Design and synthesis of novel 2-phenyl-5-(1,3-diphenyl-1H-pyrazol-4-yl)-1, 3,4-oxadiazoles as selective COX-2 inhibitors with potent anti-inflammatory activity

Article abstract of DOI:10.1016/j.ejmech.2014.04.045

A novel series of 2-phenyl-5-(1,3-diphenyl-1H-pyrazol-4-yl)-1,3,4- oxadiazoles were designed and synthesized for selective COX-2 inhibition with potent anti-inflammatory activity. Among the compounds tested, 9g (2-(3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-5-phenyl-1,3,4-oxadiazole) was found to be the most potent inhibitor of COX-2 with IC of 0.31 μM showing promising degree of anti-inflammatory activity in the carrageenan-induced rat paw edema model with ED of 74.3 mg/kg. The lead compound 9g further showed suppression of acetic acid-induced writhes comparable to that of aspirin and gastro-sparing profile superior to the aspirin. Molecular docking analysis displayed higher binding affinity of ligands towards COX-2 than COX-1.

Full text of DOI:10.1016/j.ejmech.2014.04.045

European Journal of Medicinal Chemistry 80 (2014) 167e174
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Design and synthesis of novel 2-phenyl-5-(1,3-diphenyl-1H-pyrazol-
4-yl)-1,3,4-oxadiazoles as selective COX-2 inhibitors with potent anti-
inflammatory activity
, ,
,
,
,
Sumit Bansal a 1, Manju Bala b 1, Sharad Kumar Suthar c 1, Shivani Choudhary d,
*
**
Shoumyo Bhattacharya a, Varun Bhardwaj e, Sumit Singla f, Alex Joseph c
a School of Pharmaceutical Sciences, Shoolini University, Solan, Himachal Pradesh, India
b School of Pharmaceutical Sciences, Jaipur National University, Jaipur 302017, India
c Department of Pharmaceutical Chemistry, Manipal College of Pharmaceutical Sciences, Manipal 576104, India
d Doon Valley Institute of Pharmacy and Medicine, Karnal, Haryana 132001, India
e Department of Pharmaceutical Chemistry, ASBASJSM College of Pharmacy, Bela, Ropar, Punjab 140111, India
f Department of Pharmaceutical Chemistry, Rajendra Institute of Technology & Sciences, Sirsa, Haryana, India
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 2-phenyl-5-(1,3-diphenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazoles were designed and syn-
thesized for selective COX-2 inhibition with potent anti-inflammatory activity. Among the compounds
tested, 9g (2-(3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-5-phenyl-1,3,4-oxadiazole) was found to be
Received 19 October 2013
Received in revised form
9 April 2014
Accepted 14 April 2014
Available online 15 April 2014
the most potent inhibitor of COX-2 with IC50 of 0.31 mM showing promising degree of anti-inflammatory
activity in the carrageenan-induced rat paw edema model with ED50 of 74.3 mg/kg. The lead compound
9g further showed suppression of acetic acid-induced writhes comparable to that of aspirin and gastro-
sparing profile superior to the aspirin. Molecular docking analysis displayed higher binding affinity of
ligands towards COX-2 than COX-1.
Keywords:
Pyrazole
1,3,4-Oxadiazole
COX-2
Ó 2014 Elsevier Masson SAS. All rights reserved.
Anti-inflammatory
Analgesic
Molecular docking analysis
1. Introduction
inflammatory drugs [2,4]. These findings have led to hypothesis
that selective COX-2 inhibitors might provide good anti-
The cyclooxygenase (COX) enzymes are chiefly responsible for
the production of prostaglandins, a well-known mediator of
inflammation, pain, and swelling [1]. COX enzymes exist in three
isoforms: COX-1, COX-2, and COX-3 [2]. The COX-1 is a constitutive
enzyme found in most of the cells and has important role in the
protection of gastric mucosa, platelet aggregation, and renal blood
flow. The COX-2 is an inducible isozyme; significantly expressed
during inflammation, pain, and oncogenesis [3], while COX-3, a
splice variant of COX-1, is considered as another target for anti-
inflammatory agents with improved therapeutic potency and
reduced side effects associated with the use of conventional
nonsteroidal anti-inflammatory drugs (NSAIDs). Many selective
COX-2 inhibitors such as celecoxib [5], rofecoxib [6], valdecoxib [7],
and etoricoxib [8] are marketed as new generation NSAIDs. How-
ever, because of several cardiovascular adverse effects associated
with coxibs, they have been voluntarily withdrawn from the mar-
ket [9,10]. Thus, novel COX-2 selective inhibitors having anti-
inflammatory and analgesic activities with an improved safety
profile is the need of the hour.
Many pyrazole derivatives are known to possess a wide range of
bioactivities such as anti-inflammatory [5], analgesic [11], anti-
bacterial [12], antifungal [13], and anticancer [14,15]. The recent
success of celecoxib as selective COX-2 inhibitor has grown signif-
icant attention towards pyrazole nucleus in the designing of newer
and safer anti-inflammatory agents [5]. Besides, a great variety of
similar kinds of compounds having potent anti-inflammatory
* Corresponding author. Present address: Dept. of Pharmacy, Jaypee University of
Information Technology, Waknaghat, Solan.
** Corresponding author. Dept. of Pharmaceutical Chemistry, Manipal College of
Pharmaceutical Sciences, Manipal University, Manipal 576104, India.
E-mail addresses: bansalsumit50@gmail.com (S. Bansal), sharadpune_2009@
yahoo.com (S.K. Suthar).
1
Authors hold equal contribution.
http://dx.doi.org/10.1016/j.ejmech.2014.04.045
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

168
S. Bansal et al. / European Journal of Medicinal Chemistry 80 (2014) 167e174
activity have been synthesized [16e18]. Pyrazoles bearing 1,3-
disubstituted aryl moieties are reported as effective pharmaco-
phore for selective COX-2 inhibition [19].
(EIA) kit. The activities of the tested compounds were expressed as
IC50 (concentration exhibiting 50% enzyme inhibition). The selec-
tivity index (SI values) was calculated by IC50 (COX-1)/IC50(COX-2).
The IC50 values of celecoxib against COX-1 and COX-2 were found to
Substituted 1,3,4-oxadiazoles are one of the most important
heterocyclic compounds, which have gained attention because of
their remarkable biological and pharmacological properties, such
as anti-inflammatory [20], anticancer [21], antitubercular [22],
antibacterial [23], antiviral [24], antifungal [25], and insecticidal
activities [26]. They have also attracted interest in medicinal
chemistry as bioisosteres for carboxylic acids, esters, and carbox-
amides [27]. A number of molecules based upon this monocyclic
heterocyclic template have been investigated for their anti-
inflammatory activity [28,29]. Recently, Dekhane et al. [30] re-
ported synthesis, COX-2 inhibitory potential, and anti-
inflammatory activity of pyrazole substituted 1,3,4-oxadiazoles
and 1,2,4-oxadiazoles. Encouraged by these observations and our
ongoing efforts towards developing promising biologically active
agents using a hybrid pharmacophore approach [31e33], it was
considered worthwhile to design and synthesize novel hybrid
compounds by integrating two five membered heterocyclic rings
(pyrazole and oxadiazole) into a single molecule to investigate their
COX inhibitory potential with promising anti-inflammatory
activity.
be >100 and 0.28 mM, respectively, indicating that celecoxib is a
selective COX-2 inhibitor (SI ¼ 357), while diclofenac showed non-
selectivity towards COX-1 and COX-2. The results showed that
compounds 9a, 9d, 9g, 9j, 9k, 9n, 10c, 10d, and 10g displayed potent
COX-2 inhibition with IC50 in a range of 0.31e8.7
compared to the inhibition of COX-1 with IC50 > 100 M, as listed in
Table 1. Among these compounds, 9g was found to be the most
potent COX-2 inhibitor with lowest IC50 i.e. 0.31 M. The COX-2
mM when
m
m
inhibition depends on the electronic nature of substituents on
phenyl ring of pyrazole nucleus. It was observed that compounds
possessing electron withdrawing nitro group (9g, 9n, and 10g)
demonstrated augmented COX-2 inhibitory activity, whereas
introduction of electron donating methyl or methoxy group
decreased the COX-2 activity and selectivity. Halogen substitutions
also played a significant role in COX-2 inhibition. Chloro substitu-
tion was found more favorable towards COX-2 inhibition over flu-
oro substitution. However, bromo substitution resulted into
complete loss of COX-2 selectivity. Moreover, 1,3,4-oxadiazoles
bearing phenyl ring substitution at C-2 position showed higher
inhibitory potency towards COX-2 compared to that of pyridyl ring
substitution, while 4-chlorophenyl substitution yielded least
potent inhibitors.
2. Result and discussion
2.1. Chemistry
2.2.2. In vivo anti-inflammatory activity
The synthetic route of title compounds 9aen and 10aeg is
shown in Scheme 2. The title compounds were synthesized by
oxidative cyclization of various pyrazolylaldehyde N-acylhy-
drazones 7 and 8 with iodobenzenediacetate (IBD) in dichloro-
methane by stirring at room temperature for 20e25 min [34]. The
intermediates 7aen and 8aeg were synthesized by refluxing
ethanolic solutions of formylpyrazoles 6 with acid hydrazides 2e3
and isoniazid in the presence of few drops of acetic acid for 4 h,
respectively. Formylpyrazoles 6 were prepared by previous re-
ported method of Kira et al. [35]. Acid hydrazides were synthesized
according to Scheme 1.
Progresses of the reactions were monitored by TLC using n-
hexane: ethylacetate (4:6) as solvent system. The synthesized
compounds 9aen and 10aeg were purified by column chroma-
tography using silica gel 100e200 mesh to afford pale white or
yellow products. A total of 21 compounds were synthesized with
yield in the range of 51e84%.
Ten compounds, namely 9a, 9c, 9d, 9g, 9j, 9k, 9n, 10c, 10d, and
10g were selected for in vivo anti-inflammatory activity based on
in vitro results. These named compounds were evaluated using
well-known rat carrageenan-induced foot paw edema model. Cel-
ecoxib and diclofenac were used as the reference drugs. The ED50
values of test and reference compounds were calculated after 2 h of
treatment with Carrageenan, are represented in Table 1. All of the
ED50 values were determined in three doses of 50, 100 and 200 mg/
kg body wt. of the test compounds and 25, 50 and 100 mg/kg body
wt. of the reference drugs diclofenac and celecoxib. Among the
compounds tested, 9a, 9g,10c, and 10g showed promising degree of
anti-inflammatory activity with ED50 in the range of 72.6e
125.4 mg/kg. ED50s of the reference drugs celecoxib and diclofenac
were found to be 81.7 and 110.4 mg/kg, respectively.
2.2.3. In vivo analgesic activity
Analgesic effect of the lead compound 9g was evaluated by
employing the acetic acid-induced writhing test in mice, a visceral
inflammatory analgesic test. Mice treated with 9g exhibited 67.29%
suppression of writhes compared with the control group, whereas
the reference drug aspirin produced 72.23% suppression of
writhes. Therefore, it can be stated that the lead compound 9g
possesses analgesic potential same as that of the clinically used
drug aspirin and holds a great promise for the further develop-
ment as an NSAID.
2.2. Biological evaluation
2.2.1. In vitro cyclooxygenase (COX) inhibition assay
Newly synthesized 21 compounds and reference drugs cele-
coxib and diclofenac were evaluated for their inhibitory activities
against COX-1 and COX-2 enzymes using an enzyme immunoassay
2.2.4. Gastric ulceration study
Gastric ulcers developed on the administration of the lead
compound 9g and aspirin were measured individually in terms of
their length. Gastric ulceration study indicated extraordinary
gastro-sparing effects of the lead compound 9g with ulcer-index of
22.8 ꢀ 3.2 only. Contrastingly in the same experimental conditions,
clinically used drug aspirin demonstrated ulcer-index of 81 ꢀ 8.9,
which was >3.5-fold more than that produced by 9g. These gastro-
protective effects of 9g provide evidence that it can be developed as
a potential gastro-sparing NSAID.
Scheme 1. Synthesis of acid hydrazides (2e3).

S. Bansal et al. / European Journal of Medicinal Chemistry 80 (2014) 167e174
169
Scheme 2. Synthesis of 2-Phenyl-5-(1,3-diphenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazoles (9aen, 10aeg).
2.3. Molecular docking analysis
same target. This energy difference between 9g and diclofenac
indicates higher binding affinity of 9g towards COX-2 than
diclofenac.
All the designed compounds were docked into the active sites
of COX-1 and COX-2. Results of molecular docking analysis
indicate that all the designed and subsequently synthesized
compounds were more selective towards COX-2 than COX-1.
Estimated free energy of binding of docked compounds to-
wards COX-2 was found to be between ꢁ10.15 and ꢁ8.97 kcal/
mol, whereas compounds docked to COX-1 showed estimated
free energy of binding between ꢁ5.96 and ꢁ4.20 kcal/mol. The
estimated free energy of binding of docked ligands signifies that
synthesized compounds exhibited preferential selectivity to-
wards COX-2 instead of COX-1. The most potent compound 9g
revealed by in vitro study showed minimum estimated free en-
ergy of binding among the docked compounds and it exhibited
an estimated free energy of binding of ꢁ10.15 kcal/mol towards
COX-2, whereas the estimated free energy of binding of clinically
used drug diclofenac was found to be ꢁ9.23 kcal/mol towards the
Analysis of docked complex of 9g and COX-2 proved that the
nitro group of 9g plays a crucial role in COX-2 inhibition. Moreover,
oxygen of nitro group forms hydrogen bonding with amino group
ꢀ
hydrogen of Arg-120 with a bond distance of 2.4 A (O]NeO.HeN,
ꢀ
2.4 A) (Fig. 1). Nitro group substituted phenyl ring of 9g was placed
in the hydrophobic pocket created by Val-116, Leu-117, Val-349,
Leu-359, and Leu-531 residues of COX-2 (Fig. 2). Furthermore,
phenyl ring placed on pyrazole moiety displayed hydrophobic and
van der Waal interactions with Ile-517 and Phe-518 residues of the
COX-2, while phenyl ring of oxadiazole moiety showed hydropho-
bic interaction with Val-523 residue of the target enzyme (Fig. 2).
Stereoview of docked complex of 9g into the active site of COX-1
indicates that 9g showed hydrogen, hydrophobic, and van der
Waal interactions with various residues of COX-1 viz. Leu-93, Val-
349, Leu-352, Ser-353, Tyr-355, Phe-356, Leu-357, and Pro-514

170
S. Bansal et al. / European Journal of Medicinal Chemistry 80 (2014) 167e174
Table 1
superior to the aspirin. Molecular docking analysis exhibited higher
binding affinity of 9g and other ligands towards the COX-2 than
COX-1, while 9g displaying the lowest estimated free energy of
binding for COX-2, among all the docked compounds. Taken
together, synthesis of dual pharmacophores i.e. 1,3,4-oxadiazole
and diarylpyrazole bearing hybrid molecules exhibited anti-
inflammatory and analgesic effects comparable to those of clini-
cally used drugs with better safety profile and further research is
warranted for the optimization of current pharmacophore.
Data of In vitro COX-1/COX-2 enzyme inhibition assay and in vivo anti-inflammatory
activity (ED50 mg/kg) of the synthesized compounds.
Compound
IC50
(
m
M)a
Selectiviy indexb
ED50c (mg/kg)
COX-1
COX-2
1.5
25
6.5
4.7
>100
>100
0.31
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
10a
10b
10c
10d
10e
10f
10g
Celecoxib
Diclofenac
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>67
>4
>15.4
>21
>1
125.4
ND
175.3
170.7
ND
N
74.3
ND
>1
4. Experimental section
>222
>2.6
>1.7
>18.2
>11.5
>2
>1.5
>83.3
>9
>1.9
>43
>23
>2.6
>2.4
>200
>357
0.06
38
58
ND
4.1. General experimental methods
5.5
8.7
50
67
1.2
11.1
53
2.3
4.4
39
182.5
198.7
ND
ND
167.4
ND
All the chemicals were purchased from Merck, SigmaeAldrich,
Himedia, and Spectrochem, India of analytical or reagent grade and
were used without further purification. Merck silica gel 60 F254 thin
layer pre-coated plates were used for monitoring the chemical re-
actions. Melting points of the synthesized compounds were taken
by one end open capillary tubes melting point apparatus and are
uncorrected. Infrared spectra were recorded on a Shimadzu FTIR-
8310 spectrophotometer (KBr). 1H NMR spectra were recorded on
a Bruker 400 MHz spectrophotometer and chemical shifts are re-
ND
122.8
167.4
ND
42
0.5
0.28
2.7
ND
72.6
81.7
110.4
ported in parts per million (d) downfield from an internal standard
0.18
tetramethylsilane. Mass spectra were obtained using electrospray
ionization (ESI) technique by Agilent 1100 series LC-MS instrument.
Elemental analyses were performed on Leco CHNS-932 (Leco, St.
Joseph, MI, USA).
a
Deviation from the mean is <10% of the mean value.
Selectivity index ¼ (COX-1 IC50/COX-2 IC50).
ED50 was the effective dose calculated after 2 h.
b
c
(Figs. 3 and 4) with estimated free energy of binding of ꢁ5.96 kcal/
4.2. Synthesis of acid hydrazides (2e3)
mol towards COX-1.
A solution of substituted benzoic acid 1 (0.083 mol) in 20 ml
methanol containing 2e3 ml of sulfuric acid was refluxed for 12 h
(Scheme 1). The mixture was then allowed to cool at room tem-
perature. A saturated solution of sodium bicarbonate was added to
neutralize the mixture. Prepared ester was extracted by using
dichloromethane (DCM). The DCM layer was dried using anhydrous
sodium sulfate. The solvent was removed to dryness to afford ester
which was used without further purification. To a solution of acid
methyl ester in 15 ml of ethanol, 5 ml hydrazine hydrate was added
and the resulting solution was refluxed for 16 h. Excess ethanol was
distilled off and the concentrated solution was cooled to obtain acid
hydrazide which was used without further purification.
3. Conclusions
In conclusion, a novel series of 1,3,4-oxadiazole derivatives
containing diarylpyrazole moiety were synthesized employing
hybrid pharmacophore approach. Results of in vitro COX assay
indicated that all the compounds showed considerable COX-2
selectivity over COX-1. Moreover, substitution of electron with-
drawing nitro group (9g, 9n, and 10 g) was found to be more
favorable for COX-2 inhibition, while compounds possessing elec-
tron releasing groups showed diminished COX-2 activity. Among
the compounds tested, 9g was found to be the most potent inhib-
itor of COX-2 with the highest selectivity. The in vivo carrageenan-
induced rat paw edema and mice acetic acid-induced writhing
assays further established the anti-inflammatory potential of 9g.
Additionally, the lead compound 9g showed gastro-sparing profile
Fig. 2. Stereoview of docked complex of 9g within the active site of COX-2. The amino
acid residues of COX-2 involved in hydrogen, hydrophobic and van der Waal in-
teractions with 9g, are highlighted.
Fig. 1. Docking of 9g into the active site of COX-2. The amino acid Arg-120 involved in
hydrogen bond interaction with 9g, is highlighted.

S. Bansal et al. / European Journal of Medicinal Chemistry 80 (2014) 167e174
171
evaporated in rotary evaporator and the residual mass was tritu-
rated with petroleum ether to give a solid product which was
further purified by column chromatography using solvent system
n-hexane:ethyl acetate (5:5).
4.6.1. 2-(1,3-Diphenyl-1H-pyrazol-4-yl)-5-phenyl-1,3,4-oxadiazole
(9a)
Yield 62%, mp 118e120 ꢂC. Anal. calcd. for C23H16N4O (364.40): %
C, 75.81; H, 4.43; N, 15.38. Found: %C, 75.86; H, 4.40; N, 15.41. IR
(KBr, cmꢁ1): 1616.5 (C]N), 1537.4 (C]C), 1212.6 (CeOeC). 1H NMR
(DMSO-d6, 400 MHz,
d ppm): 8.41 (s, 1H, ]CHe of pyrazole ring),
7.82e8.02 (m, 4H, Ar-H), 7.36e7.53 (m, 11H, Ar-H). ESI-MS (m/z):
364 (Mþ).
4.6.2. 2-Phenyl-5-(1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)-1,3,4-
oxadiazole (9b)
Yield 69%, mp 196e198 ꢂC. Anal. calcd. for C24H18N4O (378.43): %
C, 76.17; H, 4.79; N,14.81. Found: %C, 76.15; H, 4.82; N,14.85. IR (KBr,
cmꢁ1): 1618.2 (C]N), 1537.1 (C]C), 1209.5 (CeOeC). 1H NMR
Fig. 3. Stereoview of docked complex of 9g into the active site of COX-1. The amino
acids of COX-1 involved in hydrogen, hydrophobic and van der Waal interactions with
9g, are highlighted.
(DMSO-d6, 400 MHz,
d ppm): 8.44 (s, 1H, ]CHe of pyrazole ring),
7.96e8.06 (m, 2H, Ar-H), 7.40e7.55 (m, 10H, Ar-H), 7.12e7.14 (m,
2H, Ar-H), 2.36 (s, 1H, CH3). ESI-MS (m/z): 378 (Mþ).
4.3. Synthesis of acetophenone phenylhydrazones (5)
A mixture of appropriate ketone 4 (0.01 mol) and phenyl-
hydrazine (0.01 mol) in ethanol (10 ml) was refluxed in the pres-
ence of few drops of glacial acetic acid for 1 h. The progress of
reaction was monitored by TLC using n-hexane:ethyl acetate
(9.5:0.5) as a mobile phase. The mixture was cooled and the solid
product obtained was filtered, washed with water (3e4 times) and
recrystallized from ethanol.
4.6.3. 2-(3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-5-phenyl-
1,3,4-oxadiazole(9c)
Yield 71%, mp 132e134 ꢂC. Anal. calcd. for C23H15FN4O (382.39):
%C, 72.24; H, 3.95; N, 14.65. Found: %C, 72.28; H, 3.97; N, 14.62. IR
(KBr, cmꢁ1): 1621.7 (C]N), 1538.8 (C]C), 1219.9 (CeOeC). 1H NMR
(DMSO-d6, 400 MHz,
d ppm): 8.52 (s, 1H, ]CHe of pyrazole ring),
7.84e7.96 (m, 4H, Ar-H), 7.40e7.55 (m, 8H, Ar-H), 7.06e7.10 (m, 2H,
Ar-H). ESI-MS (m/z): 382 (Mþ).
4.4. Synthesis of 3-aryl-1-phenyl-1H-pyrazole-4-carbaldehyde (6)
4.6.4. 2-(3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-5-phenyl-
1,3,4-oxadiazole (9d)
To an ice cold dimethylformamide (10 ml), POCl3 (0.03 mol) was
added dropwise with continuous stirring over a period of 30 min.
Stirring was continued for further 60 min, keeping the reaction
Yield 54%, mp 142e144 ꢂC. Anal. calcd. for C23H15ClN4O
(398.84): %C, 69.26; H, 3.79; N, 14.05. Found: %C, 69.23; H, 3.77; N,
14.10. IR (KBr, cmꢁ1): 1608.3 (C]N), 1555.8 (C]C), 1221.5 (CeOeC),
temp at
0 5
ꢂC. Acetophenone phenylhydrazone derivative
690.1 (CeCl). 1H NMR (DMSO-d6, 400 MHz,
d ppm): 8.46 (s, 1H, ]
(0.01 mol) was then added, the reaction mixture was allowed to
attain room temperature and stirred for further 4 h. The mixture
was then refluxed for 2 h, allowed to cool and poured onto ice. A
saturated solution of sodium bicarbonate was added to neutralize
the mixture. The solid product obtained was filtered, washed with
water followed by hexane (2e3 times) and recrystallized from
methanol. The completion of reaction was monitored by TLC using
n-hexane:ethyl acetate (9:1) as a mobile phase.
CHe of pyrazole ring), 7.83e7.95 (m, 4H, Ar-H), 7.43e7.63 (m, 10H,
Ar-H). ESI-MS (m/z): 399 (Mþ).
4.6.5. 2-(3-(4-Bromophenyl)-1-phenyl-1H-pyrazol-4-yl)-5-phenyl-
1,3,4-oxadiazole (9e)
Yield 61%, mp 174e175 ꢂC. Anal. calcd. for C23H15BrN4O
(443.30): %C, 62.32; H, 3.41; N, 12.64. Found: %C, 62.35; H, 3.40; N,
12.67. IR (KBr, cmꢁ1): 1616.3 (C]N),1556.7 (C]C),1202.8 (CeOeC).
1H NMR (DMSO-d6, 400 MHz,
d ppm): 8.61 (s, 1H, ]CHe of pyr-
4.5. Synthesis of 3-aryl-1-phenyl-1H-pyrazole-4-carbaldehyde-
acylhydrazones (7aen, 8aeg)
azole ring), 7.77e7.89 (m, 4H, Ar-H), 7.39e7.59 (m, 10H, Ar-H). ESI-
MS (m/z): 443 (Mþ).
To an ethanolic solution of acid hydrazide or isoniazid
(0.01 mol), aldehyde (0.01 mol) was added and the solution was
refluxed in the presence of few drops of acetic acid for 4 h. The
progress of reaction was monitored by TLC using n-hexane:ethyl
acetate (4:6) as a mobile phase. After completion of reaction, sol-
vent was evaporated under vacuum to half of its volume, cooled to
room temperature and poured onto ice cold water. The precipitated
product thus obtained was filtered, washed with water, dried, and
recrystallized from ethanol.
4.6.6. 2-(3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-5-
phenyl-1,3,4-oxadiazole(9f)
Yield 64%, mp 143e145 ꢂC. Anal. calcd. for C24H18N4O2 (394.43):
%C, 73.08; H, 4.60; N, 14.20. Found: %C, 73.12; H, 4.63; N, 14.25. IR
(KBr, cmꢁ1): 1614.5 (C]N), 1535.5 (C]C), 1212.7 (CeOeC). 1H NMR
(DMSO-d6, 400 MHz,
d ppm): 8.49 (s, 1H, ]CHe of pyrazole ring),
7.95e7.98 (m, 2H, Ar-H), 7.45e7.62 (m, 10H, Ar-H), 6.86e6.88 (m,
2H, Ar-H), 3.76 (s, 1H, OCH3). ESI-MS (m/z): 394 (Mþ).
4.6. Synthesis of 2,5-disubstituted 1,3,4-oxadiazoles (9aen, 10aeg)
4.6.7. 2-(3-(4-Nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-5-phenyl-
1,3,4-oxadiazole (9g)
An appropriate acylhydrazone 4 (0.01 mol) was dissolved in
DCM (20 ml) and stirred. To this solution, IBD (0.11 mol) was added
in four to five potions during 5 min and the mixture was stirred for
20e25 min at room temperature (Scheme 2). The solvent was
Yield 83%, mp 105e106 ꢂC. Anal. calcd. for C23H15N5O3 (409.4): %
C, 67.48; H, 3.69; N, 17.11. Found: %C, 67.52; H, 3.71; N, 17.08. IR (KBr,
cmꢁ1): 1616.6 (C]N), 1595.5 (C]C), 1341.7 (N]O), 1211.4 (CeOe
C). 1H NMR (DMSO-d6, 400 MHz,
d ppm): 8.39 (s, 1H, ]CHe of

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S. Bansal et al. / European Journal of Medicinal Chemistry 80 (2014) 167e174
4.6.12. 2-(3-(4-Bromophenyl)-1-phenyl-1H-pyrazol-4-yl)-5-(4-
chlorophenyl)-1,3,4-oxadiazole (9l)
Yield 51%, mp 174e175 ꢂC. Anal. calcd. for C23H14BrClN4O
(477.74): %C, 57.82; H, 2.95; N, 11.73. Found: %C, 57.79; H, 2.94; N,
11.79. IR (KBr, cmꢁ1): 1616.6 (C]N), 1504.0 (C]C), 1210.9 (CeOeC),
690.4 (CeCl), 504.6 (CeBr). 1H NMR (DMSO-d6, 400 MHz,
d ppm):
8.65 (s, 1H, ]CHe of pyrazole ring), 7.82e7.92 (m, 4H, Ar-H), 7.43e
7.62 (m, 9H, Ar-H). ESI-MS (m/z): 478 (Mþ).
4.6.13. 2-(4-Chlorophenyl)-5-(3-(4-methoxyphenyl)-1-phenyl-1H-
pyrazol-4-yl)-1,3,4-oxadiazole (9m)
Yield 59%, mp 100e101 ꢂC. Anal. calcd. for C24H17ClN4O2
(428.87): %C, 67.21; H, 4.00; N, 13.06. Found: %C, 67.23; H, 4.03; N,
13.03. IR (KBr, cmꢁ1): 1608.8 (C]N),1597.9 (C]C),1245.7 (CeOeC),
684.8 (CeCl). 1H NMR (DMSO-d6, 400 MHz,
d ppm): 8.45 (s, 1H, ]
CHe of pyrazole ring), 7.99e8.05 (m, 2H, Ar-H), 7.49e7.58 (m, 9H,
Ar-H), 6.79e6.83 (m, 2H, Ar-H), 3.77 (s, 1H, OCH3). ESI-MS (m/z):
429 (Mþ).
Fig. 4. Binding orientation of 9g within the active site of COX-1.
4.6.14. 2-(4-Chlorophenyl)-5-(3-(4-nitrophenyl)-1-phenyl-1H-
pyrazol-4-yl)-1,3,4-oxadiazole (9n)
Yield 72%, mp 156e158 ꢂC. Anal. calcd. for C23H14ClN5O3
(443.84): %C, 62.24; H, 3.18; N, 15.78. Found: %C, 62.21; H, 3.17; N,
15.83. IR (KBr, cmꢁ1): 1624.6 (C]N), 1599.5 (C]C), 1343.7 (N]O),
1225.2 (CeOeC), 684.4 (CeCl). 1H NMR (DMSO-d6, 400 MHz,
pyrazole ring), 8.27e8.29 (m, 2H, Ar-H), 7.82e7.94 (m, 4H, Ar-H),
7.44e7.63 (m, 8H, Ar-H). ESI-MS (m/z): 409 (Mþ).
d
ppm): 8.42 (s, 1H, ]CHe of pyrazole ring), 8.22e8.24 (m, 2H, Ar-
4.6.8. 2-(4-Chlorophenyl)-5-(1,3-diphenyl-1H-pyrazol-4-yl)-1,3,4-
oxadiazole (9h)
H), 7.79e7.89 (m, 4H, Ar-H), 7.39e7.58 (m, 7H, Ar-H). ESI-MS (m/z):
444 (Mþ).
Yield 55%, mp 122e123 ꢂC. Anal. calcd. for C23H15ClN4O
(398.84): %C, 69.26; H, 3.79; N, 14.05. Found: %C, 69.30; H, 3.81; N,
14.10. IR (KBr, cmꢁ1): 1609.5 (C]N), 1595.1 (C]C), 1221.7 (CeOeC),
4.6.15. 4-(5-(1,3-Diphenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)
pyridine (10a)
694.6 (CeCl). 1H NMR (DMSO-d6, 400 MHz,
d ppm): 8.43 (s, 1H, ]
Yield 52%, mp 118e120 ꢂC. Anal. calcd. for C22H15N5O (365.39): %
C, 72.32; H, 4.14; N, 19.17. Found: %C, 72.29; H, 4.13; N, 19.15. IR (KBr,
cmꢁ1): 1609.5 (C]N), 1585.5 (C]C), 1212.8 (CeOeC). 1H NMR
CHe of pyrazole ring), 7.85e7.98 (m, 4H, Ar-H), 7.32e7.59 (m, 10H,
Ar-H). ESI-MS (m/z): 399 (Mþ).
(DMSO-d6, 400 MHz,
d ppm): 8.75e8.76 (m, 2H, Py-H), 8.45 (s, 1H,
]CHe of pyrazole ring), 7.84e7.99 (m, 4H, Ar-H), 7.35e7.58 (m, 8H,
4.6.9. 2-(4-Chlorophenyl)-5-(1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)-
1,3,4-oxadiazole (9i)
Ar-H). ESI-MS (m/z): 365 (Mþ).
Yield 65%, mp 120e122 ꢂC. Anal. calcd. For C24H17ClN4O
(412.87): %C, 69.84; H, 4.15; N, 13.57. Found: %C, 69.79; H, 4.16; N,
13.59. IR (KBr, cmꢁ1): 1612.6 (C]N), 1514.0 (C]C), 1215.9 (CeOeC),
4.6.16. 4-(5-(1-Phenyl-3-p-tolyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-
2-yl)pyridine (10b)
694.5 (CeCl). 1H NMR (DMSO-d6, 400 MHz,
d ppm): 8.45 (s, 1H, ]
Yield 60%, mp 108e110 ꢂC. Anal. calcd. for C23H17N5O (379.41): %
C, 72.81; H, 4.52; N, 18.46. Found: %C, 72.79; H, 4.54; N, 18.50. IR
(KBr, cmꢁ1): 1605.6 (C]N), 1570.4 (C]C), 1224.7 (CeOeC). 1H NMR
CHe of pyrazole ring), 7.94e7.97 (m, 2H, Ar-H), 7.32e7.60 (m, 9H,
Ar-H), 7.16e7.18 (m, 2H, Ar-H), 2.35 (s, 1H, CH3). ESI-MS (m/z): 413
(Mþ).
(DMSO-d6, 400 MHz,
d ppm): 8.80e8.81 (m, 2H, Py-H), 8.49 (s, 1H,
]CHe of pyrazole ring), 7.89e7.95 (m, 2H, Ar-H), 7.37e7.54 (m, 7H,
Ar-H), 7.16e7.18 (m, 2H, Ar-H), 2.36 (s, 1H, CH3). ESI-MS (m/z): 379
(Mþ).
4.6.10. 2-(4-Chlorophenyl)-5-(3-(4-fluorophenyl)-1-phenyl-1H-
pyrazol-4-yl)-1,3,4-oxadiazole (9j)
Yield 69%, mp 105e106 ꢂC. Anal. calcd. for C23H14ClFN4O
(416.83): %C, 66.27; H, 3.39; N, 13.44. Found: %C, 66.24; H, 3.40; N,
13.48. IR (KBr, cmꢁ1): 1603.2 (C]N),1594.0 (C]C), 1221.1 (CeOeC),
4.6.17. 4-(5-(3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-
oxadiazol-2-yl)pyridine (10c)
Yield 74%, mp 116e118 ꢂC. Anal. calcd. for C22H14FN5O (383.38):
%C, 68.92; H, 3.68; N, 18.27. Found: %C, 68.97; H, 3.65; N, 18.30. IR
(KBr, cmꢁ1): 1609.3 (C]N), 1592.2 (C]C), 1229.9 (CeOeC), 1066.3
1002.7 (CeF), 679.6 (CeCl). 1H NMR (DMSO-d6, 400 MHz,
d ppm):
8.52 (s, 1H, ]CHe of pyrazole ring), 7.78e7.92 (m, 4H, Ar-H), 7.42e
7.53 (m, 7H, Ar-H), 7.12e7.14 (m, 2H, Ar-H). ESI-MS (m/z): 417 (Mþ).
(CeF). 1H NMR (DMSO-d6, 400 MHz,
d ppm): 8.77e8.79 (m, 2H, Py-
H), 8.49 (s, 1H, ]CHe of pyrazole ring), 7.88e7.92 (m, 2H, Ar-H),
7.37e7.55 (m, 7H, Ar-H), 7.18e7.20 (m, 2H, Ar-H). ESI-MS (m/z):
383 (Mþ).
4.6.11. 2-(4-Chlorophenyl)-5-(3-(4-chlorophenyl)-1-phenyl-1H-
pyrazol-4-yl)-1,3,4-oxadiazole(9k)
Yield 80%, mp 168e170 ꢂC. Anal. calcd. for C23H14Cl2N4O
(433.29): %C, 63.76; H, 3.26; N, 12.93. Found: %C, 63.78; H, 3.29; N,
12.96. IR (KBr, cmꢁ1): 1617.4 (C]N), 1598.0 (C]C), 1212.6 (CeOeC),
4.6.18. 4-(5-(3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-
oxadiazol-2-yl)pyridine (10d)
Yield 59%, mp 192e194 ꢂC. Anal. calcd. for C22H14ClN5O
(399.83): %C, 66.09; H, 3.53; N, 17.52. Found: %C, 66.06; H, 3.54; N,
17.58. IR (KBr, cmꢁ1): 1600.2 (C]N),1593.7 (C]C),1245.1 (CeOeC),
686.3 (CeCl). 1H NMR (DMSO-d6, 400 MHz,
d ppm): 8.48 (s, 1H, ]
CHe of pyrazole ring), 7.87e7.98 (m, 4H, Ar-H), 7.38e7.58 (m, 9H,
Ar-H). ESI-MS (m/z): 433 (Mþ).
684.4 (CeCl). 1H NMR (DMSO-d6, 400 MHz,
d ppm): 8.83e8.85 (m,

S. Bansal et al. / European Journal of Medicinal Chemistry 80 (2014) 167e174
173
2H, Py-H), 8.43 (s, 1H, ]CHe of pyrazole ring), 7.84e7.96 (m, 4H,
Ar-H), 7.41e7.60 (m, 7H, Ar-H). ESI-MS (m/z): 400 (Mþ).
water ad libitum during the maintenance but were fasted with only
access to water at least 14e24 h prior to perform experiments.
4.6.19. 4-(5-(3-(4-Bromophenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-
oxadiazol-2-yl)pyridine (10e)
4.7.3. Toxicological study (OECD 425 guidelines)
An acute toxicity study was done and safe dose was calculated as
per OECD test guideline 425 [36].
Yield 84%, mp 174e175 ꢂC. Anal. calcd. for C22H14BrN5O
(444.28): %C, 59.47; H, 3.18; N, 15.76. Found: %C, 59.53; H, 3.22; N,
15.82. IR (KBr, cmꢁ1): 1620.9 (C]N),1573.5 (C]C),1215.9 (CeOeC),
4.7.4. Carrageenan-induced paw edema model
750.7 (CeBr). 1H NMR (DMSO-d6, 400 MHz,
d
ppm): 8.83e8.85 (m,
The anti-inflammatory activity was evaluated using in vivo rat
carrageenan-induced paw edema method as described previously
by Winter et al. [37]. The animals (Charles Foster male albino rats,
weighing 150e200 g) were divided into three groups (n ¼ 6) viz.
test, standard, and control. The test compounds and standard drug
diclofenac were suspended in 0.5% methyl cellulose and were given
orally to each group of animals. After 1 h, 0.2 mL of 1% carrageenan
in 0.9% NaCl was injected subcutaneously into the plantar surface of
the right hind paw of each rat. The paw volume was recorded using
2H, Py-H), 8.63 (s, 1H, ]CHe of pyrazole ring), 7.75e7.83 (m, 4H,
Ar-H),7.41e7.62 (m, 7H, Ar-H). ESI-MS (m/z): 444 (Mþ).
4.6.20. 4-(5-(3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-
1,3,4-oxadiazol-2-yl)-pyridine (10f)
Yield 60%, mp 140e142 ꢂC. Anal. calcd. for C23H17N5O2 (395.41):
%C, 69.86; H, 4.33; N, 17.71. Found: %C, 69.85; H, 4.31; N, 17.70. IR
(KBr, cmꢁ1): 1611.7 (C]N), 1598.5 (C]C), 1249.1 (CeOeC). 1H NMR
(DMSO-d6, 400 MHz,
d
ppm): 8.80e8.81 (m, 2H, Py-H), 8.40 (s, 1H,
a plethysmometer (UGO Basile 7140, model-7141, Biological
]CHe of pyrazole ring), 7.93e7.96 (m, 2H, Ar-H), 7.49e7.61 (m, 7H,
Ar-H), 6.82e6.84 (m, 2H, Ar-H), 3.82 (s, 1H, OCH3). ESI-MS (m/z):
395 (Mþ).
research apparatus, Italy) immediately after the carrageenan in-
jection and again 2 h later. The mean increase in paw volume was
compared with that of control group (six rats received only carra-
geenan, no test compounds) and percent inhibition values were
calculated using the formula: anti-inflammatory activity
(%) ¼ 1 ꢁ Dt/Dc ꢃ 100, where Dt represents the difference in paw
volume before and after drug was administered to the rats, while
Dc is the difference in paw volume in the control group. The ED50
obtained after 2 h of treatment with carrageenan are depicted in
Table 1. All of the ED50 values were determined at three doses of 50,
100, and 200 mg/kg body wt. for the test compounds and 25, 50,
and 100 mg/kg body wt. for the reference drugs diclofenac and
celecoxib.
4.6.21. 4-(5-(3-(4-Nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-
oxadiazol-2-yl)pyridine (10g)
Yield 70%, mp 240e241 ꢂC. Anal. calcd. for C22H14N6O3 (410.38):
%C, 64.39; H, 3.44; N, 20.48. Found: %C, 64.44; H, 3.49; N, 20.52. IR
(KBr, cmꢁ1): 1618.5 (C]N),1575.9 (C]C),1345.7 (N]O),1207.6 (Ce
OeC). 1H NMR (DMSO-d6, 400 MHz,
d ppm): 8.78e8.80 (m, 2H, Py-
H), 8.45 (s, 1H, ]CHe of pyrazole ring), 8.25e8.27 (m, 2H, Ar-H),
7.77e7.84 (m, 4H, Ar-H), 7.44e7.56 (m, 5H, Ar-H). ESI-MS (m/z):
410 (Mþ).
4.7. Biological screening
4.7.5. Acetic acid-induced writhing assay
The most active compound (9g) emerged in the anti-
inflammatory study was further tested for its analgesic potential
by employing acetic acid-induced writhing assay. The separate
groups (each group consisting of 6 mice) were administered with
saline, lead compound 9g (200 mg/kg, p.o.), and reference drug
aspirin (100 mg/kg, p.o.). After 60 min, an i.p. injection of 0.6%
acetic acid was given to the mice of all the groups. The total number
of writhes made by each mouse was counted between 5 and 10 min
after acetic acid administration and the analgesic effect was
calculated as the percentage inhibition of writhes as compared
with the control group [30,38].
4.7.1. In vitro COX inhibition assay [16]
COX-1 and COX-2 inhibition activities of tested compounds were
determined by using a COX fluorescent inhibitor Screening assay kit
consisting of ovine COX-1 and human recombinant COX-2 enzymes.
A minimum volume of DMSO was used to prepare stock solutions of
tested compounds. A 10
100 M) were added to 960
HCl pH 8.0 containing 5 mM EDTA and 2 mM phenol) with either
COX-1 or COX-2 (10 L) enzyme in the presence of 10 L heme and
10 fluorometric substrate ADHP (10-acetyl-3,7-dihydrox-
yphenoxazine). These solutions were kept at 37 ꢂC for 5 min, 10
of arachidonic acid solution (100 M) was added and the COX re-
action was stopped by the addition of 50 L of 1 M HCl after 2 min.
m
L of test solutions (0.01, 0.1, 1, 10, 50, and
m
mL supplied buffer solution (0.1 M Trise
m
m
mL
mL
m
4.7.6. Ulcerogenic assay
m
The lead compound 9g along with reference drug aspirin were
examined for gastric ulcerogenic potential. Six rats in each group,
weighing 180e200 g and fasted for 24 h were administered the lead
compound 9g and aspirin (each 200 mg/kg) by intragastric route.
Three hours after, rats were sacrificed, their stomachs were
removed, opened along the lesser curvature, and examined for the
presence of gastric ulcers. The glandular mucosa was explored and
each hemorrhagic lesion was measured individually along its
maximum length (<1 mm: rank ¼ 1; 1e2 mm: rank ¼ 2; >2 mm:
ranking according to their utmost length). The lengths of all the
lesions were summed up to provide an overall total, defined as the
ulcer-index. The results obtained are expressed as mean ꢀ SEM
[39].
Fluorescence of resorufin, produced by the reaction between PGG2
and ADHP, were measured with an excitation wavelength of
535 nm and an emission wavelength of 590 nm. The intensity of
this fluorescence is proportional to the amount of resorufin, which
is proportional to the amount of PGG2 present in the well during
the incubation. Percentage inhibition was calculated by comparison
with control value (no inhibitor). The IC50 values of tested com-
pounds against COX-1 and COX-2 (
m
M) were calculated from the
concentration
inhibition
response curve (triplicate
determinations).
4.7.2. Animals
The experimental protocol was approved by Institutional Ani-
mal Ethics Committee (IAEC) and complied with the NIH guidelines
on handling of experimental animals. The animals were kept at
room temperature (27 ꢀ 3 ꢂC) with 14:10 h light and dark cycle and
at controlled humidity conditions. They were housed under stan-
dard conditions and received autoclaved standard food pellets and
4.8. Molecular docking study
Crystal structures of COX-1 and COX-2 were downloaded from
Protein Data Bank (PDB ID: 1Q4G and 1CX2, respectively). The 3D
structures of ligands were prepared in CS Chem Draw Ultra 8.0.

174
S. Bansal et al. / European Journal of Medicinal Chemistry 80 (2014) 167e174
[14] M.I. El-Gamal, Y.S. Park, D.Y. Chi, K.H. Yoo, C.H. Oh, European Journal of Me-
dicinal Chemistry 65 (2013) 315e322.
[15] M.I. El-Gamal, H.S. Choi, K.H. Yoo, D. Baek, C.H. Oh, Chemical Biology & Drug
Design 82 (2013) 336e347.
[16] S.H. Hwang, K.M. Wagner, C. Morisseau, J.Y. Liu, H. Dong, A.T. Wecksler,
B.D. Hammock, Journal of Medicinal Chemistry 54 (2011) 3037e3050.
[17] M.A. El-Sayed, N.I. Abdel-Aziz, A.A. Abdel-Aziz, A.S. El-Azab, Y.A. Asiri, K.E. El-
Tahir, Bioorganic & Medicinal Chemistry 19 (2011) 3416e3424.
[18] M. Amir, H. Kumar, S.A. Khan, Bioorganic & Medicinal Chemistry Letters 18
(2008) 918e922.
[19] Z. Sui, J. Guan, M.P. Ferro, K. McCoy, M.P. Wachter, W.V. Murray, M. Singer,
M. Steber, D.M. Ritchie, D.C. Argentieri, Bioorganic & Medicinal Chemistry
Letters 10 (2000) 601e604.
Molecular docking analysis was performed using AutoDock tools
1.5.4. Polar hydrogens were added to the protein and Gasteiger
charges were assigned. Non-polar hydrogens were merged and
partial charges added to their parent carbon atoms. The search
ꢀ
space was defined as a grid box with dimensions 40 Å ꢃ 40 Å ꢃ 40 A
and center x ¼ 22, y ¼ 35, z ¼ 210 for 1Q4G and dimensions
ꢀ
40 Å ꢃ 40 Å ꢃ 40 A and center x ¼ 25, y ¼ 23, z ¼ 19 for 1CX2. The
Lamarckian genetic algorithm (LGA) was applied to search con-
formers that possessed lowest binding energy. Results of molecular
docking analysis were obtained as estimated free energy of binding
in kcal/mol (docking score).
[20] B. Jayashankar, K.M. Lokanathrai, N. Baskaran, H.S. Sathish, European Journal
of Medicinal Chemistry 44 (2009) 3898e3902.
[21] S. Bondock, S. Adel, H.A. Etman, F.A. Badria, European Journal of Medicinal
Chemistry 48 (2012) 192e199.
[22] S.G. Kucukguzel, E.E. Oruc, S. Rollas, F. Sahin, A. Ozbek, European Journal of
Medicinal Chemistry 37 (2002) 197e206.
[23] N.N. Farshori, M.R. Banday, A. Ahmad, A.U. Khan, A. Rauf, Bioorganic & Me-
dicinal Chemistry Letters 20 (2010) 1933e1938.
Acknowledgments
The authors thank the SAIF labs of Panjab University, Chandi-
garh, India for carrying out spectral and CHN analyses.
[24] A.A. El-Emam, O.A. Al-Deeb, M. Al-Omar, J. Lehmann, Bioorganic & Medicinal
Chemistry 12 (2004) 5107e5113.
[25] Z.N. Cui, Y.X. Shi, L. Zhang, Y. Ling, B.J. Li, Y. Nishida, X.L. Yang, Journal of
Agricultural and Food Chemistry 60 (2012) 11649e11656.
[26] Z.N. Cui, L. Yang, X.C. Li, Z. Wang, X.L. Yang, Chinese Journal of Organic
Chemistry 26 (2006) 1647e1656.
[27] J. Boström, A. Hogner, A. Llinàs, E. Wellner, A.T. Plowright, Journal of Medicinal
Chemistry 55 (2012) 1817e1830.
[28] K. Manjunatha, B. Poojary, P.L. Lobo, J. Fernandes, N.S. Kumari, European
Journal of Medicinal Chemistry 45 (2010) 5225e5233.
References
[1] E. Palaska, G. S¸ ahin, P. Kelicen, N.T. Durlu, G. Altinok, Il Farmaco 57 (2002)
101e107.
[2] N.V. Chandrasekharan, H. Dai, K.L. Roos, N.K. Evanson, J. Tomsik, T.S. Elton,
D.L. Simmons, Proceedings of the National Academy of Sciences of the United
States of America 99 (2002) 13926e13931.
[3] H.R. Herschman, Biochimica et Biophysica Acta 1299 (1996) 125e140.
[4] L. Parente, M. Perretti, Biochemical Pharmacology 65 (2003) 153e159.
[5] T.D. Penning, J.J. Talley, S.R. Bertenshaw, J.S. Carter, P.W. Collins, S. Docter,
M.J. Graneto, L.F. Lee, J.W. Malecha, J.M. Miyashiro, R.S. Rogers, D.J. Rogier,
S.S. Yu, G.D. Anderson, E.G. Burton, J.N. Cogburn, S.A. Gregory, C.M. Koboldt,
W.E. Perkins, K. Seibert, A.W. Veenhuizen, Y.Y. Zhang, P.C. Isakson, Journal of
Medicinal Chemistry 40 (1997) 1347e1365.
[29] M. Akhter, A. Husain, B. Azad, M. Ajmal, European Journal of Medicinal
Chemistry 44 (2009) 2372e2378.
[30] D.V. Dekhane, S.S. Pawar, S. Gupta, M.S. Shingare, C.R. Patil, S.N. Thore, Bio-
organic & Medicinal Chemistry Letters 21 (2011) 6527e6532.
[31] S.K. Suthar, V. Jaiswal, S. Lohan, S. Bansal, A. Chaudhary, A. Tiwari, A.T. Alex,
A. Joseph, European Journal of Medicinal Chemistry 63 (2013) 589e602.
[32] S.K. Suthar, S. Bansal, S. Lohan, V. Modak, A. Chaudhary, A. Tiwari, European
Journal of Medicinal Chemistry 66 (2013) 372e379.
[33] A. Joseph, C.S. Shah, S.K. Suthar, A.T. Alex, N. Maliyakkal, S. Moorkoth,
J.E. Mathew, Acta Pharmaceutica: a Quarterly Journal of Croatian Pharma-
ceutical Society and Slovenian Pharmaceutical Society, Dealing With All
Branches of Pharmacy and Allied Sciences 63 (2013) 397e408.
[34] R.Y. Yang, L.X. Dai, The Journal of Organic Chemistry 58 (1993) 3381e3383.
[35] M.A. Kira, M.O. Abdel-Rahman, K.Z. Gadalla, Tetrahedron Letters 10 (1969)
109e110.
[36] OECD Guidelines for the Testing of Chemicals: 425 Acute Oral ToxicityeUp-
and-Down-Procedure (UDP), OECD, 2008, pp. 1e27.
[37] C.A. Winter, E.A. Risley, G.W. Nuss, Proceedings of the Society for Experi-
mental Biology and Medicine 111 (1962) 544e547.
[38] G. Daidone, B. Maggio, D. Raffa, S. Plescia, M.L. Bajardi, A. Caruso, V.M.C. Cutuli,
M. Amico-Roxas, European Journal of Medicinal Chemistry 29 (1994) 707e
711.
[39] L. Lazzarato, C. Cena, B. Rolando, E. Marini, M.L. Lolli, S. Guglielmo, E. Guaita,
G. Morini, G. Coruzzi, R. Fruttero, A. Gasco, Bioorganic & Medicinal Chemistry
19 (2011) 5852e5860.
[6] P. Prasit, Z. Wang, C. Brideau, C.C. Chan, S. Charleson, W. Cromlish, D. Ethier,
J.F. Evans, A.W. Ford-Hutchinson, J.Y. Gauthier, Bioorganic
Chemistry Letters 9 (1999) 1773e1778.
& Medicinal
[7] J.J. Talley, D.L. Brown, J.S. Carter, M.J. Graneto, C.M. Koboldt, J.L. Masferrer,
W.E. Perkins, R.S. Rogers, A.F. Shaffer, Y.Y. Zhang, B.S. Zweifel, K. Seibert,
Journal of Medicinal Chemistry 43 (2000) 775e777.
[8] D. Riendeau, M.D. Percival, C. Brideau, S. Charleson, D. Dube, D. Ethier,
J.P. Falgueyret, R.W. Friesen, R. Gordon, G. Greig, J. Guay, J. Mancini, M. Ouellet,
E. Wong, L. Xu, S. Boyce, D. Visco, Y. Girard, P. Prasit, R. Zamboni, I.W. Rodger,
M. Gresser, A.W. Ford-Hutchinson, R.N. Young, C.C. Chan, The Journal of
Pharmacology and Experimental Therapeutics 296 (2001) 558e566.
[9] J.M. Dogne, C.T. Supuran, D. Pratico, Journal of Medicinal Chemistry 48 (2005)
2251e2257.
[10] P. McGettigan, D. Henry, Jama: the Journal of the American Medical Associ-
ation 296 (2006) 1633e1644.
[11] G. Menozzi, L. Mosti, P. Fossa, F. Mattioli, M. Ghia, Journal of Heterocyclic
Chemistry 34 (1997) 963e968.
[12] A. Tanitame, Y. Oyamada, K. Ofuji, M. Fujimoto, N. Iwai, Y. Hiyama, K. Suzuki,
H. Ito, M. Wachi, J. Yamagishi, Journal of Medicinal Chemistry 47 (2004)
3693e3696.
[13] R. Sridhar, P.T. Perumal, S. Etti, G. Shanmugam, M.N. Ponnuswamy,
V.R. Prabavathy, N. Mathivanan, Bioorganic & Medicinal Chemistry Letters 14
(2004) 6035e6040.