Synthesis, biological evaluation and docking study of a new series of di-substituted benzoxazole derivatives as selective COX-2 inhibitors and anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmc.2018.01.007

A new series of substituted-N-(3,4-dimethoxyphenyl)-benzoxazole derivatives 13a–13p was synthesized and evaluated in vitro for their COX (I and II) inhibitory activity, in vivo anti-inflammatory and ulcerogenic potential. Compounds 13d, 13h, 13k, 13l and 13n exhibited significant COX-2 inhibitory activity and selectivity towards COX-2 over COX-1. These selected compounds were screened for their in vivo anti-inflammatory activity by carrageenan induced rat paw edema method. Among these compounds, 13d was the most promising analogs of the series with percent inhibition of 84.09 and IC₅₀ value of 0.04 μM and 1.02 μM (COX-2 and COX-1) respectively. Furthermore, ulcerogenic study was performed and tested compounds (13d, 13h, 13k, 13l) demonstrated a significant gastric tolerance than ibuprofen. Molecular docking study was also performed with resolved crystal structure of COX-2 to understand the binding mechanisms of newly synthesized inhibitors in the active site of COX-2 enzyme and the results were found to be concordant with the biological evaluation studies of the compounds. These newly synthesized inhibitors also showed acceptable pharmacokinetic profile in the in silico ADME/T analyses.

Full text of DOI:10.1016/j.bmc.2018.01.007

Accepted Manuscript
Synthesis, biological evaluation and docking study of a new series of di-substi-
tuted benzoxazole derivatives as selective COX-2 inhibitors and anti-inflam-
matory agents
Avneet Kaur, Dharam P. Pathak, Vidushi Sharma, Sharad Wakode
PII:
S0968-0896(17)32275-7
https://doi.org/10.1016/j.bmc.2018.01.007
BMC 14161
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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3 January 2018
10 January 2018
Please cite this article as: Kaur, A., Pathak, D.P., Sharma, V., Wakode, S., Synthesis, biological evaluation and
docking study of a new series of di-substituted benzoxazole derivatives as selective COX-2 inhibitors and anti-
inflammatory agents, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.01.007
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Synthesis, biological evaluation and docking study of a new series of di-substituted
benzoxazole derivatives as selective COX-2 inhibitors and anti-inflammatory agents.
Avneet Kaura, Dharam P. Pathaka, Vidushi Sharmaa, Sharad Wakodea*.
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Delhi Institute of
Pharmaceutical Sciences and Research (DIPSAR), New Delhi-110017, India.
*Corresponding author
Sharad Wakode
Department of Pharmaceutical Chemistry,
Delhi Institute of Pharmaceutical Sciences and Research (DIPSAR),
Mehrauli-Badarpur Road,
PushpVihar, Sector-3,
New Delhi-110017,
India.
E-mail address: sharadwakode.dipsar@gmail.com,
avneetkaur.dipsar@gmail.com,
Contact no. +91- 9891008594
1

Abstract
A new series of substituted-N-(3,4-dimethoxyphenyl)-benzoxazole derivatives 13a-13p was
synthesized and evaluated in vitro for their COX (I and II) inhibitory activity, in vivo anti-
inflammatory and ulcerogenic potential. Compounds 13d, 13h, 13k, 13l and 13n exhibited
significant COX-2 inhibitory activity and selectivity towards COX-2 over COX-1. These selected
compounds were screened for their in vivo anti-inflammatory activity by carrageenan induced rat
paw edema method. Among these compounds, 13d was the most promising analogs of the series
with percent inhibition of 84.09 and IC50 value of 0.04µM and 1.02µM (COX-2 and COX-1)
respectively. Furthermore, ulcerogenic study was performed and tested compounds (13d, 13h,
13k, 13l) demonstrated a significant gastric tolerance than ibuprofen. Molecular docking study
was also performed with resolved crystal structure of COX-2 to understand the binding
mechanisms of newly synthesized inhibitors in the active site of COX-2 enzyme and the results
were found to be concordant with the biological evaluation studies of the compounds. These
newly synthesized inhibitors also showed acceptable pharmacokinetic profile in the in silico
ADME/T analyses.
Keywords: Benzoxazole derivatives, selective COX-2 inhibitors, anti-inflammatory activity,
ulcerogenic liability.
2

1. Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are generally used for the treatment of
inflammation and to relieve pain either associated with surgery or any clinical conditions.1
Inflammation is a multi-step and complex biological response of the body to any harmful
stimulus. It is mediated by the release of pro-inflammatory mediators such as bradykinin and
cytokines, which increases the rate of synthesis of prostaglandin.2 NSAIDs, alters the
biosynthesis of prostaglandin, by the inhibition of cyclooxygenase (COX). It occurs in two
isoforms: COX-1 and COX-2. COX-1, a constitutive isozyme, performs vital functions of gastro
and vascular protection. On the contrary, COX-2 is an inducible isozyme responsible for the
prostaglandin synthesis that triggers inflammatory responses.3 NSAIDs’ available in the market
such as aspirin (1), ibuprofen (2) and indomethacin (3) shows their anti-inflammatory effect
through nonselective inhibition of COX. The adverse effects associated with the chronic use of
these drugs are gastric bleeding,4-7 ulceration8 and kidney problems.9-10 The selective COX-2
inhibitors (coxibs) [ celecoxib (4), valdecoxib (5) and rofecoxib (6) (Figure 1)] are developed for
the treatment of inflammation have shown to produce lower gastrointestinal (GI) side effects.
However, prolonged use of few coxibs found to possess high incidence of cardiovascular
disorders, due to which valdecoxib (5) and rofecoxib (6) are withdrawn from the market11 (Figure
1). Therefore, it is imperative to come out with the scaffolds which have the anti-inflammatory
effect but reduced side effects and improved gastric safety profile.12
Among the family of heterocycles, benzoxazole is known to possess a wide range of
biological activities such as anti-inflammatory,13-17 anti-bacterial,18-20 antifungal,21 anti-
convulsant22 and analgesic activity.23-24 The literature reports reveal that benzoxazole moiety (7-
10) (Figure 1) can be a good template for COX-2 inhibitory activity, with appreciable GI safety
margins.16, 25
Prompted by the aforementioned findings, in the present study, we hereby report the
synthesis and biological evaluation of novel series of di-substituted benzoxazole incorporating
3,4-dimethoxyphenyl ring as less ulcerogenic bioisostere. Besides, an in silico studies was also
conducted to understand the binding mechanism of newly synthesized compounds on the crystal
structure of COX-2 and ADME/T analyses was performed to evaluate their suitability as an
active drug molecule. Computer aided drug design assist in designing selective and potent
3

inhibitors as well as vaccines. Among others, molecular docking approach is one of the most
rational and authentic approaches in the drug design and discovery for studying the molecular
interaction of small molecules.26-28
O
OH
O
H3C
COOH
CH3
O
N
O
CH3
OH
CH3
O
O
H3C
Cl
Ibuprofen
Asprin
Indomethacin
(1)
(2)
(3)
CH3
SO2CH3
SO2NH2
SO2NH2
O
N
O
N
O
N
CH3
F3C
Rofecoxib
Valdecoxib
Celecoxib
(6)
(5)
(4)
O
O
H2
C
H
O
R1
R2
O
N C
N
N
N
H3COOC
N
Cl
R
R
CH3
-N
,
N
R1,R2 =
CH3
OH,OCH3
R= SO2CH3
R=
(9)
(7)
(8)
O
H
N
N
N
H3COOC
S
N
H
C
Ar
Ar= 4-NO2 , 4-Bromophenyl
(10)
Figure 1. Chemical structures of non-selective NSAIDs (1-3), COX-2 selective drugs (4-6), and the reported
benzoxazole derivatives (7-10) with COX-2 activity.
4

2. Results and Discussion
2.1. Chemistry
The title compounds, N-(2-(3,4-dimethoxyphenyl)-benzoxazol-5-yl) substituted
benzamide (13a - 13p), were synthesized as outlined in scheme 1. The compound 2-(3,4-
dimethoxyphenyl)-benzoxazol-5-amine (11) was synthesized by the reaction of 2,4-
diaminophenol dihydrochloride with 3,4-dimethoxybenzoic acid in presence of polyphosphoric
acid (PPA) followed by the synthesis of the compounds (12a – 12p) by the reaction of different
substituted acid with dicyclohexylcarodiimide (DCC). Finally, the title compounds (13a - 13p)
were synthesized by refluxing benzoxazolamine (11) with their respective anhydride (12a – 12p)
in presence of glacial acetic acid (GAA) and zinc (Zn) dust. The poor percentage (%) yield of
some of the prepared compounds may be due to the following reasons: (1) The presence of
contaminants in the reactants and reagents leads to less efficient reaction; (2) The product loss,
due to incomplete extraction or other work-up procedures; (3) The volatilization of products
during workup; (4) The incursions of side reactions leading to the formation of by- products.29
The progress of the reaction was checked by thin layer chromatography (TLC). Structures of
1
prepared analogs were confirmed by elemental analysis, FTIR, H-NMR, 13C-NMR and Mass
spectrometry. The IR spectroscopic data showed the presence of –NH-CO- linkage between 1656
– 1684 cm-1 and NH stretching in the range of 3282 – 3353 cm-1 indicates the synthesis of the
compounds. The impression of IR absorption band at 1154 – 1166 cm-1 in the synthesized
compound (13b, 13c, 13d, 13l) displayed the presence of Ar – Cl group substituted at ortho, meta
and para position of prepared compounds. The presence of Ar - NO2 group in derivatives 13k –
13n displayed symmetric and asymmetric stretches in the range of 1376 – 1394 and 1509 – 1534
cm-1. In case of compound 13o, the IR absorption band at 580 cm-1 corresponds to the C-Br
stretching of aromatic-bromo derivative.
1
The H NMR spectra of all prepared compounds are in full agreement with the proposed
structures; they showed signals corresponding to aliphatic, aromatic and NH protons. All
compounds showed singlet at 3.74 – 4.13 δ ppm due to existence of dimethoxy group. The
aromatic benzylic protons (Hb’) close to benzoxazole appeared as a singlet in the range of 6.45 –
7.36 δ ppm and other aromatic proton appeared as a multiplet peaks within the range 6.84 – 8.77
δ ppm. Compound 13e – 13g showed additional singlet within the range of 2.35 – 2.59 δ ppm due
5

to the presence of methyl group. The compound 13j showed singlet at 5.09 δ ppm due to
existence of hydroxyl group. The NH protons were observed as D2O exchangeable protons. The
elemental analysis data were within ± 0.4% of the theoretical values. Finally, the 13C NMR
spectra of the synthesized compound were recorded in CDCl3 and the spectral signals were in
accordance to the proposed molecular structure. The physicochemical parameters of the
synthesized compounds are presented in Table 1.
O
O
OH
NH2 . 2HCl
O
O
N
(a)
+
HO C
O
O
H2N
H2N
(11)
O
(b)
O
O
O
R C OH
+
+
R C O C R
N C N
N C N
H
H
(12a-12p)
O
O
O
O
O
O
O
+
(c)
O
O
R C O C R
N
R
N
N
H2N
H
(11)
(12)
(13a-13p)
R= 13a = phenyl; 13b = 4-chlorophenyl; 13c = 3-chlorophenyl; 13d = 2-chlorophenyl; 13e = 4-methylphenyl; 13f =
3-methylphenyl; 13g = 2-methylphenyl; 13h = 3,5-dimethoxyphenyl; 13i = 3,4-dimethoxyphenyl; 13j = 4-
hydroxyphenyl; 13k = 4-nitrophenyl; 13l = 2-chloro-4-nitrophenyl; 13m = 2-chloro-5-nitrophenyl; 13n = 3-
nitrophenyl; 13o = 2-bromophenyl; 13p = 4-methoxyphenyl.
Scheme 1: Synthetic scheme of title compounds (13a-13p). Reagent and conditions: (a) PPA, 6-7 h 70-80 °C; (b)
Dichloromethane; (c) Zn dust, Glacial acetic acid, and Dichloromethane.
6

Table 1
Physicochemical parameters of the synthesized compounds (13a-13p)
O
O
O
O
N
R
N
H
Molecular
Melting
point °C
Percentage
yield
Comp.
13a
R
Molecular formula
weight
C22H18N2O4
374
122-125
66%
Cl
C22H17ClN2O4
C22H17ClN2O4
408
408
110-112
70%
13b
13c
100-102
62%
Cl
C22H17ClN2O4
C23H20N2O4
C23H20N2O4
408
388
388
94-96
48%
40%
42%
13d
13e
13f
Cl
H3C
138-140
130-132
H3C
C23H20N2O4
C24H22N2O6
C24H22N2O6
388
434
434
140-142
110-112
114-116
36%
28%
34%
13g
13h
13i
CH3
O
O
O
O
HO
C22H18N2O5
C22H17N3O6
390
419
141-143
122-126
50%
35%
13j
7
O2N
O2N
13k
C22H16ClN3O6
453
154-156
41%
13l
Cl

O2N
C22H16ClN3O6
C22H17N3O6
453
419
143-145
134-136
50%
48%
13m
13n
Cl
O2N
C22H17BrN2O4
C23H20N2O5
453
404
152-156
121-126
36%
42%
13o
13p
Br
O
2.2. Biological evaluation
2.2.1. In vitro cyclooxygenase (COX-1 and COX-2) inhibition assay
The in vitro enzyme assay kit used in this study consisted of ovine COX-1 and human COX-
2, so we performed the multiple sequence alignment of human and ovine COX-1 sequences using
Clustal Omega (Figure 2). The results showed 90% whole sequence identity and 100% conserved
catalytic cavity among the two proteins. With this result, we compared the ability of the
synthesized compounds to inhibit COX-1 and COX-2 using IC50 (µM) values (Table 2). The
results of the in vitro COX-1 and COX-2 inhibitory studies revealed that the synthesized
compounds potentially inhibited COX-2 (IC50 = 0.04 - 45.54 µM range) over COX-1 (IC50 = 1.02
- 58.24 µM range). Further, the selectivity index (SI) was found to be in the range of 1.27 - 25.5.
The results showed that the compounds (13d, 13h, 13k, 13l and 13n) were found to be more
potent inhibitors of COX-2 (IC50 = 0.04 - 0.93 µM range) in comparison to COX-1 (IC50 = 1.02 -
6.02 µM range) (Figure 2) among the synthesized compounds. Compound (13d) was found to be
most potent inhibitor of the series with IC50= 0.04 µM (3.75 fold higher) as compared to
celecoxib (IC50 = 0.15 µM). The most active COX-2 inhibitory benzoxazole derivatives (13d,
13h, 13k, 13l and 13n, IC50 < 1 µM), were further evaluated for their in vivo anti-inflammatory
activity.
8

Figure 2. Multiple sequence alignment of Human COX-1(H-COX-1uniport id: P23219) and Ovine COX-1(O-COX-
1 uniport id: P05979) using Clustal omega. The alignment shows 90% sequence similarity and 100% conserved
catalytic site residues (yellow highlighted).
9

Table 2
IC50 of the synthesized compounds byin vitro COX-1 and COX-2 enzymatic assay and COX-2 selectivity index (SI)
data.
IC50 (µM)a
Compound
SIb
COX-1
6.20
7.32
8.82
1.02
7.84
5.21
9.21
6.02
8.20
58.24
3.23
5.15
10.20
5.52
10.12
16.32
6.20
1.42
COX-2
1.40
1.21
1.39
0.04
1.39
1.56
4.84
0.44
2.70
45.54
0.16
0.46
7.40
0.93
2.24
10.59
0.15
1.08
4.42
6.04
6.34
25.50
5.64
3.33
1.90
13.68
3.03
1.27
20.18
11.19
1.37
5.93
4.51
1.54
41.33
1.31
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
13m
13n
13o
13p
Celecoxib
Ibuprofen
a IC50 value is the concentration of the compound required to produce 50% of inhibition of COX-1 and COX-2
respectively using enzyme immunoassay kit (Catalogue no. 560131, Cayman Chemicals, Inc., Ann Arbor, MI, USA).
bIn vitro COX-2 selectivity index(SI): (COX-1 IC50/COX-2 IC50).
10

100
90
80
70
60
50
40
30
20
10
0
Celecoxib
13n
13l
13k
13h
13d
-3
-2
-1
0
1
log concentration
Figure 3. In vitro percentage inhibition of COX-2 versus log concentration curve of most potent compounds (13d,
13h, 13k, 13l, 13n).
2.2.2. In vivo anti-inflammatory activity
Anti-inflammatory activity of the selected compounds (13d, 13h, 13k, 13l and 13n) was
assessed by the carrageenan induced rat paw edema method. The pharmacological data of
selected compounds is shown in Table 3 (Supplementary Figure S1), which clearly implies that
the synthesized compounds exhibited significant anti-inflammatory properties ranging from 45%
to 84%. Out of the five compounds, four compounds demonstrated better anti-inflammatory
activity than standard drug ibuprofen (65.90%). Compound with 2-chlorophenyl (13d) emerged
as the most promising analog of the series with percentage inhibition of 84.09%. The compounds
with 4-nitrophenyl (13k), 2-chloro-4-nitrophenyl (13l), 3,5-dimethoxyphenyl (13h) also showed
remarkable efficacy against inflammation with percent inhibition of 79.54%, 72.72%, and
68.18% respectively. On the contrary, compound (13n) exhibited weak anti-inflammatory activity
of 54.54% than ibuprofen.
11

Table 3
In vivo anti-inflammatory activity of the most potent compounds using carrageenan-induced rat paw edema method.
Increase in paw edema
Paw edema volume (ml)
(ml)
Compound
%Inhibitionb
0 h
3 h
(Mean ± SEM) a
0.66
0.67
0.71
0.71
0.57
0.7
0.74
0.81
0.8
0.07±0.02
0.14±0.02
0.09±0.02
0.12±0.01
0.20±0.02
0.44±0.05
0.15±0.02
84.09
68.18
79.54
72.72
54.54
-
13d
13h
13k
0.77
0.77
1.14
0.8
13l
13n
Control
0.65
65.90
Ibuprofen
- = not applicable
a) Values are determined after 3 h and are expressed as Mean ± SEM
b)
p < 0.05 (significant difference)
p values were compared with control group (3 h after inducing edema) (Turkey’s test). Number of animals (rats) in
each group = 5.
2.2.3. Acute ulcerogenic activity
Analogs 13d, 13h, 13k and 13l possessing in vivo anti-inflammatory activity greater than
standard drug ibuprofen were further screened for their ulcerogenic activity according to Cioli
method.30 The results (Table 4) (Supplementary Figure S2) showed that the tested compounds
showed better G.I. safety profile with severity index ranging from 0.80 to 2.10, in comparison to
standard drug ibuprofen 2.20 ± 0.44. Most potent compound (13d) showed severity index of 0.80
± 0.44 which was 2.75 folds higher in comparison to the standard drug. Hence, these compounds
may ascertain to have better safety margin on gastric mucosa than ibuprofen.
12

Table 4
In vivo ulcerogenic activity of the most active synthesized compounds in rat model.
Ulcerogenic activity (a)
Compound
(severity index) (b), (c)
(Mean ± SD)
13d (60 mg/kg)
13h (60 mg/kg)
0.80 ± 0.44
2.10 ± 0.27
1.80 ± 0.44
1.00 ± 0.50
2.20 ± 0.44
-
13k (60 mg/kg)
13l (60 mg/kg)
Ibuprofen (60 mg/kg)
Control (normal saline)
a) Number of animals in each group = 5.
b)
Severity index = Mean score of treated group – Mean score of control group.
c) p < 0.05 (significant difference).
2.3. In silico studies
2.3.1. Docking studies and ADME/T analysis
The docking study was performed using resolved crystal structure of human COX-2 (PDB
ID: 5F19). The docking results showed that among all docked molecules, hydrogen bonds with
Arg120 and π-π interaction with Tyr355 is conserved with COX-2 active site (Table 5).
Compounds 13d, 13k and 13l showed the most promising in vitro, in vivo, ulcerogenic potential
and docking score among all of the newly synthesized compounds. In addition, the hydrophobic
cloud was contributed by the Val89, Leu93, Val116, Val349, Leu359, Leu384, Tyr385, Trp387,
Phe518, Val523, Ala527 and Leu531 (Figure 4). Apart from above mentioned residues, some
other residues (Tyr385-13i; Ser530-13k,13l) also found to be involved in interaction and among
them compounds 13k and 13l found to be amongst most promising candidates of the series.
2.3.1.1 Structure Activity Relationship (SAR)
The benzoxazole moiety plays important role in interacting by H-bond with Arg120 (with N-atom
of benzoxazole ring) and π-π interaction (with both aromatic rings of benzoxazole moiety).
Docking score showed that the presence of electron withdrawing chloro group at ortho position
of
phenyl
ring
(13d),
nitro
group
at
para
position
(13k)
13

and ortho – para disubstituted chloro, nitro group on phenyl ring (13l) leads to increase in
activity. Whereas, compound with electron withdrawing nitro group at meta position (13n),
chloro group at para, meta position (13b, 13c) produces moderate activity. Unsubstituted phenyl
ring (13a) also produces significant activity. Substitution of electron donating group at ortho
(13g-methyl), meta (13f-methyl and 13i-methoxy) and para (13e-methyl, 13j-hydroxy, 13p-
methoxy) position of phenyl ring produced detrimental effect on the activity profile of
compounds. The compound 13h was found to be exception which possesses electron donating -
methyl substituent at meta position but showed remarkable in- vitro and in-vivo activity. The
compounds showed comparable docking score to celecoxib.
The celecoxib possessed van der waals interactions with amino acid Val349, Leu384,
Trp387, Leu352, Tyr385, Phe518, Val523 and Ala527 and hydrogen bond with Gln192, Ser353,
Arg513 and Phe518 (Figure 5f). The non-selective inhibitor Ibuprofen showed no H-bond with
COX-2 but salt bridge with Arg120 (Figure 5e). Finally, the Absorption, Distribution,
Metabolism, Excretion and Toxicity (ADME/T) parameters for all synthesized ligands were
calculated using Qikprop 4.0 (Supplementary Table S1). Assessment of ADME/T property is
imperative because they exclude weak or toxic molecule at an early stage of drug discovery and
development process. The desirable ADME/T properties of these compounds make them
promising candidates as COX-2 inhibitors.
14

Figure 4. Docked pose of compound (a) 13d, (b) 13h, (c) 13k, (d) 13l (e) ibuprofen, (f) celecoxib with COX-2. Blue line
indicates hydrogen bond, yellow dashed line indicates hydrophobic interaction, pink dashed line indicates pi-pi stacking and red
line indicates salt bridge.
15

Table 5
Glide score (kcal/mol), type of interactions and interacting residues of the COX-2 protein with synthesized
compounds.
Glide
Type of Interactions
Score
Comp
(kcal/
mol)
Hydrogen bonds
π –Interactions
Dist
(Å)
2.1
2.5
2.3
2.7
2.1
2.6
2.1
1.7
2.2
2.6
2.2
2.6
2.8
2.5
2.3
2.7
2.1
2.5
3.1
2.9
2.5
2.2
1.6
1.6
2.1
1.7
1.5
3.1
2.8
2.7
2.3
2.4
2.8
3.0
2.6
-
Dist
(Å)
5.3
5.2
5.3
5.4
Atom of Amino
Amino
acids
Type
Ring/Group
Benzoxazole
Benzoxazole
Ligand
acids
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Arg120
Arg120
Arg120
Arg120
Arg120
Arg120
Arg120
Arg120
Arg120
Arg120
Arg120
Arg120
Arg120
Arg120
Arg120
Arg120
Arg120
Arg120
Tyr385
Arg120
Arg120
Arg120
Arg120
Ser530
Arg120
Arg120
Ser530
Arg120
Arg120
Arg120
Arg120
Arg120
Arg120
Arg120
Arg120
-
Tyr355
Tyr355
Tyr355
Tyr355
-9.8
-9.9
-9.8
-10.6
-9.0
9.1
π-stacking
π-stacking
π-stacking
π-stacking
π-stacking
π-stacking
π-stacking
π-stacking
13a
13b
13c
13d
13e
13f
Benzoxazole Tyr355
5.3
Tyr355
Benzoxazole
5
Tyr355
5.1
5.4
5.2
5.3
5.2
5.1
5.3
Tyr355
Tyr355
Benzoxazole
Tyr355
Tyr355
Benzoxazole
Tyr355
Tyr355
-8.6
-8.1
Benzoxazole
13g
13h
Benzoxazole Tyr355
Tyr355
5.3
5.4
5.3
N
N
-9.2
-8.7
π-stacking
π-stacking
π-stacking
Benzoxazole
13i
13j
13k
Tyr355
OCH3
N
Tyr355
Benzoxazole
5.3
5.4
N
N
N
NO2
N
N
NO2
N
N
N
N
N
N
N
N
-
Tyr355
-10.4
Benzoxazole Tyr355
Tyr355
5.2
5.1
5.2
-10.5
π-stacking
Benzoxazole
13l
Tyr355
Tyr355
Benzoxazole
5.4
5.3
5.2
5.5
5.4
5.5
5.5
5.4
3.4
-8.0
-10.0
-9.0
π-stacking
π-stacking
π-stacking
13m
13n
13o
Tyr355
Tyr355
Tyr355
Benzoxazole
Tyr355
Tyr355
Benzoxazole
Tyr355
Tyr355
-8.5
-7.3
π-stacking
Benzoxazole
13p
Salt bridge
COOH
Arg120
Ibuprofen
NH2
NH2
SO2
SO2
Gln192
Ser353
Arg513
Phe518
3.5
2.0
2.8
2.6
-10.4
Celecoxib
16

3. Conclusion
Summarizing, a series of substituted-N-(3,4-dimethoxyphenyl)-benzoxazole derivatives (13a
- 13p) were successfully synthesized and evaluated for their selective COX-2 inhibitory potential
and the active compounds were further screened for their anti-inflammatory and ulcerogenic
activity. The structures of the prepared compounds were confirmed by modern analytical
techniques (AT-FTIR, 1H NMR, 13C NMR and mass spectrometry). The docking studies found to
be in line with the experimental data and the emerged SAR showed that the benzoxazole ring
plays important role in interacting with the COX-2 enzyme. Also, electron withdrawing
substitutions have favorable effects on activity of compounds in comparison to the electron
donating substitutions. Among the synthesized compounds; 13d, 13h, 13k and 13l exhibited
significant anti-inflammatory and ulcerogenic potentials. The compound 13d emerged as the
most potent compound of the series with improved gastric safety profile. This series of compound
can be taken as a lead for development of more safe and effective anti-inflammatory agents.
4. Experimental
4.1. Chemistry
The chemical and solvents used were purchased from commercial vendors and used without
purification. The melting points of the prepared analogs were determined on LAB-India MR-VIS
visual melting point apparatus and are uncorrected. The Infrared (IR) spectra were recorded on
Bruker Optics Spectrophotometer. 1H and 13C Nuclear magnetic resonance (NMR) were recorded
in CDCl3 on Bruker, Advance DPX-300 spectrometer. Tetramethylsilane (TMS) was used as
internal standard and chemical shifts (δ) were determined in parts per million (ppm). Mass
spectral data of analogs were recorded on LCMS/LCQ (Agilent, Advantage-Max) instrument,
equipped with electro spray ion (ESI) source. Elemental analyses were carried out on Flash 2000
organic elemental analyzer. The progress of the chemical reaction was monitored using thin layer
chromatography (TLC) on pre-coated plates (Merck, Germany) and compounds were purified
using column chromatography on silica gel (100 - 200 mesh). Iodine vapors and UV-visualizer
were used for detection of spots on TLC plates.
4.2. Synthesis of 2-(3, 4-dimethoxyphenyl)-benzoxazol-5-amine (11)
2-(3,4-Diimethoxyphenyl)benzoxazol-5-amine (11) was prepared by heating 2,4-
diaminophenol dihydrochloride (0.01 mol) and 3, 4-dimethoxybezoic acid (0.01 mol) in presence
17

of cyclizing agent PPA (24 gm) at 70 – 80 °C for 6 - 7 h. After the completion of reaction,
mixture was poured into ice cold water, neutralized with an excess of 10N NaOH and extracted
with toluene, dried over anhydrous sodium sulfate and evaporated under vacuum. The product
obtained was boiled with 100 mg of charcoal in ethanol and filtered. After evaporation, the crude
product obtained was recrystallized from ethanol.
4.3. General procedure for synthesis of anhydride (12a - 12p)
Mixture of substituted acid derivative (0.02 mol) and dicyclohexylcarbodiimide (0.01 mol)
was dissolved in dichloromethane (50 ml) and stirred at room temperature for 3 - 4 h. After then
solvent was separated (12a – 12p), the reaction mixture was filtered to remove the precipitated
dicyclohexylurea and the filtrate was evaporated to get the oily product (12a – 12p)
4.4. General procedure for synthesis of N-(2-(3,4-dimethoxyphenyl)-benzoxazol-5-yl)
substituted benzamide (13a - 13p)
A reaction mixture of benzoxazolamine (1) (0.012mol), respective anhydride (12a - 12p)
(0.01 mol), zinc dust (0.010 gm) and glacial acetic acid (0.01 mol) in DCM (15 ml) was refluxed
for 4-5 h with constant stirring. After completion of the reaction, mixture was poured into ice
cold water and the resultant precipitate was separated and recrystallized with ethanol.
4.4.1. N-(2-(3,4-dimethoxyphenyl)-benzoxazol-5yl)-benzamide (13a)
ATR-FTIR (cm-1): 3284.50 (NH), 2918.20 (-CH), 1680 (-CONH-), 1620.62, 1462 (Aromatic
C=C), 1210.20 (C-N), 1100 (C-O), 732.40 (oop). 1H NMR (300 MHz, CDCl3): δ 4.00 (s, 6H, 2 ×
-OCH3), 6.91-7.01 (m, 3H, Hb’ merged with He’ and Hf’), 7.31-7.33 (d, 1H, J = 6 Hz, H7 of
benzoxazole), 7.39-7.45 (t, 3H, J = 6 Hz, Hc, Hd, He), 7.51-7.54 (d, 2H, J = 9 Hz, H4 , H6 of
benzoxazole), 7.79-7.82 (d, 2H, J = 9 Hz, Hb , Hf), 8.01 (s, 1H , NH). 13C NMR (75 MHz,
CDCl3): δ 56.58, 106.52, 110.52, 112.42, 115.56, 120.72, 127.50, 131.75, 134.99, 135.97,
140.22, 151.41, 162.24, 164.85. MS (m/z): 375 [M +1] +. Anal calc for C22H18N2O4; C, 70.58; H,
4.85; N, 7.48; Found C, 70.56; H, 4.83; N, 7.43.
4.4.2. 4-Chloro-N-(2-(3,4-dimethoxyphenyl)-benzoxazol-5-yl)-benzamide (13b)
ATR-FTIR (cm-1): 3282.60 (NH), 2921.40 (-CH), 1674 (-CONH-), 1630.40, 1466 (Aromatic
C=C), 1220.20 (C-N), 1154.32 (Ar-Cl), 1112 (C-O), 734.20 (oop).1H NMR (300 MHz, CDCl3): δ
4.01 ( s, 6H, 2 × -OCH3), 6.66-6.69 (d, 1H, J = 6 Hz , He’), 6.82 (s, 1H, Hb ‘), 6.97-7.03 (d, 1H, J
18

= 9 Hz, Hf ') , 7.43-7.53 (m, 3H, Hc, Hd and H7of benzoxazole), 7.59-7.61 (d, 2H, J = 6 Hz H4,
H6 of benzoxazole), 8.00-8.04 (d, 2H, J = 12 Hz , Hf, Hb), 8.13 (s, 1H, NH). 13C NMR (75 MHz,
CDCl3): δ 56.24, 106.88, 110.90, 112.46, 112.58, 115.88, 120.86, 128.98, 130.63, 130.86,
+
135.40, 137.11, 145.60, 149.84, 150.39, 162.70, 164.80. MS (m/z): 409 [M +1] . Anal calc for
C22H17ClN2O4; C, 64.63; H, 4.19; N, 6.85; Found C, 64.62; H, 4.22; N, 6.83.
4.4.3. 3-Chloro-N-(2-(3,4-dimethoxyphenyl)-benzoxazol-5-yl)-benzamide (13c)
ATR-FTIR (cm-1): 3302.6 (NH), 2918.4 (-CH), 1662.4 (-CONH-), 1628.26, 1520.26 (Aromatic
C=C), 1482.15 (-CH), 1212.2 (C-N), 1160.20 (Ar-Cl), 1050.23 (C-O), 800.91 (oop).1H NMR
(300 MHz, CDCl3): δ 3.88 (s, 3H, -OCH3), 3.93 (s, 3H, -OCH3), 6.59 (d/s, 2H, He’ merge with
Hb’), 6.97-7.11 (m/br, 3H, H7 of benzoxazole),7.43-7.46 (d, 1H, J = 9 Hz, Hd), 7.64-7.68 (dd,
2H, J = 3Hz, H6 of benzoxazole merge with Hb and Hf), 7.77 (s, 1H, H4 of benzoxazole), 7.97
(s,1H,-NH). 13C NMR (75 MHz, CDCl3): δ 56.22, 106.28, 110.90, 112.44, 119.55, 120.86,
127.68, 130.33, 130.63, 134.40, 135.12, 141.72, 145.63, 149.79, 162.82, 164.65. MS (m/z): 409
[M +1] +. C22H17ClN2O4; C, 64.63; H, 4.19; N, 6.85; Found C, 64.61; H, 4.18; N, 6.83.
4.4.4. 2-Chloro-N-(2-(3,4-dimethoxyphenyl)-benzoxazol-5-yl)-benzamide (13d)
ATR-FTIR (cm-1): 3328.96 (NH), 2910.2 (-CH stretching), 1660.4 (-CONH-), 1625.12, 1524.45
(Aromatic C=C), 1480.19 (-CH bend), 1210.2 (C-N), 1162.50 (Ar-Cl), 1110.62 (C-O), 800.52
(oop).1H NMR (300 MHz, CDCl3): δ 3.91 (s, 6H, 2 × -OCH3), 6.62-6.75 (d/s, 2H, Hb’, He ’),
7.24-7.26 (d, 1H, J = 6 Hz, Hf ’ of benzoxazole), 7.31-7.37 (d, 1H, J = 18 Hz, Hd , H7 of
benzoxazole), 7.57-7.62 (t, 1H, J = 9Hz, He),7.72-7.74 (d, 2H, J = 6Hz, Hc, Hd), 7.84-7.97 (dd,
13
3H, H4, H6 of benzoxazole merge with Hf), 8.25 (s, 1H, -NH). C NMR (75 MHz, CDCl3): δ
56.40, 106.29, 110.98, 112.36, 112.44, 115.80, 119.68, 120.92, 127.64, 129.12, 132.30, 132.40,
+
133.68, 135.18, 141.74, 145.92, 149.99, 150.08, 162.79, 165.84. MS (m/z): 409 [M +1] .
C22H17ClN2O4; C, 64.63; H, 4.19; N, 6.85; Found C, 64.61; H, 4.18; N, 6.83.
4.4.5. N-(2-(3,4-dimethoxyphenyl)-benzoxazol-5-yl)-4-methylbenzamide (13e)
ATR-FTIR (cm-1): 3326.42 (NH), 2992.64 (-CH stretching), 1680.46 (-CONH-), 1630.12,
1474.20 (C=C), 1458.42 (-CH bend), 1100.14 (C-O), 732.42 (oop).1H NMR (300 MHz, CDCl3):
δ 2.35 (s, 3H, CH3), 3.87 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 6.80-6.82 ( d, 1H, J = 4Hz, He’),
6.89 ( s, 1H, Hb’), 6.95-6.98 ( d, 2H, J = 9Hz, Hf ‘ merge with H7 of benzoxazole), 7.47 (s, 1H,
H4 of benzoxazole), 7.51-7.53 (d, 1H, J = 4 Hz He),7.59-7.62 (d, 1H, J = 9 Hz, Hc) , 7.76-7.78
19

(d, 1H, J = 6 Hz, H6 of benzoxazole), 7.88-7.92 (d, 2H, J = 12 Hz, Hf, Hb), 8.01 (s, 1H, NH). 13C
NMR (75 MHz, CDCl3): δ 24.60, 56.22, 106.24, 110.92, 112.36, 115.88, 119.68, 127.53, 130.32,
+
136.67, 147.62, 149.82, 150.36, 162.71, 164.78. MS (m/z): 389 [M +1] . Anal calc for
C23H20N2O4; C, 71.12; H, 5.19; N, 7.21; Found C, 71.10; H, 5.18; N, 7.20.
4.4.6. N-(2-(3,4-dimethoxyphenyl)-benzoxazol-5-yl)-3-methylbenzamide (13f)
ATR-FTIR (cm-1): 3326.22 (NH), 2998.78 (-CH stretching), 1684.67 (-CONH-), 1637.24,
1475.40 (C=C), 1460.82 (-CH bend), 1110.28 (C-O) 800.52 (oop).1H NMR (300 MHz, CDCl3): δ
2.37 (s, 3H, CH3), 3.95 (s, 3H, OCH3), 4.01 (s, 3H, OCH3), 6.87- 6.92 (s/d, 2H, He’, Hf ’), 6.97-
7.00 (d, 1H, J = 9Hz, Hb’), 7.42-7.47 (t, 1H, J = 9Hz, He), 7.50-7.53 (d, 2H, J= 9Hz, Hc and H7
of benzoxazole), 7.59-7.62 (dd, 3H, J1= 3Hz, J2= 3Hz, Hf merge with H4 and H6 of
benzoxazole), 7.75 ( s, 1H, Hb), 8.02 (s, 1H, NH). 13C NMR (75 MHz, CDCl3): δ 23.98, 56.42,
106.95, 110.96, 115.48, 119.52, 120.78, 124.58, 127.44, 127.83, 128.96, 132.55, 149.89, 150.34,
162.91, 164.84. MS (m/z): 389 [M +1] +. Anal calc for C23H20N2O4; C, 71.12; H, 5.19; N, 7.21;
Found C, 71.13; H, 5.20; N, 7.20.
4.4.7. N-(2-(3,4-dimethoxyphenyl)-benzoxazol-5-yl)-2-methylbenzamide (13g)
ATR-FTIR (cm-1): 3323.42 (NH), 2989.64 (-CH stretching), 1680.48 (-CONH-), 1624.11,
1474.23 (C=C), 1458.28 (-CH bend), 1111.72 (C-O) 780.82 (oop).1H NMR (300 MHz, CDCl3): δ
2.59 (s, 3H, -CH3), 3.99 (s, 3H, -OCH3), 6.47 (s, 1H, Hb’), 6.56-6.57 (d, 2H, J= 3 Hz, Hf’, He’),
6.81- 6.88 (t, 2H, J = 12 Hz, He, Hd), 7.41-7.51 (dd, 2H, J1 = 6Hz, J2 = 6Hz, Hf and H6 of
13
benzoxazole), 7.69 (s, 1H, H4 of benzoxazole), 7.99 (s, 1H, NH). C NMR (75 MHz, CDCl3): δ
17.18, 56.58, 106.25, 110.92, 112.36, 112.44, 119.56, 125.98, 127.46, 132.15, 135.30, 141.78,
145.69, 149.84, 162.11, 164.69. MS (m/z): 389 [M +1] +. Anal calc for C23H20N2O4; C, 71.12; H,
5.19; N, 7.21; Found C, 71.11; H, 5.21; N, 7.22.
4.4.8. 3, 5-Dimethoxy-N-(2-(3,4-dimethoxyphenyl)-benzoxazol-5-yl)-benzamide (13h)
ATR-FTIR (cm-1): 3352.64 (NH), 2950.14 (-CH stretching), 1656.04 (-CONH-), 1610.11,
1455.14 (C=C), 1462.48 (-CH bend), 1111.56 (C-O), 810.02 (oop). 1H NMR (300 MHz, CDCl3):
δ 3.81 (s, 6H, 2 × -OCH3), 3.92 (s, 6H, 2 × -OCH3), 6.45 (s, 1H, Hd), 6.56-6.57 (d, 2H, J=3Hz,
He’), 6.84-6.94 (t, 2H, J= 12Hz, Hb’ merged with Hf’), 7.21 (s, 1H, Hb), 7.40-7.51 (dd, 2H,J1=
12Hz, J2= 15Hz, H4 mreged with H7 of benzoxazole), 7.67(s, 2H, Hf merged with H6 of
20

13
benzoxazole), 7.99 (s, 1H, -NH). C NMR (75 MHz, CDCl3): δ 56.40, 106.54, 110.90, 112.33,
112.49, 115.42, 119.58, 120.86, 127.56, 135.15, 141.62, 145.67, 149.82, 149.98, 153.24, 162.11,
164.87. MS (m/z): 435 [M +1] +. Anal calc for C24H22N2O6; C, 66.35; H, 5.10; N, 6.45; Found C,
66.36; H, 5.11; N, 6.46.
4.4.9. 3,4-Dimethoxy-N-(2-(3,4-dimethoxyphenyl)-benzoxazol-5-yl)-benzamide (13i)
ATR-FTIR (cm-1): 3355.42 (NH), 2950.11 (-CH stretching), 1660.04 (-CONH-), 1610.11,
1465.10 (C=C), 1464.52 (-CH bend), 1110.32 (C-O), 800.60 (oop). 1H NMR (300 MHz, CDCl3):
δ 3.94 (s, 6H, 2 × -OCH3), 4.01 (s, 6H, 2 × -OCH3), 6.83-7.03 (br, 3H, He, Hb meged with Hf’),
7.33 (s, 1H, Hb), 7.43-7.45 (d, 1H, J = 6 Hz, H7 of benzoxazole), 7.50-7.53 (d, 1H, J = 9Hz, Hf),
7.59-7.62 (d, 1H, J = 9 Hz, H6 of benzoxazole),7.75 (s, 1H, H4 of benzoxazole), 8.06 (s, 1H, -
NH). 13C NMR (75 MHz, CDCl3): δ 56.24, 103.80, 103.82, 106.80, 110.95, 112.34, 115.82,
119.54, 120.86, 135.12, 141.74, 145.68, 149.87, 150.32, 161.85, 164.85. MS (m/z): 435 [M +1] +.
Anal calc for C24H22N2O6; C, 66.35; H, 5.10; N, 6.45; Found C, 66.37; H, 5.11; N, 6.47.
4.4.10. 4-Hydroxy-N-(2-(3,4-dimethoxyphenyl)-benzoxazol-5-yl)-benzamide (13j)
ATR-FTIR (cm-1): 3330.25 (-OH), 3312.42 (NH), 2989.46 (-CH stretching), 1665.82 (-CONH-),
1615.62, 1448.52 (C=C), 1466.20 (-CH bend), 1112.50 (C-O), 820.16 (oop). 1H NMR (300 MHz,
CDCl3): δ 3.87 (s, 3H, -OCH3), 3.91 (s, 3H,-OCH3), 5.09 (s, 1H, -OH), 6.75-6.84 (dd, 2H, Hc,
He), 6.90-6.91 (d, 1H, J = 3 Hz,He’), 7.36 (d, 2H, Hb’,Hf’), 7.46-7.47 (d, 1H, J= 3Hz, H7 of
benzoxazole), 7.52-7.55 (d, 24, H6 of benzoxazole), 7.65-7.68 (d, J = 9Hz, Hb), 7.76-7.78 (d, 1H
13
J = 6 Hz, Hf), 7.94 (s, 1H, -NH). C NMR (75 MHz, CDCl3): δ 56.28, 106.28, 110.95, 112.36,
115.84, 116.18, 119.56, 128.95, 128.96, 135.10, 141.70, 145.60, 150.39, 161.20, 164.89. MS
(m/z): 391 [M +1] +. Anal calc for C22H18N2O5; C, 67.69; H, 4.65; N, 7.18; Found C, 67.72; H,
4.67; N, 7.19.
4.4.11. N-(2-(3,4-dimethoxyphenyl)-benzoxazol-5-yl)-4-nitrobenzamide (13k)
ATR-FTIR (cm-1): 3286.40 (NH), 2928.52 (-CH stretching), 1666.40 (-CONH-), 1626.10,
1528.20 (C=C), 1532.32, 1394.20 (Ar-NO2), 1488.22 (-CH bend), 1012.32 (C-O) 800.32 (oop).
1H NMR (300 MHz, CDCl3): δ 3.93 (s, 3H, -OCH3), 3.97 (s, 3H, -OCH3), 6.96-6.98 (d, 1H,
J=6Hz, He), 7.31 (s, 1H, Hb’), 7.41-7.48 (dd, 2H, He’ merged with H7 of benzoxazole), 7.80-
7.81 (t, 1H, J = 3 Hz, H4, H6 of benzoxazole), 8.00-8.09 (t, 3H, Hb, Hf merged with –NH), 8.30-
21

8.33 (d, 2H, Hc, He). 13C NMR (75 MHz, CDCl3): δ 56.26, 106.24, 110.92, 112.36, 112.40,
115.83, 119.69, 121.23, 128.44, 135.16, 140.34, 141.79, 145.60, 149.84, 150.36, 151.96, 162.19,
164.88. MS (m/z): 420 [M +1] +. Anal calc for C22H17N3O6; C, 63.01; H, 4.09; N, 10.02; Found
C, 63.02; H, 4.13; N, 10.03.
4.4.12. 2-Chloro-N-(2-(3,4-dimethoxyphenyl)-benzoxazol-5-yl)-4-nitrobenzamide (13l)
ATR-FTIR (cm-1): 3306.2 (NH), 2908.60 (-CH stretching), 1670.43 (-CONH-), 1628.21, 1528.44
(C=C), 1532.43, 1386.60 (Ar-NO2), 1474.42 (-CH bend), 1166.40 (Ar-Cl), 1114.80 (C-O),
1
780.22 (oop). H NMR (300 MHz, CDCl3): δ 3.90 (s, 6H, 2 × -OCH3), 6.90-6.92 (d, 1H, J = 6
Hz, He), 7.40-7.49 (dd, 2H, Hb’, Hf’), 7.53-7.56 (d, 1H, J = 9 Hz, H7 of benzoxazole), 7.65 (s,
1H, H4 of benzoxazole), 7.79 – 7.82 (d, 2H, H6 of benzoxazole merge with Hf), 7.94 – 7.96 (d,
1H,J = 6Hz, He’), 8.11 (s, 1H, -NH). 13C NMR (75 MHz, CDCl3): δ 56.38, 106.54, 110.92,
112.38, 115.82, 119.36, 119.56, 120.81, 124.31, 129.84, 133.26, 135.18, 138.57, 141.75, 145.76,
149.82, 150.31, 162.71, 164.82. MS (m/z): 454 [M +1] +. Anal calc for C22H16ClN3O6; C, 58.22;
H, 3.55; N, 9.26; Found C, 58.23; H, 3.57; N, 9.27.
4.4.13. 2-Chloro-N-(2-(3,4-dimethoxyphenyl)-benzoxazol-5-yl)-5-nitrobenzamide (13m)
ATR-FTIR (cm-1): 3308.80 (NH), 2915.80 (-CH stretching), 1672.21 (-CON.H-), 1630.45,
1525.84 (C=C), 1534.44, 1379.80 (Ar-NO2), 1476.20 (-CH bend), 1162.60 (Ar-Cl), 1110.40 (C-
O), 758.44 (oop). 1H NMR (300 MHz, CDCl3): δ 3.95 (s, 6H, 2 × -OCH3), 6.89-7.01 (m, 3H, He,
Hb, Hf’ ), 7.60-7.63 (d, 1H, H7 of benzoxazole), 7.74 (s, 1H, H4 of benzoxazole), 7.53-7.56 (d,
1H, J = 9 Hz, H7 of benzoxazole), 7.65 (s, 1H, H4 of benzoxazole), 7.84-7.86 (d, 2H, Hc, H6 of
benzoxazole), 8.06 (s, 1H, -NH), 8.23 (d, 1H, Hd). 13C NMR (75 MHz, CDCl3): δ 56.42, 106.24,
110.90, 112.53, 115.86, 119.58, 125.87, 129.90, 133.33, 135.17, 138.40, 141.76, 145.68, 150.61,
162.19, 164.66. MS (m/z): 454 [M +1] +. Anal calc for C22H16ClN3O6; C, 58.22; H, 3.55; N, 9.26;
Found C, 58.24; H, 3.56; N, 9.28.
4.4.14. N-(2-(3,4-dimethoxyphenyl)-benzoxazol-5-yl)-3-nitrobenzamide (13n)
ATR-FTIR (cm-1): 3289.83 (NH), 2938.40 (-CH stretching), 1670.44 (-CONH-), 1628.80,
1526.40 (C=C), 1509.14, 1392.80 (Ar-NO2), 1488.54 (-CH bend), 1018.24 (C-O), 810.12 (oop).
1H NMR (300 MHz, CDCl3): δ 4.02 (s, 3H,-OCH3), 4.13 (s, 3H, -OCH3), 6.90-6.93 (d, 2H, J=
9Hz, He’, Hb’), 6.98-7.01 (d, 1H, J= 9Hz, Hf’), 7.42-7.45 (d, 1H, J= 9Hz, H7 of benzoxazole),
7.52-7.56 (t, 1H, J= 6Hz, He ), 7.63-7.70 (dd, 2H, Hf, H6 of benzoxazole),7.74 (s, 1H, H4 of
22

benzoxazole), 8.01 (s, 1H, -NH), 8.28-8.30 (d, 1H, J = 6 Hz, Hd), 8.74 (s, 1H, Hb). 13C NMR (75
MHz, CDCl3): δ 56.20, 106.64, 110.90, 112.46, 115.78, 129.81, 135.18, 141.70, 145.59, 148.45,
162.70, 164.80. MS (m/z): 420 [M +1] +. Anal calc for C22H17N3O6; C, 63.01; H, 4.09; N, 10.02;
Found C, 63.02; H, 4.11; N, 10.04.
4.4.15. 2-Bromo-N-(2-(3,4-dimethoxyphenyl)benzoxazol-5-yl)benzamide (13o)
ATR-FTIR (cm-1): 3286.2 (NH), 2934.5 (-CH stretching), 1668.6 (-CONH-), 1624.2, 1528.1
1
(C=C), 1486.2 (-CH bend), 1016.24 (C-O), 758.22 (oop), 580 (C-Br). H NMR (300 MHz,
CDCl3): δ 3.88 (s, 6H, 2 × -OCH3), 6.77-6.79 (d, 1H, J = 6 Hz, He’), 6.89-6.91 (d, 1H, J = 6Hz,
Hf’), 7.18 (s, 1H, Hb’), 7.28-7.29 (d, 1H, H7 of benzoxazole), 7.40-7.43 (t, 1H, He), 7.46-7.66
(br, 3H, H4, H6 of benzoxazole merge with Hd),7.74-7.76 (d, 1H, J = 6 Hz, Hc),7.85-7.87 (d, 1H,
13
Hf), 8.12 (s, 1H, -NH). C NMR (75 MHz, CDCl3): δ 56.20, 106.64, 110.90, 112.85, 115.78 ,
+.
119.59,129.81,135.18, 141.70, 145.59,162.70,164.68. MS (m/z): 454 [M +1] Anal calc for
C22H17BrN2O4; C, 58.29; H, 3.78; N, 6.18; Found C, 58.30; H, 3.79; N, 6.19.
4.4.16. 4-Methoxy-N-(2-(3,4-dimethoxyphenyl)benzoxazol-5-yl)benzamide (13p)
ATR-FTIR (cm-1): 3306.22 (NH), 2899.32 (-CH stretching), 1680.72 (-CONH-), 1634.44,
1478.20 (C=C), 1472.12 (-CH bend), 1110.28 (C-O), 810.42 (oop).1H NMR (300 MHz, CDCl3):
δ 3.84 (s, 6H, 2 × -OCH3), 3.89 (s, 3H,-OCH3), 6.86-6.98 (br, 5H, He’, Hb’ merge with Hf’, Hc
and He), 7.53-7.56 (d, 1H, J = 9 Hz, H7 of benzoxazole), 7.61-7.64 (d, 1H, H6 of bezoxazole),
7.73 (s, 1H, H4 of benzoxazole), 7.80-7.84 (dd, 2H, J = 3Hz, Hb, Hf), 8.04 (s, 1H, -NH). 13C
NMR (75 MHz, CDCl3): δ 55.29, 106.34, 110.92, 112.48, 114.74, 115.85, 119.48, 120.88,
+
135.10, 141.79, 145.94, 149.80, 162.72, 164.82. MS (m/z): 419 [M +1] . Anal calc for
C23H20N2O5; C, 68.31; H, 4.98; N, 6.93; Found C, 68.32; H, 4.99; N, 6.95.
5. Biological evaluation
5.1. In vitro cyclooxygenase inhibition assay
The ability of the synthesized compounds to inhibit COX-1 and COX-2 was carried out
by enzyme immunoassay (EIA) using in vitro enzymatic assay kit (catalog no. 560131, Cayman
Chemicals Inc., Ann Arbor, MI, USA). The kit consisted of ovine COX-1 and human
recombinant COX-2. To make the results comparable for in vitro study which is to be performed
on ovine COX-1 and human COX-2, we performed the multiple sequence alignment of Human
and Ovine COX-1 sequences. The sequences of both proteins were retrieved from UniProtKB
23

(Homo sapiens uniport id: P23219) and ovine COX-1 (Ovis aries uniport id: P05979 ) and
multiple sequence analysis was performed using Clustal Omega 31.
The procedure by Consalvi et al.32 was followed to perform the in vitro activity. At the
end, the enzymatic reaction produces distinct yellow color which absorbed at 412-415 nm. The
intensity of the produced color was determined spectrophotometrically (Bio-Rad ELISA), which
is proportional to the amount of PG tracer bound to the well and is inversely proportional to the
quantity of free PG’s present in the well. The inhibitory efficacy of novel derivatives was
calculated by comparison with various control incubations. The efficiencies of the test
compounds that causes 50% inhibition of COX-2 was calculated as IC50 from the log
concentration vs. % inhibition curve.
5.2. In vivo activity
The animals (Wistar albino rats) used in the study were procured from Animal House
Center and were divided and housed in different cages at 25-28 °C, under well maintained
hygienic and environmental conditions with relative humidity of 50–65%, under 12 h light and
dark cycles. All animals were acclimatized for a week before use. All experimental work was
conducted after receiving the approval from Institutional Animal Ethics Committee (IAEC) via
protocol no. IAEC/2015-I/Prot no. 09, 10 and IAEC/2016-I/Prot no. 10, Delhi Institute of
Pharmaceutical Sciences and Research, New Delhi. All results are expressed in Mean ± SEM and
the p- values < 0.01 were statistically significant.
5.2.1. Anti-inflammatory activity
The anti-inflammatory activity of prepared benzoxazole derivatives was evaluated on
Wistar albino rat by carrageenan induced rat paw edema as described by Winter et al.33 The
animals were divided into groups consisting of five rats in each group. Prepared compounds were
administered orally (20 mg/kg b.wt.) and the volume of paw was determined
plethysmographically (Ugo-Basyl, Italy). Control group received equivalent volume of normal
saline and ibuprofen (20 mg/kg b.wt.) was administered orally to the reference group.
Carrageenan (0.1 ml, 1.0% w/v in 0.9% of normal saline) was injected after half an hour into the
sub-plantar tissue of the rat’s hind paw. The paw volume was measured at hourly interval for 3 h
(0, 1, 2 and 3 h) and the percent inhibition of edema was calculated using formula:
% inhibition = (1- (Vs / Vc)) × 100
24

Where,
Vs= paw volume i0n sample treated group
Vc= paw volume in control group.
5.2.2. Acute ulcerogenic activity
The ulcerogenic activity of the prepared analogs was performed according to Cioli et al.30
Each study group consisted of five Wistar albino rats. The animals were fasted for 18 h before the
administration of the test compound, while water was given continuously. The dose quantity was
made three times (60 mg/kg) of the administered dose for anti-inflammatory studies (20 mg/kg).
The control group received only normal saline. After 6 h of the drug administration the rats were
sacrificed, stomach was removed and opened around the greater curvature. Inner lining was
washed properly with distilled water followed by normal saline. The mucosal damage was
examined and number of ulcers and severity index was calculated on a scale of 0 - 3, where: 0 =
no lesions; 0.5 = redness; 1.0 = spot ulcers; 1.5 = hemorrhagic streaks; 2.0 = ulcers > 3 but ≤ 5;
3.0 = ulcers > 5.
5.3. In silico studies
5.3.1. Software
The in-silico experiments were performed on Fujitsu linux workstation (Xeon quad-core
E3-1220 processor). Docking and ADME analyses were carried out using LigPrep 3.0, Impact
6.3, Prime 3.6, Glide 6.3 and QikProp 4.0 modules of Maestro 9.8 (Schrödinger, LLC, New
York, NY, 2014-2). The ligand and protein interacting behavior was studied using Protein-ligand
profiler + server.34
5.3.2. Docking Study
The molecular docking study was performed using crystal structure of human COX-2 (PDBID:
5F19). The acetylated Ser530 of this structure was mutated to Ser530, as this structure is reported
as aspirin-acetylated human COX-235.This protein structure was prepared using Protein
Preparation Wizard (Impact 6.3, Schrodinger) as previously described36-38. In brief, the structure
was processed for addition of hydrogen atoms, formal charges treatment and assignment of
correct bond orders. Structures of the molecules were sketched and prepared using LigPrep 3.0
with Epik 2.8 and tautomeric state and protonation states were expanded at 7.0 ± 2.0 pH units.
The OPLS 2005 force field was used for both molecules and protein minimization. The glide grid
25

was generated by specifying the centroid of the residues His90, Thr94, Arg120, Gln192, Tyr348,
Val349, Leu352, Ser353, Gly354, Tyr355, Leu359, Phe381, Leu384, Tyr385, Trp387, Arg513,
Ala516, Ile517, Phe518, Gly519, Met522, Val523, Gly526, Ala527, Ser530 and Leu531.
Prepared and minimized small molecules were docked into the minimized protein structure using
Glide 6.3 XP docking. The 3D complex structures of all molecules were analyzed for docking
score H-bonding, salt bridge, π-π and π-cation interactions.
5.3.4. Absorption, Distribution, Metabolism, Excretion and Toxicity (ADME/T) study
The ADME/T properties are imperative to decide the role of a new molecule in drug
development process and so are considered as crucial for rational drug design. The lack of best fit
ADME/T parameters of molecules leads to its denunciation in the progressive stages of drug
development process. All the newly synthesized small molecules were analyzed for ADME/T
parameters by QikProp 4.039-42
.
The in silico module provides the vision into vital properties like stability (no. of reactive
functional groups, no of hydrogen bond donor and acceptors, no of metabolic reactions),
druggable behavior and its pharmacokinetics (molecular weight, CNS activity, violation of
Lipinski rule of five, octanol/water coefficient, brain/blood partition coefficient, aqueous
solubility, skin permeability, binding to human serum albumin, human oral absorption , IC50
value for HERG K+ channel, caco-2 cell permeability, MDCK cell permeability)43.
The drug's permeability and transporter interactions during drug discovery and
development process can be successfully accessed by the human colon adenocarcinoma (Caco-2)
and Madin-Darby canine kidney (MDCK) epithelial cell lines which are used as in vitro probes.
This study has considerable importance in predicting absorption, permeability mechanism, effect
of formulation on permeability and probability for transport mediated drug-drug interactions44.
The HERG channel which is voltage gated potassium channel encoded by Human ether-a-go-go
related gene (HERG). These channels are actively involved in cardiac action potential
repolarization. The reduced activity of HERG leads to lengthening of ventricular action potentials
and extension of the QT interval in an electrocardiogram which in turn increases the risk for fatal
ventricular arrhythmias. Therefore, screening of compounds for activity on HERG channels
during preclinical safety studies reduces the risk of failure of compounds because of QT
prolongation45.
26

Acknowledgements
The authors are thankful to Delhi Institute of Pharmaceutical Sciences and Research for providing
infrastructure and other necessary facilities. One of the author, Avneet Kaur is thankful to
Department of Science and Technology (DST), New Delhi for award of fellowship to carry out
this work (INSPIRE_CODE: IF130575, No. DST/INSPIRE Fellowship/2013/495).
Conflict of Interest
The authors have declared no conflict of interest.
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