Design, synthesis, and biological evaluation of substituted hydrazone and pyrazole derivatives as selective COX-2 inhibitors: Molecular docking study

Article abstract of DOI:10.1016/j.bmc.2011.04.027

New arylhydrazone derivatives and a series of 1,5-diphenyl pyrazoles were designed and synthesized from 1-(4-chlorophenyl)-4,4,4-trifuorobutane-1,3-dione 1. The newly synthesized compounds were investigated in vivo for their anti-inflammatory activities using carrageenan-induced rat paw oedema model. Moreover, they were tested for their inhibitory activity against ovine COX-1 and COX-2 using an in vitro cyclooxygenase (COX) inhibition assay. Some of the new compounds (2f, 6a and 6d) showed a reasonable in vitro COX-2 inhibitory activity, with IC₅₀ value of 0.45 μM and selectivity index of 111.1. A virtual screening was carried out through docking the designed compounds into the COX-2 binding site to predict if these compounds have analogous binding mode to the COX-2 inhibitors. Docking study of the synthesized compounds 2f, 6a and 6d into the active site of COX-2 revealed a similar binding mode to SC-558, a selective COX-2 inhibitor.

Full text of DOI:10.1016/j.bmc.2011.04.027

Bioorganic & Medicinal Chemistry 19 (2011) 3416–3424
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and biological evaluation of substituted hydrazone and
pyrazole derivatives as selective COX-2 inhibitors: Molecular docking study
Magda A.-A. El-Sayed a, Naglaa I. Abdel-Aziz b, , Alaa A.-M. Abdel-Aziz b,c, Adel S. El-Azab c,d
,
⇑
Yousif A. Asiri e, Kamal E. H. ElTahir f
a Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt
b Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt
c Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
d Department of Organic Chemistry, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt
e Department of Clinical Pharmacy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
f Department of Pharmacology, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
New arylhydrazone derivatives and a series of 1,5-diphenyl pyrazoles were designed and synthesized
from 1-(4-chlorophenyl)-4,4,4-trifuorobutane-1,3-dione 1. The newly synthesized compounds were
investigated in vivo for their anti-inflammatory activities using carrageenan-induced rat paw oedema
model. Moreover, they were tested for their inhibitory activity against ovine COX-1 and COX-2 using
an in vitro cyclooxygenase (COX) inhibition assay. Some of the new compounds (2f, 6a and 6d) showed
Received 3 February 2011
Revised 12 April 2011
Accepted 13 April 2011
Available online 22 April 2011
a reasonable in vitro COX-2 inhibitory activity, with IC50 value of 0.45 lM and selectivity index of 111.1. A
Keywords:
virtual screening was carried out through docking the designed compounds into the COX-2 binding site
to predict if these compounds have analogous binding mode to the COX-2 inhibitors. Docking study of the
synthesized compounds 2f, 6a and 6d into the active site of COX-2 revealed a similar binding mode to SC-
558, a selective COX-2 inhibitor.
Hydrazone and pyrazole derivatives
Selective COX-2 inhibitors
Molecular docking
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Research efforts in the discovery of COX-2 selective agents have
produced many classes of compounds having desired selectivity.8–10
Cyclooxygenase is the key enzyme in the biosynthesis of prosta-
noids, biologically active substances that are involved in several
physiological processes but also in pathological conditions, such
as inflammation.1 It is actually well-known that this enzyme exists
under two forms: cyclooxygenase-1 (COX-1) and cyclooxygenase-2
(COX-2). COX-1 is a constitutive enzyme and is responsible for the
production of cytoprotective prostaglandins in the gastrointestinal
tract (GI) and proaggregatory thromboxane in blood platelets.
However, COX-2 is an inducible enzyme which is induced in re-
sponse to the release of several proinflammatory mediators.2–4
Both enzymes are sensitive to inhibition by conventional non-
steroidal anti-inflammatory drugs (NSAIDs). Observations that
COX-1 was involved in several homeostatic processes, while
COX-2 expression was associated with inflammation and other
pathologies, such as cancer proliferation, have led to the develop-
ment of COX-2 selective inhibitors to improve the therapeutic
potency and to reduce the classical side effects associated with
the use of conventional NSAIDs.5–7
A large number of compounds with common structural features, that
is, thepresenceoftwoarylringsonadjacentcarbonsofacyclic/acyclic
moiety, has been investigated for COX-2 inhibition. Among these
compounds, rofecoxib, celecoxib, valdecoxib and etoricoxib were ap-
proved and marketed. However, rofecoxib was banned in 2004 be-
cause of cardiac toxicity. Subsequently, some of the other coxibs
have been voluntarily withdrawn from the market.11 On the other
hand, some studies have suggested that rofecoxib’s adverse cardiac
events may not be a class effect but rather an intrinsic chemical prop-
erty related to its metabolism.12 For this reason novel scaffolds with
COX-2 selective inhibitory activity needed to be found and evaluated
for their anti-inflammatory effects. A number of molecules based
upon a monocyclic heterocyclic template have been investigated for
their COX-2 inhibitory activity.8–10 However, the recent success of
pyrazole COX-2 inhibitors has highlighted the importance of these
heterocycles in medicinal chemistry.8
Thus our main objective is to design novel heterocyclic com-
pounds as specific inhibitors of COX-2 in the hope that these mol-
ecules may be further explored as powerful and novel
non-ulcerogenic anti-inflammatory lead-candidates. Our strategy
is intended to obtain potent anti-inflammatory activity with selec-
⇑
Corresponding author. Tel./fax: +20 502247496.
E-mail address: naglaabdalaziz2005@yahoo.com (N.I. Abdel-Aziz).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.04.027

M. A.-A. El-Sayed et al. / Bioorg. Med. Chem. 19 (2011) 3416–3424
3417
NH2
S
O
COOH
H2N
S
O
O
O
N
N
N
CF3
N
F
COX-2 selectivity
CF3
H3C
Br
Celecoxib (C)
SC-558 (B)
Flurbiprofen (A)
CF3
N
N
N
N
F3C
NH
N
CF3
O
O
C
CF3
Cl
CH
Cl
C
CH
CF3
O
O
N
Cl
2f
6d
6a
Figure 1. Representative examples of nonselective (A), selective (B and C) COX-2 inhibitors and the designed arylhydrazones and pyrazoles COX-2 selective inhibitors (2–6).
tive inhibition of COX-2 using traditional medicinal chemistry
techniques motivated by the comparative modeling of a COX-1
and -2 complexed with flurbiprofen (A) and SC-558 (B) (Fig. 1) to-
gether with the available pharmacophore.13,14
In view of the previous rationale and in continuation of an
ongoing program aiming at finding new structure leads with po-
tential anti-inflammatory activities and selective COX-2 inhibi-
tion,14,15 new series of hydrazone and pyrazole derivatives (2–6)
have been synthesized and evaluated their in vitro COX-1/COX-2
inhibition, and in vivo assessment as anti-inflammatory activities
(Fig. 1).
It was reported that crystal structures of COX enzymes with car-
boxylic acid-containing NSAIDs shows that the inhibitors are posi-
tioned in a similar fashion with their carboxylates coordinates to
Arg120 and their aromatic functionality projecting into the cyclo-
oxygenase active site towards Tyr38513,16 and diarylheterocycles
inhibitors of COX-2 bind in the cyclooxygenase active site above
Arg120 and insert their sulfonamide or sulfone groups into a side
pocket bordered by Val523.17–19 Computer docking technique
plays an important role in the drug design as well as in the mech-
anistic study by placing a molecule into the binding site of the tar-
get macromolecule in a noncovalent fashion.20 MOE 2008.1021 as
flexible docking program enables us to predict favorable protein–
ligand complex structures with reasonable accuracy and speed.
The docking technique will undoubtedly continue to play an
important role in drug discovery.22 So, we docked the most active
COX-2 inhibitors into COX-2 (1CX2) active site in order to predict
their binding modes, their binding affinities and orientation of
these compounds at the active site of the COX-2 enzyme.
amines with compound 1 afforded the corresponding hydrazone
derivatives 2a–f. The latter compounds have the tautomeric rela-
tion with azo-hydrazo system. The structures of compounds 2
were established on the basis of their elemental analyses and spec-
tral data. For example, the IR spectra were characterized by the
presence of strong absorption bands at 3397–3345 cmꢀ1, 1683–
1633 cmꢀ1 and 1597–1536 cmꢀ1 representing (NH), (C@O) and
(–C@N–) groups, respectively. The diketone moieties in com-
pounds 2a–f were replaced with hydrazine hydrate in refluxing
ethanol affording the corresponding pyrazole derivatives 3a–f.
The spectral and microanalytical data for compounds 3a–f were
consistent with their chemical structures. For example, their IR
spectra were characterized by the disappearance of the absorption
bands of the carbonyl groups.
2.1.2. Synthesis of compounds 4–6 (Scheme 2)
1,5-Diphenyl-3-trifluoropyrazole derivatives containing various
substituents at 4-position were synthesized as outlined in
Scheme 2. Cyclocondensation of phenylhydrazine with trifluoro-
methyl-1,3-diketone 1 afforded the corresponding 1,5-diphenylpy-
razole derivatives
4 as the major product. The NH2 in
phenylhydrazine is more nucleophilic than NH and would react
preferentially with the more reactive carbonyl group (COCF3) lead-
ing to the production of 1,5-diphenylpyrazole regioisomer as a ma-
jor product specially if the reaction is carried out in acidic
medium.23 Moreover, Vilsmeier formylation of 1,5-diphenylpyraz-
ole derivative 4 provided the corresponding aldehyde 5 which
upon treatment with different ketones afforded the respective
chalcone derivatives 6. The structures of the isolated products 4–
6 were established on the basis of their elemental and spectral
analyses.
2. Results and discussion
2.1. Chemistry
2.2. Biological activity
The designed target compounds were obtained as outlined in
2.2.1. In vivo antiinflammatory studies
Schemes 1 and 2 starting with trifluoromethyl-1,3-diketone 1.
All the newly synthesized compounds and diclofenac sodium, as
a reference drug, were subjected to in vivo anti-inflammatory stud-
ies using the well-known rat carrageenan-induced foot paw oede-
ma model. The results of anti-inflammatory activities against
carrageenan-induced rat paw oedema and ED50 (mg/kg) of the
2.1.1. Synthesis of compounds 1–3 (Scheme 1)
Claisen condensation of p-chloroacetophenone with ethyl tri-
fluoroacetate provided trifluoromethyl-1,3-dicarbonyl adduct 1 in
a good yield. Coupling the diazonium salt of different aromatic

3418
M. A.-A. El-Sayed et al. / Bioorg. Med. Chem. 19 (2011) 3416–3424
R2
R2
R3
R1
R
R3
R1
R
Cl
Cl
N2
O
F
R
Cl
NH
O
Cl
N
O
N
F
N
F
O
EtOH
F
O
HO
F
1
F
F
F
F
2a-f
N
N
F
F
Cl
F
N
NH2NH2
EtOH
NH
R2
R= Cl, R1=R2=R3= H (2a and 3a)
R2= Cl, R=R1=R3= H (2b and 3b)
R2= Br, R=R1=R3= H (2c and 3c)
R
R3
R1
R2= NO2, R=R1=R3= H (2d and 3d)
R2= OCH3, R=R1=R3= H (2e and 3e)
R=R2= H, R1=R3= CF3 (2f and 3f)
3a-f
Scheme 1. Synthesis of the designed arylhydrazone and pyrazole derivatives.
Cl
H
N
NH2
N
N
O
F
F
F
O
Cl
F
F
EtOH/ H2SO4
F
1
4
POCl3
DMF
N
N
F
F
O
N
N
Cl
N
N
F
F
F
F
F
EtOH/ NaOH
Cyclic ketone
R
Cl
F
Cl
CH
F
O
CHO
EtOH/ NaOH
O
5
R
R= H (6e)
R= Cl (6f)
n
n= 0 (6a)
n= 1 (6b)
n= NCH3 (6c)
n= NC2H5 (6d)
Scheme 2. Synthesis of the designed pyrazoles.
tested compounds were shown in (Table 1). All the tested com-
pounds showed reasonable percentages of oedema reduction in
the range of 4.0–89.7%. The major exception proved to be com-
pound 3e being inactive at the used dose. Compounds 2f, 5, 6a
and 6d showed the highest anti-inflammatory activity among the
tested compounds.
From the structure–activity relationship (SAR) viewpoint, the
anti-inflammatory activity of the series 2a–f and 6a–d such as 1-
(4-chlorophenyl)-2-(2-(3,5-bis(trifluoromethyl)phenyl)hydrazone)-4,
4,4-trifuorobutane-1,3-dione (2f), 2-((5-(4-chlorophenyl)-1-phe-
nyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)methylene)cyclopentanone
(6a) and 3-((5-(4-chlorophenyl)-1-phenyl-3-(trifluoromethyl)-1H-
pyrazol-4-yl)methylene)-1-ethylpiperidin-4-one (6d) was found
to be the highest one compared with series 3a–b,d–f and 6e–f. How-
ever, series 3a–b,d–f and 6e–f showed the least inhibitory effect
(2.6–28.8%).
In series 2 arylhydrazone derivative of substituted p-nitro-
phenyl 2d was more potent anti-inflammatory compound than
chloro or methoxyphenyl derivative 2a–b and 2e (NO2 > Cl and
OCH3). The anti-inflammation effect of the p-chlorophenyl deriva-
tive 2b is less than that of its o-chlorophenyl derivative 2a. In addi-
tion, the introduction of CF3 moiety into the phenyl derivative of
hydrazone system 2f was found to be highly effective (88.4%) than
its chlorinated one 2a–b (35.2–41.7%). As for pyrazole series 6a–d,

M. A.-A. El-Sayed et al. / Bioorg. Med. Chem. 19 (2011) 3416–3424
3419
Table 1
Table 2
Data of the in vivo anti-inflammatory activity of the designed compounds against
carrageenan-induced rat paw oedema (IP)
Data of the in vitro COX-1/COX-2 enzyme inhibition assay of the designed compounds
Compound no.
IC50
(l
M)a
SId
e
Compound no.
% Inhibition of oedemaa
ED50
COX-1b
COX-2
After 1 hb
After 2 hb
mg/kg
mmol/kg
0.668
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
3f
5
6a
6b
6c
>50c
>50
>50
>50c
>50c
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
0.16
>50
0.90
0.91
0.91
0.90
2.95
0.45
45
40
3.00
45
45
45
0.55
0.45
45
54.9
54.9
54.9
54.9
16.9
111.1
1.1
1.3
16.7
1.1
1.1
1.1
90.9
111.1
1.1
2a
2b
47.1 (0.0)
26.7
41.7 (19.3)
35.2
260
—
2c
2d
2e
2f
35.0 (0.0)
35.8 (0.0)
27.6 (0.0)
80.0 (33.0)
38.4
33.8 (13.9)
57.3 (17.9)
42.2 (3.8)
88.4 (59.0)
15.1
280
170
240
112
—
0.646
0.425
0.624
0.228
3a
3b
8.1
9.1
—
3c
3d
3e
26.3 (17.5)
4.0
52.1 (31.3)
7.0
180
—
—
0.419
NAc
NAc
3f
5
6a
6b
40.8 (3.4)
75.0 (24.1)
80.7 (37.0)
33.7
28.8 (17.3)
59.1 (40.3)
82.0 (67.0)
23.9
—
190
118
—
0.542
0.274
10
0.45
45
45
2.5
5.0
111.1
1.1
1.1
0.06
166.7
6c
6.7
20.3
—
6d
6e
6f
Diclofenac
Celecoxib
6d
6e
6f
80.0 (41.0)
1.8
1.5
89.7 (64.0)
2.6
4.4
120
—
—
0.261
0.358
Diclofenacd
88.5
89.5
114
0.30
a
a
The results are expressed as means SEM (n = 4–6) following a 200 mg/kg IP of
IC50 value is the compound concentration required to produce 50% inhibition of
the test compound.
COX-1 or COX-2 for means of two determinations and deviation from the mean is
b
Values in parenthesis are determined at 100 mg/kg IP.
No activity was observed at the used dose.
Values are determined at 100 mg/kg IP.
<10% of the mean value.
c
b
No inhibition of COX-1 up to 50 lM and precipitation of compounds observed
d
beyond this concentration.
e
c
ED50 was the effective dose calculated after 2 h.
Compounds 2a, 2d and 2e showed 31% inhibition of COX-1 at 0.5
M.
Selectivity index (COX-1 IC50/COX-2 IC50).
lM and only
25% inhibition of COX-1 at 100
l
d
compounds 6a and 6d showed very good activity (82.0% and 89.7%
reduction, respectively) more than 6b and 6c (23.9% and 20.3%
reduction, respectively), while compound 6e and 6f had slight
activity (2.6% and 4.4% reduction, respectively). These results re-
vealed the importance of the cycloalkane moiety for the anti-
inflammatory activity of series 6.
ecoxb is a selective COX-2 inhibitor. The results showed that most
of the compounds showed potent inhibition against COX-2
(IC50 ffi 0.45–10.0
lM) compared to the inhibition for COX-1
(IC50 >50 M) as listed in (Table 2). Nearly 4 of the compounds
l
It should be noted that administration of compounds 3a, 3f and
5 produced maximum inhibition of inflammation one hour after
administration of carrageenan. At 2 h after carrageenan the per-
centage inhibition of inflammation was greatly reduced as it was
clearly observed with compound 5 which showed high anti-
inflammatory activity 1 h after carrageenan. This finding could be
explained by a rapid metabolism of this compound.
(2f, 5, 6a and 6d) were found to be potent and selective similar
to celecoxib against COX-2. The effects of substituents introduced
into the hydrazones and pyrazoles 2–6 were revealed to be direc-
tional, being dependent on the electronic nature of the substitu-
ents and the ring size of the cycloalkanone fragments, that is,
introduction of electron-withdrawal group at the phenyl group of
hydrazones 2a–d enhanced COX-2-inhibiting activity with
a
ED50 of the compounds, which were shown promising degree of
anti-inflammatory activity (33.8–89.7%), were studied. Com-
pounds 2d, 3c, 5, 6a and 6d exhibited better anti-inflammatory
activity with ED50 of 170, 180, 190, 118 and 120 mg/kg, respec-
tively, as compared to diclofenac (ED50 = 114 mg/kg). In the
carrageenan-induced rat paw oedema assay model, compound 2f
was the most potent anti-inflammatory agent (ED50 = 112 mg/kg)
within this group of compounds at 2 h postdrug administration.
COX-2-selectivity (SI = 54.9). Moreover introduction of electron-
donating such as methoxy as for compound 2e resulted in lowering
the COX-2-inhibiting activity (SI = 16.9), while introduction of a
CF3 group as compound 2f showed just the opposite effects, result-
ing in a COX-2-selective inhibitor (SI = 111.1). The activities of pyr-
azole derivatives 5–6 are shown in Table 2. Among them,
compounds 5, 6a and 6d are COX-2-selective inhibitors
(SI = 90.9–111.1). Interestingly the size of cycloalkanone fragment
on the pyrazole core plays critical roles in the COX-inhibiting activ-
ity of the tested compounds. The cyclopentanone analogue 6a
showed the most potent activity, being 100 times more active than
cyclohexanone analogue 6b COX-2- inhibiting activity and has
similar activity to N-ethylpiperidone analogue 6d. This result, as
well as the previously described effect of the substitution on phe-
nyl suggests the importance of electronic effects on hydrazone
derivatives and spatial structure around the pyrazole core.
2.2.2. In vitro cyclooxygenase (COX) inhibition assay
The second target of the biological activity tests was the study
of the ability of the tested compounds to inhibit ovine COX-1
and COX-2 using an enzyme immunoassay (EIA) kit. The efficacies
of the tested compounds were determined as the concentration
causing 50% enzyme inhibition (IC50) (Table 2). As far as COX-1
inhibitory properties are concerned, all the tested compounds
showed no inhibition of COX-1 up to 50
able in vitro COX-2 inhibitory activity was observed with com-
pounds 2f, 6a, 6d with IC50 0.45 M and for compound 5 with
IC50 0.55 M. The selectivity indices (COX-1/COX-2) were calcu-
lated and compared with that of the standard COX-2 selective
inhibitor, celecoxib.
lM. Moreover, a reason-
2.3. Molecular docking results
l
l
Insights into the differences between the binding sites of COX-
1and COX-2 obtained from X-ray crystal structure data,13,24 pro-
vided useful guidelines that facilitated the design of the selective
COX-2 inhibitors. For example, the COX-2 binding site possesses
an additional 2°-pocket, that is, absent in COX-1, which is highly
In the assay system, the IC50 values of celecoxib on COX-1 and
COX-2 were determined to be >50 and 0.30 lM, indicating that cel-

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M. A.-A. El-Sayed et al. / Bioorg. Med. Chem. 19 (2011) 3416–3424
relevant to the design of selective COX-2 inhibitors. This COX-2 2°-
pocket arises due to a conformational change at Tyr355, that is,
attributed to the presence of Ile523 in COX-1 relative to Val523
having a smaller side chain in COX-2.24 It has also been reported
that replacement of His513 in COX-1 by Arg513 in COX-2 plays a
key role with respect to the H-bond network in the COX-2 binding
site. Access of ligands to the 2°-pocket of COX-2 is controlled by
histidine (His90), glutamine (Gln192), and tyrosine (Tyr355).25
Interaction of Arg513 with the bound drug is a requirement for
time dependent inhibition of COX-2.26
The level of COX-2 inhibition and anti-inflammatory activities
of compounds 2f, 6a and 6d prompted us to perform molecular
docking studies to understand the ligand–protein interactions in
detail. All the calculations were performed using MOE 2008.10
software installed on 2.0G Core 2 Duo.21,27 The crystal structures
of COX-2 enzymes complexed with SC-558 [1CX2] were used for
the docking.13 The active site of the enzyme was defined to include
residues within a 10.0 Å radius to any of the inhibitor atoms. The
automated docking program of MOE 2008.10 was used to dock
compounds 2f, 6a and 6d on the active sites of COX-2 enzymes.
The most stable docking model was selected according to the best
scored conformation predicted by the MOE scoring function. The
complexes were energy-minimized with a MMFF94 force field28
till the gradient convergence 0.05 kcal/mol was reached. To
compare orientations of the ligands, we superimposed each
compound’s best pose which as obtained by regarding the S score
in MOE. With few exceptions, all the ligands showed similar orien-
tation in the COX-2 active site and the complex formed was stabi-
lized by formation of classical and non-classical hydrogen bonds
(Fig. 2).29,30 In general, it is observed that the p-chlorophenyl frag-
ment of the compounds fitted into the cavity formed by Leu531,
Tyr385, Trp387, Ala527 and Val349 while the other phenyl moiety
fitted into the other cavity formed by Phe518, Leu352, Ser353,
Tyr355, Val523 and Arg120. As Kurumbail et al.13 mentioned, these
features are common in non-selective and selective COX-2 inhibi-
tors such as flurbiprofen and SC-558. On the other hand, it is
known that the main difference between the two COX active sites
is the replacement of Ile523 in COX-1 by Val523, a less bulky ami-
no acid. This replacement creates an adjunct pocket in the COX-2
active site which may be responsible for selectivity. Our docking
study showed that compounds 2f, 6a and 6d were oriented so that
their 3,5-ditrifluoromethylphenyl fragment (2f) and phenyl moiety
(6a, 6d) fill this adjunct pocket. This may explain why the com-
pounds show significant selective activity against COX-2 enzyme.
Compound 2f produces a deep moving into the hydrophilic pocket
of COX-2 with which the 3,5-ditrifluoromethylphenyl and hydra-
zone fragments are able to reach the hydrophilic pocket and is in-
volved in strong hydrogen bonding with His90 (NH–F; 2.91 Å),
Arg120 (NH–C@O; 2.95 Å) and Tyr355 (OH–NH; 2.91 Å) and weak
hydrogen bonding with Tyr355 (OH–NH; 3.23 Å, OH–C@O; 3.47 Å).
Such interactions are almost essential for COX-2 inhibitory activity,
as exemplified by the binding interaction of SC-558, an analogue of
Figure 2. The orientation of 2f (color red) in COX-2 active pocket (upper left panel; SC-558 is shown as yellow). The orientation of 2f (upper right panel; color red), 6a (lower
left panel; color violet) and 6d (lower right panel; color cyan) in COX-2 active pocket (H bonds are shown as green).

M. A.-A. El-Sayed et al. / Bioorg. Med. Chem. 19 (2011) 3416–3424
3421
celecoxib cocrystallized in the COX-2 active site.13 Additionally this
complex of compound 2f was shown nonclassical hydrogen bonds
of type CH–F among Val523, Phe518 and 2f. In addition; such
interaction forces the p-clorophenyl fragment to adopt a specific
orientation to be involved in hydrophobic interaction with
Trp387, Tyr385, Val349 and Leu531. The lateral pocket of COX-2
would therefore be responsible for the COX-2 selectivity of 2f
and contributed to stabilize the ligand–enzyme complexes (Fig. 2).
The complex generated by docking studies of 2f with COX-2 and
superimposition with the structure of the selective inhibitor, SC-
558, co-crystallized with COX-2, illustrated in Figure 2, shows that
compound 2f can bind in the active site of this enzyme in
approximately similar fashion as the pyrazolic prototype
(SC-558). Comparison of the interactions performed by SC-558 in
the crystal and the docked structure of 2f with COX-2 (Fig. 2)
shows that the trifluorophenyl groups of 2f hydrogen bonded to
the amino acid residue His90, similarly to the sulfonyl moiety of
the pharmacophoric sulfonamide group pertaining to SC-558.
Moreover, an additional hydrogen bond was observed between
the carbonyl oxygen and Arg120 and Tyr355. Additionally, 3,5-
ditrifluoromethylphenyl system are positioned in the same region
as p-sulfonamido-phenyl ring of SC-558, while, the p-chlorophenyl
of compound 2f is close to the p-Br-phenyl ring of SC-558, in the
aromatic region of the active site lined by aromatic amino acid res-
idues such as Tyr385 and Trp387, among others. In short, the de-
scribed interactions are typical of selective inhibitors of COX-2,
confirming the molecular design of the reported class of anti-
inflammatory hydrazone and pyrazole derivatives.8–10 Similarly,
compounds 6a and 6d can bind in the active site of this enzyme
in approximately similar fashion SC-558 with an additional non-
classical hydrogen bond as described above.
On the other hand, compounds 2f and 6a as representative
examples were modeled into the active site of COX-1 enzyme to
understand the inactivity of such compounds toward COX-1 inhi-
bition (Table 3, Fig. 3). The crystal structures of COX-1 enzymes
complexed with flurbiprofen31 [1EQH] were used for the docking
using the same protocol described above. The two compounds
could dock into the active site of COX-1 successfully. The insuffi-
cient binding of both compounds can be explained in terms of
the occurrence of very weak hydrogen bonds in which the benzoyl
carbonyl group of compound 2f formed such weak bonds with
Arg120 (3.7 Å) and Tyr355 (3.3 Å) while CF3 group of compound
6a formed similar weak bonds with Ala527 (3.14 Å) and Gly526
(3.42 Å).
3. Conclusion
The present study describes the synthesis of new arylhydrazone
derivatives and a series of 1,5-diphenyl pyrazoles. The biological
tests showed that the new compounds with anti-inflammatory
activities were less active than diclofenac against COX-1. However,
compounds 2f, 5, 6a and 6d showed reasonable inhibitory profiles
against COX-2, indicating that they are selective inhibitors for
COX-2. Moreover, the study showed that compounds 2f, 5, 6a
and 6d were the most selective among the tested compounds with
selectivity index in the range of 90.9–111.1. The binding mode of
the tested compounds inside the COX-2 active site was predicted
using a docking technique. When the results of the inhibitory
activity and docking studies are considered together, it can be sug-
gested that: (i) the arylhydrazone moiety, which fill the adjunct
pocket in the COX-2 active site, make contribution to the selectiv-
ity; is a suitable scaffold for COX-2 enzyme; (ii) pyrazoles with
appropriate substitutions which can fill the adjunct pocket and
interact with the relatively polar residues such as Gln192 and
Arg513 may be useful to propose new molecules with enhanced
selectivity towards COX-2.
Table 3
Docking results of compounds 2f, 6a and 6d into the active sites of COX-1 and COX-2
Compound
no.
Against COX-1a
Against COX-2a
No. H-bondb
(strength)c
Eint. (kcal
No. H-bondb
(strength)c
Eint. (kcal
molꢀ1
)
molꢀ1
)
d
d
2f
2 (weak)
ꢀ9.6
7 (strong)
3 (weak)
4 (strong)
4 (strong)
ꢀ61.41
6a
6d
2 (weak)
—
ꢀ6.7
ꢀ51.06
ꢀ53.88
4. Experimental
4.1. Chemistry
—
a
b
c
Details of hydrogen bonding are explained in Figures 2 and 3.
Hydrogen bondings are refered to classical and nonclassical hydrogen bonds.
Strength of hydrogen bond refered to the length of such hydrogen bond
Melting points was determined using Fisher–Johns melting
point apparatus and are uncorrected. Infrared (IR) spectra were
recorded on Mattson 5000 FT-IR spectrometer. NMR spectra were
(>3.0 Å = weak and <3.0 Å = strong).
d
Interaction energy between the ligand and receptor.
Figure 3. The orientation of 2f (left panel; color red) and 6a (right panel; color violet) in COX-1 active pocket (H bonds are shown as green).

3422
M. A.-A. El-Sayed et al. / Bioorg. Med. Chem. 19 (2011) 3416–3424
recorded on FT-NMR spectrometer (200 MHz) Gemini Varian using
DMSO-d6 and on Ultra shield Bruker (500 MHz) using CDCl3 and
TMS was used as internal standard. Mass spectra were measured
on JEOL JMS-600H spectrometer. Elemental analysis was carried
out for C, H and N at the Microanalytical Center of Cairo University.
All reagents were purchased from the Aldrich Chemical Company.
The well-known compounds, 1-(4-chlorophenyl)-4,4,4-trifuorobu-
tane-1,3-dione 123 and 432,33 were prepared following the proce-
dures reported in the literature.
139.0, 159.3, 176.7, 191.2. Anal. Calcd for C17H12ClF3N2O3 (%): C,
53.07; H, 3.14; N, 7.28. Found: C, 53.40; H, 3.40; N, 7.65.
4.1.1.6. 1-(4-Chlorophenyl)-2-(2-(3,5-bis(trifluoromethyl)phe-
nyl)hydrazone)-4,4,4-trifuorobutane-1,3-dione (2f).
Yield,
95%; mp 44–46 °C; IR (KBr)
m
max/cmꢀ1 3350 (NH), 1683, 1670
(C@O), 1597,1537 (C@N). 1H NMR (CDCl3): d 14.7 (s, 1H, NH),
7.94–7.36 (m,7H, Ar-H). 13C NMR (CDCl3): d 115.9, 117.6, 122.9,
124.7, 126.3, 126.6, 126.9, 128.7, 129.2, 131.8, 133.8, 135.5,
138.3, 139.8, 177.1, 190.5. MS m/z (%); 490.00 (0.15, M+), 423.95
(0.81), 421.95 (2.46), 222.90 (0.30), 220.95 (0.89),139.00
(100.00),. Anal. Calcd for C18H8ClF9N2O2 (%): C, 44.06; H, 1.64; N,
5.71. Found: C, 44.41; H, 2.00; N, 6.02.
4.1.1. General method for synthesis of 1-(4-chlorophenyl)-2-(2-
arylhydrazone)-4,4,4-trifuorobutane-1,3-dione (2a–f)
(Scheme 1)
A cooled sodium nitrite solution (1.5 g in 10 mL of water) was
added dropwise during 10 min to an ice-cold solution of the appro-
priate aniline in concentrated hydrochloric acid (3 mL). The reac-
tion mixture was stirred for 30 min at the same temperature and
for 2 h at room temperature to give the corresponding diazonium
salt. The solution of diazonium salt was added dropwise with stir-
ring to an ice-cold mixture of trifluoromethyl-1,3-diketone 1 (2.5 g,
0.01 mol) in sodium acetate (4.19 g, 0.05 mol) and ethanol (50 mL)
over 15 min. The stirring was continued for 30 min and the solid
obtained was collected and crystallized from ethanol to give the
corresponding hydrazones 2a–f.
4.1.2. General method for synthesis of 3-(4-chlorophenyl)-4-(2-
arylhydrazono)-5-trifuoromethyl)-4H-pyrazoles ((3a–f)
(Scheme 1)
A mixture of the appropriate hydrazone 2a–f (0.01 mol) and
hydrazine hydrate (0.03 mol) was refluxed in ethanol (20 mL) for
4 h. The reaction mixture was allowed to cool, diluted with water
and the solid obtained was crystallized from ethanol to give 3a–f.
4.1.2.1. 3-(4-Chlorophenyl)-4-(2-(2-chlorophenyl)hydrazono)-
5-trifuoromethyl)-4H-pyrazole (3a).
Yield, 65%; mp 109–
110 °C; IR (KBr)
m
max/cmꢀ1 3297 (NH), 1599, 1547 (C@N). 1H
4.1.1.1. 1-(4-Chlorophenyl)-2-(2-(2-chlorophenyl)hydrazone)-
NMR (CDCl3): d 11.55 (s, 1H, NH), 7.40–7.14 (m, 8H, Ar-H). 13C
NMR (CDCl3): d 118.6, 120.7, 122.8, 124.8, 126.1, 126.9, 127.4,
128.3, 128.5, 129.6, 130.4, 131.1, 134.4. Anal. Calcd for
4,4,4-trifuorobutane-1,3-dione (2a).
Yield, 90%; mp 55–
56 °C; IR (KBr)
m
max/cmꢀ1 3278 (NH), 1675, 1635 (C@O), 1596,
1536 (C@N). 1H NMR (CDCl3): d 14.15 (s, 1H, NH), 7.78 (d, 1H,
J = 8.0 Hz), 7.48 (d, 2H, J = 8.5 Hz), 7.40–7.34 (4H, m), 7.15–7.12
(m, 1H). 13C NMR (CDCl3): d 117.3, 122.9, 127.4, 128.5, 128.7,
129.8, 129.9, 131.9, 135.7, 137.2, 138.6, 139.6, 177.1, 190.5. MS
m/z (%); 388.00 (58.99, M+), 111.00 (60.67), 75.00 (65.17), 63.00
(76.97), 50.00 (100.00). Anal. Calcd for C16H9Cl2F3N2O2 (%): C,
49.38; H, 2.33; N, 7.20. Found: C, 49.02; H, 2.51; N, 7.80.
C16H9Cl2F3N4 (%): C, 49.89; H, 2.36; N, 14.55. Found: C, 50.10; H,
2.70; N, 15.00.
4.1.2.2. 3-(4-Chlorophenyl)-4-(2-(4-chlorophenyl)hydrazono)-
5-trifuoromethyl)-4H-pyrazole (3b).
Yield, 55%; mp 124–
126 °C; 1H NMR (CDCl3): d 7.44–7.36 (m, 8H, Ar-H), 7.34 (s, 1H,
NH). 13C NMR (CDCl3): d 120.7, 122.8, 124.9, 126.9, 127.4, 129.6,
130.3, 130.8, 131.1, 134.5. MS m/z (%); 386.20 (26.47, M+), 265.20
(41.42), 250.20 (1.47), 147.10 (75.98), 95.05 (100.00). Anal. Calcd
for C16H9Cl2F3N4 (%): C, 49.89; H, 2.36; N, 14.55. Found: C, 50.11;
H, 2.40; N, 15.00.
4.1.1.2. 1-(4-Chlorophenyl)-2-(2-(4-chlorophenyl)hydrazone)-
4,4,4-trifuorobutane-1,3-dione (2b).
Yield, 85%; mp 59–
62 °C; IR (KBr)
m
max/cmꢀ1 3245 (NH), 1673, 1640 (C@O), 1590,
1540 (C@N). 1H NMR (DMSO-d6); d 11.90 (s, 1H, NH), 8.10–7.20
(m, 8H, Ar-H). Anal. Calcd for C16H9Cl2F3N2O2 (%): C, 49.38;
H,2.33; N, 7.20. Found: C, 49.70; H, 2.00; N,7.70.
4.1.2.3. 3-(4-Chlorophenyl)-4-(2-(4-bromophenyl)hydrazono)-
5-trifuoromethyl)-4H-pyrazole (3c).
Yield, 45%; mp 110–
111 °C; 1H NMR (CDCl3): d 11.99 (s, 1H, NH), 7.49–6.65 (m, 8H,
Ar-H). 13C NMR (CDCl3): d 101.4, 119.8, 121.9, 126.4, 126.9,
127.4, 129.5, 131.3, 135.5, 144.3. MS m/z (%); 430.00 (61.45, M+),
363.00 (68.07), 108.00 (63.25), 69.00 (100.00), 50.00 (82.53). Anal.
Calcd for C16H9BrClF3N4 (%): C, 44.73; H, 2.11; N, 13.04. Found: C,
45.00; H, 2.14; N, 13.50.
4.1.1.3. 1-(4-Chlorophenyl)-2-(2-(4-bromophenyl)hydrazone)-
4,4,4-trifuorobutane-1,3-dione(2c).
Yield, 85%; mp 90–
92 °C; IR (KBr)
m
max/cmꢀ1 3340 (NH), 1680, 1665 (C@O), 1592,
1545 (C@N). 1H NMR (DMSO-d6); d 11.60 (s, 1H, NH), 7.91–
7.40(m, 8H, Ar-H). Anal. Calcd for C16H9BrClF3N2O2 (%): C, 44.32;
H, 2.09; N, 6.46. Found: C, 44.80; H,1.80; N, 6.91.
4.1.2.4. 3-(4-Chlorophenyl)-4-(2-(4-nitrophenyl)hydrazono)-5-
4.1.1.4.
4,4,4-trifuorobutane-1,3-dione (2d).
110 °C; IR (KBr)
1-(4-Chlorophenyl)-2-(2-(4-nitrophenyl)hydrazone)-
Yield, 80%; mp 109–
max/cmꢀ1 3250 (NH), 1660, 1650 (C@O), 1580,
trifuoromethyl)-4H-pyrazole (3d).
Yield, 55%; mp 105–
107 °C; IR (KBr)
m
max/cmꢀ1 3311 (NH), 1597,1545 (C@N). 1H NMR
m
(DMSO-d6); d 13.00 (s, 1H, NH), 8.00–7.20 (m, 8H, Ar-H). MS m/z
(%); 395.10 (41.43, M+), 259.90 (43.59), 152.25 (4.99), 150.20
(27.94), 110.00 (100.00). Anal. Calcd for C16H9ClF3N5O2 (%): C,
48.56; H, 2.29; N, 17.70. Found: C, 48.66; H, 2.50; N, 17.15.
1570 (C@N). 1H NMR (DMSO-d6); d 11.95 (s, 1H, NH), 8.21–
7.40(m, 8H, Ar-H). MS m/z (%); 401.00 (33.10, M++2) 399.90
(28.78, M++1), 399.00 (100.00, M+), 262.85 (27.06), 249.00 (0.78),
113.00 (10.90). Anal. Calcd for C16H9ClF3N3O4 (%): C, 48.08; H,
2.27; N, 10.51. Found: C, 47.80; H, 1.91; N, 11.00.
4.1.2.5. 3-(4-Chlorophenyl)-4-(2-(4-methoxyphenyl)hydrazon-
o)-5-trifuoromethyl)-4H-pyrazole (3e).
Yield, 40%; mp 98–
4.1.1.5.
1-(4-Chlorophenyl)-2-(2-(4-methoxyphenyl)hydra-
Yield, 75%; mp
max/cmꢀ1 3397 (NH), 1677, 1633 (C@O),
100 °C; 1H NMR (CDCl3): d 12.55 (s, 1H, NH), 7.37–6.59 (m, 8H,
Ar-H), 3.26 (s, 3H, OCH3). 13C NMR (CDCl3): d 56.1, 101.2, 119.8,
120.7, 122.0, 122.8, 126.4, 126.8, 127.3, 129.4, 129.5, 130.5,
134.4, 135.4, 14.4. MS m/z (%); 382.05 (13.11, M++2), 380.00
(40.76, M+), 350.20 (46.44), 127.50 (18.26), 125.40 (51.91),
108.05 (100.00). Anal. Calcd for C17H12ClF3N4O (%): C, 53.63; H,
3.18; N, 14.71. Found: C, 53.90; H, 2.91; N, 15.00.
zone)-4,4,4-trifuorobutane-1,3-dione (2e).
50–52 °C; IR (KBr)
m
1594, 1536 (C@N). 1H NMR (CDCl3): d 14.40 (s, 1H, NH), 7.45 (d,
2H, J = 8.5 Hz), 7.39 (d, 2H, J = 8.0 Hz), 7.34 (d, 2H, J = 8.5 Hz),
6.92 (d, 2H, J = 9.0 Hz), 3.78 (s, 3H, OCH3). 13C NMR (CDCl3): d
55.6, 115.2, 118.7, 127.2, 128.4, 128.5, 129.7, 131.8, 134.0, 136.3,

M. A.-A. El-Sayed et al. / Bioorg. Med. Chem. 19 (2011) 3416–3424
3423
4.1.2.6. 3-(4-Chlorophenyl)-4-(2-(3,5-bis(trifluoromethyl)phe-
322.20 (0.71), 320.20 (2.20), 58.10 (100.00). Anal. Calcd for
C24H21ClF3N3O (%): C, 62.68; H, 4.60; N, 9.14. Found: C, 62.77; H,
4.80; N, 9.15.
nyl)hydrazono)-5-trifuoromethyl)-4H-pyrazole (3f).
Yield,
60%; mp 122–123 °C; 1H NMR (DMSO-d6); d 13.50 (s, 1H, NH),
7.84–7.26 (m, 7H, Ar-H). Anal. Calcd for C18H8ClF9N4 (%): C,
44.42; H, 1.66; N, 11.51. Found: C, 44.00; H, 2.00; N, 12.00.
4.1.5. General method for synthesis of 3-(5-(4-(chloro)phenyl)-
1-phenyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)-1-phenylprop-
2-en-1-one derivatives 6e,f (Scheme 2)
4.1.3. Method for synthesis of 5-(4-chlorophenyl)-1-phenyl-3-
(trifluoromethyl)-1H-pyrazole-4-carbaldehyde (5) (Scheme 2)
A solution of compound 4 (16.13 g, 0.05 mole) in dimethyl
formamide (26 mL) was heated at 90 °C, phosphorous oxychloride
(10 mL, 0.06 mole) was added dropwise with stirring and the stir-
ring was continued for 7 h. A solution of phosphorous oxychloride
(10 mL, 0.06 mole) in dimethyl formamide (26 mL) were added to
the reaction mixture at room temperature and the reaction mix-
ture was heated at 90 °C for 2 h. The reaction mixture was allowed
to cooled to 0 °C by cautious addition of ice water and neutralized
with sodium hydroxide (130 mL, 2 N). The reaction mixture was
extracted with ethyl acetate, the organic layer was washed with
water, brine, dried over anhydrous sodium sulphate, the solvent
was evaporated under reduced pressure and the residue was crys-
tallized from chloroform; yield, 40%; mp 55–58 °C; 1H NMR
(CDCl3): d 9.96 (s, 1H, CHO), 8.46 (s, 1H), 7.76 (d, 2H, J = 8.5 Hz),
7.71 (d, 2H, J = 8.0 Hz), 7.46–7.32 (m, 4H). 13C NMR (CDCl3): d
119.7, 122.5, 128.1, 128.9, 129.7, 129.8, 130.2, 123.0, 135.4,
138.9, 135.2, 184.4. Anal. Calcd for C17H10ClF3N2O (%): C, 58.22;
H, 2.87; N, 7.99. Found: C, 58.40; H, 3.00; N, 8.21.
A solution of sodium hydroxide (1.2 g, 0.03 mol) in ethanol/
water (34/46 mL) was added dropwise to a stirred solution of com-
pound 5 (24.55 g, 0.07 mol) and the appropriate ketone (1.1 mol)
in ethanol (50 mL). The reaction mixture was stirred at room tem-
perature for 6 h, the solvent was evaporated under reduced pres-
sure and the residue was crystallized from 2-propanol.
4.1.5.1.
3-(5-(4-Chlorophenyl)-1-phenyl-3-(trifluoromethyl)-
(6e). Yield,
1H-pyrazol-4-yl)-1-phenylprop-2-en-1-one
40%; mp 186–188 °C; 1H NMR (CDCl3): 8.26 (s, 1H), 7.88 (d, 2H,
J = 7.0 Hz), 7.77–7.74 (m, 1H), 7.70 (d, 2H, J = 8.0 Hz), 7.56 (d, 2H,
J = 8.5 Hz), 7.51–7.47 (m, 1H), 7.42–7.32 (m, 6H), 7.30–7.27 (m,
1H). 13C NMR (CDCl3): d 117.2, 118.3, 120.7, 125.8, 126.3, 127.0,
127.3, 127.6, 128.0, 128.6, 129.1, 129.8, 131.7, 133.8, 137.0,
138.3, 151.5, 183.3, 188.9. MS m/z (%); 452.35 (45.85, M+), 132.16
(41.35), 120.15 (12.18), 106.12 (64.87), 49.65 (100.00). Anal. Calcd
for C25H16ClF3N2O (%): C, 66.31; H; 3.56; N, 6.19. Found: C, 66.45;
H, 3.20; N, 6.60.
4.1.5.2. 1-(4-Chlorophenyl)-3-(5-(4-chlorophenyl)-1-phenyl-3-
4.1.4. General method for synthesis of 2 and/or 3-((5-(4-
chlorophenyl)-1-phenyl-3-(trifluoromethyl)-1H-pyrazol-4-
yl)methylene) derivatives (6a–d) (Scheme 2)
A solution of sodium hydroxide (1.20 g, 0.03 mol) in ethanol/
water (34/46 mL) was added dropwise to a stirred solution of com-
pound 5 (24.55 g, 0.07 mol) and the appropriate cyclic alkanone
(1.1 mol) in ethanol (50 mL). The reaction mixture was stirred at
room temperature for 6 h, the solvent was evaporated under re-
duced pressure and the residue was crystallized from 2-propanol.
(trifluoromethyl)-1H-pyrazol-4-yl))prop-2-en-1-one (6f).
Yield,
40%; mp 173–175 °C; 1H NMR (DMSO-d6); d 7.81–7.40 (m, 15H,
CH@CH, Ar-H). Anal. Calcd for C25H15Cl2F3N2O (%): C, 61.62; H,
3.10; N, 5.75. Found: C, 61.90; H, 3.20; N, 5.61.
4.2. Pharmacology
4.2.1. Materials and animals
Male Sprauge–Dawley rats weighing 250 g were purchased
from local source and kept at room temperature (22 2 °C) in a
light-controlled room with an alternating 12 h light/dark cycle.
They were fasted with free access to water at least 16 h prior to
experiments. The tested compounds were prepared as suspension
in vehicle (0.5% methyl cellulose) and diclofenac sodium was used
as a standard drug. The positive control group animals received the
reference drug while the negative control received only the vehicle.
An enzyme immunoassay (EIA) kit (catalog number 560101, Cay-
man Chemical, Ann Arbor, MI, USA) was purchased and used as
manufacturer’s instructions.
4.1.4.1.
2-((5-(4-Chlorophenyl)-1-phenyl-3-(trifluoromethyl)-
1H-pyrazol-4-yl)methylene) cyclopentanone (6a).
Yield,
75%; mp 189–190 °C; 1H NMR (DMSO-d6); d 8.20–7.20 (m, 10H,
CH@C, Ar-H), 3.00–2.80 (m, 4H, 2CH2), 2.20–2.00 (m, 2H, CH2),.
Anal. Calcd for C22H16ClF3N2O (%): C, 63.39; H, 3.87; N, 6.72. Found:
C, 63.80; H, 4.01; N, 7.00.
4.1.4.2.
2-((5-(4-Chlorophenyl)-1-phenyl-3-(trifluoromethyl)-
1H-pyrazol-4-yl)methylene) cyclohexanone (6b).
Yield,
50%; mp 183–185 °C; 1H NMR (DMSO-d6); d 8.20–7.20 (m, 10H,
CH@C, Ar-H), 2.90–2.80 (m, 4H, 2CH2), 1.81–1.75 (m, 4H, 2CH2),.
Anal. Calcd for C23H18ClF3N2O (%): C, 64.12; H, 4.21; N, 6.50. Found:
C, 64.50; H, 4.30; N, 6.10.
4.2.2. Antiinflammatory activity
The anti-inflammatory activity was evaluated using in vivo rat
carrageenan-induced foot paw oedema model reported previ-
ously.34 Oedema was produced by injecting 0.2 mL of a solution
of 1% k-carrageenan in the hind paw. The rats were injected intra-
peritoneally with 1 mL suspension in 0.5% methyl cellulose of the
tested compounds and reference drug. Paw volume was measured
by water displacement with a plethysmometer (UGO BASILE) be-
fore, 1 and 2 h after treatment. The percentage was calculated by
the following equation: anti-inflammatory activity (%) = (1 ꢀ D/
C) ꢀ 100, where D represents the difference in paw volume before
and after drug was administered to the rats, and C stands for the
difference of volume in the control groups.
4.1.4.3.
1H-pyrazol-4-yl)methylene)-1-methyl
(6c).
Yield, 70%; mp 200–201 °C; 1H NMR (DMSO-d6); d
3-((5-(4-Chlorophenyl)-1-phenyl-3-(trifluoromethyl)-
piperidin-4-one
8.11–7.40 (m, 10H, CH@C, Ar-H), 3.30 (s, 2H,CH2), 3.10–2.70 (m,
7H, 2CH2, NCH3). MS m/z (%); 445.30 (47.16, M+), 255.40 (39.05),
125.45 (15.85), 113.05 (0.37), 82.05 (100.00). Anal. Calcd for
C23H19ClF3N3O (%): C, 61.96; H, 4.30; N, 9.42. Found: C, 62.05; H,
4.51; N, 10.00.
4.1.4.4.
3-((5-(4-Chlorophenyl)-1-phenyl-3-(trifluoromethyl)-
1H-pyrazol-4-yl)methylene)-1-ethyl
(6d).
8.70–7.40 (m, 10H, CH@C, Ar-H), 3.80 (s, 2H, CH2), 2.85–2.62 (q,
2H, CH2CH3), 2.60–2.40 (m, 4H, 2CH2), 1.25–1.00 (t, 3H, CH2CH3),.
MS m/z (%); 460.30 (0.24, M++1), 459.30 (0.39, M+), 458.30 (0.54),
piperidin-4-one
4.2.3. In vitro cyclooxygenase (COX) inhibition assay
Yield, 45%; mp 210–212 °C; 1H NMR (DMSO-d6); d
The ability of the test compounds to inhibit ovine COX-1 and
COX-2 was determined using an enzyme immunoassay (EIA) (kit
catalog number 560101, Cayman Chemical, Ann Arbor, MI, USA)
according to the manufacturer’s instructions.35 Cyclooxygenase

3424
M. A.-A. El-Sayed et al. / Bioorg. Med. Chem. 19 (2011) 3416–3424
catalyzes the first step in the biosynthesis of arachidonic acid (AA)
References and notes
to PGH2. PGF2 , produced from PGH2 by reduction with stannous
a
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l
l
l
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l
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4.3. Molecular docking methodology
Docking studies have been performed using MOE 2008.10. With
this purpose, crystal structure of both COX-1/flurbiprofen (a nonse-
lective-inhibitor) and COX-2/SC-558 (a selective inhibitor) com-
plexes (PDB codes: 1EQH and 1CX2, respectively) were obtained
from the Protein Data Bank in order to prepare both proteins for
docking studies. Docking procedure was followed using the stan-
dard protocol implemented in MOE 2008.10 and the geometry of
resulting complexes was studied using the MOE’s Pose Viewer
utility.
Acknowledgments
The Authors extend their appreciation to the Deanship of Scien-
tific Research at King Saud University for funding the work through
the research group project No. RGP-VPP-163.