: Avermectins and Milbemycins
Thomas Pitterna
32.7.1
Introduction
To date, three compounds from this group of chloride channel activators

(abamectin, emamectin benzoate, and milbemectin) have been commercialized in
crop protection. In addition, a development compound – lepimectin (provisionally
approved ISO common name) – has also been investigated. In this chapter, the
origin, synonyms, and physico-chemical properties of the marketed compounds
[1] are first summarized (see Tables 32.7.1 and 32.7.2), followed by details of the
compounds’ mode of action, discovery, chemistry, insecticidal activity, agronomic
use, and safety. The family of avermectins and milbemycins, which has gained
importance not only in crop protection but also in the field of animal health, has
been recently reviewed [2–7].
• Abamectin, which is isolated from the fermentation of Streptomyces avermitilis,

a naturally occurring soil Actinomycete (see Table 32.7.2), possesses strong
anthelmintic, insecticidal, and acaricidal activities [8–13]. It was introduced as an
acaricide and insecticide by Merck Sharp and Dohme Agvet (now Syngenta Crop
®
Protection AG) in 1985. Abamectin is also the active ingredient of Avicta , the
®
first seed treatment against nematodes in agriculture. Since 1997, Avicta has
been launched in several countries by Syngenta Crop Protection AG.
• Emamectin benzoate is produced by chemical synthesis from abamectin
(Table 32.7.2) [14–17]. The extreme potency of this compound against
Lepidoptera was discovered at Merck [18]. Emamectin benzoate was introduced
to the market by Novartis (now Syngenta Crop Protection AG) in 1997.
• Milbemectin is isolated from the fermentation of Streptomyces hygrocopicus, another
naturally occurring soil Actinomycete. It was introduced as an acaricide by Sankyo
Co., Ltd in 1990 (Table 32.7.2).
32.7.2
Mode of Action
The biochemical mode of action of the avermectins and milbemycins has been
discussed in several reviews [2–6]. All natural and semisynthetic avermectins
and milbemycins interact with ligand-gated chloride channels, which are located
in the nerve cells of their target organism. In particular, they act on inverte-
brate glutamate-gated chloride channels and some vertebrate and invertebrate
γ -aminobutyric acid (GABA) receptors.
Binding of the neurotransmitter, such as glutamate and GABA, renders the
channel transiently permeable to chloride ions. The avermectins and milbemycins
exert their action by potentiating the effect of the neurotransmitter, thus increasing
the influx of chloride ions into nerve cells. This results in a disruption of nerve
impulses and cell function such that invertebrates are rapidly paralyzed.
The key findings relating to the pharmacological effects of avermectins and
milbemycins on different target organisms are summarized in the following
subsections. Although, initially, Fritz et al. [19] showed that avermectins would act
as chloride channel agonists and thus open chloride channels, it was subsequently
shown that they could also act at a site different to that of the cyclodiene insecticides
[20]. Following the discovery by Cassida and colleagues that avermectins bound
to saturable, high-affinity binding sites in Drosophila melanogaster [21], it was
also shown – for several avermectin analogs – that such binding (expressed as
IC50 -values for the displacement of radiolabeled avermectin) correlated well with
the compounds’ insecticidal activities against flies. A binding site related to a
glutamate-gated channel in D. melanogaster has been found [22, 23] and transcripts
related to the same subunit (DrosGluCl-alpha) identified in other insects, such
as cat flea (Ctenocephalides felis), fall armyworm (Spodoptera frugiperda), and cotton
bollworm (Helicoverpa zea). However, no such findings have yet been reported for
spider mites.
Table 32.7.1 Names and codes of market products.
Abamectin Emamectin benzoate Milbemectin
Structure
OMe OMe
O
HO HN
O R
OMe OMe
O O O O
O O
OH
O O O O O O
O O milbemycin A3: R = CH3
H CO2H H O
R R milbemycin A4: R = C2H5
O O O O OH
OH OH
> 80% B1a: R = C2H5 > 90% B1a: R = C2H5

< 20% B1b: R = CH3 O O
< 10% B1b: R = CH3
OH OH
Common names
Abamectin, abamectine Emamectin, emamectine Milbemectin
Other names
Avermectin B1
Composition
Mixture containing >80% Mixture of emamectin B1 a Mixture of the homologs milbemycin A3 (methyl)
Avermectin B1 a and <20% (>90%) and emamectin and milbemycin A4 (ethyl) in the ratio 3 : 7
Avermectin B1 b B1 b (<10%), as their
benzoate salts
32.7 Chloride Channel Activators/New Natural Products: Avermectins and Milbemycins
Selected product names

Agrimec, Dynamec, Banlep, Denim, Proclaim Milbeknock, Ultiflora, Koromite, Matsuguard,
Vertimec, Affirm, Agri-Mek, (Syngenta) Mesa (Sankyo Agro)
1307
Avid, Clinch, Zephyr

(Syngenta)
Table 32.7.2 Properties of market products.
Property Abamectin Emamectin benzoate Milbemectin
Melting point 161.8–169.4 ◦ C 141–146 ◦ C 212–215 ◦ C

(decomp.)
Solubility in water 0.007–0.010 mg l−1 24 mg l−1 (25 ◦ C, 7.20 mg l−1 (A3 , 20 ◦ C)
(20 ◦ C) pH 7) 0.88 mg l−1 (A4 , 20 ◦ C)
Solubility in organic Toluene 350 g l−1 Toluene 20 g l−1 Benzene 143.1 g l−1
−1
solvents Cyclohexane 6 g l Cyclohexane 0.23 g l−1 n-Hexane 1.4 g l−1
(all 21 ◦ C) (all 25 ◦ C) (all 20 ◦ C)
Partition coefficient Log Pow = 4.4 ± 0.3 Log Pow = 3.0 (pH 5.1) Log Pow = 5.3 (A3 )
(pH 7.2) Log Pow = 5.0 (pH 7.0) Log Pow = 5.9 (A4 )
Log Pow = 5.9 (pH 9.0) –
Vapor pressure <3.7 × 10−3 mPa 4 × 10−3 mPa (21 ◦ C) <1.3 × 10−5 mPa
(25 ◦ C) (20 ◦ C)
Dissociation constant – pKa,1 = 4.18 –

pKa,2 = 8.71
The avermectins and milbemycins are taken up by insects and mites via contact
and ingestion; under field conditions, ingestion is the primary route of uptake
[24]. Although the maximum mortality of affected insects may occur at two to
four days after compound application, feeding ceases very quickly after treatment
as a result of irreversible paralysis; consequently, feeding damage to the crops is
prevented.
32.7.3
Discovery and Chemistry of Avermectins
The naturally occurring avermectins are a group of 16-membered macrocyclic

lactones, which are produced by fermentation from Actinomycetes from the
genus Streptomyces (Figure 32.7.1). The soil microorganism Streptomyces avermitilis
MA-4860 (NRRL 8165) was first isolated at Merck Research Laboratories in 1976
from a soil sample of Japanese origin, collected by the research team at the Kitasato
Institute [25]. From the fermentation, eight different avermectins were isolated,
consisting of four pairs of homologs. Each pair was shown to contain a major (a)
component) and a minor (b) component; these are usually produced in a ratio of
between 80 : 20 and 90 : 10.
One of these homolog pairs, avermectin B1 [i.e., a mixture of avermectins B1 a
(>80%) and B1 b (<20%)], is commonly referred to as abamectin (Figure 32.7.2).
Abamectin was found to be active against nematodes [8, 9], insects [10–12], and
mites [13], and was subsequently selected for development in crop protection. As
OMe
HO
OMe R1 A-B R2
O O A1a -OMe -CH=CH- s-butyl
B
A A1b -OMe -CH=CH- i-propyl
O O O
O R2 A2a -OMe -CH2-CHOH- s-butyl
A2b -OMe -CH2-CHOH- i-propyl

O O
OH B1a -OH -CH=CH- s-butyl
B1b -OH -CH=CH- i-propyl

O B2a -OH -CH2-CHOH- s-butyl
R1 B 2b -OH -CH2-CHOH- i-propyl
Figure 32.7.1 Structures of the naturally occurring avermectins.
a consequence, it was introduced to the market place as an agricultural pesticide

against a broad spectrum of phytophagous mites and insects in 1985. The research
group at Merck then carried out a targeted analoging program around abamectin,
with attention focused on identifying a compound that showed activity against a
broad spectrum of Lepidoptera [26–29]. The program culminated in the discovery
of emamectin [18], initially as the hydrochloride salt (code number MK-243), but
this was later developed as the benzoate salt (MK-244) for the control of Lepidoptera
in crop protection. In 1997, emamectin benzoate was introduced to the market by
®
Novartis (now Syngenta Crop Protection AG) under the trade names Proclaim
®
and Affirm .
Emamectin is prepared from abamectin in four chemical steps [14–17]; thus, it
is also a mixture of the two homologs B1 a and B1 b. The allylic hydroxy group at
C5 of the avermectins is the most reactive in the molecule, and must be protected
before any reactions on the C4 hydroxy group can be performed. Reaction with
t-butyl-dimethylchlorosilane and imidazole in N,N-dimethylformamide gives the
5-O-t-butyldimethylsilyl ether (Scheme 32.7.1).
Alternatively, the C5 hydroxy group can be protected as the 5-O-allyloxycarbonyl
derivative (not shown in Scheme 32.7.1). This is achieved by the reaction of
abamectin with allylchloroformate and tetraethylendiamine in t-butyl methyl ether.
In this case, the C5 hydroxy group can be deprotected in the last step by treatment
with sodium borohydride in ethanol, in the presence of catalytic amounts of
tetrakis(triphenylphosphine)palladium.
After protection, the C4 hydroxy group is oxidized to the ketone; this can be
achieved with dimethylsulfoxide and phenyldichlorophosphate (or oxalyl chloride)
in the presence of triethylamine. Subsequently, the reductive amination of the
ketone can be performed with methylamine, acetic acid, and sodium borohydride
in methanol. Alternatively, the ketone can be treated with heptamethyldisilazide
and zinc chloride in iso-propyl acetate, followed by a reduction of the intermediate
imine with sodium borohydride in the presence of ethanol. This transformation
OMe OMe
HO HN
OMe OMe
O O O O
23
22
O O O 25 O O O
O O
H H
R R
O O O O
CO2H
> 80% B1a: R = C2H5 OH OH
< 20% B1b: R = CH3
O O
OH OH
Abamectin (Vertimec®, Agrimec®) Emamectin Benzoate (Proclaim®, Affirm®)
OMe OMe
HO HO
OMe OMe
O O O O
23
22
O O O O O O 25
O O
H
R
O O O O
> 80% B1a: R = C2H5 OH OH
< 20% B1b: R = CH3
O O
OH OH
® ®
Ivermectin (Heartguard , Ivomec ) Doramectin (Dectomax®)
O
OMe
HN
OMe
OMe
HO
O O
O O O
O O O
O
O
H
R O O
O O
OH
> 80% B1a: R = C2H5 OH
< 20% B1b: R = CH3
O
O
N
OH HO
Eprinomectin Selamectin
Figure 32.7.2 Structures and names of avermectins.

OMe OMe
HO HO
4" 4"
OMe OMe
O O O O
O O O O O O
O O
H H
R R
> 80% B1a: R = C2H5 O O O O
TBDMS-Cl
< 20% B1b: R = CH3 OH OH
DMF, imidazole
O 5 O 5
OH O
Si
OMe
O
4" OMe
OMe
O O HN
4"
OMe
O O O O O
O
H O O O
R
O O O
H
OH R
1. MeNH2, AcOH, MeOH, NaBH3CN
PhOP(O)Cl2 or (COCl)2 or (Me3Si)2NCH3, ZnCl2, NaBH4 O O
5 OH
O
DMSO, E3N 2. MeSO3H, MeOH
O 5
Si O
emamectin OH
Scheme 32.7.1 The synthesis of emamectin.

1311
leads to the (R)-configured 4 -desoxy-4 -epi-methylamino derivative as the predom-

inant product, with only very small amounts of the 4 -(S)-isomer being formed.
Deprotection, for example, with methanesulfonic acid in methanol, completes the
synthesis of emamectin.
In addition to its use in crop protection, abamectin has been commercialized as an
antiparasitic drug for use in animals. Further avermectin derivatives – ivermectin
(Merck), doramectin (Pfizer), eprinomectin (Merck), and selamectin (Pfizer) –
were commercialized as endo- and ectoparasiticides (Figure 32.7.2; as these four
compounds are not used in crop protection, they will not be discussed in detail
here):
• Ivermectin [30], which is derived from avermectin B1 via selective hydrogenation of
the 22,23 double bond, has been used (on a commercial basis) as an anthelmintic
in farm animals since 1981. In dogs, it is used to prevent heartworm infections,
and it has also been used in human medicine to treat onchocerciasis (river
blindness) [31].
• Eprinomectin [32], 4 -desoxy-4 -acetylamino-avermectin B1 , is an advanced de-
velopment product from Merck, and is used as a broad-spectrum parasiticide
in farm animals. Quite similar to emamectin, eprinomectin is produced from
avermectin B1 by chemical synthesis.
• Doramectin [33, 34], another avermectin derivative for animal health applications,
has been developed by Pfizer. The compound has the same structure as aver-
mectin B1 a, except that a cyclohexyl group replaces the s-butyl substituent at C25.
Doramectin is produced by fermentation, using a mutant Streptomyces strain
capable of incorporating the cyclohexyl substituent at C25 from a source that is
added to the fermentation medium.
• Selamectin [34, 35] is a new experimental antiparasitic drug introduced by Pfizer.
It is a semisynthetic avermectin-monosaccharide derivative, and it is produced
from doramectin by chemical synthesis.
32.7.4
Discovery and Chemistry of Milbemycins
The discovery of milbemycins was first reported in 1974 by the research team
at Sankyo [36, 37], using the original producing strain SANK 60576 (designated
Streptomyces hygroscopicus subsp. aureolacrimosus) [38]. Numerous fermentation
products are available from this Actinomycete and its mutants; a total of 13
milbemycins was isolated from the original strain, referred to as α1 to α10 and β1
to β3 [39], although later the α1 component was named milbemycin A3 , and the
α3 component milbemycin A4 . Additional derivatives were isolated from mutant
strains [40, 41], among them milbemycin D (Figure 32.7.3).
Milbemectin, which applies to a mixture of milbemycins A3 and A4 (also referred
to as milbemycin A3 /A4 ), was launched as an acaricide under the trade name
®
Milbeknock by Sankyo in 1990. Although, to date, this is the only commercially
available milbemycin for crop-protection use, a second compound was expected
to be launched by Sankyo, referred to as lepimectin (provisionally approved ISO

common name) in 2004. Lepimectin, as a semisynthetic derivative of milbemectin,
contains A3 and A4 components, with the latter contributing the major part.
The synthesis of lepimectin (Scheme 32.7.2), as described below and reported in
the patent literature [42–44], may be modified for industrial preparation. In order to
introduce the required oxygen functionality at C13, milbemycin A3 /A4 is first pro-
tected as the 5-O-trimethylsilyl ether; a subsequent reaction with 3-chloroperbenzoic
acid results in epoxidation of the double bond between C14 and C15. The epoxide
is rearranged by treatment with a mild Lewis acid (trimethylsilyl triflate), and the
product is deprotected. In order to suitably protect the sensitive allylic C5 hydroxy
group, it is first oxidized to the ketone, after which the C13 ester substituent is in-
troduced by an acid-mediated substitution, accompanied by allylic rearrangement.
Notably, the resulting stereochemical orientation of the C13 substituent is opposite
to that found in avermectin derivatives. The sequence is completed by a reduction
of the C5 ketone with sodium borohydride, thereby restoring the functionality and
natural stereochemistry of the C5 hydroxy group.
O O O
O R N O R
O
O O O O
OH milbemycin A3: R = CH3 OH major: R = C2H5
milbemycin A4: R = C2H5 minor: R = CH3
O O
OH OH
Milbemectin Lepimectin
(Koromite®, Milbeknock®) (provisionally approved ISO common name)
O
N
O
O O
O R
O O
O O
O O O O
OH A3: R = CH3
OH OH
A4: R = C2H5
O
O O
N
OH HO OH
milbemycin D milbemycin 5-oxime Moxydectin

(Interceptor®, Milbemax®) (Cydectin®, Moxydec®)
Figure 32.7.3 Structures and names of milbemycins.

O O O
13
O
1314
O O O
R R R
TMS-Cl MCPBA O O
O O O O
OH Imidazole OH OH
O 5 O O
OH O O
Si
32 Nervous System
Si
Milbemycin A3/A4
O
O O
O
OH O O
R OH OH
O R R
TMS-OTf O MnO2
p-Tos-OH O O O O
OH
Lutidine OH OH
O
O O
O
Si OH O
O O
O
O O O 13 O
OH
N O N O
N O O
O R R
NaBH4 O
O O O
OH OH
TFA
R = CH3 ca. 80% (A4)
O R = H ca. 20% (A3) O 5
O OH
lepimectin
Scheme 32.7.2 The synthesis of lepimectin.

Table 32.7.3 Activity of abamectin against mites and insects.
Mite species LC90 (ppm)
Phyllocoptruta oleivora (citrus rust mite) 0.02

Tetranychus urticae (two-spotted spider mite) 0.03
Panonychus ulmi (European red mite) 0.04
Polyphagotarsonemus latus (broad mite) 0.05
Panonychus citri (citrus red mite) 0.24
Insect species LC90 (ppm)

Manduca sexta (tobacco hornworm) 0.02
Leptinotarsa decemlineata (Colorado potato beetle) 0.03
Heliothis virescens (tobacco budworm) 0.10
Epilachna varivestis (Mexican bean beetle) 0.40
Heliothis zea (cotton bollworm) 1.5
Spodoptera eridania (southern armyworm) 6.0
Spodoptera frugiperda (fall armyworm) 25.0
Some milbemycins have found use in animal health as anthelmintics; the first
among these was milbemycin D, launched by Sankyo in 1986 (Figure 32.7.3).
Later, in 1990, milbemycin 5-oxime – a semisynthetic derivative of milbemectin
(milbemycin A3 /A4 ) – was introduced by Sankyo and Ciba-Geigy (now Novartis
Animal Health). Another series of milbemycin analogs from S. cyanogriseus,
identified by the team at American Cyanamide, gave rise to the discovery and
development of moxidectin (a synthetic derivative of F-28249α) as an animal health
drug [45].
32.7.5
Acaricidal and Insecticidal Activity
The whole family of macrocyclic lactones, consisting of the closely related aver-
mectins and milbemycins, displays unprecedented potency against mites, insects,
and nematodes. Typical lethal concentration (LC)90 values in greenhouse trials
are often in the range 0.1 to 0.01 ppm, and in some cases even lower. The
structure–activity relationships (SARs) of this chemical class have been the subject
of many reports; selected key findings are now discussed.
Although, initially, Putter et al. [13] reported the activity of avermectin B1 a against
several important agricultural pests in 1981, the activity of abamectin against a
more complete list of mites and insects was not described by Fisher [46] until
1989. Taken together, however, these data (Table 32.7.3) indicate that abamectin is
highly potent against most important mite species, but somewhat weaker against
Panonychus citri. A very high activity of abamectin is also evident against some
Lepidoptera, though others are less sensitive, in particular Spodoptera ssp.
The first SARs relating to crop protection targets were reported by Fisher in 1984
[47], when avermectin B1 a was shown to be somewhat more active then milbe-
mycin D against Tetranychus urticae, Heliothis virescens, and Meloidogyne incognita.
Since milbemycin D can be viewed as the 22,23-dihydro-13-desoxy derivative of
avermectin B1 b, this comparison reflects the influence of the disaccharide portion
of avermectins on their activity as pesticides.
Further structure–activity information concerning the substituents on C13 of the
avermectin aglycone have been provided by Fisher [46] and Mrozik et al. [48], with
the most important conclusions having been identified as follows (Scheme 32.7.3):
• Against Tetranychus urticae, avermectin B1 monosaccharide (2) is equally active
as avermectin B1 (1). The avermectin B1 aglycone (3) is 30-fold less active.
• Surprisingly, both 13-desoxy-avermectin B1 aglycone (4) and 22,23-dihydro-13-
desoxy-avermectin B1 aglycone (5) are threefold more active than 1. In contrast,
22,23-dihydro-avermectin B1 (6) is threefold less active than abamectin.
• The monosaccharide (7) and the aglycone (8) are practically inactive against
Tetranychus urticae. Furthermore, avermectin B1 (1) is the most active acaricide
among the naturally occurring avermectins. Avermectins B2 , A1 , and A2 are more
than 10-fold weaker.
It is apparent from the results of these studies [46, 48] that the disaccharide moiety
is not essential for potent acaricidal activity. At the same time, no significant
improvement in activity against insects (Spodoptera eridania) was observed for
the aglycones, as compared to avermectin B1 (1). Further examples of aglycone
derivatives with high activity against Tetranychus urticae are the compounds shown
in Scheme 32.7.4. Whereas the fluorides (9) and (10) are structurally quite similar
to 5, the ether substituent of 11 and 12 appears to mimic the carbohydrate structure
of a monosaccharide. The introduction of this substituent confers activity to the
otherwise inactive aglycone, and, interestingly, in this case the β-isomer at C13 is
even more active than the α-isomer.
The acaricidal and insecticidal activities of milbemectin, as described by Aoki
et al. [49], are listed in Table 32.7.4. Except for effects against some insects (thrips
and some Lepidoptera), milbemectin is mainly an acaricide, which is consistent
with the SARs observed with avermectin derivatives [28]. In general, compounds
with lipophilic substituents on C13 (or unsubstituted ones) are highly active,
whereas polar substituents diminish the activity (cf. Schemes 32.7.3 and 32.7.4).
Given the relatively low toxicity of abamectin against Spodoptera ssp., the research
team at Merck embarked on a targeted screening program to improve the ac-
tivity against Lepidoptera. In 1989, Mrozik et al. [28] reported the activity of
4 -desoxy-4 -epi-amino avermectins. In a test against neonate Spodoptera erida-
nia larvae, 4 -desoxy-4 -epi-methylamino avermectin B1 (emamectin) displayed
an LC50 of 0.004 ppm, and LC90 < 0.02 ppm (in the same test, abamectin was
inactive at 0.1 ppm). Later, Fisher reported the activity of emamectin against several
important pests [50, 51]; these data are listed in Table 32.7.5. Whilst emamectin
is highly potent against Lepidoptera, it is significantly weaker against mites and
aphids.
O O
O O
H H
R R
O O O O
OH OH
LC90 = 0.01 ppm LC90 = 0.01 ppm
O O
OH OH
4 5
OMe
HO
OMe OMe
O O HO
O O O O O O HO O
O O O
H H H
R R R
O O O O O O
abamectin
OH OH OH
LC90 = 0.03 ppm LC90 = 0.03 ppm LC90 = 1.0 ppm
O O O
1 OH 2 OH 3 OH
OMe
HO
OMe OMe
O O HO
O O O O O O HO O
O O O
H H H
R R R
O O O O O O
ivermectin OH OH OH
LC90 = 0.1 ppm LC90 > 0.5 ppm LC90 > 6.25 ppm
1317
O O O
OH OH OH
6 7 8
Scheme 32.7.3 Structure–activity relationships of avermectins against Tetranychus urticae; R = C2 H5 (>80%) and CH3 (<20%).
F O F O
13 13
O O
H H
R R
O O O O
OH OH
LC90 = 0.05 ppm LC90 = 0.01 ppm
O O
OH OH
9 10
O O O O O O
O 13 O 13
O O
H H
R R
O O O O
OH OH
LC90 = 0.05 ppm LC90 = 0.01 ppm
O O
OH OH
11 12
Scheme 32.7.4 Activity of avermectin aglycone derivatives

against Tetranychus urticae; R = C2 H5 (>80%) and CH3
(<20%).
Table 32.7.4 Activity of milbemectin against mites and insects.
Species Stage LC50 (ppm)
Tetranychus kanzawai (Kanzawa spider mite) Adult 0.7

Egg 1.5
Tetranychus urticae (two-spotted spider mite) Adult 4.7
Egg 3.3
Tetranychus cinnabarinus (carmine spider mite) Adult 2.7
Egg 2.0
Panonychus citri (citrus red mite) Adult 0.05
Egg 4.6
Myzus persicae (green-peach aphid) – <10
Toxoptera aurantii (black citrus aphid) – <2.5
Spodoptera littura (common cutworm) – 2.5
Caloptilia theivora (tea leaf roller) – <10
Jansson and Dybas [5] have provided a summary of the comparative toxicities
of abamectin and emamectin benzoate against 28 arthropod pests of agricultural
importance. These data show very clearly the complementary nature of the two com-
pounds. Against all Lepidoptera, emamectin benzoate is stronger than abamectin,
whereas against Coleoptera both compounds perform equally well; against Acarina,
Table 32.7.5 Activity of emamectin against mites and insects.
Species LC90 in ppm
Manduca sexta (tobacco hornworm) 0.003

Spodoptera exigua (beet armyworm) 0.005
Spodoptera frugiperda (fall armyworm) 0.010
Leptinotarsa decemlineata (Colorado potato beetle) 0.032
Tetranychus urticae (two-spotted spider mite) 0.29
Aphis fabae (bean aphid) 19.9
Diptera, and Homoptera, however, abamectin is more effective than emamectin

benzoate.
Fisher has also described the SARs of several 4 -amino-avermectins
against Tetranychus urticae and Spodoptera eridania [46]. Thus, it is reported that
4 -desoxy-4 -amino-avermectin B1 (14) (i.e., the (S)-isomer at C4 ; Scheme 32.7.5) is
fivefold less active against S. eridania (S.e.) than 4 -desoxy-4 -epi-amino-avermectin
B1 (15) (i.e., the (R)-isomer at C4 ). Emamectin benzoate (13) has the highest
activity against S. eridania among all 4 -amino derivatives. On the other hand, all
4 -amino derivatives are less active against T. urticae than abamectin (1).
Recently, Pitterna et al. have described the synthesis and SARs of many new
avermectins against Spodoptera littoralis, Frankliniella occidentalis, and T. urticae
[52]. These authors also included safety data of the new compounds, such as acute
oral LD50 in the rat and EC50 against Daphnia magna. The structures and biological
activities of selected new avermectins are shown in Scheme 32.7.6. Notably, a high
activity against insects and mites was shown generally not to result in less-favorable
safety properties.
32.7.6
Safety and Bioavailability
Selected toxicological and ecotoxicological data [1] of abamectin, emamectin ben-

zoate, and milbemectin are listed in Table 32.7.6. Following their application, these
compounds are all degraded rapidly in the environment. In addition, photolysis on
the plant surfaces is rapid and the compounds bind very tightly to soil, where they
are rapidly degraded by soil microorganisms [1]. No leaching or bioaccumulation
was found to occur with any of these compounds.
In general, all three products are safe for target crops [53], with their impact on
the populations of predatory arthropods being less than on the target pests [54].
The latter topic has been widely studied, in particular for abamectin [55–59] and
emamectin benzoate [60–62]. For example, Hoy and Cave [57] showed abamectin to
be more toxic towards T. urticae than towards the predator Metaseiulus occidentalis.
It was also shown that field-aged residues of abamectin were safe for M. occidentalis
at only two days after application.
OMe OMe
HO HN
OMe OMe
O O O O
O O O O O O
O O
H H
R R
abamectin O O emamectin O O
0.03 ppm T.u. OH 0.25 ppm T.u. OH
8.0 ppm S.e. 0.004 ppm S.e.
O O
OH OH
1 13
OMe OMe
H2N H2N
4" 4"
OMe OMe
O O O O
O O O O O O
O O
H H
R R
C-4"-(S)-isomer O O C-4"-(R)-isomer O O
0.25 ppm T.u. OH 0.25 ppm T.u. OH
0.10 ppm S.e. 0.02 ppm S.e.
O O
OH OH
14 15
Scheme 32.7.5 Activity of 4 -amino-avermectin derivatives

against Tetranychus urticae (T.u.) and Spodoptera eridania
(S.e.), LC90 in ppm; R = C2 H5 (>80%) and CH3 (<20%).
This reduced impact on beneficial arthropods results from the uptake and
degradation behavior of the active ingredient, and leads to the compound being
less bioavailable to the beneficials than to the pests. Moreover, any surface residues
of the compounds are subject to rapid photolysis, the typical half-life being in the
range of 4–6 h as a thin film exposed to simulated sunlight [63], and less than
one day on crops in the field [64]. Despite the short half-life under sunlight, the
active ingredient is still taken up into the leaf tissue in sufficient amounts to
provide a residual activity against mites [24]. This is accompanied by a translaminar
distribution [63] such that significant amounts of compound are made available
in the parenchymal tissue, which acts as a reservoir on which the mites feed. By
lacking a true systemicity, however, the macrocyclic lactones are not distributed
into the phloem and xylem system following foliar application. Such behavior
provides the basis for a lack of residual activity of abamectin against aphids, despite
a good contact activity [65].
In summary, the rapid uptake of these compounds into the foliage after applica-
tion, combined with the fast degradation of any surface residues, means that this
OMe OMe OMe
HO HN O N
OMe OMe OMe OMe
O O O O O O O O
O O O O O O O O O O O O
O O O O
H H H H
R R R R
abamectin O O emamectin O O O O O O
0.8 ppm S.l. OH 0.05 ppm S.l. OH 0.8 ppm S.l. OH 0.05 ppm S.l. OH
3 ppm F.o. 3 ppm F.o. 0.8 ppm F.o. 0.8 ppm F.o.
0.03 ppm T.u. O 0.2 ppm T.u. O 0.01 ppm T.u. O 0.003 ppm T.u. O
221 mg/kg rat 76 mg/kg rat < 50 mg/kg rat > 200 mg/kg rat
0.34 μg/l daphnia 1 OH 0.99 μg/l daphnia 13 OH 1.7 μg/l daphnia 16 OH 2.7 μg/l daphnia 17 OH
O OMe OMe OMe OMe

HN MeO OMe HO
N OMe NC OMe OMe OMe
H
O O O O O O O O
O O O O O O O O O O O O
O O O O
H H H H
R R R R
O O O O O O O O
3 ppm S.l. OH > 3 ppm S.l. OH 3 ppm S.l. OH 0.8 ppm S.l. OH
0.8 ppm F.o. 3 ppm F.o. 0.8 ppm F.o. 3 ppm F.o.
0.01 ppm T.u. O 0.01 ppm T.u. O 0.01 ppm T.u. O 0.01 ppm T.u. O
> 50 mg/kg rat > 50 mg/kg rat > 50 mg/kg rat 50 mg/kg rat
0.8 μg/l daphnia 18 OH n.t. daphnia 19 OH 5 μg/l daphnia 20 OH n.t. daphnia 21 OH
Scheme 32.7.6 Activity of new avermectin derivatives against Tetranychus urticae (T.u.), Frankliniella occidentalis (F.o.), and Spodoptera littoralis (S.l.).
LC90 in ppm; acute oral LD50 in the rat (mg kg−1 body weight); EC50 (48 h) against Daphnia magna (μg l−1 ). R = C2 H5 (>80%) and CH3 (<20%).
1321
Table 32.7.6 Safety and environment.
Property Abamectin Emamectin benzoate Milbemectin
Acute oral LD50 , rat 18.4 mg kg−1 (in 76–89 mg kg−1 762 mg kg−1 (male)
sesame oil)
221 mg kg−1 (in water) – 456 mg kg−1 (female)
Eye irritation, rabbit Slightly irritant Severe irritant Non-irritant
Skin irritation, rabbit Non-irritant Non-irritant Non-irritant
Acute oral LD50 , bird 84.6 mg kg−1 , mallard 46 mg kg−1 , mallard 650–660 mg kg−1 ,
duck duck chicken
> 2000 mg kg−1 , 264 mg kg−1 , bobwhite 968–1005 mg kg−1 ,
bobwhite quail quail Japanese quail
LC50 (96 h), fish 3.2 μg l−1 , rainbow 174 μg l−1 , rainbow 4.5 μg l−1 , rainbow
trout trout trout
EC50 (48 h), Daphnia 0.34 μg l−1 , Daphnia 0.99 μg l−1 , Daphnia –
magna magna
LC50 earthworm (Eisenia 28 ppm (28 day) >1000 ppm (14 day) 61 ppm (14 day)
foetida)
Contact LD50 honey bee 0.002 μg/bee (48 h) 0.0039 μg/bee (48 h) 0.025 μg/bee (48 h)
family of compounds can be considered safe towards beneficial organisms under

field conditions.
32.7.7
Use in Agriculture
Today, abamectin is used worldwide in various crops, the most important being
citrus, pome fruits, vegetables, cotton, and ornamentals (Table 32.7.7). It is also used
to control most agronomically important mites, some Lepidoptera, and dipterous
leafminers, with typical application rates in the range 5.6 to 28 g a.i. ha−1 to control
mites, and 11 to 22 g a.i. ha−1 for leafminers.
As a nematicide seed treatment, abamectin controls most nematodes that
commonly occur in agricultural crops. The seed treatment formulations con-
tain abamectin in combination with an insecticide (thiamethoxam, cf. Chapter
32.2.3) and different fungicides. These products have been introduced for use in
®
soybeans, corn, and cotton under the trade name Avicta Complete.
Emamectin benzoate is used in many countries, mainly in all types of veg-
etable crop, such as brassicas, fruiting, and leafy vegetables, and also in cotton
(Table 32.7.8). Emamectin benzoate can be used to control all agronomically im-
portant Lepidoptera pests in vegetables and cotton, with typical application rates in
the range 8.4 to 16.8 g a.i. ha−1 . Additional uses include the control of pests in tea,
and of pine wood nematodes in pine trees in Japan.
Milbemectin is mainly used as an acaricide against important mites in tea and
pome fruits, and also against pine wood nematode in Japan (Table 32.7.9). The
application rates for agricultural uses range from 5.6 to 28 g a.i. ha−1 .
Table 32.7.7 Abamectin use in crop protection.
Pest species Crop(s)
Phyllocoptrura oleivora (citrus rust mite) Citrus

Tetranychus cinnabarinus (carmine spider mite) Cotton
Tetranychus pacificus (Pacific spider mite) Cotton, deciduous tree nuts
Tetranychus urticae (two-spotted spider mite) Cotton, ornamentals, pome fruits,
strawberry, vegetables
Liriomyza trifolii (serpentine leafminer) Ornamentals, vegetables
Epitrimerus pyri (pear rust mite) Pome fruits
Panonychus ulmi (European red mite) Pome fruits
Psylla pyricola (pear psylla) Pome fruits
Keiferia lycopersicella (tomato pinworm) Vegetables
Plutella xylostella (diamond-back moth) Vegetables
Solenopsis invicta (red imported fire ant) –
Table 32.7.8 Emamectin benzoate use in crop protection.
Pest species Vegetable(s)
Hellula rogatalis (cabbage webworm) Brassicas

Mamestra brassicae (cabbage armyworm) Brassicas
Pieris rapae (small white butterfly) Brassicas
Plutella xylostella (diamond-back moth) Brassicas
Spodoptera littura (rice leafworm) Brassicas
Trichoplusia ni (cabbage looper) Brassicas, cotton, fruiting vegetables, leafy
vegetables
Helicoverpa zea (corn earworm) Brassicas, cotton, fruiting vegetables, leafy
vegetables
Spodoptera exigua (beet armyworm) Brassicas, cotton, fruiting vegetables, leafy
vegetables
Spodoptera frugiperda (fall armyworm) Brassicas, cotton, fruiting vegetables, leafy
vegetables
Helicoverpa armigera (Old World cotton Cotton
bollworm)
Heliothis virescens (tobacco budworm) Cotton, fruiting vegetables, leafy vegetables
Keiferia lycopersicella (tomato pinworm) Fruiting vegetables
Liriomyza trifolii (serpentine leafminer) Fruiting vegetables
Spodoptera eridania (southern armyworm) Fruiting vegetables
Pest species Other crops
Adoxophytes sp. (smaller tea tortrix) Tea

Hormona magnamina (oriental tea tortrix) Tea
Bursaphelenchus xylophilus (pine wood Pine (trunk injection)
nematode)
Table 32.7.9 Milbemectin use in crop protection.
Pest species Crop(s)
Tetranychus urticae (two-spotted spider mite) Apples, pears, strawberries, vegetables

Polyphagotarsonemus latus (broad mite) Citrus fruits, vegetables
Tetranychus kanzawai (Kanzawa spider mite) Pears, strawberries, tea, vegetables
Acaphylla theae (pink tea rust mite) Tea
Calacarus carniatus (purple tea mite) Tea
Bursaphelenchus xylophilus (pine wood nematode) Pine (trunk injection)
References
1. Tomlin, C.D.S. (ed.) (2003) The Pesticide 12. Wright, J.E. (1984) J. Econ. Entomol., 77,
Manual: A World Compendium, 13th 1029–1032.
edn, British Crop Protection Council. 13. Putter, I., MacConnell, J.G., Preiser,
2. Turner, M.J. and Schaeffer, J.M. (1989) F.A., Haidri, A.A., Ristich, S.S., and
in Ivermectin and Abamectin (ed. W.C. Dybas, R.A. (1981) Experientia, 37,
Campbell), Springer, New York, Berlin, 963–964.
Heidelberg, pp. 73–88. 14. Mrozik, H. (1989) Patent US 4,874,749.
3. Arena, J.P. (1994) Parasitol. Today, 10, 15. Cvetovich, R. (1994) Patent US
35–37. 5,288,710.
4. Rohrer, S.P. and Arena, J.P. (1995) 16. Cvetovich, R. (1994) Patent US
Molecular Action of Insecticides on Ion 5,362,863.
Channels, ACS Symposium Series, 17. Cvetovich, R., Demchak, R., McCauley,
vol. 591, American Chemical Society, J.A., and Varsolona, R.J. (1996) Patent
Washington, DC, pp. 264–283. WO 96/22,300.
5. Jansson, R.K. and Dybas, R.A. (1998) in 18. Cvetovich, R.J., Kelly, D.H., DiMichele,
Insecticides with Novel Modes of Action: L.M., Shuman, R.F., and Gabowski,
Mechanisms and Application (eds I. E.J.J. (1994) J. Org. Chem., 59,
Ishaaya and D. Degheele), Springer, 7704–7708.
Berlin, pp. 152–170. 19. Fritz, L.C., Wang, C.C., and Gorio, A.A.
6. Rugg, D., Buckingham, S.D., Sattelle, (1997) Proc. Natl Acad. Sci. USA, 76,
D.B., and Jansson, R.K. (2005) Compr. 2062–2066.
Mol. Insect Sci., 5, 25–52. 20. Bloomquist, J.R. (1993) Comp. Biochem.
7. Sunazuka, T., Omura, S., Iwasaki, S., Physiol., 106C, 301–314.
and Omura, S. (2002) in Macrolide An- 21. Deng, Y. and Cassida, J.E. (1992) Pestic.
tibiotics (ed. S. Omura), Academic Press, Biochem. Physiol., 43, 116–122.
San Diego, CA, pp. 99–180. 22. Rohrer, S.P., Birzin, E.T., Costa, S.D.,
8. Burg, R.W., Miller, B.M., Baker, E.E., Arena, J.P., Hayes, E.C., and Schaeffer,
Birnbaum, J., Currie, S.A. et al. (1979) J.M. (1995) Insect. Biochem. Mol. Biol.,
Antimicrob. Agents Chemother., 15, 25, 11–17.
361–367. 23. Cully, D.F., Paress, P.S., Liu, K.K.,
9. Egerton, J.R., Ostlind, D.A., Blair, L.S., Schaeffer, J.M., and Arena, J.P. (1996)
Eary, C.H., Suhayda, D. et al. (1979) J. Biol. Chem., 271, 20187–20191.
Antimicrob. Agents Chemother., 15, 24. Wright, J.E., Jenkins, J.N., and Villavaso,
372–378. E.J. (1985) Southwest. Entomol., 7
10. Ostlind, D.A., Cifelli, S., and Lang, R. (Suppl.), 11–16.
(1979) Vet. Rec., 105, 168. 25. Campbell, W.C., Burg, R.W., Fischer,
11. James, P.S., Picton, J., and Riek, R.F. M.H., and Dybas, R.A. (1984) in
(1980) Vet. Rec., 106, 59. Pesticide Synthesis through Rational
References 1325
Approaches (eds P.S. Magee, G.K. Kohn, 39. Takigutchi, Y., Mishima, H., Okuda,
and J.J. Menn), American Chemical M., Terao, M., Aoki, A., and Fukuda, R.
Society, Washington, DC, pp. 5–20. (1980) J. Antibiot., 33, 1120–1127.
26. Dybas, R.A. and Babu, J.R. (1988) Proc. 40. Maruyama, F., Iwado, S., Tachibana, S.,
Brighton Crop Prot. Conf. – Pests Dis., 1, Hayashida, S., Sato, K., and Tanaka, K.
57–64. (1988) Patent JP 63–227,590.
27. Dybas, R.A., Hilton, N.J., Babu, J.R., 41. Takiguchi, Y., Ono, M., Muramatsu,
Preiser, F.A., and Dolce, G.J. (1989) in S., Ide, J., Mishima, H., and Terao, M.
Novel Microbial Products for Medicine (1983) J. Antibiot., 36, 502–508.
and Agriculture (eds A.L. Demain, G.A. 42. Saito, A. and Kobayashi, M. (1993)
Somkuti, J.C. Hunter-Cevera, and H.W. Patent JP 05,097,863.
Rossmoore), Society for Industrial 43. Saito, A. and Kobayashi, M. (1993)
Microbiology, Fairfax, VA, pp. 203–212. Patent JP 05,097,859.
28. Mrozik, H., Escola, P., Linn, B.O., Lusi, 44. Takoshiba, H., Sato, K., Yanai, T., Yokoi,
A., Shih, T.L. et al. (1989) Experientia, S., Ichinose, R., and Tanizawa, K. (1995)
45, 315–316. Patent EP 675,133.
29. Mrozik, H. (1994) in Natural and En- 45. Barden, T.C., Asato, G., Ahmed,
gineered Pest Management Agents (eds Z.H., France, D.J., Kamesvaran, V.,
P.A. Hedin, J.J. Menn, and R.M. Parker-Jackson, E., Tamura, S.Y., Tseng,
Hollingsworth), American Chemical S.-S., and Bookwalter, B.L. (1992)
Society, Washington, DC, pp. 54–73. New Series of Milbemycin Macrolides
30. Jackson, H.C. (1989) Parasitol. Today, 5, (LL-F28249) with Endectocidal, Insec-
146–156. ticidal, and Acaricidal Activity, ACS
31. Greene, B.M., Brown, K.R., and Taylor, Symposium Series, vol. 504, American
H.R. (1989) in Ivermectin and Abamectin Chemical Society, Washington, DC,
(ed. W.C. Campbell), Springer, New pp. 227–237.
York, Berlin, Heidelberg, pp. 311–323. 46. Fisher, M.H. (1997) Structure-Activity
32. Shoop, W. and Soll, M. (2002) Macro- Relationships of the Avermectins and
cyclic Lactones in Antiparasitic Ther- Milbemycins, ACS Symposium Series,
apy, CABI Publishing, Wallingford, vol. 658, American Chemical Society,
pp. 1–29. Washington, DC, pp. 221–238.
33. McArthur, H.A.I. (1998) Dev. Ind. Micro- 47. Fisher, M.H. (1984) in The Avermectins
biol. Ser., 35, 43–48. (ed. N.F. Janes), The Royal Society of
34. Conder, G.A. and Baker, W.J. (2002) Chemistry, Special Publication No. 53,
Macrocyclic Lactones in Antiparasitic pp. 53–72.
Therapy, CABI Publishing, Wallingford, 48. Mrozik, H., Linn, B.O., Eskola, P., Lusi,
pp. 30–50. A., Matzuk, A., Preiser, F.A., Ostlind,
35. Bishop, B.F., Bruce, C.I., Evans, N.A., D.A., Schaeffer, J.M., and Fisher, M.H.
Goudie, A.C., Gration, K.A.F., Gibson, (1989) J. Med. Chem., 32, 375–381.
S.P., Pacey, M.S., Perry, D.A., Walshe, 49. Aoki, A., Nishida, A., Ando, M., and
N.D.A., and Witty, M.J. (2000) Vet. Yoshikawa, H. (1994) J. Pestic. Sci., 19,
Parasitol., 91 (3-4), 163–176. S125–S131.
36. Aoki, A., Fukuda, R., Nakayabu, T., 50. Fisher, M.H. (1993) Recent Progress in
Ishibashi, K., Takeichi, C., and Ishida, Avermectin Research, ACS Symposium
M. (1974) Patent JP 4,757,058. Series, vol. 524, American Chemical
37. Mishima, H., Kurabayashi, M., Tamura, Society, Washington, DC, pp. 169–182.
C., Sato, S., Kuwano, H., and Saito, A. 51. Fisher, M.H. (1990) Pure Appl. Chem.,
(1974) Symposium Paper of the 18th 62 (7), 1231–1240.
Symposium on the Chemistry of Natural 52. Pitterna, T., Cassayre, J., Hüter,
Products, Kyoto, Japan, p. 309. O.F., Jung, P.M.J., Maienfisch, P.,
38. Okazaki, T., Ono, M., Aoki, A., and Kessabi, F.M., Quaranta, L., and
Fukuda, R. (1983) J. Antibiot., 36, Tobler, H. (2009) Bioorg. Med. Chem.,
438–441. 17, 4085–4095.
53. Wislocki, P.G., Grosso, L.S., and Dybas, 60. Kok, L.T., Lasota, J.A., McAvoy, T.J., and
R.A. (1989) in Ivermectin and Abamectin Dybas, R.A. (1996) J. Econ. Entomol., 89,
(ed. W.C. Campbell), Springer, New 63–67.
York, Berlin, Heidelberg, pp. 182–214. 61. Chukwudebe, A.C., Cox, D.L., Palmer,
54. Dybas, R.A. (1989) in Ivermectin S.J., Morneweck, L.A., Payne, L.D.
and Abamectin (ed. W.C. Campbell), et al. (1997) J. Agric. Food Chem., 45,
Springer, New York, Berlin, Heidelberg, 3689–3693.
pp. 287–310. 62. Sechser, B., Ayoub, S., and Monuir,
55. Trumble, J.T. and Alvarado Rodriguez, N. (2003) J. Plant Dis. Protect., 110,
B. (1993) Agric. Ecosyst. Environ., 43, 184–194.
267–284. 63. MacConnell, J.G., Demchak, R.J.,
56. Lasota, J.A. and Dybas, R.A. (1991) Preiser, F.A., and Dybas, R.A. (1989)
Annu. Rev. Entomol., 36, 91–117. J. Agric. Food Chem., 37, 1498–1501.
57. Hoy, M.A. and Cave, F.E. (1985) Exp. 64. Feely, W.F., Crouch, L.S., Arison, B.H.,
Appl. Acarol., 1, 139–152. Vanden Heufel, W.J.A., Colwell, L.F.
58. Grafton-Cardwell, E.E. and Hoy, M.A. et al. (1992) J. Agric. Food Chem., 40,
(1983) J. Econ. Entomol., 76, 1216–1220. 691–696.
59. Zhang, Z. and Sandersson, J.P. (1990) J. 65. Johnson, A.W. (1985) Tobacco Sci., 29,
Econ. Entomol., 83, 1783–1790. 135–138.
1327
33
New Unknown Modes of Action
33.1
Selective Feeding Blockers: Pymetrozine, Flonicamid, and Pyrifluquinazon
Peter Maienfisch
33.1.1
Introduction
The market for compounds to treat sucking pests offers many commercial op-
portunities for innovative new products with novel modes of action, high levels
of biological efficacy, and low toxicity combined with high selectivity. This is due
mainly to an urgent need for new products which show a high level of safety against
beneficial organisms, provide control of those insects that are resistant to current
treatments or newly evolving pest problems, are suitable for use in integrated pest
management (IPM) programs, and satisfy new regulatory requirements. Recently,
the two new products pymetrozine and flonicamid – both of which act as selective
feeding blockers – have entered the market place, offering attractive alternatives to
current sucking pest products such as carbamates, organophosphates, synthetic
pyrethroids, and neonicotinoids. Furthermore, pyrifluquinazon – a novel insect
behavior regulator and analog of pymetrozine – is currently under development by
Nihon Nohyaku.
33.1.2
Pymetrozine
Pymetrozine (1; developmental code CGA 215 944) is an insecticide which is highly
active and specific against sucking pests, and is the only commercial representative
of the chemical class of pyridine azomethines. Currently, pymetrozine is marketed
by Syngenta under the trademarks of ChessTM , PlenumTM , FulfillTM , RelayTM , and
SterlingTM [1–4].
33.1.2.1 Discovery
Pymetrozine (1), a pyridine azomethine, was synthesized for the first time at the
end of 1986 [3, 5]. The concept behind this discovery was primarily chemically
directed, though with a strong element of rational design [2]. In a first step,

1328 33 New Unknown Modes of Action
ring transformations of 1,3,4 oxadiazolon-3-yl ketones (2) with N-nucleophiles

were investigated; this led to a large number of new five- and six-membered
N-amino heterocycles, such as 3 and 4, respectively (Scheme 33.1.1). In the second
(intermediate) step, many transformations and derivatizations of these N-amino
heterocycles were prepared, among which was a condensation with pyridine-3
aldehyde. On examination, the resultant compounds (e.g., CGA 207 429 and
207 936) were found to possess good insecticidal activities. The third (final) step, a
broad optimization program, resulted in the discovery of pymetrozine.
An initial patent application [5] to protect the compounds of the novel
chemical class of the pyridine azomethines (e.g., 5) was filed by Ciba-Geigy (later
Novartis, now Syngenta) in 1989, and within a short period of time a variety
of further inventions (pyridine-3-carboxaldehyde N-oxides (6) [6], imidazoles (7)
[7], 3-[(N-heterocyclyl) iminomethyl]pyridines (8) [8]) were made and patented
(Table 33.1.1). In 1996, Nihon Nohyaku discovered the insecticidal activity of the cor-
responding aminoquinazolinones (9) and (10) [9–13], and evaluated NNI 0101 (20;
see Scheme 33.1.4) in official field tests in Japan. In 2006, Nihon Nohyaku requested
a common name for NNI 0101 (common name: pyrifluquinazon; IUPAC name:
1-acetyl-1,2,3,4-tetrahydro-3-[(3-pyridylmethylamino]-6-[1,2,2,2-tetrafluoro-1-(trifluo
romethyl)ethyl]quinazolin-2-one), which was registered in Japan in October 2010
(see Section 33.1.4). A 20% formulation has been developed for Japan and
China, but it is not yet on the market. Registration in other countries has been
commenced; approval of US registration by the EPA is not expected until late 2012.
33.1.2.2 Pyridine Azomethines: Structure–Activity Relationships

The general structure–activity profile for the pyridine azomethine insecticides is
shown in Table 33.1.2.
33.1.2.3 Synthesis of Pymetrozine

An economical route was developed for the preparation of pymetrozine
(1; Scheme 33.1.2). In this case, the final – quite remarkable – step involved
a one-pot procedure in which 3-pyridine carbonitrile was converted to the
corresponding aldehyde by a catalytic hydrogenation. This was subsequently
condensed with 4-amino-6-methyl-4,5-dihydro-1,2,4-triazin-3(2H)-one to give
pymetrozine in high yields [2, 5, 14–16].
33.1.2.4 Chemical and Physical Properties of Pymetrozine

The physico-chemical properties of pymetrozine, which are listed in Table 33.1.3
[1, 3, 4], favor an efficient uptake and translocation in plants [17, 18]. The systemic
behavior of pymetrozine originates mainly from xylem and, to a minor extent,
also from phloem mobility. The half-life of pymetrozine in the soil is low (range
2–29 days), indicating a rapid degradation in the environment. Pymetrozine and
its major metabolites exhibit only a low leaching potential, and were generally
found to remain in the upper soil layer, indicating a low potential for groundwater
contamination under recommended-use conditions [4].
N
R1 NH2 OHC
N N
N N
1) R3NH2 R2 N O R1 = H, R2 = t-Bu
N O N
2) 5N HCl R3
R1 CH3
R2 H3C N
3 CGA 207'429 N
N N O N
Optimization N O
H 3C O O H
1) R3NHNH2
R1 OHC N 1
2 2) 5N HCl
R2 NH2 CGA 215'944
N N
N Pymetrozine
N
N R1 = H, R2 = t-Bu
N O N
N O
R3 H
4 CGA 207'936
Scheme 33.1.1 Discovery of pymetrozine.

33.1 Selective Feeding Blockers: Pymetrozine, Flonicamid, and Pyrifluquinazon
1329
Table 33.1.1 Key inventions related to the chemical class of the pyridine azomethines.

5 Ciba-Geigy EP 314615 1989 [5]

N
R2 R3
R1 N
N
N
N O
H
N
R2 R3 H
R1 N
N
N
N O
H
On
N
R2 R3 S
R1 N
N
6 N Ciba-Geigy EP 391849 1990 [6]
N O
H
n = 0,1
N
R2 S
N
N
7 R1 Ciba-Geigy EP 604365 1994 [7]
N O
R3
N
S
8 N Ciba-Geigy WO 9518123 1997 [8]
R
R = N-attached heterocyclyl
N
9 Nihon EP 735035 1996 [9]
S1
N Nohyaku JP 11012254 1999 [10]
N
S2 EP 1097932 1999 [11]
N O WO 2004099184 2004 [12]
R
10 N Nihon JP 11158180 1999 [13]
Nohyaku
S1
R1 N
N
S2
R2 N O
R
R1, R2 = 5-6 membered heterocycle
33.1 Selective Feeding Blockers: Pymetrozine, Flonicamid, and Pyrifluquinazon 1331
Table 33.1.2 Pyridine azomethines: structure–activity relationship.
N
R2 R3 S
R1 A
N B
N Pyridyl
N X
moiety
R4
Triazinone
moiety

feature
R1 N N R1 N R5 N
N N N N
Triazinone , > >> others
N N ,
moiety N O N O N O R1 N O
H H H H
Substituent CH3 , i-Pr, t-Bu are most favorable; larger substituents decrease the insecticidal activity
R1
Substituent H > larger groups
R2, R3
Substituent H > CH3 , COR, COOR
R4
Functional C=O > C=S
group C=X
Bond A–B N=CH, NH–CH2 >> N=C(Alkyl), NH–CH(Alkyl), others
N
, , > >> other heterocycles
Pyridyl N N+ N N
moiety O−
O OR
Substituent S H is more favorable than all other substituents
33.1.2.5 Mode of Action and Resistance
33.1.2.5.1 Mode of Action In aphids, pymetrozine inhibits feeding immediately

after application, which results in death of the insect by starvation, without
producing any visible neurotoxic effects. No repellent or antifeedant effects were
observed that could explain the inhibition of feeding [19–22].
Although not a plant-sucking insect, the locust was shown to be a valid model
for studying the details of the underlying mechanisms. Locusta migratoria was
CH3
N NH2 H
COCl2 N N CH3COCH2Cl
N N O
H3C O H3C O
O H3C O
O
N
H NC
H3C H3C NH2 H3C N
N O N N
NH2NH2 · H2O N HCl N
N N N
N O N O Ra/Ni, H2 N O
H H H
1
CGA 215'944
Pymetrozine
Scheme 33.1.2 Synthesis of pymetrozine.
Table 33.1.3 Chemical and physical properties of pymetrozine.
Feature Property

Vapor pressure at 25 ◦ C <4 × 10−6 Pa
Water solubility at 25 ◦ C 290 mg l−1
pH value 6.84 (saturated solution in water)
Partition coefficient (n-octanol/water at 25 ◦ C (log Pow )) –0.18
Soil mobility Little mobility
Hydrolysis: (estimated half-life at 25 ◦ C) pH 1: 2.8 h
pH 5: 5–10 days
pH 9: stable
shown to respond to pymetrozine by displaying unique symptoms, such as lifting

and stretching of the hind legs, in addition to the feeding inhibition. Pymetrozine
enhanced the spontaneous spike discharge of the metathoracic and subesophageal
ganglia in situ at nanomolar concentrations. Similarly, it increased the spontaneous
rhythmic contractions of the isolated foregut, with maximal effects also in the
nanomolar range. The actions of pymetrozine were counteracted by biogenic amine
receptor antagonists (such as mianserin, ketanserin, propranolol) and mimicked
by serotonin, but not by dopamine and octopamine. Moreover, pymetrozine and
serotonin strongly potentiated the effects of each other. Typically, pymetrozine was
inactive at all neurotransmitter receptors present on the isolated locust neuronal
somata, and at all other examined neuronal sites [23–25].
Similar effects were also observed in Myzus persicae [24], where electrical pen-
etration graph experiments revealed that serotonin – like pymetrozine – not only
inhibited stylet penetration but also strongly enhanced the action of pymetrozine.
Biogenic amine receptor antagonists were not specifically active in the aphid. Based
on these most recently acquired data, it was concluded that pymetrozine acts via a
novel mechanism that is linked to the signaling pathway of serotonin.
33.1.2.5.2 Resistance Whilst cross-resistance between compounds in the same

chemical class is a frequently observed phenomenon, cross-resistance between
groups that differ in structural and functional characteristics is unpredictable.
In the case of the pyridine azomethine antifeedant pymetrozine, some evidence
of cross-resistance to neonicotinoids has been reported for the tobacco whitefly
(Bemisia tabaci) [26], and the glasshouse whitefly (Trialeurodes vaporariorum) [27].
This cross-resistance most likely reflects the overexpression of a cytochrome
P450-dependent mono-oxygenase that is capable of metabolizing both types of
compound, in spite of their apparent structural dissimilarities.
Investigations into the cross-resistance relationship between pymetrozine and
neonicotinoids in the brown plant hopper (Nilaparvata lugens) failed to provide any
evidence of cross-resistance in field-, laboratory-, and metabolism-based studies
[28]. Consequently, the use of pymetrozine to control neonicotinoid resistant brown
plant hoppers is just one example of how novel chemistries can provide control of
resistant populations, and also be used to prevent the development of resistance, if
applied as part of a resistance-management strategy.
33.1.2.6 Biological Activity and Use Recommendations

Whilst the key targets for pymetrozine are aphids in potatoes, vegetables, and stone
fruits, it is also active against hoppers in rice and can also be used to control the
mobile stage of whiteflies nymphs (L1) and adults in various crops. The currently
recommended pest uses after foliar spray and seedling box application are listed in
Table 33.1.4. Although the recommended rates of active ingredient (a.i.) usage vary
according to crops and pests, in most cases 100–300 g a.i. ha−1 , 10–30 g a.i. hl−1 ,
or 1–2 g a.i. per seedling box are sufficient to provide a full control of the target
pests [1, 3, 4, 29–39].
33.1.2.7 Safety Profile

The safety profile of pymetrozine is shown in Table 33.1.5. Pymetrozine has a
low acute mammalian toxicity, and an excellent safety profile for most nontarget
arthropods, birds, and fish. It is also of low risk to beneficial insects in the field,
and is therefore very well suited for use in IPM programs [1–4, 32, 40].
33.1.3
Flonicamid
Flonicamid (12; developmental codes: IKI-220, F1785) is a selective systemic

aphicide discovered by Ishihara Sangyo Kaisha, Ltd. This pyridinecarboxamide
compound belongs to the new trifluoromethylnicotinamide chemical class. During
late 2001, the FMC Corporation obtained exclusive rights to develop, market, and
distribute flonicamid in North America, much of Latin America, UK, Spain, and
Table 33.1.4 Current insecticidal spectrum of pymetrozine.
Target pests Key crop(s)
Acyrthosiphon pisum Vegetables

Aphis citricola Citrus
Aphis fabae Vegetables, potato, cotton
Aphis frangulae Potato
Aphis gossypii Vegetables, potato, citrus, tobacco, cotton
Aphis nasturtii Potato
Aphis nicotianae Tobacco
Aulacorthum solani Vegetables, potato
Bemisia tabaci Vegetables, cotton
Bemisia trifolii Vegetables
Brevicoryne brassicae Vegetables
Idiocerus clypealis Mango
Idiocerus niveosparsus Mango
Laodelphax striatellus Rice
Lipaphis erysimi Vegetables
Macrosiphum euphorbiae Vegetables, potato, tobacco, cotton
Myzus persicae Vegetables, stone fruits, potato
Nasonovia ribisnigri Vegetables
Nilaparvata lugens Rice
Phorodon humili Hop
Sogatella furcifera Rice
Trialeurodes vaporariorum Cotton
Toxoptera aurantii Citrus
Table 33.1.5 Safety profile of pymetrozine.
Acute toxicity Result
Oral LD50 , rat >5820 mg kg−1

Dermal LD50 (24 h), rat >2000 mg kg−1
Inhalation LC50 (4 h), rat >1800 mg m−3
Skin irritation, rabbit Non-irritant
Avian oral LD50 , mallard duck >2000 mg kg−1
Freshwater fish LC50 (96 h), rainbow trout > 100 mg kg−1
Freshwater invertebrate EC50 (48 h), Daphnia magna >100 mg kg−1
Algae EC50 (72 h) Scenedesmus sp. Selenastrum sp. 47.1 mg l –1 58 mg l –1
Earthworm EC50 (14 days), Eisenia foetida >1000 mg kg−1 soil
Honey bee Oral LD50 (48 h) Contact LD50 (48 h) 117 μg /bee >200 μg/bee
Portugal. In the rest of the European Union, FMC and ISK are jointly developing
this insecticide [41, 42]. Flonicamid was launched onto the world market in 2005
[43], and is now sold in many countries, such as the USA, Canada, Brazil, France,
Korea, and Japan.
33.1.3.1 Discovery of Flonicamid and the Trifluoromethylnicotinamide Insecticides

The first trifluoromethylnicotinamides possessing aphicidal activity were described
by Ishihara Sangyo Kaisha in 1994 [44]. This early patent application covered
compounds of the general structure 11, and flonicamid (12) was one of the
compounds specifically mentioned (Table 33.1.6). The good aphicidal activity of
trifluoromethylnicotinamides of type 11 triggered research activities within several
other companies, including Sumitomo, Hoechst/Aventis (now Bayer CropScience),
and Syngenta. Some early key inventions made by these companies are highlighted
in Table 33.1.6 [45–51]. Later, other companies such as Bayer CropScience and
Sankyo Agro (now Mitsui Chemicals Agro, Inc.) joined this research area. Currently,
some trifluoromethylnicotinamides of unknown structure are being evaluated in
field tests.
33.1.3.2 Trifluoromethylnicotinamides: Structure–Activity Relationships

The general structure–activity profile for the trifluoromethylnicotinamides insecti-
cides is shown in Table 33.1.7.
33.1.3.3 Synthesis of Flonicamid

Flonicamide (12) was first synthesized in 1994 [44]. Starting from the commer-
cially available 4-(trifluoromethyl)nicotinic acid (new synthetic methods for its
production have recently been described [52, 54–57]), flonicamid can be synthe-
sized in only two steps [44] (Scheme 33.1.3). An alternative route involving 1,3,5
tricyanomethylhexyhydro-1,3,5-triazine (14) as source of amino-acetonitrile has
also been used to prepare larger amounts of flonicamid [58].
33.1.3.4 Chemical and Physical Properties of Flonicamid

The physico-chemical properties of flonicamid are listed in Table 33.1.8 [41, 42].
Notably, these properties (e.g., low logP, high water solubility) favor systemic and
translaminar activity. Flonicamid shows a minimal tendency to persist due to its
fast degradation, while its moderate soil mobility is negated by its rapid metabolism
and mineralization.
33.1.3.5 Mode of Action of Flonicamid
33.1.3.5.1 Biological Mode of Action Following treatment with flonicamid, aphids

completely stop feeding within 30 min; furthermore, a concomitant reduction
in aphid honeydew production and salivation with cessation in feeding is also
observed. Other behavioral changes noted in aphids after flonicamid treatment
include a pronounced sensitivity to light, random or irregular movements, an
altered righting response, uncoordinated locomotion, as well as creased and erratic
Table 33.1.6 Early key inventions related to the chemical

class of the trifluoromethylnicotinamides.

11 CF3 X Ishihara EP 580374 1994 [44]

R1 Sangyo
N
Kaisha
R2
N
On n = 0,1; X = O,S
12 Example
CF3 O
N CN
H
N
12
Flonicamid (IKI-220, F1785)
CF3 X A
13 N
(n)
Ishihara JP 07010841 1995 [52]
H Sangyo JP 07025853 1995 [53]
N A = heterocyclyl Kaisha
CF3 X X
N
R1
14 N S Sumitomo JP 10195072 1998 [45]
H
N X = N, CR2
CF3
A
X
15 Hoechst WO 9857969 1998 [46]
N
A = 5-membered heterocyclyl Aventis WO 2000035912 [47]
On Aventis WO 2000035913 [48]
X = CH, N; n = 0,1
CF3 O R1
16 OR2 Sumitomo JP 11180957 1999 [49]
N N
HN
N
CF3 O
R1
N
17 OR2 Syngenta WO 2001009104 2001 [50]
N
On n = 0,1
CF3
A
X
18 Syngenta WO 2001014373 2001 [51]
N
A = 6-7-membered heterocyclcyl
On
X = Ch, N; n = 0,1
Table 33.1.7 Trifluoromethylnicotinamides: structure–activity relationships.
R1 O
R2
N
S
R3
N
Pyridyl Amide
moiety moiety
Structural Structure–activity relationships

feature
Pyridyl CF3 CF3 CF3

moiety
, , N >> other heterocycles
N N+ N
O−
Substituent CF3 is clearly more favorable than all other substituents
R1
Substituent S H is more favorable than all other substituents
Amide The CONR1R2 moiety can be replaced by certain five- or six-membered heterocycles; for
moiety example,
R R4 X O
O N R
O R O HN O N
N R5 O
N
N N N N
X = O,S
Substituents A broad range of substituents/groups is tolerated. R2, R3 may also both be hydrogen. Steric
R2, R3 as well as electronic features seem not to play a very important role
antennal movement. These effects are much different from those displayed by
aphids treated with neonicotinoids [41–43, 59].
33.1.3.5.2 Target Sites Although the structure of flonicamid has some similarity
to that of the neonicotinoids, it does not bind to the nicotinic acetylcholine receptor
when directly compared to nicotine and imidacloprid [42, 59, 60]. However,
flonicamid was found to be active on the A-type potassium channel currents. The
current hypothesis is that flonicamid blockade of the A-type potassium channel
in the presynaptic terminal underlies its lethal effect in insects. The loss of the
A-type potassium rectifying current would lead to the disruption of controlled
neurotransmitter release [59, 60].
CF3 O CF3 O
1) SOCl2
OH N CN
H
2) H2NCH2CN
N N
12
Flonicamid
1) SOCl2
2) N N CN
NC
N Na2CO3, H2O
19
CN
CF3 O CF3 O
aq. HCl
N CN N CN
N Cl N OH
Scheme 33.1.3 Synthesis of flonicamid.
Table 33.1.8 Chemical and physical properties of flonicamid.
Feature Property

Vapor pressure at 25 ◦ C 9.43 × 10−4 kPa
Water solubility at 20 ◦ C 5.2 g l−1
Partition coefficient (n-octanol/water at 25 ◦ C (log Pow )) 0.3
Photolysis DT50 (aqueous) 267 days
DT50 (soil) 22 days
Soil degradation DT50 <3 days
Soil mobility Moderate
Hydrolysis: (estimated half-life at 25 ◦ C) pH 5: stable
pH 7: stable
pH 9: 204 days
33.1.3.5.3 Cross-Resistance To date, laboratory studies as well as field obser-

vations, have failed to demonstrate any cross-resistance to orgnophosphate- or
carbamate-resistant populations of Myzus persicae [42] and Aphis gossypii [59].
33.1.3.6 Biological Activity and Use Recommendations

Flonicamid exhibits an excellent activity against numerous aphids of high agro-
nomical importance (Table 33.1.9). The compound is reported to be highly active
against both the larval and adult stages of aphids. In the field, the application
rates required to provide commercially acceptable control typically range from 40
to 60 g a.i. ha−1 ; however, in order to achieve a longer residual activity and/or in
Table 33.1.9 Current aphicidal spectrum of flonicamid.
Target pest Target pest
Acyrthosiphon kondoi Hyalopterus pruni

Acyrthosiphon pisum Lipaphis erysimi
Aphis craccivora Macrosiphum euphorbiae
Aphis fabae Macrosiphum rosae
Aphis glycines Myzus persicae
Aphis gossypii Myzus cerasi
Aphis pomi Myzus nicotianae
Aphis spiraecola Nasonovia ribisnigri
Aulacorthum solani Phorodon humuli
Anuraphis helichrysi Rhopalosiphum maidis
Brevicoryne brassicae Sitobion avenae
Diuraphis noxia Schizaphis graminum
Dysaphis plantaginea Therioaphis maculate
Eriosoma lanigerum Toxoptera citricidus
situations with higher pest pressures, application rates of 60 to 80 g a.i. ha−1 are
recommended [41–43, 61, 62].
Good to excellent activity has also been observed against plant bugs (Lygus spp.),
whitefly (Trialeurodes vaporariorum), thrips (Thrips tabaci), and Psylla (Cacopsylla
pyricola).
33.1.3.7 Safety Profile of Flonicamid

Like pymetrozine, flonicamid has a very favorable toxicological, environmental,
and ecotoxic profile (Table 33.1.10), and demonstrates no major negative impact on
beneficial insects and mites such as Bombyx mori, Apis mellifera, Harmonia axyridis,
and Phytoseiulus persimilis [41, 42].
Table 33.1.10 Safety profile of flonicamid.
Acute toxicity test Result
Oral LD50 , rat 884 mg kg−1 (male)

1768 mg kg−1 (female)
Dermal LD50 (acute), rat >5000 mg kg−1
Inhalation LC50 (acute), rat >4.9 mg l−1
Eye irritation Rabbit Minimally irritant
Mutagenicity/genotoxicity Negative
Avian oral LD50 (acute), mallard duck 1591 mg kg−1
Freshwater fish LC50 (96 h), rainbow trout >100 mg kg−1
Freshwater invertebrate EC50 (48 h), Daphnia magna >100 mg l−1
Algae EC50 (96 h) 119 mg l−1
33.1.4
Pyrifluquinazon
Pyrifluquinazon (20; developmental code: NNI 0101, IUPAC name: 1-acetyl-1,2,3,4-

tetrahydro-3-[(3-pyridylmethylamino]-6-[1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl]
quinazolin-2-one)) is a novel ‘‘insect-behavior regulator’’ currently under develop-
ment by Nihon Nohyaku. This compound is a close analog of pymetrozine (1), and
was registered in Japan in October 2010, but it is yet not on the market.
33.1.4.1 Discovery of Pyrifluquinazon

During the course of their investigations on methoxyacrylate fungicides, Nihon
Nohyaku replaced the methoxyacrylate pharmacophore with a carbamate group
[63]. The key intermediate 21 prepared in this research program was further
used to prepare novel heterocyclic building blocks such as the quinazolinone
derivatives 22 and 23, respectively. Treatment of the aminoquinazolinone (23) with
pyridine-3-carbaldehyde produced the imino derivative (24), which was found to
possess a high insecticidal activity against the hemipterous insect, Myzus persicae.
This lead structure represented a close analog of pymetrozine (see Section 33.1.2),
and triggered a broad optimization program. The imino moiety was reduced in order
to increase the photostability, and alkoxycarbonyl substituents then introduced at
the 1-position in the aminoquinazolinone ring to further enhance penetration of the
compounds through the cuticular layer of insects. This optimization led to R-768,
which was selected as a candidate for development due to its good insecticidal
activity against aphids and its favorable physico-chemical properties [63]. Further
modifications resulted in the discovery of pyrifluquinazon (20) [64].
Interestingly, both insecticides recently discovered by Nihon Nohyaku, pyri-
fluquinazon (20) and the diamide insecticide flubendiamide possess a heptafluoro
isopropyl substituent – a group which has only recently been applied more broadly
in insecticide research (Scheme 33.1.4).
A first patent application to protect the novel aminoquinazolinone derivatives
was filed by Nihon Nohyaku in 1989 [9], and within a few years a number
of further inventions were patented (see Table 33.1.1). Pyrifluquinzon was first
described in EP 1097932 [11], and a novel and economical process for producing
iminoquinazolinone derivatives was patented in 2005 [65].
33.1.4.2 Structure–Activity Relationship

The general structure–activity profile for the aminoquinazolinone derivatives [63,
64], which is shown in Table 33.1.11 is very similar to that of the pyridine
azomethines derivatives (see Table 33.1.2).
33.1.4.3 Synthesis of Pyrifluquinazon

The synthesis of pyrifluquinazon (20) has not yet been described in detail; however,
a possible synthesis of this compound is highlighted in Scheme 33.1.5 [9–13, 65].
N
Br
R N
H2N-R N N
O
N O N O
N OMe H
H H
21 22 R = Me 24
23 R = NH2 Lead compound
N N
CF3
Optimization
H Optimization F H
N N
Phase 1 N Phase 2 CF3 N
N O N O
Et O Me O
R-768 20
Pyrifluquinazon
Scheme 33.1.4 Discovery of pyrifluquinazon.
Table 33.1.11 Aminoquinazolinone derivatives: structure–activity relationships.
N
R2 R3 S
R1 A
N B
N X Pyridyl
moiety
R4
Aminoquinazolin-
one moiety
Structural feature Structure activity relationships
Substituent R1 i-C3 F7 > H > CF3 , OCF3 ≥ other substituents

Substituent R2, R3 H >> other groups
Substituent R4 COC3 H7 -n, other alkylcarbonyl > H > alkoxycarbonyl
Functional group C=X C=O equal to C=S
Bond A-B N=CH equal to NH–CH2
Pyridyl moiety 3-pyridyl >> phenyl, others
Substituent S H >> halogen, alkoxy, other substituents
O CF3 CF3
CF3 F F
F CH2Br
O Cl CF3 NBS CF3
CF3
NH NH
NH2
O O O O
CF3 CF3
F O N F
H2NNH2 NH2 N N
CF3 N CF3 N
N O N O
H H
CF3
AcOH, H2 CF3 F H
F H NaH, Ac2O N N
Pd/C N N CF3 N
CF3 N
N O
N O
H
O
20
Pyrifluquinazon
Scheme 33.1.5 Synthesis of pyrifluquinazon.
33.1.4.4 Chemical and Physical Properties of Pyrifluquinazon

The physico-chemical properties of pyrifluquinazon are listed in Table 33.1.12
[64]. Pyrifluquinazon has a higher logP value and a lower water solubility than
pymetrozine (Table 33.1.3), due mainly to the lipophilic nature of the 6-heptafluoro
isopropyl quinazolinone moiety and the 1-methylcarbonyl group in the amino-
quinazolinone ring, which could facilitate penetration through the cuticular layer
of insects.

As insect-behavior regulators, aminoquinazolinone derivatives such as R-768 and
pyrifluquinazon (20) act more slowly than other conventional insecticides, such
as the neonicotinoids. Typically, it takes a few days before aphids treated with
aminoquinazolinone derivatives die, although the affected aphids stop feeding
Table 33.1.12 Chemical and physical properties of pyrifluquinazon.
Feature Property
Appearance White powder

Water solubility at 20 ◦ C 12.1 ppm
Partition coefficient (n-octanol/water at 25 ◦ C (log Pow )) 3.12
immediately after treatment and become paralyzed, leading subsequently to star-

vation. In fact, this feeding inhibition is so strong that the affected aphids never
regain an ability to feed, even after being transferred to untreated plants. Although
they may be able to undergo limited reproduction before death, the offspring are
unable to feed and consequently die without growing. The mode of action of the
aminoquinazolinone derivatives [63, 64] such as pyrifluquinazon is very similar to
that of the pyridine azomethine compound pymetrozine, is but clearly different
from that of other, conventional insecticides.
33.1.4.6 Biological Activity and Safety Profile of Pyrifluquinazon
Pyrifluquinazon shows an excellent activity against a broad spectrum of hemipteran
pests and some thysanopteran pests (Table 33.1.13) [64]. No cross-resistance with
other insecticides was observed.
Like pymetrozine (1), pyrifluquinazon (20) has a favorable safety profile
(Table 33.1.14) [64]. Furthermore, pyrifluquinazon is safe towards beneficial
Table 33.1.13 Biological activity of pyrifluquinazon.
Pest species Scientific name Stage EC50 value (mg a.i. l –1 )
Aphid Myzus persicae (S)a Nymph 0.014

Myzus persicae (R)a Nymph 0.021
Aphis gossypiia Nymph 0.045
Whitefly Trialeurodes vaporariorumb Adult 0.59
Bemisia tabaci (B biotype)b Adult 0.18
Bemisia tabaci (Q biotype)b Adult 0.38
Mealybug Pseudococcus cornstockia Nymph 0.021
Planococcus kraunhiaea Nymph 0.96
Scale Pseudaulacaspis pentagonaa Nymph 0.74
Unaspis yanonensisa Nymph 0.74
Leafhopper Empoasca onukilb Adult 1.6
Thrips Scirtothrips dorsalisb Adult 0.46
a
Spray to insects and diet.
b
Spray to diet before releasing insects.
S, susceptible standard; R, resistant to organophosphates, carbamates, and pyrethroids.
Table 33.1.14 Safety profile of pyrifluquinazon.
Acute oral LD50 , rat 300–2000 mg kg−1

Mutagenicity (Ames) Negative
Avian oral LD50 , quail 1360 mg kg−1
Aquatic organism LC50 (96 h), carp 4.4 ppm
insects and nontarget arthropods. For example, effective concentration (EC)10

values of > 50 mg a.i. l−1 were measured against Harmonia axyridis (adult),
Orius sauteri (adult), Orius strigicollis (nymph), Amblyseius cumumeris (adult),
Phytoseiulus persimilis (adult), Amblyseius calfornicus (adult), Aphidoletes aphidimyza
(larva), Bombyx mori (larva), and Bombus terrestris (adult) [64]. Consequently,
pyrifluquinazon – like pymetrozine – would be expected to serve as a useful tool
for resistance management strategies and IPM programs.
References
1. Flueckiger, C.R., Kristinsson, H., Senn, PCT International Application WO

R., Rindlisbacher, A., Buholzer, H., and 2004/099,184, (Nihon Nohyaku Co.).
Voss, G. (1992) Proc. Brighton Crop Prot. 13. Uehara, M., Sakata, K., Morimoto, M.,
Conf. – Pests Dis., 1, 43–50. and Seo, A. (1999) Japan Patent Appli-
2. Kristinsson, H. (1994) Advances in the cation JP 11,158,180, (Nihon Nohyaku
Chemistry of Insect Control III, vol. 147, Co.).
Special Publication, Royal Society of 14. Pitterna, T., Siegrist, U., and
Chemistry, pp. 85–102. Szczepanski, H. (1994) European Patent
3. Kristinsson, H. (1995) Agro-Food-Ind. Application EP 613,895, (Ciba-Geigy
Hi-Tech, 6 (21), 23–26. AG).
4. Fuog, D., Fergusson, S.J., and 15. Siegrist, U. and Szezepanski, H.
Flueckiger, C. (1998) in Insecticides with (1994) European Patent Application
Novel Modes of Action (eds I. Ishaaya and EP 613,888, (Ciba-Geigy AG).
D. Degheele), Springer, New York, pp. 16. Rapold, T. and Senn, M. (1995) US
40–49. Patent Application US 94-194,185,
5. Kristinsson, H. (1989) European Patent (Ciba-Geigy AG).
Application EP 314,615, (Ciba-Geigy 17. Wyss, P. and Bolsinger, M. (1997) Pestic.
Sci., 50, 195–202.
AG).
18. Wyss, P. and Bolsinger, M. (1997) Pestic.
6. Kristinsson, H., Boeger, M., and
Sci., 50, 203–210.
Maienfisch, P. (1990) European Patent
19. Kayser, H., Kaufmann, L., Schuermann,
Application EP 391,849, (Ciba-Geigy
F., and Harrewijn, P. (1994) Proc.
AG).
Brighton Crop Prot. Conf. – Pests Dis.,
7. Szczepanski, H. and Kristinsson, H.
2, 737–742.
(1994) European Patent Application EP
20. Harrewijn, P. and Kayser, H. (1997) Pes-
604,365, (Ciba-Geigy AG).
tic. Sci., 49, 130–140.
8. Szczepanski, H., Kristinsson, H., 21. Boina, D.J., Youn, Y., and Stelinski,
Maienfisch, P., and Ehrenfreund, J. L. (2009) Abstracts of Papers, 238th
(1995) PCT International Application ACS National Meeting, August 16–20,
WO 95/18,123, (Ciba-Geigy AG). 2009, Washington, DC, Poster abstract.
9. Uehara, M., Shimizu, T., Fujioka, S., AGRO-031.
Kimura, M., and Tsubata, K. (1996) EP 22. He, Y.-P., Chen, L., Chen, J.-M., Chen,
735,035, (Nihon Nohyaku Co.). L.-Z., and Zhang, J.-F. (2010) Effects of
10. Machiya, K., Uehara, M., Shimizu, T., pymetrozine on the feeding behavior
Furuya, T., Kawaguchi, M., Abe, N., and of rice brown planthopper, Nilaparvata
Seo, A. (1999) Japan Patent Application lugens (Stal). Zhongguo Shuidao Kexue,
JP 11,012,254, (Nihon Nohyaku Co.). 24, 635–640 (AN 2011:31985).
11. Uehara, M., Watanabe, M., Kimura, 23. Kaufmann, L., Popp, B., Schuermann,
M., Morimoto, M., and Yoshida, M. F., Wiesner, P., and Kayser, H. (1998)
(2001) European Patent Application EP Pymetrozine - approaches to a novel
1,097,932, (Nihon Nohyaku Co.). mode of action. Book of Abstracts, 216th
12. Sanpei, O., Uehara, M., Niino, N., ACS National Meeting, August 23–27,
Kodama, H., and Sakata, K. (2004) Boston. AGRO-029.
References 1345
24. Kaufmann, L., Schuermann, F., Moore, S. (2000) 1999 Field trial results
Yiallouros, M., and Harrewijn, P. (2004) with pymetrozine (Fulfill) and thi-
Comp. Biochem. Physiol. C, Toxicol. amethoxam (Centric/Actara) for control
Pharmacol., 138C, 469–483. of cotton aphid (Aphis gossypii). Proceed-
25. Ausborn, J., Wolf, H., Mader, W., and ings, Beltwide Cotton Conference, vol. 2,
Kayser, H. (2005) J. Exp. Biol., 208, pp. 1335–1337.
4451–4466. 39. Polston, J.E. and Sherwood, T. (2003)
26. Gorman, K., Slater, R., Blande, J.D., Phytoparasitica, 31, 490–498.
Clarke, A., Wren, J., McCaffery, A., and 40. Sechser, B. and Reber, B. (1998) Us-
Denholm, I. (2010) Pest Manage. Sci., 66, ing a sequential testing scheme under
1186–1190. laboratory and field conditions with the
27. Karatolos, K., Denholm, I., Williamson,
bumble bee Bombus terrestris to evaluate
M., Nauen, R., and Gorman, K. (2010)
the safety of different groups of insec-
Pest Manage. Sci., 66, 1304–1307.
ticides. Proceedings of International
28. Lind, R., Slater, R., Pedroni, D., and
Conference, Ecotoxicology: Pesticides
Senn, R. (2008) International Congress
and Beneficial Organisms, October 1996,
of Entomology 2008, Proceedings,
Symposium Number 3.02, July 6–12, Cardiff, UK, pp. 166–174.
Durban. Abstract Number 1375. 41. Morita, M., Ueda, T., Yoneda, T.,
29. Flueckiger, C.R., Senn, R., and Koyanagi, T., Murai, S., Matsuo, N.,
Buholzer, H. (1992) Proc. Brighton Crop Stratmann, B., and Ruelens, P. (2000)
Prot. Conf. – Pests Dis., 3, 1187–1192. Proc. Brighton Crop Prot. Conf. – Pests
30. Follas, G. (1993) Proc. N. Z. Plant Prot. Dis., 1, 59–65.
Conf., 46, 21–23. 42. Hancock, H.G., de Lourdes Fustaino,
31. Senn, R., Sechser, B., and Flueckiger, F.M., and Morita, M. (2003) Floni-
C.R. (1994) Proc. Brighton Crop Prot. camid (F1785, IKI220): novel insecticide
Conf. – Pests Dis., 3, 1187–1192. chemistry for cotton and other crops.
32. Sechser, B., Bourgeois, F., Reber, B., Proceedings, Beltwide Cotton Confer-
and Wesiak, H. (1994) Meded. Fac. Land- ence, vol. 2, pp. 83–88.
bouwkd. Toegep. Biol. Wet. (Univ. Gent), 43. Morita, M., Ueda, T., Yoneda, T.,
59 (2B), 579–583. Koyanagi, T., and Haga, T. (2007) Pest
33. Sato, Y., Nojiri, M., and Hashino, Manage. Sci., 63, 969–973.
Y. (1996) Proc. Brighton Crop Prot. 44. Toki, T., Koyanagi, T., Morita, M.,
Conf. – Pests Dis., 1, 355–360. Yoneda, T., Kagimoto, C., and Okada,
34. Omar, H.I.H., El-Maghraby, H.M., H. (1994) European Patent Application
El-Khawalka, M.H.M., and El-Bessomy, EP 580,374, (Ishihara Sangyo Kaisha,
M.A. (1996) Alex. Sci. Exch., 17, Ltd).
271–276.
45. Sugihara, T., Tsuchiya, S., and Matsuo,
35. Mindt, G. (1997) Gesunde Pflanz., 49,
N. (1998) Japan Patent Application JP
230–234.
10,195,072, (Sumitomo Chemical Co.,
36. Allemann, D.V., Ferguson, S.,
Ltd).
Minton, B., and Ngo, N. (1997) Ful-
46. Tiebes, J., Taapken, T., Rook, B., Kern,
fill (pymetrozine) – a new approach
to sucking insect control. Proceedings, M., and Sanft, U. (1998) PCT Inter-
Beltwide Cotton Conference, vol. 2, pp. national Application WO 9,857,969,
1093–1095. (Hoechst Schering Agrevo GmbH).
37. Koenig, J.P., Lawson, D.S., White, S.M., 47. Bastiaans, H.M.M., Tiebes, J., Jans, D.,
and Dunbar, D.M. (1998) Utility of Hempel, W., Sanft, U., and Thonessen,
Fulfill 50 WG for aphid and whitefly M.-T. (2000) PCT International Ap-
management in cotton. Proceedings, plication WO 2000/035,912, (Aventis
Beltwide Cotton Conference, vol. 2, pp. CropScience GmbH).
997–999. 48. Harmsen, S., Bastiaans, H.M.M.,
38. Koenig, J.P., Lawson, D.S., Ngo, N., Schaper, W., Tiebes, J., Doller, U.,
Minton, B., Ishida, C., Lovelace, K., and Jans, D., Sanft, U., Hempel, W., and
Thonessen, M.-T. (2000) PCT Interna- 58. Kimura, T., Yoneda, T., Wakabayashi,
tional Application WO 2000/035/913, T., and Fukui, F. (1997) Japan Patent
(Aventis CropScience GmbH). Application JP 09,323,973, (Ishihara
49. Sugihara, K., Shudo, A., and Tsuchiya, Sangyo Kaisha, Ltd).
S. (1999) Japan Patent Application JP 59. Joost, H., Staetz, C., Black, B., Hayashi,
11,180,957, (Sumitomo Chemical Co., J., Kinne, L., Kelly, G., and Treacy, K.
Ltd). (2006) Proceedings, Beltwide Cotton
50. Maienfisch, P. and Farooq, S. (2001) Conference, January 4–6, San Antonio,
PCT International Application WO Texas, pp. 1583–1590.
2001/009,104, (Syngenta Participations 60. Hayashi, J.H., Kelly, G., and Kinne, L.P.
Ag). (2006) Poster presentation, Beltwide Cot-
51. Maienfisch, P. and Farooq, S. (2001) ton Conference, January 4–6, San Anto-
PCT International Application WO nio, Texas.
2001/014,373, (Syngenta Participations 61. Hancock, H.G. (2004) Field performance
Ag). of flonicamid (F1785) in cotton. Pro-
52. Haga, T., Morita, M., and Suchiibun, ceedings, Beltwide Cotton Conference,
H.B. (1995) Japan Patent Application January 5–9, San Antonio, Texas, pp.
JP 07,010,841, (Ishihara Sangyo Kaisha, 1629–1636.
Ltd). 62. Parker, R.D., Falconer, L.L., Livingston,
53. Koyanagi, T., Morita, M., Yoneda, T., S.D., and Hopkins, S.W. (2004) Cot-
and Kagimoto, C. (1995) Japan Patent ton fleahopper: evaluation of new
Application JP 07,025,853, (Ishihara insecticides and impact on cotton lint
Sangyo Kaisha, Ltd). production. Proceedings, Beltwide
54. Koyanagi, T., Yoneda, T., Kanamori, Cotton Conference, January 5–9, San
F., Kanbayashi, S., Tanimura, T., and Antonio, Texas, pp. 1731–1734.
Horiuchi, N. (1996) European Patent 63. Uehara, M., Shimizu, T., Fujioka, S.,
Application EP 744,400, (Ishihara Kimura, M., Tsubata, K., and Seo, A.
Sangyo Kaisha, Ltd). (1999) Pestic. Sci., 55, 359–362.
55. Yoshizawa, H., Sawaki, M., Miyaji, M., 64. Uehara1, M., Shimizu, T., Watanabe,
Murakami, K., Nagase, T., and Hattori, M., Sakata, K., Kimura, M., and Fujioka,
M. (2004) Japan Patent Application JP S. (2010) 112th IUPAC International
2004/010,572, (Ishihara Sangyo Kaisha, Congress of Pesticide Chemistry, Mel-
Ltd). bourne, Australia, July 4–8, 2010. Poster
56. Cottet, F., Marull, M., Lefebvre, O., and Abstract: no. 641.
Schlosser, M. (2003) Eur. J. Org. Chem., 65. Kodama, H., Sanpei, O., Abe, N., and
1559–1568. Uehara, M. (2005) PCT International
57. Schlosser, M. and Marull, M. (2003) Application WO 2005/123,695, (Nihon
Eur. J. Org. Chem., 1569–1575. Nohyaku Co.).
33.2
Acaricides with Undefined Mode of Action: Bifenazate, Cyflumetofen,
and Amidoflumet
Mark A. Dekeyser
33.2.1
Introduction
Traditionally, neurotoxic compounds have been the largest category of acaricides,

with several representatives from the organophosphate, carbamate, and pyrethroid
classes. During recent years, however, new classes of acaricides – some with as-yet
33.2 Acaricides with Undefined Mode of Action: Bifenazate, Cyflumetofen, and Amidoflumet 1347
undefined modes of action – have been developed. The details of three such
examples, namely bifenazate, cyflumetofen, and amidoflumet, are provided in this
chapter.
33.2.2
Bifenazate
Bifenazate is the common name of the first carbazate acaricide [1]. The structure
of this new class of acaricide is shown in Figure 33.2.1.
33.2.2.1 Discovery and Structure–Activity Relationship
Serendipity in the traditional screening approaches has been one of the main
resources for discovering acaricides with novel biochemical and physiological
targets [2, 3]. Based on the discovery that fungicidal phenylhydrazide compounds
also demonstrated acaricidal activity, a synthesis program was initiated at the
Research Laboratories of Chemtura in 1990.
The general structural variations for carbazate acaricides are shown in
Figure 33.2.2, while definitions of the structural variations are presented in
Table 33.2.1 [4–6].
NH O
NH
OCH3 O
Bifenazate Figure 33.2.1 Structure of bifenazate.
R1
(A)
N
N R3
R2
R1
NH (B)
NH R3
R2
R1
(C)
N R3
NH
Figure 33.2.2 General formula of carbazate acaricides.
R2 R3
A, diazene type; B, hydrazide type; C, hydrazone type.
Table 33.2.1 Definitions of general formula for carbazate acaricides.
Type R1 R2 R3 Patent
A Phenyl C1 –C4 alkoxy COOR US 5,438,123

US 5,367,093
A Phenyl C1 –C4 alkoxy PO(OR3 )2 US 5,543,404
A Fluorenyl, thienyl, pyridyl, C1 –C4 alkoxy COOR US 5,567,723
thiazolyl
B Phenyl, H C1 –C4 alkoxy, phenyl COOR US 5,367,093
B Fluorenyl, thienyl, pyridyl, C1 –C4 alkoxy COOR US 5,567,723
thiazolyl
B Phenyl C1 –C4 alkoxy PO(OR3 )2 US 5,543,404
C Phenyl C1 –C4 alkoxy R3 = aryl US 6,706,895
See Figure 33.2.2.
Following the discovery that ortho-biphenyl-substituted hydrazide compounds

[7] demonstrated acaricidal activity in the pesticide discovery screen, several
hundred – structurally diverse – biphenyl-substituted carbazate analogs were
synthesized in an optimization process using a bioassay with the two-spotted spider
mite (Tetranychus urticae) [8]. Details of the activity of ortho-biphenyl-substituted
carbazate compounds are listed in Table 33.2.2. These data indicate that
ortho-biphenyl-substituted carbazate compounds where the ester function consists
of a straight- or branched-chain alkyl group of three or four carbon atoms,
demonstrate the best biological activities in this series.
In contrast to the simple ortho-biphenyl-substituted carbazates, substituted alkyl
meta-biphenyl-carbazates were prepared via various multistep syntheses. This
allowed the introduction of different substituents, such as alkoxy, alkylthio, alkyl-
sulfonyl, to the pendant phenyl group (Scheme 33.2.1) [8].
Based on these variations, the structure–activity relationship (SAR) for
meta-substituted biphenyl carbazates can be summarized. The introduction of a
substituent para- to the pendant phenyl group improves the acaricidal activity
compared to the unsubstituted ortho- or meta-biphenyl carbazates. The most active
carbazates are those with a methoxy, ethoxy, or β-fluoro-ethoxy substituent para-
to the phenyl moiety in the meta-biphenyl carbazates (Figure 33.2.3).
From among several hundred carbazate derivatives synthesized and evaluated
for acaricidal activity, the carbazate isopropyl-2-(4-methoxy [1,1 -biphenyl]-3-yl)
hydrazine carboxylate (bifenazate) was selected for development as the most advan-
tageous [1]. The physico-chemical data of this compound are listed in Table 33.2.3.
Compound 1 in Scheme 33.2.1 was prepared from 4-hydroxybiphenyl, which is
also an intermediate for an alternate synthesis of bifenazate and its hydroxy-analogs
[11]. Several new methods of synthesis of bifenazate have been reported, namely
via arylboronic acid or ester cross-coupling reactions [12, 13], and via Lewis
acid-catalyzed electrophilic aromatic substitution reactions [14].
Table 33.2.2 Structure–activity relationships of

ortho-biphenyl-substituted carbazate compounds [9, 10].
H O
N N R
O
H
R M.p. (◦ C) % Mortality in vivo at 5 days against

T. urticae (25 ppm)
CH3 97–100 19
C2 H5 92–95 30/0a
CH2 C6 H5 101–104 17
CH(CH3 )2 104–106 42/additive, highest activity against
Sogatodes oryzicola (rice delphacid)
n–C3 H7 88–90 80
n–C4 H9 Oil 65
n–C5 H11 Oil 22
CH2 –CH=CH2 78–80 49
C(CH3 )3 92–94 68/54a/additional high activity
against S. oryzicola (rice delphacid)
–CH(CH3 )–C2 H5 80–83 72/79a/additional high activity
against S. oryzicola (rice delphacid)
a
Data for N=N–COOR derivatives.

Bifenazate demonstrates a broad-spectrum activity on phytophagous mites, such
as Tetranychus spp., Eutetranychus spp., Oligonychus spp., and Panonychus spp. [15].
Typically, bifenazate is 30- to 100-fold more active than older chemical classes
against adults, nymphs, and larvae, for example, of Tetranychus urticae (in cowpeas)
[1] (Table 33.2.4).
The activity of bifenazate remains almost constant over the temperature range
15 to 35 ◦ C, which permits its use under a wide range of field conditions [1, 15].
Bifenazate, when formulated as 50 wettable powder (WP), 24 suspension con-
centrate (SC), and 48 SC preparations, and applied at a rate of 300 to 600 g a.i. ha−1 ,
has been shown to control Panonychus ulmi in apples or Panonychus citri in citrus
fruits, for 20 to 40 days, with a rapid knockdown activity. Mites that have been
sprayed with bifenazate will become hyperactive after approximately 3 h, and will
no longer feed. The maximal lethal effect on the population is achieved after three
to four days [15, 16].
i ii
NO2 NO2 NO2

Br X-R1 OH
3 2 1
iii, iv, v, vi
NH O
NH R
X-R1 O
4
Scheme 33.2.1 Synthesis of meta-substituted biphenyl carbazates.
NH O
NH R
X-R1 O
4
X = O > S > SO2
R1 = CH3 > C2H5 > CH2CH2F > CH2C6H5 > CH(CH3)2
R = CH(CH3)2 > CH(CH3)C2H5 > C(CH3)3 > CH3 > CH2C6H5
Figure 33.2.3 Structure–activity relationships of the meta-biphenyl carbazates.
33.2.2.3 Ecobiology
Bifenazate demonstrates a remarkable and highly favorable ecobiological profile
[15–21]. Under typical conditions of use, the compound has no adverse effect on
beneficial insects, pollinating insects or beneficial predatory mites or wasps (see
Table 33.2.5).
At the recommended application rates, bifenazate is characterized as a compound
with a very low toxicity towards beneficial arthropods. In particular, when used in
Table 33.2.3 Physico-chemical data of bifenazate.
Common name Bifenazate

IUPAC name N -(4-Methoxy-bipheny-3-yl)hydrazine carboxylic acid
isopropyl ester
Development code D2341, UCC–D2341
Patent US 5 367 093; 1994
Launch 1999
Partition coefficient, log Pow at pH 7 3.4 (25 ◦ C)
Water solubility (mg l−1 ) 3.8 (20 ◦ C)
Vapor pressure (Torr) < 1 × 10−7 (25◦ C)
Hydrolysis 6.34 days at pH 4; 7.7 h at pH 7.0; 0.45 h at pH 9
Photolysis (h) 16.20 (pH 5, 25 ◦ C)
Aerobic soil metabolism (h) 7.3
Table 33.2.4 LC50 -values of bifenazate against different

development stages of T. urticae.
Life stage LC50 (ppm) at 5 days
Adult 0.3
Nympha 0.3
Larva 0.3
Egg 12
a
Second, third, and fourth stages.
integrated pest management (IPM) situations and in combination with predatory

mites, the compound is highly advantageous.
33.2.2.4 Registration Status

The superior ecobiological and toxicological properties of bifenazate, its rapid soil
dissipation, decreased application frequency, and improved efficacy compared to
other existing and available compounds, together with the first use in ornamentals,
®
led to Floramite being introduced in the United States under a reduced-risk status
in 1999 [1, 22, 23]. Soon thereafter, bifenazate was registered for use on a multitude
of crops, including apples, pears, peaches, plums, grapes, cotton, strawberries, and
hops, for which the recommended application rate was 300 to 600 g a.i. ha−1 . In
Europe, in 2005, bifenazate received a unanimous positive vote by the EU legislative
meeting for Annex I inclusion under Council Directive 91/414/EEC [16].
Currently, bifenazate is available in over 30 countries worldwide for the selective
control of spider mites, providing acaricidal activity against the eggs, larvae,
® ®
nymphs, and mites. It is marketed under the trade names Floramite , Acramite ,
®
and Mitekohne [22].
Table 33.2.5 Ecobiological properties of bifenazate against beneficial arthropods.
Organism Effect Remarks
Honey bee No adverse impact [16]

Bumble beea No adverse impact [20, 21]
Predatory mite Harmless [17–19]
Predatory bugs Harmless –
Parasitic wasps No effect on fecundity of surviving –
females (LC50 752 g a.i. ha−1 )
Lacewings Harmless With use rate 300 g a.i. ha−1 , only
3% mortality was observed.
a
By contact application.
33.2.2.5 Resistance Behavior

Preliminary results from studies on the mode of action of bifenazate in insects
have indicated that, at high concentrations, bifenazate acts on the postsynaptic
γ -aminobutyric acid (GABA) receptor in the insect nervous system. However, this
mode of action has not been confirmed in mites, and recently others have proposed a
different mode of action. Due to the uncertainty surrounding its mode of action, the
Insecticide Resistance Action Committee (IRAC) currently classifies bifenazate as
category UN, defined as compounds of unknown or uncertain mode of action [24].
As recently as 2005, bifenazate failed to demonstrate any cross-resistance with
known acaricides from the respiration inhibitor group, with known neuroactive
acaricides, or with inhibitors of respiration in either T. urticae [25–27] or P. ulmi [28].
Investigations with resistant mites (T. urticae) against pyridaben, fenpyroximate,
tebufenpyrad, and abamectin from different investigators, as well as with P. ulmi
strains resistant to dicofol, organotins, clofentezine, or hexathiazox, support this
claim. In both resistance and mode of action studies conducted between 2006 and
2009, it has been suggested that bifenazate acts as a Q0 inhibitor, while cytochrome
b was proposed as its mitochondrial target site [29–31]. It has also been reported
that spider mites pretreated with organophosphate or carbamate insecticides
antagonized the activity of bifenazate, which suggests that the co-application of
mixtures containing these insecticides with bifenazate should be avoided [32].
33.2.3
Cyflumetofen
Cyflumetofen, 2-methoxyethyl (R,S)-2-(4-tert-butylphenyl)-2-cyano-3-oxo-3-(α,α,α-

trifluoro-ortho-tolyl)propionate, is a novel acaricide which is highly active against
phytophagous mites [33]. The structure of this new class of acaricide is shown in
Figure 33.2.4.
CF3 Figure 33.2.4 Structure of cyflumetofen.

O
NC
O
O
Cyflumetofen
33.2.3.1 Discovery
Cyflumetofen belongs to the acylacetonitrile class, a new type of acaricide chemistry,
and was discovered during investigations into acaricidal benzoylacetonitrile com-
pounds. Cyflumetofen is synthesized by reacting a substituted phenylacetonitrile
starting material with a benzoyl halide, as shown in Scheme 33.2.2 [34, 35].

Cyflumetofen is a contact acaricide that is highly active against a wide spectrum of
phytophagus mites, such as Tetranychus spp. and Panonychus spp. Details of its activ-
ity against all life stages of T. urticae are listed in Table 33.2.6. Although cyflumetofen
shows little activity against lepidopteran, homopteran, and thysanopteran pests,
field trials with a 20% SC formulation of cyflumetofen at an application rate of
400–800 g a.i. ha−1 provided an effective control of spider mites for 28–31 days
O
O O
O O CN
CN
O
O
O
CF3
NaF
Cl
CF3
O
NC
O
O
O
Cyflumetofen
Scheme 33.2.2 General formula of trifluoromethanesulfonanilides.

Table 33.2.6 LC50 -values of cyflumetofen against different

development stages of T. urticae.
Life stage LC50 (ppm)
Female adult 4.8

Teliochrysalis 2.4
Deutonymph 1.9
Deutochrysalis 2.0
Protonymph 1.0
Nymphchrysalis 0.8
Larva 0.9
Egg 2.5
on apples, citrus, and pear. No phytotoxicity of cyflumetofen has been observed on

different varieties of crops following application of at least twice the normal field
usage rates [33].
33.2.3.3 Ecobiology
Cyflumetofen is a selective acaricide which provides excellent control of phy-
tophagous mites, yet is safe towards predatory mites and other organisms. In
laboratory tests, cyflumetofen has been shown as safe to a wide variety of beneficial
insects and mites, while in field tests it did not show any harmful effects to either
the honey bee Apis mellifera or the predatory mite Amblyseius californicus [33].
33.2.3.4 Registration Status

®
Otsuka launched cyflumetofen in Japan in 2007, under the trade name Danisaraba ,
for use as an acaricide on fruit trees, vegetables, tea, and ornamentals [36]. With
cyflumetofen having also been approved in South Korea, its registration is currently
under way in over 30 countries [22].
33.2.3.5 Resistance Behavior

Whilst the mode of action of cyflumetofen is effectively unknown, adult T. urticae
treated with cyflumetofen lose motor coordination after about 4 h, leading to
paralysis and death. Cyflumetofen shows no cross-resistance to existing acaricides;
neither has any cross-resistance been observed between cyflumetofen and strains
of T. urticae resistant to fluacrypyrim, mitochondrial electron transport inhibitor
(METI) acaricides, and chlorfenapyr [33].
33.2.4
Amidoflumet
Mites are pests not only of agricultural crops but also of most households. House
dust mites, which are a common cause of asthma and allergic symptoms, can
generally be controlled with pyrethroid insecticides, although benzyl benzoate has
also been employed in this role. Recent progress in fluorine chemistry, includ-
ing the development of new synthetic methods and novel fluorinating reagents,
has resulted in an increased discovery of fluorinated pesticides such that, in
2007, it was estimated that one-third of all new pesticides contained fluorine [37].
One such example is amidoflumet, methyl 5-chloro-2-[(trifluoromethyl) sulfonyl]
aminobenzoate [37]. Subsequently, the discovery by Sumitomo Chemical of the
insecticidal action of the trifluoromethanesulfonalide compounds led to the devel-
opment of 2-alkoxycarbonyl trifluoromethanesulfonanilide acaricides and, in turn,
of amidoflumet [38].
33.2.4.1 Discovery and Development

Amidoflumet, a member of sulfonanilide class, a new type of acaricide chemistry
(Figure 33.2.5), was discovered during investigations of the insecticidal trifluo-
romethanesulfonanilides, which proved to be active against houseflies, cockroaches,
and mites [38]. Amidoflumet is prepared by reacting a substituted aniline starting
material with trifluoromethanesulfonic anhydride, as shown in Scheme 33.2.3 [37].
The X-ray structure of amidoflumet shows the presence of an intramolecular
hydrogen bond between the sulfonamide H atom and the carbonyl O atom of
the ester group [39]. Details of the SARs for 2-alkoxycarbonyl trifluoromethane-
sulfonanilides acaricides against house dust mites, Tyrophagus putrescentiae and
Dermatophagoides farinae, and the cheyletid mite, Chelacaropsis moorei, are listed in
Table 33.2.7 (see also Figure 33.2.6) [40–42]. These data reveal optimal acaricidal
activity towards two species of dust mites when R1 is a halogen. Initially, amid-
oflumet was registered in Japan in 2004, to control house dust mites. Subsequently,
®
Sumitomo Chemical launched amidoflumet under the trade name Panduck [42].

Whilst the mode of action of amidoflumet is unknown, it does demonstrate
a high activity against common house dust mites, such as the mold mite, T.
CO2CH3
Cl NHSO2CF3
Amidoflumet Figure 33.2.5 Synthesis of amidoflumet.
CO2CH3 CO2CH3
(CF3SO2)2O
Cl NH2 Cl NHSO2CF3
Et3N
Amidoflumet
Scheme 33.2.3 Synthesis of amidoflumet.

Table 33.2.7 Structure–activity relationships of the trifluo-

romethanesulfonanilides (see Figure 33.2.6).
Compound Activity
R1 R2 D. farinae T. putrescentiae C. moorei

(8 mg m−2 ) (80 mg m−2 ) (80 mg m−2 )
Cl CH3 +++ +++ +++

Cl C4 H9 +++ +++ +
Cl C6 H5 +++a − NT
CH3 CH3 +++a − NT
NO2 CH3 − +++ NT
OCH3 CH3 +a − NT
CF3 CH3 − +++ NT
H CH3 +++a − NT
80 mg m−2 .
a
+++, 100% mortality; ++, >90% mortality; +, 70–90% mortality; –, no

mortality.
NT, not tested.
Figure 33.2.6 General formula of trifluoromethanesulfo-

CO2R2
nanilides.
R1 NHSO2CF3
putrescentiae, and the American house dust mite, D. farinae, at application rates
of 80 and 8 mg m−2 , respectively [37]; it also provides an excellent activity against
cheyletid mites, such as C. moorei, at 80 mg m−2 [42]. The results of laboratory
experiments using a filter paper contact method showed amidoflumet to be
superior to the standard treatments of phenyl salicylate and benzyl benzoate for
controlling D. farinae and C. moorei, and an equivalent control by each agent of
T. putrescentiae [42]. A comparison of the onset of action of amidoflumet with
phenyl salicylate and benzyl benzoate against D. farinae, using a filter paper
contact test with 800 mg m−2 of each material, showed all amidoflumet-treated
mites to be immobilized by 5 min after treatment, whereas a 30 min exposure was
required by the other two acaricides to achieve a similar result. By using an aerosol
spray method, the application of amidoflumet at 62 mg m−2 proved superior to a
65 mg m−2 treatment with the pyrethroid phenothrin, in controlling D. farinae, T.
putrescentiae, and C. moorei. Moreover, no repellency has been observed with the
various applications of amidoflumet.
Acknowledgments
The authors thanks Dr Jerry Graham (Chemtura) for providing useful reference
material and review suggestions, and Dr Changling Liu (Shenyang Research
Institute) for providing useful reference material.
References 1357
References
1. Dekeyser, M.A., McDonald, P.T., Angle, 18. James, D.G. (2002) Int. J. Acarol., 28 (2),
G.W. Jr, and Moore, R.C. (1996) Proc. 175–179.
Brighton Crop Prot. Conf. – Pests Dis., 2, 19. Kim, S.S. and Seo, S.G. (2001) Appl. En-
487–492. tomol. Zool., 36 (4), 509–514.
2. Dekeyser, M.A. (2005) Pest Manage. Sci., 20. Sterk, G. and Benuzzi, M. (2004) Cult.
51, 103–110. Protette, 33 (1), 75–77.
3. Dekeyser, M.A. and Downer, R.G.H. 21. Besard, L., Mommaerts, V., and
(1994) Pestic. Sci., 40, 85–101. Vandeven, J. et al. (2010) Pest Manage.
4. Dekeyser, M.A. and McDonald, P.T. Sci., 66, 786–793.
(1992) Uniroyal Chemical Company, 22. Anonymous (2009) Pest Projects, PJB
Inc. US Patent 5,367,093, (Prio: 20. 11. Publications, London.
1992). 23. US/EPA (1999) New Pesticide Fact
5. Dekeyser, M.A. and McDonald, P.T. Sheet. Available at: http://www.epa.
(1994) Uniroyal Chemical Company, gov/opprd001/factsheets/bifenazate.pdf
Inc. US Patent 5,438,123, (Prio: 05. 08. (accessed 13 November 2010).
1994). 24. Insecticide Resistance Action Committee
6. Dekeyser, M.A. and McDonald, P.T. (IRAC). Available at: http://www.irac-
(1995) Uniroyal Chemical Company, online.org/teams/mode-of-action (viewed
Inc. US Patent 5,543,404, (Prio: 05. 07. 13 November 2010).
1995). 25. van Leeuwen, T., Pottelberge, S., and
7. Dekeyser, M.A. (2000) Can. Chem. News, Tirry, L. (2005) Pest Manage. Sci., 61,
52 (7), 11–12. 499–507.
8. Dekeyser, M.A., McDonald, P.T., and 26. Lee, S.Y., Ahn, K.S., Kim, C.S. et al.
Angle, G.W. Jr (2003) Chimia, 57, (2004) Korean J. Appl. Entomol., 43 (1),
702–704. 43–48.
9. Dekeyser, M.A., McDonald, P.T., and 27. van Leeuwen, T., Stillatus, V., and Tirry,
Angle, G.W. Jr (1994) J. Agric. Food L. (2004) Exp. Appl. Acarol., 32 (4),
Chem., 42, 1358–1360. 249–261.
10. Dekeyser, M.A., McDonald, P.T., and 28. Pree, D.J., Whitty, K.J., and Van Driel,
Angle, G.W. Jr (1995) J. Agric. Food L. (2005) Exp. Appl. Acarol., 37, 165.
Chem., 43, 1705–1707. 29. Van Nieuwenhuyse, P., Van Leeuwen,
11. Chee, G.-L., Park, S.B., and Dekeyser, T., Khajehali, J. et al. (2009) Pest Man-
M.A. (1999) Uniroyal Chemical Com- age. Sci., 65, 404–312.
pany, Inc. US Patent 6,093,843, (Prio: 30. van Leeuwen, T., Tirry, L., and Nauen,
06. 10. 1999). R. (2006) Insect Biochem. Physiol., 36,
12. Kitamura, Y., Sakurai, A., Udzu, T. et al. 869–877.
(2007) Tetrahedron, 63, 10596–10602. 31. van Leeuwen, T., Vanholme, B., Van
13. Felpin, F.-X. and Fouquet, E. (2008) Adv. Pottelberge, S. et al. (2008) Proc. Natl
Synth. Catal., 350, 863–868. Acad. Sci. USA, 105, 5980–5985.
14. Chee, G.-L. (2006) Synth. Commun., 36, 32. van Leeuwen, T., Van Pottelberge, S.,
2151–2156. Nauen R., and Tirry, L. (2007) Pest
15. Ochiai, N., Mizuno, M., Mimori, N. Manage. Sci., 63, 1172–1177.
et al. (2007) Exp. Appl. Acarol., 43, 33. Sasama, Y., Takahashi, N., Ishii, N. et al.
181–197. (2007) Proceedings of the International
16. Grosscurt, A.C. and Avilla, L. (2005) Plant Protection Congress, vol. 1, pp.
Proceedings – British Crop Protec- 46–51.
tion Council International Congress: 34. Li, Y., Liu, Y., Zhou, Y. et al. (2009)
Crop Science & Technology, Octo- Agrochemicals, 48, 1–12.
ber 31–November 2, 2005, Glasgow, 35. Takahashi, N., Gotoda, S., Ishii, N. et al.
Scotland, UK., pp. 49–56. (2000) Otsuka Chemical Co. Patent WO
17. Kim, S.S. and Yoo, S.S. (2002) Biocon- 2002/014,263, JP 2000/244,738, (Prio:
trol, 47, 563–573. 11. 07. 2000).
36. Agropages (2007) Otsuka’s cyflumetofen 39. Kimura, M. and Hourai, S. (2005) Acta
in Japan. www.agronews.com/news Crystallogr., E61, o3944–o3945.
(viewed 13 November 2010). 40. Mori, T., Watanabe, K., and Takada,
37. Mori, T., Ujihara, K., Matsumoto, O. Y. (1996) Japan Patent JP 08,319,202,
et al. (2007) J. Fluorine Chem., 128, December 3, 1996.
1174–1181. 41. Mori, T., Takada, Y., Hatakoshi, M.,
38. Hatakoshi, M., Konishi, H., Yano, T., and Matsuo, N. (2004) Biosci. Biotechnol.
and Hirano, M. (1982) Japan Patent JP Biochem., 68, 425–427.
57,156,407, September 27, 1982 and JP 42. Mori, T., Matsuo, N., Hatakoshi, M.
198,143,547, (Prio: 24. 03. 1981). et al. (2007) Sumitomo Kagaku, 2, 4–13.
33.3
Pyridalyl: Discovery, Insecticidal Activity, and Mode of Action
33.3.1
Introduction
During the early 1990s, synthetic pyrethroid, carbamate, or organophosphorus

insecticides were the major materials available to control insect pests on agricultural
crops. Until that time, these compounds had proved to be very useful, based on their
ability to control a wide range of insect pests. However, a reduction in the efficacy
of these insecticides, as a result of the pests developing resistance to them, led to
failures in crop protection that became a serious problem worldwide. In addition,
the impact that these synthetic pesticides were having on the environment, with
regards not only to nontarget organisms but also to potential risks for human
health, were of major concern. Consequently, during recent years efforts to reduce
the use of these synthetic pesticides have been accelerated, as a global trend.
Integrated pest management (IPM) represents a means of minimizing such
resistance problems by introducing different pest control methods, other than using
synthetic pesticides, into existing pest control practices. Nevertheless, synthetic
pesticides should maintain an important role in IPM programs if such schemes are
to remain feasible for pest control. As IPM programs must be established under
various field conditions – including changes in the weather, pest species, and the
density and growth conditions of the crops – it is highly preferable that growers
should have a wide option of materials and methods available for pest control.
With efficacy in mind, however, a selective insecticidal activity against a target pest
would be a preferred characteristic of any pesticide that might be incorporated into
an IPM program.
After having taken such circumstances into consideration, a study was initiated
to identify a new insecticide that could be used to control lepidopterous pests,
and which would have an excellent insecticidal activity, a novel mode of action,
and a lower impact on nontarget organisms. This situation had arisen because
lepidopterous pests, such as Helicoverpa spp., Spodoptera spp., and Plutella xylostella,
were the major problems worldwide at the time. Consequently, specific and
efficient evaluation systems were established to detect insecticidal activity among
lepidopterous insects, in parallel with studies of various lead compounds, at the
Agricultural Chemicals Research Laboratory of Sumitomo Chemical Co., Ltd. As
a result of these investigations, several newly synthesized chemicals that demon-
strated a high insecticidal activity were first evaluated in the laboratory, and then
entered into trials to determine their efficacy for the control of lepidopterous pests
in the field. By following this approach, several compounds were identified with
excellent efficacy; ultimately, however, pyridalyl (experimental code: S-1812) was
selected as an insecticide for further development (Figure 33.3.1). Whilst, overall,
this compound showed an excellent efficacy as an insecticide, its low-level acute
toxicity to mammals, avians, and fishes was also of great benefit (Table 33.3.1) [1].
Details of the discovery of pyridalyl, together with its insecticidal activity, mode of
action, and information regarding commercial aspects, are outlined in the following
subsections.
Cl Cl
F3C O O O Cl
N
Cl
Figure 33.3.1 Structure of pyridalyl.
Table 33.3.1 Toxicological profile of pyridalyl technical.
Acute oral LD50 , rat > 5000 mg kg−1 body weight

Acute dermal LD50 , rat > 2000 mg kg−1 body weight
Eye irritation, rabbit Slight irritation
Acute inhalation LC50 , rat > 2.01 mg l−1
Skin sensitization, guinea pig Sensitizing
Ames test Negative
Avian toxicity
Bobwhite quail, LD50 1133 mg kg−1 diet
Mallard duck, LC50 > 5620 mg kg−1 diet
Fish toxicity
Carp, 96 h LC50 > 10 mg l−1
Bluegill, 96 h LC50 > 24 mg l−1
33.3.2
Chemistry
33.3.2.1 Lead Generation

The discovery of a new insecticide begins with the creation of suitable lead
compounds; these are then modified structurally to generate other compounds
with higher insecticidal activities against target insects. Although a variety of
approaches can be used to create appropriate lead compounds it should be noted
that, in the quest for pyridalyl, existing compounds were used as leads.
At the time, lepidopterous pests were important targets, mainly because they had
already developed resistance to the existing insecticides that were being applied
routinely to cotton, vegetables, and fruits. Among several compounds that had been
reported to possess biological activity, two 3,3-dichloro-2-propenyloxy compounds
(1 and 2), in which the dichloroallyl group was a common substituent, were shown
to have properties of insect growth regulation (Figure 33.3.2). This prompted the
synthesis of a series of compounds with a 3,3-dichloro-2-propenyl group, the aim
being to generate a new lead compound [2, 3].
The structure–activity relationship (SAR) of these compounds showed that the
introduction of a phenyl ether linkage could improve their insecticidal activity.
Perhaps more importantly, some of these compounds were also found to possess
insecticidal activity against lepidopteran species.
Compound 3, [4-phenoxy-2-(trifluoromethyl)phenyl 3,3-dichloro-2-propenyl
ether], also demonstrated unique lethal symptoms as well as insecticidal activity
against S. litura at 500 ppm, and these findings led to compound 3 being used as
the next lead (Figure 33.3.2) [4, 5].
33.3.2.2 Optimization of the Lead Compound to Pyridalyl

The lead compound 3 was divided into three parts for its optimization. The SAR
of the propenyl side chain revealed that the 3,3-dichloro-2-propenyl group was
essential for insecticidal activity, while the introduction of substituents at the 3- and
5-positions of the benzene ring led to a remarkable increase in such activity. These
substituent effects may indicate the importance of these positions to fix molecular
conformations in their bioactive forms. Both, the pyridine and phenyl rings were
shown to be favorable substituents and to yield highly active compounds. Notably,
their insecticidal activity was shown to be influenced by the linker between the
two aromatic rings (in particular, the length of the linkers). The linker with a 1,3-
or a 1,4-alkylene dihydroxy group caused a major increase in insecticidal activity,
and pyridalyl was successfully designed by taking these results into consideration
(Figure 33.3.3) [4, 5].
33.3.2.3 Physico-Chemical Properties

The physico-chemical properties of pyridalyl are listed in Table 33.3.2. Pyridalyl
is an odorless liquid with a vapor pressure of 6.24 × 10−8 Pa (25 ◦ C). Although
soluble in most organic solvents, it is not readily soluble in water.
O Cl
Cl
O Cl
O Cl
(R)n
3,3-Dichloro-2-propenyloxy compound 1
Cl
O (CH2)5 O Cl CF3 Cl
O O Cl
3,3-Dichloro-2-propenyloxy compound 2
Lead compound 3
(Weak activity)
Figure 33.3.2 Discovery of lead compound 3.

33.3 Pyridalyl: Discovery, Insecticidal Activity, and Mode of Action
1361
CF3 Cl
O O Cl
Lead compound 3
R Cl
3
O Cl
5
Linker R
Cl Cl
F3C O O O Cl
N
Cl
Pyridalyl
Figure 33.3.3 Optimization of lead compound 3.
Table 33.3.2 Physico-chemical properties of pyridalyl technical.
Feature Property
Physical form Liquid (20 ◦ C)

Melting point < −17◦ C
Vapor pressure 6.24 × 10−8 Pa(25◦ C)
Solubility Water: 0.15 ppb (20 ◦ C)
Organic solvents: soluble in most
33.3.3
Biological Aspects
33.3.3.1 Insecticidal Activity and Uses

The insecticidal activity of pyridalyl against the larvae of several major lepidopter-
ous pests has been determined, and is expressed as the lethal concentration (LC)50
and LC90 values in Table 33.3.3; the latter values of pyridalyl ranged from 1.53
to 13.8 mg a.i. l−1 . Pyridalyl is also highly active against the resistant population
of P. xylostella, which typically shows a high resistance towards conventional
Table 33.3.3 Insecticidal activity of pyridalyl against lepidopterous pests.
Species Stagea Test method Days after LC50 (mg LC90 (mg
treatment a.i. l –1 ) a.i. l –1 )
Cnaphalocrosis L3 Foliar spray 5 1.80 4.95

medinalis
Helicoverpa L3 Leaf dip 5 1.36 6.51
armigera
H. zea L2 Leaf dip 5 3.23 6.06
Heliothis virescens L2 Leaf dip 5 4.29 9.06
Mamestra L3 Foliar spray 5 1.98 4.92
brassicae
Spodoptera exigua L3 Leaf dip 5 0.93 1.90
S. litura L3 Foliar spray 5 0.77 1.53
Pieris rapae L2 Foliar spray 5 3.02 6.10
Plutella xylostella L3 Leaf dip 3 4.48 13.8
a
L2 and L3 indicate second and third instar larva, respectively.
insecticides, including synthetic pyrethroids, organophosphates, or benzoylpheny-

lureas (Table 33.3.4) [1]. Pyridalyl was also found to show insecticidal activity
against dipterous leafminer and thrips, such as Liriomyza spp., Thrips palmi, and
Frankliniella occidentalis. The ovicidal activity of pyridalyl compound appeared
limited, however, being observed only in P. xylostella.
In contrast to such strong insecticidal activity against lepidopterous insects, pyri-
dalyl showed minimal insecticidal activity against species of Hemiptera, Coleoptera,
and Orthoptera. Such selectivity of the compound may reasonably be regarded as
a preferred characteristic for uses in IPM programs, because some natural enemy
insects are included in those families. In fact, pyridalyl demonstrated a minimal
impact on various beneficial arthropods such as parasitic wasps, predatory insects
and mites, and pollinators (Table 33.3.5) [1, 6, 7].
Several field evaluations carried out to date have proved that pyridalyl, at
application rates of between 100 and 220 g a.i. ha−1 or 10 g a.i. hl−1 , can provide an
Table 33.3.4 Insecticidal activity of pyridalyl against

insecticide-resistant strain of P. xylostella.
Insecticide Class LC50 (mg a.i. l –1 )
Resistant strain Susceptible strain
Pyridalyl – 2.6 4.5

Cyfluthrin Synthetic pyrethroid >500 3.7
Pyrimifos methyl Organic phosphate >450 12.0
Chlorfluazuron Benzoyl phenylurea >25 3.4
Table 33.3.5 Beneficial arthropods not affected by pyridalyl at 100 mg a.i. l –1 .
Scientific name Beneficial Stagea Test method
Trichogramma japonicum Parasitic wasp Adult Foliar spray

Eretmocerus eremicus Parasitic wasp Adult/pupa Direct spray
Aphidius ervi Parasitic wasp Adult/pupa Direct spray
Chrysoperla carnea Predatory Chrysopidae L2–L3 Insect dip
Harmonia axyridis Predatory Coleoptera L2–L3 Foliar spray
Orius sauteri Predatory Hemiptera Adult/nymph Foliar spray
O. strigicollis Predatory Hemiptera Adult/nymph Foliar spray
Phytoseiulus persimilis Predatory Acarina Adult Foliar spray
Apis mellifera Pollinator Worker Direct spray
Bombus terrestris Pollinator Worker Direct spray
a
L2–L3 means second to third instar larvae.
excellent control of lepidopterous pests on vegetables or cotton. In addition, pyridalyl

has been developed for the control of Liriomyza spp. and Thrips palmi in vegetables
and ornamentals. The labeled or proposed crops in Japan, the Netherlands and the
United States are listed in Table 33.3.6. To date, no phytotoxicity has been reported
following the application of pyridalyl.

At an early stage in the optimization studies it was noted that, in treated larvae, these
compounds produced a unique symptom that resembled barn scars. Notably, such
scars appeared in larvae that had survived pyridalyl treatment, but not in larvae that
had died within several hours after treatment with higher (lethal) pyridalyl doses.
A similar situation occurred in the larvae of various lepidopterous insects treated
with pyridalyl. In order to examine the details of the barn scars, the compound was
applied topically to the thoracic dorsum of S. litura larvae [8]. Despite there being
no remarkable change at one day after treatment with lower doses of pyridalyl,
the treated site turned darker two days later, and the barn scars appeared after
ecdysis. In contrast, after receiving lethal doses of pyridalyl the larvae died within
several hours of treatment, without any conspicuous symptoms such as convulsion,
spasm, or vomiting. It was postulated that the symptoms produced by both lethal
and lower doses of pyridalyl might relate to a degeneration of cells in the larvae.
Subsequently, the effects of pyridalyl on cultured insect (Sf9) cells that had
been established from the ovary of S. frugiperda pupae, were investigated [9, 10].
Optical microscopy observations using a Trypan blue exclusion method showed
that pyridalyl had suppressed proliferation of the cells, or had at least reduced the
cell number. The ATP concentration in Sf9 cells was also decreased at 4–6 h after
pyridalyl treatment (1.0–10 μM); this effect was thought to relate to the insecticidal
activity, on the basis that the analogs of pyridalyl which failed to show any
insecticidal activity also had no significant effects on the Sf9 cells. When the effects
of pyridalyl on both the epidermal and Sf9 cells were observed using transmission
Table 33.3.6 Labeled crops and use patterns (August, 2010).
Country Crop Usage rate (g a.i. hl –1 )
Japan Cabbage 10
Chinese cabbage 10
Qing-geng-cai 10
Japanese radish 10
Broccoli 10
Tomato 10
Eggplant 10
Pimento 10
Lettuce 10
Welsh onion, leek 10
Asparagus 10
Cucumber, melon 10
Strawberry 10
Green soybeans 5–10
Peas, immature (with pods) 10
Potato 10
Sweet potato 10
Taro 10
Chrysanthemum 10
Other herbs 10
The Netherlands Eggplant (non-covered, land-based cultivation) 10
Tomato (non-covered, land-based cultivation) 10
Peppers (non-covered, land-based cultivation) 10
Chilli (non-covered, land-based cultivation) 10
USA Enclosed greenhouse commodities 60
electron microscopy (TEM) [11], the ultrastructural changes in epidermal cells

included mitochondrial swelling, dilation of the Golgi apparatus or endoplasmic
reticulum, the disappearance of microvillus structures in the apical surface facing
the cuticle, and shrinkage of the nuclei. Mitochondrial swelling and dispersal of the
polysomes was also apparent in the Sf9 cells. However, whilst such symptoms are
common in degenerating cells, they did not infer that pyridalyl had acted on any
specific cell organelle. Rather, the change in ATP concentration and appearance
of symptoms in the cells suggested that pyridalyl acted not by interfering with
cell division, inhibiting the cytoskeleton, inducing apoptosis or inhibiting nucleic
acid synthesis, but rather had acted as an inhibitor of mitochondrial respiration.
To verify this proposal, the effect of pyridalyl on mitochondrial respiration was
assessed by monitoring the oxygen consumption of a mitochondrial suspension
isolated from the flight muscle of S. litura adults. However, no effects due to
pyridalyl were detected in these isolated mitochondria [9].
Thus, the above-described symptoms and diagnoses in insects or cells suggest
that pyridalyl functions via a different biochemical mode of action compared to
any of the existing insecticides. Moreover, the lack of cross-resistance between any
existing insecticides supports such a presumption.
In a later study, the effect of pyridalyl on protein synthesis was investigated
using a variety of cell lines [12]. Notably, pyridalyl interrupted protein synthesis in
insect cell lines such as Sf9 and High 5, but not in mammalian cell lines such
as Hep G2, COS-1, and HL-60. These results also confirmed that pyridalyl did
not interrupt protein synthesis in BmN4, a cell line derived from Bombyx mori,
in which pyridalyl did not show insecticidal activity. Taken together, these results
suggest that pyridalyl disrupts a certain essential function in the cell that is specific
to lepidopteran insects (except for B. mori). It is suspected that the interruption of
protein synthesis is a subsequent phenomenon to the primal effect of pyridalyl.
33.3.4
Commercial Status
Currently, pyridalyl insecticides have been introduced to the market in Japan, the
United States, the Netherlands, and several countries in Asia, as of August 2010.
The typical trade names of PLEO™, SUMIPLEO™, and OVERTURE™ are also
used in some Asian countries and in the United States.
33.3.5
Conclusions
In Japan, a host of studies has been conducted over the past few years to establish
local IPM programs, using natural enemies, on the basis that administrations and
consumers alike are seeking to reduce their use of synthetic pesticides. From the
grower’s point of view, an IPM program may reduce the workload in terms of
pesticide applications. Indeed, under such circumstances the selective insecticidal
activity of pyridalyl should prove to be of major interest, and since 2002 it has been
largely included in such studies. Subsequently, spray programs using pyridalyl as
an insecticide, in combination with natural enemies, have been introduced on a
practical basis and are well accepted in some areas.
In other countries, however, despite the problems of insecticide resistance in lep-
idopterous pests having decreased following the introduction of Bt-gene-expressed
crops and other new insecticides, concerns are beginning to increase. Thus, the
commercialization of an insecticide with a mode of action that differs from that of
currently available insecticides is highly desirable. At present, pyridalyl undertakes
important roles in IPM and insecticide-resistance management programs, and is
contributing to agricultural production worldwide.
References
1. Saito, S., Iasayama, S., Sakamoto, N., 2. Quistad, G.B., Cerf, D.C., Kramer,
Umeda, K., and Kasamatsu, K. (2002) S.J., Bergot, J.B., and Schooley,
Proc. Brighton Crop Prot. Conf. – Pests D.A. (1985) Agric. Food Chem., 33,
Dis., 1, 33–38. 47–50.
3. Piccardi, P., Massardo, P., Bettarini, F., 7. Tillman, P.G. and Mulroony, J.E. (2002)
and Longoni, A. (1980) Pestic. Sci., 11, J. Econ. Entomol., 93, 1638–1643.
423–431. 8. Saito, S., Isayama, S., Sakamoto, N.,
4. Sakamoto, N., Saito, S., Hirose, T., and Umeda, K. (2004) J. Pestic. Sci., 29,
Suzuki, M., Umeda, K., Tsushima, 372–375.
K., and Matsuo, N. (2002) Abstracts 9. Saito, S., Sakamoto, N., and Umeda, K.
of Papers, 10th IUPAC International (2005) J. Pestic. Sci., 30, 17–21.
Congress on the Chemistry of Crop 10. Saito, S. (2005) J. Pestic. Sci., 30,
Protection, Basel, 2002, vol. 1, p. 254. 403–405.
5. Sakamoto, N., Saito, S., Hirose, T., 11. Saito, S., Yoshioka, T., and
Suzuki, M., Matsuo, S., Izumi, K., Umeda, K. (2006) J. Pestic. Sci., 31,
Nagatomi, T., Ikegami, H., Umeda, K., 335–338.
Tsushima, K., and Matsuo, N. (2003) 12. Moriya, K., Hirakura, S., Kobayashi,
Pest Manage. Sci., 60, 25–34. J., Ozoe, Y., Saito, S., and Utsumi, T.
6. Isayama, S., Saito, S., Kuroda, K., (2008) Arch. Insect Biochem. Phys., 69,
Umeda, K., and Kasamatsu, K. 22–31.
(2005) Arch. Insect Biochem. Phys., 58,
226–233.
33.4
Recent Nematicides
33.4.1
Introduction
During the late nineteenth and early twentieth centuries, the science of nematology
experienced a rapid growth. Following heavy losses of sugar beet in central Europe,
caused by the cyst nematode H. schachtii, intensive investigations were undertaken
to identify and control the life cycle of this organism. In 1881, Julius Kühn, in
Germany, demonstrated the first chemical treatment for sugar beet cyst nematode
control by applying carbon disulfide to the soil. Kühn also conducted a series of
studies on trap crops and crop rotation to break the organism’s life cycle. Of these
alternatives, crop rotation proved to be the most effective and economic method,
and today remains the most widely used field control measure for nematodes [1].
Unfortunately, crop rotation is effective only for those species with a restricted
host range; furthermore, the intensification of cropping has been made possible
through the use of chemical nematicides and the development of cultivars that are
resistant to nematode multiplication.
Yet, growers rarely rely on a single method of control, and several measures are
usually integrated into a modern nematode management strategy (Table 33.4.1).
Nonetheless, nematode populations capable of overcoming resistance in plants are
now common, and the use of nematicides has been associated with environmental
contamination and hazards to health. In the past, nematicides have included some
of the most toxic products used in agriculture, to a point where several of these
products have been withdrawn from the market [2]. Modern nematode control
strategies require environmentally safe, cost-effective, and reliable products that

meet the demands of both the grower and the consumer [3]; such products include
novel fumigants, nonfumigants, and biological nematicides.
Although biological nematicides are often not thought of as acceptable alterna-
tives to their chemical counterparts – owing to their lack of broad-spectrum activity,
inconsistent performance and slower action – a number of commercial successes
have been achieved with biologicals in the management of nematodes. The cur-
rently available commercial biological products registered for nematode control
include formulations containing Paecilomyces lilacinus or Bacillus firmus. Recently,
in the US, Bayer CropScience introduced Votivo®, the first biological nematicide
for use as a seed treatment in corn, which contained the bacterium B. firmus. The
biological mode of action of Votivo® provides protection against damage caused by
plant-parasitic nematodes to the young corn roots during their first critical weeks
of establishment [4].
Details of these new chemical compounds employed to control plant-parasitic
nematodes are reviewed in this chapter.
33.4.2
Nematicide Chemical Classes and Products
Two types of chemical product are used to manage nematodes, namely soil
fumigants and nonfumigant (or contact) nematicides, both of which can provide
an excellent control of plant-parasitic nematodes. Details of the major nematicides
used in the main markets are listed in Table 33.4.2.
Table 33.4.1 Types of nematode management practice.
Practice Description
Use of resistant Resistant variety: cultivars with resistance to nematode multiplication.

or tolerant Tolerant variety: cultivars able to tolerate nematode attack without a
varieties yield penalty
Crop rotation Crop rotation with nonhost crops is an effective way to control
plant-parasitic nematode species with a narrow host range
Fumigants These are nonselective materials that vaporize when applied to the soil.
As gases, they move up through air spaces in the soil, killing
nematodes and other microorganisms
Nonfumigant Chemical compounds that protect plants early in the growing season
chemicals allowing them to produce healthy root systems. These compounds can
be applied as foliar applications, soil applications, and/or as seed
treatments
Biologicals These are living organisms that interfere with growth and reproduction
of plant-parasitic nematodes. Some are species-specific, while others
are broad-spectrum in nature
Table 33.4.2 Major global markets and current major products.
Region Major products
USA Fumigants: chloropicrin, dazomet, 1, 3-dichloropropene, dimethyl

disulfide, metam sodium/potassium, methyl bromide
Nonfumigants: abamectin, aldicarb, cadusafos, carbofuran, fosthiazate,
oxamyl, thiodicarb
Japan Fumigants: chloropicrin, dazomet, 1, 3-dichloropropene, metam
sodium/potassium, methyl bromide
Nonfumigants: benfuracarb, cadusafos, carbosulfan, dazomet,
fosthiazate, imicyafos, oxamyl, pyraclofos
Brazil Nonfumigants: abamectin, aldicarb, cadusafos, carbofuran, terbufos,
fosthiazate
Western Europe Fumigants: methyl bromide
Nonfumigants: ethoprophos, fenamiphos, fosthiazate, iprodione,
oxamyl
33.4.2.1 Fumigants
Fumigant nematicides (Table 33.4.3) are broad-spectrum biocides that are highly
effective in controlling nematodes and increasing crop yields. Whilst the majority
of these fumigants control plant-parasitic nematodes certain ones are also effective
against fungal diseases, soil insects, and weeds.
In 1943, the discovery by Carter of the soil fumigant 1,2-dichloropropane/1,3-
dichloropropene mixture (D-D) (5) initiated the extensive use of nematicides.
Shortly afterwards, however, ethylene dibromide (EDB) (7), 1,2-dibromo-3-
chloropropane (DBCP) (4), and methyl bromide (MBr) (8) were introduced, which
allowed the growers to fumigate either at one to two weeks before planting, or at
the time of planting.
Although these nematicides allowed a single, short-term chemical tactic, and pro-
vided a much greater flexibility for the farmers, the U.S. Environmental Protection
Agency (EPA) later proclaimed such soil fumigants as unsafe, and suspended their
registration. Apparently, D-D could affect a variety of soil organisms that included
the nitrifying bacteria, the suppression of which would lead to an accumulation
of phytotoxic levels of ammonia [5]. Both, DBCP and EDB were shown to be bio-
logically (but not physically) degraded in soil [6]; consequently, having undergone
only minimal biological degradation in the upper soil levels, these nematicides
could penetrate to a lower soil level. Trace amounts of these nematicides were also
identified in the groundwater [7].
Other fumigants currently registered for use in the United States include
chloropicrin (1), 1,3-dichloropropene (1,3-D; 3), MBr (8), metam sodium (10), and
dimethyl disulfide (6). Unfortunately, all of these compounds are generally highly
phytotoxic and so must be applied before planting.
Table 33.4.3 Major fumigant products.
Compound Active ingredient Year of Main companies Trade name

number introduction (remark)
1 Chloropicrin 1908 Mitsui Chemical Chloropicrin

[Trichloro(nitro) Chemtura
methane]
2 Dazomet (3, 5-dimethyl- 1942 Agro-Kanesho Basamid
1, 3, 5-thiadiazinane- UCB
2-thione)
3 1, 3-D 1942 Dow Telone
(1, 3-dichloropropene) Agro-Kanesho
4 DBCP (1, 2-dibromo- 1954 Shell Nemagon
3-chloropropane) Dow (registration
Occidental cancelled
Petroleum 1977)
5 D-D 1943 Shell (Registration
(1, 2-dichloropropane/ cancelled)
1, 3-dichloropropene
mixture)
®
6 Dimethyl disulfide United Paladin
Phosphorus, Inc
7 EDB (ethylene 1945 – (Registration
dibromide) cancelled in
1970s )
8 MBr (methyl bromide) 1932 Arkema Brom-O-Gas
Chemtura
9 MeI (methyl iodide) 2005 Arysta Midas
10 Metam sodium (sodium 1956 Agro-Kanesho Metam
salt of UCB
methyldithiocarbamate) Nufarm
MAI
Amvac
The most recent soil fumigant to be introduced to the market, dimethyl disulfide,
is sold under the trade name Paladin®. In this case, the active ingredient occurs
naturally within the environment and is produced biologically in plants and
animals, which makes it a good replacement for MBr. Paladin® is developed and
distributed by United Phosphorus Inc. in partnership with Arkema Inc. [8].
33.4.2.2 Carbamates and Organophosphates

The earliest nonvolatile nematicides, which were identified during the late 1950s
and early 1960s, belong to the chemical classes of carbamates and organophosphates
(OPs) (Table 33.4.4) [9]. Both, carbamates and OPs target the nervous system and
Table 33.4.4 Carbamates and organophosphates used as nematicides.
Compound Active ingredient Year of in- Main Trade

no. troduction companies name
Carbamates
11 Aldicarb 1968 Bayer Temik
S O NHMe Dow
N
Me Me O
12 Carbofuran 1967 FMC Furadan
O MAI
Rallis
MeNH O
O Me
Me
13 Carbosulfan 1979 FMC Marshall
O MAI
S
(n-Bu)2N N O
Me O Me
Me
14 Oxamyl 1973 DuPont Vydate
SMe
Me2N O NHMe
N
O O
15 Thiodicarb 1977 Bayer Larvin
Me SMe
N
O
S Me
O N N
Me
O O
Me N
SMe
Organophosphates
16 Fenamiphos 1967 MAI Gharda Nemacur
O
EtO P NH-i-Pr
O
SMe
Me

Subclass: Organothiophosphates
17 Cadusafos 1988 FMC Rugby
Me
O Et
EtO P S
S Me
Et
18 Chlorethoxyphos 1995 Amvac Fortress
S
EtO P OEt
O Cl
CCl3
19 Ethoprophos 1967 Bayer Mocap
O
EtO P S-n-Pr
S-n-Pr
20 Fosthiazate 1991 Syngenta Nematrim
Me Ishihara
O Et
EtO P S
N O
S
21 Pyraclofos 1989 Sumitomo Boltage
O
EtO P S-n-Pr
O
N
N
Cl

22 Tebupirimfos 1994 Bayer Aztec

S
EtO P O-i-Pr
O
N
N t-Bu
23 Terbufos 1974 Amva Counter
S United
EtO P OEt Phosphorous
Chinese
S
companies
S-t-Bu
Subclass: Phosphonothioates
24 Imicyafos 2009 Agro-Kanesho Nemakick
O
EtO P n-PrS
N N
CN
N
are known to be inhibitors of the enzyme acetyl cholinesterase [10, 11]. Although
initially, these compounds were monitored for their insecticidal abilities, they were
later found also to have nematicidal activities.
Both, carbamates and OPs, are applied in granular or liquid form, or as seed
treatments, and rely on water to move them through the soil profile. For the
most part, they are incorporated into the top 10–15 cm of soil, either overall, strip
applied, in-the-row-, in-furrow, or as seed treatments. Two clear advantages of
these chemical classes are their relatively low phytotoxicity [9] and their improved
selectivity compared to fumigants; as a result, their application is not limited to
preplant applications. Moreover, the lower concentrations of carbamates and OPs
required to achieve a commercially acceptable control minimize the chances of
the chemicals being leached through the soil profile in the case of abnormally
heavy rainfall, or under conditions that reduce the normal degradation rates of the
pesticide.
The carbamates aldicarb (11), carbofuran (12), and oxamyl (14) are currently
registered in the U.S. as nematicides. At present, however, aldicarb and carbofuran
are both under special review by the EPA, and will be phased out during the next
few years as they do not meet the EPA’s rigorous food safety standards and hence
may pose unacceptable dietary risks [12]. In August 2010, Bayer announced that,
from 2014 onwards, it will voluntarily cease distribution of the product Temik®,
which contains the active ingredient aldicarb.
Fenamiphos (16) and ethoprophos (19), both of which are members of the OP
class, are less systemic and highly effective against nematodes [9]. Somewhat prob-
lematically, the re-registration of fenamiphos (Nemacur®, Bayer AG, Leverkusen,
Germany) in the U.S. has not been possible, mainly because, as the regulations un-
der the U.S. Food Quality Protection Act became increasingly strict, the registration
costs became prohibitively expensive. Hence, as the re-registration of Nemacur®
proved to be nonprofitable for Bayer, in 2005 it was removed from the U.S. market.
Whilst this dilemma holds true also for many nonfumigant nematicides, one
novel OP that received its first global approval in 2010 was the active ingredient,
imicyafos (24) [13]. Yet, despite the Japanese agrochemical company Agro-Kanesho
starting to sell imicyafos in a new nematicide formulation (Nemakick 1.5 G®) in
Japan, the quest for new nematicidal chemical classes that are less hazardous to
human health and the environment continues.
33.4.2.3 Abamectin
One of the newest classes of active substances to demonstrate nematicidal efficacy
has been the avermectins. These natural products are known for their anthelmintic
and insecticidal activities, but may also affect parasitic nematodes [14]. As the
avermectins have a limited downwards movement in plants when applied as a
foliar treatment, they must be applied as either granulated or liquid formulations,
or as a seed treatment to the soil. Avermectin B1 (25; abamectin; Figure 33.4.1),
which is a mixture of avermectin B1a (≥80%) and B1b (≤20%), demonstrated the
greatest efficacy as a nematicide [15–17].
OMe
HO Seed treatment
OMe nematicide
O O
23
22 Active ingredient: Abamectin
O O O 25 Year of introduction: 2006
O Main crop usage: Cotton,corn,
H
R
soybean
O O
>80% B1a: R = C2H5 Company: Syngenta
<20% B1b: R = CH3 OH
Trade name: Avicta®
OH
Avermectin B1 (25, abamectin)
Figure 33.4.1 Avermectin B1 (25; abamectin).

Of all fumigants and nonfumigants described so far, the natural product

abamectin is the least problematic from an environmental point of view, on
the basis of its physical properties. Typically, the leaching potential of abamectin
is very low, and it undergoes a rapid degradation that prevents the persistence of
residues or the contamination of groundwater. Moreover, as abamectin does not
spread well in the rhizosphere, it must be applied to the soil in order to have any
effect on nematodes [18–20].
Abamectin was the first seed treatment nematicide to be developed by Syngenta
Crop Protection AG, and is sold under the trade name Avicta® (Figure 33.4.1). It
was launched in 2006 in the USA for the treatment of cotton, while registration for
its use in corn followed in 2009 and 2010 for countries such as the USA, Brazil,
South Africa, and Argentina. The newest market for abamectin is that of soybeans,
for which registration has been granted in Brazil, Argentina, and the USA.
33.4.2.3.1 Mode of Action of Abamectin The mode of action of the avermectins
has been investigated in plant-parasitic nematodes, in terms of their gross ef-
fects on the movement and infective behavior of the parasites. Juveniles of the
root-knot nematode Meloidogyne incognita, when exposed to a 120 nM aqueous
solution of avermectin B2 a-23-ketone, initially lost movement within 10 min, but
remained responsive to touch. Although a partial recovery occurred within 30 min
of exposure, movement was lost irreversibly after 120 min [21]. A similar triphasic
response was also seen in M. incognita juveniles exposed to avermectin B1 . The
initial loss of movement in M. incognita may be reflective of the avermectins’ activity
as γ -aminobutyric acid (GABA) agonists at inhibitory synapses [15, 22]. Evidence
supporting this theory has been provided by Wright et al. [21], who found that the
GABA antagonists picrotoxin and bicuculline would counteract the effects of aver-
mectins on the locomotion of M. incognita second-stage juveniles. More recently,
however, Dent et al. [23] reported that ivermectin (22,23-dihydroavermectin B1a +
22,23-dihydroavermectin B1b ) bonded to the Caenorhabditis elegans glutamate-gated
chloride channel (GluCl), and that mutations in GluCl would confer resistance to
ivermectin in this organism. Westerholme and Rogers [24] also noted a general
acceptance that the molecular targets of the avermectin/milbemycin anthelmintics
are the GluCls, though no investigations have yet been conducted on plant-parasitic
nematodes in this respect.
33.4.2.3.2 Biological Activity of Abamectin When Applied as a Seed Treatment
During the early development of abamectin as a nematicidal seed treatment,
extensive studies were conducted on the activity of a range of seed- and soil-applied
compounds [25]. When these compounds were tested as seed dressings in a tomato
pot test against the root-knot nematode M. incognita, those with a known soil-applied
nematicidal activity generally failed to indicate a similar degree of efficacy when
applied as a seed treatment, when compared to abamectin. For example, fosthiazate
(19) (Table 33.4.4) provided some control, but lacked the same level of persistence
over a four-week infestation period, while cadusafos (17) inhibited the germination
of tomato seeds at higher loading rates. On cotton, cadusafos provided less
protection than abamectin. The evaluation of various carbamates, including aldicarb
(12) and oxamyl (15) (Table 33.4.4), also demonstrated a poor efficacy against root
knot nematodes when applied as a seed dressing on tomato seeds.
Between 2005 and 2007, Syngenta Seed Care initiated an extensive testing of
the yield benefit of Avicta® Complete Corn (insecticide/fungicides/nematicide;
nematicidal active ingredient, abamectin) across the Midwest USA. The results
showed that, over a three-year period, growers could expect a six-bushel-per-acre
average yield increase compared to treatment with Cruiser Extreme 250® (insec-
ticide/fungicide) on 85% of the corn acres planted. In a normal season, when
the crops would be subjected to higher temperatures, rainfall, and stressful pest
pressures, Avicta® Complete Corn provided a greater yield increase than in sea-
sons with less-stressful growing conditions. Overall, the application of Avicta®
Complete Corn each season provided a reliable protection and allowed plants to
thrive, despite adverse growing conditions [26].
33.4.3
Experimental Nematicides
33.4.3.1 Spirotetramat
Spirotetramat [26; Movento®, IUPAC name: (5s,8s)-3-(2,5-dimethylphenyl)-8-
methoxy-2-oxo-1-azaspiro[4.5] dec-3-en-4-yl ethyl carbonate; Figure 33.4.2], from
Bayer CropScience, is a novel active ingredient from the new chemical class of
tetramic acids. When applied to the foliage, spirotetramat penetrates the leaf
cuticle and is translocated as spirotetramat-enol via the xylem and phloem, up to
the growing shoots and down to the roots. This full ambimobility or two-way sys-
temicity (phloem and xylem transport) ensures the control of hidden and soil-living
sucking pests following foliar application, and also protects any new shoots.
The worldwide field development of spirotetramat by Bayer CropScience AG
has resulted in numerous uses against many species of whitefly, aphid, scales
(soft and armored scales), mealy bugs, psyllids, and selected thrip species in
vegetables, cotton, soybean, pome and stone fruit, grapes, hop, citrus, nut trees,
and banana. The novel mode of action of spirotetramat makes it an excellent
rotation partner with existing products for the management of aphid, whitefly, and
psyllid populations, that are frequently resistant to conventional insecticides [27].
Notably, spirotetramat demonstrates both insecticidal and nematicidal activities.
For example, when McKenry et al. conducted a field evaluation of the efficacy of
Movento® to control nematodes infesting perennial crops, a 70% reduction in the
population levels of Xiphinema americanum (American Dagger Nematode) collected
O
HN
MeO O
OEt
O
Spirotetramat (26) Figure 33.4.2 Spirotetramat (26).
from grapevines (Vitis spp.) was reported over a three-year period [28]. In 2009, as
a result of these findings, Bayer CropScience filed a patent application for the use
of tetramic acid derivatives to control nematodes. Despite the slow onset of action
of its growth-regulating insecticidal effect and a short half-life in soil, spirotetramat
can reduce the population density of soil-dwelling, plant-damaging nematodes in
both annual and perennial crops following foliar treatment [29].
33.4.3.2 Fluensulfone
Fluensulfone [27; developmental code: MCW-2, IUPAC name: 5-chloro-2-(3,4,4-
trifluorobut-3-en-1-ylsulfonyl)-1,3-thiazole] is a nematicide belonging to the
fluoroalkenyl group, which demonstrate far lower toxicities than the OP- or
carbamate-based nematicides [30, 31]. Fluensulfone has been under development
at Makhteshim Chemical Works Ltd since 2008, and its first global registration is
anticipated in 2013–2014. The submission will support the use of fluensulfone on
fruiting and cucurbit vegetables, potatoes, tobacco, and bananas against root-knot
nematodes (Meloidogyne spp.) [32].
33.4.3.2.1 Discovery The first haloalkenes possessing nematicidal activity were

described by Stauffer in 1965 [33, 34], and by FMC [35, 36] during the late 1980s
(trifluorobutenyl sulfides of types 28 and 29; Table 33.4.5). The FMC applications, in
particular, which claimed a broad-spectrum nematicidal activity on cyst nematodes,
as well as on M. incognita (the southern root-knot nematode) [35, 36], attracted
attention and triggered extensive research efforts by other companies. For example,
as early as 1989 Schering reported compounds such as 30 [37], while further FMC
compounds (e.g., 31 [38]; also claimed by Bayer [39]) and 32 [40] directed attention
towards the –CH=CF2 (difluorovinyl) group as an alternative to the previous
–CF=CF2 group. As insecticides, however, these newer compounds displayed a
broader spectrum compared to the trifluorovinyl compounds. Further examples of
insecticidal and nematicidal difluorovinyl compounds were provided by Monsanto
(33) [41] and Ciba-Geigy (34; development candidate targeting mites and aphids)
[42, 43]. ICI (later Zeneca Agrochemicals, now Syngenta) initiated its research
program in 1989, starting with compounds of the then-current FMC patents. Since,
at that time, ICI was merging with the agrochemical interests of Stauffer, it was
realized that compounds such as 28 (as disclosed in the much earlier Stauffer
patents) were also good leads. The early investigations of the series also included
examples that contained trifluorobutenesulfinyl and sulfonyl moieties, instead of
the trifluorobutenesulfanyl moiety (as in 28); these were shown to retain or even
improve the nematicidal activity [44–47].
Subsequently, attention was turned to the difluorovinyl series [48, 49] and,
following an extensive program with analogs, in 1994 an optimized candidate
emerged for evaluation, E3274 (35) [50, 51]. This compound was able to control a
broad spectrum of nematodes while having a minimal effect on insects, but was
not further developed. The first examples of nematicidal fluoroalkenyls from Bayer
were disclosed in 1987 (31) [39]. In 1996 and 1997, three more patent applications
were made by Bayer (together with Monsanto) claiming fluoroalkenyl esters
Table 33.4.5 Key examples related to the chemical class of fluoroalkenyls.
Compound General structure Company Patent Publication Reference

type application year
28 F Stauffer US 3223707 1965 [33]

N S
F
N F
29 F FMC WO 8607590 1986 [35]

N S
Cl F
N S F
30 F Schering EP 0342150 1989 [38]

N S
F
O F
31 F Bayer EP 0257484 1987 [39] [40]
FMC US 4876285 1989
( )10 F
Difluorovinyl group
32 F FMC US 4950666 1990 [38]

HO
F
O
33 F Monsanto WO 9215555 1992 [41]
+
Cl− H3N
F
34 CF3 Ciba-Geigy EP 577555 1994 [42]
F
O O ( )3
( )2 F
O
O
35 O O F Zeneca WO 9524403 1995 [50]

S S
Cl F
N
E3274
Compound General structure Company Patent Publication Reference

type application year
36 F Nihon Bayer WO 01002378 2001 [55]

Makhteshim
S S(O)n
X F
N F
n = 0,1, 2
X = halogen
Example:
O O F 27
S S
Cl F
N F
Fluensulfone (BYI 1921, MCW-2)
and amides with insecticidal and nematicidal activities [52–54]. Fluoroalkenyls

of the heterocyclyl trifluorobutenesulfanyl type, possessing nematicidal activity,
followed in 2001 in a patent application from Nihon Bayer [55]. The latter patent cov-
ered compounds of the general structure 36, while fluensulfone (27) was mentioned
specifically (Table 33.4.5). The excellent nematicidal activity of 27 triggered further
research activities, such that a series of patent applications was filed between 2002
and 2005, not only in the heterocyclyl trifluorobutenesulfanyl series [56–66] but also
in process-chemistry cases directed towards the synthesis of 27 [67, 68]. Currently,
fluensulfone [55] is undergoing worldwide development by Makhteshim [32].
33.4.3.2.2 Fluoroalkenyl Nematicides: Structure–Activity Relationships The gen-

eral structure–activity profile for the fluoroalkenyl nematicides of the fluensulfone
type is shown in Table 33.4.6. As a consequence of the extensive investigations
with the fluoroalkenes series, it became apparent that wide variations in activity
would occur as the heterocyclic portion of the molecule was varied, but that such
variability did not appear to be a simple function of, for example, the size of the
substituent on the heterocycle or the overall logP-value. In the halogenated vinyl
moiety, much less variability was tolerated, with the best moieties by far being the
trifluorobutenyls and difluorobutenyls. Although, sulfide, sulfoxide, and sulfone
can each be used as tethers, sulfide and sulfoxide would be converted to the sulfone
in vivo, via an oxidative metabolism.
The fluoroalkenyls also display an insecticidal activity that follows a different
structure–activity profile [43]. In general, the difluoroalkenyl derivatives exhibit a
broader spectrum than the trifluoroalkenyls, while the best potency is observed
with the difluoroalkenyl carboxylic acid derivatives.
Table 33.4.6 Fluoroalkenyls of fluensulfone type: structure–activity relationship.
O O F
A
HetAr HalVinyl S S
Cl F
N F
Heterocyclyl Halogenated vinyl
moiety moiety Fluensulfone (27)

feature
Halogenated F F F
vinyl moiety
F, F> F >> others
F
HetAr Nematicidal activity found with heterocycles having a wide range of
physical properties
Functional –S–, –S(O)–, –S(O)2 – > –O–
group A
33.4.3.2.3 Synthesis of Fluensulfone The synthesis of fluensulfone (27) was first

reported in 2001 by Nihon Bayer [55]; subsequently, in 2003 and 2004, two process
patent applications were made from Bayer CropScience [67, 68] (Scheme 33.4.1).
The routes proposed by Nihon Bayer and Bayer CropScience both start from
commercially available 4-bromo-1,1,2-trifluorobut-1-ene (37). In the synthesis from
Nihon Bayer, 37 is converted directly to the thiazolyl trifluorobutenesulfanyl
intermediate 39 by coupling with 2-mercaptothiazole. In contrast, in the process
route from Bayer, 37 is first converted to the dithiocarbamate (38) either in one step
by treatment with ammonium dithiocarbamate, or in two steps (albeit in higher
yield) by sequential treatment with ammonium thiocyanate and hydrogen sulfide.
The dithiocarbamate (38) is then reacted with chloroacetaldehyde diethyl acetal in
acetic acid to produce 39. Although, the route from Bayer to 39 is less concise, the
overall yield is higher [67]. In the final steps, 39 is chlorinated regioselectively onto
the thiazole ring with N-chlorosuccinimide (NCS) to afford 40 [55]; subsequent
oxidation with Oxone® then provides fluensulfone (27) [68].
The details of other synthetic methodologies to access pesticidal fluoroalkenes
have been reported by Zeneca [50, 51, 69] and Ciba-Geigy [43]. In particular,
the synthesis of 4-bromo-1,1-difluorobut-1-ene (42) at Zeneca should be noted
(Scheme 33.4.2).
33.4.3.2.4 Chemical and Physical Properties of Fluensulfone The physico-chemical

properties of fluensulfone have not yet been reported, other than its melting point
(34 ◦ C) [68]. However, it can be inferred from the molecular structure that the
logP of fluensulfone is higher than that of the OP and carbamate standards.
Fluensulfone may be somewhat volatile although, to a certain extent, this would
F
Br
F
NH4 + S− NH2
F S SH
37
S
or N
1) NH4SCN K2CO3
2) H2S, Et3N
OEt F
F Cl
H2N S OEt S S
F F
PPTS, AcOH N F
S F
38 39
NCS, CCl4
O O F F
S S oxone R S S
Cl F Cl F
N F N F
27 40
MCW-2
Fluensulfone
Scheme 33.4.1 Synthesis of fluensulfone (27).
F F F
HBr, CH2Cl2 Br Zn, H2O
Br Br Br
F F F
F F
37 41 42
Scheme 33.4.2 Synthesis of 4-bromo-1,1,-difluorobutene-1 (42).
be a desirable trait as it would be less likely to produce a variable control in soils

of different types and moisture contents. Whilst the degradation half-life (DT50 ) of
fluensulfone in soil has been reported to range from 11 to 22 days [30], this should
reduce the potential for fluensulfone to leach into the groundwater [32]. As yet, no
information is available on the biodegradation of fluensulfone, however.
33.4.3.2.5 Mode of Action of Fluensulfone Typically, fluensulfone demonstrates

a slow and irreversible nematicidal activity against second-stage juveniles (J2)
of M. javanica in vitro. Observations from a series of exposures of J2 juveniles
to fluensulfone at various concentrations, and for various periods of time, have
suggested that when the juveniles have taken up a lethal dose of fluensulfone they
become immobile and die over time. Hence, the effect is truly nematicidal, leading
to the death of the target nematodes, rather than being a temporary, nematostatic
activity. In contrast, fluensulfone was shown to be only partially effective as an

inhibitor of the hatching process, even at the highest concentrations tested [30].
The straight rod shape of J2 larva immobilized by fluensulfone differs from that
of the J2 exposed to OPs; this suggests that the nematicidal mode of action of
fluensulfone differs from that of OPs – that is, via an inhibition of acetylcholine es-
terase [30]. Although the biochemical mode of action of fluensulfone in nematodes
is unclear, one hypothesis has proposed the inhibition of medium-chain acyl CoA
dehydrogenases, which are important enzymes for mobilization of the lipids on
which nematodes rely for energy [70–72]. In insects, the difluoroalkenyl derivatives
were found to inhibit β-oxidation of fatty acids in the mitochondria [43].
33.4.3.2.6 Biological Activity and Use Recommendations Fluensulfone is reported

to exhibit a high level of activity against root-knot nematodes (Meloidogyne spp.)
(Table 33.4.7), and is planned for use on fruiting vegetables, cucurbits, potatoes,
tobacco, and bananas [30, 31]. The results of field trials conducted in 2009 indicated
that the application rate of active ingredient (a.i.) required for commercially
acceptable control should range between 2 and 4 kg a.i. ha−1 [32]. In addition,
fluensulfone is known to display good crop safety after soil incorporation.
Although ‘‘at-planting’’ applications cannot provide season-long nematode
control, they will generally protect the plants for a sufficient time to establish a
good root system.
No insecticidal effects were observed with fluensulfone. The formulation used
for field testing was a 1.5% granular and a 480 g l−1 emulsifiable concentrate (EC
Table 33.4.7 Current nematicidal spectrum of fluensulfone (27).
Target pest Common name
Demonstrated control
Meloidogyne incognita Southern root-knot nematode
Meloidogyne hapla Northern root-knot nematode
Meloidogyne javanica Javanese root-knot nematode
Globodera pallida Potato cyst nematode
Belonolaimus spp. Sting nematode, turf
Positive indications
Helicotylenchus spp. Spiral nematode, banana
Rhadopholus similis Burrowing nematode, banana
Trichodorus spp. Stubby root nematode
Ditylenchus spp. Stem nematode
Pratylenchus penetrans Root lesion nematode
Paratylenchus spp. Pin nematode
Heterodera glycines Soybean cyst nematode
Xiphinema spp. Dagger nematode
Criconemella spp. Ring nematode
48%); however, slow-release formulations are currently under development for

drench/drip irrigation applications.
33.4.3.2.7 Safety Profile and Ecotoxicity Details of the safety profile of fluensulfone
are listed in Tables 33.4.8 and 33.4.9 [31, 32]. Fluensulfone has a low acute
mammalian toxicity, and no prohibitive findings regarding either beneficial or
nontarget organisms have been reported to date. Although some of these data are
preliminary (as the compound is still under development), fluensulfone appears
to have a much lower toxicity compared to OP- and carbamate-based nematicides
that are currently available.
33.4.3.3 Benclothiaz
The nematicidal properties of compound 43 were first reported by Ciba-Geigy (now
Syngenta) in 1991 [73]. The compound is highly active against the southern root
knot nematode (M. incognita) and the soybean cyst nematode (H. glycines). In
2002, Syngenta applied for a common name for 43 (benclothiaz; developmental
code CGA 235 860, IUPAC name: 7-chloro-1,2-benzothiazole).
33.4.3.3.1 Discovery and Properties of Benclothiaz The synthesis of benclothiaz

was first reported in 1963 by Ricci et al, in relation to a synthetic methodology for
access to benzisothiazoles [74], and reappeared in 1984 in another publication [75].
In 1991, the research team at Ciba-Geigy disclosed the nematicidal properties of
benclothiaz among a series of substituted benzisothiazoles [73]; a process patent
Table 33.4.8 Safety profile of fluensulfone (27).
Oral LD50 , rat >500 mg kg –1

Dermal LD50 , rat >4000 mg kg –1
Inhalation LC50 , rat >5058 mg m –3
Skin sensitization, guinea pig Sensitizing
Table 33.4.9 Ecotoxicity profile of fluensulfone (27).
Species Result
Birds Nontoxic
Freshwater fish Low
Freshwater invertebrates Moderate
Honey bee Nontoxic
Earthworms Nontoxic
Cl Cl
Cl Cl
Cl S
t-BuSH, K2CO3 S NH2OH S 0.05 eq. PPTS
N
O DMF n -PrOH n -PrOH
O N
OH
44 45 46 43
Scheme 33.4.3 Synthesis of benclothiaz (43).
followed in 1995, when benclothiaz became a development candidate (CGA 235 860)
[76]. Additional process patent applications were filed by Mitsubishi Chemical Corp.
and Sumitomo Seika Chemicals Co., Ltd in 2002 and 2006, respectively [77, 78].
The structure–activity relationship of the analogs appears to be very narrow,
according to the biological examples disclosed [73], and the substitution pattern
found in benclothiaz was particularly prominent. To date, the physico-chemical
properties of benclothiaz have not been reported, other than its melting point (49 ◦ C)
[76]. The biological activity of benclothiaz was described in the first patent from
Ciba-Geigy [73]; typically, benclothiaz provided 90–100% control of M. incognita
on tomato plants at 0.0006% of the soil volume (drench application, sandy soil).
Benclothiaz also provided some control of H. glycines on soybean plants (drench
application, sandy soil). As yet, the mode of action of benclothiaz remains unknown,
and no data regarding its safety profile have yet been reported.
33.4.3.3.2 Synthesis of Benclothiaz The synthesis of benclothiaz (43), as reported

by Ciba-Geigy, is shown in Scheme 33.4.3 [76]. Benclothiaz is prepared in three steps
from commercially available 2,3-dichlorobenzaldehyde (44), the key transformation
being the cyclization reaction of 2-mercaptobenzaldehydeoxime (46) to afford the
benzisothiazole (43). Previously, such cyclizations were performed in the presence
of a large excess of strong acid, but this was difficult to remove during the
work-up and the reported yields were consequentially moderate [74, 79]. However,
with n-propanol as solvent, and in the presence of a catalytic amount of pyridine
p-toluenesulfonic acid (PPTS) or other strong acid, benclothiaz can be obtained on
a large scale with a high purity and a yield in excess of 90%. Further reports on the
synthesis of benclothiaz have appeared in patent applications filed by Mitsubishi
Chemical Corp. and Sumitomo Seika Chemicals [77, 78].
References
1. Singh, R.S. and Sitaramaiah, K. (1994) Control of Plant-Parasitic Nematodes, V &

Plant Pathogens – The Nematodes, Inter- R Unipress GmbH, Göttingen.
national Science Publisher, New York, 4. Bayer CropScience (2009) Press release;
p. 4. www.bayercropscience.com.
2. Burrows, P.R., Kerry, B.R., and 5. McKenry, M.V. (1980) in CRC Hand-
Perry, R.N. (1994) J. Zool. Lond., 232, book of Pest Management in Agriculture,
341–346. Section D, vol. 3 (ed. D. Pimentel), CRC
3. Slaats, B.E. (2008) Investigations on the Press, Cleveland, OH, pp. 59–73.
Efficacy of Encapsulation of the Endopar- 6. Castro, C.F. and Belser, M.O. (1966)
asitic Fungus Hirsutella rhossiliensis for Environ. Sci. Technol., 2, 779–783.
References 1385
7. Holden, P.W. (1986) Pesticides and Symposium of Nematology, August

Groundwater Quality: Issues and Problems 30–September 3, St Andrews, Scotland.
in Four States, Board on Agriculture, 20. Nordmeyer, D. and Dickson, D.W.
National Research Council, National (1981) J. Nematol., 13, 452–453.
Academy Press, Washington, DC. 21. Wright, D.J., Birtle, A.J., Corps, A.E.,
8. United Phosphorus Inc. (2010) and Dybas, R.A. (1984) Ann. Appl. Biol.,
News release; http://news.agropages. 103, 465–470.
com/News/NewsDetail––2742.htm 22. Mellin, T.N., Busch, R.D., and Wang,
(accessed 16 August 2010). C.C. (1983) Neuropharmacology, 22,
9. Heald, C.M. (1987) Classical nematode 89–96.
management practices, in Vistas on 23. Dent, J.A., Smith, M.M., Vassilatis, D.K.,
Nematology (eds J.A. Veech and D.W. and Avery, L. (2000) Proc. Natl Acad. Sci.
Dickson), E.O. Painter Printing Co., USA, 97 (6), 2674–2679.
Deleon Springs, FL, pp. 100–104. 24. Wolstenholme, A.J. and Rogers, A.T.
10. Brown, A. (2006) Mode of action of (2005) Parasitology, 131 (Suppl. 1),
insecticides and related pest control S85–S95.
chemicals for production agriculture, 25. Grimm, C., Hofer, D., Cochran, A.,
ornamentals, and turf, Pesticide Infor- Long, D., and Xing, L. (2007) Evaluation
mation Leaflet of Maryland Cooperative of insecticides for nematode control
Extension, University of Maryland. when applied as seed treatment. Pro-
11. Slaats, B.E. (2003) Seed Dressing as ceedings Beltwide Cotton Conferences,
Crop Protection to minimize the impact
New Orleans, LA, pp. 185–188.
of plant-parasitic nematodes. Disserta-
26. Klix, M., Pedersen, P., Watrin, C., and
tion, University of Bonn.
Long, D. (2010) Avicta® Complete
12. http://www.epa.gov/oppsrrd1/cumulative/
Corn – a comprehensive, effective corn
carbamate_risk_mgmt.htm.
seed treatment program. Annual Meet-
13. Bird, J. (2010) First Launch for
ing, Society of Nematologists, Boise, ID,
Agro-Kanesho’s Imicyafos in Agrow,
July 11–15, 2010.
597, p. 28.Available at: www.agrow.com.
27. Brück, E., Elbert, A., Fischer, R.,
14. Burg, R.W., Miller, B.M., Baker, F.F.,
Krueger, S., Kühnhold, J., Klueken,
and Birnbaum, J. (1979) Antimicrob.
M., Nauen, R., Niebes, J.-F., Reckmann,
Agents Chemother., 15, 361–367.
U., Schnorbach, H.-J., Steffens, R., and
15. Campbell, W.C. (ed.) (1989) Ivermectin
and Abamectin, Springer-Verlag, New van Waetermeulen, X. (2009) Crop Prot.,
York. 28, 838–844.
16. Dybas, R.A. (1989) in Ivermectin and 28. McKenry, M., Kaku, S., and Buzo, T.
Abamectin (ed. W.C. Campbell), (2009) Evaluation of Movento (Spirote-
Springer-Verlag, New York, tramat) for Efficacy Against Nematodes
pp. 287–310. Infesting Perennial Crops. Vine Lines
17. Dybas, R.A., Hilton, N.J., Babu, J.R., (University of California August 2009
Preiser, F.A., and Dolce, G.J. (1989) in issue).
Topics in Industrial Microbiology, vol. 23 29. Fischer, R. (2009) PCT International Ap-
(eds A. Demain, J. Hunter-Cevera, H. plication WO 2009/085,176, Bayer Crop-
Rossmoore, and G. Somuti), Elsevier, Science.
Amsterdam, pp. 201–210. 30. Oka, Y., Shuker, S., and Tkachi, N.
18. Barker, K.R. (1978) Determining ne- (2009) Pest Manage. Sci., 65, 1082–1089.
matode population response to control 31. Oka, Y., Berson, M., and Barazani, A.
agent, in Methods for Evaluating Plant (2008) Proceedings, 5th International
Fungicides, Nematicides and Bactericides, Congress of Nematology, July 13–18,
American Phytopathological Society, Brisbane, Australia, pp. 313–314.
pp. 114–124. 32. Everich, R. and Schiller, C. (2009)
19. Birtle, A.J., Corps, A.E., and Wright, MCW-2: A new proprietary nematicide
D.J. (1982) Biological effects of aver- from Makhteshim Chemical Works.
mectin on nematodes. 16th International USDA/IR-4 Food Use Workshop,
Summerlin, USA, September 15–16, C.R.A., Hotson, M.B., Sillars, N.C., and
2009. Dowling, A.J. (1995) PCT International
33. Brokke, M.E. (1965) US Patent Applica- Application WO 9,524,403, (Zeneca
tion US 3,223,707, (Stauffer Chemical Ltd).
Co.). 51. Bowden, M.C. and Brown, S.F. (1998)
34. Brokke, M.E. (1970) US Patent Applica- PCT International Application WO
tion US 3,513,172, (Stauffer Chemical 9,847,884, (Zeneca Ltd).
Co.). 52. Kraatz, U., Hartwig, J., Andersch, W.,
35. Cullen, T.G. and Martinez, A.J. (1986) Erdelen, C., Turberg, A., Mencke, N.,
PCT International Application WO and Ruminski, P.G. (1996) DE Patent
8,607,590, (FMC Corp.). Application DE 4,445,792, (Bayer AG &
36. Cullen, T.G., Martinez, A.J., and Vukich, Monsanto Co.).
J.J. (1988) PCT International Application 53. Kraatz, U., Andersch, W., Turberg, A.,
WO 8,800,183, (FMC Corp.). Mencke, N., Phillion, D.P., Ruminski,
37. Hübl, D., Bühmann, U., Pieroh, E., and P.G., and Yalamanchili, G. (1997) PCT
Jopplen, H. (1989) European Patent International Application WO 9,708,130,
Application EP 0,342,150, (Shering AG). (Bayer AG & Monsanto Co.).
38. Peake, C.J. (1989) US Patent Application 54. Kraatz, U., Andersch, W.,
US 4,876,285, (FMC Corp.). Wachendorff-Neumann, U., Turberg,
39. Naumann, K., Becker, B., Homeyer, B., A., Mencke, N., and Yalamanchili, G.
and Stendel, W. (1987) European Patent (1997) PCT International Application
Application EP 0,257,484, Bayer AG. WO 9,708,132, (Bayer AG & Monsanto
40. Peake, C.J., Cullen, T.G., and Martinez, Co.).
A.J. (1990) US Patent Application US 55. Watanabe, Y., Ishikawa, K., Otsu,
4,950,666, (FMC Corp.). Y., Shibuya, K., and Abe, T. (2001)
41. Ruminsky, P.G. (1992) PCT Inter- PCT International Application WO
national Application WO 9,215,555, 2001/002,378, (Nihon Bayer Agrochem
(Monsanto Co.). K.K.).
42. Maienfisch, P., Pitterna, T., and Böger, 56. Watanabe, Y., Ishikawa, K., Narabu, S.,
M. (1994) European Patent Application Gomibuchi, T., Otsu, Y., and Shibuya,
EP 577,555, (Ciba-Geigy AG). K. (2001) PCT International Applica-
43. Pitterna, T., Böger, M., and Maienfisch, tion WO 2001/066,529, (Nihon Bayer
P. (2004) Chimia, 58, 108–116. Agrochem K.K.).
44. Turnbull, M.D. (1992) European Patent 57. Kraatz, U., Gallenkamp, B., Marhold, A.,
Application EP 507,464, (ICI PLC). Wolfrum, P., Andersch, W., Erdelen, C.,
45. Turnbull, M.D. and Finney, J. (1992) Turberg, A., Hansen, O., and Harder,
European Patent Application EP A. (2001) PCT International Application
506,270, (ICI PLC). WO 2002/006,256, (Bayer AG).
46. Turnbull, M.D. (1992) European Patent 58. Kraatz, U., Gallenkamp, B., Marhold, A.,
Application EP 506,271, (ICI PLC). Wolfrum, P., Andersch, W., Erdelen, C.,
47. Turnbull, M.D. and Finney, J. (1993) Turberg, A., Hansen, O., and Harder,
PCT International Application WO A. (2002) PCT International Application
9,304,049, (ICI PLC). WO 2002/006,257, (Bayer AG).
48. Fitzjohn, S., Robinson, M.P., and 59. Kraatz, U., Gallenkamp, B., Marhold, A.,
Turnbull, M.D. (1994) PCT International Wolfrum, P., Andersch, W., Erdelen, C.,
Application WO 9,406,777, (Zeneca Turberg, A., Hansen, O., and Harder,
Ltd). A. (2002) PCT International Application
49. Fitzjohn, S., Robinson, M.P., Turnbull, WO 2002/006,259, (Bayer AG).
M.D., Smith, A.M., Salmon, R., and 60. Kraatz, U., Gallenkamp, B., Rieck, H.,
Taylor, R. (1994) PCT International Marhold, A., Wolfrum, P., Andersch,
Application WO 9,406,782, (Zeneca W., Erdelen, C., Loesel, P., Turberg,
Ltd). A., Hansen, O., and Harder, A. (2002)
50. Turnbull, M.D., Bansal, H.S., Smith, PCT International Application WO
A.M., Salmon, R., Fitzjohn, S., Godfrey, 2002/006,260, (Bayer AG).
References 1387
61. Watanabe, Y., Ishikawa, K., Otsu, Y., R., Bansal, H.S., and Williams, A.G.
and Shibuya, K. (2003) PCT Interna- (1995) PCT International Application
tional Application WO 2003/029,231, WO 9,504,727, (Zeneca Ltd).
(Bayer AG). 70. Lai, M.-T., Liu, L.-D., and Liu,
62. Watanabe, Y., Mihara, J., Yanagi, H.-W. (1991) J. Am. Chem. Soc., 113,
A., Ishikawa, K., Otsu, Y., Shibuya, 7388–7397.
K., Emoto, A., and Abe, T. (2003) 71. Dakoji, S., Shin, I., Becker, D.F.,
PCT International Application WO Stankovich, M.T., and Liu, H.W. (1996)
2003/106,436, (Bayer AG). J. Am. Chem. Soc., 118, 10971–10979.
63. Watanabe, Y., Ishikawa, K., Otsu, Y., 72. Mansoorabadi, S.O., Thibodaux, C.J.,
Shibuya, K., and Abe, T. (2003) PCT In- and Liu, H.W. (2007) J. Org. Chem., 72,
ternational Application WO 03,049,541, 6329–6342.
(Bayer AG). 73. Sutter, M. and Kunz, W. (1991) Euro-
64. Fischer, R., Alig, B., Bretschneider, pean Patent Application EP 454,621,
T., Maurer, F., Erdelen, C., Konze, J., (Ciba-Geigy AG).
Loesel, P., and Watanabe, Y. (2004) 74. Ricci, A., Martani, A., Graziani, O., and
DE Patent Application DE 10,238,725,
Oliva, M.L. (1963) Ann. Chim. (Rome),
(Bayer AG).
53, 1860–1868.
65. Watanabe, Y., Mihara, J., Yamazaki, D.,
75. Rahman, L.K.A. and Scrowston, R.M.
Otsu, Y., Shibuya, K., and Shimojo, E.
(1984) J. Chem. Soc. Perkin Trans. 1, 3,
(2005) PCT International Application
385–390.
WO 2005/003,107, (Bayer AG).
76. Kaenel, H.-R., Wegmann, A., and Neff,
66. Watanabe, Y., Mihara, J., Yamazaki, D.,
D. (1995) European Patent Application
Otsu, Y., Shibuya, K., Shimojo, E., and
EP 655,446, (Ciba-Geigy AG).
Shirakura, S. (2005) PCT International
Application WO 2005/026,139, (Bayer 77. Okano, K., Miyauchi, A., and Tanaka,
AG). T. (2002) Japan Patent Application JP
67. Straub, A. (2003) PCT International 2002/053,563, (Mitsubishi Chemical
Application WO 03,059,896, (Bayer Corp.).
CropScience AG). 78. Tanaka, M., Kagano, H., Nakayama, K.,
68. Straub, A. (2004) PCT International and Fukutome, S. (2006) Japan Patent
Application WO 04,005,268, (Bayer Application JP 2006/131,593, (Sumitomo
CropScience AG). Seika Chemicals Co., Ltd).
69. Turnbull, M.D., Willetts, N.J., Fitzjohn, 79. Mett-Cohn, O. and Tarnowski, B. (1978)
S., Kholia, P.G., Smith, A.M., Salmon, Synthesis, 58–60.
1389
34
Insecticides Affecting Calcium Homeostasis
34.1
Ryanodine Receptor Modulators: Diamides
34.1.1
Introduction
Flubendiamide [1, 2], a member of the benzenedicarboxamides or phthalic

diamides, and chlorantraniliprole [3], a member of the anthranilic diamides,
are newly developed compounds (Figure 34.1.1) that are classified as diamides, and
which possess a unique mode of action as ryanodine receptor (RyR) modulators
within the Insecticide Resistance Action Committee (IRAC) classification [4].
A novel mode of action of RyR modulators was first suggested by the char-
acteristic symptoms induced by flubendiamide, such as gradual contractions of
the insect body, followed by thickening and shortening but without convulsions
(Figure 34.1.2). Since similar symptoms were also observed in insects treated with
the plant alkaloid ryanodine, a modulator of calcium release channels, an influence
of flubendiamide on calcium channels that appeared to be involved in muscle
contraction was assumed. Details of the investigations into the mode of action of
RyR modulators are summarized in this chapter.
34.1.2
Insecticides Affecting Calcium Homeostasis
Intracellular calcium levels are widely accepted as being pivotal regulators for
specific cell functions, such as a second messenger-regulating cell functions that
include muscle contraction, neurotransmission, hormonal release, gene expres-
sion, cell growth, and cell differentiation. The precise and dynamic control of
intracellular calcium homeostasis involves a series of versatile components; more-
over, it is recognized that the functional modulation of these components can have
significant impacts on the respective physiological functions. Such regulation is
based on a precise mechanism that maintains a low cytoplasmic calcium concen-
tration (<100 nM) during the resting phase but, in response to a stimulus, can

1390 34 Insecticides Affecting Calcium Homeostasis
Flubendiamide Figure 34.1.1 Chemical structures of flubendiamide and

CH3 CH3O O chlorantraniliprole.
S
I HN CH3
O
NH
O CF3
CH3
F
CF3
Chlorantraniliprole
Br
N
O
CH3 N
Cl
NH
N
O
Cl
HN
CH3
(a) (b) (c)
(d) (e) (f)
Figure 34.1.2 Symptoms of fifth instar lar- (c) Emamectin-benzoate 5 mg a.i. l−1 , at 24 h
vae of Spodoptera litura treated by leaf dip- after application; (d) Indoxacarb 50 mg a.i.
ping [5]. (a) Flubendiamide at 100 mg a.i. l−1 , at 24 h after application; (e) Flufenox-
l−1 , at 24 h after application; (b) Cyhalothrin uron 25 mg a.i. l−1 , at 72 h after application;
at 25 mg a.i. l−1 , at 24 h after application; (f) Untreated.
mobilize calcium into the cytoplasm from the calcium stores, the sarcoplasmic
reticulum (SR)/endoplasmic reticulum (ER), and the extracellular spaces.
The RyR is also known as the ryanodine-sensitive calcium release channel;
this contributes to the calcium mobilization from intracellular calcium stores
(such as the SR). Calcium mobilization through the RyR participates in a variety
of calcium-dependent cellular responses in excitable cells such as neurons or
muscle cells over the entire animal kingdom. In muscle cells, as RyR governs
excitation–contraction coupling, any functional abnormalities of the RyR may
cause serious disorders in both muscles and the neuronal system, including
malignant hyperthermia and periodic paralysis [6, 7].
The activity of the RyR is essentially regulated by calcium via a mechanism
termed calcium-induced calcium release (CICR). In addition, growing evidence has
indicated that such receptor activity is modulated through a functional coupling
with dihydropyridine receptors (DHPRs), or accessory proteins associated with
these receptors. In contrast, numerous molecular probes may modify the activity
of the receptor.
Ryanodine, a specific modulator of the RyR, has been used as an important bio-
chemical tool to investigate the functional regulation of RyRs. Whereas, utilization
of the receptor for pharmacological targets is restricted to an example of dantro-
lene (which is a muscle relaxant and is used to treat malignant hyperthermia),
compounds that are capable of specifically activating the RyR have not (yet) been
identified [6, 7].
In the area of agrochemical research, such knowledge may imply that the com-
ponents involved in intracellular calcium regulation might also represent possible
targets for insecticides and, indeed, several research groups have investigated this
possibility [5, 8, 9]. Notably, extracts from the tropical shrub Ryania speciosa, which
affects calcium release channels, had been applied for pest control in United States
until the US Environmental Protection Agency (EPA) registration was voluntarily
cancelled in 1997 [10].
Although, to date, a synthetic organic compound that affects intracellular calcium
levels has never been developed commercially as a pesticide, flubendiamide is the
first compound to demonstrate insecticidal activity via a direct effect on intracellular
calcium homeostasis [11–15]. Following the initial reports on flubendiamide
[1, 16], attention was switched to the anthranilic diamides, a new chemical class
with an insecticidal spectrum that had been first described by the research group
at DuPont [3]. It was shown, coincidentally, that anthranilic diamides could have a
direct effect on intracellular calcium concentrations, despite their structures being
clearly different from those of the benzenedicarboxamides [17–19].
34.1.3
Mode of Action
The mode of action of flubendiamide is illustrated schematically in Figure 34.1.3.

In the resting state, intracellular calcium is stored in the SR, but can be mobilized
by the opening of calcium release channels, such as the RyR and the inositol
triphosphate receptor (IP3 R). Two experimental approaches employing calcium
imaging in isolated neurons from the lepidopterous insect Heliothis virescens,
and calcium release from the muscle membranes from the lepidopterous insect
Ca2+
Ca2+
Ca2+
Flu Ca2+
RyR
ATP
Ca2+ Ca2+ Ca2+

Ca2+
Ca2+ Ca2+ Ca2+ Ca2+
Ca2+-pump Ca2+ Ca2+ ADP + Pi
SR
Figure 34.1.3 Schematic illustration of the effect of

flubendiamide on calcium intracellular homeostasis. RyR,
ryanodine-sensitive calcium-release channel; Flu, flubendi-
amide, benzenedicarboxamide derivatives.
Spodoptera litura, showed clearly that flubendiamide caused calcium to be mobilized

from the internal stores [11, 12].
The ryanodine-sensitive calcium release channel (RyRs) is, as noted above, an
important component in the process of calcium mobilization in excitable cells.
Whilst ryanodine specifically suppressed calcium mobilization through functional
modulations of this channel [20], the calcium response induced by flubendiamide
was suppressed by ryanodine, which suggested that flubendiamide was acting on
the RyR. The mode of action of flubendiamide was further supported by evidence
that flubendiamide induced a calcium response in transfected Chinese hamster
ovary (CHO) cells expressing the RyR from Drosophila melanogaster [11]. The RyRs
are homotetramers that consist of subunits each of 450–550 kDa [21, 22]. The
putative ryanodine-binding site, which is located in the transmembrane channel
pore region (Figure 34.1.4), is sensitive to the channel conformation, and this
is reflected by changes in [3 H]ryanodine binding affinity [23–25]. Flubendiamide
was shown to potentiate the binding affinity of [3 H]ryanodine to the muscle
membrane of two lepidopterous insects, but had no significant effect on the receptor
density (Bmax ); this indicated that the compound had shifted the conformational
equilibrium of the RyR to the open state (Figure 34.1.4) [11].
Interestingly, RyR activation by flubendiamide induced a remarkable stimulation
of the Ca2+ -pumping activity of insects, the increase being much greater than was
effected by classical RyR modulators, such as ryanodine and caffeine [12]. This
characteristic property of the effect of flubendiamide is recognized as a consequence
of a specific interaction with the distinct binding site on the RyR. The binding of
[3 H]flubendiamide to the muscle membranes of lepidopterous insects was shown
to be of high affinity, and was not competitive to the classical RyR modulators.
from NH2 to COOH to COOH from NH2

Cytoplasm
TM10
TM10
TM8
TM8 Ryd Central Membrane
cavity
pyr
AG
R G Po
G V G re
lix I he
e he G lix
o r SF
P D
Lumen
Figure 34.1.4 Illustration of the interactions a specific site near the selectivity filter (SF),
between ryanodine and the RyR conduc- while its opposite end points towards the cy-
tion pathway [26]. Hypothetical model for toplasmic mouth of the conduction pathway.
ryanodine–RyR interactions. It is proposed The estimated dimensions of the ryanodine
that ryanodine (Ryd) binds to the putative molecule and the putative selectivity filter
central cavity of the RyR conduction path- were drawn to scale relative to the mem-
way with its pyrrole group (pyr) anchoring at brane bilayer. TM, transmembrane domain.
34.1.4
Selectivity and Binding Site
Flubendiamide causes selective insecticidal effects on lepidopterous insects. Typ-

ically, the distinct binding site of flubendiamide appears to be associated with an
observed selectivity between insects and mammal since ryanodine, which binds to
specific binding sites on both insect and mammalian RyRs, causes a high acute
toxicity to mammals [27, 28], whereas flubendiamide and its derivatives had no
effect on the mammalian skeletal muscle isoform, RyR1 [11].
The mode of action of the anthranilic diamides was investigated recently
[17, 18], and also has been reviewed [29, 30]. Notably, the anthranilic diamides
were reported to activate RyR, thereby releasing stored calcium from the SR, and
also to exhibit a more than 500-fold differential selectivity towards insect receptors,
compared to their mammalian counterparts.
Investigation with calcium imaging (a technique used to monitor cytoplasmic
calcium concentrations) indicated a selective activation of the RyR, whereas in
cell cultures expressing either sRyR or rabbit RyR2, flubendiamide was seen to
activate – on a selective basis – the silkworm RyR (sRyR) but not the rabbit RyR
type 2 (RyR2) [31]. Such evidence was consistent with the selective insecticidal
effect of flubendiamide.
Such diversity in the primary structure of RyRs may provide an insight into
the mechanism that underlies the selective action of flubendiamide. The primary
structure of RyR, which has been evaluated in various animal species (including
insects), shows a high homology among mammals but a low homology between
mammals and insects [26]. In agreement with evolutionary distance, the primary
structure of RyR from a lepidopterous insect retains high levels of overall homology
with Drosophila RyR, but a relatively low-level homology with mammalian RyR
[32, 33]. Whilst most of the domain structures are highly conserved among
RyRs, flubendiamide may be able to discriminate an insect-specific site of the
channel. However, a greater understanding of the selectivity of flubendiamide
will first require details of the binding domain on the RyR to be clarified. A
promising lead for determining the specific binding site of flubendiamide was
provided in an experiment that employed the combination of a photoaffinity-labeled
compound and a chimeric receptor composed of sRyR and rabbit RyR2. RyR is a
huge homotetramer composed of 400 kDa subunits that can be delineated into a
cytoplasmic domain located at N-terminal and transmembrane domains located at
the C terminus.
Whilst the cytoplasmic domain mediates interaction with numerous accessory
proteins or voltage-dependent calcium channels, the transmembrane domain in-
cludes specific binding sites of the modulators, ryanodine or caffeine. The results of
such binding experiments, employing photoaffinity-labeled compounds, suggested
that the specific binding site of flubendiamide was located at the transmembrane
domain of the receptor [31, 34]. In this experiment, the chimeric RyR (composed of
the cytoplasmic domain from sRyR and the transmembrane domain from rabbit
RyR2) showed a significant decrease in sensitivity to flubendiamide. In contrast,
the N-terminal region appeared necessary for flubendiamide to open the RyR, as a
deletion mutant that lacked the N-terminal region of the receptor did not retain any
sensitivity to flubendiamide, but did retain its sensitivity to caffeine. Such evidence
implies that the receptor–compound interaction should be mediated by specific
binding sites located at the transmembrane domain. Otherwise, any conforma-
tional change induced by the specific binding of flubendiamide would be achieved
via an interdomain interaction with the N-terminal region [31, 34].
Although this accumulated evidence is insufficient to provide a precise under-
standing of the selective effects of flubendiamide on the insect RyR, for future
studies a divergent region located at the transmembrane domain of RyR (DR-1)
should be considered as a putative binding site of the compound.
34.1.5
Conclusions
The diamides, including benzenedicarboxamides and anthranilic diamides, may

contribute to the present knowledge of the mechanism of calcium release channels
in insects and mammals. The determination of specific binding site of diamides,
and the clarification of diversity in the functional regulation of RyR, should open a
wider window for the discovery of new insecticides possessing a wide spectrum of
activity and high selectivity.
References 1395
References
1. Tohnishi, M., Nakao, H., Kohno, E., 15. Masaki, T., Yasokawa, N., Fujioka, S.,
Nishida, T., Furuya, T., Shimizu, T., Motoba, K., Tohnishi, M., and Hirooka,
Seo, A., Sakata, K., Fujioka, S., and T. (2009) Pestic. Sci., 34 (1), 37–42.
Kanno, H. (2000) European Patent 16. Tohnishi, M., Nakao, H., Kohno, E.,
Application EP 1,006,107. Nishida, T., Furuya, T., Shimizu, T.,
2. Tohnishi, M., Nakao, H., Furuya, T., Seo, A., Sakata, K., Fujioka, S., and
Seo, A., Kodama, H., Tsubata, K., Kanno, H. (1999) European Patent
Fujioka, S., Kodama, H., Hirooka, T., 919,542.
and Nishimatsu, T. (2005) J. Pestic. Sci., 17. Cordova, D., Benner, E., Sacher,
30, 354–360. M., Rauh, J., Sopa, J., Lahm, G.,
3. Lahm, G.P., Myers, B.J., Selby, T.P., and Selby, T., Stevenson, T., Flexner, L.,
Stevenson, T.M. (2004) US Patent 6, Gutteridge, S., Rhoades, D., Wu, L.,
747,047. Smith, R., and Tao, Y. (2006) Pestic.
4. Insecticide Resistance Action Committee Biochem. Physiol., 84, 196–214.
(IRAC) (2010) Mode of Action Classifica- 18. Cordova, D., Benner, E., Sacher,
tion, Version 7.0. M., Rauh, J., Sopa, J., Lahm, G.,
5. Schmitt, M., Turberg, A., Selby, T., Stevenson, T., Flexner, L.,
Londershausen, M., and Dorn, A. (1996) Gutteridge, S., Rhoades, D., Wu, L.,
Pest Manage. Sci., 48, 375–385. Smith, R., and Tao, Y. (2007) ACS
6. Oyamada, K., Oguchi, K., Saitoh, N., Symp. Ser., 948, 223–234.
Yamazawa, T., Hirose, K., Kawana, Y.,
19. Cordova, D., Benner, E., Sacher, M.,
Wakatsuki, K., Oguchi, K., Tagami, M.,
Rauh, J., Sopa, J., Selby, G., Lahm,
Hanaoka, K., Endo, M., and Iino, M.
T., Stevenson, T., Flexner, L., Caspar,
(2002) Jpn. J. Pharmacol., 88, 159–166.
T., Ragghianti, J., Gutteridge, S.,
7. Takesima, H. (2001) Biochemistry, 73 (1),
Fhoades, D., Wu, L., Smith, R., and
5–14.
Tao, Y. (2007) Pesticide Chemistry, Crop
8. Lehmberg, E. and Casida, J.E. (1994)
Protection, Public Health, Environmental
Safety, Wiley-VCH Verlag GmbH, pp.
9. Pessah, I.N. (2001) Pest Manage. Sci., 57
121–126.
(10), 941–945.
20. Zucchi, R. and Ronca-Testoni, S. (1997)
10. Environmental Protection Agency (1999)
Pharmacol. Rev., 49, 1–51.
Reregistration Eligibility Decisions
(R.E.D), Fact Sheet, EPA-000-F-99-002. 21. Imagawa, T., Smith, J., Coronado, R.,
11. Ebbinghaus-Kintscher, U., Luemmen, and Campbell, K. (1987) J. Biol. Chem.,
P., Lobitz, N., Schulte, T., Funke, C., 262, 16636–16643.
Fischer, R., Masaki, T., Yasokawa, N., 22. Ogawa, Y. and Murayama, T. (1998)
and Tohnishi, M. (2006) Cell Calcium, in The Structure and Function of Ryan-
39, 21–33. odine Receptors (eds R. Sitsapesan and
12. Masaki, T., Yasokawa, N., Tohnishi, M., A.J. Williams), Imperial College Press,
Nishimatsu, T., Tsubata, K., Inoue, K., London, pp. 5–22.
Motoba, K., and Hirooka, T. (2006) Mol. 23. Chen, S., Li, P., Zhao, M., Li, X.,
Pharmacol., 69, 1733–1739. and Zhang, L. (2002) Biophys. J., 82,
13. Luemmen, P., Ebbinghaus-Kintscher, 2436–2447.
U., Funke, C., Fischer, R., Masaki, T., 24. Wang, R., Bolstad, J., Kong, H.,
Yasokawa, N., and Tohnishi, M. (2007) Zhang, L., Brown, C., and Chen, S.
ACS Symp. Ser., 948, 235–248. (2004) J. Biol. Chem., 279, 3635–3642.
14. Masaki, T., Yasokawa, N., 25. Welch, W., Rheault, S., West, D., and
Ebbinghaus-Kintscher, U., and Williams, A. (2004) Biophys. J., 87,
Luemmen, P. (2007) Pesticide Chem- 2335–2351.
istry, Crop Protection, Public Health, 26. Puente, E., Suner, M., Evans, A.,
Environmental Safety, Wiley-VCH Verlag McCaffey, A., and Windass, J. (2000)
GmbH, pp. 137–140. Insect Biochem. Mol. Biol., 30, 335–347.
27. Jenden, D. and Fairhurst, A. (1969) Mori, Y. (2009) Biochemistry, 48,

Pharmacol. Rev., 21, 1–25. 10342–10352.
28. Usherwood, P. and Vais, H. (1995) Toxi- 32. Takeshima, H., Nishi, M., Iwabe, N.,
col. Lett., 82/83, 247–254. Miyata, T., Hosoya, T., Masai, I., and
29. Sattelle, D.B., Cordova, D., and Hotta, Y. (1994) FEBS Lett., 337, 81–87.
33. Xu, X., Bhat, M., Nishi, M., Takeshima,
Cheek, T.R. (2008) Invertebr. Neurosci., 8
H., and Ma, J. (2000) Biophys. J., 78,
(3), 107–119.
1270–1281.
30. Lahm, G.P., Cordova, D., and Barry, J.D.
34. Mori, Y., Kato, K., Kiyonaka, S.,
(2009) Bioorg. Med. Chem., 17 (12), Mori, E., Hamachi, I., Tohnishi, M.,
4127–4133. Masaki, T., Yasokawa, N., and
31. Kato, K., Kiyonaka, S., Sawaguchi, Y., Takeshima, H. (2010) Abstracts of Pa-
Tohnishi, M., Masaki, T., Yasokawa, N., per, 12th IUPAC International Congress
Mizuno, Y., Mori, E., Inoue, K., of Pesticide Chemistry, Melbourne, July
Hamachi, I., Takeshima, H., and 4–8, p. 234.
34.2
Flubendiamide
34.2.1
Introduction
Typically, a new insecticide will attract much attention in terms of its resistance
management, environmental friendliness, and degree of insecticidal activity. Today,
the research and innovation of insecticides characterized by a new mode of action
clearly ranks very highly in the area of agrochemical research, in particular because
this approach provides much more of a challenge than does research based on
‘‘patent busting.’’
A new insecticide that is not only effective at very low dosage but also has
a novel mode of action, represents the ideal approach to overcome issues per-
taining to resistance management, as well as problems relating to ecobiology,
that are inevitably associated with the ‘‘older’’ insecticides such as pyrethroids and
organophosphates (OPs) [1, 2]. Indeed, such an approach has become the screening
target for agrochemical companies worldwide.
The research program at Nihon Nohyaku Co., Ltd (NNC) began with the
innovation of isoprothiolane [3], followed by buprofezin [4], the structures of which
were relatively new. Subsequently, the quest has been continued for compounds
characterized by a novel structure and a new mode of action. The reason for
this approach is quite simple: the research capacity at NNC is smaller, but more
compact, than that of a major company. In order to compete with a leading
company in the innovation of a new compound, and its subsequent development,
the approach to conducting research must be superior: in other words, it is vital
to create an original product that will compete with those generated by other,
larger, companies. During discovery screening at NNC, active observation by the
research team is recognized as being the most important factor, as this will allow
minor changes in symptoms to be identified, as well as an evaluation by the death

(or not) of the target insects. In this way, new chemicals can be screened through
the entire process. Over the years, this approach to research has proved to be
highly successful, with a series of new products being discovered that has included
buprofezin [4] and fenpyroximate [5].
34.2.2
History of the Invention
Flubendiamide (Figure 34.2.1), as discovered by NNC, is a member of the chemical

class of 1,2-benzendicarboxamides or phthalic acid diamides, and a novel insecticide
that can be used to control lepidopterous insect pests [6]. Such an achievement has
been the culmination of accumulated research knowledge at NNC, with the lead
compound of benzenedicarboxamides initially being synthesized in 1993. Since
that time, about 2000 derivatives have been synthesized, and the structure–activity
relationships (SARs) thoroughly examined [6, 7].
Initially, the research program faced several difficulties, the most notable being
that of practical production, as flubendiamide had a complex, new chemical
structure that was not similar to that of conventional insecticides. The second
problem encountered was the comprehensive approach to the entire profile of
product safety, including an evaluation of the mode of action that would justify
the safe future use of flubendiamide under the appropriate regulations. A third
problem was to determine and understand the compound’s mode of action, and
to characterize it by biological profiling against a range of lepidopterous pests.
Yet, despite these initial drawbacks, the research program was continued such that
these issues were resolved, with flubendiamide resulting from a collaboration of
chemistry, biology, and product safety evaluation.
The global developmental of flubendiamide was accelerated by a collaboration
with Bayer CropScience (BCS) since 2001, such that the compound has now reached
the stage of global sales. Registration was achieved in Japan and India in 2007, fol-
lowed by 29 countries including USA, Brazil, China, Australia, Greece, and Spain as
of November 2010. In order to acquire further knowledge of flubendiamide, Profes-
sor Y. Mori at Kyoto University has supported NNC in the most advanced research,
relating to the mode of action, some details of which have been reported in the sci-
entific press or presented at academic meetings [8–18]. The details of the invention
of flubendiamide, and its characteristics, are described in the following subsections.
CH3 CH3 O O
S
I HN CH3
O
NH
O CF3
CH3
F
CF3
Figure 34.2.1 Chemical structure of flubendiamide.
The initial reason why flubendiamide was defined as a ‘‘new generation’’ of

chemistry was related to the compound’s novel mode of action, as had been
indicated by the characteristic symptoms induced by flubendiamide. In particular,
attention was paid to the process of muscle contraction, with attention focused in
particular on the relationship between calcium channels and the muscle contraction
process (further details on the mode of action of flubendiamide have already been
discussed in Chapter 34.1).
34.2.3
Chemistry
The second reason for defining flubendiamide as a ‘‘new-generation’’ insecticide

is based on its chemical structure, in comparison with that of other, known
insecticidal compound classes.
Initially, the research team at NNC focused their attention on the diamide-type
compound 1 (see Figure 34.2.2), which showed herbicidal activity [19], and has
been investigated previously in this respect [20]. Surprisingly, during these in-
vestigations, research, the benzenedicarboxamide derivative 2 revealed a clear,
but low-level, insecticidal activity. Thus, the compound proved attractive for two
reasons: its novel insecticidal chemical structure, and its intriguing insecticidal
symptoms. As usual in this area of research, it was necessary to improve on several
points before the compound could be considered for practical use, most notably its
low insecticidal activity, its phytotoxicity towards crops, and its instability. Typically,
the benzenedicarboxamides are characterized by three regions of their chemical
structure (see general formula 3; Figure 34.2.2): the phthaloyl moiety (part A); the
aromatic amide moiety (part B); and the aliphatic amide moiety (part C).
This complex and novel structure presented a challenge to the research group to
(i) design a facile synthetic method for flubendiamide; and (ii) establish a practical
and economical method for its manufacture.
The primary problem here was that three different groups had to be connected
regioselectively to the 1-, 2-, and 3-positions of the benzene ring in the phthaloyl
moiety. An iodine atom was introduced selectively into the 3-position of the
phthaloyl moiety by a palladium-catalyzed reaction in the presence of a specific
substituent in the 2-position. On the basis of the SAR, the introduction of lipophilic
O R1
R O O X (C)
HN N HN N R2
N
O O
N Ar
NH NH O (B)
N R3
O O
Cl (A)
Compound 1 Compound 2 General formula 3
Figure 34.2.2 Chemical structures of 1,2-benzendicarboxamide derivatives.

alkyl substituents (including fluorine atoms) appeared to increase the activity,

although a practical method of introduction was not yet available. However, when
such difficulties were overcome this led to dramatic advances in terms of a more
detailed study on the SAR, as well as the establishment of a straightforward
synthetic method that could be used to provide a variety of new derivatives.
34.2.3.1 Challenge of Chemistry

The regioselective introduction of an iodine atom into the benzene ring of the
phthaloyl moiety was the first requirement. Although, as a conventional synthetic
method, the Sandmeyer reaction is well known, at NNC a direct and facile method
was developed to substitute a hydrogen atom with an iodine atom [21]. This reaction
method, which largely avoided the generation of waste materials, was ultimately
classified as one of the best in ‘‘green chemistry’’ [22, 23]. The configuration of
the palladium catalyst, in connection with the sulfoxide of the aliphatic side chain,
may play a key role in introducing the iodine in a regiospecific manner, although
details of this reaction have still to be clarified (Scheme 34.2.1).
Another challenge was the introduction of a bulky substituent into the ben-
zene ring of the aromatic amide moiety. The insecticidal activity was improved
significantly for those compounds where the pentafluoroethyl group or heptafluo-
roisopropyl group was introduced into the 4-position of the aniline ring, which is
connected via the 1-position to the benzene ring of the phthaloyl moiety. Although,
initially, metallic copper was applied in the coupling reaction [24], no effective
synthetic method was available for this type of aniline, such that subsequently it
was synthesized by using a radical reaction (as described in Refs [25, 26]). Notably,
this radical reaction also led to significant improvements in the practical conditions,
such that ultimately an excellent synthetic method – in terms of yield and reaction
conditions – was established (Scheme 34.2.2).
The two above-mentioned synthetic methods – namely, aniline synthesis and
iodine atom introduction – represent new and potentially significant reactions in
organic synthetic chemistry that might well be applicable for the synthesis of
similar compounds in future.
34.2.3.2 Structure–Activity Relationship

The details of SARs for the three parts of benzenedicarboxamides (Figure 34.2.2),
which have been used to select flubendiamide, are quoted from the literature [8]:
• While changing the substituents of the phthaloyl moiety during the optimization
process, there was a clear tendency for the lipophilic and bulky substituents to
show good insecticidal activities. In fact, an iodine atom in the 3-position proved
to be the best substituent (though very few commercial agrochemicals exist in
which an iodine atom is incorporated into the structure).
• For the aromatic amide moiety, the heptafluoroisopropyl group is very unusual,
having never been reported as a substituent in the chemical structure of conven-
tional pesticides. Following the discovery of flubendiamide, the substituent of
the 2-position on aniline was verified. As expected, the methyl substituent gave
1400
CH3 CH3
S O S
O O O O
I N N I
1/2 S
HN I HN I HN CH3
O H2O2
34 Insecticides Affecting Calcium Homeostasis
O cat. Pd(OAc)2 O HCO2H O

CF3 CF3 CF3
H H cat. H2SO4 H
N F N F N F
O CF3 O CF3 O CF3
H3C H3 C H3 C
85%
Scheme 34.2.1 Palladium-catalyzed regioselective iodination.

CF3
(CF3)2CFI
H2N H 2N F
cat. Na2S2O4
CF3
Na2CO3 >95%
Scheme 34.2.2 Facile synthesis by radical coupling.
the best results, which suggested that a moderate bulky substituent would be
most suitable in this position.
• Finally, for the aliphatic amide moiety, the isopropyl group proved to be the
most favorable among simple alkyl groups. The introduction of a heteroatom
(especially sulfur) on the alkyl side chain led to a marked increase in insecticidal
activity. The sulfonylalkylamine moiety has also has a high novelty as an amine
in pesticide chemistry.
The unique substituents described above account not only for the high activity of
flubendiamide, but also for its categorization as a totally new chemical structure.
34.2.3.3 X-Ray Structural Analysis

The structure of flubendiamide has been established using nuclear magnetic
resonance (NMR) spectroscopy, and confirmed by single-crystal X-ray structure
analysis. Flubendiamide was shown to possess different and bulky substituents
at the 1-, 2-, and 3-positions of the benzene ring of the phthaloyl moiety while,
interestingly, the X-ray structural analysis also revealed a peculiar arrangement
of substituents. In the case of benzamide, the carbonyl moiety and the benzene
ring were coplanar. The most energetic stable three-dimensional (3-D) structure
of flubendiamide, derived from molecular modeling calculations using MOPAC97
(AM1), was seen to be a conformer in which two carbonyl moieties faced in opposite
directions, whereas the conformer actually observed in the crystal structure had
two carbonyl moieties facing in the same direction.
34.2.4
Biological Profiles
The third reason for defining flubendiamide as a ‘‘new-generation’’ insecticide is

its biological profile, with a high level of insecticidal activity but no cross-resistance
to conventional insecticides, due to the compound’s novel mode of action (as noted
above) and its selectivity. A brief outline of the biological profile of flubendiamide
is provided in the following subsections [8, 9, 13].
34.2.4.1 Activity against Lepidopterous Pests

Flubendiamide has demonstrated a broad-spectrum activity against all lepidopter-
ous pests, but proved inactive against other insect orders such as Coleoptera,
Hemiptera, and Acarina. Notably, flubendiamide provides a very high activity
against all of the ‘‘important’’ lepidopterous insect pests (see Table 34.2.1).
Table 34.2.1 Insecticidal spectrum of flubendiamide on ma-

jor insect pests in agriculture [13].
Scientific name Common name Tested stagea Days after EC50

treatment (mg a.i. l –1 )
Lepidoptera
Plutella xylostella Diamond-back moth L3 4 0.004
Spodoptera litura Tobacco cutworm L3 4 0.19
Helicoverpa armigera Old World bollworm L3 4 0.24
Agrotis segetum Turnip moth L2–3 7 0.18
Autographa nigrisgna Beet semi-looper L3 4 0.02
Pieris rapae crucivora Common cabbage worm L2–3 4 0.03
Adoxophyes honmai Smaller tea tortrix L3 5 0.38
Homona magnanima Oriental tea tortrix L4 5 0.58
Hellula undalis Cabbage webworm L3 5 0.01
Chilo suppressalis Rice stem borer L3 7 0.01
Diaphania indica Cotton caterpillar L3 3 0.02
Coleoptera
Sitophilus zeamais Maize weevil A 4 >1000
Hemiptera
Nilaparvata lugens Brown rice planthopper L3 4 >1000
Myzus persicae Green-peach aphid All stages 7 >1000
Pseudococcus comstocki Comstock mealybug L1 7 >100
Acarina
Tetranychus urticae Two-spotted spider mite All stages 4 >100
a
L2, second instar; L3, third instar; L4, fourth instar; A, adult.
Whereas, cyhalothrin (a synthetic pyrethroid) shows similar activities towards

the different developmental stages of lepidopterous pests, flubendiamide is most
effective on the larvae, has no ovicidal activity, and is less effective on adults than
on the larvae. In fact, the larvae demonstrate unique symptoms following the
ingestion of flubendiamide (see above).
With regards to the different larval stages of lepidopterous pests, flubendiamide is
most effective against the first instar larvae, followed by third and fifth instar larvae
(Table 34.2.2). The main advantage of flubendiamide over the OPs and spinosad is
that the activity of the latter two compounds is affected drastically by the larval size.
Although, even on fifth instar larvae, flubendiamide provides a very high activity
relative to conventional insecticides, from a practical viewpoint its application to
the younger stages of larvae will provide a more effective control [9, 13].
34.2.4.2 Fast-Acting Activity and Persistence

The speed of appearance of symptoms in larvae treated with flubendiamide
indicates its fast-acting activity (Figure 34.2.3). Symptoms caused by flubendiamide
were observed within a few hours after exposure, and appeared more rapidly than
Table 34.2.2 Insecticidal activity of flubendiamide on three

different larval stages of S. litura [13].
Treatment EC50 (mg a.i. l−1 , 3 days after treatmenta )
First instar Third instar Fifth instar
Flubendiamide WDG 0.033 0.19 0.51

Cyhalothrin EC 0.08 0.36 0.72
Methomyl WP 13.8 17.3 15.4
Profenophosa EC 1.38 17.3 54.8
Spinosad WDG 0.67 45.5 54.8
a
EC, emulsion concentrate; WDG, water-dispersible granule; WP, wettable
powder.
100
80
Affected rate (%)
60
40
20
0
0 1 2 3 4 5 6 7 24 48 72
Hours after exposure
Flubendiamide at 100 mg a.i. l–1
Emamectin–benzoate at 10 mg a.i. l–1
Cyhalothrin at 25 mg a.i. l–1
indoxacarb at 50 mg a.i. l–1
Flufenoxuron at 25 mg a.i. l–1
Figure 34.2.3 Change (%) in rate of symptom appearance

on fifth instar larvae of Spodoptera litura treated by leaf dip-
ping.
those of indoxacarb, emamectin-benzoate, and flufenoxuron, but more slowly than

those of cyhalothrin.
The suppression of feeding damage to plants by larvae at 24 h after exposure
to flubendiamide is also a clear indication of the compound’s rapid onset of
action (Figure 34.2.4). In larvae treated with flufenoxuron (a typical slow-acting
insecticide), the feeding damage was similar on both treated and untreated (control)
plots, whereas flubendiamide suppressed the feeding damage very rapidly, and in
(a) (b) (c)
(d) (e) (f)
Figure 34.2.4 Suppression of feeding damage on fifth in-

star larvae of Spodoptera litura at 24 h after treatment by leaf
dipping. (a) Flubendiamide at 100 mg a.i. l−1 ; (b) Fluben-
diamide at 25 mg a.i. l−1 ; (c) Cyhalothrin at 25 mg a.i. l−1 ;
(d) Flufenoxuron at 25 mg a.i. l−1 ; (e) Emamectin-benzoate
at 5 mg a.i. l−1 ; (f) Untreated.
similar fashion to cyhalothrin and emamectin-benzoate. Despite the main symptom

of flubendiamide treatment being larval body contraction, its rapid onset of activity
is demonstrated more clearly by a very quick cessation of larval feeding.
The long-lasting activity of flubendiamide on cabbage leaves has been investigated
under both glasshouse and field conditions; typically, sufficient flubendiamide
residual activity persisted on the treated leaves for more than four weeks in
both cases. Moreover, the larvicidal activity was superior to that achieved with
emamectin-benzoate, indoxacarb, pyridalyl, and spinosad, at the practical usage
rate in Japan (100 mg a.i. l−1 ; Figure 34.2.5). When the residues of flubendiamide
on treated leaves were analyzed under the same field conditions, the levels declined
quite rapidly but remained higher, for about 40 days, than the EC99 of flubendiamide
against the third instar larvae of Trichoplusia ni (Figure 34.2.6). Such data indicate
that the long-lasting activity of flubendiamide is due to its extremely high larvicidal
activity against lepidopterous pests. The controlling efficacy of flubendiamide
against many lepidopterous pests was also confirmed to persist for about two to
three weeks in many crops under field conditions, at the recommended usage rate
in Japan (100 and 50 mg a.i. l−1 ) [9, 13].
Field evaluations of flubendiamide have been conducted in many areas on
a variety of crops, including vegetables, top fruit, and cotton. Flubendiamide
Corrected mortality (%, 3–4 DAI) 100
80
60
40
20
0
0 5 9 12 19 27 37 46
Flubendiamide 100 mg a.i. l–1 Emamectin-benzoate 10 mg a.i. l–1

Indoxacarb 100 mg a.i. l–1 Pyridaryl 100 mg a.i. l–1
Spinosad100 mg a.i. l–1
Application Date: Sept. 15, 2004
Figure 34.2.5 Long-lasting activity of flubendiamide against

third and fourth instar larvae of Trichoplusia ni on cabbage
leaves under field conditions.
Flubendiamide residue on the leaf (μg cm2–1)
10
0.1
0.01
0.001
0 10 20 30 40 50
Application date: Sept. 15, 2004
Figure 34.2.6 Correlation of flubendiamide concentration on

the cabbage leaves and biological activity against Trichoplusia
ni, after foliar application. The bold horizontal line indicates
the EC99 (μg cm2−1 ) of flubendiamide against the third in-
star larvae.
demonstrated an excellent control of the major lepidopterous pests on each crop at

the recommended doses, and with an efficacy comparable to, or better, than those
of standard insecticides [9, 13]. Flubendiamide (20%, water-dispersible granule)
showed no phytotoxicity towards any of the crops tested, despite being applied at
double the recommended rate.
34.2.4.3 Cross-Resistance
Based on its unique symptoms and novel mode of action, flubendiamide would
not be expected to demonstrate any cross-resistance with conventional insecticides.
Consequently, when the activity of flubendiamide was evaluated against the larvae
of Plutella xylostella that was resistant to synthetic pyrethroids, benzoylphenylureas,
organophosphates, and carbamates, flubendiamide provided the same EC50 -values
against both the resistant and susceptible strains [8, 13]. The absence of any
cross-resistance between flubendiamide and conventional insecticides, which most
likely results from its novel mode of action, indicates that flubendiamide would fit
very well into any insecticide resistance management (IRM) program.
34.2.5
Toxicity towards Beneficial Arthropods
The application of flubendiamide to several species of beneficial arthropods, at

rates ranging from 100 to 400 mg a.i. l−1 , indicated a low level of acute toxicity
Table 34.2.3 Toxicity of flubendiamide to natural enemies [8].
Common name Scientific name Test stage Test method EC30 value
(mg a.i. l−1 )
Lady beetle Harmonia axyridis Adult Insect dipping >200

Coccinella septempunctata Adult Insect dipping >200
bruckii
Parasite wasp Encarsia formosa Adult Dry film >400
Aphidius colemani Adult Dry film >400
Cotesia glomerata Adult Dry film >100
Green lacewing Chrysoperla carnea Larva Spraying on food and >100
insect
Predatory bug Orius strigicollis Adult Spraying on food and >100
insect
Predatory midge Aphidoletes aphidimyza Larva Spraying on food and >100
insect
Predatory mite Amblyseius cucumeris Adult Spraying on food and >200
insect
Phytoseiulus persimilis Adult Spraying on food and >200
insect
Spider Pardosa pseudoannulata Adult Insect dipping >100
Misumenops tricuspidatus Adult Insect dipping >200
Table 34.2.4 Toxicological profile of flubendiamide [13].
Toxicity test Result
Acute oral LD50 , rat >2000 mg kg−1 (male and female)

Acute dermal LD50 , rat >2000 mg kg−1 (male and female)
Eye irritation, rabbit Slight-irritant
Mutagenicity (Ames test) Negative
Aquatic organisms LC50 (96 h), carp >546 μg l−1
Honeybee oral/contact LD50 (48 h) >200 μg l−1
Avian oral LD50 , bobwhite quail >2000 mg kg−1
(Table 34.2.3) [8, 13]. Hence, flubendiamide should be compatible with any planned
integrated pest management (IPM) program.
34.2.6
Toxicological Properties
The toxicological profile of flubendiamide provided in Table 34.2.4 supports the

very favorable acute toxicity of the compound. To date, flubendiamide has been
registered in Japan, USA, Greece, Australia, and other countries, which is indicative
of its safety towards mammals under practical use conditions. Data indicating that
flubendiamide had essentially no effect on the mammalian type 1 ryanodine
receptor (RyR) supported these findings [12].
34.2.7
Conclusions
Flubendiamide, as a novel class of insecticide with a unique chemical structure,

is the first synthetic compound to possess insecticidal activity via RyR modulation
[2]. To date, all of the data obtained with flubendiamide suggest that it should
be classified as a ‘‘new-generation’’ insecticide, based on its biochemical mode of
action, chemistry, and biology. Flubendiamide provides excellent activity against
a broad spectrum of lepidopterous insect pests, shows no-cross resistance to
conventional insecticides, and is also much safer against natural enemies. Taken
together, these properties suggest that flubendiamide will be very well suited to
both IRM and IPM programs.
Whilst the benzenedicarboxamide compounds (including flubendiamide)
were identified through original research conducted at NNC, the anthranilic
diamides – which in structural terms differ greatly from the benzenedicarboxam-
ides – were discovered by the research team at DuPont [27], and have essentially
the same mode of action [28]. To date, various companies have followed with patent
applications: Nissan Chemical [29] and Takeda Pharmaceutical Company [30] have
applied for patents of the related compounds of benzenedicarboxamides, while
Ishihara Sangyo Kaisha [31] has applied for a patent of the related compounds
of anthranilic diamides. Clearly, the future entry into the market of this new
generation of insecticides will intensify the competition with ‘‘conventional’’
compounds.
Acknowledgments
The authors sincerely thank their colleagues at Nihon Nohyaku Co., Ltd, who
have contributed to the research and development of flubendiamide. They also
wish to acknowledge those scientists at Bayer CropScience AG, and Prof. Yasuo
Mori at Kyoto University, for their helpful discussions of the mode of action of
flubendiamide.
References
1. Nauen, R. (2005) Proc. BCPC Int. Congr. 10. Tohnishi, M., Nakao, H., Furuya, T.,
Crop Sci. Technol., 3A-1, 123–130. Seo, A., Kodama, H., Tsubata, K.,
2. Insecticide Resistance Action Committee Fujioka, S., Kodama, H., Hirooka, T.,
(IRAC) (2005) Mode of Action Classifica- and Nishimatsu, T. (2005) Abstracts of
tion, Version 4.2.1. Papers, 230th ACS National Meeting,
3. Taninaka, K., Kurono, H., Hara, T., Washington DC, August 28–September
and Murata, K. (1976) J. Pestic. Sci., 1, 1, AGRO-General Posters 9.
115–122. 11. Luemmen, P., Ebbinghaus-Kintscher,
4. Kanno, H., Ikeda, K., Asai, T., and U., Lobitz, N., Schulte, T., Funke, C.,
Maekawa, S. (1981) Proc. Brighton Crop and Fischer, R. (2005) Abstracts of
Prot. Conf. - Pests Dis., 1, 59–66. Papers, 230th ACS National Meeting,
5. Konno, T., Kuriyama, K., Hamaguchi, Washington DC, August 28–September
H., and Kajihara, O. (1990) Proc. 1, AGRO-025.
Brighton Crop Prot. Conf. - Pests Dis., 12. Ebbinghaus-Kintscher, U., Luemmen,
2-8, 71–78. P., Lobitz, N., Schulte, T., Funke, C.,
6. Tohnishi, M., Nakao, H., Kohno, E., Fischer, R., Masaki, T., Yasokawa, N.,
Nishida, T., Furuya, T., Shimizu, T., and Tohnishi, M. (2006) Cell Calcium,
Seo, A., Sakata, K., Fujioka, S., and 39, 21–33.
Kanno, H. (2000) European Patent 13. Nishimatsu, T., Hirooka, T., Kodama,
Application EP 1,006,107. H., Tohnishi, M., and Seo, A. (2005)
7. Tohnishi, M., Nakao, H., Kohno, E., Proc. BCPC Int. Congr. Crop Sci. Tech-
Nishida, T., Furuya, T., Shimizu, T., nol., 2A-3, 57–64.
Seo, A., Sakata, K., Fujioka, S., and 14. Masaki, T., Yasokawa, N., Tohnishi, M.,
Kanno, H. (1999) European Patent Nishimatsu, T., Tsubata, K., Inoue, K.,
919,542. Motoba, K., and Hirooka, T. (2006) Mol.
8. Tohnishi, M., Nakao, H., Furuya, T., Pharmacol., 69, 1733–1739.
Seo, A., Kodama, H., Tsubata, K., 15. Seo, M., Tohnishi, M., Nakao, H.,
Fujioka, S., Kodama, H., Hirooka, T., Furuya, T., Kodama, H., Tsubata, K.,
and Nishimatsu, T. (2005) J. Pestic. Sci., Fujioka, S., Kodama, H., Nishimatsu,
30, 354–360. T., and Hirooka, T. (2007) Pesticide
9. Nishimatsu, T., Kodama, H., Kuriyama, Chemistry, Crop Protection, Public Health,
K., Tohnishi, M., Ebbinghaus, D., and Environmental Safety, Wiley-VCH Verlag
Schneider, J. (2005) International Con- GmbH, pp. 127–135.
ference on Pesticides, Book of Abstracts, 16. Masaki, T., Yasokawa, N.,
Kuala Lumpur, Malaysia, pp. 156–161. Ebbinghaus-Kintscher, U., and
34.3 Anthranilic Diamide Insecticides: Chlorantraniliprole and Cyantraniliprole 1409
Luemmen, P. (2007) Pesticide Chem- Tumas, W. (2000) Pure Appl. Chem.,

istry, Crop Protection, Public Health, 72, 1207–1228.
Environmental Safety, Wiley-VCH Verlag 24. Kuroda, K. and Ishikawa, N. (1972) J.
GmbH, pp. 137–140. Chem. Soc. Jpn, 10, 1876.
17. Masaki, T., Yasokawa, N., Fujioka, S., 25. Tordeux, M., Langlois, B., and
Motoba, K., Tohnishi, M., and Hirooka, Wakselman, C. (1990) J. Chem. Soc.
T. (2009) Pestic. Sci., 34 (1), 37–42. Perkin Trans. 1, 8, 2293.
18. Mori, Y., Kato, K., Kiyonaka, S., Mori, 26. Onishi, M., Yoshiura, A., Kohno, E.,
E., Hamachi, I., Tohnishi, M., Masaki, and Tsubata, K. (2000) European Patent
T., Yasokawa, N., and Takeshima, Application EP 1,006,102.
H. (2010) Abstracts of Paper, 12th 27. Lahm, G.P., Myers, B.J., Selby, T.P.,
IUPAC International Congress of Pes- and Stevenson, T.M. (2004) US Patent
ticide Chemistry, Melbourne, July 4–8, 6,747,047.
p. 234. 28. Cordova, D., Benner, E., Sacher, M.,
19. Tsuda, T., Yasui, H., and Ueda, H. Rauh, J., Sopa, J., Lahm, G., Selby, T.,
(1989) J. Pestic. Sci., 14, 241–243. Stevenson, T., Flexner, L., Gutteridge,
20. Tohnishi, M., Katsuhira, K., Otsuka, T., S., Rhoades, D., Wu, L., Smith, R., and
and Miura, Y. (1997) Jpn. Kokai Tokkyo Tao, Y. (2006) Pestic. Biochem. Physiol.,
Koho 09-323,974. 84, 196–214.
21. Kodama, H., Katsuhira, T., Nishida, 29. Numata, A., Maeda, K., Mita, T.,
T., Hino, T., and Tsubata, K. (2001) Miyake, T., Takii, S., and Itoh, T. (2003)
PCT International Application WO PCT International Application WO
0,183,421. 03/11,028.
22. Anastas, P. and Williamson, T. (1996) 30. Kajiwara, T. and Tanimoto, T. (2005)
ACS Symp. Ser., 626, 1–17. Japanese Patent Application 272,304.
23. Tundo, P., Anastas, P., Black, D.S., 31. Koyanagi, T., Morita, M., Nakamoto, K.,
Breen, J., Collins, T., Memoli, S., and Hisamatsu, A. (2005) PCT Interna-
Miyamoto, J., Polyakoff, M., and tional Application WO 05/077,934.
34.3
Anthranilic Diamide Insecticides: Chlorantraniliprole and Cyantraniliprole
George P. Lahm, Daniel Cordova, James D. Barry, John T. Andaloro, I. Billy Annan, Paula
C. Marcon, Hector E. Portillo, Thomas M. Stevenson, and Thomas P. Selby
34.3.1
Introduction
The anthranilic diamide insecticides were first discovered in 1999, by the research
team at DuPont [1, 2]. Their discovery had origins in the groundbreaking investiga-
tions of Tohnishi, Seo, and coworkers in the area of phthalic diamides, leading to the
discovery of flubendiamide [3–6]. Chlorantraniliprole (1; Rynaxypyr®) was discov-
ered in 2001 following the intensive evaluation of approximately 2000 analogs [7, 8],
while cyantraniliprole (2; CyazypyrTM ) was discovered in 2003 through a dedicated
effort to expand the pest spectrum by modification of physical properties, specifically
targeting a reduction in log P [9, 10]. These products differ only in the 4-substituent
of the anthranilic core, which are chloro and cyano, respectively; nonetheless, this
difference is sufficient to modify the physical properties responsible for the pest
spectra. Specifically, chlorantraniliprole largely displays excellent activity against
Lepidoptera, whereas cyantraniliprole controls a broader pest complex, including

those of the orders Hemiptera and Thysanoptera, in addition to Lepidoptera.
Cl Me N NC Me N
O O
Cl Cl
N N
N N
H N H N
Me Me
N O N O
H Br H Br
1 2
34.3.2
Discovery of Anthranilic Diamides
The discovery of the anthranilic diamides followed on from studies with the emerg-
ing class of insecticidal phthalic diamides [11] (Figure 34.3.1). The most promising
members of the phthalic diamide class, such as compound 3, were reported to
control (at an application rate of 500 ppm) three species of Lepidoptera, namely
Spodoptera litura, Plutella xylostella (diamond-back moth), and Cnaphalocrocis medi-
nalis (rice leaf roller) [3]. The key features associated with this compound include
the presence of an ortho-halo substituent (i.e., iodo) adjacent to the alkyl amide,
and the presence of the 2-methyl-4-trifluoromethyl substituent pattern on the
aniline.
Initial targets focused on amide modifications that reversed the order of con-
nectivity of one or both amides. Compounds 4 and 5, for example, reversed the
connectivity of the larger benzamide (4) and the smaller isopropyl amide (5),
respectively. Analogs were also evaluated where the aromatic substituent was var-
ied across the sites of the core anthranilic ring. In laboratory studies, compound
4 demonstrated a surprisingly strong lepidopteran activity on P. xylostella and
5 6
Me Me
4 O O Me
3 N N
H H
N O OCF3 N O CF3
H H
4 6
O
I Me
HN
HN O
5
CF3 4 6
Me
Me
3 O H
Cl N
3
O NH HN
NH O
CF3
OCF3 O
5 7
Figure 34.3.1 Lead discovery.

Spodoptera frugiperda (fall armyworm) in the range of 10 to 50 ppm. This analog

required both a reversal of the benzamide, coupled with migration of the aromatic
methyl substituent to the 6-position. The corresponding 3-methyl analog was not
active even at the highest screening rates of 250 ppm. These results also differed
significantly from the structure–activity relationships of the phthalic diamides.
In contrast, compounds of type 5, which explored a reversal of the isopropyl
amide, did not provide compounds of any significant biological activity, indepen-
dent of the location of the chloro substituent at either positions 3 or 6. To confirm
this observation as relevant to the phthalic diamide (3), the anthranilic diamides
(6) and (7) were prepared with the preferred 2-methyl-4-trifluoromethyl substituent
pattern found on the aniline of 3. As anticipated, and consistent with results for
4 and 5, compound 6 showed an increase in potency across multiple species of
Lepidoptera, whereas compound 7 was inactive. Thus, compound 6 served as a
strong lead for this new class of insecticidal anthranilic diamides.
34.3.3
®
Discovery of Chlorantraniliprole (Rynaxypyr )
At the time of the anthranilic diamide lead discovery, the research group at
DuPont had re-engineered its discovery organization so that pesticidal efficacy was
optimized concurrently with safety and environmental attributes. This required
front-loading the safety and environmental testing in parallel with insecticide
screens. The chlorantraniliprole discovery resulted from this integrated optimiza-
tion program.
Following lead compound 6, a variety of heterocyclic variants of the benzamide
were evaluated that included the pyridine (7) and pyrazole (8) (Figure 34.3.2). Both
analogs incorporate an equivalent to the 2-methyl-4-trifluoromethyl pattern of 6,
Me Me Me
O Me O Me O Me
N
N N N N
H H H N
N O CF3 N O CF3 N O
H H H CF3
6 7 8
Me Me N
O R O
Cl
N N
N N
H N H N
N O N O
H CF3 H CF3
9a R = 2-Cl 10
9b R = 3-Cl
9c R = 4-Cl
Figure 34.3.2 Evolution of structure for anthranilic diamide insecticides.

and showed a modest improvement in biological activity. The pyrazole (8) was an
especially interesting variant as compared to the six-membered rings, and offered
relative ease in the exploration of N-substituent groups. Evaluation pointed to a
general improvement in biological performance as the size of the N-substituent
was increased, and a dramatic effect for the N-phenyl pyrazoles (9a–c). Specifically,
the N-(2-chlorophenyl)-pyrazole of structure 9a produced a 200-fold increase in
insecticidal activity, whereas the meta and para analogs, 9b and c, were significantly
less active. Compound 9a was, in fact, superior to many commercial standards but
lacked sufficient soil degradation properties. The introduction of a chloropyridyl
group, as in compound 10, provided not only a twofold improvement in insecticidal
activity but also an improved soil profile over the phenyl analog.
Further optimization of the trifluoromethylpyrazole (10) to the bromopyrazole
(11) maintained excellent insecticidal activity, and also afforded an additional
improvement in the soil degradation profile (Figure 34.3.3). However, compound
11 was found to have significant mammalian toxicity with an LD50 in rats of
approximately 25 mg kg−1 body weight. During the course of preparing a tritiated
sample of compound 10, the 4-bromoanthranilic derivative 12 was prepared for
tritium exchange. This compound showed a surprising two- to fivefold increase in
insect potency – a fortuitous discovery that prompted a concerted evaluation of the
4-halo substituents, including the 4-chloro analogs 13a and b. These maintained
the high potency, with the bromo derivative 13b again showing a superior soil
degradation profile. Improved soil degradation was found with the methyl amides
14a and b, with 14b (chlorantraniliprole) providing the optimum combined set
of attributes of low mammalian toxicity, favorable soil degradation, and high
insecticidal activity across a broad spectrum of Lepidoptera, at application rates
spanning from 0.01 to 0.05 ppm in laboratory studies.
Me N Me N Br Me N
O O O
Cl Cl Cl
N N N
N N N N N N
H H H
N O N O N O
H CF3 H Br H CF3
10 11 12
Cl Me N Cl Me N
O O
Cl Cl
N N
N N N N
H Me H
N O N O
H X H X
13a X = CF3 14 X = CF3
13b X = Br 14 X = Br (chlorantraniliprole)
Figure 34.3.3 Evolution of structure and discovery of chlorantraniliprole.

34.3.4
Discovery of Cyantraniliprole (Cyazypyr™)
The cyantraniliprole discovery involved a concerted effort to expand the pest spec-
trum by modification of the physical properties, specifically targeting a reduction in
log P to increase the water solubility [9, 10]. A range of substituent patterns across
all regions of the molecule investigating lower log P groups was evaluated. The
introduction of a cyano-group at the 4-position of the anthranilic core reduced the
log P of 2.86 for chlorantraniliprole (pH 7, 20 ◦ C, shake flask) to a value of 1.91 (pH
7, 20 ◦ C, shake flask) for cyantraniliprole. This produced an increase in water sol-
ubility from approximately 1 ppm for chlorantraniliprole to approximately 15 ppm
for cyantraniliprole; this physical property change was reflected in the biological
attributes. Specifically, cyantraniliprole demonstrated control over a broader pest
complex, including pests of the orders Hemiptera, Thysanoptera, and Lepidoptera.
Details of the optimization program that surrounded this work are forthcoming.
Selected physical properties of these compounds are listed in Table 34.3.1.
34.3.5
Chemical Synthesis
A number of efficient syntheses of chlorantraniliprole and cyantraniliprole

have been developed. As a common intermediate, these compounds share the
3-bromopyrazole acid (20), the synthesis of which was previously described
[12]. The reaction of 3-chloro-2-hydrazinopyridine (15) with diethylmaleate (16)
in the presence of sodium ethoxide affords the pyrazolone (17) in 55% yield.
Subsequent treatment of 17 with phosphoryl bromide in acetonitrile at 80 ◦ C
affords the bromopyrazoline (18) in 95% yield. Oxidation to the pyrazole (19) can
be accomplished with potassium persulfate as the oxidant in 90% yield, followed
by hydrolysis to the acid 20 (Figure 34.3.4).
The 4-chloro-6-methylanthranilamide (22), which is required for the synthesis of
chlorantraniliprole, can be prepared in three steps from 2-amino-3-methylbenzoic
acid (21) by N-chlorosuccinimide (NCS) chlorination and amide formation from
the methyl ester in 54% yield (Figure 34.3.5). Coupling of the acid 20 with the
Table 34.3.1 Physico-chemical properties of chlorantraniliprole and cyantraniliprole.
Property Chlorantraniliprole Cyantraniliprole
Chemical class Anthranilic diamide Anthranilic diamide

Empirical formula C18 H14 BrCl2 N5 O2 C19 H14 BrClN6 O2
Molecular weight (g mol−1 ) 483.15 473.72
Physical form Off-white crystalline powder Solid, powder
Melting point (◦ C) 208–210 224
Water solubility (20 ◦ C; mg l−1 ) 0.9–1.0 14.2–17.2
Log Pow 2.86 at 20 ◦ C 1.9 at 22 ◦ C
N N
N CO2 Et a O b O
Cl Cl
Cl + N N
CO2 Et EtO EtO N
HN N
NH2
OH Br
15 16 17 18
N N
c O d O
Cl Cl
N N
EtO HO
N N
Br Br
19 20
Figure 34.3.4 Pathway (a): NaOEt, EtOH, reflux, 55%; Path-

way (b): POBr3 , MeCN, 83 ◦ C, 95%; Pathway (c): MeCN,
H2 SO4 , K2 S2 O8 , reflux, 90%; Pathway (d): (i) aq. NaOH,
MeOH, and (ii) aq. HCl, 91%.
Me Cl Me Cl Me N
O
a, b, c d Cl
NH2 N
NH2 N
H N
Me Me
HO O N O N O
H H Br
21 22 1
Figure 34.3.5 Pathway (a): N-chlorosuccinimide (NCS),

dimethylformamide (DMF), 100 ◦ C, 75%; Pathway (b):
Me2 SO4 , 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), CH3 CN,
0–5 ◦ C, 88%; Pathway (c): MeNH2 , CH3 CN, 60 ◦ C, 85%;
Pathway (d): MeSO2 Cl, picoline, 20, MeCN, 0–5 ◦ C, 97%.
benzamide (22), using methanesulfonyl chloride and picoline in acetonitrile, is

achieved in high yield to afford chlorantraniliprole (1) [13].
A convenient synthesis of the cyantraniliprole intermediate, anthranilic amide
(24), can be accomplished in three steps from isatoic anhydride (23) (Figure 34.3.6)
[13, 14]. Treatment with methyl amine, followed by bromination, affords the
4-bromo-6-methylanthranilamide (24) in 83% yield over the two steps. A variety of
useful methods have been developed for introduction of the cyano group [13, 15,
16]. One example includes the reaction of 24 with sodium cyanide in the presence of
zinc, Pd2 (dba)3 catalyst, and tri-tert-butylphosphine as the ligand to afford excellent
yields of 25 [15]. Coupling with the pyrazole acid (20), using methanesulfonyl chlo-
ride and picoline in acetonitrile, affords cyantraniliprole (2) in excellent yield [14].
Me Br Me NC Me
a, b c
NH NH2 NH2
Me Me
O O O N O N O
H H
23 24 25
NC Me N
d O
Cl
N 2
N N
H
Me
N O
H Br
Figure 34.3.6 Pathway (a): EtOAc, HOAc, MeNH2 ,

35–37 ◦ C, 92%; Pathway (b): Br2 , HOAc, NaOH, H2 O,
55 ◦ C, 90%; Pathway (c): Pd2 (dba)3 , t-Bu3 P, tetrahydrofu-
ran (THF), NaCN, Zn, 25 ◦ C, 95%; Pathway (d): MeSO2 Cl,
picoline, 20, MeCN, 0–5 ◦ C, 92%.
34.3.6
Mode of Action
As described above, flubendiamide and the anthranilic diamide insecticides –

chlorantraniliprole and cyantraniliprole – act at the same target, namely the ryan-
odine receptor (RyR). Thus, the Insecticide Resistance Action Committee (IRAC)
has classified these new insecticides in Group 28, Ryanodine Receptor Modulators
[17]. The action of anthranilic diamides on insect and mammalian RyRs will be
described in more detail in the following subsections.
Insects (as do vertebrate organisms) possess two type of calcium channel: (i)
voltage-gated calcium channels, which regulate the external entry of Ca2+ into the
cell; and (ii) RyR channels, which regulate the release of internal calcium stores
[18]. The name of the RyRs is derived from the plant alkaloid, ryanodine, which
locks the RyRs in the open state. Although the insecticidal properties of ryanodine
were first described during the 1940s [19], prior to the discovery and development
of diamide insecticides, there were no synthetic RyR molecules known to provide
commercial-level control of insects. The RyR consists of four identical subunits that
form a tetramer with a central pore which regulates the flow of Ca2+ (Figure 34.3.7);
a more detailed description of insect RyRs is provided in the review by Sattelle et al.
[20].
Unlike ryanodine, which locks the channel in the open state when the RyR has
been activated, anthranilic diamides activate the receptor channel directly, as shown
in Figure 34.3.8. Chlorantraniliprole was found to stimulate receptor-mediated Ca2+
release with a 50% effective concentration (EC50 ) of 40–50 nM against both native
(American cockroach, Periplaneta americana) and recombinant (fruit fly, Drosophila
melanogaster; tobacco budworm, Heliothis virescens; and hemipteran chimeric) insect
CAM CAM
RyR RyR
FK
Cytosol BP BP
FK
Ju
in
nc
iad
tin
Tr
Lumen CS
Q
2+
Ca
Figure 34.3.7 Schematic representation of long N-terminal region that extends into the
the ryanodine receptor (RyR) and impor- cytoplasm. Some of the important associ-
tant associated proteins. The receptor is a ated proteins that interact directly with the
tetramer of identical subunits; for simplic- RyR are shown. CAM (calmodulin); FKBP
ity, only two of the four subunits are shown. (FK506-binding protein); CSQ (calsequestrin);
Each subunit has four transmembrane re- Ca2+ (calcium ions). Reproduced with per-
gions in the endoplasmic reticulum mem- mission from Ref. [20]; © 2008, Springer
brane (sarcoplasmic reticulum in muscle Science+Business Media.
cells), close to the C terminus, and a very
KCI
DP-012
Caff Caff
1 μM ryanodine
100 nM
4 min
Figure 34.3.8 Characterization of the an- Ca2+ response while subsequent challenges
thranilic diamide-stimulated Ca2+ response with either DP-012 or caffeine fail to elicit
in Periplaneta americana embryonic neurons. a response. The [Ca2+ ]i increase stimulated
Repeated challenges with the RyR activator, by depolarization with KCl (100 mM) re-
caffeine (Caff; 20 mM), or DP-012 in standard mains unaffected, demonstrating that while
saline stimulate transient increases in the in- anthranilic diamides stimulate release of
tracellular Ca2+ concentration ([Ca2+ ]i ). Per- RyR-mediated Ca2+ stores, voltage-gated
fusion with ryanodine (1 μM) alone does not Ca2+ channels are unaffected. Reprinted with
affect basal [Ca2+ ]i . However, in the presence permission from Ref. [23]; © 2006, Elsevier.
of ryanodine, DP-012 stimulates a prolonged
Table 34.3.2 Activity of anthranilic diamides against recom-

binant insect RyR stably expressed in a Spodoptera frugiperda
cell line (Sf9).
Anthranilic Dipteran RyR Lepidopteran Hemipteran

diamide (D. melanogaster) RyR (H. virescens) RyR (proprietary chimera)
EC50 (μM) EC50 (μM) EC50 (μM)
Chlorantraniliprole 0.04 0.05 0.04

Cyantraniliprole 0.09 0.25 0.09
RyRs [8, 11]. While cyantraniliprole exhibits an improved potency against sucking
insects, it has a comparable potency to chlorantraniliprole when evaluated against
lepidopteran and hemipteran RyRs (Table 34.3.2) [21, 22].
As is the case with flubendiamide, anthranilic diamides bind to a site distinct
from that of ryanodine [23, 24]; this variation was confirmed using membranes
prepared from P. americana leg muscle, whereby anthranilic diamides failed to
displace [3 H]ryanodine. The biochemical characterization of this insecticidal class
has been reported using two radiolabeled anthranilic diamides: [3 H]DP-010 and,
more recently, [3 H]DP-033 [22]. Interestingly, while the binding of [3 H]DP-010 was
enhanced by the presence of ryanodine, the same was not true for [3 H]DP-033. The
reason for this disparity was unclear, though the greater aqueous solubility of the
latter probe may have played a role. Both, chlorantraniliprole and cyantraniliprole
exhibit equal potency for displacing [3 H]DP-033 from membranes of the hemipteran
insect, Perigrinus maidis (corn planthopper). As these insecticides have comparable
potencies against hemipteran RyRs, any enhanced control of sucking insects would
most likely be attributed to the improved plant systemicity of cyantraniliprole
[21, 22].
Me Cl Cl N
O O
F Cl
N N
N N
H N H N
Me
N O N O
H CF3 H Cl
DP-010 DP-033
The pore region of the RyR (amino acid residues 4475–5037 for RyR1) is
the putative binding site for ryanodine [25]. Through photoaffinity studies, and
replacement of select regions of recombinant insect RyRs, Kato et al. recently
reported that amino acid residues 4111–5084 are critical for sensitivity to phthalic
and anthranilic diamide insecticides [26]. These amino acid residues appeared to be
more critical for sensitivity to flubendiamide over the anthranilic diamide, DP-23.
The authors speculated that the diamides may regulate conformational coupling
between the cytoplasmic and transmembrane domains of the RyR.
100
Normalized response (% of max) 80
60
40
20
0
−9.0 −8.0 −7.0 −6.0 −5.0 −4.0
Anthranilic diamide concentration (log M)
Figure 34.3.9 Comparative dose–response compounds exhibit strong selectivity for in-
curves for chlorantraniliprole (red) and sect over mammalian RyRs. , Lepidopteran
cyantraniliprole (blue) on cells expressing (H. virescens); •, dipteran (D. melanogaster);
RyRs from insect (solid line) and mam- , mouse RyR1; , rat RyR2; x, human (un-
malian (dotted line) organisms. Both defined isoform).
Both chlorantraniliprole and cyantraniliprole exhibit outstanding toxicology pro-

files for nontarget organisms, with very low mammalian toxicity (Table 34.3.4).
Receptor-based studies of various natively expressed mammalian RyR isoforms
have demonstrated that chlorantraniliprole has a 300-fold to more than 2000-fold
selectivity for insect, over mammalian, RyRs [8]. Cyantraniliprole has been found
to have an even lower activity against mammalian RyRs. This compound exhibits
an almost 500-fold lower potency against the most sensitive mammalian receptor
isoform tested (mouse RyR1) compared to insect RyRs (Figure 34.3.9) [21, 22].
Consequently, target site selectivity appears to be a major factor contributing to the
low mammalian toxicity associated with the anthranilic diamide insecticides.
34.3.7
®
Biological Profile of Chlorantraniliprole (Rynaxypyr )
34.3.7.1 Insecticidal Potency and Attributes

Chlorantraniliprole provides an effective control of economically important pests,
resulting in excellent plant protection; typically, a broad range of Lepidoptera
in approximately 15 families and over 75 genera are controlled [27, 28]. Chlo-
rantraniliprole provides larval control that is generally one to two orders of
magnitude more potent against target pests when compared to commercial
standards. For example, the LC50 for Plutella xylostella is 0.05 ppm for chlo-
rantraniliprole, compared to 0.5 ppm for indoxacarb and 2.1 ppm for cypermethrin.
In addition, chlorantraniliprole shows activity on select pests in additional insect
orders (Table 34.3.3). In nonagricultural markets, chlorantraniliprole is highly
potent against most scarabaeid grubs and Listronotus maculicollis (annual bluegrass
Table 34.3.3 Insecticidal activity: chlorantraniliprole and cyantraniliprole.
Insect order Common name Scientific name EC50 (ppm)

Chlorantraniliprole Cyantraniliprole
Lepidoptera Fall armyworm Spodoptera 0.06 0.35

frugiperda
Lepidoptera Diamond-back moth Plutella xylostella 0.05 0.07
Lepidoptera Tobacco budworm Heliothis virescens 0.04 0.21
Lepidoptera Beet armyworm Spodoptera exigua 0.1 0.75
Lepidoptera Cabbage looper Trichoplusia ni 0.06 0.26
Coleoptera Colorado potato Leptinotarsa <0.1 <0.1
beetle decemlineata
Hemiptera Green-peach aphid Myzus persicae 5.0 1.1
Hemiptera Cotton/melon aphid Aphis gossypii 12.4 0.4
Hemiptera Potato leafhopper Empoasca fabae 1.5 2.0
Hemiptera Sweetpotato whitefly Bemisia 0.8 0.08
argentifolii
Thysanoptera Western flower Frankliniella – 3.1
thrips occidentalis
Orders represent insects feeding by chewing (Coleoptera, Lepidoptera), sucking (Hemiptera), and
rasping (Thysanoptera).
weevil) that attack lawn and sod grasses in the United States, and also provides an
excellent control of termites [29–31].
The field usage rates of chlorantraniliprole vary from 10 to 100 g a.i. ha−1 on
agricultural crops, and its utility has been demonstrated in many crop markets, as
well as adjacent markets that include turf, ornamentals, and home and industrial
sectors. Chlorantraniliprole is also expected to be used in seed treatments for field
and specialty crops.
Treatments with chlorantraniliprole result in plant protection through the rapid
cessation of pest feeding [32]. The affected insects are rapidly paralyzed, which re-
sults in their desiccation and susceptibility to predatory and environmental hazards.
Chlorantraniliprole is also highly potent against neonates as they hatch from the
eggs [33]. The young larvae are affected either by consuming the treated chorion,
or by taking small bites from the foliage or fruit. In addition, significant ovicidal
activity has been observed against Grapholita molesta (oriental fruit moth), Cydia
pomonella (codling moth), Pseudoplusia includens (soybean looper), and Anticarsia
gemmatalis (velvetbean caterpillar). The ovicidal effects are enhanced when the eggs
are laid on chlorantraniliprole-treated surfaces compared to applications made post
oviposition.
Chlorantraniliprole can effectively control lepidopteran and dipteran adults when
they ingest treated exudates or water droplets. Other data have shown an effect
when these insects come into contact with spray droplets, which is enhanced
with the use of adjuvants. In addition to direct toxicity, research conducted on
C. pomonella adults demonstrated that sub-lethal exposure to chlorantraniliprole

marked reductions in egg laying and subsequent egg hatch [34].
Chlorantraniliprole does not protect new growth when applied through foliar
applications; thus, the spray intervals will vary depending on the growth rate and
the production of new leaves. On a rapidly growing and expanding canopy, a spray
interval against lepidopteran species may be less than seven days under high insect
pressure. However, for mature plants with little or no new growth, residual control
may last as long as 14 – 21 days.
Chlorantraniliprole is effective at controlling foliar-feeding lepidopteran pests
when applied as a soil treatment, due to its movement through the xylem
[35–37]. In a three-year study conducted on bell peppers with Ostrinia nubi-
lalis (European corn borer), chlorantraniliprole, when applied via drip irrigation,
provided an equal or greater efficacy than standard grower programs involving foliar
sprays [38].
34.3.7.2 Mammalian and Environmental Safety

Chlorantraniliprole has an excellent toxicological profile, with low toxicity to
mammals in oral, dermal, inhalation, and eye tests (Table 34.3.4). Results were also
favorable in a range of tests to monitor mutagenicity, carcinogenicity, neurotoxicity,
and reproductive toxicity [27, 28, 39].
In addition to its low toxicity to mammals and wildlife, the ecotoxicological
profile of chlorantraniliprole is also favorable to beneficial arthropods, despite
its potent toxicity towards the target insect pests. This effect is attributed to the
minimal exposure to chlorantraniliprole of predatory and parasitic insects that do
not feed on the treated foliage. The results of extensive laboratory and field trials
have shown chlorantraniliprole to have minimal impact on populations of many
parasitic wasps (e.g., Aphelinus, Trichogramma), predatory mites (e.g., Amblyseius,
Typhlodromus), predatory insects (e.g., Coccinella, Orius, Nabis, and Chrysoperla), and
spiders [42–44]. Formulated chlorantraniliprole products have shown to produce
negligible effects on honey bees, in both laboratory toxicity studies and field tests.
In a direct contact study with the bumble bee Bombus impatiens, no mortality was
observed 72 h following exposure to chlorantraniliprole at 1.0 g L−1 [45].
Table 34.3.4 Non-insect toxicological profile of chlo-

rantraniliprole and cyantraniliprole [40, 41].
Species/measurement Chlorantraniliprole Cyantraniliprole
Acute oral LC50 , rat (mg kg−1 ) >5000 >5000

Avian, acute oral LD50 , bobwhite quail (mg kg−1 ) >2250 >2250
Aquatic. rainbow trout LC50 (mg l−1 ) >13.8 (solubility limit) >12.6
Crayfish, LC50 (mg l−1 ) >1.42 4.0
Earthworm, acute LD50 (mg kg−1 soil) >1000 >1000
34.3.7.3 Insecticide Resistance Management

Chlorantraniliprole provides a new mode of action for Insecticide Resistance
Management (IRM). Pest species known to be resistant to other insecticide
classes – such as organophosphates, carbamates, insect growth regulators, and
pyrethroids – do not exhibit any cross-resistance to chlorantraniliprole. In com-
mercial insecticide rotation programs, chlorantraniliprole is particularly valuable
for controlling resistant C. pomonella in apples, P. xylostella in crucifers, Tuta
absoluta (tomato leafminer) in tomato, Helicoverpa zea (corn earworm) in vegetable
crops, grubs in turf, shoot stem borer (Leucinodes spp.) in eggplant, and rice stem
borers (e.g., Chilo spp., Sesamia inferens, and Scirpophaga incertulas).
While cross-resistance to other nondiamide insecticides has not been identified,
a reduced efficacy has been reported in P. xylostella resistant to flubendiamide,
which is a phthalic diamide in the same IRAC mode-of-action group. Although
observations of reduced efficacy have been made in Thailand (Ban Bua Thong) and
the Philippines (Cebu Island), at present it remains unclear as to whether such
resistance is target-based or metabolic in nature.
A global IRAC Working Group has been established to develop IRM guidelines
for implementation by the IRAC teams of various countries. Currently, these global
and country teams are working in cooperation with local agricultural experts and
regulators to implement diamide IRM guidelines that are designed to help growers
prolong the benefits of these insecticides.
34.3.8
Biological Profile of Cyantraniliprole (Cyazypyr™)
Cyantraniliprole provides an effective control of a cross-spectrum of piercing-

sucking and chewing pests (Table 34.3.3). The groups for which cyantraniliprole
has broad-spectrum field efficacy include whiteflies, leaf-feeding beetles, leafmin-
ers, fruit flies, psyllids, and caterpillars [46]. Cyantraniliprole is also effective in
controlling important species of aphids, leafhoppers, planthoppers, weevils, and
thrips through population suppression [47, 48]. Although cyantraniliprole is partic-
ularly effective against the immature stages of pests [49], for certain species it also
shows excellent activity on the adult stage. Cyantraniliprole is generally effective
at usage rates of between 10 and 200 g a.i. ha−1 , depending on the pest, the crop,
the application method and its timing, and the use of adjuvants. A broad range
of pest management uses are currently under evaluation, including crops, turf,
ornamentals and animal health, as well as home and industrial sectors.
With cyantraniliprole, plant protection occurs as a result of rapid cessation of
pest feeding; typically, the affected insects are unable to feed within minutes to
hours following exposure to cyantraniliprole. While insect mortality can occur
within hours, observations of population control and field efficacy are generally
made within one to three days for biting-chewing pests, and up to five days for
piercing-sucking pests [49].
The ability of cyantraniliprole to partition into the leaf tissues provides protection
against rain wash-off, thereby enhancing residual activity. Data acquired from the
laboratory, greenhouse, and field show that cyantraniliprole has an excellent efficacy
when applied through both foliar and soil routes [49]. When applied to the plant
foliage, either via spray applications or overhead chemigation, cyantraniliprole
shows a translaminar activity that results in the control of important species of
aphids, whiteflies, and caterpillars. The use of oil adjuvants further improves leaf
penetration, which in turn enhances the level and consistency of pest control.
The physical properties of cyantraniliprole confer xylem mobility, and thus
upward translocation, in plants. When applied to the root zone, cyantraniliprole
is translocated to the above-ground parts of the plant; hence, it can be used for
soil application methods such as drip chemigation, drench, shank, nursery box,
and in-furrow liquid and granules, as well as in seed treatments. Conversely,
phloem mobility from foliar cyantraniliprole applications has not been commonly
observed.
34.3.9
Conclusions
Both, chlorantraniliprole and cyantraniliprole control insect pests through activa-

tion of insect RyRs, which represents a novel mode of action for insect control.
Both compounds are remarkably selective for insect over mammalian receptors,
and such selectivity is a key attribute of their low toxicity. The outstanding biologi-
cal properties of these compounds make them excellent crop protection agents, to
farmers and consumers alike.
References
1. Lahm, G.P., Myers, B.J., Selby, T.P., 5. Tohnishi, M., Nakao, H., Furuya, T.,
and Stevenson, T.M. (2001) Patent WO Seo, A., Kodama, H., Tsubata, K.,
01/070,671 (Chem. Abstr., 135, 272754). Fujioka, S., Kodama, H., Hirooka, T.,
2. Lahm, G.P., Selby, T.P., Freudenberger, and Nishimatsu, T. (2005) Flubendi-
J.H., Stevenson, T.M., Myers, B.J., amide, a novel insecticide highly active
Seburyamo, G., Smith, B.K., Flexner, against lepidopterous insect pests. J.
L., Clark, C.E., and Cordova, D. (2005) Pestic. Sci., 30, 354–360.
6. Seo, A., Tohnishi, M., Nakao, H.,
Insecticidal anthranilic diamides: a new
Furuya, T., Kodama, H., Tsubata, K.,
class of potent ryanodine receptor ac-
Fujioka, S., Kodama, H., Nishimatsu,
tivators. Bioorg. Med. Chem. Lett., 15,
T., and Hirooka, T. (2007) in Pesti-
4898–4906.
cide Chemistry: Crop Protection, Public
3. Tohnishi, M., Nakao, H., Kohno, E.,
Health, and Environmental Safety (eds H.
Nishida, T., Furuya, T., Shimizu, T.,
Ohkawa, H. Miyagawa, and P.W. Lee),
Seo, A., Sakata, K., Fujioka, S., and Wiley-VCH Verlag GmbH, Weinheim,
Kanno, H. (1999) European Patent pp. 127–135.
919,542 (Chem. Abstr., 131, 31808). 7. Lahm, G.P., Selby, T.P., and Stevenson,
4. Tohnishi, M., Nakao, H., Kohno, E., T.M. (2003) Patent WO 03/015,519
Nishida, T., Furuya, T., Shimizu, T., (Chem. Abstr., 138, 200332).
Seo, A., Sakata, K., Fujioka, S., and 8. Lahm, G.P., Stevenson, T.M., Selby,
Kanno, H. (2000) European Patent T.P., Freudenberger, J.H., Cordova,
1,006,107 (Chem. Abstr., 133, 17278). D., Flexner, L., Bellin, C.A., Dubas,
References 1423
C.M., Smith, B.K., Hughes, K.A., 19. Pepper, B.P. and Carruth, L.A. (1945) A
Hollingshaus, J.G., Clark, C.E., and new plant insecticide for control of the
Benner, E.A. (2007) Rynaxypyr: a new European corn borer. J. Econ. Entomol.,
insecticidal anthranilic diamide that 38, 59–66.
acts as a potent and selective ryanodine 20. Sattelle, D.B., Cordova, D., and Cheek,
receptor activator. Bioorg. Med. Chem. T.R. (2008) Insect ryanodine receptors:
Lett., 17, 6274–6279. molecular targets for novel pest con-
9. Lahm, G.P., Stevenson, T.M., Selby, trol chemicals. Invertebr. Neurosci., 8,
T.P., Freudenberger, J.H., Dubas, 107–119.
C.M., Smith, B.K., Cordova, D., 21. Selby, T.P., Lahm, G.P., Stevenson,
Flexner, L., Clark, C.E., Bellin, C.A., T.M., Hughes, K.A., Annan, I.B.,
and Hollinghaus, J.G. (2007) in Pesti- Cordova, D., Bellin, C.A., Benner,
cide Chemistry: Crop Protection, Public E.A., Wing, K.D., Barry, J.D., Currie,
Health and Environmental Safety (eds H. M.J., and Pahutski, T.F. (2008) Discovery
Ohkawa, H. Miyagawa, and P.W. Lee), of Cyazypyr™: A new cross-spectrum
Wiley-VCH Verlag GmbH, Weinheim, insecticide form the anthranilic diamide
pp. 111–120. class of ryanodine receptor activators.
10. Hughes, K.A., Lahm, G.P., Selby, T.P., Abstracts of Papers, 236th National
and Stevenson, T.M. (2004) Patent WO Meeting of the American Chemical
04/067,528 (Chem. Abstr., 141, 190786). Society, Philadelphia, PA.
11. Lahm, G.P., Cordova, D., and Barry, 22. Cordova, D. (2010) Award address. An-
J.D. (2009) New and selective ryan- thranilic diamide insecticides: selective
odine receptor activators for insect activators of insect ryanodine receptors.
control. Bioorg. Med. Chem. Lett., 17, Abstracts of Papers, 239th National
4127–4133. Meeting of the American Chemical
12. Freudenberger, J.H., Lahm, G.P., Selby, Society, San Francisco, CA.
T.P., and Stevenson, T.M. (2003) 23. Cordova, D., Benner, E.A., Sacher,
PCT International Application WO M.D., Rauh, J.J., Sopa, J.S., Lahm, G.P.,
03/016,283 (Chem. Abstr., 138, 205053). Selby, T.P., Stevenson, T.M., Flexner,
13. Bruening, J., Casalnuova, A.L., and L., Gutteridge, S., Rhoades, D.F., Wu,
Grushin, V. (2008) PCT International L., Smith, R.M., and Tao, Y. (2006)
Application WO 08/070,158 (Chem. Anthranilic diamides: a new class of
Abstr., 149, 53719). insecticides with a novel mode of action,
14. Shapiro, R. and Taylor, E.G. (2006) ryanodine receptor activation. Pestic.
PCT International Application WO Biochem. Physiol., 84, 196–214.
06/062,978 (Chem. Abstr., 145, 62887). 24. Ebbinghaus-Kintscher, U., Luemmen,
15. Annis, G.D., Bruening, J., Currie, M.J., P., Lobitz, N., Schulte, T., Funke, C.,
Dumas, D.J., and Shapiro, R. (2008) Fischer, R., Masaki, T., Yasokawa, N.,
PCT International Application WO and Tohnishi, M. (2006) Phthalic acid
08/082,502 (Chem. Abstr., 149, 152831). diamides activate ryanodine-sensitive
16. Dumas, D.J. and Casalnuova, A.L. (2008) Ca2+ release channels in insects. Cell
PCT International Application WO Calcium, 39, 21–33.
09/085,816 (Chem. Abstr., 151, 123984). 25. Callaway, C., Seryshev, A., Wang,
17. Insecticide Resistance Action Committee J.P., Slavik, K.J., Needleman, D.H.,
(2010) MOA Structures Poster (Version Cantu Iii, C., Wu, Y., Jayaraman, T.,
2), http://www.irac-online.org/wp- Marks, A.R., and Hamilton, S.L. (1994)
content/uploads/2009/09/pp_moa_structure Localization of the high and low affin-
_poster_v2.7_16oct09.pdf (accessed 20 ity [3 H]ryanodine binding sites on the
December 2010). skeletal muscle Ca2+ release channel. J.
18. Melzer, W., Herrmann-Frank, A., and Biol. Chem., 269, 15876–15884.
Luttgau, H.C. (1995) The role of Ca2 + 26. Kato, K., Kiyonaka, S., Sawaguchi, Y.,
ions in excitation-contraction coupling of Tohnishi, M., Masaki, T., Yasokawa,
skeletal muscle fibers. Biochim. Biophys. N., Mizuno, Y., Mori, E., Inoue, K.,
Acta, 1241, 59–116. Hamachi, I., Takeshima, H., and
Mori, Y. (2009) Molecular characteri- (Lepidoptera: Tortricidae) by chlo-

zation of flubendiamide sensitivity in rantranilipole, an anthranilic diamide
the lepidopterous ryanodine receptor insecticide. Pest Manage. Sci., 63,
Ca2+ release channel. Biochemistry, 48, 180–189.
10342. 35. Palumbo, J.C. (2007) Systemic efficacy
27. DuPont Altacor® Insect Control of Rynaxypyr applied through drip irri-
(2008) Technical Bulletin, DuPont gation on fall lettuce, 2006. Arthropod
Crop Protection, Wilmington, DE, Manage. Tests, 32, E25.
http://www2.dupont.com/Production_ 36. Palumbo, J.C. (2008) Systemic efficacy of
Agriculture/en_US/assets/downloads/pdfs/ Coragen applied through drip irrigation
K-14832.pdf (accessed 20 December on fall broccoli, 2007. Arthropod Manage.
2010). Tests, 33, E23.
28. DuPont Coragen® Insect Control 37. Palumbo, J.C. (2008) Systemic efficacy of
(2008) Technical Bulletin, DuPont Coragen applied through drip irrigation
Crop Protection, Wilmington, DE, on romaine lettuce, fall 2007. Arthropod
http://www2.dupont.com/Production_ Manage. Tests, 33, E36.
Agriculture/en_US/assets/downloads/pdfs/ 38. Ghidiu, G.M., Ward, D.L., and Rogers,
K-14833.pdf (accessed 20 December G.S. (2009) Control of European
2010). corn borer in bell peppers with chlo-
29. Spomer, N.A., Kamble, S.T., and rantraniliprole applied through a drip
Siegfried, B.D. (2009) Bioavailability irrigation system. Int. J. Veg. Sci., 15,
193–201.
of chlorantraniliprole and indoxacarb to
39. Bentley, K.S., Fletcher, J.L., and
eastern subterranean termites (Isoptera:
Woodward, M.D. (2010) Chlorantranilip-
Rhinotermitidae) in various soils. J.
role: an insecticide of the anthranilic
Econ. Entomol., 102, 1922–1927.
diamide class, in Hayes Handbook of
30. Yeoh, B.H. and Lee, C.Y. (2007)
Pesticide Toxicology, Ch. 102, Elsevier,
Tunneling responses of the Asian
pp. 2231–2242.
subterranean termite, Coptotermes gestroi,
40. DuPont Rynaxypyr® Insect Control
in termiticide-treated sand (Isoptera:
(2007) Technical Bulletin, DuPont
Rhinotermitidae). Sociobiology, 50,
Crop Protection, Wilmington, DE,
457–468.
http://www2.dupont.com/Production_
31. Koppenhöfer, A.M. and Fuzy, E.M.
Agriculture/en_US/assets/downloads/pdfs/
(2008) Effect of the anthranilic diamide Rynaxypyr_Tech_Bulletin.pdf (accessed 20
insecticide, chlorantraniliprole, on Het- December 2010).
erorhabditis bacteriophora (Rhabditida: 41. DuPont Cyazypyr™ Insect Control
Heterorhabditidae) efficacy against white (2010) Technical Data Sheet, DuPont
grubs (Coleoptera: Scarabaeidae). Biol. Crop Protection, Wilmington, DE.
Control, 45, 93–102. 42. Brugger, K.E., Cole, P.G., Newman,
32. Hannig, G.T., Ziegler, M., and Paula, I.C., Parker, N., Scholz, B., Suvagia,
G.M. (2009) Feeding cessation effects P., Walker, G., and Hammond, T.G.
of chlorantraniliprole, a new anthranilic (2010) Selectivity of chlorantraniliprole
diamide insecticide, in comparison with to parasitoid wasps. Pest Manage. Sci.,
several insecticides in distinct chemical 66, 1075–1081.
classes and mode-of-action groups. Pest 43. Dinter, A., Brugger, K., Bassi, A.,
Manage. Sci., 65, 969–974. Frost, N.-M., and Woodward, M.D.
33. Loriatti, C., Anfora, G., Angeli, G., (2008) Chlorantraniliprole (DPX-E2Y45,
Mazzoni, V., and Trona, F. (2009) Ef- DuPont™ Rynaxypyr®, Coragen®,
fects of chlorantraniliprole on eggs and and Altracor® insecticide) – a novel
larvae of Lobesia botrana (Denis & Schif- anthranilic diamide insecticide –
fermüller) (Lepidoptera: Tortricidae). Pest demonstrating low toxicity and low
Manage. Sci., 65, 717–722. risk for beneficial insects and predatory
34. Knight, A.L. and Flexner, L. (2007) Dis- mites, in WG Pesticides and Beneficial
ruption of mating in codling moth Organisms, Proceedings of the Meeting at
References 1425
Berlin, Germany, October 10–12, 2007, San Diego, CA, Entomological Society of
Berlin, Germany (eds H. Vogt, J.-P. America, Lanham, MD.
Jansen, E. Vinuela, and P. Medina), vol. 47. Portillo, H.E., Annan, I.B., and Marcon,
35, IOBC/wprs Bulletin, pp. 128–135 P.C. (2009) Cyazypyr™ (DPX-HGW86,
44. Preetha, G., Stanley, J., Suresh, S., Cyantraniliprole): a novel anthranilic
Kuttalam, S., and Samiyappan, R. (2009) diamide insecticide for control of white-
Toxicity of selected insecticides to Tri- flies and other important arthropod
chogramma chilonis: assessing their pests. 5th International Bemisia Work-
safety in the rice ecosystem. Phytopara- shop, November 9–12, Guangzhou,
sitica, 37, 209–215. China.
48. Stansly, P.A., Kostyk, B., and Riefer,
45. Gradish, A.E., Scott-Dupree, C.D.,
R. (2010) Effect of rate and application
Shipp, L., Harris, C.R., and Ferguson,
method of Cyazypyr (HGW86) on con-
G. (2010) Effect of reduced risk pesti-
trol of silverfleaf whitefly and southern
cides for use in greenhouse vegetable
armyworm in staked tomato, 2009.
production on Bombus impatiens (Hy- Arthropod Manage. Tests, 35, E43.
menoptera: Apidae). Pest Manage. Sci., 49. Portillo, H.E., Annan, I.B., Marcon,
66, 142–146. P.C., and Lund, A.E. (2010) Biological
46. Annan, I.B., Portillo, H.E., Lund, A.E., attributes of Cyazypyr™ (DPX-HGW86,
and Thompson, M.E. (2010) DuPont cyantraniliprole): a novel cross-spectrum
Cyazypyr™ insecticide (DPX-HGW86, anthranilic diamide insecticide, in En-
cyantraniliprole): unique product for pre- tomological Society of America National
mium pest control and agronomic plant Meeting, December 14, San Diego, CA,
protection, in Entomological Society of Entomological Society of America,
America National Meeting, December 14, Lanham, MD.
1427
Index of Common Names
a AD67 378
A-ONE® 252 Adengo® 143, 155, 156, 383
A12127 472 Aderio® 746
ABA 283 Adjust® 1172
abamectin 944, 1068, 1115, 1116, Admire® 1191, 1193
1305–1310, 1312, 1315–1320, 1322, 1323, Advantage® 1190
1352, 1369, 1374–1376 AdvantageMulti® 1190
abikoviromycin 842, 843 Advantix® 1190
abscisic acid 283, 343 AE 0001789 375, 383
AC 217 300, 1082 AE C638206 832
AC 263222 388–390 AE F070542 374
AC 322 140, 70, 72 AE F092944 81, 82
AC 90001 220 AE F099095 81, 82
Acaban® 1080 AE F107892 61, 375, 381
acaristop 1017 AE F115008 60, 62
Acatak® 640 AE F117223 1081
Accent Q® 383 AE F117233 1091, 1092
Acenit® 378 AE F122006 76, 77, 375, 382
acephate 942, 951 AE F130060 62, 64, 82
acequinocyl 946, 1082, 1097, 1099–1101 AE F130360 76, 77
acetamiprid 943, 1127, 1129, 1147, 1149, AE F140584 81, 82
1150, 1152–1154, 1167, 1169, 1171–1175, AE F147447 81, 82
1236 AE F150944 353, 355, 356
acetochlor 10, 373, 374, 377, 378, 516 AE F154851 81, 82
acetogenin 564 AE F160459 81, 82
acetoprole 1284 AE F160460 81, 82
acibenzolar-S-methyl 553, 554, 909, 910, 915, Aeris® 1195
916 Affirm® 1307, 1309, 1310
acifluoren 400 aflastatin A 842
aclonifen 269 agri-mek 1307
acramite® 1351 Agrimec® 1307, 1310
acrex 654 Agrisure Viptera™ 1035
acricid 654 Ajudazol B 565
acrinathrin 943 Akari® 1080
Act® 317 AKD 1022 1127, 1130, 1167, 1175, 1180,
Actara® 1211, 1215 1203, 1206, 1207, 1210, 1211
activity 1241 Akteur® 1191
AD-67 MON4660 374 Aktuan® 867

1428 Index of Common Names
Al Fares® 63 Artimon® 871

alachlor 10, 310, 316, 391, 401, 516 ascochlorin 568
Alanto® 1196, 1198 ASM 909, 910, 915, 917, 919
alanycarb 942 Assail® 1172, 1175
Alburin® 1181 asulam 10
aldicarb 942, 951, 1369, 1371, 1373–1375 Atlantis® OD/WG 62, 63, 65, 381
Alias® 1191 atovaquone 568
Alion™ 356, 362 atpenin A5 572
Alister® 65 atractyloside 577, 578
Aliziman® 871 atrazine 14–16, 22, 76, 228, 253, 254, 269,
All-Shine® 774, 775 377, 480, 507, 510, 516
allethrin 943 Attribut® 142, 153, 158
Alliance® 871 AU-1421 648, 653
allidochlor 312 aurachin A 565
alloxydim 455, 458 aureothin 565
alloxydim-sodium 888 aurovertin B 574, 576
Almanach® 871 Authority® 163
alpha-endosulfan 1285 Avatar® 1264
Alto® 772 Avaunt® 1264
altosid 986 avermectin B1 aglycone 1316
Alubarin® 1181 avermectin B1 a 1289, 1306, 1307, 1308,
aluminum phosphide 947 1312, 1315, 1316, 1374, 1375
Amdro® 1082 avermectin B1 b 1307
ametoctradin 550, 584, 615, 617, 621 avermectin/milbemycin 1144, 1309, 1375
ametryne 80, 480, 507, 516 avermectins B1 A1 A2 1316
amicarbazone 479, 510, 511, 515–517 avermectins B1 a/B1 b 1308
amidoflumet 1346, 1347, 1354–1356 Avicta® 1306, 1322, 1374–1376
amidosulfuron 53, 62, 63 avid 1307
Amigo® 1191 Axial® 380
aminocyclopyrachlor 278, 295, 299–303 Axiom® 318
aminopyralid 278, 281, 282, 287–294 azadirachtin 948
amiprophosmethyl 10 azamethiphos 942
amisulbrom 550, 584, 586, 613, 615, 620 azimsulfuron 51, 52, 68, 70, 71
amitraz 946, 1101 Azin® 70
amitrole 22, 198, 201 azinphos-ethyl 942
Ammate® 1264 azinphos-methyl 942, 1270
AMPA 407, 409, 412 azocyclotin 945
anilazine 556 azoxystrobin 549, 550, 586, 587, 590, 592,
anilofos 11, 70 593, 598, 601, 602, 605, 607, 612, 617
annonin VI 565 aztec 1373
ANS-118 959, 975
anthraquinone 1195 b
antimycin A, A1 567–569 B1b 1374
Anvil® 772 bacara 219
AOPP 447 bacillus firmus 1368
AOPPs 450, 453–455, 473 BAJ2740 1111
apex 986 Balance Flexx® 383
apollo/kelthane/sanmite 1017 Balance® 237, 269
apoptolidin 575 banlep 1307
Applaud® 1004 Banvel® 417
APPs CHDs, 454 Bariard® 1196, 1198
Apron XL® 904 Baroque® 1022, 1026
Archipel® 63, 65 Barrier™ 362
Arena® 1176 BAS 10501W 209, 210
BAS 145138 389–391 bioallethrin 943

BAS 490 F/500 F/505 F/520 F 586, 587 bioallethrin S-cyclopentenyl isomer 943
BAS-635 64, 66 Bion® 553
BAS600F 739 bioresmethrin 943
basamid 1370 biphenyl 552
Basta® 403 Biscaya® 1196, 1198
Battalion® 378 bispyribac 132–134
BAY DAM 4493 143, 145 bispyribac-sodium 127–129
BAY FOE5043 319 bistrifluron 945, 1003, 1004
BAY GSE 18636 144 BIT 914
BAY KRA 4145 512, 513 bitertanol 771, 776, 1195
BAY MKH 3586 510, 511, 513 bixafen 550, 630, 631, 634
BAY MKH 4340 145, 147 Blasin® 656, 660
BAY MKH 6561/6562 144 blasticidin-S 551
BAY YRC2388 322 Blizzard® 867
Baycor® 771 Blue Sky® 1191
Bayfidan® 771 BODYGUARD® 252, 255
Bayleton® 771, 776 bollgard 1036–1038
Baytan® 771, 878 boltage 1372
BeamAdmire® 1195 bongkrekic acid 577, 578
beflubutamid 205, 215, 217, 220–222 Boral® 163
Belay® 1176 borax 944
benalaxyl 901, 905 Borneo® 1022, 1026
benclothiaz 1383, 1384 boscalid 549, 550, 572, 629, 631, 633–635,
bendiocarb 942 644
benefin 439 Broadway® 380
benefin = benfluralin 10 Brom-O-Gas 1370
Benefiter® 69 bromacil 480, 508
benfuracarb 942, 1369 bromethalin 649, 657, 662
benfuresate 310, 311 bromobutide 268
benodanil 628, 630, 640 bromofenoxim 481, 507
benomyl 549 bromopropylate 948, 1060
benoxacor 253, 372, 373, 375–377, 390 bromoxynil 151, 154, 157, 273, 274, 346, 400,
bensulfuron 70 403, 481, 500, 503
bensulfuron-methyl 19, 53, 131, 264, 317, bromoxynil heptanoate/octanoate 503
320, 917 bromuconazole 783, 784
bensultap 945, 1127, 1128 BSN2060 1111
bentazone 131, 390, 481, 501, 510 Buctril® 346, 403
benthiavalicarb 807–810, 814, 815, 825 α-bungarotoxin 1240
benzoate 1305, 1318 buprofezin 946, 1004, 1005, 1396, 1397
benzobicyclon 237, 238, 243, 251, 256, 257, butachlor 10, 268
320 butamiphos 10
benzofenap 251, 267, 268, 272, 275 butoxycarboxim 942
benzoximate 948 butralin 10
Best Partner® 140 butroxydim 459
Bestguard® 1169–1172 BXN canola 405
α-BgTx 1136, 1139, 1143, 1146 BXN cotton 403
Biathlon® 64 BXN® 402, 405
Bicep II magnum® 376 BY106830 970, 971
bicuculline 1375 BYI 1921 1379
bicyclopyrone 228, 247, 248
bifenazate 948, 1083, 1097, 1346–1352 c
bifenthrin 943 14 C-DPX-JW062 1265
binapacryl 649, 653, 654, 659 14 C-glyphosate 408–410
14 C-iprovalicarb 825 CGA 215 944 1327, 1329

[14 C]metazachlor 389 CGA 246916 1169
[14 C]thiacloprid 1197 CGA 277476 79
C22T 364 CGA 279202 586
cabothiin 1180 CGA 293’343 1211
cadusafos 942, 1369, 1372, 1375 CGA 325615 344, 356–359
cafenstrole 11, 70, 309, 320 CGA 362622 79, 80
Calaris® 251, 253 CGA 43089 373
calcium phosphide 947 CGA 52232 RP 615
Calex® 1172 CGA 72662 1005
Calixin® 794 CGA 92194 373
Callisto® 250, 251 CGA 860 1384
CaLypso® 1196, 1198, 1199 CGA140408 1061
Camena® 1195 CH3 SO2 82
Camix® 251, 253, 376 Charter® 782
Cap 25® 871 CHD 447, 450, 453, 455, 466, 473
Capaz® 163 Chekker® 62, 63
CAPRENO® 143, 155, 156, 252, 382 Chess™ 1327
Capstar® 1169, 1170 Chevalier® 63, 65
captan 541, 542, 556 chinomethionat 948
cara 1017 Chinook® 1195
Caramba® 785 Chipco Tristar® 1172, 1175
carbaryl 942 chlorafenapyr 1074
carbendazime 549, 739, 741–743, 749, 757 chlorantraniliprole 947, 1102, 1389, 1390,
carbetamide 10 1409, 1411–1415, 1417–1422
carbofuran 942, 1369, 1371, 1373 chlorethoxyfos 942
carbonyl cyanide m-chlorophenylhydrazone chlorethoxyphos 1372
650 chlorfenapyr 611, 649, 653, 657, 945, 1068,
carbosulfan 942, 1369, 1371 1071, 1072, 1073, 1075, 1076, 1101, 1300,
carboxin 550, 571, 572, 627, 628, 635 1354
carfentrazone 154 chlorfenson 1060
carfentrazone-ethyl 58 chlorfenvinphos 942
carpropamid 553, 554, 842, 845, 846, 850, chlorfluazuron 945, 1003, 1363
852, 856–859, 1180, 1195, 1200 chloridazon 209, 210, 502
cartap 1127, 1128, 1143, 1180 chlorimuron ethyl 37, 38, 40–42, 45
cartap hydrochloride 945, 1171 chlorimuron-ethyl 53, 391
Casoron™ 362 chlormephos 942
Castellan® 788 chloroneb 552
CCCP 650, 653, 655, 660 chloronitriles 556
Celcius® 158 chloropicrin 944, 1369, 1370
Celero® 1176 chloropropane 1369
Celio® 379 chloropropylate 1060
Celsius® 143, 157 chlorothalonil 541, 542, 556
cerulenin 844 chlorotoluron 22, 505
Cesar® 1019 chloroxuron 480, 508
CF canola 405 chlorpropham 10, 741, 743
CF corn 404 chlorpyrifos 942, 951
CGA 293 343 1213 chlorpyrifos-methyl 942
CGA 123407 374 chlorsulfuron 19, 22, 25, 35, 36, 38, 39, 43,
CGA 133205 373, 379 50, 52, 53, 119, 120, 132, 133, 153, 154, 390
CGA 154281 373, 375 chlorthiamid 11, 345, 346
CGA 185072 374, 379 cholesterol 762
CGA 207 429 1328, 1329 chromafenozide 946, 959–963, 966,
CGA 207 936 1329 973–975, 976
Cinch® 376 coumaphos 942

Cinch® ATZ 376 coumethoxystrobin 594
Cinch® ATZ lite 376 coumoxystrobin 585, 594, 595
cinosulfuron 53 counter 1373
Ciral® 58 CPTA 202
citovaricin 575 crocacin 568
Clarity® 417 CropStar® 1195
classes 447 cruiser 1217, 1221, 1222
Clearfield® 46, 94 Cruiser® 1211
Clearfield® plus 94 Cruiser Extreme 250® 1376
Clearfield® CF 403 Crusoe® 785
clethodim 16, 17, 450, 459 cryolite 948
clinch 1307 cumyluron 372, 374
Clio® 272 Curzate® 554, 867
clodinafop 391, 451 cyanazine 480, 507
clodinafop-propargyl 374, 379, 391, 454, 457 cyanide 947
clofentezine 944, 949, 1012–1017, 1023, cyanophos 942
1026, 1101, 1115, 1116, 1352 cyantraniliprole 1409, 1415, 1417–1419
clomazone 198, 199, 372 cyazofamid 550, 584, 586, 612, 613, 615, 617,
clomeprop 917 620, 621
clopyralid 154, 278, 284, 287, 289, 290, 293 Cyazypyr® 1421
cloquintocet 471, 472 Cyazypyr™ 295, 1409, 1413
cloquintocet-mexyl 374, 379, 380, 391, 455, cycloartenol 762
470, 472 cyclodiene organochlorines 942
cloquintocet-mexyl (S) 472 cycloprothrin 943
cloquintocet-mexyl/clodinafop-propargyl
cyclosulfamuron 51, 52, 68, 70–72
391
cycloxydim 16, 458
cloransulam-methyl 100–104, 115
cycloxydim sethoxydim, 450
clothianidin 943, 1127, 1129, 1144, 1147,
Cydectin® 1313
1149, 1152–1154, 1157, 1167, 1169, 1175,
cyenopyrafen 571, 572, 947, 1082, 1092,
1176, 1178–1181, 1183, 1185, 1186, 1193,
1093, 1099, 1100
1210, 1213, 1215
cyfelmetofenc 1082
clothianidine 1150
Cyflamid® 891
Clutch® 1176
cyflufenamid 555, 887–891
CM-001 959, 975
colchicine 740, 741, 743 cyflumetofen 948, 1093, 1094, 1100, 1346,
Commando® 1191 1347, 1352–1354
complete 1322 cyfluthrin 943, 1195, 1363
Concep I 373 β-cyfluthrin 943, 1180, 1195, 1200
Concep I® 379 cyhalofop-butyl 109, 457
Concep II® 373, 379 cyhalothrin 943, 1402, 1403, 1404
Concep III® 373 λ-cyhalothrin 943, 1248, 1249
confidence Xtra® 377 γ -cyhalothrin 943
Confidence® 377 cyhexatin 945
Confidor® 1191, 1193, 1195 cymoxanil 541, 554, 555, 746, 747, 865–869
CONFIRM™ 959, 974, 975 cyometrinil 373, 379, 381
Conidor® 1191 cypermethrin 943, 1418
Connect® 1191, 1195 α-cypermethrin 943
Conquest® 1172 β-cypermethrin 943
Corbel® 794 θ -cypermethrin 943
corn 404 ζ -cypermethrin 943
Corvus® 143, 155, 156, 383 cyphenothrin 943
Cossack® 62, 63, 65 cyproconazole 552, 772
cotton 419 cyprodinil 551, 671, 889
cyprosulfamide 143, 147, 148, 155, 158, 269, desmetryne 480, 507
375, 383, 384, 394 4 -desoxy-4 -amino-avermectin B1 1319
cyrmenins 587 4 -desoxy-4 -epi-methylamino avermectin B1
cyromazine 946, 1005 1316
cytosine 1153 13-desoxy-avermectin B1 aglycone 1316
desoxynivalenol 784
d Deter® 1176
1,3-D (1,3-dichloropropene) 1370 DGR 419
2,4-D (2,4-dichlorophenoxyacetic acid) 151, Diacon 986
154, 277–281, 284, 289–291, 303, 371, 400, diafenthiuron 575, 611, 945, 1060–1064,
417 1066–1068
D-D (1,2-dichloropropane/ diazinon 942
1,3-dichloropropene) 1369, 1370 dicamba 143, 151, 154, 157, 158, 254, 272,
4,4-dimethylergosta-8,14,24(28)-trien-3ß-ol 278, 281, 284, 289, 383, 400, 416–420
765 dichlobenil 11, 342, 344–347, 349, 350, 352,
22,23-dihydro-13-desoxy-avermectin B1 356, 358, 360–363
aglycone 1316 dichlormid 372, 373, 377, 378
D2341 1083, 1351 dichloropropene 1369
d-cis-trans allethrin 943 dichlorvos/DDVP 942
d-trans allethrin 943 diclocymet 858, 1180
Daepo® 1181 diclofop 16, 17, 22, 401, 453
daimuron 70, 372, 374
diclofop-methyl 45, 455, 456
Daniemon® 1116
diclomezine 555, 749, 750, 865,
Danisaraba® 1082
875–877
danisaraba® 1354
diclosulam 100–104, 115
Danitron® 1080
dicofol 948, 1017, 1101, 1352
Dantop® 1176, 1179
dicoumarol 658
Dantotsu® 1176, 1179, 1180
dicrotophos 942
Dantotsupadan® 1180
dicyclomet 842, 846
Dantotsupadanvalida® 1180
dicyclonon 389
dazomet 1369, 1370
dieldrin 1285–1287, 1290
DBCP 1369, 1370
DBI-3204 1004 diethofencarb 548, 549, 739, 741–743
DCCD 1060, 1062, 1063 dietholate 372
DCJW 1265–1270, 1280, 1281 difenoconazole 552, 772
DCMU 750, 751 diflovidazin 944, 949, 1012–1018, 1023
DCPA = chlorthaldimethyl 10 diflubenzuron 945, 1002, 1003, 1005
DCSA 417 diflufenican 63, 65, 202, 204, 206, 217, 219,
DDT 949, 1060, 1078, 1265, 1267 221, 222
DDT methoxychlor 943 diflumetorim 550, 671–675
DE-638 109 difunon 209
Dectomax® 1310 diketonitrile 271
Degree Extra® 378 dim 447
Degree® 378 dimefluazole 615, 617
Delausdantotsu® 1180 dimefuron 480
Delegate® 1249 dimepiperate 372, 374
deltamethrin 943, 952, 1195, 1200 dimethanamid 10
demeton-S-methyl 942 dimethenamid 272, 390
DEN 447, 455, 473 dimetho-morph 810
DEN class 455 dimethoate 942, 1067, 1101
DEN herbicides 454 dimethomorph 553, 807–809, 811, 825–827,
denim 1307 831, 835, 836
DENs 447, 460, 464, 465, 469 dimethyl disulfide 1369, 1370
desmedipham 480, 500, 503 dimethylvinphos 942
dimoxystrobin 586, 590, 591, 598, edifenphos 552

605, 607, 616 EfA® 792, 878
dims 455 efrapeptin 574
Dinamic® 511, 516 El Hombre® 1191
dinitramine 10 EL-436 1086
dinitrocresol 648 EL-436 XDE-436, 1080
dinobuton 653, 654, 659 EL171 219
dinocap 550, 648–650, 653, 654, 659 Electis® 746
dinoseb 659 Elevate® 797
dinotefuran 914, 943, 1127, 1129, 1147, Eloge® 640
1167, 1181–1185, 1215 emamectin 1068, 1305, 1307, 1309, 1311,
dioxapyrrolomycin 1071, 1072, 1077 1312, 1316, 1318, 1319, 1390, 1403–1405
diphenamid 10 emamectin benzoate 944, 1306–1310, 1319,
Discover® 379 1322, 1323
disulfoton 942, 951 emamectine 1307
dithianon 556 Eminent® 777
dithiopyr 10, 439–445 empenthrin 943
diuron 15, 22, 227, 480, 500, 506, Enable® 778
516 Encore® 1191
DKN 229, 232, 233, 270, 271, 275 α-endosulfan 1285, 1293
DMO 417 Energy 1195
DNOC 648, 653, 654, 945 enestroburin 585, 606, 607
dodemorph 793 enstar 986
dodine 544, 554, 555 Envidor® 1111, 1116
Dorado® 768 Envoke® 80
doramectin 1310, 1312 (±)-epibatidin 1150, 1151
Doublestar® 320 (–)-epibatidine 1135, 1136
DP-010 1417 (–)-epibatidine (18) 1153
DP-033 1417 Epik® 1172, 1175
DP-23 1260, 1417 epingle 997
DPX JE874 586 episterol 765
DPX-3792 AKD-2023, 1082 episterone 798, 799
DPX-A8947 70, 71 EPN 942
DPX-JW062 1261, 1266 (+)-epopromycin B 365
DPX-KE459 58, 60 epopromycin A 365
DPX-KN128 1262, 1263 epopromycins 365
DPX-KQ926 893 epoxiconazole 552, 779–782
DPX-KZ 165 597 epoxyconazole 780
DPX-KZ165 593, 601 eprinomectin 1310, 1312
DPX-MP062 1262, 1263 Equip® 77, 382
DPX-MY926 213 Equip® plus 78
DR 417 Equitation Pro® 867
DR soybean 418 ergosterol 761–763, 765
drazoxolon 656, 660 Escocet® 1191
DTP 226 esfenvalerate 943
Dual II magnum® 376 esprocarb 71
dunaimycins 575 ethaboxam 555, 742, 747
dynamec 1307 ethalfluralin 10
ethametsulfuron-methyl 53, 149
e ethidimuron 480, 509
E3274 1377, 1378 ethiofencarb 942
(eburicol) 765 ethion 942
Ecomite® 1116 ethiprole 943, 1283, 1284, 1293
EDB 1369, 1370 ethirimol 548
ethofumesate 312 fenoxasulfuron 63

ethoprophos 942, 1369, 1372, 1374 fenoxycarb 944, 984–986
ethoxysulfuron 51, 52, 67–70, 382 fenpiclonil 715, 718, 721–723, 724, 726, 728
ethylene dibromide 1369 fenpropathrin 943, 1017
etofenprox 943 fenpropidin 552, 793–795
etoxazole 944, 1012, 1020, 1022–1026 fenpropimorph 552, 793, 794
etridiazole 552 fenpyroximate 564, 565, 611, 946, 1063,
Everest® 142, 151, 158 1079, 1080, 1084, 1097, 1099, 1101, 1115,
Evidence® 1191 1116, 1352, 1397–1399, 1401, 1402
Evolve® 867 fenthion 942
Exalt™ 1249 fentin acetate 550
EXP10745 586 fentrazamide 10, 140, 251, 252, 256, 309,
310, 312, 316, 318, 320–324
f fentrifanil 653, 657, 662
F-28249 1315 fenuron 480, 509
F1785 1333, 1336 fenvalerate 943, 952
F5231 163 ferimzone 653, 656, 660, 661, 1180
Faibel® 1191 Fierce® 330
FALCON ™ 959, 974, 975 fipronil 914, 943, 1068, 1283, 1284,
famoxadone 550, 567, 584, 586, 587, 1286–1290, 1292–1299
612–615, 619 FirstRate® 102
famphur 942
flamprop-m 10
Fandango® 791
flazasulfuron 53
Favilla® 1195
Flex® 163
FCCP 653, 655, 660
Flexidor™ 362
fecosterol 765, 799
Flexity® 893
fecosterone 765, 798, 799
Flexstar® 163
fenamidone 550, 584, 586, 587, 612–614,
flonicamid 944, 1327, 1333, 1335, 1336,
619, 835, 836
1337, 1339
fenaminosulf 563–565
Floramite® 1083, 1351
fenamiphos 942, 1195, 1369, 1371, 1374
florasulam 100, 101, 103–105, 113, 115, 291,
fenapanil 779
fenarimol 767, 768 380
fenazaquin 611, 670, 671, 687, 946, 1060, fluacrypyrim 585, 611, 946, 1082, 1097, 1103,
1080, 1086, 1092, 1099, 1101, 1102, 1252 1354
fenbuconazole 777–779 fluazifop 16, 17, 19
fenbutatin oxide 945 fluazifop-butyl 456
fenchlorazole-ethyl 374, 381, 385, 389, 391, fluazinam 541, 649, 650, 653, 657, 662–664
392, 393 fluazinam ferimzone 550
fenchlorazole-ethyl/fenoxaprop-ethyl 391 fluazuron 640
fenclorim 374, 391 flubendiamide 947, 1340, 1389–1393, 1396,
fenfuram 628, 630 1403–1409, 1415, 1417
fenhexamid 545, 551, 552, 796–799, 801 flubenzimine 1060
fenikan 219 flucarbazone 148–150
fenitrothion 942 flucarbazone-sodium 142, 144, 146,
fenobucarb 942 149–151, 153, 154, 157, 158
fenoxanil 554, 842, 846, 856, 858 flucetosulfuron 51, 52, 68, 72, 73
fenoxaprop 17, 23, 273, 274, 391–393 flucycloxuron 945
fenoxaprop-ethyl 374, 385, 389, 391, 392, 456 flucythrinate 943
fenoxaprop-P 386, 392, 393, 443 fludioxonil 549, 551, 715–718, 721–723,
fenoxaprop-P-ethyl 62, 381, 382, 385, 386, 725–730
392, 393, 454 fluensulfone 1377, 1379, 1381–1383
fenoxapropethyl 70 flufenacet 10, 269, 310–313, 316–319, 324
fenoxasulfone 309, 326, 330, 334, 335 flufenerim 1081, 1090, 1091, 1100, 1102
flufenoxuron 945, 1003, 1004, 1390, 1403, Froncide® 640

1404 frowncide 657, 662
flufenzine 1014 FS 236 3, 1191
flumethrin 943 fthalide 842, 845
flumetsulam 19, 100, 101, 114, 115 fucosterol 762
flumioxazin 330 fufenozide 978, 979
Flumite 200® 1014, 1018 FujiMite® 1080
flumorph 807–811, 826 Fulfill™ 1327
fluometuron 480, 506 Fullswing® 1176, 1179
fluopicolide 549, 640–643, 831–836 Fungazil® 769
fluopyram 550, 639–645 fungisterol 761
fluorochloridone 217 funiculosin 568
fluoxastrobin 586, 590, 592, 593, 597, 599, furadan 1371
605, 607, 618, 791, 792, 878 furalaxyl 901
flupoxam 11, 344, 351, 352 furametpyr 628, 631, 635, 636
flupyrsulfuron 51, 52 furathiocarb 942
flupyrsulfuron-methyl-sodium 51, 57–60 furilazole 372, 373, 378
fluquinconazole 788, 789 furosemide 1288
fluramim 658
flurazole 373, 376, 389, 390
fluridone 202, 204, 210–212, 217–219, 221, g
222, 227 GA21 411, 414
flurochloridone 202, 204, 210, 211, 219, 221, Gallery™ 362
222 Galmano® 788
FLUROX™ 1004 Gaucho Orge® 878
fluroxypyr 151, 154, 278, 287, 290, 291 Gaucho® 1191, 1193, 1195
flurtamone 202, 205, 207, 209–212, 219, 221, Gaucho® Orange 1195
222 Gavel® 746
flusilazole 772, 776 Gazel® 1172
flusulfamide 555, 865, 873–875 Gazelle® 1172
flutenzine 1013 Genesis® 1191
flutianil 887, 896, 897 gentrol 986
flutolanil 572, 628, 631, 640, 749, 750, 756 GET-STAR® 252
flutriafol 771 GF-120NF® 1239
τ -fluvalinate 943 Ghoulish® 1191
fluxapyroxad 630 gibberellins 764
fluxofenim 373, 376, 379, 389, 390 Gladium® 70
FMC4 1091 Glean® 35
Focus Shot® 251 glufosinate 156, 399–401, 403,
Focus® 1176 416–420
FOE1976 319 glufosinate ammonium 402
Folicur® 772, 776 glyphosate 5, 12, 13, 20, 21, 24, 25, 43, 46,
folpet 541, 542, 556, 814 78, 155, 163, 229, 254, 291, 300, 303, 330,
fomesafen 163 399–402, 404–418, 420
fop 447, 455 GM canola 405
foramsulfuron 51, 52, 76, 77, 382, 393 Goltix® 512
formetanate 942 GR soybean 411
fortress 1372 Granit® 783
Fortuna® 78 Grazie® 70
fosetyl 869 griseofulvin 739
fosetyl-aluminum 554, 555, 814, 833, 865, Grodyl® 63
869–873 Guardian® 378
fosetyl-sodium 869 guazatine 556
fosthiazate 942, 1369, 1372, 1375 Gulliver® 70
h Hussar 62
H 9789 220 Hussar maxx® 63, 65
[3 H]α-BgTx 1146, 1148 Hussar® 63, 381
[3 H]RH-4032 740 Hussar® OD 63
[3 H]RH-5854 740 Hussar® 62
[3 H](S)-zoxamide 740 Hussar® OF Evolution 63
[3 H]dihydropicrotoxinin 1285, 1286 Hustler® 1180
[3 H]epibatidine 1183, 1215 Huszar® 63
[3 H]imidacloprid 1152, 1214 Huzar® 63
[3 H]metalaxyl 904 hydramethylnon 946, 1082, 1097, 1098
[3 H]thiamethoxam 1214 hydrochloride 1128
[3 H]batrachatoxin-B 1267 hydroprene 944, 984–986
Hachihachi® 1081 hymexazol 548
Hachikusan® 1190, 1191
halfenprox 943 i
haliangicin 568 [125 I]α-BgTx 1136, 1143, 1147, 1183
halofenozide 946, 959–961, 965–968, 971, IAA 277–285, 303
973–976 Ichiyonmaru 70
halosulfuron 54 ICI A 5504 586
halosulfuron-methyl 378 IFT 269–272, 274, 275
haloxyfop 16, 17, 450–453 IKA 2002 1093
haloxyfop-methyl 457 IKA-2002 1091, 1093, 1100
haloxyfop-P-methyl 640 IKA-2002c 1082
Harness® 378 IKF 916 586
Harpon® 746 IKF-309 893
Hawk® 379 IKI-220 1333, 1336
Headline™ 609 ilicicolin H 568
Healseed® 773 imazalil 769, 773
Healthied® 773 imazamethabenz 25
Heartguard® 1310 imazamethabenz methyl 97
Heat® 163 imazamethabenz-methyl 89, 91, 95
HEC 5725 586 imazamox 89, 92, 95–97
heptenophos 942 imazapic 89, 95, 97, 388, 389
herbaflex 220 imazapyr 19, 37, 89, 91, 92, 95–97, 119, 132,
herculex 1035 404
Hero® 70 imazaquin 19, 40, 41, 45, 89, 95, 97
hexaconazole 772 imazasulfuron 19
hexaflumuron 945, 1003, 1004 imazethapyr 42, 89, 92, 95–97, 404
hexathiazox 1352 imazosulfuron 54, 317
hexazinone 480, 508, 516 Imex® 1191
hexythiazox 944, 949, 1012, 1013, 1015, imibenconazole 788–790
1018–1023, 1025, 1026, 1115, 1116 Imicide® 1191
HF-6305 788 imicyafos 942, 1369, 1373, 1374
HNPC-A3066 611, 1091 imidacloprid 878, 943, 1116–1118, 1127,
HNPC-A3066c 1083 1129, 1143, 1144, 1146, 1147, 1149–1155,
HNPC-C9908 149 1157, 1167, 1171, 1173, 1175, 1180,
HOE 095404 67 1183–1185, 1189–1197, 1200, 1205, 1207,
Hoe 095404 69 1211, 1214, 1232–1236, 1240, 1337
Hoe 11077 1081, 1091, 1092 iminoctadine 556
Hoestar® Super 63 imiprothrin 943
Hoganna® 874 impact 245
Horizon® 379, 867 Impact® 271, 771
Husar® 63 Impower® 1191
Huskie® 273 Imprimo® 1195
Impulse® 795 isoxaflutole 155, 156, 228, 237, 238, 268, 269,
Indar® 778 271, 318, 383, 384, 515
indaziflam 11, 344, 356, 361–363 isoxaflutole 270
indoxacarb 947, 1102, 1257, 1260, isoxathion 942
1262–1267, 1269, 1270, 1280–1282, 1300, ivermectin 1310, 1312, 1317, 1375
1390, 1403–1405, 1418 Ivomec® 1310
Infinito® 640, 833
Infinity® 273 j
ingard 1036 J101 414
Innova® 320 J163 414
Input® 795 jasmonic acid JA 910, 911
insegar 986 Java® 816
InSyst® 1172 JC-940 374
Intercept® 1191 JH114 978
Interceptor® 1313 JW062 1264
inthomycin 365
inthomycin A 364 k
inthomycin B 364 K223 SK-23 374
inthomycin C 364 K9 advantix® 1190
inthomycins 364 kadethrin 943
INTREPID™ 959, 974, 975 Kanemite® 1082
Intruder® 1172, 1175 karathane 654, 659
Invento® 814 kasugamycin 544, 551, 882
iodo-sulfuronmethyl sodium 65 KC10017 842, 844
iodosulfuron 51, 52, 63, 78 Kelion® 50 WG 74
iodosulfuron-methyl 381 Kendo® 1080
iodosulfuron-methyl-sodium 51, 57, 59–64, ketospiradox 245, 246
76, 78, 381, 382, 388, 393 Keystone® 377
iodosulfuron-sodium 143, 157, 158 KIF-230 814
ioxynil 218, 481, 500, 504 KILLAT® 959, 975
ioxynil octanoate 504 kinoprene 944, 984–986
ipconazole 785–787 Kinto TS® 782
ipfencarbazon 316, 322–324 KN128 1263, 1264
ipfencarbazone 10, 309 Knack® 996
iprobenfos 545 Kohinor® 1191
iprodione 551, 715, 1369 koromite 1307
iprovalicarb 553, 807–810, 812–814, Koromite® 1313
825–827, 831, 835, 836 KPP-856 214
IR-5878 74 kresoxim-methyl 545, 549, 586, 587, 590,
IR5878 74 591, 597, 598, 603, 604, 607, 609, 612, 616
isofenphos 942 Krismat® 80
isoprocarb 942 Kusakonto® 251
isopropyl O-(methoxyaminothio-phosphoryl)
salicylate 942 l
isoprothiolane 552, 1396 Lamardor® 792
isoproturon 22, 23, 218, 352, 480, 501, 510 laminarin 554
isopyrazam 550, 629, 634 Lamisil® 800
isotianil 554, 909, 910, 919–925 Lano 997
isoxaben 11, 342, 344, 348–350, 352, lanosterol 762, 765
360–363 larvin 1371
isoxadifen 252, 383 LAUDIS® 252
isoxadifen-ethyl 76, 77, 147, 148, 155, 158, Laudis® 255, 382
229, 254, 375, 382, 383, 393 Lecspro® 320
isoxadifenethyl 78 Legend® 1191
lenacil 480, 500, 505 Matador® 1080

lepidine 686 MATRIC® 959, 975
lepimectin 944, 1305, 1313, 1314 matsuguard 1307
leptospermone 236, 237 MB-38544 219
Leverage® 1195 MBr 1369, 1370
Lexar® 251, 253, 376 MCPA 151, 154, 218, 273
Lexus® 50DF 58 MCPP 218
Lexus® Class 58 MCW-2 1377, 1379, 1381
LGC-42153 72, 73 Me-Too-Lachlor II® 376
Liberty Link® 401, 418 mecarbam 942
Liberty™ 403 mefenacet 10, 140, 252, 310, 316–319, 324
lindane 1285, 1293 mefenoxam 901–903
linuron 15, 480, 502 mefenpyr 59
Lizetan® 1191 mefenpyr-diethyl 61–64, 143, 158, 273, 274,
LL canola 405 372, 375, 381, 382, 385, 388, 392, 393
LL corn 404 mefenpyr-ethyl 455
LL cotton 403 mefenpyrdiethyl 63, 65, 380
LL Soybean 402 meferimzone 660
loreclazole 1288 MeI 1370
LS-80707 201, 202 melanin 841
lufeneron 1004 Melody Care® 814
lufenuron 945, 1003 Melody Combi® 814
Lumax® 251, 253, 376 Melody Compact® 814
Luna® 645 Melody Duo® 814
LY 211795 731 Melody Multi® 813
LY 247356 1081 Melody Triplo® 814
LY 809460 1081, 1091, 1092 Melody WP® 814
LY 823089 1081, 1091 mepanipyrim 551, 671
LY-176771 1086 mepronil 628, 631, 640, 749, 750
LY-186054 731, 732 meptyldinocap 649, 659
LY-211795 732 Merit® 1190, 1191
merlin 237
m Merlin Flexx® 383
24-methylenedihydrolanosterol 765 Merlin® 269
MACH 2 975 mesa 1307
MACH 2™ 974 mesosulfuron 51, 52, 62, 63, 65
MACH2™ 959 mesosulfuron acid 81
Magestan® 379 mesosulfuron-methyl 51, 58, 59, 62–64, 81,
Magister® 1080 82, 375, 381, 388, 392, 393
magma 317 mesosulfuron-treated 375
maister power 143 mesosulfuronmethyl 65
MaisTer® 78 mesotrione 226, 228, 229, 232, 240, 242, 250,
Maister® 382 251, 254, 272
malathion 942, 951 mesotrione; 228
malonoben 651–653, 655 Mester® 78
Manage® 789 metabolism 253
mancozeb 541, 542, 556, 746, 813, 814 metaflumizone 947, 1270, 1273, 1276–1282
mandipropamid 553, 807–810, 818–820, metalaxyl 747, 835, 836, 871, 872, 888, 901,
822, 825–827 904–906, 1180
maneb 556 metalaxyl-M 548, 901–906
Manik® 1172, 1175 metam sodium 1369, 1370
Marathon® 1191 metam sodium/potassium 1369
marshall 1371 metamifop 455, 457
Masai® 1080 Metamitron 512
metamitron 480, 500, 504, 510, 512 MIMIC™ 959, 974, 975, 977
metazachlor 10, 389, 390 Miteclean® 1081
metazosulfuron 51, 75 mitekohne® 1351
metconazole 784–786 MK-239 1080, 1087
methabenzthiazuron 480, 509 MK-243 1309
methamidophos 942, 951, 1101 MK-244 1309
methasulfocarb 555 mocap 1372
methidathion 942, 1101 Molan® 1172
methiocarb 942, 1195 molinate 131
methiopyrsulfuron 65–67 MON 37500 58, 61
methomyl 942, 1403 MON13900 373, 378
methoprene 944, 984–986 MON19788 414
methoxyfenozide 946, 959–962, 965–968, MON4606 373
971, 973–978, 980 MON4660 378
methyl bromide 944, 1369 MON88913 414
metiram 556 Monarca® 1200
metobromuron 480, 505, 509 Monceren® 1195
metolachlor 10, 313, 330, 376, 377, 379, 390, Mongarit® 790
516, 903 Monguard® 876
metolachlor + atrazine 376 monitor® 59
metolcarb 942 monocrotophos 942
metominostrobin 586, 587, 590, 591, 598, monolinuron 480, 509
602, 605, 607, 616 monosulfuron 51, 65–67
metosulam 100, 101, 114, 115 monosulfuron-ester 51, 66,
metoxuron 480, 505, 509 67, 149
metrafenone 555, 887, 891–893 Montur® 1195
Metribuzin 512 morlin 360, 361
metribuzin 15, 154, 318, 480, 500, 504, 512, Mortal® 1172
516 Mospilan® 1172, 1175
metsulfuron-methyl 19, 22, 52, 54, 58, 149, Mothpiran® 1172
153, 154, 290, 291 Mothpyran® 1172
mevinphos 942 Movento® 1124
MG-191 390 movento® 1376
midas 1370 Movento™ 1123
Mikado 251 Movento® 1123
Mikal® 871 moxidectin 1315
Mikalix® 871 Moxydec® 1313
milbeknock 1307 moxydectin 1313
Milbeknock® 1312, 1313 MP062 1264
Milbemax® 1313 MPTA 202
milbemectin 944, 1305–1308, 1312, 1313, MTF 753 631
1315, 1316, 1319, 1322, 1324 MTI-446 1181
milbemycin 1312 mucidin 587
milbemycin A3 /A4 1312, 1313 multi 1195
milbemycin 5-oxime 1313 Muralla® 1191
milbemycin A3 1312, 1313 Musou® 140
milbemycin A3 /A4 1314, 1315 My Way® 140
milbemycin A4 1312, 1313 MY-93 374
milbemycin D 1312, 1313, 1316 myclobutanil 772, 779
milbemycins A3 1312 mycothiazole 566
milbemycins A4 1312 mycotoxins 784
milcol 656 myxalamide 565
Milcol® 660 myxothiazol 567, 568, 587
Miltrex® 891 myxothiazol A 588, 589
n novaluron 945, 1003, 1004

N-methylimidacloprid 1151, 1153 noviflumuron 945, 1003, 1004
NA 371, 373, 388–390 2-NP 653
NA-73 1018, 1020 NPC-C9908 65
NA-83 1082 NTN 15192 750–753
NAA 281 NTN 16543 751, 753, 754
naftifine 552, 800, 801 NTN 19701 751, 753, 754
Naftin® 800 NTN 33893 1191
naled 942 NTN-28 621 212
naproanilide 10 NTN-28621 211
napropamide 10, 312 NTN32692 1190
NC 21314 1014 Nustar® 772
NC 224 586
NC-129 1080 o
NC-319 378 2,3-oxidosqualene 765, 795
NC-510 1082 O-TEQ® 1200
NC-512 1092 Oberon® 1111, 1116–1118
NC620 75 Ocarina® 814
NCI-129 1080, 1085 Odena® 814
Nebijin® 874 Odyssee® 871
Nebiros 71 ofurace 901, 905
neburon 480, 509 Oh-Shine® 775
nemacur 1371 OK-5101 1082, 1093
Nemacur® 1195 oligomycin A 575, 576
nemacur® 1374 Olympus flex® 65
nemagon 1370 Olympus® 142, 154, 158
nemakick 1373 omethoate 942
nemakick 1.5 G® 1374 OMI-88 1081, 1087
nematrim 1372 OnePass® 63
Nexter® 1080 Optimum-GAT® 43, 46
NI-25 1172 Option® 360 77, 78, 382
nicosulfuron 54, 272, 383 Opus® 779, 780
nicotine 1152 Ordoval® 1019
(S)-nicotine 1141 orion 1017
(S)-(–)-nicotine 1127, 1128, 1135, 1141, Orthosulfamuron 74
1153, 1157, 1166 orthosulfamuron 51, 52, 68
nipyraclofen 1283 Ortus® 1080
Nissorun® 1019 orysastrobin 586, 590, 593, 602, 606, 607, 616
nitenpyram 943, 1127, 1129, 1147, 1149, orysastrobinc 598
1150, 1152–1154, 1167, 1169–1171 oryzalin 10, 439
Nithiazine 1203 Oryzemate® 553
nithiazine 1127, 1128, 1149–1152, 1167, Oshin® 1181, 1184
1189, 1203, 1204, 1207 Osprey® 63, 65
nitisinone 225, 226, 229, 232 Othello® 65
NK603 411, 414 oudemansin 587
NK94827 66, 149 oudemansin A 585, 588
NNI 0101 1328 oudermansin A 589
NNI-750/I-850 1004, 1079, 1080 ovation™ 352
nocodazole 740, 743 OVERTURE™ 1366
Nominee® 127 Oxabetrinil 373
norflurazon 197, 202, 203, 205, 209, 210, oxabetrinil 379, 381
217, 218, 220, 221 oxadixyl 901, 905
norflurazone 222 oxamyl 942, 1369, 1371, 1373, 1376
Norosac™ 362 oxasulfuron 51, 52, 78, 79
oxaziclomefone 140, 252 phenthoate 942

oxpoconazole 774, 775 phorate 942
oxpoconazole fumarate 775 phosalone 942
oxycarboxin 627, 628 phosmet 942
oxydemeton-methyl 942 phosphamidon 942
oxyfluorfen 400 phosphine 947
oxytetracycline 551 N-phosphonomethyl glycine 406
oxythioquinox 1060 phosphorothiolate 882
phoxim 942
p phthalide 1180
Pacifica® 63, 65 phthoxazolin A 364
paclitaxel 739 phytoalexin 872
paecilomyces lilacinus 1368 phytoalexins 872, 924
paicer 266 picloram 278, 281, 282, 284, 285, 287–290,
Paicer® 267 293, 298, 303
Paladin® 1370 pico 220
Pancho® TF 891 Picolinafen 207
Panduck® 1355 picolinafen 202, 206, 218, 220–222
Parallel® 376 picoxystrobin 586, 590, 592, 598, 604, 605,
paramite 1026 607, 617
paraquat 13, 24 picrotoxin 1285, 1289, 1375
parathion 942 picrotoxinin 1288
parathion-methyl 942 piericidin A 565
Pasada® 1191 pinoxaden 380, 447, 451, 453, 460, 468–472,
Patrol® 794 474
PBO 1062, 1072 pinoxaden acid 471, 472
PBZ 909, 910, 913, 914, 919 piperonyl butoxide 1062, 1072, 1253
PDTC-9 656 piperophos 11, 268
pefurazoate 773, 774 pirate 657
Pegasus® 1061 pirimicarb 942, 951
Pegasus™ 1061 pirimiphos-methyl 942
penconazole 771, 776 Pivot® 50 WG 74
pencycuron 548, 549, 749–751, 754–760, Plenum™ 1327
1195 PLEO™ 1366
pendimethalin 10, 218, 439 Plural® 1191
penflufen 630 Pluto 997
penoxsulam 100, 101, 109, 110, 115 Poast® 404
pentachlorobenzyl alcohol (PCBA) 842, 845 Podigrol® 768
pentanochlor 480, 507 podophyllotoxin 740
penthiopyrad 629, 631, 633 Polo® 1061
pentoxazone 71, 251 Polo™ 1061
percut 1017 polyoxin 553, 749, 756
Pergado® 819 Poncho® 1176, 1179, 1180
permethrin 943, 951, 1181, 1253 Positron® 814
Permit® 378 POSSIBLE® 252
pethoxamid 10 Powerflex® 380
PH 60-41 1257 prallethrin 943
PH 60-42 1257, 1258 Pre-Empt® 1191
PH I-9 1257 Precept® 273
PH-6042 1273 Prefix™ 345
Phantom® 1181, 1184 Premise® 1190, 1191
phenmedipham 480, 500, 503, 510 Prescribe® 1191
phenothrin 943, 1356 pretilachlor 10, 251, 268, 374, 391
phenoxan 686 primisulfuron-methyl 54, 149
Pristine® 1172 pterulone 686

Proban® 871 Pulstar® 867
probenazole 553, 554, 909, 914, 921 Puma S® 381
prochloraz 552, 769, 770 Punch® 772, 776
proclaim 1307 pymetrozine 944, 1219, 1327–1329,
Proclaim® 1309, 1310 1331–1334, 1339, 1340, 1343, 1344
procymidone 551 pyraclofos 942, 1369, 1372
prodiamine 439 pyraclonil 252
PRODIGY™ 959, 974, 975 pyraclostrobin 550, 586, 590, 592, 595, 599,
profenofos 942 602, 603, 606, 607, 609, 612, 618
profenophos 1403 pyradaben 1116
Profil® 1175 pyrafluprole 1284
Profile® 1172 pyrametostrobin 585, 594, 595
Profiler® 833 Pyramite® 1080
profoxydim 455, 459 Pyranica® 1080
Proline® 791, 792 pyraoxystrobin 585, 594, 595
prometryn 80 pyrasulfotole 156, 157, 228, 272–274
prometryne 480, 507 pyrazolate 225, 228, 237, 238, 263, 264
pronamide 741, 743 pyrazolynate 263–266, 272
Pronto Plus® 795 pyrazon 480
propachlor 10 pyrazone/chloridazon 500
propamocarb 552 pyrazophos 552
propamocarb-HCl 833 pyrazosulfuron-ethyl 19, 55, 317, 320
propanil 23, 24, 320, 507 pyrazoxyfen 225, 228, 266–268, 272
propanile 480 pyrazoynate 267
propargite 945, 1017, 1060, 1067 pyrethrins 943
propazine 480, 507 pyribaticarb 268
propetamphos 942 pyribencarb 585, 593, 594, 619
propham 10 pyributicarb 552, 800, 801
propiconazole 552, 771, 776 pyricut 671
propineb 556, 814 pyridaben 564, 565, 611, 670, 671, 946, 1017,
propisochlor 10 1084–1086, 1097, 1099, 1101, 1115, 1116,
propoxur 942 1352
propoxycarbazone 65, 148–150 pyridafol 481
propoxycarbazone-methyl 2 157 pyridalyl 948, 1358–1360, 1362–1365, 1404
propoxycarbazone-sodium 63, 142, 144, 146, pyridaphenthion 942
149, 153, 154, 158 pyridaryl 1405
propyrisulfuron 51, 68, 75 pyridate 481, 501, 510
Proquinazid 895 pyrifenox 768, 770
proquinazid 551, 887, 893, 894, 896 pyrifluquinazon 1327, 1328, 1340–1344
Proseed® 792 pyrimethanil 551, 671
Prosper® 127, 1176 pyrimidifen 946, 1081, 1087, 1089–1091,
Prosper® 200 1180 1100
Prosper® 400 1180 pyrimifos methyl 1363
prosulfuron 54, 153, 154 pyriminobac 132
Protect® 373 pyriminobac methyl 127
Proteus® 1200 pyriminobac-methyl 129–132, 134
prothioconazol 878 pyrimisulfan 139, 140, 330
prothioconazole 552, 784, 791–793, 878 pyrimorph 807, 808, 811
prothiofos 942 pyriofenone 887, 893
Provado® 1190, 1191, 1193 pyriprole 1283, 1284
Provence® 269 pyriproxyfen 944, 983–985, 987–997, 1116,
pryidaben 1080 1117, 1181
pterulinic acid 686 pyrithiobac 120, 132–134
pyrithiobac-sodium 20, 125–127 Ricestar® 70, 382

pyroquilon 554, 842, 845 Ricestar® Xtra 70
pyroxasulfone 11, 309, 312, 313, 326–335 Ridomil Gold® 904
pyroxsulam 100, 101, 112–115, 291, 380 Rimidin® 768
pyrrolnitrin 721–723 rimsulfuron 55, 383
ROMDAN™ 959, 974, 975
q rotenone 559, 566, 946, 1078–1080
Quick Bayt® 1191 Roundup Ready 2 Yield® (RR2Y) 416
Quick Strike® 1167 Roundup Ready Flex cotton RRF, 415
quinalphos 942 Roundup Ready Flex® (RRF) cotton 414
quinazoline 1097 Roundup Ready® 401
quinclorac 278, 284, 285 Roundup Ready® (RR) corn 409
quinmerac 284 Roundup® 407
quinoxyfen 544, 551, 686, 687, 719, 720, Roundup-Ready 510
731–733, 879, 889, 895 Roxam® 746
quizalofop 16, 18, 19 RR 2 yield soybean 414
quizalofop-ethyl 456 RR alfalfa 414
RR canola 405, 412, 414
r RR corn 403, 404, 411, 414
(R)-(+)- 777 RR corn (GA21) 416
R-29148 391 RR corn 2 (NK603) 414, 416
R-40244 219 RR cotton 402, 403, 410, 414, 415
R25788 373, 377 RR Flex cotton 402, 414, 419
(R)-Imazaquin 40 RR soybean 401, 402, 413, 414, 416
(R, S)-(±)-dinotefuran 1183 RR wheat 413
racer 219 RR2Y soybeans 418
Radiant® 1249 RR60 415
Radius® 318 RRF cotton 415
rampage 657 RT73 414
Rapid® 1191 Rubigan® 768
Raxil S® 878 rugby 1372
Raxil® 772, 776 Rumo® 1264
RE-40885 219 RUNNER™ 959, 974, 975
Real® 782 rutamycin 575
Redigo® 792 Rynaxypyr® 295–297, 1409, 1418
Reflex® 163
Relay™ 1327 s
Rescate® 1172 S-methoprene 1300
resmethrin 943 S 3422 206
Resolve® 273 S-1283 1025
Revus® 819 S-13 651–653, 655
RH 1965 201, 202 S-1560 1081, 1090
RH 3421 1258 S-1812 1359
RH-0345 959, 960, 975 S-3085 212, 215
RH-2485 959, 960, 975 S-metolachlor 253, 269, 308, 375, 376
RH-3421 1266–1270 S-Metolachlor + atrazine 376
RH-4032 740 S-Metolachlor + mesotrione + atrazine
RH-5849 957, 958, 960, 965–967 S-Metolachlor + mesotrione 376
RH-5854 740, 741 sa-lan 1017
RH-5992 959, 960, 973–975 Safari® 1181, 1183, 1184
RH-7281 739 saflufenacil 163
RH5992 979 Sakura® 330
Rhodax® 871 salicylic acid SA 910, 911
Riceguard® 69 SAN 380H 201, 202
SAN 548A 1081, 1092 Solomon® 1195

SAN 548a 1091 sonar 219
sanbird 237 soybean 417
Sanbird® 264 Specticle™ 356, 362
SANK 60576 1312 spectrin 835
Sanlit® 790 spinetoram 1102, 1127, 1130, 1131, 1147,
Sanmite® 1080 1240, 1247–1249, 1252, 1253
Saprol® 768 spinetoram spinosad 943
Saurus® 1172 spinosad 1068, 1127, 1130, 1131, 1147, 1239,
Saviour 70 1247–1249, 1252, 1253, 1300, 1402–1405
Scablok® 874 spinosyn A 1127, 1130, 1144, 1238,
Scenic® 792 1240–1242, 1246, 1251–1253
Score® 772 spinosyn D 1127, 1130, 1238, 1251, 1252
Screen® 373 spinosyn E 1242
Scribe® 867 spinosyn G 1245
(+) scytalone 840, 850, 851 spinosyn J 1130, 1131, 1253
sedaxane 630 spinosyn L 1130, 1131
Seed-one® 1191 spirodiclofen 947, 1108, 1109, 1111–1113,
Sekator® 62, 63 1115, 1116, 1118, 1122, 1124
Sekator® OD 63 spiromesifen 947, 1108, 1109, 1111–1118,
selamectin 1310, 1312 1122, 1124
Sencor® 512 spirotetramat 947, 1118–1125, 1376, 1377
Sentinel® 772 spiroxamine 552, 793, 795, 796
Sentricon® 1004 spiroxamine (Input®) 791
sethoxydim 16, 17, 19, 401, 404, 450, 455, SPLAT-MAT™ 1239
458, 460, 888 Sportak® 769
SF6847 655 SPY-4903 1091
Shakariki 71 squalene 762, 763, 765, 795
Sharpen® 163 SR 404
shirlan 657, 662 SR corn 404
show-ace 237 F-126I
Show-Ace® 251, 256 586, 587
Shuriken® 1181 Stalwart C® 377
Shuttle® 1082 Stalwart® 376
Sibenil™ 362 Stalwart® Xtra 376
Sibutol® 771 Staple® 127
sicanin 572 star 1195
siduron 480, 509 Starice® 382
silafluofen 943 Starkle® 1181, 1184
Sillage® 871 Starkul® 1181
silthiofam 550, 577, 578 Starmite® 1082
Silverado® 63, 65 Status® 383
simazine 16, 22, 480, 507, 510 Steadfast Q® 383
simeconazole 790, 791 Sterling™ 1327
Sinawi® 1116 Steward® 1264
Sirbel® 814 stigmast-7-enol 761
Skol® 70 stigmatellin 566–568
Smart® 251, 320 Stimo® 746
Smartstax™ 1036 Strada® WG 74
smite 1026 streptomycin 551
SN 72129 1082, 1093 strike 986
SOBERAN® 252 strobilurin 567, 615, 635
Soberan® 255, 382 strobilurin A 585, 587–589
solicam 220 strobilurins 584, 747
STS soybean 402 tartar emetic 944

STS® 401 Tata Manik® 1172
STS® soybeans 46 Tatamida® 1191
SU 8801 1081, 1087 tau-fluvalinate 1017
SU 9118 1081 TCM 142, 143, 146–150, 152–158
sulcotrione 225–228, 240, 242, 249, 250, 253, tebuconazole 552, 772, 776, 784, 792, 878,
254, 256, 257, 269, 272 1195
sulcotrionez 251 tebuconazole (Prosaro®) 791
sulfentrazone 163
tebufenozide 946, 959–961, 965–968, 971,
sulfluramid 658, 945
973, 974, 975–980
sulfodiazol 509
tebufenpyrad 611, 670, 671, 946, 1080, 1084,
sulfometuron 25
sulfometuron methyl 36, 41 1086–1088, 1100–1102, 1352
sulfometuron-methyl 19, 50, 55, 133, 149 tebufloquin 865, 879–882
sulfosulfuron 51, 52, 58, 59, 61 tebupirimfos 942, 1373
sulfotep 942 tebuthiuron 480, 509, 516
sulfoxaflor 1131, 1226, 1227, 1229–1236 Tec-Lead® 785, 787
sulfoxaflore 1130 Techlead® 785
sulfoximines 1228 teclofthalam 555
sulfuryl fluoride 944 teflubenzuron 945, 1003, 1004
Sumilarv 997 tefluthrin 943, 1195
SUMIPLEO™ 1366 tefuryltrione 246, 247, 252, 254–256
Sunrice® 70 Teldor® 797
Sunrice® Plus 69, 70 telone 1370
Sunrice® Super 69 tembotrione 155, 156, 228, 229, 246, 247,
Sunrice® WG 69
252, 254–256, 382
Sunstar® 70
temephos 942
Superseded Concep I® 373
temik 1371
Superseded Concep II® 373
temik® 1374
supra 1195
Supreme® 1172, 1175 tentoxin 576
Suprend® 80 tepraloxydim 451–453, 455, 459
surangin B 572 terbacil 480, 508
Surpass® 377 terbinafine 800, 801
SV-03 918 terbufos 942, 1369, 1373
SX 623509 807, 819 terbumeton 480, 507
SYP-1620 585, 594, 595 terbuthylazine 253, 254, 269, 480, 508, 510
SYP-3200 594 terbutryne 480, 508
SYP-3343 594 Termex® 1191
SYP-3375 594 tetrachlorvinphos 942
SYP-4155 594 tetraconazole 776–778
SYP-4903 1083 Tetradifon 945
SYP-Z048 770 tetradifon 1060
SYP-Z071 enestroburin 594
tetramethrin 943
Systhane® 772
tetrasan 1026
SZI 121 1014, 1015
TH 547 75
SZI-121 1016
thaxtomin 364
t thaxtomin A 361, 364
T138067 741 thaxtomins 361, 364
Taipan® 267 thenylchlor 10
Talendo® 896 thiacloprid 943, 1127, 1129, 1142, 1147,
Talius® 896 1153, 1157, 1167, 1171, 1185, 1189,
Tanos® 867 1196–1200
thiamethoxam 943, 1127, 1129, 1144, 1147, triadimefon 771, 776

1152–1154, 1167, 1175, 1180, 1181, 1193, triadimenol 771, 776, 878
1203, 1206, 1207, 1211–1215, 1217, triarimol 767
1219–1222, 1234, 1322 triasulfuron 22, 55, 153, 154, 390
thiangazole 566 triazamate 942
thiapronil 1082, 1091, 1093 triaziflam 11, 353–355
thiazopyr 10, 400, 439–445 triazines 272
thiencarbazone 252, 273, 382, 383 triazofenamide 342, 350, 351
thiencarbazone methyl 143, 144 triazophos 942
thiencarbazone-methyl 142, 146, 149, 150, triazoxide 555, 865, 877–879, 1195
156, 269, 394 tribenuron methyl 44
thiencarbazone-methyl 3 157 tribenuron-methyl 55, 149, 153
thifensulfuron-methyl 55, 153, 154 trichlorfon 942
thifluzamide 629, 631–633 triclopyr 278, 288–290
thiobencarb 131, 268, 326 tricyclazol 553
thiocyclam 945, 1127, 1128 tricyclazole 554, 842, 845, 1195
thiodicarb 942, 1195, 1369, 1371 tridemorph 793, 794
thiofanox 942 trietazine 480, 508
thiometon 942 trifloxystrobin 550, 586, 590, 592, 598, 602,
thiophanate-methyl 549, 739 604, 607, 611, 616
thiosultap-sodium 945 trifloxysulfuron 51, 52
thiram 556, 1180 trifloxysulfuron-sodium 51, 78–80
Thunder® 1195 triflumizole 769, 891
TI-304 1169 triflumuron 945, 1003, 1025
tiadinil 553, 554, 631, 909, 910, 917, 919, 921 trifluralin 10, 330, 439
tigrex 219 triflusulfuron-methyl 55
tiller gold 382 Trifmine® 769
Tiller® Gold 70 trifocide 654
Tilt® 771, 776 triforine 539, 768
Titaron® 1082 trigard 1005
TKM 19 61 Trimax® 1191, 1196
TMC 207 576 trimethacarb 942
TN-16 740 triticonazole 780–782
tolfenpyrad 946, 1079, 1081, 1087, 1088, tritosulfuron 51, 52, 59, 64–66
1099, 1100, 1102 Trust® 1191
tolnifanide 683, 684 Try® 879
tolylfluanid 813 TTFB 652, 653, 657
Tooler® 65 β-tubulin 757
Topas® 771, 776 tubulozole C 740
Topik® 379 Tundra® 273
TopNotch® 377 Turob® 70
topramezone 228, 229, 245, 271–275
torant 1017 u
Torant® 1015 UBH-820 220
torero 1017 UCC-D2341 1351
Tornado® 1264 ultiflora 1307
Totril® 346 Ultra® 63
toxaphene 1285 Unikat® 746
Toyocarb® 800 usnic acid 649, 658
tralkoxydim 17, 455, 458 Utopia 71
tralomethrin 943
tralopyril 1071 v
transfluthrin 943 Valbon® 814
Treevix® 163 Valiant® 871
validamycin 553, 750, 756, Winner® 1191

1180 Wipe® 63
valifenalate 807, 808, 816 Wolverine® 273
valinomycin 647
Valis® 816 x
vamidothion 942 XDE-436 1086
vaniliprole 1284 XMC 942
Vapcomere® 1172 XR-100 1081, 1091, 1092, 1100
Vaplex® 1191 XR-539 807, 817
Vectra 3D™ 1181 xylylcarb 942
Velocity® 273
Velocity® m3 143, 157 y
Venom® 1181 Yaba® 816
vermelone 840, 850, 851, 854 Yaiba® 140
vertimec 1307 Yakawide® 267
Vertimec® 1310 Yi Sha Guang® 1191
Victor 1017 YI-5301 1022, 1025
Viktor CL® 1015 yieldgard 1035
vinblastine 740, 741 Ynaxypyr® 1411
Vincare® 814 Yorel® 814
vinclozolin 551, 715, 718 YRC 2894 1196
Vip3A 1037 Yunta® 1191
VipCot™ 1037
Viptera™ 1035 z
Vitavax® 1180 ZA 1963 586
vivando® 893 zarilamide 740–742, 746
Volley® 377, 1172 Zark® 317
Volley® ATZ 377 zeal 1026
Vortex® 785, 787 zephyr 1307
Votivo® 1368 Zidua® 330
Vulcano® 142, 158 zinc phosphide 947
vydate 1371 zoom 1026
zorial 220
w Zorro® 1191
Wakil® 867 zoxamide 548, 549, 739–747, 827, 835, 836
Warrant® 1191 zoxamide® 746
widestrike 1037 Zoxium® 746
WinAdmire® 1195 ZR-512 984
WinBariard® 1200 ZR-515 984
Windantotsu® 1180 ZR-777 984
1449
Subject Index
a ACCase, see acetyl-CoA Carboxylase

abamectin 1305 ACCase-inhibiting herbicide 447ff.
– activity level against mites and insects – mechanism of resistance 453
1316 acceptable daily intake (ADI) 490
– bioavaibility 1322 acceptable operator exposure level
– biological activity in seed treatment 1375 (AOEL) 495
– ecotoxicological data 1319 acequinocyl
– environment 1322 – synthesis 1095
– mode of action 1375 acetamiprid 1127, 1149ff., 1172ff.
– physicochemical properties 1308 – chemical classification 1173
– toxicological data 1319
– efficacy on target pest 1174
– application rates 1322
– insecticidal activity 1175
– use in crop protection 1323
– physico-chemical properties 1173
ABC transporter, see ATP-binding cassette
– synthesis 1174
transporter
– water solubility 1173
abiotic stress 530f.
acetohydroxyacid synthase (AHAS), see
Abutilon theophrasti, herbicide resistance 23
acetolactate synthase (ALS)
acaricides 663, 688, 1022, 1352ff.
acetohydroxybutyrate (AHB) 31
– 2-alkoxycarbonyl
acetolactate (AL) 31
trifluoromethanesulfonanilide 1355
acetolactate synthase inhibition 114ff.
– carbamates (methiocarb) 942
– carbazate 1347f. – triazolopyrimidine herbicide 115
– inhibitors of mitochondrial electron acetolactate synthase/acetohydroxyacid
transport 1078ff. synthase (ALS/AHAS) 29ff., 417
– MET inhibitor-based 1100, 1354 – biochemistry of the target 29
– MET-I 1101 – catalytic subunit (CSU) 32ff.
– MET-II 1102 – catalyzed reaction 31
– mitochondrial ATP synthase as a target – engineered resistance to AHAS-inhibiting
1059 herbicides in crops 46
– organophosphates (ethion, omethoate etc.) – isoenzyme (AHAS I, II, and III) 29ff.
942 – molecular basis for resistance 41
– prethroid classes (acrinathrin etc.) 943 – mutation 42
– site of metabolism for MET 1099 – regulatory subunit (RSU) 32ff.
– strobilurins 611 – resistance to AHAS-inhibiting herbicide
– strobilurins and other complex III 43
inhibitors 610 acetolactate synthase/acetohydroxyacid
– tetrazines 1016 synthase (ALS/AHAS) inhibitors
– undefined mode of action 1346 – imidazolone herbicides 88

1450 Subject Index
acetolactate synthase/acetohydroxyacid adenosine triphosphate (ATP) 559, 573

synthase (ALS/AHAS) inhibitors (contd.) – export from the mitochondrion 577
– pyrimidinyl carboxylate (PC) herbicides S-adenosyl-methionine 711, 1250
120 Adenylat-cyclase 1062
– triazolopyrimidines 99 ADI see acceptable daily intake
– sulfonylaminocarbonyl-triazolones 142 ADME (absorption, distribution, metabolism,
– sulfonyl ureas 50 excretion) factors 850
acetyl-CoA Carboxylase (ACCase) 447, 1108 Adoxophyes honmai (smaller tea tortrix) 1402
– biochemistry 447 Aedes aegypti (Aa) 968ff., 1004
– chloroplastic 449 AFB5 Class of picolinate auxin herbicide
– crystal structure 452 receptors 281
– cytosolic 448, 449, 468 Agrobacterium-mediated transformation
acetyl-CoA Carboxylase (ACCase) inhibitor 1029
13ff., 379, 447ff., 1108ff. Agrotis segetum
– herbicides 236, 447ff – wireworm larvae 1180
– insecticides 1108 ff – turnip moth 1402
– mode of action 450 Agromyzidae (dipterous leafminer) 1248
acetyl-CoA Carboxylase (ACCase) resistance agrophore 589, 640
16, 453ff. AHAS, see acetolactate
– Alopecurus myosuroides 453 synthase/acetohydroxyacid synthase
– black grass plants 454 AKD 1022 1127, 1180, 1203ff.
Acetyl-CoA transcarboxylase 448 – partial hydrolysis 1180, 1210
Acetylcholine(Ach) – synthesis 1209
– affinity for active state 1132, 1151, 1153, (R) alanine 814, 815
1214, 1232 trans-2-aldehydes as sprout suppressors 530
– agonistic binding site 1135, 1144, 1150, alkoxy triazolinone 153
1153, 1183 2-alkoxycarbonyl trifluoromethanesulfon-
– neurotransmitter 1135, 1137, 1240 anilide acaricide 1355
acetylcholine-binding protein (AChBP) S-alkyl-1λ4 ,2,4,6-thiatriazine 357
1135ff., 1185 – 3,5-substituted 357
– carbamoylcholine binding 1142, 1148 alkylazine herbicides 353
– crystal structure 1137, – biology 354
1138,1139,1142,1156, 1157, 1185 – chemistry 353
– nAchR properties 1139 – synthesis 355
acetylcholinesterase (AChE) 951 4-alkynyl-2-anilinopyrimidine
– inhibitors 942 – synthesis 708
N-acetyl-glucosamine allosteric regulation 570
(poly-N-acetyl-glucosamine) 999 allylamines 767, 800
– biosynthesis 1000 – target of 766, 801
N-acetyl-phosphinothricin 434 Alopecurus myosuroides, herbicide resistance
– inactivation of L-phosphinothricin 433 13ff.
Acibenzolar-S-Methyl ALS, see acetolactate synthase/
– synthesis 915f. acetohydroxyacid synthase
– systemic acquired resistance induction Alternaria diseases 783
916 alternative oxidase (AOX) 573, 609
Actinomycetes 1131, 1238 Amaranthus powellii 20
acyl carrier protein 1249 Amaranthus retroflexus 20, 45
acyl-CoA dehydrogenase 573 Amaranthus rudis 20ff.
acyl-CoA elongase (VLCFAE) 306,335 – herbicide, multiple resistance 25
acylpicolides (benzamides) 549, 832 AMBA (mesotrione metabolite) 253
acyltransferase (AT) 1249 Amblyseius calfornicus (predatory mite)
adenosine deaminase 548 1344ff.
adenosine diphosphate (ADP) 559 Amblyseius cumumeris 1344
– carrier 577 Amblyseius spp. 1420
Subject Index 1451
American dagger nematode (Xiphinema – resistance mutation 1286

americanum) 1376 – vertebrate ionotropic 1288
American cockroach, see Periplaneta 1-aminocyclopropane-1-carboxylate synthase
americana (ACC synthase) 609
ametoctradin 613ff. aminocyclopyrachlor 278, 295
– synthesis 622 – discovery 295
A-group fungicides 548 – ecotoxicology 302
amicarbazone 510f. – herbicidal activity 300
– discovery 511 – mode of action 302
– metabolites 516 – properties 300f.
– physico-chemical properties 511 – resistance management 303
– synthesis 514 – site of action 302
– uses 515 aminomethylphosphonic acid
amide herbicides 480f. (AMPA) 405ff.
– status of reregistration process 507 aminomethylpyridine 685
amidoflumet 1346ff. 4-aminomethylpyrimidine 686
– biochemistry 1355 aminopyralid 278ff.
– biology 1355 – animal toxicity 293
– development 1355 – biology 288
– discovery 1355 – carcinogenicity/teratogenicity 294
amidoketones 979 – environmental degradation 292
amines, fungicides 793 – mechanism of crop selectivity 291
– biochemical target 793 – synthesis 288
amino acid amides 811 – utility 290
amino acid amide carbamate 812 aminopyrazole 1295
amino acid biosynthesis 30 4-aminopyridine 678
aminoacyl-tRNA 694, 699 aminopyrimidine
aminoacyl-tRNA synthetase 694 – phenylacetic acid amide 682
aminoalkyl-pyrimidines, fungicides 5-aminopyrimidine 4-carboxylic acid ester
671ff, 687 297
aminobenzotriazole herbicides 22 aminoquinazoline
1-aminocyclopropane-1-carboxylate (ACC) – phenylacetic acid amide 682
– synthase 609 aminoquinazolinone derivative 1342
N-amino triazolinone 514 – structure–activity relationship 1340f.
– synthesis 514 4-aminoquinoline 678
6-amino-5-bromo-2-cyclopropylpyrimidine- aminosulfones 807ff.
4-carboxylic acid ethyl ester – experimental compounds 817
– synthesis 299 – synthesis of 817
N-amino-carbamoyltriazolinone aminosulfonylamino phenyl uracil derivative
– synthesis 515 187
6-amino-5-chloro-2-cyclopropylpyrimidine- amisulbrom 613ff.
4-carboxylic acid ethyl ester – synthesis 621
– synthesis 299 ammonium thiosulfate (ATS) 528
6-amino-5-chloro-4-ethylpyrimidine 683 anacardic acid 653
5-amino-3-cyano-pyrazole 1294 anilides, succinate dehydrogenase inhibitors
4-amino-1,2,4-triazin-5-one 511f. 628ff.
aminoalkylpyrimidine fungicides 672ff. anilinopyrimidines (AP) 543, 672, 706
– general SAR 672 – biological activity 709
1-aminobenzotriazole (ABT) herbicides 22 – physiochemical properties 707
γ -aminobutyric acid (GABA) receptor – chemistry 706
1240f., 1284ff. – degradation 713
– cloning the insect GABA receptor 1286 – mechanism of resistance 711
– insecticide target site 1285 – metabolism 713
– non competitive antagonist (NCA) 1285ff. – mode of action 711
1452 Subject Index
anilinopyrimidines (AP) (contd.) arylhydrazone, uncoupler of oxidative

– structure activity relationship 710 phosphorylation 652ff., 660
– synthesis 706 aryloxyacetate, auxin mimics 278
Annex I (positive list of active substances, aryloxyphenoxypropionate (APP, AOPP, fop)
Directive 16, 447ff., 1108
91/414/EEC) 487, 500 – commercial herbicide 456f.
Annex II 487ff. arylpyrazole insecticides 1283
Annex III 487ff. arylsulfonyl acid amide 684
annual bluegrass weevil, see Listronotus arylurea 480f.
maculicollis Ascomycetes 585, 709
Anthonomus pomorum (apple weevil) 1198 ASM, see acibenzolar-S-methyl
anthranilic diamide 1389 AtMLO2 (Arabidopsis thaliana mildew
– discovery 1410 resistance locus o2) 912
– insecticide 1409ff. ATP synthase
anthraquinone 1195 – mitochondrial 1059ff.
ATP-binding cassette (ABC) transporter
Anticarsia gemmatalis (velvetbean caterpillar)
513, 651
1419
atrazine 23, 253, 269, 377, 500
anticodon 694
Australian Code System 7
Aonidiella aurantii (California red scale)
Authorizations Directive 492
993ff.
– specific guidance regarding water limits
Aphelinus spp. 1420
according to annexes 492
aphicide
Autographa nigrisgna (beet semi-looper) 1402
– pymetrozine 1331
Aux/IAA protein 279f.
Aphidoletes aphidimyza 1344
auxin (IAA) 277, 400, 525
Aphis gossypii (cotton/melon aphid) 1119ff.,
– weed selectivity at the site of auxin action
1338, 1419
283
Apis mellifera (honey bee) 1230, 1339, 1354
auxin agonist 278
– risk assessment 494 Auxin F-Box (AFB) protein 280
Aplysia californica acetylcholine-binding auxin herbicide 277ff.
protein (A-AChBP) 1135ff., 1185 – commercial 278
apple scab, see Venturia inaequalis – downstream effect 283
apple weevil, see Anthonomus pomorum – field resistance 285
Aquatic Guidance Document 493 – mode of action 277
Arabidopsis thaliana – molecular mode of action 277
– AtCesA gene 340, 350 – receptor 277ff.
– mutants deficient in cellulose biosynthesis auxin mimics 277ff.
342f. auxin response factor (ARF) protein 279
Argentine ant (Linepithema humile) 1298f. Avena fatua 17
armyworm (Spodoptera spp.) 1035, 1359 Avena sterilis 17
arthropod avermectin 1305ff., 1374
– nontarget 494 – acaricidal activity 1315
Arthropod Pesticide Resistance Database – bioavailability 1319
(APRD) 938 – biochemical mode of action 1306
N-aryl lactam 359 – chemistry 1308
– biology 359 – discovery 1308
– chemistry 359 – insecticidal activity 1315
aryl-1,3-dione (DEN) 460 – safety 1319
– discovery 460 – structure–activity relationship 1317
– structure–activity relationship 465 avermectin aglycone derivative
– synthesis 461ff. – activity 1318
arylacetic acid amide fungicides 678 avermectin B1 1309ff., 1374
arylaminopyridine, uncoupler of oxidative avermectin derivative 1321
phosphorylation 663 aviglycine-HCl (AVG) 529
Subject Index 1453
azelaic acid 911 bencarbazone

– physico-chemical property 71 – synthesis 190
azolones 613f. – use 189
– synthesis 618 beneficial insects 950, 951, 1363
beneficial predatory mites 1347
b benclothiaz 1383
B group fungicides 549 – discovery 1383
Bacillus firmus 1368 – property 1383
Bacillus thuringiensis (Bt) – synthesis 1384
– insect resistance 1039 benodanil 641
Bacillus thuringiensis (Bt) crop benoxacor 372ff.
– insect resistance 1040 – physico-chemical property 377
Bacillus thuringiensis (Bt) insecticidal crystal – soil degradation data 377
(Cry) protein 1029ff. – synthesis 376
– transgenic crop 1029ff. – toxicological data 377
Bacillus thuringiensis (Bt) plant 1032 bentazone 481, 501
– resistance management 1040 – and safener 390
– safety 1043 – expiration of Annex I inclusion 510
bacteria – market share 530
– natural compounds used in agriculture benthiavalicarb 807, 808ff.
interfering in protein synthesis 693 – environmental profile 810
bactericides 547, 549, 554, 555 – physiochemical properties 809
Bactrocera dorsalis (oriental fruit fly) 1239, – synthesis 815
1299 benzamide fungicides 739
BAH, see bisacylhydrazine – binding site 740
bakanae disease (Fusarium moliniforme) benzamide herbicides 348
773 – chemistry 348
Bar gene – cellulose inhibiting herbicides 348
– plant breeding 433 benzamidoxime fungicide 887
barbiturates 1289 benzenedicarboxamide insecticides 1389
– binding site Gaba A receptor 1288 – structure–activity relationship 1399
barley leaf rust (Puccinia hordei) 792 benzfendizone 172ff.
barley powdery mildew (Blumeria graminis – synthesis 175
f.sp. hordei) 719, 792 benzimidazole (BZ) fungicides 539ff., 713,
barnyardgrass 728, 729
– herbicidal activity 130f. – cross resistance 739
baseline data 937 – mode of action 548
Basidiomycetes 585, 642 – resistance 544
Bc1-complex 562 benzimidazole PPO herbizides 178, 180
– inhibitor 568, 584, 586, 595 benzimidazole uncouplers 652
– mutation 601, 635 1,2-benzisothiazol-3(2H)-one-1,1-dioxide
– resistance 601, 612, 635 (BIT), metabolite from probenazole 914
– structure 567, 585 benzisothiazole nematizide 1383
– target activity 597 benzobicyclon 237ff., 256, 320
beet armyworm, see Spodoptera exigua – mode of action 237
beet semi-looper, see Autographa nigrisgna – properties 251
beflubutamid – synthesis 257
– physiochemical properties 205 benzofenap 251, 267f.
– use 218 – application rate 268
– synthesis 221f. – biologically active metabolites 268
Bemisia argentifolii (sweet potato whitefly) benzoheteroaryl N-aminouracil derivative
1419 187
Bemisia tabaci (tobacco whitefly) 992ff., benzoheteroaryl Protox herbicides 178
1004, 1067, 1114, 1333 benzoheterocycle Protox herbicides 180
1454 Subject Index
benzoheterocycle uracil 187 – of insect nAChRs 1131ff.

benzoisoxazolone Protox herbicides 178, 180 – voltage-gated sodium channels (VGSCs)
benzonitrile herbicides 1265ff.
– biological activity 346 – see also mode of action and target site
– cellulose biosynthesis inhibitors 346 bioaccumulation factor 497
– chemistry 345 bioavailability
benzosulfonamide Protox inhibitors 187 – in the environment 491
benzotriazinone Protox inhibitors 189 bioinsecticide 1127, 1238
benzoxazine isoindoline-1,3-dione, Protox biophore 589
inhibitors 189 biotin carboxy carrier protein (BCCP) 16
benzoylphenyl urea, chitin biosynthesis biotin carboxylase (BC) 16
inhibitors 1001, 1003 f. bisacylhydrazine (BAH) 957ff.
benzyl uracil, Protox-inhibiting herbicides – Coleoptera- and Lepidoptera-specific 965
182 – commercialized 961
benzyladenine (6-BA), fruiting and growth – discovery 961
525ff. – ecotoxicological and mammalian
benzyloxyphenyl substituted heterocycle, reduced-risk profile 975f.
Protox-inhibiting herbicides 176, 177 – insect control 957ff.
berry moth (Lobesia spp.) 1263 – Lepidoptera-specific 965
beta-cyfluthrin – mode of action of insecticide 966
– combination products 1180, 1195ff. – resistance mechanism 977
beta (β)-tubulin assembly 548 – resistance potential 977
bialaphos (bilanaphos) – selective insect toxicity 973
– glutamine synthase inhibitor 423ff. – spectrum of activity of commercial
– resistance (bar) gene 432 insecticide 974
bicyclo[4.1.0]heptane-2,4-dione , herbicides – structure–activity relationship 962ff.
243 – synthesis 961ff.
bicyclo[3.2.1]octane-2.4-dione, herbicides bispyribac-sodium
243, 256 – biology 128
bicyclopyrone, herbicides 228, 247 – discovery 128
bifenazate 1097, 1346ff. – herbicide in direct-seeded rice 128
– biochemistry 1349 – physiochemical properties 127
– biology 1349 bitertanol
– discovery 1347 – seed treatment combination 1195
– ecobiological properties against beneficial – uses 776
arthropods 1352 bixafen 631ff.
– ecobiology 1350 – uses 631, 634
– physico-chemical data 1351 black Sigatoka pathogen (Mycosphaerella
– registration status 1351 fijiensis) 742, 776
– resistance behaviour 1352 black vine weevil (Otiorhynchus sulcatus)
– structure–activity relationship 1347 1300
– synthesis 1350 blasticidin S
bifenox – uses 694f.
– structure 166 – resistence 696
binapacryl Blattella germanica (German cockroach)
– pro-pesticide 659 1003f.
– structure bleaching herbicide 197ff.
binding site – phytotoxic effect 197
– AHAS-inhibiting herbicide 36 – target 197
– fungicides (target site) 548–556 Blumeria graminis f.sp. hordei (barley powdery
– GABA receptors 1288 mildew) 719, 792
– herbicides 15, 17, 18, 20, Bollgard II cotton, cry 24 b 1037
– IRAC mode of action (MoA) classification Bombus impatiens 1420
number 941–950 Bombus terrestris 1230, 1344
Subject Index 1455
Bombyx mori 1339ff. Cacopsylla pyricola 1339

boscalid cadusafos
– biological activity 631 – structure 1372
– group C fungicide 550 cafenstrole
– physiochemical properties 629 – rice herbicide 320
– synthesis 634 – structure 320
Botrytis cinerea 549 calcium homeostasis
brassinosteroid 532 – insecticide 1389ff.
Bremia lactucae (downy mildew on lettuce) calcium-induced calcium release (CICR)
871 1391
bromethalin California red scale, see Aonidiella aurantii
– rodenticide 650 canola
4-bromo-3-(5 -carboxy-4 -chloro-2 - – herbicide-resistant 404
fluorophenyl)-1-methyl-5-trifluoromethyl – transgenic glufosinate-tolerant 434
pyrazole 184 captan
5-bromo-2-cyclopropyl-6-methylpyrimidine – mode of action 538, 541
-4-carboxylic acid methyl ester carbamate insecticides
– synthesis 298 – actives, summary 542
5-bromo-2-cyclopropylpyrimidine-4-carboxylic – groups 1A and 1B, IRAC MoA 941
acid methyl ester – market share 953
– synthesis 298 – nematicides 1370, 1371
bromobutide carbamoyl triazolinone 309, 514
– active metabolite 268 – synthesis 514
bromoxynil carbamoyl tetrazolinone 310
– detoxification 400 carbamoyl-heterocycle 316ff.
– mixture with pyrasulfotole 273 carbazate acaricide 1347f.
– herbicide resistance 400ff. carbazate compound
– mode of action 403 – meta-biphenyl-substituted 1350
bromuconazole – ortho-biphenyl-substituted 1349
– synthesis 784 – structure–activity relationship 1349f.
– use 783 – synthesis 1350
Bromus spp. 142 carbendazim 739ff.
brown plant hopper (Nilaparvata lugens) carbodiimide
1333 – diafenthiuron activation 1062
brown spot (Cochliobolus miyabeanus) – insecticidal 1065
773 carbofuran 1373
BT, see Bacillus thuringiensis carbomethoxy indazole 1258
Bt maize event 1035 carbonyl cyanide m-chlorophenylhydrazone
buffer zone 494 (CCCP) 651
bumble bee 1230, 1420 carbonyl cyanide phenylhydrazone (CCP)
buprofezin 661
– structure 1005 carbonylcyanide-p-trifluoromethoxyphenyl-
butafenacil hydrazone (FCCP) 661
– synthesis 174 carboxamide 351, 539, 1258
5-tert-butylcarbamoyloxy-3-(3-trifluoromethyl) 3-carboxamide indazole insecticide 1258
phenyl-1,3-thiazolidin-4-one 359 3-carboxamide pyrazoline 1258
carboxamide/succinate dehydrogenase
c inhibitor (SDHI) 542
C group fungicides 550 carboxin
C1 group herbicide 479ff. – resistance 571, 635
C2 group herbicide 479ff. – physicochemical data 628
C3 group herbicide 479ff. – structure 572
cabbage looper (Trichoplusia ni) 1404, 1419 carboxylic acid amide (CAA) 542, 553
cabbage webworm (Hellula undalis) 1402 – biological activity 824
1456 Subject Index
carboxylic acid amide (CAA) (contd.) – melanin synthesis 554, 839ff.

– chemistry 809 cell wall biosynthesis 553
– environmental profile and fate in soil 810 cellulose biosynthesis 339ff.
– fungicide 807ff. cellulose biosynthesis inhibitor (CBI) 339ff.
– group H fungicide 553 – from different chemical substance classes
– mammalian toxicology 809 344
– market 541, 542 – natural source 361
– mechanism of resistance 825 – nitrile-type 345
– mode of action 553, 825 – property of commercialized CBI 361
– occurrence of practical resistance 544 cellulose synthase 826
– physico-chemical data 809 – CesA protein 340f., 358
carboxylic amide central nervous system (CNS) 1127
– structure–activity relationships 634 Cercospora beticola (cercospora leaf spot)
– succinate dehydrogenase (SDH) 627, 628 742, 777
carboxyltransferase (CT) 16 cereals, global production 57f.
carboxyatractyloside 577 cereal eyespot pathogen
carfentrazone-ethyl – Oculimacula acuformis 770
– use 168 – Oculimacula yallundae 770, 782ff.
carmin spider mite (Tetranychus cinnarabinus) cereal safener, enhancement of herbicide
1067, 1318ff. degradation 61
carotene 198 cereal sulfonylurea herbicide 59
– α-carotene 198ff. CesA gene 340f., 826
– β-carotene 198ff. CGA 325, 615
– γ -carotene 200 – biology 357
– biosynthesis 200 – 1λ4 ,2,4,6-thiatriazine herbicide 357
ζ -carotene desaturase (ZDS) 199ff. channel blocker
– inhibition 201 – mechanism of blockade 1290
– inhibitor 202 Chemicals Regulation Directorate 498
carotenogenic pathway chemiosmotic hypothesis 560
– genetic engineering of herbicide resistance chemiosmotic theory 647
203 cheyletid mite (Chelacaropsis moorei) 1355
carotenoid biosynthesis 197 Chilo spp. 1421
– herbicides for inhibition 204f. Chilo suppressalis (rice stem borer) 960, 979,
– higher plant 199 1038f., 1195, 1402
Carposina niponensis (peach fruit moth) 1174 chiral switch 901ff.
carpropamid Chironomus tentans (Ct) 968ff.
– chemistry and stereochemistry 857 chitin 1000
– mixtures for seed treatment 1180, 1195, – biosynthesis 1000
1200 chitin synthase (CHS) 1000
– structure 846 chitin synthesis inhibitor (CSI) 1002ff., 1092
– structure 3-D in the active site 852 – crop protection 1005
– synthesis 858 chlorantraniliprole 1389f., 1409ff.
cartap – biological profile 1418
– combination mixtures 1171, 1180 – discovery 1411
– pro-insecticide 1127 – environmental safety 1420
S-(+)-carvone – insecticidal activity 1418, 1419
– sprout inhibition 529 – insecticide resistance management 1421
cat flea (Ctenocephalides felis) 1300 – mammalian safety 1420
catalytic subunit (CSU) of AHAS 32ff. – mode of action 1415
– crystal structure 44 – non-insect toxicological profile 1420
– structure 34 – physico-chemical properties 1413
cell division – synthesis 1413
– fungicide 739 chlorfenapyr 1071ff., 1354
cell wall – biological activity 1073
Subject Index 1457
– market 1073 – persistence of active substance 493

– pro-insecticide 657 Chrysanthemum coronarium 45
– resistance 1076 Chrysoperla carnea 1046
– synthesis 1073f. Chrysoperla spp. 1420
– vapour pressure 650 cinidon-ethyl
chlorfluazuron – structure 170
– biological activity 1003 – use 170
– structure 1003 cinnamic acid amide fungicides 808f.
chloridazon – cross resistance 817
– photosynthesis inhibition 209f. 9-cis-epoxycarotinoid dioxygenase 283
– registration status in the EC 502 citrus mealybug (Planococcus citri) 1174f.,
– structure 210 1234
chloride channel activator 1305ff. classification, Resistance Action Committee
chlorimuron ethyl – fungicide 539ff.
– application rates 53 – insecticide 935
– crystal structure 37ff., 40 clodinafop
– insensivity mutants 42 – cross resistance 454
chlormequat-chloride (CCC) clodinafop-propargyl
– structure 528 – combination products 379
2-(4-chloro-3-isopropoxycarbonyl)phenyl – safener for 374
isothiazole-1,1-dioxide 183 clofentezine
6-chloro-pyrid-3-ylmethyl (CPM) 1166 – biochemistry 1014
2-chloro-1,3-thiazol-5-ylmethyl (CTM) 1166 – biology 1014
chloroacetamide herbicides 310ff. – formulation 1017
– K group herbicides 305 – mixtures 1017
– mode of action 308 – physicochemical data 1014
– resistance 313 – relative mite activity 1013
chloronicotyinyl insecticide (CNI) 849, 1116, – synthesis 1014, 1021
1155, 1190ff. clopyralid 278
chloronitrile fungicides 542 – use 284
chlorophthalim cloquintocet-mexyl 374
– structure 166 – mixtures 380
chlorophyll biosynthetic pathway 164 – synthesis 380
chloropicrin, nematicidal fumigant 1369 – toxicological data 380
chloroplast psbA gene 15 cloransulam-methyl 102
chlorosulfonylisocyanate (CSI) route 72 – crop selectivity 103
chlorothalonil – ecotoxicology 104
– group M fungicide 541 – environmental degradation 104
chlorpropham (CIPC) – detoxification by homoglutathione 103
– ripening sprout suppression 527, 529 – toxicology 104
chlorsulfuron 25, 35ff., 50, 120 – use 102
– crystal structure 38, 39 clothianidin 1127, 1149ff., 1175ff., 1210
– herbicidal target 35 – application 1177
– resistant mutants to 41 – biological activity 1184
– structure 50 – chemical classification 1176
chlorthiamid – chemistry 1177
– synthesis 346 – efficacy on target pest 1177
choline phosphotransferase (CPT) 826 – physico-chemical property 1176
Choristoneura fumiferana (Cf) 968ff. – spectrum of activity 1180
chromafenozide – synthesis 1177ff.
– physicochemical properties 959 clubroot disease 874
– synthesis 962, 963 Cnaphalocrocis medinalis (rice leaf roller)
– use 975 1410
chronic risk Cnaphalocrocis spp. (leaf folder) 1263
1458 Subject Index
Coccinella spp. 1420 – expressing Cot102 1037

Cochliobolus miyabeanus (brown spot) 773 – expressing Cry1Ac 1033
cockroach 1003, 1142ff., 1232, 1299f., 1415 – expressing Cry1F 1037
codling moth, see Cydia pomonella – expressing Cry2Abgene 1037
Coleoptera 966, 1297 – glyphosate resistance (RR, RRF) 402, 403,
Colorado potato beetle, see Leptinotarsa 414
decemlineata – glufosinate resistance (LL, DGR) 402, 403,
common cutworm, see Spodoptera litura 418
common cabbage worm (Pieris rapae crucivora) – herbicide-resistant 400, 402
1402 – lepidopteran resistant (Bt cotton) 1033,
Complex I, respiratory chain 562ff., 671. 1035
1059 – Roundup Ready 415
– experimental MET Complex I compound cotton aphid, see Aphis gossypii
1092 cotton bollworm (Helicoverpa zea) 1308
– inhibitor 562ff., 674, 687, 1078 cotton caterpillar (Diaphania indica) 1402
Complex II, respiratory chain 562ff. cotton herbicides
– inhibitor 570ff., 1082, 1092 – Pyrithiobac-Sodium 125
– MET Complex II compound 1093 – sulfonyl urea 51
Complex III, respiratory chain 562ff., 1059 – trifloxysulfuron-sodium 78
– inhibitor 568, 584, 613, 1095 coumarins
Complex IV, respiratory chain 562ff. – seed germination inhibitors 360
– binuclear copper center (CuA ) 569 – uncoupler 360, 652ff.
– copper ion (CuB ) 569 coumethoxystrobin 595
Complex V, mitochondrial ATP synthase coumoxystrobin 595
574, 1059 country group
comstock mealybug (Pseudococcus comstocki) – IRAC 936
1402 coupling site 560
convulsant, GABA-gated chloride channel CP4 expression
blockers 1284 – effect on plant resistance 414
Conyza canadensis 24f. CP4-EPSPS (5-enolpyruvyl shikimate
Conyza spp. 24, 301 3-phosphate synthase) 401ff.
copper criteria
– Bordeaux mixture – cut-off criteria 497, 498
– multisite fungicide 542 – inclusion of Active Substances in Annex I of
Coptotermes formosanus (Formosan termite) Directive 91/414/EEC 490
1299 – health criteria (EHC) monographs 482
corn – new approval criteria (EC) No. 1107/2009
– amicarbazone 510 495
– coleopteran resistant 1035 crop
– expressing Cry1Ac 1046 – commercialized herbicide-resistant 401
– GM-resistant 400 – expressing Bacillus thuringiensis Cry Proteins
– glufosinate-tolerant (LL corn) 454 1029ff.
– herbicide-resistant 403 – glufosinate-selectivity by target-based
– imidazolinone-insensitive ALS allele approach 431
(CF corn) 404 – genetically modified herbicide-resistant
– lepidopteran resistant 1035, 1044 399ff.
– roundup ready (RR corn) 416 – glyphosate-resistant 411f.
– Sethoxydim-resistant (SR) 404 – herbicide-resistant 11
corn earworm (Helicoverpa zea) 1421 – imidazolinone-tolerant 94
corn head smut (Sphacelotheca reiliana) 781 – losses due to insects, disease and weeds
corn planthopper, see Perigrinus maidis 1029
corn root worm (Diabrotica balteata) 1180 – mode of selectivity of PC herbicide 133
cotton 80, 419 – transgenic 1035
®
– bromoxynil resistance (BXN ) 402, 403 – yield 609
Subject Index 1459
crop baiting system – synthesis 1413

– fipronil 1298 cyazofamid 613ff.
crop protection – synthesis 621f.
– abamectin 1323 cyclodiene inscticides
– chitin synthesis inhibitor 1005 – dieldrin 1285
– emamectin benzoate 1323 – α-endosulfane 1285
– milbemectin 1324 – mode of action 1306
crop protection agent 706 – lindane 1285
crop resistance – resistance 1284, 1285
– engineering 417f. cyclohexanedione (CHD, dim) herbicides
crop selectivity 16ff., 447ff., 1108
– AOPP herbicides 455 – commercial herbicide 458f.
– by altered herbicide distribution 24 – discovery 455
– cloransulam-methyl mechanism 103 – mode of action 450
– diclosulam mechanism 103 – structures 458
– expression of phosphinothricin acetyl – synthesis 461
transferase 432 cycloheximide
– genetic approaches 431 – mode of action 699
– glufosinate 431 – structure 699
– metabolic transformation 130 cyclopentenone safener 387
– of acetyl-CoA carboxylase inhibitors 447, cyclosulfamuron (AC 322,140) 70f.
468, 471 – acute toxicity, fish, bird, daphnia, rat 51, 52
– of auxin herbicides 284, 291 – physico-chemical properties 72
– of cellulose biosynthesis inhibitors 346 – structure 68
– of HPPD herbicides 228 – synthesis 71
– of Protox herbicides 163, 168 – use 71
– safeners 371 Cydia molesta
– target site based 25 – pome and stone fruit 1199
crop tolerance Cydia pomonella (codling moth) 1199, 1419ff.
– by enhanced metabolic detoxification 21 – larvae 1248
– detoxification by GSTs 313 cyenopyrafen
– natural inherent 18 – acute toxicity 1082
CropLife International 545 – physiochemical data 1082
cross resistance – pro-acaricide 1092
– negative 547f. – synthesis 1093
cross-resistance relationship cyflufenamid 887f.
– zoxamide, carbendazim, and diethofencarb – cross-resistance 889
742 – discovery 887
cry gene 1031ff., 1049 – ecotoxicological property 888
crystal BT protein (Cry and Cyt protein) – mode of action 889
1030 – physicochemical properties 888
– Cry 1031ff., 1048f. – synthesis 890
Ctenocephalides felis (cat flea) 1300 – toxicological properties 888
cucumber downy mildew (Pseudoperonospora – use 890
cubensis) 814, 833, 867ff. cyflumetofen 1093, 1346ff.
cutworm 1035 – biology 1353
cyanine dye 1092 – discovery 1353
cyantraniliprole 1409ff. – mode of action 1093
– biological profile 1421 – registration status 1354
– discovery 1413 – resistance behaviour 1354
– insecticidal activity 1419 – synthesis 1094, 1353
– mode of action 1415 cyhalothrin
– non-insect toxicological profile 1420 – mode of action 943
– physico-chemical property 1413 – insecticidal activity 1403
1460 Subject Index
cymoxanil 554, 746, 865f. 1-deoxy-d-xylulose-5-phosphate (DOXP)

– discovery 865 199
– mode of action 868 Dermatophagoides farinae 1355
– physico-chemical properties 866 desmethyl triazolinone 154
– synthesis 867 desoxynivalenol (DON) 784
– use 867 destosyl pyrazolate (DTP) 225f.
cyometrinil detoxification
– germination inhibition 381 – fungicide 543, 545
– structure 373 – herbicide 14, 21, 22, 23, 24, 392, 400
cyprodinil – insecticide 1015, 1022
– cross -resistance 711 Deuteromycetes 585
– physicochemical properties 707 DHN melanin 839
– structure 706 DHN melanin biosynthesis
– use 709 – biochemical reaction mechanism of
cyprosulfamide (CSA) 148, 383 scytalone dehydratase 851
– mode of action 394 – fungi 840
– synthesis 384 – fungicide 842
– structure 375 – inhibitor of polyketide synthase 842
– toxicological data 384 – inhibitor of scytalone dehydratase 846ff.
– use 393 – inhibitor of 1,3,6,8-tetrahydroxynaphthalene
Cyrmenins reductase 845
– from Myxobacteria 587 Diabrotica balteata (corn root worm) 1180
cyst nematode 1367ff. Diabrotica spp. 1177
cytochrome bc1 complex 562 diafenthiuron 1060
cytochrome c oxidase 562ff. – activation
cytochrome-P450 monooxygenase system 21 – biological activity 1066
cytokinins – carbodiimide 1061
– fruit reduction 528 – discovery 1063
– kinetin, benzyladenin, zeatin 525 – ecotoxicology 1066
– mode of action 526 – mammalian toxicology 1066
cytosine – mode of action 1059, 1061–1063
– natural alkaloid 1153 – photochemical conversion 1062
– structure–activity relationship 1063, 1064
– synthesis 1065, 1066
d – toxicity data 1066
2,4-D, see 2,4-dichlorophenoxyacetic acid – translaminar activity 1067
D group fungicides 549ff. – use 1067
D1 protein, PS II reaction center 15 diafenthiuron carbodiimide 1061
daminocide 2,6-diamino-4-chloro-1,3,5-triazine 480
– structure 529 2,6-diamino-4-methoxy-1,3,5-triazine 480
– use 527 2,6-diamino-4-methylmercapto-1,3,5-triazine
Danaus plexippus 1045 480
DCMU, see N-3,4-dichlorophenyl- diamond-back moth, see Plutella xylostella
N -dimethylurea Diaphania indica (cotton caterpillar) 1402
DDT (1,1-bis-(p-chlorophenyl)-2,2,2- diaryl pyrazolecarboxylate 351
trichlorethane) 1060 diaryl-tetrazine 1013
Delia antiqua (onion fly) 1298 diarylamines
Delia coarctata (wheat bulb fly) 1298 – brommethalin, fentrifanil, fluazinam, 657
deltamethrin – uncoupler 651, 653
– combinations 1195, 1200 – toxicity 662
– mode of action 943 1,2-dibromo-3-chloropropane (DBCP) 1369
demethylation inhibitor (DMI) fungicide dicamba
541ff., 764ff. – DGR cotton 419
– SBI Class I 767 – engineering crop resistance 417
Subject Index 1461
– genetically modified crops 417 – oxidative phosphorylation 564,

– mode of action 289 dicyclonon
– structure 278 – dichloroacetamide safener 389
dicamba mono-oxygenase (DMO) 417 dieldrin
dicarboximide fungicides – mode of action 1286
– market share 541 – resistance 1286–1289
– mode of action 717 – structure 1285
– monitoring program 546 dietary intake
– resistance 545, 717 – pesticides residues 490
– target site 551 diethofencarb
dichlobenil 344f. – cross resistance 742
– biological activity 346 – inhibitor of microtubule assembly 739ff.
– cellulose biosynthesis inhibitor 346, 356 – structure 741
– chemistry 345 N,N-diethyl-N-[2-(4-chloro-phenylthio)ethyl]
– mode of action 347 amine) (CPTA)
– physicochemical properties 362f. – inhibitor of lycopene cyclase (LCC) 202
– synthesis 346 N,N-diethyl-N-[2-(4-methylphenoxy)ethyl]
dichlormid 372ff. amine (MPTA)
– physico-chemical properties 377 – inhibitor of lycopene cyclase (LCC) 202
– synthesis 377 diflovidazin 1012ff.
– use 373 – biochemistry 1014
2,6-dichloro-phenylazide 347 – biology 1014
2,6-dichloro-4-(trifluoromethyl)phenyl group – ecobiological profile 1015
1294 – mite growth inhibitor 1012
dichloroacetamide safener 372, 389f. – physico-chemical data 1015
2,4-dichlorobenzyl thiocyanate 741 – synthesis 1014ff.
2,4-dichlorophenoxyacetic acid (2,4-D) diflubenzuron
– engineered crop resistance 400, 417 – chitin synthesis inhibitor 1002
– mode of action 277, 278ff, – use 1003
– plant growth regulator 525 diflufenican
N-3,4-dichlorophenyl-N -dimethylurea – discovery 206
(DCMU) 750 – mode ls of active site 212
1,2-dichloropropane/1,3-dichloropropene – physicochemical data 204
mixture (D-D) 1369 – synthesis 221f.
1,3-dichloropropene (1,3-D) – use 217
– fumigant 1369 diflumetorim 671, 673ff.
diclocymet – discovery 673
– synthesis 858 – mode of action 671
– structure 846 – SAR studies 674
diclomezine – synthesis 675
– physico-chemical property 875 – toxicity 673
– synthesis 876 – use 673, 675
– use 876 Difluoromethanesulfonanilide herbicides
diclosulam 137
– crop utility 102 – biological activity 138
– environmental degradation 104 Difunon 207
– mechanism of crop selectivity 103 – structure 209
– structure 101 Digitaria sanguinalis 17
– toxicology 104 4,5-dihydro-isoxazole substituent 245
– use 102 dihydrodioxazine toxophor 607
N,N-dicyclohexylcarbodiimide (DCCD) dihydropyrazole
– ATP synthase inhibitor 574, 1060, 1062 – binding site 1267
– inhibitors of membrane transport systems – blockade of VGSC 1266
24 – mode of action 1268
1462 Subject Index
dihydropyridine receptor (DHPR) 1391 – biochemical mode of action 1072

dihydropyrone, ZDS inhibitor 201 diphenyl ether (DPE) 165, 400
1,8-dihydroxynaphthalene (DHN) 839 – herbicide 165f.
DHN melanin 840 – structures 166
DHN melanin biosynthesis inhibitors 842, diphenyl-oxazoline
845, 846, 851 – structure–activity data 1023
Dimefluazole, Qi site inhibitor 615ff. Diptera 1297
dimethomorph dipterous leafminer (Agromyzidae) 1248
– discovery 809 Directive 91/414/EEC 488, 500, 938
– mode of action 825 – new PPP regulation to replace directive
– physico-chemical data 809 91/414 495
– structure 808 – status of registration of PS II inhibitors
– synthesis 811 503ff.
– toxicological data 808 Directive 96/12/EC 493
4,6-dimethoxy-2-methylsulfonylpyrimidine dithiocarbamate fungicides 539, 541, 542
(DMSP), sulfonylurea intermediate 120 – multisite mode of action 556
2,6-dimethoxybenzamide substituent 349 dithiopyr 439ff.
dimethoxypyrimidinyl salicylic acid 122 – biology 439
– post-emergence herbicidal and ALS – environmental fate 441
inhibitory activities 122 – mode of action 442
dimethyl disulfide, fumigant 1369 – synthesis 443f.
dimethylallyl pyrophosphate (DMAPP), – toxicology 442
carotene biosynthesis 199 diuron
N-(N,N-dimethylaminosulfonyl)azoles, – PS II inhibitor 227
fungicides 615. – registration EU 480, 500, 506
– Complex III inhibitors at the Qi site 615 DKN (diketonitrile) 226ff., 270f.
dimoxystrobin – crystal enzyme complex 232
– discovery 591 – hydrolysis of isoxazol herbicides 270
– metabolic stability 605 dodine 554
– physicochemical data 598 doramectin 1310, 1312
– sales 586 – antiparasitic drug 1312
– synthesis 616 dormin 526
dinitroaniline herbicides 439 Dossier, registration 495
dinitrocresol (DNOC) 649, 654 Dossier content, information on
dinitrophenol fungicides 655, 659ff. – composition of active substance 496
– pro-pesticide 659 – low-risk 498
– uncouplers of oxidative phosphorylation – physical and chemical properties 489
653 f. downy mildew (Plasmopara viticola) 814
dinocap downy mildew on hop (Pseudoperonospora
– toxicity 649 humuli) 871
– use 648, 654, 659 downy mildew on lettuce, see Bremia lactucae
dinotefuran 1127, 1147, 1181f. drazoxolon
– application rate 1183 – structure 661
– biological activity 1184 – use 660
– chemical classification 1182 Drechslera tritici-repentis (tan spot) 791
– chemistry 1182 Drosophila melanogaster (Dm) 968ff., 1133ff.
– efficacy on target pest 1183 DTP (destosyl pyrazolate)
– insecticidal activity 1183 – active metabolite 225f.
– physico-chemical properties 1182
– receptor binding 1147 e
– spectrum of activity 1184 E group fungicides 549ff.
– synthesis 1183 E3 Ub-protein ligase 279
dione structure–activity relationship 466 EBOB 1286
dioxapyrrolomycin 1071f. EC market
Subject Index 1463
– situation of PS II inhibitors 499 5-enolpyruvyl shikimate-3-phosphate synthase

ecdysone (20-hydroxy), initiation of molting (EPSPS) 20, 406
958, 973 – inhibitor 406
ecdysone agonist insecticide 959, 979 environment
– chemistry 980 – concentration of the chemical in the relevant
ecdysone receptor (EcR) protein 958ff. compartment 491
– LBD (ligand binding domain) 969f. – fate and behavior 497
ecdysteroid 967 – fate and behavior of agricultural pesticide
– CoMFA and 4-D-QSAR analyses 964 491
– structure–activity relationship 962ff. Eotetranychus carpini f. vitis (yellow grape mite)
Echinochloa colona 24 1022
ecotoxicology, EC registration episystemicity 603
– Directive 96/12/EC 497 epopromycin
– guidance document on terrestrial 494 – inhibitor of cellulose biosynthesis derived
– requirements 492 from Streptomyces species 365
ectoparasiticide 1190, 1312 epoxiconazole (epoxyconazole) 531, 779f.
efficacy – cis stereoisomer 782
– dossier 484, 495 – physic-chemical data 780
– studies (GEP) 488 – synthesis 780, 781
electron transport at photosystem II inhibitor eprinomectin
346 – use 1312
ergosterol 761
electron transport chain
– biosynthesis 765
– alternative 572
ergosterol biosynthesis inhibitor (EBI)
– mitochondrial 562
fungicide 761
electron transport inhibitor 561
Eriophyd mite 1116
electrophysiology 1151
Erwinia amylovora 548
Eleusine indica 453
Erysiphe graminis 679, 719
Eleusine indica (L.) Gaertn. 440
Erysiphe necator 796
Elsinoe ampelina 789
ethaboxam
emamectin
– mode of action 742
– activity against mites and insects 1319,
ethephon
1321
– ethylene releaser 525
– chloride channel activator 1305 – plant growth regulating activity 527
– synthesis 1311 ethiprole
emamectin benzoate – chemical property 1293
– ecotoxicological data 1319, 1322 – discovery 1283
– environment 1322 – physico-chemical properties 1293
– physico-chemical properties 1308 – use 1283
– synthesis 1306, 1309, 1311 ethirimol
– toxicological data 1319, 1322 – cross resistance 719
– use in crop protection 1322, 1323 ethoprophos
Empoasca fabae (potato leafhopper) 1419 – use as nematicide 1374
endo-glucanase 344 ethoxysulfuron 382
α-endosulfan 1285 – ecobiological data 51
endothalic acid 528 – physico-chemical properties 69
energy conservation proteins 573 – products 70
enestroburin – toxicological data 52
– physico-chemical data 607 – use 51, 68, 69
– structure 594 ethylene 525ff.
enol ether 607 ethylene dibromide (EDB)
enol ether stilbene 588 – fumigant 1369
5-enolpyruvyl shikimate-3-phosphate etoxazole
(EPSP) 20 – biochemistry 1023
1464 Subject Index
etoxazole (contd.) – structure 984

– biology 1023, 1025 farnesyl pyrophosphate 199, 200
– mode of action 1025 FAO see The Food and Agriculture
– physic-chemical data 1025 Organisation
– resistance 1026 fatty acid
– synthesis 1022, 1024 – elongation 307
– use 1022, 1025 fatty acid and lipid biosynthesis inhibitor
Europe 326
– new regulations 494 fatty acid synthase (FAS) inhibitor 842
European and Mediterranean Plant Protection Fe(II)-containing dioxygenase 230
Organisation (EPPO) 493 feeding blocker
– risk-assessment schemes 493 – selective 1327ff.
European corn borer, see Ostrinia nubilalis fenamidone
European Food Safety Authority (EFSA) – binding site 614
European red mite (Panonychus ulmi) – mode of action 567, 614, 1095
1349ff. – resistance 612
European Union (EU) – physico-chemical data 613
– main changes in guidelines regarding the – synthesis 619
re-registration 488 S-fenamidone 614
– PS II inhibitors withdrawn from fenamiphos
re-registration 507ff. – combinations 1195
– registration, candidate for substitution – use as nematicide 1374
498 fenazaquin 671, 688, 1060, 1086, 1101
– re-registration process 482 – discovery 1086
– status of registration of PS II inhibitors – metabolism 1099
503ff. – mode of action 1060, 1086, 1092
– supranational harmonization activities – physico-chemical data 1080
483 – resistance 1101
Eutetranychus spp. 1349 – synthesis 1086
evaluation 495 fenbuconazole
– synthesis 779
f – use 777
F group fungicides 550ff. fenchlorazole-ethyl
F-box protein component (TIR1) 279 – metabolism 391
F-box protein jasmonate receptor 281 – structure and use 374
F1 F0 ATP synthase fenclorim
– inhibitors 574ff. – structure 374
– structure and function 574f. – use 374, 391
F1 F0 -ATPase 1059 fenhexamid
FAD see flavin adenine dinucleotide – biochemical target 798, 799
fall armyworm, see Spodoptera frugiperda – biology 798
famoxadone – discovery 796
– binding site 614 – physico-chemical data 797
– discovery 614 – resistance 801
– mode of action 567, 614, 1095 – synthesis 798
– resistance 612 fenoxanil
– physico-chemical data 613 – biology 845
– synthesis 619 – inhibitor of scytalone dehydratase 846, 856
farnesal – structure 846
– activity 983 fenoxaprop
– structure 984 – resistance 17,273, 393
farnesol fenoxaprop-ethyl
– activity 983 – safener action 385
Subject Index 1465
fenoxaprop-P-ethyl – seed treatment 1298

– detoxification 386, 391 – soil application 1297
– reaction with glutathione (GSH) 392 – turf and ornamental application 1300
fenoxasulfone – urban pest control application 1299
– biological activity 330 fire blight 548, 700ff.
– discovery 326 flavin adenine dinucleotide (FAD) 29ff.
– mode of action 334 flavoprotein UQ oxidoreductase 573
– use 326 flonicamid
fenoxycarb – aphicidal spectrum 1339
– discovery 984 – biological activity 1338
– mode of action 944, 984, 985 – biological mode of action 1335
– use 985 – chemical property 1335ff.
fenpiclonil – cross-resistance 1338
– discovery 723 – discovery 1335
– mode of action 718 – mode of action 1335
– physico-chemical data 726 – physico-chemical properties 1338
– synthesis 723, 724 – synthesis 1335ff.
– use 723, 728 – target site 1337
fenpyroximate – toxicological data (safety profile) 1339
– discovery 1079 – use recommendation 1338
– metabolism 1097 florasulam
– mode of action 1084 – ecotoxicology 105
– physico-chemical data 1080 – environmental degradation 105
– resistance 1101 – herbicidal activity 104
– synthesis 1084 – mechanism of crop selectivity 104
fentrazamide – metabolism 104, 113
– chemistry of analogs 321 – target activity 115
– physico-chemical property 322 – toxicology 105
– synthesis 321 – use 103
– toxicological data 322 fluacrypyrim 611, 1081, 1096, 1103, 1354
fentrifanil – physico-chemical properties 1081
– mode of action 653 – synthesis 1096
– structure 657 – use 1103
ferimzone fluazinam
– discovery 660 – agrophoric substituents 640
– mode of action 653, 661 – detoxification 650, 662
– plant defense responses 661 – discovery 662
– synthesis 661 – ecotoxicology 663
– use 656, 660 – mode of action 653, 662
fiprole insecticide – resistance 650, 663
– chemistry 1293 – synthesis 664
– structure–activity relationship 1294 – toxicology 649, 650, 662
– synthesis 1293 – use 662, 663
fipronil fluazolate 168, 169, 184, 185
– animal health use 1300 – mode of action 185
– biological properties 1297 – structure 169
– biological spectrum 1297 fluazuron 640, 641, 945
– chemical properties 1293 – mode of action 945
– crop baiting system 1298 – structure 640
– human health use 1300 flubendiamide 1389, 1391, 1393, 1397, 1398,
– insect selectivity 1293 1401, 1402, 1406,1407
– mode of action 1284 – binding site 1393
– physico-chemical properties 1293 – biological profile 1401
– resistance 1300 – chemistry 1398
1466 Subject Index
flubendiamide (contd.) flufenerim

– cross-resistance 1406 – activity 1090
– discovery 1397 – mode of action 1081, 1091, 1092
– insecticidal activity 1401, 1402, 1403 – synthesis 1090, 1091
– mode of action 1391 flufenoxuron
– persistence 1402 – discovery 1004
– selectivity 1393 – turnover 1004
– toxicity towards beneficial arthropods flufenpyr-ethyl
1406 – synthesis 176
– toxicological profile 1407 flumetsulam
flucarbazone – discovery 100
– discovery 151 – target activity 114, 115
– physico-chemical properties 144 – use 101
– synthesis 148, 149, 150, 151, 153 flumiclorac-pentyl
– use 142, 151, 157 – chemical structure 170
flucarbazone-sodium see also flucarbazone flumioxazin
– biology 151 – biological activity 188
– herbicidal characteristics 157 – structural analogs 179
– physico-chemical properties 144 flumorph
flucetosulfuron 51, 52, 68, 72, 73 – discovery 807, 810
– ecotoxigological data 51 – chemical structure 811
– physico-chemical properties 73 – physiochemical data 809
– toxicological data 52 – resistance 826
– use 51, 68, 72, 73 – toxicological data 809
fludioxonil fluopicolide
– biological activity (spectrum) 727f. – biological activity 831, 833
– discovery 721,723 – combinations 833
– mode of action 715, 730 – ecotoxicology 833
– physico-chemical data 726 – effect on spectrin-like protein distribution
– resistance 717 834
– synthesis 723, 725 – mammalian toxicity data 832
– use 723, 728, 729f. – mode of action 831, 835, 837
fluensulfone – mode of action shift 641, 642
– biological activity 1382 – physico-chemical properties 831, 832
– chemical property 1381 – spectrum of activity 833
– discovery 1377 – toxicology 832
– ecotoxicity 1383 fluopyram 639–645
– mode of action 1381 – biological activity 641, 644
– nematicidal spectrum 1382 – discovery 639
– physico-chemical properties 1381 – mode of action 643
– safety profile 1383 – physico-chemical properties 639
– synthesis 1380 – seed treatment 644
– use 1382 – synthesis 644
flufenacet 2-fluoro-4-chloro-5-alkoxy phenyl
– analog 318 imidazolididine triketone 186
– discovery 318 2-fluoro-4-chloro-5-isoxazolinylmethoxy
– K3 class herbicide 312 tetrahydroindazole 186
– mixtures with 269, 318 2-fluoro-4-chloro-5-substituted phenyl pyrrole
– mode of action 311 183
– physico-chemical property 319 fluoroalkenyl nematicides
– resistance 313 – fluensulfone type 1380
– synthesis 318 – general structures 1378
– toxicological data 319 – structure–activity relationship 1379,
– use 317 1380
Subject Index 1467
fluorochloridone 217 – use 219

fluoxastrobin 586, 590, 592, 593, 599, 603, flusilazole 772, 776
605, 607, 618, 791, 878 – biological advantage 776
– discovery 592 flusulfamide 865, 873, 874
– formulation 603 – discovery 873
– metabolism 607 – physico-chemical property 873
– mode of action 592 – soil treatment 874
– physiochemical data 599, 605 – synthesis 874
– structure 590 – use 874
– seed treatment 593 flutenzine
– synthesis 618 – chemical family 1013
flupoxam 344, 351, 352 fluthiacet-methyl
– biology 352 – mode of action 171
– discovery 351 flutianil
– mode of action 344 , 352 – morphological change of powdery mildew
– synthesis 351, 352 infection 896
flupyrsulfuron – synthesis 897
– biological advantage 52 flutolanil
flupyrsulfuron-methyl-sodium – mode of action 572
– combinations 58 – resistance 571
– ecotoxigological data 51 – physiochemical data 628
– physico-chemical properties 60 – structure 628
– toxicological data 52 fluxofenim
fluquinconazole – mode of action 390
– discovery 788 – safener 379, 389
– seed treatment 788 – seed treatment 373, 376, 379
– synthesis 788, 789 – synthesis 379
flurazole 389, 390 foliar absorption 407ff.
– combinations 390 foliar fungicide 787
– safener 389 folpet 541, 542, 556, 813, 814
fluridone 201, 202, 204, 210, 211, 217, 218, – combinations 814
221, 222, 227 – fungicide group IRAC 541, 542
– discovery 217 – multisite mode of action 556
– mode of action 201, 202, 227 fomesafen 163, 165, 190
– physico-chemical properties 204 – mode of action 163
– synthesis 221, 222 – structure 166
– toxicological data 204 – toxicology 190
– use 218 Food and Agriculture Organization (FAO)
flurochloridone 482
– mode of action 201, 202, foramsulfuron
– physico-chemical properties 204 – ecotoxigological data 51
– synthesis 221, 222 – physico-chemical property 77
– toxicological data 204 – product 78
– use 219 – toxicological data 52
fluroxypyr Formosan termite (Coptotermes formosanus)
– combinations 154, 290 1299
– mode of action 278 forosamine 1241ff.
– structure 278 – metabolism and penetration of the
flurtamone spinosyns 1252
– discovery 218 fosetyl-Al (fosetyl-aluminum) 554, 814, 869,
– chemistry 209 870, 871
– mode of action 202 – application 871
– physico-chemical properties 205 – physico-chemical property 870
– synthesis 221, 222 – synthesis 870
1468 Subject Index
fosthiazate 1369, 1372 – classification 539ff.

– nematicide 1369 – demethylation inhibitor (DMI) 541ff.,
– structure 1372 764ff.
FRAC (Fungicide Resistance Action – DHN melanin biosynthesis 842
Committee) 545 – ergosterol biosynthesis inhibitor (EBI) 761
– code list 540 – FRAC mode of action classification 539ff.
– grouping of SBI fungicide 767 – fungal respiration 548, 549, 550
– mode of action classification of fungicides – grouping of SBI fungicide 767
539ff. – history of use 539
Frankliniella occidentalis (Western flower – 1,4-hydroxyanilide 797
thrips) 1363 – importance of individual modes of action
fruit development 531 540
fruit growth 523 – lipid synthesis 552
fruit morphology 526 – market 542
fruit quality 527 – MBI-D 858
fruit ripening 525 – melanin biosynthesis inhibitor (MBI) 841
fruit storage 528 – melanin synthesis in cell wall 554
fruit thinning 527 – membrane synthesis 552
fruiting 528 – metabolic detoxification 543
fthalide 842, 845 – mitosis 739
– efficacy 845 – mode of action 547
– mode of action 842 – mode of action classification 539ff.
– structure 845 – multisite 539, 556
fufenozide – nucleic acid synthesis 548
– biological activity 978 – oxidative phosphorylation 559ff.
– synthesis 978 – phenylurea 748
fumigant 1369ff. – powdery mildew 887ff.
fungal respiration inhibitors 548, 549, 550 – resistance 543
fungi – resistance risk 539ff.
– Ascomycetes 585, 709, 841 – signal transduction 551, 715
– function of melanin and occurence 839 – sterol biosynthesis 552
– Basidiomycetes 585, 642, 841 – strobilurin 587ff.
– biosynthesis of DHN melanin 840 – thiophanate 739
– enzymes involved in sterol biosynthesis – triazole 771
766 – unknown mode of action 865ff.
– natural compounds interfering in protein – use of mixtures/alternations 547
synthesis 693 fumigants 1369ff.
– Oomycetes 541ff., 585, 614, 761, 807, 822, furametpyr
841 – metabolism 637
– phytopathogenic 585, 841 – structure 628
fungicides furilazole
– allylamines 767, 800 – soil degradation data 378
– amino acid and protein synthesis – structur 373
551, 693 – synthesis 378
– anti-Oomycetes fungicide 831ff. – toxicological data 378
– antitubulin 739 Fusarium culmorum 727ff.
– application dose rate 547 Fusarium graminearum 727ff.
– application frequency 547 Fusarium moliniforme (bakanae disease) 773
– application timing 547
– benzamidoxime 887 g
– beta (β)-tubulin assembly 549 G group fungicides 552
– cell division 739 γ -aminobutyric acid receptors 1132
– cell wall biosynthesis 553 – avermectins 1306
– class 547 – cloning 1286
Subject Index 1469
– Rdl receptors 1286 gibberellinic acid (GA) 525f.

– resistance mutations 1286 – GA3 525f.
– spinosyns 1144, 1240, 1241 – GA4 526
– milbemycins 1306 – GA7 526
GABAA 1288 GIFAP (the International Group of National
– barbiturates 1289 Associations of Manufacturers of
– benzodiazepines 1269 Agrochemical Products) 545
– ‘‘Cys-loop’’ superfamily of ligand-gated glasshouse whitefly (Trialeurodes
ionchannels (LGICs) 1132 vaporariorum) 990ff., 1004, 1333
– Drosophila melanogaster (fruit fly) 1132 Global Crop Protection Federation (GCPF)
– Caenorhabditis elegans (nematode) 1132 545
– subunits 1288 β-glucan 762
GABAC 1288 glucan synthase 826
– ‘‘Cys-loop’’ superfamily of ligand-gated glucose metabolism 872
ionchannels (LGICs) 1132 glufosinate
– insensitive to bicucculine 1288 – crop selectivity 431
– subunits 1288 – engineering crop resistance 418
GABA antagonist 1285 – genetic approach to generate
– convulsants picritoxin 1285 glufosinate-selectivity in crops by target-
– selectivity 1293 based approach 431
– structures of antagonists 1285 – modulation of herbicidal activity by
– structure of the binding site 1292 environmental condition 430
GABA receptor, see γ -aminobutyric acid – racemic (dl-homoalanin-4-yl
receptor (methyl)phosphonic acid) 428
GABA-gated cation channel 1289 d-glufosinate 435
– convulsants 1284 glufosinate resistance 401ff.
GABA-gated chloride channel blocker glufosinate-ammonium 427ff.
1284 – uptake and translocation 430
– fipronil 1283 glufosinate-resistant crop 418
– polychlorocycloalkane insecticides 1285 – LL plants 401, 402, 404, 405
Gaeumannomyces graminis 541, 787 glufosinate-tolerant canola
gene expression for detoxifying pathways – transgenic 434
387 glutamate receptor 1132
gene expression profiling experiment glutamate synthase (glutamine-2
387, 911, 920 oxoglutarate-aminotransferase, GOGAT)
– isotianil 920 424
– safener 385 glutamate-gated chloride channel (GluCl)
– systemic acquired resistance (SAR) 911 1132, 1289
gene induction 387 – avermectins 1306
gene silencing 1030 – desensitizing (GluClD) 1290
gene transformation – fipronil 1283
– Agrobacterium-mediated transformation – milbemycins 1306
1029 glutamic acid decarboxylase (GAD) 1286
– B. thuringiensis cry genes 1032ff. glutamine synthetase (GS) 424ff.
– particle bombardment 1030 – physiological effects of GS inhibition in
genetic engineering plant 429
– herbicide resistance 203 – plant nitrogen metabolism 424
genetically modified herbicide-resistant crop glutamine synthetase inhibitor 423ff.
399ff. – mode of action 428
genome glutarimide antibiotics 699
– Arabidopsis 280 glutathione (GSH) 386
geranylpyrophosphate (GPP) 199 glutathione-S-transferase (GST) 21, 386ff.
gibberellin 525f. – corn selectivity 313
– plant groth processes 526 – herbicide resistance 453
1470 Subject Index
glutathione-S-transferase (GST) (contd.) halofenozide 959–961, 966, 975, 976

– transgenic tobacco 313 – binding affinities 967
glycine-gated chloride channel 1290 – discovery 960
glyceric acid amide 821 – mammalian and ecotoxicological
glcyosyltansferases 341 reduced-risk profile 976
glycosyltransferase genes 1026 – mode of action 966, 972
glyoxylic acid derivative 822f. – resistance 977
– synthesis 823 – synthesis 961
glyphosate 20 – use 975
– applied acreage 12 halosulfuron-methyl 378
– factors that impact efficacy 407 haloxyfop 450–452
– herbicide resistance 5, 12, 21 – crystal structure of the CT
– translocation in plants 24 domain–haloxyfop complex 452
glyphosate acetyltransferase (GAT) 412 – mode of action 450
glyphosate oxidoreductase (GOX) 412 – structure 451
glyphosate resistance 402f. haloxyfop-P-methyl 641
– mechanism for engineering 411 Harmonia axyridis 1339ff.
glyphosate-resistant crop Hemiptera 1297, 1410
– development 411 Helicoverpa armigera 1031ff., 1043ff.
– disease control benefit 412 Helicoverpa spp. 1359
– RR corn 409 Helicoverpa zea 1036ff., 1308, 1421
– RR cotton 410 Heliothis virescens (tobacco budworm) 1032ff.
– RR plants 401, 402, 404, 405 – ecdysone receptor (HvEcR) ligand-binding
good laboratory practice (GLP) 488 domain (LBD) 964
good plant protection practice 546 Hellula undalis (cabbage webworm) 1402
GMO crops 394, 399 2-n-heptyl-4-hydroxyquinoline N-oxide
grape anthracnose 789 (HQNO) 567
grass control 153, herbicides 5ff.
– second-generation sulfonylureas 52 – ACCase (acetyl-CoA Carboxylase) 447
Grapholita molesta (oriental fruit moth) – ACCase-inhibiting 453
1174f., 1248, 1419 – AHAS as target 35f., 93
green-peach aphid (Myzus persicae) 1118ff., – AHAS-inhibiting 43
1332ff., 1419 – amicarbazone for corn and sugarcane 510
green-peach aphid efficacy – aryloxyphenoxypropionate 456f.
– sulfoximine analog 1229 – auxin 277ff.
greenhouse whitefly (Trialeurodes – bispyribac-sodium 128
vaporariorum) 990ff., 1004, 1333 – bleaching property 197ff.
griseofulvin 739 – C1 group (HRAC classification) 479ff., 500
– inhibitor of microtubule assembly 739 – C2 group (HRAC classification) 479, 480,
groundwater 481
– fate and behavior 498 – C3 group (HRAC classification) 479, 480,
– ground water limits according to annexes 481, 500
492 – carotenoid biosynthesis inhibition 204f.
growth 528 – cereal sulfonylurea 59
– inhibitor 525 – cotton 125
Grundwasserrichtlinie 500 – cyclohexanedione 458f.
GST gene sequences 386 – detoxification 392, 400
guanidines 555 – diphenyl ether (DPE) 165f.
– disturbing the synthesis of very long-chain
h fatty acid 305ff.
H group fungicides 553 – first-generation sulfonylurea 50
haloalkene 1377 – HRAC classification 7, 305
– discovery 1377 – 4-hydroxyphenylpyruvate dioxygenase
– nematicidal activity 1377 inhibitors (HPPD) 225
Subject Index 1471
– imidazolinone (IMI) 88ff. – Kochia scoparia 15, 19, 20, 25

– K3 class 305ff. – Lactuca serriola 19, 20
– maize sulfonylurea 76 – Lolium multiflorum 16, 21, 22,
– N class 305ff., 326 – Lolium rigidum 16, 17, 19, 21, 22, 23, 25
– photosynthesis inhibitors (PS II Compound – mechanism for engineering 400
Groups) 500 – mechanism of resistance 13, 14, 15, 18, 19,
– phytoene desaturase (PDS) 203ff. 23, 24, 25, 26
– protoporphyrinogen oxidase – multiple resistance 25
(PPO)-inhibiting 1283 – paraquar-resistant biotype 24
– pyrimidinyl carboxylate (PC) 118ff., 133 – Phalaris 23
– resistance 5, 7, 13, 203, 400 – Poa annua 15, 18,
– rice 129ff. – translocation, reduced or shifted 14, 24, 25
– second-generation SACT 154 – Salsola iberica 19
– sulfonanilide 140 – Scirpus juncoides 19
– thiocarbamate 326 – Setaria virides 17, 18
– triazolopyrimidine 115 – Sorghum halepense 18
– triketone 240ff. – Stellaria media 19
– urea 752 – target site 7, 14, 16–18, 21, 25
– use of phosphinothricin-containing – triazine 5, 6, 8, 14, 15, 16, 22, 23, 25
herbicides in agriculture and horticulture Herbicide Resistance Action Committee
431 (HRAC) 938
herbicide antidote, see safener – classification 7, 305
herbicide distribution herbicide safener, see safener
– altered 24 herbicide target
herbicide metabolism – expression of an insensitive herbicide target
– effect of safener 385 401
– phase I 385 herbicide translocation
– phase II 386 – influence of safener 388
– phase III 386 herbicide uptake
– phase IV 386 – influence of safener 388
herbicide resistance 7 herbicide-resistant canola 404
– Abutilon 23 herbicide-resistant corn 403
– Alopecurus myosuroides 13, 17, 25 herbicide-resistant cotton 402
– Amaranthus palmeri 21 herbicide-resistant crop 11
– Amaranthus powelli 15, 20 – commercialized 401
– Amaranthus retroflexus 20 – genetically modified 399ff.
– Amaranthus rudis 20, 25 herbicide-resistant cropping system 12
– atrazine resistant mutant 14, 15, 16, 22, 23 herbicide-resistant soybean 401
– Avena fatua 17 hetarylurea 480
– Avena sterilis 17 heteroarylmethylamine 684
– biochemistry 13 heterocycle
– biotypes 5, 6, 14, 16, 17, 19, 20, 21, 22, 23, – benzoheterocyclic attached 178
24, 25 – benzyl attached 181
– Chinopodium album 14, 15 – benzyloxyphenyl 176
– Conyza 24, 25 – fluoralkenyle 1379
– cross-pollinating species 25, 26 – hydroxyphenylpyruvate dioxygenase (HPPD)
– detoxification 14, 21–24 inhibitors 262ff.
– Echinochloa 12, 24 – NADH inhibitors 677, 683
– Eleusine 21 – phenoxyphenyl 176
– enhanced metabolism 19, 23, 25 – 3-(4,6-substituted benzoheterocyclyl)
– Festuca rubra 18 179
– genetic engineering 203 – PDS inhibitors 212–214, 216
– glyphosate 12 – pyridine azomethins 1330
– HRAC group classification 8 – trifluormethylnicotinamides 1336
1472 Subject Index
heterocycle (contd.) hydrogen cyanamide 528

– triketones 242, 246, 248 hydrogen tetraoxosulfate 528
– uncouplers 657, hydroprene 984
3-[(N-heterocyclyl) iminomethyl] pyridine – (S)-hydroprene 985
1328 – use 985
Heterodera glycines (soybean cyst nematode) 3-hydroxyacyl-CoA transferase 306
1383 N-hydroxy-anilinopyrimidine
Heterodera schachtii (beet cyst eelworm, – synthesis 708
sugarbeet nematode) 1367 hydroxyanilide 766, 796
Heterotermes tenuis (termite) 1297 – FRAC Group 767
hexaflumuron 1,4-hydroxyanilide fungicide 797
– discovery 1004 4-hydroxyphenylpyruvate dioxygenase (HPPD)
– structure 1003 198, 230f., 417
hexythiazox 1012, 1015, 1018, 1019, 1020, – herbicide target 225
1021, 1022, 1025 – inhibition 232
– biochemistry 1019 – mechanism 229
– biology 1019 – structure 229
– cross resistance 1015 4-hydroxyphenylpyruvate dioxygenase
– discovery 1018 inhibitor 227ff., 262ff.
– field application 1022 – herbicide 226
– physico-chemical data 1020 – Markush structure 262
– resistance 1025 – selectivity 228
– synthesis 1019, 1021 hyperecdysonism 972
high-osmolarity glycerol (HOG) pathway hyperosmolarity response 717
715
Hill reaction 480 i
histamine-gated chloride channel 1290 IGR, see insect growth regulator
histidine kinase 717 imazamethabenz-methyl, use in cereals
– Sln1p 716 96f.
homogentisic acid (HGA) 226 imazamox, use in soybeans 96f.
Homona magnanima (oriental tea tortrix) imazapic 89ff., 388
1402 – use in peanuts 96f.
Homoptera 1297 – use in sugarcane 96f.
honey bee (Apis mellifera) 1230, 1339, 1354 imazapyr 19, 89ff., 404
– risk assessment 494 – use in noncrop 96f.
Hordeum leporinum 24 imazaquin, use in soybeans 96f.
horticulture 431 imazethapyr 89ff., 404
– resistance to AHAS-inhibiting herbicide – use in maize 96f.
43 – use in soybeans 96f.
– use of phosphinothricin-containing imibenconazole 789
herbicides 431 – mode of action 789
host defense inducer 909ff. – physicochemical data 789
– FRAC classification (P code) – synthesis 790
host plant defense induction 550 imidacloprid 878, 1116, 1127, 1154f., 1189ff.
house dust mite 1355 – application rate 1193
housefly (Musca domestica) 986f., 1183, 1296 – biological activity 1184
HRAC (Herbicide Resistance Action – chemical classification 1190f.
Committee) 938 – chemistry 1192
– classification 7, 305 – efficacy on target pest 1193
human health – insecticidal efficacy 1194f.
– impact on 496 – metabolism 1191, 1235
hydramethylnon 1097 – physico-chemical property 1190f.
– synthesis 1098 – synthesis 1190ff.
hydrazone 661 imidazoles 769f., 1328
Subject Index 1473
imidazolinone (IMI) 19, 35, 404 – gibberellin biosynthesis 764

– commercial use 96 – glutamine synthetase 423ff.
– herbicide 88ff. – 4-hydroxyphenylpyruvate dioxygenase
– history 90 227ff., 262ff.
– ionization state 91 – lipid synthesis 1108ff.
– mechanism of selectivity 95 – lycopene cyclase (LCC) 202
– metabolism 96 – melanin biosynthesis (MBI) 839
– mode of action 93 – MET-I 1097ff.
– physico-chemical property 91 – MET-II 1100
– structural feature of herbicide 92 – MET-III 1100
– synthesis 89 – methionine biosynthesis 706
imidazolinone resistance 403 – microtubulin assembly (MAI) 439ff.
imidazolinone-tolerant crop 94 – mitochondrial electron transport (METI)
immunofluorescence 1000 1078ff.
immunohistochemistry 1000 – mono-oxygenase 1253
indaziflam 344ff. – NADH 671
– physico-chemical properties 363 – nucleic acid synthesis 901ff.
indazoles 1258ff. – oxidative phosphorylation 1059, 1070
indoles 1295 – peptidyl nucleoside antibiotic 1002
indolizinedione 460 – phosphorylation 561
indoxacarb 1257ff. – photosynthesis 479ff.
– action on mammalian VGSC 1269 – photosystem II (PS II) 14f., 227f.
– blockade of VGSC 1266 – phytoene desaturase 197ff.
– discovery 1260 – polyketide synthase 842
– insecticidal activity 1263 – protoporphyrinogen IX oxidase (Protox,
– mode of action 1265 PPO) 163ff., 185, 1108
– physico-chemical property 1263 – Qi inhibitor class 567
– proinsecticide action 1265 – Qo inhibitor class 567
– resistance 1270 – scytalone dehydratase 846ff.
– synthesis 1262 – squalene epoxidase 800f.
inhibition/inhibitors – sterol biosynthesis (SBI) 539, 719, 761ff.
– acetyl-CoA Carboxylase (ACCase) 13ff., – succinate dehydrogenase 628ff., 640
379, 447ff., 1108ff. – 1,3,6,8-tetrahydroxynaphthalene reductase
– acetylcholine esterase 941f., 951 845
– acetohydroxyacid synthase (AHAS/ALS) inositol hexakisphosphate (IP6) 280
29 ff. inositol synthesis 553
– auxin transport 9 insect-behavior regulator 1340
– carotinoid biosynthesis 8, 197ff. insect growth regulator (IGR) 983
– chitin synthesis 1002ff., 1092 insect molting 957ff.
– Complex I 562ff., 674, 687, 1078 – physiological and molecular basis of
– Complex II 570ff., 1092 hormone action 957
– Complex III 568, 584, 613, 1095 insect nAChR 1138, 1185, 1232ff.
– demethylation inhibitor (DMI) fungicide – selectivity for insect versus vertebrate
541ff., 764ff. nAChRs 1145
– DHP synthase 8, 10 insect resistance management (IRM) 1041ff.
– electron transport 346, 561 insect–resistance to Bt 1039
– 5-enolpyruvyl shikimate-3-phosphate insect-resistant crops 953
synthase (EPSPS) 406 insect selectivity
– ergosterol biosynthesis inhibitor (EBI) – recombinant nAChR 1147
fungicide 761 insect toxicity
– F1 F0 ATP synthase 574 – selective 973
– fatty acid and lipid biosynthesis 326 insect voltage-gated sodium channel 1265
– fatty acid synthase (FAS) 842 insecticidal crystal protein
– GABA 1284ff. – Bacillus thuringiensis 1030
1474 Subject Index
insecticides integrated pest management (IPM) 986,

– abamectin 1316 1044, 1067, 1118ff., 1239, 1327, 1358
– anthranilic diamide 1409 – diafenthiuron 1067
– bisacylhydrazine (BAH) 966ff. – spirodiclofen 1116
– calcium homeostasis 1389ff. – spiromesifen 1116
– carbodiimide 1065 integrated weed management (IWM) 7
– 3-carboxamide indazole 1258 iodosulfuron-methyl-sodium 60, 381
– 3-carboxamide pyrazoline 1258 – physico-chemical property 62
– chloronicotyinyl (CNI) 849, 1116, 1155, ionophore 648
1190 ipconazole 785
– classification 935 – physicochemical data 787
– ecdysone agonist 959, 979 – synthesis 786
– emamectin 1319 – stereoisomer 788
– fiprole 1283ff. ipfencarbazon 322
– inhibitor of mitochondrial electron – synthesis 323
transport 1078ff. iprovalicarb 807ff.
– major chemical classes 953 – environmental profile 810
– market share 953 – physicochemical data 809
– MET 1102 – resistance 825ff.
– milbemectin 1318 – synthesis 812
– mitochondrial ATP synthase as a target – toxicology 809
1059 IRAC, see Insecticide Resistance Action
Committee
– neonicotinoid 1116, 1127ff., 1156, 1165
iron sulfur centers, complex I 564
– nereistoxin analog 1127f.
iron sulfur centers, complex II 566
– new generation 1398
isoenzymes, safener-inducible 391
– oxadiazine 1260
isomerases 552, 766f., 793ff.
– polychlorocycloalkane (PCCA) 1285
isopentenyldiphosphate (IPP) 199
– pyridazine 1259
isoprenoid pathway 199
– semicarbazone 1273
isoquinolinedione 189
– semicarbazone sodium channel-blocking
isotianil 909ff.
1259
– application 921f.
– site of metabolism for MET 1099
– disease control spectrum 920
– sodium channel-blocking 1257ff. – gene expression profiling experiment
– spinosyn 1127ff. 920ff.
– thiourea 1064 – synthesis 919
– trifluoromethylnicotinamide 1335 – systemic acquired resistance 919
Insecticide Resistance Action Committee isoxaben 344ff.
(IRAC) 935ff. – biology 349
– activity 937 – physicochemical data 363
– education and communication 938 – synthesis 348
– functional team 936 isoxaben-resistance loci
– mode of action classification 942ff. – ixr1 and ixr2 350
– objectives 935 isoxadifen-ethyl 76f., 148, 382, 393
– organization 936 – synthesis 383
– project team 936 – toxicological data 383
– resistance management 1301 isoxaflutole (IFT) 156, 228ff., 268ff., 318
– structure 936 – synthesis 270
insecticide resistance management (IRM) isoxazoline herbicides 326
935, 1406, 1421 Ixodes scapularis 1300
– chlorantraniliprole 1421
– effective strategy and approved principle j
952 Japanese beetle (Popillia japonica) 975, 1300
intake calculation models 490 jasmonate 532
Subject Index 1475
jasmonic acid (JA) 531, 910 Lepidoptera 966, 1092, 1297, 1319, 1363,
juvenile hormone (IH) 957 1401, 1410
– mimics 944, 957 lepidopterous larvae 1247
juvenoids 983f. Lepidosaphes ulmi 1116
– commercialized 986 Lepimectin 1305ff.
– history of research 984 – discovery 1313
– synthetic 985 – synthesis 1314
– target insect 986 Leptinotarsa decemlineata (Colorado potato
beetle) 1004, 1276
k leptospermone 237ff.
K3 class herbicide 305ff. – discovery 236
– resistance 313 Leucine biosynthesis 29ff.
kasugamycin 696f. Leucinodes spp. (shoot stem borer) 1421
– mode of action 698 ligand-gated chloride channel
– classification 1288
– resistance 698
– structure 1288
– toxicity 697
ligand-gated chloride channel antagonist
KCS gene 312
1283ff.
3-keto reductase 552, 765f., 798f.
– structure–activity relationship 1294
3-ketoacyl-CoA reductase 306
ligand-gated ion channel (LGIC) 1132
3-ketoacyl-CoA synthase 306
Linepithema humile (Argentine ant) 1298f.
ketoacyl-ACP reductase 1248
lipid biosynthesis 450, 550ff, 1115
ketoacyl-ACP synthase 1248
– in aphids 1122
ketospiradox 245f.
– inhibition of 947, 1108ff., 1122
– structure 246
Liriomyza spp. 1363
kinase cascade 717
Lissorhoptrus spp. 1298
kinetin 525
Lissoropterus oryzophilus 1198
kinoprene 984
Listronotus maculicollis (annual bluegrass
Kitasatospora phosalacinea 427
weevil) 1418
Kochia scoparia 19ff., 45
Lithocolletis blancardella (leafminer) 1199
korrigan (kor) gene 344, 358
Lobesia spp. (berry moth) 1263
Krebs cycle 570
Locusta migratoria 1331
kresoxim-methyl 545ff., 587ff., 607
Lolium multiflorum 16ff.
– metabolic stability 598
Lolium perenne 19, 440
– physicochemical data 598
Lolium rigidum 16ff., 45
– synthesis 616
Long-chain fatty acids, uncouplers of oxidative
phosphorylation 647
l looper (Trichoplusia ni) 1404, 1419
lactofen 165, 166 loose smut on barley (Ustilago nuda) 687, 879
– structure 166 lycopene 200
Lactuca serriola, herbicide resistance 19f. lycopene cyclase (LCC) 401
Lanosterol synthase 765, 795 – inhibition 201, 202
Late blight 824 Lygus spp. 1339
leaf blast 845, 919ff.
leaf folder (Cnaphalocrocis spp.) 1263 m
leaf spot disease 672ff., 776, 791, 919 M group fungicides 541, 556
leaf stripe on barley (Pyrenophora graminea) macrocyclic lactones 1238, 1315
710, 730, 878f. macrolide 1249
leaf surface distribution–vapor phase 603 Magnaporthe grisea (rice blast) 773
leafminer (Lithocolletis blancardella) 1199 maize
leafroller (Pandemis spp.) 1263 – corn rootworm-resistant 953
lepidine 687 – expressing cry proteins 1034f.
– complex i inhibitors – foramsulfuron 78
– class 687 – imazethapyr 95
1476 Subject Index
maize (contd.) melon thrips (Thrips palmi) 992ff., 1174ff.,

– sulfonylurea 54f., 76 1363f.
– transgenic 434 membrane fluidity, change in 757
– production area 57 membrane permeability 574, 646f., 661,
maize weevil (Sitophilus zeamais) 1402 1030
4-maleylacetoacetate isomerase 226 membrane potential 646ff.
malonyl-CoA 16, 19, 448ff., 1108 membrane protein 1001
mandelic acid amide 807f., 818f. membrane synthesis 552
mandipropamid 807, 818ff. membrane transport systems, inhibitors of
– environmental profile 810 24
– physicochemical data of 809 mesosulfuron-methyl 62, 81, 375ff.
– synthesis 821f. – metabolic pathway in the soil 82
– toxicology 809 – physicochemical properties 64
– translaminar activity 825 mesotrione 226ff., 240ff., 253
Manduca sexta 1001f., 1032 – environmental properties 252
MAP protein kinase (MAPKKKs) 551, 715ff., – in planta metabolism 253
913ff. – physicochemical properties 252
Market – synthesis 254
– bentazone 530 messenger RNA (mRNA) 693
– benzoylphenyl urea’s 1006 MET, see mitochondrial electron transport
– carboxylic acid amides (CAA) 541f. metabolic degradation rate 607
– chlorfemapyr 1073 metabolic detoxification 21, 539
– dicarboximide fungicides 541 metabolic profiling 610
– dimoxystrobin 586 metabolic resistance 45, 949
– EC market, situation of PS II inhibitors metabolism
499 – amicarbazone 516
– future trends 953f. – anilinopyrimidines 713
– insecticides 953 – bisacylhydrazines 973f.
– major insecticidal classes 953 – N-dealkylation reactions 22
– pencycuron 749 – fatty acid 1108
– PS II inhibitors 510 – fenazaquin 1099
– SBI fungicides 763ff. – fenochlorazole-ethyl 391
– strobilurins 585f. – florasulam 104, 113
MBI see melanin biosynthesis inhibitors – fluoxastrobin 607
MBI-D 842ff. – furatmetpyr 637
mefenacet 310ff. – glucose 872
– analogs 318 – imidazolinones 96
– physico-chemical property 319 – MET-I inhibitor 1097
– synthesis 318 – neonicotinoids 1146, 1196f., 1234
– toxicological data 319 – plant nitrogen metabolism 424
– use in rice 316f. – selectivities 70
mefenpyr-diethyl 61, 148, 273, 375ff., 392 – spinosyns 1252f.
– synthesis 381 – strobilurins 607
– toxicological data 382 – succinate dehydrogenase inhibitor 636
melanin – sulfonylureas 69ff.
– biological occurrence and function in fungi metaflumizone 1273ff.
839 – cross-resistance potential 1281
– biosynthesis enzymes 839ff. – insecticidal activity 1276
– synthesis in cell wall 554, 839ff. – mode of action 1280
melanin biosynthesis inhibitor (MBI) 553f., metalaxyl-M 901
839ff. – absolute configuration 902
Meloidogyne incognita 1375ff. – biological activity 904
Meloidogyne spp. 1377 – cross-resistance 905
melon aphid, see Aphis gossypii – degradation 906
Subject Index 1477
– mechanism of resistance 904 milbemectin 1305ff.

– metabolism 906 – bioavailability 1319
– mode of action 904 – ecotoxicological data 1319
– physical properties 903 – insecticidal activity 1316
– synthesis 902 – physicochemical properties 1308
metamorphosis 957ff. – synthesis of 1306
metconazole 784 – toxicological data 1319
– physicochemical data 785 – use in crop protection 1324
– synthesis 786 milbemycin 1305ff.
methionine biosynthesis 711 – acaricidal activity 1315
– fungicide target 551 – bioavailability 1319
methionine biosynthesis inhibitor 706 – biochemical mode of action 1306
methionine sulfoximine (MSO), GS inhibitor – chemistry 1312
β-methoxyacrylate (MOA) 587, 1096 – insecticidal activity 1315
methoxyacrylate stilbene 588 – safety 1319
methoxyfenozide 961ff., 975 milbemycin A3 /A4 1313
– ecotoxicology 976 milbemycin D 1313
– physicochemical data 959 milbemycin 5-oxime 1313ff.
– synthesis 962 mildewicide 688, 887ff.
– toxicology 976 mildiomycin 698
3-methoxytyramine 819 mite growth inhibitors 1012ff.
methyl-benzimidazole carbamates 549 mitochondria 561ff., 1059ff., 1072
methyl bromide (MBr) 1369 mitochondrial ATP synthase 1059ff.
methyl salicylate (MeSA) 911 mitochondrial ATP export 577
methyl salicylate esterase (SABP2) 916 mitochondrial electron transport (MET) 1078
2-methyl-4-chlorophenoxyacetic acid (MCPA) – Complex I 550, 563f., 946, 1060, 1078ff.
273 – Complex II 550, 579ff.
methylchlorophenoxypropionic acid (MCPP) – Complex III 550, 564–568, 584ff., 613ff.,
218 946f., 1062, 1095ff.
1-methylcyclopropene (1-MCP) 529 – Complex IV 569f., 947
O-methyltransferase 1250f. – MET-I inhibitor 1079, 1092, 1101f.
methyltransferase, in phospholipids – MET-II acaricide 1102
biosynthesis 552 mitochondrial membrane 560ff., 1067, 1072
3-O-methyldopamine 820 – Qi site 566
METI acaricides 946 – Qo site 566
metolachlor, physicochemical properties 377 mitogen-activated protein kinases (MAPK3,
S-metolachlor 253, 375 MAPK6) 913
metominostrobin 587, 602ff. mitosis 443, 739ff.
– metabolic stability 598 mixed function oxidase (MFO) 1072
– physicochemical data 598 MOA-stilbene (MOAS) 588
– synthesis 616 mode of action
metrafenone 891 – abamectin 1305
– cross-resistance 892 – aminocyclopyrachlor 302
– ecotoxicological property 891 – anilinopyrimidines 711
– mode of action 892 – anthranilic diamides 1391f., 1415f.
– physicochemical properties 891 – auxin herbicides 277
– synthesis 892 – avermectins 1306
mevalonic acid (MVA) 199 – benzimidazole fungicides 548
microtubule-associated protein (MAP) – benzobicyclon 237
442 – bifenazate 1352f.
microtubulin assembly inhibitor (MAI) – bisacylhydrazines 957, 962, 966
439ff. – blasticidin 694
midgut 1029ff. – bromoxynil 403
1478 Subject Index
mode of action (contd.) – fluopyram 643

– carboxylic acid amide fungicides 533, – fluoxastrobin 592
825ff. – flupoxam 344, 352
– chlorantraniliprole 1415 – fluridone 201f., 227
– chloracetamide herbicides 308 – fluchloridone 201f.
– classification scheme 939ff. – fluroxypyr 278
– clofentezine 1012 – flurtamone 202
– cry proteins 1035f. – fluthiacet-methyl 171
– cyantraniliprole 1415 – flutalonil 572
– cyclodiene insecticides 1306 – fluxofenim 390
– cyclohexandione (CHD, dim) herbicides – fomesafen 163
16ff., 447ff., 1108 – fosetyl-aluminium 869ff.
– cycloheximide 699 – fungicides 550ff.
– cyflufenamid 889 – fthalide 842
– cyflumetofen 1093 – halofenozide 966, 972
– cyfluthrin 943 – haloxyfop 450
– cyhalotrin 943 – herbicide resistance 5ff.
– cymoxanil 868 – imibenconazole 789
– cyprosulfamide (CSA) 394 – imidazolinones 93
– cytokinins 526 – indoxycarb 1265
– deltamethrin 943 – IRAC classification 942ff.
– diafenthiuron 1059, 1061ff. – kasugamycin 696f.
– dicamba 289 – metalaxyl-M 904ff.
– dicarboximide fungicides 715ff. – metrafenone 892
– diclobenil 347 – milbemycins 1306f.
– 2,4-dichlorophenoxyacetic acid (2,4-D) – mildiomycin 699
277ff. – neonicotinoids 1139ff.
– dieldrin 1286 – pencycuron 756f.
– diflumetorim 671 – phenylpyrroles 715, 718, 722
– dihydropyralzole 1268 – proquinazid 895
– dimethomorph 825 – pymetrozine 1331ff.
– dithiopyr 442 – pyridalyl 1364ff.
– ethaboxam 742 – pyiridines 442
– etoxazole 1025 – pyrimidinylcarboxylates 132
– famoxadone 567, 614, 1095 – quinoxyfen 719f.
– fenamidone 567, 614, 1095 – spinosad 1240ff.
– fenazaquin 1060, 1086, 1101 – spirotetramat 1122ff.
– fenoxasulfone 334 – streptomycin 701f.
– fenoxycarb 944, 984f. – strobilurins 585
– fenpiconil 718 – tetronic acid derivatives 1115, 1118
– fenproximate 1084 – triketones 237
– fentrifanil 653 – Vip proteins 1030
– ferimzone 653, 661 mole cricket (Scapteriscus spp.) 1300
– fipronil 1284ff. molecular modeling
– flonicamid 1335 – colchicine binding site 741
– fluazinam 653, 662 – melanin biosynthesis inhibitors 850
– fluazolate 185 – protoporphyrinogen-IX-oxidase inhibitors
– fluazuron 945 183
– flubendiamide 1391 molting process 958, 972f.
– fludioxinil 715, 730 Monilinia spp. 645, 709, 774, 788, 799
– fluensulfone 1381 molting process 958, 972f.
– flufenacet 1311 Monochoria vaginalis 19
– flufenerim 1081, 1091f. Monomorium pharaonis (pharaoh ant) 1299
– fluopicolide 831, 835ff. Monoterpenes 526
Subject Index 1479
morlin – biochemical mode of action 1168

– coumarin derivative 36 – chemical structural feature 1165
morpholines 552, 767, 793f., – commercialized 1165ff.
MRL regulation 494, – control vectors 1180, 1193
mRNA (messenger RNA) 693 – five-membered ring systems 1189
MSU Resistance Database 939 – IRAC classification 1181
mucidin 587 – metabolism 1146
– extract from Oudemansiella mucida 587 – mode of action in insect 1142
Multi-site inhibitors 539, 556, 944 – open-chain-type 1169, 1185
multiple resistance 14ff., 871 – resistance 1217
Musca domestica (housefly) 986f., 1183, 1296 – ring systems 1185
Mycospaerella graminicola 612 – SAR studies 1141
Mycosphaerella fijiensis (Black Sigatoka – selectivity 1145
pathogen) 742, 776 – six-membered ring systems 1203ff.
mycotoxin 784 nereistoxin analogues 945, 1127f., 1142,
myxobacteria 561, 587 1236
Myzus persicae 1118ff., 1332ff., 1419 nervous system, transmission of electrical
impulses 1265
n neuron preparation
N class herbicide 305ff., 326 – native 1149
– resistance 313 – whole-cell voltage–clamp 1149
Nabis spp. 1420 neurotransmitters
NADH 561ff. – acetylcholine 766
NADH binding site, complex I 563 – ionotropic 1053
NADH cytochrome c reductase in lipid Nicotiana plumbaginifolia 33
peroxidation 550 nicotinamide adenine dinucleotide (NAD+ )
NADH inhibitors 671ff. 559
NADH–UQ oxidoreductase 561f., 573 (S)-nicotine 1136
naphthalene acetic acid (NAA) 525 nicotinic acetylcholine receptor (nAChR)
1,8-naphthalic anhydride (NA) 389f. 1183, 1232ff.
natural compounds – 3D models 1132ff.
– interfering in protein synthesis of fungi and – ACh binding site 1139ff.
bacteria 693 – activation of 1143
– plant growth regulator 525, 526 – agonistic action 1135, 1183
– use of phosphinothricin-containing – agonist-binding site 1135
herbicides 431 – agonists/antagonists 943, 945, 1143
nematicides 1367ff. – binding domains 1132f.
– carbamate 1371ff. – existence of diverse insect receptor subtypes
– chemical class 1368 1137
– fluoroalkenyl 1379 – human gene family 1133
– fumigant 1369 – insect selectivity in recombinant nAChRs
– in development 1376 1147
– organophosphate 1371ff. – non-competitive blocker 1135
– product 1368 – point mutation 1149
nematodes 951 – receptor binding domains 1132f.
– cyst 1367ff. – selectivity 1146f.
– root-knot 1377 – structure 1132f.
nematode management practice 1368 nicotinic pharmacophore 1140
neomenthol 530 nicotinoids 1131ff.
neonicotinoid insecticide 1116, 1127ff., Nilaparvata lugens (brown plant hopper)
1140ff., 1166 1004, 1333
– agonistic efficacy 1149 nitenpyram 1127, 1149ff., 1169
– binding assay 1143 – application 1171
– binding model 1136ff. – chemistry 1171
1480 Subject Index
nitenpyram (contd.) one-shot application 845, 849

– efficacy on target pest 1171 onion fly (Delia antiqua) 1298
– physico-chemical properties 1170 Oomycete pathogen 739
– special soil treatment 1170 Oomycetes 541ff., 585, 614, 761, 807, 822
– synthesis 1171 – anti-Oomycetes fungicide 831ff.
nitisinone, HPD inhibitor 226 operator exposure data requirement 490
nitrate reductase 609 organic arsine fungicides 749
nitrile 8f., 11, 345, 481, 503ff. organoarsenical 9
nitroenamines 1204 organochlorines 942, 953
nitroguanidine 1204 organomercurials 544
N-nitroguanidine 1166f., 1175, 1181 organophosphate (OP) 950f., 1175, 1231,
nitroimino-heterocycle 1370ff.
– hydrolytic stability 1210 organotin miticides 945
– six-membered 1205f. oriental fruit fly (Bactrocera dorsalis) 1239,
2-nitroimino-hexahydro-1,3,5-triazine 1299
– synthesis 1209 oriental fruit moth, see Grapholita molesta
4-nitroimino-1,3,5-oxadiazinane 1212 oriental tea tortrix (Homona magnanima)
– synthesis 1209 1402
2-nitroimino-1,3,5-triazinane 1208 Orius sauteri 1344
nitromethylene 1146, 1166ff. Orius spp. 1420
2-nitromethylene piperidine 1203 Orius strigicollis 1344
2-nitromethylene pyrrolidine 1204 Orthoptera 1297
2-nitromethylene-1,3-diazacycloalkane 1204 orthosulfamuron 74
3-nitropropionic acid 571 – physico-chemical properties 74
no-till practices 12f., 400 orysastrobin 593ff., 607
Noctuidae 1297 – physico-chemical properties 598
non-competitive antagonist (NCA), GABA – synthesis 617
receptors 1285 osmolarity, growth defects 716
non-competitive blocker (NCB), nAChR 940 Ostrinia nubilalis (European corn borer)
non-crop uses, imazapyr 96ff. 1034ff., 1420
non-extractable residue (NER) 81 Otiorhynchus sulcatus (black vine weevil)
non-heme Fe(II) containing oxygenase 230 1300
non-sphingolipid VLCFA biosynthesis Oudemansiella mucida 587
– in plant 306 ovicidal activity 990f., 1179, 1200
non-target-site resistance 14ff., 940 ovicidal mite activity 1013, 1022, 1174
norbornadiene (2,5-NBD) 529 oxadiazines 1260
norflurazon 202ff., 217f. – soil half life 1260
– physcochemical properties 205 – structure-activity relationship 1260
– synthesis 221f. – synthesis 1261
NPR1 gene (non-expressor of PR genes) 912 oxasulfuron, physico-chemical properties 79
nucleic acid synthesis oxazine-3,5-diones, herbicides 466
– fungicide 548 oxazole-γ -pyrone structure 687
– inhibitor 901ff. oxazolidinedione 9, 173
oxazolines, insecticides 1020ff.
o oxidase
octopaminergic agonists 946, 1062 – alternative (AOX) 573, 609
Oculimacula acuformis (cereal eyespot – mixed function (MFO) 1072
pathogen) 770 oxidative desulfurization 1061
Oculimacula yallundae (cereal eyespot oxidative metabolism 1028, 1146, 1252
pathogen) 770, 782ff. – strobilurins 585ff.
Odontotermes takensis (termite) 1297 oxidative phosphorylation 559ff., 1059
OECD databasea on pesticides 486 – disruption of the proton gradient 1070ff.
old world bollworm, see Helicoverpa armigera – disruption of ATP formation 945
Oligonychus spp. 1349 – inhibitors 1059, 1070
Subject Index 1481
– uncouplers 646f. – ecotoxicology 110

oxidative stress 573, 609 – environmental degradation 110
2,3-oxidosqualene 765 – mechanism of crop selectivity 109
oxime ethers 379 – toxicology 110
oximino amides 607 pentachlorobenzyl alcohol (PCBA) 842
oximino esters 607 peptidyl nucleoside antibiotic inhibitor 1002
oxpoconazole, synthesis 775 Perigrinus maidis (corn planthopper) 1417
oxyacetamide 9f., 310ff Periplaneta americana (American cockroach)
– physicochemical properties 319 1232, 1415
oxygenases 230f. Periplaneta americana L. 1142ff.
oxylipin pathway 387 Peronosporales 807
– plant internal signaling substances 387 peroxidase 609
– phytoprostane 387 persistent, bioaccumulative, and toxic
substance (PBT) 497
p persistent organic pollutant (POP) 497
P group fungicides 554 pesticide, fate and behavior of agricultural
P 450 monooxygenases 977 pesticides in the environment 491
Paecilomyces lilacinus 1368 Pesticide Safety Directorate 498f.
PAL gene 913 pesticides evaluation scheme of the WHO
Pandemis spp. (leafroller) 1263 (WHOPES) 482
Panonychus spp. 1349 pesticides residues, estimation of dietary
Panonychus citri (citrus red mite) 1349 intake 490
Panonychus ulmi (European red mite) 1349ff. PGR, see plant growth regulator
paralysis 951f. Phakopsora pachyrhizi (soybean rust) 413,
pat gene, plant breeding 433 585
pathogenesis-related (PR) gene 911 Phalaris minor 23
PBZ-inducible (PBZ1) gene 914 pharaoh ant (Monomorium pharaonis) 1299
PCR (polymerase chain reaction) phenols 651ff., 659ff.
– primer-introduced restriction analysis phenoxan 686, 687
(PIRA) 859 – from Polyganium sp. 687
PDS inhibitors, structural elements 216 phenoxybenzamides, phytoen desaturase
peach fruit moth, see Carposina niponensis inhibitors 203
Pectinophora gossypiella (pink bollworm) phenoxynicotinamide 206
1031ff. phenoxyphenyl, heterocycle 176
pefurazoate 773f. 4-phenoxyphenyl juvenoids 986
– physicochemical data 773 phenoxypyridine ether, herbicides 207ff.
– synthesis 774 phenoxypyridinecarbonamides, herbicides
pelargonic acid 528 206
pencycuron 549, 748ff., 1195 phenylacetic acid amide
– biology 756 – aminopyrimidine 682
– chemistry 750 – aminoquinazoline 682
– development 749 phenylalanine 406
– ecotoxicology 758 Phenylamide Fungicide Resistance Action
– discovery 750 Committee (PA-FRAC) 905
– metabolism 759 phenylamides, fungicides 543, 827, 831, 905
– mode of action 755 phenylamide resistance 905
– physico-chemical property 756 phenylcarbamate 480f.
– sensitivity to several Anastomosis groups N-phenylcarbamate (NPC) 739
(AGs) of Rhizoctonia solani 757f. phenylfuranone 207
– structure–activity relationship 753 phenylindazoles 1258
– synthesis 755f. phenylpyrazoles 943, 1283ff.
– toxicology 758 phenylpyrazoline 16
penoxsulam phenylpyridazines 481
– crop utility 109 phenylpyridazinone 209f.
1482 Subject Index
phenylpyridine 172 – status of registration under EU Directive

phenylpyridinone 210 91/414/EEC 503ff.
phenylpyrroles 544, 551, 715ff – withdrawn from re-registration in EU
– mode of action 715ff. 507ff.
– resistance 551, 718, 728 Phthalamate 9
– seed treatment 728 phthalamic acid 555
– synthesis 722 phthalic diamide 1389
phenylpyrrolidinone 210 phthalide 1180
phenylsulfonic acid 686 phthalimide 542
phenyltetrahydrophthalimide 168 physicochemical properties
phenyltetrahydropyrimidinone 211 – abamectin 1308
phenylthiadiazole[3,4a] pyridazine system – acequinocyl 1082
170f. – acetamiprid 1173
phenyltriazolopyridazine 171 – ametoctradin 613
1-phenyl-1-pyrimidinyl-hydrazine – amicarbazone 511
– synthesis 708 – aminocyclopyrachlor 301
3-phenyl-6-trifluoromethyluracil 182 – anilinopyrimidines 707
phenylurea fungicides 748 – azimsulfuron 71
phenylurea herbicides 15, 22 – azolones 61
phloem mobile insecticide 1123 – azoxystrobin 598
Phorodon humuli 1122 – beflubutamid 205
– benoxacor 377
phosphatidylcholine biosynthesis 826
– benthiovalicar 809
phosphinic acid 8
– benzobicyclon 251
phosphinothricin 426
– bifenazate 1083, 1351
– herbicidal activity 427ff.
– bispyribac-sodium 127
– uptake and translocation 428ff.
– bixafen 630
l-phosphinothricin 423ff.
– boscalid 629
– inactivation by N-acetylation 433
– bromuconazole 783
phosphinothricin-N-acetyltransferase (PAT)
– carboxin 628
413
– carboxylic acid amid fungicides 809
– crop selectivity by expression 432
– chloroantraniliprole 1413
L-phosphinothricin acetyltransferase 418
– chromofenozide 959
phosphinothricyl alanyl-leucine (phosalacine) – clofentizine 1014
427 – clothianidin 1176
phosphoenolpyruvate (PEP) 20 – cyantraniliprole 1413
phospholipids 531, 764 – cyazofamid 613
phosphonates 541, 555, 869, 872 – cyclosulfamuron 72
phosphoroamidate 439 – cyenopyrafen 1082
phosphorodithioate 9 – cyflomethofen 1082
phosphorothiolates 544 – cyflufenamid 888
phosphorylation inhibitor 561, 576 – cymoxanil 866
Photinus pyralis 1062 – cyprodinil 707
photoaffinity labeling 564, 570 – diclobenil 362f.
photodegradation, pyrethrins 951 – dichlormid 377
photorespiration 425 – diclomezine 875
photosensitizer 1062 – doflovidazin 1015
photosynthesis inhibitor 479ff., 796 – diflufenican 204
photosystem II (PS II) – dimethomorph 809
– current market share of PS II compound – dimoxystrobin 598
groups 500 – dinotefuran 1182
– inhibitor of electron transport 346 – emamectin benzoate 1308
photosystem II (PS II) inhibitor 14f., 227f. – enestrobin 607
– situation in the EC market 499 – epoxiconazole 780
Subject Index 1483
– ethiprole 1293 – metalaxyl-M 903

– ethoxysulfuron 69 – metconazole 785
– etoxazole 1025 – methoxyfenozide 959
– famoxadone 613 – metominostrobin 598
– fenazaquin 1080 – metolachlor 377
– fenamidone 613 – metrafenone 891
– fenbuconazole 778 – milbemectin 1308
– fenhexamid 797 – nitenpyram 1170
– fenpiconil 726 – norflurazon 205
– fenpyroximate 1080 – oryzastrobin 598
– fentrazamid 322 – oxadiazines 1261
– fipronil 1293 – oxasulfuron 79
– flonicamid 1338 – oxpoconazole 775
– fluacrypyrim 1081 – oxyacetamide 319
– fluorcarbazone-methyl 144 – pefurazoate 773
– flucetosulfuron 73 – pencycuron 756
– fludioxinil 726 – penflufen 630
– fluensulfone 1381 – penthiopyrad 629
– flufenacet 319 – picolinafen 205
– flumorph 809 – picostrobin 598
– fluopicolide 831f. – pinoxaden 469
– fluopyram 639 – propoxycarbazone-sodium 144
– fluoxastrobin 599, 605 – proquinazid 894
– flupysulfuron-methyl-sodium 60 – prothioconazole 792
– fluquinconazole 788 – pymetrozine 1328ff.
– fluridone 204 – pyraclostrobin 599
– flurochloridone 204 – pyridaben 1080
– flurtamone 205 – pyridalyl 1360ff.
– flutolanil 628 – pyrifluquinazon 1342
– flusulfamide 873 – pyrimethanil 709
– fluxapyroxad 630 – pyrimidifen 1081
– foramsulfuron 77 – pyriminobac-methyl 127
– foestyl-aluminium 870 – pyrimisulfan 160
– halofenozide 959 – pyriproxyfen 996
– hexathiazox 1020 – pyrithiobac-sodium 127
– hydramethylnon 1082 – quinoxyfen 733
– imibenconazole 789 – sedaxane 630
– imidacloprid 1190f. – simeconazole 790
– imidazolinones 91 – spirodiclofen 1122
– indaziflam 362f. – spiromesifen 1122
– indoxacarb 1263 – spirotetramat 1122
– iodosulfuron-methyl-sodium 62 – spiroxamine 795
– ipconazole 787 – sulcotrione 251
– iprovalicarb 809 – sulfosulfuron 61
– isopyramzam 629 – sulfoxaflor 1230
– isoxaben 362f. – tebufenozide 959
– kresoxim-methyl 598 – tebufenpyrad 1080
– lepimectin 1314 – tebufloquin 880
– mandipropamid 809 – tefuryltrione 252
– mefenacet 319 – tembotrione 252
– mefenpyr-diethyl 382 – tetraconazole 778
– mepanipyrim 707 – thiacloprid 1196f.
– mesosulfuron-methyl 64 – thiamethoxam 1213
– mesotrione 252 – thiapronil 1083
1484 Subject Index
physicochemical properties (contd.) pinoxaden 460ff.

– thiencarbazone-methyl 144 – biology 470
– thifluzamide 629 – ecotoxicological environmental profile 469
– tolfenpyrad 1081 – metabolism in planta 471
– triazoxide 877 – physico-chemical propertie 469
– trifloxystrobin 598 – selectivity 471
– trifloxysulfuron-sodium 80 – soil metabolism 472f.
– tritconazole 782 – synthesis 470
– tritosulfuron 66 – toxicological profile 469
– uncouplers of oxidative phosphorylation pinworm (Tuta spp.) 1263
652 piperazines 768ff., 522
phytoalexins 872 piperidines 793f., 552
phytoene 199 piperonyl butoxide (PBO) 1072, 1253
phytoene desaturase (PDS) 197ff. pKa, uncouplers of oxidative phosphorylation
– inhibition 201 651ff., 660f.
phytoene desaturase inhibitors 197ff. Planococcus citri (citrus mealybug) 1174f.,
– acute oral toxicity 203 1234
– application data 219ff. plant breeding, Bar and Pat gene 433
– binding site 212 plant bugs (Lygus spp.) 1339
– biology 217 plant defense inducers 540, 547, 909ff.
– enzyme activity 203 plant defense reactions 610
– physical data 203 plant defense responses 869, 872f., 912ff.,
– synthesis 218ff. 918, 924
– use pattern 217 plant engineering 1029
phytofluene 199f. plant growth regulator (PGR) 523ff., 764
phytohormone 911 – DMI fungicides 764
phytophagous mite 1349 – epoxiconazole 779
Phytophthora cinnamomi 868f. plant nitrogen metabolism, glutamine
Phytophthora infestans 833 synthetase 424
– effect on zoospores and mycelial growth plant regulator (PR) 523ff.
833 – commercialized 524
– spectrin-like protein 836 – modern agriculture 527
Phytophthora spp. 871 – new aspects 523ff.
phytoprostane 387 plant resistance, effect of CP4 expression 414
Phytoseiulus persimilis 1339ff. plant transformation 1030
phytotoxic effect Plasmodiophora brassicae 615, 874
– bleaching herbicide 197 Plasmodoriophoromycete 615
– uncouplers of oxidative phosphorylation Plasmopara viticola (vine downy mildew) 613,
649 814
picolinafen 202ff., 218 plastoquinone (PQ) 197, 226
– physicochemical properties 205 Plutella xylostella (diamond-back moth) 990,
– synthesis 221f. 1004, 1034ff., 1062ff., 1300, 1359, 1406ff.,
picolinate auxin herbicide receptor 1418ff.
– AFB5 class 273 – insecticidal activity of pyridalyl against
picoxystrobin 592, 607 insecticide-resistant strain 1363
– metabolic stability 605 Poa annua 15, 18, 440
– physicochemical properties 598 Point mutation
– synthesis 618 – EPSP synthase 21
picrotoxin (PTX) 1289f. – herbicide resistance 17f., 21, 26
piericidin 564 polyamines 531f.
Pieris rapae crucivora (common cabbage polychlorocycloalkane (PCCA) insecticide
worm) 1402 1285
pink bollworm (Pectinophora gossypiella) – cross resistance 1285
1031ff. – noncompetitive antagonist (NCA) 1285
Subject Index 1485
– acetamiprid 1175ff. proton gradient , inhibitor of oxidative

– inhibitor in DHN melanin biosynthesis phosphorylation 1070
842ff. proton transport 648f.
polyoxins 553, 1002 proton-gated chloride channel 1290
pome and stone fruit, Cydia molesta 1199 protoporphyrinogen IX oxidase (Protox, PPO)
Popillia japonica (Japanese beetle) 975, 1300 inhibitors 163ff.
positive list of active substances (Annex I) – binding studies 176, 183
487 – crystal structure 183
potato late blight 814ff., 867 – molecular modelling 176, 183
potato leafhopper, see Empoasca fabae – toxicology 190
potato powdery scab (Spongospora subterranea) PS II herbicides 25, 501ff.
874 – market 510
powdery mildew 660ff., 698, 719, 731, 770ff., – nitril 503, 507
881 – triazines 480
predatory mite (Amblyseius calfornicus) – uracil 505, 508
1344ff. – urea 501f., 508
pregnane steroids 1288 – withdrawn 505ff.
prephenate dehydroenase 215 – see also photosynthesis inhibitors
pro-fungicides 654 psbA gene 15
pro-herbicides 256 Pseudocercosporella herpotrichoides (cereal
– HPPD inhibitors 228 eyespot pathogen) 770
Pseudococcus comstocki (comstock mealybug)
– isoxazole 271
1402
– N-acetyl-phosphinothricin 434
Pseudomonas 423ff.
pro-insecticides 1065, 1072, 1095
– syringae pv. tabaci 423ff.
– nAChR 1132
Pseudoperonospora cubensis (cucumber downy
pro-safener 382
mildew) 814, 833, 867ff.
probenazole (PBZ) 909ff.
Pseudoperonospora humuli (downy mildew on
– synthesis 914
hop) 871
– systemic acquired resistance induction 914
Pseudoplusia includens (soybean looper) 1419
proline dehydrogenase 573
Psylla piri 1116
pronamide 740
Psylla spp. 1339
– tobacco tubuline binding inhibitor
pterulinic acid 687
propoxycarbazone pterulone 687
– herbicidal characteristics 157 PttMAP20 gene 347
– physico-chemical properties 144 Puccinia hordei (barley leaf rust) 792
– synthesis 148ff. Puccinia triticina (rust) 791
proquinazid 893f. putrescine 531
– cross-resistance 895 pymetrozine 1327
– discovery 894 – biological activity 1333
– ecotoxicological property 894 – chemical property 1328ff.
– mode of action 895 – discovery 1327ff.
– physical property 894 – insecticidal spectrum 1334
– product 896 – mode of action 1331
– synthesis 895 – physicochemical properties 1328ff.
– toxicological property 894 – resistance 1333
protein synthesis – safety profile 1333f.
– biosynthesis mechanism 693f. – synthesis 1328ff.
– fungicides acting on 551, 693 – use recommendation 1333
prothioconazole 791, 878 pyraclostrobin 531, 592ff., 606
– physicochemical properties 792 – metabolic stability 599
– resistance 791 – physicochemical properties 599
prothoracicotropic hormone (PTTH) 989 – translaminar movement 592
1486 Subject Index
pyraflufen-ethyl, synthesis of 185 N-(4-pyridyl)biphenyl acetic acid amide 679

pyrazogyl, synthesis of 183 2-pyridylsulfonic acid 685
pyrazolecarboxamides insecticides 1087 pyrifluquinazon 1327f., 1340
pyrazoles 173, 182, 245 – biological activity 1343
pyrazolines 1257 – chemical property 1342
– insecticidal 1273 – mode of action 1342
– isomer 1276 – physicochemical properties 1342
– safener 381 – safety profile 1343
– sodium channel blocker 1258 – synthesis 1340ff.
– structure–activity relationship 1274ff. pyrimidifen, synthesis 1089
pyrazolium herbicides 9 pyrimidine 706, 768ff.
pyrazolones 272, 275 pyrimidine carboxylic acid 295
pyrazolotriazinone 189 2 -pyrimidinecarbonyl sulfonanilide 135
pyrazolynate 263f. – rice herbicide 135
– half-lives 264 pyrimidinyl carboxylate herbicides 117ff.
– synthesis 264ff. – ALS inhibitor 120
pyrazoxyfen 225ff., 266 – discovery of herbicide 118
– synthesis 267 – mode of action 132
Pyrenophora graminea (leaf stripe on barley) – physico-chemical and toxicological
710, 730, 878f. characteristics 127
pyrethroid 941, 951, 1068, 1226, 1253, 1346 – structure–activity relationship 120
– synthetic 951, 1270 – synthesis 121
– type I 951 pyrimidinyl glycolate herbicides 122ff.
– type II 951 pyrimidinyl salicylate herbicides 117ff., 135
pyribencarb 593 pyrimidinyl thiobenzoate herbicides 19, 35
– synthesis 620 N-(4-pyrimidyl) aryl acetamide 683
Pyricularia oryzae (rice blast) 661, 694ff., 839 pyriminobac methyl 127
pyridaben 671, 1084ff., 1101 – biology 131
– discoverymm 1085ff. – discovery 129
– metabolism 1079 – ecotoxcoloies 127
– physico.chemical data 1080 – rice herbicide 129
– resistance 1101 pyrimisulfan 140
– synthesis 1085 – biology 139
pyridalyl 1358ff. – rice herbicide 139
– biological aspect 1362 – synthesis 136
– chemistry 1360 pyriproxyfen 983ff.
– discovery 1359f. – activity of optical isomers 987
– insecticidal activity 1362 – biological activity 990
– mode of action 1364 – mechanism of action 987
– physicochemical properties 1360ff. – physicochemical properties 995
– structure–activity relationship 1360 – resistance 995
– toxicological profile 1359 – stability 996
pregnane steroids 1288 – synthesis 995
– synthesis 1261 – toxicology 996
pyridazine insecticide 1259 pyrithiobac-sodium 127
pyridazinones 8, 173, 480f., 555, 875, 1085 – biology 126
pyridine azomethine 1327ff. – cotton herbicide 125
– structure–activity relationship 1328ff. – discovery 125
pyridine sulfonamide 686 – physicochemical properties 127
pyridine-3-carboxaldehyde N-oxide 1328 pyroxasulfone 309, 327
pyridinyl-ethyl benzamide 640 – biological activity 328
pyridines 8ff., 400, 439ff., 552, 767ff. – mode of action 330
N-(4-pyridyl) aryl acetamide 679 – synthesis 328
pyridyl triketone 247 pyroxsulam
Subject Index 1487
– crop utility 112 – anilinopyrimidine 711

– ecotoxicology 113 – aromatic hydrocarbons 544
– environmental degradation 113 – auxin herbicide field resistance 285
– herbicidal activity 113 – azolones 614
– mechanism of crop selectivity 112 – benzimidazole 544, 739, 882
– metabolite 113f. – benzamides 745ff.
– synthesis 102 – benzoylurea 1270
– toxicology 113 – bialaphos 432
pyrroles 651, 1073ff. – bifenazate 1352
pyruvate 19, 30, 318 – bisacylhydranzine 977
pyruvate decarboxylase 30 – blasticidin S 696
®
pyruvate transporter 560 – bromoxynil (BXN ) 402
– CAA fungicides 553, 827
q – carbamates 1270
Q cycle 566 – carboxamides 554
Qi inhibitor class 567 – carboxin 571, 635-
Qo inhibitors 531, 541, 567, 713 – clofentezine 1001f., 1015f.
quinazoline insecticides 1086 – chlorfenapyr 1076
quinazolinedione 189 – chloroacetamide herbicides 313
N-(4-quinazolinyl) aryl acetamide 683 – chlorsulfuron 41
quinolin-2-one herbicides 178 – continuous selection 543, 762
N-(4-quinolinyl) aryl acetamide 679 – cross-resistance, see cross-resistance
quinoxyfen 549, 687f., 719 – cry proteins 1039f.
– mode of action 565 – cyflufenamid 889f.
– physicochemical properties 578 – diafenthiuron 1067f.
– resistance 420 – dicarboximide fungicides 545, 551, 717
– synthesis 577 – dieldrin 1286ff.
– toxicology 577 – diethofencarb 742
– dimethoate 1101
r – DMI fungicides 552
radioligand binding assay 740, 1151 – dodine 555
rational drug design 855f. – engineering crop 46, 203, 418
Rdl gene 1286 – etoxazole 1012, 1025f.
re-registration process in the European Union – famoxadone 612
482 – fenamidone 612
– main changes in guidelines 488 – fenazaquin 1101f.
– PS II inhibitors withdrawn 507ff. – fenhexamid 798ff.
– status of registration of PS II inhibitors – fenoxprop 17, 273, 393
under EU Directive 91/414/EEC 503ff. – fenproximate 1101f.
recombinant hybrid insect 1147f. – fipronil 1300
red imported fire ant (Solenopsis invicta) 1299 – flonicamid 1338
reduced herbicide translocation, herbicide – fluazinam 650, 663
resistance 16, 24 – flubendiamide 1406
reductase in melanin biosynthesis 554 – fludioxinil 717
reductase in sterol biosynthesis 552, 765ff., – flufenacet 313
795, 798f. – flumorph 826
reregistration 488, 500 – flutolanil 571
residue data requirement 490 – fosethyl-aluminium 715
resistance 543ff., 717 – fungicides 543ff.
– ACCase-inhibiting herbicide 453 – glufosinate 401ff.
– AHAS-inhibiting herbicide 43 – glyphosate herbicide 12
– aminocyclopyrachlor resistance – halofenizide 977
management 303 – hexathiazox 1020, 1025
– aminopyrimidines 544 – imidazolinone 403
1488 Subject Index
resistance (contd.) resistance database 939

– indoxacarb 1270 resistance development 5
– induced 913 resistance management 546, 156
– iprovalicarb 825ff. – programs 546f.
– IRAC resistance management 1301 – regulatory requirement 938
– isoxaben-resistance loci (ixr1 and ixr2) 350 – shifting 762
– kasugamycin 419, 543, 698 – strategies 546, 645, 1116
– MBI-D fungicides 554, 858ff. – use of alternations 941, 956
– mechanism 543, 1100 resistance monitoring method 937
– mechanisms for engineering glyphosate resistance mutations 543ff., 857, 1286f.
resistance 411 resistance risk assessment 546
– MET inhibitor-based acaricide 1100 respirasome 562
– metabolic detoxifacation 543 respiration inhibitors 578
– metalaxyl-M 904ff. respiratory chain 562
– methamidophos 1101 Reticulitermes flavus (subterranean termite)
– methidathion 1101 1299
– metrafenone 892f. Reticulitermes speratus (termite) 1174f.
– molecular basis 41 rhamnose derivative 1245f.
– monogenic 543 – structure and insecticidal activity of
– multiple 14ff. rhamnose-modified spinosyn analog
– non-target-site 14ff., 940 1246
– organochlorines 1240, 1287 Rhizoctonia solani (rice sheath blight) 748ff.
– organomercurials 544 Rhizoglyphus echinopus 1062
– organophosphates 1226, 1270 ribosomal RNA (rRNA) 693
– paraquat 24 ribosome 693
– phenylamides 544, 827 ribulose-1,5-bisphosphate carboxylase
– phosphoro-thiolates 544 oxygenase (Rubisco) 424
– phenylpyrroles 544, 717, 728 rice blast
– polygenic 543 – Magnaporthe grisea 773
– proquinazid 895f. – Pyricularia oryzae 661, 694, 839
– prothioconazole 791 rice herbicides
– pymetrozine 1331 – azimsulfuron 70
– pyridaben 1101 – benzobicyclon 256
– pyriproxyfen 995 – bispyribac-sodium 128
– QoI fungicides 544 – cyclosulfamuron 70
– quinoxyfen 544 – dithiopyr 439
– risk analysis 546 – fentrazamide 320
– SBI –fungicides 766 – flucetosulfuron 72f.
– scytalone dehydratase inhibitors 850, 858f. – mefenacet 316
– spionsyns 1240f., 1252f. – penoxsulam 109
– streptomycin 701ff. – pyrazolate 237
– strobilurins 612f. – pyriminobac methyl 129
– succinate dehydrogenase inhibitor 636 – pyrimisulfan 139
– synthetic pyrethroids 1264, 1270 – sulfonylurea 68
– sulfonylurea 401 rice leaf roller (Cnaphalocrocis medinalis)
– systemic acquired, see systemic acquired 1410
resistance rice sheath blight (Rhizoctonia solani) 553,
– target-site 14, 940 748ff., 757, 791, 876
– tebufenpyrad 1100ff. rice stem borer (Chilo suppressalis) 960, 979,
– triazine 14, 405 1038, 1195, 1402, 1421
– triphenyltins 544 rice water weevil
– uncoupler 651 – Lissoropterus oryzophilus 1198
– zoxamide 745 – Lissorhoptrus spp. 1298
Resistance Action Group (RAG) 937 Rieske iron-sulfur protein (ISP) 566
Subject Index 1489
ripening 528 Sclerotinia sclerotiorum 698, 730

risk assessment 482ff. Sclerotium rolfsii (white mold) 876
RNA 693ff. sclerotization 973
RNA polymerase I 548 scytalone dehydratase (SD) 842
RNA polymerization 904 – active site 850
root uptake 605 – biochemical reaction mechanism 850f.
root-knot nematode 1377 – biology of inhibitor 842
rotenoid 1078 – computational investigations of the enzyme
rotenone 564, 1078f. mechanism 854
roundup ready – crystal structure analysis 850
– corn 409ff. – inhibitor complex 852
– cotton 414f. – inhibitor in DHN melanin biosynthesis
– soybean 416 846ff.
rust – inhibitor structures in the SD binding niche
– Asian 413 855
– Puccinia hordei (barley leaf rust) 792 – resistance 852f., 858
– Puccinia triticina (rust) 791 – sequence alignment 853
ryanodine receptor (RyR) 1393f., 1415 – structure-based inhibitor design 850
– modulators 1389 sediment water study 493f.
seed born diseases, Pyrenophora 878
s seed dressing 388f., 878, 1193
saccharin 150 seed treatment
Saccharopolyspora pogona 1242 – fungicides 577, 593, 607, 631ff., 644,
Saccharopolyspora spinosa 1131, 1238 660ff., 723, 726ff., 762ff., 773, 776, 780ff.,
safener 61, 148, 352, 371ff. 790ff., 878
– dichloroacetamide 372, 389f. – insecticides 1175, 1179f., 1191ff., 1211ff.,
– commercial 373ff. 1239, 1283, 1297f., 1306, 1322, 1375
– composition 496 – herbicide safener 372f.,379ff., 390
– effect on herbicide metabolism 385 selective feeding blocker 1327ff.
– effect on gene expression 385 semicarbazone
– herbicide detoxification 392 – herbicide 9
– influence on herbicide translocation 388 – insecticide 1259f., 1373ff.
– influence on herbicide uptake 388 senescence 609
– mechanisms of action 385 sensitivity monitoring 543, 826
– mode of action in agricultural practice 389 Septoria tritici 585, 612, 770ff., 791ff.
– stimulation of GST activity 390 Serine-glyoxylate-aminotransferase 424
salicylanilide 656 Sesamia inferens 1421
– uncoupler 652 Setaria faberi 17
salicylic acid (SA) 387, 531, 910 Setaria viridis 17
salicylic acid-glucoside (SAG) 914 sethoxydim-resistant (SR) corn 404
salicylic acid pathway 554 shakariki 71
Salmonella typhimurium 36 sheath blight of rice 757f.
Salsola iberica 19 shikimate-3-phosphate (S3P) 20
Sarcoplastic reticulum 1063 shikimate pathway 406
SBI see sterol biosynthesis inhibitors shoot growth inhibition 403
SBI class I, DMI fungicides 767ff. shoot stem borer (Leucinodes spp.) 1421
SBI class II, amines 767, 793ff. signal perception 912
SBI class III 767, 799 signal transduction 551, 715, 912
SBI class IV 767, 801 signaling pathway 387
SBI fungicides 541ff., 761ff. simeconazole
Scapteriscus spp. (mole cricket) 1300 – physicochemical data 790
Scirpophaga incertulas (yellow stem borer) – seed treatment 790
1421 – synthesis 791
Scirpus juncoides19 singlet oxygen 165, 198
1490 Subject Index
site-directed mutation 857 spinosyns 1127ff., 1147, 1238

Sitophilus zeamais (maize weevil) 1402 – aglycone 1241ff, 1250
sitosterol-cellodextrin 347 – biosynthesis 1249
sitosterol-β-glucoside 347 – core-modified analog (aglycone) 1241ff.
smaller tea tortrix, see Adoxophyes honmai – genetics 1249
sodium channel-blocking insecticide 1257ff. – insecticidal activity of rhamnose-modified
sodium channel modulators 943 spinosyn analog 1246
soil – metabolism 1252
– carboxylic acid amide (CAA) 810 – mode of action in insect 1144, 1240
– sulfonylurea 81 – modification involving the C9 sugar 1245
soil application – modification involving the C17 sugar 1244
– fipronil 1297 – modified at C21 1242f.
– thiamethoxam 1216 – penetration 1252
soil degradation data – semi-synthetic 1144ff., 1238ff.
– benoxacor 377 – spn genes 1250
– furilazole 378 – structure of rhamnose-modified spinosyn
soil metabolism analog 1246
– mesosulfuron-methyl 82 spirodiclofen 1108ff.
– pinoxaden 472f. – biological propertyies 1122
soil nontarget microorganism – discovery 1109
– risk 494 – development 1116
Solenopsis invicta (red imported fire ant) – physico-chemical properties 1122
1299 – synthesis 1111f.
somatic embryogenesis receptor kinase – use 1116
(SERK) 917 spiroketalamines 552, 767, 795
Sorghum halepense 18 spiromesifen 1108ff.
Southern armyworm 1280 – biological property 1122
soybean 78 – discovery 1109, 1124
– expressing of cry proteins 1034ff. – physico-chemical property 1122
– gene modified 400ff. – synthesis 1111ff.
– herbicide-resistant 401ff. – use 1116
soybean looper, see Pseudoplusia includens spirotetramat 1118ff., 1376
soybean rust (Phakopsora pachyrhizi) 413, – biological property 1122
585 – biology 1122
soybean stem weevil (Sternechus subsignanthus) – mode of action 1122
1298 – nematicidal activity 1376
spectrin-like protein – physico-chemical properties 1122
– characterization in Phytophthora infestans by – synthesis 1120f.
bioanalysis 836 spiroxamine 795
– distribution 834 – diastereomer 796
spectrum shift 1063, 1124 – synthesis 796
spermidine 531 spn genes 1250
spermine 531 Spodoptera eridania 1280
Sphacelotheca reiliana (corn head smut) 781 Spodoptera exigua (beet armyworm) 1004,
spider mite 1034, 1419
– Tetranychus cinnarabinus (carmin spider Spodoptera frugiperda (fall armyworm) 1248,
mite) 1067, 1318ff. 1280, 1411
– Tetranychus urticae (two-spotted spider mite) Spodoptera littoralis 1034
1110ff., 1348ff. Spodoptera litura (common cutworm, tobacco
spinetoram 1127ff., 1147, 1238ff. cutworm) 988, 1003, 1067, 1403ff.
– activity 1247 Spodoptera spp. (armyworm) 1359
spinosad 1127ff., 1147, 1238ff. Spongospora subterranea (potato powdery scab)
– biological activity 1239ff. 874
– primary use 1239 sprout inhibition 529
Subject Index 1491
sprout suppressant 529 – vapor pressure 604

squalene epoxidase inhibitor 522, 800f. – water solubility 609
squalene monooygenase 765f., 795 – yield enhancement 592, 609
stacking traits 416 Strobilurus tenacellus 612
Stellaria media 19, 58 Structure-activity relationship (SAR)
Sternechus subsignanthus (soybean stem weevil) – anilinopyrimidines 710
1298 – aryl-diones 466
sterol biosynthesis – avermectin 1317
– enzymes involved in fungi 764 – benzamides 743ff.
– fungicide 552 – benzenedicarboxamides 1398f.
– target of agricultural fungicide 764 – bifenazate 1347
sterol biosynthesis inhibitor (SBI) 539, 719, – biphenyl carbazates 1349
761ff. – bisacylhydrazines 962ff.
– biochemical target of SBI fungicide 763 – diafenthiuron 1063f.
– class 764 – diflumetorim 674
– Class I (DMI fungicide) 767 – diphenyl-oxazolinone 1023
– Class II (amine) 793 – ecdysteroids 962ff.
– Class III (hydroxyanilide) 796 – fiprole insecticides 1294
– Class IV (squalene epoxidase inhibitor) – N.benzyl-4-pyrimidine-amines 674
801 – N-triazolo(1,5c)pyrimidine sulfonamides
– fungicide in agriculture 761ff. 102
– grouping fungicide 767 – oxadiazines 1260
– market importance of fungicide 763 – oxazolines 1023
stilbene synthase 309 – phenylpyrazoles 1283
storage 489, 526ff. – phenylurea fungicides 753ff.
Streptomyces 424ff. – phytoene desaturase inhibitors 203
– griseus 699f. – pyridazinones 1085
– hygroscopicus 427ff. – pyridine azomethines 1328f.
– kasugaensis 696 – pyrimidinylcarboxylates 117ff.
– phosalacineus 427 – pyrimidinylsalicylates 117ff.
– phosphinothricin-producing 432f. – succinate dehydrogenase inhibitors 635
– viridochromogenes 426ff. – strobilurins 597
streptomycin 700f. strychnine-sensitive glycine receptors 1288
Streptoverticillium rimofaciens 698 subterranean termite (Reticulitermes flavus)
stress defense 530 1299
stress protectants, triazoles 530 succinate dehydrogenase (SDH) 570, 628ff.
stress tolerance, strobilurins 586, 609 succinate dehydrogenase inhibitor 628ff.,
strigolactone 532 640
strobilurins 531, 541, 567, 584ff., 1095 – biological activity and application 634, 645
– acaricide 611 – influence of structural elements on the
– bioavaibility 603 biological spectrum 642
– biokinetic behavior 596ff. – metabolism 636
– biological use pattern 598 – resistance 636
– commercial fungicide 590 – structure–activity relationship 635
– distribution 603 – structures 627ff.
– evolution as agricultural fungicides 587 succinate–UQ oxidoreductase 570
– interplay of target activity and biokinetic sugar accumulation 527
behavior 595 sugarbeet, vectors of virus diseases 1180
– physico-chemical data 598 sugarcane 51, 78ff., 80, 97, 269, 510
– resistance 612f. suicide substrate 571
– root uptake 598, 605ff. sulcotrione 225ff., 240ff.
– structure–activity relationship 595ff. – environmental properties 251
– target activity 595ff. – physicochemical properties 251
– transportation 603 – metabolism 249
1492 Subject Index
sulcotrione (contd.) – synthesis 1226f.

– synthesis 250 – toxicology 1226ff.
sulfamides, fungicides 556 sulfoximine 1226
sulfcarbamide-1-aminomethanamide 528 – acyclic 1228
sulfonamide antibiotics 702 – green-peach aphid (Myzus persicae) efficacy
sulfonamide, fluorinated 659 1229
sulfonanilide – synthesis 1227
– commercialized herbicide 140 sulfur formulation 542
– discovery 134 sweet potato whitefly, see Bemisia argentifolii
– effect of benzene ring substitution 137 synergists 496, 984
– effect of bridge moiety 136 Synthesis of
– effect of sulfonamide moiety 136 – acequinocyl 1095
– structure–activity relationship 135 – acetamiprid 1174
N-sulfonyl amino acid amide 818 – acibenzolar-S-methyl 915
sulfonylaminocarbonyl-triazolinone (SCT, – alkylazine herbizides 355
SACT) 19, 142ff. – 4-alkynyl-2-anilinopyrimidine 708
– biological profile 151ff. – amectoctradin 621
– discovery 143 – amicarbazone 514
– optimization 143 – amidoflumet 1355
– structurr-activity relationship 141 – N-amino triazolinone 514
– synthesis of 143 – aminocyclopyrachlor 299
sulfonylisocyanates, production of 52 – aminopyralid 288
sulfonylisoxazoline 309 – aminosulfones 807ff.
sulfonyltriazole 309 – amisulbrom 620
sulfonylurea (SU) 19, 50ff., 374–393 – anilinopyrimidine 706
– agricultural utility 56ff. – aryl-1,3-dione 461ff.
– behavior in the soil 81 – azolones 618
– cereal 59 – azoxystrobin 617
– degradation 81 – beflubutamid 221f.
– development 52 – bencarbazone 190
– herbicide 50ff., 78, 393 – benclothiaz 1384
– history 52 – benoxacor 376
– maize 75 – benthiovalicar 815
– metabolic fate 81 – benzfendizone 175
– resistance 401 – benzobicyclon 257
– rice 68 – bifenazate 1350
– safener 59, 374–393 – bisacylhydrazine 961ff.
– selectivity 69, 76 – boscalid 634
– synthesis 56 – bromuconazole 784
– third-generation 52 – butafenacil 174
– toxicity 51f. – carbazate 1350
sulfosulfuron 58 – carpropamid 858
– physico-chemical properties 61 – chloroantraniliprol 1413
sulfoxaflor 1226 – chlorfenapyr 1073f.
– biology 1233 – chlorthiamid 346
– chemical property 1230 – chromofenozide 962f.
– efficacy on resistant pests 1233 – clofentezine 1014, 1021
– environmental toxicology 1230ff. – cloquontocet-mexyl 380
– field use pattern 1233 – clothianidin 1177ff.
– mammalian toxicology 1230f. – cyantraniliprol 1415
– metabolism 1235 – cyazofamid 621f.
– mode of action 1232 – cyclohexandione 461
– physico-chemical properties 1228 – cyclosulfamuron 71
– structure–activity relationship 1228f. – cyenopyrafen 1093
Subject Index 1493
– cyflufenamid 890 – fluxofenim 379

– cyflometofen 1094, 1353 – foestyl-aluminium 870
– cymoxanil 867 – fufenozide 978
– cyprodinil 708 – furilazole 378
– cyprosulfamide 384 – glyoxylic acid derivatives 823
– cyzofamid 620 – halofenozide 961
– diafenthiuron 1065f. – hexathiazox 1019ff.
– dichlobenil 346 – hydomethylnon 1098
– dichlormid 377 – imibenconazole 790
– diclocymet 858 – imidacloprid 1190ff.
– diclomezine 876 – imidazolinones 89
– diflovidazin 1014ff. – indaziflam 355
– diflufenican 221f. – indoxacarb 1262
– dilfumetorim 674 – ipconazole 786
– dimethomorph 811 – ipfencarbazone 323
– dimoxystrobin 616 – iprovalicarb 812
– dinotefuran 1183 – isotianil 919
– dithiopyr 443f. – isoxaben 348
– emamectin benzoate 1306, 1309, 1311 – isoxadifen-ethyl 270
– epoxiconazole 780f. – juvenoids 985
– ethiprole 1293f. – kresoxim-methyl 616
– etoxazole 1022, 1024 – lepimectin 1313
– famoxadone 619 – mandipropamid 821f.
– fenamidone 619 – mefenacet 318
– fenazaquin 1086 – mefenpyr-diethyl 381
– fenbuconazole 779 – mepanipyrim 708
– fenhexamid 798 – mesotrione 238
– fenoxasulfone 327 – metalaxyl-M 902
– fepiclonil 723f. – metconazole 786
– fenpyroximate 1084 – methoxyfenozide 962
– fentrazamid 321 – metominostrobin 616
– ferimzone 661 – metrafenone 892
– fipronil 1293f. – nitenpyram 1171
– flonicamid 1335ff. – norflurazon 221f.
– fluacrypyrim 1096 – oryzastrobin 616
– fluazinam 664 – oxadiazines 1261
– flubendiamide 1398 – oxpoconazole 775
– fluensulfone 1380f. – pefurazoate 774
– flucarbazone-sodium 148ff., 153 – pencycuron 758
– fludioxonil 723, 725 – phenylpyrroles 722
– fluensulfone 1380 – picolinafen 221f.
– flufenacet 318 – picoxystrobin 617
– flufenerim 1090f. – pinoxaden 470
– flufenpyr-ethyl 176 – probenazole 914
– flumorph 811 – propoxycarbazone 148ff.
– fluopyram 644 – proquinazid 895
– fluoxastrobin 618 – prothioconazole 792f.
– flupoxam 351f. – pymetrozine 1328ff.
– fluquinconazole 788f. – pyraclostrobin 619
– fluridone 221f. – pyraflufen-ethyl 175
– flurochloridone 221f. – pyrazogyl 183
– flurtamone 221f. – pyrazolynate 264ff.
– flusulfamide 874 – pyrazoxyfen 267
– flutianil 897 – pyribencarb 619
1494 Subject Index
Synthesis of (contd.) tan spot (Drechslera tritici-repentis) 791

– pyridaben 1085 tanning 973
– pyridazine 1261 target site, genetic modification of 940
– pyrifluquinazon 1340ff. target-site mutation 14, 612
– pyrimethanil 708 target-site resistance 14, 940
– pyrimidifen 1089 Tarsonemid mite 1116
– pyrimisulfan 136 TCA cycle 570
– pyriproxyfen 995 tebufenozide 961ff., 975
– pyriproxyfen 995 – ecotoxicology 976
– pyroxasulfone 328 – physicochemical properties 959
– quinoxyfen 732 – synthesis 961
– simeconazole 791 – toxicology 976
– spirodiclofen 1111f. tebufenpyrad 671, 1084ff., 1100f.
– spiromesifen 1111f. – discovery 1087ff.
– spirotetramat 1120f. – metabolism 1097
– spiroxamine 796 – physico-chemical data 1080
– sulcotrione 250 – resistance 1101
– sulfoxaflor 1226f. – synthesis 1088
– sulfoximine 1227 tebufloquin 879
– tebufenozide 961 – application 881
– tebufenpyrad 1088 – physico-chemical properties 880
– tebufloquin 880 – plant disease 881
– tefuryltrione 256 – synthesis 880
– tembotrione 256 tefuryltrione 246ff.
– tetraconazole 778 – synthesis 256
– thiacloprid 1198 tembotrione 156, 228, 246ff., 382
– thiamethoxam 1211f. – synthesis 256
– thiazopyr 443ff. termite 1174f., 1297
– thiencarbazone-methyl 148ff. – Heterotermes tenuis 1297
– thifluzamide 633f. – Reticulitermes speratus 1174f.
– tiadinil 917ff. tetraconazole 776f.
– tolfenpyrad 1088 – biologically most active enantiomer 778
– topramezone 273 – physicochemical data 778
– triaziflam 355 – synthesis 778
– triazoxide 878 tetracyclic macrolide polyketide 1128
– trifloxystrobin 616 tetracycline antibiotics 551
– triticonazole 782 tetrahydroindazole, PPO herbicides 186
– valifenalate 816f. tetrahydrophthalimide, Protox herbicides
– zoxamide 744 167ff.
systemic acquired resistance (SAR) 911 tetrahydropyrazolodione 460
– biochemical change 912 tetrahydroquinoline 979
– induction by acibenzolar-S-methyl 916 1,3,6,8-tetrahydroxynaphthalene reductase
– induction by probenazole 914 (MBI-R) 842ff.
– isotianil 919 – inhibitor in DHN melanin biosynthesis
– priming 913 845
– systemic signal 911 tetramic acid 462, 1118
– tiadinil 918 – spirocyclic derivative 1119
systemic translocation 409 Tetranychus cinnarabinus (carmin spider mite)
systemicity in plant 604 1067, 1318ff.
Tetranychus spp. 1349
t Tetranychus urticae (two-spotted spider mite)
tabtoximine-β-lactam 423 1110ff., 1348ff.
take-all fungus (gaeumannomyces graminis) tetrapyrrole, mimicry 177
577, 788 tetrazines acaricides 1013ff.
Subject Index 1495
tetrazolinones, herbicides 9f., 316ff. thifluzamide 632ff.

tetrodotoxin-sensitive (TTX-S) sodium current – synthesis 633f.
1269 thiobenzamide 345
tetrodotoxin-resistant (TTX-R) sodium current thiocarbamates 9, 326, 373, 379, 541ff., 767,
1269 800
tetronic acid 462, 1110 thioesterase 1250
tetronic acid derivative 1110ff. thiophosphate 316
– biology 1114 thiourea insecticide 1064
– mode of action 1114 thioxo-oxazolidinone 614
thiacloprid 1127, 1189ff. thrips
– activity 1199 – Frankliniella occidentalis (Western flower
– application 1198 thrips) 1363
– ecotoxicological profile 1198 – Thrips palmi (melon thrips) 992ff., 1174ff.,
– efficacy on target pests 1198 1363f.
– ovicidal activity 1200 – Thrips tabaci 1339
– physico-chemical properties 1196f. – Thysanoptera 1297, 1410
– synthesis 1198 Thrips palmi (melon thrips) 992ff., 1174ff.,
thiadiazolecarboxamide fungicides 554 1363f.
thiadiazoles 8f., 552 Thrips tabaci (thrips) 1339
thiamethoxam 1127, 1180, 1203ff. Thysanoptera 1297, 1410
– acute toxicity 1219 tiadinil 632, 909ff.
– application 1215 – synthesis 917
– biological activity 1215 – systemic acquired resistance 918
– biological mode of action 1215 tick vector
– chemical property 1213 – Ixodes scapularis 1300
– ecotoxicology 1220 TIPS-EPSPS 411
– effects on beneficial arthropods 1220 TIPS-EPSPS gene 416
– foliar application 1216 TIR1/AFB protein 277ff.
– hydrolytic degradation pathway 1214 TIR1/AFB receptor family 282
– mammalian toxicology 1219 tobacco budworm, see Heliothis virescens
– mode of action 1214 tobacco cutworm (Spodoptera litura) 988,
– physical property 1213 1003, 1067, 1403ff.
– physico-chemical property 1213 tobacco whitefly, see Bemisia tabaci
– safety profile 1219 tocopherol 227
– seed treatment application 1218 tocopherol cyclase 228
– soil application 1216 tolfenpyrad 1079ff.
– target sites in the nervous system 1214 tomato late blight 824, 867
thiamine diphosphate (ThDP) 29 topramezone 245, 271f.
thiatriazine 356 – synthesis 273
– chemistry 356 tortricide 1199
1λ4 ,2,4,6-thiatriazine 357 toxicology 497
thiazolidinone 1018f. – carbamates 951
thiazolidinone carbamate 359 – carbocyclic acid amide fungicides 809
– synthesis 360 – neonicotinoids 1156
thiazolo(2,3-b)triazine, fungicides 1018 – organophosphates 951
– synthesis 443ff. – selective insect toxicity 973
– toxicology 442 – uncouplers or oxidative phosphorylation
thidiazimin 178 661
thiencarbazone-methyl (TCM) 142ff., 155, toxophore 589, 676, 1294
269, 394 2,3-trans-enoyl-CoA reductase 306
– herbicidal characteristics 157 transcription factors
– physico-chemical properties 144 – OsWRKY45 912ff.
– synthesis 148ff. – OsWRKY62 921ff.
1496 Subject Index
transcription factors (contd.) Trichogramma spp. 1420

– OsWRKY76 921ff. Trichoplusia ni (cabbage looper) 1404, 1419
transfer RNA (tRNA) 693 4,5,6-trichloro-2-trifluoromethyl
transformation benzimidazole (TTFB) 653
– Agrobacterium-mediated 1029 trifloxystrobin 592ff., 607
transgenic plants – physicochemical data 598
– Bacillus thuringiensis Cry protein 1029ff. – synthesis 616
– heterologous expression of HPPDs 226 trifloxysulfuron-sodium 79
– maize 411ff. – physico-chemical properties 80
translocation, systemic 409 trifluoromethanesulfonanilide
transpiration stream concentration factor – structure–activity relationship 1356
(TSCF) 605 – substituted 137
trehalose 429, 599 trifluoromethylnicotinamide 1336
Trialeurodes vaporariorum (glasshouse – structure–activity relationship 1335ff.
whitefly, greenhouse whitefly) 990ff., trifluoromethylnicotinamide insecticide
1004, 1067, 1333 1335
1,3,5-triazindione 481 triketone 235ff.
1,3,5-triazine 480f. – discovery 236
triazine tolerance (TT) 405 – mode of action 237
triazine resistance 14 – physical, chemical, toxicological, and
1,3,5-triazine-2,4(1H,3H)-dione 480 environmental property 251f.
1,2,4-triazinone 480f., 512 – structure–activity relationship (SAR) 239
triazole 531, 776 – synthesis 237ff.
triazole fungicides 8, 552, 584, 762–767, triticonazole 780ff.
770f., 776–793 – physicochemical properties 782
triazole pyridine triketone 248 – synthesis 782
1,2,4-triazole-3-thione 791f. tritosulfuron 64
triazolinone 172ff., 316ff. – physico-chemical properties 66
– monocyclic 145 tryptophan 425
– synthesis 150 β-tubulin assembly 548
triazolocarboxamide 351 turnip moth (Agrotis segetum) 1402
– biology 352 Tuta spp. (pinworm) 1263
– chemistry 351 two-spotted spider mite, see Tetranychus urticae
triazolopyrimidine (TP) 19, 35, 99ff. txr gene 361ff.
– AHAS inhibition 114f. Typhlodromus spp. 1420
– sulfonamides 101ff. Tyrophagus putrescentiae 1355
N-triazolo[1,5-a]pyrimidine sulfonamide tyrosine 226, 406, 854f.
111ff.
– biology 111 u
– herbicidal activity of analog 112 ubiquinol oxidase 572
– synthesis 111 ubiquinol–cytochrome c oxidoreductase
N-triazolo[1,5-c]pyrimidine sulfonamide 105 561f.
– biology 107 ubiquinone (UQ) 559ff.
– herbicidal activity of analog 108 ubiquitin 278
– synthesis 106 ubisemiquinone 564
N−triazolo[1,5-a]pyrimidine sulfonanilide UDP-glucose 341ff.
100, 114 UDP-glucose pyrophosphorylase 341
N-triazolo[1,5-c]pyrimidine sulfonanilide 100 ultraspiracle protein (USP) 960ff.
– biology 100 uncoupler 548, 561, 574, 646f., 1076
– synthesis 100 – arylhydrazone 652
triazoxide 877, 1195 – benzimidazole 652
– application 879 – classical 648
– physico-chemical property 877 – coumarin 652
– synthesis 878 – diarylamine 652
Subject Index 1497
– fungicidal 655 voltage–clamp

– mechanism of action 647 – native neuron preparation 1149
– neutral 649 – whole-cell 1149
– physico-chemical properties of voltage-gated sodium channel (VGSC) 1265
protonophoric uncoupler 652 – blockade by dihydropyrazole 1266
– protonophoric 649 – blockade by indoxacarb 1266ff.
– pyrrole 652 – insect 1265
– resistance 651 – insecticide binding site 1266
– salicylanilide class 652 – mammalian 1269
– selectivity 649 – neurotoxin binding site 1266
– toxicity 649
uncoupling protein (UCP) 647ff. w
Uniform Principle 487, 498 Weed Science Society of America (WSSA)
uracil 172, 480f. Code System 7
– benzoheterocycle uracil 187 weed selectivity
uracil heterocycle 174 – auxin action 283
urban pest control application 1299 Western flower thrips, see Frankliniella
urea herbicide 7, 360, 378, 384, 387 occidentalis
US-EPA 483ff. wheat bulb fly (Delia coarctata) 1298
usnic acid 650 white mold (Sclerotium rolfsii) 876
Ustilago maydis 571, 687f. whitefly
Ustilago nuda (loose smut on barley) – Bemisia argentifolii (sweet potato whitefly)
687, 879 1419
– Bemisia tabaci (tobacco whitefly) 992ff.,
v 1004, 1067, 1114, 1333
V75M mutants 859f. – Trialeurodes vaporariorum 990ff., 1004,
valifenalate 807ff. 1333ff.
– synthesis 816f. whole-cell voltage–clamp
valinamide 808 – native neuron preparation 1149
valine 29ff. whole-organism effect 972
valifenalate, synthesis 816 wireworm larvae, see Agrotis segetum
vapor-phase activity 790 Working Group (WG)
vegetative insecticidal protein (Vip) 1030 – IRAC 936
velvetbean caterpillar, see Anticarsia gemmatalis World Health Organization (WHO) 936
Venturia inaequalis (apple scab) 776 – pesticides evaluation scheme WHOPES
vertebrate nAChR 482
– selectivity for insect versus vertebrate WRKY transcription factor 912ff.
nAChRs 1145
very long-chain fatty acid (VLCFA) 333 x
– biological function in plant 306 x-ray structure analysis
– biosynthesis 306 – ACC-ase 452
– inhibitor of synthesis 305ff. – acetylcholine-binding protein (AchBP)
– mode of action of inhibitor 308 1135ff., 1142, 1156f., 1185
vigor effect of thiamethoxam 1220 – ADP/ATP carrier protein 575
vine downy mildew (Plasmopara viticola) – AHAS 37ff.
613, 814 – complex III 564
violaxanthin 201 – complex IV 569
Vip3A Cot102 cotton 1037 – Cry proteins 1031f.
VipCot™ cotton 1037 – EcR Heliothis virescense 971
VLCA biosynthesis in plants 306 – EPSPS 411
VLCFA elongase ((VLCFAE)-type elongating – F1Fo-ATP synthase (complex V) 574
enzyme) 306ff., 331ff. – hydroxyphenylpyruvate dioxygenase 229f.
– fiddlehead-like 310 – protoporphyrinogen IX oxidase 182
– phylogenetic tree for Arabidopsis 332 – PS II ubichinon binding niche 479
1498 Subject Index
x-ray structure analysis (contd.) z

– scytalone dehydrase 850ff. ZDS, see ζ -carotene desaturase
– succinate dehydrogenase 578 zeaxanthin 201
Xiphinema americanum 1376 zoospore 833
xylem transportation 604 – Phytophthora infestans 833
zoxamide 548, 739ff.
y – cross-resistance relationship 742
yellow grape mite (Eotetranychus carpini f. – mechanism of action 739
vitis) 1022 – metabolism 746
yellow stem borer (Scirpophaga incertulas) – resistance risk 745
1421 – structure–activity relationship 743
yieldgard rootworm 1035 – synthesis 744f.
yield-losses, crop 57, 67 – toxicology 746

Modern Crop Protection Compounds 2nd 2011

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Modern Crop Protection Compounds 2nd 2011

Uploaded by

Copyright:

Available Formats

Edited by

Wolfgang Krämer, Ulrich Schirmer,

Modern Crop Protection Compounds

Wiley-VCH (ed.) Ziska, Lewis H./Dukes, Jeffrey

Ullmann’s Agrochemicals Weed Biology and Climate

Walters, Dale Tadros, T.F. (ed.)

Plant Defense Colloids in Agrochemicals

Modern Crop Protection Compounds

Second, Revised and Enlarged Edition

Dr. Peter Jeschke British Library Cataloguing-in-Publication

© 2012 Wiley-VCH Verlag & Co. KGaA,

All rights reserved (including those of

Composition Laserwords Private Ltd.,

Printed in the Federal Republic of Germany

Print ISBN: 978-3-527-32965-6

Preface to the Second Edition

The authors have named the products/compounds preferably by their common

December 2011 Wolfgang Krämer

List of Contributors XXV

1 HRAC Classiﬁcation of Herbicides and Resistance Development 5

2 Acetohydroxyacid Synthase Inhibitors (AHAS/ALS) 29

2.1 Biochemistry of the Target and Resistance 29

2.1.1 Acetohydroxyacid Synthase (AHAS) 29

2.1.7 Engineered Resistance to AHAS-Inhibiting Herbicides in Crops 46

2.2 Newer Sulfonylureas 50

2.3 Imidazolinone Herbicides 88

2.4.3.1 Synthesis 106

2.5 Pyrimidinylcarboxylates and Sulfonanilides 117

2.5.1 Introduction 117

2.5.11.1 Biology 139

2.6 Sulfonylaminocarbonyl-Triazolinones 142

2.6.1 Introduction 142

3 Protoporphyrinogen IX Oxidase Inhibitors 163

3.1 Introduction 163

4 Herbicides with Bleaching Properties 197

4.1 Phytoene Desaturase Inhibitors 197

4.1.1 Introduction 197

4.1.2.2 Carotenoids: Properties and Functions 198

4.2 Hydroxyphenylpyruvate Dioxygenase (HPPD): The Herbicide

4.2.1 Herbicidal Mode of Action 225

4.3 Triketones 235

4.3.1 Introduction 235

4.3.7.1 Sulcotrione 249

4.4 Hydroxyphenylpyruvate Dioxygenase (HPPD) Inhibitors:

4.4.1 Introduction 262

5 New Auxin Mimics and Herbicides 277

5.1 The Molecular Mode of Action of Auxin Herbicides 277

5.1.1 Introduction 277

5.2 Aminopyralid 287

5.2.1 Introduction 287

5.2.6.1 Animal Toxicity 293

5.3 Pyrimidine Carboxylic Acids, Aminocyclopyrachlor 295

5.3.1 Introduction 295

6 Herbicides Disturbing the Synthesis of Very Long-Chain Fatty

6.1 Inhibitors of the Synthesis of Very Long-Chain Fatty Acids

6.1.1 Biochemistry 305

6.2 Chemistry and Biology of Oxyacetamides, Tetrazolinones,

6.2.1 Introduction 316

6.3 Isoxazolines 326

6.3.1 Introduction 326

6.3.3 Biological Activities of Pyroxasulfone 328

7 Inhibitors of Cellulose Biosynthesis 339

7.1 Introduction 339

8 Safeners for Herbicides 371