Design and synthesis of pyrazole–pyrazoline hybrids as cancer-associated selective COX-2 inhibitors

Article abstract of DOI:10.1002/ardp.202000116

In continuation of our previous work on cancer and inflammation, 15 novel pyrazole–pyrazoline hybrids (WSPP1–15) were synthesized and fully characterized. The formation of the pyrazoline ring was confirmed by the appearance of three doublets of doublets in ¹H nuclear magnetic resonance spectra exhibiting an AMX pattern for three protons (H_A, H_M, and H_X) of the pyrazoline ring. All the synthesized compounds were screened for their in vitro anticancer activity against five cell lines, that is, MCF-7, A549, SiHa, COLO205, and HepG2 cells, using the MTT growth inhibition assay. 5-Fluorouracil was taken as the positive control in the study. It was observed that, among them, WSPP11 was found to be active against A549, SiHa, COLO205, and HepG2 cells, with IC₅₀ values of 4.94, 4.54, 4.86, and 2.09 μM. All the derivatives were also evaluated for their cytotoxicity against HaCaT cells. WSPP11 was also found to be nontoxic against normal cells (cell line HaCaT), with an IC₅₀ value of more than 50 μM. The derivatives were also evaluated for their in vitro anti-inflammatory activity by the protein (egg albumin) denaturation assay and the red blood cell membrane stabilizing assay, using diclofenac sodium and celecoxib as standard. Compounds that showed significant anticancer and anti-inflammatory activities were further studied for COX-2 inhibition. The manifestation of a higher COX-2 selectivity index of WSPP11 as compared with other derivatives and an in vitro anticancer activity against four cell lines further established that compounds that were more selective toward COX-2 also exhibited a better spectrum of activity against various cancer cell lines.

Full text of DOI:10.1002/ardp.202000116

Received: 12 April 2020
Revised: 22 August 2020
Accepted: 11 September 2020
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DOI: 10.1002/ardp.202000116
F U L L P A P E R
Design and synthesis of pyrazole–pyrazoline hybrids as
cancer‐associated selective COX‐2 inhibitors
Wasim Akhtar1 | Akranth Marella2 | Mohammad Mumtaz Alam1
Mohemmed F. Khan1 | Mymoona Akhtar1 | Tariq Anwer3 | Farah Khan4
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Md. Naematullah4 | Faizul Azam5 | Moshahid A. Rizvi6 | Mohammad Shaquiquzzaman1
1Drug Design and Medicinal Chemistry
Laboratory, Jamia Hamdard, New Delhi, India
Abstract
2Fryer Global Regulatory Solutions and
In continuation of our previous work on cancer and inflammation, 15 novel
Services, Hyderabad, Telangana, India
3Department of Pharmacology, College
of Pharmacy, Jazan University, Gizan,
Saudi Arabia
pyrazole–pyrazoline hybrids (WSPP1–15) were synthesized and fully characterized.
The formation of the pyrazoline ring was confirmed by the appearance of three
doublets of doublets in 1H nuclear magnetic resonance spectra exhibiting an AMX
pattern for three protons (HA, HM, and HX) of the pyrazoline ring. All the synthesized
compounds were screened for their in vitro anticancer activity against five cell lines,
that is, MCF‐7, A549, SiHa, COLO205, and HepG2 cells, using the MTT growth
inhibition assay. 5‐Fluorouracil was taken as the positive control in the study. It was
observed that, among them, WSPP11 was found to be active against A549, SiHa,
COLO205, and HepG2 cells, with IC50 values of 4.94, 4.54, 4.86, and 2.09 µM. All the
derivatives were also evaluated for their cytotoxicity against HaCaT cells. WSPP11
was also found to be nontoxic against normal cells (cell line HaCaT), with an IC50
value of more than 50 µM. The derivatives were also evaluated for their in vitro anti‐
inflammatory activity by the protein (egg albumin) denaturation assay and the red
blood cell membrane stabilizing assay, using diclofenac sodium and celecoxib as
standard. Compounds that showed significant anticancer and anti‐inflammatory
activities were further studied for COX‐2 inhibition. The manifestation of a higher
COX‐2 selectivity index of WSPP11 as compared with other derivatives and an in
vitro anticancer activity against four cell lines further established that compounds
that were more selective toward COX‐2 also exhibited a better spectrum of activity
against various cancer cell lines.
4Department of Biochemistry, Jamia Hamdard,
New Delhi, India
5Department of Pharmaceutical Chemistry &
Pharmacognosy, Unaizah College of Pharmacy,
Qassim University, Unaizah, Saudi Arabia
6The Genome Biology Lab, Jamia Millia Islamia,
New Delhi, India
Correspondence
Mohammad Shaquiquzzaman, Department
of Pharmaceutical Chemistry, School of
Pharmaceutical Education and Research
(NIRF‐2019), Jamia Hamdard,
New Delhi 110062, India.
Email: shaqiq@gmail.com and
mszaman@jamiahamdard.ac.in
K E Y W O R D S
cancer, COX‐2, hybrid, inflammation, pyrazole, pyrazoline
18.1 million new cases of cancer reported in 2018.[1] In 2019, 1.76
million new cancer cases and 606,880 cancer deaths were pro-
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1
INTRODUCTION
Cancer is a generic term used for a group of diseases characterized
by an abnormal growth of cells beyond their usual boundaries,
which may spread to adjoining parts or other organs. According to
the GLOBOCAN 2018 database, cancer was the leading cause
of death globally, resulting in around 9.6 million deaths out of
jected to occur in the United States.[2] The major problems asso-
ciated with the available chemotherapeutic agents are the lack of
selectivity and the occurrence of side effects. Although FDA has
approved targeted therapies like small molecule medicines and
monoclonal antibodies such as imatinib, bortezomib, bevacizumab,
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Arch Pharm. 2020;e2000116.
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© 2020 Deutsche Pharmazeutische Gesellschaft
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and so forth for cancer, still search of a newer anticancer agent
remains a need of the hour.
cytokines, mitogens, and tumor promoters, especially in cells involved
in inflammation, pain, fever, Alzheimer's disease, osteoarthritis, or
tumor formation.[8,9] COX‐2 is released by cancer‐associated fibro-
blasts, macrophage type 2 (M2) cells, and cancer cells in the tumor
microenvironment. It induces cancer stem cell‐like activity and pro-
motes apoptotic resistance, proliferation, angiogenesis, inflammation,
invasion, and metastasis of cancer cells. It exerts most of the func-
tions through its metabolite; thus, it appears to play predominant
roles in the initiation and promotion of cancer progression.[10]
Members of the mitogen‐activated protein kinase family, epidermal
growth factor receptor, and nuclear factor‐κB are the main upstream
modulators for COX‐2 in cancer cells.[10] In some and limited
situations, COX‐2 may act as an antitumor enzyme. Earlier, excess
of COX‐2 has been reported in various cancers like lung,[11,12]
colon,[11,13,14] cervical,[15] and so on.
The functional relationship between cancer and inflammation
was first postulated by the German pathologist Virchow in 1863.[3,4]
It has now been proved beyond doubt that cell proliferation in an
environment rich in inflammatory cells promotes the neoplastic risk.
An inflammatory response to a tissue injury is self‐limiting and it
ceases once the repair is completed, but, if it remains unregulated
and chronic, it causes malignant growth and tumor initiation in the
surrounding area. This continuous production of growth factors al-
ters the genome.[5] Il'yasova et al.[6] analyzed the association be-
tween inflammatory markers, such as interleukin‐6 (IL‐6), tumor
necrosis factor‐α (TNF‐α), and C‐reactive protein (CRP), and cancer
incidence and concluded that they are more strongly associated with
the risk of cancer death. Watson et al.[7] also studied the predictive
value of inflammatory markers for cancer diagnosis and concluded
that cancer should be considered in patients with raised inflammatory
markers.
A significant work has been done in the field of development of
pyrazole‐based drug candidates (Figure 1). It is of great importance
to medicinal chemists, owing to its immense pharmacological
utilities.[16–18] Considering this, there has been much impetus to
evaluate the anticancer propensity of pyrazole in different types of
Cyclooxygenase‐2 (COX‐2) is highly inducible and can be rapidly
upregulated in response to various proinflammatory agents, including
FIGURE 1 Pyrazole‐containing drugs for cancer, inflammation, and others. NSAID, nonsteroidal anti‐inflammatory drugs

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cancers.[19–26] Celecoxib, a COX‐2 selective inhibitor and a typical
model of a pyrazole‐tethered diaryl heterocyclic small molecule, is
also reported to exhibit activity against prostate tumors in various
experimental models.[27–29]
Considering all these factors and in continuation of work going on
in our lab in the field of cancer‐associated COX‐2 inhibition, we en-
visaged the synthesis of novel pyrazole–pyrazoline derivatives with
potential anticancer and anti‐inflammatory activities. The initial syn-
thetic step involved the formation of pyrazole carbaldehyde through
the Vilsmeier–Haack reaction,[40] followed by Claisen–Schmidt con-
densation of these aldehydes with acetophenones. Resulting chalcones
on treatment with semicarbazide gave pyrazolines, the title com-
pounds, WSPP1–15.[41,42] The synthesized compounds were evaluated
for their anticancer activity using MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐
2,5‐diphenyl tetrazolium bromide) growth inhibition assay.[43,44] The in
vitro anti‐inflammatory activity was also performed by protein albu-
min denaturation assay[45] and red blood cell membrane stabilizing
assay method.[46] In continuance with our earlier reported work[47,48]
to develop more COX‐2‐selective agents for the treatment of cancer
and its associated inflammation, compounds with a good activity were
also evaluated for their COX‐2 inhibition study.[49] Finally, to provide
an insight into the possible binding site, docking studies were per-
formed for the synthesized compounds.
Pyrazoline, the reduced form of pyrazole, is also of great utility to
medical chemists, owing to its diverse biological proclivity. It is known
to have anti‐inflammatory, analgesic, antibacterial, anticancer, and so
forth activities.[30] Various works have been reported enumerating the
anticancer potential of pyrazolines (Figure 2). Ramirez‐Prada et al.[31]
synthesized quinazoline‐based dihydropyrazole compound (1) and
determined its anticancer activity against various cancer cell lines.
Yang et al.[32] reported the anticancer activity of pyrazoline (2) with an
inhibitory concentration, IC50 value of 0.04 µM against the human
melanoma cell line. George et al.[33] reported pyrazoline derivative (3),
which was found to exhibit IC50 values of 8.33, 1.67, and 10 µM
against HepG‐2, MCF‐7, and CaCo‐2 cancer cell lines, respectively.
Altintop et al.[34] synthesized a series of thiazolyl–pyrazoline deriva-
tives (4 and 5) and evaluated their anticancer potential against the
A549 cancer cell line. Minmin et al.[35] synthesized compound 6, which
showed the most potent activity against COX‐2, with an IC50 value of
0.008 μM, and the HeLa cell line, with a GI50 value of 19.86 μM.
The latest trend for the novel drug development is the usage of
hybrid molecules. Glycopeptide bleomycin obtained from Strepto-
myces verticillus is a naturally occurring highly effective anticancer
agent.[36] Considering the importance of molecular hybridization
of pyrazoline and thiazole scaffolds in the discovery of potent an-
ticancer agents, Ciftci et al. synthesized 22 new thiazolyl–pyrazoline
derivatives, and out of them, 1‐(4‐(4‐cyanophenyl)thiazol‐2‐yl)‐3‐
(4‐morpholinophenyl)‐5‐(4‐chlorophenyl)‐2‐pyrazoline (5) was found
to be potent against the MCF‐7 cell line with an IC50 value of
8.06 1.47.[37] Taking the concept of hybrid molecules into con-
templation, a number of researchers have developed many potential
anticancer hybrids.[38,39]
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2
RESULTS AND DISCUSSION
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2.1
Chemistry
The synthesis of the title compounds involved four steps (Scheme 1).
Initially, substituted acetophenones were reacted with phenylhydrazine
to give hydrazones (WSH1–3), a condensation product. During its for-
mation, the lone pair of electron present on –NH2 of phenylhydrazine
reacts with the carbonyl carbon of substituted acetophenone to give an
intermediate, which loses a water molecule to form hydrazone.
The hydrazones in the presence of dimethylformamide and
phosphorous oxychloride formed pyrazole carbaldehyde (WSP1–3)
FIGURE 2 Pyrazoline‐containing derivatives as anticancer agents

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SCHEME 1 The synthesis route for the generation of WSPP1–15. DMF, dimethylformamide; GAA, glacial acetic acid
via Vilsmeier–Haack formylation.[40] In this reaction, the reactive
formylating intermediate formed by the reaction of dimethylforma-
mide with phosphorous oxychloride acts as an electrophile. The
carbanion generated from substituted hydrazones attacks the elec-
trophilic center, which finally cyclizes and gets formylated to form
pyrazole carbaldehyde.
pair of electrons present on the nitrogen of semicarbazide attacks the
carbonyl carbon of the chalcone, resulting in the formation of an in-
termediate, which follows dehydration and cyclization reaction to give
the desired compounds. The purity of synthesized compounds was
ascertained by a single spot in thin‐layer chromatography (TLC) and a
sharp melting point.
These aldehydes were further reacted with substituted acet-
ophenones to get the corresponding chalcones (WSC1–15) through
the Claisen–Schmidt reaction, wherein the carbanion formed attacks
the carbonyl carbon of the WSPs to give an alkoxide, which finally
undergoes dehydration reaction to form the chalcone.
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2.1.1
Spectral characterization
In this series, 15 compounds (WSPP1–15) were synthesized and
their structure was characterized using elemental analysis and
spectroscopic methods. Values of elemental analysis were within
the range of 0.04%. The infrared (IR) spectra showed stretching
Finally, these chalcones were treated with semicarbazide to give
pyrazole–pyrazoline hybrids (WSPP1–15) by involving 1,4‐addition
reaction at α,β‐unsaturated carbonyl system (WSCs). Initially, the lone

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absorption bands at 3,227–3,368 cm−1 for asymmetric and sym-
metric vibrations of the amino group (–NH2) and 1,657–1,673 cm−1
for the carbonyl group (–C═O).
13C NMR spectra of compound WSPP13 showed chemical shift
values at their respective positions. The peaks at 42.99 and 52.86
were assigned to saturated carbons of pyrazoline moiety.
The compounds bearing chloro substituents WSPP4, WSPP6–10,
and WSPP14 were further confirmed by the appearance of the (M++2)
isotopic peak.
The 1H nuclear magnetic resonance (NMR) spectra showed AMX
pattern for three protons (HA, HM, and HX) of the pyrazoline ring,
which appeared as doublet of doublet (Figure 3) at δ 3.09 ppm
(J values of 17.6 and 6.4 Hz) for HA proton, δ 3.79 ppm for HM proton
(J values of 17.6 and 12.0 Hz), and δ 5.48 ppm (J values of 12.0 and
6.4 Hz) for HX proton. The splitting was observed due to transoid and
cisoid coupling of pyrazoline protons. The HX proton was more de-
shielded due to the effect of electronegative nitrogen and anisotropic
effect of pyrazole ring.
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2.2
Biology
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2.2.1
In vitro anticancer activity
All the 15 synthesized compounds (WSPP1–15) were screened for
their in vitro anticancer potential against five cell lines, that is,
MCF‐7, A549, SiHa, COLO205, and HepG2. To evaluate the
toxic effect of the synthesized compounds, the derivatives
were also tested on HaCaT cells using MTT growth inhibition
assay.[43,44] 5‐Fluorouracil (5‐FU) and cisplatin were taken as the
positive control in the study. WSPP3 and WSPP4 were found to be
inactive against all cell lines (IC50 >50 µM). All the compounds
showed much less activity against breast cancer cell line MCF‐7.
Out of the 15 tested compounds, only WSPP11 and WSPP13
showed the best activity against HepG2 cell lines, with IC50 of 2.09
and 8.56 µM, whereas IC50 values of 5‐FU and cisplatin were found
to be 19 and 0.295 µM. Among the compounds tested against SiHa
cell line, WSPP6, WSPP11, and WSPP13 exhibited a good activity,
with IC50 values of 4.54, 4.58, and 5.67, respectively, as compared
with 5.78 µM of 5‐FU, the standard. Compounds WSPP11,
WSPP13, and WSPP14 also showed appreciable results against
COLO205, with IC50 values of 4.86, 6.69, and 7.16 µM, respec-
tively. However, 5‐FU was found to have an IC50 value of 4.00 µM
against COLO205 cell line. Compound WSPP11 also showed a
good anticancer activity against A549 cell line, with an IC50 value
of 4.94 µM. Except WSPP4 and WSPP10, all compounds were
found to be nontoxic against the normal cell line HaCaT, with an
IC50 value of more than 50 µM (Table 1).
Ring A protons
This ring was unsubstituted in all the pyrazoline derivatives
(WSPP1–15). H‐4′ appeared either as a triplet in the range of δ
7.28–7.31 ppm or as a multiplet merged with aromatic protons. H‐3′
and H‐5′ also appeared either as a triplet due to coupling to H‐2′/6′
and H‐4′ or as a multiplet. The H‐2′ and H‐6′ protons appeared as a
doublet at the downfield position due to the electron‐withdrawing
effect of pyrazole nitrogen.
Ring B protons
H‐3 and H‐5 showed coupling to fluorine at the fourth position and
appeared either as a triplet or as a multiplet. Compounds bearing
chlorine at the fourth position exhibited either A2B2 pattern or such
protons appeared as a multiplet
Ring C protons
Protons of compounds bearing substitution at the fourth position
appeared either as a doublet exhibiting A2B2 pattern or as a multiplet
merged with other aromatic protons at a downfield region due to
anisotropic effects exerted by –C═N of the pyrazoline ring.
The two protons of CONH2 appeared in the range of
δ
6.42–6.54 ppm as a broad singlet and were D2O exchangeable. A singlet
that appeared around δ 8.30 ppm was assigned to the pyrazole proton.
FIGURE 3 AMX pattern representation along with representative peak for –NH2 and pyrazole proton

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TABLE 1 The in vitro anticancer activity of compounds WSPP1–15
In vitro anticancer activity, IC50 (μM)a
MCF7
A549
SiHa
COLO205
17.48 0.08
13.15 0.02
>50
HepG2
>50
HaCaT
>50
Compound no.
WSPP1
37.38 1.17
33.07 0.95
47.75 0.90
>50
17.12 0.19
16.83 0.01
>50
18.34 0.20
11.43 0.05
>50
WSPP2
WSPP3
WSPP4
WSPP5
WSPP6
WSPP7
WSPP8
WSPP9
WSPP10
WSPP11
WSPP12
WSPP13
WSPP14
WSPP15
5‐FU
>50
>50
>50
>50
>50
>50
>50
>50
45.18 0.05
>50
>50
16.77 0.01
15.30 0.01
21.94 0.23
32.01 0.04
28.96 0.03
25.45 0.03
4.94 0.05
27.27 0.09
10.27 0.03
11.50 0.01
26.25 0.10
4.35 0.01
–
17.57 0.01
4.54 0.01
32.27 0.08
>50
16.54 0.08
12.95 0.07
25.35 0.19
>50
>50
31.38 0.05
>50
>50
>50
>50
>50
34.31 0.08
>50
>50
>50
>50
>50
>50
>50
>50
38.85 0.09
4.58 0.01
>50
29.33 0.04
4.86 0.01
>50
>50
48.05 0.46
>50
27.37 0.12
>50
2.09 0.01
>50
>50
29.31 0.03
30.05 0.03
>50
5.67 0.06
6.40 0.05
>50
6.69 0.08
7.16 0.01
31.27 0.05
4.00 0.01
–
8.56 0.05
>50
>50
>50
>50
>50
2.08 0.01
–
5.78 0.04
–
19.01 0.21
0.295 0.04
20.21 0.11
–
Cisplatin
Abbreviations: 5‐FU, 5‐fluorouracil; A549, adenocarcinomic human alveolar basal epithelial cells; COLO205, colon cancer cell line; HacaT cells, normal
human keratinocytes; HepG2, liver cell line; MCF‐7, established from the pleural effusion from female suffering from a breast adenocarcinoma; SiHa,
cervical cancer.
aIC50 values are the concentrations that cause 50% inhibition of cancer cell growth (μM). Data represent the mean standard error deviation of three
independent experiments performed in triplicate.
All 15 compounds (WSPP1–15) were also evaluated for their in
vitro anti‐inflammatory activity using protein albumin denaturation
assay[45] and red blood cell membrane stabilizing assay.[46]
WSPP14 displayed a significant in vitro anti‐inflammatory activity in
a concentration‐dependent manner (Table 3).
It is well known that COX‐2 exerts most of the functions through
its metabolite in the initiation and promotion of cancer progression.[10]
Thus, four compounds, WSPP6, WSPP11, WSPP13, and WSPP14,
which showed a significant anticancer and anti‐inflammatory activity,
were further studied for COX‐2 inhibition to study the potential bio-
logical targets of the derivatives formed.
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2.2.2
In vitro anti‐inflammatory inhibition
Protein albumin denaturation assay
Mizushima and Kobayashi method[45] was used to assess the in vitro
anti‐inflammatory activity at concentrations of 25, 50, 100, and
200 μg/ml using diclofenac sodium and celecoxib as standard. Out of
the 15 synthesized compounds, only 4 compounds, that is, WSPP6,
WSPP11, WSPP13, and WSPP14, demonstrated a significant in vitro
anti‐inflammatory activity, with IC50 values of 71.11, 81.77, 76.58,
and 73.35 µg/ml as compared with standard diclofenac sodium and
celecoxib, which showed IC50 values of 55.65 and 44.81 µg/ml,
respectively (Table 2).
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2.2.3
In vitro COX inhibition
All four compounds, that is, WSPP6, WSPP11, WSPP13, and
WSPP14, were evaluated for their in vitro COX‐2 inhibitory activities
by enzyme immunoassay using celecoxib as standard.[49] WSPP11
exhibited good selectivity toward COX‐2 with a selectivity index (SI)
of 80.03 as compared with the standard celecoxib, which was found
to have an SI of 95.84 (Table 3).
Red blood cell membrane stabilizing assay
The presence of a higher COX‐2 selectivity index of WSPP11 as
compared with other derivatives and a good in vitro anticancer activity
against four cell lines, that is, A549, SiHa, COLO205, and HepG2, further
established that compounds that are more selective toward COX‐2 also
have a better spectrum of activity against various cancer cell lines.
All the compounds were also evaluated for their in vitro anti‐
inflammatory activity by red blood cell membrane stabilizing assay
method[46] at concentrations of 100 and 200 μg/ml using diclofenac
sodium and celecoxib as standard. WSPP6, WSPP11, WSPP13, and

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TABLE 2 The anti‐inflammatory activity of compounds WSPP 1–15 on the basis of protein albumin denaturation assay
% Inhibition (µg/ml)
25
50
100
200
Compound
WSPP1
IC50 (µg/ml)
10.93 0.22
13.18 0.37
11.18 0.05
13.22 0.04
13.26 0.15
12.90 0.28
12.72 0.33
12.8 0.39
11.52 0.27
12.81 0.11
9.19 0.22
13.58 0.24
11.06 0.14
12.67 0.27
11.45 0.13
7.47 0.66
11.23 0.10
40.15 0.17
53.64 0.29
29.45 0.11
30.99 0.06
51.42 0.20
54.46 0.22
38.98 0.28
33.08 0.22
34.48 0.39
37.57 0.21
59.27 0.28
35.54 0.17
58.04 0.14
56.58 0.24
36.61 0.30
67.31 0.43
59.41 0.29
72.71 0.23
77.94 0.21
60.22 0.16
63.91 0.07
75.99 0.16
78.30 0.32
71.41 0.25
65.57 0.38
66.71 0.27
69.74 0.27
81.26 0.17
67.62 0.37
80.68 0.14
79.52 0.28
68.24 0.24
92.62 0.58
75.29 0.20
100 0.00
100 0.00
100 0.00
100 0.00
100 0.00
100 0.00
100 0.00
100 0.00
100 0.00
100 0.00
100 0.00
100 0.00
100 0.00
100 0.00
100 0.00
100 0.00
100 0.00
61.45
WSPP2
48.13
81.77
76.58
50.63
47.42
63.32
73.18
70.87
65.77
43.47
69.28
44.30
45.51
67.77
54.65
44.81
WSPP3
WSPP4
WSPP5
WSPP6
WSPP7
WSPP8
WSPP9
WSPP10
WSPP11
WSPP12
WSPP13
WSPP14
WSPP15
Diclofenac sodium
Celecoxib
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2.3
Docking and molecular dynamics simulations
visualized in Biovia Discovery Studio 2020 and PyMol1.7.4
programs to study ligand–protein interactions. Moreover, the
conformation of the docked compound having the lowest binding
energy of −10.66 kcal/mol, showing maximum number of clusters,
was compared with the crystal structure of SC‐558 to exhibit an
RMSD of 1.38 Å (Figure 5). Docked WSPP11 shared similar
residues for interaction within the inhibitor binding cavity in
comparison with SC‐558.
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2.3.1
Validation of the docking protocol
A successful experiment always relies upon validation of the im-
plemented protocol. The docking methodology used in this study was,
therefore, authenticated by redocking of native co‐crystallized ligand,
1‐phenylsulfonamide‐3‐trifluoromethyl‐5‐parabromophenylpyrazole
(SC‐558), in the binding pocket of COX‐2. The root mean square
deviation (RMSD) of the best‐docked conformation of SC‐558
and X‐ray crystal structure was calculated as 0.36 Å (Figure 4),
confirming the reliability of the scoring function. However, the
standard RMSD for successful docking according to the reported
protocols is ≤2.0 Å.[50,51] AutoDock 4.2, therefore, can be
exploited to predict the molecular interaction of WSPP11 in the
inhibitor binding cavity of COX‐2.
The analysis of the docking results presented in Figure 6 clearly
reveals that WSPP11 accommodates most comfortably in the bind-
ing pocket with the help of both hydrophobic as well as hydrophilic
interactions. The H‐atom of the amide group substituted in the
pyrazoline ring serves as an interesting site for the hydrogen‐bond
interaction with Ser353. The carbon atom of the methoxy group
substituted at p‐position of the aromatic ring is supported in the
hydrophobic canal by π–alkyl interaction involving aromatics of
Phe381 and Tyr385 residues. Arg120 and Arg513 are involved in
π–cation interaction. Val523, which plays an important role in the
selectivity of COX‐2 inhibitors by providing an extra bulky site for
accommodation in the hydrophobic tunnel, plays a vital role in ac-
commodating the phenyl ring of WSPP11 and providing a platform
for π–alkyl interaction. Additional residues engaged in anchoring the
docked compound within the binding cavity constitute Pro86, Val89,
His90, Val349, Leu352, Tyr355, Leu359, Leu384, Trp387, Phe518,
Met522, Glu524, Gly526, Ala527, and Leu531.
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2.3.2
Docking of WSPP11 with COX‐2
Computational determination of the binding modes of ligands with
their targets by molecular docking is routinely employed in various
drug design and discovery programs due to its swiftness and
robustness.[52] After successful completion of the docking, each
conformer of WSPP11 obtained from 100 docking runs was

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TABLE 3 The anti‐inflammatory activity of WSPP1–15 on the basis of red blood cell membrane stabilizing assay and COX‐2 selectivity index
of WSPP6, 11, 13, and 14
% Protection
In vitro cytotoxicity assay (SI)a
COX‐1,
IC50 (μM)
COX‐2,
IC50 (μM)
SI (COX‐1/
COX‐2)
100 (µg/ml)
48.03 0.68
52.06 0.49
35.09 0.63
36.94 0.35
49.04 0.44
53.18 0.68
47.28 0.43
39.89 0.38
38.83 0.54
44.72 0.82
58.24 0.81
41.94 0.31
54.86 0.35
53.9 0.38
200 (µg/ml)
64.81 0.74
70.33 0.68
46.97 0.30
50.19 0.72
67.03 0.41
72.69 0.78
62.88 0.54
52.15 0.76
53.90 0.69
60.88 0.58
78.14 0.75
58.26 0.64
76.00 0.48
75.26 0.89
59.84 0.57
83.54 0.61
89.59 0.33
Compounds
WSPP1
nt
nt
–
WSPP2
nt
nt
–
WSPP3
nt
nt
–
WSPP4
nt
nt
–
WSPP5
nt
nt
–
WSPP6
92.172 0.05
1.45 0.014
63.56
WSPP7
nt
nt
–
WSPP8
nt
nt
–
WSPP9
nt
nt
–
WSPP10
nt
nt
–
WSPP11
87.239 0.24
1.09 0.001
nt
80.03
–
WSPP12
nt
WSPP13
135.78 1.66
114.53 0.48
nt
2.10 0.018
1.79 0.014
nt
64.65
63.98
–
WSPP14
WSPP15
43.07 0.52
70.17 0.22
77.50 0.38
Diclofenac sodium
Celecoxib
Abbreviation: nt, not tested.
nt
nt
–
24.92 016
0.26 0.002
95.84
aSelectivity index: Values are the mean standard error of the mean of three independent experiments using COX assay kits.
|
2.4
Structure–activity relationship
b. The effect of substitution on ring B, that is, “R” in Figure 3:
• Compound bearing no substitution on ring B was found to be
the most active (WSPP11 appeared as the best compound from
this series).
On the basis of the above results, the following observations
were made:
• The increase in electronegativity on ring B decreases the
activity (WSPP1 and WSPP3 were found to be more active
than WSPP6 and WSPP8, respectively).
a. Pyrazoline–pyrazole hybrids are found to have anticancer and
anti‐inflammatory activities.
FIGURE 4 A comparison of docked
SC‐558 (yellow) with the native co‐crystallized
conformation (green), demonstrating a root
mean square deviation of 0.36 Å

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FIGURE 5 The inhibitor binding pocket of
COX‐2 (depicted as a surface style) occupied
by the co‐crystallized selective COX‐2
inhibitor, SC‐558 (yellow stick), and docked
WSPP11 (blue stick) exhibiting a root mean
square deviation of 1.38 Å
|
CONCLUSION
• The increase in electronegativity on ring B and electropositivity
on ring C was found to exhibit a good effect on activity (WSPP2
was more active than WSPP7).
3
In continuation of our previous work on cancer and inflammation, 15
novel pyrazole–pyrazoline hybrids (WSPP1–15) were synthesized
and fully characterized. The formation of the pyrazoline ring was
confirmed by the appearance of three doublets of doublets in 1H
NMR spectra exhibiting AMX pattern for three protons (HA, HM, and
HX) of the pyrazoline ring. All the derivatives were screened for their
in vitro anticancer activity against five cell lines, that is, MCF‐7,
A549, SiHa, COLO205, and HepG2 cells, using the MTT growth in-
hibition assay. 5‐FU and cisplatin were taken as the positive control
in the study. Out of the 15 tested compounds, only WSPP11 and
WSPP13 showed the best activity against HepG2 cell lines, with IC50
values of 2.09 and 8.56 µM, respectively. WSPP11 was also found to
be active against A549, SiHa, and COLO205, with IC50 values of 4.94,
4.54, and 4.86 µM. All the derivatives were also evaluated for their
cytotoxicity against HaCaT cells. Except WSPP4 and WSPP10, all
compounds were found to be nontoxic against the normal cell line
HaCaT, with IC50 values of more than 50 µM. For anti‐inflammatory
activity, compounds WSPP1–15 were evaluated by protein (egg
albumin) denaturation assay and red blood cell membrane stabi-
lizing assay using diclofenac sodium and celecoxib as the standard.
Four compounds, WSPP6, WSPP11, WSPP13, and WSPP14, which
showed significant anticancer and anti‐inflammatory activities,
were further studied for COX‐2 inhibition to study the potential
biological target of the derivatives formed. The presence of a
higher COX‐2 selectivity index of WSPP11 as compared with
other derivatives and the in vitro anticancer activity against cell
lines further established that compounds that were more selective
toward COX‐2 also exhibited a better spectrum of activity against
various cancer cell lines.
c. The effect of substitution on ring C, that is, “R1” in Figure 3:
• The results indicated that methoxy‐containing derivatives were
found to be more suitable for in vitro anticancer activity
(WSPP11 was most active in the series).
• The increase in electronegativity on ring C decreases the ac-
tivity (WSPP4 and WSPP9 were more active than WSPP3 and
WSPP8, respectively).
d. Compounds were more selective toward COX‐2 enzyme; hence, it
could be established that they exhibited an anticancer activity
through inhibition of COX‐2.
FIGURE 6 The lowest energy configuration of the docking result of
WSPP11 (shown as ball and stick in magenta color) with the binding
pocket of COX‐2 (shown as green sticks). The broken line in green
represents the H‐bond interaction, orange lines denote π–cation
interactions, and purple lines represent various hydrophobic interactions

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General procedure for the synthesis of
4
EXPERIMENTAL
4.1.5
1′,3′,5‐triphenyl‐3,4‐dihydro‐1′H,2H‐[3,4′‐bipyrazole]‐
2‐carboxamides (WSPP1–15)
|
4.1
Chemistry
|
4.1.1
General
Equimolar amounts of WSC (0.01 mol) and semicarbazide (0.01 mol;
01.15 g) were refluxed in the presence of an ethanolic solution of
sodium hydroxide (0.025 mol; 1.00 g in 25 ml ethanol) for 3 hr. After
cooling, the separated solid was filtered, washed with water, and
dried to obtain pure crystals of the product.
All the chemicals and solvents used in the study were procured
from E. Merck Ltd. and S. D. Fine Chemicals, and they were used
without further purification. Melting points were recorded by an
open‐end capillary method and were uncorrected. TLC was done
with silica gel 60 and 254 (Merck) with methanol/chloroform
(1:9 v/v) as the solvent system. IR and 1H NMR spectra (see the
Supporting Information) were recorded using Bruker Alpha‐T
spectrophotometer and Bruker Avance‐400 MHz, respectively.
3‐(4‐Fluorophenyl)‐5‐[3‐(4‐methoxyphenyl)‐1‐phenyl‐1H‐pyrazol‐4‐
yl]‐4,5‐dihydropyrazole‐1‐carboxamide (WSPP1)
White flakes; mp, 265–267°C; yield, 60%; Rf, 0.78; FTIR (cm−1): 3,340
and 3,259 (NH2), 1,657 (C═O), 1,586 (C═N); 1H NMR (400 MHz,
DMSO‐d6, δ); 3.09 (dd, 1H, JAM = 15.6 Hz and JAX = 6.4 Hz, HA), 3.79 (s,
Dimethyl sulfoxide (DMSO)‐d6 was used as
a solvent and
tetramethylsilane was taken as an internal standard for NMR
spectra. 13C NMR spectrum of one representative compound
WSPP13 was recorded on Bruker Advance‐100 MHz instrument
using DMSO‐d6 as the solvent. Chemical shift values (δ) were
represented in parts per million (ppm). Coupling constants (J)
have been reported in hertz (Hz). Waters SYNAPT UPLC‐MS/MS
working on Mass Linux V4.1 software was used for mass spectra.
Elemental analysis was done using a PerkinElmer Model 240
Analyzer. Results were found to be within the range of 0.4% of
the theoretical values. % Yield refers to products obtained after
crystallization.
3H, –OCH3 merged with HM), 3.79 (dd, 1H, JMA = 15.2 Hz and JMX =
12.0 Hz, HM), 5.46 (dd, 1H, JXM = 12.0 Hz and JXA = 6.8 Hz, HX), 6.46
(bs, 2H, NH2, D2O exchangeable), 6.97 (d, 2H, J = 8.8 Hz, H‐3″, H‐5″),
7.27–7.32 (m, 3H, H‐3, H‐5, H‐4′), 7.45 (t, 2H, J = 8.4 Hz, H‐3′, H‐5′),
7.70 (d, 2H, J = 8.8 Hz, H‐2′, H‐6′), 7.86–7.89 (m, 4H, H‐2, H‐6, H‐2″, H‐
6″), 8.32 (s, 1H, pyrazole ring); electrospray ionization–mass spectro-
metry (ESI–MS) (m/z): 456 (M++1); elemental analysis (%) of
C26H22FN5O2, calculated/found: C (68.56/68.29), H (4.87/4.85), N
(15.38/15.42).
3‐(4‐Fluorophenyl)‐5‐[3‐(4‐methylphenyl)‐1‐phenyl‐1H‐pyrazol‐4‐
yl]‐4,5‐dihydropyrazole‐1‐carboxamide (WSPP2)
The InChI codes of the investigated compounds, together
with some biological activity data, are provided as Supporting
Information.
White fluffy solid; mp, 261–263°C; yield, 66%; Rf, 0.82; FTIR (cm−1):
3,360 and 3,275 (NH2), 1,665 (C═O), 1,584 (C═N); 1H NMR
(400 MHz, DMSO‐d6, δ); 2.33 (s, 3H, –CH3) 3.09 (dd, 1H, JAM = 17.6
Hz and JAX = 6.4 Hz, HA), 3.80 (dd, 1H, JMA = 17.6 Hz and JMX = 12.0
Hz, HM), 5.48 (dd, 1H, JXM = 12.0 Hz and JXA = 6.8 Hz, HX), 6.49 (bs,
2H, NH2, D2O exchangeable), 7.23 (d, 2H, J = 8.0 Hz, H‐3″, H‐5″),
7.27–7.32 (m, 3H, H‐3, H‐5, H‐4′), 7.45 (t, 2H, J = 8.0 Hz, H‐3′, H‐5′),
7.66 (d, 2H, J = 8.0 Hz, H‐2′, H‐6′), 7.86–7.89 (m, 4H, H‐2, H‐6, H‐2″,
H‐6″), 8.33 (s, 1H, pyrazole ring); ESI–MS (m/z): 440 (M++1); ele-
mental analysis (%) of C26H22FN5O, calculated/found: C (71.06/
71.42), H (5.05/5.01), N (15.94/15.97).
|
4.1.2
General procedure for the synthesis of 1‐
phenyl‐2‐(1‐phenylethylidene)hydrazines (WSH1–3)
WSH1–3 were synthesized in accordance with the earlier reported
method.[53]
|
4.1.3
General procedure for the synthesis of
1,3‐diphenyl‐1H‐pyrazole‐4‐carbaldehydes (WSP1–3)
3‐(4‐Fluorophenyl)‐5‐[3‐(4‐fluorophenyl)‐1‐phenyl‐1H‐pyrazol‐4‐yl]‐
4,5‐dihydropyrazole‐1‐carboxamide (WSPP3)
WSP1–3 were synthesized in accordance with the earlier reported
method.[53]
White fluffy solid; mp, 243–245°C; yield, 62%; Rf, 0.79; FTIR (cm−1):
3,318 and 3,256 (NH2), 1,678 (C═O), 1,536 (C═N); 1H NMR
(400 MHz, DMSO‐d6, δ); 3.10 (dd, 1H, JAM = 17.6 Hz and JAX = 6.8 Hz,
HA), 3.79 (dd, 1H, JMA = 17.6 Hz and JMX = 12.0 Hz, HM), 5.47 (dd, 1H,
JXM = 12.4 Hz and JXA = 6.8 Hz, HX), 6.52 (bs, 2H, NH2, D2O ex-
changeable), 7.24–7.30 (m, 5H, H‐3, H‐5, H‐4′, H‐3″, H‐5″), 7.43
(t, 2H, J = 8.4 Hz, H‐3′, H‐5′), 7.79–7.87 (m, 6H, H‐2, H‐6, H‐2′, H‐6′,
H‐2″, H‐6″), 8.34 (s, 1H, pyrazole ring); ESI–MS (m/z): 444 (M++1);
elemental analysis (%) of C25H19F2N5O, calculated/found: C (67.71/
67.95), H (4.32/4.30), N (15.79/15.81).
|
4.1.4
General procedure for the synthesis of 3‐
(1,3‐diphenyl‐1H‐pyrazol‐4‐yl)‐1‐phenylprop‐2‐en‐1‐
ones (WSC1–15)
WSC1–15 were synthesized in accordance with the earlier reported
method.[53]

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5‐[3‐(4‐Chlorophenyl)‐1‐phenyl‐1H‐pyrazol‐4‐yl]‐3‐(4‐fluorophenyl)‐
4,5‐dihydropyrazole‐1‐carboxamide (WSPP4)
White flakes; mp, 247–249°C; yield, 57%; Rf, 0.81; FTIR (cm−1): 3,353
and 3,251 (NH2), 1,666 (C═O), 1,587 (C═N); 1H NMR (400 MHz,
DMSO‐d6, δ); 3.10 (dd, 1H, JAM = 17.6 Hz and JAX = 6.4 Hz, HA), 3.79
3‐(4‐Chlorophenyl)‐5‐[3‐(4‐fluorophenyl)‐1‐phenyl‐1H‐pyrazol‐4‐yl]‐
4,5‐dihydropyrazole‐1‐carboxamide (WSPP8)
White fluffy solid; mp, 261–263°C; yield, 47%; Rf, 0.82; FTIR (cm−1):
3,363 and 3,294 (NH2), 1,663 (C═O), 1,583 (C═N); 1H NMR
(400 MHz, DMSO‐d6, δ); 3.11 (dd, 1H, JAM = 17.6 Hz and JAX = 6.4 Hz,
HA), 3.80 (dd, 1H, JMA = 17.6 Hz and JMX = 12.0 Hz, HM), 5.49 (dd, 1H,
JXM = 12.8 Hz and JXA = 6.4 Hz, HX), 6.51 (bs, 2H, NH2, D2O ex-
changeable), 7.24–7.30 (m, 3H, H‐4′ H‐3″, H‐5″), 7.44 (t, 2H,
J = 8.0 Hz, H‐3′, H‐5′), 7.49 (d, 2H, J = 8.4 Hz, H‐3, H‐5), 7.79–7.83
(m, 6H, H‐2, H‐6, H‐2′, H‐6′, H‐2″, H‐6″), 8.30 (s, 1H, pyrazole ring);
ESI–MS (m/z): 461 (M++2); elemental analysis (%) of C25H19ClFN5O,
calculated/found: C (65.29/65.45), H (4.16/4.14), N (15.23/15.21).
(dd, 1H, JMA = 17.6 Hz and JMX = 12.0 Hz, HM), 5.48 (dd, 1H, JXM
=
12.4 Hz and JXA = 6.4 Hz, HX), 6.53 (bs, 2H, NH2, D2O exchangeable),
7.25 (t, 3H, J = 8.8 Hz, H‐3, H‐5, H‐4′), 7.43–7.50 (m, 4H, H‐3′, H‐5′,
H‐3″, H‐5″), 7.76 (d, 2H, J = 8.4 Hz, H‐2′, H‐6′), 7.84–7.86 (m, 4H, H‐2,
H‐6, H‐2″, H‐6″), 8.34 (s, 1H, pyrazole ring); ESI–MS (m/z): 462
(M++2); elemental analysis (%) of C25H19ClFN5O, calculated/found: C
(65.29/65.82), H (4.16/4.14), N (15.23/15.45).
5‐(1,3‐Diphenyl‐1H‐pyrazol‐4‐yl)‐3‐(4‐fluorophenyl)‐4,5‐
3‐(4‐Chlorophenyl)‐5‐[3‐(4‐chlorophenyl)‐1‐phenyl‐1H‐pyrazol‐4‐yl]‐
4,5‐dihydropyrazole‐1‐carboxamide (WSPP9)
dihydropyrazole‐1‐carboxamide (WSPP5)
White fluffy solid; mp, 252–254°C; yield, 59%; Rf, 0.83; FTIR (cm−1):
3,348 and 3,265 (NH2), 1,668 (C═O), 1,534 (C═N); 1H NMR
(400 MHz, DMSO‐d6, δ); 3.07 (dd, 1H, JAM = 17.6 Hz and JAX = 5.2 Hz,
HA), 3.65 (dd, 1H, JMA = 17.2 Hz and JMX = 12.0 Hz, HM), 5.76 (dd, 1H,
JXM = 15.6 Hz and JXA = 4.4 Hz, HX), 7.10 (t, 2H, J = 8.8 Hz, H‐3, H‐5),
7.24 (t, 1H, J = 8.0 Hz, H‐4′), 7.39–7.43 (m, 5H, H‐3′, H‐5′, H‐3″, H‐4″,
H‐5″), 7.61 (d, 2H, J = 8.0 Hz, H‐2′, H‐6′), 7.67 (d, 2H, J = 8.0 Hz, H‐2″,
H‐6″), 7.74–7.77 (m, 2H, H‐2, H‐6), 7.86 (s, 1H, pyrazole ring);
ESI–MS (m/z): 426 (M++1); elemental analysis (%) of C25H20FN5O,
calculated/found: C (70.58/70.21), H (4.74/4.68), N (16.46/16.51).
White fluffy solid; mp, 264–266°C; yield, 63%; Rf, 0.77; FTIR (cm−1):
3,352 and 3,279 (NH2), 1,672 (C═O), 1,582 (C═N); 1H NMR
(400 MHz, DMSO‐d6, δ); 2.90 (dd, 1H, JAM = 16.4 Hz and JAX = 10.8
Hz, HA), 3.42 (dd, 1H, JMA = 16.0 Hz and JMX = 10.8 Hz, HM), 4.94 (dd,
1H, JXM = 10.8 Hz and JXA = 7.6 Hz, HX), 7.27 (t, 1H, J = 7.6 Hz, H‐4′),
7.40 (d, 2H, J = 8.4 Hz, H‐3, H‐5), 7.46 (t, 2H, J = 8.0 Hz, H‐3′, H‐5′),
7.49 (d, 2H, J = 8.4 Hz, H‐3″, H‐5″), 7.60 (d, 2H, J = 8.8 Hz, H‐2′, H‐6′),
7.76 (d, 2H, J = 8.4 Hz, H‐2″, H‐6″), 7.86 (d, 2H, J = 8.0 Hz, H‐2, H‐6),
8.56 (s, 1H, pyrazole ring); ESI–MS (m/z): 477 (M++2); elemental
analysis (%) of C25H19Cl2N5O, calculated/found: C (63.03/63.15), H
(4.02/3.98), N (14.70/14.66).
3‐(4‐Chlorophenyl)‐5‐[3‐(4‐methoxyphenyl)‐1‐phenyl‐1H‐pyrazol‐4‐
yl]‐4,5‐dihydropyrazole‐1‐carboxamide (WSPP6)
3‐(4‐Chlorophenyl)‐5‐(1,3‐diphenyl‐1H‐pyrazol‐4‐yl)‐4,5‐
dihydropyrazole‐1‐carboxamide (WSPP10)
White solid; mp 273–275°C; yield, 66%; Rf, 0.80; FTIR (cm−1): 3,316
and 3,246 (NH2), 1,663 (C═O), 1,587 (C═N); 1H NMR (400 MHz,
DMSO‐d6, δ); 3.13 (dd, 1H, JAM = 17.4 Hz and JAX = 6.4 Hz, HA), 3.84
White flakes; mp, 257–259°C; yield, 52%; Rf, 0.79; FTIR (cm−1): 3,326
and 3,246 (NH2), 1,668 (C═O), 1,522 (C═N); 1H NMR (400 MHz,
DMSO‐d6, δ); 3.09 (dd, 1H, JAM = 17.6 Hz and JAX = 6.4 Hz, HA), 3.79
(s, 3H, –OCH3 merged with HM), 3.79 (dd, 1H, JMA = 17.2 Hz and
JMX = 12.0 Hz, merged with –OCH3, HM), 5.48 (dd, 1H, JXM = 12.0 Hz
and JXA = 6.4 Hz, HX), 6.47 (bs, 2H, NH2, D2O exchangeable), 6.97 (d,
2H, J = 8.8 Hz, H‐3″, H‐5″), 7.28 (t, 1H, J = 7.2 Hz, H‐4′), 7.45 (t, 2H,
J = 8.0 Hz, H‐3′, H‐5′), 7.51 (d, 2H, J = 8.4 Hz, H‐3, H‐5), 7.70 (d, 2H,
J = 8.8 Hz, H‐2′, H‐6′), 7.86–7.88 (m, 4H, H‐2, H‐6, H‐2″, H‐6″), 8.33
(s, 1H, pyrazole ring); ESI–MS (m/z): 473 (M++2); elemental analysis
(%) of C26H22ClN5O2, calculated/found: C (66.17/66.32), H (4.70/
4.67), N (14.84/14.72).
(dd, 1H, JMA = 17.2 Hz and JMX = 12.0 Hz, HM), 5.51 (dd, 1H, JXM
=
12.0 Hz and JXA = 6.8 Hz, HX), 6.53 (bs, 2H, NH2, D2O exchangeable),
7.28 (t, 1H, J = 7.6 Hz, H‐4′), 7.42–7.54 (m, 7H, H‐3, H‐5, H‐3′ H‐5′, H‐
3″, H‐4″, H‐5″), 7.77 (d, 2H, J = 8.0 Hz, H‐2′, H‐6′), 7.86–7.89 (m, 4H,
H‐2, H‐6, H‐2″, H‐6″), 8.36 (s, 1H, pyrazole ring); ESI–MS (m/z): 443
(M++2); elemental analysis (%) of C25H20ClN5O, calculated/found: C
(67.95/67.75), H (4.56/4.54), N (15.85/15.61).
5‐[3‐(4‐Methoxyphenyl)‐1‐phenyl‐1H‐pyrazol‐4‐yl]‐3‐phenyl‐4,5‐
dihydropyrazole‐1‐carboxamide (WSPP11)
3‐(4‐Chlorophenyl)‐5‐[3‐(4‐methylphenyl)‐1‐phenyl‐1H‐pyrazol‐4‐
yl]‐4,5‐dihydropyrazole‐1‐carboxamide (WSPP7)
White fluffy solid; mp, 238–240°C; yield, 54%; Rf, 0.77; FTIR (cm−1):
3,360 and 3,276 (NH2), 1,663 (C═O), 1,592 (C═N); 1H NMR
(400 MHz, DMSO‐d6, δ); 3.06 (dd, 1H, JAM = 17.6 Hz and JAX = 6.4 Hz,
HA), 3.77 (dd, 1H, JMA = 17.6 Hz and JMX = 12.0 Hz, merged with
–OCH3, HM), 3.77 (s, 3H, –OCH3 merged with HM), 5.48 (dd, 1H,
JXM = 12.0 Hz and JXA = 6.4 Hz, HX), 6.42 (bs, 2H, NH2, D2O ex-
changeable), 6.94 (d, 2H, J = 8.4 Hz, H‐3″, H‐5″), 7.25 (t, 1H,
J = 7.2 Hz, H‐4′), 7.36 (t, 1H, J = 7.2 Hz, H‐4), 7.43 (t, 4H, J = 6.8 Hz,
H‐3, H‐5, H‐3′, H‐5′), 7.68 (d, 2H, J = 8.0 Hz, H‐2′, H‐6′), 7.79
(d, 2H, J = 8.4 Hz, H‐2″, H‐6″), 7.84 (d, 2H, J = 8.0 Hz, H‐2, H‐6),
8.26 (s, 1H, pyrazole ring); ESI–MS (m/z): 438 (M++1); elemental
White flakes; mp, 252–254°C; yield, 53%; Rf, 0.78; FTIR (cm−1): 3,368
and 3,309 (NH2), 1,665 (C═O), 1,593 (C═N); 1H NMR (400 MHz,
DMSO‐d6, δ); 2.31 (s, 3H, –CH3), 3.07 (dd, 1H, JAM = 17.6 Hz and
JAX = 6.4 Hz, HA), 3.78 (dd, 1H, JMA = 17.6 Hz and JMX = 12.0 Hz, HM),
5.47 (dd, 1H, JXM = 12.4 Hz and JXA = 6.8 Hz, HX), 6.45 (bs, 2H, NH2,
D2O exchangeable), 7.16–7.29 (m, 3H, H‐4′, H‐3″, H‐5″), 7.43–7.50
(m, 4H, H‐3, H‐5, H‐3′, H‐5′), 7.63 (d, 2H, J = 8.0 Hz, H‐2′, H‐6′),
7.77–7.85 (m, 4H, H‐2, H‐6, H‐2″, H‐6″), 8.30 (s, 1H, pyrazole ring);
ESI–MS (m/z): 457 (M++2); elemental analysis (%) of C26H22ClN5O,
calculated/found: C (68.49/68.69), H (4.86/4.83), N (15.36/15.33).

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analysis (%) of C26H23N5O2, calculated/found: C (71.38/71.51),
H (5.30/5.28), N (16.01/16.10).
(400 MHz, DMSO‐d6, δ); 3.11 (dd, 1H, JAM = 17.6 Hz and JAX = 6.4 Hz,
HA), 3.83 (dd, 1H, JMA = 17.6 Hz and JMX = 12.4 Hz, HM), 5.51 (dd, 1H,
JXM = 12.4 Hz and JXA = 6.4 Hz, HX), 6.53 (bs, 2H, NH2, D2O ex-
5‐[3‐(4‐Methylphenyl)‐1‐phenyl‐1H‐pyrazol‐4‐yl]‐3‐phenyl‐4,5‐
dihydropyrazole‐1‐carboxamide (WSPP12)
changeable), 7.25 (t, 1H, J = 7.2 Hz, H‐4′), 7.37–7.48 (m, 8H, H‐3, H‐4,
H‐5, H‐3′, H‐5′, H‐3″, H‐4″, H‐5″), 7.75 (d, 2H, J = 8.0 Hz, H‐2′, H‐6′),
7.80 (d, 2H, J = 7.2 Hz, H‐2″, H‐6″), 7.85 (d, 2H, J = 8.0 Hz, H‐2, H‐6),
8.31 (s, 1H, pyrazole ring); ESI–MS (m/z): 408 (M++1); elemental
analysis (%) of C25H21N5O, calculated/found: C (73.69/73.57), H
(5.19/5.17), N (17.19/17.22).
White fluffy solid; mp, 218–220°C; yield, 52%; Rf, 0.79; FTIR (cm−1):
3,362 and 3,273 (NH2), 1,664 (C═O), 1,590 (C═N); 1H NMR
(400 MHz, DMSO‐d6, δ); 2.33 (s, 3H, –CH3), 3.09 (dd, 1H, JAM
17.6 Hz and JAX = 6.4 Hz, HA), 3.81 (dd, 1H, JMA = 17.6 Hz and JMX
=
=
12.0 Hz, HM), 5.51 (dd, 1H, JXM = 8.8 Hz and JXA = 6.4 Hz, HX), 6.49
(bs, 2H, NH2, D2O exchangeable), 7.22 (d, 2H, J = 8.0 Hz, H‐3″, H‐5″),
7.27 (t, 1H, J = 7.2 Hz, H‐4′), 7.38 (t, 1H, J = 7.6 Hz, H‐4), 7.45 (t, 4H,
J = 7.2 Hz, H‐3, H‐5, H‐3′, H‐5′), 7.66 (d, 2H, J = 8.0 Hz, H‐2′, H‐6′),
7.81 (d, 2H, J = 7.2 Hz, H‐2″, H‐6″), 7.86 (d, 2H, J = 8.0 Hz, H‐2, H‐6),
8.29 (s, 1H, pyrazole ring); ESI–MS (m/z): 422 (M++1); elemental
analysis (%) of C26H23N5O, calculated/found: C (74.09/74.41), H
(5.50/5.48), N (16.62/16.55).
In vitro anticancer activity
All the cell lines, namely MCF‐7 (established from the pleural effusion
from a female suffering from a breast adenocarcinoma), A549 (ade-
nocarcinomic human alveolar basal epithelial cells), SiHa (cervical
cancer), COLO205 (colon cancer cell line), HepG2 (liver cell line), and
HaCaT cells (normal human keratinocytes), were obtained from the
National Center for Cell Science (NCCS), Pune, India; and the in vitro
anticancer activity was performed at Department of Biochemistry,
School of Chemical and Life Sciences, Jamia Hamdard, New Delhi,
India; and The Genome Biology Lab, Department of Biosciences,
Jamia Millia Islamia, New Delhi, India, using MTT growth inhibition
assay[43,44] (Table 1).
5‐[3‐(4‐Fluorophenyl)‐1‐phenyl‐1H‐pyrazol‐4‐yl]‐3‐phenyl‐4,5‐
dihydropyrazole‐1‐carboxamide (WSPP13)
White fluffy solid; mp, 220–222°C; yield, 67%; Rf, 0.82; FTIR (cm−1):
3,316 and 3,227 (NH2), 1,666 (C═O), 1,583 (C═N); 1H NMR
(400 MHz, DMSO‐d6, δ); 3.12 (dd, 1H, JAM = 17.6 Hz and JAX = 6.4 Hz,
HA), 3.83 (dd, 1H, JMA = 17.2 Hz and JMX = 12.4 Hz, HM), 5.34 (dd, 1H,
Cells in 96‐well plates were cultured in Dulbecco's modified
Eagle's medium supplemented with 10% heat‐inactivated fetal bo-
vine serum medium. Cells were maintained in a humidified incubator
containing 5% CO2 at 37°C. After the desired incubation with drugs,
cell viability was assessed by MTT assay. In brief, MTT was dissolved
in a phosphate‐buffered saline (PBS) at 5 mg/ml. The MTT solution
(0.5 mg/ml) was added to all wells and incubated at 37°C for 4 hr.
After incubation, the medium containing MTT was removed and MTT
crystals were dissolved by the addition of 100 μl DMSO. Absorbance
was measured using an ECIL microplate reader at a wavelength of
570 nm. IC50 values were determined from a graph plotted between
% cell viability (from control) versus concentration with the help of
dose–response regression equation. 5‐FU and cisplatin were taken as
the positive control. All the tests were done in at least three in-
dependent experiments. Initially, all the compounds were tested for
cytotoxicity effects against all lines at a concentration of 100 μM.
Compounds with ≤50% cytotoxicity were further evaluated for
dose–response study in a concentration range of 100–10 μM. Com-
pounds WSPP6, WSPP11, WSPP13, and WSPP14 were found to
exhibit ≤50% cytotoxicity at 10 μM concentration. Hence, con-
centrations were further decreased for these compounds and were
evaluated in the range of 1–10 μM.
JXM = 12.0 Hz and JXA = 6.4 Hz, HX), 6.54 (bs, 2H, NH2, D2O ex-
changeable), 7.25–7.31 (m, 3H, H‐4′, H‐3″, H‐5″), 7.38 (t, 1H,
J = 7.2 Hz, H‐4), 7.45 (t, 4H, J = 7.6 Hz, H‐3, H‐5, H‐3′, H‐5′),
7.81–7.89 (m, 6H, H‐2, H‐6, H‐2′, H‐6′, H‐2″, H‐6″), 8.32 (s, 1H,
pyrazole ring); 13C NMR (100 MHz, DMSO‐d6): 42.99, 52.86, 115.92,
116.14, 118.59, 125.24, 126.67, 126.97, 128.42, 128.54, 128.80,
128.84, 129.02, 129.14, 129.23, 129.96, 133.47, 139.83, 149.89,
150.17, 155.82, 162.08; ESI–MS (m/z): 426 (M++1); elemental ana-
lysis (%) of C25H20FN5O, calculated/found: C (70.58/70.39), H (4.74/
4.72), N (16.46/16.38).
5‐[3‐(4‐Chlorophenyl)‐1‐phenyl‐1H‐pyrazol‐4‐yl]‐3‐phenyl‐4,5‐
dihydropyrazole‐1‐carboxamide (WSPP14)
White fluffy solid; mp, 217–219°C; yield, 64%; Rf, 0.81; FTIR (cm−1):
3,328 and 3,246 (NH2), 1,673 (C═O), 1,592 (C═N); 1H NMR
(400 MHz, DMSO‐d6, δ); 3.09 (dd, 1H, JAM = 17.6 Hz and JAX = 6.4 Hz,
HA), 3.80 (dd, 1H, JMA = 17.6 Hz and JMX = 12.0 Hz, HM), 5.51 (dd, 1H,
JXM = 12.0 Hz and JXA = 6.4 Hz, HX), 6.52 (bs, 2H, NH2, D2O ex-
changeable), 7.25 (t, 1H, J = 7.2 Hz, H‐4′), 7.36 (t, 1H, J = 7.6 Hz, H‐4),
7.43–7.48 (m, 6H, H‐3, H‐5, H‐3′, H‐5′, H‐3″, H‐5″), 7.72–7.79 (m, 4H,
H‐2′, H‐6′, H‐2″, H‐6″), 7.84 (d, 2H, J = 8.0 Hz, H‐2, H‐6), 8.30 (s, 1H,
pyrazole ring); ESI–MS (m/z): 443 (M++2); elemental analysis (%) of
C25H20ClN5O, calculated/found: C (67.95/67.68), H (4.56/4.54), N
(15.85/15.61).
|
4.2
Biology
|
4.2.1
In vitro anti‐inflammatory activity
5‐(1,3‐Diphenyl‐1H‐pyrazol‐4‐yl)‐3‐phenyl‐4,5‐dihydropyrazole‐1‐
carboxamide (WSPP15)
Protein albumin denaturation assay
White fluffy solid; mp, 215–217°C; yield, 65%; Rf, 0.82; FTIR (cm−1):
3,362 and 3,274 (NH2), 1,663 (C═O), 1,592 (C═N); 1H NMR
The in vitro protein denaturation method described by Mizushima
and Kobayashi[45] was used for in vitro assessment of compounds

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WSPP1–15. A mixture of 1% aqueous solution of bovine serum al-
bumin (500 μl) and 1.4 ml of PBS (pH 7.4) was added separately to
100 μl of various concentrations (25, 50, 100, and 200 µg/ml) of
compounds WSPP1–15. The mixture was incubated at 37°C in an
incubator for 15 min. Then the reaction mixture was heated at
70°C for 5 min. Double‐distilled water was used as control. The reaction
mixture was cooled and the turbidity was measured at 660 nm.
Diclofenac sodium and celecoxib were used as standard. The entire
experiment was done in triplicate and average values were calculated.
The percentage inhibition of protein denaturation was calculated using
the following formula:
buffer solutions (960 ml, 0.1 M Tris‐HCl, pH 8.0 containing 5 mM
EDTA and 2 mM phenol) with either COX‐1 or COX‐2 (10 μl) en-
zyme in the presence of heme (10 μl). Prepared solutions were in-
cubated for a period of 5 min at 37°C, after which 10 μl of
arachidonic acid (100 μM solution) was added and the COX reaction
was stopped by the addition of 50 μl of 1 M HCl after 2 min. PGF2α
produced from PGH2 by reduction with stannous chloride was de-
termined by enzyme immunoassay. This assay is based on the
phenomenon of competition between prostaglandins (PGs) and
PG–acetylcholinesterase conjugate (PG tracer) for a limited amount
of PG antiserum. The amount of PG tracer that is able to bind to the
PG antiserum is inversely proportional to the concentration of PGs
in the wells, as when the concentration of PG tracer is held con-
stant, the concentration of PGs varies. This antibody–PG complex
binds to a mouse anti‐rabbit monoclonal antibody that had been
previously attached to the well. The plate is washed to remove any
unbound reagents and then Ellman's reagent, which contains the
substrate to acetylcholine esterase, is added to the well. The pro-
duct produced after this enzymatic reaction produces a distinct
yellow color that shows absorbance at 405 nm. The intensity of this
color, determined spectrophotometrically, is proportional to the
amount of PG tracer bound to the well, which is inversely propor-
tional to the amount of PGs present in the well during the incuba-
tion: Absorbance ∝ [Bound PG tracer] ∝ 1/PGs.
Percent inhibition = [(Abs. sample − Abs. control)/Abs. control]
× 100,
where Abs. control is the absorbance of the control and Abs. sample
is the absorbance of the test sample.
Red blood cell membrane stabilizing assay
The compounds were further evaluated for the anti‐inflammatory
activity using the reported method.[46] Blood from healthy volunteers
was mixed with an equal volume of 2% dextrose, 0.8% sodium citrate,
0.05% citric acid, and 0.42% sodium chloride in water. The prepared
mixture was centrifuged at 300 rpm and 10% v/v suspension was
prepared with isosaline (0.85%, pH 7.2) to get human red blood cell
(HRBC) suspension.
Percent inhibition was calculated by the comparison of com-
pound treated to various control incubations. The concentration of
the test compound causing 50% inhibition (IC50, μM) was calculated
from the concentration–inhibition response curve.
A mixture of compounds WSPP1–15 (100 and 200 µg/ml), 1 ml
phosphate buffer (0.15 M, pH 7.4), 2 ml of hyposaline (0.36%), and
0.5 ml of HRBC suspension was centrifuged. The hemoglobin content
in the supernatant solution was measured using colorimeter at
560 nm. Diclofenac sodium and celecoxib were used as standard. The
percentage of RBC membrane stabilization was determined using the
formula:
The ratio of IC50 value of COX‐1 and COX‐2 is called the se-
lectivity index of the derivatives. The higher the value, the higher will
be the selectivity of the derivatives toward COX‐2.
|
4.3
Docking and molecular dynamics simulations
% Protection = [(OD1)/(OD2)] × 100,
The three‐dimensional X‐ray crystal structure of COX‐2 in complex
with the selective inhibitor, 1‐phenylsulfonamide‐3‐trifluoromethyl‐
5‐parabromophenylpyrazole (PDB ID: 1CX2), was retrieved from
Protein Data Bank. AutoDock Tools 1.5.6 package was used to pre-
pare docking files. The two‐dimensional structure of the most potent
compound WSPP11 was drawn in ChemDraw Ultra 8.0 and con-
verted to its three‐dimensional coordinate, energy minimized by
MM2 method implemented in Chem3D Ultra 8.0, and saved as in a
pdb format. The partial atomic charges were calculated by the
Gasteiger method, nonpolar hydrogens were merged, and rotatable
bonds and torsion tree were defined. AutoGrid 4.2 was employed to
calculate a grid of 60, 60, and 60 points in x, y, and z directions having
grid spacing of 0.375 Å, and a distance‐dependent function of the
dielectric constant was instigated for the calculation of energetic
maps. AutoDock 4.2 was employed for docking simulation involving
100 independent runs by Lamarckian genetic algorithm methodology,
keeping default settings for all other parameters.[54] At the end of
docking, the best pose was analyzed for intermolecular interactions
where OD1 is the optical density of the test sample and OD2 is the
optical density of the control.
|
4.2.2
In vitro COX inhibition assay
To study the biological target, all four compounds, that is, WSPP6,
WSPP11, WSPP13, and WSPP14, were evaluated for their COX‐2
inhibition using method reported by Uddin et al.,[49] taking celecoxib
as standard.
The in vitro COX‐2 (IC50 values, μM) inhibition was evaluated
using COX (ovine) Inhibitor Screening Assay Kit (Item No. 560101),
as described earlier,[48] in accordance with the methodology de-
scribed by Uddin et al.[49] The stock solution of test compounds was
prepared in minimum volume of DMSO. Also, 10 μl of various con-
centrations of test drug solutions (0.01, 0.1, 1, 10, 50, and 100 μM in
a final volume of 1 ml) were added to a series of supplied reaction

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molecules24132413
ACKNOWLEDGMENT
The authors are thankful to Bioinformatics Facility, Jamia Hamdard,
New Delhi, for providing necessary facilities.
[22] T. Renjith, Y. Sheena, K. S. Resmi, B. Narayana, B. K. Sarojini, G.
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CONFLICTS OF INTERESTS
The authors declare that there are no conflicts of interests.
[24] S. Xuanrong, Z. Longchao, G. Mengshi, Q. Xiangjie, Z. Chenfeng, Z.
Dabu, C. Yue, Molecules 2019, 24, 624. https://doi.org/10.3390/
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ORCID
Mohammad Mumtaz Alam
http://orcid.org/0000-0001-6420-8757
[25] U. Jasmine, A. Ozair, M. J. Naim, N. Farah, M. J. Alam, A. K. Panda, M.
A. Alam, D. Vinod, J. Pharm. Bioallied Sci. 2016, https://doi.org/10.
4103/0975-7406.179425
Mymoona Akhtar http://orcid.org/0000-0003-4380-3965
Mohammad Shaquiquzzaman
http://orcid.org/0000-0002-
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Design and synthesis of pyrazole–pyrazoline hybrids as
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