Synthesis, biological evaluation and molecular modeling study of pyrazole derivatives as selective COX-2 inhibitors and anti-inflammatory agents

Article abstract of DOI:10.1016/j.bioorg.2014.05.004

A novel series of pyrazole derivatives were synthesized and evaluated in vivo for their anti-inflammatory activity in carrageenan-induced rat paw edema model. Among all compounds, 5a, and 5b showed comparable anti-inflammatory activity to Nimesulide, the

Full text of DOI:10.1016/j.bioorg.2014.05.004

Bioorganic Chemistry 56 (2014) 8–15
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, biological evaluation and molecular modeling study
of pyrazole derivatives as selective COX-2 inhibitors
and anti-inflammatory agents
a
a
a
b
Ashish Kumar Tewari a, , Ved Prakash Singh , Pratima Yadav , Garima Gupta , Amit Singh ,
⇑
Raj Kumar Goel b, Pravin Shinde c, C. Gopi Mohan c
a Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi 221 005, India
b Department of Pharmacology, IMS, Banaras Hindu University, Varanasi 221 005, India
c Amrita Centre for Nanosciences and Molecular Medicine (ACNSMM), Amrita Institute of Medical Sciences & Research Centre, Ponekkara, Kochi 682 041, Kerala State, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 January 2014
Available online 16 May 2014
A novel series of pyrazole derivatives were synthesized and evaluated in vivo for their anti-inflammatory
activity in carrageenan-induced rat paw edema model. Among all compounds, 5a, and 5b showed com-
parable anti-inflammatory activity to Nimesulide, the standard drug taken for the studies. In silico (dock-
ing) studies were carried out to investigate the theoretical binding mode of the compounds to target the
cyclooxygenase (COX-2) using Autodock 4.2.
Keywords:
Pyrazole
COX-2 inhibitors
Inflammation
Drug design
Ó 2014 Elsevier Inc. All rights reserved.
Molecular modeling
1. Introduction
[6,7]. In contrast, the COX-2 isozyme is induced by stimuli such
as mitogenes, oncogenes, growth factors, hormones and disorders
Inflammation is a multifactorial process which reflects the
response of the organism to various stimuli and is related to
many disorders such as arthritis, asthma, and psoriasis, etc [1].
Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the
most widely used therapeutics. Through their anti-inflammatory,
anti-pyretic and analgesic activities, they represent a choice of
treatment in various inflammatory diseases such as arthritis, rheu-
matisms as well as to relieve the pains of everyday life [2]. The
pharmacological effect of this class of drugs are due to inhibition
of Cyclooxegenase enzyme [3]. Cyclooxygenase is the key enzyme
in the biosynthesis of prostanoids, biologically active substances
that are involved in several physiological processes and also in
pathological conditions, such as inflammation [4]. There exist
mainly two form of enzyme COX-1, COX-2, and recently, COX-3
as well as two smaller COX-I derived proteins (partial COX-1 or
PCOX-I proteins) are also discovered [5]. Cox-1 is constitutively
expressed in the Gastrointestinal tract, kidneys, and platelets and
is involved in the regulation of physiological functions in maintain-
ing vascular homeostasis, normal renal function, and gastric muco-
sal integrity, and the autocrine response to circulating hormones
of water-electrolyte homeostasis linking its involvement to patho-
logical processes such as inflammation and various cancer types
[8–10]. Long term use of classical NSAIDs cause side-effects such
as gastrointestinal ulcer, renal dysfunction, and hepatotoxicity
(Nimesulide and more recently, lumiracoxib). Side-effects are due
to high COX-1 versus COX-2 selectivity [11–14]. Therefore, during
the last two decades medicinal chemist, develops a new class of
drug by taking the advantage of fact that COX-2 active site is 20%
larger than COX-1 active site. These molecules, termed as coxibs
(i.e., Celecoxib, Rofecoxib, Valdecoxib, and Etoricoxib) showed
reduced gastrointestinal side effects as compared to traditional
NSAIDs [15,16]. Unfortunately, some selective COX-2 inhibitory
drugs that include Rofecoxib, Valdecoxib and Celecoxib alter the
natural balance in the COX biochemical pathway, this natural
imbalance in the COX pathway are believed to be responsible for
increased incidences of high blood pressure and myocardial infarc-
tion in long term use and high dosage, that ultimately prompted
the withdrawal of these drugs from the market [17,18].
This has led intense efforts in search for potent and selective
COX-2 inhibitors which could provide anti-inflammatory drugs
with fewer side-effects. Therefore, medicinal chemists are work-
ing on various scaffolds for development of NSAIDs with
improved safety profile [19–22]. Many pyrazole derivatives are
⇑
Corresponding author.
E-mail address: tashish2002@yahoo.com (A.K. Tewari).
http://dx.doi.org/10.1016/j.bioorg.2014.05.004
0045-2068/Ó 2014 Elsevier Inc. All rights reserved.

A.K. Tewari et al. / Bioorganic Chemistry 56 (2014) 8–15
9
Fig. 1. Chemical structures of anti-inflammatory agents (Pyrazole derivative).
acknowledged to possess a wide range of bioactivities. The pyra-
zole motif makes up the core structure of numerous biologically
active compounds. Some representatives of this heterocycle
exhibit anti-viral/anti-tumor [23–25], antibacterial [26–29],
antiinflammatory [30–33], analgesic [34], fungistatic [35], and
anti-hyperglycemic activity [36,37].
this design was assessed through preliminary in vitro, in vivo anti-
inflammatory screening, and docking simulation in our attempt to
accentuate anti-inflammatory activity of compounds.
2. Results and discussion
The well known anti-inflammatory drug, Celecoxib, is pyrazole
derivative, some other examples of pyrazole derivative as NSAIDs
are Mefobutazone, Ramifenazone, Famprofazone. (Fig. 1) Uses of
few pyrazole derivatives are limited, as it is associated with serious
side-effects such as bone marrow depression, water and salt reten-
tion and carcinogenesis [38]. This gave a great impetus to speculate
for a new pyrazole derivative as anti-inflamatory agent with more
potent activity and less toxicity.
Encouraged with the above literature and as a continuation of
our research interest in the synthesis and biological activities of
novel pyrazole derivatives [39–42]. The present study aimed at
synthesis of the novel pyrazole derivative as they are the pharma-
cophore of various anti-inflammatory drugs [30,38]. The validity of
2.1. Chemistry
The compounds were synthesized as shown in Scheme 1 via a
general nucleophilic substitution, condensation and reduction
reactions. The starting compound 1 was prepared according to pre-
viously reported procedure [39]. Compound 3 was prepared in two
step procedure starting from 1. In the first step, the compound 1
was treated with excess of 1, 2 dibromoethane (5 equivalent) in
presence of K2CO3 in DMF to afforded the compound 2 in good
yield. In subsequent step compound 2 was treated with p-hydroxy-
benzaldehyde (1 equivalent) in presence of K2CO3 in DMF, leading
to formation of compound 3 in moderate yield. The intermediate 3
Scheme 1.

10
A.K. Tewari et al. / Bioorganic Chemistry 56 (2014) 8–15
Table 1
smoothly reacted with various substituted aniline, hydroxylamine
hydrogensulfate and hydrazine hydrate to provide corresponding
Schiff base 5a, 5b, 5c oxime 6 and hydrazone 7 in good yield. In
case of Schiff base there should be two products syn and anti,
but this isomerism is present only in case of 5a. Two isomer were
separated by crystallization and differentiated on basis of their
melting point and kmax absorption [43]. Melting point of syn and
anti isomer are 88, 96 °C respectively, whereas kmax of syn and anti
are 320, 331 respectively. Syn isomer is the major product whereas
anti isomer is the minor product. But in case of 5b, 5c there exist
only syn isomer, this may be due to bulkier size of substituent.
Whereas aromatic hydrazone and oxime exist only in syn isomer
because aromatic aldehydes always give syn isomer in these type
of reactions. Compound 8 was prepared by the reaction of phen-
ylpyrazolone 1 with carbon disulfide followed by methylation. Fur-
ther base catalyzed alcholysis of 8 followed by acidification gave
pyrazole-ester 9 in good yield [44–46]. The reaction of 9 with the
para methylbenzene-1-sulfonyl chloride resulted the formation
of compound 10 in moderate yield. The newly synthesized com-
pounds 1-10 were established on the basis of spectroscopic data.
1H NMR spectra of compounds were studied in CDCl3 showed char-
acteristic peak at d 5.5 ppm correspond to CH proton of pyrazole,
peak at d 2.2 ppm correspond to methyl proton of pyrazole ring.
The characteristic peaks observed at d 4.3–4.4 ppm correspond to
OCH2 of linker. 13C NMR spectrum of compounds show signals at
14.5 ppm assigned for the methyl carbon of pyrazole ring, while
signal at 66.1 and 70.0 correspond to linker carbon. The mass spec-
tra of all compounds showed molecular ion peak corresponding to
their molecular formula.
Percentage edema growth relative to control at different time intervals
(mean S.E.M.).
Group
0 min
30 min
90 min
Control
Nimesulide
3
4
5a
5b
5c
6
7
10
100
100
100
100
100
100
100
100
100
100
0
0
0
0
0
0
0
0
0
0
130.1 6.54 (30.1)
115.1 2.88 (15.1)
115.1 2.88 (15.1)
111.5 2.89 (11.5)
110.1 5.14 (10.1)
114.1 2.88 (14.1)
109.5 2.68 (9.5)
114.8 4.03 (14.8)
115.8 5.19 (15.8)
122.1 5.11 (22.1)
138.7 4.47 (38.7)
123.4 3.27* (23.4)
125.8 8.42* (25.8)
119.9 3.30*(19.9)
113.4 7.77* (13.4)
119.4 3.17* (19.4)
123.8 7.12* (23.8)
124.4 6.78* (24.4)
128.4 4.78* (28.4)
132.6 3.13* (32.6)
Table 2
Paw edema at different time interval (ml/Rat) (mean S.E.M.).
Group
0 min
30 min
90 min
Control
Nimsulide
3
4
5a
5b
5c
6
7
10
0.99 0.067
1.02 0.054
0.78 0.033
1.16 0.054
0.98 0.031
1.22 0.061
1.06 0.041
1.08 0.038
1.06 0.044
0.98 0.053
1.27 0.043**
1.16 0.065
0.89 0.041
1.29 0.061
1.06 0.038
1.35 0.056
1.20 0.048
1.25 0.054
1.21 0.058
1.19 0.058
1.36 0.070
1.26 0.038
0.98 0.044
1.39 0.068
1.11 0.045
1.46 0.068
1.31 0.057
1.38 0.058
1.31 0.061
1.29 0.061
2.2. Biological studies
2.2.1. In vivo anti-inflammatory studies
All the newly synthesized compounds and Nimesulide, as a ref-
erence drug, were subjected to in vivo anti-inflammatory studies
using the well-known rat carrageenan-induced foot paw edema
model [47]. In carrageenan stimulated peripheral inflammation
model, edema formation as inflammatory response is developed
in multiphase mechanisms as reported, thus, (i) proinflammatory
cytokines such as IL-6, IL-1b, and TNF-a are released by the periph-
eral stimulus, (ii) COX-2 mRNA and COX-2 protein are inducted
and/or upregulated, and (iii) COX-2-dependent induction/upregu-
lation of PGE2 and PGI2 causes increases of vasodilation, vascular
permeability, extravasation of plasma proteins, and leakage to
waterwithin the site to form PGG2, and then (iii) the hydroperoxide
group of PGG2 is reduced (PGG2 is subjected to the reduction of
two electrons) to form PGH2 at peroxidase site where haem
(ironeporphyrin complex) is bound and histidine groups are linked
[48–50]. The results of anti-inflammatory activities against carra-
geenan-induced rat paw edema model are shown in Tables 1 and
2. Compound 5b, 5c & 4 show maximum inhibitory effect, while
compound 6, 7 & 10 showed intermediate effect. The result of pres-
ent study had shown that compounds 5a and 5b possess maximum
inhibitory effect when compared to control group. It has been
observed that maximum percentage of paw edema growth in con-
trol group after 90 minutes was 38.7% which was found to decrease
up to 13.4, 19.4% in the group of rats treated with 5a, 5b respec-
tively, compared to Nimesulide treated group where it was found
23.4%, also the values were found statistically very significant.
Compound 4 and 5c found to possess good anti-inflammatory
property as the percentage paw edema growth was shown to be
only 19.9, 23.8% when compared to that of control group. Com-
pound 6 show moderate effect, while compound 3, 7 & 10 showed
almost negligible effects (Fig. 2). Therefore, the results showed that
5a and 5b are potent inhibitor of inflammation.
Fig. 2. Effect of different drugs on carrageenan induced rat paw edema.
2.2.2. COX assays
All the synthesized compounds were evaluated for their
anti-inflammatory activity by biochemical COX (COX-1 & COX-2)
inhibitory assay. The ratio of IC50 of COX-2 to IC50 of COX-1
(COX-2/COX-1) would suggest the selectivity of the compound
(Table 3).
2.3. Molecular docking
Molecular docking is a frequently used tool in computer-aided
structure-based rational drug design. It evaluates how small mole-
cule (substrate, inhibitor, drug or drug candidate) and the target
macromolecule (receptor, enzyme or nucleic acid) fit together. This
can be useful for developing better drug candidates and also for
the understanding the nature of the binding. Insights into the

A.K. Tewari et al. / Bioorganic Chemistry 56 (2014) 8–15
11
Table 3
respect to the H-bond network in the COX-2 binding site. Access
of ligands to the secondary pocket of COX-2 is controlled by
histidine (His90), glutamine (Gln192), and tyrosine (Tyr355) [53].
Interaction of Arg513 with the bound drug is a requirement for
time dependent inhibition of COX-2 [54].
Docking (in silico) studies were in good agreement with
pharmacological results. Compounds 4, 5a, 5c and 6 were the most
promising compounds by computational studies, which is almost
consistent with biological evaluation assays. Due to the increased
flexibility introduced by the linker group the pyrazole derivatives
show a potential higher affinity to COX-2. The results of the dock-
ing studies, docking scores, and involved amino acids interacted
ligand moieties and hydrogen bond length for each compound
and the reference native ligand are listed in Table 2, Figs. 3 and 4.
Cox-2/Cox-1 ratio of pyrazole derivatives.
Compound
Cox-1
Cox-2
IC50 (l
M)a
SIb
IC50
(
l
M)a
Celecoxib
3
4
5a
5b
5c
6
7
10
15
0.04
18.5
0.0028
0.78
0.53
0.5100
0.4400
0.5400
0.5300
5.2100
1.53
24.0
28.3
32.9
32.5
28.5
28.8
3.39
20.1
15.0
16.8
14.3
15.4
15.3
17.4
13.1
differences between the binding sites of COX-1and COX-2 obtained
from X-ray crystal structure data provided useful guidelines that
facilitated the design of the selective COX-2 inhibitors. For exam-
ple, the COX-2 binding site possesses an additional secondary
pocket, that is, absent in COX-1, which is highly relevant to the
design of selective COX-2 inhibitors. This COX-2 20 pocket arises
due to a conformational change at Tyr355, that is, attributed to
the presence of Ile523 in COX-1 relative to Val523 having a smaller
side chain in COX-2 [51,52]. It has also been reported that replace-
ment of His513 in COX-1 by Arg513 in COX-2 plays a key role with
(i) Compound 4 fits in the active site of COX-2 and form one
hydrogen bonds between H of the CH2OH group and Gln
356 with distance 2.16 Å which is consistent with the
moderate anti-inflammatory activity.
(ii) Docking study for the compound 5b indicates that the
phenyl ring of pyrazole, exhibit pi-pi interaction with
amino acids Phe367, Phe191, Trp373which is consistent
with the good anti-inflammatory activity.
Fig. 3. Interaction diagrams for compounds 4 and 5b active site of COX-2 Green lines indicate H-bonds formed between the compound and the enzyme active site residues
yellow cylinder represent interaction.
p–p
Fig. 4. Interaction diagrams for compounds 6 and 10 in active site of COX-2 Green lines indicate H-bonds, yellow cylinder represent
p–p interaction, and yellow cone
represent cation–p interaction.
Table 4
Dock scores and summary of molecular interactions of compounds after docking into COX-2 active site.
S. No.
Dock score
Type of interactions
Amino acid interactions
Interacting groups and distance
4
À6.66
À6.66
À8.93
À5.33
À5.66
À6.67
H bond
–
Gln 356, Ser 107, Lys 158, Ile 110, Tyr 108
Gly121, Tyr120, His119
Phe367, Phe191, Trp373
Arg 293, Gly245, Cys555, Asn557
Cys 44 Cys 42
Val 335, Tyr371, Gly542, Phe367, Leu338, Phe 504
ACH2OH (2.62 Å)
5a
5b
6
7
10
p
–p
H bond, cation–
–
2H bond,
p
ANH (2.28 Å)
p
–p
N & O of pyrazole ring (2.07 Å, 1.93 Å)

12
A.K. Tewari et al. / Bioorganic Chemistry 56 (2014) 8–15
(iii) The binding mode of 6 into the active site of COX-2 enzyme
TLC), solvent was evaporated under reduced pressure and the res-
idue was dissolved in chloroform. The organic layer was washed
with water and brine, dried over anhydrous Na2SO4 and concen-
trated. The residue was purified by silica gel chromatography with
chloroform/ethyl acetate as eluent to afford 2 as yellow powder.
Yellow powder (3.00 g, 79%); Rf = 0.38 (Hexane/EtOAc, 9:1); mp.
48–50 °C; 1H NMR (300 MHz, CDCl3, d ppm); 2.27 (3H, s, CH3), 3.13
(2H, t, CH2, J = 6.0 Hz,), 4.37 (2H, t, OCH2, J = 6.3 Hz,), 5.50 (1H, CH
pyrazole) 7.23–7.70 (5H, m, Ar H). MS (m/z): 281 (M++1). Anal.
Calcd for C12H13BrN2O (280.02): C, 51.26; H, 4.66; N, 9.96. Found:
C, 51.62; H, 4.52; N, 9.61.
revealed one hydrogen bonds with Gly 245 and show strong
cation–pi interaction with Arg 293. Hydrogen bond and
cation–pi interaction are responsible for moderate activity
of compound.
(iv) Finally, Compound 10 formed two hydrogen bonds, one
hydrogen bond between Tyr 371 with N of pyrazole and
another one between Val 335 with O of pyrazole with a
distance of 1.93 and 2.07 Å, respectively and also show
pi–pi interaction with amino acids Phe 195, Phe 504 (see
Table 4).
4.1.3. Synthesis of 4-[2-(5-Methyl-2-phenyl-2H-pyrazol-3-yloxy)-
ethoxy]-benzaldehyde (3)
3. Conclusion
A mixture of p-hydroxybenzaldehyde, (2.34 g, 19.25 mmol), and
anhydrous K2CO3 (4.04 g, 28.8 mmol) in 10 ml DMF was stirred for
half an hour. Compound 2 (5.41 g, 19.25 mmol) was added and
stirred at room temperature for 24 h. After completion of reaction
(evident by TLC), the solvent was concentrated, and the residue
was dissolved in chloroform. The organic layer was washed with
water and brine, dried over anhydrous Na2SO4 and concentrated.
The residue was purified by silica gel chromatography with hex-
ane/ethyl acetate as eluent to afford 3.
Yield, (3.00 g, 49%); Rf = 0.70 (Hexane/EtOAc, 9:1); mp. 88–
90 °C; 1H NMR (300 MHz, CDCl3, d ppm); 2.28 (3H, s, CH3), 4.41-
4.45 (4H, m CH2CH2), 5.57 (1H, s, CH pyrazole), 7.00-7.85 (9H, m,
ArAH) 9.90 (1H, s, CHO). 13C NMR (CDCl3, d ppm); 14.5(CH3),
66.1(OCH2) 70.0(OCH2), 86.8(C@C of pyrazole), 114.8(ArC), 121.7(ArC),
125.8(ArC), 128.7(ArC), 130.3(ArC), 131.9, (ArC), 138.5(ArC),,
148.6(ArC), 154.0(pyrazole C), 163.2(ArC), 190.5(CHO). MS (m/z):
323 (M++1). Anal. Calcd for C19H18N2O3 (322.36): C, 70.80; H,
5.59; N, 8.70%. Found: C, 70.62; H, 5.52; N, 8.61.
A series of novel pyrazole analogues were synthesized and their
anti-inflammatory activity was determined using carrageenan
mouse paw edema bioassay. In synthesized compounds, 5b exhib-
ited good anti-inflammatory activity, and optimal COX-2 inhibitory
potency (IC50 = 0.44)
pyrazole analogues interact with COX-2 active site by forming clas-
sical hydrogen bonding, interaction and cation– interaction
lM). Molecular modeling showed that
p–p
p
these interactions increase the residence time of ligand in the
active site consequently augmenting anti-inflammatory activity
of compounds.
4. Experimental
4.1. General
Melting points were determined in open capillaries on a Buchi
Melting-point apparatus and are uncorrected. All the reactions
were monitored by thin layer chromatography (TLC) on precoated
silica gel 60 F254 (mesh); spots were visualized with UV light.
Merck silica gel (60–120 mesh) was used for column chromatogra-
phy. The IR spectra were recorded on VARIAN 3300 FTIR spectro-
photometers as KBr pellets and the wave numbers were given in
4.1.4. Synthesis of {4-[2-(5-Methyl-2-phenyl-2H-pyrazol-3-yloxy)-
ethoxy]-phenyl}-methanol (4)
A mixture of compound 3 (0.20 g, 0.62 mmol), and NaBH4
(0.012 g, 0.31 mmol) in 10 ml methanol was refluxed for 15 min.
After completion of reaction (evident by TLC), the reaction mixture
was concentrated to leave residue which was crystallized from
ethanol to afford compound 4.
Yield, (0.10 g, 40%); Rf = 0.50 (Hexane/EtOAc, 9:1); mp. 120 °C;
1H NMR (300 MHz, CDCl3, d ppm); 2.28 (3H, s, CH3), 4.40–4.43
(4H, m CH2 CH2), 4.63 (2H, d, CH2, J = 5.4 Hz), 5.56(1H, s, CH pyra-
zole), 6.90–7.61(9H, m, ArAH). 13C NMR (CDCl3, d ppm); 14.4 (CH3)
62.5(OCH2), 65.9(OCH2), 70.7(OCH2OH), 87.3(C@C of pyrazole),
115.3(ArC), 120.9(ArC), 125.6(ArC), 128.0(ArC), 128.9(ArC), 135.0(ArC),
138.5(ArC), 148.0(ArC), 155.1(ArC), 157.1(ArC). MS (m/z): 325
(M++1). Anal. Calcd for C19H20N2O3 (324.37) C, 70.37; H, 6.17; N,
8.64. Found: C, 69.98; H, 6.02; N, 8.51.
cmÀ1 1H NMR (300 MHz), and 13C NMR (75 MHz) spectra were
.
recorded on a JEOL AL 300 MHz NMR spectrometer in CDCl3 solu-
tion using TMS as an internal standard. All chemical shifts were
reported in d (ppm) using TMS as an internal standard. Mass spec-
tra of the compounds were taken with JEOL SX 102/Da-600 mass
spectrometer. Elemental analysis was performed on Exter Analyti-
cal Inc. ‘Model CE-400 CHN Analyzer. The well known compounds
were prepared following the procedures reported in the literature.
4.1.1. Synthesis of 3-methyl-1-phenyl-pyrazol-5-one (1)
In a 250-ml round-bottom flask, ethyl acetoacetate (48 ml,
0.385 mol) was taken and phenyl hydrazine (41.54 ml,
0.385 mol) was added slowly with stirring. The reaction mixture
was refluxed on a heating mantle for 2 h. The completion of reac-
tion was checked via TLC. The reaction mixture was cooled and
diethyl ether was slowly added with stirring. After 10 min, light
yellow colored precipitate appeared which was filtered and
washed with ether. Pure white colored 3-methyl-1-phenyl-pyra-
zol-5-one (1) was then recrystallized with hot aqueous ethanol.
Mp = 127 °C. Yield = 60.20 g (90%). 1H NMR (300 MHz, CDCl3, d
ppm); d 2.20 (s, 3H, CH3), d 3.44 (s, 2H, CH2), d 7.18–7.87 (m,
5H, ArAH).
4.1.5. General method for synthesis of {4-[2-(5-Methyl-2-phenyl-2H-
pyrazol-3-yloxy)]-benzylidine}-aryl-amine
A mixture of compound 3 (0.5 g, 1.55 mmol), and appropriate
aryl amine (0.14 g, 1.55 mmol) was refluxed in 10 ml of dichloro-
methane (DCM) with stirring. After completion of reaction (evident
by TLC), the reaction mixture was concentrated to leave oily
residue which was crystallized from ethanol to afford compound
5a–5c.
4.1.5.1. {4-[2-(5-Methyl-2-phenyl-2H-pyrazol-3-yloxy)]-benzylidine}-
phenyl-amine (5a). Yield, (0.18 g, 36%); Rf = 0.52 (Hexane/EtOAc,
9:1); mp. 88 °C; (Major product) 1H NMR (300 MHz, CDCl3,
d ppm); 2.29 (3H, s, CH3); 4.38-4.45 (4H, m, CH2CH2), 5.57 (1H, s,
CH pyrazole), 6.97-8.39 (14H, m, ArAH). 13C NMR (CDCl3,
d ppm); 14.5(CH3), 66.0(OCH2), 70.3(OCH2), 86.8(C@C of pyrazole),
114.8(ArC), 120.8(ArC), 120.8(ArC), 125.6(ArC), 125.9(ArC), 128.7(ArC),
4.1.2. Synthesis of 5-(2-bromo-ethoxy)-3-methyl-1-phenyl-1H
pyrazole (2)
A mixture of compound 1(2.60 g, 11.2 mmol), anhydrous K2CO3
(2.31 g, 16.8 mmol) in 10 ml DMF was stirred for half an hour. 1,2-
dibromoethane (4.8 mL, 56 mmol) was added and stirred at room
temperature for 24 h. After completion of reaction (evident by

A.K. Tewari et al. / Bioorganic Chemistry 56 (2014) 8–15
13
129.1(ArC), 129.8(ArC), 130.5(ArC), 138.6(ArC), 148.7(ArC), 152.2(ArC),
4.1.8. Synthesis of 4-(bis-methylsulfanyl-methylene)-5-methyl-2-
phenyl-2,4-dihydro-pyrazol-3-one (9)
155.2(ArC), 159.4(ArC), 160.9(ArC). MS (m/z): 398 (M++1). Anal. Calcd
for C25H23N3O2(397.18) C, 75.57; H, 5.79; N, 10.58. Found: C, 73.62;
H, 5.49; N, 10.48.
In a 500-ml round-bottom flask, fitted with guard tube, anhy-
drous K2CO3 (59.48 g, 0.431 mol) was taken, and 20 ml DMF and
40 ml dry benzene were added and stirred in an ice bath for 1 h.
In another 500-ml round-bottom flask, fitted with guard tube (1)
(30.0 g, 0.172 mol) was taken and 20 ml DMF was added. It was
stirred for 15 min. Then, carbon disulfide (10.40 ml, 0.172 mol)
was added slowly with stirring. It was also stirred for 1 h in an
ice bath. Then, the content of first round bottom flask was added
to second round bottom flask with stirring and the reaction mix-
ture was stirred for 6 h. With the use of a dropping funnel, methyl
iodide (21.46 ml, 0.345 mol) was added very slowly along with
10 ml dry benzene. The addition of methyl iodide was accompa-
nied with the use of ice bath. After complete addition, the reaction
was stirred for 6 h. The completion of reaction was checked by TLC.
Reaction was worked up. Solvents were removed under pressure
through rotary evaporator and the reaction mixture was extracted
with CHCl3/H2O (200/200 9 2 ml). The CHCl3 layer was dried with
anhydrous Na2SO4 and filtered. Chloroform was removed and the
product was purified via SiO2-column chromatography. Eluent
used was 20% ethyl acetate in hexane.
4.1.5.2. {4-[2-(5-Methyl-2-phenyl-2H-pyrazol-3-yloxy)-ethoxy]-ben-
zylidine}-p-tolylamine (5b). Yield, (0.29 g, 55%); Rf = 0.54 (Hexane/
EtOAc, 9:1);mp 146 °C; 1H NMR (300 MHz, CDCl3, d ppm);; 2.29
(3H, s, CH3); 2.37 (3H, s, CH3), 4.38–4.45 (4H, m, CH2CH2), 5.57
(1H, s, CH pyrazole), 6.97–8.39 (13H, m, ArAH). 13C NMR (CDCl3,
d
ppm); 14.5(CH3), 20.9, 66.0, 70.3, 86.8(C@C of pyrazole),
114.8(ArC), 120.7(ArC), 121.8(ArC), 125.9(ArC), 128.7(ArC), 129.7(ArC),
130.0(ArC), 130.4(ArC), 135.4(ArC), 138.6(ArC), 148.7(ArC), 149.6,
154.4, 158.6, 160.8. MS (m/z): 412 (M++1). Anal. Calcd for
C26H25N3O2 (411.19) C, 75.91; H, 6.08; N, 10.21. Found: C, 75.52; H,
5.90; N, 10.01.
4.1.5.3. (4-Methoxy-phenyl)-{4-[2-(5-Methyl-2-phenyl-2H-pyrazol-
3-yloxy)-ethoxy] benzylidine}-amine (5c). Yield, (0.25 g, 67%);
Rf = 0.57 (Hexane/EtOAc, 9:1);mp 138 °C; 1H NMR (300 MHz,
CDCl3, d ppm); 2.29 (3H, s, CH3), 3.83 (3H, s, OCH3), 4.39–4.43
(4H, m CH2CH2), 5.57 (1H, s, CH pyrazole), 6.91–8.41 (13H, m,
Ar–H). 13C NMR (CDCl3, d ppm); 14.5(CH3), 55.4, 66.0, 70.3,
86.8(C@C of pyrazole), 114.7(ArC), 114.8(ArC), 121.8–122.0(ArC),
125.9(ArC), 128.7(ArC), 130.1–130.2(ArC), 132.0(ArC), 138.6(ArC),
145.1(ArC), 148.7(ArC), 155.2, 157.6, 158.0, 160.7, 177.8. MS
(m/z): 428 (M++1).). Anal. Calcd for C26H25N3O3 (427.19) C, 73.07;
H, 5.85; N, 9.84. Found: C, 72.92; H, 5.63; N, 9.71.
Mp = 46–52 °C. Yield = 40.5 g (85%). 1H NMR (300 MHz, CDCl3, d
ppm); 2.52 (s, 3H, CH3), 2.69 (s, 3H, SCH3), 2.76 (s, 3H, SCH3), 7.12–
7.99 (m, 5H, ArAH).
4.1.9. Synthesis of 3-methyl-1-phenyl-5-(toluene-4-sulfonyloxy)-1H-
pyrazole-4 carboxylic acid methyl ester (10)
A mixture of compound 9 (1.0 g, 3.9 mmol), anhydrous K2CO3
(0.85 g, 5.86 mmol) in 10 ml DMF was stirred for half an hour. After
half hour 4-methylbenzene-1-sulfonyl chloride (0.82 g, 3.9 mmol)
was added and stirred at room temperature for 24 h. After comple-
tion of reaction (evident by TLC), the reaction mixture was concen-
trated and the residue was dissolved in chloroform. The organic
layer was washed with water and brine, dried over anhydrous
Na2SO4 and concentrated. The residue was purified by silica gel
chromatography with hexane/ethylacetate as eluent to afford 10.
Yield, (0.62 g, 49.5%); mp. 88–90 °C; 1H NMR (300 MHz, CDCl3, d
ppm); 2.36 (3H, s, CH3), 2.50 (3H, s, CH3), 3.75 (3H, s, OCH3), 7.04–
7.07(2H, d, ArAH, J = 6 Hz), 7.24–7.26 (5H, m, ArAH), 7.44–
7.77(1H, s, ArAH). 13C NMR (CDCl3, d ppm); 12.1, 14.6, 21.2, 50.9,
91.1, 122.1, 124.0, 125.8, 128.7(ArC), 129.2(ArC), 129.8(ArC),
139.1(ArC), 145.3, 156.6. MS (m/z): 387 (M++1). Anal. Calcd for
4.1.6. Synthesis of 4-[2-(5-Methyl-2-phenyl-2H-pyrazol-3-yloxy)-
ethoxy]-benzaldehyde-oxime (6)
To solution of compound 3 (0.20 g, 0.62 mmol) in ethanol
(7 mL), hydroxylamine hydrogensulfate (0.143 g, 0.87 mmol), was
added and the mixture was stirred for half an hours. Sodium
hydroxide (0.004 g) in 5 mL water was added in portion over
5 min. The reaction mixture was refluxed for 4 h, after completion
of reaction (evident by TLC), the reaction mixture was cooled and
poured into HCl–water (5:1). Oxime crystal formed was filtered
and washed with water.
Yield, (0.13 g, 65%); mp 183 °C; 1H NMR (300 MHz, CDCl3, d
ppm);; 2.16 (3H, s, CH3), 4.48-4.46 (4H, m, CH2CH2), 5.78(1H, s,
CH, J = 9 Hz), 6.99–7.63 (9H, m, ArAH), 8.47(1H, s, CH). 13C NMR
(CDCl3, d ppm); 15.3(CH3), 66.0(OCH2), 70.6, 87.3(C@C of pyrazole),
114.9, 120.9, 125.6, 126.0, 127.9, 128.9, 138.4, 147.5, 147.9, 154.0,
159.0. MS (m/z): 338 (M++1). Anal. Calcd for C19H19N3O3 (337.14)
C, 67.67; H, 5.64; N, 12.46. Found: C, 67.45; H, 5.40; N, 12.36.
C19H18N2O5S (387.09) C, 59.06; H, 5.08; N, 7.91. Found: C, 60.13;
H, 4.99; N, 7.39.
4.2. Pharmacology
4.1.7. Synthesis of {4-[2-(5-Methyl-2-phenyl-2H-pyrazol-3-yloxy)-
ethoxy]-benzylidine}-hydrazine (7)
4.2.1. In vitro assays for cyclooxygenase 1 and 2
COX-1 has been isolated from Rat seminal vesicles. Recombi-
nant human COX-2 has been expressed in insect cell expression
system. These enzymes have been purified by employing conven-
tional chromatographic techniques. Enzymatic activities of COX-1
and COX-2 were measured according to the method of Copeland
et al. [55], with slight modifications using a chromogenic assay
based on the oxidation of N,N,N,N,-tetramethyl-4-phenylene dia-
mine (TMPD) during the reduction of PGG2 to PGH2 [56,57]. Briefly,
the assay mixture contained TriseHCl buffer (100 mM, Ph 8.0),
hematin (15 mM), EDTA (3 mM), enzyme (100 mg COX-1 or COX-
2) and the test compound. The mixture was pre-incubated at
25 °C for 1 min and then the reaction was initiated by the addition
of arachidonic acid and TMPD, in total volume of 1 ml. The enzyme
activity was determined by estimating the velocity of TMPD oxida-
tion for the first 25 s of the reaction by following the increase in
absorbance at 603 nm. A low rate of non enzymatic oxidation
A mixture of hydrazine hydrate (0.062 ml, 1.24 mmol), 1 ml
acetic acid in 0.5 ml water was stirred for 10 min. Compound 3
(0.02 g, 0.62 mmol) was added to reaction mixture and refluxed
for 4 h. After completion of reaction (evident by TLC), the reaction
mixture was concentrated and crystallized from ethanol to afford
compound 7.
Yield, (0.13 g, 65%); mp 220 °C.1H NMR (300 MHz, CDCl3,
d ppm); 1.2 (2H, s, NH2), 2.29 (3H, s, CH3), 4.40–4.46 (4H, m,
CH2CH2), 5.57(1H, s, CH pyrazole), 6.96–7.85 (9H, m, ArAH),
8.61(1H, s, CH). 13C NMR (CDCl3, d ppm); 14.5(CH3), 66.6(OCH2),
70.8, 87.7(C@C of pyrazole), 115.4(ArC), 121.4(ArC), 126.1(ArC),
129.2(ArC), 130.1(ArC), 132.2(ArC), 138.5(ArC), 148.5(ArC), 154.4,
163.4, 191.9. MS (m/z); 337 (M++1). Anal. Calcd for C19H20N4O2
(336.16) C, 67.86; H, 5.95; N, 16.67. Found: C, 67.43; H, 5.60; N,
16.31.

14
A.K. Tewari et al. / Bioorganic Chemistry 56 (2014) 8–15
observed in the absence of COX-1 and COX-2 was subtracted from
the experimental value while calculating the percent inhibition.
boxes were created by AutoGrid algorithm to evaluate the binding
energies on the macromolecule coordinates. Ligand PDB were pre-
pared using ChemBio3D. The grid maps representing the intact
ligand in the actual docking target site were calculated with Auto-
Grid (part of the AutoDock package). Eventually cubic grids encom-
passed the binding site where the intact ligand was embedded.
Finally, AutoDock was used to calculate the binding free energy
of a given inhibitor conformation in the macromolecular structure
while the probable structure inaccuracies were ignored in the cal-
culations. The search was extended over the whole receptor pro-
tein used as blind docking. Nimesulide (Native Ligand) in the
crystal structure was docked as reference.
4.2.2. In vivo anti-inflammatory activity
All experiments have been conducted on adult Wistar strain
albino rats of either sex, weighing between 150 and 200 g. The
animals were obtained from the Central Animal House of the Insti-
tute of Medical Sciences, B.H.U. They were kept in colony cages
under identical housing conditions at an ambient temperature of
25 2 °C and 45–55% relative humidity with a 12 h light–dark
cycle in the departmental animal room and fed on standard diet.
Animals were acclimatized for a week before use.
4.2.3. Experimental model of Inflammation
Acknowledgments
Carrageenin Induced Paw Edema in Rats: Target compounds
were suspended in aqueous solution of carboxymethyl cellulose
(CMC, 1% w/v) and administered orally at a dose level of 10 mg/kg
of NSAID drugs Nimesulide and their equivalent doses of the
target derivatives (3–7, 10). After 15 min control animals were
similarly treated with aqueous solution of carboxymethyl cellulose
(CMC, 1% w/v). One percent carrageenin suspension was prepared
as a homogeneous solution in distilled water. A volume of 0.1 ml of
carrageenin solution was injected through a 26 gauge needle into
the plantar surface of the left hind paw below the plantar aponeu-
rosis. The volume of paw was measured before and at different
intervals for 2 h after injection of carrageenin. The difference in
paw volume before and after administration of the phlogistic agent
was taken as the measure of pedal edema.
We gratefully acknowledge financial support from the UGC
(Grant Sanction No. 37-54/2009) India. The authors acknowledge
Department of Chemistry, for instrumental facilities, Department
of Pharmacology, IMS, Banaras Hindu University, Varanasi, India
for the in vitro assays and Amrita Centre for Nanosciences and
Molecular Medicine (ACNSMM), Amrita Institute of Medical Sci-
ences & Research Centre, Ponekkara, Kochi, Kerala, India for the
docking studies For the activity studies, permission was obtained
from the Animal Ethical Committee.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2014.05.
004.
4.2.4. Measurement of paw volume
The volume of hind paw of the rats up to the ankle joint was
measured plathysmographically by the mercury displacement
method. The ankle joint of the rats was marked with a skin mark-
ing pencil and the paw was dipped in the mercury, so that the
mark on the paw coincides with a prefixed line kept constant on
the syringe. The level of the mercury was every time brought to
the level of this line by adjusting the height of the displaced mer-
cury. The difference in the paw volume before and after injection
of the phlogistic agents was taken as a measure of pedal edema.
The change in paw volume was expressed in ‘‘ml’’ of mercury
displaced.
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