Synthesis, anti-inflammatory activity and COX-1/COX-2 inhibition of novel substituted cyclic imides. Part 1: Molecular docking study

Article abstract of DOI:10.1016/j.ejmech.2011.02.013

A group of cyclic imides (1-13) was designed for evaluation as selective COX-2 inhibitors and investigated in vivo for their anti-inflammatory activities using carrageenan-induced rat paw edema model. Compounds 5b, 6b, 11b, 11c, 12b and 12c were proved to be potent COX-2 inhibitors with IC₅₀ range of 0.1-1.0 μM. In vitro COX-1/COX-2 inhibition structure-activity studies identified compound 5b as a highly potent (IC₅₀ = 0.1 μM), and an extremely selective [COX-2 (SI) = 400] comparable to celecoxib [COX-2 (SI) > 333.3], COX-2 inhibitor that showed superior anti-inflammatory activity (ED ₅₀ = 104 mg/kg) relative to diclofenac (ED₅₀ = 114 mg/kg). A Virtual screening was carried out through docking the designed compounds into the COX-2 binding site to predict if these compounds have analogous binding mode to the COX-2 inhibitors. Molecular modeling (docking) study showed that the CH₃O substituents of 5b inserted deep inside the 2°-pocket of the COX-2 active site, where the O-atoms of such group underwent a H-bonding interaction with His⁹⁰ (2.43, 2.83 ), Arg⁵¹³ (2.89 ) and Tyr³⁵⁵ (3.34 ). Docking study of the synthesized compound 5b into the active site of COX-2 revealed a similar binding mode to SC-558, a selective COX-2 inhibitor.

Full text of DOI:10.1016/j.ejmech.2011.02.013

European Journal of Medicinal Chemistry 46 (2011) 1648e1655
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, anti-inflammatory activity and COX-1/COX-2 inhibition of novel
substituted cyclic imides. Part 1: Molecular docking study
,
b
,
c
d
Alaa A.-M. Abdel-Aziz a , Kamal E.H. ElTahir , Yousif A. Asiri
*
a Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
b Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, 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
a r t i c l e i n f o
a b s t r a c t
Article history:
A group of cyclic imides (1e13) was designed for evaluation as selective COX-2 inhibitors and investi-
gated in vivo for their anti-inflammatory activities using carrageenan-induced rat paw edema model.
Compounds 5b, 6b, 11b, 11c, 12b and 12c were proved to be potent COX-2 inhibitors with IC50 range of 0.1
Received 1 November 2010
Received in revised form
4 February 2011
Accepted 9 February 2011
Available online 15 February 2011
e1.0
m
M. In vitro COX-1/COX-2 inhibition structureeactivity studies identified compound 5b as a highly
M), and an extremely selective [COX-2 (SI) ¼ 400] comparable to celecoxib [COX-2
potent (IC50 ¼ 0.1
m
(SI) > 333.3], COX-2 inhibitor that showed superior anti-inflammatory activity (ED50 ¼ 104 mg/kg)
relative to diclofenac (ED50 ¼ 114 mg/kg). A Virtual screening was carried out through docking the
designed compounds into the COX-2 binding site to predict if these compounds have analogous binding
mode to the COX-2 inhibitors. Molecular modeling (docking) study showed that the CH3O substituents of
5b inserted deep inside the 2ꢀ-pocket of the COX-2 active site, where the O-atoms of such group
underwent a H-bonding interaction with His90 (2.43, 2.83 Å), Arg513 (2.89 Å) and Tyr355 (3.34 Å). Docking
study of the synthesized compound 5b into the active site of COX-2 revealed a similar binding mode to
SC-558, a selective COX-2 inhibitor.
Keywords:
Cyclic imides
COX-2 inhibitors
Anti-inflammatory activities
Molecular docking
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
consists of designing selective COX-2 inhibitors with an improved
gastric safety profile [10,11]. The improved safety profile of COX-2
Inflammation is a normal response to any noxious stimulus that
threatens the host and may vary from a localized response to
a generalized one [1]. Non-steroidal anti-inflammatory drugs
(NSAIDs) are widely used for the treatment of rheumatism diseases,
as rheumatoid arthritis, and pain [2,3]. The pharmacological effects
of NSAIDs are due to inhibition of a membrane enzyme called
cyclooxygenase (COX) which is involved in the prostaglandin
biosynthesis [4]. There are two isoforms, COX-1 and COX-2, helped
to understand the side-effects of NSAIDs [5]. The constitutive COX-1
is found in healthy populations and has mainly a physiological role
in the kidneys and the stomach. In contrast, the mainly inducible
COX-2 is involved in the production of prostaglandins mediating
pain and supporting the inflammatory process [6,7]. Classical
NSAIDs such as flurbiprofen (Fig. 1A) nonselectively inhibit both
isoenzymes and cause gastric failure like bleeding and ulcer [8,9]. In
order to prevent or decrease these side-effects, a current strategy
inhibitors may allow the use of these new agents for long-term
prophylactic use in certain chronic diseases [4,10,11]. This has led
intense efforts in search for potent and selective COX-2 inhibitors
which could provide anti-inflammatory drugs with fewer risks.
Several classes of compounds having selective COX-2 inhibitory
activity have been reported in the literature such as SC-558 [12] and
celecoxib [13] (Fig. 1B and C respectively).
On the other hand the cyclic imido group is a common protecting
group for amines and is widely used in carbohydrate chemistry. As
a known pharmacophore, the phthalimido moiety also has a wide
range of activities such as antitumor, [14,15] and anti-inflammatory
activities [16,17]. Systematic investigation of phthalimide analogs
revealed that derivatives bearing a spherical alkyl group, such as an
adamantyl group or a carboranyl group possess potent bi-direc-
tional TNF-
a (tumor necrosis factor-a) production-regulating
activity [18,19]. Recently it is reported that thalidomide directly
inhibits COX-1/COX-2 with efficacy comparable to that of the
representative drug, aspirin [20,21]. Such earlier work on the COX-
inhibiting activity of thalidomide afforded a new scaffold for small-
molecular COX inhibitors, such as compound (D), which should offer
opportunities for various kinds of structural development (Fig. 1).
* Corresponding author. Department of Medicinal Chemistry, Faculty of Phar-
macy, University of Mansoura, Mansoura 35516, Egypt. Tel.: þ966 56 2947305;
fax: þ966 1 4676220.
E-mail address: alaa_moenes@yahoo.com (A.A.-M. Abdel-Aziz).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.013

A.A.-M. Abdel-Aziz et al. / European Journal of Medicinal Chemistry 46 (2011) 1648e1655
1649
NH2
S
O
COOH
H2N
S
O
O
O
N
N
N
H3C
O
CH3
O
CF3
N
F
COX-2 selectivity
CF3
N
Fragment replacement
H3C
Br
SC-558 ( )
New scaffold f or small
molecular COX inhibitors
C
Celecoxib ( )
B
A
Flurbiprofen ( )
N-(m,m-Dimethylphenyl)
R1
R1
D
phthalimide ( )
R1
n
o
ti
a
z
i
Fragment replacement
m
i
t
p
o
d
a
e
L
N
O
O
R2
Designed cyclic
E
imides ( )
Fig. 1. Representative examples of nonselective (A) and selective (B, C and D) COX-2 inhibitors and the designed cyclic imides (E).
Thus our main objective is to design novel cyclic imides as
specific inhibitors of COX-2 in the hope that these molecules may
be further explored as powerful and novel non-ulcerogenic anti-
inflammatory lead-candidates. Our strategy is intended to obtain
potent anti-inflammatory activity with selective inhibition of COX-
2 using traditional medicinal chemistry techniques motivated by
the comparative modeling of a COX-1 and -2 complexed with A and
B together with the available pharmacophore.
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of compounds 1e13 (Scheme 1)
The preparation of target cyclic imides is shown in Scheme 1.
Classical condensation of trisubstituted aniline with acid anhy-
drides in refluxing acetic acid (method A) afforded these imides in
satisfactory yields that could sometimes be increased by adding
acetic anhydride or by heating the mixture at 160e200 ꢀC in the
absence of solvent (method B). The structures of the isolated
products 1e13 were established on the basis of their elemental and
spectral analyses.
In view of the previous rationale and in continuation of an
ongoing program aiming at finding new structure leads with
potential anti-inflammatory activities and selective COX-2 inhibi-
tion [22], new series of cyclic imides (1e13) have been synthesized
and evaluated their in vitro COX-1/COX-2 inhibition, and in vivo
assessment as anti-inflammatory activities (Fig. 1). In the present
study, the substitution pattern at the N-phenyl and the core cyclic
imides was selected so as to confer different electronic environ-
ment that would affect the lipophilicity, and hence the activity of
the target molecules. The objective of forming these hybrids is an
attempt to reach an active anti-inflammatory agent with potenti-
ated activity and selectivity toward COX-2. Moreover, in order to
investigate the contribution of the imide framework in the anti-
inflammatory activity and COX inhibition, the succinimide group
was replaced by phthalimide group substituted by a versatile
electron repelling or attracting moiety in the derivatives 4e10.
Next, we decide to replace the phthalimide ring with other cyclic
imides, providing derivatives 2, 3, 11, 12 and 13 (Scheme 1). Drug-
likeness and molecular docking methodology were used to iden-
tify the structural features required for the COX-2 inhibition
properties of these new series. However the results of this
molecular docking could support the postulation that our active
compounds may act on the same enzyme target where COX-2
inhibitors act confirming the molecular design of the reported
class of COX-2 inhibitors.
2.2. Biological activity
2.2.1. In vitro COX inhibition
It was found that the structurally simple compound, m,m-
dimethylphenylphthalimide (Fig. 1D), was obtained as a potent and
slightly COX-2-selective inhibitor. According to the above rationale,
the compounds synthesized in this work 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) are determined and are means of two determina-
tions acquired and the deviation from the mean is <10% of the mean
value. 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 0.30 mM respectively,
indicating that celecoxib is a selective COX-2 inhibitor [COX-2
(SI) > 333.3]. The results showed that most of the compounds
showed potent inhibition against COX-2 (IC50 y 0.1e1.0
compared to the inhibition for COX-1 (IC50 > 100 M) as listed in
Table 1. Nearly 6 of the compounds (5b, 6b, 11b, 11c, 12b and 12c)
mM)
m

1650
A.A.-M. Abdel-Aziz et al. / European Journal of Medicinal Chemistry 46 (2011) 1648e1655
O
R1
R1
O
H2N
R1
+
O
M ethod B: Acetic acid/Acetic anhydride, Reflux, 20h
M ethod A: Acetic acid, Reflux, 12h
R2
Cl
Cl
N
N
Cl
O
O
N
Cl
Cl
O
O
O
Cl
O
O
O
O
N
O
O
N
N
N
O
N
O
R1
R1
O
N
R1
R1
R1
R1
R1
R1
R1 R1
R1
R1
R1
R1
R1
R1
R1
R1
R1
1a-c
2a-c
3a-c
4-10a-c
11a-c
12a-c
R1
R1
a
b
c
R1= ;H, ; MeO, ; Cl
13a-c
4
5
6
7
8
R2= ; H, ; 5-NO2, ; 5-Me, ; 5-tert-But, ; 5,6-Dichloro,
9; 4,5,6,7-Tetrachloro, 10; 4,5,6,7-Tetrabromo
Scheme 1. Synthesis of the designed cyclic imide derivatives.
were found to be potent and selective similar to celecoxib against
2.2.2. In vivo anti-inflammatory studies
COX-2. Compound 5b was the most potent inhibitor in this series
All the synthesized compounds which have COX-2 inhibitory
activity were tested for anti-inflammatory activities including
compounds 1b,c and 3e12b,c. The results of anti-inflammatory
activities against carrageenan-induced rat paw edema and ED50
(mg/kg) of all tested compounds were shown in Table 1. Out of 21
compounds, 16 compounds (3e8 and 11e12) were found to possess
potent anti-inflammatory activity (60e94% reduction in inflam-
mation) and some of which shown higher inhibition in comparison
to reference drug diclofenac. In general, cyclic imides which were
substituted with chloro moiety at N-phenyl ring (R1 ¼ Cl) have
shown lower percentage of inhibition of edema when compared
with compounds containing CH3O group at the same position
(R1 ¼ CH3O). These findings may be attributed to importance of
methoxy group in COX receptor binding site. Compounds 5b
(R1 ¼ CH3O and R2 ¼ NO2), 6b (R1 ¼ CH3O and R2 ¼ CH3), 7b
(R1 ¼ CH3O and R2 ¼ tert-butyl), 11b (R1 ¼ CH3O), 11c (R1 ¼ Cl) and
12b (R1 ¼ CH3O) have shown the maximum percentage of anti-
inflammatory activity (94%, 94%, 89%, 91%, 88%, 84% respectively).
More interestingly, cyclic imides based on poly halogenated acid
anhydrides; such as compounds 8e10 and 13; were shown lower
activity compared with non halogenated derivatives this may be
attributed to very high lipid solubility and their low bio-availability.
ED50 of the compounds, which were shown promising degree of
anti-inflammatory activity (65e94%), were studied. Compounds
3b, 5b, 6b, 11b, 11c and 12b exhibited better anti-inflammatory
activity with ED50 of 123, 105, 107, 117, 115 and 120 mg/kg,
respectively, as compared to diclofenac (ED50 ¼ 114 mg/kg). In the
carrageenan-induced rat paw edema assay model, compound 5b
was the most potent anti-inflammatory agent (ED50 ¼ 105 mg/kg)
within this group of compounds at 2 h postdrug administration (IP).
This high in vivo anti-inflammatory activity shown by 5b, relative to
other compounds, is attributed to the fact that 5b inhibits both
COX-1 and COX-2 whereas the other compounds inhibit only COX-
2. Moreover predictions of ADME properties for all active
compounds [23] show that all calculated compounds comply with
with the activity (IC50 ¼ 0.1
mM) 3-fold higher than celecoxib
(IC50 ¼ 0.3 M). The effects of substituents introduced into the
m
phthaloyl moiety of compounds 4e10 were revealed to be direc-
tional, being dependent on the electronic nature of the substituents,
that is introduction of a nitro group at the 5-position (5b) enhanced
both COX-1- and COX-2-inhibiting activity with higher potency for
the latter, resulting in a COX-2-selective inhibitor (SI ¼ 400).
Moreover introduction of halogen such as chloro or bromo (8e10)
resulted in lowering the COX-2-inhibiting activity, while introduc-
tion of an electron-donating methyl group (6b) showed just the
opposite effects, resulting in a COX-2-selective inhibitor (SI ¼ >250).
The activities of some other cyclic imides analogs (11e12) are shown
in Table 1. Among them, compounds 11b and 12b are a COX-2-
selective inhibitor (SI ¼ >222). No clear-cut structureeactivity
relationships could be deduced at this stage. The activities of
N-unsubstituted phenyl cyclic imide derivatives (1ae13a) are not
shown in Table 1 and are inactive. Interestingly methoxy substitu-
ents on the N-phenyl group play critical roles in the COX-inhibiting
activity of the tested compounds. The 3,4,5,trimethoxyphenyl
analog (5b) showed the most potent activity, being 2.5 and 3 times
more active than celecoxib in COX-1 and COX-2-inhibiting activity,
respectively. The structureeactivity relationship of compounds
1b,ce13b,c indicates the introduction of 3,4,6-trichloro groups at N-
phenyl cyclic imides lowered the activity. This result, as well as the
previously described effect of the 5-substitution on phthalylimide
suggests the importance of electronic effects and spatial structure
around the nitrogen atom of the core cyclic imides. Next we inves-
tigated the COX-inhibiting activity of non-aromatic succinimide 1
and cyclic imides 2 and 13 which were less active inhibitor as
compounds 1 or inactive such as compounds 2 and 13 compared to
the phthalimide derivatives 4e10 and its analogs 11e12. Diminish-
ment of activity of such compounds 1, 2 and 13 may be explained on
the bases of non-aromatic feature of the imide core indicating the
importance of aromatic fragment of imide core for the activity.

A.A.-M. Abdel-Aziz et al. / European Journal of Medicinal Chemistry 46 (2011) 1648e1655
1651
Table 1
2.3. Docking studies
In vitro COX-1/COX-2 enzyme inhibition assay, in vivo anti-inflammatory activity
against carrageenan-induced rat paw edema and ED50 (mg/kg) of the designed
compounds.
Insights into the differences between the binding sites of COX-1
and COX-2 obtained from X-ray crystal structure data [12,24],
provided useful guidelines that facilitated the design of the selec-
tive COX-2 inhibitors. 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 [24]. It has also been reported
that replacement of His513 in COX-1 by Arg513 in COX-2 plays a key
role with respect to the H-bond network in the COX-2 binding site.
Access of ligands to the 2ꢀ-pocket of COX-2 is controlled by histi-
dine (His90), glutamine (Gln192), and tyrosine (Tyr355) [25]. Inter-
action of Arg513 with the bound drug is a requirement for time
dependent inhibition of COX-2 [26].
The level of COX-2 inhibition and anti-inflammatory activities of
compound 5b prompted us to perform molecular docking studies
to understand the ligandeprotein interactions in detail. All the
calculations were performed using MOE 2008.10 software installed
on 2.0G Core 2 Duo [27e29]. The crystal structures of COX-2
enzymes complexed with SC-558 [1CX2] were used for the docking
[12]. The active site of the enzyme was defined to include residues
within a 10.0 Å radius to any of the inhibitor atoms. The automated
docking program of MOE 2008.10 was used to dock compound 5b
on the active sites of COX-2 enzymes. The most stable docking
model was selected according to the best scored conformation
predicted by the MOE scoring function. The complexes were
energy-minimized with a MMFF94 force field [30] till the gradient
convergence 0.05 kcal/mol was reached. The compound 5b could
dock into the active site of COX-2 successfully (Fig. 2). Compound
5b 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 strong hydrogen bonding with His90 (2.43,
2.83 Å) and Arg513 (2.89 Å) and weak hydrogen bonding with Tyr355
(3.34 Å). Such interactions are almost essential for COX-2 inhibitory
activity, as exemplified by the binding interaction of SC-558, an
analog of celecoxib cocrystallized in the COX-2 active site [12]. 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, Tyr385, Val523, Leu352 and
Phe518. Moreover one of the carbonyl moieties of the imide core and
the 5-nitro group were forming weak hydrogen bond with Ala527
d
Compound
No
IC50
(
m
M)a
SIb
% Inhibition of
edemac
ED50
(mg/kg)
COX-1
COX-2
After 1 h
After 2 h
1b
1c
2b
2c
3b
3c
4b
4c
5b
5c
6b
6c
7b
7c
8b
8c
>100
>100
>100
>100
>100
>100
>100
>100
40.0
40.00
40.00
>100
>100
5.20
10.50
6.40
9.10
>2.5
>2.5
e
20
30
e
40
33
e
e
e
e
e
e
e
e
>19.2
>9.5
>15.6
>11.0
400.0
0.90
>250.0
>5.1
>10.0
>4.8
>8.1
>3.3
>2.2
>2.2
>1.5
e
72
47
43
49
87
79
84
36
60
35
59
75
0
84
73
67
70
94
79
94
65
89
60
80
65
53
45
55
e
123
175
185
172
105
122
107
183
146
195
186
187
212
229
213
e
0.10
40.00
0.40
36.1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
e
19.50
10.00
20.70
12.40
30.10
45.00
45.00
65.00
>100
0.45
1.00
0.45
1.00
>100
>100
e
9b
9c
44
42
e
10b
10c
11b
11c
12b
12c
13b
13c
Diclofenac
Celecoxib
>222.2
>100.00
>222.2
>100.00
e
86
79
86
75
36
23
88
e
91
88
84
72
34
37
89
e
117
115
120
150
e
e
e
e
114
e
>100
0.30
>333.3
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).
c
The carrageenan-induced rat paw edema assay was carried out using six
animals (male SpragueeDawley rats)/group following IP of the test compound. The
results are expressed as means ꢁ SEM (n ¼ 4e6) following a 100 mg/kg IP of the test
compounds.
d
ED50 was the effective dose calculated after 2 h.
these rules and even the diclofenac show no violation. Theoreti-
cally, these compounds should present good passive oral absorp-
tion and differences in their bioactivity can not be attributed to this
property.
Fig. 2. Left panel showed docking of compound 5b into the active site of COX-2. Hydrogen bonds are shown in green. Right panel showed alignment of 5b (yellow) and selective
inhibitor SC-558 (red) in the active site of COX-2. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

1652
A.A.-M. Abdel-Aziz et al. / European Journal of Medicinal Chemistry 46 (2011) 1648e1655
(3.64 Å) and Tyr385 (3.00 Å) respectively. The lateral pocket of COX-
2 would therefore be responsible for the COX-2 selectivity of 5b and
contributed to stabilize the ligandeenzyme complexes (Fig. 2).
The complex generated by docking studies of 5b 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 5b 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 5b with COX-2 (Fig. 2) shows that the
methoxy groups of 5b hydrogen bonded to the amino acid residue
His90 and Arg513, similarly to the sulfonyl moiety of the pharma-
cophoric sulfonamide group pertaining to SC-558. Moreover, an
additional hydrogen bond was observed between the carbonyl
Thin layer chromatography 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. Com-
pounds 1ae13a, 1c and 3b were prepared following their proce-
dures reported in the literature [15,17,31e44].
4.1.1. General procedure for the synthesis of compounds 1be13b
(method A)
A solution of 3,4,5-trimethoxyaniline (1.83 g, 10 mmol) and acid
anhydride (10 mmol) in glacial acetic acid (15 mL) was heated
under reflux for 12 h. After the evaporation of the reaction mixture
to dryness under reduced pressure, the residue was neutralized by
a solution of sodium bicarbonate (4%) until effervescence ceased.
The precipitate obtained was washed with water, dried (P2O5) and
recrystallized from an appropriate solvent.
oxygen and Ala527, while NO2 hydrogen bonded with Tyr385
.
Additionally, N-phenyl system are positioned in the same region as
p-sulfonamido-phenyl ring of SC-558, while, the aromatic ring of
imide core of compound 5b is close to the p-Br-phenyl ring of
SC-558, in the aromatic region of the active site lined by aromatic
amino acid residues such as Phe518, Tyr385 and Trp387, among
others. In short, the described interactions are typical of selective
inhibitors of COX-2, confirming the molecular design of the
reported class of anti-inflammatory imide derivatives [16,17].
4.1.1.1. 1-(3,4,5-Trimethoxyphenyl)pyrrolidine-2,5-dione (1b). Yield,
70%; mp 201e202 ꢀC (Methanol); IR (KBr, cmꢂ1
)
n
: 1773, 1718 (C]
1
O); H NMR (DMSO-d6);
d 2.77 (s, 4H, CH2eCH2), 3.71e3.76 (d, 9H,
J ¼ 22.0 Hz, 3CH3) 6.58 (s, 2H, Ar-H); 13C NMR (DMSO-d6):
d 28.91,
56.66, 60.54, 105.97, 128.92, 138.08, 153.39, 177.17; MS m/z (%): 267.2
(12.0, Mþ þ 2), 266.0 (18.0, Mþ þ 1), 264.9 (22.0, Mþ); C13H15NO5,
Cal. C, 58.86; H, 5.70; N, 5.28, Found C, 58.01; H, 6.20; N, 5.88.
3. Conclusion
4.1.1.2. cis-2-(3,4,5-Trimethoxyphenyl)-3a,4,7,7a-tetrahydro-1H-iso-
Different (substituted) cyclic imide derivatives were synthesized
and screened for COX-1/COX-2 inhibition and anti-inflammatory
activity. Compounds which showed significant COX-2 inhibition
were subjected to anti-inflammatory studies. Compound 5b exhibit
indole-1,3(2H)-dione (2b). Yield, 69%; mp 141e142 ꢀC (Methanol);
IR (KBr, cmꢂ1 : 1717, 1663 (C]O); 1H NMR (DMSO-d6)
) n d: 1.80 (s,
2H, CH2), 2.27e2.29 (d, 2H, J ¼ 11.0 Hz, CH2), 2.43e2.46 (d, 2H,
J ¼ 14.5 Hz, CHeCH), 3.68e3.73 (d, 9H, J ¼ 26.0 Hz, 3CH3), 5.94 (s,
optimal COX-2 inhibitory potency (IC50 ¼ 0.1
mM) and selectivity
2H, CH]CH), 6.49 (s, 2H, Ar-H); 13C NMR (DMSO-d6);
d 23.58, 39.13,
(SI) ¼ 400 comparable with celecoxib and better ED50 than diclo-
fenac, so it appears promising. 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 CH3O moieties of 5b insert deep
inside the COX-2 2ꢀ-pocket and forming strong hydrogen bond with
His90 (2.43, 2.83 Å) and Arg513 (2.89 Å) and weak hydrogen bonding
with Tyr355 (3.34 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 inhi-
bition. Indeed molecular docking studies further supported the
strong inhibitory activity of 5b and further help understanding the
various interactions between the ligands and enzyme active sites in
detail and thereby help to design novel potent inhibitors. The
structureeactivity relationships acquired show that appropriately
substituted cyclic imides have the necessary geometry to provide
potent and selective inhibition of the COX-2 isozyme, and to exhibit
excellent anti-inflammatory activities.
56.57, 60.52, 105.44, 128.04, 128.71, 137.94, 153.38, 179.61; MS m/z
(%): 317.8 (2.3, Mþ); C17H19NO5, Cal. C, 64.34; H, 6.03; N, 4.41, Found
C, 63.99; H, 6.81; N, 4.91.
4.1.1.3. 2-(3,4,5-Trimethoxyphenyl)isoindoline-1,3-dione (4b). Yield,
88%; mp 241e242 ꢀC (Ethanol); IR (KBr, cmꢂ1
)
n
: 1766, 1713 (C]O);
3.62 (s, 3H, CH3), 3.75 (s, 3H, 2CH3), 6.79 (s, 2H,
Ar-H), 7.08 (s, 2H, Ar-H), 7.37 (s, 2H, Ar-H); 13C NMR (DMSO-d6);
1H NMR (DMSO-d6)
d
d
56.64, 60.56, 98.01, 106.39, 123.79, 130.23, 132.00, 135.13, 153.36,
167.48; MS m/z (%): 314.6 (6.0, Mþ þ 1), 313.8 (18.0, Mþ); C17H15NO5,
Cal. C, 65.17; H, 4.83; N, 4.47, Found C, 64.89; H, 5.05; N, 5.00.
4.1.1.4. 5-Nitro-2-(3,4,5-trimethoxyphenyl)isoindoline-1,3-dionꢂe1
) n:
(5b). Yield, 87%; mp 258e260 ꢀC (Methanol); IR (KBr, cm
1778, 1728 (C]O); 1H NMR (DMSO-d6)
d 3.73 (s, 3H, CH3), 3.77 (s,
3H, 2CH3), 6.82 (s, 2H, Ar-H) 8.11e8.14 (t, 1H, J ¼ 7.5 Hz, Ar-H),
8.25e8.26 (d, 1H, J ¼ 7.0 Hz, Ar-H), 8.34e8.36 (d, 1H, J ¼ 7.5 Hz, Ar-
H); 13C NMR (DMSO-d6);
d 56.03, 60.06, 105.76, 122.75, 126.94,
127.05, 128.33, 133.46, 136.42, 137.55, 144.45, 152.89, 162.64, 165.21.
MS m/z (%): 358.5 (8.0, Mþ). C17H14N2O7, Cal. C, 56.99; H, 3.94; N,
7.82, Found C, 57.45; H, 4.33; N, 7.64.
4. Experimental
4.1. Chemistry
4.1.1.5. 5-Methyl-2-(3,4,5-trimethoxyphenyl)isoindoline-1,3-dioneꢂ1
)
n
Melting points (uncorrected) were recorded on Barnstead Elec-
trothermal 9100 melting apparatus. IR spectra were recorded on
a FT-IR PerkineElmer spectrometer. 1H NMR and 13C NMR were
recorded in DMSO-d6 and/or CDCl3 on a Bruker 500 MHz instrument
(6b). Yield, 90%; mp 222e223 ꢀC (Methanol/CH2Cl2); IR (KBr, cm
: 1774, 1707 (C]O); 1H NMR (DMSO-d6)
d 2.53 (s, 3H, CH3), 3.72 (s,
3H, CH3), 3.77 (s, 3H, 2CH3), 6.79 (s, 2H, Ar-H), 7.70e7.72 (d, 1H,
J ¼ 7.5 Hz, Ar-H), 7.78 (s, 1H, Ar-H), 7.83e7.84 (d, 1H, J ¼ 7.5 Hz, Ar-
using TMS as internal standard (chemical shifts in
d
ppm). Micro-
H); 13C NMR (DMSO-d6);
d 21.39, 56.05, 60.04, 105.69, 123.25,
analytical data (C, H, and N) were performed on PerkineElmer 240 B
analyzer and they agreed with proposed structures within ꢁ0.4% of
the calculated values. Mass spectra were recorded on a Per-
kineElmer, Clarus 600 GC/MS and Varian, TQ 320 GC/MS/MS mass
spectrometers. Solvent evaporation was performed under reduced
pressure using Buchan Rotatory Evaporator unless otherwise stated.
123.70,127.58,129.00,132.00,135.04,137.24,145.63,152.82,167.00;
MS m/z (%): 327.0 (9.0, Mþ); C18H17NO5, Cal. C, 66.05; H, 5.23; N,
4.28, Found C, 65.65; H, 5.71; N, 4.33.
4.1.1.6. 5-Tert-Butyl-2-(3,4,5-trimethoxyphenyl)isoindoline-1,3-dione
(7b). Yield, 63%; mp 145e147 ꢀC (Methanol/CH2Cl2); IR (KBr, cmꢂ1
)

A.A.-M. Abdel-Aziz et al. / European Journal of Medicinal Chemistry 46 (2011) 1648e1655
1653
n
: 1777, 1708 (C]O); 1H NMR (DMSO-d6)
d
1.38 (s, 9H, 3CH3), 3.78
a solution of sodium bicarbonate (4%) until effervescence ceased.
The precipitate obtained was washed with water, dried (P2O5) and
recrystallized from an appropriate solvent.
(s, 3H, CH3), 3.83 (s, 3H, 2CH3), 6.93 (s, 2H, Ar-H), 7.56e7.58 (d, 1H,
J ¼ 7.5 Hz, Ar-H), 7.76e7.77 (d, 1H, J ¼ 7.5 Hz, Ar-H), 7.99 (s, 1H, Ar-
H); MS m/z (%): 369.1 (22, Mþ); C21H23NO5, Cal. C, 68.28; H, 6.28; N,
3.79, Found C, 68.59; H, 5.87; N, 4.00.
4.1.2.1. cis-
indole-1,3(2H)-dione (2c). Yield, 61%; mp 159e160 ꢀC (Ethanol/DMF);
IR (KBr, cmꢂ1 : 1786,1716 (C]O); 1H NMR (DMSO-d6)
: 1.85 (s, 2H,
2-(3,4,5-Trichlorophenyl)-3a,4,7,7a-tetrahydro-1H-iso-
4.1.1.7. 5,6-Dichloro-2-(3,4,5-trimethoxyphenyl)isoindoline-1,3-
)
n
d
dione (8b). Yield, 95%; mp 197e198 ꢀC (Methanol/DMF); IR (KBr,
CH2), 2.33 (s, 2H, CH2), 2.46 (s, 2H, CHeCH), 5.97 (s, 2H, CH]CH),
cmꢂ1
)
n
: 1779, 1726 (C]O); 1H NMR (DMSO-d6)
d
3.72 (s, 3H, CH3),
8.03e8.04 (d, 2H, J ¼ 3.0 Hz, Ar-H); 13C NMR (DMSO-d6);
d 23.07,
3.77 (s, 3H, 2CH3), 6.80 (s, 2H, Ar-H), 8.29 (s, 2H, Ar-H); 13C NMR
39.15, 128.82, 128.87, 134.15, 134.42, 135.70, 177.55. MS m/z (%): 330.9
(DMSO-d6);
d
56.06, 60.06, 105.57, 125.47, 127.20, 131.54, 137.48,
(14.0, Mþ).
152.57, 165.27. MS m/z (%): 385.2 (9.0, Mþ þ 3), 384.1 (3.0, Mþ þ 1),
382.6 (24.0, Mþ); C17H13Cl2NO5, Cal. C, 53.42; H, 3.43; N, 3.66,
Found C, 53.66; H, 3.01; N, 3.90.
4.1.2.2. 6-(3,4,5-Trichlorophenyl)-5H-pyrrolo[3,4-b]pyrazine-5,7
(6H)-dione (3c). Yield, 65%; mp >300 ꢀC (Acetic acid); IR (KBr, cmꢂ1
)
n
: 1798,1745 (C]O); 1H NMR (DMSO-d6)
1H, Ar-H), 8.79 (s, 1H, Ar-H); 13C NMR (DMSO-d6);
132.18, 134.57, 141.27, 145.69, 162.71. MS m/z (%): 328.5 (2.00, Mþ).
d
7.77 (s, 2H, Ar-H), 8.61 (s,
127.44, 128.13,
4.1.1.8. 4,5,6,7-Tetrachloro-2-(3,4,5-trimethoxyphenyl)isoindoline-ꢂ1
d
1,3-dione (9b). Yield, 93%; mp >300 ꢀC (Acetic acid); IR (KBr, cm
)
n
: 1782, 1709 (C]O); 1H NMR (DMSO-d6)
d
3.70 (s, 3H, CH3), 3.76 (s,
56.76, 60.14,
3H, 2CH3), 7.06 (s, 2H, Ar-H); 13C NMR (DMSO-d6);
d
4.1.2.3. 2-(3,4,5-Trichlorophenyl)isoindoline-1,3-dione
81%; mp 291e293 ꢀC (Acetic acid); IR (KBr, cmꢂ1
O); 1H NMR (DMSO-d6)
(4c). Yield,
: 1789,1727 (C]
97.38, 125.47, 128.04, 132.50, 152.79, 170.00. MS m/z (%): 451.4 (11.0,
Mþ); C17H11Cl4NO5, Cal. C, 45.26; H, 2.46; N, 3.11, Found C, 45.00; H,
2.77; N, 2.85.
)
n
d
8.03 (s, 4H, Ar-H), 8.10e8.11 (m, 2H, Ar-H);
13C NMR (DMSO-d6);
d 124.44, 128.97, 130.20, 130.61, 132.45, 135.33,
135.86, 173.27. MS m/z (%): 326.1 (11.0, Mþ).
4.1.1.9. 4,5,6,7-Tetrabromo-2-(3,4,5-trimethoxyphenyl)isoindoline-
1,3-dione (10b). Yield, 94%; mp >300 ꢀC (Acetic acid); IR (KBr,
4.1.2.4. 5-Nitro-2-(3,4,5-trichlorophenyl)isoindoline-1,3-dione
cmꢂ1
)
n
: 1706, 1667 (C]O); 1H NMR (DMSO-d6)
d
3.73 (s, 3H, CH3),
(5c). Yield, 78%; mp 219e220 ꢀC (Methanol/DMF); IR (KBr, cmꢂ1
) n:
3.77 (s, 3H, 2CH3), 6.68 (s, 2H, Ar-H). MS m/z (%): 628.5 (19.0, Mþ);
C17H11Br4NO5, Cal. C, 32.47; H, 1.76; N, 2.23, Found C, 32.65; H, 2.00;
N, 2.50.
1788,1739 (C]O); 1H NMR (DMSO-d6)
d 8.04 (s, 2H, Ar-H) 8.23e8.26
(t,1H, J ¼ 7.5 Hz, Ar-H), 8.41e8.42 (d,1H, J ¼ 7.0 Hz, Ar-H), 8.49e8.50
(d, 1H, J ¼ 7.5 Hz, Ar-H); 13C NMR (DMSO-d6);
d 122.21, 126.02,
127.43, 128.17, 128.45, 129.10, 130.09, 132.23, 135.24, 136.51, 137.72,
4.1.1.10. 2-(3,4,5-Trimethoxyphenyl)-1H-benzo[de]isoquinoline-1,3
144.93, 160.47, 163.26. MS m/z (%): 371.8 (2.00, Mþ).
(2H)-dione (11b). Yield, 89%; mp 175e177 ꢀC (Methanol/DMF); IR
(KBr, cmꢂ1
)
n
: 1784, 1711 (C]O); 1H NMR (DMSO-d6)
d
3.72 (s, 3H,
CH3), 3.75 (s, 3H, 2CH3), 6.98 (s, 2H, Ar-H) 7.90e7.95 (m, 2H, Ar-H),
8.51e8.58 (m, 4H, Ar-H); 13C NMR (DMSO-d6);
55.98, 60.04, 96.73,
4.1.2.5. 5-Methyl-2-(3,4,5-trichlorophenyl)isoindoline-1,3-dioneꢂ1
) n:
(6c). Yield, 67%; mp 279e281 ꢀC (Methanol/DMF); IR (KBr, cm
d
1786,1727 (C]O); 1H NMR (DMSO-d6)
d 2.55 (s, 3H, CH3), 7.81e7.83
106.73, 127.22, 127.55, 130.69, 132.44, 134.41, 135.35, 152.60, 173.95.
MS m/z (%): 363.8 (8.0, Mþ); C21H17NO5, Cal. C, 69.41; H, 4.72; N,
3.85, Found C, 69.13; H, 5.21; N, 4.05.
(d, 1H, J ¼ 7.0 Hz, Ar-H), 7.93 (s, 1H, Ar-H), 7.97e7.99 (d, 1H,
J ¼ 7.5 Hz, Ar-H), 8.03 (s, 2H, Ar-H); 13C NMR (DMSO-d6);
d 21.44,
118.29, 124.31, 124.79, 126.78, 127.45, 128.02, 128.94, 131.01, 135.36,
136.05, 136.13, 147.13, 165.21, 165.35. MS m/z (%): 339.4 (2.00, Mþ),
339.4 (2.00, Mþ).
4.1.1.11. 6-(3,4,5-Trimethoxyphenyl)-5H-dibenzo[c,e]azepine-5,7
(6H)-dione (12b). Yield, 70%; mp >300 ꢀC (Ethanol); IR (KBr, cmꢂ1
)
n
: 1695, 1663 (C]O); 1H NMR (DMSO-d6)
d
3.61 (s, 3H, CH3), 3.72 (s,
4.1.2.6. 5-tert-Butyl-2-(3,4,5-trichlorophenyl)isoindoline-1,3-dione
3H, 2CH3), 6.92e6.94 (d, 2H, J ¼ 6.5 Hz, Ar-H) 6.97 (s, 2H, Ar-H),
(7c). Yield, 58%; mp >300 ꢀC (Methanol/DMF); IR (KBr, cmꢂ1
) n:
7.23e7.33 (m, 4H, Ar-H), 7.39e7.43 (m, 2H, Ar-H); 13C NMR (DMSO-
1779,1732 (C]O); 1H NMR (DMSO-d6)
1.39 (s, 9H, 3CH3),
8.03e8.11 (m, 5H, Ar-H); 13C NMR (DMSO-d6);
d 30.65, 35.71,121.25,
d
d6);
d
55.62, 60.05, 96.73, 126.82, 127.86, 129.01, 133.12, 135.49,
139.59,152.61,168.12,171.85. MS m/z (%): 389.1 (11, Mþ); C23H19NO5,
124.32, 126.78, 128.14, 128.95, 130.95, 132.78, 135.37, 136.07, 159.74,
C, 70.94; H, 4.92; N, 3.60, Found C, 71.36; H, 5.26; N, 3.81.
165.07, 165.43. MS m/z (%): 382.9 (14.0, Mþ).
4.1.1.12. 4,5,6,7,8,8-Hexachloro-2-(3,4,5-trimethoxyphenyl)-3a,4,7,7a-
4.1.2.7. 5,6-Dichloro-2-(3,4,5-trichlorophenyl)isoindoline-1,3-dꢂio1ne
) n:
tetrahydro-1H-4,7-methanoisoindole-1,3(2H)-dioꢂn1e (13b). Yield, 57%;
(8c). Yield, 55%; mp >300 ꢀC (Acetic acid); IR (KBr, cm
mp 220e221 ꢀC (Methanol/CH2Cl2); IR (KBr, cm
)
n
: 1786,1717 (C]
1787,1727 (C]O); 1H NMR (DMSO-d6)
d 8.04 (s, 2H, Ar-H), 8.63 (s,
O); 1H NMR (DMSO-d6)
d
3.71 (s, 3H, CH3), 3.75 (s, 3H, 2CH3), 4.23 (s,
51.99, 56.03,
2H, Ar-H); 13C NMR (DMSO-d6);
d 126.20, 126.73, 129.08, 130.30,
2H, CHeCH), 6.36 (s, 2H, Ar-H); 13C NMR (DMSO-d6);
d
135.19, 136.41, 138.96, 163.42. MS m/z (%): 395.9 (4.00, Mþ);
60.08, 79.02, 103.64, 104.35, 126.57, 130.65, 137.93, 153.21, 169.61. MS
m/z (%): 535.6 (2, Mþ ꢂ 1), 536.4 (1, Mþ); C18H13Cl6NO5, Cal. C, 40.33;
H, 2.44; N, 2.61, Found C, 40.64; H, 2.93; N, 3.02.
C14H4Cl5NO2 C, 42.52; H, 1.02; N, 3.54.
4.1.2.8. 4,5,6,7-Tetrachloro-2-(3,4,5-trichlorophenyl)isoindoline-1,3-
dione (9c). Yield, 61%; mp 261e263 ꢀC (Acetic acid); IR (KBr, cmꢂ1
)
4.1.2. General procedure for the synthesis of compounds 1ce13c
(method B)
n
: 1793,1732 (C]O); 1H NMR (DMSO-d6)
d 7.94 (s, 1H, Ar-H), 8.05 (s,
1H, Ar-H); 13C NMR (DMSO-d6);
d 126.64, 129.16, 129.48, 135.19,
A solution of 3,4,5-trichloroaniline (1.96 g, 10 mmol) and acid
anhydride (10 mmol) in glacial acetic acid (20 mL) was refluxed for
3 h. The solvent was removed under reduced pressure until the
volume reached ca. 5 mL. After addition of 10 mL of acetic anhy-
dride, the solution was refluxed again for 20 h. The solvent was
removed under reduced pressure. The residue was neutralized by
139.86, 160.76, 172.04. MS m/z (%): 464.1 (2.00, Mþ).
4.1.2.9. 4,5,6,7-Tetrabromo-2-(3,4,5-trichlorophenyl)isoindoline-1,3-
dione (10c). Yield, 49%; mp 299e300 ꢀC (Acetic acid); IR (KBr, cmꢂ1
)
n
: 1778, 1731 (C]O); 1H NMR (DMSO-d6) 8.04 (s, 2H, Ar-H); 13C
d
NMR (DMSO-d6);
d 120.97, 121.95, 126.12, 126.97, 129.10, 129.93,

1654
A.A.-M. Abdel-Aziz et al. / European Journal of Medicinal Chemistry 46 (2011) 1648e1655
135.21, 136.53, 138.22, 140.29, 161.13, 166.59. MS m/z (%): 642.3
4.2.2. Anti-inflammatory activity
(6.00, Mþ).
The test compounds were evaluated using in vivo rat carra-
geenan-induced foot paw edema model reported previously [45].
Male SpragueeDawley rats (250 g) were fasted with free access to
water at least 16 h prior to experiments. Edema was produced by
4.1.2.10. 2-(3,4,5-Trichlorophenyl)-1H-benzo[de]isoquinoline-1,3
(2H)-dione (11c). Yield, 70%; mp 216e217 ꢀC (Ethanol/DMF); IR
(KBr, cmꢂ1
)
n
: 1770, 1740 (C]O); 1H NMR (DMSO-d6)
d
7.91e7.94
injecting 0.2 mL of a solution of 1% l-carrageenan in the hind paw.
(m, 3H, Ar-H), 8.53e8.56 (m, 5H, Ar-H); 13C NMR (DMSO-d6);
119.01, 127.54, 129.71, 131.36, 132.44, 135.37, 160.68. MS m/z (%):
376.9 (3.00, Mþ).
Paw volume was measured by water displacement with a plethys-
mometer (UGO BASILE) before, 1 and 2 h after treatment. The
compounds were administered intraperitoneally with a 1 mL
suspension of test compounds in vehicle (0.5% methyl cellulose).
The percentage was calculated by the following equation: anti-
inflammatory activity (%) ¼ (1 ꢂ D/C) ꢂ 100, where D represents
the difference in paw volume before and after compounds was
administered to the rats, and C stands for the difference of volume
in the control groups.
4.1.2.11. 6-(3,4,5-Trichlorophenyl)-5H-dibenzo[c,e]azepine-5,7(6H)-
dione (12c). Yield, 61%; mp 283e285 ꢀC (Ethanol/DMF); IR (KBr,
cmꢂ1 : 1695 (C]O); 1H NMR (DMSO-d6)
) n d 6.93e6.94 (d, 2H,
J ¼ 6.5 Hz, Ar-H), 7.31e7.38 (m, 6H, Ar-H), 7.43e7.45 (d, 2H,
J ¼ 6.5 Hz, Ar-H); 13C NMR (DMSO-d6);
d 126.87, 127.16, 127.99,
128.16, 129.03, 134.48, 138.13, 138.62, 171.90, 172.51. MS m/z (%):
402.2 (4.00, Mþ).
4.3. Docking methodology
4.1.2.12. 4,5,6,7,8,8-Hexachloro-2-(3,4,5-trichlorophenyl)-3a,4,7,7a-
Docking studies have been performed using MOE 2008.10. With
this purpose, crystal structure of COX-2/SC-558 (a selective inhib-
itor) complex (PDB codes: 1CX2) was obtained from the Protein
Data Bank in order to prepare the protein for docking studies.
Docking procedure was followed using the standard protocol
implemented in MOE 2008.10 and the geometry of resulting
complexes was studied using the MOE’s Pose Viewer utility.
tetrahydro-1H-4,7-methanoisoindole-1,3(2H)-dione (13c). Yield, 47%;
mp 256e257 ꢀC (Acetic acid); IR (KBr, cmꢂ1 : 1795,1734 (C]O); 1H
) n
NMR (DMSO-d6)
d
4.80(s, 2H, CHeCH), 8.04 (s, 2H, Ar-H); 13C NMR
(DMSO-d6);
d 51.52, 78.70, 105.03, 125.96, 129.08, 129.18, 131,02,
133.79, 134.15, 136.73, 168.13. MS m/z (%): 549.3 (3.00, Mþ).
4.2. Pharmacology
Acknowledgments
4.2.1. In vitro cyclooxygenase (COX) inhibition assay
The ability of the test compounds listed in the Table 1 to inhibit
ovine COX-1 and COX-2 (IC50 values, mM) was determined using an
enzyme immunoassay (EIA) kit (catalog number 560101, Cayman
Chemical, Ann Arbor, MI, USA). Cyclooxygenase catalyzes the first
Our sincere acknowledgments to Chemical Computing Group
Inc, 1010 Sherbrooke Street West, Suite 910, Montreal, H3A 2R7,
Canada, for its valuable agreement to use the package of MOE
2008.10 software. We are highly appreciated Cayman Chemicals
Inc., Ann Arbor, MI, USA, for in vitro COX-1/COX-2 screening
tests.
step in the biosynthesis of arachidonic acid (AA) to PGH2. PGF2
,
a
produced from PGH2 by reduction with stannous chloride, is
measured by enzyme immunoassay (ACEÔ competitive EIA). Stock
solutions of test compounds were dissolved in a minimumvolume of
DMSO. Briefly, to a series of supplied reaction buffer solutions
References
(960
phenol) witheitherCOX-1or COX-2(10
heme (10 L) were added 10 L 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 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.
m
L, 0.1 M TriseHCl pH 8.0 containing 5 mM EDTA and 2 mM
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mL)enzymein thepresence of
m
m
m
m
m
m
PGF2a, produced from PGH2 by reductionwith 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 propor-
tionaltothe concentration of PGs in thewells 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 Ellman’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 406 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
amountof PGs present in thewell during the incubation: Absorbance
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