Pyrazole–chalcone derivatives as selective COX-2 inhibitors: design, virtual screening, and in vitro analysis

Article abstract of DOI:10.1007/s00044-019-02368-8

In the process of research and development of new drugs, in silico analyzes are widely used. They address the pharmacokinetics of the molecules in study and can predict the binding mode and affinity, using a docking software. This approach can optimize th

Full text of DOI:10.1007/s00044-019-02368-8

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02368-8
ORIGINAL RESEARCH
Pyrazole–chalcone derivatives as selective COX-2 inhibitors: design,
virtual screening, and in vitro analysis
1,2,3
Anelise F. Macarini1,2 Thales U. C. Sobrinho3 Gerusa W. Rizzi1 Rogério Corrêa
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Received: 26 February 2019 / Accepted: 21 May 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
In the process of research and development of new drugs, in silico analyzes are widely used. They address the
pharmacokinetics of the molecules in study and can predict the binding mode and affinity, using a docking software. This
approach can optimize the development of new drugs, reducing costs, time, and resources. In this study, a library of 300
pyrazole–chalcone derivatives were designed, the in silico ADMET (absorption, distribution, metabolism, excretion, and
toxicity) properties were evaluated, and a structure-based virtual screening was performed using AutoDock Vina. The
docking results exhibited that the derivatives binding mode at the COX-2 active site is similar to celecoxib, the reference
drug, and presented similar binding energy. Six compounds were synthetized and tested for in vitro inhibition of the COX-1
and COX-2 isoenzymes and the selectivity index (SI) was calculated. The compound 2a11 showed the best activity for
COX-2 (IC50COX-2 = 0.73 μM) whereas the control, celecoxib, resulted IC50COX-2 = 0.88 μM. All the other compounds
synthetized presented better potency for COX-2 inhibition than the control. Compound 2a23 exhibited the higher SI, of
280.17 (IC50COX-1 = 210.13 μM/ IC50COX-2 = 0.75 μM), while celecoxib was 246.88 (IC50COX-1 = 217.26 μM/
IC50COX-2 = 0.88 μM). These results corroborate with a possible anti-inflammatory activity and COX-2 selectivity of
the new compounds synthetized.
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Keywords Pyrazole-Chalcones Molecular docking Medicinal Chemistry COX-2 inhibition ADMET Anti-
inflammatory
Introduction
inflammatory drugs (NSAIDs) are the most used drugs to
combat inflammation having this effect due to inhibition of
the ciclooxigenases (COXs) (Khalaf et al. 2018; Umama-
heswaran et al. 2018). The main problem of NSAIDs use is
the gastrointestinal adverse effects, caused by unselective
COX inhibition (Rayar et al. 2017). There are two main
COX enzymes, COX-1, which is constitutive and partici-
pate in physiologic processes, and COX-2, that is induced
in inflammatory processes (Umamaheswaran et al. 2018).
Due to the adverse effects caused by unselective COX
inhibition, selective COX-2 inhibitors, the coxibes, were
introduced in the therapeutics (Rayar et al. 2017; Schmidt
et al. 2016). This decreased the gastrointestinal problems,
but, the coxibes presented severe cardiovascular side effects
(Hawkey 1999; Patrono and Baigent 2017; Schmidt et al.
2016). Thus, new drug development is necessary to
diminish these side effects.
Inflammation is an organism response to tissue rupture and,
also, initiate the reparation and cure process (Serhan and
Savill 2005; Sugimoto et al. 2016). The problem is when
this process stop being functional, and prolongate, becom-
ing a chronic inflammation. This can lead to irreversible
tissue damages and function loss. The nonsteroidal anti-
* Rogério Corrêa
roger@univali.br
1
Núcleo de Investigações Químico-Farmacêuticas/NIQFAR,
UNIVALI, Rua Uruguai, 458, Itajai, Santa Catarina 88302-901,
Brasil
2
Programa de Pós-Graduação em Ciências Farmacêuticas,
Universidade do Vale do Itajaí—UNIVALI, Rua Uruguai, 458,
Drug development is an expensive process and reducing
fails, as early as possible, is the main goal of the researchers
and the pharmaceutical industry (Egan et al. 2000; van de
Waterbeemd and Gifford 2003). In silico predictions can aid
Itajai, Santa Catarina 88302-901, Brasil
3
Curso de Engenharia Química, Escola do Mar, Ciência e
Tecnologia, UNIVALI, Rua Uruguai, 458, Itajai, Santa Catarina
88302-901, Brasil

Medicinal Chemistry Research
Fig. 1 Series 1 (left) and 2
(right) of the designed
compounds, where R1 is the
Topliss substituents, and R2 is
30 different possible bioactive
substituents
in this process by reducing the number of molecules syn-
thetized and tested, once many of the molecule properties
can be calculated beforehand. Tools and models available
can predict absorption, distribution, metabolism, excretion,
and toxicity (ADMET) properties, and structure-based in
silico tools, as molecular docking, can predict the possible
interactions with the target of study (Danielson et al. 2017).
ADMET tools sometimes use filters based on rules, as
the “Rule of five” introduced by Lipinski (Lipinski 2016;
Lipinski et al. 1997), and other filters that followed (Egan
et al. 2000; Ghose et al. 1999; Martin 2005; Muegge et al.
2001; Teague et al. 1999; Veber et al. 2002) that attempt to
predict oral passive absorption and bioavailability. In
addition to these, inhibition of metabolism enzymes are
important to evaluate metabolic conditions and identifica-
tion of molecules or fragments potentially toxic or proble-
matics (Baell and Holloway 2010; Brenk et al. 2008).
Another approach is the structure-based virtual screening,
using molecular docking programs, like AutoDock Vina
(Trott and Olson 2009). These programs can predict the
binding energy of the ligand–protein complex. AutoDock
Vina employs an iterated local search global optimizer, and,
among other tested programs, achieved one of the best
scoring powers (Wang et al. 2016), which substantiate its
use for Virtual Screening.
Chalcone variation can be made using three main stra-
tegies. They are: structural modification in the aryl rings,
substitution of one or both aryl rings by heteroaryl rings,
and, molecular hybridization through conjugation with
biologically active molecules (Özdemir et al. 2017). This
work contemplates the use the first and third strategy to
evaluate the activity of pyrazole–chalcone derivatives as
COX-2 inhibitors.
Material and methods
Design and in silico analysis
The pyrazole–chalcone derivatives were designed based in
the conjugation of a chalcone and a pyrazole nucleus, linked
on the aliphatic instauration, forming the backbone of the
molecules. As shown in Fig. 1 ring A of the chalcone
moiety was modified using the classical Topliss substituents
(Richter 2017; Topliss 1972). Ring B was modified using
30 different possible bioactive substituents. Also, the pyr-
azole ring was derived using, as starting materials, the
unsubstituted pyrazole ring, and the 3,5-dimethylpyrazole.
This resulted in two series of 150 compounds each. The
molecules were drawn with ACD/ChemSketch 2017.2.1
(ACD/Labs) freeware.
Bioactive small molecules tested in these analyses are
sometimes derived of natural products, as chalcones. In
nature, chalcones are precursors in the biosynthesis of fla-
vonoids, and can be isolated from superior plants (Gaonkar
and Vignesh 2017; Gomes et al. 2017; Singh et al. 2014). In
organic synthesis, there are several methods for the pre-
paration of chalcones, the most known is the
Claisen–Schmidt condensation. Chemically these com-
pounds have two aromatic rings that are linked by an ali-
phatic three-carbon chain composed by an α,β-unsaturated
ketone (Gaonkar and Vignesh 2017; Singh et al. 2014). In
terms of biological activity, they are reportedly to have anti-
inflammatory activity, as well as COX-2 selectivity
(Bandgar et al. 2010; Li et al. 2017; Singh et al. 2014;
Zhuang et al. 2017). Pyrazole derivatives are other example
of small molecules that are largely studied (Karrouchi et al.
2018; Khan et al. 2016; Li and Zhao 2014), including for its
anti-inflammatory activity (Mukarram et al. 2017; Viveka
et al. 2018) and the specific interaction with COX-2 (Shen
et al. 2017).
ADMET analysis
The online free tool SwissADME (swissadme.ch) (Daina
et al. 2017) was used to calculate physicochemical proper-
ties, lipophilicity, water solubility, pharmacokinetics, dru-
glikeness, and other chemometrics or medicinal chemistry
parameters.
Docking
The molecular docking was performed using AutoDock
Vina (Trott and Olson 2009). The ligands were assumed to
be flexible and the protein, rigid. The ligand setup was of
six poses maximum with exhaustiveness of 14, being
selected and presented the ones with better energy of
binding. The protein was obtained from Protein Data Bank
(PDB) (Berman 2000), code 3LN1 (Wang et al. 2010), and,
was prepared in Chimera 2.1 (Pettersen et al. 2004) as

Medicinal Chemistry Research
follow: chain A was isolated, charges and polar hydrogens
added, and the solvent removed. The ligands were prepared
using AutoDock Tools (Morris et al. 2009). The root mean
square deviation (RMSD) was calculated between the
crystallography celecoxib pose and the docked pose. If
RMSD < 2 Å, the docking was accepted as valid.
After the docking procedures, the poses were visualized
in Chimera 2.1, H-bonds and other non-covalent interac-
tions were accounted for and pictures saved. BIOVIA
Discovery Studio 2017 was used to produce the 2D diagram
of the docked poses.
2-bromo-1-(4-chlorophenyl)ethan-1-one (1d) Yield 70%;
mp 96 °C. 1H NMR (CDCl3, 300 MHz) δ = 5.44 (2H, s, H-
10a, b); 7.46–7.49 (2H, d, J = 8.7 Hz, H-3, 5); 7.90–7.93
(2H, d, J = 8.7 Hz, H-2, 6).
2-(3,05-dimethyl-1H-pyrazol-01-yl)-01-phenylethan-01-one
intermediates synthesis
One millimolar of 3,5-dimethylpyrazole was dissolved in
acetonitrile, and, K2CO3 (2eq.) was added. One millimolar
of the wanted substituted phenacyl bromide (1a, 1c, and 1d)
was added to the mixture. The reaction was assisted by a
microwave reactor (CEM Discovery) for 60 min at 82 °C
under reflux. After the completion of the reaction, cold
water was added to the mixture and stirred until complete
precipitation. The precipitate was filtered off and dried
overnight in vacuum desiccator with silica gel as drying
agent. The product was recrystallized with cyclohexane.
Synthesis
All reagents were purchased from Vetec® and
Sigma–Aldrich® and were analytic grade. All reactions were
monitored using thin layer chromatography (TLC),
Macherey–Nagel® pre-coated aluminium sheets 0.2 mm
silica gel with fluorescent indicator UV254, (eluent hexane:
ethyl acetate, 80:20). The synthesis was performed in three
steps, as follows.
2-(3,5-dimethyl-1H-pyrazol-1-yl)-1-phenylethan-1-one
1
(2a) Yield 67%; mp 95 °C. H NMR (CDCl3, 300 MHz)
Final products characterization was done by Infrared
(FT-IR), recorded with an IR-Prestige—21 (Shimadzu Inc.
—Tokyo, Japan); 1H and 13C NMR, that were recorded on a
Bruker ADVANCE DPT-300 (Bruker, Karlsruhe, Ger-
many); and elemental analysis that were performed in a
Perkin Elmer 2400 Series II CHNS/O System—Elemental
Analyzer (Perkin Elmer—Massachusetts, USA), operating
in three modes: CHN, CHNS, and Oxygen, in the range of
temperature from 100 to 1100 °C.
δ = 2.16 (3H, s, H-9a, b, c); 2.24 (3H, s, H-10a, b, c); 5.46
(2H, s, H-6a, b); 5.91 (1H, s, H-4); 7.48–7.53 (2H, t, J =
7.2 Hz, H-13,15); 7.60–7.63 (1H, t, J = 7.2 Hz, H-14);
7.97–7.99 (2H, d, J = 7.2Hz H-12, 16).
2-(3,5-dimethyl-1H-pyrazol-1-yl)-1-(4-methoxyphenyl)
ethan-1-one (2c) Yield 54%; mp 104 °C. 1H NMR
(CDCl3, 300 MHz) δ = 2.14 (3H, s, H-9a, b, c); 2.21 (3H, s,
H-10a, b, c); 3.74 (3H, s, H-18a, b, c); 5.39 (2H, s, H-6a, b);
5.88 (1H, s, H-4); 7.44–7.47 (2H, d, J = 8.5 Hz, H-13, 15);
7.82–7.85 (2H, d, J = 8.5 Hz, H-12, 16).
Phenacyl bromide synthesis
Substituted acetophenones (4-H, 4-Cl and 4-OCH3),
2 mmol, were dissolved in acetic acid. Pyridinium tri-
bromide (2 mmol) was added to the solution. The reaction
occurred in microwave reactor (CEM Discovery) for 30 min
at 80 °C under reflux. After the completion of the reaction,
cold water was added to the mixture and stirred until
complete precipitation. The precipitate was filtered and
dried in vacuum desiccator with silica gel as drying agent.
No purification was needed.
1-(4-chlorophenyl)-2-(3,5-dimethyl-1H-pyrazol-1-yl)ethan-1-
one (2d) Yield 70%; mp 155 °C. 1H NMR (CDCl3,
300 MHz) δ = 2.16 (3H, s, H-9a, b, c); 2.23 (3H, s, H-10a,
b, c); 5.40 (2H, s, H-6a, b); 5.90 (1H, s, H-4); 7.46–7.49
(2H, d, J = 8.4 Hz, H-13, 15); 7.90–7.93 (2H, d, J = 8.4 Hz,
H-12, 16).
4-[(1Z)-2-(3,5-dimethyl-1H-pyrazol-1-yl)-3-oxo-3-
phenylprop-1-en-1-yl]benzonitrile synthesis
2-bromo-1-phenylethan-1-one (1a) Yield 67%; mp 50 °C.
1H NMR (CDCl3, 300 MHz) δ = 5.49 (2H, s, H-9a, b);
7.48–7.53 (2H, t, J = 7.2 Hz, H-3, 5); 7.60–7.63 (1H, t, H-
J = 7.2 Hz, H-1); 7.97–7.99 (2H, d, J = 7.2Hz H-2, 6).
0.5 mmol of the respective intermediate (2a, 2c, and 2d) was
dissolved in ethanol, and an aqueous solution of K2CO3
(2eq.) was added. The substituted benzaldehyde (0.5 mmol)
was added to the mixture and it was kept stirring at room
temperature for 10 h. After the completion of the reaction,
cold water was added to the mixture and stirred until
complete precipitation. The precipitate was filtered and
dried in vacuum desiccator with silica gel as drying agent.
The product was recrystallized with cyclohexane.
2-bromo-1-(4-methoxyphenyl)ethan-1-one (1c) Yield 54%;
1
mp 72 °C. H NMR (CDCl3, 300 MHz) δ = 5,41 (2H, s,
H-11a, b); 3.78 (3H, s, H-11a, b, c); 7.44–7.47 (2H, d, J =
8.5 Hz, H-3, 5); 7.82–7.85 (2H, d, H- J = 8.5 Hz, H-2, 6).

Medicinal Chemistry Research
4-[(1Z)-2-(3,5-dimethyl-1H-pyrazol-1-yl)-3-oxo-3-phenyl-
prop-1-en-1-yl]benzonitrile (2a11) Yield: 27%; mp 120 °
C. IR (KBr cm–1): 1274 (C—N); 1319 (C—N); 1666
6.79–6.82 (2H, d, J = 8.7 Hz, H-13, 17); 7.23–7.26 (2H,d,
J = 7.8 Hz, H-19, 23); 7.41–7.46 (2H, t, J = 7.5 Hz, H-20,
22); 7.49 (1H, s, H-11); 7.53–7.55 (1H, t, J = 7.2 Hz, H-
21); 7.75–7.77 (2H, d, J = 7.5 Hz, H-14, 16). 13C NMR
(CDCl3, 75.47 MHz) δ = 10.94 (C-10); 13.73 (C-9); 106.89
(C-4); 128.45 (C-11); 129.14–137.10 (C-Ar); 139.74 (C-6);
140.66 (C-5); 150.61 (C-3); 191.98 (C-7). Anal. calcd. for
C20H17ClN2O (in %): C-71.32, H-5.09, N-8.32, O-4.75.
Found C-71.32, H-5.05, N-8.32, O-4.76.
1
(C=O); 2224 (Ar—C≡N). H NMR (CDCl3, 300 MHz) δ
= 1.92 (3H, s, 9-Ha, b, c); 2.27 (3H, s, H-10a, b, c); 5.98
(1H, s, H-4), 6.98–7.01 (2H, d, J = 8.4 Hz, H-13, 17);
7.41–7.48 (3H, m, J = 7.2 Hz, H-22, 20, H-11); 7.54–7.57
(3H, t, J = 8.4 Hz, H-14, 16); 7.75–7.77 (2H, d, J = 7.5 Hz,
H19, 23). 13C NMR (CDCl3, 75.47 MHz) δ = 11.03 (C-10);
13.67 (C-9); 107.36 (C-4); 118.22 (C-24); 133.56 (C-11);
128.57–136.98 (Ar-C); 137.39 (C-6); 140.67 (C-5); 151.04
(C-3); 191.48 (C-7). Anal. calcd. for C21H17N3O (in %): C-
77.04, H-5.23, N-12.84, O-4,89. Found C-77.06, H-5.2, N-
12.84, O-4,89.
(2Z)-3-(4-chlorophenyl)-2-(3,5-dimethyl-1H-pyrazol-1-yl)-1-
(4-methoxyphenyl)prop-2-en-1-one (2c7) Yield: 15%;
mp: 151 °C. IR (KBr cm–1): 1089 (Ar—Cl); 1112 (C—O—
1
C); 1271 (C—N); 1322 (C—N); 1689 (C=O). H NMR
(Acetone-d6, 300 MHz) δ = 1.95 (3H, s, H-9a, b, c); 2.79
(3H, s, H-10a, b, c); 3.91 (3H, s, H-26a, b, c); 4.73 (1H, s,
H-4), 5.71 (1H, s, H-11); 7.04–7.06 (d, 2H, J = 6.9 Hz, H-
13, 17); 7.16–7.18 (2H, d, J = 6.6 Hz, H-19, 23); 7.31–7.33
(2H, d, J = 6.9 Hz, H-20, 22); 8.24–8.26 (2H, d, J = 6.9 Hz,
H-14, 16). 13C NMR (Acetone-d6, 75.47 MHz) δ = 10.10
(C-10); 12.72 (C-9); 55.19 (C-26); 106.17 (C-4); 113.86 (C-
11); 128.14–132.59 (C-Ar); 134.50 (C-6); 141.78 (C-5);
149.04 (C-3); 164.57 (C-21); 188.50 (C-7). Anal. calcd. for
C21H19ClN2O2 (in %): C-68.76, H-5.22, N-7.64, O-8.72.
Found C-68.76, H-5.18, N-7.64, O-8.73.
(2Z)-2-(3,5-dimethyl-1H-pyrazol-1-yl)-3-(4-nitrophenyl)-1-
phenylprop-2-en-1-one (2a17) Yield: 31%; mp: 118 °C.
IR (KBr cm–1): 1267 (C—N); 1313 (C—N); 1342 (N—O);
1
1514 (N—O); 1668(C=O). H NMR (CDCl3, 300 MHz)
δ = 1,93 (3H, s, 9-Ha, b, c); 2.28 (3H, s, H-10a, b, c); 5.99
(1H, s, H-4), 7.06–7.09 (2H, d, J = 8.7 Hz, H- 13, 17);
7.42–7.47 (2H, t, J = 7.5 Hz, H-22, 20); 7.52 (1H, s, H-1);
7.55–7.58 (1H, t, J = 7.5 Hz, H-21); 7.76–7.77 (2H, d, J =
7.5 Hz, H19, 23); 8.11–8.14 (2H, d, J = 9 Hz, H-14, 16).
13C NMR (CDCl3, 75.47 MHz) δ = 11.05 (C-10); 13.65 (C-
9); 107.49 (C-4); 123.78 (C-11); 128.61–137.33 (Ar-C);
138.58 (C-6); 140.75 (C-5); 148.25 (C-15); 151.13 (C-3);
191.38 (C-7). Anal. calcd. for C20H17N3O3 (in %): C-69.15,
H-4.93, N-12.1, O-13.82. Found C-69.16, H-4.9, N-12.1,
O-13.83.
4-[(1Z)-3-(4-chlorophenyl)-2-(3,5-dimethyl-1H-pyrazol-1-yl)-
3-oxoprop-1-en-1-yl]benzonitrile (2d11) Yield: 29%; mp:
149 °C. IR (KBr cm–1): 1089 (Ar—Cl); 1271 (C—N); 1328
(C—N); 1664 (C=O); 2227 (Ar—C≡N). 1H NMR (CDCl3,
300 MHz) δ = 1.90 (3H, s, H-9a, b, c); 2.27 (3H, s, H-10a,
b, c); 5.98 (1H, s, H-4); 6.99–7.02 (2H, d, J = 8.4 Hz, H-13,
17a); 7.41–7.38 (2H, d, J = 8.7 Hz, H-19, 23); 7.49 (1H, s,
H-11); 7.55–7.58 (2H, d, J = 8.4 Hz, H-20, 22); 7.65–7.68
(2H, d, J = 8.4 Hz, H-14, 16). 13C NMR (CDCl3,
75.47 MHz) δ = 10.47 (C-10); 13.34 (C-9); 106.95 (C-4);
119.75 (C-11); 121.24 (C-24); 128.21-134.17 (C-Ar);
138.14 (C-6); 140.54 (C-5); 150.72 (C-3); 191.45 (C-7).
Anal. calcd. for C21H16ClN3O (in %): C-69.71, H-4.46, N-
11.61, O-4.42. Found C-69.71, H-4.43, N-11.62, O-4.43.
N-{4-[(1Z)-2-(3,5-dimethyl-1H-pyrazol-1-yl)-3-oxo-3-phenyl-
prop-1-en-1-yl]phenyl}acetamide (2a23) Yield: 30%; mp:
187 °C. IR (KBr cm–1): 1321 (C—N); 1373 (C—N); 1660
(C=O); 1674(C=O); 3319 (N—H). 1H NMR (CDCl3,
300 MHz) δ = 1.96 (3H, s, H-27a, b, c); 2.11 (3H, s, H-9a,
b, c); 2.31 (3H, s, H-10a, b, c); 6.01 (1H, s, H-4), 6.73–6.86
(d, 2H, J = 8.7 Hz, H-13, 17); 7.34–7.37 (2H, d, J = 8.4 Hz,
H-19, 23); 7.41–7.46 (2H, t, J = 7.2 Hz, H-20, 22); 7.49
(1H, s, H-11); 7.53–7.55 (1H, t, J = 7.2 Hz, H-21);
7.71–7.74 (2H, d, J = 6.9 Hz, H-14, 16); 8.08 (1H, s, NH-
24). 13C NMR (CDCl3, 75.47 MHz) δ = 10.92 (C-10);
13.77 (C-9); 24.60 (C-27); 106.74 (C-4); 119.43 (C-11);
127.44–133.22 (C-Ar); 137.46 (C-6); 140.93 (5-C); 150.33
(C-3); 168.67 (C-25); 192.34 (C-7). Anal. calcd. for
C22H21N3O2 (in %): C-73.52, H-5.89, N-11.69, O-8.9.
Found C-73.53, H-5.85, N-11.7, O-8.91.
In vitro COX-1/COX-2 inhibition assay
The COX (ovine) inhibitor screening assay kit (catalog
number 560101, Cayman Chemical, Ann Arbor, MI, USA)
was used according to the manufacturer’s instructions to
directly measure prostaglandin (PGF2α) produced by SnCl2
reduction of COX-derived prostaglandin H synthase.
In a recipient was added: assay buffer (150 µl), heme
(10 µl), COX-1 or COX-2 enzyme solution (10 µl) and
samples or the positive control (celecoxib) (10 µl). In the
negative control recipient, the inhibitors weren’t added.
Then the recipient was shaken carefully for 10 s and
(2Z)-3-(4-chlorophenyl)-2-(3,5-dimethyl-1H-pyrazol-1-yl)-1-
phenylprop-2-en-1-one (2a7) Yield: 35%; mp: 147 °C. IR
(KBr cm–1): 1089 (Ar—Cl); 1273 (C—N); 1317 (C—N);
1658 (C=O). 1H NMR (CDCl3, 300 MHz) δ = 1.93 (3H, s,
H-9a, b, c); 2.29 (3H, s, H-10a, b, c); 5.98 (1H, s, H-4),

Medicinal Chemistry Research
incubated at 37 °C for 5 min. Arachidonic acid solution
(10 µl) was added to initiate the conversion of arachidonic
acid to PGH2. The recipients were shaken for 10 s and
incubated at room temperature for exactly 2 min. The
reaction was stopped by adding acid SnCl2 solution, which
is used to reduce the PGH2 produced in the COX reaction to
the more stable PGF2α.
fit well with how ligand binding sites are formed in proteins
(Lipinski 2016).
Regarding metabolization predictions, the program ana-
lyzes if the molecules are P-glycoprotein (P-gp) substrates.
P-gp acts as an active transporter expelling molecules out of
the cell, being able to reduce or prevent absorption of drugs
and other substances (Moroy et al. 2012). As none of the
synthetized molecules resulted “yes” for P-gp substrate,
chances are good that the compounds can be absorbed. The
program also predicts inhibition of 5 CYP enzymes. The
cytochrome P450 (CYP) is the major, and most studied
phase I metabolizing enzyme family. CYPs are responsible
for metabolism of approximately 75% of all marketed drugs
(Moroy et al. 2012). The most important enzyme of the
CYP family is the CYP3A4, which alone metabolizes ~50%
of all drugs (Moroy et al. 2012). So is plausible to assume
that the synthetized compounds don’t affect the metabolism
of at least half of all drugs, being that none resulted as an
inhibitor for this enzyme. Withal, all the compounds are
inhibitors of the CYP2C9 enzyme. In the CYP family,
CYP2C9 metabolizes celecoxib, diclofenac, ibuprofen,
other nonsteroidal anti-inflammatory drugs (NSAIDs) (Daly
et al. 2018) and, possibly, the synthetized compounds.
In a 96-wells plate, where the wells are previously
attached with polyclonal anti-IgG, the previous solution
(50 µL) was added to the wells together with a PG-
acetylcholinesterase (PG-tracer) (50 µL) and a PG mono-
clonal antibody (50 µL) that attach to the well. The PGF2α
or PG-tracer complexes with the PG monoclonal antibody.
The amount of PG-tracer that is able to bind to the well will
be inversely proportional to the concentration of PGF2α in
the well. This solution was incubated for 18 h. Then, the
plates were washed, and, Ellman’s Reagent (200 µL),
which contains the substrate to acetylcholinesterase, was
added. This reaction produces color, and the intensity of it
was measured at 405 nm using an ELISA Plate Reader.
This color represents the amount of PG-tracer bound to the
well, which is inversely proportional to the amount of free
PGF2α present in the well during the incubation. Pros-
taglandin standards were used to construct a standard
curve. The absorbance was transformed to pg/ml of PGF2α
using standard curve computed on excel. To calculate the
selectivity index, the ratio the ratio of IC50COX-1/
IC50COX-2.
Docking
To the docking method be considered valid, we calculated
the RMSD (root mean square deviation) value between the
docked and crystallography pose of celecoxib. The docking
would be considered valid if RMSD ≤ 2 Å, and we obtained
0.4 Å, so the docking was considered valid. Then, virtual
screening was performed for all the 300 molecules designed
using the described method. All compounds showed good
energy binding values. Attention was taken for the series 2
compounds, as compounds 2a11 and 2a23. The best scored
compounds suggest that the 3,5-dimethylpyrazole (series 2)
and unsubstituted A ring (2a derivatives) improve binding
affinity.
The best scored molecule was 2a11, with a binding
energy of −11kcal/mol, whereas the docked celecoxib
presented a binding energy of −12.2 kcal/mol. The results
were analyzed using BIOVIN Discovery Studio and Chi-
mera 1.12. Figure 2 shows the 2D diagram of the docked
pose of compound 2a11, indicating a hydrogen bond
between a phenylalanine residue (Phe504) and the benzo-
nitrile group of ring B that stabilize the compound in the
selectivity pocket. Also, non-polar interactions stabilize the
compound in the active site (Fig. 3).
Results and discussion
ADMET properties
ADMET studies were performed, for the 300 compounds
designed, using the SwissADME online tool (swissadme.
ch). Results are shown in Table 1—we presented only the
results of the synthetized compounds (see section 3.3). In
the drug development point of view, the compounds have
favorable drug properties, predicting a favorable oral
absorption. In the filters based on rules applied, compound
2d11 showed 1 violation in the Muegge filter, due to
excessive log P value, threshold was 2 ≤ XLOGP ≤ 5. Only
in Leadlikeness filter, all compounds showed at least one
violation, and compound 2d11 showed two. These viola-
tions relate to high log P (XLOGP ≤ 3.5) for all compounds,
and molecular weight (250 ≤ MW ≤ 350) for compound
2d11. However, Tian et al. (2015) state that drug leads have
lower molecular weight and are less complex and less
lipophilic than market drugs. Also, lipophilicity does not
directly relate to the biophysics of ligand–protein complex,
being more of an experimental solubility ratio; on the other
hand, H-bond donors and acceptors and molecular weight
Figure 4 presents the second-best docked compound,
2a23, with a binding energy of −10.6 kcal/mol in the best
docked pose. Three H-bonds can be visualized, in the
selectivity pocket there is one between the nitrogen of
amide group of B ring and a leucine residue (Leu338) and

Medicinal Chemistry Research
Table 1 ADMET properties
calculated with the SwissADME
online tool, and docking results,
showing the six synthetized
compounds
Properties
Docking
Parameters
Compounds
2a11 2a17
2a23
2a7
2c7
2d11
Binding energy (kcal/mol) −11
−10.5 −10.6 −10.4 −8.6
−10
Physicochemical
Properties
Mol weight (g/mol)
Rotatable bonds
H-bond acceptors
H-bond donors
TPSAa (Å)
327.38 347.37 359.42 308.76 338.79 361.82
4
3
0
5
4
0
6
3
1
4
2
0
5
3
0
4
3
0
58.68 80.71 63.99 34.89 44.12 58.68
Lipophilicity
iLOGP
2.70
3.96
4.14
1.91
3.24
3.10
MSb
MS
MS
High
Yes
No
No
No
Yes
No
No
0
2.52
4.50
4.18
2.04
2.03
3.06
MS
MS
MS
High
No
No
No
Yes
Yes
No
No
0
3.15
4.79
4.04
2.91
3.89
3.82
MS
MS
PSc
High
Yes
No
Yes
Yes
Yes
No
Yes
0
2.70
3.96
4.31
1.91
3.24
3.10
MS
MS
MS
High
Yes
No
No
No
Yes
No
No
0
3.14
4.47
4.32
2.74
3.86
3.71
MS
MS
PS
3.71
5.99
4.80
4.14
5.52
5.01
PS
XLOGP3
WLOGP
MLOGP
Silicos-IT log P
Consensus log P
ESOL Class
Water Solubility
Pharmacokinetics
Ali Class
PS
Silicos-IT class
GId absorption
BBBe permeant
P-gpf substrate
CYP1A2 inhibitor
CYP2C19 inhibitor
CYP2C9 inhibitor
CYP2D6 inhibitor
CYP3A4 inhibitor
Lipinski violations
Ghose violations
Veber violations
Egan violations
Muegge violations
Bioavailability Score
PAINSg alerts
PS
High
Yes
No
Yes
Yes
Yes
No
No
0
High
Yes
No
Yes
Yes
Yes
No
No
0
Druglikeness
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0.55
1
0.55
0
0.55
1
0.55
1
0.55
0
0.55
0
Medicinal Chemistry
Brenk alerts
2
3
1
2
1
1
Leadlikeness violations
Synthetic accessibility
1
1
1
1
1
2
3.21
3.18
3.31
3.21
2.89
3.13
aTotal polar surface area
bModerately soluble
cPoorly soluble
dGastrointestinal
eBlood-brain barrier
fP-glycoprotein
gPan Assay Interference Compounds
the oxygen of the amide carbonyl group and an arginine
residue (Arg499). Besides these, the oxygen of the carbonyl
group of the chalcone moiety exhibit a H-bond with a serine
residue (Ser516), that is one interaction celecoxib don’t
possess, which can provide a higher selectivity and better
stability in the active site. Other non-polar interactions can
be visualized in Fig. 5.
The docked poses of the compounds synthetized were
superimposed and showed in Fig. 6. The compounds
occupy the same space, volumetrically, and geometrically,
as the reference drug, celecoxib (in blue). This can lead to
the conclusion that the compounds possibly have the same
activity as celecoxib, due to similar interactions with the
biological receptor.

Medicinal Chemistry Research
Fig. 4 Docked poses of compound 2a23 in COX-2 active site.
Hydrogen bonds are shown in green, other interactions, as, van der
Waals, pi-sigma, and alkyl, are shown in blue, purple, and pink,
respectively
Fig. 2 2D diagram of compound 2a11 in COX-2 active site. Hydrogen
bond is shown in green, other interactions, as, van der Waals, pi-
sigma, and alkyl, are shown in blue, purple, and pink, respectively
Fig. 3 3D image of compound 2a23 in COX-2 active site. Showing an
hydrogen bond in red line, and, other non-polar interactions with the
protein in lime green lines
Fig. 5 3D image of compound 2a23 in COX-2 active site. Showing
three hydrogen bonds in red lines, and, other non-polar interactions
with the protein in lime green lines
Synthesis
From the docking results, six of the 300 molecules designed
were synthetized based on the obtained results of binding
energy and structure characteristics. To achieve the com-
pounds designed, a three-step synthesis was planned. First,
a phenacyl bromide synthesis, then, its reaction with 3,5-
dimethylpyrazole, resulting in the 2-(3,5-dimethyl-1H-pyr-
azol-1-yl)-1-phenylethan-1-one intermediates, lastly, the
reaction of the intermediates with substituted benzalde-
hydes, forming the final products 4-[(1Z)-2-(3,5-dimethyl-
1H-pyrazol-1-yl)-3-oxo-3-phenylprop-1-en-1-yl]benzoni-
trile derivatives as shown in Scheme 1. As is possible to
observe, A ring groups come from the acetophenones used
to produce the phenacyl bromides, and the B ring groups
from the benzaldehydes.
Phenacyl bromides synthesis (1a, 1c, and 1d)
The substituted phenacyl bromides synthesis was done by
reaction of the chosen substituted acetophenones with pyr-
idinium tribromide in equimolar amounts, using acetic acid
as solvent. All reactions were performed under microwave
irradiation. Mean yields of 70% were obtained, without
impurities. The melting points found were 50.5, 72.5, and
96.5 °C for the unsubstituted (1a), 4-methoxy (1c) and 4-
chloro phenacyl bromide (1d), respectively. In the 1H NMR
spectra, it’s possible to observe signals of the aromatic ring
and a singlet in the 5–6 ppm region respective to the
methylene carbon.

Medicinal Chemistry Research
2-(3,5-dimethyl-1H-pyrazol-1-yl)-1-phenylethan-1-one
intermediates (2a, 2c, and 2d)
4-[(1Z)-2-(3,5-dimethyl-1H-pyrazol-1-yl)-3-oxo-3-
phenylprop-1-en-1-yl]benzonitrile synthesis (2a7, 2a11,
2a17, 2a23, 2c7, 2d11)
The intermediate compounds were synthetized using equi-
molar amounts of the phenacyl bromides and reacting it
with 3,5-dimethylpyrazole, using catalytic amounts of
K2CO3 and acetonitrile as solvent. The synthesis occurred
under microwave irradiation, and the result was a pure
product, analyzed by TLC; however, recrystallization with
cyclohexane was carried out for the determination of the
melting point. The crude yield was 60% and the recrys-
tallized 10%. Characterization was done by 1H NMR,
where was noticeable the appearing of signals referent to the
3,5-dimethylpyrazole ring, e.g., a singlet in 6 ppm referent
of the methine carbon, and two singlets in the 2–3ppm
region referent of the methoxy groups, in addition to the one
present in the phenacyl bromide spectra.
The final product was synthetized using a derivation of the
Claisen–Schmidt condensation, once the final products are
chalcone derivatives. The intermediate was dissolved in
ethanol and catalytic amounts of K2CO3 was added to the
solution. The chosen benzaldehyde was added, and the
reaction occurred at room temperature with constant
stirring.
Before choosing the K2CO3 as base, we tried the classic
strong base, NaOH, but the reaction was too fast, forming
too much impurities. The choice of a basic salt, a weaker
base, instead of the strong base used in classical
Claisen–Schmidt condensation, was due to the reactivity of
the methylene carbon in the intermediates (2a, 2c, and 2d).
The chalcone reaction occurs in some steps: first the
base, in aqueous solution, attacks the methyl group of
acetophenone—in our case the methylene carbon of the
intermediates—forming the carbanion. Then, carbanion will
attack the carbonyl group of benzaldehyde, followed by a
rearrangement and dehydration step, forming the chalcone,
and regenerating the base (Enchev and Mehandzhiyski
2017). In the pyrazole ring system, the nitrogen (atom 1)
have unshared electrons that conjugates with the aromatic
system, and its adjacent nitrogen (atom 2) unshared elec-
trons doesn’t (Faria et al. 2017). We suppose that the
methylene carbon in our intermediate is more susceptible to
the nucleophilic attack by the base, because of the two
electronegative groups next to it, which explains why the
K2CO3 was able to abstract the hydrogen and form the
carbanion. However, this carbanion is less reactive than the
one formed by the acetophenone, given its more stable.
Moreover, presents the steric hindrance factor of the 3,5-
Fig. 6 Docked poses of compounds 2a7, 2a11, 2a17, 2a23, and cel-
ecoxib superimposed in the active site of COX-2 enzyme
Scheme 1 Synthesis scheme of
the pyrazole–chalcone
derivatives. a (microwave,
80 °C, acetic acid),
b (microwave, K2CO3,
acetonitrile), and c (room
temperature, K2CO3, ethanol)

Medicinal Chemistry Research
Table 2 In vitro enzyme inhibition assay COX-1/COX-2 for
synthesized compounds and reference drug (celecoxib)
models, evaluating all the biological effects of these com-
pounds and possibly finding a hit molecule and future drug.
Compounds
IC50 COX-1 (μM)a
IC50 COX-2 (μM)a
ISb
2a11
2a23
2a17
2a7
200.64 0.96
210.13 0.40
207.76 0.82
215.48 0.60
213.40 0.88
225.86 0.92
217.72 0.22
0.73 0.02
0.75 0.04
0.78 0.02
0.84 0.004
0.84 0.006
0.86 0.002
0.88 0.002
274.85
280.17
266.36
256.52
254.05
262.63
247.41
Conclusions
In the present study, new potential COX-2 selective inhi-
bitors were designed and analyzed thorough ADMET filters
and structure-based virtual screening, using molecular
docking. The results showed that the compounds of the
series 2, with the 3,5-dimethylpyrazole ring, and that pos-
sessed polar groups that can interact through H-bonds with
the enzyme in ring A, were the best scored docked
molecules.
Six compounds (2a11, 2a23, 2a17, 2a7, 2d11, and 2c7)
were synthetized and submitted to the COX (ovine) Inhi-
bitor Screening Assay Kit to examine its capacity to
selectively inhibit COX-2 in vitro. The results found that, in
corroboration with the docking analysis, all the compounds
synthetized were more potent then celecoxib in inhibiting
COX-2, also possessing a higher selectivity index.
In conclusion, the presented compounds can be further
evaluated for in vivo models, based on the in silico and
in vitro results, and could serve as hit molecules for the
development of new COX-2 inhibitors.
2d11
2c7
Celecoxib
All compounds have a p < 0001
aIC50 corresponds to the required concentration of the compound to
produce 50% inhibition of COX-1 or COX-2 by three determinations,
the mean deviation being less than 10% in all cases. Determined with
COX (ovine) Inhibitor Screening Assay Kit (catalog N°. 560101,
Cayman Chemicals Inc., Ann Arbor, MI, USA)
bIS (selectivity index) IC50COX-1/IC50COX-2
dimethylpyrazole ring, that may difficult the carbanion
attack. This can be the reason we obtained lower yields then
the normally observed in this reaction.
In vitro COX-1/COX-2 inhibition assay
As shown in Table 2, all the compounds synthesized have
lower IC50 and better selectivity indexes (SI) than the
reference drug (celecoxib) for COX-2 inhibition. It is noted
that AutoDock Vina showed a very precise scoring power
for the designed and synthetized ligands, since the com-
pound with better binding energy was also the one that
presented the lowest IC50 for COX-2. It should be stated
that celecoxib, used as a control, is the ligand with lowest
docking energy, but obtained a less potent IC50 then the
other tested compounds.
The SI shows the ratio of IC50COX-1/IC50COX-2, where
compound 2a23 has the best SI, being greater than the
reference drug. These results corroborate the docking ana-
lysis, where the compound 2a23 showed interactions with
COX-2, outside the selectivity pocket, whereas celecoxib
did not. The reason, this compound is less potent for COX-
1, may be due to the steric effect caused by the large volume
of its substituent on the B ring. The other compounds tested
also have higher selectivity indexes than celecoxib.
Besides the anti-inflammatory effects, selectively inhi-
biting COX-2 can have a protective effect on breast cancer
risk, regarding estrogen-responsive cancer (de Pedro et al.
2015). Also, anti-angiogenesis caused by inhibition of
COX-2 were presented as an innovative strategy to a
synergetic treatment efficacy of hypoxia-responsive pro-
drugs in cancer treatment (Kim et al. 2018). This reinforce
the importance of continuing this study in cell and animal
Acknowledgements This study was financed in part by the Coorde-
nação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil
(CAPES)—Finance Code 001, and, research support fund (FAP-
UNIVALI).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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