Synthesis of novel 2-mercaptobenzoxazole based 1,2,3-triazoles as inhibitors of proinflammatory cytokines and suppressors of COX-2 gene expression

Article abstract of DOI:10.1016/j.ejmech.2014.05.012

A library of novel bis-heterocycles containing 2-mercaptobenzoxazole based 1,2,3-triazoles has been synthesized using click chemistry approach. The compound 4 exhibited the most potent in vivo anti-inflammatory activity with 66.66% and 61.11% inhibition in comparison to celecoxib which showed 72.22% and 65.55% inhibition after 3 h and 5 h respectively. The compounds 4 and 9 suppressed the COX-2 gene expression by 0.94 and 0.79 fold and exhibited a selective index (COX-1/COX-2) of 64.79 and 66.47 respectively in comparison to celecoxib (SI value of 75.56). The in silico molecular docking studies showed the interactions of these molecules with Tyr-59, Tyr-119 and Gly-121. When compared with the standard drug celecoxib, compounds 4, 5, 7, 9 and 16 did not cause any gastric ulceration.

Full text of DOI:10.1016/j.ejmech.2014.05.012

Accepted Manuscript
Synthesis of novel 2-mercaptobenzoxazole based 1,2,3-triazoles as inhibitors of
proinflammatory cytokines and suppressors of COX-2 gene expression
Saqlain Haider, Mohammad Sarwar Alam, Hinna Hamid, Syed Shafi, Abhijeet Dhulap,
Firasat Hussain, Perwez Alam, Sadiq Umar, M.A.Q. Pasha, Sameena Bano, Syed
Nazreen, Yakub Ali, Chetna Kharbanda
PII:
S0223-5234(14)00426-7
10.1016/j.ejmech.2014.05.012
EJMECH 6968
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 January 2014
Revised Date: 5 April 2014
Accepted Date: 2 May 2014
Please cite this article as: S. Haider, M.S. Alam, H. Hamid, S. Shafi, A. Dhulap, F. Hussain, P.
Alam, S. Umar, M.A.Q. Pasha, S. Bano, S. Nazreen, Y. Ali, C. Kharbanda, Synthesis of novel
2-mercaptobenzoxazole based 1,2,3-triazoles as inhibitors of proinflammatory cytokines and
suppressors of COX-2 gene expression, European Journal of Medicinal Chemistry (2014), doi: 10.1016/
j.ejmech.2014.05.012.
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ACCEPTED MANUSCRIPT
Synthesis of novel 2-mercaptobenzoxazole based 1,2,3-triazoles as inhibitors of
proinflammatory cytokines and suppressors of COX-2 gene expression.
a
a*
a
a
c
Saqlain Haider , Mohammad Sarwar Alam , Hinna Hamid , Syed Shafi , Abhijeet Dhulap ,
b
d
e
d
a
Firasat Hussain , Perwez Alam , Sadiq Umar , M. A. Q. Pasha Sameena Bano , Syed
,
a
a
a
Nazreen , Yakub Ali and Chetna Kharbanda
Gastric Ulceration
In Silico Molecular Docking
N
N
O
COX-2 Gene
Expression
X-Ray
Crystallography
N
S
Ar
N
TNF-α, IL-1β, NO
A library of 2-mercaptobenzoxazole based 1,2,3-triazoles has been synthesized with an aim
to develop new molecules with a potential to inhibit the increase in the levels of
proinflammatory mediators like such as TNF-α, IL-1β, COX-2 and NO without inducing any
gastric ulceration.

ACCEPTED MANUSCRIPT
Synthesis of novel 2-mercaptobenzoxazole based 1,2,3-triazoles as inhibitors of
proinflammatory cytokines and suppressors of COX-2 gene expression.
a
a*
a
a
c
Saqlain Haider , Mohammad Sarwar Alam , Hinna Hamid , Syed Shafi , Abhijeet Dhulap ,
b
d
e
d
a
Firasat Hussain , Perwez Alam , Sadiq Umar , M. A. Q. Pasha Sameena Bano , Syed
,
a
a
a
Nazreen , Yakub Ali and Chetna Kharbanda .
a
Department of Chemistry, Faculty of Science, Jamia Hamdard (Hamdard University) New
b
Delhi -110 062, India. Department of Chemistry, Faculty of Science, University of Delhi,
c
New Delhi -110007, India. Unit for Research and Development of Information Products-
d
CSIR, Pune -411038, India. Functional Genomics Unit, CSIR-Institute of Genomics &
e
Integrative Biology, New Delh-110007. Department of Toxicology, Faculty of Science, Jamia
Hamdard (Hamdard University) New Delhi -110 062, India.
Correspondence to Prof. Mohammad Sarwar Alam, Jamia Hamdard, New Delhi-110 062,
Email ID. msalam@jamiahamdard.ac.in. msalam5555@gmail.com
6059688(5555), Fax: +91 11 26059663
Phone: +91 11
2
1

ACCEPTED MANUSCRIPT
Abstract
A library of novel bis-heterocycles containing 2-mercaptobenzoxazole based 1,2,3-triazoles
has been synthesized using click chemistry approach. The compound 4 exhibited the most
potent in vivo anti-inflammatory activity with 66.66% and 61.11% inhibition in comparison
to celecoxib which showed 72.22% and 65.55% inhibition after 3 h and 5 h respectively. The
compounds 4 and 9 supressed the COX-2 gene expression by 0.94 and 0.79 fold and
exhibited a selective index (COX-1/COX-2) of 64.79 and 66.47 respectively in comparison to
celecoxib (SI value of 75.56). The in silico molecular docking studies showed the interactions
of these molecules with Tyr-59, Tyr-119 and Gly-121.When compared with the standard drug
celecoxib, compounds 4, 5, 7, 9 and 16 did not cause any gastric ulceration.
Key words: Cyclooxygenase, crystallography, molecular docking, antinociceptive, gene
expression
2

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1
. Introduction
The present market of the anti-inflammatory drugs is encumbered with NSAIDs associated
with adverse effects like ulceration and gastric haemorrhage [1]. A prolonged consumption of
these drugs may cause gastric injuries [2]. The pro-inflammatory mediators like TNF-α, IL-
1
β, NO and COX-2 play an imperative role in the inflammatory reactions like tissue
destruction, shock and organ failure [3-5]. The aim of this work is to develop new molecules
with a potential to inhibit the increase in the level of proinflammatory mediators such as
TNF-α, IL-1β, NO and COX-2 without inducing any gastric ulceration. Our group has
recently reported the 2-mercaptobenzothiazole based 1,2,3-triazoles as a class of anti-
inflammatory and antinociceptive agents with potent COX-2 selectivity [6]. This prompted us
to synthesize and evaluate the anti-inflammatory activity (Figure 1) of 2-
mercaptobenzoxazole based 1,2,3-triazoles which has an oxygen atom in place of the sulphur
atom in the benzoxazole nucleus.
Figure 1
2
-mercaptobenzoxazoles are the thiol derivatives (Figure 2) of simple benzoxazole
nucleus. They exist in two tautomeric forms i.e. thiol and thione [7].
Figure 2
2
-mercaptobenzoxazoles have emerged as a potent medicinal scaffold and have remained the
focus of drug discovery due to their important therapeutic values [8]. Benzoxazoles and their
derivatives are known to exhibit antibacterial, antifungal, antitumor, anti-tubercular, anti-
inflammatory and HIV-1 reverse transcriptase inhibitory activities [9-13]. 1,2,3-triazole
nucleus is present in many drugs [14] and has been found to be associated with potent
antimicrobial [15,16], anti-inflammatory [17], local anaesthetic [18], anticonvulsant [19],
anti-neoplastic [20], antimalarial [21] and antiviral activities [22]. In continuation of our
efforts to develop novel molecules for the treatment of inflammatory disorders like
osteoarthritis and crohn’s disease we have linked the 1,2,3-triazole nucleus with 2-
mercaptobenzoxazoles under one construct through a methylene linkage. We have carried out
the in silico molecular docking studies with respect to COX-2 and TNF-α target in order to
get an insight into the binding modes of these novel molecules with their active sites. The
compounds showing significant in vivo anti-inflammatory activity have been further screened
against proinflammatory cytokine mediators like TNF-α, IL-1β, COX-2 and NO using in vivo
assays. The potent anti-inflammatory molecules thus obtained were subjected to in vivo
antinociceptive activity by writhing test method followed by their gastric ulceration study. It
was observed that the compounds 4, 5, 7, 9 and 16 did not induce any gastric ulceration.
3

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Since the free radical production occurs simultaneously during inflammation therefore we
have screened our compounds for their antioxidant activity using reduced glutathione and
lipid peroxidation assays.
2
. Results and discussion
2
.1. Chemistry
A focused library of eighteen novel compounds has been synthesized. As shown in Scheme
, 2-mercaptobenzoxazole on being refluxed with propargyl bromide and potassium
1
carbonate in dry acetone for 5-7 h yielded the propargyl derivative (A). The 1,3-diploar
cycloaddition of the propargylated 2-mercaptobenzoxazole derivative (A) with different
substituted aromatic azides under click chemistry conditions resulted in the formation of
novel 2-mercaptobenzoxazole based 1,2,3-triazoles (1-18) in quantitative yields (Table 1).
Aromatic azides with various substitutions including electron withdrawing and electron
donating groups have been used. The propargylation of the 2-mercaptobenzoxazole was
1
confirmed by the presence of a signal in a range of δ 4.76-4.81 (s, 2H, CH ) in the H-NMR
2
spectra. The formation of 1,2,3-triazoles was confirmed by the resonance of the triazollyl
proton as singlet in a range of δ 8.29-8.91 for different derivatives. The structural assignment
13
was also supported by the C-NMR spectral data, which showed the C-atom signals
corresponding to triazole derivatives. The final confirmation was made by the ESI-MS
+
+
+
+
analysis which showed the presence of [M] or [M+1] or [M+2] or [M+3] ion peaks.
Compound 6 was also subjected to X-ray crystallographic study which is being reported here
for the first time.
Scheme 1 & Table 1
2
.2 In vivo anti-inflammatory activity
All the synthesized compounds have been screened for their in vivo anti-inflammatory
activity by the carrageenan-induced hind paw edema model. All the compounds showed a
time-dependent decrease in the inhibition of inflammation after 3 h and 5 h. It was found that
the compound 4 showed potent anti-inflammatory activity with 66.66% and 61.11%
inhibition after 3 h and 5 h as compared to celecoxib which showed 72.22% and 65.55%
inhibition after 3 h and 5 h respectively (Figure 3). The compound 7 exhibited 68.88%
inhibition at 3 h post-carrageenan and 55.55% inhibition, 5 h post-carrageenan administration
in comparison to celecoxib. It was observed that the compounds 5, 9 and 16 exhibited
moderate anti-inflammatory activity in comparison to the standard drug celecoxib.
Figure 3
4

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2
.3. Effect on IL-1β, TNF-α and COX-2
During the inflammatory reactions, large amounts of the proinflammatory mediators are
generated which affect the immune system by suppressing the proliferation of T and B cells,
as well as cytokine synthesis [23]. Blockade of these molecules results in a reduction of
disease severity and bone resorption [24-27]. Proinflammatory cytokines like IL-1β and
TNF-α as well as COX-2 have an important role in the perpetuation of chronic inflammation
and tissue damage during progression of inflammatory disorder. There is a significant
increase in the level of TNF-α, IL-1β and COX-2 in carrageenan induced edema rats as
compared to the control Figure 4. Administration of the selected active compounds 4, 5, 7, 9
and 16 suppressed the increase in the level of IL-1β, TNF-α and COX-2 significantly when
compared with the edema group. The compound 16 exhibited a reduction in the level of IL-
1
β to 2.66 ± 0.13 pg/ml in comparison to celecoxib which showed a reduction to 2.90 ± 0.16
pg/ml. The compound 5 supressed the level of TNF-α to 3.10 ± 0.17 pg/ml in comparison to
celecoxib which showed a reduction to 3.35 ± 0.16 pg/ml. A significant decrease in the
amount of COX-2 was also observed for the compound 5 which reduced the COX-2 level to
6
.54 ± 0.26 as compared to celecoxib which showed a reduction to 7.90 ± 0.25. The
compounds 4 (COX-1 IC = 246.20µM; COX-2 IC = 3.8µM; SI = 64.79) and 9 (COX-1
50
50
IC50 = 186.11µM; COX-2 IC50 = 2.8µM; SI = 66.47) exhibited potent selective COX-2
inhibition as compared to celecoxib (COX-1 IC = 20.40µM; COX-2 IC = 0.27µM; SI =
5
0
50
7
5.56). The gastric tolerance of these compounds may be related to their selective COX-2
inhibition (Table 2).
The structure activity relationship of the synthesized compounds has been analysed on
the basis of COX-1/COX-2 ratio (Selective Index). The SI values were found to vary
depending upon the nature and the position of the substituents on the aryl ring attached with
the 1,2,3-triazole ring. The compounds containing electron donating groups on the para
position of the aryl ring exhibited more potent anti-inflammatory activity in comparison to
the compounds containing electron withdrawing groups. The compounds 4, 16 and 15
containing the C H , OC H and C(CH ) groups respectively at the para position displayed
2
5
2
5
3 3
potent selectivity towards COX-2 with the SI values of 64.79, 46.64 and 40.06 respectively.
The COX-2 selectivity was considerably reduced by the presence of the strong electron
withdrawing NO group at the meta (SI=10.80) and para (SI=11.67) positions for the
2
compounds 1 and 2 respectively.
Amongst the halogen containing compounds the COX-2 selectivity was found to
decrease with a decrease in the size of halogen (Br>Cl>F) i.e activity was found to decrease
5

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in the order 9(SI=66.47)>7(SI=42.54)>10(SI=36.88). The para substituted halogen
containing compounds 7, 9, and 10 were found to exhibit more potent COX-2 selectivity in
comparison to their corresponding ortho substituted derivatives i.e. 8, 12 and 13. The
compounds 17 and 18 containing pyridyl and substituted pyridyl ring, respectively exhibited
a moderate COX-2 selectivity with a selectivity index of 24.26 and 20.40.
Figure 4 & Table 2
2
.4. Gene expression study on COX-2
The results of the gene expression study on COX-2 (Figure 5) showed that the compounds 4
and 9 supressed the expression of COX-2 gene by 0.94 and 0.79 fold in comparison to the
standard drug celecoxib. Thus these molecules may be used to develop new leads for the
treatment of inflammatory disorders like arthritis.
Figure 5
2
.4. Nitric oxide
Nitric oxide (NO) is an important signalling molecule, produced as part of the inflammatory
response from activated cells and macrophages [28, 29]. An increase in the NO level has been
previously reported in synovial fluids of patients suffering from rheumatoid arthritis [30]. In
the present study, increased NO levels have been detected in carrageenan group similar to
those previously reported in synovial fluids of patients with rheumatoid arthritis. Analysis of
nitrite estimation is summarised in Figure 6. A significant increase in the nitrite level was
observed in carrageenan induced edema group as compared to control. All synthesized
compounds declined the increase in the nitrite level significantly as compared to the edema
group. Compound 4 significantly supressed the increase in the NO level to 5.10 ± 0.18
ꢀ
mol/mg comparison to celecoxib which showed a reduction to 5.50 ± 0.16 ꢀmol/mg.
Figure 6
2
.5. Effect on TBARS and GSH
Lipid peroxidation has been implicated in the pathogenesis of cancer, atherosclerosis,
degenerative diseases and inflammatory arthritis [31]. During lipid Peroxidation, lipid
peroxyl radicals are produced that cause cell membrane damage. Matrix degradation arising
from cytokine-stimulated chondrocytes has been shown to be primarily due to lipid
peroxidation [32]. The effect of all the synthesized compounds on TBARS (Thiobarbituric
acid reactive substances) level was measured to demonstrate the oxidative damage on lipid
Figure 7. A significant increase in TBARS level was observed in carrageenan induced edema
group when compared to the control group. The level of TBARS was supressed to 5.10 ±
0
.23 nmole by the compound 16 whereas celecoxib reduced the TBARS level to 6.3 ± 0.16
6

ACCEPTED MANUSCRIPT
nmole. Free radical production that occurs during development of arthritis in the articular
cartilage leads to decreased GSH (Glutathione) and SOD (Super oxide dismutase) levels as a
result of their consumption during oxidative stress and cellular lysis [33-35]. The
concentration of GSH was evaluated to estimate endogenous defences against hydrogen
peroxide formation. Figure 8 shows the changes in GSH levels evaluated in the joints of the
experimental groups. A marked decrease in GSH was found in the hind paw joint of
carrageenan induced (0.58 ± 0.020 ꢀGSH/g tissue) edema rats. However the treatment with
compounds 4 (0.84 ± 0.025 ꢀGSH/g tissue) and 16 (0.84 ± 0.027 ꢀGSH/g tissue)
significantly inhibited the decrease in GSH as compared to celecoxib (0.81 ± 0.027 ꢀGSH/g
tissue).
Figure 7 & 8
2
.6. Molecular Docking studies on COX-2
In order to get an insight into the binding mode of the synthesized molecules with COX-2, all
the newly synthesized molecules have been docked against COX2 (PBD NO: 3LN1). All the
molecules were docked against COX-2 target protein separately. Figures 9a and 9b show that
the compounds 4 and 9 have purely hydrophobic interactions with the COX-2 protein. The
binding energies of the eighteen new ligands were found to be in a range of -23.07 to -48.4
kcal/mol. Compounds 4 (G score = -8.6) and 9 (G score = -8.14) were found to have the closest
binding efficiency with respect to celecoxib (G score = -11.29). The predicted binding energies
are summarized in Table 3. The QikProp program was used to predict the ADME (absorption,
distribution, metabolism and excretion) properties of the molecules. Normal mode was applied
in the program to predict the partition coefficient (log P o/w), van der Waals surface area of
polar nitrogen and oxygen atoms (PSA) and aqueous solubility (log S) properties. The results
obtained are listed in Table 4 respectively. As seen from the table the values of the calculated
properties are within the acceptable range.
Figure 9a, 9b & Table 3 & Table 4
2
.7. Molecular Docking studies on TNF-α
The binding pocket of 2AZ5 is large and without defined cervices. The binding site is mainly
hydrophobic consisting of glycine, leucine and tyrosine residues. In order to bind to this
hydrophobic large pocket, the ligands also need to be hydrophobic and of large size. Before
docking the new ligands against the generated grid, the reference ligand was separately
docked against the generated grid to validate the grid and docking methodology. Figure 10a
shows comparison between the original binding modes of reference ligand against docked
7

ACCEPTED MANUSCRIPT
binding mode as predicted by Schrodinger Glide software. Figure 10a clearly shows that
adapted Schrodinger methodology successfully predicted the binding mode with root mean
0
square deviation of 0.003 A. All the ligands were docked against TNF-α target protein
separately. Figure 10b and 10c show that the compound 8 is embedded in the pocket of the
TNF-α protein and compound 10 is involved in H-bond formation with GLY 121 residue.
The binding energies of the eighteen novel ligands was found to be in the range of -35.09 to -
4
1.4 kcal/mol. Molecules 8 (G score = -6.38) and 10 (G score = -6.77) exhibited the closest
binding efficiency with respect to reference ligand 2AZ5 (G score = -7.1). The predicted
binding energies are summarized in Table 5. The QikProp program was used to predict the
ADME (absorption, distribution, metabolism and excretion) properties of the molecules.
Normal mode was applied in the program to predict partition coefficient (log P o/w), van der
Waals surface area of polar nitrogen and oxygen atoms (PSA) and aqueous solubility (log S)
properties. The results obtained are listed in Table 6. As seen from the table the values of the
calculated properties lie within the acceptable range.
Figure 10a, 10b, 10c & Table 5 & Table 6
2
.8. In vivo antinociceptive activity
The compounds showing significant in vivo anti-inflammatory activity have been further
evaluated for their in vivo antinociceptive potential. It was found that the compounds 5 and 7
exhibited 55.55% and 51.50% inhibition (Figure 11) respectively in comparison to celecoxib
which showed 70.89% inhibition.
Figure 11
2
.9. Ulcerogenic study
The compounds showing potential in vivo anti-inflammatory and in vivo antinociceptive
activities were further tested for their gastric ulceration activity (Figure 12). When compared
with celecoxib, the compounds 4, 5, 7, 9 and 16 did not induce any gastric ulceration and
rupture of the gastric mucosal layer.
Figure 12
2
.10. Crystallographic study
Intensity data were collected at 183(2) K an Oxford Xcalibur Sapphire 3 diffractometer (a
single wavelength Enhance X-ray source with MoK radiation, λ = 0.71073 Å) [36]. The
α
selected suitable single crystals were mounted using paratone oil on the top of a glass fiber
fixed on a goniometer head and immediately transferred to the diffractometer. Pre-experiment
8

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data collection, data reduction and analytical absorption corrections [37] were performed with
the Oxford program suite [38] CrysAlisPro. The crystal structures were solved with
SHELXS-97 [38] using direct methods. The structure refinements were performed by full-
2
matrix [39] least-squares on F with SHELXL-97. All programs used during the crystal
structure determination process were present in the WINGX software [40]. The chemical
formula and ring labelling system is shown in Figure 13. Crystal data for compound 6: C16
H ClN OS, Mr, 342.80; system, triclinic; space group, P -1; unit cell dimensions, a =
1
1
4
6
8
.7407(3) Å; b = 7.4806(5) Å; c = 15.6537(9) Å; α = 78.181(5)⁰; β = 79.697(4)⁰; γ =
3
2.127(5)⁰; V = 756.00(7)Å ; Z = 2; T = 298 K; R , 0.0380; R(all), 0.0442; Gof = 1.076;
int
3
3
ꢁρmax = 0.23 e Å ; ꢁ
= -0.35 e Å . All hydrogen atoms were calculated after each cycle of
ρmin
refinement using a riding model, with C-H = 0.93 Å + U (H) = 1.2U (C) for aromatic H
iso
eq
atoms, with C-H = 0.97 Å + U (H) = 1.2U (C) for methylene H atoms. Crystallographic
iso
eq
data for the compound 6 has been deposited with the Cambridge Crystallographic Data
Center (CCDC) under the number 950223. Copies of the data can be obtained, free of charge,
on application to CCDC 12 Union Road, Cambridge CB2 1EZ, UK [Fax: þ44-1223-336033;
e-mail: deposit@ccdc.cam.ac.uk or at www.ccdc.cam.ac.uk].
Figure 13
3
. Conclusion
We have synthesized a focussed library of novel bis-heterocycles containing 2-
mercaptobenzoxazole moiety and 1,2,3-triazole nucleus conjugated through a methylene
linkage. The crystallographic study of the compound 6 confirmed the formation of final
molecule and supported the spectroscopic data. The compounds 4, 5, 7, 9 and 16 exhibiting
potent in vivo anti-inflammatory and in vivo antinociceptive activities have been further
screened for their in vivo COX-2 selectivity and proinflammatory cytokine mediators like
TNF-α, IL-1β and NO. The results of the in vivo (COX 1 / COX-2) selective index showed
that the compounds 4 and 9 exhibit a target specific inhibiton of COX-2, which is comparable
with the standard drug celecoxib. The compounds 4 and 9 supressed the COX-2 gene
expression by 0.94 and 0.79 fold which in turn shows their potential to reduce inflammation
by inhibiting the expression of COX-2 gene. The results of the in silico molecular docking
studies against COX-2 showed that hydrogen bonding and hydrophobic interactions are
responsible for the interactions of the synthesized compounds with their corresponding active
sites on the target protein. The in silico molecular docking study against TNF-α shows that
the molecule 8 interacts with Tyr-59 and Tyr-119 amino acid residues on the TNF-α protein.
The molecule 10 was found to show the interactions with Tyr-59 and Gly-121 on TNF-α
9

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protein. The compounds 4, 5, 7, 9 and 16 did not induce any gastric ulceration thus showing
their effective tolerance towards gastric mucosa. Thus these molecules may be used as leads
for the development of novel anti-inflammatory drugs which may help in the treatment of
inflammatory disorders like rheumatoid arthritis and crohn’s disease.
4
. Experimental
4
.1. Chemistry
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
Veego VMP-DS apparatus, IR spectra were recorded as potassium bromide pellets on a
Perkin Elmer 1650 spectrophotometer (USA), 1H NMR spectra was 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 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 novel and their
formation has been confirmed from the spectral data. Compound A obtained from the
t
propargylation of 2-mercaptobezoxazole was dissolved in 20 mL of Butanol:water (1:1)
solvent at ambient temperature. CuSO .5H O was charged into it and the reaction mixture
4
2
was stirred for 5 min at the room temperature. 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 reaction mixture then changed to dark yellow. After 15 min azides were added. The
reaction mixture was then allowed to stir for further 5-7 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.
4
.1.1. 2-[1-(3-Nitro-phenyl)-1H-[1,2,3]triazol-4-ylmethylsulfanyl]-benzoxazole
ο
-1
Yellowish brown crystals; yield 85% ; m. p. 210-211 C; IR (KBr): ν (cm ) 3145, 3130,
1
1
580, 1525, 1390, 924; H NMR (400 MHz, DMSO): δ 4.80 (s, 2H), 7.31-7.37 (m, 2H), 7.66
(d, 1H, J=1.6 Hz), 7.68 (d, 1H, J=2.0 Hz), 7.88 (t, 1H, J=8.0 Hz), 8.31 (dd, 1H, J=1.2 and
1
3
8
.0 Hz), 8.38 (dd, 1H, J=1.2 and 8.0 Hz), 8.71 (t, 1H, J=2.0 Hz), 9.08 (s, 1H); C NMR
(75MHz, DMSO): δ 26.57, 110.06, 115.32, 118.45, 121.14, 123.23, 124.21, 124.46, 125.95,
1
0

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+
1
30.93, 137.56, 141.66, 145.20, 148.85, 152.09, 163.96; ESI-MS: 354 (M+1) ; Anal. Calcd.
for C H N O S: C, 54.38; H, 3.14; N, 19.82; S, 9.07. Found C, 54.39; H, 3.13; N, 19.83; S,
1
6
11
5
3
9
.06.
.1.2. 2-[1-(4-Nitro-phenyl)-1H-[1,2,3]triazol-4-ylmethylsulfanyl]-benzoxazole
4
ο
-1
Yellowish brown crystals; yield 88% ; m. p. 205-206 C; IR (KBr): ν (cm ) 3155, 3127,
1
1
598, 1533, 1376, 855; H NMR (400 MHz, DMSO): δ 4.80 (s, 2H), 7.32-7.37 (m, 2H), 7.68
1
3
(
dd, 2H, J=1.6 and 7.2 Hz), 8.22 (d, 2H, J=9.2 Hz), 8.43 (d, 2H, J=6.9 Hz), 9.06 (s, 1H); C
NMR (75MHz, DMSO): δ 26.42, 109.91, 118.33, 120.45, 121.32, 124.09, 124.34, 125.30,
+
1
40.93, 141.53, 144.99, 147.01, 151.89, 163.69; ESI-MS: 354 (M+1) ; Anal. Calcd. for
C H N O S: C, 54.38; H, 3.14; N, 19.82; S, 9.07. Found C, 54.36; H, 3.12; N, 19.81; S,
16
11
5
3
9
.05.
.1.3. 2-(1-Phenyl-1H-[1,2,3]triazol-4-ylmethylsulfanyl)-benzoxazole
4
ο
-1
White powder; yield 90% ; m. p. 190-191 C; IR (KBr): ν (cm ) 3165, 3130, 1530, 1520,
1
1
354; H NMR (400 MHz, DMSO): δ 4.78 (s, 2H), 7.31-7.37 (m, 2H), 7.49 (d, 1H, J=7.6
1
3
Hz), 7.59 (d, 1H, J=7.6 Hz), 7.89 (d, 2H, J=8.0 Hz ), 8.84 (s, 1H); C NMR (75MHz,
DMSO): δ 26.56, 110.61, 118.43, 120.42, 120.55, 122.22, 123.59, 124.44, 124.60, 141.33,
+
1
41.63, 143.69, 147.31, 150.59, 163.78; ESI-MS: 309 (M+1) ; Anal. Calcd. for C H N OS:
1
6
12
4
C, 62.32; H, 3.92; N, 18.17; S, 10.40. Found C, 62.30; H, 3.91; N, 18.18; S, 10.39.
.1.4. 2-[1-(4-Ethyl-phenyl)-1H-[1,2,3]triazol-4-ylmethylsulfanyl]-benzoxazole
4
ο
-1
Yellowish white powder; yield 88% ; m. p. 195-196 C; IR (KBr): ν (cm ) 3250, 3140, 1490,
1
1
2
8
1
433, 1256; H NMR (400 MHz, DMSO): δ 1.20 (t, 3H), 2.67 (q, 2H, J=6.8 Hz) 4.77 (s,
H), 7.31-7.36 (m, 2H), 7.41 (d, 2H, J=8.0 Hz), 7.67-7.69 (m, 2H), 7.77 (d, 2H, J=8.4 Hz),
1
3
.79 (s, 1H); C NMR (75MHz, DMSO): δ 16.86, 26.30, 109.41, 117.13, 121.25, 122.22,
24.09, 124.34, 125.30, 140.93, 141.53, 142.24, 144.99, 147.01, 152.65, 163.75; ESI-MS:
1
1

ACCEPTED MANUSCRIPT
+
3
6
4
37 (M+1) ; Anal. Calcd. for C H N OS: C, 64.26; H, 4.79; N, 16.65; S, 9.53. Found C,
18 16 4
4.27; H, 4.78; N, 16.64; S, 9.54.
.1.5. 2-[1-(4-Methoxy-phenyl)-1H-[1,2,3]triazol-4-ylmethylsulfanyl]-benzoxazole
ο
-1
White powder; yield 92% ; m. p. 178-179 C; IR (KBr): ν (cm ) 3165, 3116, 1468, 1433,
1
1
356; H NMR (400 MHz, DMSO): δ 3.82 (s, 3H), 4.76 (s, 2H), 7.11 (d, 2H, J=9.2 Hz),
1
3
7
.31-7.37 (m, 2H), 7.67-7.69 (m, 2H), 7.77 (d, 2H, J=8.8 Hz), 8.73 (s, 1H); C NMR
(75MHz, DMSO): δ 25.62, 52.34, 110.61, 117.23, 118.43, 120.34, 121.35, 125.14, 126.20,
+
1
41.93, 142.13, 143.39, 146.31, 152.89, 162.79; ESI-MS: 339 (M+1) ; Anal. Calcd. for
C H N O S: C, 60.34; H, 4.17; N, 16.56; S, 9.48. Found C, 60.32; H, 4.18; N, 16.54; S,
17
14
4
2
9
.49.
.1.6. 2-[1-(3-Chloro-phenyl)-1H-[1,2,3]triazol-4-ylmethylsulfanyl]-benzoxazole
4
ο
-1
Brown crystals; yield 94% ; m. p. 203-204 C; IR (KBr): ν (cm ) 3205, 3150, 1460, 1510,
1
1
7
1
1
3
4
260; H NMR (300 MHz, DMSO): δ 4.77 (s, 2H), 7.31-7.36 (m, 2H), 7.51- 7.67(m, 4H),
1
3
.88 (d, 1H, J=7.8 Hz), 8.00 (s, 1H), 8.91 (s, 1H); C NMR (75MHz, DMSO): δ 26.72,
10.04, 118.47, 120.75, 121.06, 124.13, 124.41, 128.84, 130.78, 135.51, 137.70, 141.76,
+
44.64, 152.10, 164.08; ESI-MS: 343 (M+1) ; Anal. Calcd. for C H ClN OS: C, 56.06; H,
1
6
11
4
.23; N, 16.34; S, 9.35. Found C, 56.04; H, 3.22; N, 16.33; S, 9.34.
.1.7. 2-[1-(4-Chloro-phenyl)-1H-[1,2,3]triazol-4-ylmethylsulfanyl]-benzoxazole
ο
-1
Brown powder; yield 90% ; m. p. 196-197 C; IR (KBr): ν (cm ) 3157, 1886, 1603, 1554,
1
1
4
1
1
398, 924; H NMR (300 MHz, DMSO): δ 4.77 (s, 2H), 7.33-7.36 (m, 2H), 7.64- 7.69 (m,
1
3
H), 7.93 (d, 2H, J=9.0 Hz), 8.88 (s, 1H); C NMR (75MHz, DMSO): δ 26.65, 110.05,
18.41, 121.07, 121.72, 124.15, 124.41, 129.88, 134.59, 135.35, 141.71, 144.60, 152. 07,
+
64.12; ESI-MS: 343 (M+1) ; Anal. Calcd. for C H ClN OS: C, 56.06; H, 3.23; N, 16.34;
16
11
4
S, 9.35. Found C, 56.08; H, 3.21; N, 16.35; S, 9.33.
.1.8. 2-[1-(2-Chloro-phenyl)-1H-[1,2,3]triazol-4-ylmethylsulfanyl]-benzoxazole
4
1
2

ACCEPTED MANUSCRIPT
ο
-1
Yellow powder; yield 88% ; m. p. 190-191 C; IR (KBr): ν (cm ) 3139, 3115, 1556, 1500,
1
1
7
1
456, 1237, 1137; H NMR (300 MHz, DMSO): δ 4.79 (s, 2H), 7.32-7.35 (m, 2H), 7.53-
1
3
.75(m, 6H), 8.60 (s, 1H); C NMR (75MHz, DMSO): δ 26.76, 110.01, 118.44, 124.06,
24.35, 125.08, 127.70, 127.89, 128.45, 130.74, 134.74, 141.79, 143.30, 152.06, 164.10;
+
ESI-MS: 343 (M+1) ; Anal. Calcd. for C H ClN OS: C, 56.06; H, 3.23; N, 16.34; S, 9.35.
1
6
11
4
Found C, 56.04; H, 3.22; N, 16.33; S, 9.31.
.1.9. 2-[1-(4-Bromo-phenyl)-1H-[1,2,3]triazol-4-ylmethylsulfanyl]-benzoxazole
4
ο
-1
Brown crystals; yield 86% ; m. p. 212-213 C; IR (KBr): ν (cm ) 3154, 3116, 1548, 1507,
1
1
454, 1133, 1051; H NMR (300 MHz, DMSO): δ 4.76 (s, 2H), 7.32-7.35 (m, 2H), 7.65- 7.68
1
3
(
m, 2H), 7.78 (d, 2H, J=9.0 Hz), 7.86 (d, 2H, J=9.0 Hz), 8.87 (s, 1H); C NMR (75MHz,
DMSO): δ 26.67, 110.06, 118.43, 120.95, 121.96, 122.49, 124.14, 124.41, 132.87, 135.86,
+
+
1
41.74, 144.69, 152.11, 164.15; ESI-MS: 387 (M+2) , 388 (M+3) ; Anal. Calcd. for
C H BrN OS: C, 49.62; H, 2.86; N, 14.47; S, 8.28. Found C, 49.60; H, 2.87; N, 14.48; S,
16
11
4
8
.27.
.1.10. 2-[1-(4-Fluoro-phenyl)-1H-[1,2,3]triazol-4-ylmethylsulfanyl]-benzoxazole
4
ο
-1
White crystals; yield 92% ; m. p. 194-195 C; IR (KBr): ν (cm ) 3154, 3139, 1600, 1549,
1
1
454, 1236, 1131; H NMR (300 MHz, DMSO): δ 4.77 (s, 2H), 7.30-7.36 (m, 2H), 7.40-7.46
1
3
(
m, 2H), 7.66-7.69 (m, 2H), 7.90-7.94 (m, 2H), 8.81 (s, 1H); C NMR (75MHz, DMSO): δ
2
1
3
4
6.74, 110.06, 116.53, 118.45, 121.25, 121.72, 122.55, 122.66, 124.13, 124.40, 141.80,
+
44.57, 152.15, 164.23; ESI-MS: 327 (M+1) ; Anal. Calcd. for C H FN OS: C, 58.89; H,
1
6
11
4
.40; N, 17.17; S, 9.83. Found C, 58.88; H, 3.39; N, 17.16; S, 9.84.
.1.11. 2-(1-o-Tolyl-1H-[1,2,3]triazol-4-ylmethylsulfanyl)-benzoxazole
ο
-1
Yellowish white powder; yield 83% ; m. p. 188-189 C; IR (KBr): ν (cm ) 3144, 3124, 1555,
1
1
505, 1379, 1135, 1100; H NMR (300 MHz, DMSO): δ 2.06 (s, 3H), 4.77 (s, 2H), 7.29-7.34
1
3
(
m, 2H), 7.38 (d, 2H, J=5.7 Hz), 7.45 (d, 2H, J=5.7 Hz), 7.64-7.67 (m, 2H), 8.48 (s, 1H); C
1
3

ACCEPTED MANUSCRIPT
NMR (75MHz, DMSO): δ 17.82, 26.83, 110.03, 118.43, 124.09, 124.37, 124.52, 125.95,
1
26.83, 129.87, 131.47, 133.56, 136.35, 141.79, 143.40, 152.07, 164.28; ESI-MS: 323
+
(
M+1) ; Anal. Calcd. for C H N OS: C, 63.33; H, 4.38; N, 17.38; S, 9.95. Found C, 63.32;
17
14
4
H, 4.36; N, 17.39; S, 9.93.
4
.1.12. 2-[1-(2-Bromo-phenyl)-1H-[1,2,3]triazol-4-ylmethylsulfanyl]-benzoxazole
ο
-1
Yellowish flakes; yield 86% ; m. p. 202-203 C; IR (KBr): ν (cm ) 3124, 3125, 1587, 1505,
1
1
7
457, 1378, 1238, 1155; H NMR (300 MHz, DMSO): δ 4.78 (s, 2H), 7.29-7.36 (m, 2H),
1
3
.50-7.58 (m, 2H), 7.61-7.67 (m, 3H), 7.86 (d, 1H, J=7.8 Hz), 8.54 (s, 1H); C NMR
(75MHz, DMSO): δ 26.80, 110.03, 118.38, 118.47, 124.08, 124.36, 125.15, 128.14, 128.47,
+
+
1
31.15, 133.90, 141.82, 143.25, 152.07, 164.11; ESI-MS: 387 (M+2) , 388 (M+3) ; Anal.
Calcd. for C H BrN OS: C, 49.62; H, 2.86; N, 14.47; S, 8.28. Found C, 49.63; H, 2.85; N,
1
6
11
4
1
4.46; S, 8.29.
.1.13. 2-[1-(2-Fluoro-phenyl)-1H-[1,2,3]triazol-4-ylmethylsulfanyl]-benzoxazole
4
ο
-1
White powder; yield 90% ; m. p. 187-188 C; IR (KBr): ν (cm ) 3182, 3170, 1595, 1379,
1
1
047; H NMR (300 MHz, DMSO): δ 4.81 (s, 2H), 7.31-7.36 (m, 2H), 7.44 (d, 1H, J=7.5
1
3
Hz), 7.52-7.59 (m, 2H), 7.67-7.69 (m, 2H), 7.83 (t, 1H, J=7.8 Hz), 8.64 (s, 1H); C NMR
(75MHz, DMSO): δ 26.72, 111.02, 116.49, 118.47, 120.36, 121.27, 121.70, 122.54, 122.65,
+
1
24.10, 124.42, 141.82, 144.55, 152.18, 164.20; ESI-MS: 327 (M+1) ; Anal. Calcd. for
C H FN OS: C, 58.89; H, 3.40; N, 17.17; S, 9.83. Found C, 58.87; H, 3.38; N, 17.18; S,
16
11
4
9
.82.
.1.14. 2-[1-(3,4-Dimethyl-phenyl)-1H-[1,2,3]triazol-4-ylmethylsulfanyl]-benzoxazole
4
ο
-1
Yellow powder; yield 88% ; m. p. 211-212 C; IR (KBr): ν (cm ) 3145, 3118, 1501, 1454,
1
1
133, 1098, 1045; H NMR (300 MHz, DMSO): δ 2.27 (s, 3H), 2.30 (s, 3H), 4.76 (s, 2H),
1
3
7
.30-7.38 (m, 3H), 7.55 (d, 1H, J=8.1 Hz), 7.67 (d, 3H, J=6.6 Hz), 8.72 (s, 1H); C NMR
(75MHz, DMSO): δ 19.43, 19.86, 26.85, 110.04, 117.92, 118.45, 121.12, 121.81, 124.07,
1
4

ACCEPTED MANUSCRIPT
1
24.36, 130.58, 134.84, 137.64, 138.34, 141.83, 144.03, 152.11, 164.33; ESI-MS: 337
+
(
M+1) ; Anal. Calcd. for C H N OS: C, 64.26; H, 4.79; N, 16.65; S, 9.53. Found C, 64.24;
18
16
4
H, 4.78; N, 16.64; S, 9.51.
4
.1.15. 2-[1-(4-tert-Butyl-phenyl)-1H-[1,2,3]triazol-4-ylmethylsulfanyl]-benzoxazole
ο
-1
White powder; yield 88% ; m. p. 193-195 C; IR (KBr): ν (cm ) 3154, 3141, 1555, 1520,
1
1
453, 1378, 1133; H NMR (300 MHz, DMSO): δ 1.31 (s, 9H), 4.77 (s, 2H), 7.32-7.36 (m,
13
2
H), 7.58 (d, 2H, J=8.7 Hz), 7.65-7.69 (m, 2H), 7.77 (d, 2H, J=8.7 Hz), 8.77 (s, 1H); C
NMR (75MHz, DMSO): δ 26.83, 31.23, 34.76, 110.04, 118.44, 120.33, 121.15, 124.08,
+
1
24.37, 126.59, 134.50, 141.81, 144.11, 152.10, 152.22, 164.30; ESI-MS: 365 (M+1) ; Anal.
Calcd. for C H N OS: C, 65.91; H, 5.53; N, 15.37; S, 8.80. Found C, 65.90; H, 5.54; N,
2
0
20
4
1
5.36; S, 8.81.
.1.16. 2-[1-(4-Ethoxy-phenyl)-1H-[1,2,3]triazol-4-ylmethylsulfanyl]-benzoxazole
4
ο
-1
White powder; yield 85% ; m. p. 186-187 C; IR (KBr): ν (cm ) 3155, 3143, 1522, 1498,
1
1
457, 1390, 1251; H NMR (300 MHz, DMSO): δ 1.34 (t, 3H, J=6.6 Hz), 4.09 (q, 2H, J=6.9
Hz), 4.76 (s, 2H), 7.09 (d, 2H, J=9.0 Hz ), 7.30-7.38 (m, 2H), 7.66-7.68 (m, 2H), 7.75 (d, 2H,
1
3
J=8.7 Hz), 8.77 (s, 1H); C NMR (75MHz, DMSO): δ 14.69, 26.84, 63.91, 110.04, 115.25,
1
18.45, 121.24, 122.24, 124.07, 124.36, 130.22, 132.91, 141.82, 144.04, 152.11, 164.32;
+
ESI-MS: 353 (M+1) ; Anal. Calcd. for C H N O S: C, 61.35; H, 4.58; N, 15.90; S, 9.10.
1
8
16
4
2
Found C, 61.33; H, 4.57; N, 15.89; S, 9.11.
4
.1.17. 2-(1-Pyridin-3-yl-1H-[1,2,3]triazol-4-ylmethylsulfanyl)-benzoxazole
ο
-1
Brown powder; yield 84% ; m. p. 193-194 C; IR (KBr): ν (cm ) 3155, 3144, 1555, 1505,
1
1
453, 1131, 1095; H NMR (300 MHz, DMSO): δ 4.78 (s, 2H), 7.30-7.33 (m, 2H), 7.59-7.66
1
3
(
m, 3H), 8.29 (d, 1H, J=7.8 Hz), 8.66 (d, 1H, J=3.9 Hz), 8.29 (s, 1H), 9.10 (s, 1H); C NMR
(75MHz, DMSO): δ 26.18, 109.61, 118.01, 120.97, 123.75, 123.83, 124.02, 127.63, 133.06,
+
1
41.16, 141.24, 144.31, 149.49, 151.59, 163.49; ESI-MS: 353 (M+1) ; Anal. Calcd. for
1
5

ACCEPTED MANUSCRIPT
C H N OS: C, 58.24; H, 3.58; N, 22.64; S, 10.37. Found C, 58.22; H, 3.57; N, 22.62; S,
15
11
5
1
0.36.
.1.18. 2-[1-(2-Chloro-pyridin-3-yl)-1H-[1,2,3]triazol-4-ylmethylsulfanyl]-benzoxazole
4
ο
-1
Yellow powder; yield 85% ; m. p. 186-187 C; IR (KBr): ν (cm ) 3154, 3132, 1571, 1497,
1
1
457, 1135, 1100; H NMR (300 MHz, DMSO): δ 4.80 (s, 2H), 7.33-7.35 (m, 2H), 7.65-7.71
1
3
(
m, 3H), 8.21 (d, 1H, J=7.8 Hz), 8.63 (d, 1H, J=3.6 Hz), 8.67 (s, 1H); C NMR (75MHz,
DMSO): δ 26.63, 110.03, 118.47, 123.33, 124.16, 124.42, 124.90, 131.97, 135.84, 141.75,
+
1
43.99, 144.81, 150.20, 152.09, 163.94; ESI-MS: 344 (M+1) ; Anal. Calcd. for
C H ClN OS: C, 52.40; H, 2.93; N, 20.37; S, 9.33. Found C, 52.39; H, 2.94; N, 20.35; S,
15
10
5
9
.31.
4
4
.2. Pharmacology
.2.1. Animals
Albino Wistar rats of either sex (150–200 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
1
73- CPCSEA).
4
.2.2. Chemicals
Celecoxib, carrageenan, potassium chloride and carboxymethylcellulose along with the other
chemicals used in the different experiments were purchased from Sigma–Aldrich Chemicals
Pvt. Limited, Bangalore, India.
4
.2.3. Anti-inflammatory activity
The synthesized compounds were tested for their in vivo anti-inflammatory activity using
carrageenan-induced hind paw edema method. The rat paw edema was induced by
subcutaneous injection of 0.1 ml of 1% freshly prepared saline solution [41] of carrageenan
into the right hind paw of rats. The standard drug, celecoxib (0.05 mmoles/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
1
6

ACCEPTED MANUSCRIPT
synthesized compounds as the standard drug, 1 h before the administration of carrageenan.
The paw volumes were measured using plethysmometer [42] at interval of 3 h and 5 h.
4
.2.4. Measurement of IL-1β, TNF-α and COX-2
Levels of the proinflammatory cytokines (IL-1β and TNF-α) [43] and COX-2 [44] in the
serum have been determined by using commercially available ELISA kits (eBioscience and
Cayman, USA). Assays have been performed in duplicate in accordance to the
manufacturer’s guidelines. Cytokine concentrations were expressed as picograms of antigen
per millilitre of protein.
4
4
.2.5. COX-2 gene expression study
.2.5.1. Cell culture experiments
HeLa cells (ATCC) were seeded in 24 well plate, 24 hrs before the treatment in DMEM
containing 10% calf serum (Invitrogen). After 24 hrs cells were treated with compounds 4 &
9
(10 ꢀM) and the standard drug celecoxib (10 ꢀM) as positive control and DMSO as
negative control followed by 24 hrs of incubation of cells in CO incubator at 37 ºC and 5%
2
CO2.
4
.2.5.2. RNA extraction, reverse transcription and gene expression analysis
After 24 hrs cells were scrapped and collected in 1.5 ml micro centrifuge tubes. The total
RNA was isolated by TRI Reagent® (Molecular Research Centre). RNA quantity and quality
was determined on a NanoDrop ND-2000c spectrophotometer and integrity was checked on a
1
.5% agarose gel. Total RNA (1 ꢀg) was used to generate cDNA using an EZfirst strand
cDNA synthesis kit for RT (reverse transcription)–PCR (Biological Industries). Primers for
real-time PCR were designed for COX-2 and GAPDH using the Pearl Primer software and
are listed in Table 7.
Table 7
Reactions were run at 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and
6
0 °C for 1 min. Real-time PCR was performed on an ABI Prism 7300 Sequence Detection
System (Applied Biosystems) using the SYBR Green PCR Master Mix (Applied
Biosystems). PCR was performed in triplicate and was repeated two times for each gene and
each sample. Relative transcript quantities were calculated using the Ct method with GAPDH
as the endogenous reference gene.
4
.2.6. Estimation of thiobarbituric acid reactive substances (TBARS)
The assay of TBARS was done according to earlier method [45] adapted to microtiter plates
by bringing the final volume to 150 µL. In brief, hind paw tissue homogenate was prepared in
1
7

ACCEPTED MANUSCRIPT
o
o
0
.15 M KCl (5%w/v homogenate) and aliquots of 30 µL were incubated for 0 C and 37 C for
hr. Subsequently, 60 µL of 28% w/v TCA was added and the volume was made up to 150
1
µL by adding 60 µL of distilled water followed by centrifugation at 3000xg for 10 min. The
supernatant (125 µL) was taken and colour was developed by addition of 25 µL of 1% w/v
TBA dissolved in 0.05 N NaOH and kept in boiling water bath for 15 min. The absorbance
was read at 532 nm in a plate reader (Bio-Rad, U.S.A). The results are expressed in µmoles
5
−1
−1
of TBARS formed/hr/g tissue using a molar extinction coefficient of 1.56×10 M cm .
.2.7. Reduced glutathione (GSH)
4
GSH level was measured using the method described earlier [46]. Homogenized hind paw
tissue (10% w/v in phosphate buffer pH 7.4) was deproteinized by adding an equal volume of
o
1
0% TCA and was allowed to stand at 4 C for 2 h. The contents were centrifuged at 2000xg
for 15 min. 50 µL supernatant was added to 200 µL of 0.4 M Tris buffer (pH 8.9) containing
.02 M EDTA (pH 8.9) followed by the addition 20 µL of 0.01M DTNB. The absorbance
was read in a microplate reader at 412 nm and results are expressed as µg GSH/g tissue using
0
3
−1
−1
a molar extinction coefficient of 13.6×10 M cm .
.2.8. Measurement of Nitric oxide (NO) level
4
Animals were sacrificed and their hind paw tissues were washed with PBS (pH 7.4) and
placed on ice as described earlier [47]. 50µL of the sample was added to 100 µL of Griess
reagent and reaction mixture was incubated for about 5-10 minutes at room temperature and
protected from light. The optical density was measured at 540 nm in microplate reader
according to the reagent manufacturer’s protocol. Calculations were done after generating a
standard curve for sodium nitrite in the same buffer as used for preparation of homogenate.
4
.2.9. Antinociceptive activity
Writhing test
The writhing test in mice was carried out using the method of Koster [48]. The writhes were
induced by intraperitoneal injection of 0.6% acetic acid (v/v) (80 mg/kg). The standard drug
i.e. celecoxib was given orally at a dose 0.05 mmoles/Kg of body weight. The test
compounds were administered orally at an equimolar dosage to groups of six animals each,
3
0 min before chemical stimulus. The numbers of muscular contractions 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.
1
8

ACCEPTED MANUSCRIPT
4
.3. Ulcerogenic activity
The compounds having anti-inflammatory and antinociceptive activities comparable with
celecoxib were further tested for their ulcerogenic risk evaluation [49]. This was done at three
times higher dose in comparison to the dose used for anti- inflammatory activity, i.e. 0.15
mmoles/Kg body weight of celecoxib and the test compounds were used. Each group had
three animals which were later sacrificed after five hours of oral drug administration. When
compared with celecoxib, these compounds did not cause any gastric ulceration and
disruption of gastric epithelial cells at the above mentioned oral dose. Hence the gastric
tolerance of these compounds towards the gastric mucosa was better than that of celecoxib.
4
.4. Molecular Docking studies on COX-2
Crystallized structure of COX-2 protein with reported ligand (celecoxib) was considered for
the docking studies. For this purpose we shortlisted Protein 3LN1 from Protein Data Bank as
this includes celecoxib drug selectivity bound to binding site of COX. The structure of
protein COX-2 along with celecoxib (3LN1) was retrieved from the Protein Data Bank
(PDB). First protein structure of 3LN1 was imported in Schrodinger using Protein
Preparation Wizard. This wizard was used to optimize and minimize the protein structure
which involves removing undesirable water molecules and other defects in the target protein.
Finally a low energy and structurally correct target protein was achieved. This minimized
protein was used for further docking analysis. As the target protein has already the site for
celecoxib, the grid was generated by selecting the celecoxib ligand as the reference ligand.
Finally the grid was validated and was used for further docking with new unknown molecules
to predict their docking score. Chemical structures were drawn in maestro and geometrically
refined by LigPrep module for Ligand preparation. In this module 2-D structures were
converted into 3-D structures, which were further subjected to OPLS-2005 force field to
generate single low energy 3-D structure for each input structure. During this step chiralities
were maintained. Docking was carried using Glide software. It was carried using Extra
precision and write XP descriptor information. This generates favourable ligand poses which
were further screened through filters to examine the spatial fit of the ligands in the active site.
Ligand poses which pass through initial screening are subjected to evaluation and
minimization of grid approximation. Scoring is then carried on energy minimized poses to
generate Glide score.
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4
.5. Molecular Docking studies on TNF-α
Crystallized structure of 2AZ5 was chosen from Protein Data Bank and used as target for
molecular docking studies. 2AZ5 structure was reported with the specific ligand celecoxib
which inhibits it. 2AZ5 structure was imported in Schrodinger using Protein Preparation
Wizard. Missing hydrogen and other atoms were added using prime interface. Undesired
water molecules were removed. The protein was then optimized and minimized to give low
energy and structurally correct target protein. As the target protein had already the site for
reference ligand, the grid was generated by selecting the ligand as the reference ligand.
Finally the grid was validated and was used for further docking with new unknown ligands to
predict their docking score. Chemical structures were drawn in maestro and geometrically
refined by Lig Prep module. In this module 2-D structures were converted into 3-D
structures, which were further subjected to OPLS-2005 force field to generate single low
energy 3-D structure for each input structure. During this step chiralities were maintained.
Docking was carried out using Glide software using Extra precision and write XP descriptor
information. This generates favourable ligand poses which were further screened through the
filters to examine spatial fit of the ligand into the active site. Ligand poses which pass
through initial screening are subjected to evaluation and minimization of grid approximation.
Scoring is then carried on energy minimized poses to generate Glide score.
Acknowledgements
The authors thank Dr. G. N. Qazi, Vice-Chancellor, Jamia Hamdard for providing necessary
facilities to the Department of Chemistry. One of the authors (SH) is also thankful to Council
of Scientific and Industrial Research (CSIR), New Delhi for the award of senior research
fellowship.
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List of captions:
Figure 1: The
structural
resemblance
of
2-mercaptobenzoxazole
with
2-
mercaptobenzothiazole is shown.
Figure 2: The tautomeric forms of 2-mercaptobenzoxazole are shown.
Figure 3: The results of the in vivo antiinflammatory activity of all the synthesized
compounds by carrageenan induced hind paw edema method is shown.
Figure 4: The compounds 4, 5, 7, 9 and 16 showing potent in vivo antiinflammatory activity
have been screened for their effects on the levels of the TNF-α, IL-1β and COX-2
in the serum.
Figure 5: The compounds 4 and 9 supressed the COX-2 gene expression by 0.94 and 0.79
folds respectively.
Figure 6: All the synthesized compounds have been screened for their effect on the level of
Nitric oxide.
Figure 7: The results the antioxidant activity by the lipid peroxidation are shown.
Figure 8: The results the antioxidant activity by the glutathione method are shown.
Figure 9a: The In Silico molecular docking structure of the compound 4 against COX-2
protein is shown.
Figure 9b: The In Silico molecular docking structure of the compound 9 against COX-2
protein is shown.
Figure 10a: The docked reference ligand used against TNF-α is shown.
Figure 10b: The In Silico molecular docking structure of the compound 8 against TNF-α
protein is shown.
Figure 10c: The In Silico molecular docking structure of the compound 10 against TNF-α
protein is shown.
Figure 11: In vivo antinociceptive activity of the active compounds by writhing test method.
Figure 12: The histopathology report of the active compounds is shown.
Figure 13: The X-ray crystallographic structure of the compound 2-[1-(3-Chloro-phenyl)-
1
H-[1,2,3]triazol-4-ylmethylsulfanyl]-benzooxazole(6) is shown.
2
4

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Ar
N
N
N
O
N
(a)
O
N
(
b)
SH
S
Ar-N
O
Reflux
3
Br
S
A
N
K CO / CH COCH
Reflux
2
3
3
3
t
(a)
(b) CuSO .5H O , Sodium Ascorbate, BuOH:H O , r.t
4
2
2
Scheme 1. Synthesis of novel 2-mercaptobenzoxazole linked 1,2,3-triazoles
2
5

ACCEPTED MANUSCRIPT
Table 1. Structures of the novel 2-mercaptobenzoxazole based 1,2,3-triazole.
Compound
% Yield
Structure
O
O-
N+
1
N
8
8
5
8
N
S
N
N
O
O
N+
O-
N
N
2
S
N
N
O
N
O
S
N
N
N
3
4
5
6
9
8
9
9
0
8
2
4
N
N
N
S
N
O
O
N
N
N
S
N
O
Cl
N
N
S
N
N
O
Cl
N
N
S
N
N
7
8
9
8
0
8
O
Cl
N
N
N
S
N
O
Br
N
O
N
N
9
S
N
8
9
8
6
2
3
F
N
N
10
S
N
N
O
N
N
N
11
S
N
O

ACCEPTED MANUSCRIPT
Br
N
N
1
2
8
9
6
0
N
S
S
N
O
F
N
N
O
1
3
N
N
CH3
CH3
1
4
8
8
8
8
N
N
N
S
N
O
3
H C
CH
CH
3
3
N
N
1
5
S
N
N
O
O
N
N
S
N
N
1
6
8
8
8
5
4
5
O
N
O
N
S
N
1
7
N
N
N
N
N
N
1
8
S
Cl
N
O

ACCEPTED MANUSCRIPT
Table 2. Inhibitory activity of the 1,2,3-triazole based benzoxazolinones.
Compounds
IC (µM)
Selectivity index
COX-1/COX-2
5
0
(
COX-1)
103.52
115.23
(COX-2)
9.58
1
2
10.80
11.67
9.87
3
4
5
6
7
8
9
107.25
246.20
89.37
6.42
3.8
16.70
64.79
38.86
19.06
42.54
12.56
66.47
36.88
2.3
88.26
4.63
1.9
80.82
44.35
3.53
2.8
186.11
192.15
10
5.21
1
1
1
2
132.32
38.36
4.68
2.75
28.27
13.94
1
1
1
1
1
1
3
4
5
6
7
8
24.23
140.45
154.26
163.24
96.56
2.40
4.35
3.85
3.5
10.09
32.28
40.06
46.64
24.26
20.40
75.56
3.98
5.03
0.27
102.66
20.40
Celecoxib
Values are the means ± SEM from three independent experiments using COX assay kits (Cayman Chemicals
Inc., Ann Arbor, MI, USA).