Condensed bridgehead nitrogen heterocyclic system: Synthesis and pharmacological activities of 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole derivatives of ibuprofen and biphenyl-4-yloxy acetic acid

Article abstract of DOI:10.1016/j.ejmech.2007.09.025

Several 3,6-disubstituted-1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazoles were prepared by condensation of 4-amino-5-substituted-3-mercapto-(4H)-1,2,4-triazoles (3a,b) with various substituted aromatic acids and aryl/alkyl isothiocyanates through a one-pot reac

Full text of DOI:10.1016/j.ejmech.2007.09.025

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 2056e2066
http://www.elsevier.com/locate/ejmech
Original article
Condensed bridgehead nitrogen heterocyclic system: Synthesis and
pharmacological activities of 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole
derivatives of ibuprofen and biphenyl-4-yloxy acetic acid
*
Mohd. Amir , Harish Kumar, S.A. Javed
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University, New Delhi 110 062, India
Received 15 December 2006; received in revised form 22 September 2007; accepted 27 September 2007
Available online 6 October 2007
Abstract
Several 3,6-disubstituted-1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazoles were prepared by condensation of 4-amino-5-substituted-3-mercapto-(4H )-
1,2,4-triazoles (3a,b) with various substituted aromatic acids and aryl/alkyl isothiocyanates through a one-pot reaction. These compounds were
investigated for their anti-inflammatory, analgesic, ulcerogenic, lipid peroxidation, antibacterial and antifungal activities. Some of the synthe-
sized compounds showed potent anti-inflammatory activity along with minimal ulcerogenic effect and lipid peroxidation, compared to those of
ibuprofen and flurbiprofen. Some of the tested compounds also showed moderate antimicrobial activity against tested bacterial and fungal
strains.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Triazolo-thiadiazoles; Anti-inflammatory; Ulcerogenicity; Lipid peroxidation; Hepatotoxic effect
1. Introduction
NSAIDs have been taken with the aim of improving their
safety profile.
Currently available non-steroidal anti-inflammatory drugs
(NSAIDs) like ibuprofen, flurbiprofen, fenbufen and naproxen
exhibit gastric toxicity. Long-term use of these drugs has been
associated with gastro-intestinal (GI) ulceration, bleeding and
nephrotoxicity [1]. The GI damage from NSAIDs is generally
attributed to two factors, i.e. local irritation by the carboxylic
acid moiety common to most NSAIDs (topical effect); and de-
creased tissue prostaglandin production, which undermines the
physiological role of cytoprotective prostaglandins in main-
taining GI health and homeostasis [2,3]. The pharmacological
activity of NSAIDs is related to the suppression of prostaglan-
din biosynthesis from arachidonic acid by inhibiting cycloox-
ygenases (COXs) [2,4]. The chronic use of NSAIDs including
ibuprofen may elicit appreciable GI toxicity [5]. Therefore
synthetic approaches based upon chemical modification of
Heterocycles bearing a symmetrical triazole or 1,3,4-thia-
diazole moieties, represent an interesting class of compounds
possessing a wide spectrum of biological activities such as
anti-inflammatory [6e8], antiviral [9] and antimicrobial
[10,11] properties. It has also been reported that derivatives
of 1,2,4-triazole and 1,3,4-thiadiazole condensed nucleus sys-
tems exert diverse pharmacological activities such as anti-
inflammatory [12], antitumor [13], antifungal and antibacterial
[14]. Furthermore, literature survey revealed that modification
of the carboxyl function of representative NSAIDs resulted in
increased anti-inflammatory activity with reduced ulcerogenic
effect [15e17]. Our former studies [18,19] have shown that cer-
tain compounds bearing 1,2,4-triazole and 1,3,4-thiadiazole nu-
clei possess significant anti-inflammatory activity with reduced
GI toxicity. It was therefore considered worthwhile to replace
the carboxylic acid group of 2-(4-isobutylphenyl)propanoic
acid and biphenyl-4-yloxy acetic acid by a composite system,
which combines both the triazole and the thiadiazole nucleus
in a ring to give a compact and planar structure. The compounds
* Corresponding author. Tel.: þ91 11 26059878; fax: þ91 11
26059688x5307.
E-mail address: mamir_s2003@yahoo.co.in (Mohd. Amir).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.09.025

Mohd. Amir et al. / European Journal of Medicinal Chemistry 43 (2008) 2056e2066
2057
thus synthesized have been found to possess an interesting pro-
file of anti-inflammatory activity with significant reduction in
their ulcerogenic effect. In view of the reported antimicrobial
activity of triazolo-thiadiazoles, these compounds were also
tested for their antibacterial and antifungal activities. Two
selected compounds were also studied for their hepatotoxic
effects on rat liver.
The structure of 4-amino-3-substituted-5-mercapto-(4H )-
1,2,4-triazoles (3a,b) was confirmed by 1H NMR spectral
data and microanalysis. The 1H NMR spectra showed a down-
field D2O exchangeable singlet at d 13.19e13.58 attributed to
the SH group, while the NH2 group appeared as a singlet at
d 5.41e5.62. The absence of signals due to NH2 and SH
protons confirmed that the triazoles were converted into tria-
zolo-thiadiazoles (4aee, 5aef) by reacting with COOH group
of acids. Although in the 1H NMR spectra of triazolo-
thiadiazoles (6aed, 7aed) the signals of NH2 and SH protons
disappeared, but a singlet of alkyl/aryl amino D2O exchange-
able proton appeared in the range of d 8.20e13.84, confirming
that triazoles were converted into triazolo-thiadiazoles (6aed,
7aed) by reacting with alkyl/aryl isothiocyanates.
2. Chemistry
The acid hydrazides (1a,b) were prepared by esterification
of 2-(4-isobutylphenyl)propanoic acid and biphenyl-4-yloxy
acetic acid followed by treatment with hydrazine hydrate in ab-
solute ethanol [18]. 4-Amino-3-substituted-5-mercapto-(4H )-
1,2,4-triazoles (3a,b) were prepared following the procedure
of Reid and Heindel [20]. The acid hydrazides were allowed
to react with carbon disulphide in the presence of potassium
hydroxide in ethanol to afford the corresponding intermediate
potassium dithiocarbazinate (2a,b). This salt underwent ring
closure with an excess of 99% hydrazine hydrate to give
4-amino-3-substituted-5-mercapto-(4H )-1,2,4-triazoles (3a,b).
The resultant triazoles (3a,b) were further converted to 1,2,4-
triazolo-[3,4-b]-1,3,4-thiadiazoles 4aee, 5aef, 6aed and
7aed through one-pot reaction by condensation with aromatic
acids in the presence of phosphorus oxychloride and with aryl/
alkyl isothiocyanates in the presence of DMF, respectively, as
outlined in Scheme 1.
3. Results and discussion
3.1. Biological evaluation
3.1.1. Anti-inflammatory activity
The anti-inflammatory activity of the synthesized com-
pounds 4aee, 5a,b,def, 6aed and 7a,ced was evaluated by
carrageenan-induced paw edema method of Winter et al.
[21]. The compounds (4aee, 6aed) were tested at an equimo-
lar oral dose relative to 70 mg/kg ibuprofen and the com-
pounds (5a,b,def and 7a,c,d) were tested at an equimolar
oral dose relative to 10 mg/kg flurbiprofen. The tested
S
S- K +
H
N
H
Ar
Ar
N
NH
(2a-b)
N
H
NH2
KOH, C2H5OH
CS2, Stirring
room temp.
O
O
(1a-b)
Water,
NH2NH2.H2O
Reflux
N
N
N
Ar
RCOOH, POCl3
Reflux
N
N
S
N
Ar
SH
N
(4a-e, 5a-f)
R
N
N
NH2
RNCS, DMF
Reflux
(3a-b)
Ar
S
N
N
R
NH
(6a-d, 7a-d)
Scheme 1. Synthetic pathways to 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole derivatives of ibuprofen (4aee and 6aed) and biphenyl-4-yloxy acetic acid (5aef and
7aed).

2058
Mohd. Amir et al. / European Journal of Medicinal Chemistry 43 (2008) 2056e2066
compounds showed anti-inflammatory activity ranging from
18.17% to 80.29%, whereas standard drugs ibuprofen and flur-
biprofen showed 80.38% and 80.29% inhibition, respectively,
after 4 h (Table 1). The anti-inflammatory activity of triazolo-
thiadiazole derivatives of ibuprofen was in the range of
18.17e80.29%. It was observed that the triazolo-thiadiazole
derivatives having a 2,4-dichlorophenyl (4a) and n-butylamino
group (6a) at 6th position possess highest activity (80.29%). Re-
placement of these groups by 4-aminophenyl (4d) resulted in
slight decrease of anti-inflammatory activity (78.78%). Further
it was observed that the presence of 2,4-dichlorophenoxy methyl
moiety (4b) at C-6 showed decrease of activity (38.63%) and re-
placement of this group by 2-(2,6-dichloroanilino)benzyl group
resulted in sharp decrease of anti-inflammatory activity
(18.17%). Compounds 4c and 6b,d showed moderate activity.
The anti-inflammatory activity of triazolo-thiadiazole deriva-
tives of biphenyl-4-yloxy acetic acid was found in the range
39.38e78.78%. The compounds having 4-chlorophenyl (5b),
4-aminophenyl (5f) and 4-fluorophenylamino (7c) groups at
C-6 of triazolo-thiadiazole ring showed anti-inflammatory activ-
ity (78.78%, 76.51% and 78.18%, respectively) comparable to
that of flurbiprofen (80.29%), the standard drug. The other com-
pounds showed moderate activity. From the view point of
structureeactivity relationship (SAR), it is clear that the
triazolo-thiadiazole derivatives of ibuprofen were found to be
more active than biphenyl-4-yloxy acetic acid derivatives. The
anti-inflammatory activity of compounds 4a and 6a having
2,4-dichlorophenyl and n-butylamino groups, respectively,
was found to be the highest, being slightly less than ibuprofen,
but equivalent to flurbiprofen. In general the presence of 2,4-
dichlorophenyl, 4-chlorophenyl, n-butylamino and 4-aminophenyl
groups at C-6 of triazolo-thiadiazole ring resulted in high anti-
inflammatory activity.
triazolo-thiadiazole derivatives of biphenyl-4-yloxy acetic acid
were found to be more active than ibuprofen derivatives. In
general, the presence of n-butylamino and 4-phenylamino
groups at C-6 of triazolo-thiadiazole ring resulted in good an-
algesic activity.
3.1.3. Acute ulcerogenesis
The compounds which were screened for analgesic activity
were further tested for their acute ulcerogenic activity. Com-
pounds 4a,d and 6a,b were tested at an equimolar oral dose
relative to 210 mg/kg ibuprofen and compounds 5b,f and 7c
were tested at an equimolar oral dose relative to 30 mg/kg flur-
biprofen. The tested compounds showed low ulcerogenic ac-
tivity ranging from 0.333 ꢀ 0.10 to 1.000 ꢀ 0.31, compared
to standard drugs’ ibuprofen and flurbiprofen high severity in-
dex of 2.000 ꢀ 0.13 and 1.666 ꢀ 0.24, respectively. The max-
imum reduction in ulcerogenic activity (0.333 ꢀ 0.10) was
found in the triazolo-thiadiazole derivative of ibuprofen hav-
ing 2,4-dichlorophenyl group (4a) at C-6 of thiadiazole ring.
The triazolo-thiadiazole derivative of biphenyl-4-yloxy acetic
acid showing high anti-inflammatory activity (7c) also showed
reduction in severity index (0.666 ꢀ 0.16). The other tested
compounds also exhibited better GI safety profile as compared
to their standard reference drugs, as illustrated in Table 2.
3.1.4. Lipid peroxidation
It has been reported [22,23] that compounds showing less
ulcerogenic activity also showed reduced malondialdehyde
(MDA) content, a byproduct of lipid peroxidation. Therefore
an attempt was made to correlate the decrease in ulcerogenic
activity of the compounds with that of lipid peroxidation.
All the compounds screened for ulcerogenic activity were
also analyzed for lipid peroxidation. The lipid peroxidation
was measured as nmoles of MDA/100 mg of tissue. Ibuprofen
and flurbiprofen exhibited high lipid peroxidation 7.79 ꢀ 0.13
and 7.51 ꢀ 0.68, respectively, whereas control group showed
3.25 ꢀ 0.05. It was found that all the cyclised derivatives
showing less ulcerogenic activity also showed reduction in
lipid peroxidation (Table 2). Thus these studies showed that
synthesized compounds have inhibited the induction of gastric
mucosal lesions and the results further suggested that their
protective effect might be related to the inhibition of lipid per-
oxidation in gastric mucosa.
3.1.2. Analgesic activity
The compounds that exhibited anti-inflammatory activity
higher than 68% (4a,d, 5b,f, 6a,b and 7c) were further tested
for their analgesic activity at the same oral dose as used for the
anti-inflammatory activity. All the compounds showed moder-
ate analgesic activity in comparison to their respective
standard drugs. The compounds showed analgesic activity
ranging from 36.2% to 53.1% inhibition, whereas standard
drugs ibuprofen and flurbiprofen showed 74.15% and 69.5%
inhibition, respectively (Table 2). The analgesic activity of tri-
azolo-thiadiazole derivatives of ibuprofen was in the range of
36.2e51.4%. The compound 6a having n-butylamino group at
C-6 of triazolo-thiadiazole ring showed high activity (51.4%).
Replacement of this group by 2,4-dichlorophenyl (4a), 4-ami-
nophenyl (4d) and phenyl (6b) groups resulted in decrease of
activity (36.2%, 37.3% and 38.3%, respectively). The analge-
sic activity of triazolo-thiadiazole derivatives of biphenyl-4-
yloxy acetic acid was found in the range of 53.1e48.4%.
The compound 7c having a 4-fluorophenylamino group showed
highest analgesic activity (53.1%). Replacement of this group
by 4-chlorophenyl (5b) and 4-aminophenyl (5f) groups resulted
in a slight decrease of activity (48.1% and 48.4%, respectively).
According to structureeactivity relationship, it is clear that the
3.1.5. Hepatotoxic studies
The compounds 4a and 7c, derivatives of 2-(4-isobutylphe-
nyl)propanoic acid and biphenyl-4-yloxy acetic acid, respec-
tively, showing potent anti-inflammatory activity with
reduced ulcerogenicity and lipid peroxidation, were further
studied for their hepatotoxic effect. Both compounds were
studied for their effect on biochemical parameters (serum en-
zymes, total protein and total albumin), and liver histopatho-
logical testing was also carried out. As shown in Table 3,
activities of liver enzymes SGOT, SGPT, alkaline phosphatase,
total protein and total albumin were almost identical with con-
trol values, except for compound 7c, whose total protein and
total albumin were markedly reduced. The histopathological

Mohd. Amir et al. / European Journal of Medicinal Chemistry 43 (2008) 2056e2066
2059
Table 1
Anti-inflammatory and antimicrobial activity of the compounds (4aee and 5aef, 6aed and 7aed)
Compound
R
Ar
Anti-inflammatory activity
(% inhibition ꢀ SEM after 4 h)e
Antimicrobial activity (MIC mg/mL)
S. aureus
E. coli
C. albicans
Cl
Cl
a
4a
4b
80.29 ꢀ 1.51
200
200
200
100
Cl
Cl
a
38.63 ꢀ 3.05**
200
25
O
NH2
a
a
4c
57.57 ꢀ 1.91**
78.78 ꢀ 1.91
200
200
100
12.5
4d
12.5
200
200
H2N
Cl
a
4e
18.17 ꢀ 4.23**
50
200
N
H
Cl
b
b
c
5a
5b
65.14 ꢀ 3.03*
78.78 ꢀ 3.03
25
25
25
100
50
Cl
Cl
Cl
Cl
b
b
b
d
5c
5d
5e
25
25
25
50
50
50
50
50
Cl
50.75 ꢀ 2.73**
47.72 ꢀ 2.56**
O
NH2
25
b
a
5f
76.51 ꢀ 3.65
80.29 ꢀ 2.25
50
200
100
200
H2N
6a
CH3CH2CH2CH2e
200
50
a
a
c
6b
6c
68.17 ꢀ 3.10*
28.02 ꢀ 2.17**
100
200
25
200
200
200
F
CH3
a
6d
53.02 ꢀ 2.54**
200
50
H3C
b
b
c
c
7a
7b
CH3CH2CH2CH2e
65.90 ꢀ 3.05*
100
25
25
d
200

2060
Mohd. Amir et al. / European Journal of Medicinal Chemistry 43 (2008) 2056e2066
Table 1 (continued)
Compound
R
Ar
Anti-inflammatory activity
(% inhibition ꢀ SEM after 4 h)e
Antimicrobial activity (MIC mg/mL)
S. aureus
E. coli
C. albicans
b
b
c
c
7c
78.18 ꢀ 3.47
25
F
CH3
c
7d
39.38 ꢀ 3.45**
80.38 ꢀ 2.62
50
100
H3C
d
d
d
d
d
d
d
d
Ibuprofen
Flurbiprofen
Ketoconazole
Ofloxacin
Control
80.29 ꢀ 2.25
d
6.25
d
d
6.25
6.25
c
c
c
*p < 0.05.
**p < 0.01.
a
O
b
c
Did not show any activity.
Not tested.
d
e
Relative to their respective standard and data were analyzed by ANOVA followed by Dunnett’s multiple comparison test for n ¼ 6.
studies of the liver samples do not show any significant path-
ological changes in comparison to control group (Fig. 1). No
hepatocyte necrosis or degeneration was seen in any of the
samples.
of all the tested compounds were reported as minimal inhibi-
tory concentrations (MICs, mg/mL), and are shown in Table 1.
The results revealed that most of the newly synthesized com-
pounds exhibited promising antibacterial activities, but they
showed poor antifungal activity. Generally the test compounds
showed better activity against Gram-negative bacteria (Table 1).
Out of the compounds tested, 5a,cee (MIC, 25 mg/mL) ex-
hibited moderate antibacterial activity against Gram-positive
bacteria S. aureus, as compared to antibiotic ofloxacin (MIC,
6.25 mg/mL). It was noted that compound 4d (MIC, 12.5 mg/
mL) was 50% as active as ofloxacin (MIC, 6.25 mg/mL) against
3.1.6. Antimicrobial activity
The compounds 4aee, 5aef, 6aed and 7aed were evalu-
ated for their antimicrobial activity [32] against Staphylococ-
cus aureus representing Gram-positive bacteria, Escherichia
coli representing Gram-negative bacteria, and Candida albi-
cans representing fungi. The results of antimicrobial effect
Table 2
Analgesic, ulcerogenic and lipid peroxidation activity of selected compounds
Compound
Analgesic activitya
Ulcerogenic activity
(severity index ꢀ SEM)b
nmol MDA content ꢀ
SEM/100 mg tissueb
Pre-treatment (normal 0 h (s))
Post-treatment (after 4 h (s))
% Inhibition
4a
4d
1.810 ꢀ 0.186
0.992 ꢀ 0.076
1.156 ꢀ 0.067
1.482 ꢀ 0.153
1.217 ꢀ 0.120
1.171 ꢀ 0.084
1.860 ꢀ 0.223
e
2.467 ꢀ 0.290
1.362 ꢀ 0.139
1.706 ꢀ 0.069
2.200 ꢀ 0.162
1.843 ꢀ 0.147
1.620 ꢀ 0.088
2.513 ꢀ 0.202
e
36.2
0.333 ꢀ 0.10y
0.75 ꢀ 0.25y
0.833 ꢀ 0.24z
0.417 ꢀ 0.08 y
1.000 ꢀ 0.31y
0.667 ꢀ 0.10y
0.666 ꢀ 0.16y
0.00
5.04 ꢀ 0.53y
5.65 ꢀ 0.36z
5.82 ꢀ 0.50z
4.82 ꢀ 0.25y
5.78 ꢀ 0.97z
5.12 ꢀ 0.29
5.62 ꢀ 0.29z
3.25 ꢀ 0.05
7.79 ꢀ 0.13
7.51 ꢀ 0.68
37.3***
48.1*
48.4*
51.4**
38.3***
53.1**
e
5b
5f
6a
6b
7c
Control
Ibuprofen
Flurbiprofen
1.361 ꢀ 0.086
1.15 ꢀ 0.060
2.37 ꢀ 0.131
1.95 ꢀ 0.097
74.1*
69.5*
2.000 ꢀ 0.13
1.666 ꢀ 0.24
*p < 0.0001.
**p < 0.001.
***p < 0.01.
yp < 0.01.
zp < 0.05.
a
Relative to normal and data were analyzed by paired Student’s t-test for n ¼ 6.
b
Relative to their respective standard and data were analyzed by ANOVA followed by Dunnett’s multiple comparison test for n ¼ 6.

Mohd. Amir et al. / European Journal of Medicinal Chemistry 43 (2008) 2056e2066
2061
Table 3
Effect of compounds on serum enzymes, total proteins and total albumin
Compound
SGOT units/mLa
SGPT units/mLa
Alkaline phosphatasea
Total protein (g/dL)a
Total albumin (g/dL)a
Control
4a
7c
148.67 ꢀ 1.50
147.50 ꢀ 0.34
137.00 ꢀ 0.72*
27.67 ꢀ 0.84
28.17 ꢀ 0.83
26.50 ꢀ 0.72
13.06 ꢀ 0.25
15.18 ꢀ 0.13*
17.07 ꢀ 0.11*
1.80 ꢀ 0.01
1.89 ꢀ 0.07
0.90 ꢀ 0.09*
1.67 ꢀ 0.01
1.80 ꢀ 0.05**
0.71 ꢀ 0.07*
*p < 0.0001.
**p < 0.01.
a
Relative to control and data were analyzed by Student’s t-test for n ¼ 6.
E. coli. Compounds 4b, 5a,b, 6b and 7b,c exhibited only 25%
(MIC, 25 mg/mL) of the activity of ofloxacin against E. coli.
The other tested compounds were weakly active against both
organisms with MIC values ranging between 50 mg/mL and
200 mg/mL. All the tested compounds showed weak antifungal
activity against C. albicans (MIC values of 50e200 mg/mL),
except for compounds 4c (MIC, 12.5 mg/mL), 5c and 7a
(MIC, 25 mg/mL) that showed 50%, 25% and 25% activity, re-
spectively, as compared to the antifungal drug ketoconazole
(MIC, 6.25 mg/mL).
per million relative to tetramethylsilane (TMS); coupling con-
stants (J ) are reported in hertz, and refer to apparent peak
multiplicities, and may not necessarily be true coupling
constants. Mass spectra were measured on a Jeol SR-102
(FAB) mass spectrometer. Unless otherwise specified, all re-
actions were carried out in oven-dried glassware, and com-
mercially available starting materials were used without
further purification.
5.1. Chemistry
4. Conclusions
5.1.1. General method for synthesis of potassium
dithiocarbazinate (2a,b)
Various triazolo-thiadiazole derivatives of ibuprofen and
biphenyl-4-yloxy acetic acid were synthesized and screened
for anti-inflammatory, analgesic, ulcerogenic and antimicrobial
activities. It was interesting to note that seven cyclised
compounds 4c,d, 5b,f, 6a,b and 7c were found to have anti-
inflammatory properties comparable to their standard reference
drugs ibuprofen and flurbiprofen. When these compounds were
subjected to analgesic activity by tail immersion method in
mice, all compounds exhibited moderate to good activity. These
compounds were also tested for ulcerogenic activity and lipid
peroxidation, and showed superior GI safety profile along
with reduction in lipid peroxidation as compared with ibupro-
fen and flurbiprofen. Compound 4d demonstrated about half
the activity of ofloxacin against E. coli. The other compounds
showed moderate to weak antibacterial activity against S. au-
reus and E. coli. The synthesized compounds showed weak
antifungal activity against C. albicans, except for compound
4c that showed half of the activity of the antifungal drug (ke-
toconazole). Thus the triazolo-thiadiazole derivatives were
found having dual functional properties (anti-inflammatorye
analgesic and antimicrobial), and represent a promising class
of compounds with an interesting pharmacological profile.
Potassium hydroxide (0.03 M) was dissolved in absolute
ethanol (50 mL). The solution was cooled in ice bath and
appropriate acid hydrazide (1a,b; 0.02 M) was added with
stirring. To this carbon disulphide (0.025 M) was added in
small portions with constant stirring. The reaction mixture
was agitated continuously for 12 h at room temperature.
The precipitated potassium dithiocarbazinate was collected
by filtration, washed with anhydrous ether (100 mL) and
dried in vacuum. The potassium salt thus obtained was in
quantitative yield and was used in the next step without
further purification.
5.1.2. General method for synthesis of 4-amino-5-
substituted-3-mercapto-(4H )-1,2,4-triazoles (3a,b)
A suspension of potassium dithiocarbazinate (2a,b; 0.02 M)
in water (50 mL) and hydrazine hydrate (99%, 0.04 M) was re-
fluxed for 18e20 h with occasional shaking. The colour of the
reaction mixture changed to green with the evolution of hydro-
gen sulfide gas. A homogenous reaction mixture was obtained
during the reaction process. The reaction mixture was cooled to
room temperature and diluted with water (20 mL). On acidifi-
cation with acetic acid the required triazole was precipitated
out. It was filtered, washed thoroughly with cold water, dried
and recrystallised from ethanol. Purity of the compound was
checked by TLC using silica gel-G coated plates by using
toluene:ethylacetate:formic acid (T:E:F); (5:4:1) as solvent
system, and observed in UV light.
5. Experimental protocols
The melting points were determined in open capillary
tubes in a Hicon melting point apparatus and are uncorrected.
Elemental analysis (C, H, N, S) was performed on the CHNS
Elementar (Analysen systime, GmbH) Germany Vario EL III.
FTIR spectra were recorded as KBr pellets on a Jasco FT/IR
5.1.2.1.
4-Amino-5-[1-(4-isobutylphenyl)ethyl]-3-mercapto-
(4H )-1,2,4-triazole (3a). M.p.: 154 ꢂC, yield (%): 67%; IR
(KBr) n (cmꢁ1): 3315 (NH2), 3070 (CeH aromatic), 2958
(CeH aliphatic), 2608 (SH), 1605 (C]N); 1H NMR
(DMSO-d6) d: 0.83 (d, J ¼ 6.5 Hz, 6H, (CH3)2), 1.50 (d,
1
410 spectrometer and frequency was expressed in cmꢁ1. H
NMR spectra were recorded on a Bruker model DPX 300
NMR spectrometer. Chemical shifts (d) are expressed in parts

2062
Mohd. Amir et al. / European Journal of Medicinal Chemistry 43 (2008) 2056e2066
276 (Mþ). Anal. Calcd. (%) for C14H20N4S: C, 60.84; H,
7.29; N, 20.27; S, 11.60. Found: C, 60.89; H, 7.32; N,
20.23; S, 11.64.
5.1.2.2. 4-Amino-5-[(biphenyl-4-yloxy)methyl]-3-mercapto-(4H)-
1,2,4-triazole (3b). M.p.: 224 ꢂC, yield (%): 60%; IR (KBr)
n (cmꢁ1): 3290 (NH2), 3105 (CeH aromatic), 2948 (CeH
1
aliphatic), 2596 (SH), 1617 (C]N); H NMR (DMSO-d6)
d: 4.57 (s, 2H, OCH2), 5.62 (s, 2H, NH2), 6.83e7.58 (m,
9H, AreH), 13.29 (br s, 1H, SH); Mass m/z: 298 (Mþ).
Anal. Calcd. (%) for C15H14N4OS: C, 60.38; H, 4.73; N,
18.78; S, 10.75. Found: C, 60.43; H, 4.76; N, 18.75; S,
10.71.
5.1.3. General method for the synthesis of 6-aryl/-
aryloxymethyl/substituted benzyl-3-substituted[1,2,4]-
triazolo[3,4-b][1,3,4]thiadiazoles (4aee, 5aef)
An equimolar mixture (0.10 M) of 4-amino-5-substituted-
3-mercapto-(4H )-1,2,4-triazoles (3a,b) and aromatic acids in
phosphorus oxychloride (10 mL) was refluxed for 5 h. The re-
action mixture was cooled to room temperature and then grad-
ually poured on to crushed ice with stirring. The mixture was
allowed to stand overnight and the solid separated out was
filtered, treated with dilute sodium hydroxide solution and
washed thoroughly with cold water. The compound so ob-
tained was dried and crystallized from ethanol.
5.1.3.1. 6-(2,4-Dichlorophenyl)-3-[1-(4-isobutylphenyl)ethyl]-
[1,2,4]triazolo[3,4-b][1,3,4] thiadiazole (4a). M.p.: 96 ꢂC,
yield (%): 81%; IR (KBr) n (cmꢁ1): 3090 (CeH aromatic),
1
2958 (CeH aliphatic), 1637 (C]N); H NMR (CDCl3) d:
0.89 (d, J ¼ 6.5 Hz, 6H, (CH3)2), 1.76e1.87 (m, 4H, CH and
CH3), 2.40 (d, J ¼ 6.5 Hz, 2H, CH2), 4.68 (q, J ¼ 7.0 Hz,
1H, CH), 6.94e7.57 (m, 7H, AreH); Mass m/z: 431 (Mþ).
Anal. Calcd. (%) for C21H20Cl2N4S: C, 58.47; H, 4.67; N,
12.99; S, 7.43. Found: C, 58.51; H, 4.72; N, 12.95; S, 7.40.
5.1.3.2.
6-(2,4-Dichlorophenoxymethyl)-3-[1-(4-isobutyl-
phenyl)ethyl]-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (4b). M.p.:
102 ꢂC, yield (%): 79%; IR (KBr) n (cmꢁ1): 3101 (CeH aro-
matic), 2954 (CeH aliphatic), 1610 (C]N), 1238 (CeOeC);
1H NMR (CDCl3) d: 0.87 (d, J ¼ 6.5 Hz, 6H, (CH3)2), 1.76e
1.87 (m, 4H, CH and CH3), 2.42 (d, J ¼ 6.5 Hz, 2H, CH2),
4.59 (q, J ¼ 6.5 Hz, 1H, CH), 5.28 (s, 2H, OCH2), 6.87e7.42
(m, 7H, AreH); Mass m/z: 461 (Mþ). Anal. Calcd. (%) for
C22H22Cl2N4OS: C, 57.27; H, 4.81; N, 12.41; S, 6.95. Found:
C, 57.22; H, 4.75; N, 12.42; S, 6.92.
5.1.3.3. 6-(2-Aminophenyl)-3-[1-(4-isobutylphenyl)ethyl-[1,2,4]-
triazolo[3,4-b][1,3,4] thiadiazole (4c). M.p.: 208 ꢂC, yield (%):
75%; IR (KBr) n (cmꢁ1): 3346 (NH), 3098 (CeH aromatic),
2959 (CeH aliphatic), 1605 (C]N); 1H NMR (CDCl3) d:
0.87 (d, J ¼ 6.5 Hz, 6H, (CH3)2), 1.76e1.84 (m, 1H, CH),
1.91 (d, J ¼ 7.0 Hz, 3H, CH3), 2.43 (d, J ¼ 7.0 Hz, 2H, CH2),
4.43 (s, 2H, NH2), 4.67 (q, J ¼ 7.0 Hz, 1H, CH), 6.66e7.55
(m, 8H, AreH); Mass m/z: 377 (Mþ). Anal. Calcd. (%) for
Fig. 1. Histopathological studies of liver.
J ¼ 7.0 Hz, 3H, CH3), 1.74e1.83 (m, 1H, CH), 2.38 (d,
J ¼ 7.0 Hz, 2H, CH2), 4.30 (q, J ¼ 7.0 Hz, 1H, CH), 5.41 (s,
2H, NH2), 7.10 (d, J ¼ 7.9 Hz, 2H, AreH), 7.15 (d,
J ¼ 7.9 Hz, 2H, AreH), 13.58 (br s, 1H, SH); Mass m/z:

Mohd. Amir et al. / European Journal of Medicinal Chemistry 43 (2008) 2056e2066
2063
C21H23N5S: C, 66.82; H, 6.14; N, 18.55; S, 8.49. Found: C,
66.87; H, 6.15; N, 18.59; S, 8.46.
1H NMR (DMSO-d6) d: 5.05 (s, 4H, 2OCH2), 6.98e7.60 (m,
12H, AreH); Mass m/z: 483 (Mþ). Anal. Calcd. (%) for
C23H16Cl2N4O2S: C, 57.15; H, 3.34; N, 11.59; S, 6.63. Found:
C, 57.18; H, 3.30; N, 11.55; S, 6.66.
5.1.3.4. 6-(4-Aminophenyl)-3-[1-(4-isobutylphenyl)ethyl]-[1,2,4]-
triazolo[3,4-b][1,3,4] thiadiazole (4d). M.p.: 140 ꢂC, yield (%):
83%; IR (KBr) n (cmꢁ1): 3340 (NH), 3110 (CeH aromatic),
2959 (CeH aliphatic), 1604 (C]N); 1H NMR (CDCl3) d:
0.85 (d, J ¼ 6.5 Hz, 6H, (CH3)2), 1.75e1.84 (m, 1H, CH),
1.87 (d, J ¼ 7.0 Hz, 3H, CH3), 2.41 (d, J ¼ 6.8 Hz, 2H, CH2),
4.19 (s, 2H, NH2), 4.61 (q, J ¼ 7.0 Hz, 1H, CH), 6.66 (d,
J ¼ 8.0 Hz, 2H, AreH), 7.10 (d, J ¼ 6.7 Hz, 2H, AreH), 7.34
(d, J ¼ 6.7 Hz, 2H, AreH), 7.55 (d, J ¼ 8.0 Hz, 2H, AreH);
Mass m/z: 377 (Mþ). Anal. Calcd. (%) for C21H23N5S: C,
66.82; H, 6.14; N, 18.55; S, 8.49. Found: C, 66.86; H, 6.14;
N, 18.51; S, 8.45.
5.1.3.10. 6-(2-Aminophenyl)-3-[(biphenyl-4-yloxy)methyl]-[1,2,4]-
triazolo[3,4-b][1,3,4] thiadiazole (5e). M.p.: 248 ꢂC, yield (%):
60%; IR (KBr) n (cmꢁ1): 3315 (NH), 3119 (CeH aromatic),
1
2934 (CeH aliphatic), 1609 (C]N), 1273 (CeOeC); H NMR
(CDCl3) d: 4.59 (s, 2H, NH2), 5.21 (s, 2H, OCH2), 6.69e7.98
(m, 13H, AreH); Mass m/z: 399 (Mþ). Anal. Calcd. (%) for
C22H17N5OS: C, 66.15; H, 4.29; N, 17.53; S, 8.03. Found: C,
66.11; H, 4.32; N, 17.59; S, 8.00.
5.1.3.11. 6-(4-Aminophenyl)-3-[(biphenyl-4-yloxy)methyl]-[1,2,4]-
triazolo[3,4-b][1,3,4] thiadiazole (5f). M.p.: 112 ꢂC, yield (%):
63%; IR (KBr) n (cmꢁ1): 3340 (NH), 3126 (CeH aromatic),
2922 (CeH aliphatic), 1601 (C]N), 1286 (CeOeC); 1H
NMR (CDCl3) d: 4.40 (s, 2H, NH2), 5.10 (s, 2H, OCH2), 6.69e
7.98 (m, 13H, AreH); Mass m/z: 399 (Mþ). Anal. Calcd. (%)
for C22H17N5OS: C, 66.15; H, 4.29; N, 17.53; S, 8.03. Found:
C, 66.13; H, 4.31; N, 17.55; S, 8.01.
5.1.3.5. 6-[2-(2,6-Dichloroanilino)benzyl]-3-[1-(4-isobutylphe-
nyl)ethyl]-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (4e). M.p.:
210 ꢂC, yield (%): 78%; IR (KBr) n (cmꢁ1): 3379 (NH),
3090 (CeH aromatic), 2959 (CeH aliphatic), 1634 (C]N);
1H NMR (DMSO-d6) d: 0.84 (d, J ¼ 6.5 Hz, 6H, (CH3)2),
1.56 (d, J ¼ 7.0 Hz, 3H, CH3), 1.77e1.86 (m, 1H, CH), 2.40
(d, J ¼ 7.0 Hz, 2H, CH2), 4.29 (s, 2H, CH2), 4.54 (q,
J ¼ 7.0 Hz, 1H, CH), 6.90e7.85 (m, 11H, AreH), 9.50 (br
s, 1H, NH); Mass m/z: 536 (Mþ). Anal. Calcd. (%) for
C28H27Cl2N5S: C, 62.68; H, 5.07; N, 13.05; S, 5.98. Found:
C, 62.66; H, 5.09; N, 13.10; S, 5.94.
5.1.4. General method for the synthesis of 6-aryl/
alkylamino-3-substituted[1,2,4] triazolo[3,4-b][1,3,4]-
thiadiazoles (6aed, 7aed)
An equimolar (0.01 M) mixture of 4-amino-5-substituted-
3-mercapto-(4H )-1,2,4-triazoles (3a,b) and aryl/alkyl isothio-
cyanate in dimethylformamide (10 mL) was refluxed for
20e22 h. The reaction mixture was cooled to room tempera-
ture and then gradually poured on to crushed ice with stirring.
The mixture was allowed to stand overnight and the solid
separated out was filtered, and washed thoroughly with cold
water. The compound so obtained was dried and crystallized
from ethanol/methanol.
5.1.3.6. 3-[(Biphenyl-4-yloxy)methyl]-6-phenyl[1,2,4]triazolo-
[3,4-b][1,3,4]thiadiazole (5a). M.p.: 108 ꢂC, yield (%): 71%;
IR(KBr)n(cmꢁ1):3061(CeHaromatic),2922(CeH aliphatic),
1687 (C]N), 1272 (CeOeC); 1H NMR (CDCl3) d: 5.40 (s, 2H,
OCH2), 7.28e8.24 (m, 14H, AreH); Mass m/z: 384 (Mþ). Anal.
Calcd. (%) for C22H16N4OS: C, 68.73; H, 4.19; N, 14.57; S, 8.34.
Found: C, 68.71; H, 4.17; N, 14.55; S, 8.32.
5.1.3.7. 3-[(Biphenyl-4-yloxy)methyl]-6-(4-chlorophenyl)[1,2,4]-
triazolo[3,4-b][1,3,4] thiadiazole (5b). M.p.: 124 ꢂC, yield
(%): 69%; IR (KBr) n (cmꢁ1): 3089 (CeH aromatic), 2932
(CeH aliphatic), 1641 (C]N), 1263 (CeOeC); 1H NMR
(CDCl3) d: 5.39 (s, 2H, OCH2), 7.04e8.15 (m, 13H, AreH);
Mass m/z: 418 (Mþ). Anal. Calcd. (%) for C22H15ClN4OS:
C, 63.08; H, 3.61; N, 13.37; S, 7.65. Found: C, 63.07; H,
3.63; N, 13.39; S, 7.62.
5.1.4.1. 6-n-Butylamino-3-[1-(4-isobutylphenyl)ethyl]-[1,2,4]-
triazolo[3,4-b][1,3,4] thiadiazole (6a). M.p.: 166 ꢂC, yield
(%): 61%; IR (KBr) n (cmꢁ1): 3333 (NH), 3079 (CeH aro-
matic), 2956 (CeH aliphatic), 1611 (C]N); 1H NMR
(CDCl3) d: 0.90e0.96 (m, 9H, (CH3)2 and CH3), 1.55e1.60
(m, 5H, CH3 and CH2), 1.70e1.75 (m, 2H, CH2), 1.83e1.89
(m, 1H, CH), 2.19 (d, J ¼ 7.0 Hz, 2H, AreCH2), 3.90 (t,
J ¼ 6.9 Hz, 2H, NeCH2), 4.39 (q, J ¼ 6.9 Hz, 1H, CH),
7.11e7.69 (m, 4H, AreH), 9.62 (br s, 1H, NH); Mass m/z:
357 (Mþ). Anal. Calcd. (%) for C19H27N5S: C, 63.83; H,
7.16; N, 19.59; S, 8.97. Found: C, 63.87; H, 7.14; N, 19.61;
S, 8.94.
5.1.3.8. 3-[(Biphenyl-4-yloxy)methyl]-6-(2,4-dichlorophenyl)-
[1,2,4]triazolo[3,4-b][1,3,4] thiadiazole (5c). M.p.: 172 ꢂC,
yield (%): 59%; IR (KBr) n (cmꢁ1): 3113 (CeH aromatic),
2916 (CeH aliphatic), 1655 (C]N), 1223 (CeOeC); 1H
NMR (CDCl3) d: 5.38 (s, 2H, OCH2), 7.21e8.19 (m, 12H, Are
H). Anal. Calcd. (%) for C22H14Cl2N4OS: C, 58.29; H, 3.11; N,
12.36; S, 7.07. Found: C, 58.25; H, 3.16; N, 12.39; S, 7.05.
5.1.4.2. 3-[1-(4-Isobutylphenyl)ethyl]-6-phenylamino-[1,2,4]-
triazolo[3,4-b][1,3,4] thiadiazole (6b). M.p.: 174 ꢂC, yield
(%): 65%; IR (KBr) n (cmꢁ1): 3311 (NH), 3087 (CeH aro-
matic), 2958 (CeH aliphatic), 1601 (C]N); 1H NMR
(DMSO-d6) d: 0.85 (d, J ¼ 6.5 Hz, 6H, (CH3)2), 1.59 (d,
J ¼ 7.0 Hz, 3H, CH3), 1.79e1.85 (m, 1H, CH), 2.34 (d,
J ¼ 7.0 Hz, 3H, CH2), 4.47 (q, J ¼ 7.0 Hz, 1H, CH), 6.57e
7.53 (m, 8H, AreH), 9.24 (br s, 1H, NH). Anal. Calcd. (%)
5.1.3.9. 3-[(Biphenyl-4-yloxy)methyl]-6-[(2,4-dichlorophen-
oxy)methyl][1,2,4]triazolo[3,4-b][1,3,4] thiadiazole (5d). M.p.:
300 ꢂC, yield (%): 62%; IR (KBr) n (cmꢁ1): 3095 (CeH aro-
matic), 2932 (CeH aliphatic), 1605 (C]N), 1247 (CeOeC);

2064
Mohd. Amir et al. / European Journal of Medicinal Chemistry 43 (2008) 2056e2066
for C21H23N5S: C, 66.82; H, 6.14; N, 18.55; S, 8.49. Found: C,
66.80; H, 6.11; N, 18.52; S, 8.47.
192 ꢂC, yield: 52%; IR (KBr) n (cmꢁ1): 3233 (NH), 3091
(CeH aromatic), 2916 (CeH aliphatic), 1605 (C]N), 1267
1
(CeOeC); H NMR (DMSO-d6) d: 2.09 (s, 3H, CH3), 2.76
5.1.4.3. 6-(4-Fluorophenylamino)-3-[1-(4-isobutylphenyl)ethyl]-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (6c). M.p.: 96 ꢂC, yield
(%): 78%; IR (KBr) n (cmꢁ1): 3319 (NH), 3096 (CeH
aromatic), 2952 (CeH aliphatic), 1598 (C]N); 1H NMR
(CDCl3) d: 0.87 (d, J ¼ 6.5 Hz, 6H, (CH3)2), 1.65 (d,
J ¼ 7.0 Hz, 3H, CH3), 1.82e1.87 (m, 1H, CH), 2.41 (d,
J ¼ 7.0 Hz, 2H, CH2), 4.51 (q, J ¼ 7.0 Hz, 1H, CH), 6.89e
7.59 (m, 8H, AreH), 8.33 (br s, 1H, NH); Mass m/z: 395
(Mþ). Anal. Calcd. (%) for C21H22FN4S: C, 63.78; H, 5.61;
N, 17.71; S, 8.11. Found: C, 63.74; H, 5.67; N, 17.77; S, 8.15.
(s, 3H, CH3), 5.16 (s, 2H, OCH2), 6.72e7.47 (m, 12H, Are
H), 12.50 (br s, 1H, NH); Mass m/z: 427 (Mþ). Anal. Calcd.
(%) for C24H21N5OS: C, 67.43; H, 4.95; N, 16.38; S, 7.50.
Found: C, 67.47; H, 4.98; N, 16.35; S, 7.54.
5.2. Anti-inflammatory activity
The synthesized compounds were evaluated for their anti-
inflammatory activity using carrageenan-induced paw edema
method of Winter et al [21]. The experiment was performed
on Albino rats of Wistar strain of either sex, weighing 180e
200 g. The animals were randomly divided into groups of six.
Group I was kept as control, and received only 0.5% carboxy-
methyl cellulose (CMC) solution. Groups II and III were kept
as standard, and received ibuprofen (70 mg/kg p.o.) and flurbi-
profen (10 mg/kg p.o.), respectively. Carrageenan solution
(0.1% in sterile 0.9% NaCl solution) in a volume of 0.1 mL
was injected subcutaneously into the sub-plantar region of the
right hind paw of each rat, 1 h after the administration of the
test compounds and standard drugs. The right hind paw volume
was measured before and after 4 h of carrageenan treatment by
means of a plethysmometer. The percent anti-inflammatory ac-
tivity was calculated according to the following formula.
5.1.4.4. 6-(2,4-Dimethylphenylamino)-3-[1-(4-isobutylphenyl)-
ethyl]-[1,2,4]triazolo[3,4-b][1,3,4] thiadiazole (6d). M.p.:
140 ꢂC, yield (%): 72%; IR (KBr) n (cmꢁ1): 3297 (NH),
3116 (CeH aromatic), 2957 (CeH aliphatic), 1618 (C]N);
1H NMR (DMSO-d6) d: 0.70 (d, J ¼ 6.5 Hz, 6H, (CH3)2),
1.50 (d, J ¼ 7.0 Hz, 3H, CH3), 1.75e1.86 (m, 1H, CH), 2.21
(d, J ¼ 7.0 Hz, 5H, CH3 and CH2), 2.32 (s, 3H, CH3), 4.11
(q, J ¼ 7.0 Hz, 1H, CH), 6.91e7.62 (m, 7H, AreH), 11.01
(br s, 1H, NH); Mass m/z: 405 (Mþ). Anal. Calcd. (%) for
C23H27N5S: C, 68.12; H, 6.71; N, 17.27; S, 7.91. Found: C,
68.17; H, 6.68; N, 17.30; S, 7.87.
5.1.4.5. 6-n-Butylamino-3-[(biphenyl-4-yloxy)methyl]-[1,2,4]-
triazolo[3,4-b][1,3,4] thiadiazole (7a). M.p.: 240 ꢂC, yield
(%): 51%; IR (KBr) n (cmꢁ1): 3316 (NH), 3109 (CeH aro-
matic), 2927 (CeH aliphatic), 1659 (C]N), 1248 (CeOe
Percent anti-inflammatory activity ¼ ðVc ꢁ Vt=VcÞ ꢃ 100
where, Vt represents the mean increase in paw volume in rats
treated with test compounds, and Vc represents the mean in-
crease in paw volume in control group of rats.
1
C); H NMR (CDCl3) d: 1.21 (t, 3H, CH3), 1.61e1.65 (m,
2H, CH2), 2.06e2.13 (m, 2H, CH2), 3.64 (t, 2H, NeCH2),
4.94 (s, 2H, OCH2), 6.95 (d, 2H, AreH), 7.54e7.66 (m, 7H,
AreH), 8.20 (br s, 1H, NH); Mass m/z: 379 (Mþ). Anal.
Calcd. (%) for C20H21N5OS: C, 63.30; H, 5.58; N, 18.46; S,
8.45. Found: C, 63.33; H, 5.55; N, 18.48; S, 8.45.
5.3. Analgesic activity
Analgesic activity was evaluated by tail immersion method
[24]. Swiss albino mice divided into different groups consist-
ing of six animals each, of either sex, weighing 25e30 g, were
used for the experiment. Analgesic activity was evaluated after
oral administration of the test compounds (4a,d and 6a,b) at
an equimolar dose relative to 70 mg/kg ibuprofen, and test
compounds (5b,f and 7c) at an equimolar dose relative to
10 mg/kg flurbiprofen. Test compounds and standard drugs
were administered orally as suspension in CMC solution in
water (0.5% w/v). The analgesic activity was assessed before
and after 4 h interval of the administration of test compounds
and standard drugs. The lower 5 cm portion of the tail was
gently immersed into thermostatically controlled water at
55 ꢀ 0.5ꢂC. The time in seconds for tail withdrawal from
the water was taken as the reaction time with a cut of time
of immersion, set at 10 s for both control as well as treated
groups of animals.
5.1.4.6. 3-[(Biphenyl-4-yloxy)methyl]-6-phenylamino-[1,2,4]-
triazolo[3,4-b][1,3,4] thiadiazole (7b). M.p.: 148 ꢂC, yield
(%): 47%; IR (KBr) n (cmꢁ1): 3420 (NH), 3104 (CeH aro-
matic), 2931 (CeH aliphatic), 1657 (C]N), 1251 (CeOe
1
C); H NMR (DMSO-d6) d: 5.17 (s, 2H, OCH2), 7.12e7.63
(m, 14H, AreH), 13.84 (s, 1H, NH); Mass m/z: 399 (Mþ).
Anal. Calcd. (%) for C22H17N5OS: C, 66.15; H, 4.29; N,
17.53; S, 8.03. Found: C, 66.12; H, 4.31; N, 17.56; S, 8.01.
5.1.4.7. 3-[(Biphenyl-4-yloxy)methyl]-6-(4-fluoro phenylamino)-
[1,2,4]triazolo[3,4-b][1,3,4] thiadiazole (7c). M.p.: 242 ꢂC,
yield (%): 49%; IR (KBr) n (cmꢁ1): 3271 (NH), 3120 (CeH
aromatic), 2916 (CeH aliphatic), 1611 (C]N), 1253 (Ce
1
OeC); H NMR (CDCl3) d: 5.05 (s, 2H, OCH2), 7.29e7.83
(m, 13H, AreH), 12.57 (s, 1H, NH); Mass m/z: 417 (Mþ).
Anal. Calcd. (%) for C22H16FN5OS: C, 63.30; H, 3.86; N,
16.78; S, 7.68. Found: C, 63.27; H, 3.90; N, 16.82; S, 7.66.
5.4. Acute ulcerogenicity
5.1.4.8. 3-[(Biphenyl-4-yloxy)methyl]-6-(2,4-dimethyl phenyla-
mino)-[1,2,4]triazolo[3,4-b][1,3,4] thiadiazole (7d). M.p.:
Acute ulcerogenicity was determined according to Cioli
et al. [25]. The animals were divided into different groups

Mohd. Amir et al. / European Journal of Medicinal Chemistry 43 (2008) 2056e2066
2065
consisting of six animals in each group. Ulcerogenic activity
5.6.2. Histopathological studies of liver
was evaluated after oral administration of the test compounds
(4a,d and 6a,b) at an equimolar dose relative to 210 mg/kg ibu-
profen, and test compounds (5b,f and 7c) at an equimolar dose
relative to 30 mg/kg flurbiprofen. Control group received only
0.5% CMC solution. Food, but not water, was removed 24 h
before administration of the test compounds. After the drug
treatment the rats were fed with normal diet for 17 h and
then sacrificed. The stomach was removed and opened along
the greater curvature, washed with distilled water and cleaned
gently by dipping in normal saline. The mucosal damage was
examined by means of a magnifying glass. For each stomach
the mucosal damage was assessed according to the following
scoring system: 0.5: redness, 1.0: spot ulcers, 1.5: hemorrhagic
streaks, 2.0: ulcers > 3 but ꢄ 5, 3.0: ulcers > 5. The mean score
of each treated group minus the mean score of control group
was regarded as severity index of gastric mucosal damage.
Histopathological studies were carried out by reported
method [31]. The rats were sacrificed under light ether anes-
thesia after 24 h of the last dosage, the liver was removed
and washed with normal saline, and stored in formalin solu-
tion. Sections of 5e6 microns thickness were cut, stained
with haematoxylin and eosin, and then studied under an elec-
tron microscope (Fig. 1).
5.7. Antibacterial and antifungal activities
Antibacterial activity of the synthesized compounds was de-
termined in vitro by using cup plate method [32] against S. au-
reus (ATCC 19433) and E. coli (ATCC 25922) at 200 mg/mL,
100 mg/mL, 50 mg/mL and 25 mg/mL concentrations, respec-
tively, in the nutrient agar media. Standard antibiotic ofloxacin
was used as reference drug at 50 mg/mL, 25 mg/mL, 12.5 mg/
mL and 6.25 mg/mL concentrations. The test compounds
which showed inhibition at 25 mg/mL concentration, were fur-
ther tested at 12.5 mg/mL and 6.25 mg/mL concentrations.
Similarly, the antifungal activity of the synthesized com-
pounds was determined in vitro by cup plate method against
fungal strain C. albicans (ATCC 2091) at 200 mg/mL, 100 mg/
mL, 50 mg/mL and 25 mg/mL concentrations in sabouraud
dextrose medium. Ketoconazole was used as standard drug at
50 mg/mL, 25 mg/mL, 12.5 mg/mL and 6.25 mg/mL concentra-
tions. The test compounds which showed inhibition at 25 mg/
mL concentration, were further tested at 12.5 mg/mL and
6.25 mg/mL concentrations. Solutions of required concentra-
tions of test compounds were prepared by dissolving the com-
pounds in DMF. The minimum inhibitory concentration (MIC)
obtained for the test compounds and reference drugs are re-
ported in Table 1. The minimum inhibitory concentration
(MIC) was defined as the lowest concentration of the com-
pounds that inhibited visible growth of microorganisms on
the plate.
5.5. Lipid peroxidation
Lipid peroxidation in the gastric mucosa was determined ac-
cording to the method of Ohkawa et al. [26]. After screening
for ulcerogenic activity, the gastric mucosa was scraped with
two glass slides, weighed (100 mg) and homogenized in
1.8 mL of 1.15% ice-cold KCl solution. The homogenate was
supplemented with 0.2 mL of 8.1% sodium dodecyl sulphate
(SDS), 1.5 mL of acetate buffer (pH 3.5) and 1.5 mL of 0.8%
thiobarbituric acid (TBA). The mixture was heated at 95 ꢂC
for 60 min. After cooling the reactants were supplemented
with 5 mL of a mixture of n-butanol and pyridine (15:1 v/v),
shaken vigorously for 1 min., and centrifuged for 10 min at
4000 rpm. The supernatant organic layer was taken out and ab-
sorbance was measured at 532 nm on UV spectrophotometer.
The results were expressed as nmols MDA/100 mg tissue, us-
ing extinction coefficient 1.56 ꢃ 105 cmꢁ1Mꢁ1
.
5.6. Hepatotoxic studies
The study was carried out on Wistar albino rats of either
sex weighing 150e200 g. The animals were divided into three
groups of six rats each. Group I was kept as control and re-
ceived only vehicle (0.5% w/v solution of CMC in water),
while group II and III received compounds 4a and 7c, respec-
tively, in 0.5% w/v solution of CMC in water for 15 days. Af-
ter the treatment (15 days) blood was obtained from all the
groups of rats by puncturing the retro-orbital plexus. Blood
samples were allowed to clot for 45 min at room temperature
and serum was separated by centrifugation at 2500 rpm for
15 min and analyzed for various biochemical parameters.
Acknowledgements
The authors are thankful to Head, Department of Pharma-
ceutical Chemistry for providing laboratory facilities, Central
Drug Research Institute (CDRI) for spectral analysis of the
compounds, and Majeedia Hospital, Hamdard University for
providing necessary strains of bacteria and fungus. Authors
are also thankful to Mrs. Shaukat Shah, in-charge animal
house, Hamdard University for providing Wistar rats, and
Dr. A. Mukherjee, MD, Department of Pathology, All India In-
stitute of Medical Sciences (AIIMS), New Delhi, for carrying
out histopathological studies.
5.6.1. Assessment of liver function
Assessment of liver function such as serum glutamate oxa-
loacetate transaminase (SGOT) and serum glutamate pyruvate
transaminase (SGPT) were estimated by a reported method
[27]. The alkaline phosphatase, total protein and total albumin
were measured according to reported procedures [28e30]. All
the data obtained are shown in Table 3.
References
[1] M.B. Kimmey, J. Rheumatol. 19 (1992) 68e73.
[2] C.J. Smith, Y. Zhang, C.M. Koboldt, J. Muhammad, B.S. Zwefel,
A. Shaffer, J.J. Talley, J.L. Masferrer, K. Serbert, P.C. Isakson, Proc.
Natl. Acad. Sci. U.S.A. 95 (1998) 13313e13318.

2066
Mohd. Amir et al. / European Journal of Medicinal Chemistry 43 (2008) 2056e2066
[3] C. Hawkey, L. Laine, T. Simon, A. Beaulieu, J. Maldonado-Cocco,
E. Acevedo, A. Shahane, H. Quan, J. Bolognese, E. Mortensen, Arthritis.
Rheum. 43 (2000) 370e377.
[17] A. Kalgutar, B. Crews, S. Rowlinson, C. Garner, K. Siebert, L. Marnett,
Science 280 (1998) 1268e1270.
[18] M. Amir, S. Kumar, Eur. J. Med. Chem. 39 (2004) 535e545.
[19] M. Amir, S. Kumar, Arch. Pharm. Chem. Life Sci. 338 (2005) 24e31.
[20] J.R. Reid, N.D. Heindel, J. Heterocycl. Chem. 13 (1976) 925e932.
[21] C.A. Winter, E.A. Risley, G.N. Nus, Proc. Soc. Exp. Biol. 111 (1962)
544e547.
[4] T.D. Warner, F. Giuliano, I. Vaynovie, A. Bukasa, J.A. Mitchell,
J.R. Vave, Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 7563e7568.
[5] F.L. Lanza, Am. J. Gastroenterol. 93 (1998) 2037e2046.
[6] D.H. Boschelli, D.T. Connor, D.A. Barnemeier, J. Med. Chem. 36 (1993)
1802e1810.
[22] Y. Naito, T. Yoshikawa, N. Yoshinda, M. Kondo, Dig. Dis. Sci. 43 (1998)
30se34s.
[7] P.C. Unangst, G.P. Shrum, D.T. Connor, D.R. Dyer, D.J. Schrier, J. Med.
Chem. 35 (1992) 3691e3698.
[23] T. Pohle, T. Brzozowski, J.C. Becker, I.R. Vander Voort, A. Markmann,
S.J. Konturek, A. Moniczewski, W. Domschke, J.W. Konturek, Aliment.
Pharmacol. Ther. 15 (2001) 677e687.
[8] M. Amir, M.S.Y. Khan, M.S. Zaman, Indian J. Chem. 43B (2004)
2189e2194.
[9] S.S.P. Garoufalias, E. Tani, O. Todoulou, A.P. Valiraki, E. Filippatos,
E.D. Clercq, P.N. Kourounakis, J. Pharm. Pharmacol. 50 (1998) 117e124.
[10] X.P. Hui, C.H. Zang, Q. Wang, Q. Zhang, Indian J. Chem. 41B (2002)
2176e2179.
[24] P.A.J. Janssen, C.J.E. Niemegeers, J.G.H. Dony, Drug Res. 6 (1963)
502e507.
[25] V. Cioli, S. Putzolu, V. Rossi, S.P. Barcellona, C. Corradino, Toxicol.
Appl. Pharmacol. 50 (1979) 283e289.
[11] T. Tsukuda, Y. Shiratori, M.H. Watanade, K. Ontsuka, M. Hattori,
N. Shirai, Shimma, Bioorg. Med. Chem. Lett. 8 (1998) 1819e1824.
[12] B. Berk, E. Aktay, E. Yesilada, M. Ertan, Pharmazie 56 (2001)
613e616.
[26] H. Ohkawa, N. Ohishi, K. Yagi, Anal. Biochem. 95 (1979) 351e358.
[27] S. Reitman, S. Frankel, Am. J. Clin. Pathol. 28 (1957) 56.
[28] E.J. King, A.R.A. Armstrong, Can. Med. Assoc. J. 31 (1934) 376e381.
[29] J.G. Reinhold, in: M. Reiner (Ed.), Standard Methods in Clinical Chem-
istry, first ed. Academic Press, New York, 1953, pp. 88e90.
[30] H. Varley (Ed.), Practical Clinical Biochemistry, first ed. CBS Publishers
and Distributors, New Delhi, 1988, pp. 236e238.
[31] L.G. Luna, in: Manual of Histological Staining Methods of the Armed
Forces Institute of Pathology, third ed. Mc-Graw-Hill, New York,
1968, pp. 567e568.
[13] B.S. Holla, K.N. Poojary, B.S. Rao, M.K. Shivananda, Eur. J. Med.
Chem. 37 (2002) 511e517.
[14] S.N. Swamy, Basappa, B.S. Priya, B. Prabhuswamy, B.H. Doreswamy,
J.S. Prasad, K.S. Rangappa, Eur. J. Med. Chem. 41 (2006) 531e538.
[15] A. Kalgutar, A. Marnett, B. Crews, R. Remmel, L. Marnett, J. Med.
Chem. 43 (2000) 2860e2870.
[16] M. Duflos, M. Nourrison, J. Brelet, J. Courant, G. Le Baut, N. Grimaud,
J. Petit, Eur. J. Med. Chem. 36 (2001) 545e553.
[32] A.L. Barry, The Antimicrobial Susceptibility Test: Principle and Prac-
tices, Lea and Febiger, Philadelphia, USA, 1976, p. 180.