Synthesis, pharmacological screening and in silico studies of new class of Diclofenac analogues as a promising anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmc.2014.03.043

A novel series of 5-[2-(2,6-dichlorophenylamino)benzyl]-3-(substituted)-1, 3,4-oxadiazol-2(3H)-thione (4a-k) derivatives have been synthesized by the Mannich reaction of 5-[2-(2,6-dichlorophenylamino)benzyl]-1,3,4-oxadiazol-2(3H)- thione (3) with an appropriately substituted primary/secondary amines, in the presence of formaldehyde and absolute ethanol. Structures of these novel compounds were characterized on the basis of physicochemical, spectral and elemental analysis. The title compounds (4a-k) were screened for in vivo acute anti-inflammatory and analgesic activities at a dose of 10 mg/kg b.w. Compound 4k exhibited the most promising and significant anti-inflammatory profile while compounds 4a, 4d, 4e, 4i, and 4j showed moderate to good inhibitory activity at 2nd and 4th h, respectively. These compounds were also found to have considerable analgesic activity (acetic acid induced writhing model) and antipyretic activity (yeast induced pyrexia model). In addition, the tested compounds were also found to possess less degree of ulcerogenic potential as compared to the standard NSAIDs. Compounds that displayed promising anti-inflammatory profile were further evaluated for their inhibitory activity against cyclooxygenase enzyme (COX-1/COX-2), by colorimetric COX (ovine) inhibitor screening assay method. The results revealed that the compounds 4a, 4e, 4g and 4k exhibited effective inhibition against COX-2. In an attempt to understand the ligand-protein interactions in terms of the binding affinity, docking studies were performed using Molegro Virtual Docker (MVD-2013, 6.0) for those compounds, which showed good anti-inflammatory activity. It was observed that the binding affinities calculated were in agreement with the IC ₅₀ values.

Full text of DOI:10.1016/j.bmc.2014.03.043

Bioorganic & Medicinal Chemistry 22 (2014) 2855–2866
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, pharmacological screening and in silico studies of new
class of Diclofenac analogues as a promising anti-inflammatory
agents
a
a
b
Mahesh B. Palkar a,d, , Anuj S. Singhai , Pradeepkumar M. Ronad , A. H. M. Vishwanathswamy ,
⇑
Thippeswamy S. Boreddy c, Veeresh P. Veerapur c, Mahamadhanif S. Shaikh d, Rajesh A. Rane d,
Rajshekhar Karpoormath d,
⇑
a Department of Pharmaceutical Chemistry, K.L.E.U’s College of Pharmacy, Vidyanagar, Hubli 580 031, Karnataka, India
b Department of Pharmacology, K.L.E.U’s College of Pharmacy, Vidyanagar, Hubli 580 031, Karnataka, India
c Department of Pharmacology, Shri Siddaganga College of Pharmacy, Tumkur 572 102, Karnataka, India
d Department of Pharmaceutical Chemistry, School of Health Sciences, University of KwaZulu-Natal, Westville Campus, Durban 4000, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 5-[2-(2,6-dichlorophenylamino)benzyl]-3-(substituted)-1,3,4-oxadiazol-2(3H)-thione
(4a–k) derivatives have been synthesized by the Mannich reaction of 5-[2-(2,6-dichlorophenylami-
no)benzyl]-1,3,4-oxadiazol-2(3H)-thione (3) with an appropriately substituted primary/secondary
amines, in the presence of formaldehyde and absolute ethanol. Structures of these novel compounds
were characterized on the basis of physicochemical, spectral and elemental analysis. The title compounds
(4a–k) were screened for in vivo acute anti-inflammatory and analgesic activities at a dose of 10 mg/kg
b.w. Compound 4k exhibited the most promising and significant anti-inflammatory profile while com-
pounds 4a, 4d, 4e, 4i, and 4j showed moderate to good inhibitory activity at 2nd and 4th h, respectively.
These compounds were also found to have considerable analgesic activity (acetic acid induced writhing
model) and antipyretic activity (yeast induced pyrexia model). In addition, the tested compounds were
also found to possess less degree of ulcerogenic potential as compared to the standard NSAIDs. Com-
pounds that displayed promising anti-inflammatory profile were further evaluated for their inhibitory
activity against cyclooxygenase enzyme (COX-1/COX-2), by colorimetric COX (ovine) inhibitor screening
assay method. The results revealed that the compounds 4a, 4e, 4g and 4k exhibited effective inhibition
against COX-2. In an attempt to understand the ligand–protein interactions in terms of the binding affin-
ity, docking studies were performed using Molegro Virtual Docker (MVD-2013, 6.0) for those compounds,
which showed good anti-inflammatory activity. It was observed that the binding affinities calculated
were in agreement with the IC50 values.
Received 13 February 2014
Revised 27 March 2014
Accepted 28 March 2014
Available online 6 April 2014
Keywords:
1,3,4-Oxadiazole
Anti-inflammatory
Analgesic
Cyclooxygenase (COX)
Docking
Diclofenac
Mannich base
NSAIDs
Ulcerogenic
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
arachidonic acid by inhibiting the enzyme Cyclooxygenases (COXs)
and tromboxane synthase with a varying degree of selectivity.2 It is
Non-steroidal anti-inflammatory drugs (NSAIDs) are the most
preferred class of drugs in use for the treatment of various patho-
logical conditions such as pain, fever, inflammatory diseases and
rheumatoid arthritis.1 The pharmacological activity of NSAIDs is
related to the suppression of prostaglandin biosynthesis from
well known that COX exists in two isoforms, COX-1 and COX-2,
which are regulated differently.3 COX-1 provides cytoprotection
in the gastrointestinal (GI) tract whereas inducible COX-2 mediates
inflammation.4 COX-2 is an attractive target for medicinal chemists
as it is expressed only in few normal tissues and is greatly upreg-
ulated in inflamed tissues as well as many premalignant and
malignant tumors. Since, most of the NSAIDs in the market show
greater selectivity for COX-1 than COX-25 chronic use of NSAIDs,
including Diclofenac may elicit appreciable gastro-intestinal (GI)
irritation, bleeding and ulceration.6 The GI damage from NSAIDs
is generally attributed to two factors, local irritation by the carbox-
ylic acid moiety, common to most NSAIDs and (topical effect)
⇑
Corresponding authors. Tel.: +91 9448316466, +27 848777147; fax: +91
8362371694 (M.B.P.); tel.: +27 (0)312607179, +27 721107207; fax: +27
(0)312607792 (R.K.).
E-mail
addresses:
palkarmahesh4u@rediffmail.com,
palkar@ukzn.ac.za
(M.B. Palkar), karpoormath@ukzn.ac.za, rvk2006@gmail.com (R. Karpoormath).
http://dx.doi.org/10.1016/j.bmc.2014.03.043
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

2856
M. B. Palkar et al. / Bioorg. Med. Chem. 22 (2014) 2855–2866
decreased tissue prostaglandin production, which undermines the
physiological role of cytoprotective prostaglandins in maintaining
the GI health and homeostasis.7 Inflammation is known not only
as a symptom of great deal of common diseases but also as an early
phase of some serious diseases such as cancer, cardiac vascular dis-
eases and Alzheimer’s dementia. Therefore, the challenge still
exists for the pharmaceutical industry to develop, effective anti-
inflammatory agents with enhanced safety profile.
One of the most important strategies used to overcome NSAIDs
side effects is to modify the vital carboxylic acid group, yet main-
taining the efficacy of the drug. It has been reported that the deriv-
atization of the carboxylic functional group of some NSAIDs, for
example Flufenamic and Meclofenamic acids with a tetrazole
group, not only retained COX inhibitory activity of the parent drug,
but also introduced 5-lipooxygenase (5-LOX) inhibition. These
drugs also showed an increased anti-inflammatory activity with
reduced ulcerogenic effect.8,9 Several other heterocyclic com-
pounds, including pyrroles, thiazoles, imidazoles, thiadiazoles, di-
tert-butylphenyloxadiazoles and substituted 1,3,4-oxadiazole
derivatives have been studied and proved to be potent COX/5-LOX
inhibitors.10-14
Diclofenac is one of most widely used anti-inflammatory drug,
which suffers from a common toxicity of GI irritation, due to
non-selective inhibition of COX enzymes.15 From literature, it has
been observed that the Diclofenac moiety has been extensively
exploited by the organic and medicinal chemist to synthesize
potential therapeutic agents such as antibacterial, antitumor and
antituberculosis etc.16–18 Further, Bhandari et al.,19 have reported
the synthesis and pharmacological evaluations (anti-inflammatory,
analgesic and ulcerogenicity) of novel S-substituted phenacyl-
1,3,4-oxadiazol-2-thione and Schiff bases of Diclofenac. Recently,
El-Henawy20 reported the synthesis and molecular docking studies
of some novel Diclofenac derivatives containing phenylalanine
moiety as selective COX-2 inhibitors.
antitubercular and immunosuppressive.21–27 It has been reported
in literature that certain compounds bearing 1,3,4-oxadiazole
nucleus possess significant anti-inflammatory activity with
reduced ulcerogenic effect.28–30 The substituted 1,3,4-oxadiazole
derivatives serve both as biomimetic and reactive pharamaco-
phores, which are of considerable pharmaceutical interest. Based
on the above mentioned studies and in continuation of our
attempts31–34 to develop novel therapeutic anti-inflammatory
agents, we herein report the synthesis of a novel series of Mannich
base of 1,3,4-oxadiazole derivatives (4a–k). These compounds have
displayed a promising anti-inflammatory and analgesic profile
with a significant reduction in their ulcerogenic effect.
2. Results and discussion
2.1. Synthetic and spectral studies
The synthesis of 5-[2-(2,6-dichlorophenylamino)benzyl]-3-
(substituted methyl)-1,3,4-oxadiazol-2(3H)-thione (4a–k) deriva-
tives were achieved through convenient and efficient synthetic
route as outlined in Scheme 1. The methyl ester of Diclofenac (1)
was prepared by esterification of 2-[(2,6-dichloroanili-
no)phenyl]acetic acid (Diclofenac) in absolute methanol and few
drops of concd H2SO4 under reflux for 8 h. Methyl ester of Diclofe-
nac (1) was reacted with hydrazine hydrate (99%) in methanol
(anhydrous) and refluxed for 20 h to afford 2-[2-(2,6-dichlorophe-
nylamino)phenyl]acetohydrazide (2), which was further treated
with carbon disulphide and potassium hydroxide in methanol
(anhydrous) to give 5-[2-(2,6-dichlorophenylamino)benzyl]-1,3,4-
oxadiazol-2(3H)-thione (3). Finally, the title compounds (4a–k)
were synthesized in good yield by reacting 5-[2-(2,6-dichlorophe-
nylamino)benzyl]-1,3,4-oxadiazol-2(3H)-thione (3) with various
appropriately substituted primary/secondary amines in formalde-
hyde. This reaction is an example of the Mannich reaction.35
Structures of compound 3 and its Mannich base derivatives
(4a–4k) were established on the basis of their physicochemical
(see Table 1), spectral (UV, IR, 1H NMR, 13C NMR) and elemental
1,3,4-Oxadiazole is an imperative scaffold since several of these
derivatives are known to be associated with multiple biological
activities such as antiallergic, antibacterial, anticancer,
O
COOH
COOCH3
NH
C
NHNH2
Cl
(a)
(b)
NH
NH
Cl
Cl
Cl
Cl
Cl
2-(2-(2,6-dichlorophenylamino)
phenyl)acetic acid
(2)
(1)
methyl-2-(2-(2,6-dichlorophenyl
amino)phenyl)acetate
[Diclofenac]
2-(2-(2,6-dichlorophenylamino)
phenyl)acetohydrazide
(c)
R
N
NH
N
N
N N
6'
3'
5'
4'
1'
2'
S
S
SH
O
O
O
(d)
NH
NH
NH
1
Cl
Cl
Cl
Cl
Cl
Cl
2
3
6
5
4
(3)
(4a-k)
5-(2-(2,6-dichlorophenyl
amino)benzyl)-1,3,4-
oxadiazole-2-thiol
5-(2-(2,6-dichlorophenyl
5-[2-(2,6-dichlorophenylamino)
benzyl]-3-(substituted)-
amino)benzyl)-1,3,4-
oxadiazole-2(3H)-thione
1,3,4-oxadiazol-2(3H)-thione
Scheme 1. Synthesis of a novel series of 5-[2-(2,6-dichlorophenylamino)benzyl]-3-(substituted)-1,3,4-oxadiazol-2(3H)-thione (4a–k) derivatives. Reagents and Conditions:
(a) methanol, concd H2SO4, reflux, 2 h; (b) hydrazine hydrate (99%), absolute methanol, reflux, 20 h; (c) carbon disulphide, KOH, methanol, reflux, steam bath, 12 h; (d)
primary/secondary amine, formaldehyde, ethanol, gentle reflux, 10–15 h.

M. B. Palkar et al. / Bioorg. Med. Chem. 22 (2014) 2855–2866
2857
analysis. All the newly synthesized compounds showed acceptable
analysis of their anticipated structures, which are summarized in
experimental section. In general, the IR spectrum of compound 3
showed absorption bands ranging from 3373 to 3359 cmꢀ1 for N-
H, 1577 cmꢀ1 for C@N, 1362 cmꢀ1 for C@S and 1245 cmꢀ1 for
CAOAC groups, respectively. The absence of absorptions band of
the amidic carbonyl in the region 1690 cmꢀ1 and NHANH2 in the
region 3448–3437 cmꢀ1 indicated the formation of compound 3
from the corresponding acid hydrazide 2. This is further substanti-
ated from 1H NMR spectrum of compound 3, which showed pres-
ence of D2O exchangeable broad singlet peak at d 10.85 ppm for the
N-H protons, indicating its formation by simple cyclo-condensa-
tion process.36,37 The IR spectra of the title compounds (4a–4k)
Diclofenac) groups, respectively. The peaks around d 156.2–150.3
and 70.1–64.2 ppm accounted for fifth carbon of 1,3,4-oxadiazole
nucleus and methylene carbon of Mannich base (NACH2AN), thus
confirming the anticipated structures. The signals between d
141.2–118.0 ppm were assigned to the aromatic carbons while
the peaks of various carbons of secondary amines appeared
between d 58.0–45.0 and 40.0–20.1 ppm, respectively.
2.2. Pharmacological evaluation
The acute toxicological studies were carried out for all com-
pounds (4a–k) as per the guidelines of Organization for Economic
Co-operation and Development (OECD). The in vivo anti-inflamma-
tory (Carrageenan induced rat paw edema model) and analgesic
(acetic acid induced writhing test) activities were evaluated on
male/female albino rats and mice respectively. The most active
compounds were further evaluated for their antipyretic and
ulcerogenic activities.
showed moderately strong bands around 3377–3292 cmꢀ1
,
1606–1567 cmꢀ1 and 1324–1236 cmꢀ1, which are characteristic
of the N-H, C@N and C@S groups respectively. In 1H NMR spectra
the characteristic N-H proton (singlet) of Diclofenac ring appears
at d 9.11–8.5 ppm and the two distinctive singlets of methylene
protons (ANACH2ANA and ArACH2ACA) resonating at d 5.05–
4.42 and 3.43–3.13 ppm, respectively. Aromatic protons appeared
as multiplets between d 8.08–6.91 ppm while the protons signals
of various secondary amines were observed around d 2.97–
1.13 ppm, respectively. In 13C NMR spectrum, characteristic signals
appeared at around d 173.8–164.4 and 43.3–39.1 ppm, strongly
suggesting the presence of C@S (of 1,3,4-oxadiazole), and CH2 (of
2.2.1. Acute toxicity study
Preliminary toxicity data of all the test compounds demon-
strated high safety profile even at the upper most doses
(2000 mg/kg). The reported compounds herein are synthetic deriv-
atives of the marketed NSAID (Diclofenac), hence the dosage for
the pharmacological screening was fixed at 10 mg/kg b.w.
Table 1
Physicochemical data of a series of 5-[2-(2,6-dichlorophenylamino)benzyl]-3-(substituted methyl)-1,3,4-oxadiazol-2-(3H)-thione derivatives (4a–k)
R
N
N
6'
5'
4'
1'
2'
S
O
NH
3'
Cl
1
Cl
2
3
6
5
4
(4a-k)
Compound
R
Mp (°C)
132–134
162–164
Yield (%)
74.5
Rf valuea
0.30
Mol. formula
Mol. weight
451.30
4a
4b
N
N
O
C20H20N4Cl2O2S
C21H22N4Cl2OS
70.7
0.29
449.40
4c
184–186
66.1
0.44
C22H24N4Cl2OS
463.45
N
N
N
N
N
4d
4e
4f
176–178
190–192
198–200
182–184
71.4
75.5
74.8
71.9
0.53
0.43
0.52
0.44
C21H23N5Cl2OS
C22H25N5Cl2OS
C20H21N5Cl2OS
C22H24N4Cl2OS
464.40
478.30
450.35
463.40
N
CH3
N
C2H5
NH
4g
H3C
4h
4i
272–274
132–136
60.6
68.6
0.38
0.46
C23H20N4Cl2OS
C22H24N4Cl2OS
471.40
463.42
N
HN
Cl
4j
208–210
220–224
66.3
67.5
0.32
0.52
C22H17N4Cl3OS
C22H17N4Cl3OS
491.82
491.82
HN
HN
Cl
4k
a
All synthesized compounds were purified by flash chromatography using 200–400 mesh silica gel, eluted with hexane and dichloromethane as mobile phase and iodine
vapours as visualizing agent.

2858
M. B. Palkar et al. / Bioorg. Med. Chem. 22 (2014) 2855–2866
Table 2
10 mg/kgb.w.by carrageenan induced rat paw edema method. Car-
rageenan induced edema is a non-specific inflammation resulting
from a complex of diverse mediators. Since edema of this type is
highly sensitive to NSAIDs, carrageenan has been accepted as a
useful agent for studying anti-inflammatory screening. During
the second phase of inflammation, anti-inflammatory properties
of test agents are detected as a result of inhibition of prostaglandin
amplification. This model reliably predicts the anti-inflammatory
efficacy of the NSAIDs.38 As shown in Table 2, moderate to good
anti-inflammatory activity was observed for the synthesized com-
pounds, which exhibited 36.9–71.8% and 33.8–74.4% inhibition of
edema formation at 2nd and 4th h, respectively. Whereas the ref-
erence drug Diclofenac (13.5 mg/kg) displayed 65.5% and 66.2%
inhibition. In particular compound 4k (71.8% and 74.4%) was most
active, while compounds 4a (64.3% and 63.8%), 4d (65.1% and
64.5%), 4e (63.1% and 68.9%), 4i (65.9% and 67.5%) and 4j (59.4%
and 61.3%) displayed good to moderate inhibitory activity at 2nd
and 4th h, respectively. The anti-inflammatory activities of these
compounds were comparable to that of the standard drug Diclofe-
nac. The structure activity relationship (SAR) studies of these com-
pounds indicated that the presence of secondary amines like
morpholino, piperazino (especially N-substituted piperazine) and
chlorophenyl groups at the 3rd position of 1,3,4-oxadiazole moiety
greatly enhanced the anti-inflammatory activity, whereas com-
pounds containing piperidine group exhibited poor anti-inflamma-
tory activity. Compounds with good anti-inflammatory activity
profile were further evaluated for analgesic, antipyretic and ulcero-
genic activities.
Acute anti-inflammatory activity data of novel series of 5-[2-(2,6-dichlorophenyla-
mino)benzyl]-3-(substituted methyl)-1,3,4-oxadiazol-2(3H)-thione derivatives (4a–
k)
Compound
Paw volumea
% Inhibition of edemab
2nd h
4th h
2nd h
4th h
Control
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
2.49 0.13
0.88 0.03
1.50 0.13
1.51 0.05
0.87 0.07
0.92 0.07
1.21 0.01
1.57 0.10
1.45 0.08
0.85 0.02
1.01 0.04
0.70 0.01
0.86 0.09
2.90 0.07
1.05 0.31
1.57 0.05
1.84 0.01
1.03 0.06
0.90 0.05
1.26 0.07
1.92 0.05
1.71 0.09
0.94 0.07
1.12 0.24
0.74 0.01
0.98 0.14
—
—
64.3ꢁ
48.3ꢁ
39.4ꢁ
65.1ꢁ
63.1ꢁ
51.4ꢁ
36.9ꢁ
41.8ꢁ
65.9ꢁ
59.4ꢁ
71.8ꢁ
65.5ꢁꢁ
63.8ꢁ
45.9ꢁ
36.6ꢁ
64.5ꢁ
68.9ꢁ
56.5ꢁ
33.8ꢁ
41.0ꢁ
67.5ꢁ
61.3ꢁ
74.4ꢁ
66.2ꢁꢁ
Diclofenac
a
Values (mean SEM) are obtained using in vivo carrageenan induced rat paw
edema model, using six animals per group of male albino rats.
b
Significance was calculated by using one-way ANOVA with Dunnet ‘t’ test. The
difference in results was considered significant when P <0.05.
ꢁ
ꢁꢁ P <0.001 vs control at 10 mg/kg b.w.
P <0.001 vs control at 13.5 mg/kg b.w.
Table 3
Analgesic activity data of compounds 4a, 4b, 4d–f and 4i–k by acetic acid induced
writhing method
Compound
No. of writhes in 15 mina (mean SEM)
% Protection
Control
4a
4b
4d
4e
4f
4i
4j
29.0 1.81
6.67 0.33
7.50 0.76
6.50 0.56
5.50 0.22
7.00 0.68
6.50 0.43
6.50 0.56
5.18 0.40
6.00 0.86
—
2.2.3. Analgesic activity
77.0ꢁ
74.1ꢁ
77.6ꢁ
81.0ꢁ
75.9ꢁ
77.6ꢁ
77.6ꢁꢁ
82.2
An established and sensitive procedure to evaluate peripherally
acting analgesics is the acetic acid induced abdominal constriction
(writhing) response in mice. Table 3 summarizes the analgesic
activity of the selected compounds at a dose of 10 mg/kg b.w. It
was observed that compounds 4e (81.0%) and 4k (82.2%) exhibited
excellent activity, while compounds 4a (77.0%), 4b (74.1%), 4d, 4i,
4j (77.6%) and 4f (75.9%) displayed almost comparable analgesic
activity with that of reference drug (Diclofenac, 79.3%).
4k
Diclofenac
79.3ꢁꢁ
a
The results are expressed as mean SEM (n = 6). Significance was calculated by
using one-way ANOVA with Dunnet ‘t’ test. The difference in results was considered
significant when P <0.05.
2.2.4. Antipyretic activity
ꢁ
Promising anti-inflammatory and analgesic activity prompted
us to carry out the antipyretic activity (yeast-induced pyrexia)
for compounds 4a, 4d, 4e, 4i, 4j and 4k at a dose of 10 mg/kg
b.w. in rats. The results of antipyretic activity are presented in
Table 4. Perusal of the data strongly suggests that compounds 4e,
4i and 4k exhibited excellent antipyretic activity, while
ꢁꢁ P <0.001 vs control at 10 mg/kg b.w.
P <0.001 vs control at 19.5 mg/kg b.w.
2.2.2. Acute anti-inflammatory activity
The data presented in Table 2 shows the results of acute anti-
inflammatory activity of title compounds (4a–k) at a dose of
Table 4
Antipyretic activity data of the title compounds 4a, 4d, 4e, 4i, 4j and 4k by yeast induced pyrexia
Compound
Mean temperature in °C at intervalsb
TIc
0a
1
2
3
4
5
Control
4a
4d
4e
4i
4j
4k
38.30
38.10
38.27
39.40
38.13
38.01
39.20
39.00
38.26ꢁꢁ
37.70
38.20ꢁꢁ
37.55
38.11
38.00ꢁꢁꢁ
37.06
37.89ꢁꢁꢁ
37.15
ꢀ1.04
ꢀ3.91
ꢀ2.81
ꢀ6.60
ꢀ4.38
ꢀ3.06
ꢀ5.40
ꢀ6.70
37.13ꢁꢁꢁꢁ
37.72ꢁꢁꢁ
39.20
37.94ꢁꢁ
38.30ꢁꢁ
37.69ꢁꢁ
37.77ꢁ
38.60ꢁ
38.0ꢁꢁꢁ
37.80
37.44ꢁꢁꢁ
37.70ꢁꢁꢁ
37.79ꢁꢁꢁ
37.06ꢁꢁꢁ
37.80ꢁꢁ
37.3ꢁꢁꢁ
37.20ꢁꢁꢁ
37.10ꢁꢁ
36.78ꢁꢁꢁ
37.11ꢁꢁ
37.20ꢁꢁ
37.0ꢁꢁ
37.90ꢁꢁꢁ
37.03ꢁꢁꢁ
37.36ꢁꢁꢁ
38.10ꢁꢁ
37.6ꢁꢁꢁ
37.98ꢁ
37.74ꢁ
38.90ꢁ
38.4ꢁꢁ
Paracetamol
a
b
c
18th h After yeast injection was considered as 0 h.
Temperature was recorded hourly from 0 to 5 h after dosing.
Temperature index (TI) is the sum of mean temperature changes from the 0 h. The results are expressed as mean values (n = 6). Significance was calculated by using one-
way ANOVA with Dunnet ‘t’ test. The difference in results was considered significant when P <0.05.
ꢁ
ꢁꢁ P <0.05 vs control.
ꢁꢁꢁ P <0.01 vs control.
P <0.001 vs control.

M. B. Palkar et al. / Bioorg. Med. Chem. 22 (2014) 2855–2866
2859
compounds 4a and 4j possess reasonable to good antipyretic activ-
ity as compared to standard drug Paracetamol.
summarized in Table 6. These results indicate that the compounds
4a, 4e, 4i and 4k exhibited significant and effective inhibition
against COX-2 (67.5%, 73.16%, 81.36% and 84.14%) as compared
to the inhibition of COX-1 (34.14%, 28.36%, 19.56% and 25.04%).
The IC50 values of these compounds 4a, 4e, 4i and 4k were 15.0,
2.2.5. Ulcerogenic activity
One of the major side effects of NSAIDs is gastric irritation,
which leads to the formation of gastric ulcers. The extent of ulcero-
genic effect was evaluated for compounds 4a, 4d, 4e, 4i, 4j and 4k
at a therapeutic dose (10 mg/kg b.w.) in rat stress model. The gas-
tric ulcerogenic potential was evaluated by calculating the ulcer
index in treated and control animals. Table 5 indicates the ulcer
index range of compounds 4a (2.6–3.1), 4d (2.3–2.8), 4e (1.9–
2.4), 4i (3.6–4.1), 4j (3.2–3.5) and 4k (2.1–2.6) as compared to
the standard drugs Diclofenac (5.5–6.2), Aspirin (4.7–5.2) and Flur-
biprofen (5.9–6.6), respectively. From the results obtained it was
clearly suggests that the tested compounds cause less gastric ulcer-
ation and disruption of gastric epithelial cells at the above men-
tioned oral dose. Hence, gastric tolerance to these compounds
was found to be better than that of standard drugs.
12.5, 10.5 and 9.0
whereas for COX-1 it was greater than 50
of standard drugs the IC50 values were 0.04, 14.0
and 15.0, 6.25
respectively.
l
M, respectively against COX-2 enzyme,
M. However, in the case
M (Celecoxib)
lM (Diclofenac) against COX-2 and COX-1 enzyme,
l
l
2.4. Molecular docking studies
The in vitro COX-2 enzyme inhibition results were promising
and to further validate our experimental results, molecular docking
studies for the selected compounds 4a, 4b, 4d–f and 4i–k were car-
ried out using Molegro Virtual Docker (MVD-2013, 6.0). Figure 1
shows the native crystal structure of Diclofenac (DIF701) bound
to the active site of COX-2 obtained from Protein Data Bank
(http://www.rscb.org/pdb) with the PDB ID: 1PXX.
2.3. In vitro colorimetric COX (ovine) inhibitor assay
The crystal structure of 1PXX exists as a homodimeric assembly,
and the inhibitor DIF701 is bound to all four chains of the struc-
ture, but none of the binding sites come in the interface of two
domains or two different chains.40 Thus the docking studies were
performed on a single subunit of the homodimeric COX structure.
The PDB structure 1PXX bound to the inhibitor DIF701 shows a
true binding site for each of the subunits. An essential feature of
the binding site is the conservation of hydrogen bondings and
The synthesized compounds 4a, 4b, 4d–f and 4i–k were also
evaluated for their ability to inhibit COX-2 enzyme by in vitro col-
orimetric COX (ovine) inhibitor assay method,39 which utilizes the
peroxidase component of cyclooxygenase. The peroxidase activity
is assayed colormetrically by monitoring the appearance of oxi-
dized N,N,N0,N0-tetramethyl-p-phenylenediamine (TMPD), which
is produced during the reduction of PGG2 to PGH2, at 590 nm.
The results of in vitro COX enzyme inhibition assay studies are
the aromatic p–p stacking interactions. The active pocket consisted
of amino acid residues such as Val349, Ser530, Leu352, Tyr385,
Tyr348, Trp387, Gly526, Ala527, Met522, and Leu384. Hence to
identify other residual interactions of the tested compounds, a grid
box (include residues within a 10.0 Å radius) large enough to
accommodate the active site was constructed. DIF701 being a
known inhibitor, the center of this site was considered as the cen-
ter of search space for docking. The 2D structures of the synthe-
sized ligands were converted to energy minimized 3D structures
and were used for in silico protein–ligand docking calculations.
Docking of the synthesized compounds with the COX-2 enzyme,
exhibited the well-conserved hydrogen bonds with one or more
amino acid residues of the active pocket. From Table 6, following
conclusions can be drawn from the docking studies. For com-
pounds 4a, 4b, 4d–f and 4i–k, the MolDock score ranged from
ꢀ97.620 to ꢀ140.521 while the Moldock score of standard drug
DIF701 was ꢀ96.142. The best poses of the docked compounds
and of DIF701 are shown in Figure 2. Compound 4k displayed
Table 5
Ulcerogenic activity data of selected compounds in
comparison with standard NSAIDs
Compound
Ulcer index rangea
Control
4a
4d
4e
4i
4j
4k
Diclofenac
Aspirin
Flurbiprofen
1.3–1.6
2.6–3.1
2.3–2.8
1.9–2.4
3.6–4.1
3.2–3.5
2.1–2.6
5.5–6.2
4.7–5.2
5.9–6.6
a
The results are expressed as ulcer index values
from minimum to maximum range.
Table 6
In vitro COX-1/COX-2 inhibition data along with Moldock scores and other energy calculations of the title compounds by MVD-2013 (6.0) against 1PXX
Compound
% Inhibitiona,b
COX-2
Moldock
score
(Kcal/mol)
E-inter
(protein–ligand)
H-bonds
Heavy LE1
atoms
count
LE3
Docking
Score
(Kcal/mol) (Kcal/mol)
Rerank
score
COX-1
No. (Kcal/mol)
4a
4b
4d
4e
4f
4i
34.14 2.32 (>50)c 67.50 1.21 (15.0) ꢀ103.864
ꢀ131.169
ꢀ139.534
ꢀ129.197
ꢀ138.776
ꢀ139.512
ꢀ147.219
ꢀ134.903
ꢀ153.544
2
1
4
4
1
4
3
5
2
—
ꢀ4.7427
ꢀ2.5000
ꢀ8.4047
ꢀ8.7451
ꢀ2.5000
ꢀ7.2633
ꢀ6.6861
ꢀ8.1369
ꢀ5.0000
—
29
29
30
31
29
30
31
31
19
—
ꢀ3.5815 ꢀ0.4290 ꢀ586.846
ꢀ4.0859 ꢀ1.2531 ꢀ595.114
ꢀ12.4416
ꢀ36.3414
15.9847
ꢀ11.2921
ꢀ78.1681
ꢀ72.0776
ꢀ58.3729
ꢀ84.6778
ꢀ72.4004
—
30.86 1.55 (>50)
35.24 1.02 (>50)
28.36 1.73 (>50)
43.16 2.61 (>50)
19.56 1.17 (>50)
27.82 3.06 (>50)
25.04 1.46 (>50)
60.22 0.62 (15.0) ꢀ118.492
63.74 1.36 (14.5) ꢀ116.825
73.16 2.25 (12.5) ꢀ124.225
58.10 3.18 (12.0) ꢀ110.828
81.36 0.80 (10.5) ꢀ136.089
64.58 1.75 (13.0) ꢀ109.092
ꢀ3.8941
0.5328 ꢀ611.878
ꢀ4.0072 ꢀ0.3642 ꢀ621.682
ꢀ3.8216 ꢀ2.6954 ꢀ600.68
ꢀ4.5363 ꢀ2.4025 ꢀ633.994
ꢀ3.5190 ꢀ1.8830 ꢀ589.038
ꢀ4.5329 ꢀ2.7315 ꢀ635.878
ꢀ5.0601 ꢀ3.8105 ꢀ586.01
4j
4kꢁ
84.14 1.18 (9.0)
ꢀ140.521
Diclofenac
Celecoxib
90.64 1.15 (6.25) 48.58 1.42 (15.0)
2.8 0.35 (14) 97.94 2.72 (0.04)
ꢀ96.1429 ꢀ109.698
—
—
—
—
—
a
b
c
Values are acquired using in vitro ovine COX-1/COX-2 assay kit (Catalog No. 760 111, Cayman Chemicals Inc., Ann Arbor, MI).
Experiments were carried out in triplicate at 10 g/ml and have less than 10% error.
Values in bracket are IC50 in M.
l
l
ꢁ
Compound exhibited high binding energy (Moldock score); H bonds: hydrogen bonding energy between protein and ligand; LE1 (ligand efficiency): MolDock score divided
by heavy atoms count; LE3: Rerank score divided by heavy atoms count.

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M. B. Palkar et al. / Bioorg. Med. Chem. 22 (2014) 2855–2866
Figure 1. (A) Secondary structure (complete protein) of COX-2 enzyme (PDB ID: 1PXX) complexed with DIF701 (Diclofenac), showing its dimeric nature with two identical
subunits in each monomer; (B) chain A colored in pink (part of complete protein) complexed with DIF701.
highest number of conserved hydrogen bonds (5) with the residues
of the active site. In compounds 4d (3.2208 Å), 4e (3.2425 Å), 4f
(2.6769 Å), 4i (3.2782 Å), 4j (3.2627 Å) and 4k (3.4474 Å) promi-
nent hydrogen bonding interactions were observed between the
oxygen atom of heterocyclic ring (1,3,4-oxadiazole) and the hydro-
xyl group (OH) of the amino acid residue Ser530. While in some
compounds 4d (3.2982 Å), 4e (3.2084 Å), 4i (3.1182 Å) and 4k
(2.9650 Å) hydrogen bonding was also observed between the oxy-
gen atom of the 1,3,4-oxadiazole and OH group of Tyr385 residue.
The nitrogen atom (secondary amines) of various Mannich bases
also displayed hydrogen bonding with either OH group of Ser530
or Tyr385. The nitrogen atom in compounds 4d (p-methyl pipera-
zine), 4e (p-ethyl piperazine) and 4k (3-chlorophenyl amine) dis-
played hydrogen bonding (2.6998 Å, 2.6878 Å and 2.5210 Å) with
OH group of Ser530. However in the case of compound 4a, the
nitrogen and oxygen atoms of the morpholine ring exhibited
hydrogen bonding (2.9881 Å, 3.1514 Å) with the OH group of
Tyr385 as well as with Ser530 residues respectively. The docking
results of the active compounds showed conserved hydrogen bon-
dings with both the residues Ser530 and Tyr385, which is essential
for the inhibition of COX-2 enzyme.40 These results provide strong
evidence that the novel Mannich bases of 1,3,4-oxadiazole play a
vital role in binding, leading to an effective inhibition of COX-2
enzyme.
3. Conclusion
We herein report the synthesis, spectral studies and pharmaco-
logical evaluation of a novel series of 5-[2-(2,6-dichlorophenylami-
no)benzyl]-3-(substituted methyl)-1,3,4-oxadiazol-2(3H)-thione

M. B. Palkar et al. / Bioorg. Med. Chem. 22 (2014) 2855–2866
2861
Figure 2. Showing the compounds docked in best of its conformation into the binding site of 1PXX (A) binding mode of DIF701 (Diclofenac) forming 1H bond with Ser530 and
1H bond with Tyr385; (B) binding mode of compound 4d forming 3H bonds with Ser530 and 1H bond with Tyr385; (C) binding mode of compound 4e forming 3H bonds with
Ser530 and 1H bond with Tyr385; (D) binding mode of compound 4i forming 3H bonds with Ser530 and 1H bond with Tyr385; (E) binding mode of compound 4j forming 2H
bonds with Ser530 and 1H bond with Tyr385; (F) binding mode of compound 4k forming 4H bonds with Ser530 and 1H bond with Tyr385.
(4a–k) derivatives. From the acute toxicity experiments, these
compounds showed no toxic effects even at high doses
(2000 mg/kg b.w). Compound 4k was most promising with signif-
icant anti-inflammatory activity, while moderate to good activity
was observed for compounds 4a, 4d, 4e, 4i, and 4j. Surprisingly
these compounds also exhibited significant analgesic and antipy-
retic activity with least ulcerogenic properties. The tested com-
pounds also showed better in vitro inhibitory activity against

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M. B. Palkar et al. / Bioorg. Med. Chem. 22 (2014) 2855–2866
COX-2, when compared to COX-1 enzyme. Molecular docking stud-
ies (MVD-2013, 6.0) performed was useful in finding some possible
lead compounds among the tested series. The binding orientation
of the docked compounds almost resembled the orientation of
the known standard drug (DIF701) with the enzyme 1PXX. Further
the binding affinity/energies (MolDock score) calculated by dock-
ing studies on COX-2 enzyme were in agreement with the
observed experimental results. In conclusion, compound 4k has
presented the most encouraging results and could be further
exploited for developing new anti-inflammatory agents with better
efficacy and safety.
15944.24). IR spectra (KBr, m
max, cmꢀ1): 3448, 3437 (NH2), 3396
(N-H), 3022 (CAH), 1690 (C@O), 770 (C-Cl). 1H NMR (CDCl3, d,
ppm): 8.95 (s, 1H, CONH), 8.44 (s, 1H, N-H), 7.63–6.87 (m, 7H,
Ar-H), 4.26 (s, 2H, NH2), 2.64 (s, 2H, CH2). 13C NMR (CDCl3, d,
ppm): 181.1 (C@O), 140.4 (C-1), 134.3 (C-20), 129.8 (C-60), 128.8
(C-6), 128.5 (C-2), 127.2 (C-3, C-5), 125.3 (C-30), 122.9 (C-10),
121.2 (C-50), 119.7 (C-4), 118.4 (C-40), 35.1 (CH2). EIMS m/z:
311.3 (M+1)+.
4.2.3. Synthesis of 5-[2-(2,6-dichlorophenylamino)benzyl]-
1,3,4-oxadiazole-2(3H)-thione(3)
A mixture of compound 2 (0.005 mol, 1.55 g), KOH (0.005 mol,
0.28 g) and carbon disulphide (5 ml) in anhydrous methanol
(50 ml) was refluxed on a steam bath for 12 h (monitored by
TLC). The solution was then concentrated, cooled and acidified
with dil. HCl. The pale yellowish solid mass that separated out
was filtered, washed with ethanol, dried and recrystallized from
ethanol/water (3:1). Yield: 72.68%. Mp 162–164 °C. UV (CH3OH,
4. Experimental
4.1. Chemistry protocols
All research chemicals were purchased from Sigma–Aldrich (St.
Louis, Missouri, USA) or Lancaster Co. (Ward Hill, MA, USA) and
used as such for the reactions. Various NSAIDs used in this work
were received as a gift sample from Fourrtis (India) Lab. Pvt. Ltd,
Chennai (Tamilnadu, India). Solvents except laboratory reagent
grade were dried and purified according to the literature when
necessary. The progress of reactions and purity of compounds were
monitored by thin-layer chromatography (TLC) on pre-coated silica
gel plates procured from E. Merck and Co. (Darmstadt, Germany).
Melting points of synthesized compounds were determined in
Thermonik (Mumbai, India) melting point apparatus and are
uncorrected. UV spectra were recorded on Thermospectronic
(Rochester, NY, USA) and IR spectra were recorded on Thermo
Nicolet IR200 FT-IR Spectrometer (Madison, WI, USA) by using
KBr pellets. The 1H NMR and 13C NMR were recorded on Bruker
AVANCE 400 (Bruker, Rheinstetten/Karlsruhe, Germany) using
appropriate solvent. Chemical shifts are reported in d ppm units
with respect to TMS as internal standard. The elemental analysis
(C,H,N) of the compounds were performed on Heraus CHN rapid
analyzer. Results of elemental analysis were within 0.4% of the
theoretical values. The synthesized title compounds were purified
by using flash chromatography SP-01 (Biotage, Sweden). The anti-
inflammatory activity was carried out using digital plethysmome-
ter (Ugo-Basile, Italy) and antipyretic activity was carried out using
Elicotelethermometer (Hyderabad, India).
kmax): 267 (e: 29330.99). IR spectra (KBr, m
max, cmꢀ1): 3373, 3359
(N-H), 3086 (CAH), 1577 (C@N), 1362 (C@S), 1245 (C–O–C), 777
(C-Cl). 1H NMR (CDCl3, d, ppm): 10.85 (s, 1H, N-H of 1,3,4-oxadiaz-
ole, D2O exchangeable), 8.95 (s, 1H, N-H, D2O exchangeable), 7.85–
7.05 (m, 7H, Ar-H), 3.18 (s, 2H, CH2). 13C NMR (CDCl3, d, ppm):
170.6 (C@S), 150.8 (C-5 of oxadiazole), 136.3 (C-1), 131.1 (C-20),
130.4 (C-60), 129.7 (C-6), 129.0 (C-2), 128.1 (C-3, C-5), 126.9
(C-30), 124.5 (C-10), 122.8 (C-50), 120.2 (C-4), 119.2 (C-40), 38.4
(CH2). EIMS m/z: 353.1 (M+1)+.
4.2.4. General procedure for the synthesis of 5-[2-(2,6-
dichlorophenylamino)benzyl]-3-(substituted methyl)-1,3,4-
oxadiazol-2-(3H)-thione (4a–k)36,37
A mixture of compound 3 (0.002 mol), an appropriately substi-
tuted primary/secondary amines (0.004 mol), and 37% aqueous
formaldehyde (0.002 mol) in 20 ml of ethanol was heated under
gentle reflux for 10–15 h, and the solvent was removed under vac-
uum. The residue was dissolved in ethyl acetate, washed twice
with water, and dried (Na2SO4). Further, the title compounds were
purified by removal of the solvent followed by flash chromatogra-
phy on 200–400 mesh silica gel, eluting with hexane and dichloro-
methane. The physicochemical properties of these compounds are
summarized in Table 1.
4.2.4.1.
5-[2-(2,6-Dichlorophenylamino)benzyl]-3-(morpholi-
This was
4.2. Synthesis
nomethyl)-1,3,4-oxadiazol-2-(3H)-thione (4a).
obtained by reacting 5-[2-(2,6-dichlorophenylamino)benzyl]-
1,3,4-oxadiazole-2(3H)-thione (3, 0.704 g) and morpholine
(0.408 g) as described in the general procedure and isolated as
cream colored crystals. Yield: 74.5%. Mp 132–134 °C. UV (CH3OH,
4.2.1. Synthesis of methyl-2-[2-(2,6-
dichlorophenylamino)phenyl]acetate (1)
The methyl ester of Diclofenac was prepared as per procedure
reported earlier in the literature.31 This compound was obtained
as a white crystalline solid. Yield: 76.43%. Mp 96–98 °C. UV
kmax): 266.5 (e 14477.10). IR spectra (KBr, m
max, cmꢀ1): 3325 (N-
H), 3068 (CAH), 1576 (C@N), 1511 (C@C), 1312 (C@S), 1229
(CAOAC), 762 (C-Cl). 1H NMR (CDCl3, d, ppm): 8.50 (s, 1H, N-H),
7.86–6.96 (m, 7H, Ar-H), 4.93 (s, 2H, NACH2AN), 3.68–3.64 (s,
4H, morpholine), 3.27 (s, 2H, CH2), 2.92–2.94 (m, 4H, morpholine).
13C NMR (CDCl3, d, ppm): 166.4 (C@S), 153.2 (C-5 of oxadiazole),
145.7 (C-20), 141.2 (C-1), 130.1 (C-60), 124.7 (C-2, C-6), 124.3
(C-3, C-5), 123.6 (C-40), 122.2 (C-10), 119.9 (C-30), 119.8 (C-4), 112.6
(C-50), 68.1 (NACH2AN), 62.4, 57.1 (C-2, C-3, C-5, C-6 of morpho-
line), 39.7 (CH2). Elemental (CHN) analysis calculated for C20H20Cl2
N4O2S: C, 53.24; H, 4.48; N, 12.40 (found: C, 53.20; H, 4.50; N,
12.44).
(CH3OH, kmax): 271.5 (e: 8173.37). IR spectra (KBr, m
max, cmꢀ1):
3447 (N-H), 3082 (CAH), 1730 (C@O), 1238 (CAOAC), 783 (C-Cl).
1H NMR (CDCl3, d, ppm): 8.69 (s, 1H, N-H), 7.52–6.93 (m, 7H,
Ar-H), 3.82 (s, 3H, OCH3), 2.75 (s, 2H, CH2). 13C NMR (CDCl3, d,
ppm): 183.5 (C@O), 141.2 (C-1), 136.7 (C-20), 132.1 (C-30), 129.9
(C-2), 129.2 (C-6), 128.4 (C-3, C-5), 127.5 (C-60), 127.0 (C-10),
126.2 (C-40), 123.0 (C-4), 121.4 (C-50), 57.3 (OCH3), 34.7 (CH2).
4.2.2. Synthesis of 2-[2-(2,6-
dichlorophenylamino)phenyl]acetohydrazide (2)
Compound
1
(0.01 mol, 3.10 g) and hydrazine hydrate
(0.02 mol, 1 g) were refluxed in absolute methanol (50 ml) for
24 h (monitored by TLC). The mixture was concentrated, cooled
and poured in ice cold water. White amorphous solid thus sepa-
rated out was filtered, dried and recrystallized from ethanol and
4.2.4.2. 5-[2-(2,6-Dichlorophenylamino)benzyl]-3-(piperidin-1-
ylmethyl)-1,3,4-oxadiazol-2-(3H)-thione (4b).
This was
obtained by reacting 5-[2-(2,6-dichlorophenylamino)benzyl]-
1,3,4-oxadiazole-2(3H)-thione (3, 0.704 g) and piperdine (0.400 g)
as described in the general procedure and isolated as yellowish
water. Yield: 66.0%. Mp 156–158 °C. UV (CH3OH, kmax): 280.5 (e:

M. B. Palkar et al. / Bioorg. Med. Chem. 22 (2014) 2855–2866
2863
white crystalline solid. Yield: 70.7%. Mp 162–164 °C. UV (CH3OH,
kmax): 265.5 ( 16882.40). IR spectra (KBr,
max, cmꢀ1): 3377 (N-
oxadiazole), 136.6 (C-20), 134.0 (C-1), 131.3 (C-60), 129.7 (C-2,
C-6), 128.1 (C-3, C-5), 126.0 (C-40), 123.9 (C-10), 121.5 (C-30),
120.4 (C-4), 118.6 (C-50), 67.6 (NACH2AN), 55.3–47.2 (C2, C3, C5
and C6 of piperazine), 39.1 (CH2), 20.1 (CH3). Elemental (CHN)
analysis calculated for C22H25Cl2N5OS: C, 55.24; H, 5.28; N, 14.65
(found: C, 55.27; H, 5.24; N, 14.64).
e
m
H), 2926 (CAH), 1575 (C@N), 1505 (C@C), 1252 (C@S), 1205
(CAOAC), 781 (C-Cl). 1H NMR (CDCl3, d, ppm): 9.07 (s, 1H, N-H),
7.76–7.24 (m, 7H, Ar-H), 4.82 (s, 2H, NACH2AN), 3.16 (s, 2H,
CH2), 2.80–2.72 (m, 4H, piperidine), 2.31–2.28 (dd, 2H, piperidine),
1.84–1.79 (m, 4H, piperidine). 13C NMR (CDCl3, d, ppm): 171.3
(C@S), 152.7 (C-5 of oxadiazole), 137.1 (C-20), 134.4 (C-1), 131.6
(C-60), 130.1 (C-2, C-6), 128.9 (C-3, C-5), 125.5 (C-40), 125.4 (C-10),
123.2 (C-30), 120.3 (C-4), 118.4 (C-50), 64.6 (NACH2AN), 40.2
(CH2), 34.2-24.3 (C2, C3, C4, C5 and C6 of piperdine). Elemental
(CHN) analysis calculated for C21H22Cl2N4OS: C, 56.12; H, 4.94; N,
12.49 (found: C, 56.15; H, 4.91; N, 12.46).
4.2.4.6. 5-[2-(2,6-Dichlorophenylamino)benzyl]-3-(piperazin-1-
ylmethyl)-1,3,4-oxadiazol-2-(3H)-thione (4f).
This was
obtained by reacting 5-[2-(2,6-dichlorophenylamino)benzyl]-
1,3,4-oxadiazole-2(3H)-thione (3, 0.704 g) and piperazine
(0.404 g) as described in the general procedure and isolated as dark
cream coloured crystalline solid. Yield: 74.8%. Mp 198–200 °C. UV
(CH3OH, kmax): 266 (e 45900). IR spectra (KBr, m
max, cmꢀ1): 3350
4.2.4.3. 5-[2-(2,6-Dichlorophenylamino)benzyl]-3-[(2-methylpi-
peridin-1-yl)methyl]-1,3,4-oxadiazol-2-(3H)-thione (4c). This
was obtained by reacting 5-[2-(2,6-dichlorophenylamino)benzyl]-
1,3,4-oxadiazole-2(3H)-thione (3, 0.704 g) and 2-methylpiperdine
(0.457 g) as described in the general procedure and isolated as yel-
lowish white crystalline solid. Yield: 66.1%. Mp 184–186 °C. UV
(N-H), 2940 (CAH), 1575 (C@N), 1500 (C@C), 1324 (C@S), 1224
(CAOAC), 760 (C-Cl). 1H NMR (CDCl3, d, ppm): 9.02 (s, 1H, N-H),
8.08–7.24 (m, 7H, Ar-H), 5.01 (s, 2H, NACH2AN), 4.13 (s, 1H, N-H
of piperazine), 3.37 (s, 2H, CH2), 2.47–2.43 (dd, 4H, piperazine),
2.25–2.19 (dd, 4H, piperazine). 13C NMR (CDCl3, d, ppm): 171.5
(C@S), 154.1 (C-5 of oxadiazole), 137.2 (C-20), 133.3 (C-1), 131.6
(C-60), 130.1 (C-2, C-6), 129.3 (C-3, C-5), 125.7 (C-40), 123.2 (C-10),
121.3 (C-30), 120.0 (C-4), 118.8 (C-50), 64.2 (NACH2AN), 57.1–
48.5 (C2, C3, C5 and C6 of piperazine), 39.8 (CH2). Elemental
(CHN) analysis calculated for C20H21Cl2N5OS: C, 53.35; H, 4.69; N,
15.55 (found: C, 53.33; H, 4.72; N, 15.54).
(CH3OH, kmax): 264.5 (e 23196.30). IR spectra (KBr, m
max, cmꢀ1):
3307 (N-H), 2942 (CAH), 1578 (C@N), 1507 (C@C), 1314 (C@S),
1217 (CAOAC), 740 (C-Cl). 1H NMR (CDCl3, d, ppm): 8.81 (s, 1H,
N-H), 7.64–7.19 (m, 7H, Ar-H), 4.76 (s, 2H, NACH2AN), 3.43 (s,
2H, CH2), 2.80–2.72 (m, 1H, CH of piperidine), 1.83–1.56 (m, 8H,
piperidine), 1.37–1.35 (d, 3H, methyl protons of piperidine). 13C
NMR (CDCl3, d, ppm): 169.5 (C@S), 150.3 (C-5 of oxadiazole),
140.2 (C-20), 133.1 (C-1), 131.3 (C-60), 129.9 (C-2, C-6), 129.1
(C-3, C-5), 123.6 (C-40), 122.7 (C-10), 121.4 (C-30), 119.4 (C-4),
118.2 (C-50), 70.1 (NACH2AN), 42.1 (CH2), 40.6-26.4 (C2, C3, C4,
C5 and C6 of piperidine), 21.2 (CH3). Elemental (CHN) analysis
calculated for C22H24Cl2N4OS: C, 57.03; H, 5.21; N, 12.08 (found:
C, 57.01; H, 5.23; N, 12.10).
4.2.4.7. 5-[2-(2,6-Dichlorophenylamino)benzyl]-3-[(4-methylpi-
peridin-1-yl)methyl]-1,3,4-oxadiazol-2-(3H)-thione (4g). This
was obtained by reacting 5-[2-(2,6-dichlorophenylamino)benzyl]-
1,3,4-oxadiazole-2(3H)-thione (3, 0.704 g) and 4-methylpiperidine
(0.456 g) as described in the general procedure and isolated as light
yellow coloured crystalline solid. Yield: 71.9%. Mp 182–184 °C. UV
(CH3OH, kmax): 264.5 (e 27502.20). IR spectra (KBr, m
max, cmꢀ1):
3360 (N-H), 3069 (CAH), 1567 (C@N), 1451 (C@C), 1267 (C@S),
1197 (CAOAC), 789 (C-Cl). 1H NMR (DMSO-d6, d, ppm): 9.11 (s,
1H, N-H), 7.93–7.19 (m, 7H, Ar-H), 4.96 (s, 2H, NACH2AN), 3.59
(s, 2H, CH2), 3.12–3.04 (m, 2H, piperidine), 2.92 (s, 3H, 4-CH3 pro-
tons of piperidine), 2.86–2.83 (m, 1H, piperidine), 1.69–1.63 (m,
4H, piperidine), 1.12–1.07 (m, 2H, piperidine). 13C NMR (CDCl3, d,
ppm): 170.0 (C@S), 153.6 (C-5 of oxadiazole), 145.7 (C-20), 139.9
(C-1), 130.0 (C-60), 126.6 (C-2, C-6), 124.5 (C-3), 123.4 (C-5),
122.0 (C-40), 119.5 (C-10, C-30), 112.9 (C-4), 110.8 (C-50), 66.3
(NACH2AN), 43.3 (CH2), 33.9–29.7 (C2, C3, C4, C5 and C6 of piper-
idine), 21.3 (CH3of piperidine). Elemental (CHN) analysis calcu-
lated for C22H24Cl2N4OS: C, 57.02; H, 5.23; N, 12.08 (found: C,
57.03; H, 5.25; N, 12.06).
4.2.4.4. 5-[2-(2,6-Dichlorophenylamino)benzyl]-3-[(4-methylpi-
perazin-1-yl)methyl]-1,3,4-oxadiazol-2-(3H)-thione (4d). This
was obtained by reacting 5-[2-(2,6-dichlorophenylamino)benzyl]-
1,3,4-oxadiazole-2(3H)-thione (3, 0.704 g) and 4-methylpiperazine
(0.460 g) as described in the general procedure and isolated as
cream coloured crystalline solid. Yield: 71.4%. Mp 176–178 °C.
UV (CH3OH, kmax): 268 (e 19720). IR spectra (KBr, m
max, cmꢀ1):
3324 (N-H), 2938 (CAH), 1587 (C@N), 1504 (C@C), 1288 (C@S),
1197 (CAOAC), 756 (C-Cl). 1H NMR (DMSO-d6, d, ppm): 8.93 (s,
1H, N-H), 7.76–7.00 (m, 7H, Ar-H), 4.42 (s, 2H, NACH2AN), 3.57
(s, 4H, piperazine), 3.17 (s, 2H, CH2), 2.39 (s, 4H, piperazine), 2.13
(s, 3H, CH3). 13C NMR (CDCl3, d, ppm): 172.2 (C@S), 153.1 (C-5 of
oxadiazole), 145.6 (C-20), 140.0 (C-1), 130.0 (C-60), 125.9 (C-2, C-
6), 124.7 (C-30), 123.4 (C-40), 122.0 (C-10), 119.9, 119.5 (C-3, C-5),
112.9 (C-4), 110.9 (C-50), 68.2 (NACH2AN), 56.8, 45.4 (C2, C3, C5
and C6 of piperazine), 40.9 (CH2), 36.3 (N-CH3). Elemental (CHN)
analysis calculated for C21H23Cl2N5OS: C, 54.30; H, 4.98; N, 15.07
(found: C, 54.32; H, 5.00; N, 15.10).
4.2.4.8. 5-[2-(2,6-Dichlorophenylamino)benzyl]-3-[(N-methyl-
N-phenylamino)methyl]-1,3,4-oxadiazol-2-(3H)-thione
(4h).
This was obtained by reacting 5-[2-(2,6-dichlorophe-
nylamino)benzyl]-1,3,4-oxadiazole-2(3H)-thione (3, 0.704 g) and
N-methylaniline (0.429 g) as described in the general procedure
and isolated as light brown solid. Yield: 60.6%. Mp 272–274 °C.
4.2.4.5. 5-[2-(2,6-Dichlorophenylamino)benzyl]-3-[(4-ethylpi-
perazin-1-yl)methyl]-1,3,4-oxadiazol-2-(3H)-thione (4e). This
was obtained by reacting 5-[2-(2,6-dichlorophenylamino)benzyl]-
1,3,4-oxadiazole-2(3H)-thione (3, 0.704 g) and 4-ethylpiperazine
(0.516 g) as described in the general procedure and isolated as
vanilla coloured crystalline solid. Yield: 75.5%. Mp 190–192 °C.
UV (CH3OH, kmax): 275.0 (e 33676.50). IR spectra (KBr, mmax,
cmꢀ1): 3368 (N-H), 3053 (CAH), 1592 (C@N), 1455 (C@C), 1262
(C@S), 1173 (CAOAC), 778 (C-Cl). 1H NMR (CDCl3, d, ppm): 8.91
(s, 1H, N-H), 7.86–6.98 (m, 12H, Ar-H), 4.79 (s, 2H, NACH2AN),
3.40 (s, 2H, CH2), 2.76 (s, 3H, N-CH3).
UV (CH3OH, kmax): 266.5 (
e
31882.60). IR spectra (KBr, mmax
,
4.2.4.9. 5-[2-(2,6-Dichlorophenylamino)benzyl]-3-[(cyclohexyl-
cmꢀ1): 3359 (N-H), 2944 (CAH), 1606 (C@N), 1505 (C@C), 1272
(C@S), 1189 (CAOAC), 775 (C-Cl). 1H NMR (CDCl3, dd, ppm): 8.80
(s, 1H, N-H), 7.84-7.17 (m, 7H, Ar-H), 4.52 (s, 2H, NACH2AN),
3.34 (s, 2H, CH2), 2.81–2.77 (t, 4H, piperazine), 2.56–2.50 (t, 4H,
piperazine), 2.26 (q, 2H, CH2 of piperazine), 1.38 (t, 3H, CH3of
piperazine) .13C NMR (CDCl3, d, ppm): 173.1 (C@S), 156.2 (C-5 of
amino)methyl]-1,3,4-oxadiazol-2-(3H)-thione
(4i).
This
was obtained by reacting 5-[2-(2,6-dichlorophenylamino)benzyl]-
1,3,4-oxadiazole-2(3H)-thione (3, 0.704 g) and cyclohexylamine
(0.456 g) as described in the general procedure and isolated as
white crystalline solid. Yield: 68.6%. Mp 132–136 °C. UV (CH3OH,
kmax): 266 (e 15973.50). IR spectra (KBr, m
max, cmꢀ1): 3292 (N-H),

2864
M. B. Palkar et al. / Bioorg. Med. Chem. 22 (2014) 2855–2866
2930 (CAH), 1594 (C@N), 1503 (C@C), 1315 (C@S), 1209 (CAOAC),
755 (C-Cl). 1H NMR (CDCl3, d, ppm): 8.86 (s, 1H, N-H), 7.72–6.91
(m, 7H, Ar-H), 6.02 (s, 1H, N-H ofcyclohexylamine), 4.85 (s, 2H,
NACH2AN), 3.33 (s, 2H, CH2), 2.72–2.68 (dd, 1H, CH of cyclohexyl-
amine), 1.75–1.20 (m, 10H, CH2 of cyclohexylamine). 13C NMR
(CDCl3, d, ppm): 164.4 (C@S), 151.2 (C-5 of oxadiazole), 138.8
(C-20), 134.3 (C-1), 132.2 (C-60), 130.0 (C-2, C-6), 129.1 (C-3, C-5),
125.6 (C-40), 124.8 (C-10), 122.7 (C-30), 120.3 (C-4), 119.5 (C-50),
68.3 (NACH2AN), 55.3 (CH of cyclohexylamino group), 39.2
(CH2), 36.1–22.5 (C-2, C-3, C-5, C-6 of cyclohexylamino group). Ele-
mental (CHN) analysis calculated for C22H24Cl2N4OS: C, 57.01; H,
5.22; N, 12.09 (found: C, 56.99; H, 5.20; N, 12.10).
administered to animals orally. The control group animals received
appropriate volumes of vehicle and reference drugs. The experi-
mental animal handling procedure followed was similar for all
groups of animals.
4.3.3. Acute toxicity
The acute toxicity test was carried out as per the guidelines of
Organization for Economic Co-operation and Development
(OECD)41 to establish the effective dose of test compounds. Albino
mice of either sex weighing between 20 and 25 g were grouped
into 12 groups of six animals each, starved for 24 h with water
ad libitum prior to test. On the day of the experiment animals were
administered with different compounds to different groups in an
increasing dose of 10, 20, 100, 200, 1000 and 2000 mg/kg b.w.
orally. The animals were then observed continuously for 3 h for
general behavioral, neurological, autonomic profiles and then
every 30 min for next 3 h and finally for next 24 h or till death.
4.2.4.10.
5-[2-(2,6-Dichlorophenylamino)benzyl]-3-[(2-chlor-
ophenylamino)methyl]-1,3,4-oxadiazol-2-(3H)-thione (4j). This
was obtained by reacting 5-[2-(2,6-dichlorophenylamino)benzyl]-
1,3,4-oxadiazole-2(3H)-thione (3, 0.704 g) and 2-chloroaniline
(0.510 g) as described in the general procedure and isolated as
white pluffy crystalline solid. Yield: 66.3%. Mp 208–210 °C. UV
4.3.4. Acute anti-inflammatory activity
(CH3OH, kmax): 267.0 (
e
39418.30). IR spectra (KBr,
m
max, cmꢀ1):
In vivo acute anti-inflammatory activity was evaluated using
carrageenan-induced rat paw edema assay model42 for the com-
pounds listed in Table 2. Male albino rats (170–220 g) were fasted
with free access to water at least 12 h prior to experiments and
were divided randomly into 13 groups of six each. Control group
received 1 ml of 0.5% sodium CMC, standard group received
13.5 mg/kg b.w. of Diclofenac and test groups received 10 mg/kg
b.w. of synthesized compounds (4a–k). The rats were dosed orally,
1 h later; a subplantar injection of 0.05 ml of 1% solution of carra-
geenan in sterile distilled water was administered to the left hind
footpad of each animal. The paw edema volume was measured with
a digital plethysmometer at 0, 2nd, 4th h after carrageenan injec-
tion. Paw edema volume was compared with vehicle control group
and percent reduction was calculated as 1 ꢀ (edema volume in the
drug treated group/edema volume in the control group) ꢂ 100.
3371 (N-H), 3068 (CAH), 1599 (C@N), 1514 (C@C), 1256 (C@S),
1214 (CAOAC), 790 (C-Cl). 1H NMR (CDCl3, d, ppm): 9.43 (s, 1H,
N-H), 9.02 (s, 1H, N-H), 7.90–7.44 (m, 7H, Ar-H), 7.22–7.17 (d,
2H, Ar-H of 2-chloroaniline group), 7.08–7.02 (d, 2H, Ar-H of
2-chloroaniline group), 4.61 (s, 2H, NACH2AN), 3.29 (s, 2H, CH2).
4.2.4.11. 5-[2-(2,6-Dichlorophenylamino)benzyl]-3-[(3-chlorophe-
nylamino)methyl]-1,3,4-oxadiazol-2-(3H)-thione (4k). This was
obtained by reacting 5-[2-(2,6-dichlorophenylamino)benzyl]-
1,3,4-oxadiazole-2(3H)-thione (3, 0.704 g) and 3-chloroaniline
(0.510 g) as described in the general procedure and isolated as
white pluffy crystalline solid. Yield: 67.5%. Mp 220–224 °C. UV
(CH3OH, kmax): 267.5 (e 39545.75). IR spectra (KBr, m
max, cmꢀ1):
3369 (N-H), 2949 (CAH), 1592 (C@N), 1507 (C@C), 1236 (C@S),
1205 (CAOAC), 805 (C-Cl). 1H NMR (DMSO-d6, d, ppm): 9.66 (s,
1H, N-H), 8.98 (s, 1H, N-H), 8.19 (s, 1H, Ar-H of 3-chlorophenyl-
amine), 7.91–7.71 (m, 4H, Ar-H of Diclofenac), 7.48–7.35 (m, 3H,
Ar-H of 3-chlorophenylamine), 7.17–7.07 (m, 3H, Ar-H of Diclofe-
nac), 5.05 (s, 2H, NACH2AN), 3.20 (s, 2H, CH2). 13C NMR (CDCl3,
d, ppm): 173.8 (C@S), 155.5 (C-5 of oxadiazole), 151.2 (C-1, C-5
of chlorophenyl), 145.8 (C-3 of chlorophenyl), 142.5 (C-20), 130.3
(C-2, C-6), 126.0 (C-3, C-5), 123.6 (C-1), 122.4, 122.1, 120.4 (C-2,
C-4, C-6 of chlorophenyl), 112.7 (C-30, C-40), 111.3 (C-10, C-50),
107.2 (C-4), 65.6 (NACH2AN), 40.5 (CH2). Elemental (CHN) analysis
calculated for C22H17Cl3N4OS: C, 53.74; H, 3.48; N, 11.40 (found: C,
53.72; H, 3.49; N, 11.37).
4.3.5. Analgesic activity43
Twenty-four hours prior to actual testing a large number of
mice (20–25 g) received intraperitoneally 10 ml/kg 0.6% glacial
acetic acid. Animals were observed for writhing movements. Only
those showing one or other type of writhing movements (positive
responders) were chosen for the test on the next day. On the test
day the responders received compounds half an hour prior to gla-
cial acetic acid challenge. Compounds listed in Table 3 were orally
administered at a dose of 10 mg/kg b.w. as a suspension in 0.5%
sodium CMC. Standard drug used was Diclofenac at a dose of
19.5 mg/kg as a suspension in 0.5% sodium CMC. Each mouse
was then observed for the total number of stretching episodes or
writhings for 15 min following glacial acetic acid injection. The
mean value for each group was calculated.
4.3. Pharmacological screening
4.3.1. Animals
Albino mice of either sex weighing 20–25 g were used for acute
toxicity studies and analgesic activity. Healthy male albino adult
rats weighing 150–230 g were used for various pharmacological
screenings. Animals were procured from Venkateshwara Enter-
prises, Bangalore, India, (245/CPCSEA) and housed individually in
polypropylene cages, maintained under standard conditions of
alternating 12 h light and dark cycles at a constant temperature
(25 2 °C and 35–60% relative humidity). The pharmacological
evaluations were conducted after obtaining ethical clearance from
Animal Ethical Committee of KLEU’s College of Pharmacy, Hubli
(India). Animals were fed with standard rat pellet diet procured
from Hindustan Lever Ltd., Mumbai, India and water ad libitum.
4.3.6. Antipyretic activity44
Male albino rats weighing between 170 and 230 g were injected
subcutaneously with 2.5 ml of 20% aqueous suspension of baker’s
yeast. Rectal temperature was recorded using telethermometer
prior to and at 18 h after yeast injection. The first reading recorded
at 18th h after yeast injection was considered as 0 h. Rats develop-
ing satisfactory pyrexia (raise in body temperature 1–2 °C) were
divided into eight groups of six animals each. Thirty minutes after
the 18th h reading, animals of control group received orally 1 ml of
0.5% sodium CMC, while the standard group received 135 mg/kg
b.w. of Paracetamol and test groups received the compounds 4a,
4d, 4e, 4i, 4j and 4k at a dose of 10 mg/kg b.w. Temperatures were
recorded hourly up to 5th h after dosing. The mean temperatures
after the treatment were compared with those of 18th h and
expressed as a temperature index (TI), which was the sum of mean
temperature changes from the 0 h.
4.3.2. Preparation of test compounds
After suspending the test compounds in 0.5% aqueous solution
of sodium carboxymethyl cellulose (sodium CMC), were

M. B. Palkar et al. / Bioorg. Med. Chem. 22 (2014) 2855–2866
2865
4.3.7. Ulcerogenic activity
protein data bank, which would be considered as the probable best
accurate region as it is solved by experimental crystallographic
data.40 The docking protocol was carried out for synthesized com-
pounds/ligands as listed in Table 6 using MVD-2013 (6.0) software
using the standard operating procedures.47
Albino rats of either sex were divided into control, different
standard and various test groups of six animals each group (170–
250 g). They were starved for 48 h (water ad libitum) prior to drug
administration. Control group received only 0.5% sodium CMC
solution, standard groups were orally administered with Diclofe-
nac, Aspirin and Flurbiprofen in sodium CMC solution, at the dose
of 10 mg/kg b.w. respectively. The test compounds as listed in
Table 5 were administered orally at the dose of 10 mg/kg b.w. All
animals were sacrificed after 7 h of drug administration. Stomach
was removed and placed on saline-soaked filter paper until inspec-
tion. A longitudinal incision along the greater curvature was made
with fine scissors. The stomach was everted over the index finger
and the presence or absence of gastric irritation was determined.
The ulcer index for each group was determined according to a
previously reported method45 by counting the number of lesions
(x) in each of five size classes (y). The classes were defined as
y = 1 (pinpoint lesion), y = 2 (lesions <1 mm diameter), y = 3
(lesions 1–2 mm diameter), y = 4 (lesions 2–4 mm diameter) and
y = 5 (lesions >4 mm diameter). The ulcer index was calculated
4.6. Statistical analysis
Statistical analysis was performed by one-way analysis of vari-
ance (ANOVA) followed by Dunnett’s t-test for multiple compari-
sons of all compounds in various pharmacological assays. Data
values are expressed as Mean SEM. The significance of difference
was accepted at P <0.05.
Acknowledgements
The authors express their humble thanks to The Principal,
KLEU’s College of Pharmacy, Hubli (India) and University of KwaZ-
ulu-Natal (UKZN, South Africa) for providing necessary facilities to
carry out this work. Authors also express special thanks to Fourrtis
(India) Lab. Pvt. Ltd, Chennai (Tamilnadu, India) for providing gift
samples of NSAIDs. We are grateful to SAIF, Punjab University
(India) for providing spectral and elemental analysis. We also sin-
cerely thank Dr. Rene Thomsen for providing MVD-2013 (version:
6.0) trial license.
using
R5i = 1xi yi.
4.4. In vitro cylcooxygenase (COX) inhibition studies
The selected compounds listed in Table 6 were tested for their
ability to inhibit COX-2/COX-1 using a colorimetric in vitro COX
(ovine) inhibitor screening kit (catalog no. 760111, Cayman Chem-
icals Inc., Ann Arbor, MI, USA) using the previously established
method.39 The percentage of inhibition of COX-1 and COX-2 was
calculated by the comparison of the sample treated incubations
to control incubations. Diclofenac and Celecoxib were used as ref-
erence standards in the study.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.03.043.
References and notes
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