molecules

Article
Design and Synthesis of Novel Pyrazole-Substituted
Different Nitrogenous Heterocyclic Ring Systems as
Potential Anti-Inflammatory Agents
Eman S. Nossier 1 , Hoda H. Fahmy 2 , Nagy M. Khalifa 2,3, *, Wafaa I. El-Eraky 4
and Marawan A. Baset 4
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University,
Cairo 11754, Egypt; emy28_s@hotmail.com
2 Department of Therapeutical Chemistry, Pharmaceutical and Drug Industries Division,
National Research Centre, Giza 12622, Egypt; hh_fahmy@yahoo.com
3 Pharmaceutical Chemistry Department, Drug Exploration & Development Chair (DEDC),
College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
4 Pharmacology Department, National Research Centre, Dokki, Cairo 12622, Egypt;
wafaa_nrc@yahoo.com (W.I.E.-E.); dr.marawan@gmail.com (M.A.B.)
* Correspondence: nagykhalifa@hotmail.com or nkhalifa.c@ksu.edu.sa; Tel.: +966-14-677-343; Fax: +966-14-676-220

Academic Editor: Derek J. McPhee

Received: 24 January 2017; Accepted: 2 March 2017; Published: 24 March 2017

Abstract: With the aim of developing novel anti-inflammatory scaffolds, a new series of
pyrazole-substituted various nitrogenous heterocyclic ring systems at C-4 position were synthesized
through different chemical reactions and validated by means of spectral and elemental data.
The new obtained compounds were investigated for their anti-inflammatory activity using
the carrageenan-induced paw edema standard technique and revealed that, compound 6b showed
increased potency with % inhibition of edema 85.23 ± 1.92 and 85.78 ± 0.99, respectively,
higher than the standard reference drugs indomethacin and celebrex (72.99% and 83.76%).
Molecular modeling studies were initiated herein to validate the attained pharmacological data
and provide understandable evidence for the observed anti-inflammatory behavior.

Keywords: 1,3-diaryl pyrazole derivatives; anti-inflammatory activity; synthesis

1. Introduction
The effective and quick preparation of biologically active compounds has encouraged Researchers
to identify new strategies which could be beneficial to the pharmaceutical industry. Pyrazole analogs
are a class of bioactive nitrogenous heterocycles, playing an essential role in the medicinal chemistry
fields. Incorporation of different aryl and sulphonamides onto pyrazole nucleus have resulted in
Celecoxib (1) and Rimonabant (2) which are anti-inflammatory drugs (Figure 1). Recently, Alegaon et al.
in 2014 have reported some 1,3,4-trisubstituted pyrazole derivatives are potent anti-inflammatory
activity and COX-2 selective inhibition [1,2]. In addition, numerous reports have appeared in the
literature describing different bioactivities and good safety profiles of 1,3,4-trisubstituted pyrazole
derivatives including: anti-inflammatory [3–7], analgesic, lipid peroxidation, ulcerogenic [8,9],
antipyretic [10], antioxidant [11], antimicrobial, antiviral [12–14], anticancer [15–18], antimitotic [19],
and immunosuppressive agents [20]. In addition, some pyrazole compounds have gained great
attention as antibacterial and fungicidal isoforms of human cytosolic carbonic anhydrase I or II and
antitumor properties [21–23]. Extension of our research towards the identification of an efficient
synthesis of biologically active pyrazole compounds [24–29], we report herein the synthesis of novel
derivatives of 1,3-diaryl pyrazoles and their anti-inflammatory activities.

Molecules 2017, 22, 512; doi:10.3390/molecules22040512 www.mdpi.com/journal/molecules

 and marked decrease in compounds (4b, 5b, and 8b). However, cyclization of chalcone 2a having
4-bromophenyl at position-4 of pyrazole moiety led to a drop in anti-inflammatory activity as in
3a–9a derivatives. The cyanopyridone derivative 6b seems to be the most effective prepared
anti-inflammatory agent, revealing better activity (89.57% inhibition of edema) than both
indomethacin and celecoxib (standard drugs). Insertion of cyanoiminopyridine moiety as in 7a,b
(19.11% and 86.37% inhibition) instead of cyanopyridone in 6a,b (37.35% and 89.57 % inhibition)
displayed a little decrease in the activity. While replacement of thiopyrimidine in 9a,b (29.61% and
Molecules 2017, 22, 512 2 of 16
87.42% inhibition) with aminopyrimidine moiety in 8a,b led to a drop in the activity (17.66% and
42.78% inhibition) (Figures 1–5).

SO2NH2
Cl
Cl

O
N N
N N HN N

F3C
Cl
Celecoxib Rimonabant
(1) (2)

Figure 1. Structures of the selective COX-2 inhibitors, celecoxib and Rimonabant.

Figure 1. Structures of the selective COX-2 inhibitors, celecoxib and Rimonabant.
120.00

2. Results and Discussion

100.00

2.1. Chemistry 80.00

60.00
The reaction sequences
Molecules 2017, 22, 512 outlined in Scheme 1 was used for the synthesis 2 of 16 of the target
compounds. Application of
40.00the Claisen Schmidt condensation on substituted
biologically active pyrazole compounds [24–29], we report herein the synthesis of novel derivatives acetophenones namely,
of 1,3-diaryl pyrazoles and their anti-inflammatory activities.
4-bromoacetophenone or20.00 4-methoxyacetophenone and 1-(3-chlorophenyl)-3-(4-methoxyphenyl)-
1H-pyrazole-4-carboxaldehyde
2. Results and Discussion (1) in ethanolic sodium hydroxide solution afforded (E)-1-(4-substituted
0.00
phenyl)-3-(1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)
2.1. Chemistry prop-2-en-1-one (2a,b), which
was used as startingThe materials.
reaction sequences outlined in Scheme 1 was used for the synthesisketone
Cyclocondensation of the α,β-unsaturated 2a,b with hydrazine
of the target
hydrate in glacial acetic acid
compounds. yielded
Application the %
of the Claisen corresponding
Changecondensation
Schmidt 1h pyrazoline derivatives
on substituted acetophenones 3a,b. On the other
namely,
4-bromoacetophenone or 4-methoxyacetophenone and 1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-
hand, heating of pyrazole-4-carboxaldehyde
2a,b with thiosemicarbazide (1) in ethanolic in ethanolic
sodium hydroxideNaOH provided
solution afforded 1-thiocarbamoyl pyrazole
(E)-1-(4-substituted
derivatives 4a,b. phenyl)-3-(1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)prop-2-en-1-one
In addition, condensation of compound 2a,b with hydroxylamine (2a,b), which washydrochloride in
used as starting materials. Cyclocondensation of the α,β-unsaturated ketone 2a,b with hydrazine
refluxing ethanolhydrate
in theinpresence of sodium
glacial acetic acid yielded the hydroxide as alkaline
corresponding pyrazoline medium
derivatives 3a,b. Onafforded the corresponding
the other hand,
isoxazoline 5a,b. Reaction of α,β-unsaturated ketone 2a,b with ethyl cyanoacetate, or malononitrile
heating of 2a,b with thiosemicarbazide in ethanolic NaOH provided 1-thiocarbamoyl pyrazole
derivatives 4a,b. In addition, condensation of compound 2a,b with hydroxylamine hydrochloride in
in presence of ammonium
refluxing ethanolacetate, gave
in the presence the corresponding
of sodium 2-oxo(imino)pyridine
hydroxide as alkaline medium afforded the correspondingderivatives 6a,b,
and 7a,b, respectively. Furthermore, treatment of 2a,b with guanidineorsulfate
isoxazoline 5a,b. Reaction of α,β-unsaturated ketone 2a,b with ethyl cyanoacetate, in in
malononitrile ethanolic sodium
presence of ammonium acetate, gave the corresponding 2-oxo(imino)pyridine derivatives 6a,b, and 7a,b,
hydroxide gave respectively.
2-aminopyrimidine
Furthermore, treatment of 2a,b with guanidine sulfate in ethanolic sodium hydroxidewith thiourea in
derivatives 8a,b. Finally, treatment of 2a,b
presence of sodiumgavehydroxide
2-aminopyrimidine gavederivatives
the corresponding pyrimidine-2-thione
8a,b. Finally, treatment of 2a,b with thiourea derivative
in presence of 9a,b (Scheme 1).
sodium hydroxide gave the corresponding pyrimidine-2-thione derivative 9a,b (Scheme 1).

OCH3 OCH3 OCH3

R
R R
N N N
CN
N N N O N
Cl N N NH2 Cl Cl HN
O
S
4a,b 5a,b 6a,b

(iii) (iv) (v)

OCH3 OCH3 OCH3

R
N (i) N (ii) N
O
N N
Cl
N
O Cl Cl N N CH3
R
O

1 2a,b 3a,b

(vi) (vii) . (viii)

OCH3 OCH3 OCH3

R R R
N N N
CN N NH
N N N
Cl HN Cl N Cl N
NH NH2 S
7a,b 8a,b 9a,b

2-9: a, R = 4-C6H4-Br
b, R = 4-C6H4-OCH3

Scheme 1. Synthetic route for polysubstituted pyrazole compounds 2–9. Reagents and Conditions:
Scheme 1. Synthetic route
(i) RCOCH for polysubstituted
3 derivatives/30%Etanolic pyrazole
NaOH/r.t./12 compounds
h, 83–85%; 2–9. Reagentsh, and Conditions:
(ii) N2H4·H2O/AcOH/reflux/4–6
73–81%; (iii) NH2CSNHNH2/EtOH/NaOH/reflux/2–3 h, 71–73%; (iv) NH2OH·HCl/ethanol/NaOH/
(i) RCOCH3 derivatives/30%Etanolic NaOH/r.t./12 h, 83%–85%; (ii) N
reflux/2–3 h, 80%–85%; (v) NC-CH2COOEt/CH3COONH4+/ethanol//reflux/6 h, 75%–76%;
·H2CNCH
2 H4 (vi) O/AcOH/reflux/4–6
2CN/
h,
73%–81%; (iii) CHNH3COONH
2 CSNHNH /EtOH/NaOH/reflux/2–3
4+/ethanol/reflux/6
2 h, 70–73%; (vii) NH h, 71%–73%;
2NHNH2.H2SO4/ethanol/NaOH/reflux/5–7 h, (iv)
69–71%; NH 2 OH · HCl/
(viii) NH2CSNH2/ethanol/NaOH/reflux/6–8 h, 80–81%.
ethanol/NaOH/reflux/2–3 h, 80%–85%; (v) NC-CH2 COOEt/CH3 COONH4 + /ethanol//reflux/6 h,
75%–76%; (vi) CNCH2 CN/CH3 COONH4 + /ethanol/reflux/6 h, 70%–73%; (vii) NH2 NHNH2 .H2 SO4 /
ethanol/NaOH/reflux/5–7 h, 69%–71%; (viii) NH2 CSNH2 /ethanol/ NaOH/ reflux/6–8 h, 80–81%.
 All the newly synthesized trisubstitutedpyrazole compounds 2–9 were evaluated for their in vivo
anti-inflammatory activity using carrageenan induced rat paw edema method [30]. The primarily
antiinflammatory activity results (Table 1) revealed that, six compounds (2a, 2b, 3a, 6b, 7b, and 9b)
showed consistently excellent anti-inflammatory activity (84.39–89.57% inhibition) 4 h after the
carrageenan injection comparable to that of standard drugs indomethacin and celebrex (72.99% and
Molecules 2017, 22, 512 3 of 16
83.76%), respectively. Chalcones 2a and 2b showed approximately equal anti-inflammatory activity
higher than the reference drugs (% inhibition of edema = 85.23 ± 1.92 and 85.78 ± 0.99), respectively.
Considering
2.2. BiologicalofEvaluation
the target pyrazoles, it was noticed that the electron donating substituent (methoxy
group) at position-4 of pyrazole moiety exhibited higher activity than their congeners with 4-electron
2.2.1. In Vivo substituent
withdrawing Anti-Inflammatory
(bromo Activity
group) except for acetylpyrazoline derivatives 3a,b. Cyclization of
α,β-unsaturated ketone 2b bearing 4-methoxyphenyl at position-4 of pyrazole moiety, gave
All the newly synthesized trisubstitutedpyrazole compounds 2–9 were evaluated for their in vivo
increased activity in compounds (6b, 7b, and 9b) with little decrease in acetyl pyrazoline derivative 3b
anti-inflammatory activity using carrageenan induced rat paw edema method [30]. The primarily
and marked decrease in compounds (4b, 5b, and 8b). However, cyclization of chalcone 2a having
antiinflammatory activity results (Table 1) revealed that, six compounds (2a, 2b, 3a, 6b, 7b, and 9b)
4-bromophenyl at position-4 of pyrazole moiety led to a drop in anti-inflammatory activity as in
showed consistently excellent anti-inflammatory activity (84.39%–89.57% inhibition) 4 h after the
3a–9a derivatives. The cyanopyridone derivative 6b seems to be the most effective prepared
carrageenan injection comparable to that of standard drugs indomethacin and celebrex (72.99%
anti-inflammatory agent, revealing better activity (89.57% inhibition of edema) than both
and 83.76%), respectively. Chalcones 2a and 2b showed approximately equal anti-inflammatory
indomethacin and celecoxib (standard drugs). Insertion of cyanoiminopyridine moiety as in 7a,b
activity higher than the reference drugs (% inhibition of edema = 85.23 ± 1.92 and 85.78 ± 0.99),
(19.11% and 86.37% inhibition) instead of cyanopyridone in 6a,b (37.35% and 89.57 % inhibition)
respectively. Considering of the target pyrazoles, it was noticed that the electron donating substituent
displayed a little decrease in the activity. While replacement of thiopyrimidine in 9a,b (29.61% and
(methoxy group) at position-4 of pyrazole moiety exhibited higher activity than their congeners
87.42% inhibition) with aminopyrimidine moiety in 8a,b led to a drop in the activity (17.66% and
with 4-electron withdrawing substituent (bromo group) except for acetylpyrazoline derivatives 3a,b.
42.78% inhibition) (Figures 1–5).
Cyclization of α,β-unsaturated ketone 2b bearing 4-methoxyphenyl at position-4 of pyrazole moiety,
gave increased activity in SO compounds (6b, 7b, and 9b) with little decrease in acetyl pyrazoline
2NH2
derivative 3b and marked decrease in compounds (4b, Cl 5b, and 8b). However, cyclization of chalcone
Cl
2a having 4-bromophenyl at position-4 of pyrazole moiety led to a drop in anti-inflammatory activity
as in 3a–9a derivatives. The cyanopyridone derivative 6b seems O most effective prepared
N be the
N to
anti-inflammatory agent,Nrevealing better activity (89.57% inhibition of edema) than both indomethacin
N HN N
and celecoxib (standard drugs). Insertion of cyanoiminopyridine moiety as in 7a,b (19.11% and
86.37% inhibition) F3Cinstead of cyanopyridone in 6a,b (37.35% and 89.57 % inhibition) displayed a little
decrease in the activity.Celecoxib Cl
While replacement of thiopyrimidine inRimonabant
9a,b (29.61% and 87.42% inhibition)
(1)
with aminopyrimidine moiety in 8a,b led to a drop in the activity (17.66% (2) and 42.78% inhibition)
(Figures 2–5). Figure 1. Structures of the selective COX-2 inhibitors, celecoxib and Rimonabant.

120.00

100.00

80.00

60.00

40.00

20.00

0.00

% Change 1 h
Figure 2. Percentage of inhibition after 1 h of carrageenan injection comparable to that of standard
drugs, indomethacin and Celebrex.
Molecules
Molecules 2017,
2017, 22,
22, 512
512 44 of
of 16
16

Figure
Molecules 2017,2.
Figure Percentage
2.22, 512
Percentage of
of inhibition
inhibition after
after 11 hh of
of carrageenan
carrageenan injection
injection comparable
comparable to
to that
that of
of standard
standard4 of 16
drugs,
drugs, indomethacin
indomethacin and
and Celebrex.
Celebrex.

140
120
100
80
60
40
20
0

% Change 2 h

3. Percentage of
Figure 3.
Figure of inhibition after
after 2 h of carrageenan
carrageenan injection comparable
comparable to that
that of standard
standard
Figure 3. Percentage
Percentage of inhibition
inhibition after 22 hh of
of carrageenan injection
injection comparable to
to that of
of standard
drugs,
drugs, indomethacin and Celebrex.
drugs, indomethacin
indomethacin and
and Celebrex.
Celebrex.

160
160

140
140

120
120

100
100

80
80

60
60

40
40

20
20

00

% Change 3 h

Figure
Figure 4.
4. Percentage
Percentage of
of inhibition
inhibition after
after 333 h
h of
of carrageenan
carrageenan injection
injection comparable
comparable to
to that
that of
of standard
standard
Figure 4. Percentage of inhibition after h of carrageenan injection comparable to that of standard
drugs,
drugs, indomethacin
indomethacin and
and Celebrex.
Celebrex.
drugs, indomethacin and Celebrex.
Molecules 2017, 22, 512 5 of 16

Table 1. Anti-inflammatory activity of the tested compounds 2–9 using carrageenan-induced paw edema in rats.

%Change %Change %Change %Change

Drugs % Inhibition % Inhibition % Inhibition % Inhibition
1h 2h 3h 4h
control 100.5 ± 9.07 —- 113.8 ± 13.21 —- 102.4 ± 10.42 —- 32.05 ± 2.80 —-
2a 88.78 ± 9.70 −11.62 94.47 ± 10.13 −17.00 58.42 ± 7.31 * −42.93 4.735 ± 1.92 * −85.23
2b 77.43 ± 2.25 −22.92 82.37 ± 2.55 −27.62 36.27 ± 3.73 * −64.57 4.557 ± 0.99 * −85.78
3a 64.03 ± 4.96 * −36.26 70.22 ± 5.50 * −38.30 63.92 ± 3.59 * −37.56 5.003 ± 0.94 * −84.39
3b 91.8 ± 9.65 −8.61 98.19 ± 9.24 −13.72 48.54 ± 4.24 * −52.58 19.56 ± 2.79 −38.96
4a 96.39 ± 6.90 −4.05 103 ± 6.95 −9.51 131 ± 12.23 27.98 35.83 ± 1.50 11.80
4b 55.82 ± 4.05 * −44.43 64.57 ± 5.18 * −43.27 28.38 ± 2.86 * −72.28 7.442 ± 1.59 * −76.78
5a 73.41 ± 5.92 −26.93 78.32 ± 7.00 −31.18 66.51 ± 6.60 * −35.02 19.73 ± 2.44 −38.45
5b 100.4 ± 10.72 −0.06 107.8 ± 10.97 −5.26 53.77 ± 5.47 * −47.47 10.38 ± 1.77 * −67.62
6a 86.3 ± 2.17 −14.09 93.78 ± 2.51 −17.60 84.33 ± 6.57 −17.62 44.02 ± 5.39 37.35
6b 41.96 ± 3.82 * −58.23 61.37 ± 4.23 * −46.08 53.86 ± 4.94 * −47.39 3.345 ± 0.52 * −89.57
7a 84.47 ± 4.65 −15.92 86.53 ± 2.72 −23.97 114.6 ± 6.95 12.00 38.18 ± 5.73 19.11
7b 74.78 ± 5.38 −25.56 80.48 ± 5.51 −29.28 61.94 ± 3.79 * −39.49 4.368 ± 1.18 * −86.37
8a 60.3 ± 4.08 * −39.97 71.13 ± 6.50 * −37.50 82.85 ± 8.84 −19.07 26.39 ± 2.92 −17.66
8b 77.38 ± 6.13 −22.97 112.4 ± 10.61 −1.25 76.74 ± 6.85 −25.03 18.34 ± 2.40 * −42.78
9a 52.79 ± 5.44 * −47.45 61.83 ± 6.32 * −45.67 68.91 ± 5.30 −32.68 22.56 ± 0.81 −29.61
9b 39.06 ± 2.75 * −61.11 46.06 ± 3.05 * −59.53 62.15 ± 6.83 * −39.29 4.033 ± 0.87 * −87.42
Indomethacin 60.14 ± 6.64 * −40.1295 67.97 ± 6.03 * −40.2745 62.27 ± 8.14 * −39.17 8.655 ± 1.53 * −72.9943
Celebrex 52.39 ± 3.03 * −47.8509 58.69 ± 2.95 * −48.4341 60.25 ± 5.74 * −41.1372 5.205 ± 0.65 * −83.7597
Values represent the mean ± S.E. of six animals for each groups. * p < 0.05: Statistically significant from the control using one-way ANOVA (using Tukey as post hoc test).
Molecules 2017, 22, 512 6 of 16
Molecules 2017, 22, 512 6 of 16

60

50

40

30

20

10

% Change 4 h

Figure 5. Percentage of inhibition after 4 h of carrageenan injection comparable to that of standard

Figure 5. Percentage of inhibition after 4 h of carrageenan injection comparable to that of standard
drugs, indomethacin and Celebrex.
drugs, indomethacin and Celebrex.

2.2.2. Ulcerogenic Liability

2.2.2. Ulcerogenic Liability
Ulcerogenic liability of all prepared anti-inflammatory agents 2–9 was determined following
Ulcerogenic liability of all prepared anti-inflammatory agents 2–9 was determined following the
the reported standard method [31] using indomethacin and Celebrex (in a dose 0.28 mmol/kg) as
reported standard method [31] using indomethacin and Celebrex (in a dose 0.28 mmol/kg) as reference
reference standards. It was noticed that all compounds revealed no ulcers, like celebrex, and they are
standards. It was noticed that all compounds revealed no ulcers, like celebrex, and they are considered
considered safer than indomethacin itself which produced an ulcer count of 14 ± 1.2.
safer than indomethacin itself which produced an ulcer count of 14 ± 1.2.
2.3. Molecular Modeling Study
2.3. Molecular Modeling Study
Molecular modeling study of the highly observed anti-inflammatory active agent 6b was
Molecular modeling study of the highly observed anti-inflammatory active agent 6b was
performed herein to understand the observed pharmacological data. Docking study was initiated
performed herein to understand the observed pharmacological data. Docking study was initiated
using MOE 2008.10 program. It is used to predict the binding modes and orientation of compound
using MOE 2008.10 program. It is used to predict the binding modes and orientation of compound 6b
6b at the active site of the ATP binding site of COX-2 enzyme. The coordinates of this enzyme
at the active site of the ATP binding site of COX-2 enzyme. The coordinates of this enzyme structure
structure were obtained from the crystal structure of COX-2 with its inhibitor (PDB ID: 1CX2). The
were obtained from the crystal structure of COX-2 with its inhibitor (PDB ID: 1CX2). The root mean
root mean square difference (RMSD) between the top docking pose and original crystallographic
square difference (RMSD) between the top docking pose and original crystallographic geometry of
geometry of co-crystallized ligand SC-558 was 0.9 Å. The phenylsulphonamide moiety of this
co-crystallized ligand SC-558 was 0.9 Å. The phenylsulphonamide moiety of this SC-558 is surrounded
SC-558 is surrounded by hydrophobic residues Leu352, Tyr355, Phe518, Val523, and the backbone
by hydrophobic residues Leu352, Tyr355, Phe518, Val523, and the backbone of Ser353. Beyond this
of Ser353. Beyond this hydrophobic pocket, the sulphonamide exhibits hydrophilic interaction with
hydrophobic pocket, the sulphonamide exhibits hydrophilic interaction with His90, GIn192, and
His90, GIn192, and Arg513 [32]. Celecoxib forms three hydrogen bonds with the hydrophilic side
Arg513 [32]. Celecoxib forms three hydrogen bonds with the hydrophilic side chains (His90 and
chains (His90 and Gln192) in the side pocket and the main chain carbonyl at residue Leu338 [33].
Gln192) in the side pocket and the main chain carbonyl at residue Leu338 [33].
In Table 2, the compounds 2a, 2b, 3a, 4b, 5b, 6b, 7b and 9b with the highest and moderate
In Table 2, the compounds 2a, 2b, 3a, 4b, 5b, 6b, 7b and 9b with the highest and moderate
anti-inflammatory activity were found to have high binding energy ranging from −6.25 −to1
anti-inflammatory activity were found to have high binding energy ranging from −6.25 to −8.11 kcal·mol
−8.11 kcal·mol−1 in comparison with reference ligands SC-558 and celecoxib (−6.30 and −6.55 kcal·mol−1
in comparison with reference ligands SC-558 and celecoxib (−6.30 and −6.55 kcal·mol−1 respectively).
respectively). It was observed that most of the active compounds have arene-cation interaction
It was observed that most of the active compounds have arene-cation interaction between (Arg120
between (Arg120 and Arg513) and the newly inserted 4-methoxyphenyl moiety.
and Arg513) and the newly inserted 4-methoxyphenyl moiety.
Molecules 2017, 22, 512 7 of 16

Table 2. Docking results of the compounds 2–9 with COX-2 enzyme in comparison with the ligands,
SC-558 and celecoxib using MOE software version 2008.10.

Docking Score Amino Acid Residues

Compd. NO. Atoms of Compound Type of Bond
(Kcal/mol) (Bond Length Å)
His90 (2.6); O(CO) H-acc
2a −6.78
Arg120 (2.1); O(OCH3 ) (parent) H-acc
His90 (2.4); O(CO)
H-acc
Arg120 (2.3); O(OCH3 ) (parent)
2b −6.93 H-acc
Arg120; C6 H3 -4-OCH3 (new)
Arene-cation Arene-cation
Arg513 C6 H3 -4-OCH3 (new)
3a −7.12 Arg120 (1.8); O(OCH3 ) (parent) H-acc
Arg120 (2.1); O(OCH3 ) (parent)
H-acc
3b −5.46 Arg120; C6 H3 -4-OCH3 (new)
Arene-cation Arene-cation
Arg513 C6 H3 -4-OCH3 (new)
4a −5.22 Arg120 (2.6); O(OCH3 ) (parent) H-acc
Arg120 (2.4); O(OCH3 ) (parent)
H-acc
4b −7.24 Arg120; C6 H3 -4-OCH3 (new)
Arene-cation Arene-cation
Arg513 C6 H3 -4-OCH3 (new)
5a −5.14 Arg120 (2.7); O(OCH3 ) (parent) H-acc
Arg120 (2.1); O(OCH3 ) (parent)
H-acc
5b −6.25 Arg120; C6 H3 -4-OCH3 (new)
Arene-cation Arene-cation
Arg513 C6 H3 -4-OCH3 (new)
His90 (2.4); NH(pyridone) H-acc
6a −5.43 His90 (2.6); O(pyridone) H-acc
Arg120 (2.5); O(OCH3 ) (parent) H-acc
His90 (2.7); NH(pyridone)
H-acc
His90 (2.7); O(pyridone)
H-acc
6b −8.11 Arg120 (3.1); O(OCH3 ) (parent)
H-acc
Arg120; C6 H3 -4-OCH3 (new)
Arene-cation Arene-cation
Arg513 C6 H3 -4-OCH3 (new)
His90 (2.6); NH(iminopyridine) H-acc
7a −5.45
Arg120 (2.3); O(OCH3 ) (parent) H-acc
His90 (2.7); NH(iminopyridine)
H-acc
Arg120 (2.1); O(OCH3 ) (parent)
7b −6.98 H-acc
Arg120; C6 H3 -4-OCH3 (new)
Arene-cation Arene-cation
Arg513 C6 H3 -4-OCH3 (new)
8a −5.20 Arg120 (2.2); O(OCH3 ) (parent) H-acc
Arg120 (1.9); O(OCH3 ) (parent)
H-acc
8b −6.26 Arg120; C6 H3 -4-OCH3 (new)
Arene-cation Arene-cation
Arg513 C6 H3 -4-OCH3 (new)
9a −5.75 Arg120 (2.5); O(OCH3 ) (parent) H-acc
Arg120 (1.9); O(OCH3 ) (parent)
H-acc
9b −7.48 Arg120; C6 H3 -4-OCH3 (new)
Arene-cation Arene-cation
Arg513 C6 H3 -4-OCH3 (new)
His90 (2.2); H(NH2 ) H-acc
GIn192 (2.4); H(NH2 ) H-don
Arg513 (2.2); H(NH2 ) H-don
SC-558 −6.30 Val523; C6 H3 -4-Br Arene-Arene
Ala527; C6 H3 -4-Br Arene-Arene
Leu352; Phenylsulphonamide Arene-Arene
Ser353 Phenylsulphonamide Arene-Arene
His90 (1.9); H(NH2 ) H-acc
GIn192 (2.1); H(NH2 ) H-don
Leu338 (2.3); H(NH2 ) H-acc
Celecoxib −6.55 Val523; C6 H3 -4-OCH3 Arene-Arene
Ala527; C6 H3 -4-OCH3 Arene-Arene
Leu352; Phenylsulphonamide Arene-Arene
Ser353 Phenylsulphonamide Arene-Arene
Molecules 22, 512
2017, 2017,
Molecules 22, 512 8 of 16
8 of 16

From Figure 6, it was found that the compound 6b revealed good fitting inside the binding site
From Figure 6, it was found that the compound 6b revealed good fitting inside the binding site of
of the protein molecular surface and having minimum binding energy of −8.11−1kcal·mol−1. There
the protein molecular surface and having minimum binding energy of −8.11 kcal·mol . There were
were two hydrogen bonds linking the sidechain of His90 with NH and oxygen of pyridone moiety as
two hydrogen bonds linking the sidechain of His90 with NH and oxygen of pyridone moiety as
hydrogen acceptors (distance: 2.76 and 2.79 Å, respectively). The 4-methoxyphenyl moiety attached to
hydrogen acceptors (distance: 2.76 and 2.79 Å, respectively). The 4-methoxyphenyl moiety attached to
pyridone formed two arene-cation interactions with Arg120 and Arg513. Furthermore, one H-bond
pyridone formed two arene-cation interactions with Arg120 and Arg513. Furthermore, one H-bond
acceptor was observed between the sidechain of Arg120 and 4-methoxyphenyl linked to pyrazole
acceptor was observed between the sidechain of Arg120 and 4-methoxyphenyl linked to pyrazole
scaffold (distance: 3.19 Å). The previous results indicated that the insertion of 4-methoxyphenyl
scaffold (distance: 3.19 Å). The previous results indicated that the insertion of 4-methoxyphenyl group
group to the pyridone moiety might reinforce the combination of compound 6b and the receptor,
to the pyridone moiety might reinforce the combination of compound 6b and the receptor, which might
which might enhance the binding affinity, resulting in the increased anti-inflammatory activity of
enhance the binding affinity, resulting in the increased anti-inflammatory activity of this compound.
this compound.

Figure
Figure 6. 6.
TheTheproposed
proposedbinding
binding mode
mode ofofcompound
compound 6b6b
docked in the
docked in active site ofsite
the active COX-2; (A) and (B)
of COX-2;
showing 2D and 3D ligand-receptor interactions (hydrogen bonds are illustrated
(A,B) showing 2D and 3D ligand-receptor interactions (hydrogen bonds are illustrated as dotted as dotted purple
lines;
purple C atoms
lines; areare
C atoms colored
coloredgray, NN
gray, blue and
blue OO
and red).
red).
Molecules 2017, 22, 512 9 of 16

3. Experimental Section

3.1. General Information

Melting points were measured in open capillary tubes using a Griffin apparatus and are
uncorrected. Structures of compounds were confirmed by routine spectrometric analysis. Elemental
analyses were carried results were within ±0.4% of the theoretical values. Infrared spectra were
recorded on a 435 IR spectrophotometer (Shimadzu Bruker, Tokyo, Japan) using KBr discs. 1 H-NMR
and 13 C-NMR spectra were obtained on a Gemini 500 MHz spectrophotometer (Varian, Polo Alto,
CA, USA) or on a Bruker 500 MHz spectrophotometer, and measured in δ scale using TMS as an
internal standard. Mass Spectra were recorded on a 5988 spectrometer (Hewlett Packard, Palo Alto,
CA, USA). Analytical thin layer chromatography (TLC) was performed using silica gel aluminum
sheets, 60 F254 (E. Merck, Darmstadt, Germany) for the progress of reactions and visualization with
ultraviolet light (UV) at 365 and 254 nm.

3.1.1. 1-(4-Substitutedphenyl)-3-(1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)prop-2-
en-1-one (2a,b)
A mixture of pyrazolecarbaldehyde derivative 1 (0.01 mol) and substituted acetophenones,
namely 4-bromoacetophenone or 4-methoxyacetophenone (0.01 mol), in of 30% ethanolic NaOH
solution (40 mL) was stirred for 12 h at room temperature. The progress of reaction was monitored
by TLC. After completion, the reaction mixture was poured into acidified ice cold water of pH ~2.
The precipitated solid formed was filtered, washed with water and recrystallized from ethanol to
afford the title compounds 2a,b.
1-(4-Bromophenyl)-3-(1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)prop-2-en-1-one (2a). Yield 83%;
m.p. 235–237 ◦ C; IR (KBr, cm−1 ) ν: 1673 (C=O), 1589 (C=C); 1 H-NMR (DMSO-d6 -δ ppm): 3.81 (s, 3H,
OCH3 ); 7.09–7.99 (m, 14H, ArH + CH=CH), 9.45 (s,1H, CH of pyrazole); 13 C-NMR (DMSO-d6 -δ ppm):
55.60, 114.34, 116.62, 117.75, 125.11, 125.95, 127.67, 129.08, 129.81, 131.74, 133.85, 134.47, 135.44, 141.08,
144.32, 151.54, 159.58, 191.96; MS (EI, 70 eV): m/z (%): 492 (11) [M]+ ; Anal. Calcd. for C25 H18 BrClN2 O2
(493.78): C, 60.81; H, 3.67; N, 5.67; Found: C, 61.65; H, 3.77; N, 5.32.
1-(4-Methoxphenyl)-3-(1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)prop-2-en-1-one (2b). Yield
85%; m.p. 152–154 ◦ C; IR (KBr, cm−1 ) ν: 1680 (C=O), 1601 (C=C); 1 H-NMR (DMSO-d6 -δ ppm): 3.80,
3.85 (2s, 6H, 2OCH3 ); 7.10–8.09 (m, 14H, ArH + CH=CH), 9.43 (s,1H, CH of pyrazole); MS (EI, 70 eV):
m/z (%): 447 (11, M+ + 3), 445 (29, M+ + 1), 410 (100); Anal. Calcd. for C26 H21 ClN2 O3 (444.91): C, 70.19;
H, 4.76; N, 6.30; Found: C, 70.35; H, 4.52; N, 6.55.

3.1.2. 1-(3-(4-Substituted phenyl)-5-(1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-4,5-

dihydro-pyrazol-1-yl ethanone (3a,b)
A solution of hydrazine hydrate (0.01 mol) was added to a solution of compounds 2a,b (0.01 mol)
in glacial acetic acid (20 mL) and the mixture was refluxed for 4–6 h. The reaction mixture was cooled to
room temperature and the precipitated solid was filtered, washed with water, dried, and recrystallized
from ethanol to give the title compounds 3a,b.
1-(3-(4-Bromophenyl)-5-(1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-4,5-dihydro-pyrazol-1-yl
ethanone (3a). Yield 73%; m.p. 233–235 ◦ C; 1 H-NMR (DMSO-d6 -δ ppm): 2.28 (s, 3H, COCH3 ), 3.16
(dd, 1H, CH), 3.77 (s, 3H, OCH3 ), 3.80 (dd, 1H, CH), 5.63 (dd, 1H, CH), 7.01–8.43 (m, 12H, Ar-H), 9.68
(s, 1H, CH-pyrazole); 13 C-NMR (DMSO-d6 -δ ppm): 24.0, 43.8, 55.6, 63.9, 114.3, 116.8, 117.6, 125.4, 125.9,
126.1, 127.6, 129.1, 129.8, 130.7, 131.1, 134.4, 137.8, 140.8, 150.4, 155.2, 159.1, 160.9, 168.2; MS (EI, 70 eV):
m/z (%): 548 (43) [M]+ ; Anal. Calcd. for C27 H22 BrClN4 O2 (549.85): C, 58.98; H, 4.03; N, 10.19; Found:
C, 58.77; H, 4.37; N, 10.36.
Molecules 2017, 22, 512 10 of 16

1-(3-(4-Methoxyphenyl)-5-(1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-4,5-dihydro-pyrazol-1-yl
ethanone (3b). Yield 81%; m.p. 175–179 ◦ C; 1 H-NMR (DMSO-d6 -δ ppm): 2.27 (s, 3H, COCH3 ), 3.13
(dd, 1H, CH), 3.76, 3.77 (2s, 6H, 2OCH3 ), 3.80 (dd, 1H, CH), 5.59 (dd, 1H, CH), 6.98–8.87 (m, 12H,
Ar-H), 9.69 (s, 1H, CH-pyrazole) ppm; 13 C-NMR (DMSO-d6 -δ ppm): 24.01, 43.8, 55.6, 63.9, 114.3, 116.8,
117.6, 125.4, 125.9, 126.1, 127.6, 129.1, 129.8, 130.7, 131.1, 134.4, 137.8, 140.8, 150.4, 155.2, 159.1, 160.9,
168.2 ppm; MS (EI, 70 eV): m/z (%): 503 (11, M+ + 3), 501 (25, M+ + 1), 77 (100); Anal. Calcd. for
C28 H25 ClN4 O3 (500.98): C, 67.13; H, 5.03; N, 11.18; Found: C, 67.38; H, 4.95; N, 11.13.

3.1.3. 3-(4-Substitutedphenyl)-5-(1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-4,5-
di-hydropyrazole-1-carbothioamide (4a,b)
Thiosemicarbazide was added to a mixture of chalcone 2a,b (0.01 mol) in absolute ethanol (30 mL)
containing sodium hydroxide (1 g, 0.025 mol). The reaction mixture was heated under reflux for
2–3 h. The contents were reduced, cooled, and poured onto crushed ice containing a few drops of
hydrochloric acid (until pH ~6). The resulting precipitate was collected by filtration and recrystallized
from methanol to give the title compounds 4a,b.
3-(4-Bromophenyl)-5-(1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-4,5-di-hydropyrazole-1-
carbothioamide (4a). Yield 71%; m.p. 284–286 ◦ C; IR (KBr, cm−1 ) ν: 3414 (NH2 ), 1592 (C=C), 1067 (C=S);
1 H-NMR (DMSO-d -δ ppm): 3.24 (dd, 1H, CH), 3.78 (s, 3H, OCH ), 3.81 (dd, 1H, CH), 6.00 (dd, 1H,
6 3
CH), 7.00–8.25 (m, 14H, Ar-H + NH2 exchangeable with D2 O), 9.17 (s, 1H, CH of pyrazole); 13 C-NMR
(DMSO-d6 -δ ppm): 40.5, 55.8, 56.2, 114.5, 114.7, 116.9, 118.0, 118.5, 124.3, 125.6, 126.1, 129.6, 129.9, 130.9,
131.6, 132.0, 134.4, 141.0, 150.1, 154.1, 159.7, 176.5; MS (EI, 70 eV): m/z (%): 569 (1, M + 4), 567 (2.7,
M + 2), 565 (2.7, M+ ), 111 (100); Anal. Calcd. for C26 H21 BrClN5 OS (566.9): C, 55.09; H, 3.73; N, 12.35;
Found: C, 54.92; H, 3.52; N, 12.14.
3-(4-Methoxyphenyl)-5-(1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-4,5-dihydro-pyrazole-1-
carbothioamide (4b). Yield 73%; m.p. 223–225 ◦ C; IR (KBr, cm−1 ) ν: 3444 (NH2 ), 1594 (C=C), 1092 (C=S);
1 H-NMR (DMSO-d -δ ppm): 3.24 (dd, 1H, CH), 3.75, 3.78 (2s, 6H, 2OCH ), 3.84 (dd, 1H, CH), 5.98
6 3
(dd, 1H, CH), 6.95–8.37 (m, 12H, Ar-H), 8.18 (s, 2H, NH2 exchangeable with D2 O ), 9.17 (s, 1H, CH of
pyrazole); 13 C-NMR (DMSO-d6 -δ ppm): 42.3, 55.2, 55.3, 61.4, 114.0, 114.1, 114.2, 116.4, 118.9, 125.6,
126.5, 128.0, 128.9, 129.5, 131.1, 134.8, 134.8, 140.5, 150.4, 151.6, 159.2, 160.4, 175.6; MS (EI, 70 eV): m/z
(%): 519 (2.9, M+ + 2), 517 (7, M+ ), 369 (100); Anal. Calcd. for C27 H24 ClN5 O2 S (518.03): C, 62.60; H,
4.67; N, 13.52; Found: C, 62.52; H, 4.43; N, 13.26.

3.1.4. 4-(3-(4-Substituted phenyl)isoxazol-5-yl)-1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazole (5a,b)

A mixture of compounds 2a,b (0.01 mol) and hydroxylamine hydrochloride (0.01 mol) in ethanol
(30 mL) containing sodium hydroxide solution (0.5 g NaOH in 0.5 mL water) was refluxed for 2–3 h.
The reaction mixture was poured onto ice-water, neutralized with drops of concentrated Hydrochloric
acid, and the solid precipitate formed filtered off, washed with water, and recrystallized from methanol
to afford the desired compounds 5a,b.
4-(3-(4-Bromophenyl)isoxazol-5-yl)-1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazole (5a). Yield 85%;
m.p. 168–170 ◦ C; IR (KBr, cm−1 ) ν: 3064 (CH-Ar), 1601 (C=C); 1 H-NMR (DMSO-d6 -δ ppm): 3.77 (s, 3H,
OCH3 ), 6.59–8.88 (m, 13H, Ar-H + CH-isoxazole), 9.18 (s, 1H, CH-pyrazole); 13 C-NMR (DMSO-d6 -δ
ppm): 55.3, 113.7, 114.1, 114.2, 116.6, 118.6, 123.4, 126.5, 127.5, 128.6, 129.5, 129.7, 130.8, 131.3, 132.1,
134.1, 140.2, 151.5, 159.8, 164.3, 168.8; MS (EI, 70 eV): m/z (%): 509 (0.1, M+ + 4), 507 (0.2, M+ + 2), 505
(0.1, M+ ), 327 (100); Anal. Calcd. for C25 H17 BrClN3 O2 (506.78): C, 59.25; H, 3.38; N, 8.29; Found: C,
59.48; H, 3.27; N, 8.17.
4-(3-(4-Methoxyphenyl)isoxazol-5-yl)-1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazole (5b). Yield 80%;
m.p. 227–229 ◦ C; 1 H-NMR (DMSO-d6 -δ ppm): 3.72, 3.78 (2s, 6H, 2OCH3 ), 7.00–8.87 (m, 13H,
Ar-H + CH-isoxazole ring), 9.18 (s, 1H, CH-pyrazole); MS (EI, 70 eV): m/z (%): 461 (1.5, M+ + 4),
Molecules 2017, 22, 512 11 of 16

459 (4.8, M+ + H2 ), 327 (100); Anal. Calcd. for C26 H20 ClN3 O3 (457.91): C, 68.20; H, 4.40; N, 9.18; Found:
C, 68.41; H, 4.62; N, 9.23.

3.1.5. 6-(4-Substitutedphenyl)-4-(1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-1,2-
dihydro-2-Oxopyridine-3-carbonitrile (6a,b) and 6-(4-substitutedphenyl)-4-(1-(3-chlorophenyl)-3-
(4-methoxyphenyl)-1H-pyrazol-4-yl)-1,2-dihydro-2-iminopyridine-3-carbonitrile (7a,b)
A mixture of compounds 2a,b (0.01 mol), ethyl cyanoacetate or malononitrile (0.01 mol), and
ammonium acetate (6 g, 0.08 mol) was refluxed in ethanol (30 mL) for 6 h. The formed precipitate was
collected by filtration, washed several times with water, dried and recrystallized from ethanol to afford
the title compounds 6a,b and 7a,b, respectively.
6-(4-Bromophenyl)-4-(1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-1,2-dihydro-2-oxopyridine-
3-carbonitrile (6a). Yield 75%; m.p. 217–219 ◦ C; IR (KBr, cm−1 ) ν: 3335 (NH), 2192 (C≡N), 1687 (C=O),
1587 (C=C); 1 H-NMR (DMSO-d6 -δ ppm): 3.77 (s, 3H, OCH3 ), 6.71 (s, 1H, CH-pyridine), 6.96–8.05 (m,
13H, Ar-H + NH exchangeable with D2 O), 9.07 (s, 1H, CH of pyrazole); 13 C-NMR (DMSO-d6 -δ ppm):
55.6, 114.7, 116.6, 117.5, 117.8, 118.7, 124.6, 125.3, 127.1, 129.4, 130.0, 130.8, 131.9, 132.4, 134.6, 140.5,
150.9, 152.3, 160.0, 162.9, 167.3; MS (EI, 70 eV): m/z (%): 558 (3.7, M+ + 2), 557 (1.7, M+ + 1), 91 (100);
Anal. Calcd. for C28 H18 BrClN4 O2 (557.83): C, 60.29; H, 3.25; N, 10.04; Found: C, 60.41; H, 3.32; N, 10.23.
6-(4-Methoxyphenyl)-4-(1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-1,2-dihydro-2-oxo-pyridine-
3-carbonitrile (6b). Yield 76%; m.p. 198–200 ◦ C; IR (KBr, cm−1 ) ν: 3417 (NH), 2216 (C≡N), 1726 (C=O),
1654 (C=N), 1597 (C=C); 1 H-NMR (DMSO-d6 -δ ppm): 3.35 (s, 1H, NH exchangeable with D2 O), 3.77,
3.81 (2s, 6H, 2OCH3 ), 6.60 (s, 1H, CH-pyridine), 6.97–9.00 (m, 12H, Ar-H), 9.23 (s, 1H, CH of pyrazole);
13 C-NMR (DMSO-d -δ ppm): 55.3, 55.5, 100.2, 114.1, 114.1, 114.5, 115.7, 115.8, 116.2, 118.2, 122.6, 124.1,
6
126.6, 127.7, 129.0, 130.0, 130.3, 134.1, 140.0, 150.5, 155.2, 159.5, 160.3, 161.7, 161.9; MS (EI, 70 eV): m/z
(%): 510 (4, M+ + 2), 508 (2, M+ ), 303 (100); Anal. Calcd. for C29 H21 ClN4 O3 (508.96): C, 68.44; H, 4.16;
N, 11.01; Found: C, 68.41; H, 4.22; N, 10.88.
6-(4-Bromophenyl)-4-(1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-1,2-dihydro-2-imino-pyridine-
3-carbonitrile (7a). Yield 73%; m.p. 172–174 ◦ C; IR (KBr, cm−1 ) ν: 3335, 3195 (2NH), 2192 (C≡N), 1587
(C=C); 1 H-NMR (DMSO-d6 -δ ppm): 3.59 (s, 1H, NH exchangeable with D2 O), 3.78 (s, 3H, OCH3 ),
6.52 (s, 1H, CH-pyridine), 6.93–8.04 (m, 13H, Ar-H + NH exchangeable with D2 O), 8.89 (s, 1H,
CH-pyrazole); 13 C-NMR (DMSO-d6 -δ ppm): 55.6, 113.0, 114.1, 114.6, 115.5, 116.8, 118.3, 118.6, 123.1,
125.4, 126.7, 128.4, 129.3, 130.7, 131.2, 131.9, 132.1, 134.6, 140.8, 151.4, 159.8, 160.5, 164.2, 169.0; MS
(EI, 70 eV): m/z (%): 507 (2.8, M+ + 2), 555 (7, M+ ), 149 (100); Anal. Calcd. for C28 H19 BrClN5 O (556.84):
C, 60.39; H, 3.44; N, 12.58; Found: C, 60.48; H, 3.27; N, 12.17.
6-(4-Methoxyphenyl)-4-(1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-1,2-dihydro-2-iminopyridine-
3-carbonitrile (7b). Yield 70%; m.p. 246–248 ◦ C; IR (KBr, cm−1 ) ν: 3405, 3334 (2NH), 2198 (C≡N),
1573 (C=C); 1 H-NMR (DMSO-d6 -δ ppm): 3.26 (s, 1H, NH exchangeable with D2 O), 3.74, 3.79 (2s, 6H,
2 OCH3 ), 6.92–8.05 (m, 13H, Ar-H + CH-pyridine), 7.24 (s, 1H, NH exchangeable with D2 O), 8.88
(s, 1H, CH-pyrazole); 13 C-NMR (DMSO-d6 -δ ppm): 55.1, 55.3, 109.5, 113.9, 114.0, 114.6, 115.1, 116.4,
117.8, 118.8, 122.5, 124.5, 126.1, 128.4, 129.6, 130.0, 131.4, 134.1, 140.3, 150.0, 159.3, 160.8, 161.0, 161.5,
169.3; MS (EI, 70 eV): m/z (%): 509 (10, M+ + 2), 507 (14, M+ ), 55 (100); Anal. Calcd. for C29 H22 ClN5 O2
(507.97): C, 68.57; H, 4.37; N, 13.79; Found: C, 68.41; H, 4.42; N, 13.43.

3.1.6. 4-(4-Substitutedphenyl)-6-(1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)
pyrimidin-2-amine (8a,b)
An aqueous solution of sodium hydroxide (40%, 5 mL) was added portion wise during a period
of 3 h to a mixture of 1-substituted prop-2-en-1-ones 2a or 2b (0.01 mol) and guanidine sulphate (1.6 g,
0.01 mol) in ethanol (25 mL). After refluxing for 5–7 h, the solid product formed upon pouring onto
ice/water containing a few drops of hydrochloric acid (until pH ~6) was collected by filtration, washed
with water, then recrystallized from methanol.
Molecules 2017, 22, 512 12 of 16

4-(4-Bromophenyl)-6-(1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)pyrimidin-2-amine (8a). Yield

69%, m.p. 163–165 ◦ C; IR (KBr, cm−1 ) ν: 3406 (NH2 ), 1581 (C=C); 1 H-NMR (DMSO-d6 -δ ppm): 3.80
(s, 3H, OCH3 ), 6.58 (s, 2H, NH2 exchangeable with D2 O), 6.97–8.05 (m, 13H, Ar-H + CH-pyridine), 9.08
(s, 1H, CH-pyrazole); 13 C-NMR (DMSO-d6 -δ ppm): 55.1, 104.1, 114.0, 114.0, 116.1, 118.1, 124.0, 125.5,
126.3, 128.6, 129.9, 130.8, 131.2, 131.7, 133.9, 134.1, 140.7, 145.5, 159.4, 161.0, 162.7, 163.7; MS (EI, 70 eV):
m/z (%): 535 (1.3, M+ + 4), 533 (7.6, M+ + 2), 531 (9, M+ ), 111 (100); Anal. Calcd. for C26 H19 BrClN5 O
(532.82): C, 58.61; H, 3.59; N, 13.14; Found: C, 58.48; H, 3.27; N, 13.17.
4-(4-Methoxyphenyl)-6-(1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)pyrimidin-2-amine (8b).
Yield 71%, m.p. 224–226 ◦ C; IR (KBr, cm−1 ) ν: 3324 (NH2 ), 1577 (C=C); 1 H-NMR (DMSO-d6 -δ ppm):
3.78, 3.81 (2s, 6H, 2 OCH3 ), 6.97 (s, 2H, NH2 exchangeable with D2 O), 7.01–8.56 (m, 13H,
Ar-H + CH-pyridine), 9.06 (s, 1H, CH-pyrazole; 13 C-NMR (DMSO-d6 -δ ppm): 55.3, 55.5, 103.6, 113.3,
114.0, 114.0, 115.8, 118.1, 125.2, 125.5, 126.6, 128.1, 128.9, 130.2, 131.2, 134.1, 140.7, 150.8, 160.4, 161.2,
163.5, 163.7; MS (EI, 70 eV): m/z (%): 485 (0.01, M+ + 2), 483 (0.02, M+ ), 135 (100); Anal. Calcd. for
C27 H22 ClN5 O2 (483.95): C, 67.01; H, 4.58; N, 14.47; Found: C, 67.41; H, 4.62; N, 14.23.

3.1.7. 4-(4-Substitutedphenyl)-6-(1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)
pyrimidine-2-(1H)-thione (9a,b)
A mixture of 1-substituted prop-2-en-1-ones 2a or 2b (0.01 mol) and thiourea (0.76 g, 0.01 mol) in
ethanol (30 mL) containing (1 g, 0.025 mol) sodium hydroxide was refluxed 6–8 h. The solid product
formed upon pouring onto ice/water containing a few drops of hydrochloric acid (until pH ~6) was
collected by filtration, washed with water, then recrystallized from methanol to yield the desired
compounds 9a,b.
4-(4-Bromophenyl)-6-(1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)pyrimidine-2-(1H)-thione (9a).
Yield 81%; m.p. 180–182 ◦ C; IR (KBr, cm−1 ) ν: 3384 (NH), 1591 (C=C), 1174 (C=S); 1 H-NMR
(DMSO-d6 -δ ppm): 3.77 (s, 3H, OCH3 ), 5.23 (s, 1H, NH exchangeable with D2 O), 7.00–7.85 (m, 13H,
Ar-H + CH-thiopyrimidine), 9.25 (s, 1H, CH-pyrazole); 13 C-NMR (DMSO-d6 -δ ppm): 55.2, 113.4, 114.0,
116.3, 117.1, 118.3, 122.0, 125.5, 126.8, 128.6, 129.2, 130.2, 131.1, 131.7, 134.0, 134.1, 140.6, 150.4, 160.8,
164.4, 176.1, 181.1; MS (EI, 70 eV): m/z (%); MS m/z (%): 535 (1.3, M+ + 4), 533 (7.6, M+ + 2), 531 (9,
M+ ), 155 (100); Anal. Calcd. for C26 H18 BrClN4 OS (549.87): C, 56.79; H, 3.30; N, 10.19; Found: C, 56.48;
H, 3.27; N, 10.17.
4-(4-Methoxyphenyl)-6-(1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)pyrimidine-2-(1)-thione
(9b). Yield 80%; m.p. 233–235 ◦ C; IR (KBr, cm−1 ) ν: 3384 (NH), 1591 (C=C), 1174 (C=S); 1 H-NMR
(DMSO-d6 -δ ppm): 3.78 (br s, 6H, 2 OCH3 ), 7.00–7.85 (m, 14H, Ar-H, CH-thiopyrimidine and NH
exchangeable with D2 O), 9.26 (s, 1H, CH-pyrazole); 13 C-NMR (DMSO-d6 -δ ppm): 55.2, 55.7, 101.9,
109.4, 114.0, 114.2, 115.0, 116.3, 117.9, 125.2, 125.8, 126.6, 127.4, 128.5, 130.0, 130.8, 135.1, 150.1, 159.7,
161.0, 165.1, 176.3, 180.3; MS (EI, 70 eV): m/z (%); MS m/z (%): 563 (0.7, M+ + 2), 561 (1.6, M+ ), 310 (100);
Anal. Calcd. for C27 H21 ClN4 O2 S (501): C, 64.73; H, 4.22; N, 11.18; Found: C, 64.41; H, 4.62; N, 11.23.

3.2. Measurement of Anti-Inflammatory Activity

Compounds 2–9 were screened for their in vivo anti-inflammatory activity by the
carrageenan-induced paw edema standard method [1]. Mature Swiss male albino rats were obtained
from The Animal House, NRC, Cairo, weighing 150–200 g. Edema was induced in the left hind paw
of all rats by subcutaneous injection of 0.1 mL of 1% (w/v) carrageenan in distilled water into their
footpads. Rats were divided into six groups of six rats each. The first group was kept as control, and
was given the respective volume of the solvent (1% of tween-80 in distilled water). The other groups
were orally administered the drugs—indomethacin and celebrex (reference standards) and the tested
compoundsafter dissolution in water and 1% tween 80 in dose of 0.28 mmol/kg, 1 h before carrageenan
injection. The paw volume of each rat was measured using Vernier caliper; before carrageenan injection
Molecules 2017, 22, 512 13 of 16

and then hourly for 4 h post-administration of the drugs. The edema rate and inhibition rate of each
group were calculated as follows:

Percentage change of Edema rate (E) % = [(Vt − Vo )/Vo] × 100

Inhibition rate (I)% = [(Ec − Et)/Ec] × 100

where: Vo is the volume before carrageenan injection (mL). Vt is the volume at t hour after carrageenan
injection (mL). Ec is the edema rate of control group. Et is the edema rate of the treated group.

3.3. Ulcerogenic Liability

After five hours of measuring the anti-inflammatory activity, the rats were sacrificed by
decapitation. Their stomachs were removed, opened along the greater culvature, and the number
of ulcers was assessed by the reported standard method [2]. The separate groups which received
indomethacin and Celebrex (0.28 mmol/kg) as positive controls were used. The results were compared
with tween-80 (1% solution) treated group as negative control.

3.4. Molecular Modeling Study

All the molecular modeling calculations and docking simulation studies were performed using
Molecular Operating Environment (MOE® ) [3] 2008.10. All the interaction energies and different
calculations were automatically calculated.

3.4.1. Optimization of the Target Compound 6b

The target compound 6b was constructed into a 3D model using the builder interface of
the MOE program. After checking their structures and the formal charges on atoms by 2D depiction,
the following steps were carried out: the target compound was subjected to a conformational search.
All conformers were subjected to energy minimization, all the minimizations were performed with
MOE until a RMSD gradient of 0.01 Kcal/mole and RMS distance of 0.1 Å with MMFF94X force-field
and the partial charges were automatically calculated. The obtained database was then saved as MDB
file to be used in the docking calculations.

3.4.2. Optimization of the Enzymes Active Site

The X-ray crystallographic structure of COX-2 receptor complexed with 1-phenylsulfona-
mide-3-trifluoromethyl-5-(4-bromophenyl)pyrazole, SC-558 (PDB ID: 1CX2) [4] was obtained from the
Protein Data Bank through the internet. The enzyme was prepared for docking studies by removing
the ligand molecule SC-558 from the COX-2 receptor active site. Hydrogen atoms were added to the
system with their standard geometry. The atoms connection and type were checked for any errors
with automatic correction. Selection of the receptor and its atom potential were fixed. MOE Alpha Site
Finder was used for the active site search in the enzyme structure using all default items. Dummy
atoms were created from the obtained alpha spheres. Re-docking of co-crystalline ligand to the receptor
active site to insure the docking method was efficient and the active pocket was saved as a MOE file to
be used for docking simulation of the selected compounds.

3.4.3. Docking of the Target Molecule 6b and Celecoxib to the Receptor Active Sites
Docking of the conformation database of the target compounds was done using MOE-Dock
software. The following methodology was generally applied via loading of the enzyme active site file
and the dock tool was initiated. The program specifications were adjusted to:

- Dummy atoms as the docking site.

- Triangle matcher as the placement methodology to be used.
- London dG as scoring methodology to be used and was adjusted to its default values.
Molecules 2017, 22, 512 14 of 16

The MDB file of the ligand to be docked was loaded and dock calculations were run automatically.
The obtained poses were studied and the poses showed best ligand-enzyme interactions were selected
and stored for energy calculations. The 2D interaction and stereo view for compound 6b inside the
active site of COX-2 kinase were obtained and saved as both MOE and photo files.

4. Conclusions
In summary, we have designed and synthesized a new series of 1,3,-diaryl pyrazole derivatives
linked different nitrogenous heterocyclic ring systems at C-4 position including pyrazoles, isoxazole,
pyridines, or pyrimidines and evaluated for their anti-inflammatory activity using standard acute
carrageenan-induced paw edema method. From the obtained results, six compounds (2a, 2b, 3a, 6b,
7b, and 9b) showed consistently excellent anti-inflammatory activity (84.39–89.57% inhibition) 4 h
after the carrageenan injection comparable to that of the standard drugs indomethacin and Celebrex
(72.99% and 83.76%, respectively). The cyanopyridone derivative 6b seems to be the most effective
product, displayed better activity (89.57% inhibition of edema) than both indomethacin and celecoxib
(reference standards), and could be considered a promising selective anti-inflammatory lead for further
development of more potent anticancer agents. The structures of the newly prepared compounds were
elucidated using spectroscopic and elemental analysis.

Acknowledgments: The project was financially supported by King Saud University, Vice Deanship of Research Chairs.
Author Contributions: The listed authors contributed to this work as described in the following. Eman S. Nossier,
carried out the synthetic work, interpreted the results, and prepared the manuscript; Hoda H. Fahmy gave the
concepts of the work, interpreted the results, and prepared the manuscript; Nagy M. Khalifa interpreted the
results and cooperated in the preparation of the manuscript; and Wafaa I. El-Eraky and Marawan A. Baset carried
and interpreted the results of the biological activities. All authors read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of all the compounds are available from the authors.

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