Celecoxib analogs bearing benzofuran moiety as cyclooxygenase-2 inhibitors: Design, synthesis and evaluation as potential anti-inflammatory agents

Article abstract of DOI:10.1016/j.ejmech.2014.02.033

Novel series of celecoxib analogs endowed with benzofuran moiety 3a-e and 9a-d were synthesized and evaluated for COX-1/COX-2 inhibitory activity in vitro. The most potent and selective COX-2 inhibitors - compounds 3c, 3d, 3e, 9c and 9d - were assessed fo

Full text of DOI:10.1016/j.ejmech.2014.02.033

European Journal of Medicinal Chemistry 76 (2014) 482e493
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Celecoxib analogs bearing benzofuran moiety as cyclooxygenase-2
inhibitors: Design, synthesis and evaluation as potential anti-
inflammatory agents
,
b
Ghaneya Sayed Hassan a, Sahar Mahmoud Abou-Seri a , Gehan Kamel ,
*
Mamdouh Moawad Ali c
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo 11562, Egypt
b Pharmacology Department, Faculty of Veterinary, Cairo University, Egypt
c Biochemistry Department, Division of Genetic Engineering and Biotechnology, National Research Centre, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel series of celecoxib analogs endowed with benzofuran moiety 3aee and 9aed were synthesized
and evaluated for COX-1/COX-2 inhibitory activity in vitro. The most potent and selective COX-2 in-
hibitors e compounds 3c, 3d, 3e, 9c and 9d e were assessed for their anti-inflammatory activity and
ulcerogenic liability in vivo. The 3-(pyridin-3-yl)pyrazole derivatives 3c and 3e exhibited the highest
anti-inflammatory activity, that is equipotent to celecoxib. Furthermore, the tested compounds proved to
have better gastric safety profile compared to celecoxib. In particular, compound 3e demonstrated about
40% reduction in ulcerogenic potential relative to the reference drug. Finally, molecular docking simu-
lation of the new compounds in COX-2 active site and drug likeness studies showed good agreement
with the obtained pharmaco-biological results.
Received 20 November 2013
Received in revised form
11 February 2014
Accepted 13 February 2014
Available online 14 February 2014
Keywords:
Celecoxib analogs
Benzofuran
Ó 2014 Elsevier Masson SAS. All rights reserved.
Pyrazole
Anti-inflammatory agents
COX-1/COX-2 inhibitory activity
1. Introduction
atherosclerosis [8]. Furthermore, platelet COX-1 is the target for
novel antithrombotic agents, which affect platelet aggregation
Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used
to alleviate inflammation and pains associated with many patho-
logical conditions and are often the initial therapy for common
inflammation. NSAIDs inhibit the biosynthesis of prostaglandins
(PGs). In general, biosynthesis involves the conversion of arach-
idonic acid to prostaglandin H2 (PGH2), a reaction catalyzed by the
sequential action of cyclooxygenases (COXs) [1]. Currently, it is well
established that there are at least two distinct COX isoforms [1e3].
The constitutively expressed COX-1 isoform is produced in most
tissues and appears to be important for the maintenance of physi-
ological functions such as synthesis of cytoprotective PGs in the
gastrointestinal tract, normal renal function, vascular homeostasis
and the biosynthesis of pro-aggregatory TXA2 in blood platelets [4e
7]. However, it is now believed that COX-1 is involved in a variety of
pathologies that comprise many health problems as neuro-
inflammation, tumorgenesis, endothelial dysfunction and
through suppression of COX-1 dependant platelet-derived TXA2 [9].
Another isoform, COX-2 is rapidly induced in inflammatory cells
in response to mitogenic and proinflammatory stimuli. COX-2
makes a significant contribution to the production of inflamma-
tory PGs, and the inhibition of COX-2 attenuates the expression of
inflammatory mediators such as TNF-
a
, iNOS and IL-1
b
[10,11] and
reduces the activation of transcriptional factor NF-kB which plays a
crucial role in inflammation [12].
Traditional nonselective NSAIDs inhibit both COX enzymes and
hence down regulate PGs formation in almost all cells and tissues.
This broad inhibitory profile accounts for their anti-inflammatory
activity as well as their pronounced side effects [4e7]. It was pro-
posed that a selective inhibitor of COX-2 would be an attractive
approach for the treatment of inflammatory conditions, without
concomitant gastric and renal toxicity. Hence, a number of selective
COX-2 inhibitors such as celecoxib, rofecoxib and valdecoxib (cox-
ibs) have been developed and were approved for clinical use by
virtue of their fewer gastrointestinal side effects compared to
traditional NSAIDs. However, the long-term use of both traditional
NSAIDs and coxibs has been reported to cause significant
* Corresponding author.
E-mail address: saharshaarawy_69@yahoo.com (S.M. Abou-Seri).
http://dx.doi.org/10.1016/j.ejmech.2014.02.033
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

G.S. Hassan et al. / European Journal of Medicinal Chemistry 76 (2014) 482e493
483
cardiovascular side effects [13e15]. Celecoxib has advantages in
this respect; because it is not associated with an increased inci-
dence of cardiovascular events compared with placebo and with
nonselective NSAIDs [16]. There are, however, some characteristics
of celecoxib that could be improved. For example, celecoxib is not
effective in all patients and has some gastrointestinal side effects
[17,18]. Therefore, development of new effective celecoxib analogs
with improved gastrointestinal safety profile is still a necessity.
Celecoxib belongs to the 1,5-diaryl pyrazole class of COX-2 in-
hibitors (Fig. 1). Extensive SAR studies of such class of compounds
indicated that the phenyl sulfonamide moiety of the inhibitors was
a major determinant for COX-2 selectivity and in vivo efficacy [19e
21]. Keeping the phenyl sulfonamide moiety, two series of new
celecoxib analogs were designed in which the central pyrazole ring
was substituted with functionalized benzofuran moiety either at 5-
position 3aee or 3-position 9aed (Fig. 1). This bulky bicyclic ring
system could maximize interaction with hydrophobic residues
within COX-2 active site and enhance COX-2 selectivity, as the new
molecules will be too large to fit into the smaller COX-1 active site
[22]. Moreover, preliminary molecular docking studies showed that
the methoxy and hydroxyl substituents on benzofuran were
capable of being engaged in H-bonding with essential amino acids
in COX-2 active site and increased the binding affinities of the target
molecules (Fig. 1). In addition, benzofuran derivatives were re-
ported to possess anti-inflammatory activity [23e25] and exhibited
promising gastro-protective effect [26e28]. Hence, the
incorporation of benzofuran moiety in the designed compounds
may yield new effective anti-inflammatory agents with higher
safety profile compared to celecoxib.
Herein, we report the synthesis of celecoxib analogs endowed
with benzofuran moiety as selective COX-2 inhibitors. The synthe-
sized compounds were evaluated for their in vitro COX-1/COX-2
inhibitory properties as well as their in vivo anti-inflammatory
activity and ulcerogenic liability. Furthermore, docking simulation
and drug likeness studies were applied to identify the structural
features required for COX-2 inhibitory properties of the new de-
rivatives and to rationalize the obtained pharmaco-biological results.
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of 4-[3-substituted-5-(6-hydroxy-4-
methoxybenzofuran-5-yl)-1H-pyrazol-1-yl)]benzene sulfonamide
derivatives 3aec and their 5-(6-hydroxy-4,7-
dimethoxybenzofuran-5-yl) analogs 3d,e (Scheme 1)
The 1,3-diketones 2aee were synthesized through Claisen
condensation of 1-(6-hydroxy-4-methoxybenzofuran-5-yl)etha-
none 1a, or its 7-methoxy analog 1b with ethyl acetate, ethyl tri-
fluoroacetate or ethyl nicotinate in presence of sodium metal by
applying two methods A and B. In method A, the acetophenones 1a,
b were refluxed with excess ethyl ester for 2 h, while in method B
Fig. 1. Representative examples of selective COX-2 inhibitors and the designed pyrazoles 3aee and 9aed.
Scheme 1. Reagents and reaction conditions: i) RCOOC2H5, Na metal powder, reflux 2 h ii) H2NHNC6H4SO2NH2.HCl, 6 N HCl, ethanol, reflux 8 h.

484
G.S. Hassan et al. / European Journal of Medicinal Chemistry 76 (2014) 482e493
was expected with 3-aryl substituent [21]. Since, it was reported
[29] that 2-hydroxyphenyl chalcones underwent oxidative cycli-
zation to aurones (2-benzylidene-1-benzofuran-3-one derivatives),
accordingly, the oxidation of 4a,b with hydrogen peroxide in
alkaline medium into the corresponding 2-benzylidene furano[3,2-
f]benzofuran-3(2H)-one 5a, b instead of the a,b-epoxy ketones was
expected (Scheme 2). Subsequently, the 1,3,5-triaryl pyrazoles 9ae
d were synthesized using the dimethoxy chalcone derivatives 7ae
d as outlined in Scheme 3. Methylation of 1a with methyl iodide in
the presence of potassium carbonate in dry acetone gave 1-(4,6-
dimethoxybenzofuran-5-yl)ethanone 6. The chalcones 7aed were
prepared by conventional ClaiseneSchmidt condensation reaction
of 6 with different aldehydes in the presence of 30% sodium hy-
droxide in ethanol. Treatment of
d with 30% hydrogen peroxide under basic conditions yielded the
-epoxy ketone intermediates 8aed via WeitzeScheffer oxida-
a,b-unsaturated ketones 7ae
a,b
tion reaction [30,31]. Finally, reacting 8aed with p-sulfamoylphe-
nylhydrazine hydrochloride in boiling ethanol acidified with acetic
Scheme 2. : Reagents and reaction conditions: i) AreCHO, 30% NaOH, ethanol, 48 h ii)
30% H2O2, 4 N NaOH, methanol/acetone, 24 h.
acid,
led
to
the
formation
of
4-[5-aryl-3-(4,6-
dimethoxybenzofuran-5-yl)-1H-pyrazol-1-yl]benzenesulfonamide
derivatives 9aed.
equimolecular amounts of the reactants were heated in ethanol for
10 h. It is worthy note that higher yield and more pure compounds
were obtained in method B. Cyclocondensation of p-sulfamoyl-
phenyl hydrazine hydrochloride with 1,3-diketones 2aee in pres-
ence of hydrochloric acid and refluxing ethanol afforded the
corresponding 1,5-diarylpyrazole derivatives 3aee. This reaction is
regioselective and the 1,5-diarylpyrazoles could be generated
almost exclusively by carrying out the condensation in acidic me-
dium [21].
2.2. Pharmacobiological activity
2.2.1. In vitro cyclooxygenase (COX) inhibition assay
The target of the biological activity tests was to study the ability
of tested compounds to inhibit ovine COX-1 and human recombi-
nant COX-2 using an enzyme immunoassay (EIA) kit. The efficacies
of tested compounds were determined as the concentration
causing 50% enzyme inhibition (IC50); Table 1. All the tested com-
pounds showed no inhibition of COX-1 up to 50
potent COX-2 inhibitory activity was observed with compounds 3c,
3d, 3e, 9c and 9d with IC50 0.40, 0.52, 0.36, 0.46, 0.34 M respec-
tively. Moreover, the selectivity indices (SI values) e defined as IC50
(COX-1)/IC50 (COX-2) e were calculated and compared with that of
the standard selective COX-2 inhibitor; celecoxib. In the assay
system, the IC50 values of celecoxib on COX-1 and COX-2 were
mM. Meanwhile,
2.1.2. Synthesis of 4-[5-Aryl-3-(4,6-dimethoxybenzofuran-5-yl)-
1H-pyrazol-1-yl]benzene sulfonamide derivatives 9aed (Schemes 2
and 3)
The synthetic pathway of such group of compounds should
proceed through the a,b-epoxy ketone intermediates, initially ob-
m
tained by oxidation of the respective chalcones 4a,b as illustrated in
Scheme 2, this method was utilized when little regiochemical bias
determined to be >50 and 0.28 mM, indicating that celecoxib is a
Scheme 3. Reagents and reaction conditions: i) CH3I, anhy. K2CO3, dry acetone, reflux 24 h, ii) AreCHO, 30% NaOH, ethanol, 48 h, iii) 30% H2O2, 4 N NaOH, methanol/acetone, 24 h,
iv) H2NHNC6H4SO2NH2.HCl, ethanol, acetic acid, reflux 6 h.

G.S. Hassan et al. / European Journal of Medicinal Chemistry 76 (2014) 482e493
485
Table 1
formalin-induced foot paw edema method in rats using celecoxib
as a reference drug [32]. Mean changes in paw edema thickness
after 1/2, 1, 2 and 3 h from induction of inflammation and per-
centages of edema inhibition given by the tested compounds and
celecoxib at 50 mg/kg body weight dose level are depicted in
Table 2. All the tested compounds were found to possess potent
anti-inflammatory activity (83.6e100% reduction in inflammation
after 3 h). The 3-(pyridin-3-yl)pyrazole derivatives 3c and 3e
showed the highest in vivo activity with edema inhibition % ¼ 90
after 1/2 h to 100 after 3 h, that was equipotent to the reference
drug celecoxib (edema inhibition % ¼ 94 after 1/2 h to 100 after 3 h).
Moreover, compounds 3d, 9c and the most potent COX-2 inhibitor
9d exhibited impressive anti-inflammatory effect (edema inhibi-
tion % ¼ 91.8, 83.61 and 91.8 after 3 h, respectively). The higher
in vivo potency of pyridine containing derivatives 3c and 3e
compared to compounds 3d, 9c and 9d reflects their better phar-
macokinetic properties and higher systemic bioavailability. This
might be partly due to their enhanced aqueous solubility and in
consequence, increased rate of dissolution as a perquisite param-
eter for absorption through different biological membranes.
Data of the in vitro COX-1/COX-2 enzyme inhibition assay of the designed
compounds.
Compound No.
IC50
(
m
M)a
SIb
COX-1
COX-2
3a
3b
3c
3d
3e
9a
9b
9c
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
7.50
7
0.40
0.52
0.36
35
>6.67
>7.14
>125
>96.15
>138.90
>1.43
26
>1.93
0.46
0.34
0.28
>108.70
>147.06
>178.57
9d
Celecoxib
a
IC50 value is the compound concentration required to produce 50% inhibition of
COX-1 or COX-2 for means of two determinations and deviation from the mean is
<10% of the mean value.
b
Selectivity index IC50 (COX-1)/IC50 (COX-2).
selective COX-2 inhibitor (SI > 178.57). The results revealed that
most of the compounds showed potent inhibition against COX-2
(IC50
(IC50 > 50
:
0.34e7.50
mM) and were inactive against COX-1
2.2.3. Ulcerogenic liability
mM) as listed in Table 1. Nearly, five compounds (3c,
The ulcerogenic potential of the most potent COX-2 inhibitors
3c, 3d, 3e, 9c and 9d (50 mg/kg) was evaluated according to
Meshali’s method [33] and the ulcer index was calculated according
to Robert’s method [34]. The ulcerogenic effect was compared to
celecoxib (50 mg/kg) and the classical NSAID ibuprofen (25 mg/kg)
[35].1 From the data obtained, Table 3, it has been observed that all
the synthesized pyrazoles endowed with benzofuran moiety
caused less gastric ulceration effect (ulcer index of compound 3c:
13.82 ꢀ 0.62; 3d: 11.56 ꢀ 0.54; 3e: 10.50 ꢀ 0.63; 9c: 11.75 ꢀ 0.63
and 9d: 10.50 ꢀ 0.59) in the experimental animals, compared to
that of the standards, ibuprofen (ulcer index 22.32 ꢀ 0.86) and
celecoxib (ulcer index 16.12 ꢀ 0.86).
3d, 3e, 9c and 9d) were found to be potent and selective similar to
celecoxib against COX-2.
The structure activity data acquired showed that the pyrazole C-
3 substituent was a determinant of COX-2 inhibitory potency and
selectivity of the 5-(6-hydroxy-4-methoxybenzofuran-5-yl)pyr-
azole derivatives 3aec. The celecoxib analogue 3b with tri-
fluoromethyl substituent displayed moderate activity compared to
the reference drug with IC50 ¼ 7.0
group in 3b into methyl group in 3a led to minor decrease in COX-2
M). Conversely, a remarkable improve in
mM. Changing the trifluoromethyl
inhibition (IC50 ¼ 7.50
m
enzyme inhibitory activity as well as selectivity was observed upon
replacement of the same group with pyridin-3-yl moiety in 3c
Therefore, the potential value of these compounds as anti-
inflammatory agents is that they have highly better safety margin
on gastric mucosa than ibuprofen. These promising ulcer protective
properties of the designed pyrazole-benzofuran derivatives greatly
supported our main objective to avoid gastric injuries caused by
COX-1 inhibition. Moreover, compound 3e with best overall profile
in animal efficacy model showed about 40% reduction in ulcero-
genic potential versus celecoxib. Hence, this successful result sup-
ported the aim of the present work to develop novel series of
celecoxib analogs bearing benzofuran moiety as COX-2 inhibitors
with diminished gastrointestinal side effects.
(IC50 ¼ 0.40
mM, SI > 125.00). On the other hand, both COX-2 po-
tency and selectivity of 3b were found to increase also, by intro-
ducing an extra methoxy group on C-7 of the benzofuran moiety
(3d; IC50 ¼ 0.52
structural modification to 3c produced 3e with minor improvement
in COX-2 inhibitory effect (IC50 ¼ 0.36 M, SI > 138.90).
mM, SI > 96.15). Interestingly, applying the same
m
Considering the 5-[4-(un) substituted phenyl]-3-(4,6-
dimethoxybenzofuran-5-yl)pyrazole derivatives 9aed, it seems
that the p-substituent on the phenyl ring modulated the COX-2
inhibitory potency of these compounds. In general, molecules
possessing p-electronegative substituent 9bed were more potent
COX-2 inhibitors than the unsubstituted phenyl counterpart 9a
(CF3 > F > Cl > H). Although, compound 9b with p-chlorophenyl
moiety proved to be weak and nonselective COX-2 inhibitor
2.3. Molecular modeling
To further explore the mechanism of interaction of the newly
synthesized compounds within COX-2 active site and to account for
the obtained biological results docking analysis was carried out.
Docking of the new inhibitors and the reference drug celecoxib into
the crystal structure of COX-2 enzyme catalytic domain in complex
with the diaryl heterocyclic inhibitor SC-558 [PDB ID code 6COX]
[36] was performed using Molecular Operating Environment MOE
version 2008.10 [37]. The most stable docking model was selected
according to the best scored conformation predicted by the MOE
scoring function, Table 4.
(IC50 ¼ 26.00
mM, SI > 1.93), grafting the more electronegative
fluorine atom in 9c or trifluoromethyl group in 9d to the phenyl
ring of 9a induced COX-2 selectivity and resulted in approximately
77 and 100 fold boost in potency, respectively (9c: IC50 ¼ 0.34
mM,
SI > 108.70; 9d: IC50 ¼ 0.34
mM, SI > 147.06). The difference in
electronegativity between fluorine and carbon creates a large
dipole moment in this bond. This dipole may contribute to the
molecule’s ability to be engaged in intermolecular interactions with
COX-2 active site.
2.2.2. In vivo anti-inflammatory activity against formalin induced
rat paw edema
Anti-inflammatory activity of compounds 3c, 3d, 3e, 9c and 9d,
which showed the highest inhibitory potency and selectivity for
COX-2 isozyme in vitro, was evaluated by employing the standard
1
The animal dose of the reference drugs was calculated relative to human dose
using reported equivalent surface area dosage conversion factors which correlate
the dosage of different species [35]. Calculations were made considering the
highest adult dose of celecoxib (400 mg) and the lowest adult dose of ibuprofen
(200 mg) to demonstrate the high safety margin of the new compounds.

486
G.S. Hassan et al. / European Journal of Medicinal Chemistry 76 (2014) 482e493
Table 2
Anti-inflammatory activity of the tested compounds and celecoxib (50 mg/kg p.o.) against formalin-induced paw edema in rats.
Treatment
Increase in paw thickness (mm)
Edema inhibition %
30 min
1 h
2 h
3 h
30 min
1 h
2 h
3 h
Control
Celecoxib
0.54 ꢀ 0.018
0.03 ꢀ 0.0027a
0.05 ꢀ 0.007a
0.15 ꢀ 0.012a
0.05 ꢀ 0.006a
0.11 ꢀ 0.018a
0.18 ꢀ 0.034a
0.57 ꢀ 0.016
0
0.58 ꢀ 0.019
0.61 ꢀ 0.022
0
0
0
100
100
87.93
100
82.76
94.83
0
100
100
91.80
100
83.61
91.80
0
0
94.44
90.14
72.22
90.74
79.63
66.67
100
96.19
78.95
96.49
82.46
77.19
3c
3d
3e
9c
9d
0.02 ꢀ 0.001a
0.12 ꢀ 0.008a
0.02 ꢀ 0.003a
0.10 ꢀ 0.018a
0.13 ꢀ 0.022a
0
0
0.07 ꢀ 0.004a
0
0.05 ꢀ 0.003a
0
0.10 ꢀ 0.019a
0.10 ꢀ 0.019a
0.03 ꢀ 0.006a
0.05 ꢀ 0.01a
Results are means of five experiments ꢀ S.E.
a
Significantly different from the control value at p < 0.01.
Table 3
Ulcerogenic effect of celecoxib, 3c, 3d, 3e, 9c and 9d in rats (50 mg/kg) and ibuprofen (25 mg/kg).
Treatment
No. of animals
with ulcers
% Incidence
divided by 10
Average number
of ulcers
Average severity
(Ulcer score)
Ulcer index
Control
Celecoxib
Ibuprofen
3c
3d
3e
9c
9d
0/5
5/5
5/5
5/5
4/5
4/5
4/5
4/5
0
10
10
10
8
8
8
8
0
5
10
3
3
2
0
0
1.12
2.32
0.82
0.56
0.5
16.12 ꢀ 0.86
22.32 ꢀ 0.86
13.82 ꢀ 0.62b
11.56 ꢀ 0.54a,b
10.50 ꢀ 0.63a,b
11.75 ꢀ 0.63a,b
10.50 ꢀ 0.59a,b
3
2
0.75
0.50
a
Significantly different from celecoxib value at p < 0.01.
Significantly different ibuprofen value at p < 0.01. Results are means of five experiments ꢀ S.E.
b
Visual inspection of the protein complex reveals that COX active
involved in areneecation interaction with the guanidinium side
chain of Arg 120. The benzofuran moeity is positioned in the pri-
mary hydrophobic pocket occupied by the bromophenyl ring of SC-
558 and is stabilized by hydrophobic interaction with Leu 384, Tyr
385, Trp 387 and Phe 518 with contribution from the backbone of
Leu 531. Also, the N1-phenyl sulfonamide is oriented toward the
side pocket, where one of the sulfonamide oxygen atoms is linked
by H-bond to Arg 513, while the amide nitrogen is H-bonded to His
90 (Fig. 2BeD, Table 4).
site has three deferential regions: the carboxylate site at entrance of
COX active site which appears as a constriction composed of three
hydrophilic residues Arg 120, Tyr 355 and Glu 524 arranged to form
H-bond network. The entrance leads to a long hydrophobic channel
that extends deep into the interior of the catalytic domain. In COX-
2, this channel forks to a primary hydrophobic pocket and side
pocket. The primary pocket is defined by the amino acids Tyr 385,
Trp 387, Phe 518 and Ser 530. The side pocket (selectivity site) is
located above Arg 120, Tyr 355, Glu 524 constriction; this pocket is
bordered by Val 523 and contains the conserved residue His 90 and
the nonconserved residue Arg 513 [38].
Moreover, the pyridine ring of 3c was in van der Waal contact
with side chains of Val 116, Val 439, Tyr 355 and Leu 531 amino
acids, while its benzofuran moiety was pushed more deep in the
hydrophobic pocket compared to the same moiety in 3a and 3b.
Thus, the difference in relative position of benzofuran enabled H-
To validate the docking protocol, the co-crystallized ligand was
redocked into COX-2 active site and the docking pose was
compared with the initial pose using root mean square deviation
(RMSD). SC-558 docked almost at the same position
ꢀ
bonding between 6-hydroxy group and Val 523 (2.80 A) (Fig. 2B).
On the other hand, the docked posses of compounds 3d and 3e with
extra methoxy group on C-7 of the benzofuran moiety (Fig. 2C and
D) illustrated the potential of the 7-methoxy oxygen to be involved
in two H-bonds with the side chain hydroxyl groups of Ser 530 and
Try 385 amino acids. Several structural and functional evidences
supported that Try 385 and Ser 530 chelation is critical for inhibi-
tion of COX-2 by the clinically used NSAIDs, diclofenac, piroxicam
and nimesulide [39]. Accordingly, the lack of additional H-bond
interactions with Val 523 in case of 3c or with Ser 530 and Try 385
for the analogs 3d and 3e might be responsible for the reduced
COX-2 inhibitory activity of 3a and 3b.
ꢀ
(RMSD ¼ 0.218 A) with docking score of ꢁ14.11 kcal/mol (Table 4).
The bromophenyl ring is bound in the primary hydrophobic pocket.
The trifluoromethyl group is bound in an adjacent pocket formed
by Val 116, Val 349, Tyr 355, Leu 359 and Leu 531. The phenyl
sulfonamide moiety is inserted in the side pocket where the sul-
ꢀ
fonamide group H-bonded with His 90 and Arg 513 (2.90 and 2.70 A
respectively) Fig. 2A, such interactions are almost essential for COX-
2 inhibitory activity [36,38]. Also, the selectivity of SC-558 seems to
result from the binding of the sulfonamide group to this pocket
which is more restricted in COX-1 [38]. Similar to SC-558, celecoxib
was docked into COX-2 active site revealing the same orientation
and binding behavior (Fig. 2A, Table 4).
2.3.2. Docking of 4-[5-aryl-3-(4,6-dimethoxybenzofuran-5-yl)-1H-
pyrazol-1-yl]benzene sulfonamide derivatives 9aed
2.3.1. Docking of 4-[3-substituted-5-(6-hydroxy-4-
methoxybenzofuran-5-yl)-1H-pyrazol-1-yl)]benzene sulfonamide
derivatives 3aec and their 5-(6-hydroxy-4,7-
The docking models of the pyrazoles 9aed showed that the
compounds are typically positioned in COX-2 active site with their
substituted phenyl ring projecting up into the primary hydrophobic
pocket toward Try 385 and Trp 387. Meanwhile, the N1 phenyl
sulfonamide is directed toward the side pocket with the sulfon-
amide functionality embedded in the selectivity site forming two
H-bonding interactions with His 90 and Arg 513, Table 4. The 4,6-
dimethoxybenzofuran-5-yl) analogs 3d,e
Docking calculations showed that the new pyrazoles (3aee)
bound to COX-2 binding site with the same orientation of the co-
crystalized ligand SC-558 and celecoxib. The central pyrazole is

G.S. Hassan et al. / European Journal of Medicinal Chemistry 76 (2014) 482e493
487
Table 4
distribution, metabolism and elimination (ADME) and is used to
insure that drug-like physicochemical properties are maintained
during drug design. These simple rules state that oral bioavail-
ability is likely to occur if at least three of the following rules are
obeyed: molecular weight less than 500 Da; no more than five
hydrogen bond donors and less than 10 hydrogen bond acceptors;
and calculated octanolewater partition coefficient (c log P) not
greater than 5 [45]. Also, topological polar surface area (TPSA) and
the number of rotatable bonds have been found to be very good
descriptors of oral bioavailability of drugs. Compounds which
meet the two criteria of ten or fewer rotatable bonds and polar
surface area equal to or less than 140 Å2 are predicted to have
good oral bioavailability [44]. The calculated parameters (Table 5)
showed good bioavailability of studied compounds. Compounds
3c, 3d and 9c fulfilled all rules, similar to the clinically used drug
celecoxib. Meanwhile, compound 3e had slightly large TPSA
Docking score and bond interactions of SC-558, celecoxib and synthesized com-
pounds with amino acids of COX-2.
ꢀ
Compound Docking
score
No. of
H-bonds
Distance (A) Amino acid Molecular
involved
structure
(kcal/mol)
SC-558
Celecoxib
3a
ꢁ14.11
ꢁ16.35
ꢁ16.66
ꢁ15.23
ꢁ17.04
2
2
2
2
3
2.90
2.70
2.91
2.64
2.90
2.46
2.89
2.42
2.89
2.43
2.80
2.88
2.45
2.47
2.92
2.19
2.48
2.50
2.76
2.88
2.52
2.71
2.99
2.96
2.61
2.98
2.52
2.61
2.96
2.49
2.66
His 90
Arg 513
His 90
Arg 513
His 90
Arg 513
His 90
Arg 513
His 90
Arg 513
Val 523
His 90
Arg 513
Ser 530
Tyr 385
His 90
Arg 513
Ser 530
Tyr 385
His 90
Arg 120
Arg 513
His 90
Arg 120
Arg 513
His 90
Arg 120
Arg 513
His 90
Arg 120
Arg 513
NH of SO2NH2
O of SO2NH2
NH of SO2NH2
O of SO2NH2
N of SO2NH2
O of SO2NH2
N of SO2NH2
O of SO2NH2
N of SO2NH2
O of SO2NH2
H of 6-OH
N of SO2NH2
O of SO2NH2
O of 7-OCH3
O of 7-OCH3
N of SO2NH2
O of SO2NH2
O of 7-OCH3
O of 7-OCH3
NH of SO2NH2
O of 6-OCH3
O of SO2NH2
NH of SO2NH2
O of 6-OCH3
O of SO2NH2
NH of SO2NH2
O of 6-OCH3
O of SO2NH2
NH of SO2NH2
O of 6-OCH3
O of SO2NH2
3b
3c
3d
3e
ꢁ18.19
ꢁ18.51
4
4
2
ꢀ
(142.72 A ) and compound 9d was violated from the rule of five by
one represented by molecular weight value. Theoretically, these
compounds should present good passive oral absorption and dif-
ferences in their bioactivity cannot be attributed to these
properties.
Compounds 9d, 3e, 3c, 9c and 3d which showed the highest
inhibitory activity and selectivity for COX-2 enzyme in the in vitro
9a
9b
9c
9d
ꢁ17.84
ꢁ17.09
ꢁ18.82
ꢁ20.18
3
3
3
3
assay (IC50 ¼ 0.34, 0.36, 0.40, 0.46, 0.52
mM respectively), exhibited
anti-inflammatory activity with edema inhibition % (66.67, 90.74,
90.14, 81.84 and 72.22 respectively) after 30 min. The high potency
and the rapid onset of action of 3-(pyridin-3-yl)pyrazoles 3e and 3c
might be attributed to their enhanced aqueous solubility (miLogP:
2.61 and 2.62, respectively). Aqueous solubility governs both the
rate of dissolution of the compound and the maximum concen-
tration reached in the gastrointestinal fluid and may be the main
factor ensuring a good level of distribution of these compounds
in vivo [46]. Besides, partition coefficients of compounds 3e and 3c
were measured experimentally using reversed-phase thin layer
chromatography [47]. The determined log P values were 2.48 and
2.21, respectively, which asserted the hydrophilic characteristics of
compounds.
dimethoxy benzofuran moiety at C-3 is oriented to the carboxylate
site of the enzyme in correspondence with the trifluoromethyl
group of SC-558. This orientation involved hydrogen bonding of the
oxygen atom of the 6-methoxy group with Arg 120. Furthermore,
Ile 345, Val 349 and Leu 531 were within close van der Waal contact
with benzofuran moiety (Fig. 3AeB).
3. Conclusion
The present study reported the design and synthesis of novel
celecoxib analogs endowed with benzofuran moiety as selective
COX-2 inhibitors. The synthesized compounds were evaluated for
their COX-1/COX-2 inhibitory activity in vitro. Compounds 3c, 3d,
3e, 9c and 9d were found to be potent and selective COX-2 in-
It was interesting to note that the structural analogs 9ae
d showed strikingly different levels of activity in the enzyme assay.
The profound enhancement in COX-2 inhibitory effect of the fluo-
rophenyl and trifluoromethyl phenyl derivatives 9c and 9d sug-
gested that they bound to the enzyme in such away to maximize
their interaction with the binding site. As seen in Table 4, these
compounds had the highest binding affinities (docking
score: ꢁ18.82 and ꢁ20.18 kcal/mol). This may be attributed to the
highly electronegative nature of fluorine atom or trifluoromethyl
hibitors (IC50: 0.34e0.52
mM) and were inactive against COX-1
(IC50 > 50 M). SAR was discussed in terms of the enzyme inhibi-
m
tory activity and was supported by molecular docking simulations
and analysis of the binding modes of the new inhibitors within
COX-2 active site. The H-bonding capability seems to be the most
crucial factor affecting the activity of a series 5-(6-hydroxy-4-
methoxybenzofuran-5-yl)pyrazoles 3aec and their 5-(6-hydroxy-
4,7-dimethoxybenzofuran-5-yl) analogs 3d,e. Meanwhile, the
electronic effect (electronegativity) of p-substituent on the phenyl
ring exerted the major influence on the COX-2 inhibitory potency of
5-[4-(un) substituted phenyl]-3-(4,6-dimethoxybenzofuran-5-yl)
pyrazole derivatives 9aed. In addition, the most potent COX-2 in-
hibitors 3c, 3d, 3e, 9c and 9d were assessed for their anti-
inflammatory activity and ulcerogenic liability in vivo. Interest-
ingly, the 3-(pyridin-3-yl)pyrazole derivatives 3c and 3e exhibited
the highest anti-inflammatory activity, that is equipotent to the
reference drug celecoxib. These results suggested a contributory
role of C-3 pyridin-3-yl in improving the anti-inflammatory effi-
ciency in animal models. Moreover, the tested compounds proved
to have better gastrointestinal safety profile compared to celecoxib.
group which led to a more efficient
pep stack interaction of the
phenyl ring with Try 385 and Trp 387 [40,41]. Also, the difference in
electronegativity between fluorine and carbon creates a large
dipole moment in this bond. This dipole may contribute to the
molecule’s ability to be engaged in intermolecular interactions with
COX-2 active site [42,43].
2.4. ADME profiling
The bioavailability of the reference drug celecoxib and the most
active compounds 3cee, 9c and 9d was assessed using mipc e
Molinspiration Property Calculator [44]. In particular, we calcu-
lated the compliance of compounds to Lipinski’s “rule of five” [45].
The rule describes molecular properties important for drug’s
pharmacokinetics in the human body, including their absorption,

488
G.S. Hassan et al. / European Journal of Medicinal Chemistry 76 (2014) 482e493
Fig. 2. (A) Superimposition of SC-558 (blue) and celecoxib (purple) docked in the active site of COX-2 enzyme. (BeD) Docking poses of the potent 5-(6-Hydroxy-4-
methoxybenzofuran-5-yl) pyrazole derivative 3c (B) and 5-(6-Hydroxy-4,7-dimethoxybenzofuran-5-yl) pyrazole derivatives 3d (C) and 3e (D) within COX-2 active site. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
In particular, compound 3e demonstrated about 40% reduction in
ulcerogenic potential relative to the reference drug, which makes it
a good lead-candidate for further optimization and development of
potent and safe anti-inflammatory agents.
300 MHz and 13C spectra were run at 75.46 MHz in deuterated
chloroform (CDCl3) or dimethyl sulphoxide (DMSO-d6). Chemical
shifts are quoted in
d as parts per million (ppm) downfield from
tetramethylsilane (TMS) as internal standard. Mass spectra were
recorded using Hewlett Packard Varian (Varian, Polo, USA) and
Shimadzu Gas Chromatograph Mass spectrometer-QP 1000 EX
(Shimadzu, Kyoto, Japan). TLC were carried out using Art.DC-
Plastikfolien, Kieselgel 60 F254 sheets (Merck, Darmstadt, Ger-
many), the developing solvents were CCl4/CH3COOC2H5 (9:1) or
(4:1) and the spots were visualized at 366, 254 nm by UV Vilber
Lourmat 77202 (Vilber, Marne La Vallee, France).
4. Experimental
4.1. Chemistry
Melting points were determined by open capillary tube method
using Gallen Kamp melting point apparatus MFB-595-010M (Gallen
Kamp, London, England) and were uncorrected. Microanalysis was
carried out at the regional center for microbiology and biotech-
nology, Al-Azhar University; Organic Microanalyses Section, Cen-
tral Laboratory, National Research Center and the Micro Analytical
Center, Faculty of Science, Cairo University. Infrared Spectra were
recorded as potassium bromide discs on Schimadzu FT-IR 8400S
spectrophotometer (Shimadzu, Kyoto, Japan) and expressed in
wave number (cmꢁ1). The NMR spectra were recorded on a Varian
Mercury VX-300 NMR spectrometer. 1H spectra were run at
Compounds 1a, 1b [48], 2a [49], 2c, 2e [50], 4a, 4b, 5a, 6 [51] and
7a [52] were prepared according to reported procedures.
4.1.1. General procedure for the synthesis of 4,4,4-trifluoro-1-(6-
hydroxy-4-methoxybenzofuran-5-yl)butane-1,3-dione derivatives
(2b) and 4,4,4-trifluoro-1-(6-hydroxy-4,7-dimethoxybenzofuran-5-
yl)butane-1,3-dione (2d)
4.1.1.1. Method A. A solution of 1a or 1b (10 mmol) in ethyl tri-
fluoroacetate (50 mmol, 7.10 g, 6.0 ml) was treated with (25 mmol,

G.S. Hassan et al. / European Journal of Medicinal Chemistry 76 (2014) 482e493
489
Fig. 3. Docking poses of the most potent 3-(4,6-dimethoxybenzofuran-5-yl)pyrazole derivatives 9c (A) and 9d (B) in the active site of COX-2 enzyme.
0.57 g) powdered sodium (prepared under toluene). After the initial
reaction subsided, the mixture was refluxed for 2 h. The reaction
mixture was left to stand overnight, treated with ice-water and
extracted with ether. The aqueous solution, after being freed from
ether by a stream of air was acidified with acetic acid and extracted
with CHCl3. The chloroformic extract was filtered through anhy-
drous Na2SO4 and the filtrate was evaporated under vacuum. The
residue was crystallized from CHCl3/ether to give 2b and 2d,
respectively.
4.1.2. General procedure for the synthesis of 4-[3-substituted-5-(6-
hydroxy-4-methoxybenzofuran-5-yl)-1H-pyrazol-1-yl)]benzene
sulfonamide derivatives (3aec) and 4-[3-substituted-5-(6-hydroxy-
4,7-dimethoxybenzofuran-5-yl)-1H-pyrazol-1-yl)]benzene
sulfonamide derivatives (3d,e)
The appropriate
b-diketone derivative 2aee (10 mmol) was
added to a stirred solution of p-sulfamoylphenylhydrazine hydro-
chloride (11 mmol, 2.46 g) in ethanol (40 ml) and 6 N HCl (22 mmol,
3.7 ml). The mixture was heated to reflux and stirred for 8 h. After
cooling to room temperature, the reaction mixture was concen-
trated under vacuum. The residue was taken up with ethyl acetate
and washed with water, dried over anhydrous MgSO4, filtered, and
evaporated under vacuum. The residue was crystallized from
ethanol.
4.1.1.2. Method B. To a solution of sodium (30 mmol, 0.69 g) in
methanol (20 ml), ethyl trifluoroacetate (20 mmol, 2.84 g,
2.4 ml) was added. After 30 min, a solution of 1a or 1b
(20 mmol) in methanol (5 ml) was added. The resulting reac-
tion mixture was stirred for 10 h at 80 ꢂC then evaporated to
dryness. The obtained sodium salt was dissolved in water
(10 ml), acidified with acetic acid and extracted several times
with ethyl acetate. The organic layer was dried over anhydrous
MgSO4 and evaporated under vacuum to give 2b and 2d,
respectively.
4.1.2.1. 4-[5-(6-Hydroxy-4-methoxybenzofuran-5-yl)-3-methyl-1H-
pyrazol-1-yl]benzene sulfonamide (3a). Yield 55%, m.p. 112e113 ꢂC.
IR (KBr, cmꢁ1): 3329, 3248 (NH2), 3151 (OH), 1624 (C]N), 1338,
1153 (SO2). 1H NMR (CDCl3)
d ppm: 1.55 (s, 2H, NH2, exch. D2O), 2.72
(s, 3H, CH3), 4.22 (s, 3H, OCH3), 4.94 (s, 1H, H-4 pyrazole), 6.73 (s,
1H, H-7 benzofuran), 7.24 (d, 1H, H-3 benzofuran, J ¼ 1.8 Hz), 7.48
(d, 1H, H-2 benzofuran, J ¼ 1.8 Hz), 7.76 (d, 2H, H-2 and H-6
ArSO2NH2, J ¼ 8.7 Hz), 8.13 (d, 2H, H-3 and H-5 ArSO2NH2,
J ¼ 8.4 Hz), 10.80 (s, 1H, OH, exch. D2O). MS, m/z: 399 [Mþ]. Anal.
Calcd. for C19H17N3O5S (399.42): C, 57.13; H, 4.29; N, 10.52; S, 8.03.
Found: C, 56.81; H, 4.14; N, 10.13; S, 7.81.
4.1.1.3. 4,4,4-Trifluoro-1-(6-hydroxy-4-methoxybenzofuran-5-yl)
butane-1,3-dione (2b). Yield 50% (method A) and 60% (method
B), m.p. 180e182 ꢂC. IR (KBr, cmꢁ1): 3128 (OH), 1701, 1662 (2
C]O). 1H NMR (CDCl3)
d
ppm: 4.14 (s, 2H, COCH2CO), 4.23 (s,
3H, OCH3), 6.72 (s, 1H, H-7 benzofuran), 6.89 (d, 1H, H-3
benzofuran,
J
¼
2.4 Hz), 7.49 (d, 1H, H-2 benzofuran,
4.1.2.2. 4-[5-(6-Hydroxy-4-methoxybenzofuran-5-yl)-3-
J ¼ 2.4 Hz), 10.50 (s, 1H, OH, exch. D2O). MS, m/z: 302 [Mþ].
Anal. Calcd. for C13H9F3O5 (302.20): C, 51.67; H, 3.00. Found: C,
51.52; H, 2.72.
trifluoromethyl-1H-pyrazol-1-yl]benzene
Yield 60%, m.p. 192e193 ꢂC IR (KBr, cmꢁ1): 3336, 3244 (NH2), 3151
(OH), 1635 (C]N), 1334, 1161 (SO2). 1H NMR (DMSO-d6)
ppm: 2.49
sulfonamide
(3b).
d
(s, 2H, NH2, exch. D2O), 4.02 (s, 3H, OCH3), 6.85 (s, 1H, H-7 benzo-
furan), 7.13 (d, 1H, H-3 benzofuran, J ¼ 2.4 Hz), 7.33 (s, 1H, H-4
pyrazole), 7.56 (d, 1H, H-2 benzofuran, J ¼ 2.4 Hz), 7.84 (d, 2H, H-2
and H-6 ArSO2NH2, J ¼ 8.4 Hz), 8.06 (d, 2H, H-3 and H-5 ArSO2NH2,
4.1.1.4. 4,4,4-Trifluoro-1-(6-hydroxy-4,7-dimethoxybenzofuran-5-yl)
butane-1,3-dione (2d). Yield 55% (method A) and 63% (method B),
m.p. 216e218 ꢂC. IR (KBr, cmꢁ1): 3132 (OH), 1700, 1651 (2C]O). 1H
NMR (CDCl3)
d
ppm: 3.32 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 4.01 (s,
J ¼ 8.7 Hz), 10.01 (s, 1H, OH, exch. D2O). 13C NMR (DMSO)
d: 60.19
2H, COCH2CO), 7.15 (d, 1H, H-3 benzofuran, J ¼ 2.4 Hz), 7.86 (d, 1H,
(OCH3), 92.95 (C-7),105.18 (C-5), 105.83 (C-3), 110.18 (C-4 pyrazole),
112.15 (C-3a), 125.09 (C-3 and C-5 ArSO2NH2), 127.04 (C-2 and C-6
ArSO2NH2), 131.00 (CF3), 140.77 (C-1 ArSO2NH2), 143.45 (C-4
ArSO2NH2), 144.64 (C-5 pyrazole), 147.43 (C-2), 151.87 (C-3 pyr-
azole), 154.51 (C-6), 156.64 (C-4), 157.54 (C-7a). MS, m/z: 454
[Mþ þ 1]. Anal. Calcd. for C19H14F3N3O5S (453.39): C, 50.33; H, 3.11;
N, 9.27; S, 7.07. Found: C, 50.60; H, 3.05; N, 8.81; S, 6.93.
H-2 benzofuran, J ¼ 2.4 Hz), 11.54 (s, 1H, OH, exch. D2O). 13C NMR
(DMSO) d: 33.5 (CH2), 61.0 (OCH3), 61.1 (OCH3), 106.4 (C-3 and C-
3a), 111.5 (C-5), 114.2 (CF3), 128.7 (C-7), 145.2 (C-2 and C-4), 149.2
(C-7a), 150.9 (C-6), 204.2 (2C]O). MS, m/z: 333 [Mþ þ1]. Anal.
Calcd. for C14H11F3O6 (332.23): C, 50.61; H, 3.34. Found: C, 50.72; H,
3.14.

490
G.S. Hassan et al. / European Journal of Medicinal Chemistry 76 (2014) 482e493
Table 5
ADME of celecoxib and the newly synthesized compounds using mipc-Molinspiration Property Calculator.
Compound
MW
No. of H-bond donors
No. of H-bond acceptors
miLogP
No. of rotatable bonds
TPSA
No. of violations
Celecoxib
381.378
462.49
483.42
492.51
493.52
543.52
2
3
3
3
2
2
5
9
9
10
8
8
3.66
2.62
3.14
2.61
4.12
4.87
4
5
6
6
6
7
77.99
133.49
129.83
142.72
109.60
109.60
0
0
0
1
0
1
3c
3d
3e
9c
9d
MW: molecular weight.
miLogP: Octanol-water partition coefficient (predicted logP at Molinspiration).
TPSA: Topological polar surface area.
4.1.2.3. 4-[5-(6-Hydroxy-4-methoxybenzofuran-5-yl)-3-(pyridin-3-
yl)-1H-pyrazol-1-yl]benzene sulfonamide (3c). Yield 55%, m.p. 124e
126 ꢂC IR (KBr, cmꢁ1): 3305, 3251 (NH2), 3153 (OH), 1660 (C]N),
hydroxide (1.5 ml) and 30% hydrogen peroxide solution (2 ml). The
reaction mixture was heated to boiling during 1 h. After standing
for 24 h at room temperature, the formed precipitate was filtered
off and crystallized from chloroform-acetone mixture. Compounds
5a, b were yellow-orange crystals which had a negative ferric
chloride reaction and were insoluble in alkali.
1334, 1153 (SO2). 1H NMR (DMSO-d6)
d ppm: 2.13 (s, 2H, NH2, exch.
D2O), 4.12 (s, 3H, OCH3), 6.71 (s, 1H, H-7 benzofuran), 6.95 (d, 1H, H-
3 benzofuran, J ¼ 2.1 Hz), 7.06 (m, 1H, H-5 pyridine), 7.16 (d, 2H, H-2
and H-6 AreSO2NH2, J ¼ 8.1 Hz), 7.31 (s, 1H, H-4 pyrazole), 7.56 (d,
1H, H-2 benzofuran, J ¼ 2.1 Hz), 7.81 (d, 2H, H-3 and H-5 ArSO2NH2,
J ¼ 8.1 Hz), 8.60 (d, 1H, H-4 pyridine, J ¼ 7.5 Hz), 8.80 (d, 1H, H-6
pyridine, J ¼ 7.5 Hz), 9.40 (s,1H, H-2 pyridine),10.00 (s,1H, OH exch.
D2O). MS, m/z: 462 [Mþ]. Anal. Calcd. for C23H18N4O5S (462.48): C,
59.73; H, 3.92; N, 12.11; S, 6.93. Found: C, 59.57; H, 3.80; N, 12.59; S,
6.69.
4.1.3.1. 2-(4-Chlorobenzylidene)-4-methoxy-furano[3,2-f]benzo-
furan-3(2H)-one (5b). Yield 85%, m.p. 229e231 ꢂC. IR (KBr, cmꢁ1):
1697 (C]O). 1H NMR (DMSO- d6)
d ppm: 4.32 (s, 3H, OCH3), 6.76 (s,
1H, H-7 benzofuran), 7.30 (d, 1H, H-3 benzofuran, J ¼ 2.1 Hz), 7.50
(s, 1H, ¼CHe), 7.57 (d, 2H, H-3 and H-5 ArCl, J ¼ 9.0 Hz), 7.70 (d, 1H,
H-2 benzofuran, J ¼ 2.1 Hz), 7.98 (d, 2H, H-2 and H-6 ArCl,
J ¼ 9.0 Hz). MS, m/z: 326 [Mþ], 328 [Mþ þ 2]. Anal. Calcd. for
C18H11ClO4 (326.73): C, 66.17; H, 3.39. Found: C, 66.39; H, 3.20.
4.1.2.4. 4-[5-(6-Hydroxy-4,7-dimethoxybenzofuran-5-yl)-3-
trifluoromethyl-1H-pyrazol-1-yl]benzene
Yield 55%, m.p. 118e120 ꢂC. IR (KBr, cmꢁ1): 3356, 3255 (NH2), 3132
(OH), 1624 (C]N), 1338, 1134 (SO2). 1H NMR (DMSO-d6.D2O)
ppm:
sulfonamide
(3d).
4.1.4. General procedure for the synthesis of (E)-3-(4-substituted
phenyl)-1-(4,6-dimethoxybenzofuran-5-yl)prop-2-en-1-one (7be
d)
d
2.60 (s, 2H, NH2, exch. D2O), 3.95 (s, 3H, OCH3), 4.10 (s, 3H, OCH3),
6.85 (d, 1H, H-3 benzofuran, J ¼ 2.1 Hz), 7.01 (d, 2H, H-2 and H-6
ArSO2, J ¼ 8.4 Hz), 7.20 (s, 1H, H-4 pyrazole), 7.65 (d, 1H, H-2
benzofuran, J ¼ 2.1 Hz), 7.71 (d, 2H, H-3 and H-5 ArSO2, J ¼ 8.7 Hz),
10.51 (s, 1H, OH exch. D2O). MS, m/z: 485 [Mþ þ2]. Anal. Calcd. for
The worm solution of 1-(4,6-dimethoxybenzofuran-5-yl)etha-
none 6 (10 mmol, 2.2 g) and the appropriate aromatic aldehyde
(11 mmol) in ethanol (10 ml) was treated with sodium hydroxide
(30%, 5 ml). The obtained red solution was allowed to stand for 48 h
at room temperature, and then it was diluted with water and
acidified with acetic acid. The produced precipitate was filtered,
dried and crystallized from ethanol to give orange to red crystals
having negative ferric chloride reaction.
C
20H16F3N3O6S (483.42): C, 49.69; H, 3.34; N, 8.69; S, 6.63. Found: C,
50.01; H, 3.72; N, 8.82; S, 6.50.
4.1.2.5. 4-[5-(6-Hydroxy-4,7-dimethoxybenzofuran-5-yl)-3-(pyridin-
3-yl)-1H-pyrazol-1-yl]benzene sulfonamide (3e). Yield 65%, m.p.
154e155 ꢂC. IR (KBr, cmꢁ1): 3336, 3267 (NH2), 3124 (OH), 1643 (C]
4.1.4.1. (E)-3-(4-Chlorophenyl)-1-(4,6-dimethoxybenzofuran-5-yl)
prop-2-en-1-one (7b). Yield 76%, m.p. 152e154 ꢂC. IR (KBr, cmꢁ1):
N), 1330, 1153 (SO2). 1H NMR (DMSO-d6)
d ppm: 2.46 (s, 2H, NH2,
1685 (C]O). 1H NMR (DMSO-d6)
d ppm: 3.91 (s, 3H, OCH3), 4.09 (s,
exch. D2O), 3.85 (s, 3H, OCH3), 4.08 (s, 3H, OCH3), 6.85 (d, 1H, H-3
benzofuran, J ¼ 2.1 Hz), 7.05e7.26 (m, 1H, H-5 pyridine), 7.45 (s, 1H,
H-4 pyrazole), 7.51 (d, 2H, H-2 and H-6 AreSO2NH2, J ¼ 8.1 Hz), 7.63
(d, 2H, H-3 and H-5 ArSO2NH2, J ¼ 8.1 Hz), 7.87 (d, 1H, H-4 pyridine,
J ¼ 7.5 Hz), 8.27 (d, 1H, H-2 benzofuran, J ¼ 2.1 Hz), 8.60 (d, 1H, H-6
pyridine, J ¼ 7.5 Hz), 9.17 (s, 1H, H-2 pyridine), 9.99 (s, 1H, OH exch.
3H, OCH3), 6.77 (s, 1H, H-7 benzofuran), 6.88 (d, 1H, H-3 benzo-
furan, J ¼ 2.1 Hz), 6.98 (d, 1H, eCOeCH]CHe, J ¼ 15.9 Hz), 7.35 (d,
2H, H-3 and H-5 ArCl, J ¼ 8.7 Hz), 7.46 (d, 2H, H-2 and H-6 ArCl,
J ¼ 8.7 Hz), 7.54 (d, 1H, H-2 benzofuran, J ¼ 2.1 Hz), 7.95 (d, 1H, e
COeCH]CHe, J ¼ 15.9 Hz). MS, m/z: 342 [Mþ], 344 [Mþ þ 2]. Anal.
Calcd. for C19H15ClO4 (342.77): C, 66.58; H, 4.41. Found: C, 67.02; H,
4.47.
D2O). 13C NMR (DMSO)
d: 61.02 (OCH3), 61.81 (OCH3), 95.93 (C-7),
105.48 (C-5), 106.55 (C-3), 110.72 (C-4 pyrazole), 112.49 (C-3a),
120.21 (C-2 and C-6 ArSO2NH2), 125.82 (C-5 pyridine), 128.95 (C-3
and C-5 ArSO2NH2), 130.44 (C-1 pyridine), 134.18 (C-6 pyridine),
138.01 (C-3 pyrazole), 141.45 (C-4 ArSO2NH2), 143.06 (C-1
ArSO2NH2), 144.99 (C-5 pyrazole), 146.00 (C-2), 147.00 (C-4), 148.68
(C-4 pyridine), 149.37 (C-7a), 150.45 (C-2 pyridine), 150.95 (C-6).
MS, m/z: 493 [Mþ þ1]. Anal. Calcd. for C24H20N4O6S (492.50): C,
58.53; H, 4.09; N, 11.38; S, 6.51. Found: C, 58.53; H, 3.92; N, 11.33; S,
6.65.
4.1.4.2. (E)-3-(4-Fluorophenyl)-1-(4,6-dimethoxybenzofuran-5-yl)
prop-2-en-1-one (7c). Yield 70%, m.p. 138e140 ꢂC. IR (KBr, cmꢁ1):
1681 (C]O). 1H NMR (DMSO-d6)
d ppm: 3.78 (s, 3H, OCH3), 3.97 (s,
3H, OCH3), 6.76 (s, 1H, H-7 benzofuran), 7.00 (d, 1H, H-3 benzo-
furan, J ¼ 2.1 Hz), 7.16 (d, 1H, eCOeCH]CHe, J ¼ 16.5 Hz), 7.31 (d,
2H, H-3 and H-5 ArF, J ¼ 9.0 Hz), 7.45 (d, 1H, H-2 benzofuran,
J ¼ 2.1 Hz), 7.82 (d, 2H, H-2 and H-6 ArF, J ¼ 9.3 Hz), 7.98 (d, 1H, e
COeCH]CHe, J ¼ 15.9 Hz). MS, m/z: 326 [Mþ]. Anal. Calcd. for
C19H15FO4 (326.32): C, 69.93; H, 4.63. Found: C, 69.64; H, 4.46.
4.1.3. General procedure for the synthesis of 2-(4-(un) substituted
benzylidene)-4-methoxy-furano[3,2-f]benzofuran-3(2H)-one (5a,b)
To a solution of compound 4a or 4b (5 mmol) in a mixture
acetone (5 ml) and methanol (15 ml) was added 4 N sodium
4 .1. 4 . 3 . ( E ) - 3 - ( 4 -T r i fl u o r o m e t h y l p h e n y l ) - 1 - ( 4 , 6 -
dimethoxybenzofuran-5-yl)prop-2-en-1-one (7d). Yield 60%, m.p.

G.S. Hassan et al. / European Journal of Medicinal Chemistry 76 (2014) 482e493
491
146e148 ꢂC. IR (KBr, cmꢁ1): 1701 (C]O). 1H NMR (DMSO-d6)
d
ppm:
4.1.6. General procedure for the synthesis of 4-[5-aryl-3-(4,6-
dimethoxybenzofuran-5-yl)-1H-pyrazol-1-yl]benzene sulfonamide
derivatives (9aed)
3.97 (s, 3H, OCH3), 4.04 (s, 3H, OCH3), 6.97 (s, 1H, H-7 benzofuran),
7.07 (d, 1H, H-3 benzofuran, J ¼ 2.1 Hz), 7.13 (d, 2H, H-2 and H-6
ArCF3, J ¼ 9.3 Hz), 7.54 (d, 1H, H-2 benzofuran, J ¼ 2.1 Hz), 7.61 (d,
1H, eCOeCH]CHe, J ¼ 16.8 Hz), 7.88 (d, 2H, H-3 and H-5 ArCF3,
J ¼ 9.3 Hz), 8.30 (d, 1H, eCOeCH]CH-, J ¼ 15.9 Hz). MS, m/z: 376
[Mþ]. Anal. Calcd. for C20H15F3O4 (376.33): C, 63.83; H, 4.02. Found:
C, 63.54; H, 3.95.
The oxirane derivative 8aed (10 mmol) was added to a stirred
solution of p-sulfamoylphenylhydrazine hydrochloride (11 mmol,
2.46 g) in ethanol (10 ml) and acetic acid (0.5 ml). The reaction
mixture was stirred at reflux for 6 h. After cooling to room tem-
perature, the reaction mixture was concentrated under vacuum.
The residue was taken up with ethyl acetate. The organic layer was
washed several times with water, dried over anhydrous MgSO4 and
evaporated under vacuum. The residue was crystallized from
ethanol.
4.1.5. General procedure for the synthesis of [3-(4-substituted
phenyl)oxiran-2-yl](4,6-dimethoxybenzofuran-5-yl)methanone
(8aed)
A solution of the appropriate chalcone 7aed (10 mmol) in
acetone (10 ml) and methyl alcohol (30 ml) was mixed with 4 N
sodium hydroxide (3 ml) followed by drop wise addition of
hydrogen peroxide (30%, 5 ml). The solution was shaken and heated
to the boiling point during 1 h; then allowed to stand overnight at
room temperature, the reaction mixture became lighter in color.
4.1.6.1. 4-[3-(4,6-Dimethoxybenzofuran-5-yl)-5-phenyl-1H-pyrazol-
1-yl]benzene sulfonamide (9a). Yield 64%, m.p. 144e145 ꢂC. IR (KBr,
cmꢁ1): 3387, 3263 (NH2), 1624 (C]N), 1350, 1153 (SO2). 1H NMR
(DMSO-d6)
d ppm: 1.40 (s, 2H, NH2, exch. D2O), 4.02 (s, 3H, OCH3),
4.22 (s, 3H, OCH3), 6.95 (s, 1H, H-7 benzofuran), 7.12 (d, 1H, H-3
benzofuran, J ¼ 2.1 Hz), 7.19 (s, 1H, H-4 pyrazole), 7.26e7.40 (m, 5H,
ArH), 7.60 (d, 2H, H-2 and H-6 ArSO2NH2, J ¼ 8.7 Hz), 7.80 (d, 1H, H-
2 benzofuran, J ¼ 2.1 Hz), 7.95 (d, 2H, H-3 and H-5 ArSO2NH2,
J ¼ 8.4 Hz). MS, m/z: 474 [Mþ ꢁ 1]. Anal. Calcd. for C25H21N3O5S
(475.52): C, 63.15; H, 4.45; N, 8.84; S, 6.74. Found: C, 63.08; H, 4.28;
N, 8.84; S, 6.74.
Crystals of the a-keto epoxide deposited and extracted with diethyl
ether (3 ꢃ 5 ml), the ethereal extracts were combined and evapo-
rated. The residue was crystallized from petroleum ether to give
yellowish-white to white powder.
4.1.5.1. (4,6-Dimethoxybenzofuran-5-yl)(3-phenyloxiran-2-yl)meth-
4.1.6.2. 4-[5-(4-Chlorophenyl)-3-(4,6-dimethoxybenzofuran-5-yl)-
1H-pyrazol-1-yl]benzene sulfonamide (9b). Yield 75%, m.p. 156e
158 ꢂC. IR (KBr, cmꢁ1): 3383, 3271 (NH2), 1630 (C]N), 1334, 1153
anone (8a). Yield 64%, m.p. 102e104 ꢂC. IR (KBr, cmꢁ1): 1693 (C]
O). 1H NMR (DMSO- d6)
d
ppm: 3.80 (d, 1H, H-3 oxirane, J ¼ 2.7 Hz),
3.99 (s, 3H, OCH3), 4.07 (s, 3H, OCH3), 4.20 (d, 1H, H-2 oxirane,
J ¼ 2.7 Hz), 6.76 (s, 1H, H-7 benzofuran), 6.98 (d, 1H, H-3 benzo-
furan, J ¼ 2.1 Hz), 7.13e7.54 (m, 5H, ArH), 7.88 (d, 1H, H-2 benzo-
furan, J ¼ 2.4 Hz). MS, m/z: 324 [Mþ]. Anal. Calcd. for C19H16O5
(324.33): C, 70.36; H, 4.97. Found: C, 70.80; H, 4.51.
(SO2). 1H NMR (CDCl3)
d ppm: 1.26 (s, 2H, NH2, exch. D2O), 3.85 (s,
3H, OCH3), 4.06 (s, 3H, OCH3), 6.75 (s, 1H, H-7 benzofuran), 6.83 (d,
1H, H-3 benzofuran, J ¼ 2.1 Hz), 6.89 (s, 1H, H-4 pyrazole), 6.93 (d,
2H H-3 and H-5 ArCl, J ¼ 9.0 Hz), 6.98 (d, 2H, H-2 and H-6
ArSO2NH2, J ¼ 8.7 Hz), 7.33 (d, 2H, H-3 and H-5 ArSO2NH2,
J ¼ 8.7 Hz), 7.44 (d, 2H, H-2 and H-6 ArCl, J ¼ 9.0 Hz), 7.60 (d,1H, H-2
benzofuran, J ¼ 2.1 Hz). MS, m/z: 509 [Mþ], 511 [Mþ þ 2]. Anal.
Calcd. for C25H20ClN3O5S (509.96): C, 58.88; H, 3.95; N, 8.24. Found:
C, 59.38; H, 4.06; N, 8.58.
4.1.5.2. [3-(4-Chlorophenyl)oxiran-2-yl](4,6-dimethoxybenzofuran-
5-yl)methanone (8b). Yield 72%, m.p. 132e134 ꢂC. IR (KBr, cmꢁ1):
1701 (C]O). 1H NMR (CDCl3)
d ppm: 3.79 (s, 3H, OCH3), 3.94 (d, 1H,
H-3 oxirane, J ¼ 2.4 Hz), 4.02 (d, 1H, H-2 oxirane, J ¼ 2.4 Hz), 4.12 (s,
3H, OCH3), 6.77 (s, 1H, H-7 benzofuran), 6.86 (d, 1H, H-3 benzo-
furan, J ¼ 2.1 Hz), 7.34 (d, 2H, H-2 and H-6 ArCl, J ¼ 8.4 Hz), 7.49 (d,
1H, H-2 benzofuran, J ¼ 2.4 Hz), 8.02 (d, 2H, H-3 and H-5 ArCl,
J ¼ 8.4 Hz). MS, m/z: 358 [Mþ], 360 [Mþ þ 2]. Anal. Calcd. For
4.1.6.3. 4-[5-(4-Fluorophenyl)-3-(4,6-dimethoxybenzofuran-5-yl)-
1H-pyrazol-1-yl]benzene sulfonamide (9c). Yield 60%, m.p. 175e
177 ꢂC. IR (KBr, cmꢁ1): 3361, 3261 (NH2), 1620 (C]N), 1334, 1155
(SO2). 1H NMR (DMSO-d6)
d ppm: 1.25 (s, 2H, NH2, exch. D2O), 3.79
(s, 3H, OCH3), 4.08 (s, 3H, OCH3), 6.80 (s, 1H, H-7 benzofuran), 7.06
(d, 1H, H-3 benzofuran, J ¼ 2.1 Hz), 7.14 (d, 2H, H-3 and H-5 ArF,
J ¼ 9.3 Hz), 7.20 (d, 2H, H-2 and H-6 ArF, J ¼ 9.0 Hz), 7.41 (d, 2H, H-2
and H-6 ArSO2NH2, J ¼ 8.4), 7.60 (d, 2H, H-3 and H-5 ArSO2NH2,
J ¼ 8.4 Hz), 7.89 (d, 1H, H-2 benzofuran, J ¼ 1.8 Hz), 8.30 (s, 1H, H-4
pyrazole). MS, m/z: 493 [Mþ]. Anal. Calcd. for C25H20FN3O5S
(493.51): C, 60.84; H, 4.08; N, 8.51; S, 6.50. Found: C, 61.04; H, 3.95;
N, 8.32; S, 6.30.
C
19H15ClO5 (358.77): C, 63.61; H, 4.21. Found: C, 63.61; H, 3.96.
4.1.5.3. [3-(4-Fluorophenyl)oxiran-2-yl](4,6-dimethoxybenzofuran-
5-yl)methanone (8c). Yield 65%, m.p. 118e120 ꢂC. IR (KBr, cmꢁ1):
1701 (C]O). 1H NMR (CDCl3)
d ppm: 3.83 (s, 3H, OCH3), 3.88 (d, 1H,
H-3 oxirane, J ¼ 2.7 Hz), 3.97 (d, 1H, H-2 oxirane, J ¼ 2.7 Hz), 4.10 (s,
3H, OCH3), 6.78 (s, 1H, H-7 benzofuran), 6.88 (d, 1H, H-3 benzo-
furan, J ¼ 2.1 Hz), 7.46 (d, 2H, H-3 and H-5 ArF, J ¼ 7.8 Hz), 7.65 (d,
1H, H-2 benzofuran, J ¼ 2.4 Hz), 7.65 (d, 2H, H-2 and H-6 ArF,
J ¼ 7.8 Hz). MS, m/z: 342 [Mþ]. Anal. Calcd. For C19H15FO5 (342.32):
C, 66.66; H, 4.42. Found: C, 66.71; H, 4.30.
4 . 1. 6 . 4 . 4 - [ 5 - ( 4 -T r i fl u o r o m e t h y l p h e n y l ) - 3 - ( 4 , 6 -
dimethoxybenzofuran-5-yl)-1H-pyrazol-1-yl]benzene
(9d). Yield 58%, m.p. 185e186 ꢂC. IR (KBr, cmꢁ1): 3387, 3275 (NH2),
1620 (C]N), 1327, 1157 (SO2). 1H NMR (DMSO-d6)
ppm: 1.25 (s,
sulfonamide
d
2H, NH2, exch. D2O), 3.86 (s, 3H, OCH3), 4.02 (s, 3H, OCH3), 6.90 (s,
1H, H-7 benzofuran), 7.00 (d, 1H, H-3 benzofuran, J ¼ 2.1 Hz), 7.15
(d, 2H, H-2 and H-6 ArCF3, J ¼ 9.3 Hz), 7.30 (d, 2H, H-3 and H-5
ArCF3, J ¼ 9.3 Hz), 7.50 (s, 1H, H-4 pyrazole), 7.60 (d, 1H, H-2
benzofuran, J ¼ 1.8 Hz), 7.70 (d, 2H, H-2 and H-6 ArSO2NH2,
J ¼ 8.7 Hz), 7.85 (d, 2H, H-3 and H-5 ArSO2NH2, J ¼ 8.7 Hz). 13C NMR
4.1.5.4. {3-[4-(Trifluoromethyl)phenyl]oxiran-2-yl}(4,6-
dimethoxybenzofuran-5-yl)methanone (8d). Yield 60%, m.p. 126e
128 ꢂC. IR (KBr, cmꢁ1): 1705 (C]O). 1H NMR (CDCl3)
d ppm: 3.82 (s,
3H, OCH3), 3.93 (d, 1H, H-3 oxirane, J ¼ 2.4 Hz), 4.05 (s, 3H, OCH3),
4.20 (d, 1H, H-2 oxirane, J ¼ 2.4 Hz), 6.84 (s, 1H, H-7 benzofuran),
6.90 (d, 1H, H-3 benzofuran, J ¼ 2.4 Hz), 7.32 (d, 2H, H-2 and H-6
ArCF3, J ¼ 8.1), 7.54 (d, 1H, H-2 benzofuran, J ¼ 2.4 Hz), 7.63 (d, 2H,
H-3 and H-5 ArCF3, J ¼ 8.1 Hz). MS, m/z: 392 [Mþ]. Anal. Calcd. For
(DMSO) d: 56.06 (OCH3), 56.31 (OCH3), 89.53 (C-7), 102.06 (C-5),
105.17 (C-3), 111.19 (C-3a), 115.20 (C-4 pyrazole), 120.63 (C-2 and C-
6 ArSO2NH2), 125.58 (C-3 and C-5 ArCF3), 126.92 (CF3), 127.59 (C-2
and C-6 ArCF3), 129.09 (C-3 and C-5 ArSO2NH2), 129.92 (C-4 ArCF3),
C
20H15F3O5 (392.33): C, 61.23; H, 3.85. Found: C, 61.55; H, 3.88.

492
G.S. Hassan et al. / European Journal of Medicinal Chemistry 76 (2014) 482e493
130.02 (C-1 ArCF3), 136.47 (C-4 ArSO2NH2), 142.97 (C-1 ArSO2NH2),
144.30 (C-5 pyrazole), 146.00 (C-2), 152.00 (C-6), 156.00 (C-4),
158.00 (C-7a),160.36 (C-3 pyrazole). MS, m/z: 543 [Mþ]. Anal. Calcd.
for C26H20F3N3O5S (543.51): C, 57.46; H, 3.71; N, 7.73; S, 5.90.
Found: C, 57.90; H, 3.86; N, 7.66; S, 6.27.
the standard formalin-induced paw edema method in rats [32].
Wister albino rats were divided into seven groups of five animals
each. Thickness of the left hind paw of each rat was measured, in
millimeters using Vernier caliper, before any drug administration
(0 h). The first group was kept as negative control given 10% DMSO
aqueous solution (v/v). Celecoxib was orally administered to the
second group as a reference standard (50 mg/kg). The tested
compounds 3c, 3d, 3e, 9c and 9d in the form of 10% DMSO aqueous
solutions were given via oral route to the rest groups at a dose of
50 mg/kg body weight, treatments began 1 h before induction of
inflammation. Paw edema was induced by subcutaneous injection
of 2.5% formalin solution (0.1 ml/rat) into the right hind paw of each
rat. Paw thickness of each rat was measured after 30 min, 1, 2 and
3 h of formalin injection using Vernier caliper. The edema thickness
(mm) was calculated by subtracting the zero-hour reading from
each time reading. The anti-inflammatory activity was expressed as
percentage inhibition of edema thickness in treated animals in
comparison with the control group, Table 1
4.2. Pharmaco-biological evaluation
4.2.1. In vitro cyclooxygenase (COX) inhibition assay
The ability of the test compounds to inhibit ovine COX-1 and
human recombinant COX-2 was determined using an enzyme
immunoassay (EIA) (kit catalog number 560131, Cayman Chemical,
Ann Arbor, MI, USA) according to the manufacturer’s instructions.
Cyclooxygenase catalyzes the first step in the biosynthesis of
arachidonic acid (AA) to PGH2. PGF2a, produced from PGH2 by
reduction with stannous chloride, is measured by enzyme immu-
noassay (ACEÔ competitive EIA). Stock solutions of test compounds
were dissolved in a minimum volume of DMSO. Briefly, to a series of
supplied reaction buffer solutions (950
containing 5 mM EDTA and 2 mM phenol) with either COX-1 or
COX-2 (10 L) enzyme in the presence of heme (10 L) were added
20 L of various concentrations of test drug solutions (0, 0.01, 0.1, 1,
10 and 50 M in a final volume of 1 ml). These solutions were
incubated for a period of 10 min at 37 ꢂC, after which 10
L of AA
(100 M) solution were added and the COX reaction was stopped by
the addition of 50 L of 1 M HCl after 2 min. PGF2a, produced from
PGH2 by reduction with stannous chloride was measured by
enzyme immunoassay. This assay is based on the competition be-
tween PGs and a PG-acetyl cholinesterase conjugate (PG tracer) for
a limited amount of PG antiserum. The amount of PG tracer, that is,
able to bind to the PG antiserum is inversely proportional to the
concentration of PGs in the wells since the concentration of PG
tracer is held constant while the concentration of PGs varies. This
antibodyePG complex binds to a mouse anti-rabbit monoclonal
antibody that had been previously attached to the well. The plate is
washed to remove any unbound reagents and then Elman’s reagent,
which contains the substrate to acetylcholine esterase, is added to
the well. The product of this enzymatic reaction produces a distinct
yellow color that absorbs at 410 nm. The intensity of this color,
determined spectrophotometrically, is proportional to the amount
of PG tracer bound to the well, which is inversely proportional to
the amount of PGs present in the well during the incubation:
mL, 0.1 M TriseHCl pH 8.0
Edema inhibition% ¼ Vc ꢁ Vt=Vc ꢃ 100
m
m
Where, Vc and Vt are the thickness of edema for the control and
drug-treated animal groups.
m
m
m
4.2.3. Ulcerogenic liability
m
The ulcerogenic effect of the most active compounds 3c, 3d, 3e,
9c and 9d as well as celecoxib and ibuprofen was evaluated ac-
cording to Meshali’s method [33]. Forty adult male albino rats were
used in this study and divided into 8 groups. The animals were
fasted 18 h before drug administration. The first group received 10%
DMSO aqueous solution (v/v) and kept as control the second group
received celecoxib in a dose of 50 mg/kg, while the third group
received ibuprofen in a dose of 25 mg/kg. The other groups received
3c, 3d, 3e, 9c and 9d in a dose 50 mg/kg. Animals were fed 2 h after
administration of the drug. Rats received the given dose orally for
three successive days. Two hours following the last dose, rats were
scarified; the stomach of each rat was removed, opened along the
greater curvature and rinsed with 0.9% sodium chloride. The
stomach was stretched; by pins; on a corkboard. Examination with
a magnifying lens (10 xs) was done for the presence of ulcers and
erosions. The ulcer index was calculated according to Robert’s
method [34]. The degree of ulcerogenic effect was expressed in
term of the percentage incidence of ulcers in each group of animals
divided by 10, the average number of ulcers per stomach and the
average severity of ulcers (ulcer score) by visual observation. The
ulcer scores were: 0 ¼ no ulcer, 1 ¼ mucosal erythema only,
2 ¼ mild mucosal edema, slight bleeding or slight erosion,
3 ¼ moderate edema, bleeding ulcers or erosions, 4 ¼ sever ul-
ceration, erosions, edema and tissue necrosis. The ulcer index is the
value that result from the sum of the above three values.
m
Absorbance
a [Bound PG Tracer] a1/PGs. Percent inhibition was
calculated by the comparison of compound treated to various
control incubations. The concentration of the test compound
causing 50% inhibition (IC50, mM) was calculated from the concen-
trationeinhibition response curve (duplicate determinations).
4.2.2. In vivo anti-inflammatory activity
All the animal experiments were performed according to pro-
tocols approved by the Animal Use and Care Committee of Uni-
versity of Cairo, Egypt.
4.2.4. Statistical analysis
The results were expressed as Mean ꢀ S.E. “standard error”. The
significant difference among the groups was assessed using one
way analysis of variance (ANOVA) followed by Dunnett’s test at
p < 0.01.
4.2.2.1. Animals. Adult male Wister albino rats (120e150 g) were
used to study the anti-inflammatory activity and ulcerogenic lia-
bilities. The animals (five per cage) were maintained under stan-
dard laboratory conditions (light period of 12 h/day and
temperature 27 ꢀ 2 ꢂC), with access to food and water. The
experimental procedures were carried out in strict compliance with
the Institutional Animal Ethics Committee regulations. All experi-
ments were performed in the morning according to the guidelines
for the care of laboratory animals [53].
4.3. Molecular modeling
All molecular modeling calculation and docking studies were
carried out using Molecular Operating Environment MOE version
2008.10 [37]. The target compounds were drawn on MOE. The
structures were subjected to energy minimization using
Hamiltonian-Force Field-MMFF94x. The most stable conformers for
each compound were retained and partial charges were calculated.
4.2.2.2. Formalin induced rat paw edema. Anti-inflammatory ac-
tivity study for the prepared compounds was determined in vivo by

G.S. Hassan et al. / European Journal of Medicinal Chemistry 76 (2014) 482e493
493
W.E. Perkins, K. Seibert, A.W. Veenhuizen, Y.Y. Zhang, P.C. Isakson, Journal of
Medicinal Chemistry 40 (1997) 1347e1365.
[22] O.L. Lorens, J.J. Perez, A. Palomer, D. Mauleon, Journal of Molecular Graphics
and Modelling 20 (2002) 359e371.
[23] M. Zheng, Z. Zhang, W. Zhu, H. Liu, X. Luo, K. Chen, H. Jiang, Bioorganic and
Medicinal Chemistry 14 (2006) 3428e3437.
[24] J.M. Janusz, P.A. Young, J.M. Ridgeway, M.W. Scherz, K. Enzweiler, L.I. Wu,
L. Gan, J. Chen, D.E. Kellstein, S.A. Green, J.L. Tulich, T. Rosario-Jansen,
I.J. Magrisso, K.R. Wehmeyer, D.L. Kuhlenbeck, T.H. Eichhold, R.L. Dobson,
Journal of Medicinal Chemistry 41 (1998) 3515e3529.
[25] R. Deshpande, M. Bhagawan Raju, S. Parameshwar, S.M. Shanth Kumar,
S. Appalaraju, S. Manjunath, S.M. Yelagatti, American Journal of Chemistry 2
(2012) 115e120.
[26] G.S. Hassan, G.A. Soliman, European Journal of Medicinal Chemistry 45 (2010)
4104e4112.
[27] F.A. Ragab, G.S. Hassan, H.A. Yossef, H.A. Hashem, European Journal of Me-
dicinal Chemistry 42 (2007) 1117e1127.
[28] G.O. Dengiz, F. Odabasoglu, Z. Halici, H. Suleyman, E. Cadirci, Y. Bayir, Archives
of Pharmacal Research 30 (2007) 1426e1434.
[29] T.A. Geissman, D.K. Fukushima, Journal of the American Chemical Society 70
(1948) 1686e1689.
[30] E. Weitz, A. Scheffer, Berichte 54 (1921) 2327e2344.
[31] Y. Apeloig, M. Karni, Z. Rapoport, Journal of the American Chemical Society
105 (1983) 2784e2793.
[32] R. Turner, Screening Method in Pharmacology. Anti-inflammatory Agent 13,
Academic Press New York, London, 1965, p. 158.
[33] M. Meshali, E. El-Sabbah, A. Foda, Acta Pharmaceutica Technologica 29 (1983)
217e230.
[34] A. Robert, J.E. Nezamis, J.P. Phillips, Gastroenterology 55 (1968) 481e487.
[35] E.J. Freireich, E.A. Gehan, D.P. Rall, L.H. Schmidt, H.E. Skipper, Cancer
Chemotherapy Reports 50 (1966) 219e244.
[36] J.L. Wang, D. Limburg, M.J. Graneto, J. Springer, J.R. Hamper, S. Liao, J.L. Pawlitz,
R.G. Kurumbail, T. Maziasz, J.J. Talley, J.R. Kiefer, J. Carter, Bioorganic & Me-
dicinal Chemistry Letters 20 (2010) 7159e7163.
[37] MOE, Chemical Computing Group, Inc., Montreal, http://www.chemcomp.
com.
The X-ray crystal structure of COX-2 enzyme in complex with SC-
558, PDB ID code 6COX was recovered from RSCB protein data
bank [38]. The enzyme was prepared for docking as follows: 1) The
Co-crystallized ligand and water molecules were removed. 2) The
enzyme was 3D protonated, where hydrogen atoms were added at
their standard geometry, the partial charges were computed and
the system was optimized. Flexible ligand- rigid receptor docking of
the most stable conformers was done with MOE-DOCK using tri-
angle matcher as placement method and London dG as a scoring
function. The obtained poses were subjected to force field refine-
ment using the same scoring function. Ten of the most stable
docking models for each ligand were retained with the best scored
conformation. In order to validate the docking procedure, SC-558
was docked into the active site of 6COX. The docking results show
that the compound exhibit similar interaction reported in literature
ꢀ
with RMSD ¼ 0.218 A.
References
[1] D.A. Williams, T.L. Lemke, Non-steroidal anti-inflammatory drugs, in:
D.A. Williams (Ed.), Foye’s Principles of Medicinal Chemistry, fifth ed., Lip-
pincott Williams & Wilkins, 2002, pp. 751e793.
[2] P. Gund, T.Y. Shen, Journal of Medicinal Chemistry 20 (1977) 1146e1152.
[3] J.Y. Jouzeau, B. Terlain, A. Abid, E. Nedelec, P. Netter, Drugs 53 (1997) 563e
582.
[4] H. Yoshimura, S. Sekine, H. Adachi, Y. Uematsu, A. Mitani, N. Futaki,
N. Shimizu, Protein Expression and Purification 80 (2011) 41e46.
[5] B. Yao, J. Xu, R.C. Harris, M.Z. Zhang, American Journal of Physiology e Renal
Physiology 294 (2008) 433e439.
[6] H. Haruna, T. Shimizu, Y. Ohtsuka, Y. Yarita, T. Fujii, T. Kudo, Y. Yamashiro,
Pediatrics International 50 (2008) 1e6.
[7] S. Yoshida, M. Ujiki, X.Z. Ding, C. Pelman, M.S. Talamonti, R.H. Bell,
W. Denham, T.E. Adrian, Molecular Cancer 4 (2005) 27.
[8] M.G. Perrone, A. Scilimati, L. Simone, P. Vitale, Current Medicinal Chemistry 17
(2010) 3769e3805.
[9] P. Vitale, S. Tacconelli, M.G. Perrone, P. Malerba, L. Simone, A. Scilimati,
A. Lavecchia, M. Dovizio, E. Marcantoni, A. Bruno, P. Patrignani, Journal of
Medicinal Chemistry 56 (2013) 4277e4299.
[10] K.G. Peri, P. Hardy, D.Y. Li, D.R. Varma, S. Chemtob, Journal of Biological
Chemistry 270 (1995) 24615e24620.
[38] R.G. Kurumbail, A.M. Stevens, J.K. Gierse, J.J. McDonald, R.A. Stegeman, J.Y. Pak,
D. Gildehaus, J.M. Miyashiro, T.D. Penning, K. Seibert, P.C. Isakson,
W.C. Stallings, Nature 384 (1996) 644e648.
[39] S.W. Rowlinson, J.R. Kiefer, J.J. Prusakiewicz, J.L. Pawlitz, K.R. Kozak,
A.S. Kalgutkar, W.C. Stallings, R.G. Kurumbail, L.J. Marnett, Journal of Biological
Chemistry 46 (2003) 45763e456769.
[40] A. DerHovanessian, J.B. Doyon, A. Jain, P.R. Rablen, A.M. Sapse, Org. Lett. 1
(1999) 1359e1362.
[41] Laurent Schaeffer, in: C.,G. Wermuth (Ed.), The Role of Functional Groups in
Drug Receptor Interaction; The Practice of Medicinal Chemistry, third ed.,
Elsevier Ltd, 2008, pp. 464e479.
[11] A.L. Sampaio, J. Dalli, V. Brancaleone, F. D’Acquisto, M. Perretti, C. Wheatley,
Mediators of Inflammation 2013 (2013) 741804.
[42] P.N. Rao, Q.H. Chen, E.E. Knaus, Journal of Medicinal Chemistry 49 (2006)
1668e1683.
[12] R. Calvello, M.A. Panaro, M.L. Carbone, A. Cianciulli, M.G. Perrone, P. Vitale,
P. Malerba, A. Scilimati, Pharmacological Research 65 (2012) 137e148.
[13] L.A. Garcia-Rodriguez, S. Hernández-Diaz, Epidemiology 14 (2003) 240e246.
[14] C.H. Ruan, S.P. So, K.H. Ruan, Life Sciences 88 (2011) 24e30.
[15] M. Khan, A. Fraser, Irish Medical Journal 105 (2012) 119e121.
[16] W.B. Whyte, G. Faich, J.S. Borer, R.W. Makuch, American Journal of Cardiology
92 (2003) 411e418.
[17] N.M. Davies, A.J. McLachlan, R.O. Day, K.M. Williams, Clinical Pharmacoki-
netics 38 (2000) 225e242.
[18] J. Castellsague, N. Riera-Guardia, B. Calingaert, C. Varas-Lorenzo, A. Fourrier-
Reglat, F. Nicotra, M. Sturkenboom, S. Perez-Gutthann, Drug Safety 35 (2012)
1127e1146.
[19] A.S. Kalgutkar, B.C. Crews, S.W. Rowlinson, A.B. Marnett, K.R. Kozak,
R.P. Remmel, L.J. Marnett, Proceedings of the National Academy of Sciences of
the United States of America 97 (2000) 925e930.
[20] L. Di Nunno, P. Vitale, A. Scilimati, S. Tacconelli, P. Patrignani, Journal of Me-
dicinal Chemistry 47 (2004) 4881e4890.
[21] T.D. Penning, J.J. Talley, S.R. Bertenshaw, J.S. Carter, P.W. Collins, S. Docter,
M.J. Graneto, L.F. Lee, J.W. Malecha, J.M. Miyashiro, R.S. Rogers, D.J. Rogier,
S.S. Yu, G.D. Anderson, E.G. Burton, J.N. Cogburn, S.A. Gregory, C.M. Koboldt,
[43] Y. Harrak, G. Casula, J. Basset, G. Rosell, S. Plescia, D. Raffa, M.G. Cusimano,
R. Pouplana, M.D. Pujol, Journal of Medicinal Chemistry 53 (2010) 6560e6571.
[44] Molinspiration Cheminformatics (http://www.molinspiration.com/cgi-bin/
properties).
[45] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Advanced Drug Delivery
Reviews 46 (2001) 3e26.
[46] P.R. Duchowicz, A. Talevi, C. Bellera, L.E. Bruno-Blanch, E.A. Castro, Bioorganic
and Medicinal Chemistry 15 (2007) 3711e3719.
[47] D.B. Jack, J.L. Hawker, L. Rooney, M. Beerahee, J. Lobo, P. Patel, Journal of
Chromatography 452 (1988) 257e264.
[48] E. Spath, W. Gruber, Ber 71 (1938) 106e113.
[49] A. Schönberg, A. Sina, Journal of the American Chemical Society 72 (1950)
1611e1616.
[50] A. Schönberg, N. Badran, N.A. Starkowsky, Journal of the American Chemical
Society 77 (1955) 5430e5431.
[51] A. Schönberg, N. Badran, N.A. Starkowsky, Journal of the American Chemical
Society 75 (1953) 4992e4995.
[52] O.M. Abdel Hafez, K.H.M. Ahmed, E.E. Haggag, Molecules 6 (2001) 396e405.
[53] M. Zimmermann, Pain 6 (1983) 109e110.