Synthesis, anti-inflammatory evaluation and docking studies of some new fluorinated fused quinazolines

Article abstract of DOI:10.1016/j.ejmech.2010.07.063

A series of novel 8/10-trifluoromethyl-substituted-imidazo[1,2-c] quinazolines have been synthesized and evaluated in vivo (rat paw edema) for their anti-inflammatory activity and in silico (docking studies) to recognize the hypothetical binding motif of the title compounds with the cyclooxygenase isoenzymes (COX-1 and COX-2) employing GOLD (CCDC, 4.0.1 version) software. The compounds, 9b and 10b, were found to have good anti-inflammatory activity [around 80% of the standard: indomethacin]. The binding mode of the title compounds has been proposed based on the docking studies.

Full text of DOI:10.1016/j.ejmech.2010.07.063

European Journal of Medicinal Chemistry 45 (2010) 4904e4913
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, anti-inflammatory evaluation and docking studies of some new
fluorinated fused quinazolines
C. Balakumar a, P. Lamba b, D. Pran Kishore b, B. Lakshmi Narayana b, K. Venkat Rao b,
,
c
d
K. Rajwinder b, A. Raghuram Rao b , B. Shireesha , B. Narsaiah
*
a Centre with Potential for Excellence in Biomedical Sciences (CPEBS), Panjab University, Chandigarh 160 014, India
b Pharmaceutical Chemistry Division, University Institute of Pharmaceutical Sciences and UGC Center of Advanced Study in Pharmaceutical Sciences (UGC-CAS), Panjab University,
Chandigarh 160 014, India
c Vaagdevi College of Pharmacy, Ram Nagar, Hanamkonda-506 001, Andhra Pradesh, India
d Fluoroorganics Division, Indian Institute of Chemical Technology (IICT), Tarnaka, Hyderabad-500 007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 8/10-trifluoromethyl-substituted-imidazo[1,2-c] quinazolines have been synthesized
and evaluated in vivo (rat paw edema) for their anti-inflammatory activity and in silico (docking studies)
to recognize the hypothetical binding motif of the title compounds with the cyclooxygenase isoenzymes
(COX-1 and COX-2) employing GOLD (CCDC, 4.0.1 version) software. The compounds, 9b and 10b, were
found to have good anti-inflammatory activity [around 80% of the standard: indomethacin]. The binding
mode of the title compounds has been proposed based on the docking studies.
Received 23 April 2010
Received in revised form
22 July 2010
Accepted 29 July 2010
Available online 11 August 2010
Crown Copyright Ó 2010 Published by Elsevier Masson SAS. All rights reserved.
Keywords:
Fluorinated quinazolines
Cyclooxygenase
Anti-inflammatory activity
Docking studies
1. Introduction
COX-2 isoenzyme is evident to be over-expressed during
inflammatory conditions and was also found to exhibit a protective
Inflammation is a multifactorial process. It reflects the response
of the organism to various stimuli and is related to many disorders
such as arthritis, asthma and psoriasis which require prolonged or
repeated treatment. Cyclooxygenase (COX) and lipoxygenase (LOX)
produce two groups of arachidonic acid metabolites, prostaglandins
(COX products) and leukotrienes (LOX products), that play a key role
in inflammation. The classical nonsteroidal anti-inflammatory drugs
(NSAIDs) act via the inhibition of the COX-1 isoenzyme or the
combined inhibition of COX-1 and COX-2 isoenzymes. For example,
aspirin is a COX-1 selective inhibitor, whereas indomethacin and
naproxen are COX-1/COX-2 inhibitors. Because COX-1 is mainly
responsible for mucus formation in the gastrointestinal (GI) tract,
COX-1 inhibition is implicated for inducing GI irritation, the main
undesired side effect of such agents [1]. Another side effect, mild
bleeding diathesis also results from the selective inhibition of the
COX-1 catalyzed synthesis of the platelet aggregation factor,
thromboxane A2 [2].
role in asthma [3]. Literature reports reveal that COX isoenzymes
are the attractive molecular targets for the development of NSAIDs
[4]. Thus, inhibition of these enzymes leads to a decreased
production of prostaglandins and thromboxanes which in turn
accounts for the beneficial effects of NSAIDs (e.g. anti-inflamma-
tory, antipyretic, analgesic and cardiovascular effects) as well as
their undesirable side effect profiles (e.g. GI irritation). It has been
hypothesized that there might be other forms of the COX enzyme
yet to be discovered. The failure of COX-1 and COX-2 selective
inhibitors evoked the concept that inflammation be considered as
a multifactorial process and all biochemical pathways should be
taken into account [5].
Quinazolines are well known for their wide range of pharma-
cological activities including anti-inflammatory activity [6]. It has
been observed that a fluoro or trifluoromethyl group at a strategi-
c position on the heterocyclic ring enhances the activity of
a molecule due to its enhanced lipophilicity [7]. Imidazo[1,2-c]
quinazolines [8], l,2,4-triazolo[4,3-c]quinazolines [9], pyrroloqui-
nazolines [10], 4-phenethylaminoquinazolines [11], 2,3,6-trisub-
stituted quinazolines [12], 2-substituted aminoquinazolinones [13]
and benzothiazolylquinazolinones [14] were reported to have
* Corresponding author. Tel.: þ91 172 2534111; fax: þ91 172 2543101.
E-mail address: rrakkinepally@pu.ac.in (A. Raghuram Rao).
0223-5234/$ e see front matter Crown Copyright Ó 2010 Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.07.063

C. Balakumar et al. / European Journal of Medicinal Chemistry 45 (2010) 4904e4913
4905
significant anti-inflammatory, analgesic and antipyretic proper-
ties [15]. Some of the important fluorinated and nonfluorinated
compounds developed as cyclooxygenase (COX) inhibitors are
mentioned (Fig. 1).
The binding pockets of COX-1 [16] and COX-2 [17] were found to
be similar except one amino acid residue at position 523 where
COX-2 has a smaller valine (Val) residue, while COX-1 has larger
isoleucine (Ile) residue at that position. This minor difference in the
binding site produces a secondary pocket extending off the primary
binding site in COX-2, which is absent in COX-1. The combined
volume of the primary binding site and the secondary pocket in
COX-2 is about 25% larger (394 Å3) than the volume of the COX-1
binding site (316 Å3) [18].
In the present study we report the synthesis and evaluation of
in vivo anti-inflammatory activity of a series of novel 8/10-tri-
fluoromethyl-substituted-imidazo[1,2-c] quinazolines. Further, we
propose the molecular interactions and the binding mode of the
compounds using two targets (COX-1 and COX-2) based on in silico
docking studies.
2. Chemistry
As illustrated in Scheme 1, designed molecules were synthe-
sized from 4,4,4-trifluoro-1-phenylbutane-1,3-dione 1 which on
a reaction with malononitrile resulted in an interesting mixture of
trifunctional intermediate, 3-cyano-4-trifluoromethyl-6-phenyl-2-
F
F
O
HOOC
CH3
N
N
F
O
S
S
O
H2N
NH
F
H3C
O
F
S
H3C
O
CH3
O
H3C
(c) Ibuprofen
(b) L-745337
(a) Deracoxib
CH3
F
H3C
Cl
CH3
F3C
N
N
N
H
COOH
F
O
HOOC
S
NH2
O
(f) Flurbiprofen
(e) Lumiracoxib
(d) Celecoxib
COOH
SO2NH2
Cl
O
HN
N
Br
CH3
COOH
Cl
Cl
N
N
H3CO
CF3
(g) SC-558
(i) Diclofenac
(h) Indomethacin
H3C
O
H3C
O
S
O
O
O
O
N
Cl
S
H2N
O
O
S
N
N
H3C
O
H3C
(k) Valdecoxib
(l) Etoricoxib
(j) Rofecoxib
Fig. 1. Some important fluorinated and nonfluorinated compounds developed as cyclooxygenase (COX) inhibitors: (a) deracoxib; (b) L-745337 [COX-2(IC50:9.7
[COX-2(IC50: >30 M)]; (d) celecoxib [COX-2(IC50:1.0 M)]; (e) lumiracoxib; (f) flurbiprofen [COX-2(IC50: 6.46 M)]; (g) SC-558 [COX-2(IC50: 0.0093 M)]; (h) indomethacin [COX-2
(IC50: 0.49 M)]; (i) diclofenac [COX-2(IC50: 0.05 M)]; (j) rofecoxib [COX-2(IC50: 0.5 M)]; (k) valdecoxib [COX-2 (IC50: 0.89 M)]; (l) etoricoxib [COX-2 (IC50: 1.0 M)].
mM)]; (c) ibuprofen
m
m
m
m
m
m
m
m
m

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C. Balakumar et al. / European Journal of Medicinal Chemistry 45 (2010) 4904e4913
CF3
CN
CF3
CF3
O
CN
CN
i
O
+
+
CN
C6H5
N
H
O
C6H5
NH2
C6H5
CN
3
1
2
ii
CF3
O
C6H5
O
CF3
NH
iii
CONH2
NH2
NH
+
N
R
F3C
N
R
C6H5
H
H
C6H5
CONH2
CONH2
CONH2
6a-c
4
5a-c
iv
iv
CF3
O
N
C6H5
O
N
NH
R
NH
R
C6H5
F3C
R
O
CONH2
CONH2
a
8a-c
7a-c
v
v
b
OH
C6H5
N
CF3
N
N
N
S
c
F3C
N
R
C6H5
N
R
CONH2
CONH2
9a-c
10a-c
Scheme 1. Synthesis of 8/10-trifluoromethyl-substituted-imidazo[1,2-c] quinazoline derivatives (9aec and 10aec). Reagents and conditions: (i) methanol, reflux, 6 h; (ii) 20% KOH,
reflux, 7 h; (iii) aldehydes (a. 2-Furfuraldehyde, b. 4-Hydroxybenzaldehyde, c. Thiophene-2-carboxaldehyde), AcOH, r.t., 4 h; (iv) MnO2, dichloromethane, r.t., 2 h; (v) POCl3, 2-
chloroethylamine hydrochloride, reflux, 5 h.
(1H)-pyridone
2
and 2,6-dicyano-3-trifluoromethyl-5-phenyl
spectra of the resulting 9 and 10 showed signals around 3390 cmꢀ1
(NeH stretch) and 1135 cmꢀ1 (CeF stretch) and 1H NMR spectra
aniline 3, in different proportions. The intermediate 3 was hydro-
lyzed in basic conditions to result in 2,6-dicarboxamido-3-tri-
fluoromethyl-5-phenyl aniline 4 quantitatively and characterized
based on an earlier report [19]. Cyclization with different alde-
hydes, 4 gave a mixture of regioisomers 5aec and 6aec [20]. Each
isomer was separated by column chromatography using n-hexane/
ethyl acetate as mobile phase and characterized based on their
physical and spectral (IR, 1H NMR) data. The IR spectra of these
compounds (5 and 6) showed signals around 3434 cmꢀ1 (NeH
stretch), 1632 cmꢀ1 (C]O stretch) and 1147 cmꢀ1 (CeF stretch). 1H
NMR spectra of these compounds (5aec) gave two broad signals at
gave signals at
ance of the signal at
d
3.4 ppm and 4.2 ppm for imidazoline. Disappear-
7.8 ppm indicated the absence of cyclic amide
d
proton (CONH) due to cyclization and confirms the formation of
the title compounds.
3. Pharmacological evaluation: anti-inflammatory activity e
carrageenan-induced rat paw edema assay
The in vivo anti-inflammatory screening for the synthesized
compounds was performed by using the functional model of
carrageenan-induced rat paw edema and is presented as the
percentage inhibition of edema at the right hind paw in comparison
to the control (Table 1). Carrageenan-induced edema is a nonspe-
cific inflammation but is highly sensitive to NSAIDs. Indomethacin,
a potent NSAID was used as a reference standard. COX-2-mediated
increase in prostaglandin E2 (PG-E2) production contributes to the
severity of the inflammatory and pain responses in this model.
Wistar rats (female) weighing between 190 and 260 g,
obtained from Central Animal House, Panjab University, Chandi-
garh, India and were used in the present study. Animals were kept
in wire-mesh cages and maintained under constant environmental
d
6.6 ppm and
regioisomers (6aec) exhibited two broad signals at
7.7 ppm due to electron-withdrawing nature of CF3 group at the
ortho position to amide group.
d
7.4 ppm for amide group whereas their
d
7.0 ppm and
d
Each isomer under mild dehydrogenation using active MnO2
gave 4(3H)-quinazolones 7aec and 8aec respectively. Disappear-
ance of 1H NMR signals at
d 5.8 ppm (methine H) and d 8.2 ppm
(cyclic NH) in the product was conscipious. A facile cyclization of 7
and 8 on heating under reflux with 2-chloroethylamine hydro-
chloride in the presence of phosphorus oxychloride readily yielded
the title compounds (9aec and 10aec) in quantitative yields. The IR

C. Balakumar et al. / European Journal of Medicinal Chemistry 45 (2010) 4904e4913
4907
Table 1
The synthesized compounds were tested for their anti-inflam-
matory activity against carrageenan-induced paw edema at dose of
10 mg/kg. The percentage inhibition of edema was calculated using
the formula given below:
Anti-inflammatory data of the 8/10-trifluoromethyl-substituted-imidazo[1,2-c]
quinazoline derivatives (9aec and 10aec) on carrageenan-induced paw edema in
rats.
Treatment
Volume of paw edema (ml)*
Vc ꢀ Vt
ꢃ 100
Vc
2 h
4 h
Control (carrageenan treated)
0.89 ꢁ 0.069
0.84 ꢁ 0.016
Indomethacin (Imn)b
0.17 ꢁ 0.002a (79)
0.62 ꢁ 0.040a (31)
0.64 ꢁ 0.013a (29)
0.36 ꢁ 0.012a (61)
0.38 ꢁ 0.017a (59)
0.68 ꢁ 0.013 (25)
0.59 ꢁ 0.014a (34)
0.22 ꢁ 0.015a (75)
0.59 ꢁ 0.037a (30)
0.60 ꢁ 0.019a (28)
0.33 ꢁ 0.017a (60)
0.34 ꢁ 0.012a (57)
0.63 ꢁ 0.011 (24)
0.57 ꢁ 0.015a (33)
where Vc is the increase in paw volume of control (in the absence of
test compound) and Vt is the increase in paw volume after
administration of the test compound.
9a
10a
9b
10b
9c
10c
The given data is of five animals (female wistar rats) per group
and divided into eight groups. Group-1 referred as control (animals
received carrageenan along with the vehicle [0.5% carboxymethyl-
cellulose (CMC)]) and Group-2 received standard reference (indo-
methacin) along with the vehicle prior to the administration of
carrageenan. Group-3, 4, 5, 6, 7 and 8 received the test compounds
(9aec & 10aec) respectively 1 h prior to the administration of
carrageenan. All the test compounds were suspended in 0.5% of CMC
and administered orally (10 mg/kg) 60 min prior to the injection of
0.1 ml of freshly prepared carrageenan (1%) in physiological solution
(154 mM NaCl) into the sub-planter tissue of hind paw of each rat.
The equivalent volume of carrageenan (1%) in physiological solution
was injected into hind paw of the control. The volumewas measured
three times using water plethysmometer prior to the administration
of carrageenan, 2 h and 4 h after the injection. The increase in
volume of the paw was adopted as a measure of edema [22]. The
*Values are expressed as mean ꢁ S.E.M (n ¼ 5) and analyzed by ANOVA.
Values in parenthesis (percentage inhibition of edema), Dose ¼ 10 mg/kg.
a
Different from carrageenan group (p < 0.001).
b
Reference standard.
conditions [23 ꢁ 2 ꢂC, 12 h, light]. All animals had free access to
food and water (ad libitum), in a constant lightedark cycle. During
the course of experiment, the general behavior of animals was
normal. All the experimental protocols were approved by the
Institutional Animal Ethical Committee (IACE) and experiments
were conducted in accordance with the standard guidelines.
Carrageenan and indomethacin were procured from Sigma Chem-
ical, Co. For statistical analysis we have used GraphPad Prism 3.0
version [21].
Fig. 2. Treated groups versus paw edema (ml); (a) after 2 h, (b) after 4 h.

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C. Balakumar et al. / European Journal of Medicinal Chemistry 45 (2010) 4904e4913
antiedematous effects of the compounds were estimated as
percentage inhibition of the induced inflammation in comparison
with control. Statistical analysis was carried out using a one-way
analysis of variance (ANOVA). In all cases, post-hoc comparisons of
the means of individual groups were performed using Tukey’s test. A
significance level of p < 0.001 denoted significance in all cases.
inflammatory activity compared to other derivatives. The SAR of
the title compounds were drawn based on docking studies and
their binding mode analyses.
The final compounds (9aec and 10aec) were evaluated in silico
(docking) to recognize their hypothetical binding mode using the
X-ray crystal structure of COX-1 (PDB ID: 1PRH) and COX-2 (PDB ID:
1CX2) respectively and also to rationalize their structure activity
relationships. To investigate the ability of molecular docking to
reproduce an experimentally observed ligand-binding mode, the
co-crystallized ligand SC-558 (a selective COX-2 inhibitor) has been
used as reference ligand. It was docked back into its binding site
(Fig. 3a) of the crystal structure of the COX-2 using GOLD docking
program (CCDC, 4.0.1 version) [23]. The top docked conformations
(poses) closely resembled the co-crystallized conformation with
a root-mean-square deviation (RMSD) 0.70e1.07 of nonhydrogen
atomic positions of the ligand (SC-558).
4. Results and discussion
All the title compounds were screened for their in vivo anti-
inflammatory activity using the carrageenan-induced rat paw
edema model and exhibited protection against carrageenan-
induced edema (Fig. 2a and b). The protection ranged up to 61%,
while the reference drug (indomethacin) showed 79% at an
equivalent dose. Among all the tested compounds, 9b and 10b
with 4-hydroxyphenyl substituent showed remarkable anti-
Fig. 3. (a) Superimposition of redocked SC-558 (grey colour) with its original position (yellow colour) as co-crystal in the binding site of the crystal structure of COX-2 (PDB ID:
1CX2) isoenzyme (RMSD-0.70) showing H-bond interactions with the amino acid residues His90, Arg120, Ser353 and Arg513 respectively. (b) Binding orientation of indomethacin
within the binding site of COX-2 showing salt bridge formation between the carboxylate ion and Arg120. (c) Hypothetical binding motif and superimposition of the most active
compound 9b and SC-558 original position (yellow colour) to the crystal structure of COX-2 showing H-bonding interaction with the amino acid residues His90, Arg120 and Arg513
respectively. (d) Superimposition of the most active regioisomers 9b & 10b. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)

C. Balakumar et al. / European Journal of Medicinal Chemistry 45 (2010) 4904e4913
4909
The experimental binding mode of SC-558 was reproduced and
as shown in the Fig. 3a, the trifluoromethyl group (eCF3) formed
strong hydrogen bonding interactions with the exocyclic NH2 group
of Arg120 at a distance of 3.02527 Å. The same group (eCF3) was also
found to be located in the vicinity of Leu531, Val116, Val349, Leu359
and Tyr355. The Val523 permits the location of the phenyl-
sufonamide of the ligand (SC-558) in a cavity formed by Phe518,
Leu352, Val523, the backbone of Ser353 and Tyr355. Further sul-
phonamido group interacted with His90, Arg513 and Gln192 by
forming a hydrogen bond which is the key point of interaction for
COX-2 selectivity. In particular, sulphonamide eNH2 was found to
form hydrogen bonding with the nitrogen atom of imidazole ring of
His90 and oxygen atom of Ser353 at a distance of 2.68895 Å and
3.19485 Å respectively, whereas sulphonamide oxygen (]O)
formed hydrogen bond with eNH of Arg513. Pyrazole ring of SC-558
is found to be located within the vicinity of Tyr355 and Val349.
Sulphonyl group of the ligand was located around the residues of
Ser353, Phe518 and Val523. Bromophenyl moiety was surrounded
by Gly526 and Ala527. These residues were identified as crucial and
involved in the bindingwhich perhaps contributing tothe selectivity
of the ligand.
Similarly, from the docking studies of the indomethacin,
a nonselective COX-1/2 inhibitor, it has been observed that the
carboxylate ion formed a salt bridge with the Arg120 in COX-1 and
COX-2 isoenzymes (Fig. 4a and 3b) which gives a more generalised
anchoring point for all the classical NSAIDs thus limiting their
selectivity due to curtailed freedom of movement of the ligand. The
acetyl CH2 of indomethacin formed a strong hydrogen bond with
the phenolic OH group of Tyr355 at a distance of 1.5829 Å, whereas
the chlorophenyl group showed significant
pep stacking interac-
tion with the phenyl ring of Phe518.
Interestingly, the most active compound 9b also docked well
into the binding pocket of COX-2 (Fig. 3c). In particular, tri-
fluoromethyl group at 8th position of the compound (9b) was
Fig. 4. (a) Binding orientation of indomethacin within the binding site of COX-1 (PDB ID: 1PRH) showing salt bridge formation between the carboxylate ion and Arg120. (b)
Hypothetical binding motif and superimposition of the target compounds 9aec and 10aec to the crystal structure of COX-1 showing important H-bonding interaction with the
amino acid residues His90, Tyr355 and Ser530 respectively. (c) Binding orientation of the most active compound 9b.

4910
C. Balakumar et al. / European Journal of Medicinal Chemistry 45 (2010) 4904e4913
found to form a weak H-bond (3.55889 Å) with the exocyclic NH2
group of Arg120 compared to SC-558 and a strong H-bond with the
phenolic OH group of Tyr355 at a distance of 1.75355 Å. Phenyl ring
at 10th position was surrounded by Arg513, His90, Phe518 and
Gln92. Like sulphonamido group of SC-558, the NH2 of carbox-
amide group also found to be involved in the formation of a strong
H-bond with the nitrogen atom of imidazole ring of His90. Whereas
for regioisomer 10b (Fig. 3d), the phenyl ring at 8th position was
found to be aligned similar to trifluoromethyl group of SC-558 but
lacking the H-bond interaction with Arg120 and showed weak
hydrogen bonding with Tyr355. Further, trifluoromethyl group at
the 10th position of regioisomer (10b) formed a strong H-bond
with the nitrogen atom of imidazole ring of His90 (2.64416 Å) and
NH2 of Arg513 (2.44115 Å). The tricyclic nucleus located well within
the binding pocket. The phenolic ring of the compounds 9b and 10b
was found to be aligned with the bromophenyl ring of SC-558 and
6. Experimental protocols
Melting points were recorded in open capillaries on Casiaa
Siamea (VMP-AM) melting point apparatus and are uncorrected. IR
spectra were recorded on a Perkin Elmer FT-IR 240-C spectropho-
tometer using KBr discs. 1H NMR spectra were recorded on Bruker
Avance II 400 MHz spectrometer in DMSO-d6 using TMS as internal
standard. All the reactions were monitored by thin layer chroma-
tography (TLC) on precoated silica gel 60 F254 (mesh) (E. Merck,
Mumbai) and spots were visualized under UV light (254 nm). Silica
gel from E. Merck, Mumbai (100e200 mesh) was used for column
chromatography. All chemicals used were of analytical grade and
commercially available. The compounds 3 and 4 were synthesized
following a reported method [19].
6.1. Synthesis of quinazolinone regioisomers (5aec and 6aec)
showed significant pep stacking interaction with Phe518 similar to
indomethacin which probably account for their better anti-
inflammatory efficacy compared to other analogues (9a&c; 10a&c)
of the series. Similarly, the formation of weak H-bond with Arg120
could be reasonably suspected for their reduced activity as
compared to the indomethacin. Furan ring of compounds 9a and
10a showed hydrophobic interaction with the residues of Gly526,
Tyr385, Trp387, Phe381 and Ser530 (aligned in orientation as that
of bromophenyl moiety of SC-558). Sulphur atom of the thiophene
ring of the compounds 9c and 10c exhibited van der Waals inter-
action with the amino acid residues such as Gly526, Tyr385, Trp387,
Phe381 and Ser530 as well.
The compound 4 (2 mmol) and different aldehydes (2-furfur-
aldehyde or 4-hydroxybenzaldehyde or thiophene-2-carbox-
aldehyde) (2 mmol) were taken in acetic acid (5 ml) and stirred at
room temperature for 4 h. The reaction mixture was cooled and
poured onto crushed ice. The separated solid was filtered, washed
with water till washings were neutral to litmus and dried. The dried
product was found to contain equivalent mixture of two
regioisomers (5aec and 6aec) respectively. Each isomer was sepa-
rated by passing through column in n-hexane/ethyl acetate (3:1).
6.1.1. 2-(Furan-2-yl)-4-oxo-7-phenyl-5-(trifluoromethyl)-1,2,3,4-
tetrahydroquinazoline-8-carboxamide (5a)
It was shown that docking of indomethacin within COX-1
includes a salt bridge formation with Arg120 and significant
pe
p
Yellow solid, Yield: 49%, mp: 266 ꢂC, IR (KBr: , cmꢀ1): 3434
n
stacking interaction between phenyl ring of indole moiety and the
phenyl ring of Phe518 (Fig. 4a). Interestingly no such salt bridge
formation was observed between the test compounds (9aec and
10aec) and Arg120 (Fig. 4b). The eCF3 and the phenolic eOH group
of the most active compound 9b involved in the formation of strong
H-bond with the phenolic eOH of Tyr355 and side chain oxygen
atom of Ser530 (Fig. 4c). Hence it can be deduced that in silico
docking studies can enable us to get an insight and further guide us
to rationally design novel and potent anti-inflammatory agents
with a high degree of selectivity in future.
(NeH stretch), 3189 (aromatic CeH stretch),1695 (NeH bend),1632
(C]O stretch), 1593 (C]C ring stretch), 1392 (CeN stretch), 1197
(CeO stretch), 1147 (CeF stretch); 1H NMR (DMSO-d6,
d ppm): 5.81
(t, 1H, methine H), 6.32 (s, 1H, NH), 6.42 (d, 1H, (C-5 furan-H)), 6.61
(s, 1H, CONH2), 6.92 (t, 1H, (C-4 furan-H)), 7.10 (s, 1H, CONH2), 7.40
(s,1H, HeC(6)), 7.42e7.45 (m, 5H, AreH), 7.51 (d,1H, (C-3 furan-H)),
8.23 (d, 1H, CONH).
6.1.2. 2-(Furan-2-yl)-4-oxo-5-phenyl-7-(trifluoromethyl)-1,2,3,4-
tetrahydroquinazoline-8-carboxamide (6a)
Yellow solid, Yield: 37%, mp: 270 ꢂC, IR (KBr: , cmꢀ1): 3385
n
5. Conclusion
(NeH stretch), 3190 (aromatic CeH stretch), 1669 (NeH bend), 1511
(C]O stretch), 1475 (C]C ring stretch), 1363 (CeN stretch), 1283
A series of novel 8/10-trifluoromethyl-substituted-imidazo[1,2-
c]quinazolines (9aec and 10aec) were synthesized, spectroscopi-
cally characterized and evaluated for in vivo anti-inflammatory
activity. The compounds 9b and 10b with 4-hydroxyphenyl
substituent were found to have good anti-inflammatory activity
which is on a par with indomethacin. Further in silico docking
studies were undertaken to gain insight into the molecular inter-
actions and binding mode of the target compounds into COX
isoenzymes. All the final compounds were docked well into the
binding pocket of the target proteins (COX-1 and COX-2) and inter-
acted with the crucial amino acid residues. The 4-hydroxyphenyl
ring of the most active compounds (9b and 10b) showed significant
(CeO stretch), 1134 (CeF stretch); 1H NMR (DMSO-d6,
d ppm): 5.84
(t, 1H, methine H), 6.36 (s, 1H, NH), 6.44 (d, 1H, (C-5 furan-H)), 6.55
(t, 1H, (C-4 furan-H)), 6.98 (s, 1H, CONH2), 7.22e7.31 (m, 5H, AreH),
7.35 (s, 1H, CONH2), 7.42 (s, 1H, HeC (6)), 7.57 (d, 1H, (C-3 furan-H)),
8.41 (d, 1H, CONH).
6.1.3. 2-(4-Hydroxyphenyl)-4-oxo-7-phenyl-5-(trifluoromethyl)-
1,2,3,4-tetrahydroquinazoline-8-carboxamide (5b)
White solid, Yield: 49%, mp: 255 ꢂC, IR (KBr: , cmꢀ1): 3466
n
(OeH stretch), 3328 (NeH stretch), 3151 (aromatic CeH stretch),
1682 (N-H bend),1634 (C]O stretch),1592 (C]C ring stretch), 1147
(CeF stretch); 1H NMR (DMSO-d6,
d ppm): 5.70 (s, 1H, methine H),
p
e
p
stacking interaction with the phenyl ring of Phe518 similar to
6.41 (s, 1H, NH), 6.82 (d, 2H, AreH), 7.04 (s, 1H, CONH2), 7.11 (s, 1H,
CONH2), 7.20 (s, 1H, HeC(6)), 7.35e7.41 (m, 5H, AreH), 7.54 (d, 2H,
AreH), 8.04 (s, 1H, CONH), 9.30 (s, 1H, OH).
indomethacin which probably account for their better anti-inflam-
matory activity compared to other analogues (9a&c; 10a&c) of the
series. The docking results revealed useful information to under-
stand the interaction mode between fused quinazolines and cyclo-
oxygenase and will facilitate the next cycle of drug design (toexplore
the newer fluorinated quinazoline-based lead molecules as potent
anti-inflammatory agents). Efforts are currently being taken up to
optimize the lead structure and the results of which will be the basis
of our future research endeavor.
6.1.4. 2-(4-Hydroxyphenyl)-4-oxo-5-phenyl-7-(trifluoromethyl)-
1,2,3,4-tetrahydroquinazoline-8-carboxamide (6b)
White solid, Yield: 35%, mp: 260 ꢂC, IR (KBr: , cmꢀ1): 3366
n
(OeH stretch), 3320 (NeH stretch), 3050 (aromatic CeH stretch),
1674 (NeH bend), 1625 (C]O stretch), 1598 (C]C ring stretch),
1137 (CeF stretch); 1H NMR (DMSO-d6,
d ppm): 5.80 (s, 1H,

C. Balakumar et al. / European Journal of Medicinal Chemistry 45 (2010) 4904e4913
4911
methine H), 6.42 (s, 1H, NH), 6.84 (d, 2H, AreH), 7.0 (s, 1H, HeC(6)),
7.15 (d, 2H, AreH), 7.28 (s, 1H, CONH2), 7.3e7.4 (m, 5H, AreH), 7.50
(s, 1H, CONH2), 8.08 (s, 1H, CONH), 9.2 (s, 1H, OH).
NMR (DMSO-d6, d ppm): 6.82 (d, 2H, AreH), 7.06 (s, 1H, CONH2),
7.14 (s, 1H, CONH2), 7.20 (s, 1H, HeC(6)), 7.35e7.42 (m, 5H, AreH),
7.56 (d, 2H, AreH), 8.12 (s, 1H, CONH), 9.31 (s, 1H, OH).
6.1.5. 4-Oxo-7-phenyl-2-(thiophen-2-yl)-5-(trifluoromethyl)-
1,2,3,4-tetrahydroquinazoline-8-carboxamide (5c)
6.2.4. 2-(4-Hydroxyphenyl)-4-oxo-7-phenyl-5-(trifluoromethyl)-
3,4-dihydroquinazoline-8-carboxamide (8b)
Yellow solid, Yield: 49%, mp: 269 ꢂC, IR (KBr:
n
, cmꢀ1): 3434
White solid, Yield: 75%, mp: 331 ꢂC, IR (KBr: , cmꢀ1): 3470
n
(NeH stretch), 3189 (aromatic CeH stretch),1695 (NeH bend),1632
(C]O stretch), 1593 (C]C ring stretch), 1392 (CeN stretch), 1197
(OeH stretch), 3318 (NeH stretch), 3040 (aromatic CeH stretch),
1643 (NeH bend), 1631 (C]O stretch), 1576 (C]C ring stretch),
1440 (C]N ring stretch), 1131 (CeF stretch); 1H NMR (DMSO-d6,
(CeO stretch), 1147 (CeF stretch); 1H NMR (DMSO-d6,
d ppm): 5.90
(t, 1H, methine H), 6.96 (t, 1H, (C-4, thiophene-H)), 7.02 (s, 1H, HeC
(6)), 7.08 (d, 1H, (C-5, thiophene-H)), 7.28 (d, 1H, (C-3, thiophene-
H)), 7.37 (s, 1H, CONH2), 7.41 (d, 1H, NH), 7.42e7.47 (m, 5H, AreH),
7.52 (s, 1H, CONH2), 9.02 (d, 1H, CONH).
d ppm): 6.82 (d, 2H, AreH), 6.96 (s, 1H, HeC(6)), 7.15 (d, 2H, AreH),
7.26 (s, 1H, CONH2), 7.35e7.45 (m, 5H, AreH), 7.62 (s, 1H, CONH2),
7.83 (s, 1H, CONH), 9.2 (s, 1H, OH).
6.2.5. 4-Oxo-7-phenyl-2-(thiophen-2-yl)-5-(trifluoromethyl)-
3,4-dihydroquinazoline-8-carboxamide (7c)
6.1.6. 4-Oxo-5-phenyl-2-(thiophen-2-yl)-7-(trifluoromethyl)-
1,2,3,4-tetrahydroquinazoline-8-carboxamide (6c)
Yellow solid, Yield: 49%, mp: 259 ꢂC, IR (KBr: , cmꢀ1): 3390
n
Yellow solid, Yield: 39%, mp: 274 ꢂC, IR (KBr:
n
, cmꢀ1): 3385
(NeH stretch), 2925 (aromatic CeH stretch), 1700 (NeH bend),
1570 (C]O stretch), 1468 (C]C ring stretch), 1448 (C]N ring
stretch),1415 (CeN stretch), 1220 (CeO stretch), 1143 (CeF stretch);
(NeH stretch), 3190 (aromatic CeH stretch), 1669 (NeH bend), 1511
(C]O stretch), 1475 (C]C ring stretch), 1363 (CeN stretch), 1283
(CeO stretch), 1134 (CeF stretch); 1H NMR (DMSO-d6,
d
ppm): 6.0
1H NMR (DMSO-d6,
d ppm): 7.21 (t, 1H, (C-4 thiophene-H)), 7.35 (s,
(t, 1H, methine H), 6.97 (t, (C-4 thiophene-H)), 7.02 (s, 1H, HeC(6)),
7.06 (d, (C-5 thiophene-H)), 7.29 (d, (C-3 thiophene-H)), 7.4 (d, 1H,
NH), 7.42e7.48 (m, 5H, AreH), 7.57 (s, 1H, CONH2), 7.70 (s, 1H,
CONH2), 9.04 (d, 1H, CONH).
1H, HeC(6)), 7.36e7.40 (m, 5H, AreH), 7.59 (s, 1H, CONH2), 7.79 (s,
1H, CONH2), 7.93 (d, 1H, (C-5 thiophene-H)), 8.21 (d, 1H, (C-3
thiophene-H)), 8.36 (d, 1H, CONH).
6.2.6. 4-Oxo-5-phenyl-2-(thiophen-2-yl)-7-(trifluoromethyl)-,3,4-
dihydroquinazoline-8-carboxamide (8c)
6.2. Dehydrogenation of quinazolinones to quinazolones (7aec and
8aec)
Yellow solid, Yield: 39%, mp: 274 ꢂC, IR (KBr: , cmꢀ1): 3410
n
(NeH stretch), 2928 (aromatic CeH stretch), 1665 (NeH bend),
1520 (C]O stretch), 1467 (C]C ring stretch), 1499 (C]N stretch),
1362 (CeN stretch),1269 (CeO stretch),1132 (CeF stretch); 1H NMR
The compounds 5aec and 6aec (0.16 mmol) were dissolved in
dry dichloromethane (10 ml) independently, active MnO2 (650 mg)
was added and stirred for 2 h at room temperature. The reaction
mixture was filtered and washed with dichloromethane. The
filtrate is concentrated and the resulted residue is purified through
column of silica gel using ethyl acetate/hexane (6:4) as eluents to
give compounds 7aec and 8aec respectively.
(DMSO-d6, d ppm): 7.24 (t, 1H, (C-4 thiophene-H)), 7.35 (s, 1H, HeC
(6)), 7.35e7.39 (m, 5H, AreH), 7.79 (s, 1H, CONH2), 7.89 (s, 1H,
CONH2), 7.96 (d, (C-5 thiophene-H)), 8.24 (d, 1H, (C-3 thiophene-
H)), 8.42 (d, 1H, CONH).
6.3. Cyclization to imidazo[1,2-c]quinazolines (9aec & 10aec)
6.2.1. 2-(Furan-2-yl)-4-oxo-7-phenyl-5-(trifluoromethyl)-3,4-
dihydroquinazoline-8-carboxamide (7a)
Compounds 7aec and 8aec (4 mmol) individually in 6 ml of
phosphorus oxychloride (POCl3) was boiled for 2 h. Then 2-chlor-
oethylamine hydrochloride (6 mmol) was added individually, and
the mixture was heated under reflux for further 3 h. The excess of
POCl3 was evaporated under vacuum, the residue was triturated
with ice water and pH was adjusted to 10e11 (using an ammonia
solution). The precipitate obtained was filtered, dried and crystal-
lized from toluene to get the title compounds (9aec & 10aec).
Yellow solid, Yield: 79%, mp: 318 ꢂC, IR (KBr: , cmꢀ1): 3390
n
(NeH stretch), 2925 (aromatic CeH stretch), 1700 (NeH bend),
1570 (C]O stretch), 1468 (C]C ring stretch), 1448 (C]N ring
stretch), 1415 (CeN stretch),1220 (CeO stretch),1143 (CeF stretch);
1H NMR (DMSO-d6,
d ppm): 6.44 (d, 1H, (C-5 furan-H)), 6.62 (s, 1H,
CONH2), 6.93 (t, 1H, (C-4 furan-H)), 7.12 (s, 1H, CONH2), 7.45 (s, 1H,
HeC(6)), 7.45e7.49 (m, 5H, AreH), 7.52 (d, 1H, (C-3 furan-H)), 8.26
(d, 1H, CONH).
6.3.1. 5-(Furan-2-yl)-8-phenyl-10-(trifluoromethyl)-2,3-
dihydroimidazo[1,2-c]quinazoline-7-carboxamide (9a)
6.2.2. 2-(Furan-2-yl)-4-oxo-5-phenyl-7-(trifluoromethyl)- 3,4-
dihydroquinazoline-8-carboxamide (8a)
Yellow solid, Yield: 60%, mp: 350 ꢂC (toluene), IR (KBr: , cmꢀ1):
n
Yellow solid, Yield: 79%, mp: 327 ꢂC, IR (KBr:
n
, cmꢀ1): 3410
3390 (NeH stretch), 2917 (aromatic CeH stretch), 1696 (NeH
bend),1635 (C]O stretch),1519 (C]C ring stretch),1472 (C]N ring
stretch),1438 (CeN stretch), 1197 (CeO stretch),1144 (CeF stretch);
(NeH stretch), 2928 (aromatic CeH stretch), 1665 (NeH bend),
1520 (C]O stretch), 1467 (C]C ring stretch), 1499 (C]N stretch),
1362 (CeN stretch),1269 (CeO stretch),1132 (CeF stretch); 1H NMR
1H NMR (DMSO-d6,
d ppm): 3.40 (t, 2H, imidazoline-H), 4.22 (t, 2H,
(DMSO-d6,
d
ppm): 6.50 (d, 1H, (C-5 furan-H)), 6.91 (s, 1H, CONH2),
imidazoline-H), 6.46 (d, 1H, (C-5 furan-H)), 6.64 (s, 1H, CONH2),
6.98 (t, 2H, (C-4 furan-H)), 7.13 (s, 1H, CONH2), 7.40 (s, 1H, HeC(6)),
7.45 (d, 1H, (C-3 furan-H)), 7.49e7.52 (m, 5H, AreH).
6.95 (t, 1H, (C-4 furan-H)), 7.26e7.32 (m, 5H, AreH), 7.37 (s, 1H,
CONH2), 7.48 (s, 1H, HeC(6)), 7.56 (d, 2H, (C-3 furan-H)), 8.18 (d, 1H,
CONH).
6.3.2. 5-(Furan-2-yl)-10-phenyl-8-(trifluoromethyl)-2,3-
dihydroimidazo[1,2-c]quinazoline-7-carboxamide (10a)
6.2.3. 2-(4-Hydroxyphenyl)-4-oxo-7-phenyl-5-(trifluoromethyl)-
3,4-dihydroquinazoline-8-carboxamide (7b)
Yellow solid, Yield: 55%, mp: 360 ꢂC (toluene), IR (KBr: , cmꢀ1):
n
White solid, Yield: 74%, mp: 325 ꢂC, IR (KBr:
n
, cmꢀ1): 3456
3400 (NeH stretch), 2918 (aromatic CeH stretch), 1653 (NeH
bend), 1508 (C]O stretch), 1458 (C]C ring stretch), 1374 (C]N
ring stretch), 1338 (CeN stretch), 1283 (CeO stretch), 1135 (CeF
(OeH stretch), 3330 (NeH stretch), 3049 (aromatic CeH stretch),
1676 (NeH bend), 1644 (C]O stretch), 1588 (C]C ring stretch),
1445 (C]N ring stretch), 1360 (CeN stretch), 1135 (CeF stretch); 1H
stretch); 1H NMR (DMSO-d6,
d ppm): 3.38 (t, 2H, imidazoline-H),

4912
C. Balakumar et al. / European Journal of Medicinal Chemistry 45 (2010) 4904e4913
4.21 (t, 2H, imidazoline-H), 6.50 (d, 1H, (C-5 furan-H)), 6.98 (s, 1H,
CONH2), 7.25 (t, 2H, (C-4 furan-H)). 7.32 (s, 1H, CONH2), 7.41 (s, 1H,
HeC(6)), 7.46 (d, 1H, (C-3 furan-H)), 7.54e7.59 (m, 5H, AreH)
[25], since the two have 87% identity with strict conservation in the
active site [26]. The mouse COX-2 may thus be taken as a model for
the human COX-2 enzyme. The COX-2 protein exists in situ as
a dimer; however, the reason for dimerization is not known [24]; the
monomer structure alone has always been considered in molecular
modeling studies [27,28] assuming the interactions governing the
COX-2 inhibitor binding to be reproducible using one monomer. The
6.3.3. 5-(4-Hydroxyphenyl)-8-phenyl-10-(trifluoromethyl)-2,3-
dihydroimidazo[1,2-c] quinazoline-7-carboxamide (9b)
Brown solid, Yield: 57%, mp: 320 ꢂC (toluene), IR (KBr: , cmꢀ1):
n
3444 (OeH stretch), 3334 (NeH stretch), 3065 (aromatic CeH
stretch), 1672 (NeH bend), 1624 (C]O stretch), 1578 (C]C ring
stretch), 1443 (C]N ring stretch), 1362 (CeN stretch), 1132 (CeF
superposition of the a-carbons of the entire enzyme for all residue
pairs of the considered models (inhibited and uninhibited), yields
a RMSD ranging from 0.35 to 0.56 Å. When the superposition is
limited to the subset of residues closely surrounding the inhibitor
molecule (residues within 5 Å of the inhibitor), the RMSD ranged
from 0.35 to 0.58 Å. A visual inspection of the models revealed that
the inhibition does not produce significant changes in the confor-
mation of the residues around the inhibitors; though the confor-
mation of Arg120 appearedtobe stronglyinfluenced bythe inhibitor
interaction. In the complex SC-558/COX-2, the X-ray data do not
allow unique definition of the conformation of the sulphonamide
[25] and the conformation depends on the X-ray structure (6COX
and 1CX2). The binding of inhibitors does not greatly perturb the
resting structure [25], as is shown by the fact that the greatest RMSD
stretch); 1H NMR (DMSO-d6,
d ppm): 3.40 (t, 2H, imidazoline-H),
4.21 (t, 2H, imidazoline-H), 6.83 (d, 2H, AreH), 7.02 (s, 1H, CONH2),
7.18 (s, 1H, CONH2), 7.23 (s, 1H, HeC(6)), 7.35e7.45 (m, 5H, AreH),
7.58 (d, 2H, AreH), 9.34 (s, 1H, OH).
6.3.4. 5-(4-Hydroxyphenyl)-10-phenyl-8-(trifluoromethyl)-2,3-
dihydroimidazo[1,2-c] quinazoline-7-carboxamide (10b)
Brown solid, Yield: 59%, mp: 345 ꢂC (toluene), IR (KBr: , cmꢀ1):
n
3444 (OeH stretch), 3334 (NeH stretch), 3065 (aromatic CeH
stretch), 1672 (NeH bend), 1624 (C]O stretch), 1578 (C]C ring
stretch), 1443 (C]N ring stretch), 1362 (CeN stretch), 1132 (CeF
stretch); 1H NMR (DMSO-d6,
d
ppm): 2.88 (t, 2H, imidazoline-H),
deviation for all Ca atoms between the X-ray structures (presented
3.67 (t, 2H, imidazoline-H), 6.84 (d, 2H, AreH), 6.96 (s, 1H, HeC(6)),
7.28 (s, 1H, CONH2), 7.31e7.39 (m, 5H, AreH), 7.41 (s, 1H, CONH2),
7.56 (d, 2H, AreH), 9.28 (s, 1H, OH).
in Table 2) is 0.56 Å. On this basis, the choice of model to use as
starting point for the study does not appear to be relevant, and we
therefore used the structure 1CX2, since the molecule SC-558 (a
typical COX-2 selective inhibitor) can be used as a validation system.
6.3.5. 8-Phenyl- 5-(thiophene-2-yl)-10-(trifluoromethyl)-2,3-
dihydroimidazo[1,2-c] quinazoline-7-carboxamide (9c)
7.2. Preparation of protein
Yellow solid, Yield: 46%, mp: 322 ꢂC (toluene), IR (KBr: , cmꢀ1):
n
3340 (NeH stretch), 2923 (aromatic CeH stretch), 1670 (C]O
stretch), 1519 (C]C ring stretch), 1455 (C]N ring stretch), 1405
PDB structures (www.rcsb.org) [1CX2 (COX-2) and 1PRH
(COX-1)] were downloaded and refined, prepared using Schro-
dinger protein preparation wizard tool (Glide), which performs the
following steps: assigning of bond orders, addition of hydrogens,
optimization of hydrogen bonds by flipping amino side chains,
correction of charges, and minimization of the protein complex. All
the bound water molecules, ligands and cofactors were removed
(preprocess) from the proteins which were taken in .mae format.
The tool neutralized the side chains that are not close to the binding
cavity and do not participate in salt bridges. This step is then fol-
lowed by restrained minimization of co-crystallized complex,
which reorients side chain hydroxyl groups and alleviates potential
steric clashes. The complex obtained was minimized using
OPLS_2005 force field [29] with Polack-Ribiere Conjugate Gradient
(PRCG) algorithm. The minimization was terminated either
completion of 5000 steps (or) after the energy gradient converged
below 0.05 kcal/mol. In the absence of a co-crystallized complex of
inhibitor bound to COX-1, the binding site was defined using the
crystal coordinates of SC-558, which was superimposed to the same
frame of residues of COX-1 using DaliLite [30].
(CeN stretch), 1146 (CeF stretch); 1H NMR (DMSO-d6,
d ppm): 3.42
(t, 2H, imidazoline-H), 4.20 (t, 2H, imidazoline-H), 6.48 (d, 1H, (C-5
thiophene-H)), 6.69 (s, 1H, CONH2), 6.98 (t, 2H, (C-4 thiophene-H)),
7.16 (s,1H, CONH2), 7.40 (s,1H, HeC(6)), 7.45 (d,1H, (C-3 thiophene-
H)), 7.50e7.54 (m, 5H, AreH).
6.3.6. 10-Phenyl- 5-(thiophene-2-yl)-8-(trifluoromethyl)-2,3-
dihydroimidazo[1,2-c] quinazoline-7-carboxamide (10c)
Yellow solid, Yield: 52%, mp: 335 ꢂC (toluene), IR (KBr: , cmꢀ1):
n
3436 (NeH stretch), 2923 (aromatic CeH stretch), 1697 (NeH
bend),1632 (C]O stretch), 1516 (C]C ring stretch),1471 (C]N ring
stretch),1440 (CeN stretch),1198 (CeO stretch),1144 (CeF stretch);
1H NMR (DMSO-d6,
d ppm): 3.38 (t, 2H, imidazoline-H), 4.24 (t, 2H,
imidazoline-H), 6.51 (d, 1H, (C-5 thiophene-H)), 7.12 (s, 1H, CONH2),
7.25 (t, 2H, (C-4 thiophene-H)). 7.32 (s, 1H, CONH2), 7.42 (s, 1H, HeC
(6)), 7.47 (d, 1H, (C-3 thiophene-H)), 7.54e7.59 (m, 5H, AreH).
7. Molecular modeling study
Molecular modeling investigations were carried out using Dell
Precision work station T3400 running Intel Core2 Duo Processor,
4 GB RAM, 250 GB hard disk, and NVidia Quodro FX 4500 graphics
card. GOLD (Genetic optimization for ligand docking) module
(CCDC, 4.0.1 version) was employed for the docking studies.
7.3. Preparation of ligands
Structures of the ligands were sketched using built panel of
Maestro and taken in .mae format. LigPrep is a utility of Schrodinger
software suit that combines tools for generating 3D structures from
7.1. Selection of the COX-2 structures
Table 2
X-ray structures for COX-2 isoenzymes.
COX-2 enzyme contains 587 amino acids and one ferric heme
group [24]. The X-ray structure of mouse (Mus musculus) COX-2 has
been resolved as uninhibited, as well as in the presence of four
inhibitors (the PDB code and the resolution of the crystal data are
reported in Table 2). The amino acid sequence of COX-2 is conserved
between species with 85e90% sequence similarity. The structure of
human COX-2 is expected to be very similar to the mouse enzyme
Ligand
Resolution
PDB code
Uninhibited
Flurbiprofen (f)
SC-558 (g)
SC-558 (g)
Indomethacin (h)
Diclofenac (i)
3.0
2.5
3.0
3.0
2.9
2.9
5COX
3PGH
1CX2
6COX
4COX
1PXX

C. Balakumar et al. / European Journal of Medicinal Chemistry 45 (2010) 4904e4913
4913
1D (Smiles) and 2D (SDF) representation, searching for tautomers,
steric isomers and perform a geometry minimization of the ligands.
Molecular Mechanics Force Fields (OPLS_2005) with default
settings were employed for the ligand minimization.
CSIR, New Delhi for awarding the Senior Research Fellowship. This
work was supported by AICTE, New Delhi (RPS-8023/2006e07).
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GOLD is a ligand-docking application that utilizes a genetic
algorithm (GA) to explore ligand conformation flexibility and
orientation with partial flexibility of the protein, and satisfy ligand-
binding requirements. One advantage of GOLD over many other
docking algorithms is that it allows for both unconstrained ligand
flexibility and partial flexibility of the binding pocket thus affording
a more realistic environment for ligandereceptor associations.
For each of the 10 independent GA runs, a maximum number of
100 GA operations were performed. The standard set parameters
wereused in all the calculations. Defaultoperatorweights wereused
for crossover, mutation, and migration of 95, 95, and 10, respectively.
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vdW) were employed. Pop. Size ¼ 100; max ops ¼ 100,000; niche
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GA docking was terminated when the top three solutions were
within 1.5 Å RMSD of each other. GOLD scores recorded on each
binding mode using a fitness function that accounts for the steric
and electrostatic complementarity between the ligand and receptor.
The GOLD scoring function includesthe terms forhydrogenbonding,
vdW and intramolecular energies. The first ranked solutions of the
ligands were taken for further observation of binding orientation
and H-bond interactions.
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The authors thankfully acknowledge the Chairman, University
Institute of Pharmaceutical Sciences (UIPS) and Co-ordinator,
Centre with Potential for Excellence in Biomedical Sciences
(CPEBS), Panjab University (PU), Chandigarh for providing the
facilities. They gratefully acknowledge Mrs. Lauren Thomas, CCDC,
Cambridge, UK for providing the GOLD software. Authors are
thankful to Dr. Kanwaljit Chopra, Pharmacology Division, UIPS, PU,
for valuable help during pharmacological studies. Authors are also
thankful to Avtar Singh, Central Instrumentation Laboratory (CIL),
PU for carrying out the NMR studies. Authors (CB, BLN, PL and KR)
wish to thank University Grants Commission (UGC), New Delhi for
providing fellowship (UGC-RFSMS). Authors (DPK and KVR) thank