Design, Synthesis and Evaluation of Antiinflammatory, Analgesic and Ulcerogenicity studies of Novel S-Substituted phenacyl-1,3,4-oxadiazole-2-thiol and Schiff bases of Diclofenac acid as Nonulcerogenic Derivatives

Article abstract of DOI:10.1016/j.bmc.2007.11.014

Diclofenac sodium is being used for its anti-inflammatory actions since 28 years, but as all the NSAIDs are suffering from the deadlier GI toxicities, diclofenac sodium is also not an exception to these toxicities. The free -COOH group is thought to be re

Full text of DOI:10.1016/j.bmc.2007.11.014

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 1822–1831
Design, Synthesis and Evaluation of Antiinflammatory, Analgesic
and Ulcerogenicity studies of Novel S-Substituted
phenacyl-1,3,4-oxadiazole-2-thiol and Schiff bases of Diclofenac
acid as Nonulcerogenic Derivatives
Shashikant V. Bhandari,* Kailash G. Bothara, Mayuresh K. Raut, Ajit A. Patil,
Aniket P. Sarkate and Vinod J. Mokale
Department of Pharmaceutical Chemistry, A.I.S.S.M.S. College of Pharmacy, Near RTO, Kennedy Road, Pune 411001, India
Received 21 June 2007; revised 1 November 2007; accepted 2 November 2007
Available online 19 November 2007
Abstract—Diclofenac sodium is being used for its anti-inflammatory actions since 28 years, but as all the NSAIDs are suffering from
the deadlier GI toxicities, diclofenac sodium is also not an exception to these toxicities. The free –COOH group is thought to be
responsible for the GI toxicity associated with all traditional NSAIDs. In the present research work, the main motto was to develop
new chemical entities as potential anti-inflammatory agents with no GI toxicities. In this paper, the results of synthesis and phar-
macological screening of a series of S-substituted phenacyl 1,3,4-oxadiazoles and Schiff bases derived from 2-[(2,6-dichloroanilino)
phenyl] acetic acid (diclofenac acid) are described. The 1,3,4-oxadiazoles and diclofenac moieties are important because of their ver-
satile biological actions. In the present studies, the oxadiazole system has been functionalized onto the diclofenac acid moiety and 18
compounds in this series were synthesized. The structures of new compounds are characterized by TLC, FTIR, 1H NMR and Mass
spectral data. These compounds were tested in vivo for their anti-inflammatory activity. The compounds, which showed significant
activity (comparable to the standard drug diclofenac sodium), were screened for their analgesic activity and to check their ability to
induce ulcers by ulcerogenicity and histopathology studies. Eight new compounds, out of 18, were found to have significant anti-
inflammatory activity in the carrageenan induced rat paw oedema model, with significant analgesic activity in the acetic acid induced
writhing model with no ulcerogenicity. The compounds, which showed negligible ulcerogenic action, also showed promising results
in histopathology studies, that is, they were found to be causing no mucosal injury.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
chidonic acid by inhibiting the enzyme prostaglandin
endoperoxidase, popularly known as cyclo-oxygenase
(COX).5,6 It was discovered that COX exists in two iso-
forms, COX-1 and COX-2, which are regulated and ex-
pressed differently.7–9 COX-1 provides cytoprotection in
the gastrointestinal tract (GIT), whereas inducible
COX-2 selectively mediates inflammatory signals.10–12
Since most of the currently available NSAIDs in the
market show greater selectivity for COX-1 than COX-
2,13 chronic use of NSAIDs, including diclofenac, may
elicit appreciable GI irritation, bleeding and ulcera-
tion.14 The incidences of clinically significant GI side ef-
fects due to long term use of NSAIDs are very high
(30%) and cause some patients to abandon NSAID ther-
apy.4 GI damage from NSAID is generally attributed to
two factors. Local irritation by the direct contact of
carboxylic acid (–COOH) moiety of NSAID with GI
mucosal cells (topical effect) and decreased tissue prosta-
glandin production in tissues which undermines the
Non-steroidal anti-inflammatory drugs (NSAIDs) are
widely used for the treatment of pain and inflammation,
particularly for different types of arthritis.1–3 Among the
most popular NSAIDs worth mentioning is diclofenac
sodium, which is approved in more than 120 countries
across the globe since its introduction, 28 years ago,
and is ranked 30th among the top 200 drugs with respect
to new prescriptions.4
The pharmacological activity of NSAIDs is related to
the suppression of prostaglandin biosynthesis from ara-
Keywords: Diclofenac; 1,3,4-Oxadiazole; Anti-inflammatory; Analge-
sic; Ulcerogenicity.
*
Corresponding author. Tel.: +91 9423574082; fax: +91 20
26058208; e-mail addresses: drugdesign1@gmail.com;
drugdesign1@rediffmail.com
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.014

S. V. Bhandari et al. / Bioorg. Med. Chem. 16 (2008) 1822–1831
1823
physiological role of cytoprotective prostaglandins in
maintaining GI health and homoeostasis.5,15
proton NMR spectral data, all protons were seen
according to the expected chemical shift and integral
values. The aromatic protons appeared as multiplet
peaks within the range 6.8–7.8 d ppm, singlet signals de-
rived from hydrazide (–NH–NH2) structure appeared at
4.23 ppm. Ethylene protons resonated as singlet at
Synthetic approaches based upon chemical modification
of NSAIDs have been taken with the aim of improving
safety profile and in turn therapeutic window of this
NSAID. Several studies have described the derivatiza-
tion of the carboxylate function16–18 of representative
NSAID with less acidic azoles, viz. 1,3,4-oxadiazole,
Triazole, etc. which resulted in an increased anti-inflam-
matory activity with reduced ulcerogenicity. Further-
more, it has been reported in the literature that certain
compounds bearing 1,3,4-oxadiazole nucleus possess
significant anti-inflammatory activity.19–23 In our at-
tempt to discover new, safer and potent agents for treat-
ment of inflammatory diseases, we have replaced the
carboxylic acid group of diclofenac acid with less acidic
heterocycle, 1,3,4-oxadiazole, in order to accentuate
potency and reduce GI toxicities associated with the
parent diclofenac due to its free –COOH group. The
compounds designed so were found to possess much sig-
nificant analgesic-anti-inflammatory profile with signifi-
cant reduction in potential for ulcerogenic toxicities.24,25
1
4.86 ppm. The H NMR spectra of compounds (3a–k)
and (4a–h) displayed singlet due to –NH– groups
around 8.1, 9.4 and 10.3 ppm (probably due to their
ability to get exchanged with D2O) each signal showing
integration for one proton. For the compounds (3a–k),
the signals belonging to benzylidene group were ob-
served at aromatic region, while the signals belonging
to –NHNH2 disappeared indicating functionalization
of hydrazide to hydrazone with substituted aromatic
aldehydes.
2.2. Pharmacology
Continuing our studies on 2-[(2,6-dichloroanilino) phe-
nyl] acetic acid derivatives that are attractive candidates
as antinociceptive agents, we have designed a new series
of 2-[(2,6-dichloroanilino) phenyl] acetic acids function-
alized with different oxadiazole derivatives. In the phar-
macological study, we have investigated anti-
inflammatory and analgesic activity as well as the acute
ulcerogenicity of both oxadiazole derivatives (4a–h) and
Schiff bases (3a–k). Diclofenac, the parent compound,
was used as a reference standard. The experiments were
performed on albino rats of Wistar strain of either sex,
weighing 100–120 g. The animals were maintained at
25 2 ꢁC, 50 5% relative humidity and 12 h light/dark
cycle. The animals were fasted for 24 h prior to the
experiments and water provided ad libitum. The test
compounds were suspended in 1% aqueous carboxy
methyl cellulose (CMC) solution and administered oral-
ly to experimental animals.
2. Results and discussion
2.1. Chemistry
The synthetic route used to synthesize title compounds is
outlined in Scheme 1. Ethyl-[2-(2,6-dichloroanilino)-
phenyl]acetate (2), the starting material, was prepared
according to the method reported in the literature, using
2-[(2,6-dichloroanilino) phenyl]acetic acid (diclofenac
acid). The acid hydrazide (3) was prepared by esterifica-
tion of 2-[(2,6-dichloroanilino) phenyl]acetic acid (1) fol-
lowed by treatment with hydrazine hydrate in absolute
ethanol. The reaction of hydrazide 3 with carbon
disulfide in an alkaline medium afforded 5-[2-(2,6-dichlo-
roanilino) benzyl] 2-mercapto-1,3,4-oxadiazole (4). Vari-
ous S-substituted phenacyl-1,3,4-oxadiazoles (4a–h) were
prepared by treatment of hydrazide with various substi-
tuted acetophenones in the presence of pyridine. [2-(2,6-
dichloroanilino) benzyl carbazide] (3) was condensed
with different substituted aromatic aldehydes in ethanol
to give corresponding Schiff bases (3a–k) in very good
yields. The structures of various synthesized compounds
were assigned on the basis of different chromatographic
and spectral studies. The physical data, FTIR, 1H
NMR and Mass spectral data for all the synthesized com-
pounds are reported in experimental protocols.
2.2.1. Anti-inflammatory activity. Anti-inflammatory
activity of the synthesized compounds was evaluated
by carrageenan induced rat paw oedema model, equimo-
lar doses, that is, equivalent to 0.033 M, of diclofenac
acid sub planter injection of 0.1 mL, 1% carrageenan
produced increase in paw volume (oedema) of all the
animals of various groups. The onset of action was evi-
dent from 1 h in various test groups. The significant
(p < 0.01) reduction of rat paw oedema was observed
by most of the test compounds at 3 h compared to con-
trol group (Tables 1 and 2). Compounds with significant
anti-inflammatory profile were subjected to ulcerogenic-
ity potential test at 12 times the therapeutic doses with
additional physical (cold) stress. A thorough examina-
tion of the results of histopathological studies indicated
absence of the disruption of gastric epithelial morphol-
ogy and absence of ulcers/erosion in test group animals
compared to reference standard, diclofenac acid, and
control group animals. The results of the ulcerogenicity
studies are presented in Table 4 and results of histopa-
thological studies are depicted in Figure 1a–e.
The FTIR spectra of the Schiff bases exhibited very sim-
ilar features and showed the expected bands for the
characteristic groups which are present in the com-
pounds such as C–H and the C@N stretching vibrations
and another specific band for Ar–C–N vibrations. Com-
pounds (3a–k) have C@O stretching bands in the range
1703–1677. For 1,3,4-oxadiazole derivatives, the pres-
ence of C@N stretching band at 1666–1598 cmÀ1 is an
evidence of ring closure. The schiff bases (3a–k) and
oxadiazole (4a–h) showed N–H stretching bands at
3362–3236 and 3467–3106 cmÀ1, respectively. In the
From close inspection of the results of in vivo experi-
ments, we can conclude that the cyclization of hydrazide
moiety to oxadiazole yielded compounds with different

1824
S. V. Bhandari et al. / Bioorg. Med. Chem. 16 (2008) 1822–1831
COONa
NH
Cl
Cl
HCl
ethanol
COOH
NH
Cl
Cl
(1)
Conc.H2SO4
ethanol
COOC2H5
NH
Cl
Cl
(2)
NH2NH2.H2O
ethanol
CONHNH2
CONHN CHAr
Reflux 5-8 hrs
NH
ethanol
NH
Cl
Cl
CHO
Cl
Cl
R
(3)
(3a-k)
CS2,KOH
ethanol
N
O
N
N
N
SCH2C
O
SH
O
Pyridine
R
NH
NH
reflux
Cl
Cl
Cl
Cl
5-8hrs,
substituted
acetophenon
(4)
(4a-h)
Scheme 1.
therapeutic efficacy. In this series compounds 4b, 4c, 4e,
and 4g exhibited very significant anti- inflammatory
activity compared to standard drug diclofenac. The re-
sults of two different series of compounds, viz. 1,3,4-
oxadiazole derivatives containing substituted acetophe-
nones (4a–h) and acyclic hydrazones containing substi-
tuted aromatic aldehydes (3a–k) indicate that latter
series of compounds are more potent.
Based on findings of these preclinical results, further
studies need to be carried out to investigate the other
specifications, such as in vitro assays, chronic ulceroge-

S. V. Bhandari et al. / Bioorg. Med. Chem. 16 (2008) 1822–1831
1825
Table 1. Results of anti-inflammatory activity of Schiff bases (3a–k) against carrageenan induced rat paw oedema model in rats
Compound
Change in paw volume in (mL) after drug treatment
( SEM)
Anti-inflammatory activity (% inhibition)
1h
2h
3h
1h
2h
3h
—
Control
Diclofenac
1.77 0.08
1.97 0.05
1.84 0.06
2.02 0.06
1.98 0.03
1.67 0.02
1.82 0.01
1.48 0.03
1.81 0.01
1.94 0.05
1.62 0.03
1.68 0.02
2.01 0.02
1.94 0.07
2.27 0.04
2.15 0.05
2.33 0.06
2.29 0.04
1.97 0.02
2.18 0.00
1.84 0.03
1.95 0.02
2.28 0.05
2.00 0.03
1.98 0.02
2.32 0.01
2.31 0.05
2.57 0.04
2.14 0.05
2.61 0.06
2.58 0.04
2.26 0.02
2.50 0.01
2.16 0.02
2.66 0.01
2.62 0.04
2.33 0.05
2.28 0.02
2.65 0.01
—
—
28.69**
32.28**
31.38**
29.11**
27.21**
20.87**
16.64ns
16.28*
20.99*
13.54ns
28.20**
30.53**
48.99**
48.81**
46.96**
50.97**
51.88**
47.02**
41.35**
38.14**
43.09**
40.20**
48.75**
51.72**
71.30**
72.76**
72.88**
71.93**
73.36**
69.79**
60.34**
70.94**
68.62**
60.99**
69.39**
80.07**
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
Data analyzed by one way ANOVA followed by Dunnett’s ‘t’ test, (n = 6), *P < 0.05, **P < 0.01 significant from control; ns, not significant.
Table 2. Reults of anti-inflammatory activity of oxadiazole derivatives (4a–h) against carrageenan induced rat paw oedema model in rats
Compound
Change in paw volume in (mL) after drug treatment
( SEM)
Anti-inflammatory activity (% inhibition)
1h
2h
3h
1h
2h
3h
Control
Diclofenac
1.77 0.08
1.97 0.05
1.53 0.05
1.71 0.05
1.92 0.01
1.79 0.02
2.08 0.03
2.04 0.03
2.12 0.05
1.86 0.05
1.94 0.07
2.27 0.04
1.91 0.06
2.10 0.05
2.27 0.01
2.09 0.01
2.34 0.01
2.35 0.01
1.91 0.06
2.23 0.03
2.31 0.05
2.57 0.04
2.22 0.03
2.39 0.00
2.59 0.01
2.37 0.02
2.67 0.03
2.65 0.03
2.76 0.04
2.56 0.04
—
—
—
71.3**
28.69**
20.99*
23.80**
23.43**
30.46**
31.55**
28.34**
26.02**
17.94ns
48.99**
45.64**
52.09**
48.63**
52.26**
48.91**
49.47**
45.16**
44.53**
4a
4b
4c
4d
4e
4f
67.19**
74.23**
72.63**
60.39**
71.99**
60.39**
71.05**
69.89**
4g
4h
Data analyzed by one way ANOVA followed by Dunnett’s ‘t’ test, (n = 6), *P < 0.05, **P < 0.01 significant from control; ns, not significant.
nicity studies, toxicological studies and mechanism by
which these drugs exhibit potential analgesic, anti-
inflammatory activity.
2.2.3. Acute ulcerogenicity studies. Ulcerogenic effect of
Schiff bases (3b, 3d, 3k) and oxadiazole derivatives
(4b, 4c) with best overall profile in animal efficacy
model was evaluated for gastric ulcerogenic potential
in rat stress model at 12 times the therapeutic doses
(Table 4). When compared with diclofenac acid, these
four compounds did not cause any gastric ulceration
and disruption of gastric epithelial cells at the above-
mentioned oral doses. Hence gastric tolerance to these
compounds was better than that of diclofenac acid
indicating that the functionalization of the acidic
–COOH group of the diclofenac acid with substituted
1,3,4-oxadiazole and substituted hydrazones has re-
sulted in more potent and nontoxic New Chemical
Entities (NCEs). All above results have proved that
the rationale for selecting less acidic 1,3,4-oxadiazoles
and hydrazones was accurate and fruitful. The results
are shown in Table 4.
2.2.2. Analgesic activity. The analgesic activities of the
compounds were studied by using acetic acid induced
writhing test in mice. The compounds, which exhibited
significant anti-inflammatory activity comparable to that
of diclofenac acid, were screened for analgesic activity.
The analgesic activity was evaluated at equimolar doses
equivalent to 10 mg/kg (diclofenac acid) body weight.
These compounds presented an important analgesic pro-
file measured by the classical acetic acid induced writhing
model. From the results of acetic acid induced writhing
test, it was noticed that all compounds possess significant
analgesic activity (Table 3). The analgesic effects of (3k)
(68.66%) and (4b) (66.89%) were found to be better than
that of diclofenac sodium (64.65%).
Additionally, these studies showed that the most potent
analgesic agent from hydrazone series was (3k). While
(4b) was found to be the most active in the oxadiazole
series. When compared all 8 compounds together, the
most of the 1,3,4-oxadiazole/hydrazone derivatives were
found to have significant analgesic action.
2.2.4. Histopathological studies. The stomach specimen
of diclofenac acid treated rats was characterized by com-
plete disruption of protective mucosal layer (Fig. 1b).
Histopathological analysis also showed characteristic
features of ulceration in diclofenac acid treated group
of animals. The tissue of diclofenac acid treated rats

1826
S. V. Bhandari et al. / Bioorg. Med. Chem. 16 (2008) 1822–1831
Figure 1. Haematoxylin and eosin Immunohistochemical staining of gastric ulcers after ulcer induction in rats. As illustrated in Fig. specimen (a)
shows Intact Mucous membrane in control treated rat showing granular tissues composed of macrophages, fibroblasts and endothelial cells forming
microvessels. Congestion of mucosal blood vessels in diclofenac treated group, specimen (b). No damage was seen to mucosa of rat treated with test
compound, 3d, specimen (c), 3k, specimen (d) and 4b, specimen (e), these specimens c–e were identical to that of the control, specimen (a). Original
magnification 200·.
has shown that some epithelial cells in the ulcer margin
had proliferated and migrated over and into the ulcer
crater, which was strongly infiltrated by inflammatory
cells, fibroblasts and endothelial cells indicating com-
plete disruption of gastric epithelial layer. Scanning of
stomach specimens using electron microscope revealed
that in the rats treated with Schiff bases (3b, 3d, 3k)
and oxadiazole derivatives (4b, 4c) there was no injury
observed in stomach mucosa. As illustrated in Figure
1, specimen C which is identical to that of the control
(Fig. 1a).
3. Conclusions
Various substituted oxadiazole and benzylidene hydra-
zone derivatives were synthesized and screened for anal-
gesic, anti-inflammatory and ulcerogenic potential.
Most compounds exhibited significant analgesic and
anti-inflammatory activity. Compounds (3k) and (4b)
which have benzylidene hydrazine and oxadiazole moi-
ety showed strong analgesia in acetic acid induced writh-
ing tests. Among all the synthesized compounds,
compounds (3k) and (4b) exhibited most prominent

S. V. Bhandari et al. / Bioorg. Med. Chem. 16 (2008) 1822–1831
1827
Table 3. Results of analgesic activity of synthesized compounds
against acetic acid induced writhing tests in mice
reference (Chemical shift represented in d ppm). Mass
spectra were recorded on GC-MS QP5050ASystem
(benchtop quadrupole mass spectrophotometer). Purity
of the compounds was checked on TLC plates using sil-
ica gel G as stationary phase and iodine vapors as visu-
alizing agent. The 2-[(2,6-dichloroanilino) phenyl] acetic
acid sodium salt (diclofenac sodium) was procured from
Iatros Pharmaceuticals Ltd., Pune, India.
Compound Dose
No of writhes in 25 % inhibition
(mg/kg, p.o) min after treatment
(Mean SEM)
Control
Std.
3a
—
10
10
10
10
10
10
10
10
10
27.83 0.477**
9.83 0.60**
15.16 0.83**
11.0 0.57**
12.83 1.13**
8.66 0.55**
9.16 0.70**
13.5 0.99**
8.83 0.47**
12.5 0.76**
—
64.65
45.27
60.46
53.86
68.66
66.89
51.36
68.22
55.00
3b
3d
4.1. Hydrolysis of diclofenac sodium to 2-[(2,6-dichloro-
anilino) phenyl] acetic acid (1)
3k
4b
4c
4e
Diclofenac sodium (0.101 mol) was dissolved in ethanol
(2.5 mol); to this solution conc.H2SO4 was added drop-
wise to hydrolyse the salt to acid. The acid obtained was
filtered, dried mp 153–155 ꢁC.
4g
Data analyzed by one way ANOVA followed by Dunnett’s ‘t’ test,
(n = 6), **P < 0.01 significant from control.
4.2. Synthesis of ethyl-[2-(2,6-dichloroanilino) phenyl]
acetate (2)
Table 4. Ulcerogenic effects of synthesized compounds in comparison
with diclofenac sodium
The ethyl ester was prepared as per procedure reported
in the literature.26
Compound Dose
(mg/kg, p.o)
Ratio of ulcerated Ulcer index
animals
(mean SE)
Diclofenac
10
4/6
6/6
1.8 0.1
2.1 0.2
—
4.3. Synthesis of [2-(2,6-dichloroanilino) phenyl] acetic
acid hydrazide (3)
30
50
Not tested
3b
3d
3k
4b
4c
10
30
50
—
—
—
—
—
—
The hydrazide was prepared as per the reported proce-
dure,27 yield: 72%; mp 134–136 ꢁC. FTIR spectra of
the compound showed bands at 3325 (N–H); 2970 (C–
1
10
30
50
—
—
—
—
—
—
H); 1638 (C@O). H NMR chemical shifts at (CDCl3,
d ppm): 3.59 (s, 2 H, CH2CO); 4.14 (s, 2H, NH2);
6.82–6.98 (m, 4H, 3,4,5,6 ArH); 7.21–7.27 (m, 3H, di-
chloro ArH); 7.54 (s, 1H, NH); 8.27 (s, 1H, CONH).
Mass spectra of compound exhibited molecular ion peak
at m/z 309 (M+), other important fragments were ob-
served at 310 (M++1), 311 (M++2), 313 (M++4), 278,
250, 214.
10
20
50
—
—
—
—
—
—
10
30
50
—
—
—
—
—
—
4.4. General procedure for the preparation of Schiff bases
(3a–k)
10
30
50
—
—
—
—
—
—
The Schiff bases were prepared as per the reported meth-
od28 and purified by recrystallization from DMSO.
4.4.1. N-(Cinnamaldenyl)-[2-(2,6-dichloroanilino)benzyl
carbazide] (3a). Mp 200–202 ꢁC; yield 87%; FTIR
(KBr) cmÀ1: 3285 (NH), 1677 (C@O), 1598 (C@N),
and consistent anti-inflammatory activity. From the de-
tailed analysis of the results of histopathological studies,
we conclude that the synthesized compounds have not
only retained the anti-inflammatory profile of diclofenac
acid but also have helped in enhancing the anti-inflam-
matory activity and are devoid of the deadlier gastroin-
testinal toxicities.
1
C@C, 747 (C–Cl); H NMR; mass: m/z 424 (M+), 423
(26%), 425 (M+1) (64.6%); 1H NMR (300 MHz,
DMSO-d6), 8.6 (s, 1H, CH@N), 7.22–7.29 (m, 4H,
3,4,5,6 ArH), 7.45–7.48 (m, 3H, dichloro ArH), 7.35
(m, 3H, phenyl ring),11.87 (bs, 1H, –CONH–), 8.1 (s,
1H, –NH–).
4. Experimental
4.4.2. N-(2-Hydroxybenzylidenyl)-[2-(2,6-dichloroanili-
no)benzyl carbazide] (3b). Mp 210–212 ꢁC ; yield 70%;
FTIR (KBr) cmÀ1: 3285 (NH), 1685 (C@O), 1624
(C@N), 3293 (OH), 752 (C–Cl); mass: m/z 413 (M+),
Melting points were determined by open capillary tubes
and are uncorrected. FTIR spectra of the powdered
compounds were recorded using KBr on a Jasco FTIR
V 430+ spectrometer using Diffuse Reflectance Attach-
1
414 (M+1), 415 (M+2); H NMR (300 MHz, DMSO-
d6), 8.5 (s, 1H, CH@N), 13.0 (s, 1H, OH), 7.45–7.48
(m, 3H, dichloro ArH), 7.31–7.36 (m, 4H, phenyl ring),
7.22–7.29 (m, 4H, 3,4,5,6 ArH), 10.17 (bs, 1H,–CONH–),
9.4 (s, 1H, NH).
ment and are reported in cmÀ1 and H NMR spectra
1
were recorded on a Varian Mercury YH300 (300 MHz
FT NMR) spectrophotometer using TMS as an internal

1828
S. V. Bhandari et al. / Bioorg. Med. Chem. 16 (2008) 1822–1831
4.4.3. N-(3,4,5-Trimethoxybenzylidenyl)-[2-(2,6-dichloro-
anilino) benzyl carbazide] (3c). Mp 218–220 ꢁC; yield
65%; FTIR (KBr) cmÀ1: 3262 (NH), 1689 (C@O),
1577 (C@N), 1166 (OCH3), 782 (C–Cl); mass: m/z
FTIR (KBr) cmÀ1: 3282 (NH), 1694 (C@O), 1590
(C@N), 760 (C–Cl); mass: m/z 431 (M+), 433 (M+2);
1H NMR (300 MHz, DMSO-d6), 8.14 (s, 1H, CH@N),
6.94–7.66 (m, 4H, phenyl ring), 7.20–7.23 (m, 4H,
3,4,5,6 ArH), 7.49–7.58 (m, 3H, dichloro ArH), 10.59
(bs, 1H, –CONH–), 10.3 (s, 1H, NH).
1
487(M+), 488 (M+1), 489 (M+2); H NMR (300 MHz,
DMSO-d6), 8.9 (s, 1H, CH@N), 7.45–7.48 (m, 3H, di-
chloro ArH), 7.31–07.36 (s, 2H, phenyl ring), 7.22–
7.29 (m, 4H, 3,4,5,6 ArH), 3.91 (s, 3H, p-OCH3), 3.86–
3.88 (s, 6H, both m-OCH3), 1.58 (s, 2H, CH2), 11.21
(bs, 1H, –CONH–),10.3 (s, 1H, NH).
4.4.10. N-(4-Dimethylamino benzylidenyl)-[2-(2,6-dichlo-
roanilino) benzyl carbazide] (3j). Mp 119–121 ꢁC; yield
65%; FTIR (KBr) cmÀ1: 3263 (NH), 1696 (C@O),
1602 (C@N), 763 (C–Cl); 1367, 1454 (N(CH3)2); mass:
m/z 440 (M+), 441 (M+1), 442 (M+2); 1H NMR
(300 MHz, DMSO-d6), 8.6 (s, 1H, CH@N), 7.57 (s,
2H, phenyl ring), 7.53 (s, 2H, phenyl ring), 7.25–7.39
(m, 3H, dichloro ArH), 7.0–7.08 (m, 4H, 3,4,5,6 ArH),
3.74 (s, 2H, CH2), 10.42 (bs, 1H, –CONH–), 8.1 (s,
1H, NH), 3.25 (s, 6H, N(CH3)2).
4.4.4. N-(4-Methoxybenzylidenyl)-[2-(2,6-dichloroanili-
no)benzyl carbazide] (3d). Mp 230–232 ꢁC; yield 78%;
FTIR (KBr) cmÀ1: 3259 (NH), 1701 (C@O), 1600
(C@N), 1164 (–OCH3), 782 (C–Cl); mass: m/z
1
427(M+), 428 (M+1), 429 (M+2); H NMR (300 MHz,
DMSO-d6), 8.14 (s, 1H, CH@N), 7.45–7.48 (m, 3H, di-
chloro ArH), 7.31–7.36 (m, 4H, phenyl ring of schiff
base), 7.22–7.29 (m, 4H, 3,4,5,6 ArH), 3.91 (s, 3H, p-
OCH3), 10.05 (bs, 1H, –CONH–),8.1 (s, 1H, NH).
4.4.11. N-(4-Bromo benzylidenyl)-[2-(2,6-dichloroanili-
no)benzyl carbazide] (3k). Mp 119–121 ꢁC; yield 65%;
FTIR (KBr) cmÀ1: 3362 (NH), 1666 (C@O), 1621
(C@N), 758 (C–Cl); 556 (C–Br), mass: m/z 476 (M+),
478 (M+2), 1H NMR (300 MHz, DMSO-d6); 8.5 (s,
1H, CH@N), 7.39 (m, 3H, dichloro ArH), 7.0–7.08 (m,
4H, 3,4,5,6 ArH), 3.74 (s, 2H, CH2), 6.94–7.16 (m, 4H,
phenyl ring of Schiff base), 11.14 (bs, 1H, –CONH–),
8.1 (s, 1H, NH).
4.4.5. N-(3-Nitrobenzylidenyl)-[2-(2,6-dichloroanilino)-
benzyl carbazide] (3e). Mp 209–212 ꢁC; yield 88%; FTIR
(KBr) cmÀ1: 3357 (NH), 1680 (C@O), 1564 (C@N),
NO2 1531 (asymm) 1347 (symm), (OCH3), 735 (C–Cl);
1
mass: m/z 442 (M+), 443 (M+1), 444 (M+2); H NMR
(300 MHz, DMSO-d6), 8.6 (s, 1H, CH@N), 7.45–7.48
(m, 3H, dichloro ArH), 7.22–7.29 (m, 4H, 3,4,5,6
ArH),11.73 (bs, 1H, CONH), 6.94–7.1 (m, 4H, phenyl
ring of Schiff base), 9.4 (s, 1H, NH).
4.5. Synthesis of 5-[2-(2,6-dichloroanilino)benzyl]2-mer-
capto-1,3,4-oxadiazole (4)
4.4.6. N-(4-Fluorobenzylidenyl)-[2-(2,6-dichloroanilino)-
benzyl carbazide] (3f). Mp 180–182 ꢁC; yield 86%; FTIR
(KBr) cmÀ1: 3343 (NH), 1690 (C@O), 1602 (C@N),
1231 (C–F), 740 (C–Cl); mass: m/z 430 (M+), 431
(M+1), 432 (M+2); H NMR (300 MHz, DMSO-d6),
8.5 (s, 1H, CH@N), 7.49–7.58 (m, 3H, dichloro ArH),
7.20–7.23 (m, 4H, 3,4,5,6 ArH), 6.94–7.8 (m, 4H, phenyl
It was prepared as per the reported method.27 FTIR
spectra of the compound showed bands at 3354 (N–
H); 1525 (C–N); 1182 (C@S). 1H NMR (CDCl3, d
ppm): 4.13 (s, 2H, CH2); 7.25–7.30 (m, 4H, 3,4,5,6
ArH); 7.51–7.53 (m, 3H, dichloro ArH); 7.90 (s, 1H,
NH); 10.51 (s, 1H, SH).
1
ring
–CONH–), 10.3 (s, 1H, NH).
of
Schiff
base),
10.98
(bs,
1H,
4.6. General procedure for the preparation of S-substi-
tuted phenacyl 1,3,4-oxadiazole-2-thiol (4a–h)
4.4.7. N-(4-Hydroxybenzylidenyl)-[2-(2,6-dichloroanili-
no)benzyl carbazide] (3g). Mp 238–240 ꢁC; yield 87%;
FTIR (KBr) cmÀ1: 3257 (NH), 1681 (C@O), 1603
(C@N), 3293 (OH), 748 (C–Cl); mass: m/z 413 M+,
S-Substituted phenacyl 1,3,4-oxadiazole-2-thiol deriva-
tives were prepared as per the reported procedures.29
4.6.1. 5-[2-(2,6-Dichloroanilino) benzyl]-S-(4-nitro phena-
cyl)-1,3,4-oxadiazole-2-thiol (4a). Mp 110–112 ꢁC; yield
47%; FTIR (KBr) cmÀ1: 3370(NH), 1675, 1602, 1504,
1450 (ring stretch of oxadiazole nucleus), 1055 (C–O
stretch of oxadiazole nucleus), 1345 (symm) 1504
(asymm) NO2, 790 (C–Cl); mass: m/z 514 (M+), 515
(M+1), 516 (M+2),1H NMR (300 MHz, CDCl3); 9.4
NH (s, 1H) hump, 4.16 CH2CO (s, 2H), 6.97–7.11 (m,
4H, 2,3,5,6 ArH), 7.14–7.21 (m, 3H, dichloro ArH),
7.23–7.38 (m, 4H, phenyl ring onto oxadiazole).
1
414 (M+1), 415 (M+2); H NMR (300 MHz, DMSO-
d6), 8.9 (s, 1H, CH@N), 10.3 (bs, 1H, CONH), 8.10 (s,
1H, NH), 7.49–7.58 (m, 3H, dichloro ArH), 7.20–7.23
(m, 4H, 3,4,5,6 ArH), 6.83 (s, 2H, phenyl ring), 6.79
(s, 2H, phenyl ring), 4.08 (s, 2H, CH2),13 (s, 1H, OH).
4.4.8. N-(4-Methyl benzylidenyl)-[2-(2,6-dichloroanili-
no)benzyl carbazide] (3h). Mp 119–121 ꢁC; yield 65%;
FTIR (KBr) cmÀ1: 3257 (NH), 1683 (C@O), 1563
(C@N), 2902 (C–CH3), 752 (C–Cl); mass: m/z
411(M+), 412 (M+1), 413 (M+2); 1H NMR
(300 MHz,DMSO-d6), 8.3 (s, 1H, CH@N), 7.20–7.23
(m, 4H, 3,4,5,6 ArH), 7.49–7.58 (m, 3H, dichloro
ArH), 11.35 (bs, 1H, –CONH–), 9.4 NH (s, 1H), 6.94–
7.16 (m, 4H, phenyl ring).
4.6.2. 5-[2-(2,6-Dichloroanilino) benzyl]-S-(3-methoxy
phenacyl)-1,3,4-oxadiazole-2-thiol (4b). Mp 90–92 ꢁC;
yield 71%; FTIR (KBr) cmÀ1: 3318 (NH), 1577, 1504,
1454 (ring stretch of oxadiazole nucleus), 1065 (C–O
stretch of oxadiazole nucleus), 1150 (OCH3), 779 (C–
1
Cl); mass: m/z 499 (M+), 500 (M+1), 501 (M+2), H
4.4.9. N-(2-Chloro benzylidenyl)-[2-(2,6-dichloroanili-
no)benzyl carbazide] (3i). Mp 119–121 ꢁC; yield 65%;
NMR (300 MHz, CDCl3), 4.27 (s, 2H, CH2CO), 3.93
(s, 2H, CH2), 7.52 (m, 3H, dichloro ArH), 7.24–7.30

S. V. Bhandari et al. / Bioorg. Med. Chem. 16 (2008) 1822–1831
1829
(m, 4H, 2,4,5,6 ArH), 3.9 (s, 3H, OCH3), 4.43–5.31 NH
(bs, 1H) hump.
(C–Cl); mass: m/z 485 (M+), 486 (M+1), 487 (M+2);
1H NMR (300 MHz, CDCl3), 10.5 NH (s, 1H) hump,
7.5 (m, 3H, dichloro ArH), 7.24–7.31 (m, 4H, 2,4,5,6
ArH), 4.21 (s, 2H, CH2CO), 3.8 (s, 2H, CH2), 9.9 (s,
1H, OH).
4.6.3. 5-[2-(2,6-Dichloroanilino) benzyl]-S-(3,4-dimethoxy
phenacyl)-1,3,4-oxadiazole-2-thiol (4c). Mp 80–82 ꢁC;
yield 55%; FTIR (KBr) cmÀ1: 3068 (NH), 1577, 1500,
1454 (ring stretch of oxadiazole nucleus), 1057 (C–O
stretch of oxadiazole nucleus), 1150 (OCH3), 779 (C–
5. Pharmacology
5.1. Animals
1
Cl); mass: m/z 529 (M+), 530 (M+1), 531 (M+2); H
NMR (300 MHz, CDCl3), 7.49–7.52 (m, 3H, dichloro
ArH), 7.24–7.30 (m, 4H, 3,4,5,6 ArH), 7.11–7.13 (m,
3H, dimethoxy phenyl ring), 4.43–5.31 NH (bs, 1H)
hump, 4.15 (s, 2H, CH2CO), 3.93 (s, 2H, CH2), 2.67.
(s, 2H, OCH3), 2.68 (s, 2H, OCH3).
Swiss albino mice of either sex weighing 20–25 g and
Wistar rats weighing in the range 100–120 g were ob-
tained from National Institute of Virology, Pune, India.
All the animals were housed under standard ambient
conditions of temperature (25 2 ꢁC) and relative
humidity of 50 5%. A 12:12 h light:dark cycle was
maintained. All the animals were allowed to have free
access to water and standard palletized laboratory ani-
mal diet 24 h prior to pharmacological studies. All the
experimental procedures and protocols used in this
study were reviewed and approved by the Institutional
Animal Ethical Committee (IAEC) of College, Pune,
constituted in accordance with the guidelines of the
Committee for the Purpose of Control and Supervision
of Experiments on Animals (CPCSEA), Government of
India.
4.6.4. 5-[2-(2,6-Dichloroanilino) benzyl]-S-(3,4-dichloro
phenacyl)-1,3,4-oxadiazole-2-thiol (4d). Mp 107–110 ꢁC;
yield 73%; FTIR (KBr) cmÀ1: 3467 (NH), 1577, 1506,
1455 (ring stretch of oxadiazole nucleus), 1057 (C–O
stretch of oxadiazole nucleus), 778 (C–Cl); mass: m/z
(M+) 538, 540 (M+2), 536, 1H NMR (300 MHz,
CDCl3), 7.52–7.54 (m, 3H, dichloro ArH), 7.33–7.37
(m, 4H, 3,4,5,6 ArH), 7.24–7.3 (m, 3H, phenyl ring),
8.1 (s, 1H, NH), 4.17 (s, 2H, CH2CO), 3.82 (s, 2H, CH2).
4.6.5.
5-[2-(2,6-Dichloroanilino)benzyl]-S-(4-hydroxy
phenacyl)-1,3,4-oxadiazole-2-thiol (4e). Mp 138–140 ꢁC;
yield 76%; FTIR (KBr) cmÀ1: 3318 (NH), 1577, 1506,
1455 (ring stretch of oxadiazole nucleus), 1066 (C–O
stretch of oxadiazole nucleus), 3293 (OH), 778 (C–Cl);
mass: m/z 485 (M+), 486 (M+1) (26%), 487 (M+2)
5.2. Preparation of test compounds
After suspending the test compounds in 1.0% aqueous
solution of sodium carboxymethyl cellulose (CMC), test
samples were administered to test animals orally. The
positive and negative control group animals received
the same experimental handling as those of the test
groups except that the drug treatment, control group
animals received only appropriate volumes of vehicle
and of the reference drug, diclofenac sodium,
respectively.
1
(68.5%); H NMR (300 MHz, CDCl3), 9.4 NH (s, 1H)
hump, 4.16 CH2CO (s, 2H), 6.97–7.11 (m, 4H, 3,4,5,6
ArH), 7.14–7.21 (m, 3H, dichloro ArH), 7.23–7.38 (m,
2H, phenyl ring on oxadiazole), 7.41–7.42 (m, 2H, phe-
nyl ring on oxadiazole), 3.49 (s, 2H, CH2).
4.6.6. 5-[2-(2,6-Dichloroanilino)benzyl]-S-(3-nitro phena-
cyl)-1,3,4-oxadiazole-2-thiol (4f). Mp 120–122 ꢁC; yield
86%; FTIR (KBr) cmÀ1: 3318 (NH), 1675, 1588, 1505,
1451 (ring stretch of oxadiazole nucleus), 1055 (C–O
stretch of oxadiazole nucleus), 1505 (asymm) 1345
(symm) NO2, 790 (C–Cl); mass: m/z 514 (M+), 515
5.3. Anti-inflammatory activity
Anti-inflammatory activity was evaluated using the well-
known Carrageenan induced rat paw oedema model of
Winter et al.30 using groups of six animals each. A
freshly prepared aqueous suspension of carrageenan
(1.0% w/v, 0.1 mL) was injected in the subplanter region
of right hind paw of each rat. One group was kept as
control and the animals of the other group were pre-
treated with the test drugs, 1 h before the carrageenan
treatment. The volume was measured before and after
carrageenan treatment at the 30 min. interval with the
help of digital plethysmometer (Panlab LE 7500).
1
(M+1), 516 (M+2); H NMR (300 MHz, CDCl3), 10.5
NH (s, 1H) hump, 7.81–7.83 (m, 4H, phenyl ring),
7.43–7.5 (m, 3H, dichloro ArH), 7.24–7.31 (m, 4H,
2,4,5,6 ArH), 4.16 (s, 2H, CH2CO), 2.68 (s, 2H, CH2).
4.6.7. 5-[2-(2,6-Dichloroanilino) benzyl]-S-(4-bromo
phenacyl)-1,3,4-oxadiazole-2-thiol (4g). Mp 103–105 ꢁC;
yield 67%; FTIR (KBr) cmÀ1: 3318 (NH), 1577, 1504,
1454 (ring stretch of oxadiazole nucleus), 1066 (C–O
stretch of oxadiazole nucleus), 561 (C–Br), 777 (C–Cl);
mass: m/z 548 (M+), 550 (M+2), 546, 1H NMR
(300 MHz, CDCl3), 10.3 NH (s, 1H) hump, 7.5 (m,
3H, dichloro ArH), 7.24–7.31 (m, 4H, 2,3,5,6 ArH),
4.19 (s, 2H, CH2CO), 2.38 (s, 2H, CH2).
5.4. Analgesic activity
The analgesic activity was evaluated using the acetic
acid induced writhing method.31
4.6.8. 5-[2-(2,6-Dichloroanilino) benzyl]-S-(3-hydroxy
phenacyl)-1,3,4-oxadiazole-2-thiol (4h). Mp 145–147 ꢁC;
yield 57%; FTIR (KBr) cmÀ1: 3318 (NH), 1577, 1588,
1506, 1455 (ring stretch of oxadiazole nucleus), 1063
(C–O stretch of oxadiazole nucleus), 3293 (OH), 774
5.5. Acute ulcerogenicity studies
Acute ulcerogenicity screening was done according to
method reported by Cioli et al.32 The mucosal damage

1830
S. V. Bhandari et al. / Bioorg. Med. Chem. 16 (2008) 1822–1831
was examined by means of an electron microscope. For
each stomach specimen, the mucosal damage was as-
sessed according to the following scoring system.
viding facilities for spectral studies of compounds and
Iatros Pharmaceuticals Ltd. Pune, for providing pure
diclofenac sodium. The authors are also thankful to
Medicare pathology laboratory, Pune, for providing
facilities for histopathological studies.
Score Description
0.0
0.5
1.0
2.0
Normal (no injury, bleeding and latent injury).
Latent injury or widespread bleeding (>2 mm).
Slight injury (2–3 dotted lines).
Severe injury (continuous lined injury or 5–6
dotted injuries).
References and notes
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3.0
4.0
Very severe injury (several continuous lined
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Widespread lined injury or widened injury.
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5.7. Statistical analysis
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were expressed as mean of change in paw volume SD
and analyzed by one-way ANOVA followed by Dun-
nett’s test. Data from acetic acid induced writhing model
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National Chemical Laboratories (NCL), Pune, for pro-

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