Synthesis of novel 2-mercapto benzothiazole and 1,2,3-triazole based bis-heterocycles: Their anti-inflammatory and anti-nociceptive activities

Article abstract of DOI:10.1016/j.ejmech.2012.01.032

A focused library of novel bis-heterocycles encompassing 2-mercapto benzothiazole and 1,2,3-triazoles were synthesized using click chemistry approach. The synthesized compounds have been tested for their anti-inflammatory activity by using biochemical cyc

Full text of DOI:10.1016/j.ejmech.2012.01.032

European Journal of Medicinal Chemistry 49 (2012) 324e333
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of novel 2-mercapto benzothiazole and 1,2,3-triazole based
bis-heterocycles: Their anti-inflammatory and anti-nociceptive activities
,
a
b
c
Syed Shafi a , Mohammad Mahboob Alam , Naveen Mulakayala , Chaitanya Mulakayala ,
*
G. Vanaja d, Arunasree M. Kalle d, Reddanna Pallu d, M.S. Alam a
,
*
a Department of Chemistry, Faculty of Science, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India
b Institute of Life Science, University of Hyderabad Campus, Hyderabad 500046, India
c Department of Biochemistry, Sri Krishnadevaraya University, Anantapur 515003, India
d Department of Animal Sciences, University of Hyderabad, Hyderabad 500046, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A focused library of novel bis-heterocycles encompassing 2-mercapto benzothiazole and 1,2,3-triazoles
were synthesized using click chemistry approach. The synthesized compounds have been tested for
their anti-inflammatory activity by using biochemical cyclooxygenase (COX) activity assays and carra-
geenan-induced hind paw edema. Among the tested compounds, compound 4d demonstrated a potent
selective COX-2 inhibition with COX-2/COX-1 ratio of 0.44. Results from carrageenan-induced hind paw
edema showed that compounds 4a, 4d, 4e and 4f posses significant anti-inflammatory activity as
compared to the standard drug Ibuprofen. The compounds showing significant activity were further
subjected to anti-nociceptive activity by writhing test. These four compounds have shown comparable
activity with the standard Ibuprofen. Further ulcerogenic studies shows that none of these compounds
causing gastric ulceration.
Received 17 September 2011
Received in revised form
6 January 2012
Accepted 16 January 2012
Available online 24 January 2012
Keywords:
Benzothiazole
1,2,3-Triazole
Click chemistry
bis-Heterocycles
Anti-inflammatory activity
COX
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
synthesized with the help of Benzothiazole nucleus. These
compounds have special significance due to their remarkable
Benzothiazoles are bicyclic ring system with multiple applica-
tions. Benzothiazole ring is a part of several marine and terrestrial
natural compounds that are having useful biological activities [1].
Heterocycles containing thiazole fraction are present in many
natural products, such as bleomycin, epothilone B, lyngbyabellin
pharmacological potentialities.
Although thiazoles have been known from long time [4e6],
their varied biological features are still of great scientific interest.
Benzothiazoles shows broad spectrum of biological activities
including antitumor activity, especially phenyl-substituted benzo-
thiazoles [7e9] while condensed pyrimidobenzothiazoles and
benzothiazoloquinazolines exert antiviral activity [10]. Some of the
bis-substituted amidinobenzothiazoles have been found to be
potential anti-HIV agents [11] while some of the substituted 6-
nitro-and 6-aminobenzothiazoles [12] are reported to exhibit
potential antimicrobial activity.
and dolastatin 10 [2]. In the 1950s,
a
number of 2-
aminobenzothiazoles were intensively studied as central muscle
relaxants after the discovery of Riluzole [3]. Riluzole (6-
trifluoromethoxy-2-benzothiazolamine, PK-26124, RP-25279,
Rilutek) was found to interfere with glutamate neurotransmission
in biochemical, electrophysiological and behavioral experiments.
Since then benzothiazole derivatives have been studied extensively
and found to have diverse chemical reactivity and broad spectrum
of biological activity. A large number of therapeutic agents were
The studies of structureeactivity relationship of benzothiazoles
interestingly reveal that change of the structure of substituent
group at C-2 position commonly results the change of its bioactivity
[13]. The new class of 2-substituted aminobenzothiazoles has
shown a wide range of biological activities including antibacterial,
antitumor, anti-tubercular, carbonic anhydrase inhibitory activities
[14]. Significant anti-inflammatory activity is displayed by some
new 2-(40-butyl-30-50-dimethylpyrazol-1-yl)-6-substituted benzo-
* Corresponding authors. Tel.: þ91 9717927759/9891171278; fax: þ91 011
26059663.
E-mail addresses: shafirrl@gmail.com (S. Shafi), msalam@jamiahamdard.ac.in
(M.S. Alam).
thiazoles
and
4-butyl-1-(60-substituted-20-benzothiazolyl)-3-
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.01.032

S. Shafi et al. / European Journal of Medicinal Chemistry 49 (2012) 324e333
325
methylpyrazol-5-ones [15]. 2-Substituted 5- or 6-Benzo thiazole-
acetic acids and their derivatives have been demonstrated to be
nonsteroidal anti-inflammatory agents [16]. Some of the benzo-
thiazole derivatives have been reported to selectively inhibit
human Cyclooxygenase-2-enzyme (COX-2) [17].
reacted with propargylated 2-mercapto benzothiazole (2) in order
to obtain the novel bis-heterocycles with improved hydrophilicity
compared to the aromatic azide derived bis-heterocycles (Scheme
2). A focused library of fifteen compounds was synthesized
(Table 1) and all the synthesized bis-hetero cycles were screened
for their anti-inflammatory activity.
Similarly 1,2,3-triazoles and their derivatives have attracted
considerable attention for the past few decades due to their
chemotherapeutic value [18]. They have been considered as an
interesting component in terms of biological activity and found in
many drugs [19]. Many 1,2,3-triazoles are found to be potent
antimicrobial [20,21], analgesic [22], anti-inflammatory, local
anesthetic [23], anticonvulsant [24], anti-neoplastic [25], anti-
malarial [26] and antiviral agents [27]. Thus 1,2,3-triazoles have
emerged as powerful pharmacophores on their own right [28].
Several combinations of bis-heterocycles bearing benzothiazole
as a main constituent have been synthesized in order to obtain the
COX-2 selectivity. Some of them are selectively inhibiting COX-2
while most of them inhibiting both COX-1 and COX-2. But
majority of the NSAIDs so far developed to heal the inflammation
are causing adverse effects like gastric ulcer [29], kidney damage
[30] and some of the NSAIDs also cause hepatotoxicity [31]. Even
with selective coxibs that inhibit only COX-2 unexpected cardio-
vascular adverse effects were observed [32]. Thus there remains
a compelling need for effective NSAIDs with an improved safety
profile.
All the products were characterized by 1H NMR, 13C NMR, IR,
MALDI-MS/ESI-MS. In the 1H NMR spectra, the formation of tri-
azoles was confirmed by the resonance of H-C(5) of the triazole ring
in the aromatic region. The structure was further supported by the
13C NMR spectra, which showed the C-atom signals corresponding
to triazole derivatives. MALDI-MS/ESI-MS of all compounds showed
[M þ Na] or [M þ Na] or [M þ 1].
2.2. COX assays
All the above synthesized compounds were evaluated for their
anti-inflammatory activity by biochemical COX (COX-1 & COX-2)
inhibitory assay. The ratio of IC50 of COX-2 to IC50 of COX-1 (COX-
2/COX-1) would suggest the selectivity of the compound and hence
its gastric liability. The COX-2/COX-1 ratio of the above compounds
showed that compound 4d is a selective COX-2 inhibitor with
a ratio of 0.44 and 2-fold selective when compared to COX-1
(Table 2).
In view of the biological importance of benzothiazoles and 1,2,3-
triazoles as anti-inflammatory and anti-nociceptive agents, we aim
to conjugate these two important ligands under one construct
through a sulfur linkage. Hitherto for the first time, we report the
synthesis of benzothiazoles and 1,2,3-triazole based bis-heterocy-
cles andtheir anti-inflammatory and analgesic activities.
2.3. In vivo anti-inflammatory activity
Further the above synthesized compounds were tested for their
in vivo anti-inflammatory activity by carrageenan-induced hind
paw edema model. The results (Table 3 & Fig. 1) indicated that the
compounds 4a, 4d, 4e and 4f possessed good anti-inflammatory
activity. The anti-inflammatory activity profile of compound 4d
(77.83% inhibition at 3 h post-carrageenan and 81.13% inhibition at
5 h post-carrageenan) was better than that of standard NSAID,
Ibuprofen (69.50% inhibition at 3 h as well as 71.69% inhibition at
5 h) and showed a time-dependent increase in the inhibition of
inflammation.
Whereas compounds 4a, 4e and 4f showed the opposite profile
with a time-dependent decrease in the inhibition of inflammation
(83.30, 75.00, 75.00% inhibition at 3 h post-carrageenan and 78.11,
65.47, 59.24% inhibition at 5 h post-carrageenan respectively).
Compounds 4k and 4l showed moderate anti-inflammatory effect
comparable to Ibuprofen, at 3 h hour intervals (64.00 and 59.66%).
Compounds 4c and 4h showed weak anti-inflammatory activities
and compound 4g exerted no activity.
2. Results and discussion
2.1. Chemistry
From the in silico data it is revealed that the bis-heterocyclic
compounds encompassing 2-mercaptobenzthiazole and 1,2,3-
triazole moieties showing significant binding potential towards
COX enzyme. The designer molecules were synthesized and eval-
uated for their anti-inflammatory and analgesic activities. As
illustrated in Scheme 1 novel bis-heterocycles were synthesized by
the cycloaddition reaction of 2-(prop-2-ynylthio)benzo[d]thiazole,
2 with various azides. Towards the synthesis of compound 2, 2-
mercaptobenzothiazole was refluxed with propargyl bromide in
dry THF. Various substituted aromatic/sugar azides 3 were reacted
Further, the anti-inflammatory efficacy of the synthesized
compounds was analyzed based on the structureeactivity rela-
tionships considering the three structural components: the nature
of the group attached to 1,2,3-triazollyl ring, nature of the
substituents (functional group) and the position of substitutions on
aromatic ring.
with propargylated 2-mercaptobenzothiazole,
2 under click
chemistry reaction conditions to obtain the novel bis-heterocycles
4ae4m in quantitative yields. Aromatic azides with varying
substitutions including electron withdrawing and electron
donating groups were reacted with compound 2.
To improve the hydrophilicity of the resultant compounds;
galactosyl, glyceryl and pyridyl azides were synthesized and
First, the influence of the nature of the group attached to triazollyl
ring was easily observed as aryl substitution on triazollyl ring (4a, 4d,
R
N
N
R-N3
3
S
N
N
S
(a)
N
S
N
S
SH
S
(b)
4
1
2
(a)
(b) CuSO4.5H2O, Sodium ascorbate, tBuOH: H2O (1:1), r.t.
TEA, Dioxane, reflux
Br
Scheme 1. Synthetic approach for novel bis-heterocycles.

326
S. Shafi et al. / European Journal of Medicinal Chemistry 49 (2012) 324e333
R'
N
N
N
N3
O
R'
S
R': o-Cl, m-Cl, p-Cl, p-F, p-Br, p-NO2, m-NO2,
p-OEt, o-Me, H.
S
N3
N
(4a-4j)
O
OH
O
O
N
N
N
N
N
S
N
OH
OH
O
O
O
O
S
N
TFA
TFA
O
O
N
S
S
5
O
4l
N
S
HO
O
S
N
N
O
N3
N
O
N
S
2
OH
OH
S
N
N
S
N
S
N
O
4m
N
N
6
N3
N
S
N
N
S
N
4k
Scheme 2. Click chemistry approach for novel bis-heterocycles.
4e and 4f) showed superior activity over the galactosyl, glyceryl and
pyridyl substitutions. Regarding the substitution on the aromatic
ring, electronwithdrawing groups conferred the greater activity over
the electron donating groups. The derivatives with the electron
withdrawing groups i.e. eCl (4a), eF (4d), eBr (4e), eNO2 (4f)
substitutions showed significantly higher activities compared to
eCH3, eOEt substitutions. Compounds with fluoro and chloro
substitutionsconfersignificantlyhigheractivityat3haswellasat5h.
Regarding the position of the substituent on the aromatic ring,
halogens substituted at para position has mostly shown significant
activity. When chlorine was ortho substituted, it exerts significant
activity compared to the para substitution. When eNO2 group was
substituted at para position, it conferred greater activity than it was
substituted at meta position (significantly lost the activity).
patterns. Furthermore, the lower score acquired for PROSA e7.68
indicating the high quality of the model.
2.4.2. Docking studies on human COX-2
Docking analysis was performed on novel benzothiazole deriv-
atives to identify key amino acid residues involved in making
interactions with the human COX-2 model structure. All inhibitors
were docked successfully in the human COX-2 homology model,
showing good docking scores. The docking program AutoDock
computes binding energy, RMSD and inhibition constant (Ki) with
respect to the docked inhibitors. The AutoDock computed binding
energy; RMSD and Ki values along with H-bond interacting residues
are presented in Table 4.
The binding 3D conformations of COX-2 with bis-heterocycles
are displayed in Fig. 2. It was observed that the interaction of bis-
heterocycles with COX-2 occurred in a distinct site, formed by
Tyr334, Val335, Leu338, Leu370, Ser448, Met508, Gly512, and Ser516. All
these residues are functionally important and are found to be
conserved. Among all the benzothiazole derivatives 4e has given the
best predicted binding free energy (ꢀ12.54 kcal/mol) onto human
COX-2. The docked pose of the most potent molecule, 4e obtained
using AutoDock4 software is represented in Fig. 2. 4e showed strong
hydrogen bonding interactions, hydrophobic interactions and van
2.4. Molecular docking studies
2.4.1. In silico modeling of human COX-2
In order to further demonstrate that the anti-inflammatory
efficacy of the compounds synthesized is attributed to the inhibi-
tion of COX-2, in silico docking studies have been carried out.
Considering the limited sequence identity between human Cox-2
and the templates used for its modeling, an objective validation
gives results suggestive of reliable models. Human COX-2 model
construction starts with PSI-BLAST analysis, which was used to
select best template. Human COX-2 sequence was directlycompared
to amino acid residue sequences that possess structures experi-
mentally resolved and deposited in the Protein Data Bank [33].
Human COX-2 presented 87% of identity with 1PXX, being chosen as
a template. Alignments among sequences and structures were
carried by using Clustal-W [34]. After alignment analysis, atomic
coordinated were transferred to human COX-2 primary structure for
the construction of 3D model using Modeller 9v.6 (Fig. 2).
Procheck summary of human COX-2 showed that 100% amino
acid residues were in most favorable regions, additionally allowed
regions and generously allowed regions with 0.0% residues in the
disallowed regions. Structural differences between crystal structure
1PXX and predicted three dimensional structure of human COX-2
were calculated by superimposing both structures. The RMSD
values between the crystal structure 1PXX and homology model
der Waals interactions with Tyr334, Val335, Leu338, Leu370, Gly422
Ser448, Phe449, Asn450, Glu451, Glu465, Glu470, Glu478, Met508, Val509
,
,
Glu510 Gly512, Ala513, Pro514, and Ser516. In summary, the benzo-
thiazole derivatives studied are potent inhibitors targeting human
COX-2. We observed that the homology model of human COX-2
assisted molecular docking analysis of benzothiazole derivatives.
2.5. Analgesic activity
The results of analgesic activity are summarized in Fig. 3. Among
tested compounds, compounds 4d and 4e are showing better
activity compared to standard drug Ibuprofen, while compounds 4a
and 4f are showing comparable analgesic activity with reference to
standard drug. All data’s were analyzed by one-way ANOVA test
followed by Dunnet’s test.
2.6. Ulcerogenic studies
human COX-2 calculated for C
a traces and main chain atom were
1.25 Å. The RMSD values and small variability among experimental
structures template and the structure modeled reflect the presence
of strong restraints in most regions and emphasize a similar folding
The compounds showing potential anti-inflammatory activity
were further carried out for ulcerogenic studies. When compared
with Ibuprofen, compounds 4a, 4d, 4e and 4f did not cause any

S. Shafi et al. / European Journal of Medicinal Chemistry 49 (2012) 324e333
327
Table 1
Novel bis-heterocycles.
Sr. No.
a
Azide (3)
bis-Heterocycles (4)
Reaction time (h)
8
Yieldsa (%)
N
N3
S
N
95
93
89
S
Cl
N
Cl
N
N
S
N3
S
S
S
b
c
7
7
Cl
N
N
Cl
Cl
N
Cl
N
S
N3
N
N
N
N
N
N
F
N
S
d
e
8
6
95
97
F
N3
N
N
Br
N
S
Br
N3
S
S
N
N
NO2
N
S
f
8
93
O2N
O2N
N3
N
N
N
S
N3
S
S
g
h
i
7
8
7
92
88
94
NO2
N
N
N
N
Me
N
S
N
N
N3
Me
OEt
N
S
EtO
N3
S
N
N
N
N
S
S
S
j
8
8
92
95
N3
N
N
N
N
N
S
N
N
k
N3
N
N
N
S
O
O
O
O
N3
S
N
N
l
9
9
90
87
O
O
O
O
N
O
O
O
N
S
O
N3
S
m
N
N
O
O
N
a
Isolated yields.

328
S. Shafi et al. / European Journal of Medicinal Chemistry 49 (2012) 324e333
Table 2
4. Experimental
COX-2/COX-1 ratio of the benzothiazole derivatives.
4.1. Chemistry
Sr. No.
Compound
COX-2/COX-1
1
4a
0
2
3
4
5
6
7
8
9
4c
4d
4e
4f
4g
4k
4l
4m
14.90136
0.440456
0
0
5.212629
0
1.532059
0
7.164179
0.0028
All commercial chemicals used as starting materials and reagents
in this study were purchased from Merck (India), Spectrochem, and
Sigma Aldrich which were of reagent grade. All melting points were
uncorrected and measured using Electro-thermal IA 9100 apparatus
(Shimadzu, Japan); IR spectra were recorded as potassium bromide
pellets on a Perkin Elmer 1650 spectrophotometer (USA), 1H NMR
spectra were determined on a Bruker (200, 300 and 400 MHz)
spectrometer and chemical shifts were expressed as ppm against
TMS as internal reference. Mass spectra were recorded on MALDI-
AB4800 and 70 eV (EI Ms-QP 1000EX, Shimadzu, Japan), 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 with spectral data.
10
11
Indo methacin
Celecoxib
gastric ulceration and disruption of gastric epithelial cells at the
below mentioned oral doses. Stomach wall of Ibuprofen treated
group at low power (10ꢁ) photomicrograph showed damage of the
mucosa and the sub mucosa. Stomach wall of the same section at
high power (40ꢁ) photomicrograph showing desquamated
epithelial cells in the lumen whereas in tested compounds (4a, 4d,
4e and 4f) treated animals, no surface epithelial damage and
submucosal damage was seen. Stomach wall of 4a, 4d, 4e and 4f
treated animals showed no damage of any layer. The results are
shown in Table 5 and Fig. 4.
t
Compound 2 was dissolved in 20 mL of Butanol:water (1:1)
solvent at ambient temperature. Then was charged CuSO4$5H2O
and the reaction mixture was stirred for 5 min. Reaction mixture
was light blue in colour. Then sodium ascorbate was added at once
to the reaction mixture and allowed to stir for 15 min. Reaction
mixture colour was changed to dark yellow. After 15 min azide was
added at once. The reaction mixture was allowed to stir for further
8 h at ambient temperature. After the completion of the reaction,
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.
3. Conclusion
In the present study
mercaptobenzothiazole derived bis-heterocycles encompassing
benzothiazole-1,2,3-triazole moieties conjugated through
a
focussed library of 2-
4.1.1. 2-((1-(2-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methylthio)
benzo[d]thiazole (4a)
a sulphur linkage are synthesized and evaluated for their anti-
inflammatory actity. From the structureeactivity relationship
aromatic ring attached to triazollyl moiety are showing poten-
tial activity when compared to aliphatic/alicyclic rings. Electron
withdrawing groups when substituted on aromatic ring mostly
at the para position are exhibiting potential anti-inflammatory
activity (compounds 4a, 4d, 4e and 4f) compared to the stan-
dard drug ibuprofen without causing any ulceration. The COX
inhibitory potential of compound 4d and the ulcerogenic studies
further concludes that these kinds of molecules can be
considered as potent anti-inflammatory agents. Further chemical
and biological studies are under way.
M.P. 104e105 ꢂC; IR (KBr) (cmꢀ1): 3074, 2920, 2851, 2372, 1496,
1461, 1381, 1231, 1075, 1039, 994, 753, 719; 1H NMR (CDCl3,
400 MHz)
d
(ppm): 4.79 (s, 2H), 7.30 (t, 1H, J ¼ 7.20 Hz), 7.39e7.44
(m, 3H), 7.52e7.54 (m, 1H), 7.59e7.61 (m, 1H), 7.76 (d, 1H,
J ¼ 8.00 Hz), 7.89 (d, 1H, J ¼ 8.00 Hz), 8.09 (s, 1H); 13C NMR (CDCl3,
75 MHz):
d 27.68, 121.10, 121.50, 124.41, 125.13, 126.07, 127.68,
127.87, 128.40, 130.67, 130.72, 134.77, 135.50, 143.74, 152.99, 165.58;
MALDI-MS: 359 (Mþþ1), 381 (MþþNa); Anal. Calcd.for
C16H11ClN4S2: C, 53.55; H, 3.09; Cl, 9.88; N, 15.61; S, 17.87%, Found:
C, 53.51; H, 3.13; Cl, 9.82; N, 15.67; S, 17.82%.
Table 3
In vivo anti-inflammatory activity of novel bis-heterocycles.
Sr. No.
Compound
Change in paw edema volume (ml) after drug
Antiinflamatory activity %
treatment
inhibition
3 h
5 h
3 h
5 h
1
2
3
4
5
6
7
8
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
5
4m
6
0.10 0.051***
0.39 ꢃ 0.048
0.11 0.016***
0.32 ꢃ 0.042
0.23 ꢃ 0.055*
0.10 0.044***
0.21 0.030*
0.18 0.040*
0.41 ꢃ 0.107ns
0.30 ꢃ 0.051ns
0.35 ꢃ 0.05ns
0.36 ꢃ 0.045ns
0.23 ꢃ 0.055*
0.23 ꢃ 0.098ns
0.21 ꢃ 0.046ns
0.27 ꢃ 0.042ns
0.25 ꢃ 0.046ns
0.15 0.056**
0.53 ꢃ 0.071
83.30
35.00
50.00
77.83
75.00
75.00
11.66
47.30
38.33
46.66
64.00
59.66
63.33
53.33
56.66
69.50
e
78.11
39.60
43.39
81.13
59.24
65.47
21.50
43.39
33.96
32.07
56.03
56.60
60.37
49.05
52.83
71.69
e
0.30 ꢃ 0.051ns
0.13 0.033**
0.15 0.042**
0.15 0.042**
0.53 ꢃ 0.180ns
0.31 ꢃ 0.079ns
0.37 ꢃ 0.045ns
0.32 ꢃ 0.045ns
9
10
11
12
13
14
15
16
17
*
0.21 ꢃ 0.030
0.24 ꢃ 0.060ns
0.22 ꢃ 0.051ns
0.28 ꢃ 0.042ns
0.26 ꢃ 0.042ns
0.18 0.054**
0.60 ꢃ 0.089
Ibuprofen
Control
Data is analyzed by one way ANOVA followed by Dunnett’s ‘t’ test and expressed as mean ꢃ SEM from six observations; *** indicates P < 0.001, ** indicates P < 0.01
& * indicates P < 0.05.

S. Shafi et al. / European Journal of Medicinal Chemistry 49 (2012) 324e333
329
Fig. 1. In vivo anti-inflammatory activity of novel bis-heterocycles.
4.1.2. 2-((1-(3-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methylthio)
4.1.3. 2-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methylthio)
benzo[d]thiazole (4b)
benzo[d]thiazole (4c)
M.P. 102e104 ꢂC; IR (KBr) (cmꢀ1): 3114, 3078, 1592, 1455, 1426,
M.P. 150e152 ꢂC; IR (KBr) (cmꢀ1): 3136, 3062, 3041, 2441, 1501,
1252,1048, 790, 751, 680; 1H NMR (CDCl3, 400 MHz)
d
(ppm): 4.70 (s,
1462, 1427, 1229, 1094, 1050, 999, 831, 746, 722; 1H NMR (CDCl3,
2H), 7.25 (t, 1H, J ¼ 8.00 Hz), 7.31e7.39 (m, 3H), 7.50e7.52 (dd, 1H,
500 MHz)
d
(ppm): 4.77 (s, 2H), 7.32 (t,1H, J ¼ 8.00 Hz), 7.43e7.48 (m,
J ¼ 7.20 and 1.6 Hz), 7.66 (s, 1H), 7.70 (d, 1H, J ¼ 8.00 Hz), 7.85 (d, 1H,
3H), 7.62e7.64 (dd, 2H, J ¼ 7.0 and 1.5 Hz), 7.70 (d, 1H, J ¼ 8.00 Hz),
J ¼ 8.00 Hz), 7.99 (s, 1H); 13C NMR (CDCl3, 75 MHz):
d
27.63, 121.06,
7.91 (d,1H, J ¼ 8.5 Hz), 8.05 (s,1H); C13 NMR (CDCl3, 75 MHz):
d 27.57,
121.44, 124.37, 125.10, 126.03, 127.61, 127.83, 128.33, 130.66, 134.69,
135.44, 143.66, 152.92, 165.52; MALDI-MS: 359 (Mþþ1), 381
(MþþNa); Anal. Calcd.for C16H11ClN4S2: C, 53.55; H, 3.09; Cl, 9.88; N,
15.61; S,17.87%, Found: C, 53.49; H, 3.16; Cl, 9.79; N, 15.57; S, 17.92%.
121.20, 121.48, 121.73, 124.51, 126.18, 129.91, 134.60, 135.42, 135.51,
152.98, 165.69; MALDI-MS: 359 (Mþþ1), 381 (MþþNa), 397
(MþþK); Anal. Calcd.for C16H11ClN4S2: C, 53.55; H, 3.09; Cl, 9.88; N,
15.61; S,17.87%, Found: C, 53.47; H, 3.01; Cl,10.91; N,15.66; S,17.93%.
Fig. 2. Docking results of bis-heterocycles onto human COX-2.

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S. Shafi et al. / European Journal of Medicinal Chemistry 49 (2012) 324e333
(MþþK); Anal. Calcd.for C16H11N5O2S2: C, 52.02; H, 3.00; N, 18.96; S,
Table 4
Docking results of bis-heterocycles onto human COX-2.
17.36, Found: C, 52.11; H, 3.05; N, 18.92; S, 17.42%.
Compound
Binding energy
(Kcal/mol)
Inhibition constant
(Ki) (mm)
RMSD
(Å)
4.1.7. 2-((1-(3-Nitrophenyl)-1H-1,2,3-triazol-4-yl)methylthio)
benzo[d]thiazole (4g)
4a
4c
4d
4e
4f
4g
4k
4l
ꢀ11.86
ꢀ10.93
ꢀ11.52
ꢀ12.54
ꢀ10.65
ꢀ11.23
ꢀ11.09
ꢀ12.38
ꢀ12.25
2.56
5.56
1.25
0.36
5.12
6.96
1.99
0.97
1.06
1.65
1.12
1.09
0.56
1.59
1.38
1.16
0.69
0.93
M.P. 153e155 ꢂC; IR (KBr) (cmꢀ1): 3121, 3030, 2438, 1534, 1454,
1426, 1343, 1002, 758; 1H NMR (CDCl3, 300 MHz)
d (ppm): 4.79 (s,
2H), 7.32 (t, 1H, J ¼ 7.20 Hz), 7.45 (t, 1H, J ¼ 7.20 Hz), 7.69e7.78 (m,
2H), 7.92 (d, 1H, J ¼ 7.80 Hz), 8.12 (d, 1H, J ¼ 8.10 Hz), 8.20 (s, 1H),
8.28 (d, 1H, J ¼ 8.10 Hz), 8.55 (d, 1H, J ¼ 1.8 Hz); 13C NMR (CDCl3,
100 MHz):
d 27.42, 115.36, 121.11, 121.22, 121.53, 123.24, 124.59,
4m
126.00,126.25,130.95,135.55,137.67,145.87, 148.93, 152.96,165.46;
MALDI-MS: 369 (Mþ), 370 (Mþþ1), 392 (MþþNa); Anal. Calcd.for
C16H11N5O2S2: C, 52.02; H, 3.00; N, 18.96; S, 17.36, Found: C, 52.08;
H, 3.03; N, 19.03; S, 17.33%.
4.1.4. 2e((1-(4-Fluorophenyl)-1H-1,2,3-triazol-4-yl)methylthio)
benzo[d]thiazole (4d)
M.P. 126e128 ꢂC; IR (KBr) (cmꢀ1): 3135, 3074, 2928, 1779, 1514,
1463, 1427, 1231, 1048, 998, 834, 747; 1H NMR (CDCl3, 500 MHz)
4.1.8. 2-((1-(2-Methylphenyl)-1H-1,2,3-triazol-4-yl)methylthio)
benzo[d]thiazole (4h)
d
(ppm): 4.77 (s, 2H), 7.19 (t, 2H, J ¼ 8.5 Hz), 7.32 (t,1H, J ¼ 7.5 Hz), 7.44
M.P. 103e105 ꢂC; IR (KBr) (cmꢀ1): 3244, 3115, 2920, 2517, 1544,
(t, 1H, J ¼ 7.5 Hz), 7.64e7.67 (m, 2H), 7.77 (d, 1H, J ¼ 8.0 Hz), 7.91 (d,
1H, J ¼ 8.5 Hz), 8.02 (s,1H); 13C NMR (CDCl3, 75 MHz):
d 27.45,116.41,
1455, 1426, 1324, 1263, 1048, 760; 1H NMR (CDCl3, 300 MHz)
116.72, 121.08, 121.28, 121.35, 122.37, 122.48, 124.38, 126.06, 133.37,
135.37, 144.80, 152.83, 163.94, 165.60; MALDI-MS: 365 (MþþNa),
381 (MþþK); Anal. Calcd.for C16H11FN4S2: C, 56.12; H, 3.24; F: 5.55;
N, 16.36; S, 18.73%, Found: C, 56.08; H, 3.19; N, 16.27; S, 18.65%.
d
(ppm): 2.13 (s, 3H), 4.78 (s, 2H), 7.26e7.46 (m, 6H), 7.76 (d, 1H,
J ¼ 8.10 Hz), 7.81 (s, 1H), 7.88 (d, 1H, J ¼ 8.10 Hz); ESI-MS: 339
(Mþþ1), 361 (MþþNa); Anal. Calcd. for C17H14N4S2: C, 60.33; H,
4.17; N, 16.55; S, 18.95%, Found: C, 60.36; H, 4.25; N,16.52; S, 18.97%.
4.1.5. 2-((1-(4-Bromophenyl)-1H-1,2,3-triazol-4-yl)methylthio)
benzo[d]thiazole (4e)
4.1.9. 2-((1-(4-Ethoxyphenyl)-1H-1,2,3-triazol-4-yl)methylthio)
benzo[d]thiazole (4i)
M.P. 148e150 ꢂC; IR (KBr) (cmꢀ1): 3126, 3058, 2394, 1496, 1458,
M.P. 110e112 ꢂC; IR (KBr) (cmꢀ1): 3107, 3030, 2922, 2382, 1515,
1426, 1050, 999, 821, 746, 722; 1H NMR (CDCl3, 200 MHz)
d (ppm):
1457, 1426, 1305, 1248, 1051, 996, 825, 752; 1H NMR (CDCl3,
4.77 (s, 2H), 7.32 (t, 1H, J ¼ 7.42 Hz), 7.45 (t, 1H, J ¼ 7.13 Hz),
400 MHz)
d
(ppm): 1.43 (t, 3H, J ¼ 7.20 Hz), 4.06 (q, 2H, J ¼ 7.20 Hz),
7.55e7.61 (m, 4H), 7.77 (d, 1H, J ¼ 7.44 Hz), 7.92 (d, 1H, J ¼ 7.85 Hz),
4.77 (s, 2H), 6.97 (dd, 2H, J ¼ 7.20 & 2.00 Hz), 7.31 (t,1H, J ¼ 8.00 Hz),
7.43 (t, 1H, J ¼ 7.60 Hz), 7.55 (dd, 2H, J ¼ 7.20 and 2.00 Hz), 7.76 (d,
1H, J ¼ 7.60 Hz), 7.91 (d, 1H, J ¼ 8.00 Hz), 7.97 (s, 1H); 13C NMR
8.06 (s, 1H); 13C NMR (CDCl3, 75 MHz):
d 27.56, 121.20, 121.49,
121.96,122.48,124.52,126.19,132.88,135.54,135.90,152.98, 165.69;
MALDI-MS: 403 (Mþ), 405 (Mþþ2); Anal. Calcd.for C16H11BrN4S2: C,
47.65; H, 2.75; Br, 19.81, N, 13.89; S, 15.90%, Found: C, 47.57; H, 2.69;
Br, 19.89, N, 13.91; S, 16.02%.
(CDCl3, 75 MHz): d 14.68, 27.72, 63.88, 115.20, 121.13, 121.26, 121.47,
122.19,124.40,126.09,130.22,135.50,144.42,153.01,159.22,165.83;
MALDI-MS: 369 (Mþþ1), 391 (MþþNa); Anal. Calcd.for
C18H16N4OS2: C, 58.67; H, 4.38; N, 15.21; S, 17.40%, Found: C, 58.62;
H, 4.45; N, 15.29; S, 17.52%.
4.1.6. 2-((1-(4-Nitrophenyl)-1H-1,2,3-triazol-4-yl)methylthio)
benzo[d]thiazole (4f)
M.P. 178e180 ꢂC; IR (KBr) (cmꢀ1): 3106, 3030, 2381, 1596, 1523,
4.1.10. 2-((1-Phenyl-1H-1,2,3-triazol-4-yl)methylthio)benzo[d]
thiazole (4j)
1474, 1407, 1333, 1009, 1050, 852; 1H NMR (CDCl3, 200 MHz)
M.P. 98e100 ꢂC; IR (KBr) (cmꢀ1): 3114, 3067, 1592, 1499, 1419,
d
(ppm): 4.79 (s, 2H), 7.33 (t,1H, J ¼ 7.86 Hz), 7.46 (t,1H, J ¼ 8.48 Hz),
1378, 1238, 1046, 996, 754, 684; 1H NMR (CDCl3, 400 MHz)
d (ppm):
7.78 (d, 1H, J ¼ 7.16 Hz), 7.90e7.94 (m, 3H), 8.20 (s, 1H), 8.35e8.41
(dd, 2H, J ¼ 9.13 and 2.0 Hz); 13C NMR (CDCl3, 75 MHz):
d 27.28,
4.77 (s, 2H), 7.31 (t, 1H, J ¼ 7.80 Hz), 7.38e7.51 (m, 4H), 7.67 (d, 2H,
120.48, 121.05, 121.22, 121.41, 124.58, 125.48, 126.22, 135.49, 141.00,
J ¼ 7.20 Hz), 7.76 (d, 1H, J ¼ 7.80 Hz), 7.9 (d, 1H, J ¼ 8.10 Hz), 8.06 (s,
145.97, 147.16, 152.85, 165.44; MALDI-MS: 392 (MþþNa), 408
1H); 13C NMR (CDCl3, 75 MHz):
d 27.68, 120.60, 121.17, 121.50,
Fig. 3. Analgesic activity of bis-heterocycles by writhing test.

S. Shafi et al. / European Journal of Medicinal Chemistry 49 (2012) 324e333
331
Table 5
J ¼ 7.20 Hz), 4.27e4.29 (m, 1H), 4.37e4.44 (m, 1H), 4.55e4.60 (m,
2H), 4.69 (s, 2H), 5.43 (d, 1H, J ¼ 8.0 Hz), 7.30 (t, 1H, J ¼ 7.8 Hz), 7.42
(t, 1H, J ¼ 7.5 Hz), 7.75 (d, 1H, J ¼ 8.1 Hz), 7.81 (s, 1H), 7.90 (d, 1H,
Histopathology report of ulcerogenic activity.
Group
Surface epith. Sub. mucosal Deep mucosal Muscular layer
J ¼ 8.1 Hz); 13C NMR (CDCl3, 75 MHz):
d 24.24, 24.76, 25.75, 25.89,
damage
damage
damage
damage
4a
4d
4e
4f
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
27.81, 50.49, 67.09, 70.23, 70.66, 71.04, 96.13, 108.96, 109.79, 121.01,
121.53, 124.09, 124.25, 125.96, 135.45, 143.47, 153.06, 165.86;
MALDI-MS: 491 (Mþþ1), 513 (MþþNa), 529 (MþþK); Anal. Calcd.
For C22H26N4O5S2, 53.86; H, 5.34; N, 11.42, S, 13.07%, Found: C,
53.77; H, 5.38; N, 11.39; S, 13.12%.
e
e
e
Control
Ibuprofen þþþ
e
þþþ
e, No damage; þþþ, indicates high degree of damage.
4.1.13. 2-(1-((2,2-Dimethyl-1,3-dioxalan-4yl)-1H-1,2,3-triazol-4yl-)
methylthio)benzo[d]thiazole (4m)
Semisolid; IR (CHCl3) (cmꢀ1): 3090, 2935, 2363, 1426, 1233,
124.46, 126.15, 128.82, 129.73, 135.54, 136.96, 144.80, 153.03,
165.78; ESI-MS: 325 (Mþþ1), 347 (MþþNa), 363 (MþþK); Anal.
Calcd.for C16H12N5S2: C, 59.23; H, 3.73; N, 17.27; S, 19.77%, Found: C,
59.29; H, 3.66; N, 17.31; S, 19.82%.
1098, 988, 931, 793, 758; 1H NMR (CDCl3, 300 MHz)
d (ppm): 1.31
(s, 3H), 1.40 (s, 3H), 3.56e3.61 (m, 1H), 3.92e4.03 (m, 1H),
4.25e4.35 (m, 2H), 4.39e4.47 (m, 1H), 4.61 (d, 2H, J ¼ 3.0 Hz),
7.22 (t, 1H, J ¼ 7.8 Hz), 7.35 (t, 1H, J ¼ 8.1 Hz), 7.66e7.74 (m, 2H),
7.81 (d, 1H, J ¼ 8.1 Hz); 13C NMR (CDCl3, 75 MHz):
d 23.80,
4.1.11. 2-((1-(Pyridine-3yl)-1H-1,2,3-triazol-4-yl)methylthio)benzo
[d]thiazole (4k)
24.88, 27.60, 52.17, 65.86, 73.50, 110.70, 121.04, 121.40, 124.32,
126.01, 135.42, 143.87, 152.95, 165.81; MALDI-MS: 463
(Mþþ1), 385 (MþþNa); Anal. Calcd.for C16H18N4O2S2: C, 53.02; H,
5.01; N, 15.46; S, 17.69%, Found: C, 53.07; H, 4.95; N, 15.41; S,
17.65%.
M.P. 136e138 ꢂC; IR (KBr) (cmꢀ1): 3131, 3058, 2920, 2437, 1584,
1498, 1419, 1233, 1009, 806, 755, 703;1H NMR (CDCl3, 400 MHz)
d
(ppm): 4.71 (s, 2H), 7.19e7.2 (m, 3H), 7.35e7.48 (m, 2H), 7.69 (d,1H,
J ¼ 7.60 Hz), 7.84 (d,1H, J ¼ 7.60 Hz), 8.03 (d,1H, J ¼ 8.00 Hz), 8.07 (s,
1H); 13C NMR (CDCl3, 75 MHz):
27.46,121.21,121.49,124.55,126.22,
d
4.1.14. 3-(4-((Benzo[d]thiazol-2-ylthio)methyl)-1H-1,2,3-triazol-1-yl)
propane-1,2-diol (6)
128.08, 135.53, 145.51, 150.26, 152.94, 165.55; MALDI-MS: 348
(MþþNa), 364 (MþþK); Anal. Calcd.for C15H11N5S2: C, 55.36; H, 3.41;
N, 21.52; S, 19.71%, Found: C, 55.29; H, 3.46; N, 21.61; S, 19.77%.
(Gly-OH): semisolid; IR (CHCl3) (cmꢀ1): 3303, 3298, 3039, 2913,
1396, 1266, 1109, 972, 922, 793, 758; 1H NMR (CDCl3, 300 MHz)
d
(ppm): 3.33e3.41 (m, 2H), 3.77e3.80 (m, 1H), 4.27e4.33 (m, 1H),
4.1.12. Table entry (4l)
M.P. 123e125 ꢂC; IR (KBr) (cmꢀ1): 2978, 2926, 2455, 1455, 1425,
4.36e4.41 (m, 1H), 4.75 (s, 2H), 7.34 (t, 1H, J ¼ 6.0 Hz), 7.46 (t, 1H,
J ¼ 6.0 Hz), 7.78 (d, 1H, J ¼ 6.0 Hz), 7.83 (s, 1H), 7.93 (d, 1H,
J ¼ 6.0 Hz); MALDI-MS: 323 (Mþþ1), 345 (MþþNa)Anal. Calcd.for
1372,1258,1079,1005, 801, 755;1H NMR (CDCl3, 300 MHz)
1.23 (s, 3H), 1.28 (s, 3H), 1.30 (s, 3H), 1.45 (s, 3H), 4.12 (d, 2H,
d
(ppm):
Fig. 4. Histopathology report of ulcerogenic activity. As illustrated in above figure, the high and low power micrograph of the Specimen. 4e, 4d, 4a, 4f, Control Stom 10ꢁ. Low
power photomicrograph of stomach wall from corresponding 4e, 4d, 4a, 4f, Control, group animal showing intact mucosal layer of the stomach. (HE x 100). 4e, 4d, 4a, 4f, Control
Stom 40ꢁ. High power photomicrograph of stomach wall from 4e, 4d, 4a, 4f, Control group animal showing intact surface lining epithelium and superficial glands in the stomach
mucosa. (HE ꢁ 400). The stomach wall from Ibuprofen group animal at 10ꢁ showing superficial ulceration of the mucosa. Desquamated epithelial cells are seen in the lumen and at
40ꢁ showing the desquamated epithelial cells in the lumen. The control group animal showing intact mucosal layer of the stomach and at 40ꢁ showing intact surface lining
epithelium and superficial glands in the stomach mucosa.

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S. Shafi et al. / European Journal of Medicinal Chemistry 49 (2012) 324e333
C13H14N4O2S2: C, 48.43; H, 4.38; N, 17.38; S, 19.89%, Found: C, 48.51;
H, 4.35; N, 17.41; S, 19.96%.
4.2.2.4. Statistical analysis. Results are expressed as the
mean ꢃ SEM, and different groups were compared using one way
analysis of variance (ANOVA) followed by Tukey Kramer test for
multiple comparisons.
4.1.15. (2S,3S,4S,5R)-6-((4-(Benzo[d]thiazol-2-ylthio)methyl)-1H-
1,2,3-triazol-1yl)methyl)-tetrahydro-2H-pyran-2,3,4,5-tetraol (5)
semisolid; IR (KBr) (cmꢀ1): 2978, 2926, 2455, 1455, 1425, 1372,
4.2.3. In silico inhibitor and enzyme interactions
1258, 1079, 1005, 801, 755; 1H NMR (CDCl3, 300 MHz)
d
(ppm): 4.12
4.2.3.1. Molecular modeling. Primary sequence of human Cyclo-
oxygenase (COX-2) was obtained from NCBI gene bank with
accession number (gi: 181254). PSI-BLAST [39] was used for
template data mining. Mouse Diclofenac bound COX-2 PDB: 1PXX
[40], which shows 87% identity, was chosen as template. The COX-2
three-dimensional model was constructed by using crystal atomic
coordinates of free 1Pxx at a resolution of 2.9Å. Fifty models were
constructed by using Modeller 9v.6 [41] where protein tertiary
structure models were chosen for their fulfillment of spatial
restraints, taking into account loops energy minimization con-
ducted by default parameters [42]. Predicted human COX-2 model
evaluation, i.e geometry, stereochemistry, and energy distributions
in the models was performed using PROSA to analyze packing and
solvent exposure characteristics and PROCHECK for additional
analysis of stereochemical quality [43,44]. In addition, RMSD was
(d, 2H, J ¼ 7.20 Hz), 4.27e4.29 (m,1H), 4.37e4.44 (m,1H), 4.55e4.60
(m, 2H), 4.74 (s, 2H), 5.38 (d, 1H, J ¼ 8.1 Hz), 7.33 (t, 1H, J ¼ 7.8 Hz),
7.42 (t,1H, J ¼ 7.8 Hz), 7.74 (d, 1H, J ¼ 8.1 Hz), 7.83 (s,1H), 7.89 (d,1H,
J ¼ 8.1 Hz); MALDI-MS: 411 (Mþþ1), 433 (MþþNa), 449 (MþþK);
Anal. Calcd.for C16H18N4O5S2: C, 46.82; H, 4.42; N, 13.65; S, 15.62%,
Found: C, 46.77; H, 4.37; N, 13.61; S, 15.69%.
4.2. Pharmacology
4.2.1. Assays for cyclooxygenase 1 and 2
COX-1 has been isolated from Ram seminal vesicles. Recombi-
nant human COX-2 has been expressed in insect cell expression
system. These enzymes have been purified by employing conven-
tional chromatographic techniques. Enzymatic activities of COX-1
and COX-2 were measured according to the method of Copeland
et al. (1994) [35], 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 (Egan et al.,
Pagels et al., 1983) [36]. Briefly, the assay mixture contained
calculated by overlap of C
a traces and backbones onto the template
crystal structure through SPDB viewer [45]. The protein structures
were visualized and analyzed on Delano Scientific’s PYMOL http://
pymol.sourceforge.net/.
4.2.3.2. Molecular docking. AutoDock 4.0 program [46] was used for
docking calculations combined with Lamarckian genetic algorithm
(LGA) to search for the preferable conformation. The dimensions of
the box were set to 60 ꢁ 60 ꢁ 60 with grid spacing of 0.375 Å. During
the docking experiments, the receptor was kept rigid and ligand was
set flexible. The maximum number of energy evaluations was set to
2.5 ꢁ 107, and the rest of docking parameters were used the default
values. 50 independent docking runs were performed to find the
preferable conformation. The docking results were clustered
according to the root mean square deviation (RMSD) of 2.0 Å. The
structures with the relative lower binding free energy and the most
cluster members were chosen, best docking conformation.
TriseHCl buffer (100 mM, Ph 8.0), hematin (15
mM), EDTA (3 mM),
enzyme (100 g COX-1 or COX-2) and the test compound. The
m
mixture was pre-incubated at 25 ꢂC 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 non-
enzymatic oxidation observed in the absence of COX-1 and COX-2
was substracted form the experimental value while calculating
the percent inhibition.
4.2.2. In vivo anti-inflammatory activity
4.2.2.1. Animals. 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 (regis-
tration number 173-CPCSEA).
4.2.3.3. In silico modeling of human COX-2. Considering the limited
sequence identity between human COX-2 and the templates used
for its modeling, an objective validation gives results suggestive of
reliable models. Human COX-2 model construction starts with PSI-
BLAST analysis, which was used to select best template. Human
COX-2 sequence was directly compared to amino acid residue
sequences that possess structures experimentally resolved and
deposited in the Protein Data Bank [33]. Human COX-2 presented
87% of identity with 1PXX, being chosen as a template. Alignments
among sequences and structures were carried by using Clustal-W
[34]. After alignment analysis, atomic coordinated were trans-
ferred to human COX-2 primary structure for the construction of 3D
model using Modeller 9v.6 (Fig. 2).
4.2.2.2. Drugs. Ibuprofen, Indomethacin, Celecoxib, (Ranbaxy Pvt.
Ltd) and Carrageenan (Merck co.) were used in the biological
assays.
4.2.2.3. Anti-inflammatory assay. The synthesized compounds
were assessed for their anti-inflammatory activity using carra-
geenan-induced hind paw edema method. The rat paw edema was
induced by subcutaneous injection of 0.1 ml of 1% freshly prepared
saline solution of carrageenan into the right hind paw of rats
(Winter et al., 1962) [37]. The standard drug Ibuprofen (10 mg/kg/
p.o) 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 the synthesized
compounds at the equimolar dosage of the standard drug, 1 h
before the administration of carrageenan. The paw volumes were
measured using plethysmometer (Ferreira, 1979) [38] at interval of
3 h and 5 h.
Procheck summary of human COX-2 showed that 100% amino
acid residues were in most favorable regions, additionally allowed
regions and generously allowed regions with 0.0% residues in the
disallowed regions. Structural differences between crystal structure
1PXX and predicted three dimensional structure of human COX-2
were calculated by superimposing both structures. The RMSD
values between the crystal structure 1PXX and homology model
human COX-2 calculated for C
a traces and main chain atom were
1.25 Å. The RMSD values and small variability among experimental
structures template and the structure modeled reflect the presence
of strong restraints in most regions and emphasize a similar folding
patterns. Furthermore, the lower score acquired for PROSA e7.68
indicating the high quality of the model.

S. Shafi et al. / European Journal of Medicinal Chemistry 49 (2012) 324e333
333
(h) M.R. Shiradkar, K.K. Murahari, H.R. Gangadasu, T. Suresh, C.A. Kalyan,
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Acknowledgements
The authors thank Dr. G. N. Qazi, Vice-Chancellor Jamia Ham-
dard for providing necessary facilities. The authors are also thankful
to Hamdard National Foundation (HNF) for providing Hamdard
centenary research fellowship to SS and Hamdard National
fellowships to MA. CM thanks CSIR for awarding fellowship.
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