Synthesis, Biological Activity, and Molecular Modeling Studies of Pyrazole and Triazole Derivatives as Selective COX-2 Inhibitors

Article abstract of DOI:10.1155/2020/6393428

Series of diaryl-based pyrazole and triazole derivatives were designed and synthesized in a facile synthetic approach in order to produce selective COX-2 inhibitor. These series of derivatives were synthesized by different reactions like Vilsmeier-Haack reaction and click reaction. In vitro COX-1 and COX-2 inhibition studies showed that five compounds were potent and selective inhibitors of the COX-2 isozyme with IC₅₀ values in 0.551-0.002 μM range. In the diarylpyrazole derivatives, compound 4b showed the best inhibitory activity against COX-2 with IC₅₀ = 0.017 μM as one of the N-aromatic rings was substituted with sulfonamide and the other aromatic ring was unsubstituted. However, when the N-aromatic ring was substituted with sulfonamide and the other aromatic ring was substituted with sulfone (compound 4d), best COX-2 selectivity was achieved (IC₅₀ = 0.098 μM, SI = 54.847). In the diaryltriazole derivatives, compound 15a showed the best inhibitory activity in comparison to all synthesized compounds including the reference celecoxib with IC₅₀ = 0.002 μM and SI = 162.5 as it could better fit the extra hydrophobic pocket which is present in the COX-2 enzyme. Moreover, the docking study supports the obtained SAR data and binding similarities and differences on both isozymes.

Full text of DOI:10.1155/2020/6393428

Hindawi
Journal of Chemistry
Volume 2020, Article ID 6393428, 14 pages
https://doi.org/10.1155/2020/6393428
Research Article
Synthesis, Biological Activity, and Molecular Modeling Studies of
Pyrazole and Triazole Derivatives as Selective COX-2 Inhibitors
Mohyeddin Assali ,1 Murad Abualhasan ,1 Hadeel Sawaftah,1 Mohammed Hawash ,1
and Ahmed Mousa2
1Department of Pharmacy, Faculty of Medicine and Health Sciences, An-Najah National University, P. O. Box 7,
Nablus, State of Palestine
2Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, An-Najah National University, P. O. Box 7,
Nablus, State of Palestine
Correspondence should be addressed to Mohyeddin Assali; m.d.assali@najah.edu
Received 5 October 2019; Revised 15 February 2020; Accepted 20 February 2020; Published 24 March 2020
Academic Editor: Maria Grazia Perrone
Copyright © 2020 Mohyeddin Assali et al. )is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Series of diaryl-based pyrazole and triazole derivatives were designed and synthesized in a facile synthetic approach in order to
produce selective COX-2 inhibitor. )ese series of derivatives were synthesized by different reactions like Vilsmeier–Haack
reaction and click reaction. In vitro COX-1 and COX-2 inhibition studies showed that five compounds were potent and selective
inhibitors of the COX-2 isozyme with IC50 values in 0.551–0.002 μM range. In the diarylpyrazole derivatives, compound 4b
showed the best inhibitory activity against COX-2 with IC50 � 0.017 μM as one of the N-aromatic rings was substituted with
sulfonamide and the other aromatic ring was unsubstituted. However, when the N-aromatic ring was substituted with sul-
fonamide and the other aromatic ring was substituted with sulfone (compound 4d), best COX-2 selectivity was achieved
(IC50 � 0.098 μM, SI � 54.847). In the diaryltriazole derivatives, compound 15a showed the best inhibitory activity in comparison
to all synthesized compounds including the reference celecoxib with IC50 � 0.002 μM and SI � 162.5 as it could better fit the extra
hydrophobic pocket which is present in the COX-2 enzyme. Moreover, the docking study supports the obtained SAR data and
binding similarities and differences on both isozymes.
conditions such as inflammation, hyperalgesia, and cancer
[6, 7]. In early 1990, the structures of both COX enzymes
were defined and they revealed that both isoforms are 67%
identical in amino acid sequences. However, the most
substantial difference between both isoforms is the presence
of valine (Val523) in COX-2 instead of isoleucine (Ile523) in
COX-1 which allows 25% greater available space binding
region in COX-2 in comparison to COX-1 [8].
Nonsteroidal anti-inflammatory drugs (NSAIDs) are
among the most prescribed medications for the relief of pain
and inflammation [9]. )eir pharmacological mechanism is
the inhibition of both COX enzymes which decreases
prostaglandin synthesis to exhibit their anti-inflammatory
activity [10]. Since they inhibit the constitutive COX-1
enzyme beside the COX-2, their chronic use will lead to
1. Introduction
Cyclooxygenase (COX) enzymes catalyze the biosynthesis of
prostaglandin H2 from arachidonic acid which was released
by the action of phospholipase A2 [1]. Prostaglandin H2 is
the precursor for the formation of other prostaglandins,
prostacyclin, and thromboxane that play major roles in
various important physiological and pathological responses
[2, 3]. COX are membrane-bound enzymes with the exis-
tence of two major isoforms: COX-1 and COX-2 [4]. COX-1
is a constitutive enzyme that is involved in the production of
essential prostaglandins which maintains the regular func-
tions in the body especially gastrointestinal and cardio-
vascular systems [5], whereas COX-2 is an inducible enzyme
that was overexpressed in various pathophysiological

2
Journal of Chemistry
unwanted side effects such as gastric ulcer, liver, and kidney
toxicities [11]. On the other hand, the selective inhibition of
the inducible COX-2 isozyme will provide a useful thera-
peutic approach with less produced side effects in com-
parison to the classical NSAIDs. In this context, various
selective COX-2 inhibitors (COXIBs) were developed and
marketed such as rofecoxib [12], celecoxib [13], valdecoxib
[14], and etoricoxib [15]. )ese drugs were used extensively
for the treatment of various inflammation conditions with
less gastric ulceration such as rheumatoid arthritis and
osteoarthritis [16]. However, different studies reported that
the chronic treatment of selective COX-2 inhibitors showed
potential cardiovascular side effects due to the decrease in
the production of the protective antiaggregatory prostacy-
clin (PGI2) while leaving the biosynthesis of prothrombotic
thromboxane A2 unaffected [17]. )erefore, various
COXIBs were withdrawn from the market, which increased
the demand for the synthesis of novel COX-2 inhibitors.
Studying the structures of most selective COX-2 inhibitors
revealed the presence of diaryl substitution on carbocyclic or
heterocyclic core (Figure 1). Moreover, structure-activity
relationship (SAR) studies of COXIBs showed that a COX-2
selectivity raised due to a hydrophobic interaction with an
extra hydrophobic region and polar interactions with a
secondary polar pocket that are found in COX-2 isozyme
[18]. Moreover, the presence of electron withdrawing group
in one of the aryl substitutions in the para position is more
preferable than corresponding electron donating group [18].
Various previous studies were implemented to synthesize
selective COX-2 inhibitors by using different methods
[18–22]. )e reported syntheses are commonly complex
which makes the production costs more expensive and
beyond the reach of an ordinary person. )erefore, there is a
need for new, effective, selective, cheaper, and safer COX-2
inhibitors. Herein, we aim to use a facile synthesis of series of
COX-2 inhibitors based on pyrazole or triazole core (Fig-
ure 1). Moreover, pyrazole and triazole heterocycles showed
interesting biological activity especially as anti-inflamma-
tory behavior and they have been used to synthesize novel
compounds as selective COX-1 inhibitors [23, 24]. In ad-
dition, they could synthesize easily with a high yield in one
step reaction through Vilsmeier–Haack reaction or click
reaction, respectively [25–27]. )en, the in vitro inhibitory
COX-1 and COX-2 were investigated for the synthesized
derivatives.
chromatography was used. )e used column was XTERRA
®
MS C18, 5 μm, 4.6 × 250 mm cartridge column, and the
mobile phase was acetonitrile : water (80 : 20). Chromato-
graphic separation and determination of % purity of the
eluted peaks for the tested compounds were performed using
high-performance liquid chromatography (Waters-1525,
Singapore) instrument with a binary HPLC pump and
Waters-2298 Photodiode Array Detector.
2.2. General Synthesis of Hydrazone Derivatives. An amount
of acetic acid glacial was added to a mixture of acetophenone
and phenyl hydrazine derivatives in 20 ml ethanol (EtOH),
and the reaction was refluxed overnight at 70°C. Ethanol was
removed under reduced pressure and the produced solid
product was used without further purification for pyrazole
synthesis.
2.3. General Synthesis of Pyrazole Derivatives.
Vilsmeier–Haack reagent was prepared by adding phosphorus
oxychloride (POCl3) dropwise at 0°C to a stirred dime-
thylformamide (DMF). )en, the synthesized hydrazone so-
lution in 1 ml DMF was added dropwise to Vilsmeier–Haack
reagent. )e reaction was refluxed for 5-6 h at 70°C. Later, the
reaction was poured on cold distilled water (DW) mixed with
concentrated solution of sodium bicarbonate. )en, the so-
lution was filtered by suction filtration. )e precipitate was
taken and purified by flash chromatography.
2.3.1. Synthesis of 3-(4-(Methylsulfonyl)Phenyl)-1-Phenyl-
1H-Pyrazole-4-Carbaldehyde (4a). To synthesize hydrazone
3a: 4′-(methyl sulfonyl)acetophenone (198.2 mg, 1.0 mmol)
with phenyl hydrazine (113 μl, 1.15 mmol) and glacial acetic
acid (86 μl, 1.5 mmol) was used to obtain a pure orange
product (Yield 97%, 280 mg, 0.97 mmol). Rf: 0.3 (Hexane :
EtOAc 1 :1). To synthesize pyrazole 4a: hydrazone 3a (0.3 g,
0.92 mmol) with POCl3 (1.5 ml, 15.8 mmol) in DMF (813 μl,
10.50 mmol) was used to obtain a pure white product (Yield
80%, 240 mg, 0.73 mmol). Rf: 0.3 (Hexane : EtOAc 1 :1). 1H
NMR (500 MHz, CDCl3): δ 10.05 (s, 1H, CHO), 8.56 (s, 1H,
CH pyrazole), 8.15 (d, 2H, J � 9.0 Hz, Ph’H-2, Ph’H-6), 8.04
(d, 2H, J � 9.1 Hz, Ph’H-3, Ph’H-5), 7.78 (d, 2H, J � 7.5 Hz,
Ph H-2, Ph H-6), 7.51 (t, 2H, J � 7.5 Hz, Ph H-3, Ph H-5),
7.41 (t, 1H, J � 7.5 Hz, Ph H-4), 3.08 (s, 3H, SO2CH3). 13C
NMR (125 MHz, CDCl3): δ 184.9, 151.0, 141.5, 138.9, 136.6,
136.5, 130.2, 129.9, 128.4, 127.7, 122.9, 119.8, 43.9. HRMS
(ESI, m/z): calcd. for C17H14N2O3S [M + H]+ 327.0803,
found 327.0798. % purity of 99% (by HPLC).
2. Materials and Methods
2.1.General. NMR analysis was measured by Bruker Avance
500 spectrometer at Jordan University. TMS was used as
internal standard and chemical shifts were expressed in ppm
on δ scale and coupling constant (J) was measured in Hz.
Unilab microplate reader 6000 was used to read the plate for
COX (human) Inhibitor Screening Assay Kit. High-reso-
lution mass spectra data (HRMS) were collected using a
Shimadzu LCMS-IT-TOF using ESI (+) method at Doping
and Narcotics Analysis Laboratory of the faculty of phar-
macy, Anadolu University, Turkey. To determine the purity
of the all synthesized COX-2 inhibitors, reverse phase
2.3.2. Synthesis of 4-(4-Formyl-3-Phenyl-1H-Pyrazol-1-yl)
Benzenesulfonamide (4b). To synthesize hydrazone 3b:
acetophenone (272.1 μl, 2.33 mmol) with 4-sulfonamide
phenyl hydrazine HCl (600 mg, 2.68 mmol) and glacial acetic
acid (116 μl, 2.03 mmol) was used to obtain a pure orange
product (Yield 96%, 650 mg, 2.25 mmol). Rf: 0.3 (Hexane :
EtOAc 1 :1). To synthesize pyrazole 4b: hydrazone 3b (1.0 g,
3.46 mmol) with POCl3 (2.77 ml, 29.63 mmol) in DMF
(1.53 ml, 19.75 mmol) was used to obtain a white product

Journal of Chemistry
3
O
O
CI
NH2
S
S
O
O
F
N
N
N
N
F
O
O
O
F
S
O
Rofecoxib
Celecoxib
Etoricoxib
R′
R′
N
N
N
N
R′
N
O
N
O
O
H
N
N
R
R
R
(1)
(2)
(3)
Figure 1: Structure of some selective COX-2 inhibitors and target compounds (1–3).
(Yield 94%, 1.07 g, 3.27 mmol). Rf: 0.3 (Hexane : EtOAc 1 :1).
1H NMR (500 MHz, CDCl3): δ 10.02 (s, 1H, CHO), 8.59 (s,
1H, CH pyrazole), 8.00 (d, 2H, J � 8.3 Hz, Ph H-2, Ph H-6),
7.89 (d, 2H, J � 8.4 Hz, Ph H-3, Ph H-5), 7.78 (d, 2H,
J � 6.0 Hz, Ph’ H-2, Ph’ H-6), 7.49–7.43 (m, 5H, J � 7.5 Hz,
Ph’ H-3, Ph’ H-5, Ph’H-4, SO2NH2). 13C NMR (125 MHz,
CDCl3): δ 185.0, 159.9, 141.6, 141.2, 132.3, 131.8, 131.0,
130.6, 129.8, 129.6, 129.0, 128.9, 128.7, 128.2, 123.0, 119.9,
119.5. HRMS (ESI, m/z): calcd. for C16H13N3O3S [M + H]+
328.0756, found 328.0750. % purity of 97% (by HPLC).
hydrazone 3d: 4′-(methyl sulfonyl) acetophenone (308 mg,
1.55 mmol) with 4-sulfonamide phenyl hydrazine HCl
(400 mg, 1.79 mmol) and glacial acetic acid (133 μl,
2.33 mmol) was used to obtain a pure orange product (Yield
88%, 500 mg, 1.36 mmol). Rf: 0.6 (Hexane : EtOAc 1 : 2). To
synthesize pyrazole 4d: hydrazone 3d (0.6 mg, 1.48 mmol)
with POCl3 (1.16 ml, 12.68 mmol) in DMF (655 μl,
8.46 mmol) was used to get a pure white product (Yield 43%,
260 mg, 0.64 mmol). Rf : 0.6 (Hexane : EtOAc 1 : 2).1H NMR
(500 MHz, DMSO): δ 10.05 (s, 1H, CHO), 8.62 (s, 1H, CH
pyrazole), 8.12–7.89 (m, 8H, Ph – Ph’), 7.23 (s, 2H, SO2NH2),
3.08 (s, 3H, SO2CH3). 13C NMR (125 MHz, DMSO): δ 184.9,
160.4, 151.5, 151.0, 148.1, 142.5, 141.7, 140.9, 138.7, 137.1,
136.5, 130.0, 128.2, 127.7, 127.5, 123.3, 123.0, 120.0, 119.1,
43.9. HRMS (ESI, m/z): calcd. for C17H15N3O5S2 [M + H]+
406.0531, found 406.0526. % purity of 96% (by HPLC).
2.3.3. Synthesis of 4-(3-(4-Bromophenyl)-4-Formyl-1H-Pyr-
azol-1yl)Benzenesulfonamide (4c). To synthesize hydrazone
3c: 4-bromoacetophenone (464 mg, 2.33 mmol) with 4-
sulfonamide phenyl hydrazine HCl (600 mg, 2.68 mmol) and
glacial acetic acid (116 μl, 2.03 mmol) was used to obtain a
pure red product (Yield 85%, 730 mg, 1.9 mmol). Rf: 0.6
(Hexane : EtOAc 1 : 2). To synthesize pyrazole 4c: hydrazone
3c (0.6 g, 1.6 mmol) with POCl3 (1.28 ml, 13.7 mmol) in
DMF (708 μl, 9.14 mmol) was used to obtain a pure white
product (Yield 92%, 600 mg, 1.48 mmol). Rf: 0.6 (Hexane :
2.3.5. Synthesis of 4-(4-Formyl-1-Phenyl-1H-Pyrazol-3-yl)
Benzenesulfonamide (4e). To synthesize hydrazone 3e: 4-
acetyl benzene sulfonamide (400 mg, 2.0 mmol) with phenyl
hydrazine (226 μl, 2.3 mmol) and glacial acetic acid (172 μl,
3.0 mmol) was used to get a pure red product (Yield 95%,
550 mg, 1.9 mmol). Rf: 0.5 (Hexane : EtOAc 1 :1). To syn-
thesize pyrazole 4e: hydrazone 3e (0.5 g, 1.9 mmol) with
POCl3 (1.52 ml, 16.3 mmol) in DMF (841 μl, 10.86 mmol)
was used to obtain a white product (Yield 32%, 200 mg,
0.61 mmol). Rf: 0.5 (Hexane : EtOAc 1 :1). 1H NMR
(500 MHz, DMSO): δ 9.95 (s, 1H, CHO), 9.27 (s, 1H, CH
pyrazole), 7.95 (d, 2H, J � 8.6 Hz, Ph’ H-2, Ph’ H-6), 7.87 (d,
2H, J � 7.9 Hz, Ph’ H-3, Ph’ H-5), 7.72 (d, 2H, J � 7.9 Hz, Ph
H-2, Ph H-6), 7.52 (t, 3H, J � 7.6 Hz, Ph H-3, Ph H-5, Ph
H-4), 7.39 (s, 2H, SO2NH2). 13C NMR (125 MHz, DMSO): δ
185.1, 160.4, 152.6, 148.9, 139.0, 136.3, 135.4, 131.4, 130.2,
1
EtOAc 1 : 2). H NMR (500 MHz, DMSO): δ 9.32 (s, 1H,
CHO), 7.56 (s, 1H, CH pyrazole), 7.45–7.42 (m, 2H, Ph H-2,
Ph H-6), 7.27–7.16 (m, 6H, Ph H-3, Ph H-5, Ph’H-2, Ph’H-3,
Ph’H-5, Ph’H-6), 6.97–6.94 (m, 2H, SO2NH2). 13C NMR
(125 MHz, DMSO): δ 184.9, 160.4, 152.1, 142.3, 140.9, 136.8,
132.0, 131.1, 130.7, 128.2, 127.5, 123.4, 123.1, 119.9, 119.0.
HRMS (ESI, m/z): calcd. forC16H12N3O3SBr [M + H]+
405.9861, found 405.9855. % purity of 97% (by HPLC).
2.3.4. Synthesis of 4-(4-Formyl-3-(4-(Methylsulfonyl)Phenyl)-
1H-Pyrazol-1-yl)Benzenesulfonamide (4d). To synthesize

4
Journal of Chemistry
129.6, 128.2, 126.6, 126.2, 122.7, 119.7. HRMS (ESI, m/z):
calcd. for C16H13N3O3S [M + H]+ 328.0756, found 328.0750.
% purity of 96% (by HPLC).
obtained (yield 85.3%, 490 mg, 2.47 mmol). )e character-
ization of 6a was adequate to the literature [28, 29].
(2) Synthesis of 1-Azido-4(Methylsulfonyl) Benzene (6b).
Following the general synthesis of azide derivatives, com-
pound 6b was obtained (yield 88%, 420 mg, 2.13 mmol). )e
characterization of 6b was adequate to the literature [30].
2.3.6. Synthesis of 1-(4-Bromophenyl)-3-(4-(Methylsulfonyl)
Phenyl)-1H-Pyrazole-4-Carbaldehyde (4f). To synthesize
hydrazone 3f: 4′-(methyl sulfonyl) acetophenone (400 mg,
2.0 mmol) with 4-bromo phenyl hydrazinum chloride
(514 mg, 2.3 mmol) and glacial acetic acid (172 μl, 3.0 mmol)
was used to get a pure brown product (Yield 91%, 670 mg,
1.8 mmol). Rf: 0.3 (Hexane : EtOAc 1 :1). To synthesize
pyrazole 4f: hydrazone 3f (0.6 g, 1.6 mmol) with POCl3
(1.31 ml, 14.0 mmol) in DMF (720 μl, 9.33 mmol) was used to
have a pure yellow product (Yield 55%, 360 mg, 0.88 mmol).
Rf: 0.3 (Hexane : EtOAc 1 :1). 1H NMR (500 MHz, DMSO): δ
9.96 (s, 1H, CHO), 9.38 (s, 1H, CH pyrazole), 8.17 (d, 2H,
J � 7.9 Hz, Ph’ H-2, Ph’ H-6), 8.01 (d, 2H, J � 7.6 Hz, Ph’ H-3,
Ph’ H-5), 7.93 (d, 2H, J � 10.1 Hz, Ph H-2, Ph H-6), 7.73 (d,
2.4.2. General Synthesis of Triazoles (Click Reaction). A
mixture of sodium ascorbate (1 eq.) and CuSO4 anhydrous (1
eq.) in 4 ml distilled was added to a mixture of azide de-
rivative (1 eq.) and 4-ethynyl-α,α,α-triflurotoluene 7 (1.2 eq)
in 4 ml DCM, and the reaction was left on stirrer overnight.
After that, the reaction was diluted with 70 ml DCM and
washed with 50 ml DW. )e solvent was removed under
reduced pressure and the remaining crude was purified by
flash chromatography on silica gel.
(1) Synthesis of 4-(4-(4-(Trifluoromethyl)Phenyl)-1H-1,2,3-
Triazol-1-yl)Benzenesulfonamide (8a). Following the general
procedure of click reaction, 4-azidobenzenesulfonamide 6a
2H, J � 8.6 Hz, Ph H-3, Ph H-5), 3.22 (s, 3H, SO2CH3). 13
C
(150 mg, 0.76 mmol), 4-ethynyl-α,α,α-triflurotoluene
7
NMR (125 MHz, DMSO): δ 189.1, 155.8, 146.2, 142.8, 141.4,
137.7, 134.5, 132.3, 127.9, 126.3, 125.7, 48.8. HRMS (ESI, m/
z): calcd. For C17H13N2O3SBr [M + H]+ 404.9909, found
404.9903. % purity of >99% (by HPLC).
(149.0 μl, 0.91 mmol), sodium ascorbate (150.6 mg,
0.76 mmol), and CuSO4 anhydrous (121.3 mg, 0.76 mmol)
were used. )e crude was purified by flash chromatography
on silica gel, eluting with Hexane : EtOAc (2 :1), and a pure
white solid product was obtained (Yield 36%, 100 mg,
0.27 mmol). Rf: 0.3 (Hexane/EtOAc 2 :1).1H NMR
(500 MHz, DMSO): δ 9.61 (s, 1H, CH triazole), 8.18 (dd, 4H,
J � 5.8 Hz, J � 2.8 Hz, Ph H-2, Ph H-6, Ph H-3, Ph H-5), 8.09
(d, 2H, J � 8.5 Hz, Ph’ H-3, Ph’ H-5), 7.91 (d, 2H, J � 8.2 Hz,
Ph’ H-2, Ph’ H-6), 7.55 (s, 2H, SO2NH2). 13C NMR
(125 MHz, DMSO): δ 146.7, 144.6, 139.0, 134.5, 129.2, 128.9,
128.1, 126.5, 126.4, 125.8, 123.6, 121.6, 120.8. HRMS (ESI, m/
z): calcd. for C15H11N4O2F3S [M + H]+ 369.0633, found
369.0628. % purity of 99% (by HPLC).
2.3.7. Synthesis of 3-(4-(Methylsulfonyl)Phenyl)-1-(4-Nitro-
phenyl)-1H-Pyrazole-4-Carbaldehyde (4g). To synthesize
hydrazone 3g: 4′-(methyl sulfonyl)acetophenone (400 mg,
2.0 mmol) with 4-Nitro phenyl hydrazine (352 mg,
2.3 mmol) and glacial acetic acid (172 μl, 3.0 mmol) was used
to have a pure orange product (Yield 88%, 590 mg,
1.76 mmol). Rf: 0.3 (Hexane : EtOAc 1 :1). To synthesize
pyrazole 4g: hydrazone 3g (0.6 mg, 1.8 mmol) with POCl3
(1.45 ml, 15.4 mmol in DMF (794 μl, 10.28 mmol) was used
to have a pure brown product (Yield 99%, 663 mg,
1.8 mmol). Rf: 0.3 (Hexane : EtOAc 1 :1). 1H NMR
(500 MHz, DMSO): δ 10.03 (s, 1H, CHO), 9.57 (s, 1H, CH
pyrazole), 8.37 (d, 2H, J � 6.7 Hz, Ph H-3, Ph H-5), 8.26 (d,
2H, J � 7.9 Hz, Ph H-2, Ph H-6), 8.20 (d, 2H, J � 8.5 Hz, Ph’
H-2, Ph’ H-6), 8.02 (d, 2H, J � 7.9 Hz, Ph’ H-3, Ph’ H5), 3.16
(s, 3H, SO2NH2). 13C NMR (125 MHz, DMSO): δ 184.9,
151.9, 146.5, 143.2, 141.8, 137.6, 136.1, 130.0, 127.7, 125.9,
123.7, 120.3, 43.9. HRMS (ESI, m/z): calcd. for C17H13N3O5S
[M + H]+ 372.0654, found 372.0649. % purity of 96% (by
HPLC).
(2) Synthesis of 1-(4-(Methylsulfonyl)Phenyl)-4-(4-(Tri-
fluoromethyl)Phenyl)-1H-1,2,3-Triazole (8b). Following the
general procedure of click reaction, 1-azido-4(methyl sul-
fonyl) benzene 6b (150 mg, 0.76 mmol), 4-ethynyl-
α,α,α-triflurotoluene
7 (149.0 μl, 0.91 mmol), sodium
ascorbate (150.6 mg, 0.76 mmol), and CuSO4 anhydrous
(121.3 mg, 0.76 mmol) were used. )e crude was purified by
flash chromatography on silica gel, eluting with Hexane :
EtOAc (3 : 2). A pure white solid product was obtained
(Yield 88%, 420 mg, 2.13 mmol). Rf: 0.4 (Hexane/EtoAc 3 :
2).1H NMR (500 MHz, DMSO): δ 9.62 (s, 1H, CH triazole),
8.23 (d, 2H, J � 8.9 Hz, Ph’ H-3, Ph’H-5), 8.18 (dd, 4H,
J � 8.5 Hz, J � 8.2 Hz, Ph H-2, Ph H-6, Ph H-3, Ph H-5), 8.87
(d, 2H, J � 8.2 Hz, Ph’ H-2, Ph’ H-6), 3.28 (s, 3H, SO2CH3).
13C NMR (125 MHz, DMSO): δ 146.8, 141.1, 140.3, 134.4,
129.6, 129.2, 128.9, 126.5, 126.4, 125.7, 121.7, 121.0, 44.0.
HRMS (ESI, m/z): calcd. forC16H12N3O2F3S [M + H]+
368.0681, found 368.0675. % purity of >99% (by HPLC).
2.4. Synthesis of Triazole Derivatives
2.4.1. General Synthesis of Azide Derivatives. A solution of
the amine (1 eq.) is dissolved in 4 M HCl (5 ml) at 0°C, 5 M
sodium nitrite (NaNO2) (1.2 eq.). )en, the solution was
added dropwise and stirred for about 10 min. )en, 5 M
solution of sodium azide (NaN3) (1.5 eq.) was added to
reaction dropwise. )e reaction was stirred for 30 min at RT.
A solid product was obtained after evaporation and was used
without further purification.
2.4.3. Synthesis of 2-Hydroxyethyl 4-Methylbenzenesulfonate
(11). A mixture of ethylene glycol 9 (5 ml, 89.72 mmol) and
triethylamine (6.25 ml, 44.86 mmol) in 6 ml DCM was
stirred for 30 min. )en, tosyl chloride 10 (8.55 g,
44.80 mmol) was added dropwise for about 30 min. )e
(1) Synthesis of 4-Azidobenzenesulfonamide (6a). Following
the general synthesis of azide derivatives, compound 6a was

Journal of Chemistry
5
reaction was stirred overnight. )e reaction was diluted with
70 ml DCM and washed with 1 M HCl (50 ml). )e solvent
was removed under reduced pressure and semisolid product
was obtained. )e crude was purified by flash chromatog-
raphy using Hexane : EtOAc (1 :1). An oily pale yellow
product was obtained (Yield 8.5%, 1.65 g, 7.63 mmol). Rf: 0.4
(500 MHz, DMSO): δ 8.12 (d, 2H, J = 8.5 Hz, Ph H-2, Ph
H-6), 7.94 (d, 2H, J = 8.5 Hz, Ph H-3, Ph H-5), 7.53 (s, 2H,
SO2NH2), 4.45 (t, 2H, J = 4.9 Hz, CH2O), 3.66 (t, 2H,
J = 4.9 Hz, CHN3).13C NMR (125 MHz, DMSO): δ 165.0,
148.7, 132.6, 130.4, 127.7, 126.6, 125.5, 64.6, 49.8.
1
(Hexane/EtOAc 1 :1). H NMR (500 MHz, CDCl3): δ 7.79
2.4.6. Synthesis of 2-(4-(4-(Trifluoromethyl)Phenyl)-1H-
1,2,3-Triazol-1-yl)Ethyl 4-(Methylsulfonyl)Benzoate (15a).
Following the general procedure of click reaction, 2-azi-
(d, 2H, J � 8.4 Hz, H2OTs), 7.34 (d, 2H, J � 7.9 Hz, H2OTs),
4.11 (t, 2H, J � 4.3 Hz, CH2O), 3.79 (t, 2H, J � 4.6 Hz, CH2O),
2.42 (s, 3H, CH3), 2.00 (bs, 1H, OH). 13C NMR (125 MHz,
CDCl3): δ 145.1, 132.6, 130.0, 128.0, 71.7, 60.7, 21.7.
doethyl
4-(methylsulfonyl)benzoate
14a
(240 mg,
(174.5 μl,
0.89 mmol), 4-ethynyl-α,α,α-triflurotoluene
7
1.1 mmol), sodium ascorbate (176.3 mg, 0.89 mmol), and
CuSO4 anhydrous (142 mg, 0.89 mmol) were used. )e crude
was purified by flash chromatography on silica gel, eluting
with DCM : MeOH (20 :1). A pure white solid product was
obtained (Yield 36%, 140 mg, 0.32 mmol). Rf: 0.4 (DCM/
2.4.4. Synthesis of 2-Azidoethanol (12). Sodium azide (0.3 g,
4.65 mmol) was added to a solution of monotosylated
ethylene glycol 11 (1 g, 4.2 mmol) in 5 ml ethanol, and the
reaction was refluxed overnight on 70°C. )en, the reaction
was diluted with diethyl ether and washed with water three
times. )en, the organic layers were collected and a pure pale
yellow oily product was obtained after evaporation (Yield
1
MeOH 20 :1). H NMR (500 MHz, DMSO): δ 8.84 (s, 1H,
CH triazole), 8.10 (d, 2H, J � 8.6 Hz, Ph’ H-3, Ph’ H-5), 8.02
(dd, 4H, J � 6.4 Hz, J � 1.8 Hz, Ph H-2, Ph H-6, Ph H-3, Ph
H-5), 7.78 (d, 2H, J � 8.2 Hz, Ph’ H-2, Ph’ H-6), 8.86 (t, 2H,
J � 4.6 Hz, CH2O), 4.74 (t, 2H, J � 4.9 Hz, CH2N), 3.28 (s, 3H,
SO2CH3). 13CNMR (125 MHz, DMSO): δ 164.7, 145.6, 135.1,
134.0, 130.6, 127.9, 126.1, 123.6, 64.2, 49.3, 43.6. HRMS (ESI,
m/z): calcd. forC19H16N3O4F3S [M + H]+ 440.0892, found
440.0885. % purity of 98% (by HPLC).
1
96%, 350 mg, 4.02 mmol). Rf: 0.6 (DCM/MeOH 20 :1). H
NMR (500 MHz, CDCl3): δ 3.73 (t, 2H, J � 4.1 Hz, CH2OH),
3.40 (t, 2H, J � 4.7 Hz, CH2N3), 2.25 (bs, 1H, OH). 13C NMR
(125 MHz, CDCl3): δ 61.8, 53.5.
2.4.5. General Procedure of Esterification Reaction. A mix-
ture of acid derivative (1 eq.), 2-azidoethanol 12 (1.5 eq.), 1-
(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochlo-
ride (EDC) (2 eq.), and dimethylamino pyridine (DMAP)
(1.5 eq.) in 6 ml of DCM was put on stirrer under argon and
the reaction was left overnight. )en, the reaction was di-
luted with 70 ml DCM and washed with 50 ml 1 M HCl. )e
organic layer was collected and evaporated under reduced
pressure. )e remaining crude was purified by flash chro-
matography on silica gel.
(1) Synthesis of 2-Azidoethyl 4-(Methylsulfonyl)Benzoate
(14a). Following the general procedure for esterification
reaction, 4-(methylsulfonyl) benzoicacid 13a (150 mg,
0.75 mmol), 2-azidoethanol 12 (98 mg, 1.125 mmol), EDC
(287.55 mg, 1.5 mmol), and DMAP (138.0 mg, 1.125 mmol)
in 6 ml DCM were used. )en, the crude product was pu-
rified using flash chromatography with a mobile phase of
Hexane : EtOAc (1 :1). A pure white solid product was ob-
tained (Yield 69%, 140 mg, 0.52 mmol). Rf: 0.4 (Hexane/
EtOAc 1 :1).1H NMR (500 MHz, DMSO): δ 8.17 (d, 2H,
J = 7.9 Hz, Ph H-2, Ph H-6), 8.07 (d, 2H, J = 8.2 Hz, Ph H-3,
Ph H-5), 4.47 (t, 2H, J = 4.9 Hz, CH2O), 3.67 (t, 2H,
J = 5.2 Hz, CH2N3), 3.24 (s, 3H, SO2CH3). 13C NMR
(125 MHz, DMSO): δ 164.9, 145.4, 134.1, 130.6, 128.0, 64.8,
49.8, 43.7.
2.4.7. Synthesis of 2-(4-(4-(Trifluoromethyl)Phenyl)-1H-
1,2,3-Triazol-1-yl)Ethyl
4-Sulfamoylbenzoate
(15b).
Following the general procedure of click reaction, 2-azi-
doethyl 4-sulfamoylbenzoate 14b (280 mg, 1.04 mmol), 4-
ethynyl-α,α,α-triflurotoluene 7 (203.57 μl, 1.24 mmol), so-
dium ascorbate (206 mg, 1.04 mmol), and CuSO4 anhydrous
(166 mg, 1.04 mmol) were used. )e crude was purified by
flash chromatography on silica gel, eluting with Hexane :
EtOAc (1 :1), and a pure white solid product was obtained
(Yield 48%, 220 mg, 0.5 mmol). Rf: 0.4 (Hexane/EtOAc 1 :1).
1H NMR (500 MHz, DMSO): δ 8.86 (s, 1H, CH triazole),
8.07-8.03 (m, 4H, Ph H-2, Ph H-6, Ph H-3, Ph H-5), 7.92-
7.90 (m, 2H, Ph’ H-3, Ph’ H-5), 7.80–7.77 (m, 2H, Ph’ H-2,
Ph’ H-6), 7.52 (bs, 2H, SO2NH2), 4.86 (t, 2H, J � 4.0 Hz,
CH2O), 4.73 (t, 2H, J � 3.9 Hz, CH2N). 13C NMR (125 MHz,
DMSO): δ 164.9, 148.7, 145.5, 135.1, 132.4, 130.4, 128.6,
126.5, 123.7, 64.0, 49.4. HRMS (ESI, m/z): calcd. for
C18H15N4O4F3S [M + H]+ 441.0844, found 441.0839. %
purity of 98% (by HPLC).
2.5. In Vitro COX-1 and COX-2 Inhibition Assay. )e COX-1
and COX-2 inhibitory activities were tested on COX (human)
Inhibitor Screening Assay Kit (supplied by Cayman chemicals,
Ann Arbor, MI, USA). )e preparation of the reagents and the
testing procedure were performed according to the manu-
facturer recommendations. In brief, various concentrations of
the inhibitors and celecoxib (concentration range
100 μM–0.001 μM) dissolved in a minimum quantity of
dimethylsulfoxide (DMSO) were incubated with a mixture of
COX-1 or COX-2 enzyme, and heme dissolved in the reaction
buffer. )e reaction was initiated by adding 50 μl of
(2) Synthesis of 2-Azidoethyl 4-Sulfamoylbenzoate (14b).
Following the general procedure for esterification reaction,
4-sulfamoylbenzoicacid 13b (150 mg, 0.75 mmol), 2-azi-
doethanol 12 (97.4 mg, 1.12 mmol), EDC (287.6 mg,
1.5 mmol), and DMAP (138.0 mg, 1.125 mmol) in 6 ml DCM
were used. )e crude product was purified using flash
chromatography with a mobile phase of DCM : MeOH (9 :
1). A pure white solid product was obtained (Yield 49%,
1
100 mg, 0.37 mmol). Rf: 0.4 (DCM : MeOH 9 :1). H NMR

6
Journal of Chemistry
arachidonic acid followed by incubation at 37°C for exactly 30
seconds. )en, the reaction was stopped by adding 30 μl of
stannous chloride solution to each reaction tube followed by
incubation for 5 min at room temperature. )e produced
PGF2a in the samples by COX reactions was quantified via
enzyme-linked immunosorbent assay (ELISA). )e 96-well
plate was covered with plastic film and incubated for 18 h at
room temperature on an orbital shaker. After incubation, the
plate was rinsed five times with the washed buffer followed by
the addition of Ellman’s reagent (200 μl) and incubated for
about 60–90 min at room temperature until the absorbance of
Bo well is in the range 0.3–0.8 at 405 nm. )e plate was then
read by an ELISA plate reader Unilab microplate reader 6000.
)e inhibitory percentage was measured for the different tested
concentrations against the control. IC50 was calculated from
the concentration inhibition response curve and the selectivity
index (SI) was calculated by dividing the IC50 COX-1 on the
IC50 COX-2. Celecoxib was used as positive standard drug in
the study. )e COX testing procedure was done in duplicate.
compounds (3a–3g) were generated by reacting com-
pounds of acetophenone derivatives with different phenyl
hydrazine derivatives in ethanol (EtOH) using acetic acid
glacial as an acid catalyst. After that, hydrazones were
reacted with Vilsmeier–Haack reagent which was produced
by reacting dimethyformamide (DMF) with phosphorus
oxychloride (POCl3) [40]. Different pyrazoles were pro-
duced as shown in Scheme 1. All synthesized pyrazoles
were purified by flash chromatography. )e structures of
these compounds were confirmed by high-resolution mass
1
spectrometry (HRMS), and H NMR, 13C NMR spectral
data, and % purity of the synthesized compounds were
determined by HPLC.
3.1.2. Synthesis of Triazole Derivatives. Click chemistry was
introduced in 2001 by Sharpless and co-workers. It com-
prises a series of nearly perfect chemical reactions that were
involved in many synthetic approaches with a variety of
starting materials and reagents [26, 27, 41]. )e synthesis of
triazole compounds (8a and 8b) was achieved by converting
the amino group of sulfanilamide or 4-(methyl sulfonyl)
aniline to their corresponding azide. )is conversion was
achieved by reacting the amino group with HCl, NaNO2, and
NaN3 to synthesize compounds 6a or 6b, respectively, as
shown in Scheme 2. )ese prepared azides were subjected to
cycloaddition with 4-Ethynyl-α,α,α-trifluorotoluene (7)
using anhydrous copper sulfate in the presence of sodium
ascorbate in DCM/H2O (1 :1) providing the corresponding
triazoles (8a and 8b) as shown in Scheme 2. All synthesized
compounds were purified by flash chromatography. )e
chemical structure and % purity of each compound were
2.6. Molecular Modeling Studies. )e predicted protein-li-
gand interactions were identified by doing molecular docking
with human COX-1 and human COX-2 crystal structures
(PDB codes 3KK6 [31] and 5KIR [32], respectively). While
building the docking grids, crystal structure of COX-1 was
chosen from a highly close protein-ligand complex of cele-
coxib, 3KK6 [31]. Crystal structure for COX-2 was chosen by
analyzing crystal evaluation. Results were analyzed for 5KIR
and compared to a highly similar COXIB member containing
˚
crystal structure, 6COX [33] (e.g., resolutions were 2.7 A and
˚
2.8 A, respectively). Both structures were used for docking
1
confirmed by H NMR, 13C NMR, HRMS, and HPLC.
since the active site residues (Figures S1–S3) were conserved
within crystal source organisms. Generated docking results
)e synthesis of the triazole derivatives with spacer was
achieved firstly through the synthesis of bifunctional spacer
12 by selective tosylation of ethylene glycol followed by a
nucleophilic substitution using sodium azide to obtain the
corresponding spacer 12 as shown in Scheme 3. )e syn-
thesized spacer 12 was esterified with 4-(methyl sulfonyl)
benzoic acid (13a) or 4-sulfamoyl benzoic acid (13b) using
EDC as a coupling agent and DMAP as a catalyst to syn-
thesize 14a or 14b, respectively, as shown in Scheme 4. In a
final step, a click reaction was performed with the synthe-
sized azide derivatives and 4-ethynyl-α,α,α-trifluorotoluene
(7) in the presence of anhydrous copper sulfate and sodium
ascorbate in DCM : H2O (1 :1) as shown in Scheme 4,
obtaining the final triazole derivatives 15a and 15b. All
synthesized compounds were purified by flash chromatog-
raphy. )e chemical structure and % purity of each com-
pound were confirmed by 1H NMR, 13C NMR, HRMS, and
HPLC.
˚
with 5KIR and 6COX were highly close (RMSD < 1 A) based
on the heavy atoms of the ligands. We decided to report the
docking results obtained with 5KIR for COX-2 because the
binding orientations were more relevant due to shared car-
bonyl moiety. )e docking results of pyrazole and triazole
derivatives were visualized at the COX-1 and COX-2 active
sites. )e structures were drawn by using Maestro 11.1
Graphical User Interface [34]. )e ligands were prepared by
LigPrep [35] routine (pH 7.0 2.0) to prepare the ligands and
parameterize the atom types and the protonation states with
OPLS2005 force field. )e same force field was used for
proteins, and predicted positions of the missing side chains
were added with Protein Preparation Wizard [36]. )e mo-
lecular docking studies were done with Glide 7.4 [37] to
predict the protein-ligand interactions. )e grids were gen-
erated, and docking simulations were done in single-precision
mode (GlideScore SP). )e binding modes were evaluated
with PLIP 1.4.4 [38], and the resulting poses were visualized
with PyMOL 2.3 [39].
3.2. In Vitro COX-1 and COX-2 Inhibition Assay. All syn-
thesized compounds were tested for inhibition assay on
COX-1 and COX-2 enzymes using the COX-1 (human)
Inhibitor Screening Assay Kit and COX-2 (human) Inhibitor
Screening Assay Kit (Cayman Chemical Company, Ann
Arbor, MI, USA). Serial concentrations in the range
(100 μM–0.001 μM) were used for each compound in the
3. Results and Discussion
3.1. Chemistry
3.1.1. Synthesis of Pyrazole Derivatives. Pyrazole derivatives
(4a–4g) were synthesized in two steps. Firstly, hydrazone

Journal of Chemistry
7
R′
R′
O
R
(b)
(a)
+
NH
H
N
2
O
N
N
N
H
R′
N
H
R
Acetophenone
derivative
Phenyl hydrazine
derivative
R
1a R′=SO2CH3
1b R′=H
1c R′=Br
2a R=SO2NH2
2b R=H
2c R=Br
3a R=H, R′=SO2CH3
3b R=SO2NH2, R′=H
3c R=SO2NH2, R′=Br
4a R=H, R′=SO2CH3
4b R=SO2NH2, R′=H
4c R=SO2NH2, R′=Br
1d R′=SO2NH2
2d R=NO2
3d R=SO2NH2, R′=SO2CH3
3e R=H, R′=SO2NH2
4d R=SO2NH2, R′=SO2CH3
4e R=H, R′=SO2NH2
4f R= Br, R′=SO2CH3
4g R=NO2, R′=SO2CH3
3f R=Br, R′=SO2CH3
3g R=NO2, R′=SO2CH3
Scheme 1: Synthesis of pyrazole derivatives (4a–4g). Reagents and conditions: (a) acetic acid, ethanol, and reflux; (b) dimethyl formamide,
POCl3, and reflux.
O
R
S
O
O
O
R
R
S
S
(a)
(b)
N
O
O
N
N3
H2N
N
F3C
5a R=NH2
5b R=CH3
6a R=NH2
6b R=CH3
8a R=NH2
8b R=CH3
Scheme 2: Synthesis of triazole derivatives (8a and 8b). Reagents and conditions: (a) HCl, NaNO2, and NaN3; (b) 4-ethynyl-
α,α,α-trifluorotoluene 7, CuSO4, sodium ascorbate, and DCM : H2O (1 :1).
(a)
(b)
HO
N3
TsO
OH
OH
OH
9
11
12
Scheme 3: Synthesis of spacer 12. Reagents and conditions: (a) tosyl chloride 10, triethylamine, and DCM; (b) sodium azide, ethanol, and
reflux.
CF3
N
N
O
N
O
O
S
O
O
O
OH
O
O
R
N3
(a)
(b)
OH
+
S
R
O
N3
O
12
S
13a R=CH3
13b R=NH2
14a R=CH3
14b R=NH2
15a R=CH3
15b R=NH2
O
R
Scheme 4: Synthesis of triazole compounds (15a and 15b). Reagents and conditions: (a) EDC, DMAP, and DCM; (b) 4-ethynyl-
α,α,α-trifluorotoluene 7, CuSO4, sodium ascorbate, and DCM : H2O (1 :1).

8
Journal of Chemistry
Table 1: In vitro activity on COX-1 and COX-2 with selectivity
assay kit to determine the half-maximal inhibitory con-
centration (IC50) and compared with the IC50 of celecoxib.
Moreover, the selectivity index (SI) values were measured as
IC50 (COX-1)/IC50 (COX-2). )e results are shown in
Table 1.
index.
IC50 COX-1
Compound
IC50 COX-2 (μM) Selectivity index
(μM)
4a
4b
4c
4d
4e
4f
4g
8a
8b
15a
15b
17.507 1.050
0.263 0.016
18.646 1.119
5.375 0.323
21.306 1.278
3.438 0.206
0.012 0.001
0.145 0.009
0.075 0.005
0.325 0.020
0.273 0.016
1.386 0.083
0.017 0.001
1.305 0.078
0.098 0.006
0.551 0.033
1.902 0.114
1.460 0.088
3.324 0.199
0.226 0.014
0.002 0.000
0.131 0.008
0.004 0.000
12.631
15.471
14.288
54.847
38.668
1.902
)e compounds 4a–4g with a core structure of het-
erocyclic pyrazole showed COX-2 inhibitory activity with
IC50 value 1.902–0.017 μM. It was noticed that compounds
in this series with sulfonamide substitution (4b–4e) at either
the N-aromatic ring or the aromatic ring at position 4
showed better COX-2 selectivity in compression with
compounds that did not have sulfonamide substituent.
However, the compound 4b with sulfonamide substituent
on the N-aromatic ring and the other aromatic ring which
was unsubstituted had better COX-2 inhibition activity
(IC50 � 0.017 μM) compared with compounds 4c and 4d in
which the other ring was substituted by sulfone or bromide.
However, when the N-aromatic ring was substituted with
sulfonamide and the other aromatic ring was substituted
with sulfone (compound 4d), they showed better COX-2
selectivity among the synthesized pyrazole library
(SI � 54.847). In addition, this compound showed a high
COX-2 inhibition activity (IC50 � 0.098 μM). It was noticed
that nitro substitution on the N-aromatic ring showed more
inhibition towards COX-1 (compound 4g).
In the case of triazole derivatives (compounds 8a, 8b,
15a, and 15b), triazole compounds have N-aromatic ring
either directly attached to the core or through a spacer. )e
results showed that the presence of a spacer improved the
selectivity of inhibition towards COX-2 (compounds 15a
and 15b). )e presence of a spacer in the synthesized
compounds makes it better fit the extra hydrophobic pocket
which is present in the COX-2 enzyme. )is is a possible
explanation for the increased inhibition activity of these
compounds compared to compounds without spacer.
Moreover, results showed that sulfone substitution on the
aromatic ring had a better COX-2 inhibition activity than
compounds with sulfonamide substituted either in both
spacer and without spacer compounds (Compounds 8b and
15a). Compound (15a) which has a spacer and sulfone
substitution on one ring and CF3 on the other ring showed
the best COX-2 inhibition activity among the whole syn-
thesized library (IC50 � 0.002 μM). Moreover, this com-
pound had a two-fold increase in inhibition activity
compared to celecoxib. )e results also showed that com-
pounds without spacer were more selective towards COX-1
(compounds 8a and 8b). A possible explanation is that these
compounds fit better to COX-1 enzyme.
0.008
0.044
0.332
162.500
2.084
369.750
Celecoxib 1.479 0.089
Structure-activity relationship (SAR) data were supported
with predicted binding data, retrieved by molecular docking
studies with human COX-1 (PDB code 3KK6 [31]) and
human COX-2 (PDB code 5KIR [32]). )e findings obtained
by molecular modeling study support the SAR data and
binding similarities and differences on both isozymes.
Results of COX-1 binding analysis show shared inter-
action pattern with amino acids located at the active site. van
der Waals (vdW) interactions with amino acids were ob-
served with ILE-89 LEU-93, VAL-116, and TYR-355 amino
acids and the substituted or unsubstituted phenyl groups
located at the right hydrophobic pocket of the active site.
Additional vdW interactions were observed at the left
binding pocket, LEU-352, and ILE-523 amino acids. π-π
interactions were also seen with TYR-355 amino acid and
central core of the ligands. Hydrogen bonds were identified
with pyrazole or triazole central cores of the ligands and side
chain of ARG-120 amino acid. )e existence of the sulfone
fragment on the molecules supports the hydrogen bonds
with SER-530 of COX-1. Most of the pyrazole derivatives are
having undesired steric limitations for having optimal
binding and resulting with very limited IC50 values. )e
position of the aldehyde group of 4b is shifted compared to
4a and 4e, and this showed better inhibition profile com-
pared to both ligands, which is related with an undesired
interaction between ALA-527 amino acid and aldehyde
group of both ligands. Replacing hydrogen at the para
position of 4b with a larger bromine atom also resulted with
a decrease in COX-1 inhibitory profile, but 4f was affected
less due to the formation of halogen bond with guanidino
group of ARG-83 amino acid. Another hydrophilic inter-
action—a hydrogen bond—was seen between sulfonamide
group of 4d and guanidino group of ARG-86 amino acid.
)e most active pyrazole containing COX-1 inhibitor was 4g
due to having smaller volume and more suitable positioning
of the negatively charged nitro group in the same location.
)e same trend can also be observed with the triazole series
and pyrazole series with an ester spacer. Aldehyde group on
the heterocyclic group was cut out and trifluoromethyl
containing para-substituted phenyl groups of 8a, 8b, 15a,
and 15b form a more favored positioning at the same lo-
cation. )is showed increased potency against COX-1
3.3. Molecular Modeling Study. COX-2 isozyme has an
additional secondary binding pocket, which does not exist in
COX-1 isozyme and thought to be related with selective
COX-2 inhibition. )e formation of that pocket is related
with the conformational differentiations in both isozymes,
caused by the replacement of ILE-532 amino acid in COX-1
with VAL-532 in COX-2 [42]. Compounds 4d and 15a
showed higher COX-2 selectivity index (IC50COX-1/
IC50COX-2), compared to the other molecules in the series.

Journal of Chemistry
9
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
Figure 2: Continued.

10
Journal of Chemistry
(i)
(j)
(k)
Figure 2: Predicted binding orientations of (a) 4a, (b) 4b, (c) 4c, (d) 4d, (e) 4e, (f) 4f, (g) 4g, (h) 8a, (i) 8b, (j) 15a, and (k) 15b visualized in
COX-1 active site (PDB code 3KK6 [31]); ligands are shown in orange and amino acids are shown in blue. Only the interacting residues and
their interaction patterns are shown.
isozyme, where the whole molecule fits in a better orien-
tation for triazole derivatives, 8a and 8b, but ester spacer
carrying triazole derivatives show differed binding mode in
COX-1 active site due to the size and flexibility of the spacer,
which showed slight decrease in COX-1 inhibitory effect.
Interacting residues and interacting locations are visualized
in Figure 2.
)e fragments causing steric issues and unfavored
binding orientation have resulted in a decreased COX-2
inhibitory profile. When the bromine atom is taken out from
4c and 4e, which causes unfavored contacts with TYR-385,
the resulting compound 4b shows increased binding affinity
for COX-2. Other structurally close analogues of 4b in the
molecule series are 4a and 4e which showed less favored
interactions. )is is due to the positional change of the
aldehyde group which caused less steric issues and unfa-
vored contact with ALA-527. Nitro group of 4g was noticed
to form undesired contact with the phenyl group of TYR-
385. Compound 4d showed better inhibitory profile because
of the hydrogen bond formed with SER-530 amino acid.
Triazole derivatives also showed similar binding pattern to
pyrazoles, but due to the absence of a polar group on the
heterocyclic group, they have not showed better binding
profile compared to the pyrazoles. Ester spacer containing
triazole derivatives changed the binding mode of 15a and
15b and resulted in the most active compound in the series,
15b. )e strong change in the inhibition profile is due to
having favored binding in the hydrophobic pocket between
LEU-96 and VAL-116 amino acids, without having hy-
drogen bond restrictions with the polar backbone atoms
of ILE-517 and PHE-518. All acquired binding modes of
COX-2 inhibitors: celecoxib, rofecoxib, pyrazole derivatives,
and triazole derivatives, are shown in Figure 3.
Celecoxib [43] and SC-558 [33] were chosen as
reference ligands to identify the COX-2 binding mode of
the synthesized compounds. As can be seen, all syn-
thesized compounds bind to the secondary pocket of
COX-2. )e phenyl group of each compound is forming
vdW interactions with the surrounding amino acids,
LEU-252, PHE-518, and VAL-523. COX-2 inhibition
data showed that all molecules bind to the COX-2 en-
zyme with inhibition values in the range of micro or
nanomolar. Both methylsulfonyl and aminosulfonyl
functional groups fall in this region and help positioning
the ligands in that binding domain of COX-2. Both
groups make hydrogen bonds with the imidazole group
of HIS-90 and guanidino group of ARG-513 amino acids.
Additionally, aminosulfonyls form hydrogen bonds with
backbone atoms of ILE-517 and PHE-518 amino acids,
which is in agreement with previously published COX-2
binding modes of coxibs, such as celecoxib, SC-558, and
rofecoxib.

Journal of Chemistry
11
(a)
(c)
(e)
(g)
(b)
(d)
(f)
(h)
Figure 3: Continued.

12
Journal of Chemistry
(i)
(j)
(k)
(l)
(m)
Figure 3: Crystal binding mode of (a) rofecoxib, and predicted binding orientations of (b) celecoxib, (c) 4a, (d) 4b, (e) 4c, (f) 4d, (g) 4e, (h)
4f, (i) 4g, (j) 8a, (k) 8b, (l) 15a, and (m) 15b visualized in COX-2 active site (PDB code 5KIR [32]); ligands are shown in orange and amino
acids are shown in green. Only the interacting residues and their interaction patterns are shown.
inhibitory activity of COX-2 in comparison to celecoxib with
IC50 of 0.002 μM. Furthermore, SAR and docking studies
showed the importance of the spacer availability in the
inhibitor structure because the spacer placed the aromatic
ring in the favored position for hydrophobic binding in the
COX-2 enzyme. More interestingly, the two aryl groups on
the heterocyclic cores (pyrazole or triazole) are not vicinal in
contrast to the most known COX-2 inhibitors. Further study
is needed as a continuation of this work to synthesize a
variety of pyrazole and triazole compounds with a different
spacer length and variety of substitutions to evaluate their
4. Conclusion
A facile synthesis was achieved to synthesize a variety of
COX-2 inhibitors based on pyrazole and triazole rings.
Pyrazole derivatives were successfully synthesized using
Vilsmeier–Haack reaction, whereas triazole derivatives
without or with spacer were successfully synthesized me-
diating click reaction. )e pyrazole derivatives 4b and 4d
showed potent and good COX-2 selective inhibitory activity
with IC50 of 0.017 and 0.098 μM, respectively. Regarding the
triazole derivatives, compound 15a showed the most potent

Journal of Chemistry
13
´
[7] J.-B. Meric, S. Rottey, K. Olaussen et al., “Cyclooxygenase-2 as
a target for anticancer drug development,” Critical Reviews in
Oncology/Hematology, vol. 59, no. 1, pp. 51–64, 2006.
[8] A. L. Blobaum and L. J. Marnett, “Structural and functional
basis of cyclooxygenase inhibition,” Journal of Medicinal
Chemistry, vol. 50, no. 7, pp. 1425–1441, 2007.
role in COX-2 inhibitory activity and selectivity. Moreover,
in vivo anti-inflammatory and cardiotoxicity studies will be
managed to support the obtained in vitro results.
Data Availability
[9] A. S. Mehanna, “NSAIDs: chemistry and pharmacological
actions,” American Journal of Pharmaceutical Education,
vol. 67, no. 2, p. 63, 2003.
)e data used to support the findings of this study are
available from the corresponding author upon request.
[10] G. Dannhardt and W. Kiefer, “Cyclooxygenase inhib-
itors—current status and future prospects,” European Journal
of Medicinal Chemistry, vol. 36, no. 2, pp. 109–126, 2001.
[11] J. Meyer-Kirchrath and K. Schror, “Cyclooxygenase-2 inhi-
bition and side-effects of non-steroidal antiinflammatory
drugs in the gastrointestinal tract,” Current Medicinal
Chemistry, vol. 7, no. 11, pp. 1121–1129, 2000.
Conflicts of Interest
)e authors declare that there are no conflicts of interest
regarding the publication of this paper.
Acknowledgments
[12] P. Prasit, Z. Wang, C. Brideau et al., “)e discovery of
rofecoxib, [MK 966, VIOXX , 4-(4′-methylsulfonylphenyl)-
®
)e authors would like to thank Dr. Abdurrahman Olgac for
3-phenyl-2(5H)-furanone], an orally active cyclooxygenase-2
inhibitor,” Bioorganic & Medicinal Chemistry Letters, vol. 9,
no. 13, pp. 1773–1778, 1999.
¨
giving the opportunity and the assistance to use Schrodinger
software in his molecular modeling research laboratory/
Department of Pharmaceutical Chemistry—Faculty of
Pharmacy at Gazi University, Turkey, as well as Mrs. Ghada
Shubietah from the Language Center/Faculty of Humanities
at An-Najah National University for her effort in editing the
paper.
[13] T. D. Penning, J. J. Talley, S. R. Bertenshaw et al., “Synthesis
and biological evaluation of the 1,5-diarylpyrazole class of
cyclooxygenase-2 inhibitors: identification of 4-[5-(4-meth-
ylphenyl)-3-
(trifluoromethyl)-1H-pyrazol-1-yl]benzene-
sulfonamide (SC-58635, celecoxib),” Journal of Medicinal
Chemistry, vol. 40, no. 9, pp. 1347–1365, 1997.
[14] J. J. Talley, D. L. Brown, J. S. Carter et al., “4-[5-Methyl-3-
Supplementary Materials
phenylisoxazol-4-yl]- benzenesulfonamide, Valdecoxib:
a
potent and selective inhibitor of COX-2,” Journal of Medicinal
Chemistry, vol. 43, no. 5, pp. 775–777, 2000.
)e supplementary material is supplementary figures re-
garding the molecular modeling study. Figure S1: phar-
macophore features of SC-558 and rofecoxib. Figure S2:
binding orientation of SC-558 (cyan) in 6COX and the
docking result of SC-558 (orange) in 5KIR. Figure S3: se-
quence alignment results of COX-2 sequence in Homo sa-
piens (human) and Mus musculus (mouse). (Supplementary
Materials)
[15] R. W. Friesen, C. Brideau, C. C. Chan et al., “2-Pyridinyl-3-(4-
methylsulfonyl)phenylpyridines: selective and orally active
cyclooxygenase-2 inhibitors,” Bioorganic
&
Medicinal
Chemistry Letters, vol. 8, no. 19, pp. 2777–2782, 1998.
[16] P. L. McCormack, “Celecoxib: a review of its use for symp-
tomatic relief in the treatment of osteoarthritis, rheumatoid
arthritis and ankylosing spondylitis,” Drugs, vol. 71, no. 18,
pp. 2457–2489, 2011.
[17] J. A. Cairns, “)e coxibs and traditional nonsteroidal anti-
inflammatory drugs: a current perspective on cardiovascular
risks,” Canadian Journal of Cardiology, vol. 23, no. 2,
pp. 125–131, 2007.
[18] G. Carullo, F. Galligano, and F. Aiello, “Structure-activity
relationships for the synthesis of selective cyclooxygenase 2
inhibitors: an overview (2009–2016),” MedChemComm,
vol. 8, no. 3, pp. 492–500, 2017.
[19] B. J. Al-Hourani, S. K. Sharma, M. Suresh, and F. Wuest,
“Novel 5-substituted 1H-tetrazoles as cyclooxygenase-2
(COX-2) inhibitors,” Bioorganic & Medicinal Chemistry
Letters, vol. 22, no. 6, pp. 2235–2238, 2012.
[20] K. R. A. Abdellatif, M. A. Abdelgawad, M. B. Labib, and
T. H. Zidan, “Synthesis, cyclooxygenase inhibition, anti-in-
flammatory evaluation and ulcerogenic liability of novel
triarylpyrazoline derivatives as selective COX-2 inhibitors,”
Bioorganic & Medicinal Chemistry Letters, vol. 25, no. 24,
pp. 5787–5791, 2015.
References
[1] W. L. Smith, Y. Urade, and P.-J. Jakobsson, “Enzymes of the
cyclooxygenase pathways of prostanoid biosynthesis,”
Chemical Reviews, vol. 111, no. 10, pp. 5821–5865, 2011.
[2] D. Engblom, M. Ek, S. Saha, A. Ericsson-Dahlstrand,
P.-J. Jakobsson, and A. Blomqvist, “Prostaglandins as in-
flammatory messengers across the blood-brain barrier,”
Journal of Molecular Medicine, vol. 80, no. 1, pp. 5–15, 2002.
[3] F. Fitzpatrick, “Cyclooxygenase enzymes: regulation and
function,” Current Pharmaceutical Design, vol. 10, no. 6,
pp. 577–588, 2004.
[4] R. M. Garavito and D. L. DeWitt, “)e cyclooxygenase iso-
forms: structural insights into the conversion of arachidonic
acid to prostaglandins,” Biochimica et Biophysica Acta,
vol. 1441, no. 2-3, pp. 278–287, 1999.
[5] N. S. Kirkby, M. H. Lundberg, L. S. Harrington et al.,
“Cyclooxygenase-1, not cyclooxygenase-2, is responsible for
physiological production of prostacyclin in the cardiovascular
system,” Proceedings of the National Academy of Sciences,
vol. 109, no. 43, pp. 17597–17602, 2012.
[6] T. Grosser, Y. Yu, and G. A. FitzGerald, “Emotion recollected
in tranquility: lessons learned from the COX-2 saga,” Annual
Review of Medicine, vol. 61, no. 1, pp. 17–33, 2010.
[21] M. Azami Movahed, B. Daraei, S. Shahosseini,
M. Esfahanizadeh, and A. Zarghi, “Design, synthesis, and
biological evaluation of new pyrazino [1, 2-a] benzimidazole
derivatives as selective cyclooxygenase (COX2) inhibitors,”
Archiv der Pharmazie, vol. 352, no. 2, Article ID 1800265,
2019.

14
Journal of Chemistry
[22] B. Jiang, X. Huang, H. Yao et al., “Discovery of potential anti-
inflammatory drugs: diaryl-1,2,4-triazoles bearing N-hy-
droxyurea moiety as dual inhibitors of cyclooxygenase-2 and
5-lipoxygenase,” Organic & Biomolecular Chemistry, vol. 12,
no. 13, pp. 2114–2127, 2014.
[23] M. G. Perrone, P. Vitale, A. Panella, C. G. Fortuna, and
A. Scilimati, “General role of the amino and methylsulfamoyl
groups in selective cyclooxygenase (COX)-1 inhibition by 1,4-
diaryl-1,2,3-triazoles and validation of a predictive pharma-
cometric PLS model,” European Journal of Medicinal
Chemistry, vol. 94, pp. 252–264, 2015.
interaction profiler,” Nucleic Acids Research, vol. 43, no. W1,
pp. W443–W447, 2015.
¨
[39] Schrodinger (LLC), Ge PyMOL Molecular Graphics System,
V. Schro¨dinger, LLC, New York, NY, USA, 2010.
[40] M. M. A. Hawash, D. C. Kahraman, F. Eren, R. Cetin Atalay,
and S. N. Baytas, “Synthesis and biological evaluation of novel
pyrazolic chalcone derivatives as novel hepatocellular carci-
noma therapeutics,” European Journal of Medicinal Chemis-
try, vol. 129, pp. 12–26, 2017.
[41] C. W. Tornøe, C. Christensen, and M. Meldal, “Peptido-
triazoles on solid phase: [1, 2, 3]-triazoles by regiospecific
copper (I)-catalyzed 1, 3-dipolar cycloadditions of terminal
alkynes to azides,” Ge Journal of Organic Chemistry, vol. 67,
no. 9, pp. 3057–3064, 2002.
[24] M. G. Perrone, P. Vitale, P. Malerba et al., “Diarylheterocycle
core ring features effect in selective COX-1 inhibition,”
ChemMedChem, vol. 7, no. 4, pp. 629–641, 2012.
[42] E. A. Meade, W. L. Smith, and D. L. DeWitt, “Differential
inhibition of prostaglandin endoperoxide synthase (cyclo-
oxygenase) isozymes by aspirin and other non-steroidal anti-
inflammatory drugs,” Journal of Biological Chemistry, vol. 268,
no. 9, pp. 6610–6614, 1993.
[43] J. L. Wang, D. Limburg, M. J. Graneto et al., “)e novel
benzopyran class of selective cyclooxygenase-2 inhibitors.
Part 2: the second clinical candidate having a shorter and
favorable human half-life,” Bioorganic & Medicinal Chemistry
Letters, vol. 20, no. 23, pp. 7159–7163, 2010.
[25] K. Singh, S. Ralhan, P. K. Sharma, and S. N. Dhawan,
“Vilsmeier-Haack reaction on hydrazones: a convenient
synthesis of 4-formylpyrazoles,” Journal of Chemical Research,
vol. 2005, no. 5, pp. 316–318, 2005.
[26] H. C. Kolb, M. G. Finn, and K. B. Sharpless, “Click chemistry:
diverse chemical function from a few good reactions,”
Angewandte Chemie International Edition, vol. 40, no. 11,
pp. 2004–2021, 2001.
[27] L. Liang and D. Astruc, “)e copper (I)-catalyzed alkyne-
azide cycloaddition (CuAAC) “click” reaction and its appli-
cations. An overview,” Coordination Chemistry Reviews,
vol. 255, no. 23-24, pp. 2933–2945, 2011.
[28] R. Kumar, L. Vats, S. Bua, C. T. Supuran, and P. K. Sharma,
“Design and synthesis of novel benzenesulfonamide con-
taining 1,2,3-triazoles as potent human carbonic anhydrase
isoforms I, II, IV and IX inhibitors,” European Journal of
Medicinal Chemistry, vol. 155, pp. 545–551, 2018.
[29] B. L. Wilkinson, L. F. Bornaghi, T. A. Houston et al., “Car-
bonic anhydrase inhibitors: inhibition of isozymes I, II, and IX
with triazole-LinkedO-glycosides of benzene sulfonamides,”
Journal of Medicinal Chemistry, vol. 50, no. 7, pp. 1651–1657,
2007.
[30] F. Wuest, X. Tang, T. Kniess, J. Pietzsch, and M. Suresh,
“Synthesis and cyclooxygenase inhibition of various (aryl-
1,2,3-triazole-1-yl)-methanesulfonylphenyl derivatives,” Bio-
organic & Medicinal Chemistry, vol. 17, no. 3, pp. 1146–1151,
2009.
[31] G. Rimon, R. S. Sidhu, D. A. Lauver et al., “Coxibs interfere
with the action of aspirin by binding tightly to one monomer
of cyclooxygenase-1,” Proceedings of the National Academy of
Sciences, vol. 107, no. 1, pp. 28–33, 2009.
[32] B. J. Orlando and M. G. Malkowski, “Crystal structure of
rofecoxib bound to human cyclooxygenase-2,” Acta Crys-
tallographica Section F Structural Biology Communications,
vol. 72, no. 10, pp. 772–776, 2016.
[33] R. G. Kurumbail, A. M. Stevens, J. K. Gierse et al., “Structural
basis for selective inhibition of cyclooxygenase-2 by anti-
inflammatory agents,” Nature, vol. 384, no. 6610, pp. 644–648,
1996.
¨
[34] Maestro S (LLC), Schrodinger Release 2017-1, Maestro, S.,
LLC, New York, NY, USA, 2017.
¨
[35] LigPrep S (LLC), Schrodinger Release 2017-1, LigPrep, S., LLC,
New York, NY, USA, 2017.
¨
¨
¨
[36] Schrodinger (LLC), Schrodinger Release 2017-1: Schrodinger
¨
Suite 2017-1 Protein Preparation Wizard, Schrodinger, LLC,
New York, NY, USA, 2017.
[37] Glide S (LLC), Schro¨dinger Release 2017-1, Glide, S., LLC, New
York, NY, USA, 2017.
[38] S. Salentin, S. Schreiber, V. J. Haupt, M. F. Adasme, and
M. Schroeder, “PLIP: fully automated protein-ligand