PREPERATION AND BIOLOGICAL SCREENING OF...

22

CHAPTER-III

PREPERATION AND BIOLOGICAL SCREENING

OF TRAIZOLES

(C) Triazolo Thiadiazoles

(D) 3,4-disubstituted traizoles and their derivatives

bearing inh and pyrazinamide moieties.

23

A) Preperation and Biological screening of Triazoles

The history of 1,2,4-Triazoles is less than a century old. It begins with

the work carried by Bladin who synthesized the first representatives.

He coined the name triazole for this class of compounds. Until

recently well over 20,000 triazoles are known but practical

applications have been very few. Although most triazoles are readily

prepared by different synthetic routes and stored, expensive starting

materials and sensitive intermediates appear to have discouraged

industrial synthesis and wide applications.

Triazoles1

3 nitrogen atoms present in five membred ring structures describe an

important class of hetrocylices called the triazoles. These are of 2

types of structures the v-triazoles or 1,2,3-triazoles (1) and s-triazoles

or 1,2,4-triazoles (2).

The carbon nitrogen ring system C2H3N3 was first named as triazoles

by Bladdin , who described its derivatives in 1885. Later on, because

of various applications, triazoles took special attention particularly by

the chemical industry. The stable 1,2,4-Triazole neucleus may be

considered as aromatic as shown by its chemical behavior as it is

stabilized by resonance as shown below.

NN

N

(1)

N

NH

N

(2)H

24

1,2,4-triazole analogues having substitution at 3 and 4.

5-Mercapto triazoles form a special class of triazole derivatives among

triazoles, because of tautomerism exhibited by them. The labile H

atom can be present on S-or N atom. Previous studies of IR, NMR and

UV spectra have provided strong evidence that mercapto triazoles are

present predominantly in the thione form (4) [3]. Due to liability of

hydrogen, both N and S substitution may be incorporated by

modifying the reaction parameters. These mercapto triazoles have

been reported to show all the characteristic reactions of a nucleophilic

species.

The following are some of the reactions of mercapto triazole derivatives

N

N

N

H+

N

N

N

H+

N

N

N

H+

N

NH

N

N

N

N

R

SHR

(3)

N

N

N

R

SR

(4)

Scheme : Nucleophilic reactivity of thiols.

R SH

RSRRSSR + RSSR + RSH

RSSCI2) RCI RSSR

SCl2

RSSR

1) NaOH

RSSCOClClSCOCl

Na2S3O6

RSSO3Na+

RSO2R

SO2Cl2

RSClCl2 -50Cin HCl

RSO2NH2RSO2ClNH3

25

1,2,4-triazole-5-thione derivatives

Among the substituted triazoles, aminotriazoles constitute class

because of their widespread biological activities e,g, 3-aminotriazole,

better known as amizol was the first triazole which was used as an

important herbicide. Aminotriazoles may have a C-amino group or an

N-amino group.

OO2N CHO

N

N

N

NH2

OO2N CH

HN

N

N

N

(1.1)

N

N

N

NH2

2 ClCOOC2H5

N

N

N

NHCOOC2H5

N

N

N

NH2.HCl

(1.2)

26

Biological Action and Uses of Triazoles

Disubstituted-2,4-dihydro-3H-1,2,4-triazole-5-thiones and their

derivatives have gained lot of interest in the last decade due to their

biological, industrial and agricultural importance. A well known

example is that of fluconazole, an antifungal drug for the treatment of

fungal diseases & recently it is highlighted that s-triazoles possess a

variety of biological properties such as anti-fungal [12-14], diuretic

[15], antibacterial [16,17], hypoglycemic, anti-tubercular,

antidepressant, anti-amoebic, antibiotic, anti-inflammatory, anti-

carcinogenic, hypnotic, sedatives, plant growth regulators and

insecticidal. Some pyridyl and pyrimidyl substituted mercaptotriazoles

(5) are known to posses antithyroid property. N-

aminomercaptotriazoles (6) are known to posses potent bactericidal

and fungicidal properties. Some disubstituted mercaptotriazoles (7)

and (8) have been tested for antifungal activity. The highest activity

against all species was shown by 8 (R = 4-IC6H4). Acylation of some

aminotriazoles gives triazoleamides (9) which specifically .

27

N

N

N

R SH

R

R=2-,3-,4-PyridylR'=H, CH3

(5)

N

N

N

SH

NH2

(6)

N

N

N

N

R SR2

R1

R=2,6-Me2C6H3OCH2R1=4-MeOC6H4,PhCH2

R2=Me(7)

N

N

NH

S

R

R=Ph,4-BrC6H4,4-1C6H4,2-,3-,4-MeC6H4

(8)

Cl OH

N

N

NH

R2 NHCOR3

R2=H, MeS, NH2, MeSO2R3=2-ClC6H4CO, EtCO, Me2CH2CO

PhOCH2CO(9)

N

N

NH

RC6H4 S

R=H, p-Me, p-MeO, p-Cl, m-Br, p-BrR1=H(10)

NHR1

N

N

N

RC6H4 SCH2R3

R=H, o,-m-, p-Me, p-MeOR3=CO2Me

(11)

NH2

N

N

NR2

S

R=Halo, C1-6 alkyl, CF3r1 & R2=C1-6 alkyl

(12)

R1Rn

N

N

N

SH

N

Cl(13)

28

inhibited rubella virus 4-Amino-1,2,4-triazoles and their derivatives

(10) and (11) have been prepared and found to possess bactericidal

and/or fungicidal activity. Certain 4-alkylsubstituted traizoles (12)

have been reported to inhibit reserpine induced ptosis in mice with an

ED50 of 0.27mg/kg. Certain substituted 5-(4-pyridly)-3-mercepto-

1,2,4-triazoles e.g. (13) have been used as additives.

N-benzylated aminotriazole (14) is a s-triazole possesing useful activity

of inhibiting acid fading dyestuff. s-triazole derivatives of 4-

(phenylureido)- are used as defoliants (15)

A new series of condensation polymers of s-triazoles have been

synthesized , having properties of nylon. These are fibre forming and

used to make threads which possess very strong nature and attraction

for dyestuffs. The polyaminotriazoles are fused polymers and are

prepared by

Reaction of dihydrazides with a small quantity of hydrazine at high

temperature. The name refers to the rearranging linkage, the s-triazole

N

N

N

R R

NHCH2C6H5

(14)

N

NNNHCONH

X

(15)

r.(NH3NHCORCONHNH2) H2NNHCO RCONHNH2 + 2H2O (1.3)

N N

N

NH2

R

n-1

29

deravitives [16] and [17] can be used for stabilization of thermoplastic

polymers.

Few important reports on biological activity.

Dubey et al2 prepared Bis-triazole derivatives and screened them for

antifungal activity.

Mohan et al3 synthesized certain Triazolo thiadiazine derivatives and

screened them for antimicrobial activity.

Gupta et al4 synthesized certain triazolo thiadiazoles and evaluated

their anti-inflammatory & anti-microbial activity.

N

N

N

R1 Z(CH2)m

R1=H, OH, C1-6 alkoxy or alkythio,CO2H, SH

R2=H, OH, SH, NH, O, S, CH2m = 1-20, n=1,2

(16)

R2

n

N

N

N

R R1

R=H, NH2, SH, C1-12 alkylthioR1=CO2R3

R3=H, C1-8 alkyl(17)

H

N

N

N

NH2

HS N SH

NN

NH2

NH

N

S NN

NO2

30

Bennur et al5 synthesized certain deravitives of triazoles and

evaluated them for anti-microbial property.

Bennur et al6 synthesized certain compounds of triazole series and

carried out screening of microbial inhibition activity.

Fikry et al7 synthesized certain cyclohexyltriazoles and evaluated their

microbial inhibition activity.

N

N

N

R

HN

S

R1

OR2

OR3

R = CH3R1 = C2H5R2 = CH3R3 = CH3

N

N

N

R

NH2

SH

N

N

N

S

N

R1

RR =R1 = H

CH2CH2HN

O

O

HOOCN

N

N

S

O N NH

31

Sen et al8 synthesized thiazolo triazoles and evaluated them against

their filarial property.

Radhakrishnan et al9 synthesized certain 5 membered ring

compounds containing oxygen, sulpher and nitrogen and their

deravitives and evaluated their bacterial property against different

organisms.

Shigare et al10 synthesized certain dihydro-pyridino triazoles and

thidiazoles and screened them for microbial resistance activity.

N

N

N

S

O

N

Cl

HN NO2C6H5

N

H2CNH

NN

SH

32

Talwar et al11 observed preparation and biological activities of s-

triazole derivative reactions of 3,4,5- tri substituted triazoles, few

derivatives have shown marked activities against fungi.

Gadaginamath et al12 reported preparation of 1,3,4-oxadiazolyl /2,5-

dimethyl pyrrolyl/ 1,2,4-triazolmethoxybisbenzylpiperazine-2,5-

diones and screened them for anti-microbial activity.

Saad13 synthesized some pyridyloxymethyl oxadiazoles, thiadiazoles

and traizoles.

N

N N

HS

H3C

R1

NH

R

CH3

N N

N SH

R1

R = 2 - ClR1 = 4 - Cl

N

NHN

S H2CO CH2

HN

NH

O

O

CH2 OCH2N

NHN

S

N

OCH2N

NN

N=CHC6H5

SH

33

Sayeed et al14 synthesized 4-benzyl-1 (2H)-phthalazinone derivatives

and evaluated them for anti-bacterial and antifungal property.

Kudari et al15 synthesized certain bis 5 membered hetero derivatives

containing oxygen sulpher and nitrogen and evaluated them for anti-

microbial property.

Bennur et al16 synthesized certain 5 membered Oxadiazoles,

Thiadiazoles and triazoles.

Mohan et al17 synthesized certain thiazolo triazole derivatives and

screened them for antimicrobial and diuretic activity.

N

N

N

C6H5

S CH2 CH2 COOHH2CH2CHOOC

N

N

N

HSNHCH2

N

NN

SH

N

N

N

R2

R1 R3

R1= C6H5, XCH2R2= C6H5, NH2R3= NH2, SHX= S, SO2

34

Udupi et al18 synthesized certain 3-substituted 4-(n-pyridyl

carboxamido)5-mercapto symmetrical triazole deravitives and

screened their anti-tubercular activity.

Kataky et al19 synthesized certain s-triazolo thiadiazoles

Master et al20 synthesized 3-substituted pyrimido anthraquinone

related fused s-triazolo, tetrazolo and pyrazoline derivatives.

Cl

N

NN

S

N

N

N

HN

Ar SH

CO NAr = Phenyl2 - Chlorophenyl4 - Aminophenyl

NO2

N

NN

N

S

R1

R1=4 - NO2 - Ph

N

N

N

H3C

N

O

R

R = Cl, OCH3, CH3

O

O

35

Kalluraya et al21 synthesized some 1,2,4-triazole starting from thymol

such as 3-(2-isopropyl-5-methyl) phenoxy methyl-4-amino-5-

mercepto-1,2,4-triazole. This was then employed in the synthesis of

some N-Bridged heterocycles and were screened for antibacterial and

anti-fungal activities.

Where R = Phenyl, furyl, thymoxymethyl, p-chlorophenoxymethyl,

anilinomethyl, o-toluidinomethyl, p-toluidinomethyl, o-

chloroanilinomethyl, etc.

Chande et al22 synthesized certain s-triazoles bearing organo

phosphorus type deravitives and screened them for insecticidal

activity.

Rao et al23 carried out fusion of 3-alkyl triazoles having mercapto at

5th position with acetyl coumarin and its derivatives. which have been

shown to exhibit remarkable anthelmintic activity and also coumarin

derivatives with heterocyclic system at position - 3 exhibit a promising

biological activity.

N

N

N

OH2CAr

N

S

R

N

N

N

OH2C

N

S

O

Ar

N

N

N

NH

S

S

CH2 S P

S

OCH3

OCH3

36

Krishnan et al24 synthesized s-triazolo[4,3-a] quinoxalines and studied

the different methods for the synthesis of such type of compounds and

also the biological activities associated with them.

Yi et al25 carried out the studies on the condensation of heterocyclic

compounds. They synthesized s-triazolo thiadiazoles. They also

reported the fragmentation pattern of the mass spectra and

antibacterial activity of several representative compounds screened

against B. subtili and E. coli.

Udupi et al26 carried out the preparation and screening of biological

activity of s-triazole deravitives. They synthesized condensed

deravitives containing triazole and thiazolodine ring. Mannich bases

have been prepared and evaluated anti-tubercular, anti-bacterial,

anti-fungal and anti-inflammatory activity.

N

N OR1

N

N

R

R = MeOC6H4R1 = CH3 or C2H5

N

N

N

Ar S

N NC

O

N

N

N

N CH2

Ar1 S

HN C

O

N

R

Ar = Phenyl4 - Chlorophenyl4 - Aminophenyl

Ar1 = PhenylR = 2-amino-4-Nitrobenzoic acid

4-Amino Salicylic acid

37

Udupi et al27 carried out the synthesis and biological activity of 3-

pyridyl-4-[N-substituted phenyl caboxamido]-5-mercapto-1,2,4-

triazoles and screened them for microbial inhibition activity.

Udupi et al28 carried out their studies on anti-tubercular agents. They

synthesized s-triazolo thiadiazolidines exhibiting significant anti-

tubercular activity. They also reported the anti-inflammatory activity

for some of the compounds synthesized.

Udupi et al29 carried out their studies on the synthesis and biological

activity of 1,2,4-Triazole derivatives containing triazolo-thiadiazole and

triazole-thiadiazolidine ring system. They reported the anti-tubercular,

anti-bacterial and anti-fungal activity of some of the compounds

synthesized.

NN

NN

SH

N C Ar

O

Ar = Phenyl2 - Aminophenyl4 - Nitrophenyl4 - Chlorophenyl

N C O

N

Ar = Phenyl4 - Chlorophenyl4 - AminophenylPhenoxymethyl

CH2

SN

NN

Ar

N

N

N

Ar

N

S

N

N

N

N

Ar

N

S

SON

Ar = Phenyl4 - Chlorophenyl3 - methyl phenoxymethyl

38

Udupi et al30 synthesized and carried out the screening of s-triazolo

deravitivies bearing ibuprofen moiety. Some compounds have shown

significant antiinflamatory and antibacterial properties.

Mohan et al31 carried out their studies on synthesis and bioactivity of

s-triazolo thiadiazoles and s-triazolo thiadiazines and s-triazolo

thiadiazino guinoxaline. They reported the microbial inhibition

property of some of the deravitives synthesized.

N

N

N

Ar

HN C CH

O CH3

S.CH2COOH

CH2 CH

CH3

CH3

N

N

N

Ar

N C

S

CH

CH3

CH2 CH

CH3

CH3

Ar = Phenyl4 - Nitrophenyl4 - AminophenylPhenoxymethyl

39

(i) CICH2COOH, NaOAc; (ii) RICOCH2Br, K2CO3,

(iii) C6H5CHOHCOC6H5; KOH; (iv) 2,3-Dichloroquinoxaline, NaOAc;

(v) CS2, KOH; (vi) ArCOOH, POCI3; (vii) ArCHO

Isloor et al32 synthesized s-triazolo thiadiazoles and reported their

characterization. They also carried out the screening of the

compounds for anti-cancer activity on Hep G2 cell lines. The

Thiadiazole with napthyloxy methyl and flurophenyl group as

substitutent showed excellent anti-proliferative effect.NN

NR

N

S

NH

N

3-substituted-6-(3-substituted-1H-pyrazol-4-yl)[1,2,4]triazolo[3,4-b][1,3,4] thiadiazole

40

Ilango33 and Velentina synthesized series of s-triazolo thiadiazoles.

The compounds were evaluated for antimicrobial, antifungal and

antioxidant activity by DPPH and nitric oxide methods. Few

compounds exhibited significant antifungal activity with MIC value at

6.25 µg/ml. All the compounds showed moderate to good antioxidant

activity by both the methods.

Karale at el34 reported synthesis and biological screening of some

halogenated thiadiazoles and triazoles. They reported preparation of

the deravitives by conventional method and ultrasound irradiation

method. Some of the prepared deravitives were screened for their

antimicrobial, antiviral and antioxidant properties. None of the

deravitives did not show any activity however some of the synthesized

compounds showed potent antioxidant activity. Few compounds were

also tested for percent superoxide dismutase activity but none of them

exhibited high percentage of SOD activity.

NH

H2C

Cl Cl

N

NN

N

S

Ar

41

Subramani et al35 reported the study on triazole thiadiazoles involving

their preparation and screening for various biological activities

R2

R3

R4

R1

ONH

HN

HN

Ar

O

S

Conc. H2SO41. conventional2. ultrasound

1N NaOH1. conventional2. ultrasound

R3

R2

R1

O

R4

N N

SNHAr

R3

R2

R1

O

R4

N N

NSH

Ar

HO

HO

HO

N N

N S

N

Ar

Ara) 2-OH-C6H4b) 3-OH-C6H4c) 4-OH-C6H4d) 2-NO2-C6H4e) 3-NO2-C6H4f) 4-NO2-C6H4g) 2-Cl-C6H4h) 3-Cl-C6H4i) 4-N(CH3)2-C6H4j) 3,4,5-(OCH3)3-C6H2

42

EXPERIMENTALIII A Triazolo Thiadiazoles

Scheme I

Br COOH

NH2

(1) Br

I [A]

CH3OH, H2SO4

Br COOCH3

NH2

(1) Br

NH2.NH2H2O (99%)

Br CONH.NH2

NH2

(2) Br

KOH, CS2

Br

Br

CONH.NH

NH2

C S-K+

S

(3)

NH2.NH2 H2O (99%)

Br

Br

NH2

N

NN

S-K+

NH2

(4)

Dil HCl

(Yield 70%)

Br

Br

NH2

N

NN

SH

NH2

43

(B)Br COOH

NH2

Br(1)

H2N NH C NH.NH2

S

(2)

I) for 1 1/2 hours

II) NaHCO3 5%

(Yield 85%)

Br

Br

NH2

N

NN

SH

NH2

(II)

Br

Br

NH2

N

NN

SH

NH2

Z-COOH, POCl3

Br

Br

NH

N S

N Z

NN

(T1 - T28)

44

Preparation of Triazole (T0) (Method A)

I. Preparation of 3,5-dibromo anthranilic acid hydrazide

Dibromo deravitive of methyl ester of anthranilic acid (0.01 mol) in

ethyl alcohol (30ml) and NH2-NH2. H20 (0.03 mol) was reflexed for 8

hours. Cooled , and the product obtained was filtered and it was

crystallized from the solvent ethyl alcohol. The thin layer

chromatography was used to confirm the purity using mobile phase

n-hexane and ethyacetate . 8:2 v/v (Yield 88%).

II. Preparation of potassium dithiocarbazinate salt(PDCS):

A mixture of 3,5-Dibromo anthranilic acid hydrazide (0.01 mol), KOH

(0.03 mol) were added to 70ml anhydrous ethyl alcohol and CS2 was

stirred for 12-13 hours. The solid product was filtered dried, washed

with ether and directly used for the preparation of triazole

III. Synthesis of triazole (T0).

The above dithiocarbazinate and NH2NH2. H20 in the ratio of 1:3

was heated at 155°C till H2S gas was evolved. The product was added

to water and acedified using hydrochloric acid 35% so as to obtain the

required triazole and was purified by crystallization using ethyl

alcohol. (M.P 78-800C, Yield: 70%).

45

Method B

The well triturated mixture of 3,5-dibromo anthranilic acid and

NH2NH C=S NH NH2 in equimolor proportion was fused for 2 hours.

Then cooled to room temperature , washed with NAHCO3 5% solution

to remove unreacted acid and again washed with water the dried

compound was recrystallized from ethyl alcohol. And thin layer

chromatography used to confirm its purity using mobile phase N-

hexane and ethyacetate 7:3 v/v (M.P. 78-80°C, Yield 85%).

Preparation of triazolothiadiazoles (T1 – T28)

The required triazolo thiadiazoles were prepared by heating equimolar

Mixture of triazole and carboxylic acid in presence of POCl3 (10 ml)

for more than 7 hours. POCl3 which is present in excess was removed

by distillation under vaccum. The product obtained was dissolved in

water and treated with NAHCO3 to remove the unreacted acid. The

product thus obtained was washed with water and purified by

crystallization using ethyl alcohol. Thin layer chromatography was

used to establish its purity. The same procedure was used for the

preparation of other compounds. (T1-T28)

46

Table 1: Data showing Characterization of 3-(2’-amino-3’,5’-dibromo phenyl)-6-substituted (3,4-b) (1,3,4) triazole thiadiazolesand bis-triazolo thiadiazoles

Sr.No. Code Z Mol. Formula M.P.

(°C)Yield(%)

01. T1 2-Amino-3,5-dibromophenyl C15H8N6Br4S 170 80

02. T2 2-(2’,6’-dichloro anilino)benzyl C22H14N6Br2SCl2 110 70

03. T3 4–Isobutyl phenyl ethyl C21H21N5Br2S 80 74

04. T4 2–Chloro pyridinyl C14H7N6Br2SCl 140 72

05. T5 Pyridinyl C14H8N6Br2S 246 74

06. T6 Diphenyl methyl C22H15N5Br2S 170 76

07. T7 2–Chloro benzyl C16H10N5Br2SCl 60 70

08. T8 Penta fluoro phenyl C15H4N5Br2SF5 110 78

09. T9 3,5–dinitrophenyl C15H7O4N7Br2S 120 76

10. T10 4–Chloro phenyl C15H8N5Br2SCl 158 72

11. T11 2–Chloro phenyl C15H8N5Br2SCl 140 70

12. T12 1–Naphthyloxy methyl C20H13N5Br2S 142 78

13. T13 2,4–Dichloro phenoxy methyl C16H9ON5Br2SCl2 120 80

14. T14 4–Chloro phenoxy methyl C16H10ON5Br2SCl 102 82



17. T17 2–Naphthoxy methyl C20H13N5Br2S 134 76

18. T18 2–Amino-4-nitro phenyl C15H9O2N7Br2S 170 70

19. T19 4–Amino phenoxy methyl C16H12ON7Br2S 160 72

20. T20 2–Methyl phenoxy methyl C17H13ON5Br2S 118 80

21. T21 4–Methyl phenoxy methyl C17H13ON5Br2S 120 76

22. T22 Phenoxy methyl C16H11ON5Br2S 98 69

23. T23 4–Bromo phenoxy methyl C16H10ON5Br2S 128 74

24. T24 4–Nitro phenoxy methyl C16H10O3N6Br2S 170 76

25. T25 2–( 2’,3’–dimethyl anilino)phenyl C24H18N6Br2S 140 78

Br

Br

NH2

N S

NN

N Z

47

Bis-Triazolo thiadiazoles

Sr.No.

Code Structure Mol. FormulaM.P.(°C)

Yield(%)

26. T26 C18H8N10Br4S2 170 70

27. T27 C20H12N10Br4S2 110 75

28. T28 C20H12O2N10Br4

S2102 68

Br

Br

NH2

N S

NN

N N

NS

NN

H2N

Br

Br

Br

BrNH2

N S

NN

N CH2CH2 NNS

NN

H2NBr

Br

Br

Br

NH2

N S

NN

N CH CH N

NS

NN

H2N

Br

Br

OH OH

48

Characterization of Triazolo Thiadiazoles

Spectral Data of Parent Triazole (T0)

IR (KBr) CM-1:IR Spectrum of parent triazole (T0) i.e. 3(2-amino-3,5-dibromophenyl)-

substituted s-triazole showed its characteristic absorption bands in

the following region. 3465 and 3349 (NH2 groups), 3074 (Ar.C-H Str),

1612 (C=N), 1571, 1531, 1451 (C=C rig Str.), 1303 (C=S), 879

(substituted phenyl ring) 690 (C-Br).

1H NMR : ( ppm)

The NMR data of parent triazole (T0) exhibited its signals as below δ3.04 (1H, s, SH), 3.65-3.89 (2H, s, NH2),6.17 (2H, s, 2H of N-NH2)

7.94-9.02 (2H, m, Ar-H)

Br

Br

NH2

N SH

NH2

N N

(T0)

51

Spectral Data of Triazolo Thiadiazolo (TT9 or T-9)

IR (KBr) CM-1:The IR data TT9 showed its characteristic absorption bands in the

following region. 3472, 3370 (NH2 groups), 3073 (Ar-C-H-Str) 1599

(C=N), 1572, 1532, 1508 (C=C ring Str.) 1532 and 1348 (NO2). The

peak at 1303 for C=S of parent triazole (T0) disappeared. 870 and 787

(substituted phenyl ring) 681 (C-S) 616 (C-Br).

1H NMR : ( ppm)

The NMR data of TT9 exhitited its peaks at δ 3.86 (2H, s, NH2) 7.128-

9.01 (7H, m, 5H of ar-H and 2H of NH2)

Br

Br

NH2

N

N

S

N

N NO2

H2N

52

13C NMR13C NMR data of TT9 gave the peaks signals for its 15 magnetically

different environment carbon atoms as indicated below.

Sr. No. Ppm values Assignment1 146.185 C12 166.289 C23 141.505 C34 139.856 C135 138.809 C86 134.185 C67 133.158 C48 130.406 C119 129.903 C910 123.892 C1211 122.696 C1412 115.97 C1513 111.167 C1014 107.458 C515 119.731 C7

Mass Spectrum

The Triazolo thiadiazole derivative (TT9, Mol Wt. 513) showed a

molecular ion peak at m/e 513. The daughter ion m/e 278 is the base

peak, remaning important daughter ions peaks are observed at M/z

304, 149, 105.

Br

Br

NH2

N

N

S

N

N NO2

H2N16

7

2

3

5

4

813

109

1211

1415

56

Spectral data of TT9 or T9

57

IR (KBr) CM-1:IR spectrum of TT10 showed its characteristic absorption bands in the

following region. 3472, 3246-(NH2), 3073 (Ar.C-H Str) 2947-2851 (C-H

Str of OCH2) asymmetric & symmetric) 1638 (NH bending), 1613

(C=N), 1592, 1558, 1507 (C=C ring Str) 1558 and 1341 (NO2) 1457

and 1341 (C-H deformation of oxymethyl group asym and sym) 1277 (-

O-), 869 (p-substituted benzene ring), 828 (1,4-disubstituted phenyl

ring) 705 (Carbon-Sulpher), 680 (C-Br). The peak at 1303 for C=S of

parent triazole To disappeared.

Mass Spectrum of TT10The triazolo thiadiazole (T10, Mol. Wt. 524) showed the peak for

parent ion at m/e 524. The other major daughter ion peaks are

observed at m/e 339, 218, 138, 97, 89 and 59. The base peak is

identified at m/e 59.

Br

Br

N S

NN

N C OCH2 NO2

60

Spectral Data of TT11 or T11

IR (KBr) CM-1:

The IR spectrum of TT11 showed its characteristic absorption bands

in the following region. 3473, 3371 (NH2), 32427 (NH), 3073 (aromatic

(C-H str), 2950 and 2843 (C-H str of CH3 asymmetic and symmetric),

1640 (N-H deformation), 1613 (C=N), 1575, 1507, 1457, ring

Stretching C=C 1433, 1366 (Substituted phenyl ring) 725 (1,2-

disubstituted phenyl ring) 680 (C-S) 608, (C-Br). The peak at 1303 for

C=S of parent triazole T0 disappeared.

1H NMR : ( ppm)

The 1H NMR spectrum for TT11 exhibited peaks as shown below.

δ 2.23 and 2.35 (2x3H, s, 2xCH3] 4.982-5.30 (NH2, bb, 2H of NH2)

7.061-8.41 (9H, m, 9H of Ar-H) 9.14 (1H, s, NH).

Br

Br

NH2

N

N

S

N

N

HN

CH3

CH3

61

13CNMR of TT11 (T11):13C NMR Spectrum of TT11 gave the signals for its 23 magnetically

different environmental carbon atoms as indicated below:

Sr. No. Ppm values Assignment1 165.333 and 164.994 C4 & C5

2 145.245 C3

3 145.245 C8 & C10

4 137.845 C11

5 134.938 C9

6 132.216 C17 & C18

7 129.465 C12 & C6

8 127.174 C19 & C20

9 121.766 C22 & C23

10 118.780 C9 & C16

11 116.226 C13

12 111.345 C14

13 106.506 C15 & C21

14 30.501 C2

15 25.491 C1

Mass SpectrumThe Triazolo thiadiazole derivative (TT11 molecular wt. 572) showed a

parent ion peak at m/e 572, other major daughter ions are observed

at m/e 294, 219, 131, 96.8, 78.7.

Br

Br

NH2

N

N

S

N

N

HN

CH3

CH31

2

46

3

57

8

18

19

21

23

2022

10

11

13

1512

14

1617

9

66

Spectral Data for TT 1 or T-1

IR (KBr) CM-1:IR spectrum for TT1 showed its characteristic absorption bands in the

following region. 3365.57 (Amino group), 3063 (Ar-C-H Str) 1595.98

(C=N) 1584.54, 1563, 1495 (C=C ring Str) 1371 (C-N). The peak at

1303 for C=S of parent triazole T0 disappeared. 874 (substituted

phenyl ring) 724 (mono substituted phenyl ring) 699.35 (C-S) 630 (C-

Br).

Spectral Data for TT2 or T2

IR (KBr) CM-1:

IR spectrum for triazole (TT2) exhibited its peaks at 3445.62 (broad

peak NH2 hydrogen bonded) 3069.38 (Ar-C-H str) 2950 (C-H

Streching of CH2 group) 1601.88 (C=N) 1555.16, 1518.84, 1474.35

(C=C ring Str) 1440.49 and 1345 (C-H bending of CH group) 1314, (C-

N) the peak at 1303 for (C=S) of parent triazole T0 disappeared, 780

Br

Br

NH2

N

N

S

N

N CH

Br

Br

NH2

N

N

S

N

N CH2

Cl

67

and 750 (substituted phenyl ring) 679.5 (C-S) 660.86 (C-Br) 544.78

(C-Cl).

70



following region. 3445.43 (Broad peak NH2 hydrogen bonded) 3070

(Ar-C-H Str.) 2950 and 2858.80 (C-H Streching of methyl and OCH2

groups), 1607.48 (C=N) 1586.35, 1555.63, 1508.40 (C=C ring Str),

1435 and 1340 (C-H bending of CH3 and OCH2), 1308 (C-N). The

peak at 1303 for C=S of parent triazole T0 disappeared, 1121.03 (C-O-

C) 875.38 (Substituted phenyl ring) 816.20 (1,4-disubstituted phenyl

ring), 659.17 (C-Br).


IR (KBr) CM-1:IR spectrum for TT3 exhibited its peaks at 3467.18 and 3353.96

(Amine), 3070 (Ar-C-H str), 1616 (C=N), 1593, 1567, 1524.63 and

1488 (C=C ring Str) 1402 (C-N). The peak at 1303 for C=S of parent

triazole T0 disappeared 875 (Substituted phenyl ring) 843

(disubstituted phenyl ring) 679.63 (Carbon-Sulpher), 655.97 (C-Br)

555.36 (C-Cl).

Br

Br

NH2

N

N

S

N

N CH2O CH3

Br

Br

NH2

N

N

S

N

N Cl

73


IR (KBr) CM-1:IR spectrum for TT4 showed its characteristic absorption bands in

the following region.3460.87 (Broad peak NH2, hydrogen bending),

3092 (Ar-C-H streching), 1624 (Carbon=Nitrogen) 1590, 1580, 1541

(Carbon=Carbon ring Streching) 1541.92 and 1344.64 (Nitro group)

1437.74 (Carbon-Nitrogen). The peak at 1303 for C=S of parent

triazole T0 disappeared 895.98 and 881.15 (substituted phenyl rings)

698.5 (C-S), 663.87 (C-Br).


IR (KBr) CM-1:IR spectrum for TT5 showed its characteristic absorption bands in the

following region.3445.42 (NH2) 3069.25 (Ar and heteroaryl C-H str),

1640 (N-H bending), 1622 (C=N) 1583, 1555.51, 1507.55

(Carbon=Carbon ring Streching) 1402 (Carbon-Nitrogen). The peak at

1303 for C=S of parent triazole T0 disappeared. 869.29 (substituted

phenyl ring) 698.74 (C-S) 678 (C-Br), 546.85 (C-Cl).

Br

Br

NH2

N

N

S

N

N

NO2

NO2

Br

Br

NH2

N

N

S

N

N

N

Cl

76



following region. 3420.85 (Broad peak NH2), 3067.69 (Ar-C-H str) (a)

2953.14, 2867.28 (b) 2928.81 and 2830 (Carbon-Hydrogen Streching

of CH3 and CH2 groups both asym and symm) 1596.86, 1555.84,

1508.35, 1462.80 (C=C ring Str.) 1314.23 (C-N). The peak at 1303 for

C=S of parent Triazole T0 disappeared, 870 (substituted phenyl ring).

848 (disubstituted phenyl ring), 691.78 (Carbon-Sulpher), 595.16 (C-

Br).



following region. 3471 and 3368 (NH2), 3071 (Ar-C-H str) 1601.90

(C=N, and C=C), 1362 (C-N). The peak at 1303 for C=S of parent

triazole T0 disappeared, 871 (substituted phenyl ring) 681.93 (C-S),

666 (C-Br).

Br

Br

NH2

N

N

S

N

N CH

CH3

CH2 CH

CH3

CH3

Br

Br

NH2

N

N

S

N

N

H2N Br

Br

79

Biological Activites

A. Antimicrobial activity

The antimicrobial evaluation performed at the department of medical

microbiology, Navodaya Medical College Hospital and Research

Centre, Raichur.

The invitro antibacterial activity36 of all 28 synthesized compounds

was carried out by well diffusion method (T1 to T28).

Anti microbial property of pharmaceutical substances were tested by

well diffusion technique by punching template on Muller Hinton agar.

Well size of 6 mm was made on agar with a holding capacity of 50 µl.

Three standard of bacterial strains viz.. E coli (ATCC NO8739),

P aeruginosa (ATCC NO 9027) and S aureus (ATCC NO 6538) were used

for this purpose. Inoculum size of these standard strains were

matched with 0.5 Mac Farlands comparator to get 1.5 x 105

organisms/ml. Lawn culture is made on Muller Hinton agar plate with

standard strain. Known quantity of each sample was dissolved in

1000 µl DMSO (Dimethyl sulphoxide) solvent in a sterile screw capped

Bijou bottle. 50 µl of solvent dissolved sample was charged in to the

wells of inoculated Muller Hinton Agar. Incubation of plates was done

for 12 hours at 37°C and later looked for zone of inhibition around the

well. The diameter of inhibitory zone was measured and recorded.

80

The result is presented in Table No.2. The inhibition zone was

recorded in millimeter and concentration of the test and standard

compounds were taken at 10 µg/ml.

Table No. 2

Sr.No. Sample No.

Zone of Inhibition (mm)

Staphylococcusaureus E.Coli Pseudomonas

aeruginosa

1 T1 38 22 00

2 T2 26 24 00

3 T3 22 26 00

4 T4 24 24 00

5 T5 22 26 00

6 T6 22 24 00

7 T7 00 22 00

8 T8 22 22 00

9 T9 24 24 00

10 T10 -- -- --

11 T11 00 26 00

12 T12 00 24 00

13 T13 00 26 00

14 T14 00 28 00

15 T15 00 30 00

16 T16 00 24 12

17 T17 -- -- --

18 T18 00 24 22

19 T19 00 22 24

81

20 T20 00 24 24

21 T21 00 20 20

22 T22 00 26 24

23 T23 00 28 28

24 T24 -- -- --

25 T25 -- -- --

26 T26 00 24 24

27 T27 00 20 22

28 T28 00 28 24

29 Ciprofloxacin 30 28 27

30 Gentamycin 34 30 35

31 Tobramycin 30 32 34

Results and Discussion

Among the Triazole derivatives screened for antibacterial activity, it

was observed that majority of the triazole deravitives exhibited

moderate to excellent activity against organisms E.coli in comparison

with standard drugs Ciprofloxacin, Gentamycin and Tobramycin. We

found that the compounds like T3, T5, T11, T13, T14, T15, T22, T23

and T28 exhibited equipotent activity with the standard Ciprofloxacin

and rest of the deravitives exhibited slightly better activity against all

the bacteria. The compounds T10, T7, T24 and T25 did not show any

activity against all the pathogenic organisms. It was noticed that T1

exhibited excellent antibacterial property with respect to

Staphylococcus aureus. Zone of inhibition was found to be more than

82

the standard drugs and rest of the compounds like T2 to T6, T8, T9

showed moderate activity and remaining derivatives showed no

activity. Surprisingly the analogs T1 to T6, T8, T9 which showed

activity against Staphylococcus aureus and E.coli did not show

antibacterial activity against Pseudomonas aeruginosa when compared

with the standard drugs. The compounds T11, T12, T13, T14, T15

showed activity only against E.coli and no activity was observed with

Staphylococcus aureus and Pseudomonas aeruginosa. But the

compounds T16, T18 to T23, T26 to T28 showed activity against

Pseudomonas aeruginosa and E.coli and no activity against

Staphylococcus aureus. The compound T23 showed almost the same

activity with the reference drug Ciprofloxacin.

The evaluation indicates that the derivatives are much active

against organism E.coli.

The general observation on structure activity relationship revealed

that the triazole thiadiazole derivatives with the substituents at 6th

position like 2-amino-3,4-dibromo phenyl, 4-isobutyl phenylethyl-2-

chlorophenyl, 2,4-dichlorophenylmethyl, 4-chlorophenyl methyl, 2-

chlorophoxymethyl, phoxymethyl, 4-bromo phenoxy methyl and a bis

derivative bearing –CHOH showed much significant activity –CHOH in

this series of compounds. It is quite clear that compounds having

electron withdrawl at 6th position, contribute much in enhancing the

antibacterial activity. Thus the study revealed no specific correlation

between activity and structure for antibacterial activity, it can be

83

observed from the study that in general electron attracting groups

substituted at 6th position of triazolo thiadiazole ring play a vital role

in the antibacterial property of the compounds, Hence if this series of

compounds are suitably modified they may yield much better

compounds with enhanced activity. After carrying out the toxicity

studies the better ones can be recommended for antibacterial

infections as antibacterial drugs in several forms like powder,

ointment, gel, lotion etc. for topical use. Therefore the detailed

investigation in this regard is quite fruitful.

84

Anti Fungal Activity36

The invitro antifungal property of the Triazoles synthesized was

performed at Maratha Madals Nathaji Rao Dental college and

Research Centre, Belgaum, recognized by Rajiv Gandhi University of

Health Sciences, Bangalore as nodal centre for carrying out biological

evaluation.

Preparation of Sub-Culture

The subculture was prepared by using standard reported method &

kept in incubater for 48 hours at room temperature.

Preperation of test compounds :

The triazole thiadiazole compounds solution were prepared in DMSO

So to get 75, 50, 25 & 10 µg/ml solutions and used for testing against

Aspergillus Fumigatus. The testing procedure adopted was similar to

that of antibacterial testing (Diffusion Method). Dimethyl sulphoxide

was used as control.

The Triazolo thiadiazole series of compounds (T0, T1 to T28) were

screened for antifungal activity by agar diffusion method. The selected

few compounds having significant activity were further screened for

their antifungal activity following the MIC (Minimum inhibition

concentration) procedure against the organism. Aspergillus Fumigatus

ATCC No. 13073. Flucanazole as reference. The data displayed in

Table No. 3

85

Table 3: Antifungal Activity of Triazoles

Sr. No. CompoundsConcentration (µg/ml)

75 50 25 101 T-1 740mm 740mm 740mm 38mm

2 T-2 38mm 36mm 32mm 30mm

3 T-3 40mm 40mm 40mm 35mm

4 T-4 34mm 34mm 32mm 28mm

5 T-5 28mm 26mm 26mm 23mm

6 T-6 36mm 34mm 34mm 32mm

7 T-7 32mm 26mm 26mm 18mm

8 T-8 36mm 34mm 34mm 34mm

9 T-9 34mm 28mm 28mm 23mm

10 T-10 36mm 33mm 33mm 30mm

11 T-11 34mm 32mm 32mm 28mm

12 T-12 30mm 30mm 30mm 28mm

13 T-13 34mm 32mm 32mm 30mm

14 T-14 34mm 30mm 30mm 26mm

15 T-15 740mm 740mm 38mm 34mm

16 T-16 38mm 36mm 36mm 34mm

18 T-18 24mm 23mm 23mm 20mm

19 T-19 34mm 32mm 32mm 30mm

20 T-20 24mm 18mm 15mm 11mm

21 T-21 22mm 19mm 14mm 10mm

22 T-22 28mm 25mm 21mm 18mm

23 T-23 26mm 22mm 20mm 17mm

24 T-24 28mm 23mm 21mm 19mm

25 T-25 35mm 29mm 24mm 20mm

26 T-26 32mm 28mm 26mm 20mm

27 T-27 24mm 20mm 15mm 11mm

28 T-28 26mm 24mm 21mm 20mm

29 TO 30mm 30mm 25mm 22mm

86

Minimum Inhibition concentration (MIC) Method:

1. 9 dilutions of each drug have to be done with BHI for MIC.

2. In the initial tube 20microliter of drug was added into the

380microliter of BHI broth.

3. For dilutions 200microliter of BHI broth was added into the next

9 tubes separately.

4. Then from the initial tube 200microliter was transferred to the

first tube containing 200microliter of BHI broth. This was

considered as 10-1 dilution.

5. From 10-1 diluted tube 200microliter was transferred to second

tube to make 10-2 dilution.

6. The serial dilution was repeated up to 10-9 dilution for each

drug.

7. From the maintained stock cultures of required organisms,

5microliter was taken and added into 2ml of BHI (brain heart

infusion) broth.

8. In each serially diluted tube 200microliter of above culture

suspension was added.

9. The tubes were incubated for 24 hours and observed for

turbidity

87

Table 4: Antifungal Activity by MIC Method

Sr.no. Compounds

Concentration (µg/ml)

500 250 125 62.5 31.25 16 8 4 2 1

1 T-1 S S S S S S S R R R

2 T-2 S S S S S S S S S R


4 T-4 S S S S S S R R R R





9 T-12 S S S S S R R R R R

10 T-13 S S S S S S S S R R

11 T-14 S S S S S S S S R R



14 TO S S S S S S S R R R

88

Results and Discussion:

Parent triazole (T0) and its derivatives 3-(2’-amino-3’,5’-

dibromophenyl)-6-substituted-triazolo thiadiazoles (T1-T28) have been

evaluated for their antifungal activity following standard procedure as

described above. The screening results indicated that the triazolo

thiadiazole derivatives derived from the parent triazole (T0) possess

excellent antifungal property when compared with the choosen

reference . The deravitivies of the series except T20, T21, T27 and T7

posses significant activity. Some compounds exhibited equipotent and

others superior activity, even at low conc of 10 µg/ml. Further it is

interesting to observe that active ones have better activity than the

parent Triazole (T0).

The presence of NCS link present in the fused system like Triazolo

thiadiazole, generally enhances the antifungal activity of the

compounds. Hence the condensed derivatives like triazolo thiadiazoles

have exhibited better activity than the parent compound 5-mercapto

triazole (T0). The observation on the result revealed that the

substitutents on the 6th position of triazolo thiadiazole system have no

specific influence on the activity. It is evident by the fact that both

electron withdrawing and releasing groups on the aromatic ring at 6th

position of triazolo thiadiazole system have exhibited enhanced

antifungal activity. It is also clear from the study of the antifungal

activity that the triazolo thiadiazole derivatives have better activity

89

incomparison with the bis derivatives obtained by the condensation

with aliphatic dicarboxylic acids.

The MIC study on this series of compounds revealed that the

antifungal activity of triazolo thiadiazole derivatives have better

activity than the parent triazole T0 even at low concentrations.

Thus the antifungal screening results support the fact that molecular

modification of mercapto triazole to 3,6-disubstituted triazolo

thiadiazole yields comparatively enhanced activity. This is in

accordance with the fact that the condensation of 2 different

heterocyclic moieties to get a fused heterocyclic system enhances the

biological activity of the resulted compounds.

90

Anti Tubercular Activity

The in-vitro anti-tubercular activity of the triazolo thiadiazoles was

carried out at Maratha Mandals Nathaji Rao Dental college and

Research Centre, Belgaum recognized by Rajiv Gandhi University of


evaluation.

The evaluation of antitubercular activity was performed using the

standard strain of mycobacterium tuberculosis H37 RV and

middlebrook 7H-9 broth referring the standard procedure.

The growth of M.tuberclosis strain (100000 organisms/ml) was

measured after a period of 3 weeks

Antibiotic standards used include

Streptomycin 7.5 µg/ml

Pyrazinamide 7.5 µg/ml

91

Table 5: Anti-tuberculosis Activity of Triazoles

Sr.No. Compounds

Concentration

5µg/ml 10µg/ml 15µg/ml

01 T-0 (ATB35) R S S

02 T-11 (ATB36) R S S

03 T-18 (ATB37) R S S

04 T-8 (ATB38) R R R

05 T-22 (ATB39) R S S

06 T-24 (ATB40) R R R

07 T-14 (ATB41) R R R

08 T-21 (ATB42) R S S

09 T-13 (ATB43) R R R

10 T-5 (ATB44) S S S

S- Sensitive

R- Resistance


The parent s-triazole (T0) and some of its triazolo thiadiazole

derivatives [3-[2’-amino-3’,5’-dibromophenyl)-6-substituted triazolo

thiadiazoles] like T5, T8, T10, T13, T14, T21, T22 and T24 were screened

against Mycobacterium H37, at cons of 25,10 & 5 µg/ml.

The derivatives T5 (ATB44) showed anti-tubercular activity at all the 3

concentrations 5,10 & 25 µg/ml. the compound T0, T10, T18, T21 and

T22 showed activity at 10 and 25 µg/ml and found to be inactive at 5

92

µg/ml. However the derivatives T8, T13, T14 and T24 were inactive to the

organism at all the 3 concentrations.

Among the compounds screened for anti-tubercular activity T5 which

has the pyridyl substituent at 6th position of triazolo thiadiazole ring

system has shown activity at 5, 10 and 25 µg/ml. The compound T5

possesses comparable activity with the standard drugs Streptomycin

and Pyrazinamide. However the parent triazole T0 and some of its

active derivatives T10, T18, T21 and T22 showed the activity only at 10

and 25 µg/ml concentrations. Perhaps the heterocyclic moiety as

substituent at 6th position of triazolo thiadiazole ring system of T5 may

be responsible for its enhanced antiburcular property.

93

Anthelmintic property of representative Triazolo thiadiazoles

Anthelmintic activity of representative compounds of triazolo

thiadiazole series was performed in the dept of pharmacology, NET

Pharmacy College, Raichur. The required earthworms of almost equal

size (8cm + 1cm) were procured from University of Agricultural

Sciences, Raichur.

Procedure

Albendazole solution of 0.1, 0.2, 0.5 % in DMF was prepared using

normal saline and taken as reference and 25 ml each of the solution

was poured in to different pertidishes. Similarly 0.1, 0.2, 0.5 %

solutions of the synthesized triazolo thiadiazole deravitives were made

in ethyl alcohol and DMF, normal saline solution was used as control ,

and poured into petridishes. 6 earthworms of equal size for each

petridish at room temperature are used for the study. The time

required for paralysis and death of earthworms are noted to calculate

the Anthelmintic activity.

94

Table 6: Data Showing Anthelmintic Activity of triazoleThiadiazoles

Sr.No.

CompoundCode % Con.

Time in min

Paralysis Death Average

01. Alcohol Alive

02. DMF Alive

03. Albendazole(Std)

0.1 09 12

8.6 min0.2 07 08

0.5 06 06

04. T1

0.1 05 12

100.2 05 10

0.5 07 08

05. T30.1 07 15

14.60.2 07 150.5 05 14

06. T8

0.1 06 11

9.30.2 04 09

0.5 03 08

07. T90.1 06 13

110.2 06 110.5 04 09

08. T130.1 5 12

09.030.2 4 080.5 2 08

09. T21 0.1 7 19

180.2 7 18

0.5 4 17

10. T24

0.1 07 10

8.60.2 07 09

0.5 05 07

95

Results & Discussions

The aim of our work on the study of anthelmintic property of Triazolo

thiadiazole derivatives is mainly to know whether such analouges

exhibit potent activity or not. Few representative compounds like

triazolo thiadiazole series were screened for their anthelmintic activity.

Earth warms of almost equal size were used for the study.

Albandazole (concentration 0.1%, 0.2% and 0.5%) was used as a

reference drug for comparison.

Among the compounds T1, T3, T8, T9, T13, T21 and T24 screened for

anthelmintic activity, T1, T8, T9 and T24 showed significant activity

and remaining compounds exhibited weak to moderate activity. it can

be concluded that a few generalizations can be derived from the

comparative study on the evaluation of active compounds. The

aryl/aryloxy substitutent at the 6th position of triazolo thiadiazole

fused ring system plays vital role in enhancing the potency. The

observed enhancement in the property is mainly due to the presence

of electron withdrawing groups like Br, F, NO2 etc in the aryl/aryloxy

ring at 6th position of triazolo thiadiazole fused ring system.

96

Anticancer Activity of Triazoles by MTT Assay Procedure

The anticaner activity37 of representative compounds of triazole

thiadiazoles series was carried out in the department of Microbiology

at Maratha Mandals, Nathaji Rao Dental college & Research Centre,

Belgaum recognized by Rajiv Gandhi Univeristy of Health Sciences as

Nodal Research Centre for biological activities.

The literature survey reveals that triazole derivatives are active against

many tumer cell lines. The present work describes the preperation of

new triazolo thiadiazole derivatives which posses cytotoxic activity.

The anticancer activity was carried out using the following cell lines in

the present work.

1) Hela cell line (Human cervix)

2) A-549 Human:lungs: carcinoma

3) MDA-MB Human; Adeno carcinoma; mammary gland

4) HT-29-Human: colorectal adeno carcinoma.

During the present work triazolo thiadizole derivatives, T3, T6, T8, T9,

T13, T14, T18, T22, T24 and T27 were screened for anti proliferative

activity following microculture tetrazolium assay (MTT Assay).

Procedure

1) MTT Solution Preparation: 10 mg in 10 ml of Hank’s balanced

solution.

2) Cell culture: A) The cell lines were maintained in 96 wells

microtiterplate. Containing 5% of mixture of

Gentamycin, Pencillin (100 units/ml) and

97

Streptomycin (100 µg/ml) in presence of 5%

CO2 at 37°C for 3-4 days.

B) After 3-4 days remove the supernatumt

and replace MEM media with Hank’s

balanced solution supplemted with

Gentamycin, Pencillin and Streptomycin,

incubate overnight.

Cytotoxicity assay

Invitro growth inhibition effect of ten representative Triazolo

thiadiazoles derivatives was assessed by Colorometric or

spectrophotometric determination.

98

Table 7: MTT Assay Results (IC50 Values) of Triazoles &Thiadiazoles

Sr.No

CompoundCode

Hela Cell Line(HumanCervix) A-549-Human Lung Carcinoma

10 μl 20μl 30μl Average 10μl 20μl 30μl Average

1 T3(ATC1) 28.12 25.1 24.09 25.77 27.62 24.65 23.16 25.14

2 T6(ATC3) 84.18 65.41 55.93 68.41 118.6 128.26 128.38 125.08

3 T8(ATC10) 116.8 117.71 126.23 120.24 117.6 118.82 127.36 121.26

4 T9(ATC2) 31.71 29.09 28.69 29.83 32.12 28.96 27.99 29.69

5 T13ATC9) 39.83 32.76 32.12 34.9 74.9 70.66 38.85 61.47

6 T14ATC7) 69.45 53.55 45.15 56.05 72.28 38.36 37.36 49.33

7 T18ATC8) 25.06 24.89 23.24 24.39 73.69 70.66 36.86 60.2

8 T22ATC5) 35.31 34.18 33.05 34.18 74.98 36.12 34.58 48.56

9 T24ATC6) 75.78 41.48 40.63 52.63 35.98 34.26 32.83 34.35

10 T27ATC4) 85.59 41.2 36.76 54.51 36.46 31.84 29.88 32.72

Table 8

Sr.No

CompoundCode

MDA-MB Human Adino CarcinomaMammary Gland

HT-29 Human Colorectal AdenoCarcinoma

10μl 20μl 30μl Average 10μl 20μl 30μl Average

1 T3(ATC1) 96.83 38.38 36.44 57.21 28.24 27.48 26.86 27.522 T6(ATC3) 85.92 32.42 31.68 50.006 98.08 92.48 91.58 94.043 T8(ATC10) 85.69 84.76 84.04 84.83 108.62 104.84 103.84 105.764 T9(ATC2) 75.88 74.14 73.96 74.66 104.44 102.86 102.18 103.165 T13ATC9) 116.12 108.94 36.64 87.23 98.68 36.44 31.86 55.666 T14ATC7) 112.54 36.46 35.84 61.61 88.86 40.28 36.82 55.327 T18ATC8) 108.62 40.28 38.64 49.08 102.42 39.12 36.88 59.478 T22ATC5) 98.64 34.44 32.64 62.51 28.82 26.86 24.26 26.649 T24ATC6) 102.58 36.48 34.88 57.98 32.62 31.56 30.98 31.7210 T27ATC4) 100.08 96.74 29.68 75.5 96.62 92.86 38.54 76.006

99

Figure 1: Graph Showing IC 50 Values of Triazolo thiadiazole compounds

I C 5 0 v a lu e s a g a in s tH e la C e l l l in e s ( H u m a n C e r v ix )

) 1(AT

C3T

) 3(AT

C6T

) 10(AT

C8T

) 2(AT

C9T

) 9(AT

C13T

) 7(AT

C14T

) 8(AT

C18T

) 5(AT

C22T

) 6(AT

C24T

) 4(AT

C27T

0

5 0

1 0 0

1 5 0

T r ia z o lo t h ia d ia z o le c o m p o u n d s

IC50

valu

es

I C 5 0 v a lu e s a g a in s tA -5 4 9 -H u m a n L u n g C a rcim o m a

) 1(AT

C3T

) 3(AT

C6T

)10

(ATC

8T

) 2(AT

C9T

) 9(AT

C13T

) 7(AT

C14T

) 8(AT

C18T

) 5(AT

C22T

) 6(AT

C24T

) 4(AT

C27T

0

50

100

150

Tria zo lo th ia dia zo le co m po u n ds

IC50

valu

es

I C 5 0 v a lu e s a g a in s tM D A -M B H u m a nA de n o C a rcin o m a m a m m a ry G la n d

) 1(AT

C3T

) 3(AT

C6T

) 10(AT

C8T

) 2(AT

C9T

) 9(AT

C13T

) 7(AT

C14T

) 8(AT

C18T

) 5(AT

C22T

) 6(AT

C24T

) 4(AT

C27T

0

20

40

60

80

100

Tria zo lo th ia dia zo le co m po u n ds

IC50

valu

es

IC 50 valu e s again s t Hu m anC olore ctal A de n o C arcin om a

) 1(A

TC3T

) 3(A

TC6T

)10

(ATC

8T

) 2(A

TC9T

) 9(A

TC13T

) 7(A

TC14T

) 8(A

TC18T

) 5(A

TC22T

) 6(A

TC24T

) 4(A

TC27T

0

50

100

150

Triazolo th iadiazole com pou n ds

IC50

valu

es

100


The compounds screened for anti-cancer activity exhibited

encouraging cytotoxicity activity against all the four cell lines namely

(1) Hela cell line (Human cervix)

(2) A-549 Human:lungs: carcinoma

(3) MDA-MB Human; Adeno carcinoma; mammary gland

(4) HT-29-Human: colorectal adeno carcinoma

Except the compound T8 all other compounds exhibited significant

anti-cancer activity against Hela cell lines (Human cervix). Majority of

the active compounds namely T9, T13, T14, T18, T24 contain an electron

withdrawing group on the aromatic ring substituted at 6th position of

triazolothiadiazole ring system.

The compounds T3, T9, T13, T14, T18, T22, T24 and T27 exhibited

significant activity against A-549 Human lung carcinoma. Except T6

and T18 all the above triazolo thiadiazoles screened for anti-cancer

activity showed moderate activity against (MDC-MB Human

adinocarcinoma) (Mammary gland).

The compounds T3, T22 and T24 exhibited significant activity, while the

compounds T13, T14, T18 & T27 exhibited moderate activity and T6, T8,

T9 were found to be inactive against HT-29, Human Colorectal Adeno

carcinoma.

The detailed analysis of the results suggests that compounds T3, T9,

T13, T18 & T22 posses potent anti cancer activity against Hela cell lines

101

(Human cervix). The Triazolo thiadiazole derivatives, T3, T9, T24 & T27

are potent against A-549 Human lung carcinoma. The compounds T3,

T22, T24 showed potent activity against HT-29 Human Colorectal Adeno

carcinoma. The results indicated that molecular modification involving

condensation of 1,2,4-Triazole ring with 1,3,4-thiadiazole ring resulted

in producing the compounds with potent anticancer activity.

102

B) 3,4-Disubstituted Traizoles and their derivatives bearing

INH and Pyrazinamide Moieties.

Scheme:1

N CONH.NH2alc KOH, CS2

N CONH NH C

S

S-K+

Ar CONHNH2N

N

NN

S-K+

NH C Ar

O

dil HCl

NN

NN

SH

NH C Ar

O

(TI1 - TI11)

103

Scheme: 2

NN

NN

SH

NH C Ar

O

N CONH.NH2

NN

NN

NH C Ar

O

NHNH C

O

N

TIT1 - TIT11

Scheme: 3

NN

NN

SH

NH C Ar

O

NN

NN

NH C Ar

O

NH C

O

TIP1 - TIP11

N

N CONH2

N

N

104

Synthesis of potassium dithiocarbazinate from INH

The mixture of KOH (8.5 g, 0.15 mol) in dry ethyl alcohol (125 ml),

CS2 (11.2 g, 0.15 mol) and INH (0.1 mol) was agitated for 16 hrs. To

the resulting solution dry ether (250 ml) was added to the resultant

solution and precipitated product was filtered . It was washed with

ether and dried. The product thus obtained in quantitative yield was

used directly.

Synthesis of 3-pyridyl-4-(N-substituted phenyl carboxamido)-5-

mercapto-1,2,4-triazole derivatives (TI1 – TI11).

Potassium dithiocarbazinate obtained above and aryl/aryloxy acid

hydrazides were heated in equimolar proportion in at 170 – 1800C for

5-6 hrs, when profuse evolution of Hydrogen gas was observed. The

cooled mixture was diluted with water & acidified with HCL to obtain

the required triazole. Other compounds were obtained by similar

method. Purity is checked by TLC using mobile phase CHCl3 :

CH3COOCH5 (1:1 v/v) and silica gel G plates. The characterization

data of the compounds is presented in table No 9.

105

Synthesis of 3-pyridyl-4(N-substituted phenyl carboxamido) 5-

substituted- 1,2,4–Triazoles (TIT1–TIT11 and TIP1–TIP11)

The mixture of 3-pyridyl-4(N-substituted phenyl carboxamido)-5-

mercapto 1,2,4-triazole and INH / pyrazinamide was heated in

equimolar proportion in an oil bath to get a homogenous mixture.

Heating was continued at the same temp (1450–1500C) for another 1

hr. The product obtained was purified by crystallization from ethyl

alcohol same method was followed for other compounds and their

purity was checked by TLC. The characterization data of the

compounds is presented in table No10 & 11.

106

Table No 9 : Characterization data of 3-Pyridyl (aryl/aryloxy

carboxamido)-5-mercapto-1,2,4-triazoles

Sr.No Code Ar mol. formula m.p.

(0C)Yield(%)

01 TI14–Nitro phenyl C14H10O3N6S 218-

220 74

02 TI22–Methoxyphenoxymethyl

C16H15O3N5S 202 80

03 TI3 4–Chloro phenyl C14H10ON5SCl 276 82

04 TI42–Chlorophenoxymethyl

C15H12O2N5SCl 246 72

05 TI54–Chlorophenoxymethyl

C15H12O2N5SCl 128 78

06 TI6 Benzyl C15H13ON5S 222 76

07 TI72–Naphthyloxymethyl

C19H15O2N5S 236 82

08 TI84–Bromophenoxymethyl

C15H12O2N5SBr2 214 70

09 TI9 2–Amino phenyl C14H12ON6S 220 76

10 TI101–Naphthyloxymethyl

C19H12O2N5S 240 72

11 TI11 Phenyl C14H11ON5S 282 78

NN

NN

SH

NH C Ar

O

107

Table No. 10: Characterization data of Hydrazino – Triazolo

Derivatives (bearing INH moiety)


(0C)Yield(%)

01 TIT1 4–Nitro phenyl C20H15O4N9 110 80

02 TIT22–Methoxy phenoxymethyl

C22H20O4N8 138 82

03 TIT3 4–Chloro phenyl C20H15O2N8Cl 118 74

04 TIT42–Chloro phenoxymethyl

C21H17O3N8Cl 210 80

05 TIT54–Chloro phenoxymethyl

C21H17O3N8Cl 120 80

06 TIT6 Benzyl C21H20O2N8 180 8207 TIT7 2–Naphthyloxy methyl C25H20O3N8 154 78

08 TIT84–Bromo phenoxymethyl

C21H17O3N8Br 140 74

09 TIT9 2–Amino phenyl C20H17O2N9 132 7510 TIT10 1–Naphthyloxy methyl C25H20O3N8 148 7911 TIT11 Phenyl C20H16O2N8 206 80

NN

NN

NH C Ar

O

NHNH C

O

N

108

Table No.11: Characterization data of Triazole derivatives

(bearing Pyrazinamide moiety)


(0C)Yield(%)

01 TIP1 4-Nitrophenyl C19H13O4N9 140 74

02 TIP22–Methoxy phenoxymethyl

C21H18O4N8 160 76

03 TIP3 4–Chlorophenyl C19H13O2N8Cl 208 69

04 TIP42–Chlorophenoxymethyl

C20H15O3N8Cl 120 76

05 TIP54–Chlorophenoxymethyl

C20H15O3N8Cl 180 74

06 TIP6 4–Benzyl C20H16O2N8 180 8007 TIP7 2–Naphthyloxy methyl C24H18O3N8 140 75

08 TIP82–Bromo phenoxymethyl

C20H15O3N8Br 220 70

09 TIP9 2–Amino phenyl C19H15O2N9 168 7010 TIP10 1–Naphthyloxy methyl C24H18O3N8 126 7111 TIP11 Phenyl C19H14O2N8 220 76

NN

NN

NH C Ar

O

NHCO

N

N

109

3,4-Disubstituted Triazoles and their Derivatives BearingINH and Pyrazinamide Moieties.HT3 or TI3

IR (KBr) CM-1:

IR spectrum of the mercapto triazole (HT3) showed its characteristic

absorption bands in the following region. 3152 (NH), 1690 (C=0), 1594

(C=N), 1594, 1481 (C=C ring str), 1410 (C-N), 1264 (C=S), 834 (1,4–

disubstituted phenyl ring), 684 (C-S), 616.82 (C-Cl)

1H NMR : ( ppm)

The 1HNMR spectrum of the mercapto triazole (HT3) exhibited the

peaks at

δ 13.76 (1H,s,SH), 10.90 (1H,s,CONH), 7.55-8.84 (84,m,4H of Ar-H

and 4H of pyridyl)

The broad peak at 3.39 is due to water.

NN

NN

NH

SH

C

O

Cl

112

Spectra Data for HT1 or TI1

IR (KBr) CM-1:IR spectrum of the mercapto triazole (HT1) showed its characteristic

absorption bands in the following region. 3250-3100 (a broad peak

NH), 1680 (C=0), 1603.88 (C=N), 1603, 1517 (C=C ring str), 1344

(C=N), 1517 & 1345 (NO2), 1270 (C=S), 829 (1,4-disubstituted phenyl

ring), 697.80 (C-S), 616.80 (C-Cl).

Spectral Data for HT5 or TI5

IR (KBr) CM-1:IR spectrum of the mercapto triazole HT5 exhibited its peak at 3174.94

(NH), 3050 (aromatic C-H str), 2932 (C-H str of OCH2 ---), 1661 (C=0),

1594.64, 1491 (C=C ring str), 1408 (C-N), 1491 & 1339 (C-H bending

of OCH2 ---), 1281 (C=S), 1094 (C-O-C), 822 (1,4-disubstituted phenyl

ring), 708 (C-S), 610 (C-Cl).

NN

NN

NH

SH

C

O

NO2

NN

NN

NH

SH

C CH2O

O

Cl

115

Spectral Data for HT6 or TI6

IR (KBr) CM-1:IR spectrum of HT6 showed its characteristic absorption bands in the

following region. 3250-3135 (NH), 3035 (aromatic C-H str), 1698

(C=0), 1587 (C=N), 1587, 1547, 1488 (C=C ring str), 1411 (C-N), 1273

(C=S), 832 (p-substituted benzene ring), 699 (C-S), 616 (C-Cl).

Spectral Data for HTI3 or TIT3

IR (KBr) CM-1:The IR spectrum of HTI3 obtained by the replacement of –SH group by

INH moiety showed its characteristic absorption bands in the following

region. 3153 (NH), 1691.53 (C=0), 1594.51 (C=N), 1594 & 1481.52

(C=C ring str), 1353 (C-N), 824 (1,4-disubstituted phenyl ring), 616 (C-

Cl).

1H NMR : ( ppm)

The 1HNMR spectrum for HTI3 exhibited the peaks as below

δ 12.27 (1H, s, NHCO of phenyl), 10.74 (1H, s, NHCO of pyridyl) 7.60-

8.86 (12H,m,8H of pyridyl & 4h of Ar-H), 3.34 (1H,s,NH of NH-NH-

CO).

NN

NN

NH

SH

NN

NN

NH C

O

Cl

NH.NH C

O

N

119


IR (KBr) CM-1:The IR spectrum of HTI5 showed its characteristic absorption bands in

the following region. 3158 (NH), 2932.11 & 2840 (C-H str of OCH2),

1691 & 1661 (C=0), 1599 (C=N), 1580, 1491 (C=C ring str), 1416 &

1342 (C-H bending of OCH2), 822 (p-substituted benzyne ring) 588 (C-

Cl).

1H NMR : ( ppm)

The 1HNMR spectrum for the mercapto triazole derivative HTI5

exhibited the peaks at

δ 4.66 (1H, s, NH) 5.15 (2H,s,OCH2), 7.02 to 7.86 (12H,m,Ar-H), 8.86

(1H, s, NH of CONH), 10.24 (H,s,NH of CONH pyridyl).

NN

NN

NH C CH2O

O

NH.NH C

O

N

Cl

120

13C NMR Spectra of HTI5 or TIT5

13C NMR spectrum of HTI5 gave the signal for its 21 magnetically

different environmental carbon atoms as indicated.

C8-168.903C16-167.597C2-165.068C1-157.381C5&C6-152.258 & 151.469C19&C20-149.06 and 148.706C13-138.854C4,C7,C18,C21-130.109C3-126.131C10&C17-122.313 and 121.702C11,C15 and C12,C14-117.745C9-60.441

Mass SpectrumThe mercapto triazole derivative HTI5 has the mol.wt 464. The

molecular ion peak of HTI5 could not be seen as the spectra is taken

in the range 50 to 4000. However important daughter ion peaks are

obtained at m/e 365, 257 113 and 74. The peak at m/e 257 is the

base peak.

NN

NN

NH C CH2O

O

NH.NH C

O

N

Cl

1 23

45

6 7 8 9 1011

12

13

14

15

16 17

18 19

2021

125


IR (KBr) CM-1:IR spectrum of the mercapto triazole derivative HTI6 showed its

characteristic absorption bands in the following region.

3422(NH), 3034(Ar-CH str), 2861(C-H str of CH2), 1699(C=0),

1587(C=N), 1564.96, 1519.62 & 1488 (C=C ring str), 1488-1359 (C-H

bending of CH2), 832-869(substituted phenyl and heterocyclic ring)

NN

NN

NH C CH2

O

NH.NH C

O

N

127

Spectral Data for HTP1 or TIP1

IR (KBr) CM-1:The IR spectrum of the mercapto triazole HTP1 showed its

characteristic absorption bands in the following region..

3316 (NH), 3086 (Ar-CH Str), 1690-1635 (C=O) of CONH groups), 1603

(C=N), 1521, 1491 (C=C ring str.), 1521 and 1345 (NO2), 777 and 804

(substituted phenyl ring heterocyclic rings)

1H NMR : ( ppm)

The 1H NMR spectrum of the mercapto triazole HTP1 exhibited the

peaks at

δ 6.592-9.19 (13,m, 2H of two CONH groups 4H of Ar-H, and 4H of

pyridyl and 3H of pyrazine), (The broad peak at 3.41 is due to water)

Mass spectrumThe mercapto triazole derivative HTP1 has mol wt 431

The parent ion peak at m/e 431 corresponds to molecular wt of the

compound.

The peak at m/e 311.1 is the base peak prominent.

The other daughter ion peaks are observed at m/e 341, 266, 166, 122.

NN

NN

NH C

O

NHCON

N

NO2

128

NN

NN

NH C

O

N+-OH

NO2

m/e 341

NN

NN

N+

N-OH

NO2

Base Peak m/e 311.1

NN+

NN

NH

m/e 266

C

O

N

N

NN

NN

N+-OH

m/e 177.1

H

CO

m/e 166

O2N NH2

NO2

m/e 122

132


IR (KBr) CM-1:IR spectrum of the mercapto triazole HTP3 showed its characteristic

absorption bands in the following region.

3434 and 3159 (NH), 3084 (Ar.CH-Str), 1689 (C=N), 1594, 1517, 1479

(C=C ring str) 1264 (C-O bend), 1094 (C-O-C), 833 (1.4-disubstituted

phenyl ring), 653 (C-Cl)

1H NMR : ( ppm)

The 1H NMR spectrum of the mercapto triazole HTP3 exhibited its

peaks at δ 7.61 to 9.21 (11 H,m, 3H of pyrazine 4H of pyridyl and 4H

of Ar-H), 10.66 (1H,s, NH of CONH pyrazine), 12.31 (1H,S, NH of

CONH p-chloro phenyl)

NN

NN

NH C

O

NH-CON

N

Cl

133

13C NMR Spectra of HTP3 or TIP3

13C NMR spectrum of HTP3 gave the signal for its 19 magnetically


C8, C15, 169.247 and 168.13

C1, C2-165.41 and 164.97

C5-150.97

C6-148.76

C16,C19-147.7 and 145.46

C17, C18-143.96

C12-132.18

C11,C13-130 and 129.58

C10,C14-127.784

C3,C4,C7,C9-121.71, 121.09, 120.45, 119.81

NN

NN

NH C

O

NH C

O

N

Cl

1 23

45

6 7 8 910

11

12

13

14

16

17 18

19

15

134

Mass spectrum of HTP3

The mercapto triazole derivative HTP3 (mol.wt 420)

The molecular ion peak of HTP3 was observed at m/e 420. The base

peak was observed at m/e 177, other daughter ion peaks were

observed at m/e 363, 210, 177.

NN

NN

N+ C CN

O

NH C

O

Cl

M/e 363

NN

NN

N+ OH

NH C

O

Cl

M/e 330

NNH

NN

N+ OH

Base peak M/e 177

139


IR (KBr) CM-1:The IR spectrum of the mercapto triazole HTP5 showed its

characteristic absorption bands in the following region.3446 (NH),

3060 (Ar-CH str), 2932 and 2850 (C-H str of OCH2 asymmetric and

symmetric), 1691 (C=O), 1598 (C=N), 1581,1491(C=C ring str), 1491,

1368 (C-H bending of –OCH3 asymmetric and symmetric), 1076 (C-O-

C), 822 (1,4-disubstituted phenyl ring), 754 (Mono substituted

heterocyclic rings), 588 (C-Cl)

1H NMR : ( ppm)

The 1H NMR spectrum of the mercapto triazole HTP5 exhibited the

peaks as below

5.16 (2H,s,-OCH2), 6.86, 8.86 (11H,m, 4H of pyridyl 3H of pyrazine

and 4H of Ar-H) 9.58 (1H,s,NH of CONH pyrazine), 10.26(1H,s,NH of

CONH of p-chloro phenoxy methyl)

NN

NN

HN C CH2O

O

Cl

N-CHOHN

N

140

13C NMR spectrum of HTP5

13C NMR spectrum of HTP5 gave the signal for its 20 magnetically


C8-168.38

C16-167.85

C13-167.099

C1 & C2-157.10 & 156.55

C18-151.77

C19&C20-150.97 & 150.49

C5&C6-148.56 & 148.22

C17-138.35

C4&C7-129.60

C3&C10-125.88 and 125.42

C11&C15-121.82 & 121.20

C12&C14-117.23 & 116.90

C9-60.04

NN

NN

HN C CH2O

O

Cl

NHN

N

123

45

6 7

8 910

11 12

13

1415

16

17

18

1920C

O

144


IR (KBr) CM-1:The IR spectrum of the mercapto triazole derivative HTP6 showed its

characteristic absorption bands in the following

region.3400,3250(NH), 2804(C-H str), 1699 (C=O), 1587(C=O),

1547,1518,1477(C=C ring str) 1477 & 1352 (C-H bending), 1082(C-O-

C), 738 & 831 (phenyl and heterocyclic rings)

NN

NN

HN C CH2

O

NHCON

N

146

Anti-bacterial Activity of 3,4-Disubstituted Triazoles and their

derivatives

The invitro antibacterial screening was carried out at the Microbiology

department at , Navodaya Medical College Hospital and Research

Centre, Raichur.

Following the same procedure as described in Chapter-III. The result

is presented in Table No. 12.

Sr.No. Sample No.

Zone of Inhibition (mm)

Staphylococcusaureus E.coli Pseudomonas

aeruginosa

1 TI1 28 22 00

2 TI2 22 30 00

3 TI3 24 28 00

4 TI4 22 32 00

5 TI5 26 22 00

6 TI6 00 26 00

7 TI7 24 24 00

8 TI8 30 22 24

9 TI9 00 00 00




147

Table 13

Sr.No. Sample No.

Zone of Inhibition (mm)Staphylococcus

aureus E.Coli Pseudomonasaeruginosa

1 TIT1 24 22 00

2 TIT2 28 30 00

3 TIT3 00 24 00

4 TIT4 22 30 00

5 TIT5 24 22 22

6 TIT6 28 00 00

7 TIT7 00 22 24

8 TIT8 00 00 00




Table 14

1 TIP1 00 24 00

2 TIP2 26 24 00

3 TIP3 32 22 24

4 TIP4 22 00 00

5 TIP5 24 00 26

6 TIP6 32 26 00

7 TIP7 00 00 00

8 TIP8 24 22 00




148


3,4-Disubstituted triazoles and their derivatives bearing

INH/Pyrazinamide at 5th position of Triazole moiety were screened for

antibacterial activity among the parent 3,4-disubstituted triazoles

TIT2 and TIT6 exhibited significant activity. The compounds TIT1,

TIT4 and TIT5 showed moderate activity and rest of the compounds

did not exhibit activity.

The two compounds TIT2 and TIT4 showed much better activity

against E.coli than Staphylococcus aureus. The activity of the

remaining compounds was Nil to moderate. The inhibition of the

compounds against Pseudomonas aeruginosa was found to be

moderate for TIT5 and TIT7. No activity was observed with the rest of

the compounds.

149

Antifungal Activity of Triazolo derivatives bearing

INH/Pyrazinamide

The invitro antifungal activity of the Triazolo derivatives bearing

INH/Pyrazinamide compounds synthesized was carried at Maratha

Madals Nathaji Rao G. Halgekar Institute of Dental Sciences and



evaluation.

Table 15: Antifungal Activity of Triazolo Derivatives bearing INH

Sr. no. Compounds Concentration (µg/ml)

75 50 25 10

1 TIT-1 30mm 18mm R R

2 TIT-2 20mm R R R

3 TIT-3 18mm 16mm R R4 TIT-4 R R R R

5 TIT-5 R R R R

6 TIT-6 18mm R R R

7 TIT-7 28mm 28mm 28mm 10mm8 TIT-8 34mm 30mm 27mm 22mm

Table 16: Antifungal Activity By MIC Method

Sr.no.

Compounds Concentration (µg/ml)

500 250 125 62.5 31.25 16 8 4 2 1

1 TIT S S S S S S S R R R

2 TIT-8 S S S S S S R R R R

150

Table 17:Antifungal Activity of Triazolo Derivatives bearing Pyrazinamide

Sr. no. Compounds Concentration (µg/ml)

75 50 25 10

1 TIP-1 24mm 24mm 22mm 16mm

2 TIP-2 26mm 24mm 24mm 22mm3 TIP-3 33mm 30mm 22mm 18mm




7 TIP-7 29mm 29mm 25mm 20mm8 TIP-8 25mm 24mm 22mm 22mm

Table 18: Antifungal Activity By MIC Method

Sr.no.

Compounds Concentration (µG/ML)

500 250 125 62.5 31.25 16 8 4 2 1

1 TIP-3 S S S R R R R R R R

2 TIP-6 S S S S S S S R R R

151


3-substituted-4-amino triazoles and their derivatives (TI1 - TI8) and

their derivatives bearing INH (TIT1 - TIT8) and bearing pyrazinamide

(TIP1 - TIP8) at 5th position of parent triazole of TI to TI8 series were

subjected for invitro antifungal studies. The results indicated that the

compounds like TI2, TI3, TI5 and TI6 exhibited equipotent activity in

comparison with the standard drug rest of the compounds exhibited

moderate activity. Surprisingly all the compounds of the series except

TI4 have shown much superior activity at concentrations 25, 50, 75

µg/ml. The mercapto group of the triazoles (TI1 to TI8) at 5th position

was replaced by INH and pyrazinamide. The INH bearing triazole

derivatives (TIT1 - TIT8) showed that except TIT8 other compounds of

the series do not posses antifungal activity worth mentioning. TIT8

showed equipotent activity even at lower concentration of 10 µg/ml.

The triazole derivatives bearing pyrazinamide (TIP1 - TIP8) showed

that among this series of compounds TIP2, TIP6 and TIP8 exhibited

excellent activity. In comparison it can be said that except TIP which

possesses moderate activity rest of the compounds of the series

showed significant activity.

152

Anti-tubercular activity of Triazoles bearing INH/Pyrazinamide

The in-vitro anti-tubercular activity38 of the triazolo thiadiazoles was

carried out at Maratha Mandals Nathaji Rao Dental college and



evaluation. The compounds were screened for anti-tubercular activity

following the same procedure as described in Chapter III.A.

Table: 19 Anti-tubercular activity of Triazoles bearingINH/Pyrazinamide

Sr. No. CompoundsConcentration

5mg/ml 10mg/ml 15mg/ml

01 TI-3 (ATB1) S S S





06 TI-8 (ATB6) R R R

07 TI-7 (ATB7) R R R

S- Sensitive

R- Resistance

153

Table: 20 Anti-tubercular activity of Triazoles bearing INH



01 TIT-3 (ATB8) S S S



04 TIT-4 (ATB11 ) S S S



07 TIT-10 (ATB14) S S S

Table: 21 Anti-tubercular activity of Triazoles bearingPyrazinamide



01 TIP-11 (ATB15) R R R

02 TIP-3 (ATB16) S S S

03 TIP-1 (ATB17) R S S



06 TIP-5 (ATB20) R S S

07 TIP-8 (ATB21) R R R

08 TIP-7 (ATB22) R R R

154


The parent mercapto triazoles (TI1, TI3, TI4-TI8), their derivatives

obtained by the replacement of mercepto group by INH TIT1 (TIT3,

TIT4-TIT6, TIT8 and TIT10) and the derivatives obtained by the

replacement of mercapto group by pyrazinamide (TIP1, TIP3, TIP4-TIP8

and TIP11) were screened for their antimycobacterial activity following

the standard procedure. The introduction of INH moiety at the 5th

position by replacing mercapto group has improved the activity. All

the compounds showed equipotent activity. The mercapto triazoles TI7

and TI8 which failed to show anti-tubercular activity showed potent

activity when the molecules were modified. The resulted new

compounds TIT8 and TIT10 showed much significant activity than their

parent mercapto triazoles. However the introduction of Pyrazinamide

moiety at the 5th position of parent triazole (TI) series has not

produced any substantial increase in the activity. Infact at lower

concentration (5µg/ml) the activity was not observed in few

compounds like TIP7 and TIP8.

155

The Anthelmintic activity of 3,4-disubstituted triazoles

The Anthelmintic activity of representative compounds of 3,4-

disubstituted traizoles series was carried out in the department of

pharmacology, NET Pharmacy College, Raichur as described in

Chapter III-A.

Table 22

Sr.No.

CompoundCode % Con. Time in min

Paralysis Death Average

1. TI2

0.1 06 12

10.60.2 06 10

0.5 04 10

2. TI5

0.1 7 13

12.60.2 5 13

0.5 5 11

3. TI6

0.1 5 10

8.60.2 5 09

0.5 5 07

4. TI7

0.1 06 12

090.2 06 08

0.5 05 07

5. TIT7

0.1 07 11

9.30.2 07 09

0.5 06 08

6. TIT8

0.1 04 13

110.2 04 11

0.5 03 09

7. TIP6

0.1 06 14

12.60.2 05 12

0.5 05 12

8. TIP70.1 06 12

9.30.2 04 090.5 07 09

156

Result and Discussions

3-pyridyl-4-(substituted phenyl carboxamido)-5-mercapto 1,2,4-

triazole derivatives (TI series) and their derivatives obtained by the

replacement of 5-mercepto group by isoniazid/pyrazinamide (TIT and

TIP series) were screened for their anthelmintic activity. Few selected

representative compounds from each series like TI2, TI5, TI6, TI7,

TIT7, TIT8, TIP6 and TIP7 were evaluated. The compounds of all the 3

types of triazole derivatives showed significant activity almost

comparable with the standard Albandazole. Thus the preliminary

results indicate that these compounds posses great potential to serve

as promising candidate for further development of anthelmintic agents

with improved potency.

157

Analgesic & anti Inflammatory activity of Triazolo ThiadiazolesLiterature survey showed that the Triazolo Thiadiazoles also exhibit

medium to excellent anti-inflammatory39&40 and analgesic activity41.

Hence a few representative compounds synthesized were screened for

anti-inflammatory and analgesic activity was carried out at the

department of pharmacology. NET pharmacy college Raichur and the

required animals (Albino Rats).

ProcedureRat paw oedema model (Carrageenan induced) :

Anti inflammatory activity was carried out using albino rats weighing

100-150 gms which were maintained on standard pallet diet and free

access to water was provided.

12 groups eachi having six almino rats were treated as below

Group 1: Norma control (treated with 0.2 ml of 2% gum acacia p.o)

Group 2 : Reference drug (54 mg/ kg p.o)

We have selected diclofenac sodium as reference for the study. Each

triazolo compound selected for study is treated as a separate group

(200 mg/ kg in 2% gun acacia p.o.)

Test compounds, reference drug and normal control were

administered to the rats half hour before the injection of 0.1 ml of 1%

carrageenan suspension in normal saline. The carrageenan

suspension was injected into the sub-planner region of the left hind

paw and the right hind paw served as reference. The oedema volume

of the injected paws were measured by plethysmograph mercury

displacement method.

Percentage reduction in the oedema volume was calculated .

The data is presented in the table 23.

158

Analgesic activity

Determination of acute toxicity (LD50):The lethal dose of the synthesized compounds was determined using

albino mice weighing (20-30 grams) which were maintained under

standard husbandary conditions. The procedure followed as per the

OECD guidelines No 420. 1/5th of the lethal dose was treated as

effective dose ( Therapeutic dose).

The analgesic activity was performed by following Eddy’s hot plate

method described by woolfe and Mac Donald.

Six groups of Albino mice in two phases containing 6 animals of

albino mice weighing 20-25 gms were used the animals were fasted for

18 hours without food and water. The animals with a basel reaction

time of less than 8 seconds were selected for the study the protocol is

given below.

Group 1: (2% of gum acacia) control

Group 2: reference (10 mg/kg sub cutanius)

We have used pentazocin as reference standard . the triazolo

compounds selected for study were given to other groups at 200

mg/kg body weight p.o

The data of analgesic activity were presented in table No 24.

159

Table: 23 Antiinflammatory Activity of Triazolo-Thiadiazoles(IIIA)

And Triazole derivatives bearing INH (III B)

Result: The compounds screened showed that T1 possess moderate

activity and rest of the compounds are inactive.

Sl. No Treatment

Percentage inhibition of rat’s hind paws Oedema atdifferent time intervals

30min 1 hr 2 hr 4 hr 6 hr 8 hr

01 Control 0.00 0.00 0.00 0.00 0.00 0.00

02 DiclofenacSodium 18.47 19.94 19.81 36.76 38.08 36.36

03 T2 2.10 8.42 3.60 0.00 0.79 0.82

04 T3 4.34 9.47 9.00 2.94 0.79 2.47

05 T21 26.08 33.68 23.42 6.61 1.58 1.65

06 T25 1.08 3.15 3.60 0.00 0.00 0.82

07 T27 14.13 18.94 18.01 6.61 4.76 0.00

08 TI1 7.60 26.31 13.51 11.76 11.90 23.96

09 TI3 17.4 32.65 21.65 4.42 4.78 5.00

10 TI4 32.60 37.89 20.73 8.82 0.00 3.30

11 TI5 11.96 28.43 27.94 4.43 4.68 4.96

12 TI8 7.62 34.75 39.85 37.86 0.81 5.01

160

Table 24: Analgesic Activity of Triazolo-Thiadiazoles (III-A)And Triazole derivatives bearing INH (III-B)

Result: The compounds exhibited weak to moderate analgesic activity.

Sl.No Treatment

Mean +- S.E.M

0 hr 30min 1 hr 2 hr 4 hr 6 hr 8 hr

01 Control

02 Pentazocin 6.24+0.889 12.87+1.33 12.91+1.32 13.87+0.279

13.65+0.85

11.83+1.04

11.83+1.04

03 T2 5.00+ 0.36 7.33+0.80 4.50+0.43 3.66+0.21 5.00+0.58 4.66+0.42 6.16+0.91

04 T3 4.50+0.43 5.16+0.70 3.50+0.43 4.16+0.60 5.16+0.94 4.83+0.94 3.66+0.33

05 T21 4.66+0.49 7.33+0.94 4.33+0.33 4.00+0.52 5.00+0.52 5.83+0.60 4.66+0.66

06 T25 3.83+0.48 6.16+0.79 4.50+0.67 4.00+0.45 5.66+0.33 4.83+0.87 4.00+0.36

07 T27 4.50+0.43 5.33+0.71 3.66+0.42 4.16+0.48 3.83+0.17 3.83+0.48 4.16+0.40

08 TI1 5.16+0.60 5.00+0.73 3.33+0.42 3.83+0.48 4.50+0.72 3.50+0.22 3.60+0.49

09 TI3 5.83+0.60 7.16+0.48 4.66+0.49 4.33+0.61 5.50+0.56 4.16+0.48 5.50+1.06

10 TI4 6.00+0.58 3.50+0.43 3.80+0.31 3.10+0.40 3.33+0.33 3.33+0.33 3.33+0.33

11 TI5 5.66+0.56 6.83+0.83 4.83+0.70 4.50+0.88 4.50+0.43 4.33+0.49 4.66+0.61

12 TI8 5.33+0.49 5.16+0.40 3.33+0.67 3.16+0.17 4.33+0.49 3.00+0.28 5.10+1.17

161

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