Synthesis, anti-inflammatory, analgesic and COX-1/2 inhibition activities of anilides based on 5,5-diphenylimidazolidine-2,4-dione scaffold: Molecular docking studies

Article abstract of DOI:10.1016/j.ejmech.2016.03.011

The design, synthesis and pharmacological activities of a group of 5,5-diphenylimidazolidine-2,4-dione bearing anilide, phenacyl and benzylidene fragments 2-27 were reported. The prepared 5,5-diphenylimidazolidine-2,4-dione derivatives were evaluated in vivo for anti-inflammatory, analgesic activities and in vitro for COX-1/2 inhibition assay. Among the tested compounds, derivatives 5, 9, 10, 13, and 14 showed significant and potent anti-inflammatory and analgesic activities almost equivalent to reference drug celecoxib. In COX-1/2 inhibition assay, compounds 5, 9, 10 and 14 showed high COX-2 inhibitory activity (IC₅₀ = 0.70 μM, 0.44 μM, 0.61 μM and 0.41 μM; respectively) and selectivity index (SI) range of 142-243 comparable to celecoxib [COX-2 (SI) > 333]. These potent COX-2 inhibitors 9, 10, 13, and 14 were docked into the active site pocket of COX-2 to explore the binding mode and possible interactions of these ligands.

Full text of DOI:10.1016/j.ejmech.2016.03.011

Accepted Manuscript
Synthesis, anti-inflammatory, analgesic and COX-1/2 inhibition activities of anilides
based on 5,5-diphenylimidazolidine-2,4-dione scaffold: Molecular docking studies
Alaa A.-M. Abdel-Aziz, Adel S. El-Azab, Laila A. Abouzeid, Kamal Eldin H. ElTahir,
Naglaa I. Abdel-Aziz, Rezk R. Ayyad, Abdulrahman M. Al-Obaid
PII:
S0223-5234(16)30186-6
10.1016/j.ejmech.2016.03.011
EJMECH 8436
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 January 2016
Revised Date: 2 March 2016
Accepted Date: 3 March 2016
Please cite this article as: A.A.-M. Abdel-Aziz, A.S. El-Azab, L.A. Abouzeid, K.E.H. ElTahir, N.I. Abdel-
Aziz, R.R. Ayyad, A.M. Al-Obaid, Synthesis, anti-inflammatory, analgesic and COX-1/2 inhibition
activities of anilides based on 5,5-diphenylimidazolidine-2,4-dione scaffold: Molecular docking studies,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.03.011.
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Graphical Abstract
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Synthesis, anti-inflammatory, analgesic and COX-1/2
inhibition activities of anilides based on
5,5-diphenylimidazolidine-2,4-dione scaffold:
Molecular docking studies
Alaa A.-M. Abdel-Aziz, Adel S. El-Azab, Laila A. Abouzeid, Kamal Eldin H. ElTahir,
Naglaa I. Abdel-Aziz , Rezk R. Ayyad, Abdulrahman M. Al-Obaid
Compounds 5, 9, 10 and 14 showed potential anti-inflammatory, analgesic and COX-2 inhibitory activities
compared with celecoxib.
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1
Synthesis, anti-inflammatory, analgesic and COX-1/2 inhibition
activities of anilides based on 5,5-diphenylimidazolidine-2,4-dione
scaffold: Molecular docking studies
Alaa A.-M. Abdel-Aziza,b*, Adel S. El-Azaba,c, Laila A. Abouzeidd, Kamal Eldin H. ElTahire,
Naglaa I. Abdel-Azizb, Rezk R. Ayyadf, Abdulrahman M. Al-Obaida
Department of Pharmaceutical Chemistrya, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
Department of Medicinal Chemistryb, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
Department of Organic Chemistryc, Faculty of Pharmacy, Al-Azhar University, Cairo 11884, Egypt
Department of Pharmaceutical Organic Chemistryd, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
Department of Pharmacologye, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
Department of Pharmaceutical Chemistryf, Faculty of Pharmacy, Al-Azhar University, Cairo 11884, Egypt
Abstract——The design, synthesis and pharmacological activities of a group of 5,5-diphenylimidazolidine-2,4-
dione bearing anilide, phenacyl and benzylidene fragments 2-27 were reported. The prepared 5,5-
diphenylimidazolidine-2,4-dione derivatives were evaluated in vivo for anti-inflammatory, analgesic activities and
in vitro for COX-1/2 inhibition assay. Among the tested compounds, derivatives 5, 9, 10, 13, and 14 showed
significant and potent anti-inflammatory and analgesic activities almost equivalent to reference drug celecoxib. In
COX-1/2 inhibition assay, compounds 5, 9, 10 and 14 showed high COX-2 inhibitory activity (IC50 = 0.70
µM,
0.44 M, 0.61 M and 0.41 M; respectively) and selectivity index (SI) range of 142-243 comparable to celecoxib
µ
µ
µ
[COX-2 (SI) >333]. These potent COX-2 inhibitors 9, 10, 13, and 14 were docked into the active site pocket of
COX-2 to explore the binding mode and possible interactions of these ligands.
Keywords: Imidazolidine-2,4-dione; synthesis: anti-inflammatory activity; COX-1/2 inhibition;
molecular docking study.
1. Introduction
———
* Corresponding author. Tel.: 00966-53-5991127; fax: 00966-1-4676220; e-mail: alaa_moenes@yahoo.com

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Non-steroidal anti-inflammatory therapeutics, such as aspirin, flurbiprofen, ibuprofen and indomethacin,
are considered as important compounds that used for the treatment of inflammatory disorders and pain
[1-5]. The clinical uses of these drugs are almost showed gastrointestinal implication including peptic
ulcer [1-5]. The mode of action of such group of drugs is through inhibition of cyclooxygenase enzymes
which are important for biosynthesis of the prostanoids [6-8]. These cyclooxygenase enzymes are of two
types, COX-1 is concerned with kidneys and the stomach physiological roles, while COX-2 has a main
role in production of prostaglandins which are responsible for inflammation and pain [9-12]. Ibuprofen
and flurbiprofen (A and B; Figure 1) are examples of non-steroidal anti-inflammatory drugs which are
non selective inhibitors for both COX-1 and COX-2, but bind more tightly to COX-1 [13,14]. On the
other hand, several compounds possessing pyrazole core structure have been reported in the literature as
selective COX-2 inhibitors such as celecoxib (C; Figure 1) [15-19]. These compounds showed an
improvement of gastric protection compared with non-steroidal anti-inflammatory drugs [15-19].
Moreover, imidazolidine-2,4-diones are an interesting compounds which possess structural features
required for several potential biological activities, for examples COX-1/2 inhibition (D and E; Figure 1),
anti-inflammatory (F and G; Figure 1), anticonvulsant, antitumor, antihyperlipidemic and
phosphodiesterase-5
inhibiting
activities
[20-28].
Interestingly,
compounds
of
5,5-
diphenylimidazolidine-2,4-dione substituted with methylsulfonyl fragment at the 4-position of one of
phenyl ring (E; Figure 1) were prepared and investigated their selective activity as COX-2 inhibitors
[25,26]. The results of this study indicated that N-3 substituent of imidazolidine-2,4-dione core structure
is important for COX-2 inhibitory activity [25,26].
As part of our efforts to design novel anti-inflammatory agents with COX-2 inhibiting activity and on
continuation of our previous reports [29-33], we now describe the synthesis, anti-inflammatory,
analgesic and COX-1/2 inhibitory activities of 5,5-diphenylimidazolidine-2,4-dione bearing anilide,
phenacyl and benzylidene fragments 2-27 (H and I; Figure 1), involving various substituents such as H,

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MeO, F, SO2NH2 and 3,4,5-trimethoxy groups. The rationale for evaluation the anti-inflammatory
activity and selective COX-2 inhibition of these 5,5-diphenylimidazolidine-2,4-diones (H and I; Figure
1) was the following: (i) design the structure-activity relationship for compounds incorporating anilides
with diversely substituent groups; (ii) recognize the effectiveness of the acetamide versus the
propanamide derivatives; (iii) Thus, in addition to the anilides 2-17, derivatives of phenacyl 18-20,
benzylideneacetohydrazides 23-25, as well as 4-methoxybenzylideneamino derivative 27 at the 3-
position of 5,5-diphenylimidazolidine-2,4-dione core were also included in the study in order to cover
the most relative analogues. Furthermore, molecular docking is an important technique used in drug
design through flexible alignment into the binding site of the target enzyme, and prediction the unique
binding ability for each ligand at the active site pocket of the enzyme [30-34]. The promising lead
compounds were docked into the binding sites of both COX-1 and COX-2 to explore their
complementarity with the specified binding pockets.
2. Results and discussion
2.1. Chemistry
The synthetic methods used to synthesize compounds 2-27 were shown in Schemes 1 and 2. The
structures of the target products 2-27 were confirmed by elemental, spectral analyses and X-ray of
compound 26 [35].
2.1.1. Synthesis of compounds 2–20 (Scheme 1)
5,5-Diphenylimidazolidine-2,4-dione (1) was reacted with N-aryl-2-chloroacetamide [34] in dry acetone
in the presence of potassium carbonate for 24 hrs (Scheme 1) or N-aryl-3-chloropropanamide [34] in dry
DMF in the presence of potassium carbonate at 50οC for 24 hrs to afford N-aryl-2-(2,5-dioxo-4,4-
diphenylimidazolidin-1-yl)acetamides (2-10) or N-aryl-3-(2,5-dioxo-4,4-diphenylimidazolidin-1-
yl)propanamides (11-17) in satisfactory yields. Compounds 18-20 (Scheme 1) were prepared according
to our previous report [22].

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2.1.2. Synthesis of compounds 21-27 (Scheme 2)
Compounds 21-25 were synthesized by the reaction of 5,5-diphenylimidazolidine-2,4-dione (1) with
ethyl 2-bromoacetate in dry acetone in the presence of potassium carbonate at room temperature to
afford ethyl 2-(2,4-dioxo-5,5-diphenylimidazolidin-1-yl)acetate (21) in 97 % yield. The later compound
21 was reacted with hydrazine hydrate in methanol at room temperature to give the corresponding 2-
(2,4-dioxo-5,5-diphenylimidazolidin-3-yl)acetohydrazide (22) in 90% yield. Additionally, N-arylidene-
2-(2,4-dioxo-5,5-diphenylimidazolidin-3-yl)acetohydrazides (23-25) were obtained in satisfactory yields
by refluxing compound 22 in methanol with benzaldehyde derivatives (Scheme 2).
On the other hand, compound 1 was heated with hydrazine hydrate at reflux temperature for 12 hrs to
give 3-amino-5,5-diphenylimidazolidine-2,4-dione (26) in 86 % yield (Scheme 2). The structure of
compound 26 was confirmed by X-ray crystallography analysis according to our previous report [35].
Furthermore
3-amino-5,5-diphenylimidazolidine-2,4-dione
(26)
was
reacted
with
4-
methoxybenzaldehyde to afford 3-(4-methoxybenzylideneamino)-5,5-diphenylimidazolidine-2,4-dione
(27).
2.2. Biological activity
2.2.1. In vivo anti-inflammatory activity
Twenty three compounds 2-20, 23-25 and 27 along with the reference drug celecoxib were subjected to
in vivo anti-inflammatory evaluation by employing carrageenan-induced rat foot paw oedema method
(Table 1) [29-33,36]. Most of the tested compounds showed an effective ED50 which was measured after
2 h of treatment with carrageenan (Table 1), at which maximum percentage of inhibition of carrageenan-
induced oedema was reached. ED50 values were measured with three doses of 50, 100 and 200 mg/kg in
the tested compounds while it was 25, 50 and 100 mg/kg in the reference drug celecoxib. The results of
anti-inflammatory activity showed that compounds 3, 4, 5, 7, 8, 9, 10, 12, 13, 14, 16, and 17 have
potential anti-inflammatory activity with ED50 of 75-129 mg/kg, while compounds 19 and 20 showed

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moderate anti-inflammatory activity with ED50 of 156 mg/kg and 154 mg/kg respectively. Compounds
5, 9, 10 and 14 were the most active among the tested compounds with ED50 values of 97 mg/kg, 75
mg/kg, 90 mg/kg and 75 mg/kg, respectively, which are almost similar to celecoxib (ED50= 86.4
mg/kg).
Structure activity relationship for the anti-inflammatory activity mentioned above with derivatives 2-27
(Table 1) indicated that : (i) derivatives of substituted 4-methoxy (8, 13 and 17), 3,4,5-trimethoxy (10)
and 4-sulfonamide (5, 9 and 14) belong to this category of effective anti-inflammatory agents, while
unsubstituted derivatives (2, 6, 11 and 15) have insignificant biological activity; (ii) 4-fluorophenyl
derivatives (3, 7, 12 and 16) showed lower anti-inflammatory activity compared with methoxylated
derivatives (4, 8, 10, 13 and 17), as these findings may be attributed to the electronic effect at receptor
binding site; (iii) propanamide derivatives (12-17) showed a little increase in anti-inflammatory activity
compared with acetamide derivatives (3-10); (iv) anilides (3-17) are more effective as anti-inflammatory
agents than the corresponding phenacyl (18-20) and benzylidene derivatives (23-25 and 27). For
example the 4-fluorophenyl derivatives (3: ED50= 129 mg/kg, 7: ED50= 120 mg/kg, 12: ED50= 123
mg/kg and 16: ED50= 122 mg/kg) and 4-methoxyphenyl derivatives (4: ED50= 120 mg/kg, 8: ED50= 109
mg/kg, 13: ED50= 98 mg/kg and 17: ED50= 112 mg/kg), showed different anti-inflammatory activities as
expressed by their ED50. More interestingly, the 4-sulfonamidobenzyl derivative (9: ED50= 75 mg/kg)
and 4-sulfonamidophenyl derivative (14: ED50= 75 mg/kg) incorporating different anilides (acetamide
and propanamide), showed similar activity, suggesting a quite similar mode of action on the target
enzyme. In regard to methoxylated derivatives, the 3,4,5-trimethoxy derivative (10: ED50= 90 mg/kg)
was more active compared to the corresponding 4-methoxy derivative (8: ED50= 109 mg/kg) (Table 1).
2.2.2. In vivo analgesic evaluation

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Compounds that showed in vivo anti-inflammatory activity with ED50 <200 mg/kg were selected for
evaluation the analgesic activity using hot plate technique after 60 minutes of intraperitoneal injection at
58oC [33,37,38]. The analgesic activity of the tested compounds was compared with known reference
drug celecoxib. The results of the analgesic activity of the tested compounds revealed that the highest
analgesic activity was observed for compounds 5, 8, 9, 10, 13, 14, and 16 with ED50 values of 66.2,
67.3, 54.6, 68.5, 71.1, 40.3 and 75.0 mg/kg respectively, when compared with celecoxib (ED50 of 74.1
mg/kg). It is clear that compounds 9 (54.6 mg/kg) and 14 (40.3 mg/kg) are the most potent analgesic
agents that are almost two folds as celecoxib (74.1 mg/kg). On the other hand, compounds 3, 4, 7, 12
and 17 (ED50 of 119.0, 86.0, 90.9, 80.1 and 122.0 mg/kg, respectively) have shown variable lower
analgesic activity relative to celecoxib (74.1 mg/kg).
2.2.3. In vitro COX inhibition assay
According to the previous rationale (Figure 1) and the anti-inflammatory activity of the designed
compounds (Table 1), the most active compounds as anti-inflammatory agents with ED50 <200 mg/kg,
are subjected to in vitro COX-1/2 inhibition assay using an ovine COX-1/2 assay kit (Catalog No.
560101, Cayman Chemicals Inc., Ann Arbor, MI, USA). Accordingly, fourteen compounds were
selected for in vitro evaluation, IC50 (µM) was determined [29-33,39], and the selectivity index (SI
value) was calculated as IC50 (COX-1) / IC50 (COX-2). The results of COX inhibition assay indicated
that some of the tested compounds had a reasonable potent inhibitory activity against COX-2 (IC50
0.41-1.32
compounds 19 and 20 with IC50 values of 70.5
and 17 were found to be potent and selective inhibitors to COX-2 with IC50 values of 0.7
0.44 M, 0.61 M, 0.99 M, 0.41 M, 1.32 M, respectively (Table 2).
Interestingly, anilides with sulfonamide moiety such as compounds 9 (IC50 = 0.44
>227.2) and 14 (IC50 = 0.41 M, COX-2 (SI) >243.9) were the most potent inhibitors among the tested
µ
M), while all tested compounds showed no inhibition for COX-1 (IC50 > 100
M as listed in Table 2. Compounds 5, 8, 9, 10, 13, 14,
M, 1.3 M,
µM) except
µ
µ
µ
µ
µ
µ
µ
µ
µM, COX-2 (SI)
µ

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compounds, nearly similar to celecoxib (IC50 = 0.30 µM, COX-2 (SI) >333.3). The electronic effect of
the substituents located on the arylacetamide and arylpropanamide fragments of compounds 3-5, 7-10,
12-14, 16 and 17 were approved that the introduction of a sulfonamide moiety such as compounds 5, 9
and 14 enhanced COX-2-inhibiting activity with selectivity index of >142.8, >227.2 and >243.9,
respectively (Table 2). More interestingly, introduction of methoxy groups such as compounds 8 (SI=
>76.9), 10 (SI= >163.9), 13 (SI= >101.0) and 17 (SI= >75.7) resulted in increasing the COX-2 inhibition
activity when compared with anilides having fluoro moiety such as compounds 7 (SI= >13.8), 12 (SI=
>9.2) and 16 (SI= >9.2), as the latter compounds 7, 12 and 16 showed sharp decrease of COX-2
inhibition. On the other hand, compound 10 with the function; 3,4,5-trimethoxybenzylacetamide (SI=
>163.9) showed higher COX-2 inhibition activity when compared with 4-methoxybenzylacetamide 8
(SI= >76.9). On the other hand, the COX-2 inhibitory activity of phenacyl analogues such as compounds
19 (SI= 2.7) and 20 (SI= 2.8) was sharply decreased while COX-1 inhibition activity was increased
which may be explained on the bases of the anilide fragments favour COX-2 inhibition and phenacyl
fragments favour COX-1/2 inhibition (Table 2).
The following SAR was observed for the inhibition of the COX-2 isoform mentioned above with
derivatives 3-20 (Table 2): (i) It may be observed that both sulfonamide and methoxy derivatives belong
to this category which are effective as COX-2 inhibitors, while fluoro derivatives seem to have been of
little importance for the biological activity; (ii) anilide derivatives are more effective as COX-2
inhibitors than the corresponding phenacyl derivatives; (iii) phenacyl derivatives showed higher COX-1
inhibiting activity when compared with COX-1 inhibition effect of anilide derivatives. The results of
COX-2 inhibition assay indicated that N-substituted 5,5-diphenylimidazolidine-2,4-dione scaffold that
contain 4-sulfonamidoanilide moieties (compounds 9 and 14) and 3,4,5-trimethoxyanilide moiety (10)
showed preeminent COX-2 inhibition.
2.3. Molecular docking studies

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The differences between the binding sites of COX-1and COX-2 obtained from X-ray crystal structure
indicated that the COX-2 binding site possesses an additional 2º-pocket which is obscure in COX-1.
Entrance of ligands to the 2º-pocket of COX-2 is controlled by histidine (His90), glutamine (Gln192), and
tyrosine (Tyr355) [40,41]. It has also been reported Arg513 in COX-2 plays a key role with respect to the
H-bond network in the COX-2 binding site.
To explain the COX-inhibiting activity of 5,5-diphenylimidazolidine-2,4-dione analogues and their
selectivity to COX-2 enzyme, molecular docking studies were performed using MOE 2007.09 program
[42]. The present work based on the comparative study of docking the 5,5-diphenylimidazolidine-2,4-
diones in both COX-2 and COX-1 enzyme pockets. The most active synthesized compounds were
docked into both COX-2 enzyme pocket in complex with SC-558 (PDB entry 1CX2) [18] to evaluate
their binding ability, and the binding active pocket of COX-1 to explain their inconsequential
recognition at the COX-1 binding site of the protein in complex with celecoxib (PDB entry 3KK6) [43].
Compounds 14 (IC50 = 0.41 µM), 9 (IC50 = 0.44 µM) and 10 (IC50 = 0.61 µM) were the most active
analogues and showed the highest recognition at the COX-2 binding site which was in agreement with
their selectivity index and percentage inhibition of the COX-2 enzymology assay (Table 2, Figures 2 and
3). These three selective analogues showed inconsequential recognition at the binding site of COX-1
binding pocket (Figure 4). The results of molecular docking study using COX-2 enzyme (PDB entry
1COX-2) showed that compound 14 (Figure 2) has unique binding configuration by performing
hydrogen bond between carbonyl oxygen of imidazolidine-2,4-dione ring and Arg120, while amide
carbonyl oxygen hocked by stable hydrogen bond with the conserved residue His90. Additionally the
crucial sulfonamide group held tightly at the rare end of the binding site groove through hydrogen
binding to both Arg513 and Ile517. However, the amino acid Tyr355 squashed away from the carbonyl
oxygen of imidazolidine-2,4-dione core and lost the formation of significant recognition that was due to
the bulkiness of the 5,5-diphenyl groups. It remains facing in the opening cleft of the groove with

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smooth stretching of the propanamide bridge in a zigzag form that overall balance the coherence
between ethyl linkage and the surrounding hydrophobic amino acid residues Val523 and Gly526.
Interestingly, molecular docking of compound 9 (Figure 2) showed that the central amide group at the
bridge between the two essential phenyl and imidazolidine-2,4-dione rings have proper recognition with
the most crucial active site key amino acids namely; His90 and Tyr355. The terminal sulfonamide
fragment remains the strategic function that well established recognition with the groove wall residues
through hydrogen bonding to both Arg513 and Ala516. Imidazolidine-2,4-dione was inaccessible as it was
horizontally embedded (Angle 114.4ο) nearby the amide group and the two phenyl groups prevent the
surrounding residues from reaching its promising dicarbonyl oxygen. Alignment of both compounds 14
and 9 in the pocket of COX-2 enzyme was performed which indicated that both compounds have the
same binding behaviour toward COX-2 active site (Figure 3). On the other hand, molecular docking of
compound 10 (Figure 2) showed that the carbonyl oxygen and N-amide fragments balanced the
configuration to allow interaction with the two crucial active site key amino acids namely; His90 and
Tyr355. It was noticeable that the 3,4,5-trimethoxyphenyl ring was unfavourably wide leading to
deformation of the electrostatic envelop around the groove. While 5-methoxy group substitution was
protruded out of the groove wall, the 4-methoxy oxygen group was directed away from the key amino
acid residues the groove wall. The 3-methoxy oxygen was able to perform stable hydrogen bond with
non-conserved amino acid in the narrow groove; Gln192. In comparing compounds 13 and 14, it was
clear that 4-sulfonamide group is the most vital pharmacophoric group that showed specific recognition
with the narrow groove amino acids and replacement of this group with 4-methoxy group (compound
13) abolish the hydrogen bonding interaction and in turn showed lower COX-2 selectivity; Table 2. This
molecular docking study for compound 13 also showed that the carbonyl oxygen of propanamide chain
showed proper hydrogen bonding with the key amino acid; His90, due the advantage of the presence of

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CH2-CH2 spacer adjacent to imidazolidine-2,4-dione core that was able to protrude nearby the crucial
amino acid; Arg120 to perform a stable hydrogen bond (Figure 2).
Furthermore, compounds 9 and 10 as representative examples were docked into the active site of COX-1
enzyme to understand the inactivity of such compounds toward COX-1 enzyme (Figure 4). Structural
bulkiness of compounds 9 and 10 were the most crucial features that lead to selective binding at the
COX-2 wide binding active site rather than COX-1 binding site. Compound 9 has improper binding due
to size incompatibility represented by the 5,5-diphenyl system that lay at the opening of the groove and
the presence of three methoxy groups at the far terminal forced the active site amino acids to produce
marked unfavourable configurational changes (RMSD > 1.5Å). Similarly, compound 10 has no binding
interaction at COX-1 binding site because of the incompatibility between the narrow groove and the
bulkiness of the proposed compound and also the phenyl group was squeezed out of the groove (Figure
4).
3. Conclusion
A series of 5,5-diphenylimidazolidine-2,4-dione bearing anilide, phenacyl and benzylidene derivatives
2-27 were designed, synthesized and evaluated their in vivo anti-inflammatory, analgesic activities and
in vitro COX-1/2 inhibitory activity. Compounds 5, 9, 10, 13 and 14 showed potent anti-inflammatory
and analgesic activities equivalent to reference drug celecoxib. Moreover, compounds 5, 9, 10 and 14
showed high COX-2 inhibitory activity (IC50 = 0.70
µM, 0.44 µM, 0.61 µM and 0.41 µM; respectively)
and selectivity index (SI) range of 142-243 compared to celecoxib [COX-2 (SI) >333].
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. The presence of terminal
4-sulfonamidophenyl moiety in compounds 9 and 14 enhanced the high recognition at the active site
minor groove. Additionally; keeping the amide bridge with two carbon spacers around the amide

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function was the most crucial factor controlling highest COX-2 selectivity. On contrary, these two
analogues showed inconsequential recognition at the binding site of COX-1 binding pocket.
4. Experimental
4.1. Chemistry
Melting points (uncorrected) were recorded on Barnstead 9100 Electrothermal melting apparatus. IR
spectra were recorded on a FT-IR Perkin-Elmer spectrometer. 1H NMR and 13C NMR were recorded in
DMSO-d6 on Bruker 500 and 125 MHz instrument, respectively, using TMS as internal standard
(chemical shifts in ppm). Mass spectra were recorded on an Agilent 6320 Ion Trap mass spectrometers.
Elemental analysis was carried out for C, H and N using Perkin-Elmer 240 analyzer and agreed with
proposed structures within ±0.4% of the theoretical values. Compounds 2-5 [44-46], 18-20 [22], 21 [47],
22 [47] and 26 [35] were prepared according to their reported procedure.
4.1.1. General procedure for the synthesis of compounds 2-10.
A mixture of 5,5-diphenylimidazolidine-2,4-dione (1) (1.0 mmol, 0.252 gm), N-aryl-2-chloroacetamide
(1.2 mmol) and anhydrous potassium carbonate (2.0 mmol, 0.276 gm) in dry acetone (10 mL) was
stirred at room temperature for 24 hrs. The reaction mixture was filtered off, the solvent was removed
under reduced pressure and the solid obtained was washed with water, dried and re-crystallised from an
appropriate solvent.
4.1.1.1. 2-(2,5-Dioxo-4,4-diphenylimidazolidin-1-yl)-N-phenylacetamide (2) [44].
White powder, Mp 245-247οC ( Lit. 246-247οC) [44], 88% yield (CH3H2OH/CH2Cl2); IR (KBr, cm-
1
1
)
ν
: 3198 (NH), 1772, 1717 (C=O); H NMR (500 MHz, DMSO-d6):
δ
4.33 (s, 2H), 7.06- 7.09 (t,
13
1H, J = 7.0 Hz), 7.31-7.45 (m, 12H), 7.59-7.60(d, 2H, J = 8.0 Hz), 9.71 (s, 1H), 10.43 (s, 1H); C
NMR (125 MHz, DMSO-d6):
δ 41.02, 69.50, 119.09, 123.50, 126.56, 126.95, 128.16, 128.45,
128.81, 138.55, 139.61, 154.96, 164.54, 173.40; C23H19N3O3: m/z (385.4).
4.1.1.2. 2-(2,5-Dioxo-4,4-diphenylimidazolidin-1-yl)-N-(4-fluorophenyl)acetamide (3) [45].

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White powder, Mp 271-273οC ( Lit. 272-273οC) [45], 89% yield (CH3OH/CH2Cl2); IR (KBr, cm-1)
ν
: 3220 (NH), 1779, 1720 (C=O); C23H18FN3O3: m/z (403.2).
4.1.1.3. 2-(2,5-dioxo-4,4-diphenylimidazolidin-1-yl)-N-(4-methoxyphenyl)acetamide (4) [45].
White powder, M.p 288-290οC (Lit. 294-296οC) [45], 81% yield (CH3OH/CH2Cl2); IR (KBr, cm-1)
ν
: 3196 (NH), 1770, 1716 (C=O); C24H21N3O4: m/z (415.4).
4.1.1.4. 2-(2,5-dioxo-4,4-diphenylimidazolidin-1-yl)-N-(4-sulfamoylphenyl)acetamide (5) [46].
White powder, Mp 280-282οC ( Lit. 285-287οC) [46], 85% yield (CH3H2OH); IR (KBr, cm-1)
ν:
3205 (NH), 1770, 1717 (C=O); 1H NMR (500 MHz, DMSO-d6):
δ 4.36 (s, 2H), 7.37-7.42 (m, 4H),
7.43-7.46 (t, 8H, J = 8.5 Hz), 7.73- 7.75 (d, 2H, J = 8.5 Hz), 7.79-7.81 (d, 2H, J = 9.0 Hz), 9.75 (s,
1H), 10.75 (s, 1H); 13C NMR (125 MHz, DMSO-d6):
δ
41.11, 69.57, 118.72, 126.58, 126.81,
126.91, 128.19, 128.40, 128.48, 138.70, 139.55, 141.35, 154.87, 165.19, 173.32; C23H20N4O5S: m/z
(464.6).
4.1.1.5. N-benzyl-2-(2,5-dioxo-4,4-diphenylimidazolidin-1-yl)acetamide (6).
White powder, Mp 239-240οC, 89% yield (CH3OH/CH2Cl2); IR (KBr, cm-1)
ν: 3277 (NH), 1773,
1
1717 (C=O); H NMR (500 MHz, DMSO-d6):
δ 4.14 (s, 2H), 4.32-4.33 (d, 2H, J = 5.5 Hz), 7.24-
7.41 (m, 15H), 8.72-8.73 (d, 1H, J = 5.5 Hz), 9.63 (s,1H); 13C NMR (125 MHz, DMSO-d6):
δ
42.80, 40.82, 69.95, 127.05, 127.38, 127.47, 127.71, 128.46, 128.63, 128.81, 128.91, 128.95,
139.38, 140.14, 140.47, 155.48, 166.30, 173.89; C24H21N3O3: m/z (399.3).
4.1.1.6. 2 2-(2,5-dioxo-4,4-diphenylimidazolidin-1-yl)-N-(4-fluorobenzyl)acetamide (7).
White powder, M.p 244-245οC, 90% yield (CH3H2OH/CH2Cl2); IR (KBr, cm-1)
ν: 3344, 3294 (NH),
1
1763, 1714 (C=O); H NMR (500 MHz, DMSO-d6):
δ
4.14 (s, 2H), 4.32 (s, 2H), 7.17-7.41 (m,
40.34, 41.61, 69.47, 115.03 (d,
14H), 8.73 (s, 1H), 9.63 (s, 1H); 13C NMR (125 MHz, DMSO-d6):
δ
J = 25.4 Hz), 126.97, 128.13, 128.41, 129.23, 135.12, 139.64, 154.99, 162.20, 165.85, 173.40;
C24H20FN3O3: m/z (417.4).

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4.1.1.7. 2-(2,5-dioxo-4,4-diphenylimidazolidin-1-yl)-N-(4-methoxybenzyl)acetamide (8).
White powder, M.p 197-199οC, 83% yield (CH3H2OH/CH2Cl2); IR (KBr, cm-1)
ν: 3278 (NH), 1771,
1717 (C=O); 1H NMR (500 MHz, CDCl3/DMSO-d6): δ 3.74 (s, 3H), 4.13 (s, 2H), 4.25-4.26 (d, 2H,
J = 7.0 Hz), 6.85-6.87 (dd, 2H, J = 3.0, 6.0 Hz), 7.19-7.21 (d, 2H, J = 8.5 Hz), 7.32-7.39 (m, 5H),
7.43-7.44 (d, 3H, J = 7.5 Hz), 8.22 (s, 2H); 13C NMR (125 MHz, DMSO-d6):
δ 41.77, 42.49, 54.92,
69.36, 113.59, 126.99, 127.93, 128.20, 128.53, 128.62, 130.63, 139.60, 154.93, 158.29, 165.59 (d, J
= 19.0 Hz), 173.31; C25H23N3O4: m/z (429.5).
4.1.1.8. 2-(2,5-dioxo-4,4-diphenylimidazolidin-1-yl)-N-(4-sulfamoylbenzyl)acetamide (9).
White powder, M.p 239-241οC, 73% yield (CH3H2OH); IR (KBr, cm-1)
ν: 3291 (NH), 1772, 1740,
1718 (C=O); 1H NMR (500 MHz, DMSO-d6): δ 4.17 (s, 2H), 4.40 (s, 2H), 7.34-7.46 (m, 14H), 7.79
13
(s, 2H), 8.84 (s, 1H), 9.64 (s, 1H); C NMR (125 MHz, DMSO-d6):
δ
40.86, 42.42, 69.96, 126.20,
127.05, 127.45, 127.99, 128.93, 128.99, 140.10, 143.20, 143.57, 155.47, 166.55, 173.33;
C24H22N4O5S: m/z (478.4).
4.1.1.9. 2-(2,5-dioxo-4,4-diphenylimidazolidin-1-yl)-N-(3,4,5-trimethoxybenzyl)acetamide (10).
White powder, M.p 271-273οC, 87% yield (CH3OH); IR (KBr, cm-1)
(C=O); 1H NMR (500 MHz, CDCl3/DMSO-d6):
ν: 3271 (NH), 1771, 1716
δ
3.66 (s, 3H), 3.76 (s, 6H), 4.15 (s, 2H), 4.27-4.28
(d, 2H, J = 5.0 Hz), 6.55 (s, 2H), 7.31-7.36 (m, 6H), 7.41-7.42 (d, 4H, J = 7.5 Hz), 8.62 (s, 1H), 9.53
(s, 1H); 13C NMR (125 MHz, DMSO-d6):
δ
40.38, 42.51 (d, J = 15.3 Hz), 55.63, 59.91, 69.44 (d, J =
12.3 Hz), 104.15, 126.94, 127.98 (d, J = 30.6 Hz), 134.32, 136.27, 139.5, 152.73, 154.99 (d, J = 9.3
Hz), 165.87 (d, J = 9.9 Hz), 173.38; C27H27N3O6: m/z (489.2).
4.1.2. General procedure for the synthesis of compounds 11-17.
A mixture of 5,5-diphenylimidazolidine-2,4-dione (1) (1.0 mmol, 0.252 gm), N-aryl-3-
chloropropanamide (1.2 mmol) and anhydrous potassium carbonate (2.0 mmol, 0.276 gm) in dry DMF
(10 mL) was stirred at 50οC for 24 hrs. The reaction mixture was filtered off, the solvent was removed

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under reduced pressure and the precipitate obtained was washed with water, dried and re-crystallised
from an appropriate solvent.
4.1.2.1. 3-(2,5-dioxo-4,4-diphenylimidazolidin-1-yl)-N-phenylpropanamide (11).
White powder, M.p 297-299οC, 89% yield (CH3OH/CH2Cl2); IR (KBr, cm-1)
1721 (C=O); 1H NMR (500 MHz, DMSO-d6):
ν: 3190 (NH), 1769,
δ
1.68-1.70 (d, 3H, J = 7.0 Hz), 4.77-4.79 (d, 1H, J =
7.5 Hz), 7.07 (s, 1H), 7.32-7.42 (m, 12H), 7.57-7.59 (d, 2H, J = 8.0 Hz), 9.68 (s, 1H), 10.02 (s, 1H);
13C NMR (125 MHz, DMSO-d6):
δ
15.42, 50.73, 69.24, 120.16, 124.04, 127.29, 127.55, 128.57,
128.60, 128.92, 128.94, 129.16, 139.15, 140.17, 140.39, 155.54, 168.03, 173.81; C24H21N3O3: m/z
(399.3).
4.1.2.2. 3-(2,5-dioxo-4,4-diphenylimidazolidin-1-yl)-N-(4-fluorophenyl)propanamide (12).
White powder, M.p 289-290οC, 92% yield (CH3OH/CH2Cl2); IR (KBr, cm-1)
1733 (C=O); 1H NMR (500 MHz, DMSO-d6):
7.5 Hz), 7.04-7.08 (t, 2H, J = 8.5 Hz), 7.31- 7.43 (m, 12H), 9.55 (s, 1H), 9.95 (s, 1H); 13C NMR (125
ν: 3271 (NH), 1774,
δ
1.67-1.69 (d, 3H, J = 7.5 Hz), 4.76-4.77 (d, 1H, J =
MHz, DMSO-d6):
δ 14.81, 50.01, 68.67, 114.96 (d, J = 22.0 Hz), 121.32 (d, J = 7.5 Hz), 126.80,
127.76, 127.86, 128.19, 139.70 (d, J = 34.1 Hz), 154.92, 157.21, 167.24, 173.14; C24H20FN3O3: m/z
(417.3).
4.1.2.3. 3-(2,5-dioxo-4,4-diphenylimidazolidin-1-yl)-N-(4-methoxyphenyl)propanamide (13).
White powder, M.p 261-262οC, 87% yield (CH3H2OH/CH2Cl2); IR (KBr, cm-1)
ν: 3277 (NH), 1770,
1
1716 (C=O); H NMR (700 MHz, DMSO-d6):
δ
1.69 (s, 3H), 3.72 (s, 3H), 4.77 (s, 1H), 7.37-7.53
15.42, 50.59, 55.64, 69.25,
(m, 14H), 9.62 (s, 1H), 9.82 (s, 1H); 13C NMR (176 MHz, DMSO-d6):
δ
114.29, 121.89, 127.05, 127.31, 127.56, 128.55, 128.92, 132.22, 140.40, 155.58, 156.14, 167.58,
173.81; C25H23N3O4: m/z (429.6).
4.1.2.4. 3-(2,5-dioxo-4,4-diphenylimidazolidin-1-yl)-N-(4-sulfamoylphenyl)propanamide (14).

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15
White powder, M.p 249-251ο, 79% yield (CH3H2OH); IR (KBr, cm-1)
ν
: 3204 (NH), 1770, 1717
(C=O); 1H NMR (500 MHz, DMSO-d6):
Hz), 7.28- 7.42 (m, 12H), 7.79-7.83 (dd, 4H, J = 3.5, 9.0 Hz), 9.32 (s, 1H), 10.68 (s, 1H); 13C NMR
δ 1.62- 1.64 (q, 3H, J = 3.5 Hz), 4.70-4.74 (q, 1H, J = 6.5
(125 MHz, DMSO-d6):
δ 20.84, 54.61, 70.19, 119.11, 126.55, 126.75, 127.98, 128.46, 139.02,
139.89, 141.37, 155.96, 159.59, 167.79, 174.78; C24H22N4O5S: m/z (478.3).
4.1.2.5. N-benzyl-3-(2,5-dioxo-4,4-diphenylimidazolidin-1-yl)propanamide (15).
White powder, M.p 291-292ο, 84% yield (CH3H2OH/CH2Cl2); IR (KBr, cm-1)
ν: 3280 (NH), 1776,
1
1718 (C=O); H NMR (500 MHz, DMSO-d6):
δ
1.55-1.61 (q, 3H, J = 6.5 Hz), 4.29-4.35 (m, 2H),
13
4.54-4.58 (d, 2H, J = 6.5 Hz), 7.24-7.42 (m, 15H), 8.76 (s, 1H), 9.30 (s, 1H); C NMR (125 MHz,
CDCl3/DMSO-d6):
δ 21.91, 42.78, 54.85, 70.68, 127.04, 127.39, 127.63, 128.47, 128.70, 128.82,
128.96, 139.34, 140.43, 156.62, 169.29, 175.40; C25H23N3O3: m/z (413.5).
4.1.2.6. 3-(2,5-dioxo-4,4-diphenylimidazolidin-1-yl)-N-(4-fluorobenzyl)propanamide (16).
White powder, M.p 210-211ο, 88% yield (CH3H2OH/CH2Cl2); IR (KBr, cm-1)
1719 (C=O); 1H NMR (500 MHz, CDCl3/DMSO-d6):
ν: 3289 (NH), 1777,
δ
1.59-1.61 (d, 3H, J = 7.5 Hz), 4.28-4.34 (m,
2H), 4.64-4.68 (q, 1H, J = 7.5 Hz), 7.01-7.05 (t, 2H, J = 8.5 Hz), 7.25-7.43 (m, 12H), 8.42-8.44 (t,
1H, J = 6.0 Hz), 9.51 (s, 1H); 13C NMR (125 MHz, CDCl3/DMSO-d6):
δ
15.12, 42.34, 49.68, 69.27,
115.19 (d, J = 21.3 Hz ), 127.43 (d, J = 35.0 Hz), 128.28, 128.64, 129.29 (d, J = 7.5 Hz), 135.56 (d,
J = 3.4 Hz), 140.26 (d, J = 6.3 Hz), 155.53, 160.68, 162.61, 169.08, 173.61; C25H22FN3O3: m/z
(431.3).
4.1.2.7. 3-(2,5-dioxo-4,4-diphenylimidazolidin-1-yl)-N-(4-methoxybenzyl)propanamide (17).
White powder, M.p 260-262ο, 78% yield (CH3H2OH/CH2Cl2); IR (KBr, cm-1)
ν: 3271 (NH), 1773,
1
1717 (C=O); H NMR (500 MHz, DMSO-d6):
δ 1.54-1.56 (d, 3H, J = 6.5 Hz), 3.73 (s, 3H), 4.22-
4.26 (q, 2H, J = 6.0 Hz), 4.53-4.54 (d, 1H, J = 6.5 Hz), 6.89-6.91 (m, 2H), 7.18-7.20 (d, 2H, J = 8.5
Hz), 7.33-7.42 (m, 10H), 9.30 (s, 1H), 11.10 (s, 1H); 13C NMR (125 MHz, DMSO-d6):
δ
21.41,

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16
41.79, 54.38, 55.03, 70.18, 113.74, 126.55, 127.97, 128.45, 128.54, 130.75, 139.92, 156.02, 158.30,
168.62, 174.83; C26H25N3O4: m/z (443.5).
4.1.3. General procedure for the synthesis of compounds 23-25 and 27.
A solution of 2-(2,4-dioxo-5,5-diphenylimidazolidin-3-yl)acetohydrazide (22) (1.0 mmol, 0.324 gm) or
3-amino-5,5-diphenylimidazolidine-2,4-dione (26) (1.0 mmol, 0.267 gm) and an appropriate
benzaldehyde (1.1 mmol) in dry methanol (10 mL) was refluxed for 6 hrs. The reaction mixture was
cooled and the precipitate obtained was filtered off, dried and re-crystallised from an appropriate
solvent.
4.1.3.1. N'-Benzylidene-2-(2,5-dioxo-4,4-diphenylimidazolidin-1-yl)acetohydrazidene (23).
White powder, M.p >350ο, 94% yield (CH3OH); IR (KBr, cm-1)
ν: 3229 (NH), 1773, 1713 (C=O);
1H NMR (500 MHz, DMSO-d6):
δ
4.24 (s, 0.34H), 4.62 (s, 1.66H), 7.37-7.46 (m, 13H), 7.71-7.72 (t,
13
2H, J = 3.0 Hz), 8.05 (s, 0.67H), 8.20 (s, 0.33H), 9.70 (s, 1H), 11.71 (s, 1H); C NMR (125 MHz,
DMSO-d6):
δ 40.48, 69.54, 101.35, 104.83, 108.09, 123.16, 127.94, 128.18, 143.98, 147.95, 148.97,
155.07, 166.97, 173.45; C24H20N4O3: m/z (412.6).
4.1.3.2. 2-(2,5-Dioxo-4,4-diphenylimidazolidin-1-yl)-N'-(4-fluorobenzylidene)acetohydrazide (24).
White powder, M.p >350ο, 91% yield (CH3OH/CH2Cl2); IR (KBr, cm-1)
(C=O); 1H NMR (500 MHz, DMSO-d6):
ν: 3198 (NH), 1776, 1698
δ
4.22 (s, 0.34H), 4.61 (s, 1.66H), 7.26-7.29 (t, 2H, J = 9.0
Hz), 7.37-7.43 (m, 10H), 7.77-7.80 (q, 2H, J = 6.0 Hz), 8.04 (s, 0.67H), 8.19 (s, 0.33H), 9.69 (s, 1H),
11.70 (s, 1H); 13C NMR (125 MHz, DMSO-d6):
δ
40.58, 70.03, 116.31 (d, J = 22.5 Hz), 127.44,
127.48, 128.90 (d, J = 33.8 Hz), 129.65 (d, J = 7.5 Hz), 131.01, 140.07 (d, J = 7.5 Hz), 143.73,
155.51, 167.79, 173.99; C24H19FN4O3: m/z (430.3).
4.1.3.3.
2-(2,5-Dioxo-4,4-diphenylimidazolidin-1-yl)-N'-(4-methoxybenzylidene)acetohydrazide
(25).

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17
White powder, M.p 315-316ο, 95% yield (CH3OH); IR (KBr, cm-1)
ν
: 3234 (NH), 1773, 1717
(C=O); 1H NMR (500 MHz, DMSO-d6):
(m, 11H), 7.76 (s, 2H), 9.68 (s, 1H), 10.58 (s, 1H); 13C NMR (125 MHz, DMSO-d6):
δ 3.77 (s, 3H), 4.70 (s, 2H), 6.80-7.09 (m, 2H), 7.38-7.76
δ
54.66, 55.85,
69.70, 111.10, 115.38, 118.68, 127.01, 128.07, 128.28, 137.65, 139.52, 154.66, 154.90, 164.52,
173.49; C25H22N4O4: m/z (442.5).
4.1.3.4. 3-((4-Methoxybenzylidene)amino)-5,5-diphenylimidazolidine-2,4-dione (27).
White powder, M.p 315-316ο, 97% yield (CH3OH); IR (KBr, cm-1)
(C=O); 1H NMR (500 MHz, CDCl3):
7.33-7.56 (m, 10H), 7.78-7.80 (d, 2H, J = 8.5 Hz), 9.30 (s, 1H); 13C NMR (125 MHz, CDCl3):
ν: 3188 (NH), 1720, 1700
δ
3.85 (s, 3H), 6.94-6.96 (d, 2H, J = 8.5 Hz), 7.28 (s, 1H),
δ
55.42, 68.47, 114.23, 125.86, 127.03, 128.77, 128.89, 130.32, 138.80, 154.20, 159.34, 162.65,
169.80; C23H19N3O3: m/z (385.5).
4.2. Biological assay
4.2.1. Materials and animals
Male Sprague–Dawley rats weighing 250 g and male albino Swiss mice (25 g body weight) were
obtained from the animal house of the College of Pharmacy at King Saud University, Riyadh, Saudi
Arabia. These animals were kept at room temperature (22±2oC) in a light-controlled room with an
alternating 12 h light/dark cycle. Animals were allowed to become acclimatized to laboratory conditions
before experimentation and allowed free access to standard food and water. The tested compounds were
prepared as suspension in vehicle (0.5% methyl cellulose) and celecoxib was used as a standard drug.
The positive control group animals received the reference drug while the negative control received only
the vehicle. All procedures were performed with the approval of the Institutional Animal Care and Use
Committee. An enzyme immunoassay (EIA) kit (catalog no. 560101, Cayman Chemicals Inc., Ann
Arbor, MI, USA) was used in the in vitro cyclooxygenase inhibitory study.
4.2.2. In vivo anti-inflammatory screening

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The anti-inflammatory of the tested compounds were evaluated using in vivo rat carrageenan-induced
foot paw oedema model reported previously [29-33,36]. Male Sprauge–Dawley rats (250 gm body
weight) were fasted with free access to water at least 16 h prior to experiments. Oedema was produced
by injecting 0.2 mL of a solution of 1% λ-carrageenan in the hind paw. Paw volume was measured by
water displacement with a plethysmometer (UGO BASILE) before, 1 and 2 h after treatment. The tested
compounds and reference drug celecoxib were administered intraperitoneally with a 1 mL suspension of
test compound in vehicle (0.5% methyl cellulose). 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 drug. The
ED50 was measured after 2 hours of treatment with carrageenan, at which maximum percentage of
inhibition of carrageenan-induced oedema was reached along with celecoxib. Values reported as mean
±S.E.M. and significant differences were calculated using ANOVA.
4.2.3. Analgesic screening
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 58oC [33,37,38]. The mouse was watched
carefully for the time in seconds in which it displays nociceptive responses exhibited as licking or
blowing its front paws. 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 (5-200 mg/kg, ip). The reaction 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.
4.2.3. 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 [29-

ACCEPTED MANUSCRIPT
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33,39]. Briefly, to a series of supplied reaction buffer solutions (960
µL, 0.1M Tris–HCl pH8.0
containing 5
of heme (10
and 100 M in a final volume of 1mL). These solutions were incubated for a period of 5 min at 37ºC
after which 10 L of Arachidonic Acid (100 M) solution were added and the COX reaction was stopped
by the addition of 50 L of 1M HCl after 2 min. PGF2α, produced from PGH2 by reduction with stannous
µM EDTA and 2µM phenol) with either COX-1or COX-2 (10µL) enzyme in the presence
µL) were added 10µL of various concentrations of test drug solutions (0.01, 0.1, 1, 10, 50,
µ
µ
µ
µ
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 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 antibody–PG 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 410nm. 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: Absorbance α
[Bound PG Tracer] α1/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,
was calculated from the concentration–inhibition response curve (three determinations).
4.3. Molecular docking methodology
µM)
Molecular docking studies were performed with MOE 2007.09, software available from Chemical
Computing Group Inc., 1010 Sherbrooke Street West, Suite 910, Montreal, QC. The methodology used
for molecular docking protocol was similar to our previous reports [29-33].
4.3.1. Structural preparation of the new compounds

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Initial structures for the new molecules were constructed using the 2D/3D molecule builder module in
MOE. The compute module was used calculating the MMFF94x force field partial charges to each
molecule followed by optimal energy minimization.
4.3.2. Enzyme structure
The crystallographic structure of COX-2 in complex with SC-558 (PDB entry 1CX2) was obtained from
protein data bank [18]. The crystallographic structure of COX-1 enzyme in complex with celecoxib
(PDB entry 3KK6) was obtained from protein data bank [43]. Both enzymes were refined by adding the
missing hydrogens and removing of water molecules that present away from the active site. The ligand–
protein contacts (LPC) were determined to evaluate and compromise the degree of complementarity of
the original ligand and the new enzyme inhibitors.
4.3.3. Docking experiment
The lowest energy molecule was prepositioned using the crystal structure of the reference ligand as a
template for the initial placement at the enzyme-binding domain using a flexible alignment module with
iteration limit: 2000 and the energy cutoff: 10 to obtain the optimal alignment between the enzyme
ligand and the tested compound. Once prepositioned, enzyme ligand was deleted and each molecular
structure was optimally energy minimized in the enzyme-binding pocket. The interaction of the new
compound was studied to explore the conserved amino acid residues that are involved in the compound
recognition.
Acknowledgments
The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for
funding the work through the research group project No. RGP-VPP-163.

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Figure Legends

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Figure 1. Representative examples of non-selective COX inhibitors (A & B), selective COX-2 inhibitors (C, D & E),
reported anti-inflammatory imidazolidine-2,4-dione (F & G) and the designed imidazolidine-2,4-dione (H & I).
Figure 2. Figure 2. 2D interactions of compounds 14 (upper left panel), 9 (upper right panel), 10 (middle left panel), 8
(middle right panel) and 13 (lower panel) with the active site of COX-2. Hydrogen bonds are shown in as dotted
arrows.
Figure 3. 3D alignment of compounds 9 (violet) and 14 (blue) in the active site of COX-2 enzyme
.
Figure 4. 2D interactions of compounds 9 (right panel) and 10 (left panel) with the active site of COX-1. Hydrogen bonds are
shown in as dotted arrows.
Table 1. In vivo anti-inflammatory activity against carrageenan-induced rat paw oedema and analgesic activity represented
by ED50 (mg/kg) of the designed compounds.
Table 2. In vitro COX-1/COX-2 enzyme inhibition assay of the designed compounds represented as IC50 (µM)a.
Scheme 1. Synthesis of the designed imidazolidine-2,4-dione bearing anilide and phenacyl derivatives 2-20.
Scheme 2. Synthesis of the designed imidazolidine-2,4-dione bearing benzylidene derivatives 23-27.

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Figure 1. Representative examples of non-selective COX inhibitors (A & B), selective COX-2 inhibitors (C, D & E),
reported anti-inflammatory imidazolidine-2,4-dione (F & G) and the designed imidazolidine-2,4-dione (H & I).

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Asp
515
Leu
531
Gly
519
Gly
526
Ala
527
Arg
120
Ile
517
His
90
Leu
352
Ala
516
Val
523
Ser
353
Ser
530
O
Phe
518
O
O
N
Pro
514
N
H
NH
O
Arg
513
Tyr
355
Val
349
Figure 2. 2D interactions of compounds 14 (upper left panel), 9 (upper right panel), 10 (middle left panel), 8 (middle right
panel) and 13 (lower panel) with the active site of COX-2. Hydrogen bonds are shown in as dotted arrows.

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Figure 3. 3D alignment of compounds 9 (violet) and 14 (blue) in the active site of COX-2 enzyme
.

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Figure 4. 2D interactions of compounds 9 (right panel) and 10 (left panel) with the active site of COX-1. Hydrogen bonds are
shown in as dotted arrows.