Antimicrobial Activity of 8-Substituted Fluoro Benzothiazolidine Triazoles

Antimicrobial Activity Of 8-Substituted Fluoro
Benzothiazolidine Triazoles
By
Manoj V. Vyavahare
Dissertation Submitted to the

Rajiv Gandhi University Of Health Sciences, Karnataka, Bangalore
In partial fulfillment
of the requirements for the degree of
Master of Pharmacy
in
Pharmaceutical Chemistry
Under the guidance of

S. M. Hipparagi
Department of Pharmaceutical Chemistry

K.L.E. Society’s College of Pharmacy,
Bangalore-10.
2006
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K. L. E. Society’s College of Pharmacy
Bangalore-560010
DECLARATION BY THE CANDIDATE

I hereby declare that
this dissertation/thesis entitled
“Antimicrobial Activity Of 8-Substituted Fluoro

Benzothiazolidine Triazoles”
is a bonafide and genuine research work carried out by
me under the guidance of
Prof. S. M. Hipparagi
Date:
Place:
Manoj V. Vyavahare
Bangalore-560010
CERTIFICATE BY THE GUIDE
This is to certify that the dissertation entitled

is a bonafide and genuine research work carried out

by
Manoj V. Vyavahare
in partial fulfillment
of the requirement for the degree of
Master of Pharmacy
Date: Prof. S. M. Hipparagi,

Place: Dept. of Pharmaceutical chemistry,
K.L.E. Society’s college of Pharmacy,
Bangalore-560010.
Bangalore-560010
ENDORSEMENT BY THE HOD,

PHARMACEUTICALCHEMISTRY

is a bonafide research work done

by
Manoj V. Vyavahare
under the guidance of

Prof. S. M. Hipparagi
Date: Prof. Y. D. Satyanarayana,

Place: Vice-Principal and H.O.D.,
Dept. of Pharmaceutical chemistry,
K.L.E. Society’s college of pharmacy,
Bangalore-560010.
Bangalore-560010
ENDORSEMENT BY THE PRINCIPAL/HEAD OF THE

INSTITUTION

is a bonafide research work done

by
Manoj V. Vyavahare
under the guidance of
Prof. S. M. Hipparagi.
Date: Prof. B. G. Desai

Place: Principal and H.O.D.,
Dept. of Pharmaceutical Technology,
K.L.E. Society’s college of Pharmacy,
Bangalore-560010.
ACKNOWLEDGEMENT
First and foremost indebt to my mother and father Sau. Madhuri and Shri.
Vishwanath Vyavahare, for all their hard work to bring me up with a high
quality education. and I would like to give special thanks to my respected elder
brother Mr.Amol Vyavahare for his superb guidance right from my childhood.
It gives me immense pleasure to acknowledge, the help rendered to me by a
host of a people, to whom I owe gratitude for successful completion of my
M.Pharm.
I take this opportunity to express my sincere thank to our Respected
Principal Prof. B. G. Desai for his constant enduring support anytime needed.
The research work embodied in dissertation has been carried out under
supervision of my esteemed and most respected guide Mr. S. M. Hipparagi my
greatest debt of gratitude is to him for his continous encouragement, valuable
suggestions, dynamic guidance, evereadiness to elucidate problems, constant
motivation to act with quality, supportive in times of stress and blessings on me
throughout the dissertation work.
I sincerely thanks to respected Mr. Y. D. Satyanarayana, Vice
principal and HOD, Dept. of Pharmacutical Chemistry, Dr. S. S. Karki, Dr.
Sonal Dubey, Mrs. Vanitha S., Mr. Santosh Butle and other faculty members of
KLE Society’s college of Pharmacy, Bangalore for their valuable help and
guidance during the course of my research work.
I am highly indebted to my grateful thanks to Dr. LVG Nargund, Principal,
Nargund college of pharmacy, Bangalore, for their valuable guidance in
completion of project work.
I would like to express my sincere thanks to Mrs Preethy madam for rendering
me the permission to use the Microbiology Laboratory in carrying out
antimicrobial activity studies.
K.L.E.S. Bangalore 1
I wish to express my special thanks to the authorities of Organic chemistry and
NMR research centre, IISc, Bangalore for providing the, NMR Spectra and
elemental analysis of my synthesized compounds and special thanks to my best
friend Ms. Aarti, project Assistant , NMR research centre, IISc, Bangalore for
giving expedite NMR report and guidance in NMR interpretation.I am
grateful to authorities at Sun Pharma, Baroda for providing Mass Spectra.
I than sincerely thank to Mr. Satish, librarian, and Lingu for their
help during project.
I will never forget the care and affection bestowed upon me by my colleagues
Prashant, Kanchan, Vivek, Sreekanth, Rana, Parmeet, Gajanan Who made me
stay in k.l.E.S. a memorable one.
A word of thanks to non-teaching staff: Birader Sir, Sharnappa, and other
members of college for providing all the help when required.
I would be failing in my duties if I do not thank my beloved friends for their
constant support in every endeavor of mine and provided me with necessary
stimulus for keeping the driving force integrated for successful completion of the
project.I would like to give special thanks to my best friend Pravin Zambad,
Shailesh, Prashant, Sushant, Amit, Prasad, Aashish, Divakar, Sameer, Sanit,
Raosaheb, Ranjeet who have always supported and offered me helping hand
when ever necessary and for always being there in the times when ever needed. I
cherish every moment I am associated with.
Mere words and acknowledgement are not enough to express the valuable
help and encouragement rendered by all people. I finally conclude with a saying
that “thanking may just be a formality, but if done inwardly, it surely reflects
your noblest thoughts within”.
Date:
Place : (Manoj V. Vyavahare)
LIST OF ABBREVIATION USED
0
C = Degree centigrade
cm-1 = per centimeter
CDCl3= Deuteriated Chloroform.
CHCl3= Chloroform
CH3OH= Methanol
CHN= Carbon Hydrogen Nitrogen
DMF= Dimethyl Formamide
DMSO= Dimethyl sulfoxide
FT-IR= Fourier Transform Infrared
g = Gram
KBr= Potassium Bromide
MIC= Minimum Inhibitory Concentration
ml = Milli Liter
Mol = Mole
µg = Microgram
m = Micro Mol
NMR= Nuclear Magnetic Resonance
ppm= parts per million
Sl.No. = Serial Number
TLC= Thin layer Chromatography
TMS= Tetra methyl Silane
% = Percentage
ABSTRACT
Review shows that benzothiazole and triazole are found to possess various
activities such as antimicrobial, antitumor, anthelmintic, anti-inflammatory etc.
The objective of this research work is to synthesize various compounds and their
derivatives, and characterized by various spectral and elemental analysis and
screened for antimicrobial activities.
We synthesized 7- Chloro-6-fluoro-benzothiazol-2-ylamine from 3-Chloro-4-
fluoro phenylamine. Then we synthesized 7-chloro-6-fluoro- benzothiazol-2-yl-
hydrazine from 7- Chloro-6-fluoro-benzothiazol-2-ylamine then we proceeded for
8-chloro-7-fluoro-benzo[4,5]thiazolo [2,3-c] [1,2,4]triazole.
We tried to prepare various derivatives by replacing the chlorine atom by
different amines from 8-chloro-7-fluoro-benzo[4,5]thiazolo [2,3-c] [1,2,4]triazole.
(Scheme 1). But we failed to do so then we replaced the chlorine by amine from
7- Chloro-6-fluoro-benzothiazol-2-ylamine and proceed to final compound
(Scheme II) as described in scheme 1.
Various compounds and their derivatives prepared were confirmed by IR, NMR,
MASS.
Antimicrobial study shows that synthesized compounds have moderate activity.
Keywords: Benzothiazole, triazole.
TABLE OF CONTENTS
1. Introduction Page No. 1-6
2. Objectives Page No. 7
3. Review of Literature Page No. 6-45
4. Methodology Page No. 46-91
4.1 Scheme Page No. 46-51
4.2 Experimental Page No. 52-77
4.3 Spectral data Page No. 65-78
4.4 Pharmacological Screening Page No. 79-85
5. Results &Discussion Page No. 86-88
6. Conclusion Page No. 89
7. Summary Page No. 90-91
8. Bibliography Page No. 92-104
9. Annexure Page No. 105
LIST OF TABLES
SL.
No. Title Page no.
1 Codes and corresponding R of different derivatives 50-51
Analytical data of synthesized compounds

2 60-63
Physical properties of various compounds and

3 derivatives 64
Infra red spectral study of the synthesized compounds

4 65-67
1
5 H NMR Spectral data of synthesized compounds 68
6 Antibacterial activity of 8-substituted-7-fluoro- 85
benzo[4,5]thiazolo[2,3-c][1,2,4]triazole.
LIST OF FIGURES
S. No. Figure Name P. No.
IR spectrum of 7-Chloro-6-fluoro-benzothiazole-
1 2-ylamine. 69
IR spectrum of 7-chloro-6-fluoro- benzothiazol-

2 2-yl-hydrazine. 69
IR spectrum of 8-chloro-7-fluoro- 70
3 benzo[4,5]thiazolo [2,3-c] [1,2,4]triazole.
4 IR spectrum of 2-amino-6-fluoro-7(2- 70
nitrophenylamine)benzothiazole.
5 IR spectrum of (7-fluoro-benzo[4,5]thiazolo[2,3- 71
c][1,2,4]triazol-8-yl)-2-phenylamine.
c][1,2,4]triazol-8-yl)-4-nitrophenylamine.
c][1,2,4]triazol-8-yl)-2-nitrophenylamine.
c][1,2,4]triazol-8-yl)-4-chlorophenylamine.
c][1,2,4]triazol-8-yl)-4-fluorophenylamine.
10 IR spectrum of (7-fluoro-benzo[4,5]thiazolo[2,3-
c][1,2,4]triazol-8-yl)-2-methylphenylamine. 73
1
11 H-NMR spectrum of 7-Chloro-6-fluoro- 74
benzothiazole-2-ylamine.
1
12 H-NMR spectrum of 7-chloro-6-fluoro- 74
benzothiazol-2-yl-hydrazine.
13 D2O exchange-NMR spectrum of 7-chloro-6- 75

fluoro- benzothiazol-2-yl-hydrazine.
1
14 H-NMR spectrum of 8-chloro-7-fluoro- 75
benzo[4,5]thiazolo [2,3-c] [1,2,4]triazole.
1
15 H-NMR spectrum 2-amino-6-fluoro-7(2- 76
nitrophenylamine)benzothiazole.
16 D2O exchange-NMR spectrum of 2-amino-6- 77

fluoro-7(2-nitrophenylamine)benzothiazole.
17 EI-MS of 8-chloro-7-fluoro-benzo[4,5]thiazolo 78
[2,3-c] [1,2,4]triazole.
COPYRIGHT
Declaration by the Candidate
I hereby declare that the Rajiv Gandhi University of Health Sciences,
Karnataka shall have the rights to preserve, use and disseminate this
dissertation / thesis in print or electronic format for academic / research
purpose.
Date:
Place: Manoj V. Vyavahare.
© Rajiv Gandhi University of Health Sciences, Karnataka
INTRODUCTION
Humankind has been subject to infection by microorganism since before the dawn
of recorded history. One presumes that mankind has been searching for suitable
therapy for nearly as long. This was a desperately difficult enterprise given the
acute nature of most infections and the nearly total lack of understanding of their
origins prevalent until the last century. Although one can find indications in old
medical writing of folkloric use of plant and animal preparations, soybean curd,
moldy bread and cheese, counter infection with other microbes, the slow
development of public health measures, and an understanding of the desirability
of personal cleanliness, these factors were erratically and inefficiently applied and
often failed. Until after the discovery of bacteria 300 years ago, and subsequent
understanding of their role in infection about 150 years ago, there was no hope for
rational therapy.
The modern anti-infective era opened with the discovery of the sulfonamides in
France and Germany in 1936 as an offshoot of Paul Ehrlich’s earlier
achievements in treating infections with organometallics and his theories of vital
staining.
Microbes of soil origin remain to this day the most fruitful source of antibiotics,
although the specific means employed for their discovery are infinitely more
sophisticated today than those employed 50 years ago. Initially extracts of
fermentation were screened simply for their ability to kill pathogenic
microorganisms in vitro. Those that did were pushed along through ever more
complex pathogenic and toxicologic tests in attempts to discover clinically useful
agents. Today many thousands of such extracts of increasingly exotic microbes
are tested each week and the test now include sophisticated assays for agents
operating through particular biochemical mechanism or possessing desirable
properties. The impact of genomics is expected to have very substantial impact on
this effort. As a consequence of this work mankind has now many choices for
powerful, effective and specific therapy for some of its most ancient and common
bacterial infections1.
Antimicrobial agents
Initially the term ‘chemotherapeutic agent’ was restricted to synthetic compounds,
but now since many antibiotics and their analogues have been synthesized, this
criterion has become irrelevant; both synthetically and microbiologically
produced drugs need to be include together. However it would be more
meaningful to us e the term Antimicrobial agent (AMA) to designate synthetic as
well as naturally obtained drugs that attenuate microorganisms2.
Basis of Antimicrobial Action
Various antimicrobial agents act by interfering with (1) cell wall synthesis, (2)
plasma membrane integrity, (3) nucleic acid synthesis, (4) ribosomal function,
and (5) folate synthesis.
Biochemical Basis of Antimicrobial Action
Bacterial cells grow and divide, replicating repeatedly to reach the large numbers
present during an infection or on the surfaces of the body. To grow and divide,
organisms must synthesize or take up many types of biomolecules. Antimicrobial
agents interfere with specific processes that are essential for growth and/or
division. They can be separated into groups such as inhibitors of bacterial and
fungal cell walls, inhibitors of cytoplasmic membranes, inhibitors of nucleic acid
synthesis, and inhibitors of ribosome function. Antimicrobial agents may be either
bactericidal, killing the target bacterium or fungus, or bacteriostatic, inhibiting its
growth. Bactericidal agents are more effective, but bacteriostatic agents can be
extremely beneficial since they permit the normal defenses of the host to destroy
the microorganisms.
Sites of action of different antimicrobial agents. PABA, paraminobenzoic acid;
DHFA, dihydrofolic acid; THFA, tetrahydrofolic acid3.
Inhibition of Bacterial Cell Wall Synthesis
Bacteria are classified as Gram-positive and Gram-negative organisms on the
basis of staining characteristics. Gram-positive bacterial cell walls contain
peptidoglycan and teichoic or teichuronic acid, and the bacterium may or may not
be surrounded by a protein or polysaccharide envelope. Gram-negative bacterial
cell walls contain peptidoglycan, lipopolysaccharide, lipoprotein, phospholipid,
and protein. The critical attack site of anti-cell-wall agents is the peptidoglycan
layer. This layer is essential for the survival of bacteria in hypotonic
environments; loss or damage of this layer destroys the rigidity of the bacterial
cell wall, resulting in death.
Outer wall of Gram-positive and Gram-negative species and deail of porin

channels of Gram-negative bacteria.
Antimicrobial agents diffuse easily through the loose outer wall of Gram-positive
but must go through the narrow channels of the Gram-negative species.
IMPORTANCE OF FLUORINE
Introduction of Fluorine
Since the mid 1950 progress in organic fluorine chemistry has been rapidly
translated into useful application in medicinal and biochemistry.
Advance in the area have been accelerated by the development of new technique
and reagents for the site-selective introduction of fluorine into development
specifically for the preparation of drugs. Fluorocarbon emulsion continues to
show promise as blood substitutes, and several important techniques in medical
diagnosis.
Fluorine in bioactive molecules
The incorporation of fluorine in drug molecule as a mean of increasing
therapeutic efficacy is based on several considerations,
1. Fluorine, the second smallest substituent, closely mimics hydrogen with respect
to steric requirements at enzyme receptor sites ( Vander waal’s radii F=1.35Aº ;
H=1.2Aº )
2. The strong electron withdrawing inductive effect of fluorine can significantly
influence reactivity and stability of functional groups and the reactivity of
neighboring reaction centers .
3. The substitution of hydrogen by fluorine at or near a reactive site frequently
causes inhibition of metabolism because of the high C-F bond energy.
4. The replacement of hydrogen by fluorine usually increase lipid solubility, there
by enhancing the rate of absorption and transport of drug invivo.
5. Some time the presence of fluorine instead of hydrogen actually block an
essential biochemical reaction : the fluorine behave as a deceptor group eg. 5-
fluorouracil.
In recent year the heterocyclic compounds are very much used as an antimicrobial
agent. Benzothiazole is a heterocyclic compound containing benzene ring and a
pentane ring include one nitrogen and one sulfur. Different substitutions on
benzothiazole moiety on different position are found to posses different activity,
for example if substitution on 2 position on benzothiazole by any phenyl ring are
used as an anticancer drug4, if substitution on 2,5,6 position is used as anti-
inflammatory agent. Substitution of phenyl imidazole ring used as a anthelmintic
activity5. Substitution of 4-acetamidophenyl sulphonamide on 2 position used as
antitubercular activity6. Substitution of 4-aminophenylsulphonamide on 2 position
and halo compounds on 6 position are used as anticonvulsant activity7.
Substitution on 2 position by 4-aminophenyl and on 6 position by methoxy group
used in Alzheimer’s disease8.
Similarly triazole is also a heterocyclic moiety possess three nitrogen atom in a
pentane ring and used as antimicrobial agent. Some derivatives of triazole are
used as antimicrobial9 , anticancer10, antitubercular11, anthelmintic12 agents.
Present work is on the combination of benzothiazole and triazole moieties. Both
moieties have different activity so we made the combination of both as
benzothizolidinetriazole.
AIMS AND OBJECTIVES
1. To establish the method of synthesis for the proposed compounds.
2. To synthesize the title compounds by appropriate methods.
3. To carry out the preliminary tests such as physical constant determination,
solubility, TLC.
4. To confirm the structures of the synthesized compounds by IR, 1H NMR,
Mass spectra.
5. To evaluate the proposed compounds for their antimicrobial activity.
REVIEW OF LITERATURE
Chemistry of benzothiazole is as old as recorded history. The compounds
encompassing benzothiazole moiety are of great interest and have been
extensively used in pharmaceutical chemistry and agriculture division.
Heterocycles bearing a benzothiazole ring residue are reported to show
anticancer, anti-inflammatory, analgesic, muscle-relaxant, sedative,
antitubercular, diuretic, antimicrobial, anticonvulsant, antiallergic, antimalerial,
antiviral, antioxidant, CNS depressant, and plant growth regulatory activity etc.
In addition, benzothiazole forms an important pharmacaphore in fungicidal,
herbicidal and insecticidal, agents. The use of benzothiazole derivatives is
voluminous and depicted below.
Antimicrobial activity
Udapudi and Mahajanshetty13 in 1986 synthesized some 2-arylamino-4,5,6,7-and
2-substiuted 5,5-dimethyl-7-oxo-4,5,6,7-tetrahydrobenzothiazoles for their
antimicrobial activity. All the compounds were screened in vitro for their
antifungal activity against Candida albicans and Aspergillus niger, and for
antibacterial activity against Staphylococus aureus and Eschirichia coli.
Compounds 3 in general displayed better activity than 1 and 2.
N
R
S
1
R=-C10H7NH-, -C10H7NH-, p-NO2C6H4NH-
C6H5CH2NH-, 4-Cl-2-CH3-C6H3NH-,
4-Br-2-CH3-C6H3NH-
2-arylamino-4,5,6,7-tetra-hydrobenzothiazoles
N
R
S
O
2
R= C6H5NH-, C6H5CH2NH-, O-Cl-C6H4NH-
p-Br-C6H4NH-, O-OCH3-C6H4NH-,
NH2-, CH3-, NH2NH-, C6H5-
2- substituted 5,5-dimethyl-7-oxo-4,5,6,7-tetra-hydrobenzothiazoles
HBr
N
R
S
O
3
R= C6H5NH-, C6H5CH2NH-, O-Cl-C6H4NH-

p-Br-C6H4NH-, O-OCH3-C6H4NH-,
NH2-, CH3-, NH2NH-, C6H5-
S. B. Bawsar14 in 1996 synthesized 8-[(6’-substituted-1’,3’-benzothiazole-2’-
yl)aminomethyl]-substituted hydroxycoumarins for their antimicrobial activity.
R N
S NH
CH2
O O R4
R3
R1 R2
R= Cl, R1= CH2COOCH3, R2= H, R3=H, R4= OH
T.Usha Rani and V.M.Reddy15 in 1997 reported ‘One-pot’ synthesis of 6-
hydroxy-5-undecyl-2-N(substitutedamino)benzo[2,3-d]thiazol-4,7-diones from
bromoembelin. Compounds were evaluated for their fungicidal and bactericidal
activity against Bacillus substilis, Escherichia coli, Proteus vulgaris, Bacillus
polymixia, Fusarium oxysporum and Curvularia lunata.
O O
N C 11H23 N
C11H23
NHR NHAr
S S
HO HO
O O
R=H,CH3,COCH3 Ar=2,6-dinitrophenyl,benzyl,
2-carboxyphenyl.
Hafez M.El-shaaer et al16 carried out the synthesis of the biologically active novel
systems derived from reaction of 3-formylchromones with three types of amino
derivatives, 6-R2-2-aminobenzothiazoles, 6-amino-2-R3-thiobenzothiazoles and
hydrazide derivatives(derived from cyanoacetic, isonicotine,salicylic and gallic
acids). They studied antimicrobial activity against bacteria Staphylococcus aureus
29/58, Bacillus substilis 18/66, E.coli 326/71; yeast:candidaalbicans; moulds:
Microsporum gypseum, Aspergillus nigar; and against typical and atypical
mycobacteria: Mycobacterium tuberculosis (H37 Rv), Mycobacterium kansasii
(PFG 8). They reported the hereditary bleaching effect on the plastid system of
Euglena gracilis, a unique phenomenon of the biological activity of chromone
derivatives. The bleaching test on E. gracilis is used for detecting extranuclear
mutations.
O
R1 S
CH=N R2
O N
R1= 6-CH3, 6-Cl, 6-Cl, 6-Cl

R2= 4-Cl, H, 4-Cl, H
Shastry et al17 in 2003 synthesize some newer cogners of benzothuiazole by
incorporating the sulphonamide and azetidinone moieties in single framework.
They synthesized the various 6-fluoro-7-(substituted)-2-[p-(arylidene)anilino
sulphonamido] benzothiazoles and these compounds were screened for their
antimicrobial activities. Some compounds showed good antibacterial activity
against E. coli, Staphylococus aureus and Salmonella typhi.
R1
N
NHO2S N Cl
S
F
R O
R R1
Anilino p-N,N-dimethylamino phenyl

Anilino m-Nitrophenyl
6-Fluoro-7-(substituted)-2-[p-(3’-chloro-2’-oxo-4’-substitutedaryl-1’-
azetidinyl)benzene sulphonamido]benzothiazoles.
R1
N
S
NHO2S N
S
F
R O
R R1
Anilino p-N,N-dimethylamino phenyl

p-Nitroanilino p-Chloro phenyl
6-fluoro-7-(substituted)-2-[p-(2’-substitutedphenyl-4’-thiazolidinone-3’-
yl)benzene sulphonamido]benzothiazoles.
Recently Andrea Latrofa et al18 in 2005 reported synthesis of N-cycloalkenyl-2-
acylalkylidine-2,3-dihydro-1,3-benzothiazoles and these compounds shows
interesting activity against gram positive and gram negative bacteria. The
preliminary structure-activity relationship suggested the lipophilicity as an
important property modulating the activity of the reported compounds, together
with the observation that the antibacterial activity disappear on replacing the
hydrogen atom of the following structure (R’=H) within alkyl or acyl group.
O
S R2
N R1
( )n
( Structural modifications-- )
structural modifications (---) of the N-cycloalkenyl-2acylalkylidene-2,3-
dihydro-1,3-benzothiazole.
Antifungal activity
K.Raghava Raju and P.S . Rao19 in 1985 synthesized new benzothiazole
derivative using lichen metabolites and screened for antifungal activity. The
following synthesized compound exhibited strong fungicidal activity against
phytopathogenic fungi viz Drechslera rostrata and Alternaria alternata at 200
g/ml.
HO
N
O
O
S
5-(-2-benzothiazolyl benzylidine)-4-hydroxy-3-phenyl-2(5H)furanone.
Ram lakhan and Babban J.Ral20 in 1986 synthesized five new 2-methyl-3-
[(substituted)benzothiazol-2’-yl]-4(3H)-quinazolinone. Three of them were tested
for their antifungal activity against agricultural fungi by the food poison technique
and the activity was compared with that of Dithan M-45.
X
O N
Y
N S
N CH3
No. X Y
1 5-NO2 H
2 6-NO2 H
3 4-CH3O 7-Cl
4 4-NO2 6-Cl
5 4-Cl 6-NO2
Bujdakova H et al21.in 1993 compared anti-candida activity of 6-amino-2-n-
pentylthiobenzothiazole(I), benzylester of (6-amino-2-benzothiazolylthio)acetic
acid(II) and of 3-butylthio-(1,2,4-triazolo)-2,3-benzothiazole(III) with that of 2-
mercaptobenzothiazole(IV). I and II exhibited good activity against the
C.albicans yeast form, similar to IV.
Antibacterial activity
Gurupadaiah, et al22 in 1998 synthesized 6-fluoro 7-[aniline /morpholino/
piperazino]-2-[N-p-tolylsulphonimido]benzothiazoles by cyclization of 4-fluoro-
3-chloro aniline with potassium thiocynate to give 2-amino-6-fluoro-7-chloro-
benzothiazole, followed by treatment with p-tolylsulphonyl chloride and amino
compounds, and their derivatives have been characterized by their analytical and
spectral properties. Some of these have been found to exhibit antimicrobial
activity.
N
NHSO2 CH3
S
F
R
R= Substituted aromatic and aliphatic amines.
Javed et al23 in 2004 synthesized substituted benzothiazoles having thioureido
group at the 2 position. They have synthesized the series of 2-[(4’-
halophenyl)thioureido]-6-substituted benzothiazoles. All the synthesized
copmpounds were tested in-vitro for their antibacterial activity against S. aureus
(Gram positive) and E. coli (Gram negative) bacteria. Among the compounds
tested following two compounds were found to be most potent against S. aureus
and and E. coli.
N
NH C NH
R S
S R1
R R1
CH3 F
OCH3 Br
Antitumor activity
Masao Yoshida et al24 showed based on 2-methyl-4-nitro-2H-pyrazole-3-
carboxalic acid[2-(cyclohexanecarbonylamino)benzothiazol-6-yl]amide (1),
which shows selective cytotoxicity against tumorigenic cell lines, 2,6-dichloro-N-
[2-(cyclopropanecarbonylamino)benzothiazol-6-yl]benzamide (2) was designed
and synthesized as a biologically stable derivative containing no nitro group. The
O N
NH
N
NH S
N
O
NO2
highly potent derivative (2) exhibited excellent in vivo inhibitory effect on tumor
growth. Selected regions of interest for SAR evaluation of screening hit
compound 1
Cl O N
NH
NH S R
Cl
R2
R= O
O
R2= Cyclo propyl. (2)
Malcolm et al4 in 1994 prepared a series of polyhydroxylated 2-
phenylbenzothiazole by demethylation of the precursor methoxylated 2-
phenylbenzothiazole. The compound inhibit WiDr human colon tumor cells and
MCF-7 human mammary tumor cells in vitro with IC50 value in the low micro
molar range. But the activity against MCF-7 cells is not related to estrogen
receptor binding affinity.
N R2
R1
S
R1 and R2= H, OMe or (OMe)2.
Dong-Fang Shi et al25 in 1996 synthesized a new series of 2-(4-
aminophenyl)benzothiazoles substituted in the phenyl ring and benzothiazole
moiety by simple, high-yielding routes. The molecule shows potent inhibitory
activity in vitro in the nanomolar range against a panel of human breast cancer
cell lines, but is inactive (IC50>30 M) against other cell types.
N
NH2
S
S
Mei-Sze Chua et al26 in 1999 synthesized 2-(4-acylaminophenyl)benzothiazoles
and investigated the role of acetylation in the antitumor activities of parent
amines. They synthesized N-acyl derivatives of arylamine, and in vitro studies
N-oxidation C-oxidation
( when R=Me)
R
N
C-oxidation NH2
S
N-oxidation(and conjugation)
N-acetylation(then oxidation
S-oxidation and conjugation)
confirmed N-acetylation and oxidation as the main metabolic transformations of
2-(4-aminophenyl)benzothiazoles, with the predominant process being dictated by
the nature of 3’-substituent.
G. Wells et al27 in 2000 synthesized a series of new antitumor agent, the
benzothiazole substituted quinol ethers and esters via the hypervalent iodine
mediated oxidation of hydroxylated 2-phenylbenzothiazoles. The products were
found to be active in vitro against human colon and breast cancer cell lines.
N
O
S RO
(R= acyl, alkyl)
Hutchinson et al28 in 2000 focused on the crucial role of oxidative metabolism
mediated by the cytochrome P450 isoform CYP1A1, resulting in the formation of
the 6-hydroxy metabolite for better understanding of biological mechanism of
action of the following lead compound(X= Me, R= H). In order to circumvent this
undesirable hydroxylation, they synthesized range of mono- and di-fluorinated 2-
(4-amino-3-methylphenyl)benzothiazoles (X=Me, R=F) which might divert
metabolism elsewhere in the molecule and lead to a new generation of more
potent and efficacious antitumor agents.
N
R NH2
S
X
X= H, Me, Cl, Br, I
R=H,F
Dong-Fang Shi et al29 in 2001 have synthesized a series of sulfamate salt
derivatives of the potent and selective 2-(4-aminophenyl)benzothiazoles and their
evaluation as potential prodrug for parentral administration was carried out. The
salt were sparingly soluble under aqueous conditions (pH 4-9), and degradation of
the active free amine was shown to occur under a strongly acidic conditions. The
salts were found to be markedly less active than their parent amines against
sensitive human tumor cell lines in vitro.
R
N
NHSO3
S Y
a (R=H,Y=Na or NH4)
b (R=Me, Y=Na)
C (R=Cl, Y=Na)
d (R=I, Y=Na)
Ian Hutchinson et al30 in 2002 synthesized a series of water-soluble L-lysine- and
L-alanyl-amide prodrugs of the lipophilic antitumour 2-(4-
aminophenyl)benzothiazoles . The prodrugs exhibited the required pharmaceutical
properties of good water solubility (in weak acid) and stability at ambient
temperature and degradation to free base in vivo. The lysyl-amide of 2-(4-amino-
3-methylphenyl)-5-fluorobenzothiazole (NSC 710305) has been selected for
phase 1 clinical evaluation.
R1 O
N NH2
R2 NH
H (CH2)4NH2
S
R1=CH3 R2=5-F
Paola vicini et al31 in 2003 synthesized three new series of benz[d] isothiazole,
benzothiazole and thiazole, schiff’s base and tested in vitro with the aim of
identifying novel and lead compound active against emergent and reemergent
human and cattle infection diseases (AIDS, hepatitis B and C, TB, bovine viral
diarrhoea) or against drug resistant cancers (leukemia, carcinoma, melanoma,
MDR tumors). The benzo (d) isothiazole compound show a marked cytotoxicity
(CC50 = 4-9 M) against CD4+ lymphocytes ( MT-4) that were used to support
HIV-1 growth. For this reason, the most cytotoxic compound of this series were
evaluated for that antiproliferative activity against panel of human cells lines
derived from hematological and solid tumors. The results highlighted that all the
benzo(d) isothiazole derivatives inhibited the growth of leukemia cell lines.
R
N N
N CH R'
S N CH R'
R S
a. R=H; R’=C6H5 a. R=H; R’=C6H4
b. R=H; R’=2-ClC6H4 b. R=H; R’=3-ClC6H4
c. R=F; R’=3-ClC6H4 c. R=4-OCH3C6H4; R’=C6H5
N CH R'
R
N
S
a. R=H; R’=3-ClC6H4 b. R=H; R’=4-OCH3C6H4 c. R=H; R’=5-NO2-2-Furanyl.
Structures of the benzo[d]isothiazole, benzothiazole and thiazole Schiff bases.
Based on 2-methyl-4-nitro-2yl-pyrazole-3-carboxylic acid [2-(cyclohexane
carbonyl amino) benzothiazol-6-yl] amide, which shows selective cytotoxicity
against tumour cell lines, Yoshida et al32 in 2005 synthesized 2-6-dichloro-N-[2-
(cyclopropane carbonyl amino)benzothiazole-6-yl]benzamide synthesized and
tested as a biologically stable derivatives containing no nitro group. One of the
compound showed excellent in vivo inhibitory activity on tumor growth.
N
Cl O NH
S
NH
O
Cl
Anti-inflammatory activity
Jin Wada et al33 in 1973 reported the synthesis of 2,5,6-trisubstituted
benzothiazoles compounds with an acetic acid function at the 5 or 6 position and
investigated for their anti-inflammatory activity. It was found that presence of an
acetic acid function was important for anti-inflammatory activity and also that 2-
substituted 5-benzothiazole acetic acid were better than 2-substituted 6-
benzothiazole acetic acid in anti-inflammatory activity.
HOOCH2C N
R
S
R=C6H5, 2-HOC6H4, 3,4-(CH3O)2C6H3, 2-Pyridyl
2-substituted 5-benzothiazoleacetic acid
N
R
HOOCH2C S
R=C6H5, 2-HOC6H4, 4-CH3 , 4-(CH3)2NC6H4
6-Benzothiazoleacetic 2-Substituted acid
Hiroki Miyanatsu et al34 in 1974 synthesized compounds of 5-benzothiazole--
alkylacetic acid with substitutions at 2 position and /or the alkyl function and
HO2CH2C N
S
screened for their pharmacological activities. They found that methyl group  to
the acetic acid function of 2-phenyl-5-benzothiazoleacetic acid lowered toxicity
and enhanced anti-inflammatory activity relative to that phenylbutazone
employed as a standard.
Anthelmintic activity
Nadkarni et al5 in 2000 have synthesized the substituted phenyl imidazo [2,1-
b]benzothiazoles and tested for their anthelmintic activity.
N
N
F S
Cl R=2-F, 3-F, 3-CF3
Anti tubercular activity;
Bhusari et al6 in 2000 have synthesized 2-(4-acetamidophenyl sulphonamido)
benzothiazoles and 2-(4-aminophenyl) benzothiazoles containing different
functional groups and have been established on the basis of their elemental
analysis and spectral (IR 1HNMR and MASS) data. All compounds have been
screened for their antitubercular activity in vitro.
CH3
N
NHO2S NHCOCH3
S
2-(4-acetamidophenyl sulphonamido)benzothiazole
CH3
N
NHO2S NH2
S
2-(4-aminophenyl sulphonamido)benzothiazole
Anticonvulsant activity.
Siddiqui et al7 in 2004 synthesized a series of new sulphonamide derivatives
having benzothiazole nucleus by treating 2-(4-aminophenylsulphonamido)-6-
halo/alky benzothiazoles with alkylisothiocyanates. The identity of compounds
were confirmed on the basis of their spectral data. The synthesized compounds
were evaluated for their anticonvulsant activity.
N
NHO 2 S NHCNHR2
S
R1 S
R1= CH3, OCH3, Cl, Br, F R2= CH3, C2H5
Plant Growth Regulator
Dusan Loos et al35 in 1999 have synthesized 3-alkoxycarbonylmethyl-6-bromo-
and 3-alkoxycarbonylmethyl-6-nitro-2-benzothiazolones by reaction of
alkylesters of halogenoacetic acids with 6-bromo-2-benzothiazolones and 6-nitro-
2-benzothiazolones respectively. The compounds were tested for plant growth
stimulating activity on wheat (Triticum aestivum).
Br S O2N S
O O
N N
OMe OMe
O O
Alzheimer’s Disease;
Majo et al8 in 2003 synthesized a novel and convergent palladium catalyzed 2-
arylbenzothiazole. The key step in the synthesis is a Suzuki biaryl coupling of 2-
bromobenzothiazole with aryl boronic acids to provide a variety of 2-
arylbenzothiazole derivatives in good yield. The synthetic utility of this
methodology is demonstrated by the synthesis of 2-(4-aminophenyl)-6-
methoxybenzothiazole, a PET prove precursor for the in vivo imaging of
Alzheimer’s disease.
X S
Ar
N
X= OMe, Ar= H
Antidiabetic activity
Pattan et al36 in 2005 have synthesized a new series of 2-amino[5’(4-
sulphonylbenzylidine)-2,4-thiazolidinedione]-7-chloro-6-fluorobenzothiazole. He
confirmed the compounds by UV-Vis, IR and NMR spectroscopy. And the
compounds were screened for their antidiabetic activity on albino rats.
N O
NHO2S
S NH
F S
Cl
O
Zandt et al37 in 2005 discovered and synthesized a novel series of 3-[(4,5,7-
Trifluorobenzothiazol-2-yl)methyl]indole-N-acetic Acid (Lidorestate) and
Congeners as highly potent and selective inhibitor of Aldose reductase for
treatment of chronic diabetic complications.
F
N
R F
S
N
O F
R=morphilino OH
R=phenoxy
Xiangdong Su et al38 in 2006 synthesized the benzothiazole derivatives a selective
inhibitors of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) have
considerable potential as treatments for metabolic diseases, such as diabetes
mellitus type 2 or obesity. They reported a series of novel benzothiazole
derivative and their inhibitory activities against 11β-HSD1 from human hepatic
microsomes measured using a radioimmunoassay (RIA) method.
O H
Cl
Ar S N N
O
S
Ar= 2,5-Dichloro phenyl
Ar= 4-n-propyl phenyl
Antiviral activity
Sting Akerfeldt39 in 1970 studied the effect of various heterocyclic ring system on
mice infected with various influenza virus strains, they found that 2-
aminobenzothiazole, when administered intraperitoneally to mice, gave a
protection to the animals quite comparable to that of aminoadamantane. They also
studied structure activity relationship to investigate whether certain structural
changes could improve the antiviral effect of compound. They found remarkable
sensitivity to variations in structure, out of 17 benzothiazoles only the 2-amino-
and 2-amino-4-chloro derivatives showed significant protective effect at the dose
levels tested.
Leuketriene D4 Antagonist and 5-Lipooxygenase Inhibitors.
Musser et al40 in 1987 synthesized the series of novel benzoheterocyclic
[(methoxyphenyl)amino]oxoalkanoic acid esters. These compounds were tested as
inhibitors of rat plymorphonuclear leukocytes 5-lipooxygenase (LO) in vitro and
as inhibitors of leukoetriene D4 (LTD4) and ovalbumin (OA) induced
bronchospasm in the guinea pig (GP) in vivo. In general, inhibitory activity
against 5-LO, LTD4, and OA was broadest for benzthiazole-containing analogues
(benzothiazole, benzimidazole, benzoxazole, benzofuran). They found 4-[[3-(2-
benzothiazolyl)-phenyl]hydroxyamino]-4-oxobutaonic acid methyl ester was most
potent and had IC50 of 0.36 M.
R1
N OR2
n
N
Y O O
X
X= S, Y= CH20, R1= OH, R2=CH3, n= 2
Antiallergic activity
Wade et al41 in 1983 synthesized 2- or 3-carboxy-4H-pyrimido [2,1-b]-benzazol-
4-ones by reacting 2-aminobenzothiazole, 2-aminobenzoxazole and 2-amino-1-
methylbenzimidazole with dimethyl aminofumarate (DMAF) or diethyl
ethoxymethylenemalonate (DEEM) respectively. Subsequent derivatization of
these carboxylic acids gave the corresponding tetrazolylcarboxamides and
tetrazoles. These acidic compounds were tested in the rats passive cutaneous
anaphylaxis (PCA) assay as a potential antiallergic agents. Incorporation of an
acidic functionality [carboxylic acid, N-(1H-tetrazole-5-yl)carboxamide, or
tetrazole] at either the 2- or 3- position of the 4H-pyrimado[2,1-b]benzazole-4-
one ring system gives good antiallergenic property.
N N X
CONH N
R= COOH, N
N
N N N N N
H H 2
R
3
O
X= O, S, NCH3
4H-pyrimado[2,1-b] benzazole-4-one ring system
Cox-2 Inhibitor
Paramshivappa et al42 in 2003 synthesized a series of 2-[[2-alkoxy-6-
pentadecylphenyl)methyl]thio]-1H-benzimidazoles/benzothiazoles and
benzoxazoles from anacardic acid and screened for their ability to inhibit human
cyclooxygenase-2 enzyme (COX-2). They found that 5-(methoxy)-2-[(2-hydoxy-
6-pentadecylphenyl)-methyl]-thio]-1H-benzimidazole is 384-fold and 2-[(2-
methoxy-6-pentadecylphenyl)-methyl]-thio]-benzothiazole is more than 470-fold
selective towards COX-2 campare to COX-1.
H
OH N
CH2 S
N OMe
C15H31
5-(methoxy)-2-[(2-hydox-6-pentadecylphenyl)-methyl]-thio]-1H-benzimidazole
OMe S
CH2 S
N
C 15H31
2-[(2-methoxy-6-pentadecylphenyl)-methyl]-thio]-benzothiazole
Muscle relaxant
Trapani et al43 in 1996 synthesized a series of substituted imidazo[2,1-

b]benzothiazoles and evaluated for their affinity at the central benzodiazepine
receptors. They found that substitution at the 7-position generally resulted in a
decreased ligand affinity where as a significant increase was observed for 5-
substituted compounds.
R4
R3 S
N
R2 N
COR
R1
Compound R R1 R2 R3 R4
a OCH3 H H H H
b OCH2C6H5 H H H H
c OC2H5 H H Cl H
d NH2 H H H H
Platelet ADP receptor (P2Y12) antagonist
Scarborough et al44 synthesized novel non-nucleoside tricyclic platelet ADP
receptor (P2Y12) antagonists by condensing various 2-aminobenzothiazoles with
chlorosulfonylacetyl chloride.
Photosynthesis-inhibiting activity in spinach chloropasts
In continuation of synthesis of photosynthetic inhibitors Eva Sidoova et al45 in
1999 have reported another group of compounds namely 3-(2-alkyl sulfanyl-6-
benzothiazolyl-aminomethyl)-2-benzoxazolethiones. The inhibition of
photosynthetic electron transport in spinach chloroplast was monitored by
reduction of DCPIP in presence of the studied compounds. The tested compounds
exhibited increased photosynthetic inhibition activity.
O
S S S R
N
CH2-NH N
R= CH3, C2H5, -(CH2)-CH3, -CH2-CH2-CH2,

-CH2CH=CH2, -(CH2)3-CH3, -(CH2)4CH3
Polyglutamine aggregation inhibitors of Huntington’s disease
Benzothiazole derivatives have been shown previously to be effective in treating
neurodegenerative disorders such as amyotrophic leteral sclerosis46. For example,
riluzole (2-amino-6-trifluoromethoxybenzothiazole), a potent antagonist of
glutamate release, has been reported to slow down disease progression in
amytrophic leteral sclerosis patients47.
N
NH2
F3CO S
Riluzole
The identification of benzothiazoles as polyglutamine aggregation inhibitors is
highly relevant because they are very promising candidates for future drug
development. The few structures of most effective compounds in the in vitro
screen having IC50 values in the range of 1-11M are as follows48.
H2N
N
NH2
H2N S
Benzothiazol-2,5,6-triamine (IC50, M = 3.5  1.2)
N N
H2N S S NH2
[6,6’] Bibenzothiazolyl-2,2’-diamine (IC50, M = 2.2  0.2)
Triazole
Triazole refers to either one of a pair of isomeric chemical compounds with
molecular formula C2H3N3, having a five-membered ring of two carbon atoms
and three nitrogen atoms.
The two isomers are: 1,2,3-Triazole and 1,2,4-Triazole.
N N
N N
N N
1H-1,2,3-Triazole 1H-1,2,4-triazole
1,2,4-Triazoles can be prepared synthetically using the Einhorn-Brunner reaction in
which an imide is reacted with an alkyl hydrazine to form a mixture of isomeric
R3
NH R3
O O R3 NH2
N N N N
+
R1 NH R2
R1 N R2 R1 N R2
1,2,4-triazoles.
1,2,4-Triazole derivatives find use in a wide variety of applications, most notably as
antifungal such as flucanazole and itraconazole. The triazole plant protection
fungicides include epoxiconazole, tridimenol, procinazole, cyproconazole,
tebuconazole, and flusilazole49.
N
N
N
HO
N
N
N
F F
Flucanazole
Antimicrobial activity
Jag Mohan et al50 in 1996 reported the synthesis of 2,5-disubstituted thiazolo [3,2-
b]-s-triazoles and the isomeric 3,5-disubstituted triazolo [2,3-c]-s-triazoles and
evaluated for their antimicrobial activity against Gram-positive and Gram-
negative bacteria. They have also studied diuretic, antifungal activity of some of
the compounds.
Cl N N
N S
Balakrishna Kalluraya et al9 in 1996 synthesized 3-(2-isopropyl-5-
methyl)phenoxymethyl-4-amino-5-mercapto-1,2,4-triazole, their N-bridged
heterocycles and Mannich bases of 1,3,4-oxadiazole starting from thymol. Some
of the newly synthesized compounds were screened for their antibacterial and
antifungal activity.
N N
Ar OH2C N SH
NH2
Andotra et al51 in 1996 synthesized some trisubstituted aryl-1,2,4-triazolo[1,5-a]-
s-triazine-5,7(1H,6H)-dithiones and were evaluated for antibacterial and
antifungal activity against Aspergillus flavous, Fusarium solani, S. aureus and E.
coli.
R1 N N NH
N N S
R2O H
OR3
compound R1 R2 R3
a C2H5 CH3 CH3
b C2H5 C2H5 C2H5
c C2H5 C2H5 CH3
d n-C3H7 CH3 CH3
Reddy et al52 in 1997 reported the one-pot synthesis of new 3-aryl-7-hydroxy-8-
undecyl-benzo [2’,3’-d]-thiazolo [2,3-d]-1,2,4-triazol-6,9-diones from
bromoembelin. These compounds have been screened for their antimicrobial
activity. A few of them have been found to exhibit moderate activity.
O
H23C11
N N
RO S N Ar
O
R= H Ar= Phenyl, 4-methylphenyl, 3-methylphenyl,
4-methoxyphenyl, 4-nitrophenyl.
Tsuruoka et al53 in 1997 synthesized the series of novel thiazole-containing
triazole and evaluated for their antifungal activity against variety of clinically
isolated pathogenic fungi in vitro and against systemic candidosis in vivo. Among
these compounds, (+/-)-1-(2,4-diflurophenyl)-1-[4-(2,4-diflurophenyl)thiazol-2-
yl]-2-(1H-1,2,4-triazol-1-yl)ethanol (ER24161) showed most potent and well
balanced in vitro activities and excellent in vivo efficacy. They also achieved an
enantioselective synthesis of the more potent enantiomer of ER-24161.
Tsuruoka et al54 in 1998 synthesized the series of novel thiazole-containing
triazole and evaluated for their antifungal activity against variety of clinically
isolated pathogenic fungi in vitro and against systemic candidosis in vivo. In
particular ,(2R,3R)-3-[4-(4-cynophenyl)thiazol-2-yl]-2-(2,4-diflurophenyl)-1-(1H-
1,2,4-triazole-1-yl)-2-butanol (ER-30346) showed potent and well balanced in
vitro activities and potent in vivo efficacy, and a good safety profile.
Jag Mohan and Anupama55 in 1999 given the facile synthesis of 2,3-diaryl-cis-
3,3a-dihydropyrazolo [3’,4’: 4,5] thiazolo [3,2-b]-s-triazoles. The antibacterial
and antifungal activities of the system have also been evaluated.
R
R
N
N
N N
N S
OMe
Cl
R= NO2, H
Nuray Ulusoy et al56 in 2001 synthesized ethyl 5-(2-furyl)-4-ethyl-1,2,4-triazole-
3-mercaptoacetate, 5-(2-furyl)-4-ethyl-1,2,4-triazole-3-mercaptoacetic acid
hydrazide and a series of new N-alkylidene/arylidene-5-(2-furyl)-4-ethyl-1,2,4-
triazole-3-mercaptoacetic acid hydrazides and evaluated for in vitro antibacterial
activity against Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis
ATCC 12228, Klebsiella pneumoniae ATCC 4352, Pseudomonas aeruginosa
ATCC 1539, Escherichia coli ATCC 8739, Shigella flexneri, Salmonella typhi,
Proteus mirabilis and antifungal activity against Candida albicans ATCC 10231
using the disk diffusion and microdilution methods. Some compound showed
antibacterial activity against some bacteria.
N N
SCH2CONHN CHR
O N
C2H5
R= C6H4OCH3(3-), C6H4OCH3(4-), C6H4NO2(4-),C6H3(OH)Br(2,5-).
Shivaram Holla et al57 in 2005 synthesized 6H-5-aryl-9-halo-1,2,4-triazolo [3’,4’
:3,4]-1,2,4-triazino[5,6-b]-indoles and 5H-1-aryl-5-halo-1,2,4-triazolo[4’,3’:2,3] -
1,2,4-triazino[5,6-b]-indoles. All the newly synthesized compounds were
evaluated for their antibacterial activity against B. subtilis, E. coli, P. aeruginosa
and S. aureus.
X N R
N
N N N
H
N X N
N
N
R N N N
H
X= Cl, Br R= H, p-N(CH3), m,p-O2CH2 X= Cl, Br R= H, p-N(CH3), m,p-O2CH2

6H-5-aryl-9-halo-1,2,4-triazolo [3’,4’ :3,4]- 5H-1-aryl-5-halo-1,2,4-triazolo [4’,3’:2,3] –
1,2,4-triazino [5,6-b]-indoles 1,2,4-triazino [5,6-b]-indoles
In order to improve the antibacterial and anticancer activities of
triazolothiadiazine Shivaram Holla et al58 in 2006 studied the substitution effect at
C3 position by arylomethyl and anilinomethyl moeties. In this regard they
synthesized the series of 7-arylidine-6-(2,4-dichlorophenyl)-3-
aryloxymethyl/anilinomethyl-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine. Newly
synthesized compounds were tested for their antimicrobial activities.
N N
R
XCH2 N S
R1
N
Cl
F
Cl
X= O, R= 2-Cl, 4-Cl
R1= 4-Cl, 3,4(OCH2)
Sztanke et al59 in 2006 synthesized 7-(4-methylphenyl)-3-methylthio-5H-6,7-
dihydroimidazo[2,1-c][1,2,4]triazole by the alkylation of the 7-(4-methylphenyl)-
2,5,6,7-tetrahydroimidazo[2,1-c][1,2,4]triazol-3(H)-thione with methyl iodide.
This compound showed superior activity to ampicillin.
7-(4-methylphenyl)-3-methylthio-5H-6,7- dihydroimidazo[2,1-c][1,2,4]triazole
Anthelmintic Activity
Bhusari et al12 in 2000 reported the synthesis and anthelmintic activity of new 8-
bromo-9-chloro-1,3-benzothiazolo-[5,1-b]-1,3,4-triazoles. The anthelmintic
activity for all the newly synthesized compounds was carried out in vitro against
earthworms Perituma posthuma.
N N
Br S N
R H
CH3
R= NH CH3 NH
,
,
NO2
NH
N
,
O
Jayachandran et al60 in 2006 synthesized the various 8-fluoro-9-substituted (1,3)-
benzothiazolo(5,1-B)-1,2,4-triazoles. These compounds were examined for their
anthelmintic activity against earthworm (Perituma posthuma).
Antitubercular activity
The earlier study indicated that thiol grouping at position 5 enhances the
antitubercular properties of the triazoles. In view of this R. H. Udupi and A.R.
Bhat11 in 1996 synthesized some new 4-(N-pyridylcarboxamide)-5-mercapto-3-
substituted-1,3,4-triazoles. These compounds were tested against Mycobacterium
tuberculosis.
N N
Ar SH
N
NH CO N
R= C6H5, CH3,H
R1= o, m, p-Nitro, o, m, p-Chloro, aniline, morpholine.
N R
N
F S N
H
NH
R1
Ar= Phenyl, 2-chlorophenyl, 2-hydroxyphenyl, 1-Naphthyloxy methyl.
Vera Klimesova et al10 in 2004 synthesized series of 3-benzylsulfanyl
derivatives of 1,2,4-triazole and 4-methyl-1,2,4-triazole by alkylation of starting
triazole-3- thiol with appropriately substituted benzyl halide. All members of the
set were evaluated for in vitro antimycobacterial activity against Mycobacterium
tuberculosis, M. avium, and two strains of M. kansasii. The activities were
expressed as the minimum inhibitory concentration. The compounds exhibited
only a moderate or slight antimycobacterial activity. Minimum inhibitory
concentrations fall into a range of 32->1000 µmol/l. The most active substances
bear two nitro groups or a thioamide group on the benzyl moiety. As regards the
cytotoxicity effect, the evaluated compounds can be considered as moderately
toxic.
N
N
N R1
S
R
R= H, CH3 R1= H, 4-Cl, 3-Cl, 4-F
Anticonvulsant activity
Gitto et al61 in 2003 synthesized new cyclofunctionalized 2,3-benzodiazepines
characterized by a triazolone or triazindione ring fused on the “c” edge of the
heptatomic nucleus. These compounds were evaluated as potential anticonvulsant
agents, and some of them proved to be more potent noncompetitive 2-amino-3-(3-
hydroxy-5-methylisoxazole-4-yl)propionate (AMPA). In particular, 8,9-
dimethoxy-6-(4-bromophenyl)-11H-[1,2,4]triazolo[4,5-c][2,3]benzodiazepine-
3(2H)-one was almost 10-fold more active than GYKI-52466 and 3.5 fold more
active than talampanel.
H
N N
MeO
N O
N
MeO
Br
8,9-dimethoxy-6-(4-bromophenyl)-11H-[1,2,4]triazolo[4,5-
c][2,3]benzodiazepine-3(2H)-one
Antitumor activity
Yaseen A. Al-Soud, Najim A. Al-Masoudi62 in 2004 synthesized a series of new
1,4-disubstituted piperazines bearing substituted 1H-1,2,4-triazoles and/or methyl
group as potential antiAIDS and/or antitumor, via the cycloaddition of the 1,4-bis-
N-methylcyano- and 1-N-methylcyano-4- methyl-piperazines with different
reactive cumulenes.
R2
N
R1 N
N
N N
N
N R1
N
R2
R1= Me, Et, Me, (C2H2)5
R2=Et, isopr., (C2H2)5
1,4-bis-(1,5-dialkyl-1H-[1,2,4]triazol-3-ylmethyl)piperazine
COX-2 inhibitor activity
Latifeh Navidpour et al63 in 2006 designed and synthesized a new type of 4,5-
diaryl-4H-1,2,4-triazole, possessing C-3 thio and alkylthio (SH, SMe or SEt)
substituents, for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors
with in vivo anti-inflammatory activity. The compound, 3-ethylthio-5-(4-
uorophenyl)-4-(4-methylsulfonylphenyl)-4H-1,2,4-triazole, exhibited a high in
vitro selectivity (COX-1 IC50 = 20.5 nM; COX-2 IC50 = 1.8 nM; SI = 11.39)
relative to the reference drug celecoxib (COX-1 IC50 = 3.7 nM; COX-2 IC50 =
2.2 nM; SI = 1.68) and also showed good anti-inflammatory activity compared to
celecoxib in a carrageenan-induced rat paw edema assay.
R1= F, R2= 4-CH3SO2-phenyl, R3= CH2CH3.
3-ethylthio-5-(4-uorophenyl)-4-(4-methylsulfonylphenyl)-4H-1,2,4-triazole
Synthesis of some biologically active compounds containing triazole nucleus.
1. K. T. Potts and S. Husain64 in 1971 reported the synthesis of thiazolo[2,3-c]-s-
triazole by cyclizing 4-methyl-2-thiazolylhydrazine with formic, acetic, or
propionic acid under refluxing for 6-8 hours.
S Formic, Acetic or S
NHNH2 N
Propionic acid
N N N
R Reflux R
R1
R= CH3, Ph R1= SH
2. Shishoo et al65 in 1981 synthesized 1,2,4-triazolo-[4,3-c]thieno[3,2-e]pyrimidines
by refluxing 4-hydrazinothieno[2,3-d]pyrimidines with triethyl orthoformate.
NHNH2 N N
R1 R1
N CH(OC 2 H5 )3 N
S N R3
Reflux S N R3
R2 R2
R1= R2= (CH2)4, CH3 R3= CH3, CH2C6H5
3. C. O. Kangani amd H. E. Master66 in 1996 synthesized 4-aryl-1-methyl-5(4H)
oxo-1,2,4-triazolo-[4,3-a]pyrimido[4,5-a]anthraquinones by treating 3-aryl-2-
hydrazino-4-3-(3H)-oxopyrimido[4,5-a]anthraquinones with acetic anhydride.
NHNH2 N N
H3C
O N N R O N N R
O
acetic
O
anhydride,
O reflux
O
R= H, CH3, OCH3,Cl
4. Manisha V Deshmukh67 in 1998 synthesized 5-methyl[1,2,4]triazolobenzothiazole
by condensing 2-hydrazino-4-methylbenzothiazole with formic acid.
S S
HCOOH
NHNH2
N
N Reflux N
N
CH3 CH3
5. Fouad Bentiss et al68 in 2000 reported the reaction of aromatic nitriles with
hydrazine dihydrochloride in the presence of hydrazine hydrate in ethylene glycol
under microwave irradiation, give 3,5-disunstituted 4-amino-1,2,4-triazoles.
NH2
HO-CH2-CH2-OH N N Ar N Ar
Ar C N Ar Ar
NH2-NH2-2HCl N N N N
NH2NH2, H2O H H
Aromatic nitrile 1,2-dihydro-1,2,4,5-tetrazine 3,5disubstituted4-
amino-1,2,4-triazole
6. Katritzky et al69 in 2000 given a novel one-step synthesis of thiazolo[3,2-b]1,2,4-
triazoles from the reacion of chalcones with bis(1,2,4-triazolyl)sulfoxide.
O
R2
O
N N S Tolune N O
N N
R1 R2 + N
N N 90oC N S R1
7. Michael Ciesielski et al70 in 2005 described a novel copper-mediated oxidative
heterocyclization of hydrazones yielding the corresponding 1,2,4-triazoles.
N R 2CuCl2
N NH CH N N
DMF, 90-140 C N
-2CuCl R
-2HCl
8. Kapratwar et al71 in 2005 synthesized 3’-hydroxy and 3’-mercapto-1,2,4-
triazolo[4’,5’:1,5]-1,2,4-triazolo[3,4-b]benzothiazole by heating 3-hydrazino-
1,2,4-triazolo[3,4-b]benzothiazole independently with urea and carbon disulphide
in the presence of alkali.
OH
S N S N
N Urea, N N
N N N
NHNH2
3’-hydroxy-1,2,4-triazolo[4’,5’:1,5]
3-hydrazino-1,2,4-triazolo[3,4-b]benzothiazoloe
-1,2,4-trazolo[3,4-b] benzothiazole
CS2,
KOH
SH
S N
N N
N N
3’-mercapto-1,2,4-triazolo[4’,5’:1,5]
-1,2,4-trazolo[3,4-b] benzothiazole
METHODOLOGY
SCHEME-I
1. Synthesis of 7-chloro-6-fluoro-benzothiazole-2-ylamine from 3-chloro-4-
fluoro-phenylamine
NH2
N
KSCN ; Br2 NH2
F CH3COOH S
F
Cl Cl
(M1)
2. Snthesis of 7-chloro-6-fluoro-benzothiazol-2-yl-hydrazine from 7-chloro-6-fluoro-
bnzothiazole-2-ylamine
N
NH2NH2H2O N
NH2
NHNH2
F S HCl; Ethylene glycol
S
F
Cl
Cl
(M2)
3. Synthesis of 8-Chloro-7-fluoro-benzo[4,5] thiazolo [2,3-c][1,2,4]triazole
from 7-chloro-6-fluoro-benzothiazol-2-yl-hydrazine
N
N CH(OC2H5)3 N
NHNH2 N
S Xylene S
F F
Cl Cl
(M3)
4. Synthesis of different derivatives of 8-substituted-7-fluoro-benzo[4,5]
thiazolo[2,3-c][1,2,4]triazole from 7-chloro-6-fluoro-benzothiazole-2-
ylamine
N
N
N A 22, B72 , C73 , D74
S No Reaction
F
Cl E75, F76, G77, H78
SCHEME-2
1. Synthesis of 7-substituted-6-fluoro-benzothiazole-2ylamine from 7-chloro-
6- fluoro-benzothiazole-2-ylamine
N
DMF N
NH2
NH2
S R
F S
F
Cl
R
(M4)
2 .Synthesis of 7-substituted-6-fluoro- benzothiazol-2-yl-hydrazine from 7-
substituted-6-fluoro-benzothiazole-2-ylamine
N NH2NH2.H2O N
NH2 NHNH2
S HCl S
F F
Ethylene glycol
R R
2. Synthesis of 8-substituted-7-fluoro-benzo[4,5] thiazolo [2,3-
c][1,2,4]triazole from 7-substituted-6-fluoro- benzothiazol-2-yl-hydrazine
N CH(OC2H5)3 N
N
NHNH2 N
S Xylen
F S
e F
R
R
(MV 1-9)
Table-1 Codes and corresponding R of different derivatives
code R
HN
MV1
HN NO2
MV2
HN
MV3
O2N
HN Cl
MV4
HN F
MV5
HN OMe
MV6
HN
MV7
H3 C
HN
MV8
OH
HN
MV9
H2N
EXPERIMENTAL
Identification and characterization,
The compounds synthesized were identified and characterized by following
methods such as:
 Melting point determination
 Thin layer chromatography
 Infra red spectroscopy
 Nuclear magnetic resonance spectroscopy.
Melting point determination: The melting point of an organic compound was
determined by Thiel’s melting point tube (capillary tube method). The
determination of melting point is the most important and easy way of
differentiating this physical constant of one compound from other.
Thin layer chromatography (TLC) : TLC is an important method for synthetic
chemistry to infer the formation of compound based on the Rf value since
different compound will have different Rf values. It also helps in confirming the
progress of the reaction.
Infra red spectroscopy (IR) : IR is the most important tools for determining the
various functional group and the possible chemical structure. The important
advantage of IR over other technique is that it gives fingerprints (1300-650 cm-1 )
information about the structure (functional group, bonding with each other) of
molecules easily. No two compounds have identical fingerprint region.
This technique is based upon the molecular vibration of the compound such that
each and every bond will vibrate at the different frequency and this vibration
frequency correspond to the IR frequency. Thus IR spectra of each and every
bond will be formed. FTIR spectra were recorded in KBr powder on a Jasco V410
FTIR spectrometer by diffuse reflectance technique.
Nuclear magnetic resonance spectroscopy: The introduction between matter
and electromagnetic forces can be observed by subjecting a substance
simultaneously to two magnetic forces, one stationary and other varying at some
radio frequency. At a particular combination of fields, energy is observed by the
sample and absorption can be observed as a change in single developed by a radio
frequency detector and amplifier. This energy of absorption can be related to a
magnetic dipolar nature of a spinning nucleus. This technique is known as
Nuclear Magnetic Resonance. This technique is useful in assuming the structure
of the molecule. 1H- NMR spectra were measured in CDCl3 and d6-DMSO on a
Bruker Ultraspec 500MHz/ AMX400MHz spectrometer.
The experimental work comprises of,
SCHEME-1
1. Method of preparation of 7- Chloro-6-fluoro-benzothiazole-2-
22
ylamine.
To glacial acetic acid (40 ml) precooled to 5 were added 40 g (2.4 mol) of
potassium thiocyanate and 7.25g (.05 mol) of fluorochloroaniline. The mixture
was placed in freezing mixture of ice and salt and mechanically stirred while 6 ml
of bromine in 24 ml of glacial acetic acid was added from a dropping funnel at
such a rate that the temperature does not rise beyond 0c. After all the bromine
has been added (105min), the solution was stirred for an addition 2 hour at 0c
and at room temperature for 10 hours. It was allow to stand overnight during
which an orange precipitate settled at the bottom, water (30 ml) was added
quickly and slurry was heated at 85c on a steam bath and filtered hot. The orange
residue was placed in a reaction flask and treated with 10 ml of glacial acetic acid,
heated again to 85c and filtered hot. The combined filtrate was cooled and
neutralized with concentrated ammonia solution to pH 6 when a dark yellow
precipitate was collected. Recrystallization from ethanol and water mixture.
2. Method of preparation of 7-chloro-6-fluoro- benzothiazol-2-yl-
hydrazine.79
Concentrated HCl (10 ml) was added dropwise with stirring to hydrazine hydrate
(10 ml) at 5-10c, to it ethylene glycol (22 ml) and 7-chloro-6-fluoro-
benzothiazol-2-ylamine (0.01 mol) were added and charged in microwave for 6
minutes. On cooling solid separated out, which was filtered and washed with
water and recrystallized from ethanol and water mixture.
3. Method of preparation of 8-chloro-7-fluoro-benzo[4,5]thiazolo [2,3-c]

[1,2,4]triazole65
To 7-chloro-6-fluoro-benzothiazole-2-yl-hydrazine (1g, 0.0045M),
triethylorthoformate (1.33g, 0.009M) and 60ml of xylene was added and refluxed
for 4-5 hours. Then xylene was distilled off up to 2/3rd of its volume, cooled it and
filtered, recrystallised from acetonitrile.
General method of prepration of different derivatives of 8-
Chloro-7-fluoro-benzo[4,5] thiazolo [2,3-c][1,2,4]triazole
A. Method of preparation Synthesis of (7-Fluoro-benzo[4,5]thiazolo[2,3-
c] [1,2,4]triazol-8-yl)-phenyl-amine 18
To 8-Chloro-7-fluoro-benzo[4,5] thiazolo [2,3-c][1,2,4]triazole(1g, 0.0044M )
equimolar quantity of phenylamine (0.40gm, 0.0044M) and 20ml of DMF was
added and refluxed for 15hrs, the reaction was checked in an interval of every
3hrs by TLC, After 15 hrs there was no any change in Rf value between starting
material and the reaction mixture.
B. Method of preparation of Synthesis of (7-Fluoro-benzo[4,5]thiazolo[2,3-c]
[1,2,4]triazol-8-yl)-4-nitrophenyl-amine 72
To a suspension of 8-Chloro-7-fluoro-benzo[4,5] thiazolo [2,3-
c][1,2,4]triazole(1g, 0.0044M ) in 20 ml of CH3 CN , (0.60 gm, 0.0044 M) of 4-
nitrophenylamine was added. The reaction mixture was stirred at room
temperature for 2 hrs, after 2 hrs the TLC is checked but there was no any change
in RF value between starting material and reaction mixture, the same reaction
mixture is refluxed for 6 hrs but there was no any changed in Rf value.
C. Method of preparation Synthesis of (7-Fluoro-benzo[4,5]thiazolo[2,3-c]
[1,2,4]triazol- 8- yl)-phenyl-amine.73
A mixture of 8-Chloro-7-fluoro-benzo[4,5] thiazolo[2,3-c][1,2,4]triazole(1g,
0.0044M ) , phenylamine (0.40 g, 0.0044M), anhydrous
potassium carbonate (0.90g) and copper oxide (0.064g)was refluxed for 3hrs
using an oil bath.the excess of phenyl amine was removed by distillation.
Decolourising carbon and water was added to the residual solution.the precipated
was filtered with suction when cold. The product was dried in air and recrytallised
from boiling ethanol. The TLC and melting point was found to be same as starting
material.
D. Method of preparation Synthesis of (7-Fluoro-benzo[4,5]thiazolo[2,3-c]
[1,2,4]triazol-8- yl)-phenyl-amine.74
Phenylamine (0.40 g, 0.0044M) was added slowly to 8-Chloro-7-fluoro-
benzo[4,5] thiazolo[2,3-c][1,2,4]triazole(1g, 0.0044M ) in acetone (20 ml) with
constant stirring within 5 hrs at 0 to 5C. sodium carbonate solution was added to
neutralize HCl evolved during the reaction. Finally the contents were poured into
crushed ice (100 g). The separated was filtered out out , washed with water, dried
and recrystallised from ethanol. The TLC and melting point was found to be same
as starting material
E. Method of preparation Synthesis of (7-Fluoro-benzo[4,5]thiazolo[2,3-c]
To a mixture of 8-Chloro-7-fluoro-benzo[4,5] thiazolo[2,3-c][1,2,4]triazole(1g,
0.0044M ) and phenylamine ( 0.45g,0.0049M) in 20 ml of dioxon, 0.26g of
tetraethylammonium bromide was added with stirring and the reaction mixture
was refluxed for 3 hrs., the reaction mixture was cooled and poured into crushed
ice and allowed to stand for 2 hrs. The TLC and melting point was found to be
same as starting material.
F. Method of preparation Synthesis of (7-Fluoro-benzo[4,5]thiazolo[2,3-c]
0.0044M ) and N-methyl piperazine (0.88 g, 0.0088 M) in pyridine was
refluxed for 20 hrs. The solvent was removed under vacuum and then water was
added. The solid separated was filtered and washed with water and recrystallized
from acetonitrile. The TLC and melting point was found to be same as starting
material.
G. Method of preparation Synthesis of (7-Fluoro-benzo[4,5]thiazolo[2,3-c]
0.0044M ), tetraethyl ammonium bromide (0.92 g, 0.0044 M), diethylamine (0.32
g, 0.0044 M), CH3CN (30 ml) and phenylamine (0.40g,0.0044M) were refluxed at
100C for 30 min on perfect anhydrous conditions and then it was concentrated to
half the volume at reduce pressure. The residue obtained was poured into ice
water at 5C with vigorous stirring. The resulting mixture is left at room
temperature for 5 hrs. Product was collected washed with water, dried and
recrystallised with acetonitrile. The TLC and the melting point was found to be
same as starting material.
H.Method of preparation of(7-Fluoro-benzo[4,5]thiazolo[2,3-c] [1,2,4]triazol-
8- yl)-phenyl-amine.78
To 8-Chloro-7-fluoro-benzo[4,5] thiazolo[2,3-c][1,2,4]triazole(1g, 0.0044M )
equimolar quantity of phenylamine (0.40g, 0.0044M) and 20ml of DMF was
added and refluxed for 15hrs, the reaction was checked in an interval of every
3hrs by TLC, after 15 hrs there was no any change in Rf value between starting
material and the reaction mixture.
SCHEME-2
1. Synthesis of 7-substituted-6-fluoro-benzothiazole-2-ylamine .
To the 7-chloro-6-fluoro-benzothiazole-2-ylamine (1 g, 0.005M), phenylamine
(0.48 gm, 0.0052M) and 20ml of DMF was added and refluxed for 3-4 hrs, the
reaction was checked by TLC. Then reaction mixture was poured into crushed ice
and stirred vigorously for 30 minutes, and set aside for overnight. Filtered under
vacuum and recrystallised with ethanol:water mixture.
2. Synthesis of 7-substituted-6-fluoro- benzothiazol-2-yl-hydrazine
Concentrated HCl (11.8 g, 0.31M) was added dropwise with stirring to hydrazine
hydrate (10.3 g, 0.20 M) at 5-10c, to it ethylene glycol (20-25 ml) and 7-
substituted-6-fluoro-benzothiazol-2-ylamine (0.012 mol) were added and charged
in microwave for 6 minutes. On cooling solid separated out, which was filtered
and washed with water and recrystallized from ethanol:water mixture.
3. Synthesis of 8-substituted-7-fluoro-benzo[4,5] thiazolo [2,3-
c][1,2,4]triazole
To 7-substituted-6-fluoro- benzothiazol-2-yl-hydrazine (0.0045M),
triethylorthoformate(0.009M) and 60ml of xylene was added and refluxed for 4-5
hours. Then xylene was distilled off up to 2/3rd of its volume, cooled it and
filtered, recrystallised from acetonitrile.
TABLE-2 Analytical data of synthesized compounds
Sr. Molecular Nature of % Molecular Molec

No Structure Compound Yield Formula ular
Weigh
t
N
NH2 Color less
S
M1 F powder 95 C7H4ClFN2S 202
Cl
N
M2 NHNH2 Color less
S
F Crystals 90 C7H5ClFN3S 218
Cl
N
M3 N White 60 C8H3ClFN3S 228
N
powder
S
F
Cl
N
N
N
Light
S
MV F yellow 70 C14H9FN4S 284
NH
1 powder
N
N
N
Yellowish –
S
MV F brown 60 C14H8FN5O2 329
NH
2 powder S
NO2
N
N
N
Buff C14H8FN5O2 329
S
MV F powder 72 S
NH
3 NO2
N
N
N
Whitish
S
MV F yellow 65 C14H8ClFN4 318
NH
4 powder S
Cl
N
N
N
Brown
S
MV F powder 70 C14H8F2N4S 302
NH
5
N
N
N
S
F Gray 80 C15H11FN4O 314
MV NH
6 powder S
OMe
N
N
N
White
S
F powder 75 C15H11FN4S 298
MV NH
7 CH3
N
N
N
Yellowish
S
MV F white 80 C14H9FN4O 300
NH
8 powder S
OH
N
N
N
S
MV F Brown 75 C14H10FN5S 299
9 NH
NH2 powder
Table-3 Physical properties of various compounds and derivatives
Code Melting point
No. Solubility Mobile phase for TLC (oC)
M1 Ethanol, Chloroform:Methanol 198-200
Chloroform,
DMSO
M2 Chloroform Chloroform:Methanol 242-244
M3 DMSO Chloroform:Methanol 308-310
MV1 DMSO Chloroform:Methanol 294-295
Spectral Data
TABLE-4 Infra red spectral study of the synthesized compounds
Compound Spectral peaks Molecular nature

-1
(cm )
3479, 3290 -NH2 str (symm, asymm)

3092 -C-H str (aromatic)
1654 -C-S str
M1
1451 -C=N str
1215 -C-F (aromatic)
683 -C-Cl (aromatic)
3318 -NH2 str

3201 -NH str
M2
1650 -C-S str
1451 -C=N str

1604 -C-S str
M3 1500 -C=N str
M4 3475 -NH str
1645 -C-S str
1454 -C=N str
1196 -C-F str
3476 -NH str

MV1 1649 -C-S str
1455 -C=N str
3477 -NH str

1596 -C-S str
MV2 1546 -C-N=O str asymm
1401 -C-N=O str symm
1455 -C=N str
3477 -NH str

MV3 1596 -C-S str
1546 -C-N=O str asymm
1401 -C-N=O str symm
1455 -C=N str
3481 -NH str
MV4 1503 -C-S str
1382 -C=N str
3477 -NH str

1546 -C-S str
MV5
1453 -C=N str
3248 -NH str

2991 -CH-CH str (aliphatic)
MV7
2913 -CH-CH str (aliphatic)
1527 -C-S str
1453 -C=N str
TABLE-5 1H NMR Spectral data of synthesized compounds
Compound
Chemical shift value Proton nature
7.55 d, 1H, Ar-H

M1 7.38 d, 1H, Ar-H
5.34 s, 2H, -NH
9.24 s, 2H, -NH

3.32 s, 1H, -NH
M2 7.84 d, 1H, Ar-H
7.45 d, 1H, Ar-H
9.56 s, 1H, Ar-H

M3 8.55 d, 1H, Ar-H
8.27 d, 1H, Ar-H
12.63 s, 1H, -NH

M4 7.66 s, 2H, -NH
7.43-8.60 m, 6H, Ar-H
Fig: 1 IR of 7- Chloro-6-fluoro-benzothiazol-2-ylamine (M1)
Fig: 2. IR of 7-chloro-6-fluoro- benzothiazol-2-yl-hydrazine
Fig. 3. IR spectrum of 8-chloro-7-fluoro-benzo[4,5]thiazolo [2,3-c] [1,2,4]triazole.
Fig 4 IR of 7-(2-nitrophenylamine)-6-fluoro-benzothiazole-2-ylamine
Fig 5. IR spectrum of (7-fluoro-benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-8-yl)-
2-phenylamine.

4- nitrophenylamine
2- nitrophenylamine
Fig 8 IR spectrum of (7-fluoro-benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-8-yl)-

4-chlorophenylamine.
4-fluorophenylamine.

2-methylphenylamine.
Fig 11 1H NMR of 7- Chloro-6-fluoro-benzothiazol-2-ylamine
Fig 12 1H NMR of 7-chloro-6-fluoro- benzothiazol-2-yl-hydrazine
Fig 13 D2O-Exchange NMR of 7-chloro-6-fluoro- benzothiazol-2-yl-
hydrazine
Fig 14 1H-NMR spectrum of 8-chloro-7-fluoro-benzo[4,5]thiazolo [2,3-c]

[1,2,4]triazole.
Fig: 15 1H NMR of 7-(2-nitrophenylamine)-6-fluoro-benzothiazole-2-
ylamine
Fig 16 D2O-Exchange of 7-(2-nitrophenylamine)-6-fluoro-benzothiazole-2-
ylamine
Fig. 17 EI-MS of 8-chloro-7-fluoro-benzo[4,5]thiazolo [2,3-c] [1,2,4]triazole.
ANTIMICROBIAL ACTIVITY
The antimicrobial drugs occupy uniqueness in the history of medicine. During
the entire proceeding history of medicine, less than a handful of drugs had been
submitted to systemic laboratory investigation. The exponential development in
the antibacterial field is the inevitable result of the momentum created by
sulfonamides and penicillins. The term microbe is sometimes applied only to the
unicellular microphytes. However in its broader sense it includes multicellular
microphytes and microbes as well therefore antimicrobial studies include
antibacterial, antifungal, antiprotozoal and antimycotic studies. After the
development of desired new drug molecules, with different structure, an
antimicrobial screening programme is necessary to uncover the interesting
activity of the compounds. The inhibition of the microbial growth under
standardized may be utilized for demonstrating the therapeutic efficacy of the
synthesized compounds.
The following two are the methods available for screening the antimicrobial
substances.
1. Turbidimetric/photometric/tube dilution method.
2. Agar diffusion/cup-plate/cylinder plate method.
Turbidimetric method
By this method the minimum concentration of the antimicrobial substance
required to inhibit the growth of microbial culture in a uniform bacteriological
fluid medium, containing the specified antimicrobial substance, which otherwise
is favorable for the rapid growth is determined. The minimum concentration
required for inhibiting the growth in called minimum inhibitory concentration
(MIC).
A graded concentration of the antimicrobial substance in sterile fluid nutrient
media is prepared. All of them are inoculated with a loop of specific
microorganism. A positive control, a negative control and a blank is also
maintained. They are incubated at 37 C for 24 hours or necessary conditions
depending on the organism chosen. Among the different concentration of the
substance, the least one, which inhibited the growth of the microorganism, is
noted visually or by measuring the percentage transmittance or absorbance at 530
nm against a blank.
Agar diffusion method
This method gives the extent of growth of the microorganism, inoculated into a
solid nutrient agar bed by the antimicrobial substance. The test substance is kept
in a cup made of agar bed, diffuses into it and inhibits the growth of
microorganism. The diameter of the zone of inhibition is measured in comparison
with suitable drug substance, is considered as potency of that substance. The
diameter of zone of inhibition is directly proportional to the concentration of the
drug substances added into the cup, thickness of the agar bed, diffusion
coefficient of the antimicrobial substance into the agar cup, sensitivity of the
microorganism to the test substance and the temperature. The appropriate media is
sterilized and cooled to 42 C, incubated with the test organism, mixed uniformly
and poured into petri plates and cooled. Bores are made into it; specified test
solution is added and left at room temperature for 30 minutes. Incubate at 37 c
for 24 hours. The zone of inhibition is measured in nm.
Methods of obtaining pure cultures
 The streak plate method of obtaining pure culture involves spreading
bacteria across a sterile solid surface such as agar plate so that the
progeny of a single cell can be picked up from the surface and transfer to
a sterile medium.
 The pour plate method of obtaining pure culture involves serial dilution,
transferring to melted agar, a specific volume of the dilution containing a
few organisms and picking up cells from colony of agar80,81.
Culture media
 In nature, microbes grow on natural media or the nutrients available in
water, soil and living or dead organic matters.
 In laboratory, microbes grow in artificial media.
1. Defined synthetic media: Consists of known quantities of nutrients.
2. Complex media: Consist of nutrients of reasonably well-known
composition that vary in composition from batch to batch.
Most routine laboratory culture make use of peptones (general purpose
media) or digested meat or fish proteins. Other cultures used are yeast
extracts, casein, hydrolysate, serum, whole blood or heated whole blood
(enriched media)82,83.
3. Diagnostic media.
Types of media
 Selected media: They encourage growth of some organism and inhibit the
others.
 Differential media: They allow the growth of different kinds of colonies to
grow on the same plate to be distinguished from one another.
 Enriched media: They provide a nutrient that fastens the growth of an
organisms.
Material and method
Method followed; Agar diffusion method.
Requirements: Petri plates, glass syringes, cork borers, inoculation loop, cotton.
Working procedure
Stock solution of the synthesized compounds and standard drug used were
prepared in dimethyl sulfoxide taken in the concentration of 100g/ml.
Microorganism used
Standard cultures of Bacillus subtilus, Staphylococcus aureus, Escherichia
coli and species were obtained from Microbiology Department, K.L.E.S. College
of Pharmacy, Bangalore. Staining technique and bio-chemical reaction identified
the microorganism. The microorganisms, which were maintained by sub
culturing, were used at regular intervals in nutrient agar medium.
Preparation of Inoculum
The suspension of all the organisms were prepared as Mac-Farland
Nephelometer standard (Baily and Scott 1990). A 24 hours old culture was used
for the preparation of baterial suspension. Suspension of organisms were made in
sterile isotonic solution of sodium chloride and turbidity was adjusted51,52.
Sl. No Ingredients Weight in gm

1. Beef extract 4.0
2. Peptone 5.0
3. Agar 20.0
4. Distilled water 1000 mL
PH 5.4
Preparation of assay media
The above mentioned quantities of different ingredients were accurately
weighed and dissolved in appropriate amount of distilled water. Media so
prepared was sterilized by autoclaving at 121 C for 15 minutes.
Procedure
The Petri plate were washed thoroughly and sterilized in hot air oven at 160 C
for one hour. 30 mL of sterile nutrient agar medium was poured into sterile Petri
dishes and allow to solidify. The petri plates were incubated at 37 C for 24
hoursto check for sterility. The medium was seeded with the organism by spead
platemethod using sterile cotton swabs. Bores were made on the medium using
sterileborer and 0.1 mL of the Cefazolin at a contration of 100g/mL was taken as
standard reference. A control having only DMSO in the cup was maintained in
each plate.
The petri plates were kept in refrigerator at 4 C for 15 minutes, allowing
diffusion to take place. Agar diffusion, the petri plate were incubated at 37 C for
24 hours and zone of inhibition were observed and measured using a scale.
Antimicrobial activity of all the compounds was carried out against all four
microorganisms. The zone of inhibition are as shown in table 5.
The media was used for both sub culturing and also for estimating antibacterial
activity.
Table- 6 Antibacterial activity of 8-substituted-7-fluoro-benzo[4,5] thiazolo
[2,3- c][1,2,4]triazole
Code Dose Zone of inhibition
No. (µg/ml)
E. S. B.
coli aureus subtilus
M3 +++ +++ +++
MV1 ++ ++ ++
MV2 +++ +++ +++
MV3 +++ +++ +++
MV4 100 ++++ +++ ++++
MV5 ++++ +++ ++++
MV6 + - +
MV7 + - +
MV8 ++ ++ ++
MV9 + + +
Standard 100 ++++ ++++ ++++
(Sulfadiazine)
Legends of table,
++++ : Very good, +++ : Good, ++ : Moderate, + : Least, - : No activity.
RESULT
Chemistry:
Results are summarized in tables and schemes, show the details of the synthetic
strategy adopted for the synthesis of these compounds. The product 2-
aminobenzothiazole is a versatile starting material for a number of synthesis.
Firstly we prepared 7- Chloro-6-fluoro-benzothiazole-2-ylamine from 3-chloro-4-
fluorophenylamine by reacting the potassium thiocynate with bromine solution in
glacial acetic acid according to the litrature.
After preparing 7- Chloro-6-fluoro-benzothiazole-2-ylamine (M1), it was reacted
with hydrazine hydrate in presence of conc. HCl to give 7-chloro-6-fluoro-
benzothiazol-2-yl-hydrazine (M2), then this compound was cyclized by
triethylorthoformate to give 8-Chloro-7-fluoro-benzo[4,5] thiazolo [2,3-
c][1,2,4]triazole (M3). We tried to synthesize different derivatives by replacing
the chlorine at 8th position by various aromatic amines, for this we adopted
various procedures as described in experimental section, but we failed to replace
the chlorine. This may be due to the stearic hindrance of bulky group.
Then we replaced the chlorine atom at 7th position of 7- Chloro-6-fluoro-
benzothiazole-2-ylamine (M1) by reacting it with various aromatic amines in
DMF. The product obtained from this reaction was proceeded further to get 8-
substituted-7-fluoro-benzo[4,5] thiazolo [2,3-c][1,2,4]triazole (MV1-9) as
mentioned above. The different compounds were recrystallised by suitable
solvents.
The different compounds were confirmed by TLC, melting point, IR, NMR, CHN
analysis and MASS spectroscopy.
In 7- Chloro-6-fluoro-benzothiazole-2-ylamine(M1) IR vibrations were seen at
3479-3290 cm-1 for NH2, 3092 for aromatic CH, 1654 for C-S, 1451 for C=N,
1215 for C-F aromatic, 683 for C-Cl aromatic. In 1 H NMR there are well resolved
resonance peak at 7.55 (d, 1H, Ar-H), 7.38 (d, 1H, Ar-H), 5.34 (s, 2H, NH) 
ppm, which confirm for their authenticity.
In the IR spectra of the compound, for 7-chloro-6-fluoro- benzothiazol-2-yl-
hydrazine (M2) there are bands at 3318 for –NH2, 3201 for –NH, 3068 for
aromatic CH, 1650 for C-S, 1451 for C=N, 1202 for C-F (aromatic), 689 for C-Cl
(aromatic). Similarly 1H NMR resonance peak at 9.24 (s, 2H, NH), 3.32 (s, 1H,
NH), 7.84 (d, 1H, Ar-H), 7.45 (d, 1H, Ar-H). The NH peaks were also confirmed
by taking the D2O-exchange NMR in which two NH peaks were missing at 9.24
and 3.32  ppm.
In the IR spectra of 8-Chloro-7-fluoro-benzo[4,5] thiazolo [2,3-c][1,2,4]triazole
(M3) peaks were observed at 3101 for CH aromatic, 1604 for C-S, 1500 for C=N,
1232 for C-F aromatic, 645 for C-Cl aromatic and 1H NMR resonance peak at
9.56 (s, 1H, Ar-H), 8.55 (d, 1H, Ar-H), 8.27 (d, 1H, Ar-H). The EI-MS of the
compound M3 shows the molecular ion peak at m/z 227 ( spectra no. 17) by
which we have confirmed for its authenticity.
In 7- substituted-6-fluoro-benzothiazole-2-ylamine (M4) IR vibrations were seen
at 3475 for –NH, 3091 for CH aromatic, 1645 for C-S, 1454 for C=N, 1196 for
C-F aromatic and 1H NMR resonance peak at 12.63 (s,1H, -NH), 7.66 (s, 2H, -
NH), 7.43-8.60 (m, 6H, Ar-H). The NH peaks were confirmed by taking the D2O-
exchange NMR in which two NH peaks were missing at 12.63 and 7.66  ppm.
DISSCUSION
Benzothiazoles and triazoles are known for their good antimicrobial properties.
Antimicrobial studies were carried out and the results obtained is discussed
below.
Antimicrobial activity of 8-chloro-7-fluoro-benzo[4,5]thiazolo [2,3-c]
[1,2,4]triazole (100 g/ml) was carried out by agar diffusion method and the
average radius of zone of inhibition was recorded. Among the derivatives
screened the following observations were made in comparision with the standard
Sulfadiazine (100 g/ml)
 Of the derivatives synthesized, good degree of antibacterial activity was
shown by some of the derivatives.
 4-chloro (MV4) and 4-flurphenylamine (MV5) derivatives showed better
antibacterial activity as compared to other derivatives.
 Derivative with 2-nitro and 4-nitrophenylamine substitution also showed
good antibacterial property.
 4-chloro (MV4) and 4-flurphenylamine (MV5) derivatives showed better
antibacterial activity towards both gram positive and gram negative
organism.
 Presence of methyl and methoxy group on phenylamine ring seems to no
impact in improving the antibacterial activity.
 Overall none of the compounds showed higher activity than the standard.
CONCLUSION
 The objective of the present work was to synthesize, purify,
characterize and evaluate the antimicrobial activity of novel 8-
substituted benzothiazolidinetriazoles.
 The yield of different synthesized compounds were found to be in the
range of 60-80% and the characterization was done by melting point
and TLC. Characteristic IR bands show several functional vibrational
modes which confirm the completion of reaction. Some structures
were confirmed by 1H NMR, D2O exchange NMR and Mass spectral
studies.
 All the ten test compounds showed antibacterial activity against both
gram positive and gram negative bacteriae in a concentration of 100
g/ml. Best result in terms of antimicrobial activity, were shown by 4-
chloro (MV4) and 4-flurphenylamine (MV5) derivatives for both gram
positive and gram negative organisms.
 Some compounds showed less antibacterial activity such as 4-methoxy
(MV6) and 2-methylphenylamine (MV7) derivatives.
SUMMARY
 Section I gives the brief description of the activity of antimicrobial agents and
importance of fluorine. This section also gives description about applicability
of various derivatives of benzothiazole and triazoles as pharmacological
agents.
 Section II deals with the objective of the entire research work of this
dissertation by explaining the need to develop newer compound as
antimicrobial activity.
 In section III review of literature has been discussed with a special reference
to various benzothiazoles and triazoles as antimicrobial and pharmacological
agents.
 In section IV a detailed method of synthesis of 8-Chloro-7-fluoro-benzo[4,5]
thiazolo[2,3-c][1,2,4]triazole and its various derivatives along with their
purification, physical constants has been given. All compounds were
synthesized in good yields and high purity. The compounds were
characterized by subjecting to various spectral studies such as IR, 1H NMR,
EI-MS and elemental analysis. Antimicrobial methods have also been given
here.
 Section V deals with the result of the 8-Chloro-7-fluoro-benzo[4,5]thiazolo
[2,3-c][1,2,4]triazole and 8-substituted-7-fluoro-benzo[4,5]thiazolo[2,3-
c][1,2,4]triazole compounds chemistry and its antimicrobial activity.
 In section VI, the discussion made on antimicrobial result of newly
synthesized compounds.
 Section VII deals with the conclusion of the synthesized 8-Chloro-7-fluoro-
benzo[4,5]thiazolo[2,3-c][1,2,4]triazole.
 Section VIII deals with the summary of the entire research work of this
dissertation.
 Section IX deals with the various references that led to the formation of this
dissertation.
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ANNEXURE
Paper accepted for the second international symposium on drug discovery and
process research (DDPR) at Belgaum on 11 Feb. 2006.
“Antimicrobial activity of 8-substituted benzothiazolidine triazoles”

Antimicrobial Activity of 8-Substituted Fluoro Benzothiazolidine Triazoles

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Antimicrobial Activity Of 8-Substituted Fluoro

Dissertation Submitted to the

Under the guidance of

Department of Pharmaceutical Chemistry

DECLARATION BY THE CANDIDATE

“Antimicrobial Activity Of 8-Substituted Fluoro

is a bonafide and genuine research work carried out by

me under the guidance of

CERTIFICATE BY THE GUIDE

This is to certify that the dissertation entitled

“Antimicrobial Activity Of 8-Substituted Fluoro

is a bonafide and genuine research work carried out

Date: Prof. S. M. Hipparagi,

ENDORSEMENT BY THE HOD,

This is to certify that the dissertation entitled

“Antimicrobial Activity Of 8-Substituted Fluoro

is a bonafide research work done

under the guidance of

Date: Prof. Y. D. Satyanarayana,

ENDORSEMENT BY THE PRINCIPAL/HEAD OF THE

“Antimicrobial Activity Of 8-Substituted Fluoro

is a bonafide research work done

Date: Prof. B. G. Desai

Place : (Manoj V. Vyavahare)

cm-1 = per centimeter

CDCl3= Deuteriated Chloroform.

CHN= Carbon Hydrogen Nitrogen

DMF= Dimethyl Formamide

DMSO= Dimethyl sulfoxide

FT-IR= Fourier Transform Infrared

KBr= Potassium Bromide

MIC= Minimum Inhibitory Concentration

NMR= Nuclear Magnetic Resonance

ppm= parts per million

Sl.No. = Serial Number

TLC= Thin layer Chromatography

TMS= Tetra methyl Silane

activities such as antimicrobial, antitumor, anthelmintic, anti-inflammatory etc.

derivatives, and characterized by various spectral and elemental analysis and

screened for antimicrobial activities.

We synthesized 7- Chloro-6-fluoro-benzothiazol-2-ylamine from 3-Chloro-4-

fluoro phenylamine. Then we synthesized 7-chloro-6-fluoro- benzothiazol-2-yl-

hydrazine from 7- Chloro-6-fluoro-benzothiazol-2-ylamine then we proceeded for

8-chloro-7-fluoro-benzo[4,5]thiazolo [2,3-c] [1,2,4]triazole.

We tried to prepare various derivatives by replacing the chlorine atom by

different amines from 8-chloro-7-fluoro-benzo[4,5]thiazolo [2,3-c] [1,2,4]triazole.

7- Chloro-6-fluoro-benzothiazol-2-ylamine and proceed to final compound

(Scheme II) as described in scheme 1.

Antimicrobial study shows that synthesized compounds have moderate activity.

Keywords: Benzothiazole, triazole.

1. Introduction Page No. 1-6

2. Objectives Page No. 7

3. Review of Literature Page No. 6-45

4. Methodology Page No. 46-91

4.1 Scheme Page No. 46-51

4.2 Experimental Page No. 52-77

4.3 Spectral data Page No. 65-78

4.4 Pharmacological Screening Page No. 79-85

5. Results &Discussion Page No. 86-88

6. Conclusion Page No. 89

7. Summary Page No. 90-91

8. Bibliography Page No. 92-104