1,3,4-Oxadiazole/thiadiazole and 1,2,4-triazole derivatives of biphenyl-4-yloxy acetic acid: Synthesis and preliminary evaluation of biological properties

Article abstract of DOI:10.1016/j.ejmech.2008.01.039

A series of 1,3,4-oxadiazole/thiadiazole and 1,2,4-triazole derivatives of biphenyl-4-yloxy acetic acid were synthesized in order to obtain new compounds with potential anti-inflammatory activity, analgesic activity and lower ulcerogenic potential. All co

Full text of DOI:10.1016/j.ejmech.2008.01.039

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 2688e2698
http://www.elsevier.com/locate/ejmech
Original article
1,3,4-Oxadiazole/thiadiazole and 1,2,4-triazole derivatives of
biphenyl-4-yloxy acetic acid: Synthesis and preliminary
evaluation of biological properties*
*
Harish Kumar, Sadique A. Javed, Suroor A. Khan, Mohammad Amir
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University, Hamdard Nagar, New Delhi 110 062, India
Received 15 July 2007; received in revised form 10 January 2008; accepted 18 January 2008
Available online 8 February 2008
Abstract
A series of 1,3,4-oxadiazole/thiadiazole and 1,2,4-triazole derivatives of biphenyl-4-yloxy acetic acid were synthesized in order to obtain new
compounds with potential anti-inflammatory activity, analgesic activity and lower ulcerogenic potential. All compounds were evaluated for their
anti-inflammatory activity by the carrageenan induced rat paw edema test method. The compounds possessing potent anti-inflammatory activity
were further tested for their analgesic, ulcerogenic and antioxidant activities. Out of all tested compounds, the compounds 3, 7, 17 and 20, showed
significant reduction in rat paw edema induced by carrageenan treatment. These compounds showed significant analgesic effect and at an equi-
molar oral doses relative to flurbiprofen were also found to be non-gastrotoxic in rats. Compound 17 was evaluated as the lead compound having
more anti-inflammatory activity (81.81%) than the reference drug (79.54%), low ulcerogenic potential and protective effect on lipid peroxidation.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: 1,3,4-Oxadiazole/thiadiazoles; 1,2,4-Triazole; Anti-inflammatory; Analgesic; Ulcerogenic; Lipid peroxidation
1. Introduction
selectivity for COX-1 than COX-2 [3e5]. Therefore chronic
use of NSAIDs may elicit appreciable GI irritation, bleeding
and ulceration. The incidence of clinically significant GI
side effects is high (over 30%) and causes some patients to
abandon NSAID therapy [6]. Thus the discovery of COX-2
provided the rationale for the development of drugs devoid
of GI disorders while retaining clinical efficacy as anti-inflam-
matory agents. But the recent reports showed that selective
COX-2 inhibitors (coxibs) could lead to adverse cardiovascu-
lar effects [7]. Therefore, development of novel compounds
having anti-inflammatory and analgesic activity with im-
proved safety profile is still a necessity.
Synthetic approaches based upon NSAIDs’ chemical modi-
fication have been taken with the aim of improving their safety
profile. Literature survey revealed that derivatization of the
carboxylate function of NSAIDs resulted in retained anti-
inflammatory activity with reduced ulcerogenic potential [8e
11]. It has also been reported in literature that certain com-
pounds bearing 1,3,4-oxadiazole/thiadiazole and 1,2,4-triazole
nucleus possess significant anti-inflammatory activity [12e15].
Non-steroidal anti-inflammatory drugs (NSAIDs) are
widely used to treat the sign and symptoms of inflammation,
particularly arthritic pain. NSAIDs exert their anti-inflamma-
tory effect mainly through inhibition of cyclooxygenases
(COXs), the key enzyme in prostaglandin (PG) biosynthesis
from arachidonic acid (AA). There are at least two COX
isoforms COX-1 and COX-2 [1,2]. Constitutive COX-1 is re-
sponsible for providing cytoprotection in gastrointestinal
(GI) tract whereas inducible COX-2 mediates inflammation.
Traditional NSAIDs such as aspirin, diclofenac, flurbiprofen
and ibuprofen are non-selective; however, they show greater
*
Part of the work was presented at the International Conference on Emerg-
ing Trends in Chemical Sciences (ICETCS-2007) held in January 2007 at
Mumbai, India.
* Corresponding author. Tel.: þ91 11 26059878; fax: þ91 11
26059688x5307.
E-mail address: mamir_s2003@yahoo.co.in (M. Amir).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.01.039

H. Kumar et al. / European Journal of Medicinal Chemistry 43 (2008) 2688e2698
2689
As reported [16], during the study of the pharmacological prop-
erties of a large number of substituted phenyl alkanoic acids, the
most potent were found to be substituted 2-(4-biphenylyl)
propionic acids and among these, flurbiprofen was discovered
as possessing the most favourable therapeutic profile as an
anti-inflammatory agent (Fig. 1). Furthermore, the substitution
of the carboxylic acid moiety by the bioisosteric groups such
as 1,3,4-oxadiazole/thiadiazole and 1,2,4-triazole has also
been reported [17,18]. In view of these observations and in con-
tinuation of our research programme on the synthesis of 5-mem-
bered heterocyclic compounds of aryl alkanoic acid derivatives
[19e21], we report herein the synthesis of some newer, more
potent analogues of biphenyl-4-yloxy acetic acid in which the
CHeCH3 group of 2-(4-biphenylyl)propionic acid has been re-
placed by OCH2 group. Thus we have designed and synthesized
a series of 1,3,4-oxadiazole, 1,2,4-triazole and 1,3,4-thiadiazole
derivatives, which have been found to possess an interesting pro-
file of anti-inflammatory and analgesic activity with significant
reduction in their ulcerogenic risks in the stomach.
CSNH and CONH protons were observed as singlets at
d 9.50 and d 10.00, respectively, confirming the formation of
thiosemicarbazide. The mass spectrum of compound 14
showed molecular ion peak Mþ at m/z 405 corresponding to
molecular formula C23H23N3O2S. The thiosemicarbazides 9
and 12 were oxidatively cyclised to 5-[(biphenyl-4-yloxy)-
methyl]-2-alkyl/arylamino-1,3,4-oxadiazole 15 and 16, by
elimination of H2S using iodine and potassium iodide in etha-
nolic sodium hydroxide. The IR spectra of oxadiazole 15
showed absorption peak at 1624 cmꢀ1 due to C]N streching
1
vibration. The structure was further supported by its H NMR
spectrum, which showed a multiplet at d 6.88e7.61 for nine
aromatic protons. The disappearance of CONH and CSNH sin-
glet signals of thiosemicarbazide and appearance of an NH
signal at d 10.09 confirms the formation of oxadiazole ring.
Mass spectra of compound 15 showed molecular ion peak
Mþ at m/z 323 corresponding to molecular formula
C19H21N3O2. The thiosemicarbazides 9e14 on heating with
4 N NaOH in ethanol underwent smooth cyclisation through
dehydration to afford 5-[(biphenyl-4-yloxy)methyl]-4-alkyl/
aryl-3-mercapto-(4H )-1,2,4-triazoles 17e22. The structure
of triazole 17 was confirmed by its IR spectrum, which dis-
played absorption peak at 1614 cmꢀ1 due to C]N and at
2604 cmꢀ1 corresponding to SH group. The formation of tria-
2. Chemistry
The hydrazide 1 was prepared by esterification of biphenyl-
4-yloxy acetic acid followed by treatment with hydrazine
hydrate in absolute ethanol. Various 5-[(biphenyl-4-yloxy)-
methyl]-2-substituted-1,3,4-oxadiazoles 2e8 were prepared
by treatment of hydrazide with appropriate aromatic acids in
the presence of phosphorus oxychloride (Scheme 1). The
structures of compounds were established on the basis of their
elemental analysis and spectral data. The IR spectrum of com-
pound 7 showed absorption peak at 1620 cmꢀ1 due to C]N
1
zole ring was further supported by its H NMR spectrum,
which showed a triplet of NCH2 protons at d 4.12. A singlet
of SH proton was also observed at d 11.29. The mass spectra
of compound 17 showed molecular ion peak Mþ at m/z 339
corresponding to molecular formula C19H21N3OS.
5-[(Biphenyl-4-yloxy)methyl]-2-alkyl/aryl
amino-1,3,4-
thiadiazoles 23e28 were obtained by cyclisation of 9e14 by
treating with cold concentrated sulphuric acid. The IR spec-
trum of compound 23 showed absorption peak at 1608 cmꢀ1
1
streching vibrations. Its H NMR spectrum revealed a quartet
and doublet at d 3.94 and d 1.61 for CH and CH3 protons, re-
spectively. Furthermore the appearance of a singlet for (CH3)2
group at d 0.90, doublet for CH2 group at d 2.46 and a multiplet
for CH group at d 1.81e1.91 confirmed the presence of an iso-
butyl group attached to the phenyl ring. A multiplet of aro-
matic protons was observed at d 6.90e7.81. The OCH2
protons were observed as a singlet at d 4.92. The mass spec-
trum of compound 7 showed molecular ion peak Mþ at m/z
412 corresponding to molecular formula C27H28N2O2. Treat-
ment of compound 1 with various alkyl/aryl isothiocyanates
in ethanol gave corresponding N1[2-(biphenyl-4-yloxy)etha-
noyl]-N4-alkyl/aryl-thiosemicarbazides 9e14 (Table 1). The
structure of thiosemicarbazide 14 was confirmed by its IR
spectrum which displayed absorption peaks at 3124 cmꢀ1 for
NH, 1683 cmꢀ1 for C]O and 1063 cmꢀ1 corresponding to
1
due to C]N streching vibrations. In the H NMR spectrum
of compounds the singlets of CSNH and CONH of thiosemi-
carbazide had disappeared, and a multiplet was obtained in
the aromatic region at d 7.20e7.96 for nine aromatic protons.
The NH proton was observed as a singlet at d 9.97 confirming
the structure of the compound. The mass spectra of compound
23 showed molecular ion peak Mþ at m/z 339 corresponding to
molecular formula C19H21N3OS.
3. Pharmacological results and discussion
The anti-inflammatory activity of the synthesized com-
pounds 2e8, 15e28 was evaluated by the carrageenan
induced paw edema method of Winter et al. [23]. The com-
pounds were tested at an equimolar oral dose relative to
10 mg/kg flurbiprofen. The percentage inhibition was calcu-
lated both after 3 and 4 h, and since it was found to be more
after 4 h, this was made the basis of discussion. The tested
compounds showed anti-inflammatory activity ranging from
15.90% to 81.81% (Table 2), whereas standard drug
flurbiprofen showed 79.54% inhibition after 4 h. The anti-
inflammatory activity of 1,3,4-oxadiazole derivatives 2e8,
15, 16 was in the range of 15.90% to 79.54%. The oxadiazole
derivative 7 having 2-(4-isobutylphenyl)ethyl group showed
1
C]S stretching vibrations. The H NMR spectrum showed
a multiplet at d 6.92e7.53 for 12 aromatic protons. The
CH3
COOH
CH3
COOH
F
2-(4-Biphenylyl)propionic acid
Fig. 1.
Flurbiprofen

2690
H. Kumar et al. / European Journal of Medicinal Chemistry 43 (2008) 2688e2698
O
O
NH2
N
H
(1)
RCOOH
POCl3
RNCS
Abs. C2H5OH
N
N
O
O
H
N
H
N
O
R
O
N
H
R
S
(2-8)
Conc. H2SO4
Stirring
(9-14)
C2H5OH
4N NaOH
Reflux
N
N
KI/I2
Reflux
C2H5OH
O
5N NaOH
S
R
N
N
N
N
R
O
N
R
SH
(23-28)
O
O
(17-22)
(15-16)
Scheme 1. Synthesis of target compounds.
activity equivalent to the standard drug flurbiprofen (79.54%),
whereas when this group was replaced by a phenyl group (2),
activity was found to be minimal (15.90%). It was observed
that oxadiazole derivatives having p-chlorophenyl (3), o-chlor-
ophenyl (4), 2,4-dichlorophenyl (5) and p-fluorophenylamino
(16) groups at second position showed decreasing order of
anti-inflammatory activity (77.27%, 63.63%, 57.57% and
52.27%, respectively). The rest of the oxadiazole derivatives
showed weak activity.
The anti-inflammatory activity of 1,2,4-triazole derivatives
17e22 was found between 18.18% to 81.81%. The highest ac-
tivity (81.81%) was found in the triazole derivative 17 having
n-butyl group at position 4. When this group was replaced by
p-fluorophenyl group (20), the activity was slightly decreased
and found equivalent to standard drug ibuprofen (79.54%). It
was observed that replacement of 4-fluorophenyl group by
4-chlorophenyl group (18) resulted in minimum activity
(18.18%). The rest of the triazole derivatives showed moderate
to weak anti-inflammatory activity. The 1,3,4-thiadiazole de-
rivatives 23e28 showed anti-inflammatory activity between
27.27% and 63.63%. It was observed that thiadiazole deriva-
tives having p-chlorophenylamino (24), p-bromophenylamino
(25) and 2,4-dichlorophenylamino (28) groups at second posi-
tion showed good activity viz. 54.54%, 63.63% and 56.81%,
respectively. The rest of the thiadiazole derivatives showed
weak anti-inflammatory activity.
analgesic activity (72.8%) was shown by 1,3,4-oxadiazole de-
rivative 7, which also has significant anti-inflammatory activity
(79.54%). The remaining compounds showed reduced analgesic
activity. These compounds were further screened for their acute
ulcerogenic activity. The compounds were tested at an equimo-
lar oral dose relative to 30 mg/kg flurbiprofen. The tested com-
pounds showed significant reduction in ulcerogenic activity
ranging from 0.66 ꢁ 0.10 to 1.00 ꢁ 0.34, whereas standard
drug flurbiprofen showed high severity index of 1.66 ꢁ 0.24.
The maximum reduction in ulcerogenic activity (0.66 ꢁ 0.10)
was found in compound 17 having the n-butyl group at position
4 of triazole ring. The rest of the compounds also showed better
GI safety profile as compared to flurbiprofen, as illustrated in
Table 3. Thus the results showed that substitution of the
carboxylic group by the bioisosteric groups 1,3,4-oxadiazole/
thiadiazole and 1,2,4-triazole, has resulted in significant anti-
inflammatory and analgesic activities along with reduced
ulcerogenic potential.
It has been reported that compounds showing less ulcero-
genic activity also showed reduced malondialdehyde (MDA)
content, a byproduct of lipid peroxidation [22]. Therefore, an
attempt was made to correlate the decrease in ulcerogenic ac-
tivity of the compounds with that of lipid peroxidation. All
the compounds screened for ulcerogenic activity were also an-
alyzed for their effect on lipid peroxidation. Lipid peroxidation
is measured as nanomoles of malondialdehyde (MDA)/100 mg
of gastric mucosa tissue. Flurbiprofen exhibited maximum tis-
sue lipid peroxidation 7.51 ꢁ 0.68, whereas control group
showed 3.25 ꢁ 0.05. It was found that all the cyclised deriva-
tives showing less ulcerogenic activity also showed reduction
in lipid peroxidation (Table 3). Thus these studies showed
that the 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 peroxidation in the gastric mucosa.
The compounds 3, 7, 17 and 20 which showed comparable or
equal anti-inflammatory activity to that of standard reference
drug, were further tested for their analgesic activity at an equi-
molar oral dose relative to 10 mg/kg flurbiprofen (Table 3).
The compounds showed analgesic activity ranging from
16.9% to 72.8%, whereas the standard drug flurbiprofen showed
69.5% inhibition. It was noted that compound 17, a triazole
derivative showing the highest anti-inflammatory activity, also
exhibited significant analgesic activity (58.2%). The highest

H. Kumar et al. / European Journal of Medicinal Chemistry 43 (2008) 2688e2698
2691
Table 1
Value of R, yields and melting points of the synthesized compounds
O
N
N
N
N
H
N
H
N
O
O
O
N
H
R
N
R
SH
X
R
S
X = O: 2-8 & 15,16; S: 23-28
Yield (%)
9-14
17-22
Compound
R
M.P (ꢂC)
134
2
51
50
3
4
180
102
Cl
Cl
63
47
61
58
Cl
5
6
7
98
58
Cl
Cl
Cl
O
144
8
66
122
H3CO
9
CH3CH2CH2CH2e
67
63
146
182
10
Cl
11
12
69
59
134
174
Br
F
CH3
13
14
55
62
130
104
CH3
H3C
15
16
17
CH3CH2CH2CH2NHe
62
64
65
>300
98
N
H
F
CH3CH2CH2CH2e
148
18
65
190
(continued on next page)
Cl

2692
H. Kumar et al. / European Journal of Medicinal Chemistry 43 (2008) 2688e2698
Table 1 (continued)
Compound
R
Yield (%)
71
M.P (ꢂC)
216
19
20
Br
74
68
188
174
F
CH3
21
CH3
22
23
24
25
61
72
65
63
170
172
H3C
CH3CH2CH2CH2NHe
238
N
H
Cl
Br
>300
N
H
26
27
60
58
242
178
N
F
H
CH3
N
H
CH3
28
43
274
N
H3C
H
Two compounds, 7 and 17, showing potent anti-inflamma-
tory and analgesic activities with reduced ulcerogenicity and
lipid peroxidation, were further studied for their hepatotoxic
effect. Both compounds were studied for their effect on bio-
chemical parameters (serum enzymes, total protein and total
albumin) and histopathology of liver. As shown in Table 4
both compounds showed significant reduction in SGOT level
in comparison to that of control. Compound 17 also showed
significant reduction in SGPT (22.67 IU/ml) and increase in
alkaline phosphatase level (16.76 g/ml) in comparison to that
of control. The difference in enzyme level in biochemical
studies of compounds 7 and 17 is insignificant. This is further
confirmed by the histopathological studies of liver samples
(Figs. 2,3), which have not shown any significant pathological
changes in comparison to control group (Fig. 4). No hepato-
cyte necrosis or degeneration was seen in any of the samples.
as potential anti-inflammatoryeanalgesic agents with minimum
ulcerogenic potential and lipid peroxidation. In conclusion, this
preliminary investigation showed that the carboxylic group of
biphenyl-4-yloxy acetic acid can be cyclised into 1,3,4-oxadia-
zole/thiadiazole and 1,2,4-triazole nuclei having significant
biological activities. Among the synthesized compounds 7 and
17 possessed the most prominent and consistent activity with
maximum reduction of gastrointestinal toxicity, minimum lipid
peroxidation, and no hepatocyte necrosis or degeneration.
Therefore this series has opened new doors for possible modifi-
cations of the pharmacophoric requirements of NSAIDs and fu-
ture exploitations.
5. Experimental protocols
5.1. Chemistry
4. Conclusion
Chemicals were purchased from Merck Chemical Com-
pany, S. D. Fine (India) and Qualigens (India). Melting points
were determined in open capillary tubes and are uncorrected.
IR (KBr) spectra were recorded on a Nicolet, 5PC FTIR spec-
trometer (nmax in cmꢀ1) and 1H NMR spectra were recorded in
We have described the preparation of 1,3,4-oxadiazole, 1,2,4-
triazole and 1,3,4-thiadiazole derivatives of biphenyl-4-yloxy
acetic acid. Several of these compounds have been evaluated

H. Kumar et al. / European Journal of Medicinal Chemistry 43 (2008) 2688e2698
2693
Table 2
Anti-inflammatory activity of the synthesized compounds
Compound
Paw Volume
0 h (Basal)
% Inhibition ꢁ SEMa
After 3 h
Potency
After 3 h
After 4 h
After 4 h
2
0.35 ꢁ 0.014
0.39 ꢁ 0.012
0.36 ꢁ 0.015
0.34 ꢁ 0.009
0.34 ꢁ 0.010
0.36 ꢁ 0.017
0.34 ꢁ 0.015
0.31 ꢁ 0.014
0.32 ꢁ 0.021
0.32 ꢁ 0.009
0.36 ꢁ 0.016
0.36 ꢁ 0.013
0.37 ꢁ 0.011
0.36 ꢁ 0.014
0.35 ꢁ 0.011
0.32 ꢁ 0.014
0.34 ꢁ 0.015
0.37 ꢁ 0.010
0.37 ꢁ 0.012
0.33 ꢁ 0.015
0.35 ꢁ 0.017
0.33 ꢁ 0.011
0.33 ꢁ 0.011
0.71 ꢁ 0.014
0.50 ꢁ 0.014
0.54 ꢁ 0.018
0.54 ꢁ 0.013
0.65 ꢁ 0.017
0.46 ꢁ 0.016
0.67 ꢁ 0.018
0.68 ꢁ 0.029
0.53 ꢁ 0.015
0.43 ꢁ 0.017
0.73 ꢁ 0.017
0.66 ꢁ 0.019
0.48 ꢁ 0.015
0.55 ꢁ 0.015
0.55 ꢁ 0.011
0.64 ꢁ 0.021
0.55 ꢁ 0.016
0.53 ꢁ 0.014
0.69 ꢁ 0.019
0.66 ꢁ 0.022
0.54 ꢁ 0.024
0.44 ꢁ 0.016
0.77 ꢁ 0.013
0.71 ꢁ 0.014
0.49 ꢁ 0.015
0.52 ꢁ 0.017
0.53 ꢁ 0.014
0.65 ꢁ 0.016
0.45 ꢁ 0.015
0.67 ꢁ 0.018
0.68 ꢁ 0.029
0.53 ꢁ 0.016
0.40 ꢁ 0.016
0.72 ꢁ 0.019
0.66 ꢁ 0.017
0.46 ꢁ 0.016
0.54 ꢁ 0.014
0.55 ꢁ 0.013
0.64 ꢁ 0.022
0.54 ꢁ 0.017
0.53 ꢁ 0.014
0.68 ꢁ 0.018
0.64 ꢁ 0.021
0.54 ꢁ 0.024
0.42 ꢁ 0.019
0.77 ꢁ 0.016
18.18 ꢁ 4.23
75.00 ꢁ 2.53
59.09 ꢁ 3.60
56.06 ꢁ 3.25
29.54 ꢁ 2.34
77.27 ꢁ 1.92
25.00 ꢁ 1.91
15.90 ꢁ 2.56
52.27 ꢁ 2.81
75.00 ꢁ 2.73
15.90 ꢁ 2.73
31.81 ꢁ 3.45
75.00 ꢁ 2.79
56.81 ꢁ 1.94
54.54 ꢁ 2.62
27.27 ꢁ 2.65
52.27 ꢁ 2.17
63.63 ꢁ 1.92
27.27 ꢁ 2.73
25.00 ꢁ 1.94
56.81 ꢁ 2.55
75.00 ꢁ 2.53
e
18.18 ꢁ 4.23**
77.27 ꢁ 1.91
0.22
0.97
0.79
0.71
0.36
1.00
0.32
0.20
0.65
1.02
0.24
0.39
1.00
0.74
0.67
0.34
0.67
0.80
0.37
0.37
0.71
1.00
e
3
4
63.63 ꢁ 3.79**
57.57 ꢁ 3.45**
29.54 ꢁ 2.27**
79.54 ꢁ 1.91
5
6
7
8
15
25.00 ꢁ 1.91**
15.90 ꢁ 2.56**
52.27 ꢁ 2.79**
81.81 ꢁ 2.17
16
17
18
19
18.18 ꢁ 2.25**
31.81 ꢁ 3.71**
79.54 ꢁ 3.19
20
21
59.09 ꢁ 2.73**
54.54 ꢁ 3.20**
27.27 ꢁ 2.76**
54.54 ꢁ 2.62**
63.63 ꢁ 1.92**
29.54 ꢁ 2.79**
29.54 ꢁ 2.17**
56.81 ꢁ 2.16**
79.54 ꢁ 2.25
22
23
24
25
26
27
28
Flurbiprofen
Control
e
a
Relative to standard and data were analyzed by ANOVA followed by Dunnett’s multiple comparison test for n ¼ 6; **p < 0.01.
CDCl3/DMSO-d6 on a Bruker DRX-300 (300 MHz FT NMR)
spectrometer using TMS as internal reference (Chemical shift
in d ppm). Mass spectra were recorded using Jeol SX-102
spectrometer. The purity of various synthesized compounds
was checked by TLC and elemental analysis. Spectral data
(1H NMR, IR and mass) of the synthesized compounds were
in full agreement with the proposed structures.
phosphorus oxychloride (10 ml) was refluxed for 4e6 h. The
reaction mixture was slowly poured over crushed ice and
kept overnight. The solid thus separated out was filtered,
treated with dilute NaOH, washed with water and recrystal-
lised from ethanol.
5.1.2.1. 5-[(Biphenyl-4-yloxy)methyl]-2-phenyl-1,3,4-oxadia-
zole (2). IR (KBr) n cmꢀ1: 2934 (CeH), 1612 (C]N), 1232
1
(CeOeC). H NMR (CDCl3) d (ppm): 7.27e7.61 (m, 14H,
5.1.1. Synthesis of 2-(biphenyl-4-yloxy) acetic
acid hydrazide (1)
Compound (1) was prepared by the procedure given in the
literature [20].
aromatic), 5.29 (s, 2H, OCH2). MS: m/z 328 (Mþ). Anal.
Calcd. for C21H16N2O2: C, 76.81; H, 4.91; N, 8.53. Found:
C, 76.72; H, 4.99; N, 8.49.
5.1.2.2. 5-[(Biphenyl-4-yloxy)methyl]-2-(4-chlorophenyl)-1,3,-
5.1.2. Synthesis of 5-[(biphenyl-4-yloxy)methyl]-
2-substituted-1,3,4-oxadiazoles (2e8)
A mixture of 2-(biphenyl-4-yloxy) acetic acid hydrazide
(0.001 M) and the appropriate aromatic acid (0.001 M) in
4-oxadiazole (3). IR (KBr)
n
1
cmꢀ1
:
2929 (CeH),
1621 (C]N), 1239 (CeOeC). H NMR (CDCl3) d (ppm):
7.27e8.17 (m, 13H, aromatic), 4.72 (s, 2H, OCH2).
Table 3
Analgesic, ulcerogenic and lipid peroxidation activities of selected compounds
Compound
Analgesic Activitya
Ulcerogenic activity
Nanomoles of MDA
(severity index ꢁ SEM)b content ꢁ SEM/100 mg tissueb
Pre-treatment/normal 0 h (s) Post-treatment/after 4 h (s) % Inhibition Potency
(normal)
3
1.18 ꢁ 0.12
1.40 ꢁ 0.150
1.34 ꢁ 0.136
1.80 ꢁ 0.189
1.38 ꢁ 0.143
2.42 ꢁ 0.117
2.12 ꢁ 0.144
2.35 ꢁ 0.180
1.95 ꢁ 0.097
e
16.9**
72.8***
58.2***
30.5***
69.5***
e
0.24
1.04
0.83
0.43
1.00
1.000 ꢁ 0.34
0.750 ꢁ 0.17*
0.666 ꢁ 0.10*
0.833 ꢁ 0.25
1.666 ꢁ 0.24
0.00
5.88 ꢁ 0.38
7
5.28 ꢁ 0.45**
5.19 ꢁ 0.22**
5.34 ꢁ 0.37**
7.51 ꢁ 0.68
17
20
Flurbiprofen 1.15 ꢁ 0.060
Control
e
3.25 ꢁ 0.05
a
Relative to normal and data were analyzed by paired student’s t-test for n ¼ 6; ***p < 0.0001, **p < 0.001.
b
Relative to standard and data were analyzed by ANOVA followed by Dunnett’s multiple comparison test for n ¼ 6; **p < 0.01, *p < 0.05.

2694
H. Kumar et al. / European Journal of Medicinal Chemistry 43 (2008) 2688e2698
Table 4
Effect of selected compounds on serum enzymes, total proteins and total albumin
Compound
SGOT,a units/ml
SGPT,a units/ml
Alkaline phosphatasea
Total protein,a g/dl
Total albumin,a g/dl
Control
7
148.67 ꢁ 1.50
27.67 ꢁ 0.84
29.17 ꢁ 0.75
22.67 ꢁ 0.56**
13.06 ꢁ 0.25
12.82 ꢁ 0.16
16.76 ꢁ 0.15**
1.80 ꢁ 0.01
1.92 ꢁ 0.06
1.82 ꢁ 0.06
1.67 ꢁ 0.01
1.72 ꢁ 0.16
1.71 ꢁ 0.05
141.09 ꢁ 1.65**
138.00 ꢁ 1.13**
17
a
Relative to control and data were analyzed by ANOVA followed by Dunnett’s multiple comparison test, for n ¼ 6; **p < 0.01.
MS: m/z 362 (Mþ), 364 (Mþ þ 2). Anal. Calcd. for
C21H15ClN2O2: C, 69.52; H, 4.17; N, 7.72. Found: C, 69.59;
H, 4.19; N, 7.68.
6.90e7.81 (m, 13H, aromatic), 4.92 (s, 2H, OCH2), 3.94 (q,
J ¼ 7.1 Hz, 1H, CHCH3), 2.46 (d, J ¼ 6.5 Hz, 2H, CH2),
1.82e1.91 (m, 1H, CH2CH), 1.61 (d, J ¼ 7.1 Hz, 3H, CH3),
0.90 [d, J ¼ 6.5 Hz, 6H, (CH3)2]. MS: m/z 412 (Mþ). Anal.
Calcd. for C27H28N2O2: C, 78.61; H, 6.84; N, 6.79. Found:
C, 78.57; H, 6.75; N, 6.72.
5.1.2.3. 5-[(Biphenyl-4-yloxy)methyl]-2-(2-chlorophenyl)-1,3,
4-oxadiazole (4). IR (KBr) n cmꢀ1: 2924 (CeH), 1610
1
(C]N), 1231 (CeOeC). H NMR (CDCl3) d (ppm): 7.25e
8.07 (m, 13H, aromatic), 4.65 (s, 2H, OCH2). MS: m/z 362
(Mþ), 364 (Mþ þ 2). Anal. Calcd. for C21H15ClN2O2: C,
69.52; H, 4.17; N, 7.72. Found: C, 69.57; H, 4.23; N, 7.67.
5.1.2.7. 5-[(Biphenyl-4-yloxy)methyl]-2-[1-(6-methoxy-2-naph-
thyl)ethyl]-1,3,4-oxadiazole (8). IR (KBr) n cmꢀ1: 2927 (CeH),
1
1609 (C]N), 1243 (CeOeC). H NMR (CDCl3) d (ppm):
7.04e7.78 (m, 15H, aromatic), 4.96 (s, 2H, OCH2), 4.13 (q,
J ¼ 6.6 Hz, 3H, CHCH3), 3.92 (s, 3H, OCH3), 1.69 (d,
J ¼ 6.6 Hz, 3H, CH3). MS: m/z 436 (Mþ). Anal. Calcd. for
C28H24N2O3: C, 77.05; H, 5.54; N, 6.42. Found: C, 77.12; H,
5.55; N, 6.39.
5.1.2.4. 5-[(Biphenyl-4-yloxy)methyl]-2-(2,4-dichlorophenyl)-
1,3,4-oxadiazole (5). IR (KBr) n cmꢀ1: 2938 (CeH), 1589
1
(C]N), 1223 (CeOeC). H NMR (CDCl3) d (ppm): 6.90e
8.02 (m, 12H, aromatic), 4.90 (s, 2H, OCH2). MS: m/z 397
(Mþ), 399 (Mþ þ 2), 401 (Mþ þ 4). Anal. Calcd. for
C21H14Cl2N2O2: C, 63.49; H, 3.55; N, 7.05. Found: C,
63.57; H, 3.45; N, 7.09.
5.1.3. Synthesis of N1[2-(biphenyl-4-yloxy)ethanoyl]-N4-
alkyl/aryl-thiosemicarbazides (9e14)
A mixture of 2-(biphenyl-4-yloxy) acetic acid hydrazide
(0.1 M), corresponding alkyl/aryl isothiocyanate (0.1 M) and
ethanol (50 ml) was refluxed for 2e9 h on a water bath. It
was then concentrated, cooled and kept overnight refrigerated.
The solid thus separated out was filtered, dried and recrystal-
lised from methanol/acetone.
5.1.2.5. 5-[(Biphenyl-4-yloxy)methyl]-2-(2,4-dichlorophenoxy-
methyl)-1,3,4-oxadiazole (6). IR (KBr) n cmꢀ1: 2922 (CeH),
1
1605 (C]N), 1238 (CeOeC). H NMR (CDCl3) d (ppm):
6.86e7.81 (m, 12H, aromatic), 4.69 (s, 4H, 2OCH2). MS:
m/z 427 (Mþ), 429 (Mþ þ 2), 431 (Mþ þ 4). Anal. Calcd.
for C22H16Cl2N2O3: C, 61.84; H, 3.77; N, 6.56. Found: C,
61.77; H, 3.75; N, 6.59.
5.1.3.1. N1[2-(Biphenyl-4-yloxy)ethanoyl]-N4-(n-butyl)-thiose-
micarbazide (9). IR (KBr) n cmꢀ1: 3242 (NH), 2947 (CeH),
1673 (C]O), 1248 (CeOeC), 1083 (C]S). 1H NMR
(DMSO-d6) d (ppm): 10.26 (br s, 1H, CONH), 9.76 (br s,
1H, CSNH), 9.23 (br s, 1H, NH), 6.93e7.58 (m, 9H,
5.1.2.6. 5-[(Biphenyl-4-yloxy)methyl]-2-[1-(4-isobutylpheny-
l)ethyl]-1,3,4-oxadiazole (7). IR (KBr) n cmꢀ1: 2956 (CeH),
1
1620 (C]N), 1211 (CeOeC). H NMR (CDCl3) d (ppm):
Fig. 2. Compound 7; section of liver showing portal triad structures (400ꢃ).
Fig. 3. Compound 17; section of liver showing portal triad structures (400ꢃ).

H. Kumar et al. / European Journal of Medicinal Chemistry 43 (2008) 2688e2698
2695
2H, NHCSNHAr), 6.99e7.56 (m, 13H, ArH), 4.61 (s, 2H,
OCH2), 2.59 (s, 3H, CH3). MS: m/z 391 (Mþ). Anal. Calcd.
for C22H21N3O2S: C, 67.50; H, 5.41; N, 10.73. Found: C,
67.59; H, 5.49; N, 10.79.
5.1.3.6. N1[2-(Biphenyl-4-yloxy)ethanoyl]-N4-(2,4-dimethyl-
phenyl)-thiosemicarbazide (14). IR (KBr) n cmꢀ1: 3124
(NH), 2922 (CeH), 1683 (C]O), 1241 (CeOeC), 1063
(C]S). 1H NMR (CDCl3) d (ppm): 10.00 (br s, 1H,
CONH), 9.50 (br s, 1H, CSNH), 8.90 (br s, 1H, ArNH),
6.92e7.53 (m, 12H, aromatic), 4.69 (s, 2H, OCH2), 2.95 (s,
3H, p-CH3), 2.25 (s, 3H, o-CH3). MS: m/z 405 (Mþ). Anal.
Calcd. for C23H23N3O2S: C, 68.12; H, 5.72; N, 10.26. Found:
C, 68.19; H, 5.79; N, 10.19.
5.1.4. Synthesis of 5-[(biphenyl-4-yloxy)methyl]-2-alkyl/
arylamino-1,3,4-oxadiazoles (15,16)
A suspension of corresponding N1[2-(biphenyl-4-yloxy)e-
thanoyl]-N4-alkyl/aryl-thiosemicarbazides (9 and 12) (0.002
M) in ethanol (50 ml) was dissolved in 5 N aq. sodium hy-
droxide solution (1 ml) with cooling and stirring resulting in
the formation of clear solution. To this 5% iodine in potassium
iodide solution was added dropwise with stirring till the colour
of iodine persisted at room temperature. The reaction mixture
was then refluxed for 3e5 h on water bath. It was then concen-
trated, cooled and the solid separated out was filtered, dried
and recrystallised from ethanol.
Fig. 4. Control; section of liver showing portal triad structures (400ꢃ).
aromatic), 4.58 (s, 2H, OCH2), 3.97 (t, J ¼ 7.0 Hz, 2H,
NCH2), 1.46e1.53 (m, 4H, CH3CH2CH2), 0.98 (t,
J ¼ 7.0 Hz, 3H, CH3). MS: m/z 357 (Mþ). Anal. Calcd. for
C19H23N3O2S: C, 63.86; H, 6.44; N, 11.76. Found: C,
63.93; H, 6.49; N, 11.82.
5.1.3.2. N1[2-(Biphenyl-4-yloxy)ethanoyl]-N4-(4-chlorophenyl)-
thiosemicarbazide (10). IR (KBr) n cmꢀ1: 3246 (NH), 2958
(CeH), 1683 (C]O), 1255 (CeOeC), 1099 (C]S). 1H
NMR (DMSO-d6) d (ppm): 10.33 (br s, 1H, CONH), 9.80 (br
s, 1H, CSNH), 9.67 (br s, 1H, ArNH), 7.01e7.63 (m, 13H, ar-
omatic), 4.68 (s, 2H, OCH2). MS: m/z 411 (Mþ). Anal. Calcd.
for C21H18ClN3O2S: C, 61.24; H, 4.40; N, 10.20. Found: C,
61.29; H, 4.49; N, 10.15.
5.1.4.1. 5-[(Biphenyl-4-yloxy)methyl]-2-n-butylamino-1,3,4-
oxadiazole (15). IR (KBr) n cmꢀ1: 3401 (NH), 2916 (Ce
H), 1624 (C]N), 1251 (CeOeC). 1H NMR (DMSO-d6)
d (ppm): 10.09 (br s, 1H, NH), 6.88e7.61 (m, 9H,
aromatic), 5.29 (s, 2H, OCH2), 4.12 (t, J ¼ 7.0 Hz, 2H,
NCH2), 1.50e1.56 (m, 4H, CH3CH2CH2), 1.22 (t,
J ¼ 7.0 Hz, 3H, CH3). MS: m/z 323 (Mþ). Anal. Calcd. for
C19H21N3O2: C, 70.57; H, 6.54; N, 12.99. Found: C, 70.83;
H, 6.59; N, 13.05.
5.1.3.3. N1[2-(Biphenyl-4-yloxy)ethanoyl]-N4-(4-bromophenyl)-
thiosemicarbazide (11). IR (KBr) n cmꢀ1: 3238 (NH), 2965
(CeH), 1686 (C]O), 1264 (CeOeC), 1085 (C]S). 1H
NMR (DMSO-d6) d (ppm): 10.38 (br s, 1H, CONH), 9.86 (br
s, 1H, CSNH), 9.73 (br s, 1H, ArNH), 7.11e7.76 (m, 13H, ar-
omatic), 4.79 (s, 2H, OCH2). MS: m/z 456 (Mþ). Anal. Calcd.
for C21H18BrN3O2S: C, 55.26; H, 3.94; N, 9.21. Found: C,
55.31; H, 3.98; N, 9.17.
5.1.4.2. 5-[(Biphenyl-4-yloxy)methyl]-2-(4-fluorophenylamino)-
1,3,4-oxadiazole (16). IR (KBr) n cmꢀ1: 3336 (NH), 2923
1
(CeH), 1606 (C]N), 1241 (CeOeC). H NMR (DMSO-
d6) d (ppm): 10.17 (br s, 1H, NH), 7.00e8.04 (m, 13H, ar-
omatic), 5.33 (s, 2H, OCH2). MS: m/z 361 (Mþ). Anal.
Calcd. for C21H16FN3O2: C, 69.80; H, 4.46; N, 11.63.
Found: C, 69.87; H, 4.45; N, 11.69.
5.1.3.4. N1[2-(Biphenyl-4-yloxy)ethanoyl]-N4-(4-fluorophenyl)-
thiosemicarbazide (12). IR (KBr) n cmꢀ1: 3258 (NH), 2969
(CeH), 1695 (C]O), 1267 (CeOeC), 1106 (C]S). 1H
NMR (DMSO-d6) d (ppm): 10.38 (br s, 1H, CONH), 9.87
(br s, 1H, CSNH), 9.75 (br s, 1H, ArNH), 7.16e7.84 (m,
13H, aromatic), 4.68 (s, 2H, OCH2). MS: m/z 395 (Mþ).
Anal. Calcd. for C21H18FN3O2S: C, 63.79; H, 4.55; N,
10.63. Found: C, 63.68; H, 4.49; N, 10.57.
5.1.5. Synthesis of 5-[(biphenyl-4-yloxy)methyl]-4-alkyl/
aryl-3-mercapto-(4H )-1,2,4-triazoles (17e22)
A suspension of corresponding N1[2-(biphenyl-4-yloxy)e-
thanoyl]-N4-alkyl/aryl-thiosemicarbazides (0.002 M) in etha-
nol (50 ml) was dissolved in 4 N aq. sodium hydroxide
solution (2 ml) resulting in the formation of clear solution.
The reaction mixture was refluxed for 4e6 h on water bath,
concentrated, cooled and filtered. The pH of the filtrate was
adjusted between 5 and 6 with acetic acid and kept aside for
5.1.3.5. N1[2-(Biphenyl-4-yloxy)ethanoyl]-N4-(2-methylphenyl)-
thiosemicarbazide (13). IR (KBr) n cmꢀ1: 3241 (NH), 2929
(CeH), 1670 (C]O), 1250 (CeOeC), 1100 (C]S). 1H
NMR (CDCl3) d (ppm): 8.82 (br s, 1H, CONH), 8.30 (br s,

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H. Kumar et al. / European Journal of Medicinal Chemistry 43 (2008) 2688e2698
1e2 h. The solid separated out was filtered, washed with wa-
ter, dried and recrystallised from ethanol.
stirring to cooled conc. sulphuric acid (10 ml) during
10 min. The mixture was further stirred for another 5 h in
ice bath. It was then poured over crushed ice with stirring.
Solid separated out was filtered, washed with water, dried
and recrystallised from ethanol.
5.1.5.1. 5-[(Biphenyl-4-yloxy)methyl]-4-n-butyl-3-mercapto-
(4H )-1,2,4-triazole (17). IR (KBr) n cmꢀ1: 2949 (CeH), 2604
(SH), 1614 (C]N), 1234 (CeOeC). 1H NMR (CDCl3) d (ppm):
11.29 (br s, 1H, SH), 7.04e7.56 (m, 9H, aromatic), 5.13 (s, 2H,
OCH2), 4.12 (t, J ¼ 7.2 Hz, 2H, NCH2), 1.82e1.87 (m, 2H,
CH3CH2CH2), 1.39e1.47 (m, 2H, CH3CH2), 0.97 (t, J ¼ 7.2 Hz,
3H, CH3). MS: m/z 339 (Mþ). Anal. Calcd. for C19H21N3OS: C,
67.23; H, 6.24; N, 12.38. Found: C, 67.30; H, 6.25; N, 12.49.
5.1.6.1. 5-[(Biphenyl-4-yloxy)methyl]-2-n-butylamino-1,3,4-
thiadiazole (23). IR (KBr) n cmꢀ1: 3234 (NH), 2931 (CeH),
1
1608 (C]N), 1242 (CeOeC). H NMR (CDCl3) d (ppm):
9.97 (br s, 1H, NH), 7.20e7.96 (m, 9H, aromatic), 4.97 (s,
2H, OCH2), 3.56 (t, J ¼ 6.0 Hz, 2H, NCH2), 1.54e1.60 (m,
2H, CH3CH2CH2), 1.31e1.38 (m, 2H, CH3CH2), 0.92 (t,
J ¼ 7.1 Hz, 3H, CH3). MS: m/z 339 (Mþ). Anal. Calcd. for
C19H21N3OS: C, 67.23; H, 6.24; N, 12.38. Found: C, 67.32;
H, 6.19; N, 12.45.
5.1.5.2. 5-[(Biphenyl-4-yloxy)methyl]-4-(4-chlorophenyl)-3-
mercapto-(4H )-1,2,4-triazole (18). IR (KBr) n cmꢀ1: 2912
1
(CeH), 2690 (SH), 1604 (C]N), 1234 (CeOeC). H NMR
(CDCl3) d (ppm): 13.61 (br s, 1H, SH), 6.76e7.85 (m, 13H,
aromatic), 4.81 (s, 2H, OCH2). MS: m/z 393 (Mþ), 395
(Mþ þ 2). Anal. Calcd. for C21H16ClN3OS: C, 64.04; H,
4.09; N, 10.67. Found: C, 63.97; H, 4.01; N, 10.69.
5.1.6.2. 5-[(Biphenyl-4-yloxy)methyl]-2-(4-chlorophenylamino)-
1,3,4-thiadiazole (24). IR (KBr) n cmꢀ1: 3390 (NH), 2938
(CeH), 1638 (C]N), 1260 (CeOeC). 1H NMR
(DMSO-d6) d (ppm): 10.07 (br s, 1H, NH), 6.93e7.67
(m, 13H, aromatic), 5.45 (s, 2H, OCH2). MS: m/z 393
(Mþ), 395 (Mþ þ 2). Anal. Calcd. for C21H16ClN3OS: C,
64.04; H, 4.09; N, 10.67. Found: C, 63.98; H, 4.03; N,
10.61.
5.1.5.3. 5-[(Biphenyl-4-yloxy)methyl]-4-(4-bromophenyl)-3-
mercapto-(4H )-1,2,4-triazole (19). IR (KBr) n cmꢀ1: 2932
1
(CeH), 2600 (SH), 1611 (C]N), 1244 (CeOeC). H NMR
(CDCl3) d (ppm): 13.85 (br s, 1H, SH), 6.89e7.67 (m, 13H,
aromatic), 4.93 (s, 2H, OCH2). MS: m/z 438 (Mþ), 440
(Mþ þ 2). Anal. Calcd. for C21H16BrN3OS: C, 57.54; H,
3.68; N, 9.59. Found: C, 57.59; H, 3.74; N, 9.69.
5.1.6.3. 5-[(Biphenyl-4-yloxy)methyl]-2-(4-bromophenylamino)-
1,3,4-thiadiazole (25). IR (KBr) n cmꢀ1: 3287 (NH), 2923
1
(CeH), 1624 (C]N), 1234 (CeOeC). H NMR (CDCl3)
5.1.5.4. 5-[(Biphenyl-4-yloxy)methyl]-4-(4-fluorophenyl)-3-
mercapto-(4H )-1,2,4-triazole (20). IR (KBr) n cmꢀ1: 2912
d (ppm): 9.79 (br s, 1H, NH), 6.89e7.89 (m, 13H, aromatic),
5.03 (s, 2H, OCH2). MS: m/z 438 (Mþ), 440 (Mþ þ 2). Anal.
Calcd. for C21H16BrN3OS: C, 57.54; H, 3.68; N, 9.59.
Found: C, 57.29; H, 3.71; N, 9.67.
1
(CeH), 2554 (SH), 1607 (C]N), 1238 (CeOeC). H NMR
(CDCl3) d (ppm): 13.78 (br s, 1H, SH), 6.88e7.70 (m, 13H,
aromatic), 4.92 (s, 2H, OCH2). MS: m/z 377 (Mþ), 378
(Mþ þ 1). Anal. Calcd. for C21H16FN3OS: C, 66.83; H,
4.27; N, 11.13. Found: C, 66.91; H, 4.21; N, 11.19.
5.1.6.4. 5-[(Biphenyl-4-yloxy)methyl]-2-(4-flourophenylamino)-
1,3,4-thiadiazole (26). IR (KBr) n cmꢀ1: 3301 (NH), 2923 (Ce
H), 1606 (C]N), 1241 (CeOeC). 1H NMR (CDCl3) d (ppm):
9.79 (br s, 1H, NH), 6.91e7.65 (m, 13H, ArH), 5.41 (s,
2H, OCH2). MS: m/z 377 (Mþ). Anal. Calcd. for
C21H16FN3OS: C, 66.83; H, 4.27; N, 11.13. Found: C, 66.94;
H, 4.32; N, 11.06.
5.1.5.5. 5-[(Biphenyl-4-yloxy)methyl]-4-(2-methylphenyl)-
3-mercapto-(4H )-1,2,4-triazole (21). IR (KBr) n cmꢀ1: 2926
1
(CeH), 2570 (SH), 1587 (C]N), 1239 (CeOeC). H NMR
(CDCl3) d (ppm): 13.84 (br s, 1H, SH), 6.82e7.51 (m, 13H,
aromatic), 4.85 (s, 2H, OCH2), 2.18 (s, 3H, CH3). MS: m/z
373 (Mþ). Anal. Calcd. for C22H19N3OS: C, 70.75; H, 5.13;
N, 11.25. Found: C, 70.82; H, 5.19; N, 11.34.
5.1.6.5. 5-[(Biphenyl-4-yloxy)methyl]-2-(2-methylphenylamino)-
1,3,4-thiadiazole (27). IR (KBr) n cmꢀ1: 3338 (NH), 2939
5.1.5.6. 5-[(Biphenyl-4-yloxy)methyl]-4-(2,4-dimethylphenyl)-
1
(CeH), 1619 (C]N), 1244 (CeOeC). H NMR (CDCl3)
3-mercapto-(4H )-1,2,4-triazole (22). IR (KBr) n cmꢀ1: 2932
1
(CeH), 2568 (SH), 1609 (C]N), 1248 (CeOeC). H NMR
d (ppm): 8.47 (br s, 1H, NH), 7.02e7.70 (m, 13H, aromatic),
5.32 (s, 2H, OCH2), 2.18 (s, 3H, CH3). MS: m/z 373 (Mþ).
Anal. Calcd. for C22H19N3OS: C, 70.75; H, 5.13; N, 11.25.
Found: C, 70.72; H, 5.09; N, 11.30.
(CDCl3) d (ppm): 11.33 (br s, 1H, SH), 6.84e7.51 (m, 12H,
aromatic), 4.86 (s, 2H, OCH2), 2.37 (s, 3H, p-CH3), 2.16 (s,
3H, o-CH3). MS: m/z 387 (Mþ), 388 (Mþ þ 1). Anal. Calcd.
for C23H21N3OS: C, 71.29; H, 5.46; N, 10.84. Found: C,
71.37; H, 5.51; N, 10.79.
5.1.6.6. 5-[(Biphenyl-4-yloxy)methyl]-2-(2,4-dimethylphenyl-
amino)-1,3,4-thiadiazole (28). IR (KBr) n cmꢀ1: 3427 (NH),
2922 (CeH), 1611 (C]N), 1203 (CeOeC). 1H NMR
(DMSO-d6) d (ppm): 8.01 (br s, 1H, NH), 6.95e7.66 (m,
12H, aromatic), 5.40 (s, 2H, OCH2), 2.26 (s, 3H, p-CH3),
2.22 (s, 3H, o-CH3). MS: m/z 387 (Mþ). Anal. Calcd. for
5.1.6. Synthesis of 5-[(biphenyl-4-yloxy)methyl]-2-alkyl/aryl
amino-1,3,4-thiadiazoles (23e28)
Corresponding N1[2-(biphenyl-4-yloxy)ethanoyl]-N4-alkyl/
aryl-thiosemicarbazide (0.001 M) was added gradually with

H. Kumar et al. / European Journal of Medicinal Chemistry 43 (2008) 2688e2698
2697
C23H21N3OS: C, 71.29; H, 5.46; N, 10.84. Found: C, 71.34; H,
5.50; N, 10.89.
thermostatically controlled water at 55 ꢁ 0.5 ꢂC. The time in
seconds for tail withdrawal from the water was taken as the
reaction time with a cut-off time of immersion, set at 10 s
for both control as well as treated groups of animals.
5.2. Biological evaluation
5.2.4. Acute ulcerogenicity
5.2.1. Animals
Acute ulcerogenicity was determined according to the
method of Cioli et al. [25]. The animals were allocated into
different groups consisting of six animals in each group. Ul-
cerogenic activity was evaluated after oral administration of
the test compounds at an equimolar dose relative to 30 mg/
kg flurbiprofen. Control group received only 0.5% carboxy-
methyl cellulose 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 dam-
age was examined by means of a magnifying glass. For each
stomach the mucosal damage was assessed according to the
following scoring system:
Adult Wistar strain rats of either sex, weighing 150e200 g
were used for anti-inflammatory, ulcerogenic and lipid perox-
idation activities, whereas Swiss albino mice weighing
25e30 g were used for analgesic activity. The animals were
allowed food and water ad libitum except during the experi-
ments. They were housed in a room at 25 ꢁ 2 ꢂC, and
50 ꢁ 5% relative humidity with 12 h light/dark cycle. The an-
imals were randomly allocated into groups at the beginning of
all the experiments. The experimental protocol was approved
by the animal ethics committee of Hamdard University. All
the test compounds and the reference drug were administered
orally, suspended in 0.5% carboxymethyl cellulose (CMC)
solution.
5.2.2. Anti-inflammatory activity
0.5: redness, 1.0: spot ulcers, 1.5: hemorrhagic streaks, 2.0:
ulcers >3 but ꢄ5, 3.0: ulcers >5. The mean score of each
treated group minus the mean score of control group was re-
garded as severity index of gastric mucosal damage.
The synthesized compounds were evaluated for their anti-
inflammatory activity using the carrageenan induced hind
paw edema method [23]. The animals were randomly allo-
cated into groups of six animals each and were fasted for
24 h before the experiment with free access to water. Control
group received only 0.5% carboxymethyl cellulose solution.
Standard drug flurbiprofen was administered orally at a dose
of 10 mg/kg. The test compounds were administered orally
at an equimolar oral dose relative to 10 mg/kg flurbiprofen.
Into the sub plantar region of the right hind paw of each rat,
0.1 ml of 1% carrageenan solution in saline was injected sub-
cutaneously, 1 h after the administration of the test compounds
and standard drug. The right hind paw volume was measured
before and after 3 and 4 h of carrageenan treatment by means
of a plethysmometer. The percent edema inhibition was calcu-
lated from the mean effect in the control and treated animals
according to the following equation:
5.2.5. Lipid peroxidation
Lipid peroxidation in the gastric mucosa was determined
according 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 the 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 absorbance was measured at 532 nm on UV spectropho-
tometer. The results were expressed as nmol MDA/100 mg tis-
Percent edema inhibition ¼ ðVc ꢀ Vt=VcÞ ꢃ 100
sue, using extinction coefficient 1.56 ꢃ 105 cmꢀ1 Mꢀ1
.
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.
5.2.6. Hepatotoxic studies
The study was carried out on Wistar albino rats of either
sex weighing 150e200 g. Animals were divided in to three
groups, six rats in each. Group 1 was kept as control and re-
ceived only vehicle (0.5% w/v solution of carboxymethyl cel-
lulose in water), rest of the groups received test compounds, at
an equimolar oral dose relative to 10 mg/kg flurbiprofen in
0.5% w/v solution of carboxymethyl cellulose in water once
in a day for 15 days. After the treatment (15 days) blood
was obtained from all 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 cen-
trifugation at 2500 rpm for 15 min and analyzed for various
biochemical parameters.
5.2.3. Analgesic activity
Analgesic activity was evaluated by tail immersion method
[24]. Swiss albino mice allocated into different groups consist-
ing of six animals in each, of either sex, weighing 25e30 g
were used for the experiment. Analgesic activity was evalu-
ated after oral administration of the test compounds at an equi-
molar dose relative to 10 mg/kg flurbiprofen. Test compounds
and standard drugs were administered orally as suspension in
carboxymethyl cellulose 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

2698
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Acknowledgements
The authors are thankful to the Head of the Department,
Pharmaceutical Chemistry for providing laboratory facilities,
Central Drug Research Institute (CDRI) for spectral analysis
of the compounds. Authors are also thankful to Mrs. Shaukat
Shah, in-charge, animal house, Jamia Hamdard, for providing
Wistar rats and Dr. A. Mukherjee, M.D., Department of Pa-
thology, All India Institute of Medical Sciences (AIIMS),
New Delhi, for carrying out histopathological studies.
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