REVIEW OF LITERATURE Pyrazole compounds and their ...shodhganga.inflibnet.ac.in/bitstream/10603/48928/8/08_chapter_3.pdf · REVIEW OF LITERATURE Pyrazole compounds and their pharmacological

136

REVIEW OF LITERATURE

Pyrazole compounds and their pharmacological interest

Pyrazole1-48 is the name given by “LUDWIG KNORR” to this class of compounds

in 1883. The simple doubly unsaturated compound containing two nitrogen and three

carbon atoms in the ring, with the nitrogen atoms neighboring, is known as pyrazole. The

reduction products, named as are other rings of five atoms, are pyrazoline and

pyrazolidine. Several pyrazoline substitution products are used in medicine. Many of

these are derivatives of 5-pyrazolone. Some can be related to 3,5-pyrazolidindione.

NH

N

NH

NH

NH

NH

NH

NHO

NH

NHO

O

Pyrazole Pyrazoline Pyrazolidine 5-Pyrazolinone 3,5-Pyrazolidinedione

For a long time no pyrazole derivative had been found in nature, but in 1959 β-(1-

pyrazolyl) alanine was isolated from the seeds of water melons (Citurllus lanatus) (L.

Fowden).

N

N

CH2

COOH

CNH2

H

137

Chemistry: Pyrazole is a colourless solid, m.p. 70°C. This high value (compared

with 1-alkyl or aryl substituted pyrazoles) is due to intermolecular hydrogen bonding

which results in a dimmer. Pyrazole is a tautomeric substance; the existance of

tautomerism cannot be demonstrated in pyrazole itself, but it can be inferred by the

consideration of pyrazole derivatives.

Pyrazole exhibits aromatic properties, e.g., it is readily halogenated, nitrated

and sulphonated; the group enters at position 4. The following resonating structures are

possible for pyrazole.

Pyrazole is feebly basic and forms salts with inorganic acids; the imino hydrogen

may be replaced by an acyl group. Pyrazole is very resistant to oxidizing and reducing

agents, but may be hydrogenated catalytically, first to pyrazoline, and then to

pyrazolidine. Both of these compounds are stronger bases than pyrazole.

NNH

4 3

5 21 NH

N

43

21

5

N

N N

NH

H

NNH

NN

NN

H H

NNH

..

-:

+ -: +

:-

+

NNH

NN

NNHH H

H2

Catalyst

H2

Catalyst

pyrazoline pyrazolidine

138

Pharmacological interest : Pyrazole derivatives constitute an interesting class of organic

compounds, which are associated with diverse chemical and pharmacological properties.

Pyrazolines have received considerable attention in recent years. Pyrazoline derivatives

occupy a unique place in field or medicinal chemistry due to a wide range or biological

activities exhibited by them.

Their pharmacological and biological activity listed below:-

1. Anti-inflammatory

2. Analgesic

3. Antipyretic

4. Diuretic

5. Bovine anaplasmosis

6. Treatment of rheumatic disorders.

7. Antibacterial activities

8. Antimicrobial

9. Antidepressant

10. Anticonvulsant

11. Anti-tumour

12. Antiparasitic activity

13. Antimalerial

14. Antimycotics

15. Antidiabetic

Several derivatives of these systems find use in medicine described as follows:

N

CHCH2

CH2

NH

Pyrazoline

N

N

H

Pyrazole

139

Phenylbutazone: use as analgesic, anti-inflammatory & antipyretic

Forbisen : used in bovine anaplasmosis.

Oxyphenbutazone : used in the treatment of inflammation of the eyes.

Sulphinpyrazone: It having a better therapeutic index as a uricosuric agent

N

C6H

5

C6H

5N

O

O

Phenylbutazone

CH3CH

2CH

2CH

2

NNN

N O

Ph

Me

Me Me

MeO

Ph

N C6H

5N

O

CH3CH

2CH

2CH

2O

OH

Oxyphenbutazone

NN

O

O

Ph

Ph

Sulphinpyrazone

PhSOCH2CH

2

140

Feprazone : used in the treatment of rheumatic disorders

Phenyl Butazone : used as antithrombotic.

NN

H9C4O

O

Phenazone: Phenazone is well known for its analgesic and antipyretic actions.

Topically, it is known for its local anaesthetic and styptic actions and solutions having

5% are used locally as ears drops.

Analgin : used as analgesic and antipyretic

NN

O

O

Feprazone

C = CHCH2

CH3

CH3

NN

O

CH3

CH3

C6H

5

Phenazone

NN

O

CH3

N

CH3

C6H

5

CH3

CH2SO

3Na

Analgin

141

Pyrazole derivatives : have found application in the agrochemical field as insecticides.

o,o-Diethyl o-(3-methyl-5-pyrazolyl) phosphate (a) & o,o-diethyl o-(3-methyl-5-

pyrazolyl) phosphorothioate (b).

5-Pyrazolone derivatives : have found many applications as cotton azo dyes because,

even if they were more expensive intermediates.

1. Rajeev Jain and Seema Gupta1 have reported the synthesis and anti-bacterial activity

of sulphonamoylazo purazoles.

RHNO2S

NN

NH - Ph

NO2

H3C

N = N

NH

N

Me

OP

XEtO

EtO

a) X = O

b) X = S

OH

NaO2C

N = N N = N

NN

CO2Na

OH

SO3Na

142

2. Salem A. Basif Hassan M. Faidallah and Seham Y. Hassan2 have reported synthesis

and anti-bacterial activity of substituted pyrazoles.

3. A.S. Gajare S.B. Bhawsar and M.S. Shingare3 have reported synthesis and anti-

microbial activity of pyrazoles.

4. V.V. Mulwad & A.S. Hegde4 have reported synthesis of conmarinyl Azopyrozoles.

NN

R

SO2NH

2

Cl Cl

NCl O

O

NN

R R1

O O

R

N = N

NN

C6H

5

H3C

CH3

R1

143

5. Biplab De & G.V.S. Ramasharma5 have reported analgesic activity of 5-

oximidazolyl-Amino pyrozole-4-carboxaldehydes.

6. P.B. Heda & D.J. Ghiya6 have reported synthesis of 4-Iodopyrazoles and their anti-

microbial activity.

7. Yogesh Goel Rajeh Kumar7 et al have reported microwave assisted synthesis & anti-

fungal activity of substituted pyrazoles.

O

NNNH

N

CHN

NCH3

R3

R2

NNOH

R I

N

CH3

NN

CHO

R

144

8. K. Nagarajan et., al8 reported synthesis of Nitroimidazole for anti-ameobic activity.

9. H.C. Mutraja et., al9 reported prolonged bacteriostatic action in-vivo.

R = Methyl, Ethyl, Phenyl

10. R. Weismann et., al10 were reported for Insecticidal properties.

N

NN

N

O2N

Me

R

R1R2

NN

NN

N = N

NH SO2

NH2

SO2NH

2H

3C

H

OR

NN

CH(CH3)2

OCON(CH3)2

H3C

NN

Ph

OCON(CH3)2

H3C

145

11. C.W. Noell & C.C. Chen11 were reprted for anti-leukemic property.

12. J.B. Wright et., al.,12 were reported for hypoglycemic activity.

13. E.L. Enderson J.E. Casey L.C. Greene et., al.,13 were reported for 3-amino-4-phenyl

pyrazole for muscle relaxant activity.

14. D.M. Baily14 claimed that anti-depressant and analgesic activities for series of

pyrazole derivatives.

n = 2; NRR1 = MeNH

n = 3; NRR1 = NH2, MeNH, NEt2

NN

H

CONH2

H3C

H3C

N - N = N

NN

CO - NH2

CH3

H3C

NN

H

H2N Ph

NN

(CH2)nN

Ph Ph

R

R1

146

15. S.P. Singh et. al.,15 were synthesised substituted pyrazolyl pyrazole.

Qu = Methoxy Quinoline

16. Vinod K. Ahluwalia, Vijay K. Garg, Alka Gupta16 were reported Phenyl substituted

Pyrazoles.

17. Krishna A. Rao, Jaywant N. Gadre et., al.,17 synthesised and reported Pyrozolo-

Pyrididone.

N N

CH3

Qu

H3C

N

N

R

O

NN

Ph

H3C COCH

2COCH

3

OH

NC

NNN

H R

O

147

18. D. Bhaskar Reddy, N. Subbha Reddy and T. Balaji18 were synthesised 1,3-substituted

Pyrazoles.

i) KOH / CH3OH 00C

ii) C6H5SO2Cl, C6H5N 900 – 1000C 2hr.

iii) C6H5COCl KOH / CH3OH, 900 – 1000C 2hr.

R, R1 = H

R, R1 = Ar – SO2 –

R = C6H5 CO – R1 = Ar = SO2 -

19. Vishnu J. Ram and Mahendra Nath19 synthesised and reported Diamino substituted

Pyrazole.

20. Synthetic Methods.

a) From Dicarbonyl compounds

1,3-dicarbonyl (Symmetrical compound, acetyl acetone) reacts with Hydrazine / Phenyl

hydrazine forms various substituted Pyrazoles.

NN

ArSO2

H

NN

R

R1

Ar - CH2 - CH + NH

2NH

2

CN

CN

NN

NH2

H2N

Ar

HHydrazine

Benzyl malononitrile 3,5-diamino,4-Benzyl Pyrazole

148

21. H.M. Virupakshaiah et., al.,20 synthesised substituted Pyrazole moiety using ethyl

ethoxy methyleno acetate and phenyl hydrazine in presence of ethanol and refluxed

for 3 hours.

22. Hussein A. Emam, et., al.,21 synthesised and reported the following compounds.

CH3 - C - CH

2 - C - CH

3

NH2NH2

-H2O

Ph - NH - NH2

O O NN

H

H3C

CH3

NN

Ph

H3C

CH3

-H2O

3,5-Dimethyl Pyrazole

3,5-Dimethyl-1-Phenyl Pyrazole

COOC2H

5

NH2

Ph

CN

OC2H

5

H5C

2OOC

H

Ph - NHNH2

NN

+

NN CN

R - C

O

Ph

NN CONH

2

R - C

O

Ph

NN N = CHO

2C

2H

5

R - C

O

Ph

149

23. Nada M. Abunada & et.al22 have reported that synthesis and antimicrobial evaluation

of some new pyrazole, pyrazoline and chromeno[3,4-c] pyrazole derivatives.

OO

N

N

Ar

Ar`

Ar = 4-FC6H4, 2,4-Cl2C6H3 Ar` = 4-NO2C6H4

24. Shiv P. Singh & et al.23 have reported that synthesis of 1,3,5- trisubstituted pyrazoles

as potential antimicrobial agents.

N NR

R1R2

R = Ph R1= C6H3Cl2 R2 = C6H5, p-C6H4OCH3, p-

C6H4CH3

25. Krishnadatt Sharma and P.S.Fernandes24 have reported that synthesis of 1,3-

substituted pyridazinones, pyrazolo [3,4-d] pyridazines and related compounds as

antibacterial agents.

NN

O

N NH

F

O

O

CH3

150

26. Vipin Kumar Singh & et al.25 have reported that synthesis some new 1-aryl-methyl -

4-substituted phenyl-6-imino-4,7-dihydro-1,3-oxazino (5,4-d) pyrazoles as potential

fungicides.

N

O

NN

R

CH3

NHCH3

R = C6H5 X= H, p-NO2, m-NO2, p-OCH3, m-OCH3

27. Vijai N. Pathak & et al.26 have reported that synthesis of three series of new

pyrazoles and related derivatives and few compounds have been screened for their

antimicrobial activity.

X2

X3

X1

NN

R1

O2S

X2

X1

NN

R1

CO

R1 = C6H5 X1 = Cl, Br, F X2 = 4-Cl X3 = H

151

28. R.A.Shastri & et al.27 have reported that synthesis and antimicrobial activity of 1H -

3(2`- hydroxyl substituted) phenyl 5(6``-methoxy) naphthyl pyrazolines.

NN

OCH3

COC2H5

OH

R1

R2

R3

R1= H, CH3 R2 = H, CH3 R3 = H, CH3, Cl

29. D. H. Vyas & et al.28 have reported that synthesis some pyrazoline derivatives and

their Antitubercular and antimicrobial activity.

Br

H3CO

NN C

N

O

Br

Hb

Ha

R

Hx

Br

H3CO

NN C

O

Br

Hb

Ha

R

Hx

Cl

R= C6H5, 3-Br-C6H4, 2-Cl-C6H4, 4-Cl-C6H4

152

30. Vineeta Sareen & et al.29 have reported that synthesis and antifungal activities of

some new tetrahydropyrazolo [3,4-c] pyrazoles.

NN

NN

CH3

R1

NO2

NO2

R

NN

NO

CH3

NO2

NO2

R

R = H, 2-OH, 4-F, 2-Cl R1 = C6H5, COCH3

31. Mohd.Amir & et al.30 have reported that synthesis and antibacterial activity of some

new 1-substituted 3,5-diphenyl-4-(arylazo) pyrazoles.

N

NN

N N

Ph

Ph

C O

R

R

NN

N N

Ph

Ph

C SNH2

R= o-Cl, p-Cl, o-COOH, p-OCH3, p-CH3, p-Br

32. Freddy H. Havaldar and Sushil Kumar Mishra31 have reported that synthesis of 1-

(3`-bromo-4`-methoxybenzoyl)-4-formyl-3-(substituted phenyl) pyrazoles and their

antibacterial and antiinflammatory activity.

153

N N

CHO

C

O

H3CO

Br

Ar

33. V.H.Bhaskar & et al.32 have reported that synthesis of 4,5-disubstituted -3-methyl -

1,3a,4,5-tetrahydropyrazolo[3,4-c] pyrazoles and their antiinflammatory activity.

NH

NN

N

CH3

R

R1 R2

R3

O2N

NO2

R1, R2, R3 = H R = H, Cl, OH,

34. P.R.Solanki and K.K.Wadodkar33 have reported that conventional heating and

microwave promoted synthesis of some substituted isoxazoles and pyrazoles as potent

antibacterial agents.

NH

N

R

CH3

R = 5- CH3C6H3OH, C6H4OH, 4-CH3C6H3OH

154

35. G.K. Nataraja and et al.34 have reported that synthesis of naphtha [2,1-b]

furopyrazoles as antimicrobial agents.

N N

R

O

N

R1

R = H, C6H5, 4-NO2-C6H4 R1 = H, CH3, OCH3, Cl, NO2

36. Hitesh Patel & et al.35 have reported that synthesis and antimicrobial activity of

pyrazolo [3,4-d] pyrimidines.

NN

N

NH

SH

R

CH3

Cl

NN

N

NH

OH

R

CH3

Cl

37. Anand Kumar Tengli & et al.36 have reported that microwave assisted synthesis of

pyrazoline derivatives and their antiangiogenic and antioxidant activities.

NN

COCH3

R1

R2

R1= p-OH Phenyl R2 = H, Br,

155

38. H.S. Joshi et al 37. have synthesized 1-substituted 3-aryl-5-(3'-bromophenyl)-

pyrazolines and evaluated for their anti-cancer, anti-tubercular and anti-microbial

activities.

NN

Br

C CH3O

R

R= Aryl

39. S.P. Hiremath et al 38. have synthesized 5-hydrazino-10-substituted-7H-indolo [2,3-c]

isoquinolines and 1-(10-substituted-7-H-indolo [2,3-c] isoquinolin-5-yl)-3,5-

disubstituted pyrazoles , 3-methyl pyrazol-5-ones and 3-5-disubstituted pyrazolines

and screened for their antimicrobial, analgesic and anti-inflammatory activities.

NNH

N N

CH3CH3

R

NNH

N N

CH3

R

O

NNH

N N

R1

R

H5C6

R = CH3, Br R1 = C6H5, p- H3COC6H4

156

40. Kirpa Shanker et al 39. reported the synthesis and cardiovascular activity, of

substituted pyrazolines and evaluated for their cardiovascular activity and toxicity.

NN R

C SNHR1

R = C6H5, o-(OH)-C6H4 R1= H, Cl, CH3, OCH3

41. L.V.G. Nargund, et al 40.have synthesized fluorinated phenyl styryl ketones and N-

phenyl -5-substituted aryl-3-P-(fluorophenyl) pyrazolines and pyrazoles and

evaluated for anti-inflammatory activity in vitro.

NNH R

FR1

NN R

FR1

R = H, OH, N(CH3), Cl R1 = H, OCH3

157

42. Shrinivas Rastogi et al41. have reported synthesis of 4,5-trans/cis-1-acetyl-3,4,5-

triarylpyrazolines and evaluated for anti-implantation activity.

NN

H

H

H3CO

C

O

CH3

O

N

43. V.P. Vaidya, B. Shivaramaholla et al.42 have synthesized arylfuryl ∆2-pyrazolines and

evaluated for their antimicrobial analgesic and anthelmintic activities.

NH

NO

NO2

H3CO

R

R= H, 4-Cl, 4-OCH3

44. Y.K. Agarwal et al. 43 have reported synthesized of new benzofuran 1,3,5-

trisubstituted pyrazoline derivatives of paracetamol and evaluated for their

antitubercular, antimicrobial and anti-inflammatory activities.

R

N NO

O

O

NH

CH3

O

R= OH, OCH3, NO2, Cl

158

45. Chirag Sharma & et al.44 have reported that synthesis and biological activity of some

novel ethoxyphthalimide derivatives of pyrazolo [3,4-c] pyrazoles.

N

O

O

O

NN

CH3

NN

C ON

X

R = H, OCH3, N(CH3)2, Cl

46. K.Mogilaiah & et al.45 have reported that synthesis and antibacterial activity of 3-aryl

-4-formyl-1-[3-(3-chlorophenyl)-1,8-naphthylpyridin-2-yl] pyrazoles.

N N N N

CHO

Cl

Ar

Ar = C6H5, p-ClC6H4, p-BrC6H4, p-OHC6H4, p-NO2C6H4

47. D B Arun Kumar & et al.46 have reported that synthesis and antimicrobial

investigation of some phenyl pyrazoles derivatives of benzofurans.

O

R1

N N R2

R1 = H, CH3 R2 = H, NO2

159

48. Kamal M. Dawood & et al.47 have reported that synthesis of pyrazolyloxadiazole

derivatives and their Anticancer activity.

NH

N

O N

NNHPh

Ph

O

CN

49. Shashikant R. Pattan & et.al48 have reported that synthesis and evaluation of some

novel substituted 1,3,4-oxadiazole and pyrazole derivatives for Antitubercular

activity.

O

N NH

CH3

CH2

O

R

OH

COOH

OH

NHR=

160

A BRIEF REVIEW OF THIAZOLIDINONES

CHEMISTRY OF THIAZOLIDINONES:

Introduction and Scope:

Thiazolidinones are the derivatives of thiazolidine, which belongs to an important

group of heterocyclic compounds. Thiazolidinones, with carbonyl group at 2, 4 or 5 have

been subject to extensive study in the recent past.

N

S O

H

12

34

5

N

S

HO

12

34

5

N

S

H

O1

2

34

5

1 2 3

Numerous reports have appeared in the literature, which highlight their chemistry

and use. Diverse biological activities such as bactericidal, pesticidal, fungicidal,

insecticidal, anticonvulsant, anti-tuberculosis, anti-inflammatory, antithyroidal,

potentiation of pentobarbital induced of sleeping time, etc., have been found to be

associated with thiazolidinone derivatives. In recent years several new methods for the

preparation of thiazolidinone derivatives and reactions have been reported in the

literature. Thiazolidinones, in the presence of various reagents, undergo different types

of reactions to yield other heterocyclic compounds, e.g., thiazole, benzothiophenes,

triazinones etc. These advances warrant reviewing the chemistry and biological

properties of various 4-thiazolidinones.

161

Preparation of Thiazolidinones:

4-Thiazolidinones:

1) B. B. Subudhi, P. K. Panda, B. K. Tosh, S. Sahu and P. Majhi49 have reported

Synthesis and Biological Activity Evaluation of Some Azetidinone and

Thiazolidinone Derivatives of Coumarins

OOH

CH3

N N

S

OH

O

2) Samia Bouzroura, Yamina Bentarzi, Rachedine Kaoua,Bellara Nedjar-Kolli,

Sophie Poulain-Martini and Elisabet Dunach50. Have reported A convenient one

pot preparation of 4-thiazolidinones from Enaminolactones.

O

NH

NN

S

CH3

O

O

162

3) P. Shanmugapandiyan , K.S. Denshing, R. Ilavarasan, N. Anbalagan, R. Nirmal51

have reported Synthesis and biological activity of 2-(thiazolidin-4-one) phenyl]-

1h-phenylbenzimidazoles and 2-[4-(azetidin-2-One)-3-Chloro-4- phenyl] -1h-

phenyl benzimidazoles.

N

NH

N

SO

4) Wilson Cunico, Claudia R.B. Gomes and Walcimar T. Vellasco Jr52. have

reported chemistry and biological activities of 1,3-thiazolidin-4-ones.

N

N

N S

Cl

Br

O

O

i-Pr

5) Sanjeeva R. Cherkupally, Chandrashekar R. Dasari1, Yakub Vookanti1 and

Nagaraj Adki53 have reported Synthesis and antimicrobial study of bis-

[thiadiazol-2-yltetrahydro-2H-pyrazolo[3,4-d][1,3]thiazole]methanes.

OO

N

S

N

S N

N

CH3

CH3

NH2

NH2

163

6) Firke SD, Firake BM, Chaudhari RY and Patil VR54 have reported Synthetic and

pharmacological evaluation of some pyridine containing thiazolidinones.

N O CH2 C NH N

N S

O

CH3

O

7) Navin B. Patel and Sarvil D. Patel55 have reported synthesis and in vitro

antimicrobial study of Schiff base and thiazolidinones of 1-cyclopropyl-6-fluoro-

7-(4-(2,3-dichlorophenyl)piperazin-1-yl)-4-quinolones.

N

N N

NH N

SF

Cl

Cl

O OO

8) K.H.Patel and A.G.Mehta56 have reported Synthesis of novel azetidinone and

thiazolidinones derivatives and evaluation of their antimicrobial efficacy.

Where Ar: (a) phenyl, (b) 4-methoxy phenyl (c) 4-hydroxy phenyl (d)

2-hydroxy phenyl (e) 4-methyl phenyl (f) 3,4-methylenedioxy phenyl (g) 4-

hydroxy-3-methoxy phenyl (h) 3,4-diethoxy phenyl.

N

S N

SO

Ar

164

9) K. M. Mistry and K. R. Desai57 have reported Synthesis of novel heterocyclic 4-

thiazolidinone derivatives and their antibacterial activity.

10) M. C. Sharma, N.K. Sahu, D.V.Kohali, S.C.Chaturvedi, Smita Sharma58, have

reported QSAR, synthesis and biological activity studies of some thiazolidinones

derivatives.

11) Sunila T. Patil , Dr.Parloop A. Bhatt59 have reported synthesis and biological

evaluation of some novel 2-(4-substituted phenyl)-3-(4 substituted phenyl)-5-

methyl thiazolidin-4-ones .

N

SN S

N

N

O CH3

CH3

CH3

R

R =NO2,SO2, CH3

N

NHN

S

CH3

OO

R

R =2-hydroxy-4-methoxy,4-chlorophenyl,2-nitrophenyl

N

SO

CH3

R'R

R = F, Br,NO2

R' = OH, NO2, OCH3, Cl

165

12) Sayeed Mukhtar, Mujeebur Rahman V.P. Wajid Husain Ansari, Guy Lemière,

Alex De Groot and Roger Dommisse60 have reported bifunctional derivative of

p,p'-dichlorochalcone. part II. synthesis of a novel compound 2-[2-

carboxymethylthio-2-(4-chlorophenyl)ethyl]-2-(4-chlorophenyl)-4-thiazolidinone

13) Raga Basawaraj, Amith. L, VijayKumar.T,Havangirao.Mand Upendra.C.H61.

have reported Synthesis and antitubercular activities of azetidinone and

thiazolidinone derivatives from 5-chloro-3-methylbenzofuran

O N

CH3

N S

CH3

CH3

O

R

R= C6H5, C6H4OH, C6H4Cl

14) Devappa S. Lamani, K. R. Venugopala Reddy Govinda Raju Reddy K. B, Sahana

K. N, Smitha .N.C, Anand, Pradeep. S.M, Nithin, Kenchappa, Achuthanada62

have reported A simple and efficient carbodiimide mediated one-pot synthesis of

novel 2-(2- hydroxynaphthalen-1-yl)-3-phenyl-1,3-thiazolidin-4-one derivatives:

a potent antimicrobial agent

OH

S

N

R

O

R= H, CH 3, OCH3, NO2, COOH

C CH2S NH

CH

S

CH2 COOH

ClCl

O

166

15) Divyesh Patel, Premlata Kumari, Navin Patel63 have reported Synthesis,

characterization and biological evaluation of some thiazolidinone derivatives as

antimicrobial agents.

16) S.Ramachandran, P.Shanmugapandiyan64 have reported Synthesis,

characterisation, antimicrobial evaluation of Thiazolidine-4-one derivatives.

17) Aysel Gursoy, Nalan Terzioglu65 reported the Synthesis and isolation of new

regioisomeric 4-thiazolidinones and their anticonvulsant activity.

R = CH3, C2H5, C3H5, C3H7, 4-CH3C6H4, C6H5, 4-BrC6H4, 4-ClC6H4, 4-FC6H4

R = C6H5, 4-BrC6H4, 4-ClC6H4, 4-FC6H4

N

N SNH

N

O

CH3

O

S

N

CH3

O

R

O

NH

N

SO

CH3

Ar

O

Ar= Phenyl, 4-Nitro phenyl, 3-Nitro phenyl, 3,4-dimethoxy phenyl

N

S

N

RO

R= Cl,H, OH

N

N SNH

SN

O

NCH3

O

RO

CH3

167

18) Handan Altints, Oznur Ates, Seher Birteksoz, Gulten Otuk, Meltem Uzun, Dilek

Satana66 have reported Synthesis of mannich bases of some 2,5-disubstituted 4-

thiazolidinones and evaluation of their antimicrobial activities.

19) Paola Vicini, Athina Geronikaki, Kitka Anastasia, Matteo Incertia and Franca

Zania67 have reported Synthesis and antimicrobial activity of novel 2-

thiazolylimino-5-arylidene-4-thiazolidinones.

S

N

N S

NH

CH

R

R = 4-OH, 4-OCH3, 3-OCH3, 4-Cl etc

20) Chandrakant G. Bonde and Naresh J. Gaikwad68 have reported Synthesis and

preliminary evaluation of some pyrazine containing thiazolines and

thiazolidinones as antimicrobial agents.

N

N

OCH2CONH N

N S

O

R

R= 4-Chloro-2-nitrophenyl, 4-Chlorophenyl, 2,4-Dichlorophenyl, n-Butyl etc

R=CH3, C2H5, C6H5

R' = NH O NH NH N CH3

CH3

CH3

N

SN

H5C2OCOH2C

S

NH O

R

CH2-R'

168

21) Zuhal Turgut, Cigdem Yolacan, Feray Aydogan, Emine Bagdatli and Nuket

Ocal69 have reported Synthesis of new pyrazolothiazole derivatives from 4-

thiazolidinones.

X= a, CH3; b, Cl; c, OC6 H5; d, OCH3; e, OC2 H5 etc

22) K.G. Desai, J.P. Raval and K.R. Desai70 reported the Neat Reaction Technology

for the Synthesis of 4-Oxothiazolidines derived from 2-SH-benzothiazole and

antimicrobial screening of some synthesized 4-thiazolidinones.

R = 4-NO2, 3,4,5-(OCH3)3, 2-OH, 3-OH, 4-OH, 2-OCH3,

4-OCH3, 2-Cl, 3-Cl, 4-Cl

N

S

SCH2CONH

S

N

O

H R

CH3 CH3

CH3

N

S

O

X

169

23) Maqbool Ahmed, Ranjana Sharma, Devendra P. Nagda, Jawahar L. Jat, and

Ganpat L. Talesara71 reported the Synthesis and antimicrobial activity of

succinimido(2-aryl-4-oxo-3-{[(quinolin-8-yloxy)acetyl]amino}-1,3-thiazolidin-5-

yl)acetates.

R = H, Cl, OCH3, CH3, N(CH3)2

24) P. Kohli, S. D. Srivastava and S. K. Srivastava72.have reported Synthesis and

biological activity of mercaptobenzoxazole based thiazolidinones and their

arylidenes.

N

O S C CH2 NH NAr1

CHAr2

CH3O

Ar1=C6H5, ClC6H4, NO2C6H4

Ar2=C6H5, ClC6H4, NO2C6H4, OCH3C6H4

N

ONH

N

O

S

O

R

R

O

170

AZETIDINONES OF PHARMACOLOGICAL INTEREST

Azetidinone is a 4 membered cyclic amide, which is present in the clinically

useful penicillins and cephalosporins.

A review of literature describes the diverse biological activities associated with the

azetidinone derivatives. A brief review of structure activity relationship has been

presented below.

1) M.M.J. Vijay Kumar, L. Shankarappa , H.Shameer, E. Jayachandran, G.M.

Sreenivasa73 have reported N-Substituted-3-chloro-2-azetidinones: Synthesis

and characterization of novel anthelmintic agents.

2) S.Jubie, B.Gowramma, Nitin K.Muthal, R.Kalirajan, S.Gomathi and

K.Elango74.have reported Synthesis and antimicrobial evaluation of some 2-

azetidinone derivatives.

NHO

N

SF

Cl

NH C

N

H3CO

OH

O

Cl

O

MeO N

Ar

ClO

Ar= C6H4Cl, C6H3Cl2,C6H4OMe etc

171

3) Rajiv Dua, S.K.Sonwane, S.K.Srivastava, and S.D.Srivastava75 have reported

Greener and Expeditious Synthesis of 2-azetidinone derivative from 2-mercapto

benzothiazole and their pharmacological screening of the synthesized compounds

using microwave irradiation.

N

S

NH N

Ar

ClO

Ar= Various substituted aromatic aldehydes

4) Rajasekaran A, Periasamy M and Venkatesan S76 have reported Synthesis,

characterization and biological activity of some novel azetidinones.

N

HNH2COC NO

Cl

5) Ameya A. Chavan and Nandini R. Pai77 have reported Synthesis and biological

activity of N-Substituted-3-chloro-2-azetidinones.

N

S

NH C CH2 NH

R

ClO

O

HOOC

R=3-BrC6H4, 4-OHC6H4, C6H5, 4-OCH3C6H4

172

6) Seema Kanwar and S.D. Sharma78 have reported Thienopyrimidines as heteryl

moiety in 2-azetidinones: Synthesis of 4-heteryl-2-azetidinones.

S

NH

N NR

O

O

R= C6H4OCH3, C6H4CH3

7) Shailesh J. Parmar, Ishwar J. Patel79 have reported Synthesis and biological

evaluation of some novel optically active 3-chloro-1-[4-({4-[(S)-(4-

chlorophenyl)(phenyl)methyl]-1-piperazinyl}acetyl)phenyl]-4-aryl-2-azetidinone

derivatives.

8) Mrunmayee P. Toraskar, Vilasrao J. Kadam , Vithal M. Kulkarni80 have reported

synthesis and antifungal activity of some Azetidinones.

R

NN

N Cl

O

O

ClR= H, 2-Cl, 4-Cl, 2-OCH3

N

N

N

CH2CONH N

Ar

ClO

Ar=ClC6H4, N(CH3)2C6H4, OHC6H4

173

9) Anand. K.Halve, Poonam Gour, Rakesh Dubey, Deepti Bhadauria and Bhuwan

Bhaskar81 have reported Synthesis and antimicrobial screening of 2’-hydroxy-5’-

(phenylazo)-N-(1”,3”-diketo-phenylamino)-3-chloro azetidin-2-ones.

N N CH3

N NH NH

Cl CH2

O O

R

R'

R= H, o-Cl, m-Cl, p-cl

R'= H, o-Cl, p-Cl

10) V.K Pandey, V.D. Gupta, Mrinalini Upadhyay, Mridula Upadhyay, V.K Singh

and Meenal Tandon82 have reported Synthesis, characterization and biological

activity of 1,3,4 substituted-2- azetidinones.

OC NH N

Ph

OPhO

R'

R

R= CH2CH3,C6H5

R'= N

O

O

HNOC

N

HNOC OH

HNOC

174

11) B.C.Revanasiddappa, E.V.S.Subrahmanyam, D.Satyanarayana83 have reported

synthesis and biological studies of some novel 2-azetidinones.

12) Levinine and Narayanan84 synthesized an adamantine derivative and tried

its activity against influenza virus A-PR8 and hepatitis virus MHV3 and obtained

encouraging results.

13) Piffer and Testa85 synthesized a number of azetidinones and tested for anti-

inflammatory action. The following type has been synthesized and they showed

anti-inflammatory activity.

14) Tandon M, Kumar P, Tandon P, Bhalla TN and Bharatwaj JP 86 synthesis of

new azetidinones and tested for pharmacological effects and toxicity in rats and

mice. Moderate anti-inflammatory and analgesic activities were observed with

low toxicity. Some derivatives produced in in vitro inhibition of proteolysis.

N

O

N

C NH NAr

ClO

O

Ar= C6H5, 4-OCH3, 3,4,5-(OCH3)3, 4-(CH3)2N, Furfural

N

OClCl

R1N(CH

2) O

(CH2) R

mn

175

15) Wei and Bell87 have reported synthesis a number of azetidinones and tested

them for anticonvulsant activity against electroshock and metrazole induce

convulsions.

16) Sema Tan, Vildan Adar Guner, Aysun Ergene88. have reported Antimicrobial

activity of 4-substituted –styryl-2-azetidinones.

R1

N

O

R3

R3

R2

R1 = Ph, H

R2 = H. OMe, Me, Cl

R3 = Ph, Cl

B2

Cl NO

B2

Cl NO

Cl

B2 = Benzoyl

176

17) Deepak Kumar1, F.B.Bux1, and Arun Singh89. have reported synthesis and

biological activity of Azetidinone.

N

N

H2C C NH NR

O

O

O

Ph

R= C6H5, 2-OH-C6H4, 4-OCH3-C6H4, 4-CH3-C6H4

18) Ujjwal Sahoo, A.K.Seth, A.Sen, Dhanya B, J.Patel and R.Chawla90. have

reported Synthesis and characterization of certain novel azetidinone derivatives as

antibacterial and antifungal agents.

NH NAr

OC6H5O

O

Br

H3CO

Ar= phenyl, 4-hydroxyphenyl, 4-chlorophenyl,4-methoxyphenyl,2-furyl

19) Bhupendra Mistry, Smita Jauhari91. Have reported Synthesis and characterization

of some quinoline based azetidinones and thiazolidinones as antimicrobial agents.

N

NN

NN

O

Cl

R= (Substituted Ar. amines)

R

177

20) Bijo Mathew, Githa Elizebeth Mathew, Nirmal Mathew, M.

Vijayabaskaran92 have reported Synthesis, characterisation of some 2-azetidinone

derivatives from 2-aminopyridine and evaluation of their antimicrobial activity.

N

N

R3

R2

R1

Cl

O

R1= H,OH

R2= H, OCH3

R3= H, N(CH3), Cl, OCH3, OH

21) Dinesh R. Panchasara and Subhash Pande93 have reported Synthesis and

biological activity of 3-chloro-1-(4-perimidine methylcarbonyl amino)-4-phenyl-

azetidin-2-one.

22) K.G.Desai and K.R.Desai94 have reported rapid and efficient synthesis of

some biological active 2-azetidinones under microwave irradiation.

N

N

CH3

H2C C NH N

ClO

OR

R= C6H5, 4-OH-C6H4,4-OCH3-C6H4, 4-Cl-C6H4

N

S

S CH2 C NH N

O Cl

O

R

R= 4-NO2, 2-OCH3,2-OH, 3-OH, 4-OH, 2-Cl,3-Cl,4-Cl

178

23) Modha J.J, Parmer J.M, Datta N.J and Parekh H.H95. have reported the

synthesis of some 4-aryl-1-(p-acetamidophenoxy) acetamido-3-chloro-2-

azetidinones as potential antimicrobial agents starting from ethyl-p-

acetamidophenoxyacetate.

CO NH NO CH2

R

ClO

NHCOCH3

Compounds with chloro, nitro, hydroxy or methoxy substituents were

reported to potentiate the antimicrobial activities.

24) Girija S. Singh, Elbert Mbukwa, and Tshepo Pheko96 reported the Synthesis

and antimicrobial activity of new 2-azetidinones from N-(salicylidene)amines and

2-diazo-1,2-diarylethanones.

25) Ishwar K. Bhat, Sunil K. Chaithanya, P. D. Satyanarayana and Balakrishna

Kalluraya97 reported the synthesis and antimicrobial study of some azetidinone

derivatives with the para-anisidine moiety.

R = 4-chlorophenyl, 4-nitrophenyl, 4-hydroxyphenyl,

4-dimethylaminophenyl, phenyl, 3-furyl

OCOCHPh2

N

Ph

PhO

CHPh2

OCH3

NH CH2 C NH N

OR

ClO

179

RESEARCH ENVISAGED AND PLAN OF WORK

Objective of the present work

The literature survey reveals that 2-amino benzthiazole were reported to posses

various pharmacological activities including anticancer, anti-inflammatory,

antitubercular, antioxidant, antimicrobial, anticonvulsant, and analgesic activities.

2-substituted Benzothiazole with substitution at 7th position has been reported to

be associated with various pharmacological activities.

In continuation of this work on benzthiazole, above observations promoted us to

synthesise the title compounds with presumption that incorporation of amino moiety

would produce new compounds with potent biological activities.

180

Steps Involved In the Plan of Work

• Syntheis of 2-amino benzthiazoles

• Synthesis of 3-methyl-5-phenyl pyrazolone

• Synthesis of Schiff’s base moiety

(a)Synthesis of thiazolidinones

(b) Syntheis of azetidinones

(a) synthesis of title compounds (ATZ 1-8)

(b) Syntheis of title compounds (ATZ 9-12)

Identification and characterization

Melting point, Rf values, Solubility and Spectral studies

The present work was characterized by IR, NMR and Mass spectral analysis

Pharmacological evaluation

� Evaluation of (In-vitro)Anti-inflammatory activity

� Evaluation of (In-vitro)Anti-diabetic activity

� Evaluation of Anti-oxidant activity

� Anti-bacterial activity

� Anti-fungal activity

181

SYNYHETIC SCHEME

F

Cl

NH2

S

N

Cl

F

NH2

N

N

O

C6H5

H3C

Methanol

reflux 5-6 hrs

S

N

Cl

F

N

N

N

H3C

C6H5

Chloroacetyl chlorideTriethylamine

Thioglycolic acid

Dioxane

Dioxane

S

N

Cl

F

N

N

N

H3C

C6H5

S

N

Cl

F

N

N

N

H3C

C6H5

S

O

Cl

O

S

N

NH

F

N

N

N

H3C

C6H5S

O

S

N

NH

F

N

N

N

H3C

C6H5

Cl

O

RR

R= o-phenylene diamine, pyrollidine, piperazine,diphenyl amine, diethyl amine,dimethyl amine, naphthylamine,tyrosine.

R= o-toludine, o-anisidine,phenyl ethylamine, morpholine

KSCN, Gla..CH3COOH

Br2/ NH3

Primary and secondary amines

DMF, Reflux for 2hrs

Scheme I Scheme II

182

EXPERIMENTAL WORK

Materials and Methods

The following experimental methods were used for the characterization of the

syntheised compounds.

� Melting points of the synthesized compounds were determined in open

capillary tubes and are uncorrected.

� IR spectra were recorded on ABB BOMEM FTIR spectrometer using

potassium bromide pellets.

� 1H-NMR spectra of the compounds in deutiriated dimethyl sulfoxide was

recorded on BRUKER Av 400 spectrometer.

� Mass spectra were recorded on GCMS QP 5000 Shimadzu.

Thin layer chromatography was performed using pre-coated aluminium plates,

coated with silica gel GF254 [E.Merck]. n-butanol :chloroform: benzene in the ratio of

1:4:1 was used as the eluent. The spots were visualized in the iodine chamber.

Methods of Synthesis

Step 1: Synthesis of 2-amino-6-fluoro-7-chloro (1,3) benzothiazole.

To glacial acetic acid (20ml) cooled below room temperature were added 8gm

(0.08mol) of potassium thiocyanate and 1.45g (0.01 mol) of fluoro chloro aniline. The

mixture was placed in a water bath and stirred with magnetic stirrer while 1.6ml of

bromine in 6ml of glacial acetic acid was added from a dropping funnel at such a rate that

the temperature never rises beyond room temperature. After all the bromine was added

(105min), the solution was stirred for 2 hours below room temperature and at room

183

temperature for 10 hours, it was then allowed to stand overnight, during which period an

orange precipitate settle at the bottom, water (6ml) was added quickly and slurry was

heated at 850C and filtered hot. The orange residue was placed in a reaction flask and

treated with 10ml of glacial acetic acid heated again to 850C and filtered hot. The

combined filtrate was cooled and neutralised with ammonia solution to the pH range 6.0

A dark yellow precipitate was collected. Recrystalised from benzene, ethanol of (1:1)

after treatment with animal charcoal gave yellow crystals of 2-amino-6-fluoro-7-chloro-

(1,3)-benzothiazole.

Step 2: Preparation of 3-methyl-1-phenyl-5-Pyrazolone

In 250 ml R.B flask, to 6.7ml of phenyl hydrazine hydrochloride, 9.3 gm of

sodium acetate crystal, 6.5ml of ethylacetoacetate, add 10 ml of ethanol and reflux the

mixture for 1 hour on water bath and kept aside for overnight. The seperated product was

recrystallised from toulene and wash with water to get 3-methyl-1-phenyl-5-Pyrazolone.

184

Step 4: Preparation of 7-chloro-6-fluoro-N-[(3E)-5-methyl--2-phenyl-2,4-dihydro-

3H-pyrazol-3-ylidene]-1,3-benzothiazol-2-amine.

2-amino-6-fluoro-7-chloro-benzothiazole treated with 3-methyl-1-phenyl-5-

pyrazolone in presence of methanol and refluxed for 6 hrs, then the mixture is

concentrated to remove methanol. The remaining solution is cooled and poured into ice in

small portions.

The solid separated was filtered off, dried and recrystallised with benzene and

ethanol. The product 7-chloro-6-fluoro-N-[(3E)-5-methyl--2-phenyl-2,4-dihydro-3H-

pyrazol-3-ylidene]-1,3-benzothiazol-2-amine is to be taken for next step.

SCHEME- I

Synthesis of Thiazolidinones

7-chloro-6-fluoro-N-[(3E)-5-methyl--2-phenyl-2,4-dihydro-3H-pyrazol-3-

ylidene]-1,3-benzothiazol-2-amine and thioglycolic was refluxed in presence of 1,4

dioxane. The reaction mixture was cooled and triturated with 10% sodium bicarbonate

solution. The separated solid was filtered and washed with excess of water and then

recrystallized from alcohol to get 9-(7-chloro-6-fluoro-1,3-benzothiazol-2-yl)-3-methyl-

1-phenyl-6-thia-1,2,9-triazaspiro[4.4]non-2-en-8-one.

185

Synthesis of 9-(7-chloro-6-fluoro-1,3-benzothiazol-2-yl)-3-methyl-1-phenyl-6-

thia-1,2,9-triazaspiro[4.4]non-2-en-8-one

0.0025 mol of 9-(7-chloro-6-fluoro-1,3-benzothiazol-2-yl)-3-methyl-1-phenyl-6-

thia-1,2,9-triazaspiro[4.4]non-2-en-8-one was treated with equimolar quantities of

various substituted aromatic amines and refluxed for 2hrs in presence of N,Nٰ-dimethyl

formamide (DMF). The mixture was cooled and poured in to crushed ice. The solid

separated was filtered off, dried and crystallized from alcohol and benzene.

SCHEME- II

Synthesis of 3-chloro-1-(7-chloro-6-fluoro-1,3-benzothiazol-2-yl)-7-methyl-5-

phenyl-1,5,6 triazaspiro[3.4]oct-6-en-2-one.:

7-chloro-6-fluoro-N-[(3E)-5-methyl--2-phenyl-2,4-dihydro-3H-pyrazol-3-

ylidene]-1,3-benzothiazol-2-amine (0.01 mol) in 1,4-dioxane (50ml) was added to well-

stirred mixture of chloroacetyl chloride (0.95 ml, 0.012 mol) and triethylamine (1.08 ml,

0.02 mol) at 0o C. The reaction mixture was then stirred for 18 - 20 hrs and kept aside for

186

3 days at room temperature to get 3-chloro-1-(7-chloro-6-fluoro-1,3-benzothiazol-2-yl)-

7-methyl-5-phenyl-1,5,6 triazaspiro[3.4]oct-6-en-2-one.

Synthesis of Synthesis of 3-chloro-1-(7-chloro-6-fluoro-1,3-benzothiazol-2-yl)-

7-methyl-5-phenyl-1,5,6 triazaspiro[3.4]oct-6-en-2-one derivatives

0.0025 mol of 3-chloro-1-(7-chloro-6-fluoro-1,3-benzothiazol-2-yl)-7-methyl-5-

phenyl-1,5,6-triazaspiro[3.4]oct-6-en-2-one was treated with equimolar quantities of

various aromatic amines and refluxed for 2 hours in presence of N,Nٰ-dimethyl

formamide (DMF). The mixture was cooled and poured in to crushed ice. The solid

separated was filtered off, dried and recrystallized from alcohol and benzene.

187

LIST OF SYNTHESISED COMPOUNDS

S.NO

COMPOUND

CODE

STRUCTURE AND CHEMICAL NAME

1

ATZ 1

2

ATZ 2

9-(5-fluoro-4-(naphthylene-2-yl-amino)benzo[d]thiazol-2-yl) 3-methyl-1-phenyl-6-thia-1,2,9-triazaspiro[4,4]non-2-

en-8-one

3

ATZ 3

188

4

ATZ 4

5

ATZ 5

6

ATZ 6

189

7

ATZ 7

8

ATZ 8

9

ATZ 9

3-chloro-1-(6-fluoro-7-(phenethylamino)benzo[d]thiazol-

2-yl)-7-methyl-5-phenyl-1,5,6-triazaspiro[3,4]oct-6-en-2-

one

190

10

ATZ 10

11

ATZ 11

12

ATZ 12

191

VARIOUS AMINES USED

COMPOUND CODE AMINES USED QUANTITY TAKEN

ATZ 1 Piperazine 0.43gm

ATZ 2 Naphthylamine 0.71gm

ATZ 3 Tyrosine 0.9gm

ATZ 4 o-Phenylene diamine 0.54gm

ATZ 5 Diphenyl amine 0.84gm

ATZ 6 Dimethyl amine 0.25ml

ATZ 7 Diethyl amine 0.5ml

ATZ 8 Pyrolidine 0.41ml

ATZ 9 β-Phenyl ethyl amine 1.25ml

ATZ 10 Anisidine 1.23ml

ATZ 11 Morpholine 0.86ml

ATZ 12 Toluidine 1.08ml

192

IDENTIFICATION AND CHARACTERIZATION

Introduction

The identification and characterization of the prepared compounds were carried

out by the following procedure to ascertain that all the prepared compounds have

different chemical nature than the respective parent compounds ie., Melting Point,

Solubility, Thin layer chromatography, Ultra violet -visible spectroscopy [U.V-Vis],

Infrared spectroscopy [I.R], Nuclear Magnetic resonance spectroscopy [N.M.R.] and

Mass spectroscopy.

1. Melting Point Determination

The melting points of the organic compounds were determined by open capillary

tube method.

Melting point is a valuable criterion of purity for an organic compound as a pure

crystal is having definite and sharp melting point. The synthesized compounds showed a

minute change in melting point after recrystallisation.

2. Solubility:

The solubility of synthesized compounds were tested in arious solvents .The

solubility characters were listed

3. Thin Layer Chromatography

Chromatography is an important technique to identify the formation of new

compounds and also to determine the purity of the compound. The Rf value is

characteristic for each of the compound.

193

a. Preparation of chromatoplate :

Cleaned and dried glass plates were taken. Uniform slurry of silica Gel-G in

alcohol was prepared. The slurry was then poured into the chamber of the TLC

applicator, which was fixed and the thickness was set to 0.5mm. Glass plates were

moved under the applicator smoothly to get a uniform coating of slurry on the plates.

The plates were dried first at room temperature and then kept in an oven for

activation at 1100C for 1 hour.

b. Preparation of solvent system and saturation of chamber :

The solvent system used for the development of chromatogram was prepared

carefully by mixing n-Butanol: Ethyl acetate: Benzene [1:4:1]

c. Application of sample :

The solution of the parent compounds and its target molecule were taken in small

bored capillary tube and spotted at 2 cm from the base end of the plate. After spotting the

plate were allowed to dry at room temperature and plates were transferred to

chromatographic chamber containing solvent system for development.

d. Development of Chromatogram :

Plates were developed by ascending technique when solvent front had reached a

distance of 10-12cm, they were taken out and dried at room temperature.

e. Detection of spots.

The developed spots were detected by exposing them to iodine vapours.

194

f. Calculation of Rf Values :

The Rf values of compounds were calculated using the formula.

Distance travelled by the sample Rf value = Distance travelled by the solvent front

In all the cases the distance travelled by the sample was found to be different from

that of the parent compound spotted along with it. Thus it confirms the fact that the

compounds formed were entirely different from that of the parent compound. Since,

the sample gave a single spot; the compounds were taken to be free from impurities.

The Rf value of compounds were reported.

195

Table No.3.1:ANALYTICAL DATA OF THE SYNTHESIZED COMPOUNDS

Sl.

No

Compound

Code MOL. FORM Mol. Wt M.P °°°°C

%

Yield

1 ATZ 1 C23H23OS2N6F 482.60 87 31.5%

2 ATZ 2 C29H22OS2N5F 539.65 116 28.9%

3 ATZ 3 C28H24O4S2N5F 577.65 110 25.1%

4 ATZ 4 C25H21OS2N6F 508.60 118 13.4%

5 ATZ 5 C31H24OS2N5F 565.68 120 29.5%

6 ATZ 6 C21H20OS2N5F 441.54 90 82%

7 ATZ 7 C23H24OS2N5F 469.60 117 76%

8 ATZ 8 C23H22OS2N5F 467.58 113 73%

9 ATZ 9 C27H23OSN5FCl 520.02 103 75%

10 ATZ 10 C26H21O2SN5FCl 521.99 107 83%

11 ATZ 11 C23H21O2SN5F 485.96 96 72%

12 ATZ 12 C26H21OSN5FCl 505.99 160 76%

196

Table No.3.2: SOLUBILITY DATA OF THE SYNTHESIZED COMPOUNDS

Compound

code Water Acetone DMF DMSO Ethanol Methanol Chloroform Ether

Ethyl

acetate

ATZ 1 - + + ++ + + + + -

ATZ 2 - + + ++ + + + + -

ATZ 3 - + + ++ + + + + -

ATZ 4 - + + ++ + + + + -

ATZ 5 - + + ++ + + + + -

ATZ 6 - + + ++ + + + + -

ATZ 7 - + + ++ + + + + -

ATZ 8 - + + ++ + + + + -

ATZ 9 - + + ++ + + + + -

++ = freely soluble, + = sparingly soluble, - = insoluble

197

Table No.3.3: TLC DATA OF THE SYNTHESIZED COMPOUNDS

Sl. No Compound

Code Solvent system for developing

Proportion of

Components Rf Value

1 ATZ 1 n-Butanol: Ethyl acetate: Benzene 1:4:1 0.91












198

SPECTRAL STUDIES

Ultra Violet Spectra:98-101

Molecular absorption in the UV-vis region of spectrum is characteristic of

structures of the molecules. The UV-vis scanning of the compounds was carried and

3-chloro-4-fluoroaniline exhibited λmax at 265nm. The UV-vis spectra of 2-amino-6-

fluoro-7-chloro benzothiazole exhibited λmax 303 and 288nm. This clearly indicates that

the bathochromic shift of the compounds.

IR Spectra:

The IR spectrum peaks gives an idea about the probable structure of the

compound IR region ranges between 4000-666 cm-1. Quanta radiation from this spectrum

region corresponds to energy differences between different vibrational levels of

molecules.

The compounds were recorded on SHIMADZU FTIR-8400S spectrophotometer

shows different vibration levels of molecules by using KBr pellet technique.

199

Table No.3.4:IR SPECTRAL DATA OF SYNTHESIZED COMPOUNDS

Sl. No. Compound

code

Ar-NH

cm-1

ArC=C

cm-1

Cyclic

C=N cm-1

C-F

cm-1

C-Cl

cm-1

Ar-C=O

cm-1

C-S

cm-1

1 ATZ 1 1314 1542 1630 1073 - 1702 1199

2 ATZ 2 1333 1541 1642 1062 - 1702 1187

3 ATZ 3 1332 1539 1651 1061 - 1651 1186

4 ATZ 4 1312 1543 1632 1071 - 1658 1198

5 ATZ 5 1302 1589 1648 1066 - 1648 1191

6 ATZ 6 1335 1544 1644 1067 - 1707 1191

7 ATZ 8 1336 1544 1644 1067 - 1698 1191

8 ATZ 9 1299 1539 1604 1067 749 1643 1190

9 ATZ 10 1356 1536 1595 1068 755 1650 1160

10 ATZ 12 1306 1451 1630 1070 753 1690 1196

200

1H-NMR SPECTRA

NMR spectroscopy enables us to record differences in magnetic properties of the

various magnetic nuclei present, and to deduce in the large measure about the position of

these nuclei are within the molecule. We can deduce how many different kinds of

environment are there in the molecules and also which atoms are present in neighbouring

groups.

The proton NMR spectra enable us to know different chemical and magnetic

environments corresponding to protons in molecules.

The samples are analyzed on BRUKER 300 MHz spectrometer.

201

Table No.3.5:1H-NMR SPECTRAL DATA

S.

No

Compound

Code

No of

Protons Hydrogen δδδδ (ppm) Multiplity Solvent

1. ATZ-2 22

-2H-Ar-H -1H-2CH3

-1H-NH- -12H-

Naphthyl

6.5-8.5 2.97 8.43 7.48

Multiplet Singlet Singlet Singlet

DMSO

2. ATZ-3 24 11H-Ar-H -1H-2CH3

-2H-CH2-

6.5-7.5 2.33 2.99

Multiplet Singlet Singlet

DMSO

3. ATZ-6 20

7H-Ar-H -3H-1CH3

-1H-CH2-

6.7-7.9 3.93 3.80


DMSO

4. ATZ-7 24 7H-Ar-H -3H-1CH3

-3H-CH2-

6.8-8.5 2.33 3.00


DMSO

5. ATZ-8 22

7H-Ar-H -1H-1CH3

-1H-CH2- -1H-

pyrrolidine-

6.6-8.2 2.37 2.33

3.14

Multiplet Singlet Singlet Singlet

DMSO

6. ATZ-9 22

12H-Ar-H -1H-1CH3

-2+H-CH2-

6.5-8.24 2.37 3.03


DMSO

7. ATZ-11 20

7H-Ar-H -1H-1CH3

-1-morpholine -1H-NH-

6.5-7.9 2.99

3.03


DMSO

8. ATZ-12 20 7H-Ar-H -2H-1CH3

-1H-1NH2

6.7-8.5 2.50 8.43


DMSO

202

MASS SPECTROSCOPY

Mass spectroscopy enables us to know;

a) Relative molecular masses (molecular weights) with very high accuracy, from this

exact molecular formula can be deduced.

b) To detect within the molecule the places at which it prefers fragmentation, from this

we can deduce the presence of recognizable groups within the molecule.

c) As a method of identifying analytes by comparison of their mass spectra with

libraries of digitalised mass spectra of known compounds.

Spectral data

Compound code: ATZ 4

Molecular Formula: C25H21OS2N6F

Calculated Molecular Weight: 508.6

Observed Molecular Weight: 508.0

M+ ion peak= M/Z peak = 508.0

Mass Spectrum of the compound (ATZ-4) peak= 508 for which the mol.wt of

ATZ-4 (9-(4-(2-aminophenylamino)-5-fluorobenzo[d]thiazol-2-yl)-3-methyl-1-phenyl-6-

thia-1,2,9-triazaspiro[4.4]non-2-en-8-one) was 508.

203

EVALUATION OF PHARMACOLOGICAL ACTIVITY

Evaluation of Anti-inflammatory activity102-103

Introduction

The synthesized compounds are screened for anti-inflammatory activity by using

inhibition of albumin denaturation technique which was studied according to Muzushima and

Kabayashi with slight modification.

Experimental protocol

The standard drug and test compounds were dissolved in minimum amount of

dimethyl sulfoxide (DMSO) and diluted with phosphate buffer (0.2 M, pH 7.4). Final

concentration of DMSO in all solutions was less than 2.0%. Test solution (1 ml) containing

different concentrations of drug was mixed with 1 ml of 1% mM Bovine albumin solution in

phosphate buffer and incubated at 270±10C in incubator for 15 min. Denaturation was

induced by keeping the reaction mixture at 600±10C in water bath for 10 min. After cooling

the turbidity was measured at 660 nm (UV-Visible Spectrophotometer SL-159, Elico India

Ltd.). Percentage of inhibition of denaturation was calculated from control where no drug

was added. The Diclofenac was used as standard drug.

% of inhibition =

� Preparation of Phosphate buffer solution:

• Dissolve 6.8 gm of potassium dihydrogen orthophosphate into 250 ml of distilled water.

• Dissolve 2 gm sodium hydroxide into 250 ml of distilled water. Take 173.5 ml of this

solution and add it into above prepared 250 ml solution of potassium dihydrogen

orthophosphate.

• Make up the volume up to 1000 ml with distilled water.

204

• pH of the solution was measured with digital pH meter and adjusted by using 0.2M

NaOH.

� Preparation of 0.01mM of albumin solution:

• Dissolve 0.660gm of Bovine albumin into the 100ml of freshly prepared phosphate

buffer solution.

� Preparation of test solution (stock solution):

• Dissolve 5mg of synthesized drug into the 5ml of DMF solution.

• Preparation of 0.25 mg solution: Take 0.25ml of solution from the above prepared

stock solution into the test tube. Add 1ml of albumin solution and make up the volume

up to 10ml with phosphate buffer.







205

Table No.3.6: ANTI-INFLAMMATORY ACTIVITY (In Vitro)

S.No Name of the

compounds

Absorbance value

(Mean ± SE)

Inhibition of

denaturation (in %)

01 Control 0.083 -

02 Diclofenac

Sodium 0.153 84.33%

03 ATZ 1 0.131 57.08%

04 ATZ 2 0.125 50.06%

05 ATZ 3 0.128 54.427%

06 ATZ 4 0.123 48.19%

07 ATZ 5 0.121 46.42%

08 ATZ 6 0.129 55.12%

09 ATZ 7 0.126 50.57%

10 ATZ 8 0.130 49.42%

11 ATZ 9 0.133 58.72%

12 ATZ 10 0.124 49.3%

13 ATZ 11 0.127 52.97%

14 ATZ 12 0.12 44.5%

206

EVALUATION OF ANTI-DIABETIC ACTIVITY104-105

Introduction

Diabetes mellitus (DM) is a chronic metabolic disorder which affects a significant

portion of the population worldwide. DM is a group of metabolic diseases characterized by

hyperglycemia, hypertriglyceridemia and hypercholesterolemia, results from defects in

insulin secretion, its action or both. Both type 1 and type 2 diabetes are known to be

multifactorial diseases caused by a combination of genetic (inheritance) and environmental

(diet and lifestyle) factors.

Non-insulin dependent diabetes mellitus (NIDDM) is a multifactorial disease, which

is characterized by hyperglycemia and lipoprotein abnormalities. These traits are

hypothesized to damage cell membranes, which results in excess generation of reactive

oxygen species. NIDDM has also been associated with an increased risk for developing

premature atherosclerosis due to an increase in triglycerides (TG) and low density

lipoproteins (LDL), and decrease in high-density lipoprotein levels (HDL). Treatments

include (1) agents which increase the amount of insulin secreted by the pancreas, (2) agents

which increase the sensitivity of target organs to insulin and (3) agents which decrease the

rate at which glucose is absorbed from the gastrointestinal tract. Two groups of oral

hypoglycemic drugs, sulphonylureas and biguanides have been used in the treatment if DM.

They act by lowering blood glucose thereby delaying or preventing the onset of diabetic

complications.

Carbohydrates are the major constituents of human diet and polysaccharides are one

of the main components of carbohydrates that mainly play a role in the energy supply. One

207

therapeutic approach for treating diabetes is to decrease the post-prandial hyperglycemia.

This is done by retarding the absorption of glucose through the inhibition of the

carbohydrate-hydrolysing enzymes α-glucosidase and α-amylase in the digestive tract.

Inhibitors of these enzyme delay carbohydrate digestion and prolong overall carbohydrate

digestion time, causing a reduction in the rate of glucose absorption and consequently

blunting the postprandial plasma glucose rise.

The dietary carbohydrates should first be broken down to monosaccharides by some

gastrointestinal enzymes, since only monosaccharides can be absorbed from intestinal lumen.

α-Glucosidase and α-amylase are the key enzymes involved in the digestion of

carbohydrates. α-Amylase degrades complex dietary carbohydrates to oligosaccharides and

disaccharides that are ultimately converted into monosaccharides by α-glucosidase. Liberated

glucose is then absorbed by the gut and results in postprandial hyperglycemia. The inhibition

of enzymes involved in the digestion of carbohydrates can significantly decrease the

postprandial increase of blood glucose after a mixed carbohydrate diet by delaying the

process of carbohydrate hydrolysis and absorption. The control of postprandial

hyperglycemia is an important strategy in the management of diabetes mellitus, especially

type II diabetes, and reducing chronic complications associated with the disease. Therefore,

such enzyme inhibitors can be useful in the treatment of type II diabetes. There are several

reports of established enzyme inhibitors such as acarbose, miglitol, voglibose, nojirimycin

and 1-deoxynojirimycin and their favorable effects on blood glucose levels after food uptake.

On the other hand, enzyme inhibitors may also act as effective anti obesity agents.

208


Method: α-amylase inhibition activity

� Reagents:

α – Amylase, Potato starch, Phosphate buffer, Sodium Chloride, DMSO, Sodium

Potassium Tartrate, 3, 5-dinitro salicylic acid, Sodium hydroxide.

� Standard: Acarbose

� Procedure:

The α–amylase inhibitory activity for all derivatives was determined based on the

colorimetric method using acarbose as the reference compound. The starch solution

(0.5%w/v) was obtained by stirring and boiling 0.25g of potato starch in 50ml distilled water

for 15min. The enzyme solution (0.5 units/ml) was prepared by mixing 0.001g of alpha

amylase in 100ml of 20mM phosphate buffer (pH-6.9) containing 6.7mM sodium chloride.

All the derivatives (stock solution 100mg/100ml) were dissolved and made up with DMSO.

And from stock solution different concentrations (two fold dilutions) 50, 100, 200, 400 and

800µg/ml were prepared. The color reagent was a solution containing 96mM 3, 5-dinitro

salicylic acid (20ml), 5.31mM Potassium Tartrate in 2M sodium hydroxide (8ml) and

distilled water (12ml). The 1ml of each dilution of all derivatives and 1ml enzyme solution

were mixed in a tube and incubated at 25°C for 30min. From the above 1ml of this mixture,

1ml of starch solution and the tube incubated at 25°C for 3min. Then 1ml of the color reagent

was added and the closed tube placed into 85°C water bath. After 15min, the reaction

mixturewas removed from the water bath, cooled and diluted with 9ml distilled water. The

generation of maltose was quantified by the reduction of 3, 5-dinitro salicylic acid to 3-

amino-5-nitro salicylic acid the reaction is detectable at absorbance 540nm. Acarbose

209

solution was used as positive control. The readings were compared with the controls,

containing buffer instead of sample extract. The percentage inhibition of alpha amylase was

assessed by following formulae:

[Abs540 (Control) - Abs540 (Test)]

% inhibition = X 100

[Abs540 (Control)]

210

Table No.3.7: ANTI-DIABETIC ACTIVITY OF SYNTHESIZED COMPOUNDS

(α- amylase inhibitory activity)

COMPOUND

CODE

% Alpha amylase inhibition

IC50 values

(µg/ml) 50

µg/ml

100

µg/ml

200

µg/ml

400

µg/ml

800

µg/ml

ATZ1 28.53 40.06 57.53 71.70 84.34 160

ATZ2 32.2 40.29 53.35 67.96 76.68 180

ATZ3 18.42 34.64 52.93 69.62 80.65 190

ATZ4 21.39 30.25 42.39 51.05 63.43 260

ATZ5 16.5 27.22 34.0 43.56 55.49 330

ATZ6 41.52 55.04 73.0 82.28 95.5 120

ATZ7 23.19 36.98 50.21 60.42 75.15 200

ATZ8 31.5 46.9 59.97 68.50 73.31 130

ATZ9 39.54 50.66 70.6 84.13 94.4 100

ATZ10 23.19 36.98 50.21 60.42 75.15 200

ATZ11 38.53 50.06 57.53 61.70 74.34 140

ATZ12 21.39 20.25 32.39 41.05 63.43 260

Acarbose 43.63 55.5 74.2 88.13 96.8 63

211

EVALUATION OF ANTI-OXIDANT ACTIVITY106-108

Introduction

Free radicals are reactive atom or group of atoms that has one or more unpaired

electrons, especially one that is produced in the body by natural biological processes or

introduced from outside (as in tobacco smoke, toxins or pollutants) and that can damage cells

proteins and DNA by altering their chemical structure. Free radicals are generally considered

as a fragment of molecules and which are extremely reactive and short valid. They are

produced continuously in cells either as accidental by products of metabolism or deliberately

during phagocytosis. Free radicals can be formed in three ways:-

� By the hemolytic cleavage of a covalent bond of a normal molecule, with each

fragment retaining one of the paired electrons.

� By the addition of a single from a normal molecule.

� By the addition of a single electron to a normal molecule.

The later, electron transfer, is a far more common process in biological systems, than

the other two. The most important free radicals in biological system are radical derivative of

oxygen.

In recent times, studies show that the free radical process known as lipid peroxidation

plays a radical and causative role in the pathogenesis of atherosclerosis, cancer, myocardial

infarction and also in ageing. Free radical oxidative interactions promote tissue injury in

conditions like brain trauma, ischemia, and toxicity and also in neurodegenerative diseases

such as parkinsonison’s disease, Alzheimer’s, dementia, and multiple sclerosis are now well

documented.

212

Ascorbic acid, α-tocopherol, probucol, sylibin, gnaphalin and several other

compounds have been proved to possess antioxidant activity.

The human body possesses several defense systems against free radicals although it

produces free radicals continuously, which comprise of enzymes and radical scavengers.

These are called “first-line antioxidant defense system” but are not completely efficient. The

“second-line defense systems” are constituted of repair systems of biomolecules, which are

damaged by the attack of free radicals, due to the increased use of antioxidants in therapy

An antioxidant is a molecule capable of slowing or preventing the oxidation of other

molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an

oxidizing agent. Oxidation reactions can produce free radicals, which start chain reactions

that damage cells. Antioxidants terminate these chain reactions by removing free radical

intermediates, and inhibit other oxidation reactions by being oxidized themselves.

Anti-oxidant compounds in food play an important role as a health protecting factor.

Scientific evidence suggests that antioxidants reduce the risk for chronic diseases including

cancer and heart disease. Primary sources of naturally occurring antioxidants are whole

grains, fruits and vegetables. Plant sourced food antioxidants like vitamin C, vitamin E,

carotenes and flavonoids have been recognized as having the potential to reduce disease risk.

Most of the antioxidant compounds in a typical diet are derived from plant sources and

belong to various classes of compounds with a wide variety of physical and chemical

properties.

The main characteristics of anti-oxidant are its ability to trap free radicals. Highly

reactive free radicals and oxygen species are present in biological systems from a wide

variety of sources. These free radicals may oxidize nucleic acids, proteins, lipids or DNA and

213

can initiate degenerative disease. Antioxidant compounds like polyphenols and flavonoids

scavenge free radicals such as peroxide, hydrogen peroxide or lipid peroxyl and thus inhibit

the oxidative mechanisms that lead to degenerative diseases.

There are a number of clinical studies suggesting that the antioxidants in fruits,

vegetables, tea and red wine are the main factors for the observed efficacy of these foods in

reducing the incidence of chronic diseases including heart disease and some cancers.


� Method: Hydrogen Peroxide Scavenging Method

� Reagents: Hydrogen peroxide, DMSO, Phosphate buffer saline ( pH-7.4)

� Standard: Ascorbic acid

� Procedure:

All the compounds and the standard were dissolved in DMSO as a solvent - stock

solution (100µg/100ml) and from stock solution various concentrations (two fold dilutions)

of 50, 100, 200, 400 and 800µg/ml were prepared in different volumetric flasks. To each

solution 2 ml hydrogen peroxide was added and the volume was made to 10 ml with

phosphate buffer saline (pH-7.4).A control solution was prepared with DMSO in phosphate

buffer saline without drug. The absorbance at 230nm was recorded using U.V

spectrophotometer against blank (Phosphate buffer saline). The % inhibition by hydrogen

peroxide scavenging activity was calculated using the following formula:

Percentage inhibition = X 100

214

Table No.3.8: ANTI-OXIDANT ACTIVITY OF THESYNTHESIZED COMPOUNDS

(Hydrogen Peroxide Scavenging Method)

Compound

No.

%Inhibition

IC50

1mg/ml 2mg/ml 3mg/ml 4mg/ml 5mg/ml

ATZ 1 23.1 35.9 48.53 53.9 68.9 3.37

ATZ 2 1.83 2.99 10.58 19.9 28.4 8.21

ATZ 3 18.8 29.5 50.1 59.7 65.4 4.67

ATZ 4 2.3 10.5 16.3 28.9 36.5 6.6

ATZ 5 10.23 13.36 24.6 28.3 49.3 5.22

ATZ 6 4.6 9.7 15.7 19.7 26.9 9.35

ATZ 7 3.1 7.9 12.3 17.9 24.6 9.95

ATZ 8 11.8 19.1 25.7 31.8 42.5 7.29

ATZ 9 14.4 20.2 29.5 34.8 47.4 6.83

ATZ 10 19.4 27.4 38.5 46.4 57.3 6.04

ATZ 11 20.9 31.7 45.2 56.5 66.9 5.06

ATZ 12 7.4 14.8 39.3 47.2 57.8 4.25

ASCORBIC ACID

39.6 45.7 53.9 59.5 68.3 2.55

215

EVALUATION OF ANTI-MICROBIAL ACTIVITY109-118

Introduction

The chemical substances which act against the microorganism are known as

“antimicrobial agents”, whereas the substances which act against bacteria are called anti-

bacterial agents and those which act against fungi are called anti-fungal agents.

Antimicrobial agents can be obtained from both natural and synthetic methods. The

production of these synthetic agents is a lengthy and expensive process.

The modern era of antimicrobial chemotherapy began in 1929 with Fleming’s

discovery of powerful bactericidal substance penicillin, and Domagk’s discovery of synthetic

sulfonamides with broad antimicrobial activity in 1935.

Antimicrobial agents produced by microorganisms that kill or inhibit the

microorganism are known as antibiotics. A more broadened definition included any chemical

of natural origin (from any type of cell) which has the effect on the growth of other type

cells. Since most clinically-useful antibiotics are produced by microorganisms and are used

to kill or inhibit infections of bacteria.

The increasing incidence of systematic fungal infections in hospitalized patients,

coupled with the shortage of effective and safe antifungal agents have stimulated renewed

research interest in search for broad spectrum antifungal agents.

Currently, soil microbes remain as the richest source of new antibiotic agents. In

1990, the world consumed literally tons of antibiotics valued in excess of 7 billion dollars

more than half of these antibiotics were of the beta lactam type.

216

Antibacterial activity;

The following conditions must be accomplished for the determination of proper

antibacterial activity.

� There should be intimate contact between the test organism and substance

to be evaluated.

� Microorganism should be provided with the required condition for growth.

� Measurement of activity should be done correctly.

� Aseptic environment should be maintained.

� Condition should be maintained unchanged throughout the study.

Various methods with their own advantages and limitations have been used from time

to time to evaluate the antimicrobial activity of the drug. The antimicrobial activity can be

evaluated by the following techniques.

� Agar streak dilution method

� Serial dilution method

� Agar diffusion method

o Cup plate method

o Cylinder method

o Paper disc method

� Turbidimetric method

In the present study, the well diffusion method was used to evaluate the antimicrobial

activities of the synthesized compounds in vitro. The well diffusion method is one the

methods that may be used for determining the relative effectiveness of the antibacterial

217

activity. The results obtained by this method depend not only on the toxicity of the

antimicrobial agent but also on its liability to diffuse through the medium.

The standard antibiotics used in the present study were ciprofloxacin.

Sensitive microorganisms are

� Gram –ve: E.coli and various species of Salmonella, Shigella, Enterobacter,

Campylobacter and Neisseria. Ciprofloxacin is more effective than norfloxacin against

Pseudo aeroginosa, values of MIC range from 0.5 to 6µg/ml.

� Gram +ve (less sensitive): Streptococci, Staphylococci and Histeria species.

In the present study the following bacteria were used

� Bacillus subtilis (ATCC 6051)

� Staphylococcus aureus (ATCC 12600)

� Klebsiella pneumonia (ATCC 13883)

� Escherichia coli (ATCC 11775)

The antibacterial activity of compounds (ATZ 1-12) was studied by well diffusion

method. Compounds were used in the concentration of 25µg/ml, 50µg/ml, 75µg/ml and

100µg/ml using a solvent DMSO. Ciprofloxacin 50µg/ml was used as standards.

Media used : Nutrient Agar (Composition

Sl. No INGREDIENTS QUANTITY

1. Beef Extract 3 g

2. Peptone 5 g

3. Sodium Chloride 5 g

4. Agar 15 g

5. Distilled Water Up to 1000mL

218

The zone of inhibition of various concentrations of the synthesized compounds

against gram positive and gram negative bacteria was measured and were tabulated.

Materials and Methods:

Media used

Nutrient Agar 1.5%

Media Sterilization

All the culture media were sterilized by autoclaving at 15 lbs/inch2 corresponding to

20min.

Method

Agar streak dilution method

Preparation of agar plates with different concentrations of test compounds

5µg/ml stock solution of the test compound were made using DMSO as the solvent.

From these stock solutions, required quantities of drug solutions were mixed with the known

quantities of molten sterile agar media aseptically to provide the following concentrations 25,

50 and 100µg/ml.

About 20ml of the media containing the drug was dispensed into each sterile petri-

dish (diameter about 10cm). Then the media were allowed to get solidified.

Streaking of microorganisms

Microorganisms were then streaked one by one on the agar plates aseptically. After

streaking, all the plates were incubated in the incubator, set at 37± 10C for 24 hrs. Then the

plates were observed for the growth of the microorganisms.

219

Preliminary Screening

All the compounds possess potent to moderately potent activity against gram-positive

and Gram-negative bacteria.

The antibacterial activities are performed by disc plate method ( disc diffusion

technique). The fresh culture of bacteria are obtained by inoculating bacteria nutrient broth

media and incubated at 37 ± 2˚ C for 18 – 24 hours. This culture mixed with nutrient agar

media and poured into petri-dishes by following aseptic techniques. After solidification of

the media, the plates were placed in a refrigerator for 2 hours. After two hours of cold

incubation, four discs are made at equal distance by using sterile wattman paper (5 mm

diameter).

Dip these discs in to different concentratons.. Dimethyl sulphoxide was used as a

control. After introduction of standard drugs and synthesized compounds, the plates were

placed in a refrigerator for 2hrs for proper dipping of drug into the media. After 2hrs the plate

were placed in an incubator and maintained at 370±20C for 18-24 hours. After the incubation

period, over mean the petri-plates were observed for zone of inhibition by using vernier

scale. The results evaluated by comparing the zone of inhibition shown by the synthesized

compounds with standard drug. The results are tabulated in the Table No.7.2

220

Table No.3.9: ANTI-BACTERIAL ACTIVITY OF SYNTHESISED COMPOUNDS

Compound no.

ANTI-BACTERIAL ACTIVITY (mean zone of inhibition in mm)

Bacillus subtilis Staphylococcus aureus Klebsiella pneumonia Escherichia coli

50

µg/ml

100

µg/ml

50

µg/ml

100

µg/ml

50

µg/ml

100

µg/ml

50

µg/ml

100

µg/ml

ATZ 1 9 10 8 9 9 11 7 9

ATZ 2 6 9 8 11 8 9 6 8

ATZ 3 8 11 9 13 7 9 9 11

ATZ 4 8 12 7 10 8 10 8 9

ATZ 5 7 11 13 15 7 9 11 13

ATZ 6 6 9 9 13 9 12 9 11

ATZ 7 9 10 8 10 9 11 8 9

ATZ 8 8 11 14 16 8 9 7 8

ATZ 9 8 10 7 9 9 10 9 14

ATZ 10 9 11 14 16 9 11 7 11

ATZ 11 7 9 13 15 5 6 10 12

ATZ 12 5 8 6 9 6 7 6 11

STANDARD 13 19 15 21 9 12 12 14

CONTROL 8 9 12 13 8 9 8 11

221

ANTI-FUNGAL ACTIVITY

Introduction:

The antifungal activity of the synthesized compounds was studied by disc diffusion

method.

The standard drug selected for antifungal activity was Fluconazole. It is orally active

broad spectrum antifungal agent.

The antifungal activity of the synthesized compounds was studied against the

following organisms.

1. Aspergillus niger (ATCC 9029)

2. Aspergillus flavus (ATCC 46645)

Compounds were screened at the concentrations 150, and 200µg/ml and using a

solvent system consisting of DMSO. The standard used was Fluconazole 150 and 200µg/ml

against both the organisms.

The disc diffusion method was employed for the screening of antifungal activity.

Materials and Methods

Media used

Potato Dextrose Agar Medium

Sl.No INGREDIENTS QUANTITY

1. Beef Extract 3 g

2. Peptone 5 g

3. Sodium Chloride 5 g

4. Agar 15 g

5. Potato 200 g

6. Distilled Water Up to 1000mL

222

Microorganisms used

1. Aspergillus niger (ATCC 9029)

2. Aspergillus flavus (ATCC 46645)

Media Sterilization

All the culture media were sterilized by autoclaving at 15 lbs/inch2 corresponding to

20 min.

Method

Agar streak dilution method

Preparation of agar plates with different concentrations of test compounds

5mg/ml stock solution of the test compounds were made using DMSO as the solvent.

From this stock solution, required quantities of drug solutions were mixed with the known

quantities of molten sterile agar media aseptically to provide the following concentrations

150 and 200µg/ml.

About 20ml of the media containing the drug was dispensed into each sterile petri-

dishes (diameter about 10cm). Then the media were allowed to get solidified.

Streaking of microorganisms

Microorganisms were then streaked one by one on the agar plates aseptically. After

streaking, all the plates were incubated in the incubator, set at 37± 10C for 48 hrs. Then the

plates were observed for the growth of the microorganisms.

223

Preliminary Screening

Disc diffusion method:

The synthesized compounds are screened against two selected fungal strains

Aspergillus niger and Aspergillus flavus by using diffusion method. The 48 hours old fungal

culture inoculated into nutrient broth by following aseptic techniques and incubated for 48

hours at 370± 20C in an incubator. This culture mixed with well sterilized and cooled media

like Potato-dextrose agar media and poured into petriplates. After solidification five discs

are made at equal distance by using sterile swattmann filter paper (5 mm in diameter). Into

these place different concentrations of standard drug and synthesized compounds along with

control (N, N’- Dimethyl Sulphoxide) are introduced.

After introduction of standard drug and compounds,these plates are placed in a

refrigerator at 80 – 50C for 2hrs for proper diffusion after 2hr the petriplates are transferred to

incubator and maintained at 370± 20C for 24-36 hours. After the incubation period, the plates

were observed for zone of inhibition by using vernier scale. Results are evaluated by

comparing the zone of inhibition shown by the synthesized compounds with standard drug.

224

Table No.3.10: ANTI FUNGAL ACTIVITY OF SYNTHESIZED COMPOUNDS

Sl.

No

Name of the

compounds

Mean zone of inhibition (in mm)*

Aspergillus flavus Aspergillus niger

150µg/ml 200µg/ml 150µg/ml 200µg/ml

1 Fluconazole 11 13 15 17

2 Control 6 9 7 10

3 ATZ-1 11 13 10 12

4 ATZ-2 5 7 4 6

5 ATZ-3 8 11 6 11

6 ATZ-4 5 7 5 6

7 ATZ-5 6 8 4 7

8 ATZ-6 7 14 8 14

9 ATZ-7 4 7 9 12

10 ATZ-8 6 9 6 9

11 ATZ-9 7 13 9 13

12 ATZ-10 7 9 8 12

13 ATZ-11 9 11 10 12

14 ATZ-12 8 9 6 9

225

RESULTS AND DISCUSSION

1. Anti - Inflammatory Activity

Compounds synthesised were screened for the anti inflammatory activity at the

concentrations of 100 and 150 µg/mL by inhibition of protein denaturation method using

Diclofenac sodium as standard.

Among the compounds screened for anti inflammatory activity ATZ-9, ATZ-1, ATZ-

6, ATZ-3 and ATZ-11 had shown better activity.

2. Anti - Diabetic Activity

Compounds were screened for the anti-diabetic activity by α-amylase inhibitory

activity at 50, 100, 200, 400 and 800µg/ml using Acarbose as standard. The breakdown of 3,

5-dinitro salicylic acid to 3-amino,5-nitro salicylic acid was measured at 540nm for all the

concentrations of each compound. The % Inhibitions of the compounds at various

concentrations is calculated from their absorbance values.

Among the screened compounds, ATZ-9, ATZ-6, ATZ-8, ATZ-11 and ATZ-1 had

shown the promising activity against the standard.

3. Anti - Oxidant Activity

Compounds synthesized were screened for anti oxidant activity. These compounds

are screened for the activity at 1, 2, 3, 4 and 5 µg /ml using Ascorbic acid as standard. The %

inhibition of the compounds at various concentrations is calculated from their absorbance

values. Among the screened compounds, ATZ 1, ATZ 12, ATZ 3, ATZ 11 and ATZ 5 had

shown the better activity against the standard.

226

4. Anti-Bacterial Activity

Compounds synthesized were screened for anti bacterial activity using disc plate

method at concentrations 50 and 100µg/ml using gram + ve and gram – ve strains. Such as

Bacillus subtilis, Staphylococcus aureus, Klebsiella pneumonia, Escheria coli.

Among the synthesized screened compounds, ATZ-10, ATZ-11, ATZ-5 and ATZ-6

had shown promising anti bacterial activity against the standard.

5. Anti-Fungal Activity

Compounds synthesized were screened for anti fungal activity using disc plate

method at concentrations 150 and 200µg/ml and the strains used for the screening are

Aspergillus flavus, Aspergillus niger.

Among the synthesized screened compounds, ATZ-1, ATZ-6, ATZ-9 and ATZ-11

had shown the better activity against the standard.

227

SUMMARY AND CONCLUSION

In the present study, novel benzothiazole substituted pyrazolo-thiazolidinone and

pyrazolo-azetidinone moieties were synthesized and are characterized by IR, NMR and Mass

spectral data.

All the synthesized compounds were screened for anti-inflammatory, anti-diabetic,

anti-oxidant, anti-bacterial and anti-fungal activities.

Anti-inflammatory activity was screened for synthesized compounds using

Diclofenac as standard. Compounds ATZ-9, ATZ-1, ATZ-6, ATZ-3 and ATZ-11 had shown the

better anti-inflammatory activity.

Evaluation of anti-diabetic activity was screened for synthesized compounds using

Acarbose as standard. Compounds ATZ-9, ATZ-6, ATZ-8, ATZ-11 and ATZ-1 had shown

the significant anti-diabetic activity.

Evaluation of anti-oxidant activity was screened for synthesized compounds using

Ascorbic acid as standard. Compounds ATZ-1, ATZ-12, ATZ-3, ATZ-11 and ATZ-5 had

shown the significant anti-oxidant activity.

Anti-bacterial activity of the synthesized compounds were tested against the micro-

organisms, E.coli, B,subtilis, S.aureus and K.pneumonia using Ciprofloxacin as a standard.

Compounds ATZ-10, ATZ-11, ATZ-5 and ATZ-6 had shown promising anti bacterial

activity against the standard.

Anti-fungal activity of the synthesized compounds were tested against the micro-

organisms, A.flavus and A,niger using Fluconazole as a standard. Compounds ATZ-1, ATZ-

6, ATZ-9 and ATZ-11 had shown the better activity against the standard.

228

From this study, it may be concluded that benzothiazole substituted with piperazinyl,

-N(CH3)2, -N(CH2CH2(C6H5) on seventh position enhances the anti-microbial, anti-

inflammatory, anti-diabetic, anti-oxidant and anti mitotic activities and hence the study

would deserve for future investigation and derivatisation.

229

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