Analogue-based design, synthesis and molecular docking analysis of 2,3-diaryl quinazolinones as non-ulcerogenic anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmc.2011.06.019

In our effort to identify potent gastric sparing anti-inflammatory agents, a series of methyl sulfanyl/methyl sulfonyl substituted 2,3-diaryl quinazolinones were designed by analogue-based design strategy and synthesized for biological evaluation. Subsequ

Full text of DOI:10.1016/j.bmc.2011.06.019

Bioorganic & Medicinal Chemistry 19 (2011) 4520–4528
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Analogue-based design, synthesis and molecular docking analysis of 2,3-diaryl
quinazolinones as non-ulcerogenic anti-inflammatory agents q
⇑
E. Manivannan , S. C. Chaturvedi
School of Pharmacy, Devi Ahilya Vishwavidyalaya, Ring Road, Indore 452017, MP, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In our effort to identify potent gastric sparing anti-inflammatory agents, a series of methyl sulfanyl/
methyl sulfonyl substituted 2,3-diaryl quinazolinones were designed by analogue-based design strategy
and synthesized for biological evaluation. Subsequently, the compounds were evaluated for both
cyclooxygenase inhibitions by ovine COX assay and carrageenan-induced rat paw edema assay. All the
methyl sulfonyl substituted quinazolinones were exhibited promising anti-inflammatory activity.
In particular, 6-bromo-3-(4-methanesulfonyl-phenyl)-2-phenyl-3H-quinazolin-4-one (18), 7-chloro-
3-(4-methanesulfonyl-phenyl)-2-phenyl-3H-quinazolin-4-one (19), 3-(4-methanesulfonyl-phenyl)-2-
(4-methoxy-phenyl)-3H-quinazolin-4-one (21) and 6-bromo-3-(4-methanesulfonyl-phenyl)-2-(4-meth-
oxy-phenyl)-3H-quinazolin-4-one (22) emerged as the most active compounds in the series. The results
of ulcerogenic activity assay suggest that these compounds are gastric safe compared to indomethacin.
The molecular docking analysis was performed to understand the binding interactions of these
compounds to COX-2 enzyme. The results from the present investigation suggests that 2,3-diaryl quinaz-
olinones as a promising template for the design of new gastric safe anti-inflammatory agents, which can
be further explored for potential anti-inflammatory activity.
Received 18 April 2011
Revised 4 June 2011
Accepted 8 June 2011
Available online 15 June 2011
Keywords:
Analogue-based design
2,3-Diaryl quinazolinones
COX-1
COX-2
Anti-inflammatory activity
Ulcerogenic activity
Molecular docking
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
heteroaryl-ethers have been developed as pharmacophore for
anti-inflammatory activity.12 Soon after the discovery of some
Non-steroidal anti-inflammatory drugs (NSAIDs) are among the
most widely prescribed medicines for the treatment of pain, fever,
inflammation and arthritis.1 The classical NSAIDs produce their
therapeutic benefit by inhibiting cyclooxygenase enzymes and
thus reducing the formation of inflammatory mediator prostaglan-
dins.2 The discovery of existence of two distinct isoforms of COX
demonstrated that the side effects of NSAIDs such as gastric irrita-
tion, ulcer and gastric perforation are mainly due to inhibition of
physiological enzyme COX-1 along with the desired blockade of
COX-2 enzyme.3–6 The structure of COX-1 and COX-2 enzymes
and their inhibitors have been thoroughly investigated for more
than two decades of research. This led to the development of
many of selective COX-2 inhibitors, which are almost free of gastric
side effects. Recent studies on this field show that COX-2 inhibitors
are attractive molecular target for the development cancer
chemotherapy7–9 and neurological diseases such as Parkinson10
and Alzheimer’s diseases.11
selective COX-2 inhibitors namely, celecoxib, rofecoxib, valdecoxib
and parecoxib as gastric safe anti-inflammatory drugs, researchers
focused extensively on tricyclic template based COX-2 inhibi-
tors.13–16 Variations of the central ring in tricyclic COX-2 inhibitors
have already generated a number of potent derivatives with
thiazole, triazole, imidazole, isoxazoline, pyridine, pyranone, pyri-
dazone, pyrimidine, pyran, indole, benzothiazole, benzotriazole,
benzimidazole, and benzopyran central ring scaffold.17–30 In the
present investigation, we were focused on designing novel COX-2
inhibitors based on quinazolinone template as influenced by the
facts such as limited citations as bicyclic central scaffolds as
COX-2 inhibitors and recognition of quinazolinone heterocycle in
a number of natural anti-inflammatory alkaloids like rutaecar-
pine31–33 and tryptanthrin.34–36
2. Results and discussion
2.1. Analogue-based design
Selective COX-2 inhibitors belonging to different structural
classes such as vicinal diaryl heterocycles, diaryl carbocyles and
A series of anti-inflammatory compounds possessing 2,3-diaryl
quinazolinones were designed based on ‘analogue-based’ design
strategy with the application of quantitative structure–activity
relationship analysis. The selection of diaryl quinazolinone
pharmacophore was motivated by its good compliance with both
q
A part of the work was presented in European Drug Discovery conference
Miptech-2009, Basel, Switzerland.
⇑
Corresponding author. Tel.: +91 731 2100605, mobile: +91 8982677476.
E-mail address: drmanislab@gmail.com (E. Manivannan).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.019

E. Manivannan, S. C. Chaturvedi / Bioorg. Med. Chem. 19 (2011) 4520–4528
4521
geometrical and surface properties necessary for COX-2 inhibition.
In search of structural similarity, the design foresaw replacement
of the ‘central scaffold’ A with a bicyclic structure quinazolinone,
and followed by introduction of reasonable diversity in the rest
of the structures B, C, D and E according to Figure 1. The specific
structural features were selected for designing the new anti-
inflammatory compounds with reasonable justification. The choice
of quinazolinone heterocycle as a central core was fundamental
due to its presence in the potent anti-inflammatory natural alka-
loid rutaecarpine, and tryptanthrin. The introduction of ‘diaryl’
structural motif was due to its recognition as pharmacophore10
for cyclooxygenase inhibition and as most of the tricyclic COX-2
selective inhibitors celecoxib, rofecoxib, valdecoxib and parecoxib
constituted by this chemical feature. Additional –SO2CH3 group
was based on fact that either a para-SO2CH3 or a para-SO2NH2 sub-
stituent on one of the phenyl rings is necessary for selectivity that
the group inserts into a secondary side pocket present in the COX-2
enzyme active site.12 Electron withdrawing group on central core
was introduced since the addition of small lipophilic electron with-
drawing groups found to increase the COX inhibitory activity as it
interacts with the aromatic amino acid residues of COX isozymes.
Substituents on phenyl ring were decided on the basis of quantita-
tive structure–activity relationship of various bicyclic analogs with
good COX-2 inhibitory potency.19–23
2.2. Shape based searching of the designed molecules
Shape complementarity between the protein and ligand plays
a crucial role in the binding of ligand to receptor. Since the basic
skeleton or scaffold of the molecule influences overall steric
shape of the molecule it is important to evaluate whether the
template selected permits proper orientation of the diaryl groups
to enable the binding of the ligand to enzymes active site. With
this understanding, the designed molecules were probed for
shape similarity using shape query SC558, a structurally analo-
gous potent COX-2 inhibitor extracted from pdb file 1cx2
employing PHASE module of molecular modeling software Schrö-
dinger. The shape similarity of the designed molecules to SC558
is given by similarity index which can have values from 0 to 1.
All the designed molecules exhibited similarity index values
greater than 0.5 which suggest that molecules are capable of
achieving good binding affinity to COX-2 enzyme. The molecular
alignment of the designed analogs to the shape query SC558 is
shown in Figure 2.
O
O
N
N
N
H
N
N
O
TRYPTANTHRIN
RUTAECARPINE
SO2CH3
C
O
N
DEWG
B
N
R
A
E
COMPOUNDS (9-24)
SO2CH3
SO2NH2
C
N
D
N
O
F3C
B
B
A
A
O
CH3
CELECOXIB
ROFECOXIB
Figure 1. Analogue-based design of anti-inflammatory quinazolinones.

4522
E. Manivannan, S. C. Chaturvedi / Bioorg. Med. Chem. 19 (2011) 4520–4528
benzoic acid. This method has the advantage of being a one-pot
synthesis and gave significantly good yields.
The physicochemical properties were calculated by standard
methods and the structures of the synthesized compounds were
confirmed by IR, NMR, Mass and elemental analysis. The IR spec-
trum of the substituted quinazolin-4-one compounds (9–24)
showed characteristics prominent peaks in at 1680–1710 cmꢀ1
(C@O of 4-keto-quinazolinone), which further confirms the forma-
tion of the title compounds. The compounds were also showed dis-
appearance of bands at 1600–1650 cmꢀ1 (N–H deformation) and
3460–3500 cmꢀ1 (N–H stretching) due to conversion of free amino
group into cyclic nitrogen. Halogen containing quinazolin-4-ones
showed the characteristic absorption bands at 590–760 cmꢀ1
(C–X stretching). The conversion of methylsulfanyl (–SCH3) to
methylsulphonyl (–SO2CH3) was detected by the d shift from
2.47 to 3.34 ppm for CH3 protons in the NMR spectrum.
The –OCH3 protons appeared in the chemical shift range of
3.6–3.8 ppm. Other aromatic protons appeared as multiplets in d
7.1 to 8.5 ppm. Further the mass spectrum [FAB-MS] showed a
molecular ion peak (M+) for benzoxazin-4ones and quinazolin-4-
ones respectively. In the ESI-MS spectra, molecular ion [M+] peaks,
which appeared at different intensities, confirmed the molecular
weights of the synthesized compounds. Molecular ion peaks were
the base peaks for most of the compounds. Appearance of an
isotope peak [M++2] as intense as the molecular ion peak has
confirmed shows the presence of halogen atom in compounds.
All the synthesized compounds were also characterized by elemen-
tal analysis and its experimental values coincided with the
calculated values and errors lay within 0.3% of the calculated
values.
Figure 2. The molecular alignment of the designed analogs to the shape query
SC558.
2.3. Chemistry
A set of 2,3-diaryl-3H-quinazolin-4-ones that possess a SCH3 or
SO2CH3 substituent at the para position of one of the aryl rings
with or without a methoxy substituent on the other aryl ring, along
with H, F, Cl and Br substituents on central quinazolin-4-one ring
were designed and synthesized for COX-2 inhibitory potential.
The synthetic strategy adopted for the synthesis of title com-
pounds has been depicted in Scheme 1. Sixteen analogues of 2,3-
diaryl-3H-quinazolin-4-ones 9–24 were synthesized. The starting
materials, substituted 2-phenyl-3H-benzoxazin-4-ones 1–8, were
synthesized using the known procedure.37 Various anthranilic
acids (unsubstituted, 4-chloro, 4-fluoro and 5-bromo derivatives)
were reacted with acid chlorides in pyridine at 25 °C, correspond-
ing 2-phenyl-benzoxazin-4-ones were obtained as crystalline
products. The subsequent reflux with 4-methylthio aniline in gla-
cial acetic acid gave para –SCH3 substituted 2,3-diaryl-3H-quinaz-
olin-4-ones 9–16. Further oxidation of these compounds with
‘oxone’ in tetrahydrofuran and methanol mixture yielded 4-methyl
sulfonyl substituted 2,3-diaryl-quinazolin-4-ones 17–24. The syn-
thesis of benzoxazin-4-ones involves acylation of both the amine
and acid functions of the appropriate anthranilic acid by the acid
chloride followed by ring closure of the intermediate affected via
an intramolecular nucleophilic displacement of the corresponding
2.4. In vitro cyclooxygenase inhibitory activity
Biological evaluation of title compounds with regard to COX-1
enzyme inhibition, the methyl sulfanyl/methyl sulphonyl substi-
tuted 2,3-diaryl-3H-quinazolin-4-ones 9, 13 and 17–24 proved to
be slightly active, with percentage inhibition values ranging from
0 to 30% at a concentration of 20 lM. Among all the screened
compounds, the compounds substituted with chlorine and methyl
sulphonyl groups on 2,3-diaryl-3H-quinazolin-4-ones 19 proved to
be practically inactive as they have shown 0% inhibition at 20 lM
concentration. In case of COX-2 enzyme inhibition, all the screened
compounds showed modest to better activity with percentage
inhibition values ranging from 40% to 100% at a concentration of
20 lM. The 2,3-diaryl-3H-quinazolin-4-ones substituted with
bromo and methyl sulphone 18 and chloro methyl sulphonyl 19
O
R1
R2
H2N
R1
R2
COOH
NH2
O
Cl
a.
+
+
R3
N
SCH3
O
A
B
C
R3
1-8
b.
SCH3
SO2CH3
O
N
O
N
R1
R2
R1
R2
c.
N
N
R3
R3
9-16
17-24
Scheme 1. Reagents and conditions: (a) dry pyridine, 25 °C, 1–2 h; (b) CH3COOH, reflux, PTSA; (c) oxone, THF, MeOH, 2–3 h.

E. Manivannan, S. C. Chaturvedi / Bioorg. Med. Chem. 19 (2011) 4520–4528
4523
possess a higher COX-2 inhibitory activity with a percentage of 88–
100% maximum. The in vitro COX-1 and COX-2 inhibitory activity
results (Table 1) indicate that overall the methyl sulphonyl substi-
tution on all the compounds increase the COX-2 selectivity in vary-
ing magnitude depending upon the presence of other aromatic
substitution. Comparable COX-2 selectivity with reference com-
pound celecoxib was observed when 2,3-diaryl-3H-quinazolin-4-
ones ring substituted with Br, and Cl groups.
methyl sulphonyl (–SO2CH3) at R4. Halogen atoms or methoxy
groups at R1 and R3 position of 2,3-diaryl-3H-quinazolin-4-ones
appears to have a positive effect on the anti-inflammatory potency
of these compounds.
2.6. Ulcerogenic activity
Although the activities of many synthesized compounds were
comparable to the reference drugs, the gastric toxicity experiments
were only performed with most potent compounds of substituted
2,3-diaryl-3H-quinazolin-4-ones series 18, 19. The most active
compounds of the series and indomethacin were given orally to
rats and they were sacrificed after treatment for determining the
ulcerogenic index of the said compounds. None of the compounds
showed any ulcerogenic effects (Table 3) in the gastric mucosa at a
total dose of 100 mg/kg body weight in fasted rats. From the results
of the histopathological studies, it is evident that in all the groups
treated with compounds 18, 19, the structure of gastric mucosa is
quite normal. Capillary dilatation was observed at the most super-
facial region in few parts of the gastric mucosa. The cross section
also showed that the surface epithelium was continuous and
normal. Gastric ulcers were observed in all the animals treated
with 10 mg of Indomethacin. The superficial regions of the gastric
mucosa of these animals showed multiple areas of erosions and
hemorrhage. Erosions were not observed beyond the muscularis
mucosa. The layers other than the superfacial mucosa appeared
normal. The gland beneath and around the ulcer areas were dilated
and distorted. The glands in this region were also dilated.
2.5. In vivo anti-inflammatory activity
All the compounds were screened for in vivo anti-inflammatory
activities at 50 mg/kg body weight dose level with carrageenan-in-
duced rat paw edema using indomethacin as reference drug. The
paw edema induced by subplantar injection of carrageenan was
more prominent in the group treated with carboxymethyl cellulose
sodium (CMC-Na:Vehicle) where indomethacin exerted 58.16%
anti-inflammatory effect after 180 min. Among the tested com-
pounds of 2,3-diaryl-3H-quinazolin-4-ones series (Table 2), 15,
18, 19, 21, and 22 exhibited more than 45% edema inhibition and
decreased the difference in paw thickness comparable to that of
a group received indomethacin (p <0.05). The anti-inflammatory
effect of compound 22 has comparable effect of indomethacin at
10 mg/kg dose. All test compounds of the series, anti-inflammatory
activity was retained up to six hours unlike indomethacin, the
activity was started decreasing after 4 h. The efficacy of test com-
pounds was comparable to that of indomethacin, but with a longer
duration of action. This phenomenon may partly be due to the low
systemic bioavailability of test compounds following oral dosing,
due to efficient first-pass metabolism and some degree of intestinal
metabolism. 2,3-Diaryl-3H-quinazolin-4-ones 15, 18, 19, 21 and 22
were identified as potent anti-inflammatory compounds. Among
them, 15 is the only compound with methylsulfanyl (–SCH3) at
R4 substitution position and the rest of the compounds have
2.7. Molecular docking
With the aim of getting insights into the structural basis for its
activity, 2,3-diaryl-3H-quinazolin-4-ones (compounds 9–24) were
docked into the active site of COX-2 enzyme. The Glide docking
Table 1
In vitro COX-1 and COX-2 enzyme inhibition data for substituted 2,3-diaryl-3H-quinazolin-4-ones
R4
O
R1
N
R2
N
R3
Compound
Substitution
R3
Percentage inhibitiona
Physical properties
Molecular volume
R1
R2
R4
COX-1 (20
l
M)
COX-2 (20
lM)
Selectivity COX-1/COX-2
TPSA
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
H
Br
H
H
H
Br
H
H
H
Br
H
H
H
Br
H
H
—
H
H
Cl
F
H
H
Cl
F
H
H
Cl
F
H
H
Cl
F
H
H
H
H
OCH3
OCH3
OCH3
OCH3
H
H
H
H
OCH3
OCH3
OCH3
OCH3
—
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SO2CH3
SO2CH3
SO2CH3
SO2CH3
SO2CH3
SO2CH3
SO2CH3
SO2CH3
—
12
—
—
—
30
—
—
—
13
4
0
15
24
7
12
29
3
40
—
—
—
56
—
0.3
—
—
—
0.536
—
34.897
34.897
34.897
34.897
44.131
44.131
44.131
44.131
69.039
69.039
69.039
69.039
78.273
78.273
78.273
78.273
—
305.736
323.622
319.272
310.667
331.282
349.167
344.818
336.213
319.039
336.924
332.575
323.97
344.585
362.47
358.121
349.516
—
—
—
—
—
42
100
88
63
51
66
72
64
94
0.310
0.04
—
0.238
0.471
0.106
0.167
0.453
0.032
Celecoxib
—
a
Values represents means of two determinations acquired using an ovine COX-1/COX-2 assay kits, nd - COX-1/COX-2 Inhibitory activity not determined.

4524
E. Manivannan, S. C. Chaturvedi / Bioorg. Med. Chem. 19 (2011) 4520–4528
Table 2
In vivo anti-inflammatory activity data and docking results for substituted 2, 3-diaryl-3H-quinazolin-4-ones
Compound
Percentage edema inhibition
Docking score
90 min
180 min
270 min
360 min
GLIDE XP SCORE
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
29 1.73⁄
30 0.27⁄⁄
31 1.26⁄
29 1.46⁄⁄
30 0.47⁄⁄
32 1.45
32 0.69⁄⁄⁄
33 1.82⁄
32 0.69⁄
36 1.04⁄
34 1.34⁄
35 0.14⁄
38 0.44⁄⁄⁄
34 1.35⁄⁄
33 1.37
34 0.48⁄
35 1.36⁄
34 0.48⁄
39 1.44⁄⁄
38 1.77⁄⁄
39 1.46
38 1.33⁄
39 0.24⁄
39 1.54⁄
40 1.25⁄
40 1.89⁄⁄
42 1.81
47 1.08⁄⁄
40 1.89⁄
39 0.68⁄
49 1.16⁄
45 0.82⁄⁄
41 1.92
46 1.36⁄⁄
54 1.83⁄
43 1.54⁄
39 1.14⁄⁄
13 0.24⁄
ꢀ7.4254
ꢀ3.38017
—
ꢀ7.70532
ꢀ7.11189
ꢀ3.40755
—
ꢀ7.83864
ꢀ4.93843
—
36 1.27⁄⁄⁄
31 1.45⁄⁄
30 1.82⁄
38 1.03⁄
32 0.23⁄
30 1.63⁄
35 1.53⁄
39 1.23⁄
37 1.75⁄
31 1.27⁄
41 0.48⁄
43 1.76⁄⁄⁄
37 1.22⁄⁄
36 1.55⁄
44 1.78⁄
42 0.46⁄⁄
39 0.16⁄⁄
42 0.26
40 0.64⁄⁄
37 1.44⁄
34 1.02⁄⁄⁄
38 1.47
—
ꢀ8.67037
ꢀ9.32301
—
41 0.64⁄⁄⁄
38 1.35⁄⁄
34 1.72
49 0.18⁄⁄
41 1.47⁄⁄
36 0.28⁄⁄
49 0.28⁄
—
ꢀ9.4872
Indomethacin
58 1.26⁄⁄
—
Significance levels ⁄p <0.05, ⁄⁄p <0.01 and ⁄⁄⁄p <0.001 as compared with the respective control.
All the results were expressed as means SEM (standard error of estimate).
Table 3
of SC558. It can be observed from the Figures 3 and 4 that the
docked ligand does not form hydrogen bonding interactions with
either Arg 120, Tyr 355 or His 90. The high scoring ligands (com-
pounds 20, 21 and 24) are the ones with either fluoro substitution
in 7th position of quinazolinone ring or 4 methoxy phenyl substi-
tution of 2nd position of the quinazolinone ring. However,
compound 17, which adopts a favorable disposition as that of the
SC-558 is among the low scoring ligands for no conceivable
reasons. Compound 16 which is non-sulfonyl methyl top scoring
ligand adopts favorable disposition as that of the SC-558 by align-
ing its thiomethyl group with sulphonamide group of the SC-558
(Figs. 5 and 6). The rest of the compounds does not adopts a favor-
able disposition as that of SC-558 is a resultant of dubious docking
brought about by spatial constraints in the active site. Comparison
of the enzyme inhibitory potency data with glide score suggest
that docking results concurs with the biological evaluation results
moderately in case of compounds with methyl sulphonyl groups
since only one compound 17 is predicted poorly. In case of non sul-
phonyl methyl compounds, no meaningful conclusion could be
made owing to availability of only two data points. Though the
docking results were proven to be reasonably fair, it must also be
noted that the compounds 18 and 19 were left out by the program
were the most potent inhibitors of COX-2 in the series. The greater
potency of these compounds indicates a greater flexibility of the
COX-2 enzyme.
Ulcerogenic activity of the most active 2,3-diaryl-3H-quinazolin-4-ones and indo-
methacin in rats
Compound
Dose (mg/kg)
Ulcer Lesion index (cm)
18
19
100
100
10
0
0
Indomethacin
1.42 0.32
11.48 0.6
50
algorithm could not insert six compounds (11, 15, 18, 19, 22 and
23) into the active site of COX-2 enzyme owing to spatial con-
straints. It may also be observed that the studied compounds are
bulkier than crystal bound ligand SC-558. The active site of the en-
zyme has opened up enough to accommodate a smaller molecule
like SC-558 could not accommodate diaryl bicyclic compounds
with halogen atoms of larger van der Waal’s radii. Only four com-
pounds 17, 20, 21 and 24 among docked compounds adopt a dispo-
sition exactly matching that of SC558 (Figs. 3 and 4). In particular,
the unsubstituted/substituted phenyl moiety is inserted inside the
COX-2 hydrophobic pocket while the methyl sulfonyl group is
positioned into the selectivity pocket as the corresponding moiety
3. Conclusion
In conclusion, we describe herein the analogue-based design of a
series 2,3-diaryl quinazolinones possessing methyl sulfanyl/methyl
sulfonyl pharmacophore for in vitro and in vivo biological evalu-
ation as COX-2 inhibitors. All the methyl sulfonyl substituted
quinazolinones exhibited promising anti-inflammatory activity.
In particular, 6-bromo-3-(4-methanesulfonyl-phenyl)-2-phenyl-
3H-quinazolin-4-one (18), 7-chloro-3-(4-methanesulfonyl-phenyl)
-2-phenyl-3H-quinazolin-4-one (19), 3-(4-methanesulfonyl-
phenyl)-2-(4-methoxy-phenyl)-3H-quinazolin-4-one (21) and
6-bromo-3-(4-methanesulfonyl-phenyl )-2-(4-methoxy-phenyl)-
3H-quinazolin-4-one (22) emerged as the most active compounds
in the series. The results of ulcerogenic activity assay suggest that
these compounds are gastric safe compared to indomethacin. The
GLIDE based molecular docking results are reasonably fair in under-
standing the binding interactions and in vitro anti-inflammatory
Figure 3. Alignment of docking poses of compounds 17, 20, 21 and 24 of 2,
3-diaryl-3H-quinazolin-4-ones with SC-558.

E. Manivannan, S. C. Chaturvedi / Bioorg. Med. Chem. 19 (2011) 4520–4528
4525
Figure 4. Alignment of docking poses of compounds 17, 20, 21 and 24 of 2,3-diaryl-3H-quinazolin-4-ones with SC-558 in active site of COX-2 enzyme.
rendered visible by exposing to UV light and iodine vapor. Melting
points were recorded using an electrothermal melting point appa-
ratus and are uncorrected. IR spectra were recorded using Shima-
dzu FTIR or Jasco FT/IR-470 PLUS. NMR spectral study was
carried out using Brucker DRX 400. The FAB mass spectra were re-
corded on JEOL SX 102/DA-6000. Microanalytical data were ob-
tained using a Carlo-Erba CHNS-O EA 1108 Elemental Analyser.
Elemental analyses observed for all the newly synthesized com-
pounds were within the limits of accuracy ( 0.3%). Topological po-
lar surface area (TPSA) and molecular volume were calculated
using the web-based program Molinspiration.
4.2. Synthesis
4.2.1. Synthesis of substituted 2,3-diaryl-3H-quinazolin-4-ones
(9–16)
Equimolar amount of substituted 2-phenyl-3H-benzoxazin-4-
ones (0.02 mol) and 4-thiomethyl aniline (0.02 mol) in glacial ace-
tic acid was refluxed for 2–4 h. A catalytic amount of PTSA was
added to the reaction mixture. After completion of the reaction,
the reaction mixture was cooled to room temperature and poured
into crushed ice. The product formed was filtered; vacuum dried
and crystallized using absolute ethanol.
Figure 5. Alignment of docking pose of compound 16 of 2,3-diaryl-3H-quinazolin-
4-ones with SC-558.
activity of these compounds to COX-2 enzyme. The present investi-
gation suggests that 2,3-diaryl quinazolinones as a promising tem-
plate for the design of new gastric safe anti-inflammatory agents,
which can be further explored for anti-inflammatory activity. Fur-
ther studies and related analogs will be reported in due course.
4.2.1.1. 3-(4-Methylsulfanyl-phenyl)-2-phenyl-3H-quinazolin-
4-one (9). Yield, 75%; mp 226–228 °C; Rf = 0.54 mobile phase tol-
uene–ethyl acetate (7:3); IR (KBr, cmꢀ1): 1684 (C@O stretch); 1620
(C@N stretch); 1H NMR (DMSO): d 2.47 (s, 3H, S–CH3), 7.27–7.32
(m, 3H), 7.54–7.69 (m, 6H), 7.90–7.93 (m, 3H), 8.47 (br d,
J = 8.1 Hz, 1H); FAB-MS m/z [M+H]+: 345; Elemental Anal. Calcd
for C21H16N2OS: C, 73.23; H, 4.68; N, 8.13. Found: C, 73.26; H,
4.65; N, 8.15.
4. Experimental
4.1. Materials
All commercially available solvents and reagents were used
without further purification. The starting materials were procured
from Merck and Sigma Aldrich or prepared using known proce-
dures. Column chromatographic separations were carried out by
gradient elution with hexane-ethyl acetate mixture, unless other-
wise mentioned and silica gel (60–120 mesh) used. TLC was per-
formed on E-Merck pre-coated 60 F254 plates and the spots were
4.2.1.2.
6-Bromo-3-(4-methylsulfanyl-phenyl)-2-phenyl-3H-
quinazolin-4-one (10). Yield: 72%, mp 219–220 °C; Rf = 0.48 mo-
bile phase toluene–ethyl acetate (7:3); IR (KBr, cmꢀ1): 1672 (C@O
stretch); 1622 (C@N stretch); 1H NMR (DMSO): d 2.49 (s, 3H, S–
CH3), 7.28–7.31 (d, 2H), 7.54–7.67 (m, 5H), 7.77–7.82 (dd, 1H),
7.89–7.91 (d, 2H), 8.09 (s, 1H), 8.42 (br d, J = 8.1 Hz, 1H); FAB-MS

4526
E. Manivannan, S. C. Chaturvedi / Bioorg. Med. Chem. 19 (2011) 4520–4528
Figure 6. Alignment of docking poses of compound 16 of 2,3-diaryl-3H-quinazolin-4-ones with SC-558 in active site of COX-2 enzyme.
m/z [M+H]+: 423; Elemental Anal. Calcd for C21H15BrN2OS: C,
59.58; H, 3.57; N, 6.62. Found: C, 59.60; H, 3.56; N, 6.65.
7.54 (m, 1H), 7.82–7.83 (m, 2H), 8.48 (br d, J = 8.1 Hz, 1H); FAB-MS
m/z [M+H]+: 453; Elemental Anal. Calcd for C22H17BrN2O2S: C,
58.28; H, 3.78; N, 6.18. Found: C, 58.27; H, 3.81; N, 6.20.
4.2.1.3.
7-Chloro-3-(4-methylsulfanyl-phenyl)-2-phenyl-3H-
4.2.1.7.
7-Chloro-2-(4-methoxy-phenyl)-3-(4-methylsulfanyl-
quinazolin-4-one (11). Yield: 81%; mp 234–236 °C; Rf = 0.44 mo-
bile phase toluene–ethyl acetate (7:3); IR (KBr, cmꢀ1): 1680 (C@O
stretch); 1620 (C@N stretch); 1H NMR (DMSO): d 2.49 (s, 3H,
S–CH3), 7.28–7.32 (m, 3H), 7.55–7.67 (m, 5H), 7.90–7.93 (m, 3H),
8.63 (br d, J = 8.1 Hz, 1H); FAB-MS m/z [M+H]+: 380; Elemental
Anal. Calcd for C21H15ClN2OS: C, 66.57; H, 3.99; N, 7.39. Found:
C, 66.55; H, 3.98; N, 7.41.
phenyl)-3H-quinazolin-4-one (15). Yield: 72%; mp 122–124 °C;
Rf = 0.44 mobile phase toluene–ethyl acetate (7:3); IR (KBr,
cmꢀ1): 1670 (C@O stretch); 1618 (C@N stretch); 1H NMR (DMSO):
d 2.51 (s, 3H, S–CH3), 3.69 (s, 3H, –OCH3), 7.04–7.42 (m, 7H), 7.50–
7.53 (m, 1H), 7.82–7.83 (m, 2H), 8.39 (br d, J = 8.1 Hz, 1H); FAB-MS
m/z [M+H]+: 409; Elemental Anal. Calcd for C22H17ClN2O2S: C,
64.62; H, 4.19; N, 6.85. Found: C, 64.64; H, 4.22; N, 6.89.
4.2.1.4.
7-Fluoro-3-(4-methylsulfanyl-phenyl)-2-phenyl-3H-
4.2.1.8.
7-Fluoro-2-(4-methoxy-phenyl)-3-(4-methylsulfanyl-
quinazolin-4-one (12). Yield: 70%; mp 238–240 °C; Rf = 0.42 mo-
bile phase toluene–ethyl acetate (7:3); IR (KBr, cmꢀ1): 1682 (C@O
stretch); 1622 (C@N stretch); 1H NMR (DMSO): d 2.47 (s, 3H,
S–CH3), 7.13–7.19 (m, 3H), 7.56–7.67 (m, 5H), 7.91–8.08 (m, 3H),
8.46 (br d, J = 8.1 Hz, 1H); FAB-MS m/z [M+H]+: 363; Elemental
Anal. Calcd for C21H15FN2OS: C, 69.59; H, 4.17; N, 7.73. Found: C,
69.63; H, 4.21; N, 7.76.
phenyl)-3H-quinazolin-4-one (16). Yield: 69%; mp 122–124 °C;
Rf = 0.39 mobile phase toluene–ethyl acetate (7:3); IR (KBr,
cmꢀ1): 1672 (C@O stretch); 1622 (C@N stretch); 1H NMR (DMSO):
d 2.49 (s, 3H, S–CH3), 3.76 (s, 3H, –OCH3), 7.10–7.42 (m, 7H), 7.48–
7.52 (m, 1H), 7.81–7.84 (m, 2H), 8.41 (br d, J = 8.1 Hz, 1H); FAB-MS
m/z [M+H]+: 393; Elemental Anal. Calcd for C22H17FN2O2S: C,
67.33; H, 4.37; N, 7.14. Found: C, 67.35; H, 4.35; N, 7.15.
4.2.1.5. 2-(4-Methoxy-phenyl)-3-(4-methylsulfanyl-phenyl)-3H-
quinazolin-4-one (13). Yield: 65%; mp 122–124 °C; Rf = 0.40 mo-
bile phase toluene–ethyl acetate (7:3); IR (KBr, cmꢀ1): 1677 (C@O
stretch); 1620 (C@N stretch); 1H NMR (DMSO): d 2.48 (s, 3H, S–
CH3), 3.78 (s, 3H, –OCH3), 7.06–7.43 (m, 8H), 7.50–7.54 (m, 1H),
7.81–7.83 (m, 2H), 8.46 (br d, J = 8.1 Hz, 1H); FAB-MS m/z
[M+H]+: 375; Elemental Anal. Calcd for C22H18N2O2S: C, 70.57; H,
4.85; N, 7.48. Found: C, 70.61; H, 4.88; N, 7.51.
4.2.2. Oxidation of 4-methylsuphanyl substituted 2,3-diaryl-3H-
quinazolin-4-ones (17–24)
A solution of oxone in H2O (6 ml) and methanol (4 ml) was
added dropwise to a solution of 4-methyl sulfanyl compounds
(0.015 mol) in THF (10 ml) at 25 °C with stirring. The reaction
was allowed to proceed for 2–3 h prior to addition of H2O
(10 ml) and extraction with CH2Cl2 (4 ꢁ 30 ml). The organic layer
was separated and dried over anhydrous Na2SO4. The solvent
was removed in vacuo, and the residue was purified by silica gel
column chromatography.
4.2.1.6. 6-Bromo-2-(4-methoxy-phenyl)-3-(4-methylsulfanyl-
phenyl)-3H-quinazolin-4-one (14). Yield: 68%; mp 122–124 °C;
Rf = 0.41 mobile phase toluene–ethyl acetate (7:3); IR (KBr,
cmꢀ1): 1668 (C@O stretch); 1620 (C@N stretch); 1H NMR (DMSO):
d 2.50 (s, 3H, S–CH3), 3.74 (s, 3H, –OCH3), 7.08–7.46 (m, 7H), 7.52–
4.2.2.1.
3-(4-Methanesulfonyl-phenyl)-2-phenyl-3H-quinazo-
lin-4-one (17). Yield: 61%, mp 256–258 °C; Rf = 0.51 mobile phase
toluene–ethyl acetate (7:3); IR (KBr, cmꢀ1): 1722 (C@O stretch);

E. Manivannan, S. C. Chaturvedi / Bioorg. Med. Chem. 19 (2011) 4520–4528
4527
1624 (C@N stretch); 1H NMR (DMSO): d 3.34 (s, 3H, SO2CH3),
7.15–7.41 (m, 2H), 7.53–7.68 (m, 5H), 7.82–7.86 (m, 1H), 7.90–
7.96 (m, 3H), 8.32 (br d, J = 8.1 Hz, 1H); FAB-MS m/z [M+H]+:
377; Elemental Anal. Calcd for C21H16N2O3S: C, 67.00; H, 4.28; N,
7.44. Found: C, 67.02; H, 4.30; N, 7.47.
m/z [M+H]+: 425; Elemental Anal. Calcd for C22H17FN2O4S: C,
62.25; H, 4.04; N, 6.60. Found: C, 62.26; H, 4.07; N, 6.61.
4.3. Biological evaluation
4.3.1. In vitro COX-1 and COX-2 inhibitory assay
4.2.2.2. 6-Bromo-3-(4-methanesulfonyl-phenyl)-2-phenyl-3H-
quinazolin-4-one (18). Yield: 54%; mp 248–251 °C; Rf = 0.49 mo-
bile phase toluene–ethyl acetate (7:3); IR (KBr, cmꢀ1): 1688 (C@O
stretch); 1625 (C@N stretch); 1H NMR (DMSO): d 3.31 (s, 3H,
SO2CH3), 7.16–7.41 (m, 2H), 7.54–7.68 (m, 4H), 7.82–7.86 (m,
1H), 7.91–7.96 (m, 3H), 8.34 (br d, J = 8.1 Hz, 1H); FAB-MS m/z
[M+H]+: 455; Elemental Anal. Calcd for C21H15BrN2O3S: C, 55.39;
H, 3.32; N, 6.15. Found: C, 55.42; H, 3.33; N, 6.17.
The in vitro ability of substituted 2,3-diaryl-3H-quinazolin-4-
ones and celecoxib to inhibit the COX-1 and COX-2 isozymes was
carried out using cayman colorimetric COX (ovine) inhibitor
screening assay kit (Catalog No. 760111) supplied by Caymen
chemicals, USA. The calculations were performed as per the kit
guidelines.38
4.3.2. Animals
Animals were assigned into several groups randomly and each
group consists of six animals. The animals were kept in appropriate
cages at temperature controlled (25 2 °C) rooms, under a 12 h
light and dark cycle and they were fed standard rodent pellet. All
the animals were acclimatized for a week before the experiment.
Standard ethical guidelines of Committee for the Purpose of Con-
trol and Supervision of Experiments on Animals (CPCSEA), Ministry
of Social Justice and Empowerment, Government of India were
followed.
4.2.2.3. 7-Chloro-3-(4-methanesulfonyl-phenyl)-2-phenyl-3H-
quinazolin-4-one (19). Yield: 75%; mp 230–232 °C; Rf = 0.54 mo-
bile phase toluene–ethyl acetate (7:3); IR (KBr, cmꢀ1): 1672 (C@O
stretch); 1620 (C@N stretch); 1H NMR (DMSO): d 3.32 (s, 3H,
SO2CH3), 7.18–7.42 (m, 2H), 7.56–7.68 (m, 4H), 7.82–7.86 (m,
1H), 7.92–7.98 (m, 3H), 8.34 (br d, J = 8.1 Hz, 1H); FAB-MS m/z
[M]+: 410; Elemental Anal. Calcd for C21H15ClN2O3S: C, 61.39; H,
3.68; N, 6.82. Found: C, 61.41; H, 3.71; N, 6.86.
4.2.2.4. 7-Fluoro-3-(4-methanesulfonyl-phenyl)-2-phenyl-3H-
quinazolin-4-one (20). Yield: 66%; mp 262–264 °C; Rf = 0.47 mo-
bile phase toluene–ethyl acetate (7:3); IR (KBr, cmꢀ1): 1672 (C@O
stretch); 1618 (C@N stretch); 1H NMR (DMSO): d 3.32 (s, 3H,
SO2CH3), 7.12–7.39 (m, 2H), 7.53–7.64 (m, 4H), 7.82–7.86 (m,
1H), 7.90–7.94 (m, 3H), 8.34 (br d, J = 8.1 Hz, 1H); FAB-MS m/z
[M+H]+: 395; Elemental Anal. Calcd for C21H15FN2O3S: C, 63.95;
H, 3.83; N, 7.10. Found: C, 63.96; H, 3.84; N, 7.12.
4.3.3. Dose preparation
The test compounds and the standard drugs were administered
orally in the form of a suspension in 1% or 0.5% carboxy methylcel-
lulose sodium (CMC-Na).
4.3.4. In vivo anti-inflammatory activity carrageenan-induced
rat paw edema assay method
Anti-inflammatory activity was determined using carrageenan-
induced foot paw edema assay method in rats using indomethacin
10 mg/kg as standard drug.39 The test compounds were adminis-
tered orally at a dose of 50 mg/kg. Edemas were produced by
injecting 0.1 ml of a solution of carrageenan in the hind paw.
Paw volumes were measured using the mercury displacement
technique with the help of a plethysmograph immediately before
and 90, 180, 270 and 360 min after carrageenan injection. The per-
centage of edema inhibition was calculated using the following
formula.
4.2.2.5. 3-(4-Methanesulfonyl-phenyl)-2-(4-methoxy-phenyl)-
3H-quinazolin-4-one (21). Yield: 68%; mp 234–235 °C; Rf = 0.42
mobile phase toluene–ethyl acetate (7:3); IR (KBr, cmꢀ1): 1676
(C@O stretch); 1620 (C@N stretch); 1H NMR (DMSO): d 3.26 (s,
3H, SO2CH3), 3.60 (s, 3H, –OCH3), 7.14–7.48 (m, 8H), 7.56–7.60
(m, 1H), 7.82–7.84 (m, 2H), 8.51 (br d, J = 8.1 Hz, 1H); FAB-MS m/
z [M+H]+: 408; Elemental Anal. Calcd for C22H18N2O4S: C, 65.01;
H, 4.46; N, 6.89. Found: C, 65.03; H, 4.46; N, 6.88.
Percentage of edema inhibition ¼ 100½1 ꢀ ðA ꢀ XÞ=ðB ꢀ YÞꢂ
4.2.2.6. 6-Bromo-3-(4-methanesulfonyl-phenyl)-2-(4-methoxy-
phenyl)-3H-quinazolin-4-one (22). Yield: 60%; mp 253–255 °C;
Rf = 0.40 mobile phase toluene–ethyl acetate (7:3); IR (KBr,
cmꢀ1): 1672 (C@O stretch); 1624 (C@N stretch); 1H NMR (DMSO):
d 3.24 (s, 3H, SO2CH3), 3.71 (s, 3H, –OCH3), 7.18–7.52 (m, 7H), 7.55–
7.61 (m, 1H), 7.82–7.84 (m, 2H), 8.52 (br d, J = 8.1 Hz, 1H); FAB-MS
m/z [M+H]+: 485; Elemental Anal. Calcd for C22H17BrN2O4S: C,
54.44; H, 3.53; N, 5.77. Found: C, 54.46; H, 3.55; N, 5.78.
Where, X is the mean paw volume of rats before the administra-
tion of carrageenan and test compounds or reference drugs; A and
B is the mean paw volume of rats after the administration of carra-
geenan in the test group and control group, respectively; Y is the
mean paw volume of rats before the administration of carrageenan
in the control group.
4.3.5. Ulcerogenic activity
Albino rats of Wister strain weighing 150–200 g of either sex
were distributed at random in groups of 10 animals each. Test
compounds were administered by oral route at a dose of 100 mg/
kg once daily for four consecutive days. The other group of animals
was administered with vehicle alone (control) and or indometha-
cin (reference) at dose of 10, 50 mg/kg for inter-assay comparison.
The animals were killed under deep ether anesthesia and stomachs
were removed. Then the abdomen of each rat was opened through
great curvature and examined under dissecting microscope for le-
sions or bleedings. In macroscopic examination, the severity of the
mucosal damage (ulcerogenic index) was graduated by means of
scores.40
4.2.2.7. 7-Chloro-3-(4-methanesulfonyl-phenyl)-2-(4-methoxy-
phenyl)-3H-quinazolin-4-one (23). Yield: 70%; mp: 217–218 °C;
Rf = 0.42 mobile phase toluene–ethyl acetate (7:3); IR (KBr, cmꢀ1):
1680 (C@O stretch); 1622 (C@N stretch); 1H NMR (DMSO): d 3.20
(s, 3H, SO2CH3), 3.72 (s, 3H, –OCH3), 7.18–7.52 (m, 7H), 7.55–7.61
(m, 1H), 7.84–7.86 (m, 2H), 8.51 (br d, J = 8.1 Hz, 1H); FAB-MS m/
z [M+H]+: 441; Elemental Anal. Calcd for C22H17ClN2O4S: C,
59.93; H, 3.89; N, 6.35. Found: C, 59.96; H, 3.87; N, 6.34.
4.2.2.8. 7-Fluoro-3-(4-methanesulfonyl-phenyl)-2-(4-methoxy-
phenyl)-3H-quinazolin-4-one (24). Yield: 62%; mp 248–250 °C;
Rf = 0.41 mobile phase toluene–ethyl acetate (7:3); IR (KBr,
cmꢀ1): 1686 (C@O stretch); 1620 (C@N stretch); 1H NMR (DMSO):
d 3.18 (s, 3H, SO2CH3), 3.69 (s, 3H, –OCH3), 7.09–7.49 (m, 7H), 7.54–
7.60 (m, 1H), 7.84–7.86 (m, 2H), 8.52 (br d, J = 8.1 Hz, 1H); FAB-MS
In microscopic evaluation, the tissue pieces were placed in 10%
buffered formaldehyde solution for one week in room temperature
to fix the tissue. After washing, the tissues were dehydrated in

4528
E. Manivannan, S. C. Chaturvedi / Bioorg. Med. Chem. 19 (2011) 4520–4528
ascending degrees of ethanol, cleared in xylene and embedded in
paraffin pastilles serial sections of 5 m were cut and stained with
for financial support in the form of JRF and Career Award for Young
Teachers (CAYT) Scheme.
l
hematoxylin-eosin. Sections that showed effects of compounds
and reference drugs were selected, examined and photographed
by using light microscope.
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Statistical significance of anti-inflammatory activity of the com-
pounds on rat paw edema model was analyzed using one-way
analysis of variance (ANOVA) followed by Dunnett’s multiple com-
parison test. A significance level of P <0.05 was considered as
acceptable in all cases. All the results were expressed as mean-
s
SEM (standard error of estimate).
4.4. Molecular docking
Docking studies of synthesized compounds listed in Table 2
were performed using crystal structure of COX-2 enzyme (PDB
ID: 1CX2) obtained from the RCSB Protein Data Bank, which houses
the selective COX-2 inhibitor SC-558 in its active site.41 The struc-
ture of the chain ‘A’ was chosen as the target for docking studies.
All the experiments were performed using the program GLIDE
(Grid-based Ligand Docking with Energetics) module version 4.5,
Schrödinger, LLC, New York, NY, 2007 (Schrodinger Inc.). Coordi-
nates of the full-length substrate-complexed tetramer were pre-
pared for Glide 4.0 calculations by running the protein
preparation wizard. The p-prep script produces a new receptor file
in which all residues are neutralized except those that are rela-
tively close to the ligand (if the protein is complexed with a ligand)
or form salt bridges. The impref script runs a series of restrained
impact energy minimizations using the Impact utility. Minimiza-
tions were run until the average root mean square deviation
(rmsd) of the non-hydrogen atoms reached 0.3 A°.
Glide uses two boxes that share a common centre to organize its
calculations: a larger enclosing box and a smaller binding box. The
grids themselves are calculated within the space defined by the
enclosing box. The binding box defines the space through which
the centre of the defined ligand will be allowed to move during
docking calculations. It provides a measure of the effective size
of the search space. The only requirement on the enclosing box is
that it be large enough to contain all ligand atoms, even when
the ligand centre is placed at an edge or vertex of the binding
box. Grid files were generated using the cocrystallized ligand at
the centre of the two boxes. The size of the binding box was set
at 20 A° in order to explore a large region of the protein. The
three-dimensional structures of the compounds were constructed
using the Maestro interface. The initial geometry of the structures
was optimized using the OPLS-2005 force field performing 1000
steps of conjugate gradient minimization. The compounds were
subjected to flexible docking using the pre-computed grid files.
For each compound the 100 top-scored poses were saved and ana-
lyzed and only the best scoring poses were selected for the study.
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Acknowledgments
Author E.M. thankful to University Grants Commission and All
India Council for Technical Education, New Delhi, Govt. of India