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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
15. T15 2–Chloro phenoxy methyl C16H10ON5Br2SCl 100 80
16. T16 3–Chloro phenoxy methyl C16H10ON5Br2SCl 98 72
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)
49
50
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
53
54
55
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
58
59
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
62
63
64
65
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).
68
69
70
Spectral Data for TT8 or T8
IR (KBr) CM-1:IR spectrum of TT8 showed its characteristic absorption bands in the
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).
Spectral Data for TT3 or T3
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
71
72
73
Spectral Data for TT4 or T4
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).
Spectral Data for TT5 or T5
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
74
75
76
Spectral Data for TT6 or T6
IR (KBr) CM-1:IR spectrum of TT6 showed its characteristic absorption bands in the
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).
Spectral Data for TT7 or T7
IR (KBr) CM-1:IR spectrum of TT7 showed its characteristic absorption bands in the
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
77
78
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
3 T-3 S S S S S S S S S R
4 T-4 S S S S S S R R R R
5 T-6 S S S S S S S R R R
6 T-8 S S S S S S S S S R
7 T-10 S S S S S S R R R R
8 T-11 S S S S S S S 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
12 T-15 S S S S S S R R R R
13 T-16 S S S S S S S R 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
Health Sciences, Bangalore as nodal centre for carrying out biological
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
Results and Discussion
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
Results & Discussions
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)
Sr.No Code Ar mol. formula m.p.
(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)
Sr.No Code Ar mol. formula m.p.
(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
110
111
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
113
114
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
116
117
118
119
Spectral Data for HTI5 or TIT5
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
121
122
123
124
125
Spectral Data for HTI6 or TIT6
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
126
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
129
130
131
132
Spectral Data for HTP3 or TIP3
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
different environmental carbon atoms as indicated.
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
135
136
137
138
139
Spectral Data for HTP5 or TIP5
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
different environmental carbon atoms as indicated.
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
141
142
143
144
Spectral Data for HTP6 or TIP6
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
145
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
10 Ciprofloxacin 30 28 27
11 Gentamycin 34 30 35
12 Tobramycin 30 32 34
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
9 Ciprofloxacin 30 28 27
10 Gentamycin 34 30 35
11 Tobramycin 30 32 34
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
9 Ciprofloxacin 30 28 27
10 Gentamycin 34 30 35
11 Tobramycin 30 32 34
148
Results and Discussion
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
Research Centre, Belgaum recognized by Rajiv Gandhi University of
Health Sciences, Bangalore as nodal centre for carrying out biological
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
4 TIP-4 32mm 26mm 24mm 18mm
5 TIP-5 24mm 23mm 20mm 20mm
6 TIP-6 34mm 34mm 30mm 30mm
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
Results & Discussions
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
Research Centre, Belgaum recognized by Rajiv Gandhi University of
Health Sciences, Bangalore as nodal centre for carrying out biological
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
02 TI-1 (ATB2) S S S
03 TI-6 (ATB3) S S S
04 TI-4 (ATB4) S S S
05 TI-5 (ATB5) 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
Sr. No. CompoundsConcentration
5µg/ml 10µg/ml 15µg/ml
01 TIT-3 (ATB8) S S S
02 TIT-1 (ATB9) S S S
03 TIT-6 (ATB10) S S S
04 TIT-4 (ATB11 ) S S S
05 TIT-5 (ATB12) S S S
06 TIT-8 (ATB13) S S S
07 TIT-10 (ATB14) S S S
Table: 21 Anti-tubercular activity of Triazoles bearingPyrazinamide
Sr. No. CompoundsConcentration
5µg/ml 10µg/ml 15µg/ml
01 TIP-11 (ATB15) R R R
02 TIP-3 (ATB16) S S S
03 TIP-1 (ATB17) R S S
04 TIP-6 (ATB18) S S S
05 TIP-4 (ATB19) S S S
06 TIP-5 (ATB20) R S S
07 TIP-8 (ATB21) R R R
08 TIP-7 (ATB22) R R R
154
Results and Discussion
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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