Synthesis of novel triazole based benzoxazolinones: Their TNF-α based molecular docking with in-vivo anti-inflammatory, antinociceptive activities and ulcerogenic risk evaluation

Article abstract of DOI:10.1016/j.ejmech.2013.10.032

A library of novel bis-heterocycles containing benzoxazolinone based 1,2,3-triazoles has been synthesized using click chemistry approach. The compound 3f exhibited potent selective COX-2 inhibition of 59.48% in comparison to standard drug celecoxib (66.36% inhibition). The compound 3i showed significant (p < 0.001, 50.95%), TNF-α inhibitory activity as compared to indomethacin (p < 0.001, 64.01%). The results of the carrageenan induced hind paw oedema showed that compounds 3a, 3f, 3i, 3o, and 3e exhibited potent anti-inflammatory activity in comparison to Indomethacin. The molecular docking studies revealed that 3i exhibits strong inhibitory effect due to the extra stability of the complex because of an extra π-π bond. The histopathology report showed that none of the compounds caused gastric ulceration.

Full text of DOI:10.1016/j.ejmech.2013.10.032

European Journal of Medicinal Chemistry 70 (2013) 579e588
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of novel 1,2,3-triazole based benzoxazolinones: Their TNF-
based molecular docking with in-vivo anti-inflammatory,
antinociceptive activities and ulcerogenic risk evaluation
a
a
a
,
a
a
c
*
Saqlain Haider , M. Sarwar Alam , Hinna Hamid , Syed Shafi , Amit Nargotra ,
c
a
d
a
a
Priya Mahajan , Syed Nazreen , Arunasree M. Kalle , Chetna Kharbanda , Yakub Ali ,
b
b
Aftab Alam , Amulya K. Panda
a
Department of Chemistry, Faculty of Science, Jamia Hamdard (Hamdard University), New Delhi 110 062, India
Product Development Cell, National Institute of Immunology, New Delhi 110067, India
Discovery Informatics, Indian Institute of Integrative Medicine, Jammu 180001, India
Department of Animal Sciences, University of Hyderabad, Hyderabad 500046, India
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 May 2013
Received in revised form
A library of novel bis-heterocycles containing benzoxazolinone based 1,2,3-triazoles has been synthe-
sized using click chemistry approach. The compound 3f exhibited potent selective COX-2 inhibition of
59.48% in comparison to standard drug celecoxib (66.36% inhibition). The compound 3i showed signif-
8
October 2013
icant (p < 0.001, 50.95%), TNF- inhibitory activity as compared to indomethacin (p < 0.001, 64.01%). The
a
Accepted 12 October 2013
Available online 22 October 2013
results of the carrageenan induced hind paw oedema showed that compounds 3a, 3f, 3i, 3o, and 3e
exhibited potent anti-inflammatory activity in comparison to Indomethacin. The molecular docking
studies revealed that 3i exhibits strong inhibitory effect due to the extra stability of the complex because
Keywords:
of an extra
ulceration.
p
e
p
bond. The histopathology report showed that none of the compounds caused gastric
1,2,3-Triazole
Click chemistry
Molecular docking
Benzoxazolinone
Ó 2013 Elsevier Masson SAS. All rights reserved.
Carrageenan cyclooxygenase
1. Introduction
antimicrobial activities [3e12]. Zoxazolamine is a Benzoxazole
analogue and is mainly used as skeletal muscle relaxant [13].
Non-steroidal anti-inflammatory drugs (NSAIDs) are still used
Some benzoxazolinone derivatives have been shown to inhibit
iNOS and constitute an important class of non-aminoacid based
NOS inhibitors [14]. On the other hand 1,2,3-triazoles and their
derivatives have emerged as powerful pharmacophores [15] and
have remained at the centre of interest due to their chemothera-
peutic value [16]. They are found in many drugs and have been an
interesting component in terms of their biological activity [17].
Many 1,2,3-triazoles have been found to possess potent antimi-
crobial [18,19], analgesic [20], anti-inflammatory, local anaesthetic
[21], anticonvulsant [22], antineoplastic [23], antimalarial [24]
and antiviral activity [25]. Considering the biological importance
of benzoxazolinone and 1,2,3-triazoles as anti-inflammatory and
antinociceptive agents, we aim to conjugate these two important
ligands under one construct through a methylene linkage. We
herein reporting, the design and synthesis of benzoxazolinone and
for the treatment of many rheumatic diseases but they cause many
adverse side effects, the most important being the gastric injuries
that might later cause gastric ulceration leading to death [1]. Drugs
with selective COX-1 vs. COX-2 inhibition mechanisms [2] have of
late been used as anti-inflammatory agents.
Benzoxazolinone is a cyclic isostere of the coumarin nucleus.
Benzoxazolinone and their derivatives are an important class of
biologically active molecules. Their importance is due to their
diverse application in the field of pharmaceuticals and in chemical
systems. 2-benzoxazolinones have been shown to exhibit anal-
gesic, anti-inflammatory, antineoplastic, anticonvulsant and
1
,2,3-triazole based unsymmetrical bis-heterocycles and their
in vivo anti-inflammatory and analgesic activities for the first time
along with the TNF- based molecular docking.
*
Corresponding author. Tel.: þ91 11 26059688x5555; fax: þ91 11 26059663.
E-mail addresses: msalam5555@gmail.com, msalam@jamiahamdard.ac.in
(
M.S. Alam).
a
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.10.032

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S. Haider et al. / European Journal of Medicinal Chemistry 70 (2013) 579e588
2
. Results and discussion
2.1. Chemistry
Different benzoxazolinone derivatives
1 were synthesized
by refluxing substituted ortho-aminophenols with 1,1-carbonyl
dimidazole in dry THF under nitrogen atmosphere [26,27]. As
shown in the Scheme 1, different substituted benzoxazolinone
derivatives were refluxed with propargyl bromide in the presence
of potassium carbonate in dry acetone for 7e10 h to yield prop-
argylated benzoxazolinone derivatives 2. The 1,3-diploar cycload-
dition between propargylated benzoxazolinone derivatives and
substituted aromatic azides under click chemistry conditions pro-
duced novel unsymmetrical bis-heterocycles 3ae3v in quantitative
yields. Different aromatic azides with various substitutions
including electron withdrawing and electron donating groups have
been used. The propargylation of the different benzoxazolinone
Fig. 1. In-vivo anti-inflammatory activity of novel bis-heterocycles.
derivatives was confirmed by the presence of a signal at
d
4.69e
2.2. In vivo anti-inflammatory activity
4
.73 (s, 2H, CH ) along with another signal at 3.43e3.47 (s,1H, CH)
2
d
1
corresponding to terminal alkyne, in the H NMR spectra. The for-
All the synthesized compounds have been tested for their in vivo
anti-inflammatory activity by carrageenan-induced hind paw
oedema model. The results obtained indicate that the compound 3f
exhibited potent anti-inflammatory activity with 81.39% and
80.62% inhibition after 3 h and 5 h as compared to indomethacin
which showed 79.06% and 82.25% inhibition after 3 h and 5 h
respectively (Fig 1). The compound 3a exhibited 74.00% inhibition
mation of 1,2,3-triazoles was confirmed by the resonance of the
proton in the triazollyl ring at a d 9.0e9.2 as a singlet. The structure
13
was further supported by the C NMR spectra, which showed the
C-atom signals corresponding to triazole derivatives. MALDIeMS/
þ
ESIeMS of all compounds showed [M ] or [M þ K] or [M þ Na] or
[M þ 1].
O
O
N
O
(
a)
Ar-N3
(b)
O
O
O
Reflux
N
H
N
R1
R1
R1
N
N
Ar
N
1
2
3a-3v
Compound
R1
Ar
4-C H Cl
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
s
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
5-CH3
5-CH3
5-CH3
5-CH3
5-CH3
5-CH3
5-CH3
5-CH3
5-Cl
6
4
4-C H NO
2
6
4
3-C H NO
2
6
4
4-C H OC H
6
4
2
5
2-C H Cl
6
4
4-C H F
6
4
4-C H NO
6
4
2
3-C H NO
6
4
2
4-C H Br
6
4
2-C H Cl
6 4
4-C H F
6 4
2-C H F
6
4
-C H N
5 4
4-C H Cl
6
4
4-C H Cl
6
4
5-Cl
5-Cl
5-Cl
3-C H NO
6 4 2
2-C H F
6 4
-C H N
5 4
3-C H NO
6 4 2
4-C H OC H
6 4 2 5
4-C H OCH
6 4 3
-C H N
5 4
H
H
H
H
t
u
v
(
a)
t
(
b) CuSO4.5H2O, Sodium Ascorbate, BuOH:H2O, r.t.
Br
K CO / CH COCH
3
2
3
3
Scheme 1. Synthesis of novel benzoxazolinone based 1,2,3-triazoles.

S. Haider et al. / European Journal of Medicinal Chemistry 70 (2013) 579e588
581
at 3 h post-carrageenan and 76.74% inhibition 5 h post-carrageenan
Table 2
XP Docking and MMGBSA results of molecules for TNF-a.
administration as compared to indomethacin. Whereas the
compounds 3i and 3e showed a time-dependent increase in the
inhibition of inflammation (73.25% and 71.76% inhibition at 3 h
post-carrageenan and 74.80% and 72.86% inhibition at 5 h post-
carrageenan respectively). The compound 3o showed comparable
anti-inflammatory activity with 67.44% and 65.11% inhibition after
Test Compound
XP glide score
MMGBSA energy
Co-crystallized Ligand
Rolipram
Indomethacin
ꢀ7.67
ꢀ5.66
ꢀ5.39
ꢀ5.87
ꢀ5.38
ꢀ5.33
ꢀ5.15
ꢀ4.76
ꢀ65.348
ꢀ69.445
ꢀ55.975
ꢀ63.048
ꢀ65.004
ꢀ55.522
ꢀ63.353
ꢀ53.364
3
3
3
3
e
o
f
3
h and 5 h respectively.
The structure activity relationship of the synthesized com-
i
pounds is analysed on the basis of the nature of the substituents on
the benzoxazolinone ring along with the position and nature of
substitutions on aryl group attached to the 1,2,3-triazollyl ring. The
compounds having substituted benzoxazolinone ring system
exhibited more potent anti-inflammatory activity as compared to
those having unsubstituted benzoxazolinone ring. The compounds
3a
3). The compound 3i showed significant (p < 0.001), TNF-a inhib-
itory activity with 50.95% inhibition as compared to the standard
drug indomethacin which exhibited 64.01%, (p < 0.001) inhibition.
3
t-3v conferred reduced activity whereas 3s did not show any
significant anti-inflammatory activity. It has been observed that
compounds 3a, 3f, 3i, 3k, 3o and 3n having weak electron with-
drawing halogen atoms on the aromatic ring attached to the tri-
azollyl ring conferred greater activity in comparison to compounds
2
.5. Docking studies on TNF-a
Docking studies of five compounds were carried out on the
selected protein target 2AZ5 (TNF- dimer). There was no apparent
a
3
2
b, 3c, 3g, 3h, 3p and 3s having para or meta substituted NO group.
difference in the dock scores of these five molecules, which
certainly is not the only criterion for predicting the affinity of a
ligand with the target protein (Fig 4). But, from the interaction
figures of these compounds with the protein, it was observed that
the compound 3i is oriented in the binding site in such a fashion
This is further substantiated by the docking studies where the glide
scores of halogen containing compounds are better than that of
nitro containing compounds (Table 2).Compounds 3d, 3t and 3u
containing the electron donating groups (OCH , OC H ) as well as
3 2 5
compounds 3v, 3m and 3r containing pyridyl substitutions showed
significant loss in the anti-inflammatory activity.
that it favours the possibility of two
pep interactions on the two
benzene rings with Tyr59 (chain A of the dimer) and Tyr119 (chain
B of the dimer) respectively. There is no H-bonding observed in the
interaction and the dock score is also in the range of ꢀ5, thereby
indicating that these are not very strong inhibitors of the protein.
The comparative strong inhibitory effect of the compound 3i
among others could be ascertained due to the extra stability of the
2.3. COX assay
The compounds exhibiting significant in vivo anti-inflammatory
activity were evaluated for their anti-inflammatory activity by
biochemical selective COX-2 inhibitory assay. Compounds 3a, 3f, 3i,
complex because of an extra
ures, it seems that Tyr59, Tyr119 and Tyr151 plays an important role
pep bond. From the interaction fig-
3
o and 3e showed significant COX-2 inhibition as compared to the
standard drug celecoxib (Fig 2). The COX-2 selectivity of these
compounds and their gastric liability may be related to their se-
lective percentage COX-2 inhibition (Table 1). The compounds 3a
in the binding affinity of the ligand. The compound 3e exhibited
(
Table 2) a glide score of ꢀ5.87 in comparison to the standard drug
indomethacin (ꢀ5.39) and the co-crystallized ligand which showed
a glide score of ꢀ7.67. Hence, these important parameters should be
considered while designing inhibitors of this protein.
(
COX-1 IC50 ¼ 389.2
m
M; COX-2 IC50 ¼ 5.6
M; COX-2 IC50 ¼ 2.4 M; SI ¼ 72.8) and 3i (COX-1
M; COX-2 IC50 ¼ 2.78 M; SI ¼ 67.7) exhibited potent
m
M; SI ¼ 69.5), 3f (COX-1
IC50 ¼ 174.72
m
m
m
IC50 ¼ 188.20
m
selective COX-2 inhibition as compared to celecoxib (COX-1
2.6. Analgesic activity
IC50 ¼ 25.74
m
M; COX-2 IC50 ¼ 0.32 M; SI ¼ 80.43). The COX-1/
m
COX-2 Selective Index (SI value) of the compounds 3a, 3f and 3i
shows the selective nature of these compounds towards COX-2
inhibition as compared to celecoxib.
The compounds showing significant anti-inflammatory activ-
ity in comparison to the standard drug indomethacin were
further tested for their antinociceptive activity by the writhing
test and tail immersion method. The results of the writhing test
(Fig 5) indicate that compound 3a exhibited potent
2.4. TNF-a assay
The compounds showing significant in-vivo anti-inflammatory
activity were further screened for their in-vitro TNF-a activity (Fig
Table 1
Inhibitory activity of the 1,2,3-triazole based benzoxazolinones.
Compounds
IC50
COX-1)
389.2
174.72
188.20
207.36
111.34
138.25
25.74
(
m
M)
Selectivity index (SI) COX-1/COX-2
(
(COX-2)
3
3
3
3
3
3
a
f
i
e
o
q
5.6
2.4
2.78
3.6
2.7
69.5
72.8
67.7
57.6
41.23
44.59
80.43
3.1
0.32
Celecoxib
Values are the means ꢁ SEM from three independent experiments using COX assay
kits (Cayman Chemicals Inc., Ann Arbor, MI, USA).
Fig. 2. In-vitro COX-2 activity of novel bis-heterocycles.

582
S. Haider et al. / European Journal of Medicinal Chemistry 70 (2013) 579e588
antinociceptive activity in comparison to the standard drug
indomethacin. The antinociceptive activity of the active com-
pounds has been evaluated using both chemical and thermal
methods of nociception. These methods are used to detect the
central and peripheral mechanisms of analgesia. Acetic acid
induced writhing test is used for detecting both central and pe-
ripheral analgesia, whereas tail flick test is sensitive to centrally
acting analgesia. Intraperitoneal administration of acetic acid re-
leases prostaglandins and sympathomimetic system mediators
like PGE2 and PGF2a and their levels are increased in the peri-
toneal fluid of the acetic acid induced mice [28]. Thermal induced
nociception indicates narcotic involvement [29]. Thermal noci-
ceptive tests are more sensitive to opioid
m receptors and non-
thermal tests are to opioid receptors [30,31].
k
Fig. 3. In-vitro TNF-a inhibitory activity of novel bis-heterocycles.
2.7. Ulcerogenic studies
antinociceptive activity with 41.83% inhibition as compared to the
standard drug indomethacin which caused 44.69% inhibition.
The results of the tail immersion (Table 3) method demonstrate
that the compounds 3a and 3f (p < 0.01) showed significant
The compounds showing potential anti-inflammatory and
antinociceptive activity were further tested for their gastric ulcer-
ation activity (Table 4 & Fig 6). When compared with Indomethacin,
Fig. 4. Docking results of the synthesized compounds onto TNF-a.

S. Haider et al. / European Journal of Medicinal Chemistry 70 (2013) 579e588
583
expressed as ppm against TMS as internal reference. Mass spectra
were recorded on MALDI-AB4800 and 70eV (EI Ms-QP 1000EX,
Shimadzu, Japan) and Column Chromatography was performed on
(Merck) Silica gel 60 (particle size 0.06e 0.20 mm). All compounds
prepared in this paper are new and confirmed from spectral data.
t
Compound 2 was dissolved in 20 mL of Butanol:water (1:1)
4 2
solvent at ambient temperature. CuSO .5H O was charged into it
and the reaction mixture was stirred for 5 min. Reaction mixture
became light blue in colour. Then sodium ascorbate was added to
the reaction mixture and stirred for 15 min. The colour of the re-
action mixture changed to dark yellow. After 15 min azide was
added at once. The reaction mixture was allowed to stir for further
7e10 h at ambient temperature. After the completion of the reac-
tion, monitored by TLC, reaction mixture was quenched with water
and extracted with ethyl acetate. Combined organic layers were
dried over anhydrous sodium sulphate, filtered and concentrated
under reduced pressure to obtain the final product.
Fig. 5. In-vivo antinociceptive activity by Writhing test method.
compounds 3a, 3e, 3f, 3i, and 3o did not induce any gastric ulcer-
ation and rupture of the gastric mucosal layer.
4
.1.1. 3-[1-(4-Chloro-phenyl)-1H-[1,2,3]triazol-4-ylmethyl]-6-
methyl-3H-benzooxazol-2-one(3a)
Yellowish brown crystals; yield 85%; m. p. 180e181 C; IR (KBr):
3
. Conclusion
ꢂ
ꢀ1
1
We have synthesized a focussed library of novel bis-heterocycles
n
(cm ) 3178, 3045, 1503, 1441, 1238, 1175, 1047, 825; H NMR
(300 MHz, DMSO):
2.32 (s, 3H), 5.17 (s, 2H), 7.01 (d, 1H, J ¼ 7.5 Hz),
7.19 (d, 2H, J ¼ 8.1 Hz), 7.66 (d, 2H, J ¼ 8.4 Hz), 7.92 (d, 2H,
encompassing benzoxazolinone-1,2,3-triazole moieties conjugated
through a methylene linkage and evaluated them for their anti-
inflammatory, antinociceptive activity and ulcerogenic risk evalu-
ation. The compounds 3e, 3i, 3a, 3f and 3q exhibited potent anti-
inflammatory and antinociceptive activity without exhibiting any
gastric ulceration. The selective COX-2 inhibitory potential of
compound 3f (COX-1/COX-2 SI ¼ 72.8) along with other molecules
concludes that these molecules can be considered as potent anti-
inflammatory agents as predicted by their COX-1/COX-2 selective
d
13
J ¼ 8.7 Hz), 8.92 (s, 1H); C NMR (75 MHz, DMSO):
d 21.35, 37.48,
109.73, 110.66, 112.46, 122.21, 122.58, 124.67, 128.66, 130.32, 132.62,
þ
133.50, 135.72, 142.53, 143.13, 156.15; ESIeMS: 341(M þ 1) ; Anal.
Calcd. for C
17 13 4 2
H ClN O : C, 59.92; H, 3.85; N, 16.44. Found C, 59.89;
H, 3.83; N, 16.42.
4.1.2. 6-Methyl-3-[1-(4-nitro-phenyl)-1H-[1,2,3]triazol-4-
index. The results of in-vivo TNF-
compound 3i has comparable TNF-
standard drug Indomethacin. From the molecular docking studies it
was found that the compound 3i is oriented in the binding site in
such a manner that it favours the possibility of two
teractions on the two benzene rings with Tyr59 (chain A of the
dimer) and Tyr119 (chain B of the dimer) respectively.
a
activity indicated that the
inhibition similar to the
ylmethyl]-3H-benzooxazol-2-one(3b)
ꢂ
ꢀ1
)
a
Yellow crystals; yield 88%; m. p. 194e195 C; IR (KBr):
n
(cm
1
3176, 3068, 1759, 1597, 1441, 1242, 1172, 854, 748; H NMR
(300 MHz, DMSO):
2.32 (s, 3H), 5.21 (s, 2H), 7.02 (d,1H, J ¼ 5.4 Hz),
7.19e7.21 (m, 2H), 8.20 (d, 2H, J ¼ 6.8 Hz), 8.44 (d, 2H, J ¼ 6.8 Hz),
d
pep in-
13
9.12 (s, 1H); C NMR (75 MHz, DMSO): d 22.34, 36.98, 110.73,
112.64, 113.45, 122.61, 123.68, 125.65, 129.77, 131.52, 133.63, 134.46,
þ
1
36.75, 143.56, 144.15, 157.25; ESIeMS: 390 (M þ K) ; Anal. Calcd.
4
. Experimental
for C17
13 5 4
H N O : C, 58.12; H, 3.73; N, 19.93. Found C, 58.15; H, 3.80;
N, 19.95.
4.1. Chemistry
4
.1.3. 6-Methyl-3-[1-(3-nitro-phenyl)-1H-[1,2,3]triazol-4-
ylmethyl]-3H-benzooxazol-2-one(3c)
Yellow crystals; yield 86%; m. p. 203e204 C; IR (KBr):
All the chemicals and reagents used in this study were purchased
from Merck (India), Spectrochem, and Sigma Aldrich. All melting
points were uncorrected and measured using Electro-thermal IA
ꢂ
ꢀ1
)
n
(cm
3234, 3143, 1756, 1533, 1172, 1045, 809, 737; H NMR (300 MHz,
DMSO):
2.32 (s, 3H), 5.21 (s, 2H), 7.02 (d, 1H, J ¼ 5.9 Hz), 7.18e7.21
(m, 2H), 7.88 (t, 1H, J ¼ 6.1 Hz), 8.32 (d, 1H, J ¼ 6.2 Hz), 8.39 (d, 1H,
1
9100 apparatus (Shimadzu, Japan); IR spectra were recorded as
d
potassium bromide pellets on a Perkin Elmer 1650 spectropho-
tometer (USA), 1H NMR spectra was determined on a Bruker (200,
13
J ¼ 6.0 Hz), 8.70 (t, 1H, J ¼ 1.5 Hz), 9.14 (s, 1H); C NMR (75 MHz,
3
00 and 400 MHz) spectrometer and chemical shifts were
DMSO):
d
22.40, 37.38, 110.11, 111.32, 123.15 127.13, 128.49, 129.32,
Table 3
In-vivo antinociceptive activity by tail immersion method.
Group
Dose
Basal reaction time (min)
Reaction time (min)
30
60
120
Control
Indomethacin
2 ml/kg
0.05 mmol/kg
e
e
e
e
e
2.00 ꢁ 0.23
2.08 ꢁ 0.22
1.90 ꢁ 0.20
2.28 ꢁ 0.14
2.30 ꢁ 0.21
2.48 ꢁ 0.20
2.44 ꢁ 0.15
2.22 ꢁ 0.20
3.12 ꢁ 0.11
2.18 ꢁ 0.11
2.62 ꢁ 0.26
3.08 ꢁ 0.24
3.10 ꢁ 0.28
2.88 ꢁ 0.13
2.54 ꢁ 0.05
3.68 ꢁ 0.16
2.44 ꢁ 0.18
2.74 ꢁ 0.11
3.22 ꢁ 0.14
3.24 ꢁ 0.20
2.78 ꢁ 0.07
2.38 ꢁ 0.11
3.54 ꢁ 0.15
2.56 ꢁ 0.14
2.88 ꢁ 0.09
3.34 ꢁ 0.12
3.36 ꢁ 0.15
2.64 ꢁ 0.06
*
**
**
3
3
3
3
3
e
i
a
f
q
*
*
*
*
**
**
**
Data is analysed by one way ANOVA followed by Dunnett’s ‘t’ test and expressed as mean ꢁ SEM from six observations.
**p < 0.001, **p < 0.01 & *p < 0.05.
*

584
S. Haider et al. / European Journal of Medicinal Chemistry 70 (2013) 579e588
Table 4
J ¼ 6.6 Hz), 5.15 (s, 2H), 7.00e7.11 (m, 3H), 7.19 (d, 2H, J ¼ 6.6 Hz),
13
Histopathology report of ulcerogenic activity.
7
.75 (d, 2H, J ¼ 8.4 Hz), 8.78 (s, 1H); C NMR (75 MHz, DMSO):
Group
Surface
Epith. Damage
Sup.
Mucosal
damage
Deep
mucosal
damage
Muscular
layer damage
d
15.01, 21.35, 37.52, 64.00, 109.75, 110.64, 115.72, 122.17, 122.39,
124.65, 128.69,132.58, 142.54, 142.65, 154.11, 159.05; ESIeMS: 350
þ
(
M) ; Anal. Calcd. for C19
H
18
N
4
O
3
: C, 65.13; H, 5.18; N, 15.99. Found
Control
Indomethacin
e
þ
e
e
e
e
e
e
þ
e
e
e
e
e
e
e
e
e
e
e
e
e
þ
e
e
e
e
e
C, 65.11; H, 5.15; N, 15.97.
3
3
3
3
3
e
i
a
f
4
.1.5. 3-[1-(2-Chloro-phenyl)-1H-[1,2,3]triazol-4-ylmethyl]-6-
methyl-3H-benzooxazol-2-one(3e)
Brown crystals; yield 85%; m. p. 173e174 C; IR (KBr):
ꢂ
ꢀ1
)
n
(cm
3542, 3146, 1751, 1678, 1440, 1172, 1061, 832; H NMR (300 MHz,
DMSO):
2.33 (s, 3H), 5.20 (s, 2H), 7.02 (d, 1H, J ¼ 8.1 Hz), 7.20e7.22
m, 2H), 7.5e7.6 (m, 3H), 7.76 (d, 1H, J ¼ 7.8 Hz), 8.68 (s, 1H);
NMR (75 MHz, DMSO): 21.35, 37.36, 109.80, 110.66, 124.64, 126.57,
1
q
e, No damage; þþþ, indicates high degree of damage.
d
1
3
(
C
d
1
32.12, 134.34, 135.46, 136.24, 141.34, 142.34, 155.31; ESIeMS: 351
128.68, 128.68, 128.87, 128.93, 131.00, 132.21, 132.60, 134.79, 141.83,
þ
þ
(
M þ 1) ; Anal. Calcd. for C17
H
13
N
5
O
4
: C, 58.12; H, 3.73; N, 19.93.
142.52, 154.10; MALDIeMS: 341 (M þ 1) ; Anal. Calcd. for
Found C, 58.10; H, 3.72; N, 19.90.
C
17
H13ClN
4 2
O : C, 59.92; H, 3.85; N, 16.44. Found C, 59.90; H, 3.83; N,
16.42%.
4
.1.4. 3-[1-(4-Ethoxy-phenyl)-1H-[1,2,3]triazol-4-ylmethyl]-6-
methyl-3H-benzooxazol-2-one(3d)
White crystals; yield 85%; m. p. 175e176 C; IR (KBr):
4.1.6. 3-[1-(4-Fluoro-phenyl)-1H-[1,2,3]triazol-4-ylmethyl]-6-
ꢂ
ꢀ1
n
(cm
)
methyl-3H-benzooxazol-2-one(3f)
Brown crystals; yield 93%; m. p. 181e182 C; IR (KBr):
3685, 3132, 1768, 1445, 1236, 1172, 1046, 835; H NMR (300 MHz,
1
ꢂ
ꢀ1
3
478, 3147, 2977, 1756, 1615, 1446, 1178, 1048, 804; H NMR
n
(cm
)
1
(
300 MHz, DMSO):
d
1.34 (t, 3H, J ¼ 6.3 Hz), 2.32 (s, 3H), 4.08 (q, 2H,
Fig. 6. Haematoxylin and eosin immunohistochemical staining of gastric ulcers after ulcer induction in rats.

S. Haider et al. / European Journal of Medicinal Chemistry 70 (2013) 579e588
585
DMSO):
d
2.32 (s, 3H), 5.17 (s, 2H), 7.02 (d,1H, J ¼ 7.8 Hz), 7.19 (d, 2H,
Calcd. for C17
H, 4.02; N, 17.26%.
4 2
H13FN O : C, 62.96; H, 4.04; N, 17.28. Found C, 62.93;
J ¼ 7.5 Hz), 7.44 (t, 2H, J ¼ 8.7 Hz), 7.90e7.94 (m, 2H), 8.88 (s, 1H);
13
C NMR (75 MHz, DMSO): 21.34, 37.49, 109.73, 110.65, 117.03,
d
1
17.34, 122.73, 122.85, 122.97, 124.66, 128.66, 132.61, 133.49, 142.54,
4.1.12. 3-[1-(2-Fluoro-phenyl)-1H-[1,2,3]triazol-4-ylmethyl]-5-
þ
142.98, 154.10, 163.76; ESIeMS: 324 (M) ; Anal. Calcd. for
methyl-3H-benzooxazol-2-one(3l)
ꢂ
ꢀ1
)
C
17
H13FN
4
O
2
: C, 62.96; H, 4.04; N, 17.28. Found C, 62.94; H, 4.02; N,
Yellow crystals; yield 90%; m. p. 200e201 C; IR (KBr):
n
(cm
1
17.26%.
3138, 3027, 1769, 1496, 1253, 1192, 1092 829; H NMR (300 MHz,
DMSO):
2.33 (s, 3H), 5.20 (s, 2H), 6.95 (d, 1H, J ¼ 7.8 Hz), 7.17 (s,
1H), 7.24 (d, 1H, J ¼ 8.1 Hz), 7.63e7.67 (m, 3H), 8.31 (d, 1H,
d
4.1.7. 5-Methyl-3-[1-(4-nitro-phenyl)-1H-[1,2,3]triazol-4-
13
ylmethyl]-3H-benzooxazol-2-one(3g)
Brown crystals; yield 90%; m. p. 166e167 C; IR (KBr):
J ¼ 8.4 Hz), 9.00 (s, 1H); C NMR (75 MHz, DMSO):
d 21.52, 37.33,
ꢂ
ꢀ1
n
(cm
628, 3067, 1774, 1519,1381, 1246, 1082, 855; H NMR (300 MHz,
DMSO):
2.33 (s, 3H), 5.21 (s, 2H), 6.95 (d, 1H, J ¼ 8.4 Hz), 7.17e7.25
m, 2H), 8.21 (d, 2H, J ¼ 8.4 Hz), 8.44 (d, 2H, J ¼ 8.4 Hz), 9.12 (s, 1H);
)
109.79, 110.43, 122.87, 123.22, 131.02, 133.91, 140.49, 141.81, 143.25,
1
þ
3
150.26, 151.92, 152.29, 154.33, 164.50; ESIeMS: 324 (M) ; Anal.
d
Calcd. for C17
4 2
H13FN O : C, 62.96; H, 4.04; N, 17.28. Found C, 62.95;
(
H, 4.02; N, 17.26%.
1
3
C NMR (75 MHz, DMSO):
d 21.52, 37.41, 109.81, 110.38, 121.22,
1
22.92, 123.24, 126.03, 131.00, 133.92, 141.15, 143.70, 147.24, 154.24;
4.1.13. 5-Methyl-3-(1-pyridin-3-yl-1H-[1,2,3]triazol-4-ylmethyl)-
þ
ESIeMS: 352.1 (M þ 1) ; Anal. Calcd. for C17
H
13
N
5
O
4
: C, 58.12; H,
3H-benzooxazol-2-one(3m)
ꢂ
ꢀ1
)
3
.73; N, 19.93. Found C, 58.09; H, 3.72; N, 19.91%.
Brown crystals; yield 88%; m. p. 229e230 C; IR (KBr):
n
(cm
1
3
145, 3067, 1772, 1623, 1595, 1240, 1132, 1043, 851; H NMR
(300 MHz, DMSO):
2.33 (s, 3H), 5.20 (s, 2H), 6.94 (d,1H, J ¼ 7.8 Hz),
7.20e7.24 (m, 2H), 7.43 (t, 1H, J ¼ 7.5 Hz), 7.53e7.60 (m, 2H), 7.82 (t,
4.1.8. 5-Methyl-3-[1-(3-nitro-phenyl)-1H-[1,2,3]triazol-4-
d
ylmethyl]-3H-benzooxazol-2-one(3h)
Yellow crystals; yield 83%; m. p. 209e210 C; IR (KBr):
ꢂ
ꢀ1
13
n
(cm
668, 3243, 1776, 1535, 1494, 1250, 1194, 805; H NMR (300 MHz,
DMSO):
2.33 (s, 3H), 5.21 (s, 2H), 6.95 (d, 1H, J ¼ 8.1 Hz), 7.17 (s,
H), 7.24 (d, 1H, J ¼ 8.1 Hz), 7.88 (t, 1H, J ¼ 8.1 Hz), 8.33 (d, 1H,
J ¼ 8.1 Hz), 8.41 (d,1H, J ¼ 1.2 Hz), 8.71 (t,1H, J ¼ 2.1 Hz), 9.15 (s,1H);
)
1H, J ¼ 7.5 Hz), 8.74 (s, 1H); C NMR (75 MHz, DMSO):
d 21.50,
1
3
37.26, 109.77, 110.45, 117.47, 123.18, 124.99, 125.14, 125.89, 126.05,
þ
d
131.05, 140.46, 142.40, 152.59, 154.25, 155.91; ESIeMS: 307 (M) ;
1
13 5 2
Anal. Calcd. for C16H N O : C, 62.53; H, 4.26; N, 22.79. Found C,
62.51; H, 4.24; N, 22.76%.
13
C NMR (75 MHz, DMSO):
d 21.51, 37.46, 109.81, 110.37, 115.25,
1
22.93, 123.23, 123.68, 126.56, 130.99, 132.99, 133.91, 137.50, 140.50,
4.1.14. 3-[1-(4-Chloro-phenyl)-1H-[1,2,3]triazol-4-ylmethyl]-5-
þ
1
C
43.51, 148.96, 154.25; ESIeMS: 352.1 (M þ 1) ; Anal. Calcd. for
methyl-3H-benzooxazol-2-one(3n)
ꢂ
ꢀ1
17
H
13
N
5
O
4
: C, 58.12; H, 3.73; N, 19.93. Found C, 58.08; H, 3.69; N,
Yellow crystals; yield 83%; m. p. 205e206 C; IR (KBr):
n
(cm
)
1
19.90%.
3135, 30216, 1754, 1495, 1246, 1134, 1088, 895; H NMR (300 MHz,
DMSO):
2.33 (s, 3H), 5.18 (s, 2H), 6.94 (d, 1H, J ¼ 7.5 Hz), 7.16e7.25
(m, 2H), 7.66 (d, 2H, J ¼ 7.8 Hz), 7.93 (d, 2H, J ¼ 8.1 Hz), 8.94 (s, 1H);
d
4.1.9. 3-[1-(4-Bromo-phenyl)-1H-[1,2,3]triazol-4-ylmethyl]-5-
13
methyl-3H-benzooxazol-2-one(3i)
Yellow crystals; yield 90%; m. p. 215e216 C; IR (KBr):
C NMR (75 MHz, DMSO): d 21.51, 37.43, 109.78, 110.39, 122.23,
ꢂ
ꢀ1
)
n
(cm
122.53, 123.20, 130.31, 131.02, 133.50, 135.72, 140.48, 143.17, 154.24;
1
þ
3
(
7
135, 3028, 1767, 1625, 1385, 1248, 1183, 1018, 835; H NMR
300 MHz, DMSO):
2.33 (s, 3H), 5.17 (s, 2H), 6.94 (d, 1H, J ¼ 7.8 Hz),
.16 (s, 1H), 7.23 (d, 1H, J ¼ 8.1 Hz), 7.79 (d, 2H, J ¼ 9.0 Hz), 7.86 (d,
ESIeMS: 341 (M þ 1) ; Anal. Calcd. for C17
4 2
H13ClN O : C, 59.92; H,
d
3.85; N, 16.44. Found C, 59.88; H, 3.82; N, 16.42%.
13
2
H, J ¼ 9.0 Hz), 8.94 (s, 1H); C NMR (75 MHz, DMSO):
d
21.51,
4.1.15. 5-Chloro-3-[1-(4-chloro-phenyl)-1H-[1,2,3]triazol-4-
37.43, 109.79, 110.40, 115.08, 122.46, 123.21, 123.68, 131.02, 133.89,
ylmethyl]-3H-benzooxazol-2one(3o)
Yellow crystals; yield 83%; m. p. 225e226 C; IR (KBr):
3206, 3123, 1780, 1610, 1248, 1092, 827; H NMR (300 MHz, DMSO):
þ
ꢂ
ꢀ1
(cm )
1
36.16, 140.53, 143.20, 156.45; MALDIeMS: 424 (M þ K) ; Anal.
Calcd. for C17 13BrN : C, 53.00; H, 3.40; N, 14.54. Found C, 52.98;
H, 3.38; N, 14.51%.
n
1
H
4
O
2
d
5.22 (s, 2H), 7.2 (dd, 1H, J ¼ 1.8 and 8.4 Hz), 7.41 (d, 1H, J ¼ 8.7 Hz),
7
.53 (d, 1H, J ¼ 2.1 Hz), 7.67 (d, 2H, J ¼ 8.7 Hz), 7.92 (d, 2H,
13
4
.1.10. 3-[1-(2-Chloro-phenyl)-1H-[1,2,3]triazol-4-ylmethyl]-5-
methyl-3H-benzooxazol-2-one(3j)
Brown crystals; yield 83%; m. p. 141e142 C; IR (KBr):
J ¼ 8.7 Hz), 8.94 (s, 1H); C NMR (75 MHz, DMSO):
d 37.15, 109.87,
111.07, 121.75, 122.11, 128.05, 129.83, 131.98, 133.03, 135.22, 140.73,
ꢂ
ꢀ1
þ
n
(cm
271,3035, 1761, 1622, 1435, 1249 1036, 823; H NMR (300 MHz,
DMSO):
2.33 (s, 3H), 5.20 (s, 2H), 6.94 (d, 1H, J ¼ 7.5 Hz), 7.16 (s,
H), 7.23 (d, 1H, J ¼ 8.1 Hz), 7.5e7.7 (m, 3H), 7.77 (d, 1H, J ¼ 7.5 Hz),
.71 (s, 1H); 13C NMR (75 MHz, DMSO):
21.50, 37.33, 109.78,
)
142.40, 153.41; ESIeMS: 360 (M) ; Anal. Calcd. for C16
H10Cl
2
N
4
O
2
:
1
3
C, 53.21; H, 2.79; N, 15.51. Found C, 53.18; H, 2.76; N, 15.49%.
d
1
8
4.1.16. 5-Chloro-3-[1-(3-nitro-phenyl)-1H-[1,2,3]triazol-4-
d
ylmethyl]-3H-benzooxazol-2-one(3p)
ꢂ
ꢀ1
110.54, 123.17, 126.66, 128.89, 128.96, 131.01, 132.23, 133.83, 134.81,
Yellow crystals; yield 80%; m. p. 163e164 C; IR (KBr):
n
(cm
)
þ
þ
1
1
40.46, 141.80, 154.24; MALDIeMS: 341 (M þ 1) , 363 (M þ Na) ;
Anal. Calcd. for C17 13ClN : C, 59.92; H, 3.85; N, 16.44. Found C,
9.89; H, 3.83; N, 16.41%.
3250, 3182, 1781, 1540, 1484, 1352, 1256, 823; H NMR (300 MHz,
DMSO):
5.25 (s, 2H), 7.21 (dd, 1H, J ¼ 2.1 and 8.7 Hz), 7.42 (d, 1H,
J ¼ 8.7 Hz), 7.53 (d,1H, J ¼ 2.1 Hz), 7.89 (t,1H, J ¼ 8.10 Hz), 8.33e8.41
H
4
O
2
d
5
1
3
(
m, 2H), 8.70 (t, 1H, J ¼ 2.1 Hz), 9.15 (s, 1H); C NMR (75 MHz,
4.1.11. 3-[1-(4-Fluoro-phenyl)-1H-[1,2,3]triazol-4-ylmethyl]-5-
DMSO): 37.69, 110.36, 111.60, 122.64, 122.96, 123.71, 126.56,
d
methyl-3H-benzooxazol-2-one(3k)
White crystals; yield 92%; m. p. 177e178 C; IR (KBr):
3
126.59, 128.57, 132.99, 137.49, 140.14, 143.21, 149.68, 153.91; ESIe
ꢂ
ꢀ1
þ
n
(cm
154, 3104, 1768, 1623, 1428, 1247, 1104, 843; H NMR (300 MHz,
DMSO):
2.33 (s, 3H), 5.17 (s, 2H), 6.95 (d, 1H, J ¼ 7.8 Hz), 7.17 (s,
H), 7.23 (d, 1H, J ¼ 8.1 Hz), 7.42e7.47 (m, 2H), 7.91e7.95 (m, 2H),
.89 (s, 1H); 13C NMR (75 MHz, DMSO):
21.51, 37.42, 109.78,
)
MS: 371 (M) ; Anal. Calcd. for C16
H10ClN
5
O
4
: C, 51.70; H, 2.71; N,
1
18.84. Found C, 51.67; H, 2.69; N, 18.82%.
d
1
8
4.1.17. 5-Chloro-3-[1-(2-fluoro-phenyl)-1H-[1,2,3]triazol-4-
ylmethyl]-3H-benzooxazol-2-one(3q)
White crystals; yield 82%; m. p. 159e160 C; IR (KBr):
3152, 3109, 1780, 1618, 1492, 1245, 1112, 1048, 846; H NMR
d
ꢂ
ꢀ1
)
110.40, 117.04, 122.69, 126.66, 122.88, 122.99, 123.20, 131.02, 133.49,
n
(cm
þ
1
133.90, 140.47, 143.01, 154.25; MALDIeMS: 347 (M þ Na) ; Anal.

586
S. Haider et al. / European Journal of Medicinal Chemistry 70 (2013) 579e588
(
300 MHz, DMSO):
J ¼ 8.7 Hz), 7.56e7.82 (m, 4H), 8.75 (s, 1H); C NMR (75 MHz,
DMSO): 37.47, 109.94, 111.43, 114.68, 121.66, 123.54, 124.61, 132.22,
d
5.24 (s, 2H), 7.20 (d, 1H, J ¼ 7.5 Hz), 7.40 (d, 2H,
4.2. Pharmacology
13
d
4.2.1. Animals
1
35.98, 137.12, 141.53, 143.56, 145.25, 155.35; ESIeMS: 345
Albino Wistar rats of either sex (150e200 g) were obtained from
Central Animal House, Hamdard University, New Delhi. The animals
were kept in cages at the room temperature and fed with food and
water ad libitum. Fourteen hours before the start of the experiment
the animals were sent to lab and fed only with water ad libitum.
The experiments were performed in accordance with the rules of
Institutional Animals Ethics Committee (registration number 173-
CPCSEA).
þ
(
M þ 1) ; Anal. Calcd. for C16
4 2
H10ClFN O : C, 55.75; H, 2.92; N, 16.25.
Found C, 55.73; H, 2.89; N,16.23%.
4
3
.1.18. 5-Chloro-3-(1-pyridin-3-yl-1H-[1,2,3]triazol-4-ylmethyl)-
H-benzooxazol-2-one(3r)
ꢂ
ꢀ1
)
White crystals; yield 86%; m. p. 196e197 C; IR (KBr):
n
(cm
1
3
066, 3012, 1788, 1611, 1488, 1244, 1096, 807; H NMR (300 MHz,
5.24 (s, 2H), 7.19e7.22 (m, 1H), 7.41 (d,1H, J ¼ 8.4 Hz), 7.55
s, 1H), 7.61 (dd, 1H, J ¼ 8.4 and 5.1 Hz), 8.31 (d, 1H, J ¼ 8.1 Hz), 8.69
DMSO):
d
(
(
4.2.2. Drugs
13
s, 1H), 9.01 (s, 1H), 9.12 (s, 1H); C NMR (75 MHz, DMSO):
d
37.46,
Indomethacin, Carrageenan, Carboxymethylcellulose, were
purchased from SigmaeAldrich Chemicals Pvt. Limited, Bangalore,
India.
109.77, 110.45, 117.47, 123.79, 124.16, 124.69, 126.05, 131.05,
þ
1
32.65 140.46, 142.40, 143,62 153.59; ESIeMS: 328 (M þ 1) ; Anal.
Calcd. for C15 10ClN : C, 54.97; H, 3.08; N, 21.37. Found C, 54.95;
H, 3.07; N, 21.36%.
H
5 2
O
4.2.3. Anti-inflammatory activity
The synthesized compounds were tested for their anti-
inflammatory activity using carrageenan-induced hind paw
oedema method. The rat paw oedema was induced by subcutane-
ous injection of 0.1 ml of 1% freshly prepared saline solution of
carrageenan into the right hind paw of rats [32]. The standard drug,
indomethacin (0.05 mmol/kg) was given orally as a positive control.
The control group was administered orally with 0.9% of 0.1 ml of
saline solution only. The test groups were administered orally with
equimolar dosage of the synthesized compounds as the standard
drug, 1 h before the administration of carrageenan. The paw vol-
umes were measured using plethysmometer [33] at interval of 3 h
and 5 h.
4
.1.19. 3-[1-(3-Nitro-phenyl)-1H-[1,2,3]triazol-4-ylmethyl]-3H-
benzooxazol-2-one(3s)
Yellow crystals; yield 86%; m. p. 207e208 C; IR (KBr):
ꢂ
ꢀ1
n
(cm
173, 3090, 1781, 1533, 1439, 1248, 1081, 816; H NMR (300 MHz,
DMSO):
5.24 (s, 2H), 7.12e7.24 (m, 2H), 7.36 (t, 2H, J ¼ 7.8 Hz), 7.88
)
1
3
d
(
1
t, 1H, J ¼ 8.4 Hz), 8.30e8.41 (m, 2H), 8.70 (t, 1H, J ¼ 2.1 Hz), 9.15 (s,
13
H); C NMR (75 MHz, DMSO): d 37.66, 109.78, 111.27, 116.55,
123.93, 124.33, 125.68, 127.56, 131.99, 132.66, 136.51, 140.50, 143.51,
þ
þ
1
C
2
48.96, 155.25; ESIeMS: 336 (M) , 338 (M þ 2) ; Anal. Calcd. for
16 11 5 4
H N O : C, 56.98; H, 3.29; N, 20.76. Found C, 56.96; H, 3.27; N,
0.74%.
4
.1.20. 3-[1-(4-Ethoxy-phenyl)-1H-[1,2,3]triazol-4-ylmethyl]-3H-
benzooxazol-2-one(3t)
Yellow crystals; yield 88%; m. p. 175e176 C; IR (KBr):
4.2.4. COX assay
Recombinant human COX-2 has been expressed in insect cell
expression system. The enzymes have been purified by using con-
ventional chromatographic techniques. Enzymatic activities of
COX-2 was measured according to the method of Copeland [34],
with slight modifications using a chromogenic assay based on the
oxidation of N,N,N,N,-tetra methyl-p-phenylene diamine (TMPD)
during the reduction of PGG2 to PGH2 [35]. Briefly, the assay
mixture contained TriseHCl buffer (100 mM, Ph 8.0), haematin
(15 mM), EDTA (3 mM), enzyme (100 mg COX-2) and the test
ꢂ
ꢀ1
n
(cm
172, 3123, 1774, 1521, 1488, 1252, 1050, 802; H NMR (300 MHz,
DMSO):
1.34 (t, 3H, J ¼ 6.9 Hz), 4.08 (q, 2H, J ¼ 6.9 Hz), 5.18 (s, 2H),
)
1
3
d
7
8
.08e7.21 (m, 4H), 7.34 (t, 2H, J ¼ 8.7 Hz), 7.75 (d, 2H, J ¼ 9.0 Hz),
.78 (s, 1H); 13C NMR (75 MHz, DMSO):
d 15.01, 37.54, 64.00, 110.15,
115.73, 122.18, 122.43, 122.92, 124.41, 130.19, 131.08, 142.45, 146.61,
þ
148.96, 154.00, 159.05; ESIeMS: 336 (M) ; Anal. Calcd. for
18 16 4 3
C H N O
: C, 64.28; H, 4.79; N, 16.66. Found C, 64.27; H, 4.77; N,
ꢂ
16.64%.
compound. The mixture was pre-incubated at 25 C for 1 min and
then the reaction was initiated by the addition of arachidonic acid
and TMPD, in total volume of 1 ml. The enzyme activity was
determined by estimating the velocity of TMPD oxidation for the
first 25 s of the reaction by following the increase in absorbance at
603 nm. A low rate of nonenzymatic oxidation observed in the
absence of COX-2 was subtracted from the experimental value
while calculating the percent inhibition.
4
.1.21. 3-[1-(4-Methoxy-phenyl)-1H-[1,2,3]triazol-4-ylmethyl]-3H-
benzooxazol-2-one(3u)
Yellow crystals; yield 88%; m. p. 169e170 C; IR (KBr):
ꢂ
1
ꢀ1
n
(cm
215, 3137, 1765, 1486, 11241, 1044, 830; H NMR (300 MHz,
DMSO): 3.81 (s, 3H), 5.19 (s, 2H), 7.10e7.21 (m, 4H), 7.35 (t, 2H,
)
3
d
13
J ¼ 7.8 Hz), 7.77 (d, 2H, J ¼ 9.0 Hz), 8.81 (s, 1H); C NMR (75 MHz,
DMSO):
d
37.53, 56.02, 110.17, 115.32, 122.19, 124.08, 130.34,
þ
132.09, 143.17, 160.11; ESIeMS: 322 (M) ; Anal. Calcd. for
4.2.5. Molecular docking studies on TNF-a
C
17
H
14
N
4
O
3
: C, 63.25; H, 4.38; N, 17.38. Found C, 63.23; H, 4.35; N,
All the computational studies were carried out in the Schro-
dinger suite 2010 molecular modelling software. The 2D structure
of the ligands (3a, 3e, 3f, 3i and 3 ) was built in the maestro window
17.36%.
ꢂ
4
.1.22. 3-(1-Pyridin-3-yl-1H-[1,2,3]triazol-4-ylmethyl)-3H-
[36]. The structures were then converted to their respective 3D
structures, with various conformers, tautomers and ionization
states using the Ligprep and Confgen modules [37e40]. The 3D
benzooxazol-2-one(3v)
Brown crystals; yield 90%; m. p. 179e180 C; IR (KBr):
ꢂ
ꢀ1
n
(cm
182, 3106, 1757, 1584, 1439, 1151, 1022, 811; H NMR (300 MHz,
DMSO):
5.23 (s, 2H), 7.12e7.24 (m, 2H), 7.35 (t, 2H, J ¼ 6.3 Hz),
)
1
ꢂ
3
crystal structure 2AZ5 [41], solved at a resolution of 2.10 A , was
d
downloaded from Protein Data Bank for carrying out the docking
studies. The crystal structure constitutes four chains A,B,C and D.
Chains A and B were homologous to chains C and D and hence,
chains A and B were used for docking studies, while chains C and D
were discarded for computational purpose. The coordinates of TNF-
Alpha in complex with this ligand were obtained from protein data
bank. The protein was prepared for docking using the protein
7
1
1
.62 (d, 1H, J ¼ 4.5 Hz), 8.30 (d, 1H, J ¼ 7.5 Hz), 8.69 (s, 1H), 8.99 (s,
H), 9.12 (s, 1H); 13C NMR (75 MHz, DMSO):
22.94, 124.44, 124.99, 128.44, 131.04, 141.78, 142.44, 143.17,
d 37.48, 110.15, 110.19,
þ
1
C
2
50.26, 153.99; ESIeMS: 294 (M þ 1)
;
Anal. Calcd. for
15 11 5 2
H N O : C, 61.43; H, 3.78; N, 23.88. Found C, 61.42; H, 3.76; N,
3.85%.

S. Haider et al. / European Journal of Medicinal Chemistry 70 (2013) 579e588
587
preparation wizard. In the pre-process step hydrogen’s were added
to the protein with assigning bond order, creating disulfide bond
and water residues were removed beyond 5 A from the heteroat-
tail. The selected rats were then divided into six groups of six rats
each. The control group was administered orally with 0.9% of 0.1 mL
of saline solution only. The standard drug, i.e. indomethacin and the
test compounds given orally at a dose of 0.05 mmol/kg of body
weight. After administration of the drugs, the reaction time was
measured at 30, 60 and 120 min. The basal reaction time was
calculated as the reaction time prior to the drug administration.
ꢂ
om. Further, the interactions of water residues with protein and
heteroatom were checked and only those water residues were kept,
which were interacting with protein as well as heteroatom. Then
the heteroatom was extracted and protein was refined by assigning
H-bonds and minimization at OPLS 2005 force field. A grid was
generated at active site, identified on the bases of already co-
crystallised ligand to the receptor using receptor grid generation
module. Docking studies were then carried out with the co-crys-
tallized ligand, in order to confirm the protocol. The conformation
of the co-crystallized ligand matched best with the docked
conformation using extra precision (XP) docking algorithm of Glide
module.
4.2.8. Ulcerogenic activity
The test compounds having anti-inflammatory & analgesic ac-
tivities comparable with the indomethacin were further tested for
their ulcerogenic risk evaluation [46]. This was done at three times
higher dose in comparison to the dose used for anti-inflammatory
activity, i.e. 0.15 mmol/kg body weight of indomethacin and the test
compounds were used. Each group had three animals which were
later sacrificed. When compared with indomethacin, these com-
pounds did not cause any gastric ulceration and disruption of
gastric epithelial cells at the above mentioned oral dose. Hence
gastric tolerance towards the test compounds was better than that
of indomethacin.
4.2.6. TNF-
a assay
Macrophages cells were grown in RPMI 1640 containing 10%
FBS, 1 M HEPES, 2 mM glutamine, 100 U/mL penicillin and 100 mg/
mL streptomycin was obtained by passage through a stainless
mesh. Supernatants of cell culture collected from form macro-
phages cells seeded with varying concentrations of compounds was
assayed for the pro-inflammatory cytokine levels using e-Bio-
Acknowledgements
sciences ELISA kits. For this, macrophages were pre-incubated with
The authors thank Dr. G.N. Qazi, Vice-Chancellor, Jamia Ham-
dard for providing necessary facilities to the department of chem-
istry to carry out this work. One of the authors (SH) is also thankful
to CSIR for awarding the senior research fellowship.
6
cytochalasin D (2.5
mM/1 ꢃ 10 cells) for 30 min before the start of
the experiment and continued till the end. Cytokine levels were
measured according to the protocol suggested by the manufacturer.
Cells were cultured in 96-well plates at 2 ꢃ 106 cells/mL and cy-
tokines were then measured from the supernatants by ELISA. The
assay was performed according to the manufacturer’s instruction
Appendix A. Supplementary data
with multipoint analysis [42]. Briefly, 100
mL of diluted capture
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.10.032.
antibody was added to each well in a 96 well plate and was allowed
ꢂ
to adhere overnight for 4 C. Plates were washed and then blocked
with 1 ꢃ PBS supplemented with 10% FBS for 1 h at room tem-
perature. After washing, serial dilutions of the standard and sam-
ples were prepared in the plates and were then incubated for 2 h at
room temperature. Then, plates were washed and working detector
solution (including detector antibody and avidinehorse radish
peroxidase reagent) was added into each well. Plates were then
sealed and incubated for 1 h at room temperature. After washing,
References
[1] M.M. Wolfe, D.R. Lichtenstein, G. Singh, N. Engl. J. Med. 340 (1999) 1888.
[2] G. Dannhardt, W. Kiefer, Eur. J. Med. Chem. 36 (2001) 109e126.
[3] L. Jund, J. Corse, A.S. King, H. Bayne, K. Mihrag, Phytochemistry 10 (1971)
2971.
[4] S.L. El-Ansary, E.I. Aly, M.A. Halem, Egypt J. Pharm. Sci. 33 (1992) 379.
[
[
[
5] J. Sam, J.L. Valentine, J. Pharm. Sci. 58 (1969) 1043e1054.
6] R.L. Clark, A.A. Pessolano, J. Am. Chem. Soc. 80 (1953) 1662e1664.
7] R.S. Varma, W.L. Nobles, J. Pharm. Sci. 57 (1968) 39e44.
100
mL of tri-methyl benzidine (TMB) substrate was added into each
well. Stop solution (2 N H
2
SO ) was finally added after incubation in
4
[8] N. Gökhan, H. Erdo gꢀ an, B.C. Tel, R. Demirdamar, Eur. J. Med. Chem. 31 (1996)
625e628.
the dark for 30 min at room temperature. The absorbance was read
at 450 nm. The result was analysed using softmax program and
values determined against the standard provided by the
manufacturer.
[9] M.P. Vaccher, D. Lesieur, C. Lespagnol, J.P. Bonte, J. Lamar, M. Beaughard,
G. Dureng, Farmaco Ed. Sci. 41 (1986) 257e269.
[
10] R.S. Varma, A. Kapoor, Indian J. Chem. 18B (1979) 200e204.
[11] D.D. Erol, H. Erdo gꢀ an, N. Yulu gꢀ , Pharm. Belg. 44 (1989) 334e336.
[
12] V. Kalcheva, Z. Mincheva, P. Andreeva, Arzneim. Forsch. 40 (1990) 1030e
034.
13] Hansch, Sammes, Taylor, Compr. Med. Chem. 6 (1989) 964.
1
4
4
.2.7. Antinociceptive activity
.2.7.1. Writhing test. The writhing test in mice was carried out
[
[14] Shankaran, K.L. Donnelly, S.K. Shah, J.L. Humes, S.G. Pacholok, S.K. Grant,
B.G. Green, M. MacCoss, Bioorg. Med. Chem. Lett. 7 (1997) 2887e2892.
using the method of Koster [43]. The writhes were induced by
intraperitoneal injection of 0.6% acetic acid (v/v) (80 mg/kg). The
standard drug i.e. indomethacin was given orally at a dose
[15] (a) Y. Bourne, H.C. Kolb, Z. Radi, K.B. Sharpless, P. Taylor, P. Marchot, Proc. Natl.
Acad. Sci. U S A 101 (2004) 1449e1454.
[16] (a) Y.S. Sanghvi, B.K. Bhattacharya, G.D. Kini, S.S. Matsumoto, S.B. Larson,
W.B. Jolley, R.K. Robins, G.R. Revankar, J. Med. Chem. 33 (1990) 336e344;
0
.05 mmol/kg of body weight. The test compounds were admin-
istered orally at an equimolar dosage to groups of six animals each,
0 min before chemical stimulus. The numbers of muscular con-
(b) H. Wamhoff, in: A.R. Katritzky, C.W. Rees (Eds.), Comprehensive Hetero-
cyclic Chemistry, vol. 5, Pergamon, Oxford, 1984, p. 669;
(c) M. Journet, D. Cai, J.J. Kowal, R.D. Larsen, Tetrahedron Lett. 42 (2001)
9117e9118.
3
tractions were counted over a period of 20 min after acetic acid
injection. The data represents the total number of writhes observed
during 20 min and is expressed as writhing numbers.
[17] (a) D.R. Buckle, C.J.M. Rockell, J. Chem. Soc. Perkin Trans. (1982) 627e630;
(
b) D.R. Buckle, D.J. Outred, C.J.M. Rockell, H. Smith, B.A. Spicer, J. Med. Chem.
26 (1983) 251e254;
c) D.R. Buckle, C.J.M. Rockell, H. Smith, B.A. Spicer, J. Med. Chem. 29 (1986)
262e2267;
d) R. Alvarez, S. Velázquez, A. San Felix, S. Aquaro, E.D. Clercq, C.F. Perno,
A. Karlsson, J. Balzarini, M.J. Camarasa, J. Med. Chem. 37 (1994) 4185e4194.
[18] M.D. Chen, S.J. Lu, G.P. Yuag, S.Y. Yang, X.L. Du, Heterocyclic Comm. 6 (2000)
21e426.
(
2
(
4.2.7.2. Tail immersion method. In the present study analgesia was
assessed by employing tail immersion method [44]. Prior to the
experiment the animals were screened for the sensitivity test by
4
immersing the tail of the rats gently in hot water maintained at
[19] E.A. Sherement, R.I. Tomanov, E.V. Trukhin, V.M. Berestovitskaya, Russ. J. Org.
ꢂ
5
5 C [45]. The animals flicking their tail from hot water in 5 s were
Chem. 40 (2004) 594e595.
selected for the study in order to avoid any thermal injury to the
[20] A. Allais, J. Meier, (Roussel-UCLAF) Ger. Offen., 1969, p. 1815467.

588
S. Haider et al. / European Journal of Medicinal Chemistry 70 (2013) 579e588
[
21] K.M. Banu, A. Dinakar, C. Ananthanarayanan, Indian J. Pharm. Sci. 4 (1999)
[36] Schrödinger, Maestro, Version 9.2, LLC, New York, NY, 2011.
[37] Schrödinger, ConfGen, Version 2.3, LLC, New York, NY, 2011.
[38] Schrödinger, LigPrep, Version 2.5, LLC, New York, NY, 2011.
[39] K.S. Watts, P. Dalal, R.B. Murphy, W. Sherman, R.A. Friesner, J.C. Shelley,
J.Chem. Inf. Model. 50 (2010) 534e546.
202e205.
[
[
22] R. Meier, EP. 199262; eidem, US. 4789680, 1986.
23] A. Passannanti, P. Diana, P. Barraja, F. Mingoia, A. Lauria, G. Cirrincione,
Heterocycles 48 (1998) 1229e1235.
[
[
[
[
[
[
[
[
[
[
[
24] M. Jilino, F.G. Stevens, J. Chem. Soc. Perkin Trans. 1 (1998) 1677e1684.
25] G.D. Diana, J.J. Nitz, EP. 566199, 1993.
[40] I. Chen, N. Foloppe, J.Chem. Inf. Model. 50 (2010) 822e839.
[41] M.M. He, A.S. Smith, J.D. Oslob, W.M. Flanagan, A.C. Braisted, A. Whitty,
M.T. Cancilla, J. Wang, A.A. Lugovskoy, J.C. Yoburn, A.D. Fung, G. Farrington,
J.K. Eldredge, E.S. Day, L.A. Cruz, T.G. Cachero, S.K. Miller, J.E. Friedman,
I.C. Choong, B.C. Cunningham, Science 310 (2005) 1022e1025.
[42] F. Malik, J. Singh, K. Anamika, A.S. Krishan, K.S. Naresh, S. Surjeet, K.K. Maharaj,
K. Arun, B. Aruna, G.N. Qazi, Life Sci. 80 (2007) 1525e1538.
[43] R. Koster, M. Anderson, E.J. Beer, Fed. Proc. 18 (1959) 412e416.
[44] L.C. Distasi Costa, M. Mendacolli, S.J.L. Kirzawa, M. Games, C.G. Trolin,
J. Ethnopharmacol. 24 (1988) 205.
26] R.J. Nachmann, J. Heterocyclic Chem. 19 (1982) 1545e1547.
27] A. Bali, R. Ohri, P.K. Deb, Eur. J. Med. Chem. 49 (2012) 397e405.
28] R. Deraedt, S. Joughney, F. Delevakee, M. Falhour, Eur. J. Pharmacol. 51 (1980) 17e24.
29] S.E. Besra, R.M. Sharma, A. Gomes, J. Ethnopharmacol. 54 (1996) 1e6.
30] F. Abbott, S.N. Young, Pharmacol. Biochem. Behav. 31 (1988) 855e860.
31] S. Furst, K. Gyires, J. Knoll, Drug Res. 4 (1988) 552e557.
32] C.A. Winter, E.A. Risley, G.W. Nuss, Proc. Soc. Exp. Biol. Med. 111 (1962) 544e547.
33] S.H. Ferreira, J. Pharm. Pharmacol. 31 (1979) 648.
34] R.A. Copeland, J.M. Williams, J. Giannaras, S. Nurnberg, M. Covington, D. Pinto,
S. Pick, J.M. Trzaskos, Proc. Natl. Acad. Sci. U S A 91 (23) (1994) 11202e11206.
35] (a) W.R. Pagels, R.J. Sachs, L.J. Marnett, D.L. Dewitt, J.S. Day, W.L. Smith,
J. Biol.Chem. 258 (1983) 6517e6523;
[45] A.K. Agrahari, M. Khaliquzzama, S.K. Panda, J. Curr. Pharm. Res. 1 (2010)
8e11.
[46] S. Haider, S. Nazreen, M.M. Alam, H. Hamid, M.S. Alam, J. Ethnopharmacol. 143
(2012) 236e240.
[
(
b) R.W. Egan, J. Paxton Jr., F.A. Kuehl, J. Biol. Chem. 251 (1976) 7329e7335.