Design, synthesis and pharmacological screening of novel nitric oxide donors containing 1,5-diarylpyrazolin-3-one as nontoxic NSAIDs

Article abstract of DOI:10.1016/j.ejmech.2009.06.035

Various substituted 1,5-diarylpyrazol-3-one derivatives were synthesized and screened for analgesic, anti-inflammatory activities, ulcerogenic potential and for their ability to release nitric oxide. Most compounds exhibited significant analgesic and anti

Full text of DOI:10.1016/j.ejmech.2009.06.035

European Journal of Medicinal Chemistry 44 (2009) 4622–4636
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and pharmacological screening of novel nitric oxide donors
containing 1,5-diarylpyrazolin-3-one as nontoxic NSAIDs
*
Shashikant V. Bhandari , Sudarshan C. Dangre, Kailash G. Bothara, Ajit A. Patil, Aniket P. Sarkate,
Deepak K. Lokwani, Suraj T. Gore, Bhavana J. Deshmane, Vyankatesh T. Raparti, Chetan V. Khachane
AISSMS College of Pharmacy, Near RTO, Kennedy Road, Pune 411001, Maharashtra, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Various substituted 1,5-diarylpyrazol-3-one derivatives were synthesized and screened for analgesic,
anti-inflammatory activities, ulcerogenic potential and for their ability to release nitric oxide. Most
compounds exhibited significant analgesic and anti-inflammatory activities. It was interesting to note
that out of ten compounds, 7j (59.64%) was found to have anti-inflammatory activity greater than the
standard drug Indomethacin (57.89%), whereas compound 7b (57.89%) was found to be equipotent to
that of standard, Indomethacin. The pharmacological studies suggested that the presence of 4-nitro and
2-methoxy on phenyl ring at C5 of pyrazole has a significant anti-inflammatory activity and 4-chloro
substitution on same phenyl ring was found to have decreased activity. However only a phenyl
substituted derivative was found to have most potent activity. Compound 7j containing plane phenyl at
C5 of pyrazole was found to have significant analgesic activity (56.86%) in acetic acid induced writhing
model. Compounds 7d and 7i having 4-chloro substituted phenyl ring showed least analgesic activity
(10.78%) and (6.86%) respectively. The compounds also showed significantly reduced GI-ulcerogenicity
and gastroprotective results in histopathological studies i.e. they were found to be causing no mucosal
injury. All the synthesized compounds were found to exhibit significant nitric oxide releasing activity, in
both in vitro and in vivo models. Molecular docking studies served to be an important tool for the study
of binding of compounds with that of a COX-2 enzyme. The results of the docking studies were found to
endorse the result of experimental work. Thus, the rationale used to design the NCEs was found to
produce the promising results as anticipated. Therefore it can be said that the strategy employed can
serve as an important tool in future for the design and development of novel therapeutic agents of
various categories too.
Received 20 September 2008
Received in revised form
6 June 2009
Accepted 29 June 2009
Available online 4 July 2009
Keywords:
1,5-Diarylpyrazolin-3-one
Nitric oxide
Anti-inflammatory
Analgesic
Non-ulcerogenic
Vasodilatory
Docking
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
the currently used NSAIDs, exists in two isoforms (COX-1 and COX-2)
[6]. While COX-1 plays a cytoprotective role [7], COX-2, induced at
The NSAIDs are among the most widely used of all therapeutic
agents. They are useful in the treatment of rheumatoid arthritis and
some inflammatory diseases. However, long term use of the NSAIDs
has been associated with gastrointestinal (GI) toxicities viz. ulcera-
tion, bleeding and nephrotoxicity [1]. Therefore investigation of new
anti-inflammatory agents is still a major challenge [2–4]. Pyr-
azolinone is an important pharmacophore which exhibits wide-
spread pharmacological properties such as analgesic, antipyretic,
antiphlogistic, antirheumatic, antiarthritic and uricosuric activities
[5]. The prostaglandin (PG) synthase (cyclooxygenase), the key
enzyme of inflammatory process, and an important target of most of
the time of injury, causes inflammation, pain, and fever [8]. Thus,
conventional non-steroidal anti-inflammatory drugs (NSAIDs),
being inhibitors of both, exhibit anti-inflammatory activity along
with gastrointestinal (GI) toxicity on extended use [9]. But, the
selective COX-2 inhibitors, viz. Nimesulide [10], Celecoxib [11]
Rofecoxib [12] Valdecoxib [13], and Etoricoxib [14], treat the chronic
rheumatoid and osteoarthritis without causing GI damage. A few
COX-2 inhibitors have also been studied for the treatment of cancer
[15] and Alzheimer’s disease [16]. However a mild cardiac toxicity
associated with COX-2 inhibitors (COXIBs) has raised a cautionary
flag on this research [17]. So, it is desirable to discover safe, potent,
selective and patient-acceptable COX-2 inhibitors to completely
abandon the use of steroidal and narcotic drugs.
Recent strategies adopted to minimize the side effects of NSAIDs
include the use of the dual LOX/COX inhibitors and the use of hybrid
* Corresponding author. Tel.: þ91 9423574082; fax: þ91 20 26058208.
E-mail address: drugdesign1@gmail.com (S.V. Bhandari).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.06.035

S.V. Bhandari et al. / European Journal of Medicinal Chemistry 44 (2009) 4622–4636
4623
approach i.e. molecule made up of non-selective or selective COX
inhibitors together with a vasodilator (nitric oxide releasing)
function [18,19]. Recent data revealed serious cardiovascular side
effects associated with selective COX-2 inhibitors like COXIBs [17].
The strategy involving the use of hybrid molecules made up of non-
selective COX-2 inhibitors together with a nitric oxide donating
moiety constitutes one of the most promising approaches, because
nitric oxide supports several endogenous GI defence mechanisms,
including increase in mucus, bicarbonate secretions, increase in
mucosal blood flow and inhibition of the activation of pro-inflam-
matory cells. Moreover, because of the beneficial cardiovascular
effects of NO, such drugs are expected to be devoid of the potential
adverse cardiovascular effects associated with the use of selective
COX-2 inhibitors. Among those NO-NSAIDs that came into clinical
trials are Nitroaspirin, Nitronaproxene, Nitroketoprofen, Nitro-
ibuprofen, etc. [20].
There are several reviews which are mainly focused on the
molecular and functional bases of the inhibition of COX enzymes by
non-selective and COX-2 selective inhibitors [21,22]. Therefore as
an attempt to optimize the pharmacophore requirement for potent,
nontoxic NO-NSAIDs, we thought worth optimizing structural
requirement for COX-2 binding pocket and to get rid of cardiovas-
cular toxicities associated with COXIBs by incorporating nitric oxide
(NO) releasing function (vasodilator) onto COX-2 selective
pharmacophore.
In this paper we report structure optimization, design, synthesis
and pharmacological screening of some 1,5-diarylpyrazolin-3-one
pharmacophore containing compounds. We have carried out dock-
ing studies using Glide 4.5 [23], docking programme to identify the
binding modes of synthesized derivatives required for the potential
analgesic and anti-inflammatoryactivities. The crystal structure was
downloaded from RCSB protein data bank (PDB ID: 1CX2) [24].
2. Results and discussion
2.1. Chemistry
The synthetic route used to synthesize title compounds is out-
lined in Scheme 1 and Scheme 2. The
a-acylated esters (2a–2j) could
be synthesized, in high yields, by the treatment of ethyl acetoacetate
(1a) or diethylmalonate (1b) with the appropriate acylchloride in the
presence of magnesium/ethanol in anhydrous toluene. Further pyr-
azolinones (3a–3j) were easily and cleanly synthesized from (2a–2j)
by treatment with hydrazine HCl (98%) in ethanol at room temper-
ature. In next step 4-(3-oxo-5-substituted phenyl-2,3-dihydropyr-
azol-1-yl)benzoic acids (4a–4j) were synthesized by refluxing
4-chloro benzoic acid with (3a–3j) for 4 h. The intermediates 4-(3-
oxo-5-substituted phenyl-2,3-dihydropyrazol-1-yl)benzohydrazides
(5a–5j) were further synthesized from (4a–4j) by refluxing thionyl
chloride and hydrazine hydrate for 10 h. Refluxing (5a–5j) with
O
OH
O
COCl
O
O
R1
NH
NH2NH2. HCl (98%)
R1
N
H3C
Mg, EtOH,
CCl4, Toluene
H
R1
EtOH
H3C
OEt
COOEt
2a-e
3a-e
1a
EtOH
Cl
COOH
Reflux 4 h
O
O
NH
O
R1
N
NH
NH
N
R1
R1
SOCl2, Reflux 4h
N
ClCOCH2Cl
Reflux 10 h
NH2NH2, Reflux 6h
6a-e
CONHNHCOCH2Cl
4a-e
5a-e
CONHNH2
COOH
AgNO3, CH3CN
Stirring 3 h
O
NH
R1
N
7a-e
CONHNHCOCH2ONO2
Compound code
1a
Ethyl acetoacetate
-R1
7a
7b
7c
7d
7e
-4-NO2
Ethyl acetoacetate -2-OCH3
Ethyl acetoacetate
Ethyl acetoacetate
Ethyl acetoacetate
-2-OH
-4-Cl
-H
Scheme 1. Synthesis of (7a–7e) using ethyl acetoacetate.

4624
S.V. Bhandari et al. / European Journal of Medicinal Chemistry 44 (2009) 4622–4636
O
O
OH
O
O
O
C2H5O
R1
COCl
NH
R1
C2H5O
N
NH2NH2. HCl (98%)
EtOH
R1
H
C2H5O
OEt
Mg, EtOH,
COOEt
2 f-j
3 f-j
CCl4, Toluene
1b
EtOH
Cl
COOH
O
Reflux 4h
O
O
O
C2H5O
R1
O
NH
O
N
C2H5O
R1
C2H5O
R1
NH
NH
N
SOCl2, Reflux 4h
N
ClCOCH2Cl
Reflux 10 h
NH2NH2, Reflux 6h
6 f-j
CONHNHCOCH2Cl
5 f-j
4 f-j
CONHNH2
COOH
AgNO3, CH3CN
Stirring 3 h
O
O
C2H5O
R1
NH
N
7 f-j
CONHNHCOCH2ONO2
Compound code
1b
-R1
-4-NO2
-2-OCH3
-2-OH
-4-Cl
7f
7g
7h
7i
Diethyl malonate
Diethyl malonate
Diethyl malonate
Diethyl malonate
Diethyl malonate
7j
-H
Scheme 2. Synthesis of (7f–7j) using diethylmalonate.
chloroacetylchloride in the presence of dry benzene and ethylamine
yielded N0-(2-chloroacetyl)4-(3-oxo-5-substituted phenyl-2,3-
dihydropyrazol-1-yl)benzohydrazides (6a–6j). Finally the terminal
chloro-function in (6a–6j) is then converted to target compounds
(7a–7j) with AgNO3 and acetonitrile. The structures of various
synthesized compounds were assigned on the basis of different
chromatographic and spectral studies. The physical data, FTIR, 1H
NMR and mass spectral data for all synthesized compounds are
reported in experimental protocols.
NO2 in the range of 1260–1512. In the proton NMR spectral data, all
protons were seen according to the expected chemical shift and
integral values. The aromatic protons appeared as multiplet peaks
within the range 7.4–8.2
secondary amine of pyrazolinone ring appeared at 6.2
protons resonated as singlet at 4.6 ppm.
d
ppm, singlet signals derived from
d
ppm. Ethyl
d
The detailed results of various physicochemical studies are
presented in experimental protocols.
The FTIR spectra of the final compounds exhibited very similar
features and showed the expected bands for the characteristic
groups which are present in the compounds such as C–H and the
C]N stretching vibrations and another specific stretch for two
Aromatic rings in the range of 3010–3083. Compounds (7a–7j) have
C]O stretching bands in the range 1678–1783. Similarly the
compounds also exhibit the symmetric and asymmetric stretch for
2.2. Pharmacology
In the pharmacological studies, we have investigated anti-
inflammatory and analgesic activities as well as the acute
ulcerogenicity of 1,5-diarylpyrazolin-3-ones derivatives (7a–7j).
Indomethacin was used as a reference standard. The experiments
were performed on albino rats of Wistar strain of either sex,

S.V. Bhandari et al. / European Journal of Medicinal Chemistry 44 (2009) 4622–4636
4625
weighing 120–140 g. The animals were maintained at 25 ꢀ 2 ꢁC,
50 ꢀ 5% relative humidity and 12 h light/dark cycle. The animals
were fasted for 24 h prior to the experiments and water provided
ad libitum. The test compounds were suspended in 1% aqueous
carboxymethyl cellulose (CMC) solution and administered orally to
experimental animals.
because of the presence of NO releasing moiety vasodilation,
mucous production and in turn gastrointestinal protection occur.
Thus, such functionalization of free –COOH of Diclofenac to pyr-
azolinone ring and presence of additional NO releasing function
have resulted into more potent and nontoxic New Chemical Entities
(NCEs). The results of ulcerogenicity studies are presented in Table 3.
2.2.1. Anti-inflammatory activity
2.2.4. Histopathological studies
Anti-inflammatory activity of the synthesized compounds was
evaluated by carrageenan induced rat paw oedema model, at
equimolar doses, to that of 6 mg/kg p.o of Indomethacin [25].
Subplantar injection of 0.1 mL,1% carrageenan produced increase in
paw volume (oedema) in all the animals of various groups. The
onset of action was evident from 1 h in various test groups. The
significant reduction (P < 0.01) of rat paw oedema was observed by
most of the test compounds at 3 h compared to control group
(Table 1).
The stomach specimen of diclofenac acid treated rats was char-
acterized by complete disruption of protective mucosal layer (Fig. 1
specimen b). Histopathological analyses also showed characteristic
features of ulceration in diclofenac acid treated group of animals.
The tissue of diclofenac acid treated rats has shown that some
epithelial cells in the ulcer margin had proliferated and migrated
over and into the ulcer crater, which was strongly infiltrated by
inflammatory cells, fibroblasts and endothelial cells indicating
complete disruption of gastric epithelial layer. Scanning of stomach
specimens using electron microscope revealed that in the rats
treated with 1,5-diarylpyrazolinone derivatives (7a, 7b, 7g, 7j) there
was no injury observed in stomach mucosa. As illustrated in Fig. 1,
specimens c–f which are identical to that of the control, specimen a.
2.2.2. Analgesic activity
The analgesic activities of the compounds were studied by using
acetic acid induced writhing test in mice. The analgesic activity was
evaluated at equimolar doses equivalent to 6 mg/kg p.o (Indo-
methacin) body weight. These compounds exhibited an important
analgesic profile measured by the classical acetic acid induced
writhing model. From the results of acetic acid induced writhing
test, it was noticed that all compounds exhibited significant anal-
gesic activity (Table 2). The analgesic effects of (7j) (56.86%) were
found to be better than that of Indomethacin (54.90%).
2.2.5. Vasorelaxing activity
In isolated Wistar rat aorta rings, compounds 7a–7j competi-
tively inhibited norepinephrine-induced contraction effects, causing
a shift to the right of the norepinephrine concentration response
curves. EC50
(mg/mL) values were calculated from the cumulative
concentration (dose) response curves. In order to prove the
involvement of nitric oxide in the relaxation process, the abilities of
the test compounds to release NO were ensured by assessing nitric
oxide releasing properties of synthesized compounds in phosphate
buffer, pH 7.4, in the presence of L-cysteine, relative to nitric oxide
released from standard sodium nitrite solution (Table 4).
2.2.3. Acute ulcerogenicity studies
Compounds with significant anti-inflammatory profile were
subjected to ulcerogenicity potential test at 12 times the thera-
peutic dose of diclofenac with additional physical (cold) stress for
2 h at ꢂ20 ꢁC. A thorough examination of the results of histo-
pathological studies indicated the absence of the disruption of
gastric epithelial morphology and absence of ulcers/erosion in test
group animals compared to reference standard, Diclofenac acid,
and control group animals. The results of the ulcerogenicity studies
are presented in Table 3 and results of histopathological studies are
depicted in Fig. 1a–f.
Ulcerogenic effect of 1,5-diarylpyrazolin-3-one derivatives (7a,
7b, 7g, 7j) in animal efficacy model was evaluated for gastric
ulcerogenic potential in rat stress model. When compared with
Diclofenac acid, these four compounds did not cause any gastric
ulceration and disruption of gastric epithelial cells at the above-
mentioned oral doses. Hence gastric tolerance to these compounds
was better than that of Diclofenac acid. This led us to conclude that
3. Docking studies
The molecular docking tool, Glide (Schro¨dinger Inc. USA) soft-
ware was used for ligand docking studies in the enzyme (COX-2)
binding pocket (PDB Code 1CX2) [26].
Glide is one of the most accurate docking programmes available
for ligand–protein, protein–protein binding studies. Glide was
found to produce least number of inaccurate poses and 85% of
Glides binding models had an RMSD of 1.4 Å or less from native co-
crystallized structures [27]. Glide is validated software designed for
calculating the accurate binding interaction energies of the 3D
structures of a known receptor protein with ligand or another
protein molecule [28].
Table 1
Results of anti-inflammatory activity of title compounds (7a–7j) against carrageenan induced rat paw oedema model in rats.
Mean reduction in paw volume (mL) after treatment with test compounds (mean ꢀ SEM) Anti-inflammatory activity (% inhibition)
Compounds
1 h
1.868 ꢀ 0.07
2 h
3 h
1 h
2 h
3 h
Control
Indomethacin
1.922 ꢀ 0.07
0.808 ꢀ 0.07
1.402 ꢀ 0.03
0.85 ꢀ 0.05
1.556 ꢀ 0.01
1.704 ꢀ 0.04
1.614 ꢀ 0.07
1.388 ꢀ 0.08
1.33 ꢀ 0.02
1.412 ꢀ 0.02
1.724 ꢀ 0.04
0.814 ꢀ 0.08
1.71 ꢀ 0.04
–
–
–
0.888 ꢀ 0.04
1.436 ꢀ 0.03
0.846 ꢀ 0.06
1.444 ꢀ 0.04
1.764 ꢀ 0.06
1.504 ꢀ 0.08
1.35 ꢀ 0.04
0.7224 ꢀ 0.09
1.3 ꢀ 0.05
52.68**
23.56**
55.08**
22.99**
5.88ns
19.78**
27.80**
31.01**
27.80**
3.20ns
58.33**
27.08**
55.72**
19.27**
11.45ns
16.14**
28.12**
30.72**
26.56**
10.41ns
57.81**
57.89**
23.97**
57.89**
16.37*
5.84ns
20.46**
22.22**
25.14**
21.05**
6.43ns
59.64**
7a
7b
7c
7d
7e
7f
7g
7h
7i
0.728 ꢀ 0.08
1.43 ꢀ 0.03
1.616 ꢀ 0.04
1.368 ꢀ 0.05
1.332 ꢀ 0.05
1.288 ꢀ 0.02
1.354 ꢀ 0.04
1.604 ꢀ 0.05
0.69 ꢀ 0.07
1.296 ꢀ 0.01
1.356 ꢀ 0.04
1.8126 ꢀ 0.07
0.86 ꢀ 0.08
7j
54.01**
Data analyzed by one-way ANOVA followed by Dunnett’s test, (n ¼ 6), *P < 0.05, **P < 0.01 significant from control; ns, not significant.

4626
S.V. Bhandari et al. / European Journal of Medicinal Chemistry 44 (2009) 4622–4636
Table 2
Results of analgesic activity of title compounds (7a–7j) against acetic acid induced writhing tests in mice.
Compounds
Dose (mg/kg, p.o) (0.00001911 mol/kg)
No. of writhes in 25 min after treatment (mean ꢀ SEM)
% Inhibition
Control
Standard
–
20.4 ꢀ 0.74
9.2 ꢀ 0.37
11.2 ꢀ 0.58
9.80 ꢀ 0.66
17 ꢀ 0.83
18.2 ꢀ 1.02
9.4 ꢀ 0.6
–
6.0
54.9**
45.09**
51.96**
16.66*
10.78ns
53.92**
45.9**
50.98**
10.78ns
6.86ns
56.86**
7a
7b
7c
7d
7e
7f
7g
7h
7i
7.41
7.16
6.93
7.24
6.66
8.62
8.37
8.13
8.44
15.73
11.2 ꢀ 0.86
10 ꢀ 0.7
18.2 ꢀ 1.15
19 ꢀ 0.83
8.8 ꢀ 0.66
7j
Data analyzed by one-way ANOVA followed by Dunnett’s test, (n ¼ 6), **P < 0.01 significant from control.
The Glide docking programme approximated
a
complete
It is well established and accepted fact that number of good van
der Waals interactions decides the binding affinity for any ligand
with receptor/enzyme protein. Therefore we have analyzed the
binding modes and abilities, taking into consideration the number
of vdw contacts.
The designed compounds 7a–7e and 7i showed good vdw
contacts i.e. 470, 455, 437, 426, 411 and 593 respectively, as
compared to that of a standard Indomethacin and Celecoxib
showing good vdw of 414 and 394 respectively. While the ugly
vdw contacts were found to be 3, 5, 1, 3, 6 and 2 for designed
compounds 7a–7f respectively, as compared to that of a stan-
dard Indomethacin and Celecoxib showing ugly vdw of 2 and 1
respectively.
These contacts reveal the importance of functional group which
can form good vdw interaction with the receptor and accordingly
the ligand molecules (NCE’s) can be designed and synthesized.
Standard drug Indomethacin as well as molecules 7c and 7e
docked into COX-2 binding pocket is shown in Figs. 2–5.
It was indicated from docking results that all designed and
synthesized ‘1,5-diarylpyrazolin-3-one’ derivatives show common
binding mode i.e. hydrogen bonding in the binding pockets of COX-
2 enzyme (PDB Code 1CX2) when compared with standard Cele-
coxib and Indomethacin. Thus when compounds were docked into
COX-2 enzyme, Celecoxib shows hydrogen binding with Arg120
and His90 whereas Indomethacin was docked by showing binding
to Arg120. Also all 1,5-diarylpyrazolin-3-one derivatives show
a common hydrogen binding to Arg120 and His90 indicating the
binding affinity of compounds to COX-2 enzyme.
systematic search of the conformational, orientational and posi-
tional space of the docked ligand molecules into the receptor
(protein) binding pocket [29].
The results of molecular docking studies of 1,5-diarylpyrazolin-
3-one containing compounds are presented in (Table 5).
The docking results were evaluated based on following aspects.
3.1. G score (Glide Score)
The Gscore of the standard compounds Celecoxib, SC-558, Indo-
methacin, Ibuprofenwere found to be ꢂ11.37, ꢂ8.36, ꢂ7.37 and ꢂ4.38
respectively. While the G score for the designed series of compounds
7a–7c and 7h were found to be ꢂ9.10, ꢂ9.37, ꢂ9.26 and ꢂ8.90
respectively. While the G score for compounds 7d–7h and 7j were
found to be ꢂ8.41, ꢂ8.38, ꢂ8.10, ꢂ8.00, ꢂ7.80 and ꢂ7.77 respectively.
The close analysis of those results suggests that the designed
series of compounds have G score comparable with that of a stan-
dard compound Celecoxib and is more when compared with the
Indomethacin, Ibuprofen and COX-2 selective prototype SC-558.
3.2. H-bond
H-bond is one of the most widely used alternative parameters
for the evaluation of docking results. It is an influential parameter
in deciding selective binding of compounds. The H-bond for
designed compounds 7a–7e was found to be 2, 1, 3, 2 and 2
respectively. While for standard Indomethacin, SC-558, Celecoxib,
Ibuprofen it was found to be 1, 2, 2, and 1 respectively.
From the above studies it was found that the functional group
–CONHNHCOCH2ONO2 moiety forms good H-bond interaction
revealing its importance in the compound.
4. Conclusions
Various substituted 1,5-diarylpyrazolin-3-one derivatives were
synthesized and screened for analgesic, anti-inflammatory and
ulcerogenic potential. Most compounds exhibited significant anal-
gesic and anti-inflammatory activities. Compound (7j) which is
benzene analogue shows strong analgesia in acetic acid induced
writhing tests. Among all the synthesized compounds, compounds
(7b) and (7j) exhibited most prominent and consistent anti-
inflammatory activity. From the detailed analysis of the results of
histopathological studies, we conclude that the synthesized
compounds have not only retained the anti-inflammatory profile of
Indomethacin but also have helped in enhancing the anti-inflam-
matory activity and are devoid of the deadlier gastrointestinal
toxicities. Also, the results of docking studies were found to be
comparable with that of an experimental work. Thus, the strategy
employed can serve as an important tool in future for the design,
synthesis and development of novel NSAIDs with greater selec-
tivity, potency and minimal side effects.
3.3. Contacts (Van der Waals (vdw) Interaction)
The contacts are represented in the form of
ꢃ Good vdw Contact.
ꢃ Bad vdw Contact.
ꢃ Ugly vdw Contact.
Table 3
Ulcerogenic effects of synthesized compounds in comparison with diclofenac acid.
Compounds
Dose (mg/kg, p.o)
Ratio of ulcerated
animals
Ulcer index
(mean ꢀ SEM)
Diclofenac
24
6/6
–
–
–
–
2.3 ꢀ 0.2
7a
7b
7g
7j
33.70
34.84
40.71
38.27
–
–
–
–

S.V. Bhandari et al. / European Journal of Medicinal Chemistry 44 (2009) 4622–4636
4627
Fig. 1. Haematoxylin and eosin immunohistochemical staining of gastric ulcers after ulcer induction in rats. As illustrated in figure specimen (a) shows intact mucous membrane in
control treated rat showing granular tissues composed of macrophages, fibroblasts and endothelial cells forming microvessels. Congestion of mucosal blood vessels in diclofenac
treated group, specimen (b). No damage was seen to mucosa of rat treated with test compounds – 7a specimen (c), 7b specimen (d), 7g specimen (e), and 7j specimen (f), these
specimens c–f were identical to that of the control, specimen (a). Original magnification 200ꢄ.
Table 5
Results of extra precision docking studies of 1,5-diarylpyrazolin-3-one series of
compounds (7a–7j) along with selective COX-2 inhibitors.
Sr. No. Title
GScore Energy Hbnd Good vdw Bad vdw Ugly vdw
Table 4
1
2
3
4
5
6
7
8
Celecoxib
7b
7c
7a
7i
7d
7e
SC-558
7f
7g
7h
7j
ꢂ11.37 ꢂ43.7
ꢂ9.37 ꢂ38.7
ꢂ9.26 ꢂ34.2
ꢂ9.10 ꢂ41.7
ꢂ8.10 ꢂ35.7
ꢂ8.41 ꢂ32.7
ꢂ8.38 ꢂ33.7
ꢂ8.36 ꢂ34.0
ꢂ8.10 ꢂ34.7
ꢂ8.00 ꢂ37.8
ꢂ7.80 ꢂ32.4
ꢂ7.77 ꢂ31.00
4
1
3
2
2
2
2
2
1
2
1
2
1
1
394
455
437
470
593
426
411
398
490
414
427
438
414
220
10
37
28
29
41
26
23
10
10
23
11
10
8
1
5
1
3
2
3
6
4
3
6
5
6
2
3
EC50 and nitric oxide releasing properties of the compounds (7a–7j).
Compound code
EC50
% NO releasea (%)
7a
7b
7c
7d
7e
7f
7g
7h
7i
49.11
66.85
17.82
21.32
26.74
28.18
63.09
70.79
50.18
35.48
0.39
0.32
0.69
0.66
0.51
0.49
0.35
0.27
0.37
0.46
9
10
11
12
13
14
Indomethacin ꢂ7.37 ꢂ33.10
Ibuprofen
ꢂ4.38 ꢂ33.15
7j
8
a
Percentage of NO released (n ¼ 2) relative to a theoretical maximum release of
1 mol NO/mol of test compound; determined by Griess reagent in the presence of
Hbnd ¼ No. of hydrogen bonds. Bad vdw ¼ No. of bad van der Waals contacts. Ugly
vdw ¼ No. of ugly van der Waals contacts.
5 mM L-cysteine, at pH 7.4.

4628
S.V. Bhandari et al. / European Journal of Medicinal Chemistry 44 (2009) 4622–4636
Fig. 2. Molecule celecoxib docked into COX-2 binding pocket.
5. Experimental
plates using silica gel G as stationary phase and was visualised
using iodine vapours or under UV chambers.
5.1. Synthetic studies
5.2. General procedure for the preparation of a-acylated (i) ethyl
All the reactions were carried out with dry, freshly distilled
solvents under anhydrous conditions, unless otherwise noted.
Melting points were determined by open capillary tubes and were
uncorrected. FTIR spectra of the powdered compounds were
recorded using KBr on a Jasco FTIR V 430þ spectrophotometer
using Diffuse Reflectance Attachment and are reported in cmꢂ1 and
1H NMR spectra were recorded on a Varian Mercury YH300
(300 MHz FT NMR) spectrophotometer using TMS as an internal
acetoacetate derivatives (2a–2e) (ii) diethylmalonate derivatives
(2f–2j) [30]
A mixture of ethyl acetoacetate (1a,12.0 mmol) or diethylmalonate
(1b, 12.0 mmol), magnesium (12.1 mmol), ethanol (40.0 mmol), CCl4
(0.3 mL) and anhydrous toluene (30 mL) was stirred at room
temperature for 30 min. The mixture was refluxed for 1 h, and then
cooled to 0–5 ꢁC. The acylating agent (12.1 mmol) was added dropwise
to the solution at 0–5 ꢁC over 30 min, and the reaction mixture was
stirred at room temperature for 1 h. The resulting mixture was re-
cooled to 0–5 ꢁC and washed with cold 5% aq. HCl solution (25 mL),
reference (Chemical shift represented in d ppm). Mass spectra were
recorded on GC–MS QP5050A System (benchtop quadrupole mass
spectrophotometer). Purity of the compounds was checked on TLC
Fig. 3. Molecule Indomethacin docked into COX-2 binding pocket.

S.V. Bhandari et al. / European Journal of Medicinal Chemistry 44 (2009) 4622–4636
4629
Fig. 4. Molecule 7c docked into COX-2 binding pocket (showing H-bond interaction with the amino acid residues).
satd NaHCO3 solution (25 mL) and brine (25 mL). The organic layer
was dried over anhydrous MgSO4 and concentrated under reduced
pressure to give pale yellow liquids (2a–2j).
(CH, Ar),; mass: m/z 264 (Mþ), 265 (M þ 1, 15.3%), 266 (M þ 2, 11%);
1H NMR (300 MHz, CDCl3),1.75 (s, 3H, CH3),14.88 (s,1H, OH), 3.21 (q,
2H, CH2),1.41 (t, 3H, CH3), 7.32–7.63 (m, 4H, ArH), 3.73 (s, 3H, OCH3).
5.2.1. Ethyl-3-hydroxy-2-(4-nitrobenzoyl)but-2-enoate (2a)
bp 205–207 ꢁC; yield 85%; FTIR (KBr) cmꢂ1: 3498 (Aliphatic OH),
1737 (C]O of ketone), 1798 (C]O, ester), 1315, 1456 (NO2), 2925
(CH alkane), 3035 (CH, Ar); mass: m/z 279 (Mþ), 280 (M þ 1, 15.3%),
5.2.3. Ethyl-3-hydroxy-2-(2-hydroxybenzoyl)but-2-enoate (2c)
bp 245–247 ꢁC; yield 78%; FTIR (KBr) cmꢂ1: 3476 (Aliphatic OH),
1745 (C]O of ketone), 1753 (C]O, ester), 2930 (CH alkane), 3050
(CH, Ar), 3542 (Phenolic OH); mass: m/z 250 (Mþ), 251 (M þ 1, 14%),
281 (M þ 2, 2.2%); 1H NMR (300 MHz, CDCl3,
d
ppm), 1.65 (s, 3H,
252 (M þ 2, 4%); 1H NMR (300 MHz, CDCl3,
d ppm),1.71 (s, 3H, CH3),
CH3), 14.12 (s, 1H, OH), 4.19 (q, 2H, CH2), 1.7 (t, 3H, CH3), 7.32 (d, 2H,
ArH), 8.07 (d, 2H, ArH).
15.01 (s, 1H, OH), 4.19 (q, 2H, CH2), 1.5 (t, 3H, CH3), 7.02–7.47 (m, 4H,
ArH) 8.98 (s, 1H, Phenolic OH).
5.2.2. Ethyl-3-hydroxy-2-(2-methoxybenzoyl)but-2-enoate (2b)
bp 210–212 ꢁC; yield 87%; FTIR (KBr) cmꢂ1: 3487 (Aliphatic OH),
1739 (C]O of ketone), 1729 (C]O, ester), 2947 (CH alkane), 3085
5.2.4. Ethyl-3-hydroxy-2-(4-chlorobenzoyl)but-2-enoate (2d)
bp 250–252 ꢁC; yield 67%; FTIR (KBr) cmꢂ1: 3480 (Aliphatic OH),
1791 (C]O of ketone), 1711 (C]O, ester), 2895 (CH alkane), 3044
Fig. 5. Molecule 7e docked into COX-2 binding pocket.

4630
S.V. Bhandari et al. / European Journal of Medicinal Chemistry 44 (2009) 4622–4636
(CH, Ar), 669 (C–Cl); mass: m/z 268.5 (Mþ), 269.5 (M þ 1, 14.2%),
5.3.1. 5-(4-Nitrophenyl)-1,2-dihydropyrazol-3-one (3a)
270.5 (M þ 2, 32.8%); 1H NMR (300 MHz, CDCl3,
d
ppm), 1.84 (s, 3H,
mp 221–223 ꢁC; yield 80%; FTIR (KBr) cmꢂ1: 3385 (NH), 1629
(C]O,), 3087 (CH, Ar), 1453, 1345 (NO2); mass: m/z 205 (Mþ), 206
CH3), 14.34 (s, 1H, OH), 3.79 (q, 2H, CH2), 1.9 (t, 3H, CH3), 7.28 (d, 2H,
ArH), 7.51 (d, 2H, ArH).
(M þ 1, 11.2%); 1H NMR (300 MHz, CDCl3,
d ppm), 8.3 (s, 1H, NH),
7.81 (s, 1H, NH), 5.86 (s, 1H, CH), 7.59–7.86 (m, 4H, ArH).
5.2.5. Ethyl-2-benzoyl-3-hydroxy but-2-enoate (2e)
bp 279–281 ꢁC; yield 90%; FTIR (KBr) cmꢂ1: 3499 (Aliphatic OH),
1711 (C]O of ketone), 1735 (C]O, ester), 2873 (CH alkane), 3077
(CH, Ar), 1022 (C–O); mass: m/z 234 (Mþ), 235 (M þ 1, 4%); 1H NMR
5.3.2. 5-(2-Methoxyphenyl)-1,2-dihydropyrazol-3-one (3b)
mp 170–171 ꢁC; yield 82%; FTIR (KBr) cmꢂ1: 3356 (NH), 1759
(C]O,), 3062 (CH, Ar); mass: m/z 190 (Mþ), 191 (M þ 1, 11.2%); 1H
(300 MHz, CDCl3,
d
ppm), 1.54 (s, 3H, CH3), 14.77 (s, 1H, OH), 3.7 (q,
NMR (300 MHz, CDCl3, d ppm), 8.0 (s, 1H, NH), 7.87 (s, 1H, NH), 5.36
2H, CH2), 1.8 (t, 3H, CH3), 7.49–7.81 (m, 5H, ArH).
(s, 1H, CH), 7.59–7.86 (m, 4H, ArH), 3.73 (s, 3H, OCH3).
5.2.6. Ethyl-3-ethoxy-3-hydroxy-2-(4-nitrobenzoyl)acrylate (2f)
bp 270–272 ꢁC; yield 92%; FTIR (KBr) cmꢂ1: 3489 (Aliphatic OH),
1739 (C]O of ketone), 1729 (C]O, ester), 1175 (C–O), 2917 (CH
alkane), 3054 (CH, Ar), 1455, 1328 (NO2); mass: m/z 309 (Mþ), 310
5.3.3. 5-(2-Hydroxyphenyl)-1,2-dihydropyrazol-3-one (3c)
mp 147–149 ꢁC; yield 66%; FTIR (KBr) cmꢂ1: 3387 (NH), 3545
(Phenolic OH), 1665 (C]O), 3021 (CH, Ar); mass: m/z 176 (Mþ), 177
d ppm), 7.98 (s, 1H, NH),
7.80 (s, 1H, NH), 5.53 (s, 1H, CH), 6.57–7.16 (m, 4H, ArH), 8.73 (s, 1H,
Phenolic OH).
(M þ 1, 10.6%); 1H NMR (300 MHz, CDCl3,
(M þ 1,15%), 311 (M þ 2, 2%); 1H NMR (300 MHz, CDCl3,
d
ppm),1.33
(t, 3H, CH3), 4.12 (q, 2H, CH2), 15.0 (s, 1H, OH), 3.9 (q, 2H, CH2), 2.1 (t,
3H, OCH3), 7.49–7.81 (m, 4H, ArH).
5.3.4. 5-(4-Chlorophenyl)-1,2-dihydropyrazole-3-one (3d)
5.2.7. Ethyl-3-ethoxy-3-hydroxy-2-(2-methoxybenzoyl)acrylate (2g)
bp 277–279 ꢁC; yield 94%; FTIR (KBr) cmꢂ1: 3508 (Aliphatic OH),
1739 (C]O of ketone), 1729 (C]O, ester), 1066 (C–O), 2908 (CH
alkane), 3087 (CH, Ar); mass: m/z 294 (Mþ), 295 (M þ 1, 16%), 296
mp 227–229 ꢁC; yield 65%; FTIR (KBr) cmꢂ1: 3379 (NH), 1703
(C]O), 3087 (CH, Ar) 747 (C–Cl); mass: m/z 194.5 (Mþ), 195.5
(M þ 1, 9.9%), 196.5 (M þ 2, 32%); 1H NMR (300 MHz, CDCl3,
d ppm),
8.17 (s, 1H, NH), 7.93 (s, 1H, NH), 5.21 (s, 1H, CH), 7.22–7.36 (m, 4H,
ArH).
(M þ 2, 1.3%); 1H NMR (300 MHz, CDCl3,
d ppm), 15.5 (s, 1H, OH),
3.89 (q, 2H, CH2), 1.43 (t, 3H, CH3), 6.96–7.47 (m, 4H, ArH), 3.77 (s,
3H, OCH3), 1.23 (t, 3H, CH3), 4.11 (q, 2H, CH2).
5.3.5. 5-Phenyl-1,2-dihydropyrazol-3-one (3e)
mp 187–189 ꢁC; yield 70%; FTIR (KBr) cmꢂ1: 3369 (NH), 1613
(C]O), 3067 (CH, Ar); mass: m/z 160 (Mþ), 170 (M þ 1, 9.9%); 1H
5.2.8. Ethyl-3-ethoxy-3-hydroxy-2-(2-hydroxybenzoyl)acrylate (2h)
bp 217–219 ꢁC; yield 86%; FTIR (KBr) cmꢂ1: 3482 (Aliphatic OH),
1709 (C]O of ketone), 1749 (C]O, ester), 1076 (C–O), 3533, 3509,
(Phenolic OH), 2918 (CH alkane), 3066 (CH, Ar); mass: m/z 280 (Mþ),
NMR (300 MHz, CDCl3,
d ppm), 7.90 (s, 1H, NH), 7.73 (s, 1H, NH),
4.98 (s, 1H, CH), 7.30–7.52 (m, 5H, ArH).
281(M þ 1,15%), 282(M þ 2,2.4%); 1HNMR(300 MHz, CDCl3,
d
ppm),
5.3.6. Ethyl-5-(4-nitrophenyl) 3-oxo-2,3-dihydro-1H-pyrazol-4-
carboxylate (3f)
15.56 (s,1H, OH), 3.92 (q, 2H, CH2),1.43 (t, 3H, CH3), 6.96–7.47 (m, 4H,
ArH), 8.73 (s, 1H, Phenolic OH), 1.23 (t, 3H, CH3), 4.11 (q, 2H, CH2).
mp 170–171 ꢁC; yield 88%; FTIR (KBr) cmꢂ1: 3364 (NH), 1613
(C]O), 2943 (CH, Aliphatic), 3067 (CH, Ar), 1076 (C–O); mass: m/z
277 (Mþ), 278 (M þ 1, 14.3%), 279 (M þ 2, 1.2%); 1H NMR (300 MHz,
5.2.9. Ethyl-3-ethoxy-3-hydroxy-2-(4-chlorobenzoyl)acrylate (2i)
bp 236–238 ꢁC; yield 80%; FTIR (KBr) cmꢂ1: 3458 (Aliphatic OH),
1719 (C]O of ketone), 1732 (C]O, ester), 1116 (C–O), 2948 (CH
alkane), 3032 (CH, Ar), 683 (C–Cl); mass: m/z 298.5 (Mþ), 299.5
(M þ 1, 15.1%), 300.5 (M þ 2, 32%); 1H NMR (300 MHz, CDCl3,
CDCl3,
d ppm), 8.09 (s, 1H, NH), 7.79 (s, 1H, NH), 7.33–7.82 (m, 4H,
ArH), 4.19 (q, 2H, CH2), 1.73 (t, 3H, CH3).
5.3.7. Ethyl-5-(2-methoxyphenyl) 3-oxo-2,3-dihydro-1H-pyrazol-
4-carboxylate (3g)
d
ppm), 1.60 (t, 3H, CH3), 3.26 (q, 2H, CH2), 14.60 (s, 1H, OH), 4.08 (q,
2H, CH2), 1.76 (t, 3H, CH3), 6.91–7.36 (m, 4H, ArH).
mp 217–219 ꢁC; yield 84%; FTIR (KBr) cmꢂ1: 3345 (NH), 1643
(C]O), 2927 (CH, Aliphatic), 3036 (CH, Ar), 1177 (C–O); mass: m/z
262 (Mþ), 263 (M þ 1, 14.4%), 264 (M þ 2, 1.9%); 1H NMR
5.2.10. Ethyl-2-benzoyl-3-ethoxy-3-hydroxyacrylate (2j)
bp 291–294 ꢁC; yield 85%; FTIR (KBr) cmꢂ1: 3439 (Aliphatic OH),
1703 (C]O of ketone), 1746 (C]O, ester), 1106 (C–O), 2938 (CH
alkane), 3082 (CH, Ar) mass: m/z 264 (Mþ), 265 (M þ 1, 15.3%), 266
(300 MHz, CDCl3, d ppm), 8.1 (s, 1H, NH), 7.90 (s, 1H, NH), 6.72–
7.23 (m, 4H, ArH), 3.29 (q, 2H, CH2), 1.53 (t, 3H, CH3), 3.59 (s, 3H,
OCH3).
(M þ 2, 1.1%); 1H NMR (300 MHz, CDCl3,
d ppm), 1.56 (t, 3H, CH3),
3.65 (q, 2H, CH2),15.19 (s,1H, OH), 4.13 (q, 2H, CH2),1.76 (t, 3H, CH3),
7.09–7.86 (m, 5H, ArH).
5.3.8. Ethyl-5-(2-hydroxyphenyl) 3-oxo-2,3-dihydro-1H-pyrazol-4-
carboxylate (3h)
mp 223–225 ꢁC; yield 76%; FTIR (KBr) cmꢂ1: 3348 (NH), 1613
(C]O), 2937 (CH, Aliphatic), 3067 (CH, Ar), 1123 (C–O); mass: m/z
248 (Mþ), 249 (M þ 1, 13.2%); 1H NMR (300 MHz, CDCl3,
d ppm),
5.3. General procedure for the preparation of 5-phenyl 1,2-
dihydropyrazol-3-one (3a–3j) [30]
8.03 (s, 1H, NH), 7.83 (s, 1H, NH), 6.72–7.23 (m, 4H, ArH), 3.29 (q, 2H,
CH2), 1.53 (t, 3H, CH3), 9.53 (s, 1H, Phenolic OH).
To a stirred solution of the
a
-acylated ethyl acetoacetate
5.3.9. Ethyl-5-(4-chlorophenyl) 3-oxo-2,3-dihydro-1H-pyrazol-4-
carboxylate (3i)
(5.0 mmol) in ethanol (10 mL), hydrazine 0.38 g (7.5 mmol, 98%)
was added dropwise at room temperature. The reaction mixture
was stirred at room temperature for 16 h. The mixture was
concentrated under reduced pressure, and the residue was treated
with diethyl ether (10 mL). The resulting solid was filtered and
washed with diethyl ether and recrystallized from 10% ethanol in
diethyl ether to give white crystals.
mp 288–290 ꢁC; yield 84%; FTIR (KBr) cmꢂ1: 3341 (NH),
1673 (C]O), 2951 (CH, Aliphatic), 3091 (CH, Ar), 1158 (C–O),
747 (C–Cl); mass: m/z 266.5 (Mþ), 267.5 (M þ 1, 13.2%), 268.5
(M þ 2, 32%); 1H NMR (300 MHz, CDCl3,
d ppm), 8.21 (s, 1H,
NH), 7.89 (s, 1H, NH), 7.22–7.24 (m, 4H, ArH), 3.29 (q, 2H, CH2),
1.53 (t, 3H, CH3).

S.V. Bhandari et al. / European Journal of Medicinal Chemistry 44 (2009) 4622–4636
4631
5.3.10. Ethyl-3-oxo-5-phenyl-2,3-dihydro-1H-pyrazol-4-
carboxylate (3j)
3022 (CH, Ar), 1720 (acid C]O), 1376, 1498 (NO2); mass: m/z 397
(Mþ), 398 (M þ 1, 21.7%), 399 (M þ 2, 3.5%); 1H NMR (300 MHz,
mp 257–259 ꢁC; yield 84%; FTIR (KBr) cmꢂ1: 3385, (NH), 1711
(C]O), 1094 (C–O), 2940 (CH, Aliphatic), 3033 (CH, Ar); mass: m/z
232 (Mþ), 233 (M þ 1, 13.2%), 234 (M þ 2, 1.5%); 1H NMR (300 MHz,
CDCl3, d ppm), 4.11 (q, 2H, CH2), 1.43 (t, 3H, CH3), 7.92 (s, 1H, NH),
6.61–7.81 (m, 8H, ArH), 11.86 (s, 1H, acid OH).
CDCl3,
d
ppm), 7.99 (s, 1H, NH), 7.68 (s, 1H, NH), 7.30–7.53 (m, 5H,
5.4.7. 4-(5-(2-Methoxyphenyl)-4-(ethoxycarbonyl)-3-oxo-2,3-
dihydropyrazol-1-yl) benzoic acid (4g)
ArH), 3.29 (q, 2H, CH2), 1.53 (t, 3H, CH3).
mp. 280–282 ꢁC; yield 82%; FTIR (KBr) cmꢂ1: 3338, 879 (NH),
1736 (C]O), 1529 (C]N), 1027 (C–O), 3543 (OH), 2988 (CH, Alkyl),
3011 (CH, Ar), 1719 (acid C]O); mass: m/z 382 (Mþ), 383 (M þ 1,
5.4. General procedure for the preparation of 4-(3-oxo-5-phenyl-
2,3-dihydropyrazol-1-yl)benzoic acid (4a–4j) [31]
22.1%), 384 (M þ 2, 3.7%); 1H NMR (300 MHz, CDCl3,
d ppm), 3.93 (q,
Equimolar amounts of intermediate 3 (1 g, 0.003 M) and 4-
chloro benzoic acid (0.46 g 0.003 M) were refluxed in ethanol
(10 mL) for 4 h. After completion of 3 h from starting of reflux
NaHCO3 (0.252 g, 0.003 M) was added in reaction mixture to
increase rate of forward reaction. After 4 h the reaction mixture was
cooled, ethanol was evaporated to yield product 4.
2H, CH2), 1.62 (t, 3H, CH3), 8.07 (s, 1H, NH), 6.39–7.52 (m, 8H, ArH),
12.02 (s, 1H, acid OH), 3.22 (s, 1H, OCH3).
5.4.8. 4-(5-(2-Hydroxyphenyl)-4-(ethoxycarbonyl)-3-oxo-2,3-
dihydropyrazol-1-yl) benzoic acid (4h)
mp. 215–217 ꢁC; yield 75%; FTIR (KBr) cmꢂ1: 3313, 815 (NH),
1729 (C]O), 1534 (C]N), 1170 (C–O), 3511 (OH), 2952 (CH, Alkyl),
3045 (CH, Ar), 1719 (acid C]O), 3505, 3158 (OH); mass: m/z 368
(Mþ), 369 (M þ 1, 21.3%), 370 (M þ 2, 3.3%); 1H NMR (300 MHz,
5.4.1. 4-(5-(4-Nitrophenyl)-3-oxo-2,3-dihydropyrazol-1-yl)benzoic
acid (4a)
mp. 207–209 ꢁC; yield 78%; FTIR (KBr) cmꢂ1: 3299, 826 (NH),
1763 (C]O), 1590 (C]N), 1090 (C–O), 3410 (OH), 1491, 1374 (NO2),
3033 (CH, Ar), 1702 (acid C]O); mass: m/z 325 (Mþ), 326 (M þ 1,
CDCl3, d ppm), 4.32 (q, 2H, CH2), 1.53 (t, 3H, CH3), 8.15 (s, 1H, NH),
6.97–7.70 (m, 8H, ArH), 12.32 (s, 1H, acid OH), 9.23 (s, 1H, Phenolic
OH).
18.6%), 327 (M þ 2, 1.5%); 1H NMR (300 MHz, CDCl3,
d ppm), 6.14 (s,
1H, CH), 8.03 (s,1H, NH), 7.21–8.1 (m, 8H, ArH),12.79 (s,1H, acid OH).
5.4.9. 4-(5-(2-Chlorophenyl)-4-(ethoxycarbonyl)-3-oxo-2,3-
dihydropyrazol-1-yl) benzoic acid (4i)
5.4.2. 4-(5-(2-Methoxyphenyl)-3-oxo-2,3-dihydropyrazol-1-
yl)benzoic acid (4b)
mp. 289–291 ꢁC; yield 74%; FTIR (KBr) cmꢂ1: 3387, 821 (NH),
1752 (C]O), 1544 (C]N), 1186 (C–O), 3468 (OH), 2943 (CH, Alkyl),
3033 (CH, Ar), 1724 (acid C]O), 709 (C–Cl); mass: m/z 386.5 (Mþ),
387.5 (M þ 1, 20.9%), 388.5 (M þ 2, 32%); 1H NMR (300 MHz, CDCl3,
mp. 222–224 ꢁC; yield 80%; FTIR (KBr) cmꢂ1: 3267, 816 (NH),
1756 (C]O), 1564 (C]N), 1120 (C–O), 3437 (OH), 3052 (CH, Ar),
1702 (acid C]O); mass: m/z 310 (Mþ), 311 (M þ 1, 18.7%), 312
d
ppm), 3.92 (q, 2H, CH2), 1.53 (t, 3H, CH3), 7.96 (s, 1H, NH), 6.81–
(M þ 2, 2.6%); 1H NMR (300 MHz, CDCl3,
d
ppm), 6.14 (s, 1H, CH),
7.70 (m, 8H, ArH), 12.19 (s, 1H, acid OH).
8.13 (s, 1H, NH), 7.32–8.19 (m, 8H, ArH), 11.65 (s, 1H, acid OH), 3.12
(s, 1H, OCH3).
5.4.10. 4-(4-(Ethoxycarbonyl)-3-oxo-5-phenyl-2-3-
dihydropyrazol-1-yl) benzoic acid (4j)
5.4.3. 4-(5-(2-Hydroxyphenyl)-3-oxo-2,3-dihydropyrazol-1-
yl)benzoic acid (4c)
mp. 277–279 ꢁC; yield 87%; FTIR (KBr) cmꢂ1: 3385, 826 (NH),
1756 (C]O), 1581 (C]N), 1090 (C–O), 3498 (OH), 2987 (CH, Alkyl),
3032 (CH, Ar), 1721 (acid C]O); mass: m/z 352 (Mþ), 353 (M þ 1,
mp. 233–235 ꢁC; yield 68%; FTIR (KBr) cmꢂ1: 3333, 842 (NH),
1655 (C]O), 1582 (C]N), 1220 (C–O), 3489 (OH), 3081 (CH, Ar),
1725 (acid C]O), 3540, 3558, (OH); mass: m/z 296 (Mþ), 297
20.9%), 354 (M þ 2, 3.2%); 1H NMR (300 MHz, CDCl3,
d ppm), 4.03
(q, 2H, CH2), 1.72 (t, 3H, CH3), 8.19 (s, 1H, NH), 6.31–7.64 (m, 9H,
ArH), 11.96 (s, 1H, acid OH).
(M þ 1, 18.2%), 298 (M þ 2, 1.5%); 1H NMR (300 MHz, CDCl3,
d ppm),
6.68 (s, 1H, CH), 7.98 (s, 1H, NH), 6.78–7.67 (m, 8H, ArH), 8.83 (s, 1H,
Phenolic OH), 11.57 (s, 1H, acid OH).
5.5. General procedure for the preparation of 4-(3-oxo-5-phenyl-
5.4.4. 4-(5-(4-Chlorophenyl)-3-oxo-2,3-dihydropyrazol-1-yl)
benzoic acid (4d)
2,3-dihydropyrazol-1-yl)benzohydrazides (5a–5j)
mp. 236–238 ꢁC; yield 63%; FTIR (KBr) cmꢂ1: 3371, 828 (NH),
1740 (C]O), 1594 (C]N), 1204 (C–O), 3502 (OH), 3052 (CH, Ar),
1718 (acid C]O), 645 (C–Cl); mass: m/z 314.5 (Mþ), 315.5 (M þ 1,
The compounds (5a–5j) were prepared as per procedure
reported in the literature [18].
17.5%), 316.5 (M þ 2, 32%); 1H NMR (300 MHz, CDCl3,
d
ppm), 6.85
5.5.1. 4-(5-(4-Nitrophenyl)-3-oxo-2,3-dihydropyrazol-1-
yl)benzohydrazide (5a)
(s,1H, CH), 8.11 (s,1H, NH), 6.87–7.69 (m, 8H, ArH), 8.83,11.21 (s,1H,
acid OH).
mp. 201–203 ꢁC; yield 76%; FTIR (KBr) cmꢂ1: 3367, 829 (NH),
1754 (C]O), 1564 (C]N), 1120 (C–O), 3052 (CH, Ar), 1302, 1436
(NO2); mass: m/z 339 (Mþ), 340 (M þ 1, 17.6%), 341 (M þ 2, 2.6%); 1H
5.4.5. 4-(3-Oxo-5-phenyl-2,3-dihydropyrazol-1-yl) benzoic
acid (4e)
NMR (300 MHz, CDCl3,
d ppm), 6.04 (s, 1H, CH), 8.08 (s, 1H, NH),
mp. 242–245 ꢁC; yield 68%; FTIR (KBr) cmꢂ1: 3367, 816 (NH),
1756 (C]O), 1564 (C]N), 1120 (C–O), 3427 (OH), 3052 (CH, Ar),
1702 (acid C]O); mass: m/z 380 (Mþ), 381 (M þ 1, 17.6%), 382
8.21 (s, 1H, NH), 4.35 (s, 2H, NH2), 7.02–7.95 (m, 8H, ArH).
5.5.2. 4-(5-(2-Methoxyphenyl)-3-oxo-2,3-dihydropyrazol-1-
yl)benzohydrazide (5b)
(M þ 2, 2.2%); 1H NMR (300 MHz, CDCl3,
d ppm), 6.71 (s, 1H, CH),
7.88 (s, 1H, NH), 6.31–7.59 (m, 9H, ArH), 11.38 (s, 1H, acid OH).
mp. 212–215 ꢁC; yield 78%; FTIR (KBr) cmꢂ1: 3355, 836 (NH),
1739 (C]O), 1555 (C]N), 1026 (C–O), 3066 (CH, Ar); mass: m/z
324 (Mþ), 325 (M þ 1, 18.7%), 326 (M þ 2, 2.3%); 1H NMR
5.4.6. 4-(5-(4-Nitrophenyl)-4-(ethoxycarbonyl)-3-oxo-2,3-
dihydropyrazol-1-yl) benzoic acid (4f)
(300 MHz, CDCl3,
d ppm), 6.04 (s, 1H, CH), 8.11 (s, 1H, NH), 8.38 (s,
mp. 283–286 ꢁC; yield 85%; FTIR (KBr) cmꢂ1: 3343, 811 (NH),
1732 (C]O), 1578 (C]N), 1020 (C–O), 3410 (OH), 2961 (CH, Alkyl),
1H, NH), 4.35 (s, 2H, NH2), 3.77 (s, 3H, OCH3), 6.84–7.70 (m, 8H,
ArH).

4632
S.V. Bhandari et al. / European Journal of Medicinal Chemistry 44 (2009) 4622–4636
5.5.3. 4-(5-(2-Hydroxyphenyl)-3-oxo-2,3-dihydropyrazol-1-
yl)benzohydrazide (5c)
Ar); mass: m/z 366 (Mþ), 367 (M þ 1, 20.9%), 368 (M þ 2, 2.9%); 1H
NMR (300 MHz, CDCl3,
d ppm), 1.49 (t, 3H, CH3), 3.87 (q, 2H, CH2),
mp. 219–221 ꢁC; yield 64%; FTIR (KBr) cmꢂ1: 3358, 812 (NH), 1656
(C]O),1544 (C]N),1098 (C–O), 3084 (CH, Ar), 3531, 3504 (OH); mass:
m/z 310 (Mþ), 311 (M þ 1, 17.6%), 312 (M þ 2, 2.3%); 1H NMR (300 MHz,
8.08 (s, 1H, NH), 8.41 (s,1H, NH), 4.35 (s, 2H, NH2), 6.68–7.53 (m, 8H,
ArH).
CDCl3, d ppm), 6.04 (s,1H, CH), 8.17 (s,1H, NH), 8.56 (s,1H, NH), 4.35 (s,
2H, NH2), 8.75 (s, 3H, Phenolic OH), 7.12–7.91 (m, 8H, ArH).
5.6. General procedure for the preparation of N0-(2-chloroacetyl)-4-
(3-oxo-5-phenyl-2,3-dihydropyrazol-1-yl) benzohydrazides (6a–6j)
5.5.4. 4-(5-(4-Chlorophenyl)-3-oxo-2,3-dihydropyrazol-1-
yl)benzohydrazide (5d)
The compounds (6a–6j) were prepared as per procedure
reported in the literature [18].
mp. 224–226 ꢁC; yield 61%; FTIR (KBr) cmꢂ1: 3367, 829 (NH),
1754 (C]O), 1564 (C]N), 1120 (C–O), 3052 (CH, Ar), 702 (C–Cl);
mass: m/z 328.5 (Mþ), 329.5 (M þ 1, 17.5%), 330.5 (M þ 2, 32.2%); 1H
5.6.1. N0-(2-chloroacetyl)-4-(5-(4-nitrophenyl)-3-oxo-5-phenyl-
2,3-dihydropyrazol-1-yl) benzohydrazide (6a)
NMR (300 MHz, CDCl3, d ppm), 6.04 (s, 1H, CH), 8.0 (s, 1H, NH), 8.29
(s, 1H, NH), 4.35 (s, 2H, NH2), 6.89–7.67 (m, 8H, ArH).
mp. 288–290 ꢁC; yield 74%; FTIR (KBr) cmꢂ1: 3367, 837 (NH),
1756 (C]O), 1576 (C]N), 1130 (C–O), 3032 (CH, Ar), 2859 (CH,
alkane), 1376, 1402 (NO2), 707 (C–Cl); mass: m/z 415.5 (Mþ), 416.5
(M þ 1, 21.7%), 417.5 (M þ 2, 33.4%); 1H NMR (300 MHz, CDCl3,
5.5.5. 4-(3-Oxo-5-phenyl-2,3-dihydropyrazol-1-yl)benzohydrazide
(5e)
mp. 231–233 ꢁC; yield 66%; FTIR (KBr) cmꢂ1: 3367, 829 (NH),
1754 (C]O), 1564 (C]N), 1120 (C–O), 3052 (CH, Ar); mass: m/z 294
(Mþ), 295 (M þ 1, 17.5%), 296 (M þ 2, 1.9%); 1H NMR (300 MHz,
d ppm), 6.14 (s, 1H, CH), 4.04 (s, 2H, CH2), 8.29 (s, 2H, NH), 8.05 (s,
1H, NH), 6.89–7.71 (m, 8H, ArH).
CDCl3,
d
ppm), 6.04 (s, 1H, CH), 8.11 (s, 1H, NH), 8.51 (s, 1H, NH), 4.35
5.6.2. N0-(2-chloroacetyl)-4-(5-(2-methoxyphenyl)-3-oxo-2,3-
dihydro-1H-pyrazol-1-yl)benzohydrazide (6b)
(s, 2H, NH2), 6.98–7.87 (m, 8H, ArH).
mp. 264–266 ꢁC; yield 75%; FTIR (KBr) cmꢂ1: 3377, 877 (NH),
1765 (C]O), 1572 (C]N), 1090 (C–O), 3088 (CH, Ar), 2879 (CH,
alkane), 747 (C–Cl); mass: m/z 400.5 (Mþ), 401.5 (M þ 1, 20.9%),
5.5.6. Ethyl-1-(4-(hydrazinecarbonyl)phenyl)-5-(4-nitrophenyl)-3-
oxo-2,3-dihydro-1H-pyrazol-4-carboxylate (5f)
mp. 211–213 ꢁC; yield 83%; FTIR (KBr) cmꢂ1: 3267, 829 (NH), 1754
(C]O), 1564 (C]N), 1120 (C–O), 2910 (CH, Alkyl), 3052 (CH, Ar), 1387,
1532 (NO2); mass: m/z411 (Mþ), 412 (M þ 1, 21%), 413 (M þ 2, 3.6%); 1H
402.5 (M þ 2, 32.3%); 1H NMR (300 MHz, CDCl3,
d ppm), 6.14 (s, 1H,
CH), 4.04 (s, 2H, CH2), 8.31 (s, 2H, NH), 8.09 (s, 1H, NH), 6.82–7.58
(m, 8H, ArH), 3.54 (s, 3H, OCH3).
NMR (300 MHz, CDCl3, d ppm), 1.14 (t, 3H, CH3), 3.34 (q, 2H, CH2), 8.07
(s, 1H, NH), 8.48 (s, 1H, NH), 4.35 (s, 2H, NH2), 7.09–7.88 (m, 8H, ArH).
5.6.3. N0(2-chloroacetyl)-4-(5-(2-hydroxyphenyl)-3-oxo-2,3-
dihydro-1H-pyrazol-1-yl)benzohydrazide (6c)
5.5.7. Ethyl-1-(4-(hydrazinecarbonyl)phenyl)-5-(4-
methoxyphenyl)-3-oxo-2,3-dihydro-1H-pyrazol-
4-carboxylate (5g)
mp. 267–269 ꢁC; yield 63%; FTIR (KBr) cmꢂ1: 3354, 828 (NH),
1736 (C]O), 1582 (C]N), 1030 (C–O), 3045 (CH, Ar), 2898 (CH,
alkane), 5437, 5402 (OH), 723 (C–Cl); mass: m/z 386.5 (Mþ),
387.5 (M þ 1, 21.3%), 388.5 (M þ 2, 34.9%) 1H NMR (300 MHz,
mp. 254–256 ꢁC; yield 80%; FTIR (KBr) cmꢂ1: 3341, 831 (NH),1698
(C]O), 1592 (C]N), 1098 (C–O), 2929 (CH, Alkyl), 3090 (CH, Ar);
mass: m/z 396 (Mþ), 397 (M þ 1, 22.1%), 398 (M þ 2, 3.3%); 1H NMR
CDCl3,
d ppm), 6.14 (s, 1H, CH), 4.04 (s, 2H, CH2), 8.19 (s, 2H,
NH), 8.11 (s, 1H, NH), 6.93–7.63 (m, 4H, ArH), 9.56 (s, 1H,
Phenolic OH).
(300 MHz, CDCl3, d ppm), 1.49 (t, 3H, CH3), 3.83 (q, 2H, CH2), 7.79 (s,
1H, NH), 8.37 (s, 1H, NH), 4.35 (s, 2H, NH2), 6.89–7.73 (m, 4H, ArH),
3.12 (s, 3H, OCH3).
5.6.4. N0-(2-chloroacetyl)-4-(5-(2-chlorophenyl)-3-oxo-2,3-
dihydro-1H-pyrazol-1-yl)benzohydrazide (6d)
5.5.8. Ethyl-1-(4-(hydrazinecarbonyl)phenyl)-5-(4-
hydroxyphenyl)-3-oxo-2,3-dihydro-1H-pyrazol-
4-carboxylate (5h)
mp. 252–254 ꢁC; yield 59%; FTIR (KBr) cmꢂ1: 3388, 833 (NH),
1706 (C]O), 1596 (C]N), 1139 (C–O), 3092 (CH, Ar), 2873 (CH,
alkane), 747, 664 (C–Cl); mass: m/z 405.5 (Mþ), 406.5 (M þ 1,
mp. 243–245 ꢁC; yield 74%; FTIR (KBr) cmꢂ1: 3354, 867 (NH),
1798 (C]O), 1590 (C]N), 1165 (C–O), 2976 (CH, Alkyl), 3087 (CH,
Ar), 3540, 3530 (OH); mass: m/z 382 (Mþ), 383 (M þ 1, 20.9%), 384
64.2%); 1H NMR (300 MHz, CDCl3,
d ppm), 6.14 (s, 1H, CH), 4.04 (s,
2H, CH2), 8.52 (s, 2H, NH), 7.97 (s, 1H, NH), 6.93–7.59 (m, 8H, ArH).
(M þ 2, 3.3%); 1H NMR (300 MHz, CDCl3,
d
ppm), 1.51 (t, 3H, CH3),
5.6.5. N0-(2-chloroacetyl)-4-(3-oxo-5-phenyl-2,3-dihydro-1H-
pyrazol-1-yl)benzohydrazide (6e)
3.82 (q, 2H, CH2), 8.13 (s, 1H, NH), 8.43 (s 1H, NH), 4.35 (s, 2H, NH2),
7.02–7.92 (m, 8H, ArH), 9.12 (s, 1H, Phenolic OH).
mp. 189–190 ꢁC; yield 65%; FTIR (KBr) cmꢂ1: 3387, 839 (NH),
1706 (C]O), 1581 (C]N), 1098 (C–O), 3032 (CH, Ar), 2869 (CH,
alkane), 713 (C–Cl); mass: m/z 370.5 (Mþ), 371.5 (M þ 1, 19.8%),
5.5.9. Ethyl-5-(4-chlorophenyl)-1-(4-(hydrazinecarbonyl)phenyl)-
3-oxo-2,3-dihydro-1H-pyrazol-4-carboxylate (5i)
372.5 (M þ 2, 32.3%); 1H NMR (300 MHz, CDCl3,
d ppm), 6.14 (s, 1H,
CH), 4.04 (s, 2H, CH2), 8.42 (s, 2H, NH), 8.13 (s, 1H, NH), 6.98–7.67
(m, 9H, ArH).
mp. 239–241 ꢁC; yield 71%; FTIR (KBr) cmꢂ1: 3363, 808 (NH),
1707 (C]O), 1586 (C]N), 1097 (C–O), 2954 (CH, Alkyl), 3090 (CH,
Ar), 708 (C–Cl); mass: m/z 400.5 (Mþ), 401.5 (M þ 1, 20.9%), 402.5
(M þ 2, 32.3%); 1H NMR (300 MHz, CDCl3,
d
ppm), 1.59 (t, 3H, CH3),
5.6.6. Ethyl-1-(4-(2-(2-chloroacetyl)hydrazinecarbonyl)phenyl)-5-
(4-nitrophenyl)3-oxo-2,3-dihydro-1H-pyrazol-4-carboxylate (6f)
mp. 265–267 ꢁC; yield 81%; FTIR (KBr) cmꢂ1: 3369, 837 (NH),
1756 (C]O), 1576 (C]N), 1130 (C–O), 3032 (CH, Ar), 2859 (CH,
alkane), 1387, 1498 (NO2), 747 (C–Cl); mass: m/z 459.5 (Mþ), 460.5
(M þ 1, 22.1%), 461.5 (M þ 2, 32.4%); 1H NMR (300 MHz, CDCl3,
3.82 (q, 2H, CH2), 8.1 (s, 1H, NH), 8.29 (s, 1H, NH), 4.35 (s, 2H, NH2),
6.82–7.71 (m, 8H, ArH).
5.5.10. Ethyl-1-(4-(hydrazinecarbonyl)phenyl)-3-oxo-5-phenyl-
2,3-dihydro-1H-pyrazol-4-carboxylate (5j)
mp. 261–263 ꢁC; yield 84%; FTIR (KBr) cmꢂ1: 3376, 829 (NH),
1754 (C]O), 1564 (C]N), 1120 (C–O), 2987 (CH, Alkyl), 3052 (CH,
d ppm), 1.42 (t, 3H, CH3), 3.84 (q, 2H, CH2), 8.07 (s, 1H, NH), 8.24 (s,
2H, NH), 7.01–7.84 (m, 8H, ArH).

S.V. Bhandari et al. / European Journal of Medicinal Chemistry 44 (2009) 4622–4636
4633
5.6.7. Ethyl-1-(4-(2-(2-chloroacetyl)hydrazinecarbonyl)phenyl)-5-
5.7.3. 3-(2-(4-(5-(2-Hydroxyphenyl)-3-oxo-2,3-dihydropyrazol-1-
(2-methoxyphenyl)3-oxo-2,3-dihydro-1H-pyrazol-4-
carboxylate (6g)
yl)-benzoyl)hydrazinyl)-2-oxoethylnitrate (7c)
mp. 190–192 ꢁC; yield 71.83%; FTIR (KBr) cmꢂ1: 3375, 848 (NH),
1684 (C]O),1595 (C]N),1091 (C–O),1488,1302 (NO2), 3540, 3500,
3158 (Phenolic OH); mass: m/z 413 (Mþ), 414 (M þ 1, 19.9%), 415
mp. 168–160 ꢁC; yield 78%; FTIR (KBr) cmꢂ1: 3382, 807 (NH),
1706 (C]O), 1566 (C]N), 1130 (C–O), 3032 (CH, Ar), 2923 (CH,
alkane), 689 (C–Cl); mass: m/z 444.5 (Mþ), 445.5 (M þ 1, 24.4%),
(M þ 2, 3.6%); 1H NMR (300 MHz, CDCl3,
d ppm), 6.14 (s, 1H, CH),
446.5 (M þ 2, 33.3%); 1H NMR (300 MHz, CDCl3,
d
ppm), 1.69 (t, 3H,
4.04 (s, 2H, CH2), 8.47 (s, 2H, NH), 8.0 (s, 1H, NH), 6.79–7.59 (m, 8H,
ArH), 9.56 (s, 1H, Phenolic OH). Anal. Calcd for C18H15N5O7: C, 52.30;
H, 3.66; N, 16.94. Found: C, 52.43; H, 3.73; N, 17.12.
CH3), 3.92 (q, 2H, CH2), 8.10 (s, 1H, NH), 8.43 (s, 2H, NH), 6.87–7.62
(m, 8H, ArH), 3.65 (s, 3H, OCH3).
5.6.8. Ethyl-1-(4-(2-(2-chloroacetyl)hydrazinecarbonyl)phenyl)-5-
(2-hydroxyphenyl)3-oxo-2,3-dihydro-1H-pyrazol-4-
carboxylate (6h)
5.7.4. 3-(2-(4-(5-(4-Chlorophenyl)-3-oxo-2,3-dihydropyrazol-1-
yl)-benzoyl)hydrazinyl)-2-oxoethylnitrate (7d)
mp. 218–220 ꢁC; yield 55%; FTIR (KBr) cmꢂ1: 3327, 842 (NH),
1783 (C]O), 1598 (C]N), 1092 (C–O), 1412, 1262 (NO2), 664 (C–Cl);
mass: m/z 431.5 (Mþ), 432.5 (M þ 1, 19.9%), 433.5 (M þ 2, 32.3%); 1H
mp. 176–178 ꢁC; yield 72%; FTIR (KBr) cmꢂ1: 3387, 887 (NH),
1745 (C]O), 1596 (C]N), 1180 (C–O), 3091 (CH, Ar), 2846 (CH,
alkane), 3548 (OH), 697 (C–Cl); mass: m/z 430.5 (Mþ), 431.5 (M þ 1,
NMR (300 MHz, CDCl3,
d ppm), 6.14 (s, 1H, CH), 3.98 (s, 2H, CH2),
22%), 432.5 (M þ 2, 32.3%); 1H NMR (300 MHz, CDCl3,
d
ppm), 1.39
8.29 (s, 2H, NH), 7.79 (s, 1H, NH), 6.89–7.70 (m, 8H, ArH). Anal. Calcd
for C18H14ClN5O6: C, 50.07; H, 3.27; N, 16.22. Found: C, 50.13; H,
3.31; N, 16.31.
(t, 3H, CH3), 4.04 (q, 2H, CH2), 8.07 (s, 1H, NH), 8.38 (s, 2H, NH),
6.92–7.62 (m, 8H, ArH), 9.79 (s, 1H, Phenolic OH).
5.6.9. Ethyl-1-(4-(2-(2-chloroacetyl)hydrazinecarbonyl)phenyl)-5-
(4-chlorophenyl)3-oxo-2,3-dihydro-1H-pyrazol-4-carboxylate (6i)
mp. 188–190 ꢁC; yield 70%; FTIR (KBr) cmꢂ1: 3372, 817 (NH),
1716 (C]O), 1584 (C]N), 1131 (C–O), 3082 (CH, Ar), 2859 (CH,
alkane), 689, 747 (C–Cl); mass: m/z 448.5 (Mþ), 449.5 (M þ 1,
5.7.5. 2-Oxo-2-(2-(4-(3-oxo-5-phenyl-2,3-dihydropyrazol-1-yl)-
benzoyl)hydrazinyl)ethylnitrate (7e)
mp. 208–210 ꢁC; yield 73%; FTIR (KBr) cmꢂ1: 3397, 844 (NH),
1692 (C]O), 1594 (C]N), 1095 (C–O), 1495, 1294 (NO2); mass: m/z
397 (Mþ), 398 (M þ 1, 19.9%), 399 (M þ 2, 3.1%); 1H NMR (300 MHz,
23.3%), 450.5 (M þ 2, 65.1%); 1H NMR (300 MHz, CDCl3,
d
ppm), 1.69
CDCl3, d ppm), 6.14 (s, 1H, CH), 4.04 (s, 2H, CH2), 8.3 (s, 2H, NH), 8.05
(s, 1H, NH), 6.92–7.61 (m, 9H, ArH). Anal. Calcd for C18H15N5O6: C,
54.41; H, 3.81; N, 17.63. Found: C, 54.13; H, 3.69; N, 17.48.
(t, 3H, CH3), 3.97 (q, 2H, CH2), 8.01 (s, 1H, NH), 8.2 (s, 2H, NH), 6.99–
7.71(m, 8H, ArH).
5.6.10. Ethyl-1-(4-(2-(2-chloroacetyl)hydrazinecarbonyl)phenyl)-
3-oxo-5-phenyl-2,3-dihydro-1H-pyrazol-4-carboxylate (6j)
5.7.6. Ethyl-1-(4-(2-(2-(nitrooxy)acetyl)hydrazinecarbonyl)
phenyl)-5-(4-nitrophenyl)-3-oxo-2,3-dihydropyrazol-4-carboxylate
(7f)
mp. 196–198 ꢁC; yield 82%; FTIR (KBr) cmꢂ1: 3342, 839 (NH),
1726 (C]O), 1587 (C]N), 1142 (C–O), 3042 (CH, Ar), 2888 (CH,
alkane), 667 (C–Cl); mass: m/z 414.5 (Mþ), 415.5 (M þ 1, 23.3%),
mp. 240–242 ꢁC; yield 67%; FTIR (KBr) cmꢂ1: 3383, 829 (NH),
1713 (C]O), 1599 (C]N), 1019 (C–O), 1492, 1399 (NO2); mass: m/z
514 (Mþ), 515 (M þ 1, 25.5%), 516 (M þ 2, 2.6%); 1H NMR (300 MHz,
416.5 (M þ 2, 33.1%); 1H NMR (300 MHz, CDCl3,
d ppm), 1.74 (t, 3H,
CH3), 3.82 (q, 2H, CH2), 8.12 (s, 1H, NH), 8.39 (s, 2H, NH), 6.90–7.68
(m, 9H, ArH).
CDCl3, d ppm), 1.71 (t, 3H, CH3), 4.02 (q, 2H, CH2), 3.79 (s, 2H, CH2),
8.03 (s, 1H, NH), 8.5 (s, 2H, NH), 7.02–7.81 (m, 8H, ArH). Anal. Calcd
for C21H18N6O10: C, 49.03; H, 3.53; N, 16.34. Found: C, 49.1; H, 3.57;
N, 16.36.
5.7. General procedure for the preparation of 2-oxo-2-(2-(4-(3-
oxo-5-phenyl-2,3-dihydropyrazol-1-yl) benzoyl)hydrazinyl)ethyl
nitrate (7a–7j)
5.7.7. Ethyl-1-(4-(2-(2-(nitrooxy)acetyl)hydrazinecarbonyl)
phenyl)-5-(2-methoxyphenyl)-3-oxo-2,3-dihydropyrazol-4-
carboxylate (7g)
The compounds (7a–7j) were prepared as per procedure
reported in the literature [18].
mp. 263–265 ꢁC; yield 73%; FTIR (KBr) cmꢂ1: 3385, 838 (NH),
1677 (C]O), 1598 (C]N), 1098 (C–O), 1491, 1374 (NO2); mass: m/z
499 (Mþ), 500 (M þ 1, 24.4%), 501 (M þ 2, 4.7%); 1H NMR (300 MHz,
5.7.1. 2-(2-(4-(5-(4-Nitrophenyl)-3-oxo-2,3-dihydropyrazol-1-yl)-
benzoyl)hydrazinyl)-2-oxoethylnitrate (7a)
CDCl3, d ppm), 1.62 (t, 3H, CH3), 3.98 (q, 2H, CH2), 4.02 (s, 2H, CH2),
7.89 (s, 1H, NH), 8.39 (s, 2H, NH), 6.99–7.7 (m, 8H, ArH), 3.65 (s, 3H,
OCH3). Anal. Calcd for C21H19N5O9: C, 51.96; H, 3.95; N, 14.43.
Found: C, 51.92; H, 3.89; N, 14.42.
mp. 210–212 ꢁC; yield 47%; FTIR (KBr) cmꢂ1: 3399, 826 (NH),
1763 (C]O), 1590 (C]N), 1090 (C–O), 1491, 1374 (NO2); mass: m/z
442 (Mþ), 443 (M þ 1, 19.9%), 444 (M þ 2, 3.9%); 1H NMR (300 MHz,
CDCl3),
d
ppm, 6.14 (s, 1H, CH), 4.04 (s, 2H, CH2), 8.42 (s, 2H, NH),
5.7.8. Ethyl-1-(4-(2-(2-(nitrooxy)acetyl)hydrazinecarbonyl)phenyl)
-5-(2-hydroxyphenyl)-3-oxo-2,3-dihydropyrazol-4-carboxylate (7h)
mp. 230–232 ꢁC; yield 70.25%; FTIR (KBr) cmꢂ1: 3537, 836 (NH),
1678 (C]O), 1599 (C]N), 1163 (C–O), 1512, 1282 (NO2), 3237 (OH);
mass: m/z 485 (Mþ), 486 (M þ 1, 25.1%), 487 (M þ 2, 4.7%); 1H NMR
8.05 (s, 1H, NH), 6.92–7.68 (m, 8H, ArH). Anal. Calcd for
C18H14N6O8: C, 48.87; H, 3.19; N, 19.00. Found: C, 48.84; H, 3.11; N,
18.9.
5.7.2. 3-(2-(4-(5-(2-Methoxyphenyl)-3-oxo-2,3-dihydropyrazol-1-
yl)-benzoyl)hydrazinyl)-2-oxoethylnitrate (7b)
(300 MHz, CDCl3, d ppm), 1.49 (t, 3H, CH3), 3.90 (q, 2H, CH2), 4.03 (s,
2H, CH2), 7.82 (s, 1H, NH), 8.35 (s, 2H, NH), 6.77–7.48 (m, 8H, ArH),
9.12 (s, 1H, Phenolic OH). Anal. Calcd for C22H21N5O9: C, 52.91; H,
4.24; N, 14.02. Found: C, 52.94; H, 4.25; N, 14.01.
mp. 192–194 ꢁC; yield 63%; FTIR (KBr) cmꢂ1: 3385, 840 (NH),
1677 (C]O), 1598 (C]N), 1098 (C–O), 1491, 1374 (NO2); mass: m/z
427 (Mþ), 428 (M þ 1, 21%), 429 (M þ 2, 3.5%); 1H NMR (300 MHz,
CDCl3,
d
ppm), 6.14 (s, 1H, CH), 4.18 (s, 2H, CH2), 8.32 (s, 2H, NH),
5.7.9. Ethyl-1-(4-(2-(2-(nitrooxy)acetyl)hydrazinecarbonyl)phenyl)
-5-(4-chlorophenyl)-3-oxo-2,3-dihydropyrazol-4-carboxylate (7i)
mp. 218–220 ꢁC; yield 65%; FTIR (KBr) cmꢂ1: 3323, 820 (NH),
1705 (C]O), 1593 (C]N), 1088 (C–O), 1482, 1213 (NO2), 677 (C–Cl);
8.12 (s, 1H, NH), 7.09–7.89 (m, 8H, ArH), 3.54 (s, 3H, OCH3). Anal.
Calcd for C19H17N5O7: C, 53.40; H, 4.01; N, 16.39. Found: C, 53.28; H,
3.99; N, 16.37.

4634
S.V. Bhandari et al. / European Journal of Medicinal Chemistry 44 (2009) 4622–4636
mass: m/z 503.5 (Mþ), 504.5 (M þ 1, 23.2%), 505.5 (M þ 2, 32.4%);
7. Pharmacology
1H NMR (300 MHz, CDCl3,
d ppm), 1.59 (t, 3H, CH3), 3.74 (q, 2H,
CH2), 3.89 (s, 2H, CH2), 8.03 (s, 1H, NH), 8.21 (s, 2H, NH), 6.99–7.64
(m, 8H, ArH). Anal. Calcd for C21H18ClN5O8: C, 50.06; H, 3.60; Cl,
7.04; N, 13.90. Found: C, 50.09; H, 3.62; N, 13.94.
7.1. Animals
Swiss albino mice of either sex weighing 20–25 g and Wistar
rats weighing in the range 100–120 g were obtained from, National
Center for Cell Science (NCCS), Pune, India. All the animals were
housed under standard ambient conditions of temperature
(25 ꢀ 2 ꢁC) and relative humidity of 50 ꢀ 5%. A 12:12 h light:dark
cycle was maintained. All the animals were allowed to have free
access to water and standard palletized laboratory animal diet 24 h
prior to pharmacological studies. All the experimental procedures
and protocols used in this study were reviewed and approved by
the Institutional Animal Ethical Committee (IAEC) of College, Pune,
constituted in accordance with the guidelines of the Committee for
the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA/257, CPCSEA/300), Government of India [33].
5.7.10. Ethyl-1-(4-(2-(2-(nitrooxy)acetyl)hydrazinecarbonyl)
phenyl)-3-oxo-5-phenyl-2,3-dihydro-1H-pyrazol-
4-carboxylate (7j)
mp. 200–202 ꢁC; yield 87%; FTIR (KBr) cmꢂ1: 3396, 845 (NH), 1687
(C]O),1590 (C]N),1088 (C–O),1475,1393 (NO2); mass: m/z 469 (Mþ),
470 (M þ 1, 23.2%), 471 (M þ 2, 4.2%); 1H NMR (300 MHz, CDCl3,
dppm),
1.71 (t, 3H, CH3), 3.69 (q, 2H, CH2), 4.0 (s, 2H, CH2), 7.98 (s, 1H, NH), 8.21
(s, 2H, NH), 7.1–7.82 (m, 9H, ArH). Anal. Calcd for C21H19N5O8: C, 53.73;
H, 4.08; N, 14.92. Found: C, 53.74; H, 4.10; N, 14.96.
6. Molecular docking studies
7.2. Preparation of test compounds
All the compounds were constructed using standard fragment
library of Maestro 8.0 and geometry optimized by Macromodel
programme (Schro¨dinger, LLC) using the Optimized Potentials for
Liquid Simulations-all atom (OPLS-AA) force field 2005 [32].
The X-ray crystal structure of COX-2 enzyme (PDB ID: 1CX2) was
obtained from the RCSB Protein Data Bank (PDB) was used in order to
getthedetailedsightsofligand–proteinstructureinthis study. Water
molecules of crystallization were removed from the complex and the
protein was optimized for docking using the protein preparation
wizard and refinement utility provided by Schro¨dinger LLC 8.0.
All the docking calculations were performed using ‘‘Standard
Precision’’ (SP) mode of Glide 4.5 programme and the 2005
implementation of OPLS-AA force field. The binding site, for which
the various energy grids were calculated and stored, was defined in
terms of two concentric cubes; the binding box, which must
contain the center of any acceptable ligand pose, and the enclosing
box, which must contain all ligand atoms of an acceptable pose.
Cubes with an edge length of 12 Å and centered at the midpoint of
the longest atom–atom distance in the respective co-crystallized
ligand defined the binding box in the protein. The large enclosing
box was also defined in terms of the co-crystallized ligand: an edge
length of 20 Å was used. Poses with an RMSD of less than 0.3 Å
were used for optimization. The scale factor for van der Waals radii
was applied to those atoms with absolute partial charges less than
or equal to 0.15 (scale factor of 0.8) and 0.25 (scale factor of 1.0)
electrons for ligand and protein, respectively. The best docked
structure was chosen using a Glide score (Gscore) function. The
Gscore is a modified and extended version of the empirically based
Chemscore function. Another scoring function used by Glide is
Emodel, which itself derived from a combination of Gscore, energy
in coloumbs, van der Waals and the strain energy of the ligand.
Besides this energy, contacts which include good, bad and ugly van
der Waals interactions were also taken into consideration for the
evaluation of the docked complexes. GScore is calculated as given
Eq. (1).
After suspending the test compounds in 1.0% aqueous solution
of sodium carboxymethyl cellulose (CMC), test samples were
administered to test animals orally. The positive and negative
control group animals received the same experimental handling as
those of the test groups except that the drug treatment, control
group animals received only appropriate volumes of vehicle and of
the reference drug, Indomethacin, respectively.
7.3. Anti-inflammatory activity
Anti-inflammatory activity was evaluated using the well known
carrageenan induced rat paw oedema model of Winter et al. [34]
using groups of six animals each. A freshly prepared aqueous
suspension of carrageenan (1.0% w/v, 0.1 mL) was injected in the
subplantar region of right hind paw of each rat. One group was kept
as control and the animals of the other group were pretreated with
the test drugs, 1 h before the carrageenan treatment. The volume
was measured before and after carrageenan treatment at the
30 min interval with the help of digital plethysmometer (Panlab LE
7500, USA).
7.4. Analgesic activity
The analgesic activity was evaluated using the acetic acid
induced writhing method [35].
7.5. Acute ulcerogenicity studies
Acute ulcerogenicity screening was done according to method
reported by Cioli et al. [36]. The mucosal damage was examined by
means of an electron microscope. For each stomach specimen, the
mucosal damage was assessed according to the following scoring
system.
Gscore ¼ a ꢄ vdw þ b ꢄ Coul þ Libo þ H-bond þ Metal
7.5.1. Score description
þ BuryP þ RotB þ Site
(1)
0.0 Normal (no injury, bleeding and latent injury).
0.5 Latent injury or widespread bleeding (>2 mm).
1.0 Slight injury (2–3 dotted lines).
2.0 Severe injury (continuous lined injury or 5–6 dotted
injuries).
where, vdw: van der Waals energy; Coul: Coulomb energy; Lipo:
Lipophilic contact term; H-Bond: Hydrogen-bonding term; Metal:
Metal-binding term; BuryP: Penalty for buried polar groups; RotB:
Penalty for freezing rotatable bonds; Site: Polar interactions at the
active site; and the coefficients of vdw and Coul are: a ¼ 0.065,
b ¼ 0.130.
3.0 Very severe injury (several continuous lined injuries).
4.0 Widespread lined injury or widened injury/erosion.

S.V. Bhandari et al. / European Journal of Medicinal Chemistry 44 (2009) 4622–4636
4635
The mean score of each treated group minus the mean score of
7.9. Statistical analyses
control group was regarded as severity index of gastric mucosal
damage. Data are expressed as mean ulcer score ꢀ SEM; data
analyzed by one-way ANOVA followed by Dunnett’s test to deter-
mine the significance of the difference between the standard group
and rats treated with the test compounds. The differences in results
were considered significant when P was found to be <0.01.
Data obtained for each set of anti-inflammatory model were
expressed as mean of change in paw volume ꢀ SD and analyzed by
one-way ANOVA followed by Dunnett’s test. Data from acetic acid
induced writhing model were expressed as mean of number of
writhes ꢀ SEM and analyzed by one-way ANOVA followed by
Dunnett’s ‘t’ test. Level of significance was set to P < 0.05. All
statistical calculations were performed using evaluation version of
Graph PadÒ Prism 3.0 (USA) statistical software [44].
7.6. Histopathological studies [37–40]
For the histopathological study, rats were sacrificed 4 h after the
cold stress and their stomach specimens were removed and put
into 10% formalin solution. A longitudinal section of stomach along
the greater curvature, which included the ulcer base and both sides
of the ulcer margin, was taken and fixed in 10% formalin for 24 h at
4 ꢁC and embedded in white solid paraffin. Morphological exami-
nation was performed with Haematoxylin and Eosin staining to
analyze histological changes and examined under electron micro-
scope. The disturbances in GI epithelial morphology were closely
analyzed and recorded in the form of images. The results of the
same are depicted in Fig. 1.
Acknowledgments
The authors are thankful to The Principal Incharge, Dr. A. R.
Madgulkar, AISSMS College of Pharmacy, Pune, for providing
laboratory facilities. The authors are also thankful to Prof. N.S.
Vyawahare, Head, Department of Pharmacology, for his continuous
support and guidance throughout the project. The authors also
wish to thank National Center for Cell Science (NCCS), Pune, Shi-
madzu Analytical Center and Dept. of Chemistry, University of
Pune, National Chemical Laboratories (NCL), Pune, for providing
facilities for spectral studies of compounds. The authors are also
thankful to Satav pathology laboratory, Pune, for providing facilities
for histopathological studies.
7.7. Vasorelaxing activity [41,42]
To determine a possible vasodilatory mechanism of action, the
compounds were tested on isolated aortae of male normotensive
Wistar rats (250–350 g). The rats were killed by cervical dislocation
under light ether anaesthesia and bled. The aortae were immedi-
ately excised, freed of extraneous tissues and prepared as multiple-
ring preparations. Then the vessels were suspended, under a pre-
load of 2 g, in 10 mL organ baths, containing Tyrode solution
(composition of saline in mM: NaCl 136.8; KCl 2.95; CaCl2 1.80;
MgSO4$7H2O 1.05; NaH2PO4 0.41; NaHCO3 11.9; Glucose 5.5),
thermostated at 37 ꢁC and continuously bubbled with a mixture of
O2 (95%) and CO2 (5%). Changes in tension were recorded by means
of an isometric transducer (BIOPAC System, Inc, MP 35).
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