Structure-based design of phthalimide derivatives as potential cyclooxygenase-2 (COX-2) inhibitors: Anti-inflammatory and analgesic activities

Article abstract of DOI:10.1016/j.ejmech.2014.12.039

A group of 30 cyclic imides (1-10a-c) was designed for evaluation as a selective COX-2 inhibitor and investigated in vivo for anti-inflammatory and analgesic activities. Compounds 6a, 6b, 7a and 7b exhibit optimal COX-2 inhibitory potency (IC₅₀ Combining double low line 0.18, 0.24, 0.28 and 0.36 μM; respectively) and selectivity index (SI) range of 363-668. In vitro COX-1/COX-2 inhibition structure-activity studies identified compound 6a as a highly potent (IC₅₀ Combining double low line 0.18 μM), and an extremely selective [COX-2 (SI) Combining double low line 668] comparable to celecoxib [COX-2 (SI) > 384], COX-2 inhibitor that showed superior anti-inflammatory activity (ED₅₀ Combining double low line 54.0 mg/kg) relative to diclofenac (ED₅₀ Combining double low line 114 mg/kg). Molecular Docking study of the synthesized compound 6a into the active site of COX-2 revealed a similar binding mode to SC-558, a selective COX-2 inhibitor. Docking study showed that the methoxy moeities of 6a inserted deep inside the 2°-pocket of the COX-2 active site, where the O-atoms of such groups underwent an H-bonding interaction with His⁹⁰ (3.02 g.,), Arg⁵¹³ (1.94, 2.83 g.,), and Gln¹⁹² (3.25 g.,).

Full text of DOI:10.1016/j.ejmech.2014.12.039

European Journal of Medicinal Chemistry 92 (2015) 115e123
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structure-based design of phthalimide derivatives as potential
cyclooxygenase-2 (COX-2) inhibitors: Anti-inflammatory and
analgesic activities
,
Amer M. Alanazi a, Adel S. El-Azab a b, Ibrahim A. Al-Suwaidan a, Kamal Eldin H. ElTahir c,
, e, *
Yousif A. Asiri d, Naglaa I. Abdel-Aziz e, Alaa A.-M. Abdel-Aziz a
a Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
b Department of Organic Chemistry, Faculty of Pharmacy, Al-Azhar University, Cairo 11884, Egypt
c Department of Pharmacology, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
d Department of Clinical Pharmacy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
e Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A group of 30 cyclic imides (1e10a-c) was designed for evaluation as a selective COX-2 inhibitor and
investigated in vivo for anti-inflammatory and analgesic activities. Compounds 6a, 6b, 7a and 7b exhibit
Received 19 September 2014
Received in revised form
22 December 2014
Accepted 23 December 2014
Available online 24 December 2014
optimal COX-2 inhibitory potency (IC50 ¼ 0.18, 0.24, 0.28 and 0.36
(SI) range of 363e668. In vitro COX-1/COX-2 inhibition structureeactivity studies identified compound
6a as a highly potent (IC50 ¼ 0.18 M), and an extremely selective [COX-2 (SI) ¼ 668] comparable to
celecoxib [COX-2 (SI) 384], COX-2 inhibitor that showed superior anti-inflammatory activity
mM; respectively) and selectivity index
m
>
(ED50 ¼ 54.0 mg/kg) relative to diclofenac (ED50 ¼ 114 mg/kg). Molecular Docking study of the syn-
thesized compound 6a into the active site of COX-2 revealed a similar binding mode to SC-558, a se-
lective COX-2 inhibitor. Docking study showed that the methoxy moeities of 6a inserted deep inside the
2ꢀ-pocket of the COX-2 active site, where the O-atoms of such groups underwent an H-bonding inter-
action with His90 (3.02 Å), Arg513 (1.94, 2.83 Å), and Gln192 (3.25 Å).
Keywords:
Phthalimides
COX-2 inhibitors
Anti-inflammatory activity
Analgesic activity
Molecular docking
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
responsible for maintaining homeostasis (gastric and renal integ-
rity) [7e9]. Whereas COX-2 induces inflammatory conditions and is
Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most
important class of widely used therapeutics for the treatment of
inflammation and pain [1,2]. The clinical use of traditional NSAIDs
for the treatment of inflammation and pain is often accompanied
by adverse gastrointestinal effects [1e3]. The pharmacological ef-
fects of NSAIDs are due to inhibition of a membrane enzyme called
cyclooxygenase (COX) which is involved in the prostaglandin
biosynthesis [4e13]. There are two isoforms, COX-1 and COX-2
which share the same substrates, produce the same products and
catalyze the same reaction using identical catalytic mechanisms,
but differ in inhibitor selectivity [4e9]. The isoform, COX-1 has
mainly a physiological role in kidneys and the stomach, and is
involved in the production of prostaglandins mediating pain
[10,11]. Inhibition of COX-1 is responsible for the adverse gastro-
intestinal and renal effects of NSAIDs while the inhibition of COX-2
accounts for NSAIDs' therapeutic effects. All classical NSAIDs, such
as aspirin and indomethacin are non selective inhibitors for both
COX-1 and COX-2, but bind more tightly to COX-1. In order to
prevent or decrease these side effects, a current strategy consists of
designing selective COX-2 inhibitors with an improved gastric
safety profile [14,15]. Several classes of compounds possessing se-
lective COX-2 inhibitory activity have been reported in the litera-
ture such celecoxib (A) and SC-558 (B) (Fig. 1) [16,17].
On the other hand, cyclic imides such as phthalimides possessed
structural features which conferred potential biological activity and
pharmaceutical use [18e21]. The various classes of cyclic imides
have received great attention due to their COX-1/2 inhibition, anti-
inflammatory, antihyperlipidemic and antitumor activities
[18e23]. Apart from biological activities; imide derivatives are
* Corresponding author. Department of Pharmaceutical Chemistry, College of
Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia.
E-mail address: alaa_moenes@yahoo.com (A.A.-M. Abdel-Aziz).
http://dx.doi.org/10.1016/j.ejmech.2014.12.039
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

116
A.M. Alanazi et al. / European Journal of Medicinal Chemistry 92 (2015) 115e123
Fig. 1. Representative examples of selective COX-2 inhibitors (A, B and C) and the designed cyclic imides (D).
useful in the reactions involving condensation, alkylation, acyla-
tion, and cyclocondensation [24].
We have recently reported on the synthesis and COX-2-
inhibiton of N-substituted cyclic imides [21,22] in which com-
pound C (Fig. 1) was proved to be potent COX-2 inhibitors with IC50
substituted benzyl amine with an acid anhydride in refluxing ace-
tic acid afforded the designed cyclic imides in satisfactory yields.
The structures of the isolated products 1e10a-c were established
on the basis of their spectral analyses.
value of 0.10
mM and an extremely selective [COX-2 (SI) ¼ 400] [21].
2.2. Biological activity
Accordingly, we now describe the synthesis, COX-1/2 inhibition,
anti-inflammatory and analgesic activities of a group of cyclic im-
ides 1e10a-c bearing 3,4,5-timethoxybenzyl, 4-methoxybenzyl, or
4-fluorobenzyl fragments, in conjunction with various substituents
(H, Me, NO2, Cl and t-butyl) at the cyclic imide core. The rationale
for testing of these cyclic imides (Fig.1, D) as COX-inhibitors was the
following: (i) compare the efficacy of the 3,4,5-trimethoxybenzyl
and 4-methoxybenzyl versus the 4-fluorobenzyl for the inhibitory
power against various isoforms, such as COX-1 and COX-2, in
compounds incorporating the same scaffold (i.e., succinimide;
phthalimide, etc.); (ii) delineate the structureeactivity relationship
(SAR) for the inhibition of these COX isoforms with compounds
incorporating cyclic imides with diversely substituted scaffolds.
Thus, in addition to the monocyclic succinimide (1a-c), derivatives
of tetrahydrophthalimide (2a-c), phthalimide (3a-c) as well as
phthalimide substituted with various moieties at the benzene core
(such as methyl-, tert-butyl-, dichloro-, tetrachloro- and nitro-
groups) of types 4-8a-c were also included in the study. Further-
more, derivatives incorporating the heterocyclic pyrazine-2,3-
dicarboximide (9a-c) or the bulkier naphthalene-1,10-
dicarboximide (10a-c) moieties were also included in the study,
in order to explore as much chemical space as possible.
2.2.1. COX inhibition
According to the aforementioned rationale, the synthesized
compounds are evaluated for their ability to inhibit COX-1 and COX-
2 using an ovine COX-1/COX-2 assay kit (Catalog No. 560101,
Cayman Chemicals Inc., Ann Arbor, MI, USA). IC50 (mM) is deter-
mined which is the means of two determinations acquired and the
deviation from the mean is <10% of the mean value [21,22,25]. The
selectivity index (SI values) was defined as IC50(COX-1)/IC50(COX-
2). In the assay system, the IC50 values of celecoxib on COX-1 and
COX-2 were determined to be > 100 and 0.26
indicating that celecoxib is a selective COX-2 inhibitor [COX-2
(SI) > 384.6]. The results showed that some of the tested com-
pounds had potent inhibition against COX-2 (IC50 y 0.18e8.5
compared to the inhibition for COX-1 (IC50 y 130.8 e >100 M) as
listed in Table 1. Nearly seven of the tested compounds (6a, 6b, 7a,
7b, 8b, 9a, and 10a) were found to be potent and selective against
COX-2. Interestingly, methoxy substituents on the N-benzyl group
play critical roles in the COX-inhibiting activity of the tested com-
pounds compared with fluoro derivatives.
mM respectively,
mM)
m
Compound 6a was the most potent inhibitor in this series with
the COX-2 inhibiting activity (IC50 ¼ 0.18
m
M, COX-2 (SI) ¼ 668.3), 2-
fold higher than celecoxib (IC50 ¼ 0.26
m
M, COX-2 (SI) > 384.6). The
2. Results and discussion
effects of substituents introduced into the phthaloyl moiety of
compounds 3e8 were revealed to be directional, being dependent
on the electronic nature of the substituents, that is introduction of a
nitro group at the 5-position (compound 6a) enhanced COX-2-
2.1. Chemistry
The preparation of target cyclic imides is shown in Scheme 1.
Classical condensation of 3,4,5-trimethoxybenzyl amine or 4-
inhibiting activity, resulting in
(SI ¼ 668.3). Moreover, introduction of two halogen atoms such as
a COX-2-selective inhibition

A.M. Alanazi et al. / European Journal of Medicinal Chemistry 92 (2015) 115e123
117
Scheme 1. Synthesis of the designed substituted cyclic imides 1e10.
dichloro derivative (compounds 7a-b) resulted in increasing the
COX-2-inhibitory activity, while introduction of an electron-
donating methyl or tert-butyl groups such as compounds 4 and 5
showed just the opposite effects, resulting in sharp decrease of a
COX-2 inhibition. The activities of some other cyclic imides analogs
(compounds 9e10) are shown in Table 1, among them, compounds
9a and 10a-b are a COX-2-selective inhibitor (SI¼ >5.8e13.7).
Diminishment of activity of compounds 1 and 2 may be explained
on the bases of non-aromatic feature of the imide scaffold indi-
cating the importance of aromatic fragment of imide core for the
activity. More interestingly, fluoro substituents on the N-benzyl
moiety showed loss or lower of COX-2 inhibition (compounds
1e10c) when compared with methoxy substituents (compounds
1e10a-b).
succinimide derivatives (1a and 1b), the tetrahydrophthalimides
(2a and 2b), the tetrachlorophthalimides (8a and 8b) or the het-
erocyclic pyrazineimides (9a and 9b), show different inhibition
profiles where the non-aromatic imides are much weaker in-
hibitors compared to the corresponding aromatic imides. For the
two different methoxy derivatives, generally the 3,4,5-trimethoxy
derivatives were slightly more inhibitory compared to the corre-
sponding 4-methoxy derivatives, but the differences of activity
were rather small (Table 1). It may be noted that diverse cyclic
imide scaffolds lead to a highly efficient COX-2 inhibitors and the
scaffolds leading to the best inhibition were just the 5-
nitrophthalimide and 5,6-dichlorophthalimide (compounds 6a-b
and 7a) which in fact possessed higher COX-2-selective inhibition
(SI ¼ 430e668) when compared with lead compound C reported
earlier (SI ¼ 400) [21].
The following SAR was observed for the inhibition of the COX-2
isoform mentioned above with derivatives 1e10a-c (Table 1): (i)
The COX-2 isoform was well inhibited by many of the investigated
compounds, such as 4a, 6a, 6b, 7a, 7b, 8b, 9a, 10a, and 10b, which
2.2.2. Anti-inflammatory activity
Anti-inflammatory activity of twenty one compounds, which
showed the specificity for COX-2 enzyme in the in vitro assay
including compounds 1a, 2a, 3-4a-c, 5a-b, 6a-c, 7a-b, 8a-b, and
9e10a-b, was evaluated by employing the well-known rat
carrageenan-induced foot paw oedema model (Table 2) [21,22,26].
The ED50 was measured after 2 h of treatment with carrageenan
(Table 2), at which maximum percentage of inhibition of
carrageenan-induced oedema was reached along with diclofenac
sodium and celecoxib, as a reference drugs. All of the ED50 values
were determined using three doses of 50, 100 and 200 mg/kg in the
test compounds and 25, 50 and 100 mg/kg in the reference drugs
diclofenac and celecoxib. Out of twenty one compounds, 10
showed IC50
showed more inhibitory than the standard drug celecoxib (IC50 of
0.26 M). It may be observed that both 3,4,5-trimethoxy and 4-
(mM) ranging between 0.18 and 24.0, some of them
m
methoxybenzyl derivatives belong to this category of effective
COX-2 inhibitors, while 4-fluorobenzyl seems to have been of little
importance for the biological activity. For example the 3,4,5-
trimethoxy and 4-methoxybenzyl pairs incorporating the same
scaffolds (5-nitrophthalimide and 5,6-dichlorophthalimide) 6a/6b
and 7a/7b, show similar inhibitory activity against COX-2 isoform,
suggesting a quite similar binding mode to the enzyme. However
other pairs differ considerably in their activity. For example the

118
A.M. Alanazi et al. / European Journal of Medicinal Chemistry 92 (2015) 115e123
Table 1
Table 2
In vitro COX-1/COX-2 enzyme inhibition assay.
In vivo anti-inflammatory activity against carrageenan-induced rat paw oedema and
analgesic activity represented by ED50 (mg/kg) of the designed compounds.
Compound No
IC50
(
m
M)a
SIb
Compound No
ED50; mg/kg (mmol/kg)
AIa
COX-1
COX-2
Analgesicb
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
6b
6c
7a
7b
7c
8a
8b
8c
>100
>100
>100
>100
>100
>100
>100
110
75.1
>100
>100
70.6
>100
>100
33.8
40.1
68.6
24.0
36.1
74.0
32.2
29.1
>100
0.18
0.24
31.1
0.28
0.36
>100
30.7
4.1
>100
>1.3
>1.0
>1.0
>1.4
>1.0
>1.0
>3.0
2.7
1a
2a
3a
3b
3c
4a
4b
4c
5a
5b
6a
6b
6c
7a
7b
8a
180.0
171.0
149.0
161.7
170.0
130.0
153.0
172.0
147.0
137.0
54.0
95.0
125.0
109.9
106.9
90.0
NDc
80.5
97.0
79.3
96.1
27.6
17.0
35.0
57.4
51.2
54.7
52.9
40.5
106.3
88.0
119.0
71.0
115.6
>100
1.7
>4.2
>2.8
>1.4
>3.1
>3.4
>1.0
668.3
494.2
>3.2
430.0
363.3
>1.0
>3.3
>24.4
>1.0
>11.8
3.0
>100
>100
>100
>100
>100
120.3
118.6
>100
120.4
130.8
>100
>100
>100
>100
>100
68.0
127.4
67.0
75.0
143.5
103.0
119.0
159.3.0
115.0
129.0
83.4
8b
9a
9b
10a
10b
Celecoxib
a
Anti-inflammatory activity represented by ED50 which was the effective dose
9a
9b
9c
8.5
38.7
>100
7.3
17.1
>100
calculated after 2 h.
117.1
b
Analgesic activity represented by ED50 which was the effective dose calculated
>100
>100
>100
>100
40.0
0.25
>100
>1.0
>13.7
>5.8
>1.0
400.0
after 60 min.
10a
10b
10c
C
Diclofenac
Celecoxib
c
Not determined.
0.1
4.2
0.26
contributed to the presence of methoxybenzyl fragments. Special
high analgesic activity was displayed by compounds 6a-c, which
exhibited potent analgesic activity almost more than three times
(17e35 mg/kg) as that of celecoxib (71.0 mg/kg). However, com-
pounds 2a, 3a-b, 9b and 10b (ED50 of 125.0, 109.9, 106.9, 106.3 and
119.0 mg/kg, respectively) appeared to have lower analgesic ac-
tivity in comparison with reference drug celecoxib.
0.06
>384.6
a
IC50 value is the compound concentration required to produce 50% inhibition of
COX-1or COX-2 for means of two determinations using an ovine COX-1/COX-2 assay
kit (catalog no. 560101, Cayman Chemicals Inc., Ann Arbor, MI, USA) and deviation
from the mean is <10% of the mean value.
b
Selectivity index (COX-1 IC50/COX-2 IC50).
2.3. Docking studies
compounds (4a, 6a, 6b, 6c, 7a, 7b, 8b, 9a, 10a and 10b) were found
to possess potent anti-inflammatory activity (ED50 of
54.0e130.0 mg/kg). Compound 6a showed the highest anti-
inflammatory activity among the tested compounds with ED50 of
54.0 mg/kg compared to diclofenac sodium (ED50 ¼ 114 mg/kg).
In general, cyclic imides containing 4-methoxybenzyl moiety
such as compounds 6b, 7b, 8b and 10b have shown higher anti-
inflammatory activity when compared with compounds contain-
ing 4-fluorobenzyl fragment at the same position (6c, 8b, 9b and
10b). These findings may be attributed to the importance of
methoxybenzyl fragment in COX receptor binding site. Briefly, in
the carrageenan-induced rat paw oedema assay model, compound
6a was the most potent anti-inflammatory agent (ED50 ¼ 54.0 mg/
Insights into the differences between the binding sites of COX-1
and COX-2 obtained from X-ray crystal structure data, provided
useful guidelines that facilitated the design of the selective COX-2
inhibitors [30e33]. For example, the COX-2 binding site possesses
an additional 2ꢀ-pocket that is absent in COX-1, which is highly
relevant to the design of selective COX-2 inhibitors. This COX-2 2ꢀ-
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 [34,35]. It has also been re-
ported 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 2ꢀ-pocket of COX-2 is controlled by
histidine (His90), glutamine (Gln192), and tyrosine (Tyr355) [34,35].
Interaction of Arg513 with the bound drug is a requirement for time
dependent inhibition of COX-2 [34,35].
kg) within this group of compounds at
administration.
2 h post-drug
2.2.3. Analgesic activity
To understand the COX-inhibiting behavior of the synthesized
compounds, automated docking studies were carried out using
MOE 2008.10 software installed on 2.3G Core i7 [36]. The scoring
function and a number of hydrogen bondings formed with the
surrounding amino acids are used to predict their binding modes,
binding affinities in the active sites of COX-2 enzyme. The level of
COX-2 inhibition of compound 6a prompted us to perform molec-
ular docking studies to understand the ligandeprotein interactions
in detail. For this study, the crystal structures of COX-2 enzymes
complexed with SC-558 [1CX2] were used for the docking [37]. The
The analgesic activity of the same selected compounds was
evaluated using the hot plate technique in mice after 60 min of
intraperitoneal injection at 58 ꢀC using celecoxib as a reference
drug [27e29]. A comparative study of the analgesic activity of the
test compounds relative to the reference drug at 60 min revealed
the following: the highest analgesic activity was recorded for
compounds 6a-c, 7a-b, 8a-b and 9a with ED50 of 27.6, 17.0, 35.0,
57.4, 51.2, 54.7, 52.9 and 40.5 mg/kg when compared with celecoxib
(ED50 of 71.0 mg/kg) after 60 min, respectively, this might be

A.M. Alanazi et al. / European Journal of Medicinal Chemistry 92 (2015) 115e123
119
active site of the enzyme was defined to include residues within a
10.0 Å radius to any of the inhibitor atoms and the scoring functions
for docked compounds were calculated from minimized ligande-
protein complexes. The most stable docking model was selected
according to the best scored conformation predicted by the MOE
scoring function. The compound 6a could dock into the active site
of COX-2 successfully (Fig. 2). Compound 6a produces a deep
moving into the hydrophilic pocket of COX-2 with which the
methoxy groups are able to reach the hydrophilic pocket and is
involved in hydrogen bonding with His90 (3.02 Å), Arg513 (1.94,
2.83 Å), and Gln192 (3.25 Å). Such interactions are almost essential
for COX-2 inhibitory activity, as exemplified by the binding inter-
action of SC-558, an analog of celecoxib cocrystallized in the COX-2
active site [37]. In addition; such interaction forces the imide core
to adopt a specific orientation at the top of the channel. This moiety
is involved in hydrophobic interaction with Trp387, Val523, Leu352
and Phe518. Moreover the two carbonyl moieties of the imide core
and the 5-nitro group were forming hydrogen bonds with Ala527
(3.03 Å), Val349 (2.70 Å), Gly526 (2.08 Å) and Tyr385 (2.84 Å)
respectively. The lateral pocket of COX-2 would therefore be
responsible for the COX-2 selectivity of 6a and contributed to sta-
bilize the ligandeenzyme complexes (Fig. 2).
are typical of selective inhibitors of COX-2, confirming the molec-
ular design of the reported class of anti-inflammatory imide de-
rivatives [21,22].
3. Conclusion
A group of 30 cyclic imides was synthesized and screened for
COX-1/COX-2 inhibition, anti-inflammatory and analgesic activities.
Compounds which showed significant COX-2 inhibition were sub-
jected to anti-inflammatory and analgesic studies. It was detected
that both 3,4,5-trimethoxybenzyl and 4-methoxybenzyl de-
rivatives were highly efficient COX-2 selective enzymes inhibitors
compared to the 4-fluorobenzyl incorporating the same scaffold.
Compounds 6a, 6b, 7a and 7b exhibit optimal COX-2 inhibitory
potency (IC50 ¼ 0.18, 0.24, 0.28 and 0.36
mM; respectively) and
selectivity (SI) 363e668] comparable with celecoxib and better
ED50 than diclofenac.
Molecular docking studies further help in understanding the
various interactions between the ligands and enzyme active sites in
detail and thereby help to design novel potent inhibitors. It is clear
that the methoxy moieties of 6a inserts deep inside the COX-2 2ꢀ-
pocket and forming hydrogen bonds with His90 (3.02 Å), Arg513
(1.94, 2.83 Å), and Gln192 (3.25 Å). This result was corroborated by
molecular docking studies with COX-2 inhibition, which showed
that this compound presents the pharmacophoric requisites for
COX-2 inhibition. Indeed molecular docking studies further sup-
ported the strong inhibitory activity of 6a and further help under-
standing the various interactions between the ligands and enzyme
active sites in detail and thereby help to design novel potent
inhibitors.
The complex generated by docking studies of 6a with COX-2 and
superimposition with the structure of the selective inhibitor, SC-
558, co-crystallized with COX-2, illustrated in Fig. 2, shows that
compound 6a 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 6a with COX-2 (Fig. 2) shows that the
methoxy groups of 6a hydrogen bonded to the amino acid residue
Gln192, His90 and Arg513, similarly to the sulfonyl moiety of the
pharmacophoric sulfonamide group pertaining to SC-558. More-
over, additional hydrogen bonds were observed among the
carbonyl oxygen and Val349 and Ala527, while NO2 moiety hydrogen
bonded with Gly526 and Tyr385. Additionally, N-benzyl system are
positioned in the same region as p-sulfonamido-phenyl ring of SC-
558, while, the aromatic ring of imide core of compound 6a is close
to the p-Br-phenyl ring of SC-558, in the aromatic region of the
4. Experimental
4.1. Chemistry
Melting points (uncorrected) were recorded on Barnstead 9100
Electrothermal melting apparatus. IR spectra were recorded on an
FT-IR PerkineElmer spectrometer. 1H NMR were recorded in
DMSO-d6 on Bruker 500 and 700 MHz instruments using TMS as
active site lined by aromatic amino acid residues such as Phe518
Tyr385 and Trp387, among others. In short, the described interactions
,
internal standard (chemical shifts in
d
ppm) and 13C NMR were
Fig. 2. Left panel showed docking of compound 6a into the active site of COX-2. Hydrogen bonds are shown in red. Right panel showed alignment of 6a (magenta) and selective
inhibitor SC-558 (green) in the active site of COX-2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

120
A.M. Alanazi et al. / European Journal of Medicinal Chemistry 92 (2015) 115e123
recorded in DMSO-d6 on Bruker 125 and 176 MHz instruments
using TMS as internal standard (chemical shifts in ppm). Mass
(s, 3H), 3.72 (s, 3H), 4.67 (s, 2H), 6.66 (s, 2H), 7.77e7.90 (m, 3H); 13
C
d
NMR: 21.30 (CH3), 41.02 (CH2-Benzylic), 55.80 (2CH3O), 59.92
(CH3O),104.87,123.09,123.60,128.85,131.84,132.39,134.81,136.79,
145.48, 152.86, 167.71 (C]O), 167.82 (C]O); C19H19NO5: m/z
(341.9). Anal. Calcd: C, 66.85; H, 5.61; N, 4.10. Founded: C, 66.34; H,
5.58; N, 4.03.
spectra were recorded on a Agilent 6320 Ion Trap mass spectrom-
eters. Elemental analysis was carried out for C, H and N at the
Research Centre of College of Pharmacy, King Saud University and
the results are within 0.4% of the theoretical values. Solvent
evaporation was performed under reduced pressure using Buchan
Rotatory Evaporator unless otherwise stated. Thin layer chroma-
tography was performed on precoated (0.25 mm) silica gel GF254
plates (E. Merck, Germany), compounds were detected with
254 nm UV lamp. Silica gel (60e230 mesh) was employed for
routine column chromatography separations. Compounds 1b-c, 2b,
3b-c, 6b-c, 9b and 10b-c were prepared according to their reported
procedure [20,38e47].
4.1.1.6. 2-(4-Methoxybenzyl)-5-methylisoindoline-1,3-dione
White powder, M.p 130e132ꢀ, 80% yield (CH2Cl2); IR (KBr, cmꢁ1
1765, 1701 (C]O); 1H NMR (DMSO-d6):
2.47 (s, 3H), 3.71 (s, 3H),
(4b).
) n:
d
4.67 (s, 2H), 6.87e6.88 (d, 2H, J ¼ 7.5 Hz), 7.23e7.24 (d, 2H,
J ¼ 7.5 Hz), 7.62e7.64 (d, 1H, J ¼ 7.5 Hz), 7.70 (s, 1H), 7.74e7.76 (d,
1H, J ¼ 7.5 Hz); 13C NMR: 21.31(CH3), 42.01(CH2-Benzylic), 55.03
(CH3O), 113.91, 123.04, 123.56, 128.74, 128.92, 131.86, 134.80, 145.45,
158.56, 167.61 (C]O), 167.70 (C]O); C17H15NO3: m/z (282.0). Anal.
Calcd: C, 72.58; H, 5.37; N, 4.98. Founded: C, 71.80; H, 5.30; N, 4.52.
4.1.1. General procedure for the synthesis of compounds 1e10
A solution of substituted benzyl amine (10 mmol) and an acid
anhydride (10 mmol) in glacial acetic acid (15 mL) was heated
under reflux for 4 h. After the evaporation of the reaction mixture
to dryness under reduced pressure, the residue was neutralised by a
solution of sodium bicarbonate (4%) until effervescence ceased. The
precipitate obtained was washed with water, dried and re-
crystallised from an appropriate solvent.
4.1.1.7. 2-(4-Fluorobenzyl)-5-methylisoindoline-1,3-dione
White crystals, M.p 125e126ꢀ, 92% yield (CH2Cl2/Hexane); IR (KBr,
cmꢁ1 : 1767, 1702 (C]O); 1H NMR (DMSO-d6):
2.46 (s, 3H), 4.72
(4c).
)
n
d
(s, 2H), 7.03e7.07 (q, 2H, J ¼ 8.5 Hz), 7.32e7.35 (q, 2H, J ¼ 8.5 Hz),
7.55e7.57 (d, 1H, J ¼ 7.5 Hz), 7.63 (s, 1H), 7.69e7.70 (d, 1H,
J ¼ 7.5 Hz); 13C NMR: 21.38 (CH3), 39.09 (CH2-Benzylic), 115.02,
115.19, 122.90, 123.47, 128.86, 129.61, 129.67, 131.84, 132.62, 132.64,
134.56, 145.18, 160.50, 162.45, 167.34 (C]O), 167.44 (C]O);
4.1.1.1. 1-(3,4,5-Trimethoxybenzyl)pyrrolidine-2,5-dione
(1a).
White crystals, M.p 122e123ꢀ, 89% yield (CH2Cl2/CH3OH); IR (KBr,
C16H12FNO2: m/z (270.0). Anal. Calcd: C, 71.37; H, 4.49; N, 5.20.
cmꢁ1
)
n
: 1770, 1700 (C]O); 1H NMR (DMSO-d6):
d
2.69 (s, 4H), 3.62,
Founded: C, 70.92; H, 4.16; N, 5.11.
(s, 3H), 3.74 (s, 6H), 4.47 (s, 2H), 6.55 (s, 2H); 13C NMR: 28.06 (2CH2),
41.47 (CH2-Benzylic), 55.83 (2CH3O), 59.90 (CH3O), 105.13, 131.97,
136.85, 152.79, 177.60 (2C]O); C14H17NO5: m/z (280.0). Anal. Calcd:
C, 60.21; H, 6.14; N, 5.02. Founded: C, 60.96; H, 7.01; N, 4.19.
4.1.1.8. 5-(tert-Butyl)-2-(3,4,5-trimethoxybenzyl)isoindoline-1,3-
dione (5a). White powder, M.p 119e120ꢀ, 87% yield (CH2Cl2); IR
(KBr, cmꢁ1 : 1765, 1704 (C]O); 1H NMR (DMSO-d6):
) n d 1.33 (s, 9H),
3.61 (s, 3H), 3.86 (s, 6H), 4.68 (s, 2H), 6.61 (s, 2H), 7.79e7.872 (q, 3H,
J ¼ 7.5, 8.0 Hz); 13C NMR: 30.69 (3CH3-tert-Butyl), 35.39 (C-tert-
Butyl), 41.08 (CH2-Benzylic), 55.83 (2CH3O), 59.92 (CH3O), 104.96,
119.97, 123.09, 128.98, 131.34, 131.80, 132.40, 136.81, 152.86, 158.21,
167.61 (C]O), 167.91 (C]O); C22H25NO5: m/z (384.0). Anal. Calcd:
C, 68.91; H, 6.57; N, 3.65. Founded: C, 68.82; H, 6.91; N, 3.63.
4.1.1.2. 2-(3,4,5-Trimethoxybenzyl)-3a,4,7,7a-tetrahydro-1H-iso-
indole-1,3(2H)-dione (2a). White crystals, M.p 147e148ꢀ, 93% yield
(CH2Cl2/CH3OH); IR (KBr, cmꢁ1 : 1777, 1704 (C]O); 1H NMR
) n
(DMSO-d6):
d
2.19e2.22 (d, 2H, J ¼ 15 Hz), 2.44e2.47 (d, 2H,
J ¼ 14.5 Hz), 3.23 (s, 2H), 3.61 (s, 3H), 3.71 (s, 6H), 4.48 (s, 2H), 5.91
(s, 2H), 6.57 (s, 2H); 13C NMR: 23.12 (2CH2), 38.63(2CH), 41.58 (CH2-
Benzylic), 55.75 (2 CH3O), 59.91(CH3O), 103.82, 127.89, 131.66,
136.46, 152.81, 180.03 (2C]O); C18H21NO5: m/z (332.0). Anal. Calcd:
C, 65.24; H, 6.39; N, 4.23. Founded: C, 65.80; H, 7.28; N, 4.30.
4.1.1.9. 5-(tert-Butyl)-2-(4-methoxybenzyl)isoindoline-1,3-dione
(5b). White powder, M.p 106e107ꢀ, 79% yield (CH2Cl2); IR (KBr,
cmꢁ1 : 1765, 1715 (C]O); 1H NMR (DMSO-d6):
) n d 1.32 (s, 9H), 3.70
(s, 3H), 4.68 (s, 2H), 6.86e6.87 (d, 2H, J ¼ 8.5 Hz), 7.22e7.24 (d, 2H,
J ¼ 8.5 Hz), 7.78e7.79 (d, 1H, J ¼ 8.0 Hz), 7.83e7.84 (d, 2H,
J ¼ 3.5 Hz); 13C NMR: 30.68 (3CH3-tert-Butyl), 35.38 (C-tert-Butyl),
40.24 (CH2-Benzylic), 55.01 (CH3O), 113.88, 119.94, 123.01, 128.73,
128.87, 128.98, 131.27, 131.79, 158.17, 158.55, 167.46 (C]O), 167.77
(C]O); C20H21NO3: m/z (324.1). Anal. Calcd: C, 74.28; H, 6.55; N,
4.33. Founded: C, 73.34; H, 7.03; N, 4.12.
4.1.1.3. 2-(4-Fluorobenzyl)-3a,4,7,7a-tetrahydro-1H-isoindole-
1,3(2H)-dione (2c). White crystals, M.p 75e77ꢀ, 78% yield (CH2Cl2/
CH3OH); IR (KBr, cmꢁ1 : 1767, 1695 (C]O); 1H NMR (DMSO-d6):
) n
d
2.17e2.18 (t, 2H, J ¼ 4.5 Hz), 2.20e2.17 (t, 2H, J ¼ 4.5 Hz), 3.18e3.20
(t, 2H, J ¼ 3.0 Hz), 4.46 (s, 2H), 5.85e5.86 (t, 2H, J ¼ 3.0 Hz),
6.85e6.87 (d, 2H, J ¼ 8.5 Hz), 7.10e7.12 (d, 2H, J ¼ 8.5 Hz); 13C NMR:
23.08 (2CH2), 38.55 (2CH), 40.98 (CH2-Benzylic), 113.76, 127.71,
128.10, 128.65, 158.47, 179.82 (2C]O); C15H14FNO2: m/z (260.0).
Anal. Calcd: C, 69.49; H, 5.44; N, 5.40. Founded: C, 69.89; H, 5.31; N,
5.30.
4.1.1.10. 5-(tert-Butyl)-2-(4-fluorobenzyl)isoindoline-1,3-dione (5c).
White powder, M.p 80e82ꢀ, 77% yield (CH2Cl2/CH3OH); IR (KBr,
cmꢁ1 : 1772, 1717 (C]O); 1H NMR (DMSO-d6):
) n d 1.34 (s, 9H), 4.74
(s, 2H), 7.09 (s, 2H), 7.33e7.34 (t, 2H, J ¼ 5.5 Hz), 7.79e7.84 (t, 3H,
J ¼ 6.0 Hz); 13C NMR: 30.73 (3CH3-tert-Butyl), 35.37 (C-tert-Butyl),
40.07 (CH2-Benzylic), 115.09, 115.26, 119.97, 123.01, 128.96,129.52,
129.59, 131.20, 131.78, 132.77, 158.15, 160.47, 162.41, 167.35 (C]O),
167.66 (C]O); C19H18FNO2: m/z (312.1). Anal. Calcd: C, 73.30; H,
5.83; N, 4.50. Founded: C, 72.89; H, 5.74; N, 4.50.
4.1.1.4. 2-(3,4,5-Trimethoxybenzyl)isoindoline-1,3-dione
(3a).
White powder, M.p 138e140ꢀ, 90% yield (CH2Cl2/CH3OH); IR (KBr,
cmꢁ1 : 1766, 1708 (C]O); 1H NMR (DMSO-d6):
) n d 3.61 (s, 3H), 3.72
(s, 6H), 4.69 (s, 2H), 6.617 (s, 2H), 7.86e7.88 (d, 4H, J ¼ 13.5 Hz); 13
C
NMR: 41.11(CH2-Benzylic), 55.83 (2CH3O), 59.93 (CH3O), 104.95.
123.20, 131.50, 132.31, 134.52, 136.80, 152.86, 167.77 (2C]O);
C
18H17NO5: m/z (328.0). Anal. Calcd: C, 66.05; H, 5.23; N, 4.28.
4.1.1.11. 5-Nitro-2-(3,4,5-trimethoxybenzyl)isoindoline-1,3-dione
Founded: C, 66.81; H, 5.42; N, 4.19.
(6a). Yellow crystals, M.p 155e156ꢀ, 71% yield (CH2Cl2/CH3OH); IR
(KBr, cmꢁ1 : 1778,1719 (C]O); 1H NMR (DMSO-d6):
) n d 3.64 (s, 3H),
4.1.1.5. 5-Methyl-2-(3,4,5-trimethoxybenzyl)isoindoline-1,3-dione
3.77 (s, 6H), 4.72 (s, 2H), 6.67 (s, 2H), 8.07e8.08 (d, 1H, J ¼ 8.0 Hz),
8.19e8.20 (d, 1H, J ¼ 7.5 Hz), 8.28e8.29 (d, 1H, J ¼ 8.0 Hz); 13C NMR:
41.70 (CH2-Benzylic), 55.90 (2CH3O), 59.92 (CH3O), 105.25, 105.48,
(4a). White crystals, M.p 140e141ꢀ, 88% yield (CH2Cl2); IR (KBr,
cmꢁ1 : 1768, 1700 (C]O); 1H NMR (DMSO-d6):
) n d 2.46 (s, 3H), 3.61

A.M. Alanazi et al. / European Journal of Medicinal Chemistry 92 (2015) 115e123
121
123.20, 126.96, 128.32, 131.72, 133.63, 136.12, 137.00, 144.34, 152.91,
163.19 (C]O), 165.91 (C]O); C18H16N2O7: m/z (372.9). Anal. Calcd:
C, 58.07; H, 4.33; N, 7.52. Founded: C, 57.35; H, 4.59; N, 7.50.
4.1.1.19. 6-(4-Fluorobenzyl)-5H-pyrrolo[3,4-b]pyrazine-5,7(6H)-
dione (9c). White crystals M.p 105e106ꢀ, 67% yield (CH2Cl2/
CH3OH); IR (KBr, cmꢁ1 : 1772, 1707 (C]O), 1615 (C]N); 1H NMR
) n
(DMSO-d6):
d C
4.80 (s, 2H), 7.59 (s, 2H), 7.89 (s, 2H) 8.17 (s, 2H); 13
NMR: 40.44 (CH2-Benzylic), 126.19, 129.46, 133.44, 134.68, 134.94,
150.92, 166.90 (C]O), 167.46 (C]O). Anal. Calcd for C13H8FN3O2: C,
60.70; H, 3.14; N, 16.34. Founded: C, 60.77; H, 3.21; N, 16.31.
4.1.1.12. 5,6-Dichloro-2-(3,4,5-trimethoxybenzyl)isoindoline-1,3-
dione (7a). Yellow crystals, M.p 200e202ꢀ, 95% yield (CH2Cl2/
CH3OH); IR (KBr, cmꢁ1 : 1774, 1711 (C]O); 1H NMR (DMSO-d6):
) n
d
3.61 (s, 3H), 3.72 (s, 6H), 4.69 (s, 2H), 6.61 (s, 2H), 8.19 (s, 2H); 13
C
4.1.1.20. 2-(3,4,5-Trimethoxybenzyl)-1H-benzo[de]isoquinoline-
NMR: 41.47 (CH2-Benzylic), 55.85 (2CH3O), 59.93 (CH3O), 104.90,
125.34, 131.58, 131.87, 136.82, 137.29, 152.87, 166.01 (2C]O);
1,3(2H)-dione (10a). White crystals, M.p 170e171ꢀ, 79% yield
(CH2Cl2); IR (KBr, cmꢁ1 : 1771, 1718 (C]O); 1H NMR (DMSO-d6):
) n
C
18H15Cl2NO5: m/z (396.0). Anal. Calcd: C, 54.57; H, 3.82; N, 3.54.
d
3.614 (s, 3H), 3.716 (s, 6H), 5.18 (s, 2H), 6.699 (s, 2H), 7.87e7.93 (m,
Founded: C, 54.50; H, 3.09; N, 3.47.
2H), 8.52e8.56 (m, 4H); 13C NMR: 43.12 (CH2-Benzylic), 55.78
(2CH3O), 59.84 (CH3O), 105.27, 118.94, 121.92, 127.17, 127.46, 129.64,
130.88, 131.29, 132.36, 132.99, 134.39, 135.29, 136.65, 152.67, 160.60
(C]O), 163.48 (C]O); C22H19NO5: m/z (378. 0). Anal. Calcd: C,
70.02; H, 5.07; N, 3.71. Founded: C, 70.03; H, 5.68; N, 3.60.
4.1.1.13. 5,6-Dichloro-2-(4-methoxybenzyl)isoindoline-1,3-dione
(7b). White crystals, M.p 154e155ꢀ, 81% yield (CH2Cl2/CH3OH); IR
(KBr, cmꢁ1 : 1771, 1705 (C]O); 1H NMR (DMSO-d6):
) n d 3.75 (s, 3H),
4.69 (s, 2H), 6.88 (s, 2H), 7.25 (s, 2H), 8.21 (s, 2H); 13C NMR: 41.23
(CH2-Benzylic), 55.55 (CH3O), 114.40, 125.83, 128.72, 129.55, 132.01,
137.81, 159.15, 166.38 (2C]O). Anal. Calcd for C16H11Cl2NO3: C,
57.17; H, 3.30; N, 4.17. Founded: C, 57.08; H, 3.21; N, 4.09.
4.2. Biological assay
4.2.1. In vitro cyclooxygenase (COX) inhibition assay
The in vitro ability of test compounds and celecoxib to inhibit
the COX-1 and COX-2 isozymes was carried out using Cayman
colorimetric COX (ovine) inhibitor screening assay kit (kit catalog
number 560101, Cayman Chemical, Ann Arbor, MI, USA) according
to the manufacturer's instructions [21,22,25]. Briefly, to a series of
4.1.1.14. 5,6-Dichloro-2-(4-fluorobenzyl)isoindoline-1,3-dione (7c).
White crystals, M.p 165e166ꢀ, 84% yield (CH2Cl2/CH3OH); IR (KBr,
cmꢁ1 : 1771, 1736 (C]O); 1H NMR (DMSO-d6):
) n d 4.76 (s, 2H), 7.15
(s, 2H), 7.37 (s, 2H), 8.19 (s, 2H); 13C NMR: 41.03 (CH2-Benzylic),
115.74, 115.86, 125.87, 130.14, 130.19, 132.06, 132.93, 132.95, 137.83,
161.29, 162.67 (C]O), 166.39 (C]O). Anal. Calcd for C15H8Cl2FNO2:
C, 55.58; H, 2.49; N, 4.32. Founded: C, 55.57; H, 2.06; N, 4.21.
supplied reaction buffer solutions (960
containing 5 M EDTA and 2 M phenol) with either COX-1or COX-
2 (10 L) enzyme in the presence of heme (10 L) were added 10
of various concentrations of test drug solutions (0.01, 0.1, 1, 10, 50,
and 100 M in a final volume of 1 mL). These solutions were
incubated for a period of 5 min at 37 ꢀC after which 10
L of
Arachidonic Acid (100 M) solution were added and the COX re-
action was stopped by the addition of 50 L of 1 M HCl after 2 min.
mL, 0.1 M TriseHCl pH8.0
m
m
m
m
mL
m
4.1.1.15. 4,5,6,7-Tetrachloro-2-(3,4,5-trimethoxybenzyl)isoindoline-
1,3-dione (8a). Yellow powder, M.p 205e206ꢀ, 89% yield (CH3OH);
m
IR (KBr, cmꢁ1 : 1778, 1719 (C]O); 1H NMR (DMSO-d6):
) n d 3.63 (s,
m
3H), 3.75 (s, 6H), 4.70 (s, 2H), 6.64 (s, 2H); 13C NMR: 42.33 (CH2-
Benzylic), 56.45 (2CH3O), 60.43 (CH3O), 105.64, 128.59, 129.04,
131.94, 138.55, 153.38, 163.89 (2C]O). Anal. Calcd for
m
PGF2a, produced from PGH2 by reduction with stannous chloride
was measured by enzyme immunoassay. This assay is based on the
competition between PGs and a PG-acetylcholinesterase 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 pro-
portional to the concentration of PGs in the wells since the con-
centration 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
Ellman's reagent, which contains the substrate to acetylcholine
esterase, is added to the well. The product of this enzymatic reac-
tion produces a distinct yellow color that absorbs at 410 nm. The
intensity of this color, determined spectrophotometrically, is pro-
portional to the amount of PG tracer bound to the well, which is
inversely proportional to the amount of PGs present in the well
C
18H13Cl4NO5: C, 46.48; H, 2.82; N, 3.01. Founded: C, 47.09; H, 2.18;
N, 3.47.
4.1.1.16. 4,5,6,7-Tetrachloro-2-(4-methoxybenzyl)isoindoline-1,3-
dione (8b). White crystals, M.p 207e209ꢀ, 97% yield (CH3OH); IR
(KBr, cmꢁ1 : 1775,1709 (C]O); 1H NMR (DMSO-d6):
) n d 3.73 (s, 3H),
4.70 (s, 2H), 6.89- 6.91 (d, 2H, J ¼ 9.0 Hz), 7.27e7.29 (d, 2H,
J ¼ 8.5 Hz); 13C NMR: 41.15 (CH2-Benzylic), 54.83 (CH3O), 125.84,
127.96, 128.16, 128.41, 138.16, 139.60, 143.30, 163.22 (2C]O);
C
16H9Cl4NO3: m/z (403.5). Anal. Calcd: C, 47.44; H, 2.24; N, 3.46.
Founded: C, 47.49; H, 2.20; N, 3.47.
4.1.1.17. 4,5,6,7-Tetrachloro-2-(4-fluorobenzyl)isoindoline-1,3-dione
(8c). White powder, M.p 230e231ꢀ, 91% yield (CH3OH); IR (KBr,
during the incubation: Absorbance
a [Bound PG Tracer] a1/PGs.
cmꢁ1 : 1774, 1716 (C]O); 1H NMR (DMSO-d6):
) n d 4.72 (s, 2H),
Percent inhibition was calculated by the comparison of compound
treated to various control incubations. The concentration of the test
7.02e7.03 (d, 2H, J ¼ 8.5 Hz), 7.32e7.35 (t, 2H, J ¼ 7.5 Hz); 13C NMR:
40.92 (CH2-Benzylic), 115.04, 115.21, 127.95, 128.51, 129.96, 130.03,
131.60, 138.63, 162.89 (2C]O); C15H6Cl4FNO2: m/z (391.1). Anal.
Calcd: C, 45.84; H, 1.54; N, 3.56. Founded: C, 46.22; H, 1.05; N, 3.59.
compound causing 50% inhibition (IC50
,
mM) was calculated from
the concentrationeinhibition response
curve (duplicate
determinations).
4.1.1.18. 6-(3,4,5-Trimethoxybenzyl)-5H-pyrrolo[3,4-b]pyrazine-
4.2.2. Anti-inflammatory screening
5,7(6H)-dione (9a). White powder, M.p > 300ꢀ, 61% yield (CH2Cl2/
The test compounds were evaluated using in vivo rat
carrageenan-induced foot paw oedema model reported previously
[21,26]. Male SpraugeeDawley rats (250 gm body weight) were
fasted with free access to water at least 16 h prior to experiments.
CH3OH); IR (KBr, cmꢁ1 : 1705 (C]O), 1629 (C]N); 1H NMR
) n
(DMSO-d6):
d 3.68 (s, 3H), 3.78 (s, 6H), 4.20 (s, 2H), 6.55 (s, 2H),
8.186 (s, 2H); 13C NMR: 42.48 (CH2-Benzylic), 55.68 (2CH3O), 59.89
(CH3O), 104.57, 134.96, 136.33, 152.68, 169.27 (C]O), 172.97 (C]O);
Oedema was produced by injecting 0.2 mL of a solution of 1% l-
C
16H15N3O5: m/z (330.0). Anal. Calcd: C, 58.36; H, 4.59; N, 12.76.
carrageenan in the hind paw. Paw volume was measured by water
displacement with a plethysmometer (UGO BASILE) before, 1 and
Founded: C, 58.70; H, 4.12; N, 12.16.

122
A.M. Alanazi et al. / European Journal of Medicinal Chemistry 92 (2015) 115e123
2 h after treatment. The compounds were administered intraperi-
toneally with a 1 mL suspension of test compound in vehicle (0.5%
methyl cellulose). Diclofenac and celecoxib were used as a standard
drugs. The positive control group animals received the reference
drug while the negative control received only the vehicle. The
percentage was calculated by the following equation: anti-
inflammatory activity (%) ¼ (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.
Acknowledgments
The authors extend their appreciation to the Deanship of Sci-
entific Research at King Saud University for funding the work
through the research group project No. RGP-VPP-163.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.12.039.
4.2.3. Analgesic screening
References
Male albino Swiss mice (25 gm body weight) were divided into
various groups. Each mouse was initially placed on a hot plate
thermostatically maintained at 58 ꢀC [27e29]. The mouse was
watched carefully for the time in seconds in which it displays
nociceptive responses exhibited as licking or blowing its front
paws. This time was considered as the control reaction time. A cut-
off time of 60 s was used to avoid damage to the paws. To test the
analgesic activity of the compounds each group of mice was treated
with one dose of the test compounds (5e200 mg/kg, ip). The re-
action time was then retested at 15, 30 and 60 min after injection
(each animal acted as its own control). The percentage changes in
the reaction were then calculated. The ED50 for each compound was
then calculated by linear regression.
[1] S. Fiorucci, R. Meli, M. Bucci, G. Cirino, Biochem.. Pharmacol. 62 (2001) 1433.
[2] D.A. Williams, T.L. Lemke, Non-steroidal Anti-inflammatory Drugs, in: Foye's
Principles of Medicinal Chemistry, fifth ed., Lippincott Williams& Wilkins,
2002, pp. 751e793.
€
[3] G. Dannhardt, W. Kiefer, G. Kramer, S. Maehrlein, U. Nowe, B. Fiebich, Eur. J.
Med. Chem. 35 (2000) 499.
[4] B. Balaji, S. Hariharan, D.B. Shah, M. Ramanathan, Eur. J. Med. Chem. 86 (2014)
469.
[5] P. Sarnpitak, P. Mujumdar, C. Morisseau, S.H. Hwang, B. Hammock,
V. Iurchenko, S. Zozulya, A. Gavalas, A. Geronikaki, Y. Ivanenkov, M. Krasavin,
Eur. J. Med. Chem. 84 (2014) 160.
[6] S. Bansal, M. Bala, S.K. Suthar, S. Choudhary, S. Bhattacharya, V. Bhardwaj,
S. Singla, A. Joseph, Eur. J. Med. Chem. 80 (2014) 167.
[7] J.R. Vane, Y.S. Bakhle, R.M. Botting, Ann. Rev. Pharmacol. Toxicol. 38 (1998)
9704.
[8] O. Laneuville, D.K. Breuer, D.L. DeWitt, T. Hla, C.D. Funk, W.L. Smith,
J. Pharmacol. Exp. Ther. 271 (1994) 927.
[9] D.A. Kujubu, B.S. Fletcher, B.C. Varnum, R.W. Lim, H.R. Herschman, J. Biol.
Chem. 266 (1991) 12866.
4.3. Docking methodology
[10] L.J. Crofford, J. Rheumatol. 24 (Suppl. 49) (1997) 15.
[11] K. Seibert, Y. Zhang, K. Leahy, S. Hauser, J. Masferrer, W. Perkins, L. Lee,
P. Isakson, Proc. Natl. Acad. Sci. 91 (1994) 12013.
[12] M.A.-A. El-Sayed, N.I. Abdel-Aziz, A.A.-M. Abdel-Aziz, A.S. El-Azab, Y.A. Asiri,
K.E.H. ElTahir, Bioorg. Med. Chem. 19 (2011) 3416.
Molecular modeling studies were performed with MOE 2008.10,
software available from Chemical Computing Group Inc., 1010
Sherbrooke Street West, Suite 910, Montreal, QC.
[13] M.A.-A. El-Sayed, N.I. Abdel-Aziz, A.A.-M. Abdel-Aziz, A.S. El-Azab,
K.E.H. ElTahir, Bioorg. Med. Chem. 20 (2012) 3306.
4.3.1. Selection of protein crystal structure
[14] N.S. Buttar, K.K. Wang, Mayo Clin. Proc. 75 (2000) 1027.
[15] C. Michaux, C. Charlier, Mini-Reviews Med. Chem. 4 (2004) 603.
[16] 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,
W.E. Perkins, K. Seibert, A.W. Veenhuizen, Y.Y. Zhang, P.C. Isakson, J. Med.
Chem. 40 (1997) 1347.
[17] 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) 644.
[18] T. Noguchi, R. Shimazawa, K. Nagasawa, Y. Hashimoto, Bioorg. Med. Chem.
Lett. 12 (2002) 1043.
[19] H. Sano, T. Noguchi, A. Tanatani, Y. Hashimoto, H. Miyachi, Bioorg. Med. Chem.
13 (2005) 3079.
[20] A.A.-M. Abdel-Aziz, Eur. J. Med. Chem. 42 (2007) 612.
[21] A.A.-M. Abdel-Aziz, K.E.H. ElTahir, Y.A. Asiri, Eur. J. Med. Chem. 46 (2011)
1648.
[22] I.A. Al-Suwaidan, A.M. Alanazi, A.S. El-Azab, A.M. Al-Obaid, K.E. ElTahir,
A.R. Maarouf, M.A. Abu El-Enin, A.A.-M. Abdel-Aziz, Bioorg. Med. Chem. Lett.
23 (2013) 2601.
Ligand-bound crystallographic structures of cyclooxygenase
(COX-2) is available in the Protein Data Bank (http://www.rcsb.org/
pdb/home/home.do). In this study, COX-2 complexed with SC-558
(1CX2) was evaluated and selected for docking. The errors of the
protein were corrected by the structure preparation process in
MOE. The first step in the generation of suitable protein structures
is the assignment of hydrogen positions on the basis of default
rules. All bound waters and cofactors contained in the PDB file have
been removed. Finally, partial charges (the Gasteiger methodology)
were calculated, and the active site of the ensemble has been
defined as the collection of residues within 10.0 Å of the bound
inhibitor and comprised the union of all ligands of the ensemble. All
atoms located less than 10.0 Å from any ligand atom were
considered.
4.3.2. Preparation of the ligand
[23] A.A.-M. Abdel-Aziz, A.S. El-Azab, S.M. Attia, A.M. Al-Obaid, M.A. Al-Omar,
H.I. El-Subbagh, Eur. J. Med. Chem. 46 (2011) 4324.
The ligand coordinates were built using the builder tool of the
MOE program. Next, the correct atom types (including hybridiza-
tion states) and correct bond types were defined, hydrogen atoms
were added, charges were assigned to each atom, and finally the
structures were energy minimized (MMFF94x, gradient: 0.01) [48].
The energies of ligand structures were previously minimized using
the semi-empirical AM1 method [49] with MOE program [36].
[24] K.C. Nicolaou, C.J.N. Mathison, Angew. Chem. 44 (2005) 5992.
[25] M.J. Uddin, P.N. Praveen Rao, E.E. Knaus, Bioorg. Med. Chem. 12 (2004) 5929.
[26] C.A. Winter, E.A. Risely, G.W. Nuss, Proc. Soc. Exp. Biol. 111 (1962) 544.
[27] L. Ohlsson, Acta Pharmacol. Toxicol. (Copenh). 9 (1953) 322.
[28] G.E. Leighton, M.A. Johnson, K.G. Meecham, R.G. Hill, J. Hughes, Br. J. Phar-
macol. 92 (1987) 915.
[29] H.A. Abdel-Aziz, K.A. Al-Rashood, K.E.H. ElTahir, G.M. Suddek, Eur. J. Med.
Chem. 80 (2014) 416.
[30] S.M. Sondhi, R. Rani, P. Roy, S.K. Agrawal, A.K. Saxena, Bioorg. Med. Chem. Lett.
19 (2009) 1534.
[31] L.M. Lima, P. Castro, A.L. Machado, C.A. Fraga, C. Lugnier, V.L. de Moraes,
E.J. Barreiro, Bioorg. Med. Chem. 10 (2002) 3067.
[32] J. Grover, V. Kumar, V. Singh, K. Bairwa, M.E. Sobhia, S.M. Jachak, Eur.J. Med.
Chem. 80 (2014) 47e56.
[33] M.J. Uddin, A.V. Elleman, K. Ghebreselasie, C.K. Daniel, B.C. Crews, K.D. Nance,
T. Huda, L.J. Marnett, ACS. Med. Chem. Lett. 5 (2014) 1254.
[34] O. Llorens, J.L. Perez, A. Palomer, D. Mauleon, Bioorg. Med. Chem. Lett. 9
(1999) 2779.
[35] R.M. Garavito, D.L. DeWitt, Biochem. Biophys. Acta 1441 (1999) 278.
[36] MOE, 10 of Chemical Computing Group Inc, 2008.
4.3.3. Docking experiment
The docking experiment on COX-2 (1CX2) was carried out by
superimposing the energy minimized ligand on SC558 in the PDB
file 1CX2, after which SC-558 was deleted. The default Triangle
Matcher placement method was used for docking. GBVI/WSA dG
scoring function which estimates the free energy of binding of the
ligand from a given pose was used to rank the final poses. The
ligandeenzyme complex with lowest S_score was selected.

A.M. Alanazi et al. / European Journal of Medicinal Chemistry 92 (2015) 115e123
[37] R.G. Kurumbail, A.M. Stevens, J.K. Gierse, J.J. McDonald, R.A. Stegeman, J.Y. Pak, (1992) 501.
123
D. Gildehaus, J.M. Miyashiro, T.D. Penning, K. Seibert, P.C. Isakson,
W.C. Stallings, Nature 384 (1996) 644.
[38] F. Chimenti, S. Vomero, Farm. Ed. Sci. 30 (1975) 884.
[39] K. Yoshiaki, T. Masumi, A. Kazuo, Chem. Pharm. Bull. 47 (1999) 529.
[40] B.B.C. Costa, R. Correa, M.M. De Souza, J.B. Pretto, J.V. Ardenghi, F. De Campos-
Buzzi, F.V. Cechinel, Zeitschrift fuer Naturforschung C: J. Biosci. 62 (2007) 201.
[41] J. Kim, S.H. Hong, Org. Lett. 16 (2014) 4404.
[45] M.P. Costi, D. Tondi, M. Rinaldi, D. Barlocco, G. Cignarella, D.V. Santi, C. Musiu,
I. Pudu, G. Vacca, P. La Colla, Eur. J. Med. Chem. 31 (1996) 1011.
[46] J. Zhang, M. Senthilkumar, S.C. Ghosh, S.H. Hong, Angew. Chem. 49 (2010)
6391.
[47] X.-F. Wu, S. Oschatz, M. Sharif, A. Flader, L. Krey, M. Beller, P. Langer, Adv.
Synthesis Catal. 355 (2013) 3581.
[48] T.A. Halgren, J. Comp. Chem. 17 (1996) 490.
[42] H. Takahashi, Acta Crystallogr. Sect. E Struct. Rep. Online 68 (2012) o2457.
[43] H. Tsubouchi, K. Tsuji, H. Ishikawa, Synlett 1 (1994) 63.
[49] M.J.S. Dewar, E.G. Zoebisch, E.F. Healey, J.J.P. Stewart, J. Am. Chem. Soc. 107
(1985) 3902.
[44] T. Takata, Y.Z. Menceloglu, T. Endo, J. Polym. Sci. Part A: Polym. Chem. 30