Pharmacological screening for anti-inflammatory, analgesic activity of pyrazolyl derivatives along with molecular docking studies

Article abstract of DOI:10.1007/s00044-011-9901-0

The pyrazole derivatives were synthesized and pharmacologically evaluated for analgesic (tail flick) and anti-inflammatory (based on carrageenan-induced paw edema) activities. Compound 4k showed high potency as an anti-inflammatory agent after 3 and 4-h time intervals (P<0.001) equipotent to indomethacin. They were devoid of ulcerogenic potential when administered at a dose of 30 mg/kg. The compounds, which showed less ulcerogenic action, also showed reduced malondialdehyde content (MDA), which is one of the byproduct of lipid peroxidation. Further docking studies of titled compounds was done to understand key interactions responsible for observed inhibition of COX enzyme. The most active compound 4k was found to have -11.192 kcal/mol, as the free energy of binding. Various other key interactions between the synthesized molecules and active site of COX-2 enzyme, responsible for the obtained pharmacological results were also reported. Most of the active compounds were docked well into the active sites of the receptor. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9901-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3646–3655
DOI 10.1007/s00044-011-9901-0
ORIGINAL RESEARCH
Pharmacological screening for anti-inflammatory, analgesic
activity of pyrazolyl derivatives along with molecular docking
studies
•
Darpan Kaushik Rajnish Kumar
•
•
Suroor Ahmed Khan Gita Chawla
Received: 15 April 2011 / Accepted: 9 November 2011 / Published online: 29 November 2011
Ó Springer Science+Business Media, LLC 2011
Abstract The pyrazole derivatives were synthesized and
pharmacologically evaluated for analgesic (tail flick) and
anti-inflammatory (based on carrageenan-induced paw
edema) activities. Compound 4k showed high potency as
an anti-inflammatory agent after 3 and 4-h time intervals
(P \ 0.001) equipotent to indomethacin. They were devoid
of ulcerogenic potential when administered at a dose of
30 mg/kg. The compounds, which showed less ulcerogenic
action, also showed reduced malondialdehyde content
(MDA), which is one of the byproduct of lipid peroxida-
tion. Further docking studies of titled compounds was done
to understand key interactions responsible for observed
inhibition of COX enzyme. The most active compound 4k
was found to have -11.192 kcal/mol, as the free energy of
binding. Various other key interactions between the syn-
thesized molecules and active site of COX-2 enzyme,
responsible for the obtained pharmacological results were
also reported. Most of the active compounds were docked
well into the active sites of the receptor.
Introduction
A strong linkage between antiepileptic activity and anti-
nociception is already proven for many established anti-
convulsant drugs which have long been used in pain
management, particularly in chronic neuropathic pain. For
example, carbamazepine, phenytoin, lamotrigine and fel-
bamate are clinically effective in the treatment of trigem-
inal neuralgia, valproate, and topiramate are useful for
migraine prophylaxis (Rogawski, 2006); (Rogawski and
Loscher, 2004), and gabapentin and carbamazepine as well
as phenytoin are used in the treatment of post-herpetic
neuralgia and diabetic neuropathy, respectively (Wiffen
et al., 2005); (Taylor, 1996); (Collins et al., 2000). The
mechanisms underlying the analgesic action of antiepi-
leptic drugs might include an enhancement of the GAB-
Aergic neurotransmission or effects on neuronal voltage-
gated sodium and/or calcium channels (Rogawski, 2006).
Whereas, antiepileptic drugs are efficacious in controlling
neuropathic pain, but their activity in models of acute pain
is still unclear. Only negligible effects of lamotrigine,
felbamate and gabapentin against an acute noxious stimu-
lus were determined in the rat tail flick test (Hunter et al.,
1997). Retigabine did not show analgesic effects in this test
(Blackburn-Munro and Jensen, 2003). Tiagabine, a GABA
uptake inhibitor, produced dose-dependent anti-nocicep-
tion in the hot-plate test in mice (Giardina et al., 1998)
whereas, lamotrigine and gabapentin were not efficacious
in producing result (Laughlin et al., 2002). Voltage-gated
calcium channel blockers (gabapentin and ethosuximide)
and voltage-gated sodium channel blockers (phenytoin and
carbamazepine) have been shown to be potent local anal-
gesics to ameliorate acute thermal nociception.
Keywords Anti-inflammatory ꢀ Docking ꢀ Pyrazole ꢀ
SC-558 ꢀ Ulcerogenic index
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-011-9901-0) contains supplementary
material, which is available to authorized users.
D. Kaushik (&) ꢀ S. A. Khan ꢀ G. Chawla
Medicinal Chemistry Division, Faculty of Pharmacy,
Jamia Hamdard, New Delhi, India
e-mail: darpkaush@yahoo.com
Hence, in light of above literature, we thought it
worthwhile to characterize anti-nociceptive properties of
R. Kumar
Department of Pharmaceutical Chemistry, UIPS, Chandigarh, India
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Med Chem Res (2012) 21:3646–3655
3647
our previously reported anticonvulsant (Kaushik et al.,
2010) and also investigated these pyrazole derivatives for
anti-inflammatory activity because of structural similarity
with tricyclic COX-2 selective inhibitor, i.e., SC-558 as
depicted in Fig. 1, but as most of NSAIDs are commonly
associated with serious adverse gastrointestinal side effects
such as gastro-duodenal erosions and ulcerations, therefore,
it deemed to be desirable to test these derivative for their
protective effect on gastric mucosa.
maintained. All the synthesized test compounds were sus-
pended in 30% polyethylene glycol (PEG 200).
Analgesic activity
Analgesic activity was evaluated using the tail immersion
method (Kendall et al., 1982). Each compound was
administered intraperitoneally at the dose of 20 mg/kg. The
analgesic activity was assessed at 30 min interval of
administration. Tail of each mice was gently immersed into
thermostatically controlled water at 55°C. The parameter
measured in test samples was the time that elapsed between
immersion and the attempt to withdraw the tail from hot
water for control as well as treated group of animals.
Experimental protocol
Chemistry
Anti-inflammatory activity
The compounds were synthesized as given in Scheme 1
(Kaushik et al., 2010) by refluxing the 5-chloro-3-methyl-
1-phenyl-1H-pyrazole-4-carboxaldehyde (2) with respec-
tive substituted acid hydrazide (3a–o), which in turn were
synthesised from the acids via its esterification followed by
its nucleophilic substitution reaction, i.e., hydrazinolysis
with hydrazine hydrate. The carboxyaldehyde was syn-
thesised from 3-methyl-1-phenyl-2-pyrazolin-5-one (1) by
Vilsmeir Haack reaction, while later was synthesised by
condensation of phenylhydrazine with ethylacetoacetate.
The characterization details of the synthesised compounds
are given in the supplementary data.
This activity was performed by the following procedure of
Winter et al. (1962) on group of six animals. A freshly
prepared suspension of carrageenan (1.0% w/v, 0.1 ml)
was injected in subplanter aponeurosis of left hind paw of
each rat. One group each was kept as control, standard and
the animals of the other groups were pretreated with the
test drugs suspended in vehicle given orally 1 h before the
carrageenan treatment. The volume was measured 0, 3, and
4 h of carrageenan treatment with the help of plethys-
mometer. The percent anti-inflammatory activity was cal-
culated according to the formula given below:
% Anti-inflammatory activity ¼ ½1 ꢁ Vt=Vcꢂ ꢃ 100
Pharmacology
whereas Vt represents the mean increase in paw volume in
rats treated with test compounds, and Vc represents the
mean increase in paw volume in control group of rats.
The experiments were performed on Albino rats of Wistar
strains and Albino mice of either sex, weighing 150–180
and 20–25 g, respectively. The Institutional Animal Ethics
Committee (IAEC) reviewed and approved all the animal
procedures adopted. The animals were housed at an
ambient temperature of 25 2°C, in groups as required
per metabolic cages and allowed free access to chow pel-
lets and water. The light/dark cycle of 12:12 h was
Acute ulcerogenesis
Acute ulcerogenesis test was done according to Cioli et al.
(1979). Albino rats have been divided into different groups
consisting of six animals in each group. Ulcerogenic
activity was evaluated after p.o. administration of test
compounds or indomethacin at the dose of 30 mg/kg.
Control rats received p.o. administration of vehicle (sus-
pension of 30% PEG 200). Food but not water was
removed 24 h before administration of the test compounds.
After the drug treatment, the rats were fed normal diet for
17 h and then sacrificed. The stomach was removed and
opened along the greater curvature, washed with distilled
water and cleaned gently by dipping in saline. The mucosal
damage was examined by means of a magnifying glass. For
each stomach, the mucosal damage was assessed according
to the following scoring system (Wiliheme and Gdynia,
1972).
O
F3C
HN
H3C
N
N
Ar
n
N
Br
N
Cl
N
SO2NH2
Fig. 1 Close structural similarity between COX-2 selective inhibitor
SC-558 and the titled compounds
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Med Chem Res (2012) 21:3646–3655
Scheme 1 Synthetic route to
the synthesized compounds
NHNH2
O
O
O
Ar
+
H3C
OC2H5
Ethyacetoacetate
OH
n
Phenylhydrazine
Aromatic acid
Reflux
Ethanol, Conc. Sulphuric acid
CH3
O
N
N
Ar
OC2H5
n
O
Ethyl ester of aromatic acid
(1)
3-Methyl-1-phenyl-pyrazolin-4-one
NH2NH2.H2O
CH3
O
N
N
Ar
NHNH2
+
n
CHO
Cl
5-Chloro-3-methyl-1-phenyl-pyrazole-4-carbaldehyde
Aromatic acid hydrazide
(3a-o)
(2)
GAA, Reflux
O
HN
N
H3C
Ar
n
N
Cl
N
(4a-o)
the reactant were supplemented with 5 ml of the mixture of
n-butanol: pyridine (15:1 v/v), shaken vigorously for 1 min
and centrifuged for 10 min at 4,000 rpm. The supernatant
organic layer was taken out and absorbance was measured
at 532 nm on UV spectrophotometer. The results were
expressed as nmol MDA/100 mg tissue, using extinction
0.5
1.0
1.5
2.0
3.0
Redness
Spot ulcers
Hemorrhagic streaks
Ulcers \3, but B5
Ulcers [5
coefficient 1.56 9 105 cm-1 M-1
.
The mean score of each treated group minus the mean
score of control group was regarded as severity index of
gastric mucosal damage.
Statistical data
Data are expressed as mean SEM, Student’s t test was
applied to determine the significance of the difference
between the standard group and rats treated with the test
compounds. The difference in result was considered sig-
nificant when P \ 0.05.
Lipid peroxidation
Lipid peroxidation in the gastric mucosa was determined
according to the method of Ohkawa et al. (1979). After
screening for ulcerogenic activity, the gastric mucosa was
scrapped with two glass slides, weighed (100 mg) and
homogenized in 1.8 ml of 1.15% ice cold KCl solution.
The homogenate was supplemented with 0.2 ml of 8.1%
sodium dodecyl sulfate (SDS), 1.5 ml of acetate buffer
(pH 3.5) and 1.5 ml of 0.8% thiobarbituric acid (TBA).
The mixture was heated at 95°C for 60 min. After cooling,
Molecular docking studies
Molecular modeling study was carried out using
IntelÒCoreTMi3CPUM350@2.27 GHz platform with the
help of MOE 2008.10 (MOE, 2008).
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Med Chem Res (2012) 21:3646–3655
3649
Selection of COX structures
MOE docking
The ligand docking studies were performed using the
crystal structure of COX-2 (doi:10.2210/pdb1cx2/pdb)
which contains SC-558, a selective inhibitor of COX-2 in
co-crystallized form in the active site of the receptor. The
COX-2 enzyme contains 587 amino acids and one ferric
heme group. A number of X-ray structures of mouse (Mus
musculus) COX-2 has been resolved as unsubstituted as
well as in the presence of different inhibitors. The amino
acid sequence of the COX-2 is conserved between species
with 85–90% similarity. Due to this 87% identity along
with strict conservation of active sites, the mouse enzyme
is expected to be very similar to the human (Kurumbail
et al., 1996), and can be used as model for human COX-2
enzyme.
The docking studies were performed using dock simulation
as incorporated in the MOE 2008.10. dock workflow is
composed of various stages. The 3D structures of mole-
cules were subjected to the conformational analysis in
which different conformations of the ligands were gener-
ated from the 3D conformer by applying a collection of
preferred torsional angles to the rotatable bonds. These
conformations were then used to generate different poses
of ligands using triangular matcher placement method,
which is a process of generating poses by aligning ligand
triplets of atoms on triplets of alpha spheres representing
location of tight packing in the receptor site points. During
each iteration, a random triplet of alpha sphere centres is
used to determine the pose. The pose generated using the
placement methodology was rescored using London dG
scoring function which estimates the free energy of binding
of the ligand from a given conformer. In order to further
improve the outcomes, the poses generated were refined
using MMFF94x forcefield (Halgren, 1996). During this,
solvation effects were treated by using reaction field
functional form for the electrostatic energy term. The final
energy of the molecules was calculated using generalized
Born solvation model (GB/VI) and the poses were finally
assigned a score based on the free energy in kJ/mol.
Preparation of protein
The coordinates from the X-ray crystal structure of COX-2
were used in this simulation study was obtained from
Protein Data Bank (PDB) (www.rscb.org) (Pouplana et al.,
2002). The downloaded structure was refined and prepared
for docking using protonate 3D application in MOE
2008.10. It includes addition of hydrogen atoms which is
necessary to ensure the correct ionisation and tautomer
state of the amino acid residue in the receptor structure.
Partial charges were assigned to the structure using
MMFF94x forcefield. Protonate 3D assign protonation
state from a discrete collection of states by optimising the
titration free energy of all titratable groups in the context of
an all-atom model of a macromolecular structure. All the
bound ligands and water molecules were removed from the
active site of the receptor.
Results and discussion
Analgesic activity
Some of the compound possess significant and structure
dependent activity on the laboratory animals. Analgesic
activity of the title compounds was checked by using
thermal stimulus technique against the standard drug dic-
lofenac at the dose of 20 mg/kg. Results are presented as
mean average reaction time (s) of control and treated as
depicted in Table 1 and Fig. 2a. Compounds 4e, 4h, 4j–m,
4o showed moderate but significant activity compared to
the standard drug. Remaining compounds showed very less
but significant activity.
Preparation of ligands
The 3D structures of the synthesised compounds were
constructed using ChemDrawUltra 8.0 running on an
IntelÒCoreTMi3CPUM350@2.27 GHz platform and were
subjected to energy minimization using molecular
mechanics (MM2). The minimization is continued until the
root mean square (RMS) gradient value reaches a value
p-Substituted aryl ring of acid hydrazone part play some
role in binding because the unsubstituted ring was found to
have the least activity. p-Substituted electron withdrawing
group contributed more to the activity as seen from the
data, that as the electronegativity of the substituent
increased, the activity also enhanced little, but with elec-
tron releasing substituent, the activity is partially increased,
secondly, the important notice was that all the compound
which have one more atom in between phenyl ring and
carbonyl group found to have more or less similar and
enhanced activity whether p-substitution was present or
˚
smaller than 0.001 kcal/mol A. The Hamiltonian approxi-
mations Austin model 1 (AM1) (Dewar et al., 1985)
method available in the MOPAC module (Stewart, 1990)
of Chem3D is adopted for re-optimization until the root
mean square (RMS) gradient attains a value smaller than
˚
0.001 kcal/mol A. Unless otherwise indicated, all param-
eters were kept default. The energy minimized structures
were saved as.mol2 format and further used for docking
employing dock module in MOE 2008.10.
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Med Chem Res (2012) 21:3646–3655
Table 1 Analgesic activity of novel pyrazoles (4a–o) by tail immersion method
O
HN
N
Ar
H3C
n
N
Cl
N
Compd. code
Ar
n
Mean average reaction time (s)
Mean SEM
% Increase in analgesia (against control)
Control treated
Treated
4a
Phenyl
0
0
0
0
1
1
0
0
0
0
1
2
1
0
0
3.40 0.215**
2.30 0.230*
2.80 0.100**
1.98 0.124**
2.05 0.211*
2.15 0.125**
2.60 0.115**
2.15 0.189**
2.10 0.165**
1.98 0.150**
2.00 0.116**
2.03 0.114*
2.01 0.186**
2.01 0.124*
2.01 0.153**
2.15 0.164**
4.20 0.159**
3.40 0.190*
5.40 0.159**
3.63 0.111***
6.01 0.233***
4.65 0.140**
5.91 0.153**
5.60 0.152**
5.23 0.185*
5.70 0.163**
5.83 0.114***
6.08 0.158**
5.93 0.149**
5.20 0.180*
5.48 0.207**
18.26 0.183***
22.40
48.00
92.80
83.33
193.1
116.3
127.3
160.4
149.0
187.8
191.5
199.5
195.0
158.7
172.6
749.3
4b
4-Methylphenyl
4-Nitrophenyl
4-Pyridyl
4c
4d
4e
4-Chlorophenyl
1-Naphthyl
4f
4g
4-Methoxyphenyl
4-Chlorophenyl
2-Chlorophenyl
4-Flurophenyl
Phenoxy
4h
4i
4j
4k
4l
Isobutylphenyl
Phenyl
4m
4n
4-Bromophenyl
2,4-Dichlorophenyl
–
4o
Diclofenac
n = 6; dose = 20 mg/kg (i.p.); * P \ 0.05, ** P \ 0.01, *** P \ 0.001. Data was analyzed by unpaired Student’s t test
absent compared to remaining other compounds. Thus, it
can be inferenced from this study that lead requires one
more atom between carbonyl group and phenyl ring, and to
have p-substituted electron withdrawing group to have
better analgesic activity.
and is devoid of apparent systemic effects. Moreover, the
experimental model exhibits a high degree of reproduc-
ibility (Winter et al., 1962). Carrageenan-induced edema
is a biphasic response. The first phase is mediated through
the release of histamine, serotonin and kinins, whereas the
second phase is related to the release of prostaglandin
and other slow-reacting substances which peak at 3–4 h
(Vinegar et al., 1969). Indomethacin was used as a standard
drug at the dose level of 10 mg/kg. Results are presented
as % inhibition SEM as summarized in Table 2 and
Fig. 2b, compared with the control using ANOVA fol-
lowed by Bonferronni multiple comparison tests at the time
intervals of 3 and 4 h. Compound 4k had showed more
percentage reduction in paw volume after 3 and 4 h time
Anti-inflammatory testing
The anti-inflammatory activity of the synthesised com-
pounds was carried out using carrageenan-induced rat paw
edema. Carrageenan-induced hind paw edema is the
standard experimental model of acute inflammation.
Carrageenan is the phlogistic agent of choice for testing
anti-inflammatory drugs, as it is not known to be antigenic
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Med Chem Res (2012) 21:3646–3655
3651
Fig. 2 a Bar diagram of
analgesic activity of compounds
4(a–o). b Bar diagram of anti-
inflammatory activity of
compounds 4(a–o)
intervals (P \ 0.001), which was found to be comparable
to the standard drug. The compound 4k was also exhibited
rapid onset and long duration of action. Moreover, edema
suppressant effect of the compounds 4c–d, 4f–h, 4m were
found to be moderately potent and significant after 4 h
interval and were not statistically significant at 3 h indi-
cating late onset of action of these compounds, while those
compounds which were statistically insignificant even at
(P \ 0.05) after the 3 h were considered to less active.
Compounds 4e and 4o were found statistically insignificant
inhibition. The results of the study suggested that signifi-
cantly potent compounds were found to have late onset of
action. The compounds 4k and 4b exhibited maximum and
minimum inhibition of 73.42 and 29.11%, respectively,
after 4 h in comparison to standard drug indomethacin
which showed an inhibition of 77.21% in the carrageenan-
induced rat paw edema (acute) model. The inhibition of
some the test drugs were comparable to the standard drug
indomethacin. Two pyrazole derivatives (4h and 4k) were
found to be almost equipotent with indomethacin
throughout the study.
Acute ulcerogenesis
The compounds (4h and 4k), which showed anti-inflam-
matory activity comparable to that of the standard drug
indomethacin, were screened for their ulcerogenic activity
in Wistar rats (Cioli et al., 1979; Naito et al., 1998). The
tested compounds 4h and 4k showed significant reduction
in ulcerogenic activity with values of 0.41 0.080 and
0.25 0.118, respectively, compared to the standard drug
indomethacin which showed high severity index of
4.50 0.316. This confirmed that these derivatives did not
cause any gastric ulceration as results are depicted in
Table 3.
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Med Chem Res (2012) 21:3646–3655
Table 2 Anti-inflammatory activity of some pyrazole derivatives (4a–o) by carragenan induced hind paw edema method
Compound
code
Initial paw
volume
Final paw volume
Difference after
3 h
% Inhibition SEM
3 h
3 h
4 h
4 h
4 h
Contol
0.53 0.03
0.54 0.04
0.51 0.02
0.48 0.05
0.55 0.02
0.53 0.02
0.53 0.06
0.55 0.02
0.54 0.03
0.50 .004
0.52 0.05
0.50 0.06
0.51 .004
0.50 0.06
0.64 0.04
0.59 0.04
0.59 0.01
0.58 0.04
0.65 0.02
0.61 0.02
0.58 0.05
0.62 0.03
0.60 0.02
0.56 0.04
0.59 0.04
0.56 0.05
0.58 0.05
0.56 0.05
0.80 0.04
0.60 0.05
0.69 0.01
0.67 0.05
0.67 0.02
0.67 0.03
0.61 0.05
0.67 0.02
0.65 0.03
0.60 0.04
0.60 0.04
0.57 0.05
0.61 0.03
0.57 0.05
0.11 0.01
0.05 0.008
0.08 0.01
0.09 0.01
0.09 0.008
0.08 0.008
0.04 0.01
0.06 0.008
0.05 0.01
0.06 0.008
0.07 0.01
0.05 0.008
0.07 0.01
0.05 0.01
0.26 0.02
0.06 0.01
0.18 0.008
0.18 0.01
0.12 0.01
0.14 0.01
0.07 0.008
0.11 0.008
0.11 0.01
0.09 0.008
0.08 0.01
0.07 0.01
0.10 0.01
0.06 0.01
Indomethacin
48.48 3.03***
21.21 3.83***
15.15 3.83***
12.12 3.03**
24.24 3.03**
57.57 3.85
77.21 1.96***
30.38 1.26***
29.11 1.60***
54.43 1.96***
46.83 2.77***
70.88 1.26
4a
4b
4c
4d
4e
4f
42.42 3.03
55.69 1.26***
58.23 1.69***
63.29 1.26***
67.09 2.53*
4g
4h
4j
51.52 3.83
42.42 3.03
36.36 6.21
4k
4m
4o
48.48 3.03***
36.36 6.21
73.42 1.69***
60.76 2.33***
74.68 1.60
51.51 3.83
n = 6; dose = 10 mg/kg; * P \ 0.05, ** P \ 0.01, *** P \ 0.001(compared to standard). Data was analyzed by ANOVA, followed by
Bonferroni multiple comparison test
Lipid peroxidation
ligand SC-558 (a selective COX-2 inhibitor) was docked
into the active site using dock module of MOE 2008.10 as
depicted in Table 4. The highest scored pose was having a
close resemblance with the co-crystallized confirmation
with root mean square deviation of 0.729. As shown in
Fig. 3a, the trifluromethyl group (–CF3) of SC-558 is
forming a strong hydrogen bonding interaction with
Arg120 and this group is located in the vicinity of Val349,
Leu531, Leu359, and Tyr355. Bromo group was found to
be surrounded by Leu352, Gly526, Try385, and Trp387.
Similarly, sulphanamido group showed interactions with
Arg513, His90 apart from lying in vicinity of Ala526,
Gln192, Ser353 and Ala527. In the case of docking of
indomethacin (non-selective COX-1/COX-2 inhibitor), it
has been observed that the carbonyl group of carboxylic
acid was forming a hydrogen bond with Arg120 at a dis-
It has been reported in literature that compounds showing
less ulcerogenic activity also showed reduced malondial-
dehyde (MDA) content, a byproduct of lipid peroxidation
(Naito et al., 1998). Therefore, an attempt was made to
correlate the decrease in ulcerogenic activity of the com-
pounds with that of lipid peroxidation. Both the compounds
screened for ulcerogenic activity were also analyzed for
lipid peroxidation.
The lipid peroxidation is measured as nmol of MDA/
100 mg of tissue. The indomethacin (standard drug)
showed lipid peroxidation (21.98 11.14), whereas the
control group showed 3.86 0.19. It was found that both
showing less ulcerogenic index along with reduced lipid
peroxidation level as depicted in Table 3. Thus, these
studies showed that previously synthesized compounds
have inhibited the induction of gastric mucosal lesions and
the results further suggested that their protective effect
might be related to the inhibition of lipid peroxidation in
the gastric mucosa.
˚
tance of 2.80 A, indicating the importance of Arg120 as
one of the key residue involved in binding of all classes of
COX inhibitors. Amide carbonyl was found to form
˚
hydrogen bond with Ser530 at a distance of 3.0 A. The
chlorophenyl group of indomethacin was found in the
vicinity of Tyr385, Gly526, Met522, Leu384 and Ala527.
Methoxy group attached to indole nucleus was influenced
with His90 and Ser353 as depicted in Fig. 3b.
Molecular docking studies
Molecular docking of the synthesised compounds in the
active site of COX-2 was performed in order to get further
insight into the nature of interaction between the synthes-
ised compounds and the active site of the receptor to
rationalize the obtained biological results. To establish the
ability of molecular docking to reproduce an experimen-
tally observed ligand-binding mode, the co-crystallized
Docking studies of the synthesised compounds has
identified the key interactions of the compounds with the
receptor. Docking of compound 4h having docked score of
-4.587 has shown interaction between Tyr355 with acid
hydrazone carbonyl as well as with azomethine nitrogen at
2
˚
a distance of 2.45 and 2.69 A, respectively. N -nitrogen of
pyrazole nucleus also showed interaction with Ser530 at a
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Med Chem Res (2012) 21:3646–3655
3653
Table 3 Ulcerogenic index and
Lipid peroxidation of the potent
anti-inflammatory compounds
(4h and 4k)
Compound code Observation
Score Severity index
nmol MDA content
SEM/100 mg tissue
Control
Normal
0
0.0
3.86 0.19**
Normal
0
Normal
0
Normal
0
Normal
0
Normal
0
Indomethacin
1 Ulcer ? H.S. ? redness
2 Ulcers ? spot ulcers ? redness
H.S. ? redness ? spot ulcer
4 Ulcers ? H.S. ? redness
5 Ulcers ? H.S.
5 Ulcers ? H.S. ? redness
5.0
4.5
3.0
5.0
4.5
5.0
4.5 0.316**
21.98 11.14**
Total score
27
4h
Normal
Normal
Redness
Redness
Redness
Normal
0.0
0.0
0.5
0.5
0.5
0.0
1.5
0.5
0.5
0.0
0.5
0.5
0.5
2.5
0.25 0.118** 4.51 1.11*
Total score
4k
Redness
Redness
Normal
Redness
Redness
Redness
0.41 0.080** 5.76 0.10**
n = 6; dose = 10 mg/kg;
* P \ 0.05, ** P \ 0.01,
*** P \ 0.001(compared to
standard). Data was analyzed by
ANOVA, followed by
Bonferroni multiple comparison
test
Total score
respectively whereas the phenoxy oxygen moiety was
˚
showing interaction with His90 at a distance of 2.77 A.
Table 4 Docking scores (kcal/mol) of the docked ligands into active
site of COX-2
The phenyl ring of phenoxy group lies in vicinity of
Ala516, Asp515, Ser353 and Tyr355, while phenyl
attached to N1-nitrogen of pyrazole lies in pocket sur-
rounded by Ser530, Tyr385, Leu352, Tyr355, Gln192, and
Phe518. The further structural details of compounds (4h
and 4k) in receptor site are given in Fig. 3b (C–F).
Compound
S
E_conf E_place
E_score1 E_refine
4h
4k
-4.5875 0.4136 -78.0677 -11.1566 -4.5875
-11.1924 2.0834 -92.6962 -11.0704 -11.1924
Indomethacin -11.9930 2.6307 -92.0469 -12.8779 -11.9930
sc-558 -28.1909 1.0000 -81.0361 -12.4685 -28.1909
S final score, E_conf energy of conformer, E_place score from the
placement stage, E_score1 from rescoring stage, E_refine score from
refinement stage
Conclusion
˚
bond distance of 2.82 A. The chloro phenyl attached to
In summary, two derivatives, i.e., 4h and 4k of all tested
compounds exerted potent anti-inflammatory activity that
is mainly related to the inhibition of COX enzyme as the
result are very good for the second phase of inhibition and
also results are consolidated through further docking
studies of the titled compounds to understand the key
interactions responsible for observed inhibition of COX
enzyme. The compound 4h was found to shown interaction
with Tyr355 and Ser530 while compound 4k interacted
same carbonyl lying in the pocket of Gln192, Ser353,
His90, Ile517 and Ala516 Phenyl group attached to
N1- nitrogen of pyrazole nucleus was surrounded by
Gly526, Tyr385, Ala527, Val349, Leu352 and Trp387.
Docking studies of compound 4k with a docked score of
-11.192, which was almost equal to indomethacin
revealed interaction between acid hydrazone carbonyl with
˚
His90 and Arg513 with a bond distance of 2.86 and 3.16 A,
123

3654
Med Chem Res (2012) 21:3646–3655
Fig. 3 a Key interaction of SC-558 (selective COX-2 inhibitor)
(A) and indomethacin (B) with the COX-2 enzyme active site.
b Docking of compound 4h (C) and 4k (E) in the active site of COX-2
(hydrogen bonds are shown with arrow) and key interaction with the
receptor active site of 4h (D) and 4k (F)
123

Med Chem Res (2012) 21:3646–3655
3655
epileptic drugs in rat experimental models of acute and chronic
pain. Eur J Pharmacol 324:153–160
with His90 and Arg513 with a docking score of -11.1924
which was comparable to indomethacin. Most of the active
compounds were found to be docked well into the active
sites of the receptor. These interactions of the docked
ligand with the receptor can be used further in designing
compounds with better potency and more selectivity
toward COX-2 inhibition. The titled compounds were
found to be non ulcerogenic as indicated by their antiulcer
activity. Thus, keeping in view the whole study, it can be
concluded that these pyrazolyl derivatives can be possibly
used as lead molecules for developing new safer and potent
NSAIDs.
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Acknowledgments The authors are thankful to Hamdard Univer-
sity, New Delhi for generation of FT-IR and elemental analysis data.
The authors are really grateful to CDRI, Lucknow for the generation
of FABMS and to IIT, New Delhi and Hamdard University, New
Delhi for providing the 1H NMR spectra. The authors also record their
appreciation of extraordinary assistance of Lalit kumar of Pharma-
cology Dept., Jamia Hamdard.
Conflict of interest None.
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