Cutting Edge Organic Synthesis and Chemical Biology of Bioactive Molecules The Shape of Organic Synthesis To Come Yuichi Kobayashi

Cutting Edge Organic Synthesis and
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Yuichi Kobayashi Editor
Cutting-Edge
Organic Synthesis
and Chemical
Biology of Bioactive
Molecules
The Shape of Organic Synthesis to Come
Cutting-Edge Organic Synthesis and Chemical
Biology of Bioactive Molecules
Yuichi Kobayashi
Editor
Cutting-Edge Organic
Synthesis and Chemical
Biology of Bioactive
Molecules
The Shape of Organic Synthesis to Come
Editor
Yuichi Kobayashi
Department of Biotechnology
Tokyo Institute of Technology
Yokohama, Japan
ISBN 978-981-13-6243-9 ISBN 978-981-13-6244-6 (eBook)

https://doi.org/10.1007/978-981-13-6244-6
© Springer Nature Singapore Pte Ltd. 2019

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of
the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,
broadcasting, reproduction on microfilms or in any other physical way, and transmission or information
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The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication
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The publisher, the authors, and the editors are safe to assume that the advice and information in this book
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Singapore
Preface
The purpose of organic synthesis is to provide target compounds by selective meth-

ods with high optical purity. Many transition metal-catalyzed asymmetric reactions
and reactions catalyzed by organocatalysts have been developed to efficiently reach
the goals. Reducing and oxidizing reagents that are highly chemoselective and/or
environmentally friendly are also indispensable tools toward the purpose. On the
other hand, analytical techniques have advanced to allow swift determination of the
efficiency of reactions and the structures. For example, LC-MS analysis equipped
with a chiral column could determine the chirality of the structure. Such new reac-
tions and the advances in analytical techniques produced new power for organic
synthesis to foray into fields, which used to be considered out of organic synthesis.
As a consequence, many interdisciplinary areas have emerged, attracted attention of
chemists, and rapidly grown.
This book was planned to grasp the latest and excellent achievements in organic
synthesis, which are expected to be grown more in the future. On the other hand, the
background of each chapter is explained. I hope that the chapters will be understood
easily by researchers, especially young, and stimulate research in the future.
Chapters “Microbial Fraction Library: A Screening Source for Drug Discovery”,
“Efficient Total Synthesis of Ōmura Natural Products”, “Enantioselective Total
Synthesis of the Antitumor Polycyclic Natural Products FR182877 and Taxol”,
“Synthetic Approaches on the Pluramycin-Class Antibiotics”, “Recent Progress
Toward the Total Synthesis of Duocarmycins A and SA, Yatakemycin, and PDE-I
and –II”, “Structure-Activity Relationship Studies of Maitotoxin Based on Chemical
Synthesis”, “Substitution of Allylic Picolinates with Various Copper Reagents and
Synthetic Applications”, “Total Synthesis of Ingenol” present isolation, elucidation,
and organic syntheses of several compounds that are naturally occurring. Organic
synthesis and biosynthesis of metabolites of polyunsaturated fatty acids are
described in chapters “Strategies for the Synthesis of Anti-inflammatory Metabolites
of Unsaturated Fatty Acids” and “Biosynthesis, Biological Functions, and Receptors
of Leukotriene B4 and 12(S)-Hydroxyheptadecatrienoic Acid”, respectively. Chapter
“Synthesis of Classical/Nonclassical Hybrid Cannabinoids and Related Compounds”
deals with natural and unnatural cannabinoids and would be helpful to design new
v
vi Preface
medical cannabinoids. Chapters “Exploring Bioactive Marine Natural Products and

Identification of Their Molecular Targets”, “Target Protein Chemical Modification”,
“Target Identification of Bioactive Compounds by Photoaffinity Labeling Using
Diazido Probes” explain chemical biology focusing on tools that are designed based
on organic reaction and/or interaction with target proteins.
The project of publishing this book began with an invitation of Mr. S. Koizumi,
Springer Japan. I sincerely acknowledge Ms. A. Komada of Springer and Ms.
M. Shimoda in our laboratory for checking the chapters carefully.
Yokohama, Japan  Yuichi Kobayashi

Contents
1 Microbial Fraction Library: A Screening Source for Drug

Discovery�� 1
Toshihiko Nogawa, Julius Adam V. Lopez, and Hiroyuki Osada
2 Efficient Total Synthesis of Ōmura Natural Products�� 21
Toshiaki Sunazuka, Tomoyasu Hirose, and Satoshi Ōmura
3 Enantioselective Total Synthesis of the Antitumor Polycyclic
Natural Products FR182877 and Taxol�� 49
Masahisa Nakada
4 Synthetic Approaches on the Pluramycin-Class Antibiotics�� 75
Yoshio Ando, Kei Kitamura, Takashi Matsumoto,
and Keisuke Suzuki
5 Recent Progress on the Total Synthesis
of Duocarmycins A and SA, Yatakemycin, and PDE-I
and PDE-II�� 101
Juri Sakata and Hidetoshi Tokuyama
6 Structure-Activity Relationship Studies of Maitotoxin Based
on Chemical Synthesis �� 125
Tohru Oishi
7 Substitution of Allylic Picolinates with Various Copper
Reagents and Synthetic Applications �� 145
Yuichi Kobayashi and Miwa Shimoda
8 Total Synthesis of Ingenol�� 171
Tyler F. Higgins and Jeffrey D. Winkler
9 Strategies for the Synthesis of Anti-inflammatory Metabolites
of Unsaturated Fatty Acids�� 193
Yuichi Kobayashi and Masao Morita
vii
viii Contents
10 Biosynthesis, Biological Functions, and Receptors

of Leukotriene B4 and 12(S)-Hydroxyheptadecatrienoic Acid�� 233
Toshiaki Okuno and Takehiko Yokomizo
11 Synthesis of Classical/Nonclassical Hybrid Cannabinoids
and Related Compounds �� 247
Thanh C. Ho and Marcus A. Tius
12 Exploring Bioactive Marine Natural Products
and Identification of Their Molecular Targets�� 291
Masayoshi Arai
13 Target Protein Chemical Modification�� 305
Hiroyuki Nakamura
14 Target Identification of Bioactive Compounds
by Photoaffinity Labeling Using Diazido Probes�� 335
Suguru Yoshida and Takamitsu Hosoya
List of Figures
Fig. 1.1 Screening strategy of interesting secondary metabolites................. 2

Fig. 1.2 Morphological profiles of HeLa and srcts-NRK cancer
cells treated with pyrrolizilactone.................................................... 5
Fig. 1.3 Concept of the fraction library......................................................... 7
Fig. 1.4 Culture condition and preparation of crude
extracts and fractions....................................................................... 8
Fig. 1.5 A basic concept of 2D separation for the preparation
of fractions....................................................................................... 9
Fig. 1.6 Mass distribution of fractions.......................................................... 9
Fig. 1.7 General activity profile of the fraction library evaluated
at 100 μg/mL.................................................................................... 10
Fig. 1.8 NPPlots and unusual alignments...................................................... 11
Fig. 1.9 NPPlots and specific metabolite group of Streptomyces sp.
RK88–1355...................................................................................... 11
Fig. 1.10 Structure and putative biosynthetic pathway of verticilactam......... 12
Fig. 1.11 Structures and putative biosynthetic pathway of spirotoamides...... 13
Fig. 1.12 Structures of octaminomycins A and B........................................... 14
Fig. 1.13 NPPlot of Streptomyces sp. RK88–1355 and new
antimycin-related metabolites.......................................................... 15
Fig. 1.14 Structural diversity of the antimycin class of metabolites............... 16
Fig. 1.15 Variety of the functional group at C-3 position............................... 17
Fig. 2.1 Our strategy...................................................................................... 22
Fig. 2.2 Structures of novel Ōmura natural products.................................... 23
Fig. 2.3 Structures of pyripyropenes............................................................. 24
Fig. 2.4 Biosynthesis of pyripyropene A....................................................... 24
Fig. 2.5 Structures of pyripyropene analogues.............................................. 26
Fig. 2.6 Structure-activity relationships of pyripyropenes............................ 27
Fig. 2.7 Structure of PP8201 (Afidopyropen)............................................... 27
Fig. 2.8 Structures of arisugacins.................................................................. 28
Fig. 2.9 Computer simulation of arisugacin A 8 docking with AChE........... 30
Fig. 2.10 Structures of lactacystin and salinosporamide A............................. 31
ix
x List of Figures
Fig. 2.11 Regulatory functions of the eukaryotic proteasome and

target for lactacystin......................................................................... 31
Fig. 2.12 Structures of lactacystin analogues.................................................. 32
Fig. 2.13 Inhibitory mode of macrosphelide A on cell adhesion.................... 33
Fig. 2.14 Structures of macrosphelides A and B............................................. 34
Fig. 2.15 Structures of madindolines A 41 and B 42...................................... 36
Fig. 2.16 The mode of action of madindoline A............................................. 39
Fig. 2.17 Structures of neoxaline 59 and oxaline 60....................................... 40
Fig. 2.18 Collaboration between the natural products and the
organic synthesis for drug discovery................................................ 44
Fig. 3.1 Structures of (−)-FR182877 (1) and (−)-FR182876........................ 50
Fig. 3.2 X-ray crystal structure of 29............................................................ 58
Fig. 3.3 Immunostaining images obtained using control (I),
paclitaxel (10 nM) (II), and 31 (200 μM) (III) (left) and
the mitotic indexes (right)................................................................ 59
Fig. 3.4 Structure of (−)-taxol....................................................................... 60
Fig. 4.1 Pluramycins...................................................................................... 77
Fig. 4.2 Hedamycin and conformation of α-C-glycosyl vancosamine.......... 78
Fig. 4.3 Synthetic challenges and lability of the pluramycins...................... 78
Fig. 4.4 Strategies for the skeletal construction of the
pluramycin aglycon.......................................................................... 79
Fig. 4.5 The rare deoxyamino sugars on pluramycins.................................. 84
Fig. 4.6 Platforms for installing bis-C-glycoside.......................................... 90
Fig. 4.7 Further utilities of bis-C-glycosyl monocycles................................ 92
Fig. 5.1 Structures of duocarmycins and the related compounds.................. 103
Fig. 5.2 Proposed mechanism of DNA alkylation......................................... 103
Fig. 5.3 Structures of PDE-I (5) and PDE-II (6)........................................... 104
Fig. 5.4 Proposed structure of yatakemycin (40) by
Igarashi and coworkers.................................................................... 109
Fig. 6.1 Structure of maitotoxin (MTX)........................................................ 126
Fig. 6.2 Structure of brevetoxin B (BTXB)................................................... 126
Fig. 6.3 Hypothesis of mode of action.......................................................... 127
Fig. 6.4 Partial structures of MTX................................................................ 127
Fig. 6.5 Structure of artificial ladder-shaped polyether ALP7B.................... 139
Fig. 7.1 Synthetic targets using the allylic substitution................................. 152
Fig. 7.2 Synthetic targets of the quaternary carbon-forming
allylic substitution............................................................................ 159
Fig. 8.1 (a) Ingenol 1 alongside a 3-D model that highlights the
highly contorted trans-intrabridgehead stereochemistry,
(b) trans- and cis-intrabridgehead stereochemistry in
bicyclo[4.4.1]undecanes, and (c) the Paquette ingenane
analog with cis-intrabridgehead stereochemistry............................. 172
List of Figures xi
Fig. 8.2 (a) Taxol and (b) retrosynthetic approach to

AB system of taxol........................................................................ 173
Fig. 8.3 Taxol AB ring system retrosynthetic analysis via
intramolecular dioxenone photocycloaddition-fragmentation...... 174
Fig. 8.4 (a) Ingenol in which the tricyclic core is highlighted in
blue and (b) retrosynthetic analysis for the synthesis of the
tricyclic core 19............................................................................. 175
Fig. 8.5 (a) Oxygen functionality and unsaturation previously
installed. (b) Oxygen functionality and unsaturation yet
to be installed................................................................................ 177
Fig. 8.6 Models depicting rationale for stereochemistry of
(a) 1,2-addition and (b) cyclization............................................... 185
Fig. 9.1 ω-3 Fatty acids and their metabolites............................................ 194
Fig. 9.2 Metabolites of arachidonic acid.................................................... 195
Fig. 9.3 The substructure of the metabolites in this section....................... 198
Fig. 9.4 RvE1 analogues synthesized by Kobayashi/Ogawa...................... 203
Fig. 9.5 Structural difference of the aspirin-triggered (AT)
and endogenous metabolites.......................................................... 203
Fig. 9.7 Naturally occurring RvE3............................................................. 213
Fig. 9.10 Structural similarity between 5,18-DiHETE and resolvin E2....... 226
Fig. 9.11 Purification of compounds by using a recycle HPLC:
YMC, LC-forte/R, normal phase (silica gel) column
with eluents specified.................................................................... 228
Fig. 10.1 Biosynthesis pathways of leukotriene B4 (LTB4) and LTC4 ......... 235
Fig. 10.2 LTB4 inactivation pathways........................................................... 236
Fig. 10.3 Structure of LTB4 and related fatty acids...................................... 238
Fig. 10.4 PGH2 biosynthesis pathway.......................................................... 240
Fig. 10.5 Biosynthesis and metabolism pathway
of 12-HHT and TxA2 .................................................................... 242
Fig. 11.1 Structural classification of cannabinoids with
representative compounds............................................................. 249
Fig. 11.2 Three pharmacophores and retrosynthetic analysis
of classical tricyclic cannabinoids................................................. 250
Fig. 11.3 By-products detected in the condensation of olivetol with
(+)-p-mentha-2,8-dien-1-ol........................................................... 252
Fig. 11.4 Structures of optically active monoterpenes commonly
used in cannabinoid synthesis....................................................... 253
xii List of Figures
Fig. 11.5 Examples of a classical cannabinoid, a nonclassical

cannabinoid, and a hybrid cannabinoid......................................... 263
Fig. 11.6 Structure of some fused multicyclic cannabinoids........................ 272
Fig. 11.7 Structure of some heterocyclic cannabinoids and
a phenanthrene-derived cannabinoid............................................. 277
Fig. 12.1 Chemical structures of previously discovered
bioactive marine natural products................................................. 294
Fig. 12.2 Inhibition of protein complex formation by furospinosulin-1
(A) and structure of nucleotide probe (B)..................................... 296
Fig. 12.3 Chemical structures of furospinosulin-1 probe
and dummy probe.......................................................................... 297
Fig. 12.4 Expected mechanism of action of furospinosulin-1...................... 298
Fig. 12.5 Summary of the phage display method......................................... 299
Fig. 12.6 Chemical structure (A) and activity (B) of the
dictyoceratin probes...................................................................... 300
Fig. 12.7 Strategy of target identification using M. smegmatis
transformed with genomic DNA library....................................... 302
Fig. 13.1 (a) Pre-translational protein modifications that require
genetic manipulation and (b) posttranslational protein
modification with small chemicals................................................ 306
Fig. 13.2 The photoaffinity functional groups and their
reactive intermediates.................................................................... 308
Fig. 13.3 A series of α-D-mannoside photoaffinity probes conjugated
with an azide (1), a diazirine (2), and a benzophenone (3)........... 309
Fig. 13.4 Typical chemical modification reactions of proteins
at lysine residues........................................................................... 310
at cysteine residues........................................................................ 311
at tyrosine residues........................................................................ 312
Fig. 13.7 Other amino acid residue-specific chemical modifications........... 317
Fig. 13.8 Typical trifunctional chemical probes for
target identification........................................................................ 318
Fig. 13.9 The photoaffinity labeling/photo-cross-linking process
from the non-covalent binding to the detection and identification
of the tagged proteins.................................................................... 318
Fig. 13.10 Post-photoaffinity labeling modification of a
saccharide-binding protein, concanavalin A (ConA).................... 319
Fig. 13.11 The affinity labeling modification followed by the
hydrazone/oxime exchange reaction............................................. 320
Fig. 13.12 Schematic illustration of LDT chemistry for labeling
endogenous proteins in living native cells..................................... 321
List of Figures xiii
Fig. 13.13 Schematic illustration of the strategy for the quenched

ligand-directed tosylate (Q-LDT)-mediated construction of
turn-on fluorescent biosensors....................................................... 321
Fig. 13.14 A fluorescent dye transfer strategy for identifying the
target protein of marinopyrrole A.................................................. 322
Fig. 13.15 Schematic illustration of ligand-directed acyl imidazole
(LDAI) chemistry for selective protein modification.
Lg ligand, Nu nucleophilic amino acid.......................................... 322
Fig. 13.16 Schematic of “turn-on” fluorescent labeling of a target protein
by the O-NBD method. A small bifunctional O-NBD unit
is contained in the ligand. Lg ligand............................................. 323
Fig. 13.17 Ligand-tethered-DMAP-catalyzed acyl transfer
reaction for lectin.......................................................................... 324
Fig. 13.18 Acyl transfer reaction for lectin catalyzed by DMAP................... 324
Fig. 13.19 Catalytic covalent modification of a specific side chain of a
substrate peptide using coiled-coil assembly to drive localization
of a dirhodium metallopeptide. Label subscripts represent
the position, on a helical-wheel model, of key residues that
deviate from the parent E3 or K3 sequences................................. 325
Fig. 13.20 Covalent modifications of various E3gX peptides with the
styryl-diazo reagent 8 by dirhodium metallopeptide
catalysts (K3a,eRh2)...................................................................... 326
Fig. 13.21 LDRP system for target-selective (A) modification and
(B) oxidative inactivation.............................................................. 327
Fig. 13.22 EGFR knockdown using Ru-gefitinib in A431 cells..................... 327
Fig. 13.23 Structures of [Ru(bpy)3]2+-conjugated with the GU40C
peptoid (RuGU40C) and RIP1 (RuRIP1) for CALI targeting
to VEGF and the 26S proteasome, respectively............................ 328
Fig. 13.24 Selective isolation and modification of target proteins................. 330
Fig. 14.1 Chemical methods for target identification................................... 337
Fig. 14.2 Typical photoreactive groups used for PAL probes....................... 338
Fig. 14.3 Typical detectable groups used for PAL probes............................ 339
Fig. 14.4 Diazido probe method................................................................... 340
Fig. 14.5 Diazido PAL probes developed by other groups........................... 341
Fig. 14.6 Photoreaction of an equimolar mixture of azides 1a and 1b......... 342
Fig. 14.7 Cerivastatin (4) and diazido probe photovastatin CAA1 (5)......... 343
Fig. 14.8 1BnTIQ (10) and diazido probe 11............................................... 344
Fig. 14.9 Dantrolene (18) and PAL probes 19 and 20.................................. 346
Fig. 14.10 Synthesis of biaryl-type diazido compounds using
borylated building block 21........................................................... 347
Fig. 14.11 Synthesis of aryl azides 24 by the formal C–H azidation............. 348
Fig. 14.12 Diazido building blocks prepared from diazido
compounds 14, 27, and 28............................................................. 350
xiv List of Figures
Fig. 14.13 Other bifunctional PAL probe candidates of dantrolene............... 351

Fig. 14.14 Bifunctional PAL probe and unit bearing
(ethynyldifluoromethyl)diazirinyl group....................................... 352
Fig. 14.15 Bifunctional PAL probe library..................................................... 353
Fig. 14.16 Bifunctional PAL probe 53 of palmitic acid (54).......................... 353
Fig. 14.17 Minimalist photo-crosslinkers....................................................... 354
Fig. 14.18 Minimal all-in-one PAL unit......................................................... 354
Fig. 14.19 Commercially available building blocks for bifunctional
PAL probe synthesis...................................................................... 354
List of Schemes
Scheme 2.1 Retrosynthetic analysis of pyripyropene A�� 25

Scheme 2.2 Total synthesis of pyripyropene A�� 26
Scheme 2.3 Total synthesis of arisugacin A (part 1)�� 28
Scheme 2.4 Total synthesis of arisugacin A (part 2)�� 29
Scheme 2.5 Total synthesis of lactacystin�� 32
Scheme 2.6 Total synthesis of macrosphelide A�� 35
Scheme 2.7 Strategy for a combinatorial synthesis of macrosphelide
analogues�� 35
Scheme 2.8 Asymmetric oxidative ring closure of tryptophol�� 36
Scheme 2.9 First-generation strategy for total synthesis of
madindolines�� 37
Scheme 2.10 Second-generation strategy for total synthesis of
madindolines�� 38
Scheme 2.11 The synthesis of the indoline spiroaminal framework
of neoxaline (part 1)�� 41
Scheme 2.12 The synthesis of the indoline spiroaminal framework
of neoxaline (part 2)�� 41
Scheme 2.13 Synthesis of cyclization precursor�� 42
Scheme 2.14 Construction of indoline spiroaminal�� 43
Scheme 2.15 Total synthesis of neoxaline�� 44
Scheme 3.1 Sorensen’s and Evans’ total syntheses of (−)-
FR182877 (1)�� 51
Scheme 3.2 Construction of the AB-ring moiety by the
stereoselective IMDA reaction�� 51
Scheme 3.3 Construction of the CD-ring moiety by the
stereoselective IMHDA reaction�� 52
Scheme 3.4 Transformation of 2 to 7 via the stereoselective
IMDA-IMHDA reaction cascade�� 52
Scheme 3.5 Stereoselective reduction of 7 (Table 3.1)�� 53
Scheme 3.6 Preparation of 10�� 53
Scheme 3.7 The palladium-catalyzed 7-exo-trig cyclization
of 10a and 10b (Table 3.2)�� 54
xv
xvi List of Schemes
Scheme 3.8 Possible pathway from 11 to 13a and energetic difference

between 13a and 13b calculated by the PM3 method�� 55
Scheme 3.9 Isomerization of allylic alcohol 11 to α-methyl ketone 13a
(Table 3.3)�� 55
Scheme 3.10 Enantioselective total synthesis of (−)-FR182877�� 56
Scheme 3.11 Structures of (−)-FR182877 (1) and 17, and retrosynthetic
analysis of 17�� 57
Scheme 3.14 Preparation of 17 and 29–32�� 58
Scheme 3.15 Formation of the eight-membered ring of taxol in the
convergent synthesis�� 61
Scheme 3.16 Intramolecular B-alkyl Suzuki-Miyaura coupling
reactions of 33 and 34�� 62
Scheme 3.17 Retrosynthetic analysis of (−)-taxol�� 63
Scheme 3.18 Preparation of the A-ring fragment 41-I from 43�� 63
Scheme 3.19 Enantioselective preparation of 47-I from 46-I via
organocatalysis�� 63
Scheme 3.20 The SAD of 49-I, 50-I, and 50-Br�� 64
Scheme 3.21 Transformation of 51-Br to 41-Br via 52�� 65
Scheme 3.22 Attempted aldol reaction and Nozaki-Hiyama-type
reaction and envisioned reliable stereoselective coupling
of 41 and 54 to afford 55�� 65
Scheme 3.23 Preparation of 54 via the baker’s yeast reduction of 56�� 65
Scheme 3.24 Assembly of the A- and C-ring fragments and the
intramolecular B-alkyl Suzuki-Miyaura coupling
reaction of 60�� 66
Scheme 3.25 Formation of 63 by palladium-catalyzed alkenylation
of methyl ketone 62�� 67
Scheme 3.26 Preparation of methyl ketone 68 via 1,5-hydride shift�� 68
Scheme 3.27 Formal total synthesis of (−)-taxol
via the palladium-catalyzed alkenylation of 68�� 68
Scheme 3.28 Kuwajima successful epimerization of 75 and our
failed example�� 69
Scheme 3.29 Transformation of 73 to Nicolaou’s advanced synthetic
intermediate 79�� 70
Scheme 4.1 Synthesis of O-methyl kidamycinone�� 80
Scheme 4.2 Total synthesis of γ-indomycinone�� 80
Scheme 4.3 Total synthesis of (S)-espicufolin�� 80
Scheme 4.4 Friedel–Crafts cyclization in the synthetic study of
kidamycin�� 81
Scheme 4.6 Total synthesis of AH-1763 IIa�� 82
Scheme 4.7 Total synthesis of BE-26554A and heraclemycin B�� 82
List of Schemes xvii
Scheme 4.8 Total synthesis of (R)-espicufolin�� 83

Scheme 4.9 Synthesis of pluraflavin A aglycon�� 84
Scheme 4.11 Baer and Georges synthesis�� 85
Scheme 4.12 Brimble synthesis�� 85
Scheme 4.13 McDonald synthesis�� 86
Scheme 4.14 Suzuki synthesis�� 86
Scheme 4.15 Kahne degradation�� 87
Scheme 4.16 Giuliano synthesis�� 87
Scheme 4.17 Nicolaou synthesis�� 88
Scheme 4.18 McDonald synthesis�� 89
Scheme 4.19 Parker synthesis�� 89
Scheme 4.20 Doi and Takahashi synthesis�� 89
Scheme 4.21 Suzuki synthesis�� 90
Scheme 4.22 O→C-Glycoside rearrangement�� 91
Scheme 4.23 Bis-C-glycosidation of monocyclic platforms�� 92
Scheme 4.24 Reverse polarity strategy for construction
of bis-C-glycoside�� 93
Scheme 4.25 Synthetic study of kidamycin�� 94
Scheme 4.26 Synthetic study of pluraflavin A�� 94
Scheme 4.27 Total synthesis of isokidamycin�� 95
Scheme 4.28 Total synthesis of isokidamycin�� 96
Scheme 4.29 Total synthesis of saptomycin B�� 98
Scheme 5.1 Copper-mediated intramolecular aryl amination�� 105
Scheme 5.2 Synthesis of indoline 13�� 105
Scheme 5.3 Preparation of left-hand segment 18�� 106
Scheme 5.4 Terashima’s synthesis of right-hand segment 22�� 106
Scheme 5.5 Preparation of right-hand segment 28�� 107
Scheme 5.6 Fukuyama’s total synthesis of (+)-duocarmycin A (1)�� 107
Scheme 5.7 Fukuyama’s total synthesis of (+)-duocarmycin SA (2)�� 108
Scheme 5.8 Synthesis of the left-hand segment of the putative
yatakemycin (40)�� 110
Scheme 5.9 Boger’s synthesis of proposed yatakemycin (40)�� 111
Scheme 5.10 Initial structural revision of yatakemycin�� 111
Scheme 5.11 Boger’s synthesis of revised left-hand segment 60�� 112
Scheme 5.12 The second structural revision of yatakemycin�� 112
Scheme 5.13 Boger’s total synthesis of (+)-yatakemycin (4)�� 113
Scheme 5.14 Synthesis of the middle segment 73�� 114
Scheme 5.15 Synthesis of left-hand segment 60�� 114
Scheme 5.16 Boger’s second-generation total synthesis of
(+)-yatakemycin (4)�� 115
Scheme 5.17 Synthesis of middle segment 87�� 116
Scheme 5.18 Synthesis of amino alcohol 93�� 116
Scheme 5.19 Synthesis of left-hand segment 100�� 117
xviii List of Schemes
Scheme 5.20 Synthesis of right-hand segment 105�� 118

Scheme 5.21 Fukuyama’s total synthesis of (+)-yatakemycin (4)�� 118
Scheme 5.22 Tokuyama’s synthetic strategy based on their
copper-mediated double amination�� 119
Scheme 5.23 Preparation of the substrate for the double
amination reaction�� 120
Scheme 5.24 Result of the initial trial of the double
arylamination�� 120
Scheme 5.25 Proposed reaction pathway�� 121
Scheme 5.26 Tokuyama’s first-generation synthesis of
PDE-II (6)�� 121
Scheme 5.27 Tokuyama’s synthesis of PDE-I (5)�� 122
Scheme 5.28 Tokuyama’s second-generation synthesis of
PDE-II (6)�� 122
Scheme 6.1 Convergent method via two-rings construction (1):
α-cyano ether method�� 128
Scheme 6.2 Convergent method via two-rings construction (2)�� 129
Scheme 6.3 Synthetic methods of 6/6/6-tricyclic ether systems
based on Achmatowicz reaction�� 130
Scheme 6.4 Convergent synthesis of the WXYZ ring (1)�� 130
Scheme 6.5 Convergent synthesis of the WXYZ ring (2)�� 131
Scheme 6.6 Convergent synthesis of the WXYZA′B′C′ ring�� 133
Scheme 6.7 Synthesis of the QRS ring�� 134
Scheme 6.8 Synthesis of the C′D′E′F′ ring�� 135
Scheme 6.9 Synthesis of the LMNO and ent-LMNO rings�� 136
Scheme 6.10 Synthesis of the NOPQR(S) ring�� 138
Scheme 7.1 Allylic substitutions using prim- and sec-allylic esters�� 146
Scheme 7.2 Allylic substitution of secondary allylic esters
published by 2007�� 147
Scheme 7.3 Picolinoxy (Pic) leaving group for allylic substitution�� 148
Scheme 7.4 A double activation of the Pic group�� 149
Scheme 7.5 Preliminary results using various secondary allylic
esters with phenylmetal reagents: Table 7.1�� 149
Scheme 7.6 Allylic substitution of enantioenriched picolinates
with Grignard-based copper reagents�� 150
Scheme 7.7 Substitution with PhLi-based copper reagents:
Table 7.2�� 150
Scheme 7.8 Allylic substitution with RLi-based copper reagents�� 151
Scheme 7.9 1,4-Addition reactions of classical copper reagents
to enone�� 152
Scheme 7.10 Synthesis of sesquichamaenol�� 152
Scheme 7.11 Synthesis of equol�� 153
Scheme 7.12 Synthesis of the inhibitor of ACAT�� 154
Scheme 7.13 Synthesis of the bioactive form of loxoprofen�� 155
List of Schemes xix
Scheme 7.14 Synthesis of trans-2,6-disubstituted cyclohexanones�� 156

Scheme 7.15 Allylic substitution with alkynyl reagents�� 157
Scheme 7.16 Synthesis of the Stork’s prostaglandin intermediate 61�� 158
Scheme 7.17 Quaternary carbon-forming allylic substitution with
Ph and n-Bu copper reagents: Table 7.3�� 158
Scheme 7.18 Formation of enantioenriched quaternary carbons
with Ph copper reagents derived from PhMgBr or PhLi�� 159
Scheme 7.19 Synthesis of mesembrine�� 160
Scheme 7.20 Synthesis of the verapamil intermediate�� 161
Scheme 7.21 Synthesis of LY426965�� 162
Scheme 7.22 Construction of a quaternary carbon on a
cyclohexane and possible targets�� 162
Scheme 7.23 Construction of a quaternary carbon: Table 7.4�� 163
Scheme 7.24 Conformation-controlled allylic substitution�� 164
Scheme 7.25 Synthesis of cyclobakuchiols A and B�� 165
Scheme 7.26 Synthesis of anastrephin�� 166
Scheme 7.27 Synthesis of axenol�� 167
Scheme 8.1 Baldwin’s dioxenone intermolecular

photocycloaddition-fragmentation�� 173
Scheme 8.2 Synthesis of trans-bicyclo[5.3.1]undecane�� 174
Scheme 8.3 Synthesis of the tricyclic core of the ingenane
ring system�� 175
Scheme 8.4 Synthesis of 3-oxygenated ingenane diterpenes�� 176
Scheme 8.5 Synthesis of the first biologically active
ingenane analog�� 177
Scheme 8.6 Elaboration of the A and B rings of the ingenane skeleton�� 178
Scheme 8.7 Retrosynthetic analysis for D ring incorporation�� 179
Scheme 8.8 Attempted elimination of the mesylate of the C-14
hydroxyl produced an unexpected transannular
aldol reaction�� 180
Scheme 8.9 Incorporation of the D ring cyclopropane of ingenol�� 181
Scheme 8.10 First total synthesis of ingenol�� 181
Scheme 8.11 Completion of the first total synthesis of ingenol�� 182
Scheme 8.12 Tanino and Kuwajima’s original approach
to ingenane synthesis�� 184
Scheme 8.13 Synthesis of ingenane skeleton 79 via key
Me3Al-mediated rearrangement�� 184
Scheme 8.14 Functionalization of A and B rings of ingenol�� 185
Scheme 8.15 Completion of Tanino and Kuwajima’s ingenol synthesis�� 186
Scheme 8.16 Ring-closing metathesis approach to the synthesis
of ingenol�� 187
Scheme 8.17 Improved ring-closing metathesis toward
the synthesis of ingenol�� 187
Scheme 8.18 Functionalization of the A ring of ingenol�� 188
xx List of Schemes
Scheme 8.19 Completion of Wood’s ingenol synthesis�� 189

Scheme 8.20 Baran’s retrosynthetic analysis of ingenol�� 190
Scheme 8.21 Cyclase phase for the Baran synthesis of ingenol�� 190
Scheme 8.22 Oxidase phase and completion of Baran’s
ingenol synthesis�� 191
Scheme 9.1 Construction of conjugated olefins�� 196
Scheme 9.2 Synthesis of E-iodo olefins�� 197
Scheme 9.3 Synthesis of RvE1 methyl ester by Petasis/Serhan�� 199
Scheme 9.4 Improved synthesis of RvE1 by Schwartz�� 199
Scheme 9.5 Synthesis of key intermediates�� 200
Scheme 9.6 Synthesis of the three intermediates 36, 40, and 45�� 201
Scheme 9.7 Synthesis of RvE1 by Kobayashi/Ogawa�� 202
Scheme 9.8 Improved synthesis of RvD3 by Petasis/Serhan�� 204
Scheme 9.9 Synthesis of PD1 and (18R)-PD1 (AT-PD1)
by Petasis/Serhan�� 205
Scheme 9.10 Preparation of intermediates 60 and 61�� 205
Scheme 9.11 Synthesis of PD1 by Kobayashi/Ogawa�� 206
Scheme 9.12 Preparation of intermediates 70 and 72�� 206
Scheme 9.13 Synthesis of the PD1 intermediates by Spur�� 207
Scheme 9.14 Synthesis of PD1 by Hansen�� 207
Scheme 9.15 Synthesis of 10-epi-PD1 methyl ester by Balas�� 208
Scheme 9.16 Synthesis of MaR1 and (7S)-isomer by Inoue/Arita
using Julia-Kocienski coupling�� 209
Scheme 9.17 Synthesis of MaR1 by Kobayashi/Ogawa
via Suzuki-Miyaura coupling�� 210
Scheme 9.18 Synthesis of MaR1 by Spur via Sonogashira coupling�� 210
Scheme 9.19 Synthesis of MaR1 by Hansen via Sonogashira coupling�� 211
Scheme 9.20 Three approaches to RvD4�� 212
Scheme 9.21 Synthesis of RvD4 intermediates 127 and 129�� 212
Scheme 9.22 Synthesis of RvD4 by Kobayashi/Morita�� 213
Scheme 9.23 Kinetic stability of a dihydroxy acid�� 213
Scheme 9.24 Synthesis of RvE3 by Inoue/Arita�� 214
Scheme 9.25 Synthesis of RvE3 by Kobayashi�� 215
Scheme 9.26 Synthesis of MaR2 by Spur�� 215
Scheme 9.27 Synthesis of RvD1 and RvD2 by Spur�� 216
Scheme 9.28 Synthesis of RvD2 by Rizzacasa�� 216
Scheme 9.29 Synthesis of RvD1 by Kobayashi/Morita�� 217
Scheme 9.30 Approaches to RvE2�� 218
Scheme 9.31 Synthesis of the intermediates for synthesis of RvE2�� 219
Scheme 9.32 Synthesis of RvE2 by Inoue/Arita�� 219
Scheme 9.33 Synthesis of RvE2 by Kobayashi/Ogawa�� 220
Scheme 9.34 Ozonolysis of E- and Z-allylic alcohol derivatives
(R = Et, i-Pr, C5H11)�� 220
Scheme 9.35 Synthesis of (14R,15S)-diHETE by Kobayashi�� 221
List of Schemes xxi
Scheme 9.36 Synthesis of RvE2 by Spur�� 221

Scheme 9.37 Synthesis of RvE2 by Shuto�� 221
Scheme 9.38 Synthesis of RvD6 by Spur�� 222
Scheme 9.39 Approaches to RvD5�� 222
Scheme 9.40 Synthesis of RvD5 by Spur�� 223
Scheme 9.41 Synthesis of RvD5 by Kobayashi/Ogawa/Morita�� 223
Scheme 9.42 Synthesis of (14S,20R)-dihydroxy-DHA by Inoue/Arita�� 224
Scheme 9.43 Synthesis of (17R,18S)-epoxy-(12S)-hydroxy-EPA
by Inoue/Arita�� 224
Scheme 9.44 Synthesis of (14S,21R)-DiHDHA by Kobayashi/
Morita/Hong�� 225
Scheme 9.45 Synthesis of Maresin-like 1 by Kobayashi et al.�� 226
Scheme 9.46 Retrosynthesis of (5S,18R)-DiHETE�� 226
Scheme 9.47 Synthesis of HHTE and 12S-HHT�� 227
Scheme 11.1 Synthesis of (−)-Δ8- and (−)-Δ9-THCs
from (−)-verbenol�� 251
Scheme 11.2 Synthesis of (−)-Δ8- and (−)-Δ9-THCs
from (+)-p-mentha-2,8-dien-1-ol�� 251
Scheme 11.3 Synthesis of AM-411�� 253
Scheme 11.4 Synthesis of AMG-3�� 253
Scheme 11.5 Synthesis of HU-210�� 254
Scheme 11.7 Synthesis of ajulemic acid�� 255
Scheme 11.8 Synthesis of nabilone�� 255
Scheme 11.9 Condensation of resorcinols with diacetates 40 and 41�� 256
Scheme 11.10 Synthesis of AM-993 and AM-994�� 257
Scheme 11.11 Synthesis of AM-2389 and canbisol�� 257
Scheme 11.12 Synthesis of 9β-hydroxymethyl-HHC from HU-210�� 257
Scheme 11.14 Synthesis of a 9-ketocannabinoid bearing a non-bulky
C3 side chain�� 259
Scheme 11.15 Synthesis of a series of hexahydrocannabinols
via a common intermediate�� 259
Scheme 11.16 Structures of (−)-CP-47,497 and (−)-CP-55,244
and the synthesis of (−)-CP-55,940�� 260
Scheme 11.17 Synthesis of KLS-13019�� 261
Scheme 11.18 Synthesis of PRS-211,375�� 261
Scheme 11.21 Synthesis of (−)-6β-hydroxymethyl-Δ9-THC�� 263
Scheme 11.22 C6-diastereoselective synthesis of hybrid
cannabinoids�� 264
Scheme 11.23 Total synthesis of hybrid cannabinoid AM-919
and derivatives�� 265
xxii List of Schemes
Scheme 11.24 Synthesis of hybrid cannabinoid 130 via hetero-

Diels−Alder reaction�� 267
Scheme 11.25 Preparation of the aliphatic aldehyde 125�� 267
Scheme 11.26 Preparation of resorcinol 31�� 268
Scheme 11.28 Synthesis of aliphatic aldehyde 140�� 269
Scheme 11.29 Synthesis of acetophenone 141�� 270
Scheme 11.31 Synthesis of C3-adamantyl hybrid cannabinoids via
oxymercuration-demercuration�� 271
Scheme 11.32 Synthesis of C3-adamantyl hybrid cannabinoids via
hetero-Diels−Alder cyclization�� 271
Scheme 11.33 Synthesis of tetracyclic analogs of Δ8-THC�� 272
Scheme 11.34 Synthesis of rotationally restricted
tetrahydrocannabinol ethers�� 273
Scheme 11.35 Synthesis of some cannabinoid quinones�� 274
Scheme 11.36 Synthesis of cannabinoid quinones via photooxygenation�� 274
Scheme 11.37 Synthesis of cannabinol via von Pechmann condensation�� 275
Scheme 11.38 Synthesis of cannabilactones via Suzuki
coupling reaction�� 275
Scheme 11.39 Synthesis of C-ring cannabinoid lactones�� 276
Scheme 11.40 Asymmetric synthesis of levonantradol�� 277
Scheme 11.41 Synthesis of a pentacyclic hybrid cannabinoid�� 278
Scheme 11.42 Structure of rimonabant and the synthesis
of chromenopyrazoles�� 279
Scheme 11.43 Synthesis of chromenopyrazolediones�� 280
Scheme 11.44 Synthesis of chromenoisoxazoles�� 281
Scheme 11.45 Structures of resorcinol−AEA hybrids and the
synthesis of representative compound CB-25�� 281
Scheme 11.46 Synthesis of O-2220�� 282
Scheme 11.47 Structures of 2-AG and the synthesis of a resorcinol−2-
AG hybrid�� 282
Scheme 13.1 The photoredox mechanism of the tyrosine residue-specific
modification reaction catalyzed by ruthenium complex
([Ru(bpy)3]2+)�� 313
Scheme 13.2 PTAD-based chemical modification�� 314
Scheme 13.3 Luminol-based tyrosine residue-specific chemical
modification�� 315
Scheme 13.4 MAUra-based tyrosine residue-specific chemical
modification�� 315
Scheme 13.5 Strain-promoted cycloaddition of 1,2-quinones
converted from tyrosine residues specifically oxidized
by mushroom tyrosinase�� 316
Scheme 14.1 Photoreaction of triazido compound 2�� 342
List of Schemes xxiii
Scheme 14.2 Synthesis of photovastatin CAA1 (5)�� 343

Scheme 14.3 PAL experiment using photovastatin CAA1 (5) and
recombinant HMGR�� 344
Scheme 14.4 Synthesis of diazido PAL probe 11�� 345
Scheme 14.5 PAL experiment using diazido probe 11 in subcellular
fractions of rat whole brain homogenates�� 345
Scheme 14.6 Facile synthesis of various diazido building blocks
bearing connecting groups (CGs)�� 348
Scheme 14.7 Synthesis of diazido compounds 14 and 25�� 349
Scheme 14.8 Synthesis of diazido compounds 27 and 28�� 349
Scheme 14.9 Photoreaction of diazirine 40a�� 351
Scheme 14.10 Photoreaction of diazirine 40b�� 351
Scheme 14.11 Synthesis of 3-aryl-3-(azidodifluoromethyl)diazirine
unit 48�� 352
Scheme 14.12 Photoreactions of aryl(azidodifluoromethyl)diazirine 48�� 352
List of Tables
Table 1.1 Pros/cons of biological and chemical screenings�� 2

Table 1.2 Pros/cons of natural products and synthetic compound�� 3
Table 3.1 Stereoselective reduction of 7 (Scheme 3.5)�� 53
Table 3.2 The palladium-catalyzed 7-exo-trig cyclization of 10a and
10b (Scheme 3.7)�� 54
Table 3.3 Isomerization of allylic alcohol 11 to α-methyl ketone 13a
(Scheme 3.9)�� 55
Table 6.1 Inhibitory activity of the synthetic specimens against
Ca2+ influx induced by MTX�� 138
Table 7.1 Results of Scheme 7.5: Preliminary results using various
secondary allylic esters with phenylmetal reagents�� 149
Table 7.2 Results of Scheme 7.7: Substitution of rac-12a with
PhLi-based copper reagents�� 150
Table 7.3 Results of Scheme 7.17: Quaternary carbon-forming
allylic substitution�� 158
Table 7.4 Results of Scheme 7.23: Feasibility study of the quaternary
carbon construction�� 163
xxv
Chapter 1
Microbial Fraction Library: A Screening
Source for Drug Discovery
Toshihiko Nogawa, Julius Adam V. Lopez, and Hiroyuki Osada
Abstract Natural products are an important source for drug screening because of
their diverse chemical properties and biological activities. However, there are sev-
eral drawbacks in this field such as the duplicate isolation of previously reported
metabolites and the inherent difficulty of obtaining a pure compound with signifi-
cant bioactivity, which requires considerable amount of work, time, and resources.
Researchers are continuously devising ways to efficiently discover and isolate novel
compounds by developing new screening and sample preparation methods. In this
chapter, the current situation of microbial product research is described by introduc-
ing screening and sample preparation methods with focus on fraction libraries
including our own strategy and discoveries.
Keywords Natural product · Microbial metabolite · Fraction library · Screening ·

Antimycin · Biological activity
1 Introduction
Natural products have been used as a source for the screening of drugs and drug
leads [1]. In particular, microbial secondary metabolites not only have extensive
structural diversity but also various biological activities, many of which have been
developed as drugs, pesticides, and agrochemicals [2–4]. They also have an impor-
tant role in chemical biology studies as bioprobes, which are chemical tools for the
investigation of biological functions [5, 6]. Although the versatility of natural prod-
ucts holds promise for drug discovery research, in reality, it accompanies several
challenges. A major difficulty lies in the sample screening and the isolation and
purification of compounds from natural sources such as microbial culture broths
and plant extracts.
T. Nogawa · J. A. V. Lopez · H. Osada (*)

RIKEN Center for Sustainable Resource Science, Chemical Biology Research Group,
Wako, Saitama, Japan
e-mail: hisyo@riken.jp
© Springer Nature Singapore Pte Ltd. 2019 1

Y. Kobayashi (ed.), Cutting-Edge Organic Synthesis and Chemical Biology
of Bioactive Molecules, https://doi.org/10.1007/978-981-13-6244-6_1
2 T. Nogawa et al.
The screening methods of natural products are divided into two approaches: bio-
logical and chemical screenings (Fig. 1.1, Table 1.1). Biological screening is based
on a biological activity test, for example, antimicrobial activity or cytotoxicity
against tumor cells, and focuses on searching biologically active compounds. Using
a biological screening throughout the separation process of natural sources, such as
a microbial broth, to isolate metabolites with specific activity is called activity-
guided isolation. The emergence of high-throughput screening (HTS) technology
Natural sources
(broths/extracts)
Biological Chemical
screening screening
For interesting activity For novel structure
Phenotypic Target-based
LC/MS NMR
screening screening
Fig. 1.1 Screening strategy of interesting secondary metabolites
Table 1.1 Pros/cons of biological and chemical screenings

Screening methods Pros Cons
Biological Phenotypic Discovery of activity with unknown Necessity to identify
screening screening mode of action the molecular target
Confirmation of activity against Low throughput
whole organism
Target-based Easy to apply to HTS Uncertainty on the
screening specific activity
Focus on specific activity Unexpected side
effects/off-target effects
Chemical LC/MS Applicable for most of hydrophobic Difficult to apply to
screening compounds polar compounds
Applicable for complex mixtures by
LC separation
High sensitivity Not applicable to
High throughput non-ionized
compounds
NMR Applicable for most compounds Low sensitivity
Obtainable precise structural No separation system
information such as specific like LC
functional groups Low throughput
1 Microbial Fraction Library: A Screening Source for Drug Discovery 3
has greatly improved the speed of biological screening in terms of the large number
of samples that can be tested in a single run. Nevertheless, such large number of
samples does not guarantee the discovery of first-in-class drug leads. For example,
a synthetic compound library created via combinatorial chemistry is commonly
applied to HTS to secure a large set of compounds; however, this does not assure
structural diversity compared to natural products and may contain unsuitable com-
pounds as drug candidates. In addition, not all samples can be easily applied to the
HTS format, especially natural products, because it is difficult to maintain a large
number of purified and high-quality sample set (Table 1.2) [7, 8]. On the other hand,
chemical screening is a structure-oriented approach that focuses on properties that
give information about compound structure such as UV and IR absorption spectra,
molecular weight, and other spectroscopic data. The corresponding spectroscopic
techniques are spectrophotometry, liquid chromatography/mass spectrometry (LC/
MS), and nuclear magnetic resonance (NMR). The major drawback of this approach
is the discovery of new compounds without significant bioactivity. Moreover,
whether implementing biological or chemical screening, the isolation process
entails several steps such as extraction, partition, and possibly many types of chro-
matography including high-performance liquid chromatography (HPLC) on nor-
mal- and reversed-phase modes, to obtain a pure compound. Also, the limited
amount of isolated compounds oftentimes hinders their full structural and bioactiv-
ity characterization. Clearly, the whole process from raw sample to pure compound
takes a lot of time, work, and resources.
These issues have caused drug discovery research to shift from natural products
to synthetic compound libraries prepared by combinatorial chemistry. However,
these synthetic compound libraries fail to represent the entire chemical space, which
is defined as “all possible small organic molecules, including those present in bio-
logical systems” [9]. Using statistics to compare chemical properties, natural prod-
ucts were significantly similar to developed drugs, while the distribution of synthetic
compound libraries was limited, suggesting that natural products are more suitable
for drug screening. Accordingly, many of the drugs developed in the past 30 years
are natural products or synthetic compounds based on or inspired by natural products
Table 1.2 Pros/cons of natural products and synthetic compound

Sources Pros Cons
Natural Structural diversity Time-consuming work for isolation
product and purification of compounds
Variety of biological activity Re-isolation of known compounds
Discovery of unexpected structures and Difficulty to secure a large set of
biological activity compound library for HTS
Synthetic Simple structures and low molecular Relatively low structural diversity
compound weight
Easy to secure a large set of compound
library for HTS
Focused library can be prepared for
specific structures and biological activity
4 T. Nogawa et al.
[1]. Therefore, natural products continue to be a vital source and may be more pro-
ductive sources for drug screening [10] once we can find a way to overcome the
challenges mentioned above.
In this chapter, screening methods based on bioactivity and chemical structure
are described in brief followed by a discussion on natural product fraction libraries
and our own strategy and findings.
2 Biological Screening
Biological activity screening has been a major way of discovering bioactive natural
products, and many of medicinally important compounds have been discovered in
this manner [11]. It is mainly categorized into phenotypic and target-based screen-
ings. Phenotypic screening focuses on the identification of compounds or molecules
that cause changes to the phenotypes of cells, tissues, or whole organisms without
prior knowledge of any specific targets. Another form of phenotypic screening is
high-content screening (HCS), wherein multiple information such as spatial distri-
bution and morphology can be recorded in different timescales using automated
imaging [12]. Meanwhile, target-based screening operates on a putative or known
target such as a protein and searches for compounds that will induce an effect on it
[13–15].
Between 1999 and 2008, 23% and 37% of first-in-class drugs approved by the
US Food and Drug Administration (FDA) were products of target-based and phe-
notypic screenings, respectively [13]. Later on, Eder et al. reported the contrary
that between 1999 and 2013, 69% and 7% of FDA-approved first-in-class drugs
were from target-based and phenotypic approaches, respectively [16]. However,
the authors pointed out that phenotypic screening is not inferior to but rather a
complement to target-based screening and further suggested it to be a new disci-
pline [15, 16].
In this point of view and in our search for new anticancer compounds, we have
developed a phenotypic screening system based on cell morphology changes and
created a database called MorphoBase [17, 18]. MorphoBase contains the data of
morphological changes of various cancer cell lines treated with over 200 well-
characterized drugs. It is anticipated that a new molecular mechanism of action will
be revealed upon detection of a unique cell morphology induced by a new agent in
comparison with existing profiles in the database. In the course of our phenotypic
screening, pyrrolizilactone – a novel fungal metabolite characterized by a unique
ketone-linked pyrrolizidinone and decalin structure – was found. By using
MorphoBase, pyrrolizilactone was classified as a proteasome inhibitor, and this was
later confirmed by a proteome-based profiling analysis (Fig. 1.2) [19, 20]. Based on
these facts, biological screening is an integral part of natural product research.
1 Microbial Fraction Library: A Screening Source for Drug Discovery 5
Fig. 1.2 Morphological profiles of HeLa and srcts-NRK cancer cells treated with
pyrrolizilactone
3 Chemical Screening
Nowadays, LC/MS and NMR spectrometers are routine analytical instruments.

Upgrades are continually being developed to acquire higher sensitivity and resolu-
tion. In minutes, one can obtain precise structural information of a trace amount of
compound by a single analysis of a complex mixture with no separation and purifi-
cation, which is suitable for the fast screening of structurally interesting compounds.
Each technique has its advantages and disadvantages (Table 1.1) [21].
LC/MS is a method of choice due to its high sensitivity and resolution and the
separation that LC provides. With the development of ultrahigh-performance liquid
chromatography (UHPLC), higher throughput was achieved by utilizing specialized
columns that can withstand high pressures resulting to higher resolution and shorter
run times. For example, a typical 30-min HPLC run can be done in 5 min or less
with UHPLC. Various mass spectrometers are available for LC/MS such as quadru-
pole (Q), ion trap (IT), time-of-flight (Tof), Fourier-transform (FT), as well as tan-
dem or hybrid systems which include triple quadrupole (TQ or QqQ), quadrupole +
time of flight (QTof), and quadrupole + ion trap (QTrap). In addition, different ion
sources can be used such as electron impact (EI), electrospray ionization (ESI),
atmospheric pressure chemical ionization (APCI), and matrix-assisted laser desorp-
tion/ionization (MALDI), in which ESI is the preferred option for sample screening
because it can ionize most compounds in the liquid form leading to high compatibil-
ity with typical LC systems [22, 23]. In our laboratory, we use a UHPLC-ESI-QqQ
to generate mass data for compound libraries and an LC/ESI-QTof for high-
resolution measurements in both MS and MS/MS modes, which afford accurate
estimation of the chemical formula of molecular and fragment ions, respectively.
The determination of the exact molecular formula is the initial and a critical step in
the structure elucidation of a compound. Some issues associated with LC/MS are
non-ionizable and highly polar compounds, although ambient mass spectrometry
(AMS) with dual ionization source and hydrophilic interaction chromatography
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FIRST ORDER: CHONDROPTERYGII.
Skeleton cartilaginous. Body with medial and paired fins, the
hinder pair abdominal. Vertebral column generally heterocercal, the
upper lobe of the caudal fin produced. Gills attached to the skin by
the outer margin, with several intervening gill-openings: rarely one
external gill-opening only. No gill-cover. No air-bladder. Two, three,
or more series of valves in the conus arteriosus. Ova large and few
in number,[33] impregnated and, in some species, developed within a
uterine cavity. Embryo with deciduous external gills.[34] Males with
intromittent organs attached to the ventral fins.[35]
This order, for which, also, the name Elasmobranchii has been
proposed (by Bonaparte), comprises the Sharks and Rays and
Chimæras, and is divided into two suborders: Plagiostomata and
Holocephala.
FIRST SUB-ORDER: PLAGIOSTOMATA.

From five to seven gill-openings. Skull with a suspensorium and
the palatal apparatus detached. Teeth numerous.
The Plagiostomes differ greatly among each other with regard to
the general form of their body: in the Sharks or Selachoidei the body
is elongate, more or less cylindrical, gradually passing into the tail;
their gill-openings are lateral. In the Rays, or Batoidei, the gill-
openings are always placed on the abdominal aspect of the fish; the
body is depressed, and the trunk, which is surrounded by the
immensely developed pectoral fins, forms a broad flat disk, of which
the tail appears as a thin and slender appendage. Spiracles are
always present; the number of gill-openings is constantly five; no
anal fin; dorsal fins, if present, situated on the tail. However, some of
the Rays approach the Sharks in having the caudal portion less
abruptly contracted behind the trunk.
Fossil Plagiostomes are very numerous in all formations. Some
of the earliest determinable fish remains are believed to be, or are,
derived from Plagiostomes. Those which can be referred to any of
the following families will be mentioned subsequently: but there are
others, especially fin-spines, which leave us in doubt to which group
of Plagiostomes their owners had any affinity, thus Onchus from the
upper Silurian, continuing to carboniferous formations;
Dimeracanthus, Homocanthus, from the Devonian; Oracanthus,
Gyracanthus, Tristychius, Astroptychius, Ptychacanthus,
Sphenacanthus, etc., from carboniferous formations; Leptacanthus,
from the coal to the Oolite; Cladacanthus, Cricacanthus, Gyropristis,
and Lepracanthus, from the coal measures; Nemacanthus,
Liacanthus, from the Trias; Astracanthus, Myriacanthus,
Pristacanthus, from the Jurassic group.
A. Selachoidei: Sharks.
The elongate cylindrical body, generally terminating in a more or
less pointed snout, and passing into a powerful and flexible tail,
blade-like at its extremity, gives to the Sharks a most extraordinary
power of swimming, with regard to endurance as well as rapidity of
motion. Many, especially the larger kinds, inhabit the open ocean,
following ships for weeks, or pursuing shoals of fishes in their
periodical migrations. Other large-sized sharks frequent such parts
of the coast as offer them abundance of food; whilst the majority of
the smaller kinds are shore fishes, rarely leaving the bottom, and
sometimes congregating in immense numbers. The movements of
sharks resemble in some measure those of snakes, their flexible
body being bent in more than one curve when moving.
Sharks are most numerous in the seas between the Tropics, and
become scarcer beyond, a few only reaching the Arctic circle; it is
not known how far they advance southwards towards the Antarctic
region. Some species enter fresh waters, and ascend large rivers,
like the Tigris or Ganges, to a considerable distance. The pelagic as
well as the shore species have a wide geographical range. Very few
descend to a considerable depth, probably not exceeding 500
fathoms. There are about 140 different species known.
Sharks have no scales like those of other fishes; their
integuments are covered with calcified papillæ which, under the
microscope, show a structure similar to that of teeth. If the papillæ
are small, pointed, and close set, the skin is called “shagreen;” rarely
they are larger, appearing as bucklers or spines, of various sizes.
These fishes are exclusively carnivorous, and those armed with
powerful cutting teeth are the most formidable tyrants of the ocean.
They have been known to divide the body of a man in two at one
bite, as if by the sweep of a sword. Some of the largest sharks,
however, which are provided with very small teeth, are almost
harmless, feeding on small fishes only or marine invertebrates.
Others, particularly of the smaller kinds, commonly called “Dog-
fishes,” have short or obtuse teeth, and feed on shells or any other
animal substance. Sharks scent their food from a distance, being
readily attracted by the smell of blood or decomposing bodies.
In China and Japan, and many other eastern countries, the
smaller kinds of sharks are eaten. Sharks’ fins form in India and
China a very important article of trade, the Chinese preparing from
them gelatine, and using the better sorts for culinary purposes. The
fins are obtained not exclusively from Sharks but also from Rays,
and assorted in two kinds, viz. “white and black.” The white consist
exclusively of the dorsal fins, which are on both sides of the same
uniform light colour, and reputed to yield more gelatine than the other
fins. The pectoral, ventral, and anal fins pass under the
denomination of black fins; the caudal fin is not used. One of the
principal places where shark fishery is practised as a profession is
Kurrachee. Dr. Buist, writing in 1850 (“Proc. Zool. Soc.” 1850, p.
100), states that there are thirteen large boats, with crews of twelve
men each, constantly employed in this pursuit; that the value of the
fins sent to the market varies from 15,000 to 18,000 rupees; that one
boat will sometimes capture at a draught as many as one hundred
sharks of various sizes; and that the number total of sharks captured
during the year amounts probably to not less than 40,000. Large
quantities are imported from the African coast and the Arabian Gulf,
and various ports on the coast of India. In the year 1845–46, 8770
cwt. of sharks’ fins were exported from Bombay to China.
First Family—Carchariidæ.
Eye with a nictitating membrane. Mouth crescent-shaped, inferior.
Anal fin present. Two dorsal fins, the first opposite to the space
between pectoral and ventral fins, without spine in front.
Carcharias.—Snout produced in the longitudinal axis of the body;
mouth armed with a series of large flat triangular teeth, which have a
smooth cutting or serrated edge. Spiracles absent. A transverse pit on
the back of the tail, at the root of the caudal fin.
This genus comprises the true Sharks, common in the tropical,
but less so in the temperate seas. Between thirty and forty different
species have been distinguished, of which one of the most common
is the “Blue Shark” (Carcharias glaucus). Individuals of from twelve
to fifteen feet are of very common occurrence, but some of the
species attain a much larger size, and a length of 25 and more feet.
Fishes of this genus or of closely allied genera (Corax, Hemipristis)
are not uncommon in the chalk and tertiary formations.
Galeocerdo.—Teeth large, flat, triangular, oblique, serrated on
both edges, with a deep notch on the outer margin. Spiracles small. A
pit on the tail, above and below, at the root of the caudal fin. Two
notches on the under caudal border, one of them at the end of the
spine.
Fig. 112.—Dentition of the Blue Shark
(Carcharias glaucus); the single teeth are of the
natural size.
Three species, of which one (G. arcticus) is confined to the arctic
and sub-arctic oceans. The others inhabit temperate and tropical
seas, and all attain to a very large size.
Galeus.—Snout produced in the longitudinal axis of the body;
teeth equal in both jaws, rather small, flat, triangular, oblique, serrated
and with a notch. Spiracles small. No pit at the commencement of the
caudal fin, which has a single notch on its lower margin.
These are small sharks, commonly called “Tope.” The species
found on the British coast is spread over nearly all the temperate and
tropical seas, and is common in California and Tasmania. It lives on
the bottom, and is very troublesome to fishermen by constantly
taking away bait or driving away the fishes which they desire to
catch.
Zygæna.—The anterior part of the head is broad, flattened, and
produced into a lobe on each side, the extremity of which is occupied
by the eye. Caudal fin with a single notch at its lower margin. A pit at
the root of the caudal fin. Spiracles none. Nostrils situated on the front
edge of the head.
The “Hammerheads,” or Hammerheaded Sharks, have a
dentition very similar to that of Carcharias, and although they do not
attain to the same large size, they belong to the most formidable
fishes of the ocean. The peculiar form of their head is quite unique
among fishes; young examples have the lateral extension of the skull
much less developed than adults. Five species are known, which are
most abundant in the tropics. By far the most common is Zygæna
malleus, which occurs in nearly all tropical and sub-tropical seas.
Specimens of this species may be often seen ascending from the
clear blue depths of the ocean like a great cloud. Cantor found in a
female, nearly 11 feet long, thirty-seven embryons.—Hammerheads
have lived from the cretaceous epoch.
Mustelus.—The second dorsal fin is not much smaller than the
first. No pit at the root of the caudal, which is without distinct lower
lobe. Snout produced in the longitudinal axis of the body. Spiracles
small, behind the eyes. Teeth small, numerous, similar in both jaws,
obtuse, or with very indistinct cusps, arranged like pavement.
The “Hounds” are small Sharks, abundant on the coasts of all the
temperate and tropical seas; two of the five species known occur on
the coasts of Europe, viz. M. lævis and M. vulgaris. Closely allied as
these two species are, they yet show a most singular difference, viz.
that a placenta is developed in the uterus for the attachment of the
embryo in M. lævis (the Γαλεὁς λεȋος of Aristotle, to whom this fact
was already known); whilst the embryons of M. vulgaris are
developed without such placenta (see J. Müller, “Abhandl. Ak. Wiss.”
Berl. 1840). The Hounds are bottom fish, which feed principally on
shells, crustaceans, and decomposing animal substances.
Several other genera belong to the family Carchariidæ, but it will

be sufficient to mention their names:—Hemigaleus, Loxodon,
Thalassorhinus, Triænodon, Leptocarcharias, and Triacis.
Second Family—Lamnidæ.
Eye without nictitating membrane. Anal fin present. Two dorsal
fins; the first opposite to the space between pectoral and ventral fins,
without spine in front. Nostrils not confluent with the mouth which is
inferior. Spiracles absent or minute.
All the fishes of this family attain to a very large size, and are
pelagic. But little is known of their reproduction. The first appearance
of this family is indicated by Carcharopsis, a genus from
carboniferous formations, the teeth of which differ from those of
Carcharodon only by having a broad fold at the base. In the chalk
and tertiary formations almost all the existing genera are
represented; and, besides, Oxytes, Sphenodus, Gomphodus, and
Ancistrodon, which are known from teeth only, have been considered
generically distinct from the living Porbeagles.
Lamna (Oxyrhina).—The second dorsal and anal are very small.
A pit at the root of the caudal, which has the lower lobe much
developed. Side of the tail with a prominent longitudinal keel. Mouth
wide. Teeth large, lanceolate, not serrated, sometimes with additional
basal cusps. On each side of the upper jaw, at some distance from the
symphysis, there is one or two teeth conspicuously smaller than the
others. Gill-openings very wide. Spiracles minute.
Fig. 113.—Upper and lower

tooth of Lamna.
Of the “Porbeagles,” three species have been described, of which

the one occurring in the North Atlantic, and frequently straying to the
British coasts (L. cornubica), is best known. It attains to a length of
ten feet, and feeds chiefly on fishes; its lanceolate teeth are not
adapted for cutting, but rather for seizing and holding its prey, which
it appears to swallow whole. According to Pennant it is viviparous;
only two embryoes were found in the female which came under his
observation. Haast has found this species also off the coast of New
Zealand.
Carcharodon.—The second dorsal and anal are very small. Pit
at the root of the caudal, which has the lower lobe well developed.
Side of the tail with a prominent longitudinal keel. Mouth wide. Teeth
large, flat, erect, regularly triangular, serrated. On each side of the
upper jaw, at some distance from the symphysis, there is one or two
teeth conspicuously smaller than the others. Gill-openings wide.
One species only is known (C. rondeletii), which is the most
formidable of all Sharks. It is strictly pelagic; and appears to occur in
all tropical and sub-tropical seas. It is known to attain to a length of
40 feet. The tooth figured here, of the natural size, is taken from a
jaw 20 inches wide in its transverse diameter (inside measure), each
half of the mandible measuring 22 inches.[36] The whole length of
the fish was 36½ feet.
Carcharodon teeth are of very common occurrence in various
tertiary strata, and have been referred to several species, affording
ample evidence that this type was much more numerously
represented in that geological epoch than in the recent fauna. Some
individuals attained to an immense size, as we may judge from teeth
found in the Crag, which are 4 inches wide at the base, and 5 inches
long, measured along their lateral margin. The naturalists of the
“Challenger” expedition have made the highly interesting discovery
that teeth of similar size are of common occurrence in the ooze of
the Pacific, between Polynesia and the west coast of America. As we
have no record of living individuals of that bulk having been
observed, the gigantic species to which these teeth belonged must
have become extinct within a comparatively recent period. Nothing is
known of the anatomy, habits, and reproduction of the surviving
species, and no opportunity should be lost of obtaining information
on this Shark.
Fig. 114.—Tooth of
Carcharodon rondeletii.
Odontaspis.—The second dorsal and anal are not much smaller
than the first dorsal. No pit at the root of the caudal. Side of the tail
without keel. Mouth wide. Teeth large, awl-shaped, with one or two
small cusps at the base. Gill-openings of moderate width.
Large Sharks from tropical and temperate seas; two species.

Alopecias.—The second dorsal and anal very small. Caudal fin of
extraordinary length, with a pit at its root. No keel on the side of the
tail. Mouth and gill-openings of moderate width. Teeth equal in both
jaws, of moderate size, flat, triangular, not serrated.
This genus consists of one species only, which is known by the
name of “Fox-shark” or “Thresher.” It is the most common of the
larger kinds of Sharks which occur on the British coasts; and seems
to be equally common in other parts of the Atlantic and
Mediterranean, as well as on the coasts of California and New
Zealand. It attains to a length of fifteen feet, of which the tail takes
more than one half; and is quite harmless to man. It follows the
shoals of Herrings, Pilchards, and Sprats in their migrations,
destroying incredible numbers. When feeding it uses the long tail in
splashing the surface of the water, whilst it swims in gradually
decreasing circles round a shoal of fishes, which are thus kept
crowded together, falling an easy prey to their enemy. Statements
that it has been seen to attack Whales and other large Cetaceans,
rest upon erroneous observations.
Selache.—The second dorsal and anal very small. A pit at the
root of the caudal fin, which is provided with a lower lobe. Side of the
tail with a keel. Gill-openings extremely wide. Teeth very small,
numerous, conical, without serrature or lateral cusps.
Also this genus consists of one species only, the “Basking Shark”
(Pélerin of the French). It is the largest Shark of the North Atlantic,
growing to a length of more than thirty feet. It is quite harmless if not
attacked; its food consisting of small fishes, and other small marine
animals swimming in shoals. On the west coast of Ireland it is
chased for the sake of the oil which is extracted from the liver, one
fish yielding from a ton to a ton and a-half. Its capture is not
unattended with danger, as one blow from the enormously strong tail
is sufficient to stave in the sides of a large boat. At certain seasons it
is gregarious, and many specimens may be seen in calm weather
lying together motionless, with the upper part of the back raised
above the surface of the water; a habit from which this Shark has
derived its name. The buccal and branchial cavities are of
extraordinary width, and, in consequence of the flabby condition of
those parts, the head presents a variable and singular appearance in
specimens lying dead on the ground. This peculiarity, as well as the
circumstance that young specimens have a much longer and more
pointed snout than adult ones, has led to the erroneous opinion that
several different genera and species of Basking Shark occur in the
European seas. The branchial arches of Selache are provided with a
very broad fringe of long (five to six inches) and thin gill-rakers,
possessing the same microscopical structure as the teeth and
dermal productions of Sharks. Similar gill-rakers have been found in
a fossil state in the Crag of Anvers in Belgium, proving the existence
of this Selachian type in the tertiary epoch. Nothing is known of the
reproduction of this fish. The latest contributions to its history are by
Steenstrup in “Overs. Dansk. Vidensk. Selsk., Forhandl.” 1873, and
by Pavesi in “Annal. Mus. Civ. Geneva,” 1874 and 1878.
Third Family—Rhinodontidæ.
No nictitating membrane. Anal fin present. Two dorsal fins, the
first nearly opposite to the ventrals, without spine in front. Mouth and
nostril near the extremity of the snout.
This small family comprises one species only, Rhinodon typicus,
a gigantic Shark, which is known to exceed a length of fifty feet, but
is stated to attain that of seventy. It does not appear to be rare in the
western parts of the Indian Ocean, and possibly occurs also in the
Pacific. It is one of the most interesting forms, not unlike the Basking
Shark of the Northern Seas, having gill-rakers like that species; but
very little is known of its structure and mode of life. It is perfectly
harmless, its teeth being extremely small and numerous, placed in
broad bands; it has been stated to feed on tang, an observation
which requires confirmation. The snout is very broad, short, and flat;
the eyes are very small. A pit at the root of the caudal fin which has
the lower lobe well developed; side of the tail with a keel. A
characteristic figure of this fish has been given by A. Smith in his
“Illustrations of the Zoology of South Africa,” Plate 26, from a
specimen which came ashore at the Cape of Good Hope.
Fig. 115.—Dentition of Notidanus indicus. a,

teeth in function; b, teeth in reserve; u, upper, and l,
lower, tooth, of natural size.
Fourth Family—Notidanidæ.
No nictitating membrane. One dorsal fin only, without spine,
opposite to the anal.
Notidanus.—Dentition unequal in the jaws: in the upper jaw one
or two pairs of awl-shaped teeth, the following six being broader, and
provided with several cusps, one of which is much the strongest.
Lower jaw with six large comb-like teeth on each side, beside the
smaller posterior teeth. Spiracles small, on the side of the neck. No pit
at the root of the caudal fin. Gill-openings wide, six in number in
Hexanchus, seven in Heptanchus.
Four species are known, distributed over nearly all the tropical
and sub-tropical seas; they attain to a length of about fifteen feet.
Fossil teeth belonging to this type have been found in Jurassic and
later formations (Notidanus and Aellopos).
Fifth Family—Scylliidæ.
Two dorsal fins, without spine: the first above or behind the
ventrals; anal fin present. No nictitating membrane. Spiracle always
distinct. Mouth inferior. Teeth small, several series generally being in
function.
Scyllium.—The origin of the anal fin is always in advance of that
of the second dorsal. Nasal cavity separate from the mouth. Teeth
small, with a middle longer cusp, and generally one or two small
lateral cusps arranged in numerous series. Eggs similar to those of
the Rays (Fig. 79, p. 167).
The fishes of this genus are of small size, and commonly called
“Dog-fishes.” They are coast fishes, living on the bottom, and feeding
on Crustaceans, dead fishes, etc. None of the eight species known
have a very wide distribution, but where they occur they are
generally sufficiently abundant to prove troublesome to fishermen.
They inhabit most parts of the temperate and tropical seas. On the
British coasts two species are found, the “Larger” and “Lesser
spotted Dog-fish,” Scyllium canicula and Scyllium catulus, which are
said to be more plentiful among the Orkney Islands than elsewhere.
They are scarcely ever brought to market; but the fishermen of some
localities do not disdain to eat them. Their flesh is remarkably white,
a little fibrous, and dry. In the Orkneys they are skinned, split up,
cleaned, and then spread out on the rocks to dry for home
consumption. The skins are used for smoothing down cabinet-work.
It would be worth while to apply the fins of these and other Sharks,
which are so extensively used in China for making gelatine soups, to
the same purpose in this country, or to dry them for exportation to
the East. Most of the species of Dog-fishes are spotted, and those of
the allied genera, Parascyllium and Chiloscyllium, very handsomely
ornamented.
Closely allied to Scyllium is Pristiurus, from the coasts of Europe,
which is provided with a series of small flat spines on each side of
the upper edge of the caudal fin.
Fossil forms of Dog-fishes are not scarce in the Lias and Chalk:
Scylliodus, Palæoscyllium, Thyellina, Pristiurus.
Ginglymostoma.—The second dorsal fin opposite to, and
somewhat in advance of, the anal. Eyes very small; spiracle minute
and behind the eye. Nasal and buccal cavities confluent. The nasal
valves of both sides form one quadrangular flap in front of the mouth,
each being provided with a free cylindrical cirrhus. The fourth and fifth
gill-openings are close together. The teeth stand either in many series,
each having a strong median cusp and one or two smaller ones on
each side (Ginglymostoma), or they stand in a few (three) series only,
the foremost only being in function, and each tooth having a convex,
finely and equally serrated margin (Nebrius).
Four species from the tropical parts of the Atlantic and Indian
Oceans, attaining to a length of some 12 feet. Pelagic.
Stegostoma.—The first dorsal above the ventrals, the second in
advance of the anal, which is very close to the caudal. Tail, with the
caudal fin, exceedingly long, measuring one-half of the total length.
Eyes very small, spiracle as wide as, and situated behind, the orbit.
Nasal and buccal cavities confluent. Snout very obtuse; upper lip very
thick, like a pad, bent downwards over the mouth, with a free
cylindrical cirrhus on each side. Teeth small, trilobed, in many series,
occupying in both jaws a transverse flat subquadrangular patch. The
fourth and fifth gill-openings are close together.
The single species (St. tigrinum) for which this genus has been
formed, is one of the commonest and handsomest sharks of the
Indian Ocean. Young individuals keep generally close to the coasts,
whilst the adult, which are from 10 to 15 feet long, are not rarely met
in the open ocean. The colour is a brownish yellow, ornamented with
black or brown transverse bands, or with snuff-coloured rounded
spots; hence this shark is frequently mentioned by the names of
“Zebra-Shark” or “Tiger-Shark.”
Fig. 116.—Chiloscyllium trispeculare, from North-western Australia.

Chiloscyllium.—The first dorsal fin above or behind the ventrals.
Anal fin placed far behind the second dorsal, and very close to the
caudal. Spiracle very distinct, below the eye. Nasal and buccal
cavities confluent. Nasal valve folded, with a cirrhus. Teeth small,
triangular, with or without lateral cusps. The two last gill-openings
close together.
“Dog-fishes,” from the Indian Ocean, of small size. Four species

are known, of which one, Ch. indicum, is one of the commonest
shore-fishes on the coasts of this region, extending from the
southern extremity of the African Continent to Japan.
Fig. 117.—Confluent nasal and buccal cavities of the
same fish.
Crossorhinus.—The first dorsal behind the ventrals, the second
in advance of the anal, which is very close to the caudal. Tail rather
short. Eyes small. Spiracle a wide oblique slit, behind and below the
eye. Nasal and buccal cavities confluent. Head broad, flat, with the
snout very obtuse; mouth wide, nearly anterior. A free nasal cirrhus;
sides of the head with skinny appendages. Anterior teeth rather large,
long and slender, without lateral lobes, the lateral tricuspid, smaller,
forming a few series only. The fourth and fifth gill-openings close
together.
Three species are known from the Australian and Japanese
coasts. They are evidently ground-sharks, which lie concealed on
the bottom watching for their prey. In accordance with this habit their
colour closely assimilates that of a rock or stone covered with short
vegetable and coralline growth—a resemblance increased by the
frond-like tentacles on the side of the head. This peculiarity of the
integuments, which is developed in a yet higher degree in Pediculati
and Lophobranchs, is not met with in any other Selachian. These
Sharks grow to a length of 10 feet.
Sixth Family—Hybodontidæ.
Two dorsal fins, each with a serrated spine. Teeth rounded,
longitudinally striated, with one larger, and from two to four smaller
lateral cusps. Skin covered with shagreen.
Extinct. From carboniferous, liassic, and triassic formations.
Several genera have been distinguished; and if Cladodus belongs to
this family, it would have been represented even in the Devonian.
Fig. 118.—Spine of Hybodus

subcarinatus.
Seventh Family—Cestraciontidæ.
No nictitating membrane. Two dorsal fins, the first opposite to the
space between pectoral and ventral fins; anal fin present. Nasal and
buccal cavities confluent. Teeth obtuse, several series being in
function.
Fig. 119.—Jaws of Port Jackson Shark,

Cestracion philippi.
Fig. 120.—Upper jaw of the same, half natural
size.
This family is one of particular interest, because representatives
of it occur in numerous modifications in primary and secondary
strata. Their dentition is uniformly adapted for the prehension and
mastication of crustaceous and hard-shelled animals. The fossil
forms far exceeded in size the species of the only surviving genus;
they make, their appearance with Ctenoptychius in the Devonian;
this is succeeded in the coal-measures by Psammodus,
Chomatodus, Petrodus, Cochliodus, Polyrhizodus, etc.; in the Trias
and Chalk by Strophodus, Acrodus, Thectodus, and Ptychodus. Of
the 25 genera known, 22 have lived in the periods preceding the
Oolitic.
Cestracion (Heterodontus).—Each dorsal fin armed with a
spine in front; the second in advance of the anal. Mouth rather narrow.
Spiracles small, below the posterior part of the eye. Gill-openings
rather narrow. Dentition similar in both jaws, viz. small obtuse teeth in
front, which in young individuals are pointed and provided with from
three to five cusps. The lateral teeth are large, padlike, twice as broad
as long, arranged in oblique series, one series being formed by much
larger teeth than those in the other series.
Fig. 121.—Cochliodus contortus.
Fig. 122.—Cestracion galeatus, Australia.

Four species are known from Japan, Amboyna, Australia, the
Galapagoes Islands, and California; none exceed a length of 5 feet.
The egg has been figured on p. 168 (Fig. 80).
Eighth Family—Spinacidæ.
No membrana nictitans. Two dorsal fins; no anal. Mouth but
slightly arched; a long, deep, straight, oblique groove on each side of
the mouth. Spiracles present; gill-openings narrow. Pectoral fins not
notched at their origin.
The oldest representative of this family (Palæospinax) occurs at
Lyme Regis; its skin is granular; each dorsal fin possesses a spine;
the teeth in the jaws are dissimilar—the upper being multicuspid,
longitudinally ribbed as in Hybodus, the lower smooth and tricuspid.
Drepanophorus and Spinax primævus occur in Cretaceous
formations of England and the Lebanon.
Centrina.—Each dorsal fin with a strong spine. Trunk rather
elevated, trihedral, with a fold of the skin running along each side of
the belly. Teeth of the lower jaw erect, triangular, finely serrated; those
of the upper slender, conical, forming a group in front of the jaw.
Spiracles wide, behind the eye.
One species, Centrina salviani, from the Mediterranean and
neighbouring parts of the Atlantic; of small size.
Acanthias.—Each dorsal fin with a spine. Teeth equal in both
jaws, rather small; their point is so much turned aside that the inner
margin of the tooth forms the cutting edge. Spiracles rather wide,
immediately behind the eye.
The two species of “Spiny Dog-fishes,” A. vulgaris and A.
blainvillii, have a very remarkable distribution, being found in the
temperate seas of the Northern and Southern Hemispheres, but not
in the intermediate tropical zone. They are of small size, but occur at
times in incredible numbers, 20,000 having been taken in one scene
on the Cornish coast. They do much injury to the fishermen by
cutting their lines and carrying off their hooks.
Centrophorus.—Each dorsal fin with a spine which, however, is
sometimes so small as to be hidden below the skin. Mouth wide.
Teeth of the lower jaw with the point more or less inclined backwards
and outwards. Upper teeth erect, triangular, or narrow, lanceolate, with
a single cusp. Spiracles wide, behind the eye.
Eight species are known from the southern parts of the European
seas, and one from the Moluccas; they do not appear to exceed a
length of five feet. According to the observations of E. P. Wright,
some of the species at least live at a considerable depth, perhaps at
a greater depth than any of the other known Sharks. The Portuguese

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Cutting Edge Organic Synthesis and Chemical Biology of Bioactive Molecules The Shape of Organic Synthesis To Come Yuichi Kobayashi

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Cutting Edge Organic Synthesis and

Chemical Biology of Bioactive

PRINCIPLES OF ORGANIC SYNTHESIS, 3RD EDITION Raymond

Organic Chemistry from Retrosynthesis to Asymmetric

Organic Synthesis Using Biocatalysis 1st Edition

Mechanochemical Organic Synthesis 1st Edition Davor

Routes to Essential Medicines A Workbook for Organic

Green Sustainable Process for Chemical and

Total Synthesis of Natural Products At the Frontiers of

Chemical biology: enabling approaches for understanding

ISBN 978-981-13-6243-9 ISBN 978-981-13-6244-6 (eBook)

© Springer Nature Singapore Pte Ltd. 2019

The purpose of organic synthesis is to provide target compounds by selective meth-

medical cannabinoids. Chapters “Exploring Bioactive Marine Natural Products and

Yokohama, Japan  Yuichi Kobayashi

1 Microbial Fraction Library: A Screening Source for Drug

10 Biosynthesis, Biological Functions, and Receptors

Fig. 1.1 Screening strategy of interesting secondary metabolites................. 2

Fig. 2.11 Regulatory functions of the eukaryotic proteasome and

Fig. 8.2 (a) Taxol and (b) retrosynthetic approach to

Fig. 11.5 Examples of a classical cannabinoid, a nonclassical

Fig. 13.13 Schematic illustration of the strategy for the quenched

Fig. 14.13 Other bifunctional PAL probe candidates of dantrolene............... 351

Scheme 2.1 Retrosynthetic analysis of pyripyropene A������������������������������� 25

Scheme 3.8 Possible pathway from 11 to 13a and energetic difference

Scheme 4.8 Total synthesis of (R)-espicufolin��������������������������������������������� 83

Scheme 5.20 Synthesis of right-hand segment 105���������������������������������������� 118

Scheme 7.14 Synthesis of trans-2,6-disubstituted cyclohexanones��������������� 156

Scheme 8.1 Baldwin’s dioxenone intermolecular

Scheme 8.19 Completion of Wood’s ingenol synthesis��������������������������������� 189

Scheme 9.36 Synthesis of RvE2 by Spur������������������������������������������������������� 221

Scheme 11.24 Synthesis of hybrid cannabinoid 130 via hetero-

Scheme 14.2 Synthesis of photovastatin CAA1 (5)��������������������������������������� 343

Table 1.1 Pros/cons of biological and chemical screenings��������������������������� 2

Toshihiko Nogawa, Julius Adam V. Lopez, and Hiroyuki Osada

Keywords Natural product · Microbial metabolite · Fraction library · Screening ·

T. Nogawa · J. A. V. Lopez · H. Osada (*)

© Springer Nature Singapore Pte Ltd. 2019 1

For interesting activity For novel structure

Fig. 1.1 Screening strategy of interesting secondary metabolites

Table 1.1 Pros/cons of biological and chemical screenings

Table 1.2 Pros/cons of natural products and synthetic compound

Nowadays, LC/MS and NMR spectrometers are routine analytical instruments.

FIRST SUB-ORDER: PLAGIOSTOMATA.

Several other genera belong to the family Carchariidæ, but it will

Fig. 113.—Upper and lower

Of the “Porbeagles,” three species have been described, of which

Large Sharks from tropical and temperate seas; two species.

Fig. 115.—Dentition of Notidanus indicus. a,

Fig. 116.—Chiloscyllium trispeculare, from North-western Australia.

“Dog-fishes,” from the Indian Ocean, of small size. Four species

Fig. 118.—Spine of Hybodus

Fig. 119.—Jaws of Port Jackson Shark,

Fig. 122.—Cestracion galeatus, Australia.

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Yokohama, Japan  Yuichi Kobayashi

1 Microbial Fraction Library: A Screening Source for Drug

10 Biosynthesis, Biological Functions, and Receptors

Scheme 2.1 Retrosynthetic analysis of pyripyropene A�� 25

Scheme 4.8 Total synthesis of (R)-espicufolin�� 83

Scheme 5.20 Synthesis of right-hand segment 105�� 118

Scheme 7.14 Synthesis of trans-2,6-disubstituted cyclohexanones�� 156

Scheme 8.19 Completion of Wood’s ingenol synthesis�� 189

Scheme 9.36 Synthesis of RvE2 by Spur�� 221

Scheme 14.2 Synthesis of photovastatin CAA1 (5)�� 343

Table 1.1 Pros/cons of biological and chemical screenings�� 2