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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.




K. L. E. Society’s College of Pharmacy 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


“Antimicrobial Activity Of 8-Substituted Fluoro

Benzothiazolidine Triazoles”

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.







K.L.E.S. Bangalore 1

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.





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

0C = 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

2

Analytical data of synthesized compounds

60-63

3

Physical properties of various compounds and

derivatives

64

4

Infra red spectral study of the synthesized compounds

65-67

5

1H NMR Spectral data of synthesized compounds

68

6

Antibacterial activity of 8-substituted-7-fluoro-

benzo[4,5]thiazolo[2,3-c][1,2,4]triazole.

85





LIST OF FIGURES

S. No.

Figure Name

P. No.

1

IR spectrum of 7-Chloro-6-fluoro-benzothiazole-

2-ylamine.

69 2

IR spectrum of 7-chloro-6-fluoro- benzothiazol-

2-yl-hydrazine.

69

3

IR spectrum of 8-chloro-7-fluoro-

benzo[4,5]thiazolo [2,3-c] [1,2,4]triazole.

70

4

IR spectrum of 2-amino-6-fluoro-7(2-

nitrophenylamine)benzothiazole.

70

5

IR spectrum of (7-fluoro-benzo[4,5]thiazolo[2,3-

c][1,2,4]triazol-8-yl)-2-phenylamine.

71

6


c][1,2,4]triazol-8-yl)-4-nitrophenylamine.

71

7


c][1,2,4]triazol-8-yl)-2-nitrophenylamine.

72

8


c][1,2,4]triazol-8-yl)-4-chlorophenylamine.

72

9


c][1,2,4]triazol-8-yl)-4-fluorophenylamine.

73





10


c][1,2,4]triazol-8-yl)-2-methylphenylamine.

73

11

1H-NMR spectrum of 7-Chloro-6-fluoro-benzothiazole-2-ylamine.

74

12

1H-NMR spectrum of 7-chloro-6-fluoro- benzothiazol-2-yl-hydrazine.

74

13

D2O exchange-NMR spectrum of 7-chloro-6-

fluoro- benzothiazol-2-yl-hydrazine.

75

14

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

75

15

1H-NMR spectrum 2-amino-6-fluoro-7(2- nitrophenylamine)benzothiazole.

76

16

D2O exchange-NMR spectrum of 2-amino-6-fluoro-7(2-nitrophenylamine)benzothiazole.

77

17

EI-MS of 8-chloro-7-fluoro-benzo[4,5]thiazolo

[2,3-c] [1,2,4]triazole.

78





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.

S

NR

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





S

NR

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

S

NR

O

HBr

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.

O

R2R3

R4

R1

CH2O

NH

N

S

R

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.

S

N

O

O

OH

C11H23NHR

S

N

O

O

OH

C11H23NHAr

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

OCH=N

S

N

R1R2

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.

S

NNHO2S

RF

N

O

R1

Cl

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.





S

NNHO2S

RF

NS

O

R1

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.

( Structural modifications-- )

structural modifications (---) of the N-cycloalkenyl-2acylalkylidene-2,3-

dihydro-1,3-benzothiazole.

N

S

R1

R2O

( )n





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.

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.

S

N

OO

OH

N

N

CH3

O

S

N

X

Y





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.





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.

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

S

NNHSO2

RF

CH3

S

N

RNH C NH

R1S





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

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.

O

O

NN

NO2

N

SNH

NH

R

OCl

Cl

N

SNH

NH

OO

R2

R=





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.

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

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.

S

N R2R1

S

NNH2

S

NNH2

R

C-oxidation

S-oxidation

N-oxidation C-oxidation( when R=Me)

N-oxidation(and conjugation)N-acetylation(then oxidation

and conjugation)





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.

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.

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

S

NO

RO(R= acyl, alkyl)

S

N

X

NH2R

X= H, Me, Cl, Br, IR=H,F





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.

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=CH3 R2=5-F

S

NNHSO3

R

Y

a (R=H,Y=Na or NH4)b (R=Me, Y=Na)C (R=Cl, Y=Na)d (R=I, Y=Na)

S

NR1

NHR2

ONH2

H (CH2)4NH2





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.

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

a. R=H; R’=3-ClC6H4 b. R=H; R’=4-OCH3C6H4 c. R=H; R’=5-NO2-2-Furanyl.

S

NN

R

CH R'S

N

N

R

CH R'

SN

NR

CH R'





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.

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.

Cl

Cl

NH

O

S

NNH

O

N

SR

HOOCH2C





R=C6H5, 2-HOC6H4, 3,4-(CH3O)2C6H3, 2-Pyridyl

2-substituted 5-benzothiazoleacetic acid

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

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.

S

NN

Cl

F

R

R=2-F, 3-F, 3-CF3

S

NHO2CH2C

N

SR

HOOCH2C





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.

S

NNHO2S

CH3

NHCOCH3

2-(4-acetamidophenyl sulphonamido)benzothiazole

S

NNHO2S

CH3

NH2

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.

S

NNHO2S

R1

NHCNHR2

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).

N

SO

Br

OMe

O

N

SO

O2N

OMe

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.

N

SXAr

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.

S

N

FCl

NHO2S

S NH

O

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.

N

S

NF

F

F

R

OH

O

R=morphilinoR=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.

S

NNCl

S

O

O

Ar

H

Ar= 2,5-Dichloro phenylAr= 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.

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.

X

NY

N OR2

O On

R1

X= S, Y= CH20, R1= OH, R2=CH3, n= 2





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.

5-(methoxy)-2-[(2-hydox-6-pentadecylphenyl)-methyl]-thio]-1H-benzimidazole

X

NN

OR

2

3

NH

NN

NCONH

NH

NN

NR= COOH,

OHCH2

C15H31

SN

NH

OMe





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.

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

CH2

C15H31

OMeS

N

S

S

NN

CORR1

R2

R3R4





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.

N

OS

CH2-NH N

S S R

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.

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 1-11M are as follows48.

Benzothiazol-2,5,6-triamine (IC50, M = 3.5 1.2)

S

NNH2

F3CO

S

NNH2

NH2

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.

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

1,2,4-triazoles.

S

NN

S NH2NH2

NN

N

N

NN

NHR1

O

R2

ONH

R3 NH2 NN

NR1 R2

NN

NR1 R2

R3 R3

+





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.

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.

NN

N

NN

NOH

F F





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.

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.

N

S

N

N

Cl

NN

N SHNH2

OH2CAr

N

N

N

N

NHR1

HS

S

R2O 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.

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

NN

NS

O

OArRO

H23C11





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= NO2, H

N

N

N

S

NN

RR

ClOMe





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.

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.

NN

NOSCH2CONHN

C2H5

CHR





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.

X= O, R= 2-Cl, 4-Cl

R1= 4-Cl, 3,4(OCH2)

NH

X

N

NN

NN

R

X= Cl, Br R= H, p-N(CH3), m,p-O2CH25H-1-aryl-5-halo-1,2,4-triazolo [4’,3’:2,3] –1,2,4-triazino [5,6-b]-indoles

NNNN

H

XN

N

R

X= Cl, Br R= H, p-N(CH3), m,p-O2CH26H-5-aryl-9-halo-1,2,4-triazolo [3’,4’ :3,4]-1,2,4-triazino [5,6-b]-indoles

NSN

NN

XCH2

Cl

F

Cl

R1

R





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.

NN

NSBrR H





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.

R= C6H5, CH3,H

R1= o, m, p-Nitro, o, m, p-Chloro, aniline, morpholine.

NH CH3 NH

CH3

NHNO2

NO

R= ,

,

,

NN

NNH

SHAr

CO N





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.

R= H, CH3 R1= H, 4-Cl, 3-Cl, 4-F

NN

N

S R1R

NN

NH

SNH

F

R

R1





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.

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.

NN

NHN

O

Br

MeO

MeO





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.

NN

NN

N

R2

R1

NN

NR1

R2





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.

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.

N

S

R

NHNH2S

N NN

RR1

Formic, Acetic orPropionic acid

Reflux





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.

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.

N

SNHNH2

CH3

S

NNN

CH3

HCOOH

Reflux

NN R

NHNH2

O

O

O N R

O

O

O N

NNCH3

acetic

anhydride,reflux

S N

N

R2

R1

R3

NHNH2

S N

N

NNR1

R2 R3

CH(OC2H5)3

Reflux





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.

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.

7. Michael Ciesielski et al70 in 2005 described a novel copper-mediated oxidative

heterocyclization of hydrazones yielding the corresponding 1,2,4-triazoles.

NArNN

N NAr Ar

H HN N

NNH2

ArArC

HO-CH2-CH2-OH

NH2-NH2-2HCl

NH2NH2, H2O

N NHN

CHR

NNN

R

2CuCl2

DMF, 90-140 C-2CuCl-2HCl

R1 R2

ON

N

N S

NNN

O

N

N N

S R1

R2O

+Tolune

90oC





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.

S

N

NN

NHNH2

S

N

NN

NN

OH

3-hydrazino-1,2,4-triazolo[3,4-b]benzothiazoloe 3’-hydroxy-1,2,4-triazolo[4’,5’:1,5]-1,2,4-trazolo[3,4-b] benzothiazole

S

N

NN

N

N

SH

3’-mercapto-1,2,4-triazolo[4’,5’:1,5]-1,2,4-trazolo[3,4-b] benzothiazole

Urea,

CS2,KOH





METHODOLOGY

SCHEME-I

1. Synthesis of 7-chloro-6-fluoro-benzothiazole-2-ylamine from 3-chloro-4-

fluoro-phenylamine

(M1)

2. Snthesis of 7-chloro-6-fluoro-benzothiazol-2-yl-hydrazine from 7-chloro-6-fluoro-

bnzothiazole-2-ylamine

(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

(M3)

S

N

FCl

NH2

NH2NH2H2O

S

N

FCl

NHNH2HCl; Ethylene glycol

FCl

NH2

KSCN Br2

CH3COOH S

NNH2

FCl

;

S

NNHNH2

FCl

CH(OC2H5)3

S

NNN

ClF

Xylene





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

SCHEME-2 1. Synthesis of 7-substituted-6-fluoro-benzothiazole-2ylamine from 7-chloro-

6- fluoro-benzothiazole-2-ylamine

S

NNH2

ClF S

N

RF

NH2R

DMF

(M4)

2 .Synthesis of 7-substituted-6-fluoro- benzothiazol-2-yl-hydrazine from 7-

substituted-6-fluoro-benzothiazole-2-ylamine

S

N

RF

NH2

NH2NH2.H2O

S

N

RF

NHNH2HCl

Ethylene glycol

S

NNN

ClF No Reaction

A22, B72, C73, D74

E75, F76, G77, H78





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

S

NNN

FR

S

N

RF

NHNH2

Xylene

CH(OC2H5)3

(MV 1-9)

Table-1 Codes and corresponding R of different derivatives

code R

MV1 HN

MV2 HN NO2

MV3 HN

O2N

MV4 HN Cl

MV5 HN F





MV6 HN OMe

MV7 HN

CH3

MV8 HN

OH

MV9 HN

NH2





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-

ylamine.22

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 0c. After all the bromine





has been added (105min), the solution was stirred for an addition 2 hour at 0c

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 85c 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 85c 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 5-10c, 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 CH3CN , (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 5C. 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

100C 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 5C 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 5-10c, 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.

No

Molecular

Structure

Nature of

Compound

%

Yield

Molecular

Formula

Molec

ular

Weigh

t

M1

S

NNH2

FCl

Color less

powder

95

C7H4ClFN2S

202

M2

S

N

FCl

NHNH2

Color less

Crystals

90

C7H5ClFN3S

218

M3

S

NNN

ClF

White

powder

60

C8H3ClFN3S

228





MV

1

S

NNN

FNH

Light

yellow

powder

70

C14H9FN4S

284

MV

2

S

NNN

FNH

NO2

Yellowish –

brown

powder

60

C14H8FN5O2

S

329

MV

3

S

NNN

FNH

NO2

Buff

powder

72

C14H8FN5O2

S

329





MV

4

S

NNN

FNH

Cl

Whitish

yellow

powder

65

C14H8ClFN4

S

318

MV

5

S

NNN

FNH

F

Brown

powder

70

C14H8F2N4S

302





MV 6

S

NNN

FNH

OMe

Gray

powder

80

C15H11FN4O

S

314

MV7

S

NNN

FNH

CH3

White

powder

75

C15H11FN4S

298

MV

8

S

NNN

FNH

OH

Yellowish

white

powder

80

C14H9FN4O

S

300

MV9

S

NNN

FNH

NH2

Brown

powder

75

C14H10FN5S

299





Table-3 Physical properties of various compounds and derivatives

Code

No.

Solubility

Mobile phase for TLC

Melting point

(oC)

M1 Ethanol,

Chloroform,

DMSO

Chloroform:Methanol 198-200

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

(cm-1) Molecular nature

M1

3479, 3290

3092

1654

1451

1215

683

-NH2 str (symm, asymm)

-C-H str (aromatic)

-C-S str

-C=N str

-C-F (aromatic)

-C-Cl (aromatic)

M2

3318

3201

3068

1650

1451

1202

689

-NH2 str

-NH str

-C-H str (aromatic)

-C-S str

-C=N str

-C-F (aromatic)

-C-Cl (aromatic)

M3

3101

1604

1500

1232

645

-C-H str (aromatic)

-C-S str

-C=N str

-C-F (aromatic)

-C-Cl (aromatic)





M4

3475

3091

1645

1454

1196

-NH str

-C-H str (aromatic)

-C-S str

-C=N str

-C-F str

MV1

3476

3097

1649

1455

1215

-NH str

-C-H str (aromatic)

-C-S str

-C=N str

-C-F (aromatic)

MV2

3477

3104

1596

1546

1401

1455

1193

-NH str

-C-H str (aromatic)

-C-S str

-C-N=O str asymm

-C-N=O str symm

-C=N str

-C-F (aromatic)

MV3

3477

3104

1596

1546

1401

1455

1193

-NH str

-C-H str (aromatic)

-C-S str

-C-N=O str asymm

-C-N=O str symm

-C=N str

-C-F (aromatic)





MV4

3481

3101

1503

1382

698

-NH str

-C-H str (aromatic)

-C-S str

-C=N str

-C-Cl (aromatic)

MV5

3477

3085

1546

1453

1193

-NH str

-C-H str (aromatic)

-C-S str

-C=N str

-C-F (aromatic)

MV7

3248

3039

2991

2913

1527

1453

-NH str

-C-H str (aromatic)

-CH-CH str (aliphatic)

-CH-CH str (aliphatic)

-C-S str

-C=N str





TABLE-5 1H NMR Spectral data of synthesized compounds

Compound

Chemical shift value

Proton nature

M1

7.55

7.38

5.34

d, 1H, Ar-H

d, 1H, Ar-H

s, 2H, -NH

M2

9.24

3.32

7.84

7.45

s, 2H, -NH

s, 1H, -NH

d, 1H, Ar-H

d, 1H, Ar-H

M3

9.56

8.55

8.27

s, 1H, Ar-H

d, 1H, Ar-H

d, 1H, Ar-H

M4

12.63

7.66

7.43-8.60

s, 1H, -NH

s, 2H, -NH

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.

Fig 6. IR spectrum of (7-fluoro-benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-8-yl)-

4- nitrophenylamine





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

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

4-chlorophenylamine.





Fig 9 IR spectrum of (7-fluoro-benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-8-yl)-4-fluorophenylamine.

Fig 10 IR spectrum of (7-fluoro-benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-8-yl)-

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 100g/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 100g/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

Zone of inhibition

Code

No.

Dose

(µg/ml) E.

coli

S.

aureus

B.

subtilus

M3 +++ +++ +++

MV1 ++ ++ ++

MV2 +++ +++ +++

MV3 +++ +++ +++

MV4 ++++ +++ ++++

MV5 ++++ +++ ++++

MV6 + - +

MV7 + - +

MV8 ++ ++ ++

MV9

100

+ + +

Standard

(Sulfadiazine)

100 ++++ ++++ ++++

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 1H 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”




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