Synthesis of novel celecoxib analogues by bioisosteric replacement of sulfonamide as potent anti-inflammatory agents and cyclooxygenase inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.05.029

Two series of celecoxib analogues having 1,5-diaryl relationship were synthesized. The key strategy of the molecular design was oriented towards exploring bioisosteric modifications of the sulfonamide moiety of celecoxib. First series (2a-2i) of celecoxib

Full text of DOI:10.1016/j.bmc.2013.05.029

Bioorganic & Medicinal Chemistry 21 (2013) 4581–4590
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of novel celecoxib analogues by bioisosteric replacement of
sulfonamide as potent anti-inflammatory agents and cyclooxygenase
inhibitors
Nisha Chandna a,b, Satish Kumar a, Pawan Kaushik c, Dhirender Kaushik c, Somendu K. Roy d,
Girish K. Gupta e, Sanjay M. Jachak d, Jitander K. Kapoor b, Pawan K. Sharma a,
⇑
a Department of Chemistry, Kurukshetra University, Kurukshetra 136119, India
b Department of Chemistry, National Institute of Technology, Kurukshetra 136119, India
c Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 136119, India
d Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER), Sector-67, SAS Nagar, Mohali 160062, Punjab, India
e Department of Pharmaceutical Chemistry, Maharishi Markandeshwar College of Pharmacy, Maharishi Markandeshwar University, Mullana, Ambala 133207, Haryana, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of celecoxib analogues having 1,5-diaryl relationship were synthesized. The key strategy of the
molecular design was oriented towards exploring bioisosteric modifications of the sulfonamide moiety of
celecoxib. First series (2a–2i) of celecoxib analogues bearing cyano functionality in place of sulfonamide
moiety was synthesized by the reaction of appropriate trifluoromethyl-b-diketones (5a–5i) with 4-
hydrazinylbenzonitrile hydrochloride (4) in ethanol. Cyano moiety of pyrazoles 2 was then converted
into corresponding carbothioamides 3 by bubbling H2S gas in the presence of triethylamine. All the syn-
thesized compounds (2a–2i and 3a–3i) were screened for their in vivo anti-inflammatory (AI) activity
using carrageenan-induced rat paw edema assay. COX-1 and COX-2 inhibitory potency was evaluated
through in vitro cyclooxygenase (COX) assays. Compounds 2a, 2b, 2c, 2e and 3c showed promising AI
activity at 3–4 h after the carrageenan injection that was comparable to that of the standard drug indo-
methacin. Although compounds 3d, 3e and 3f exhibited more pronounced COX-2 inhibition but they also
inhibit COX-1 effectively thus being less selective against COX-2. Three compounds 2a, 2f and 3a were
found to have a COX profile comparable to the reference drug indomethacin. However 2e, 3b, 3c and
3i compounds were the most potent selective COX-2 inhibitors of this study with 3b showing the best
COX-2 profile. In order to better rationalize the action and the binding mode of these compounds, docking
studies were carried out. These studies were in agreement with the biological data.
Received 8 March 2013
Revised 27 April 2013
Accepted 17 May 2013
Available online 25 May 2013
Keywords:
1,5-Diarylpyrazoles
Bioisostere
Carbothioamide
Sulfonamide
Anti-inflammatory activity
Cyclooxygenase inhibitors
Inflammation
NSAIDs
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
inflammation. Since the discovery of aspirin serious efforts have
been devoted to the development of NSAIDs. Main target of NASIDs
Inflammation is a defensive response of body which induces
physiological adaptations to minimize tissue damage and to re-
move the pathogenic infections. A complex series of cellular and
modular events including dilation of arterioles, venules and capil-
laries with increased vascular permeability and exudation of fluids
containing plasma proteins as well as migration of leukocytes into
inflammatory area are involved in this mechanism. A chronic
inflammation is however an important contributory factor in mor-
bidity and mortality. Such inflammatory disorders include rheu-
matoid arthritis, osteoarthritis, inflammatory bowl diseases,
retinitis, multiple sclerosis, psoriasis and atherosclerosis.1,2
is Cyclooxygenase (COX) which is one of the key enzymes3 in-
volved in the metabolism of arachidonic acid to prostaglandins
(PGs) which are important mediators of inflammation.4 COX is a
membrane-bound heme protein which exists in two distinct iso-
forms, a constitutive form COX-1 and an inducible form COX-2.5
Both COX-1 and COX-2 catalyze the same biochemical reaction
but are clearly different in terms of amino acid sequence, tissue
distribution and physiological function. COX-1 is a housekeeping
enzyme present in platelets and all tissues and produces PGs in-
volved in physiological functions such as gastric mucosal cytopro-
tection, renal homeostasis and platelet aggregation.6 Differently,
COX-2 is predominantly found in brain, kidney and endothelial
cells and activated by pro-inflammatory stimuli resulting in the re-
lease of PGs involved in inflammatory process.7 Indiscriminate
inhibition of both isoforms of COX associated with the conven-
tional NSAIDs has been associated with well-known side effects
Non-steroidal anti-inflammatory drugs (NSAIDs) are amongst
the most widely used therapeutics for the treatment of pain and
⇑
Corresponding author. Tel.: +91 94164 57355; fax: +91 1744 238277.
E-mail address: pksharma@kuk.ac.in (P.K. Sharma).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.029

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N. Chandna et al. / Bioorg. Med. Chem. 21 (2013) 4581–4590
at the gastrointestinal level as well as at the renal level.8 These
common side effects of NSAIDs have been ascribed to their ability
of inhibiting COX-1 and thus developing COX-2 selective inhibi-
tors, commonly called coxibs, was considered to be a logical strat-
egy for designing NSAIDs without the adverse events.9 The
development of multibillion dollar drugs like celecoxib10 (1) which
has shown excellent anti-inflammatory activity with reduced GI
side effects enthused the medicinal chemists all over the world
to search for new structurally different anti-inflammatory drugs.
A sulfonamide group and vicinal diaryl pattern are integral constit-
uents of many such coxibs.
isomeric pyrazole derivatives.21 19F NMR spectrum of isolated
product 2a displayed a singlet at d ꢀ62.5 which is characteristic
of CF3 located at C3 of pyrazole ring. 13C NMR spectrum gave fur-
ther credence to our assignment as it displayed a quartet due to
the carbon bearing CF3 group at d 144.5 (2JC–F = 38.2 Hz) which is
in characteristic region for the C3-carbon of pyrazole ring bearing
CF3 group.21 Had the isolated product been undesired regioisomer-
ic pyrazole that is, 1,3-diarylpyrazole 6a it would have entailed a
signal at d ꢀ60 to ꢀ57 ppm in 19F NMR and at d 133–137 ppm in
13C NMR. Earlier it has been reported by us18 and others21–24 that
two regioisomeric pyrazoles are formed by reaction of hydrazines
with the unsymmetrical trifluoromethyl-b-diketones. However,
exclusive formation of 1,5-diarylpyrazole under the current reac-
tion conditions is in consonance with the observation of Penning
et. al.,10 that 1,5-diarylpyrazoles could be generated almost exclu-
sively by using hydrochloride salt of the aryl hydrazine in refluxing
ethanol or MTBE. The other derivatives (2b–i) were synthesized in
good yields following the same procedure.
A perusal of literature reveals that the synthesis of new drugs
by using bioisosteric approach is gaining momentum.11–13 It has
also been reported that carbothioamide and sulphonamide groups
can act as effective bioisosteres of an amide group.14,15
Appreciation of these findings coupled with our ongoing inter-
est16–18 in the synthesis of novel heterocyclic analogues as anti-
inflammatory agents led us to explore the bioisosteric replacement
of the sulfonamide group of the most celebrated drug celecoxib (1)
in search of new compounds with potent AI activity and selective
COX-2 profile. We report herein design and synthesis of two novel
series of compounds 2 and 3 (Fig. 1) analogous to the celecoxib
class of vicinal diarylpyrazoles with bioisosteric replacement of
the sulfonamide group with a cyano and carbothiomide moiety.
Both the novel series were screened for their in vivo anti-inflam-
matory activities in rats and in vitro COX assays.
With cyanophenylpyrazoles 2 in hand, we attempted to achieve
the synthesis of corresponding carbothioamide bearing diarylpy-
razoles 3a–3i translating into a bioisosteric replacement of a sul-
fonamide moiety to
a cabothioamide moiety as outlined in
Scheme 1. Attempts were made to convert cyanopyrazole 2a into
the corresponding carbothioamide 3a by passing H2S gas through
the solution in pyridine in the presence of triethylamine following
a literature procedure.25 After completion of reaction as observed
by TLC monitoring and IR, reaction mixture was worked up and
crystallized from ethanol to obtain the carbothiomide 3a only in
20% isolated yield. Suspecting that low yield might be a result of
poor crystallization, we changed the solvent of crystallization to
methanol, acetone, acetonitrile and aqueous DMF, but failed to
get any significant improvement in yield. These failures prompted
us to try different reaction conditions for the synthesis of 3a. Pass-
ing H2S gas through a solution of cyanophenylpyrazole 2a in chlo-
roform in the presence of triethylamine as base resulted only in an
incomplete reaction with the separation of some crystalline salts
which did not dissolve even after prolonged purging of H2S gas.
Using mixture of chloroform and ethanol (1:1) as the reaction sol-
vent and passing H2S gas under basic conditions which should be
maintained during the reaction with the help of triethylamine,
we obtained desired pyrazole 3a in 76% yield. Other analogues
3b–3i were prepared similarly in 75–85% yield. Spectral data (1H
NMR, 13C NMR, 19F NMR, IR and mass) of the newly synthesized
compounds were in full agreement with the proposed structures.
2. Results and discussion
2.1. Chemistry
The basic requirement for the synthesis of these pyrazoles was
the availability of the 4-hydrazinylbenzonitrile hydrochloride (4).
The treatment of 4-aminobenzonitrile with sodium nitrite and
hydrochloric acid generated diazonium salt solution which on
reduction with stannous chloride dihydrate gave 4.19 The hydra-
zine 4 was then treated with unsymmetrical trifluoromethyl-b-
diketones20 in an effort to synthesize 1,5-diarylpyrazoles (2) as
outlined in Scheme 1.
Accordingly, a solution of 4-hydrazinylbenzonitrile hydrochlo-
ride (4) and 4,4,4-trifluoro-1-phenylbutane-1,3-dione (5a) in etha-
nol was refluxed on a water bath for 3 h following the procedure of
Penning et al.,10 for the synthesis of celecoxib. In our case the reac-
tion was incomplete after 3 h forcing us to prolong the refluxing
while monitoring the progress of the reaction by thin layer chro-
matography (TLC). The reaction completed in 7 h and after work-
up afforded a solid material in 85% yield which was conclusively
characterized as 1,5-diarylpyrazole 2a as exclusive product on
the basis of its spectral data. 19F NMR and 13C NMR spectroscopy
has been used as an elegant tool in such cases to study the position
of CF3 on the pyrazole ring so as to distinguish between the regio-
2.2. Biological evaluation
2.2.1. In vivo anti-inflammatory activity
All synthesized compounds 2a–2i and 3a–3i were evaluated for
their in vivo anti-inflammatory activity by carrageenan-induced
rat paw edema method.26 The protocol of animal experiments
Modification of
Sulfonamide Moiety
NC
H2NSC
H2NO2S
N
N
N
N
N
N
CF3
CF3
CF3
R
R
1
2
3
H3C
(Celecoxib)
Modification of
4-methylphenyl
Figure 1. Structure of celecoxib (1) and schematic design of its analogues.

N. Chandna et al. / Bioorg. Med. Chem. 21 (2013) 4581–4590
4583
CN
CN
N
CN
O
O
Ethanol
+
+
R
CF3
N
R
N
F3C
N
.HCl
NHNH2
CF3
R
5
4
6
2
CN
CSNH2
H2S/triethylamine
Ethanol/CHCl3
N
N
N
R
N
R
CF3
CF3
2
3
2,3,6
a
b
c
d
e
f
g
h
i
R
Ph
4-CH3-Ph
4-F-Ph 4-Cl-Ph 4-Br-Ph 4-OCH3- Ph
2-naphthyl 2-thienyl
2-furyl
Scheme 1. Synthesis of 1,5-diarylpyrazoles 2 and 3.
has been approved by the Institutional Animal Ethics Committee
(IAEC). Each test compound was dosed orally (50 mg/kg body
weight) 30 min prior to induction of inflammation by carrageenan
injection. Indomethacin was used as a reference anti-inflammatory
drug and anti-inflammatory activity was calculated at hourly inter-
vals up to 4 h after injection and presented in Table 1 as the mean
paw volume (mL) as well as the percentage anti-inflammatory
activity (%AI).
(90.39%). Interestingly two of the compounds 2a and 2c were
found to be as potent as the reference drug indomethacin after
4 h of edema induction. 1,5-dairylpyrazole 2b, the closest relative
of celecoxib having 4-methylphenyl group at C-5 of pyrazole
showed excellent anti-inflammatory activity however less than
the corresponding 4-fluorophenyl (2c) or simple phenyl (2a) ana-
logue. Considering the retention of AI activity by celecoxib ana-
logues of this study possessing
a cyano moiety instead of
Amongst nine cyano compounds 2a–2i tested, four compounds
(2a, 2b, 2c and 2e) showed excellent AI activity (P82% inhibition)
when measured 3 h after the carrageenan injection compared to
that of the standard drug indomethacin (84.97%) whereas the
remaining compounds have shown good activity with 57–69%
inhibition after 3 h. These four compounds also showed apprecia-
ble AI activity (P80%) after 4 h as compared to indomethacin
sulfonamide group, it can be concluded that cyano group is acting
as a bioisostere of the sulfonamide group in this series. Having po-
tent AI activities for some of the cyanophenylpyrazoles 2, corre-
sponding carbothioamide analogues were also evaluated for AI
activities hoping that a carbothioamide might be a better bioiso-
stere of the sulphonamide group.
Amongst nine cabothioamides 3a–3i tested (Table 2), only one
compound 3c bearing a 4-fluorophenyl substituent at C-5 of pyra-
zole displayed consistently excellent AI activity up to 4 h that was
comparable to that of the standard drug indomethacin. Com-
pounds 3b–3f showed moderate AI activity (>60%) 3 h after carra-
geenan injection. In general, within the series 3a–3i, compounds
with a halogen substituent on the C-5 phenyl (3c, 3d and 3e)
showed better activity as compared to those bearing non-halogen
substituents.
Table 1
Anti-inflammatory (AI) activity of compounds 2a–2i in carrageenan-induced rat paw
edema assay
Compound
Volume of edemaa (mL) and %AIb
1 h
2 h
3 h
4 h
Control
Indomethacin
0.99 0.12
0.04 0.02⁄⁄
(95.95)
1.96 0.23
0.19 0.03⁄⁄
(90.30)
1.73 0.22
0.26 0.02⁄⁄
(84.97)
1.77 0.33
0.17 0.03⁄⁄
(90.39)
A comparison of the two series 2a–2i and 3a–3i reveals that the
compound with 4-fluorophenyl substituent in the cyano series 2c
retains its significant activity even on transforming the cyano
group to carbothioamide group in 3c. These results are in accor-
dance with our earlier observations17,18 that the compounds with
halogen substituents show better AI activity.
2a
2b
2c
2d
2e
2f
0.08 0.05⁄⁄
(91.91)
0.37 0.11⁄⁄
(81.12)
0.27 0.09⁄⁄
(84.39)
0.16 0.09⁄⁄
(90.96)
0.34 0.06⁄⁄
(65.65)
0.25 0.09⁄⁄
(87.24)
0.30 0.05⁄⁄
(82.65)
0.35 0.06⁄⁄
(80.22)
0.20 0.03⁄⁄
(79.79)
0.37 0.29⁄⁄
(81.12)
0.27 0.01⁄⁄
(84.39)
0.17 0.12⁄⁄
(90.39)
Considering the fact that carrageenan-induced paw edema as-
say is a biphasic event27 involving the release of histamine and
serotonin as the mediators of inflammation in the first phase which
normally lasts for about 2 h after the carrageenan injection27 fol-
lowed by the second phase which generally operates between 2
and 4 h after the carrageenan injection and involves prostaglandins
as the mediators for the inflammation,27 any AI activity in the sec-
ond phase of this biphasic event can be attributed to the inhibition
of prostaglandin synthesis. A careful study of the results show
excellent AI activity of 2a, 2b, 2c, 2e and 3c in the second phase
of the biphasic carrageenan edema assay indicating the capability
of the tested compounds to inhibit prostaglandin synthesis. This
can be attributed to their ability to bind cyclooxygenase (COX),
an enzyme responsible for the synthesis of prostaglandins. Consis-
tently excellent AI activity of 2a, 2b, 2c, 2e and 3c up to 4 h
0.73 0.29
(26.26)
0.46 0.23⁄⁄
(76.53)
0.56 0.29⁄⁄
(67.63)
0.62 0.28⁄⁄
(64.97)
0.21 0.04⁄⁄
(78.78)
0.22 0.08⁄⁄
(88.77)
0.28 0.05⁄⁄
(83.81)
0.24 0.05⁄⁄
(86.44)
0.55 0.20
(44.44)
0.29 0..19⁄⁄
(85.20)
0.52 0.20⁄⁄
(69.94)
0.80 0.22⁄⁄
(54.80)
2g
2h
2i
0.15 0.06⁄⁄
(84.84)
0.63 0.11⁄⁄
(67.85)
0.70 0.12⁄⁄
(59.53)
0.92 0.12⁄⁄
(48.02)
0.18 0.07⁄⁄
(81.81)
0.56 0.10⁄⁄
(71.42)
0.73 0.11⁄⁄
(57.80)
0.78 0.13⁄⁄
(55.93)
0.20 0.01⁄⁄
(79.79)
0.41 0.14⁄⁄
(79.08)
0.56 0.19⁄⁄
(67.63)
0.68 0.16⁄⁄
(61.58)
⁄Significantly different compared to respective control values, P <0.05.
⁄⁄Significantly different compared to respective control values, P <0.01.
a
Values are expressed as mean SEM and analyzed by ANOVA.
Values in parentheses (percentage anti-inflammatory activity, % AI).
b

4584
N. Chandna et al. / Bioorg. Med. Chem. 21 (2013) 4581–4590
Table 2
COX-2 inhibition as well as IC50 as compared to the direct cele-
coxib analogue 3b bearing a methyl substituent, however, these
compounds also exhibited better COX-1 inhibition thus possess-
ing reduced selectivity index. Compound 3i possessing a furyl
moiety in place of p-toluyl substitutent was found to exhibit less
COX-2 potency (74%) as compared to 3b, however it showed sig-
nificant COX-2 selectivity index (>3.31). Compounds 2e and 3c
were also found to show moderate COX-2 inhibition (P70%) as
well as COX-2 selectivity. On the other hand three compounds
2a, 2f and 3a were found to be COX-1 selective like
indomethacin.
Based upon the results, it can be concluded that all the com-
pounds showing potent COX-2 selectivity (3b, 3i, 2e and 3c) also
displayed significant AI activity after 3 to 4 h of carrageenan injec-
tion whereas the reverse was not true as some of the compounds
showing promising AI activity, failed to significantly inhibit COX-2.
Anti-inflammatory (AI) activity of compounds 3a–3i against carrageenan-induced rat
paw edema assay
Compound
Volume of edemaa (mL) and% AIb
1 h
2 h
3 h
4 h
Control
Indomethacin
0.99 0.12
0.04 0.02⁄⁄
(95.96)
1.99 0.23
0.19 0.03⁄⁄
(90.45)
1.73 0.22
0.26 0.02⁄⁄
(84.97)
1.74 0.33
0.17 0.03⁄⁄
(90.23)
3a
3b
3c
3d
3e
3f
0.17 0.04⁄⁄
(82.83)
0.5 0.09⁄⁄
(74.87)
0.87 0.14⁄⁄
(49.71)
0.87 0.11⁄⁄
(50)
0.26 0.03⁄⁄
(73.74)
0.41 0.04⁄⁄
(79.39)
0.69 0.04⁄⁄
(60.12)
0.84 0.01⁄⁄
(51.72)
0.25 0.12⁄⁄
(74.75)
0.29 0.05⁄⁄
(85.43)
0.49 0.02⁄⁄
(71.68)
0.3 0.01⁄⁄
(82.76)
0.10 0.02⁄⁄
(89.89)
0.45 0.12⁄⁄
(77.39)
0.64 0.17⁄⁄
(63.01)
0.67 0.12⁄⁄
(61.49)
0.41 0.1⁄⁄
(54.55)
0.30 0.08⁄⁄
(84.92)
0.47 0.02⁄⁄
(72.83)
0.69 0.03⁄⁄
(60.34)
0.21 0.05⁄⁄
(78.79)
0.44 0.09⁄⁄
(77.89)
0.59 0.05⁄⁄
(65.89)
0.82 0.09⁄⁄
(52.87)
2.3. Docking studies
3g
3h
3i
0.07 0.02⁄⁄
(92.93)
0.54 0.06⁄⁄
(72.86)
0.81 0.05⁄⁄
(53.18)
0.94 0.09⁄⁄
(45.98)
0.14 0.03⁄⁄
(85.86)
0.53 0.11⁄⁄
(73.37)
0.74 0.01⁄⁄
(57.23)
0.67 0.13⁄⁄
(61.49)
Significant anti-inflammatory activity and COX inhibitory po-
tency of novel celecoxib analogues prompted us to perform molec-
ular docking studies to understand the ligand-protein interactions
and COX-1/COX-2 selectivity in detail. Automated docking studies
were carried out using Molegro Virtual Docker, the scoring func-
tions and hydrogen bonds formed with the surrounding amino
acids are used to predict their binding modes and their binding
affinities at the active site of cyclooxygenase (COX-I & II) enzyme.
Molecular docking studies of compounds 2a–2i and 3a–3i were
carried out using Molegro Virtual Docker 2010.4.129 in order to
rationalize biological activity results. Compounds 2e, 3b–3f, 3i ex-
hibit relatively more binding affinity for COX-2 having more dock-
ing score than (Fig. 2) celecoxib (the original ligand) as presented
in Tables 4 and 5, and Figures 2–17 (Figs 6–17 see Supplementary
data).
0.17 0.04⁄⁄
(82.83)
0.45 0.06⁄⁄
(77.39)
0.72 0.01⁄⁄
(58.38)
0.7 0.07⁄⁄
(59.77)
⁄Significantly different compared to respective control values, P <0.05.
⁄⁄Significantly different compared to respective control values, P <0.01.
a
Values are expressed as mean SEM and analyzed by ANOVA.
Values in parentheses (percentage anti-inflammatory activity, %AI).
b
suggests that these compounds do not get easily metabolized in
the system.
Compounds 2a–2i and 3a–3f, 3h–3i were further evaluated for
their inhibitory potency against COX-1 and COX-2.
2.2.2. In vitro COX-1 and COX-2 inhibition assay
In order to investigate whether the anti-inflammatory effect ob-
served in the carrageenan-induced rat paw edema assay is caused
by inhibition of COX enzyme (one of the mechanisms for anti-
inflammatory activity), compounds were screened by using en-
zyme-immuno assay (EIA) kit (Cayman Chemical Company, USA).
The COX-1 and COX-2 inhibitory activity of compounds were eval-
uated by method as described by Gautam et al.28 The COX-1 and
COX-2 inhibitory effect of the synthesized compounds 2a–2i and
Table 3
In vitro COX-1 and COX-2 inhibition assay for compounds 2a–2i and 3a–3i
Compound
R
Percentage of COX
inhibition at 30
Ma
COX
Inhibition
(IC50
SIc
l
lM)
COX-1
COX-2
COX- COX-
1
2
2a
2b
2c
2d
2e
2f
4-Ph
97.0 3.43
—
1.11
>30
NT
NT
NT
<0.04
NT
NT
3a–3f, 3h–3i were evaluated at 30 lM. Indomethacin and Cele-
4-CH3-Ph
4-F-Ph
4-Cl-Ph
4-Br-Ph
4-OCH3-
Ph
—
—
—
46.3 1.96 NT
—
—
coxib (selective COX-2 inhibitor) were used as positive controls
in the study (Table 3). Further IC50 values and COX-2 selectivity
were determined for compounds exhibiting potent inhibition as-
NT
NT
NT
13.4 0.77 70.2 0.75 >30
99.9 0.45 60.7 2.90 1.28
19.22 >1.56
say. IC50
(l
M) values were determined as the mean of three deter-
>30
<0.04
minations acquired and the deviation from the mean was found to
be less than 5% of the mean value. The selectivity index (SI values)
was defined as IC50 (COX-1)/IC50 (COX-2).
2g
2-
20.7 2.81
—
—
NT
NT
NT
NT
Naphthyl
2-Thienyl
2-Furyl
4-Ph
2h
2i
—
—
NT
NT
NT
NT
Compounds (2e–2f, 3a–3f) were found to be potent against
40.3 3.84 NT
3a
3b
3c
3d
3e
3f
97.9 0.13 68.1 1.23 1.42
23.56 0.06
7.07 >4.24
17.43 >1.72
COX-2 (P60% inhibition) at 30
3f exhibited the highest inhibition of COX-1 (P80% inhibition)
comparable with indomethacin (98.23%) at 30 M. IC50 values
lM. Compounds 2a, 2f, 3a, 3d–
4-CH3-Ph 44.3 2.51 83.7 0.67 >30
4-F-Ph
4-Cl-Ph
4-Br-Ph
4-OCH3-
Ph
—
73.4 0.88 >30
l
86.9 1.81 99.9 2.99 1.11
86.1 0.12 96.6 0.48 2.35
99.2 0.73 98.9 0.91 1.62
1.44
2.87
1.94
0.77
0.82
0.84
were determined for all the compounds showing potent inhibi-
tion of COX-1 and COX-2 (Table 3). Our assumption of using car-
bothioamide moiety as a bioisostere of sulphonamide moiety was
vindicated as compound 3b, the direct bioisosteric analogue of
celecoxib, was found to be the most potent and selective COX-2
3h
3i
2-Thienyl 39.8 0.23 56.9 0.93 NT
2-Furyl
NT
NT
>3.31
—
20.1 2.09 74.21 1.54 >30
98.2 0.33 51.0 0.34 0.18
13.0 0.63 95.6 0.48 >30
9.07
>30
0.15
Indomethacinb
Celecoxibb
inhibitor (IC50 = 7.07 lM, SI = >4.24) of all the tested target com-
>200
pounds. In general compounds bearing carbothioamide moiety
(series 3) were found to be exhibit better COX-2 potency as com-
pared to their corresponding cyano analogues (series 2). Within
the carbothiamide series, the compounds bearing bromo, chloro
or methoxy substituents (3d–3f) were found to exhibit better
NT – not tested.
— Not inhibiting COX.
a
Values are expressed as mean SEM of three determinations (n = 3).
Positive control used.
Selectivity index (COX-1 IC50/COX-2 IC50
b
c

N. Chandna et al. / Bioorg. Med. Chem. 21 (2013) 4581–4590
4585
Figure 4. Binding mode of compound (2e) into the binding site of COX-1 pocket. It
has Moldock score ꢀ92.15 and forms 4 hydrogen bonds shown as green dotted lines
(Table 4), showing one hydrogen bond between –N– of pyrazole moiety with Tyr
355 of distance 2.26 Å, one hydrogen bond between substituted –N– of pyrazole
moiety with Tyr 355 of distance 3.00 Å, one hydrogen bond between –N– of
pyrazole moiety with Arg 120 of distance 2.80 Å and one hydrogen bond between
substituted –N– of pyrazole moiety with Arg 120 of distance 2.79 Å, respectively.
Figure 2. Binding mode of celecoxib into the binding site of cyclooxygenase-I
(COX-1) pocket. It has Moldock score ꢀ108.56 and forms 3 hydrogen bonds shown
as green dotted lines (Table 4), showing one hydrogen bond between –O– of SO2
with His 43 of distance 2.91 Å; one hydrogen bond between –O– of SO2 with Gln 44
of distance 3.13 Å; one hydrogen bond between –N– of NH2 with Glu 465 of
distance 3.08 Å.
drug with the COX-2. The complexes of compounds 3b–3f, 3i with
COX-2 (Table-5 and Figs. 5, 7, 9, 11, 13, 15 & 17) showed mainly
interactions due to CSNH2. In view of this, it can be depicted that
both groups are bioisosteres of each other.
Compound 2e was found to dock into the active site of COX-1
with docking score of ꢀ92.15. It formed four hydrogen bonds
two with Tyr 355 and two with Arg 120 into the active site of
COX-1, N of pyrazole of 2e binds with Tyr 355 and Arg 120 with
a bond length of 2.26 and 2.80 Å, respectively; bonding distances
of substituted N of pyrazole of 2e with Tyr 355 and Arg 120 was
Celecoxib reveals docking score ꢀ112.88 for COX-2 and forms
five interactions shown as green dotted lines (Fig. 3), showing
two hydrogen bonds between –O– of SO2NH2 moiety with His 90
and Arg 513 of distances 2.86 and 3.51 Å, respectively, two hydro-
gen bonds between N of SO2NH2 moiety with Gln 192, Leu 352 of
distances 3.36 Å and 2.90 Å, respectively, and N of pyrazole binds
with Tyr 355 with a bond length of 3.20 Å. It was a clear indication
that the sulfonamide ring had significant role in binding of the
Figure 3. Binding mode of celecoxib into the binding site of cyclooxygenase-II
(COX-2) pocket. It has Moldock score ꢀ112.88 and forms 5 hydrogen bonds shown
as green dotted lines (Table 5), showing one hydrogen bond between –O– of SO2
with His 90 of distance 2.86 Å; one hydrogen bond between –O– of SO2 with Arg
513 of distance 3.51 Å; one hydrogen bond between –N– of pyrazole with Tyr 355
of distance 3.20 Å; one hydrogen bond between of substituted –N– of NH2 with Gln
192 of distance 3.36 Å and one hydrogen bond between –N– of NH2 with Leu 352 of
distance 2.90 Å, respectively.
Figure 5. Binding mode of compound (2e) into the binding site of COX-2 pocket. It
has Moldock score ꢀ122.72 and forms 1 hydrogen bond shown as green dotted line
(Table 5), showing one hydrogen bond between –N– of CN with Ser 530 of distance
3.03 Å.

4586
N. Chandna et al. / Bioorg. Med. Chem. 21 (2013) 4581–4590
found 3.00 and 2.79 Å, respectively (Fig. 4). The compound could
dock successfully into the COX-2 active site as well. The docking
score of interaction obtained for 2e was ꢀ122.72 and this score
may be attributed to its one hydrogen bonding interactions with
Ser 530. The bonding distance between N of C„N of 2e with Ser
530 was 3.03 Å (Fig. 5). All three compounds 2e, 3b and 3c have
found high moldock scores for COX-2 ꢀ122.72, ꢀ122.37 and
ꢀ128.48, respectively as compared to standard drug celecoxib
(ꢀ112.88) and their moldock scores for COX-1 were ꢀ92.15,
ꢀ104.02 and ꢀ107.19. The scoring function of 2e, 3b and 3c
with COX-2 suggests them as the preferred ligand for COX-2
than COX-1. The interaction with amino acid Ser 530 is impor-
tant for enzyme inhibitory activity and is well explained by
the binding interaction of aspirin with COX-1/COX-2.30 In case
of complex of 3b with COX-2, the two hydrogen bonding inter-
action with Ser 530 was found which could be a probable reason
for the observed better selectivity of 3b towards COX-2
inhibition.
Interestingly, In case of complex of 3b with COX-2, the hydro-
gen bonding interaction with Tyr 385 was also found to be present
which is further responsible for showing good COX-2 IC50 as it was
known that Tyr 385 is responsible for the abstraction of 13-pro-S-
hydrogen from arachidonic acid.31 However, 3c showed binding
interaction with Try 385 in case of its COX-1 complex which could
be the probable reason for showing COX-2 IC50 less than 3b.
Compounds 3d–3f showed less selectivity for COX-2 that might
be attributed due to their good interaction and dock score for both
COX-2 and COX-1 (Tables 4 and 5 and Figs. 10–15 see Supplemen-
tary data). However, the significant selectivity of 3i can be ex-
plained on the basis of its three hydrogen bonding interaction
with Ser 530 for COX-230 as shown in Table 5 and Figure 17 (see
Supplementary data).
Table 4
Docking score and bond interactions of celecoxib and synthesized compounds with amino acids of COX-1.
Compound
Celecoxib
Dock score (ꢀ)
108.56
No. of interactions
3
Distance (Å)
Amino acids involved
Molecular structure
2.91
3.13
3.08
2.26
3.00
2.80
2.79
2.96
3.49
3.10
3.10
2.90
2.64
3.43
3.10
2.70
2.85
3.10
2.98
His 43
Gln 44
O of SO2
O of SO2
N of NH2
N of Pyrazole
Substituted N of Pyrazole
N of Pyrazole
Substituted N of Pyrazole
N of NH2
N of NH2
N of NH2
N of NH2
N of NH2
N of NH2
N of Pyrazole
N of NH2
N of NH2
N of NH2
Glu 465
Tyr 355
Tyr 355
Arg 120
Arg 120
Met 522
Tyr 385
Ser 530
Ser 530
Leu 352
Gln 192
Tyr 355
Ser 353
Leu352
Gln 192
Ser 353
Met 522
2e
92.15
4
3b
3c
104.02
107.19
1
2
3d
3e
108.01
108.77
1
4
3f
3i
108.66
114.82
3
1
N of NH2
N of NH2
Table 5
Docking score and bond interactions of celecoxib and synthesized compounds with amino acids of COX-2.
Compound
Celecoxib
Dock score (ꢀ)
112.88
No. of interactions
5
Distance (Å)
Amino acids involved
Molecular structure
2.86
3.51
3.20
3.36
2.90
3.03
2.70
2.60
3.50
3.10
3.23
2.88
3.12
3.30
3.23
2.88
3.12
3.30
2.90
2.60
3.42
3.10
3.19
3.29
3.10
3.08
3.02
2.42
His 90
O of SO2
O of SO2
Arg 513
Tyr 355
Gln 192
Leu 352
Ser 530
Tyr 385
Ser 530
Ser 530
Val 344
Phe 518
Gln 192
Leu 352
Tyr 355
Phe 518
Gln 192
Leu 352
Tyr 355
Tyr 385
Ser 530
Ser 530
Val 344
Gln 192
Tyr 355
Leu 352
Ser 530
Ser 530
Ser 530
N of Pyrazole
N of NH2
N of NH2
N of CN
S of C@S
S of C@S
N of NH2
N of NH2
S of C@S
N of NH2
N of NH2
N of Pyrazole
S of C@S
N of NH2
N of NH2
N of Pyrazole
N of NH2
N of NH2
S of C@S
2e
3b
122.72
122.37
1
4
3c
3d
3e
128.48
127.59
128.03
4
4
4
S of C@S
3f
3i
128.36
120.28
3
3
S of C@S
N of Pyrazole
N of NH2
N of NH2
S of C@S
N of NH2

N. Chandna et al. / Bioorg. Med. Chem. 21 (2013) 4581–4590
4587
4.2.1. 4-[5-Phenyl-3-(trifluoromethyl)-1H-pyrazol-1-
3. Conclusion
yl]benzonitrile (2a)
Mp 78–80 °C, yield 85%; IR (KBr) cmꢀ1: 2230 (C„N stretch); 1H
NMR (300 MHz, CDCl3): d 7.67 (d, 2H, J = 8.4 Hz, Ar), 7.47 (d, 2H,
J = 8.4 Hz, Ar), 7.39–7.43 (m, 3H, Ar), 7.10–7.28 (m, 2H, Ar), 6.79
(s, 1H, pyrazole C4-H); 13C NMR (75.5 MHz, CDCl3): d 145.1,
Eighteen analogues of celecoxib consisting of bioisosteric
replacement of the sulphonamide moiety with cyano and carbo-
thioamide moiety and having 1,5-diaryl relationship 2a–2i and
3a–3i were designed, synthesized and evaluated for their in vivo
anti-inflammatory activity and in vitro COX (COX-1 and COX-2)
inhibitory activity. Compounds 2a, 2b, 2c and 2e showed excellent
AI activity (P82% inhibition) and 3b–f showed consistently good
AI activity (P60% inhibition) when measured 3 h after the carra-
geenan injection compared to that of the standard drug indometh-
acin (84.97%). Compound 3b showed potent AI activity and highest
potent COX-2 selective inhibition. Similarly 2e, 3c and 3i displayed
good AI activity and showed potent COX-2 selective inhibition.
However compounds 2a, 2f, 3a exhibited significant AI activity
and COX-1 IC50 10- to 15-fold less than that of indomethacin.
Molecular docking study further helped in supporting the observed
COX-1 and COX-2 selectivity.
2
144.5 (q, JCF = 38.2 Hz, C-3), 142.4, 133.1, 129.7, 129.1, 128.9,
1
128.7, 125.5 120.9 (q, JCF = 269.5 Hz, C3-CF3), 119.2, 117.8, 111.9,
106.8; 19F NMR (CDCl3, 282.4 MHz): d ꢀ62.5 (C3-CF3); m/z 314
([M+H]+, C17H10F3N3H+calcd. 314).
4.2.2. 4-[5-(4-Methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-
yl]benzonitrile (2b)
Mp 108–110 °C (Lit. mp 107–109 °C)32, yield 87%; IR (KBr)
cmꢀ1: 2230 (C„N stretch); 1H NMR (300 MHz, CDCl3): d 7.67
(d, 2H, J = 8.4 Hz, Ar), 7.47 (d, 2H, J = 8.4 Hz, Ar), 7.21 (d, 2H,
J = 7.8 Hz, Ar), 7.12 (d, 2H, J = 7.8 Hz, Ar), 6.76 (s, 1H, pyrazole
C4-H), 2.40 (s, 3H, CH3); 13C NMR (75.5 MHz, CDCl3): d 145.2,
2
145.1, 144.5 (q, JCF = 38.2 Hz, C-3), 143.5, 142.6, 139.9, 133.0,
1
129.8, 128.7,126.4, 125.7, 125.5, 120.9 (q, JCF = 269.5 Hz, C3-
4. Experimental protocols
CF3), 117.9, 115.7, 111.9, 106.5, 21.3 (CH3); 19F NMR (CDCl3,
282.4 MHz): d ꢀ62.5 (C3-CF3); m/z 328 ([M+H]+, C18H12F3N3H+
calcd. 328).
Melting points were determined in open capillaries in electrical
apparatus and are uncorrected. IR spectra were recorded on a Buck
Scientific IR M500 instrument. The 1H NMR, 13C NMR and 19F NMR
spectra were recorded on a Bruker instrument at 300, 75.5 and
282.4 MHz, respectively. The d values are given in ppm relative
to tetramethylsilane as internal standard (for 1H and 13C NMR).
Mass spectra (DART-MS) were recorded on a JEOL-AccuTOF JMS-
T100LC Mass spectrometer having a DART (Direct Analysis in Real
Time) source in ES+ mode. Exchangeable (ex) protons were de-
tected by disappearance of peaks upon D2O addition. The purity
of the compounds was checked by 1H NMR, thin layer chromatog-
raphy (TLC) and HPLC. Iodine or UV lamp was used as a visualizing
agent for TLC. The purity of synthetic compounds (2a–2i and 3a–
3i) was determined by HPLC, detected at kmax 254 nm on X-Bridge
4.2.3. 4-[5-(4-Fluorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-
yl]benzonitrile (2c)
Mp 90–92 °C, yield 85%; IR (KBr) cmꢀ1: 2230 (C„N stretch); 1H
NMR (300 MHz, CDCl3): d 7.69 (d, 2H, J = 8.4 Hz, Ar), 7.46 (d, 2H,
J = 8.7 Hz, Ar), 7.24 (m, 2H, Ar), 7.15 (m, 2H, Ar), 6.78 (s, 1H, pyra-
1
zole C4-H); 13C NMR (75.5 MHz, CDCl3): 163.3 (d, JCF = 251.4 Hz),
2
144.5 (q, JCF = 38.2 Hz, C-3), 143.63, 142.26, 133.2, 130.8 (d,
1
3JCF = 9.0 Hz), 125.5, 124.8, 124.7, 120.9 (q, JCF = 269.5 Hz, C3-
2
CF3), 116.4 (d, JCF = 21.9 Hz), 112.2, 106.9; 19F NMR (CDCl3,
282.4 MHz): d - 62.5 (C3-CF3); m/z 332 ([M+H]+, C17H9F4N3H+ calcd.
332).
column (RP-18, 250 ꢁ 4.5 mm2, 5
lm) eluting with water (0.1%
4.2.4. 4-[5-(4-Chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-
yl]benzonitrile (2d)
acetic acid) and acetonitrile (20:80) and it was in the range of
97.11–99.8%. All the newly synthesized compounds (2a–2i, 3a–
3i) were subjected to microanalysis and gave satisfactory analyti-
cal results (within 0.4% of the calculated values).
Mp 125–127 °C, yield 89%; IR (KBr) cmꢀ1: 2230 (C„N stretch);
1H NMR (300 MHz, CDCl3): d 7.70 (d, 2H, J = 8.4 Hz, Ar), 7.46 (d, 2H,
J = 8.1 Hz, Ar), 7.39 (d, 2H, J = 8.1 Hz, Ar), 7.18 (d, 2H, J = 8.4 Hz, Ar),
6.79 (s, 1H, pyrazole C4-H); 13C NMR (75.5 MHz, CDCl3): d 144.5 (q,
2JCF = 38.2 Hz, C-3), 143.9, 142.2, 136.1, 133.2, 130.1, 129.5, 127.0,
4.1. Synthesis of 4-hydrazinylbenzonitrile hydrochloride (4)
1
125.5, 120.9 (q, JCF = 269.5 Hz, C3-CF3), 117.7, 112.3, 106.9; 19F
To a cooled (ꢀ5 to 0 °C) stirred suspension of 4-aminobenzoni-
trile (50 g, 423 mmol) in concentrated hydrochloric acid (550 mL)
was added dropwise aqueous sodium nitrite solution (31.5 g,
457 mmol in 200 mL water). To this diazotised solution cooled
(0 °C) solution of tin (II) chloride dihydrate (477 g, 2.1 mol) in con-
centrated hydrochloric acid (370 mL) was added while stirring and
maintaining the temperature below 0 °C. The resulting solution
was further stirred for 15 min. White precipitates so formed were
collected by filtration, washed with diethylether and crystallized
from aqueous ethanol. Mp 234–236 °C (Lit. mp 235–237 °C),19
yield 60 g (84%).
NMR (CDCl3, 282.4 MHz): d - 62.5 (C3-CF3); m/z 348 ([M+H]+,
C
17H9ClF3N3H+ calcd. 348).
4.2.5. 4-[5-(4-Bromophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-
yl]benzonitrile (2e)
Mp 130–132 °C, yield 90%; IR (KBr) cmꢀ1: 2230 (C„N stretch);
1H NMR (300 MHz, CDCl3): d 7.70 (d, 2H, J = 8.4 Hz, Ar), 7.55 (d, 2H,
J = 8.4 Hz, Ar), 7.46 (d, 2H, J = 8.4 Hz, Ar), 7.12 (d, 2H, J = 8.4 Hz, Ar),
6.80 (s, 1H, pyrazole C4-H); 13C NMR (75.5 MHz, CDCl3): d 144.5 (q,
2JCF = 38.2 Hz, C-3), 143.9, 142.2, 133.2, 132.5, 130.3, 127.5, 125.5,
1
124.3, 120.9 (q, JCF = 269.5 Hz, C3-CF3), 117.7, 112.3, 106.9; 19F
NMR (CDCl3, 282.4 MHz): d ꢀ62.5 (C3-CF3); m/z 392 ([M+H]+,
4.2. General procedure for the synthesis of pyrazoles (2a–2i) in
ethanol
C
17H9BrF3N3H+ calcd. 392).
To a warm ethanolic solution (10 mL) of 4-hydrazinylbenzonit-
rile hydrochloride 4 (350 mg, 2.0 mmol) was added appropriate tri-
fluoromethyl-b-diketone 5a–5i (2.0 mmol) while stirring and the
contents were refluxed for 7 h. The progress of the reaction was
monitored by TLC. When the reaction was completed, excess sol-
vent was evaporated to reduce the volume to one half whereupon
reaction mixture was allowed to cool to 20–25 °C and the solid
thus obtained was filtered to obtain 2a–2i (85–93%).
4.2.6. 4-[5-(4-Methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-
1-yl]benzonitrile (2f)
Mp 132–134 °C, yield 87%; IR (KBr) cmꢀ1: 2230 (C„N stretch);
1H NMR (300 MHz, CDCl3): d 7.67 (d, 2H, J = 8.4 Hz, Ar), 7.48 (d, 2H,
J = 8.7 Hz, Ar), 7.16 (d, 2H, J = 8.7 Hz, Ar), 6.92 (d, 2H, J = 8.4 Hz, Ar),
6.73 (s, 1H, pyrazole C4-H), 3.86 (s, 3H, OCH3); 13C NMR (75.5 MHz,
2
CDCl3): d 145.0, 144.5 (q, JCF = 38.2 Hz, C-3), 142.6, 133.0, 130.2,
1
125.4, 120.9 (q, JCF = 269.5 Hz, C3-CF3), 117.9, 114.5, 111.8,

4588
N. Chandna et al. / Bioorg. Med. Chem. 21 (2013) 4581–4590
106.3, 55.4 (OCH3); 19F NMR (CDCl3, 282.4 MHz): d ꢀ62.5 (C3-CF3);
m/z 344 ([M+H]+, C18H12F3N3OH+ calcd. 344).
4.3.2. 4-[5-(4-Methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-
yl]benzenecarbothioamide (3b)
Mp 142–144 °C, yield 84%; IR (KBr) cmꢀ1: 3418 (SH stretch),
3148 (NH stretch); 1H NMR (300 MHz, DMSO-d6): d 9.99 (s, ex,
1H, NH/SH), 9.61 (s, ex, 1H, NH/SH), 7.94 (d, 2H, J = 8.4 Hz, Ar),
7.38 (d, 2H, J = 8.4 Hz, Ar), 7.16–7.20 (m, 5H, Ar, pyrazole C4-H),
2.31 (s, 3H, CH3); 13C NMR (75.5 MHz, DMSO-d6): d 199.0, 145.6,
4.2.7. 4-[5-(Naphthalen-2-yl)-3-(trifluoromethyl)-1H-pyrazol-1-
yl]benzonitrile (2g)
Mp 128–130 °C, yield 90%; IR (KBr) cmꢀ1
: 2230 (C„N
stretch); 1H NMR (300 MHz, CDCl3): d 7.82–7.91(m, 4H, Ar),
7.65 (d, 2H, J = 8.7 Hz, Ar), 7.56–7.62 (m, 2H, Ar), 7.51 (d, 2H,
J = 8.7 Hz, Ar), 7.22 (dd, 1H, J = 8.7, 1.2 Hz, Ar), 6.91(s, 1H, pyra-
2
142.4 (q, JCF = 37.8 Hz, C-3), 141.7, 141.2, 139.6, 129.8, 129.1,
1
128.7, 125.9, 125.4, 121.8 (q, JCF = 268.8 Hz, C3-CF3), 106.4, 21.3
zole C4-H); 13C NMR (75.5 MHz, CDCl3): d 144.5 (q, JCF = 38.5 Hz,
(CH3); 19F NMR (DMSO-d6, 282.4 MHz): d ꢀ60.8 (C3-CF3); m/z
2
C-3), 142.5, 133.3, 133.1, 133.0, 128.9, 128.6, 128.2, 127.9, 127.6,
127.2, 127.9, 127.6, 127.2, 125.9, 125.6, 125.4, 120.9 (q,
1JCF = 269.5 Hz, C3-CF3), 117.9, 111.9, 107.1; 19F NMR (CDCl3,
282.4 MHz): d ꢀ62.5 (C3-CF3); m/z 364 ([M+H]+, C21H12F3N3H+
calcd. 364).
362 ([M+H]+, C18H14F3N3SH+ calcd. 362).
4.3.3. 4-[5-(4-Fluorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-
yl]benzenecarbothioamide (3c)
Mp 115–117 °C, yield 78%; IR (KBr) cmꢀ1: 3417 (SH stretch),
3133 (NH stretch); 1H NMR (300 MHz, DMSO-d6): d 10.0 (s, ex,
1H, NH/SH), 9.52 (s, ex, 1H, SH/NH), 7.89 (d, 2H, J = 8.7 Hz, Ar),
7.37 (d, 2H, J = 8.7 Hz, Ar), 7.22–7.28 (m, 2H, Ar), 7.06–7.12 (m,
2H, Ar), 6.77 (s, 1H, pyrazole C4-H); 13C NMR (75.5 MHz, CDCl3):
4.2.8. 4-[5-(Thiophen-2-yl)-3-(trifluoromethyl)-1H-pyrazol-1-
yl]benzonitrile (2h)
Mp 114–116 °C, yield 93%; IR (KBr) cmꢀ1: 2230 (C„N stretch);
1H NMR (300 MHz, CDCl3): d 7.73 (d, 2H, J = 8.7 Hz, Ar), 7.56 (d, 2H,
J = 8.7 Hz, Ar), 7.45 (dd, 1H, J = 0.9 Hz, 5.1 Hz, Ar), 7.06 (dd, 1H,
J = 3.9 Hz, 5.1 Hz, Ar), 6.95 (dd, 1H, J = 0.9 Hz, 3.9 Hz, Ar), 6.84 (s,
1H, pyrazole C4-H); 13C NMR (75.5 MHz, CDCl3): d 144.5 (q,
2JCF = 38.2 Hz, C-3), 143.5, 142.2, 138.6, 133.1, 128.9, 128.7, 128.4,
1
2
d 201.0, 163.2 (d, JCF = 251.4 Hz), 143.9 (q, JCF = 38.2 Hz, C-3),
3
141.6, 138.7, 130.8 (d, JCF = 8.3 Hz), 127.9, 125.0, 124.9, 124.8,
1
2
123.8 (q, JCF = 269.5 Hz, C3-CF3), 116.2 (d, JCF = 21.9 Hz), 106.3;
19F NMR (DMSO-d6, 282.4 MHz): d ꢀ60.8 (C3-CF3); m/z 366
([M+H]+, C17H11F4N3SH+ calcd. 366).
1
127.9, 126.1, 120.9 (q, JCF = 269.5 Hz, C3-CF3), 117.8, 112.6,
107.2; 19F NMR (CDCl3, 282.4 MHz): d ꢀ62.5 (C3-CF3); m/z 320
4.3.4. 4-[5-(4-Chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-
yl]benzenecarbothioamide (3d)
([M+H]+, C15H8F3N3SH+ calcd. 320).
Mp 144–146 °C, yield 85%; IR (KBr) cmꢀ1: 3410 (SH stretch),
3140 (NH stretch); 1H NMR (300 MHz, DMSO-d6): d 10.01 (s, ex,
1H, NH/SH), 9.62 (s, ex, 1H, SH/NH), 7.95 (d, 2H, J = 8.7 Hz, Ar),
7.50 (d, 2H, J = 8.7 Hz, Ar), 7.33–7.41 (m, 4H, Ar), 7.27 (s, 1H, pyra-
zole C4-H); 13C NMR (75.5 MHz, DMSO-d6): d 199.0, 144.3, 142.5 (q,
2JCF = 37.8 Hz, C-3), 140.9, 139.8, 134.7, 131.2, 129.4, 128.8, 127.7,
4.2.9. 4-[5-(Furan-2-yl)-3-(trifluoromethyl)-1H-pyrazol-1-
yl]benzonitrile (2i)
Mp 108–110 °C, yield 92%; IR (KBr) cmꢀ1: 2230 (C„N stretch);
1H NMR (300 MHz, CDCl3): d 7.78 (d, 2H, J = 8.7 Hz, Ar), 7.56 (d, 2H,
J = 8.7 Hz, Ar), 7.47 (d, 1H, J = 1.8 Hz, Ar), 6.92 (s, 1H, pyrazole C4-H)
6.48 (dd, 1H, J = 3.6 Hz, 1.8 Hz, Ar), 6.35 (d, 1H, J = 3.6 Hz Ar),; 13C
1
125.4, 121.7 (q, JCF = 268.8 Hz, C3-CF3), 106.9; 19F NMR: (DMSO-
2
NMR (75.5 MHz, CDCl3): d 157.5, 144.5 (q, JCF = 38.2 Hz, C-3),
d6, 282.4 MHz):
d
ꢀ60.8 (C3-CF3); m/z 382 ([M+H]+,
143.5, 142.2, 138.6, 133.1, 128.9, 128.7, 128.4, 127.9, 126.1, 120.9
C
17H11ClF3N3SH+ calcd. 382).
1
(q, JCF = 269.5 Hz, C3-CF3), 117.8, 112.6, 107.2; 19F NMR (CDCl3,
282.4 MHz): d ꢀ62.5 (C3-CF3); m/z 304 ([M+H]+, C15H8F3N3OH+
4.3.5. 4-[5-(4-Bromophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-
yl]benzenecarbothioamide (3e)
calcd. 304).
Mp 154–156 °C, yield 85%; IR (KBr) cmꢀ1: 3342 (SH strech),
3168 (NH stretch); 1H NMR (300 MHz, DMSO-d6): d 10.01 (s, ex,
1H, NH/SH), 9.62 (s, ex, 1H, SH/NH), 7.95 (d, 2H, J = 8.7 Hz, Ar),
7.62 (d, 2H, J = 8.7 Hz, Ar), 7.40 (d, 2H, J = 8.7 Hz, Ar), 7.28 (d, 2H,
J = 8.7 Hz, Ar), 7.26 (s, 1H, pyrazole C4-H); 13C NMR (75.5 MHz,
4.3. Representative procedure for the synthesis of target
pyrazoles (3a–3i)
Added 1 mmol of triethylamine to a solution of 2 (0.3 g,
2 mmol) in ethanol and chloroform (1:1, 10 mL). Dry H2S gas in a
steady stream was passed through the solution for 7–8 h. The pro-
gress of the reaction was monitored with the help of TLC and IR.
Basic condition (pH 8–9) of reaction was maintained with the help
of triethylamine which was checked repeatedly via pH paper. On
completion of reaction, solvents were evaporated completely to
obtain a semisolid followed by addition of ice cold water (15 mL)
to it. Hazy reaction mixture thus obtained was neutralized by aq
acetic acid resulting in the formation of a crude solid product
which was filtered, dried and crystallized from ethanol to get pure
3a–3i (75–85%).
2
DMSO-d6): d 199.0, 145.6, 142.5 (q, JCF = 37.8 Hz, C-3), 141.7,
141.2, 139.6, 129.8, 129.1, 128.7, 125.7, 125.4, 121.8 (q,
1JCF = 268.8 Hz, C3-CF3), 106.4; 19F NMR (DMSO-d6, 282.4 MHz): d
ꢀ60.8 (C3-CF3); m/z 428 ([M+H]+, C17H11BrF3N3SH+ calcd. 428).
4.3.6. 4-[5-(4-Methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-
1-yl] benzenecarbothioamide (3f)
Mp 170–172 °C, yield 79%; IR (KBr) cmꢀ1: 3418 (SH strech),
3194 (NH stretch); 1H NMR (300 MHz, DMSO-d6): d 10.00 (s, ex,
1H, NH/SH), 9.61 (s, ex, 1H, NH /SH), 7.94 (d, 2H, J = 8.4 Hz, Ar),
7.39 (d, 2H, J = 8.4 Hz, Ar), 7.25 (d, 2H, J = 8.4 Hz, Ar), 7.14 (s, 1H,
pyrazole C4-H), 6.97 (d, 2H, J = 8.7 Hz, Ar), 3.77 (s, 3H, OCH3); 13C
NMR (75.5 MHz, DMSO-d6): d 199.0, 166.4, 145.5, 142.4 (q,
2JCF = 37.8 Hz, C-3), 141.3, 139.5, 130.8, 128.7, 125.4, 123.6, 121.8
4.3.1. 4-[5-Phenyl-3-(trifluoromethyl)-1H-pyrazol-1-
yl]benzenecarbothioamide (3a)
1
Mp 122–124 °C, yield 76%; IR (KBr) cmꢀ1: 3371 (SH stretch),
3163 (NH stretch); 1H NMR (300 MHz, DMSO-d6): d 10.00 (s, ex,
1H, NH/SH), 9.62 (s, ex, 1H, NH/SH), 7.94 (d, 2H, J = 8.4 Hz, Ar),
7.32–7.43 (m, 7H, Ar),7.23 (s, 1H, pyrazole C4-H); 13C NMR
(q, JCF = 268.8 Hz, C3-CF3), 121.0, 114.8, 106.1, 55.7(OCH3); 19F
NMR (DMSO-d6, 282.4 MHz): d - 60.8 (C3-CF3); m/z 378 ([M+H]+,
C
18H14F3N3OSH+ calcd. 378).
2
(75.5 MHz, DMSO-d6): d 199.0, 145.5, 142.5 (q, JCF = 37.8 Hz, C-
4.3.7. 4-[5-(Naphthalen-2-yl)-3-(trifluoromethyl)-1H-pyrazol-1-
yl]benzenecarbothioamide (3g)
3), 141.2, 139.7, 129.8, 129.4, 129.3, 128.9, 128.7, 125.4, 121.8 (q,
1JCF = 268.8 Hz, C3-CF3), 106.7; 19F NMR (DMSO-d6, 282.4 MHz): d
ꢀ60.8 (C3-CF3); m/z 348 ([M+H]+, C17H12F3N3SH+ calcd. 348).
Mp 182–184 °C, yield 81%; IR (KBr) cmꢀ1: 3348 (SH strech),
3140 (NH stretch); 1H NMR (300 MHz, DMSO-d6): d 9.98 (s, ex,

N. Chandna et al. / Bioorg. Med. Chem. 21 (2013) 4581–4590
4589
1H, NH/SH), 9.59 (s, ex, 1H, SH/NH), 8.04 (s, 1H, Ar), 7.89–7.94 (m,
5.2. In vitro COX-1 and COX-2 inhibition assay
5H, Ar), 7.55–7.59 (m, 2H, Ar), 7.43 (d, 2H, J = 8.4 Hz, Ar), 7.29–
7.35(m, 2H, Ar, pyrazole C4-H); 13C NMR (75.5 MHz, DMSO-d6): d
199.1, 145.5, 142.6 (q, JCF = 37.8 Hz, C-3), 142.3, 141.2, 139.7,
COX-1 and COX-2 inhibitory activity was determined using COX
(ovine) inhibitor screening assay EIA kit according to manufac-
turer’s instructions33 and the same kit to determine the COX-1
and COX-2 inhibitory activity has been used previously by several
research groups.29,34–37 COX catalyzes the first step in the biosyn-
2
133.2, 132.9, 129.1, 128.8, 128.7, 128.1, 127.8, 127.4, 126.3,
1
125.3, 121.8 (q, JCF = 268.8 Hz, C3-CF3), 107.1; 19F NMR (DMSO-
d6, 282.4 MHz): d ꢀ60.8 (C3-CF3); m/z 398 ([M+H]+, C21H14F3N3SH+
calcd. 398).
thesis of AA to PGH2. The PGF2a produced from PGH2 by reduction
with stannous chloride is measured by EIA. The compounds were
4.3.8. 4-[5-(Thiophen-2-yl)-3-(trifluoromethyl)-1H-pyrazol-1-
yl]benzenecarbothioamide (3h)
dissolved in dimethylsulfoxide (DMSO). The enzyme COX-1 and
COX-2 (10
the supplied reaction buffer solution {950
containing 5 mM ethylenediamine tetraacetate (EDTA) and
lL), heme (10
l
L) and samples (20
lL) were added to
Mp 130–132 °C, yield 85%; IR (KBr) cmꢀ1: 3302 (SH strech), 3155
(NH stretch); 1H NMR (300 MHz, DMSO-d6): d 10.05 (s, ex, 1H, NH/
SH), 9.68 (s, ex, 1H, SH/NH), 8.01 (d, 2H, J = 8.4 Hz, Ar), 7.66–7.68 (m,
1H, Ar), 7.53 (d, 2H, J = 8.4 Hz, Ar), 7.32 (s, 1H, pyrazole C4-H), 7.21–
7.22 (m, 1H, Ar), 7.09–7.12 (m, 1H, Ar); 13C NMR (75.5 MHz, DMSO-
d6): d 199.1, 142.4 (q, 2JCF = 37.8 Hz, C-3), 140.8, 140.6, 139.7, 129.8,
l
L, 0.1 M Tris–HCl, pH
8
2 mM phenol). The mixture of these solutions were incubated for
a period of 10 min at 37 °C, after that COX reactions were initiated
by adding AA (10
The COX reactions were stopped by addition of HCl (1 M, 50
after 2 min and then saturated stannous chloride (100 L) was
added and again incubated for 5 min at room temperature. The
PGF2 formed in the samples by COX reactions was quantified by
l
L, making final concentration 100
l
M) solution.
lL)
1
129.6, 128.8, 128.3, 126.9, 126.4, 121.7 (q, JCF = 268.8 Hz, C3-CF3),
l
106.1; 19F NMR (DMSO-d6, 282.4 MHz): d ꢀ60.9 (C3-CF3); m/z 354
([M+H]+, C15H10F3N3S2H+ calcd. 354).
a
EIA. The pre-coated 96-well plate was incubated with samples
for 18 h at room temperature. After incubation, the plate was
washed to remove any unbound reagent and then Ellman’s reagent
4.3.9. 4-[5-(Furan-2-yl)-3-(trifluoromethyl)-1H-pyrazol-1-
yl]benzenecarbothioamide (3i)
Mp 118–120 °C, yield 83%; IR (KBr) cmꢀ1: 3302 (SH strech),
3140 (NH stretch); 1H NMR (300 MHz, DMSO-d6) d: 10.05 (s, ex,
1H, NH/SH), 9.68 (s, ex, 1H, SH/NH), 8.03 (d, 2H, J = 8.4 Hz, Ar),
7.78 (d, 1H, J = 1.5 Hz, Ar), 7.54 (d, 2H, J = 8.4 Hz, Ar), 7.31 (s, 1H,
pyrazole C4-H), 6.59 (dd, 1H, J = 3.3 Hz, 1.5 Hz, Ar), 6.45 (d, 1H,
J = 3.3 Hz, Ar); 13C NMR (75.5 MHz, DMSO-d6): d 199.2, 145.1,
(200 lL), which contains substrate to acetyl cholinesterase, was
added and incubated for 60–90 min (until the absorbance of Bo
well is in the range 0.3–0.8 A.U.) at room temperature. The plate
was then read by an ELISA plate reader at 410 nm.
The percentage of inhibition of COX-1 and COX-2 was calcu-
lated by the comparison of the sample treated incubations to con-
trol incubations. Indomethacin and celecoxib were used as
reference standards in the study.
2
142.4 (q, JCF = 37.8 Hz, C-3), 141.2, 140.5, 136.5, 128.8, 125.6,
1
128.8, 125.6, 121.7 (q, JCF = 268.8 Hz, C3-CF3), 112.4, 105.2; 19F
NMR (DMSO-d6, 282.4 MHz): d ꢀ60.9 (C3-CF3); m/z 338 ([M+H]+,
C
15H10F3N3OSH+ calcd. 338).
6. Docking methodology
5. Pharmacological assay
Docking study was carried out for the target compounds using
Molegro virtual docker version 2010. MolDock scoring function is
used by MVD program is defined by:
5.1. Carrageenan-induced rat paw edema assay
Escore ¼ Einter þ Eintra
where, Escore ¼ MolDock score:
Einter ¼ ligand ꢀ Protein interaction
Male Wistar albino rats weighing 200–250 g were used
throughout the study. They were kept in the animal house under
standard conditions of light and temperature with free access to
food and water. Food was withdrawn 12 h before and during
experimental hours. The animals were randomly divided into
groups each consisting of six rats. One group of six rats was kept
as control and received tween 80 (95:5) while another group re-
ceived the standard drug indomethacin ip. Other groups of rats
were administered the test compounds at a dose of 50 mg/kg body
weight orally. A mark was made on the left hind paw just beyond
the tibiotarsal articulation, so that every time the paw was dipped
up to the fixed mark and constant paw volume was ensured. Paw
volumes were measured using a plethysmometer (model 7140,
Ugo Basile, Italy). Thirty minutes after administration of test com-
pounds and standard drug, 0.1 mL of 1% w/v of carrageenan sus-
pension in normal saline was injected into sub planter region of
the left hind paw of all the animals. The initial paw volume was
measured within 30 s of the injection and remeasured again 1, 2,
3, and 4 h after administration of carrageenan. The edema was ex-
pressed as an increase in the volume of paw and the percentage of
edema inhibition for each rat and each group was obtained as
follows:
Eintra ¼ internal energy of the ligand:
The molecules/ligands were built using Chemdraw 8.0. The 2D
structure was then converted into 3D which was saved as MDL
MolFile (⁄.mol2). Crystal structure of both COX-2 and COX-1(PDB
codes: 1CX2 and 1EQG, respectively) was obtained from the Pro-
tein Data Bank in order to prepare both proteins for docking stud-
ies. Compounds were docked into the active sites using Molegro
Virtual Docker 2010.4.1 software using the standard protocol.30
Acknowledgements
The authors are thankful to Sophisticated Analytical Instrument
Facility, Central Drug Research Institute, Lucknow and IIT Delhi for
Mass spectra and F-19 NMR, respectively. One of the authors Nisha
Chandna is thankful to the Council of Scientific and Industrial Re-
search (CSIR), New Delhi, India for the award of Senior Research
Fellowship.
% inhibition ¼ ðVt ꢀ V0Þ control ꢀ ðVt
ꢀ V0Þ tested compound=ðVt ꢀ V0Þ control ꢁ 100
A. Supplementary data
Where Vt = volume of edema at specific time interval and V0 = vol-
ume of edema at zero time interval.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.05.029.

4590
N. Chandna et al. / Bioorg. Med. Chem. 21 (2013) 4581–4590
18. Sharma, P. K.; Chandna, N.; Kumar, S.; Kumar, P.; Kumar, S.; Kaushik, P.;
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