Synthesis, biological evaluation and docking study of N-(2-(3,4,5-trimethoxybenzyl)benzoxazole-5-yl) benzamide derivatives as selective COX-2 inhibitor and anti-inflammatory agents

Article abstract of DOI:10.1016/j.bioorg.2018.07.007

A series of N-(2-(3,4,5-trimethoxybenzyl)-benzoxazole-5-yl)benzamide derivatives (3a–3n) was synthesized and evaluated for its in vitro inhibitory activity against COX-1 and COX-2. The compounds with considerable in vitro activity (IC₅₀ < 1 μM), were evaluated in vivo for their anti-inflammatory and ulcerogenic potential. Out of the fourteen newly synthesized compounds; 3b, 3d, 3e, 3h, 3l and 3m were found to be most potent COX-2 inhibitors in in vitro enzymatic assay with IC₅₀ in the range of 0.14–0.69 μM. In vivo anti-inflammatory activity of these six compounds (3b, 3d, 3e, 3h, 3l and 3m) was assessed by carrageenan induced rat paw edema method. The compound 3b (79.54%), 3l (75.00%), 3m (72.72%) and 3d (68.18%) exhibited significant anti-inflammatory activity than standard drug ibuprofen (65.90%). Ulcerogenic activity with histopathological studies was performed, and the screened compounds demonstrated significant gastric tolerance than ibuprofen. Molecular Docking study was also performed with resolved crystal structure of COX-2 to understand the interacting mechanisms of newly synthesized inhibitors with the active site of COX-2 enzyme and the results were found to be in line with the biological evaluation studies of the compounds.

Full text of DOI:10.1016/j.bioorg.2018.07.007

Accepted Manuscript
Synthesis, Biological Evaluation and Docking Study of N-(2-(3,4,5-Trimethox-
ybenzyl)Benzoxazole-5-yl) Benzamide Derivatives as Selective COX-2 Inhib-
itor and Anti-inflammatory Agents
Avneet Kaur, Dharam P. Pathak, Vidushi Sharma, Balasubramanian
Narasimhan, Prateek Sharma, Rajani Mathur, Sharad Wakode
PII:
S0045-2068(18)30442-5
DOI:
https://doi.org/10.1016/j.bioorg.2018.07.007
YBIOO 2426
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 May 2018
5 July 2018
6 July 2018
Please cite this article as: A. Kaur, D.P. Pathak, V. Sharma, B. Narasimhan, P. Sharma, R. Mathur, S. Wakode,
Synthesis, Biological Evaluation and Docking Study of N-(2-(3,4,5-Trimethoxybenzyl)Benzoxazole-5-yl)
Benzamide Derivatives as Selective COX-2 Inhibitor and Anti-inflammatory Agents, Bioorganic Chemistry (2018),
doi: https://doi.org/10.1016/j.bioorg.2018.07.007
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Synthesis,
Biological
Evaluation
and
Docking
Study
of
N-(2-(3,4,5-
Trimethoxybenzyl)Benzoxazole-5-yl) Benzamide Derivatives as Selective COX-2 Inhibitor
and Anti-inflammatory Agents
Avneet Kaura, Dharam P. Pathaka, Vidushi Sharmaa, Balasubramanian Narasimhanb, Prateek
Sharmaa, Rajani Mathura, Sharad Wakodea*.
a
Department of Pharmaceutical Chemistry, Delhi Institute of Pharmaceutical Sciences and
Research (DIPSAR), Mehrauli-Badarpur Road, PushpVihar, Sector-3, New Delhi-110017,
India.
b Faculty of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak- 124001, 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 series of N-(2-(3,4,5-Trimethoxybenzyl)-benzoxazole-5-yl)benzamide derivatives (3a-3n) was
synthesized and evaluated for its in vitro inhibitory activity against COX-1 and COX-2.The
compounds with considerable in vitro activity (IC50 < 1 µM), were evaluated in vivo for their
anti-inflammatory and ulcerogenic potential. Out of the fourteen newly synthesized compounds;
3b, 3d, 3e, 3h, 3l and 3m were found to be most potent COX-2 inhibitors in in vitro enzymatic
assay with IC50 in the range of 0.14 – 0.69 µM. In vivo anti-inflammatory activity of these six
compounds (3b, 3d, 3e, 3h, 3l and 3m) was assessed by carrageenan induced rat paw edema
method. The compound 3b (79.54%), 3l (75.00%), 3m (72.72%) and 3d (68.18%) exhibited
significant anti-inflammatory activity than standard drug ibuprofen (65.90%). Ulcerogenic
activity with histopathological studies was performed, and the screened compounds
demonstrated significant gastric tolerance than ibuprofen. Molecular Docking study was also
performed with resolved crystal structure of COX-2 to understand the interacting mechanisms of
newly synthesized inhibitors with the active site of COX-2 enzyme and the results were found to
be in line with the biological evaluation studies of the compounds.
Keywords: N-(3,4,5-trimethoxybenzyl)benzoxazole, COX-1, COX-2, Anti-inflammatory
activity, Ulcerogenic activity, Histopathology
2

1. Introduction
Cyclooxygenase (COX) also known as prostaglandin-endoperoxide synthase (PTGS) is an
enzyme which plays an important role in the formation of prostanoids, including thromboxanes
and prostaglandins such as prostacyclin. Three isoforms of this enzyme are present named as
COX-1, COX-2 and COX-3 [1-2]. COX-1 exists naturally. It is a constitutive isozyme, present in
many tissues and involved in regulation of physiological functions in maintaining homeostasis,
renal function, and gastric mucosal integrity, and the autocrine response to circulating hormones
[3-4]. On contrary, COX-2 isozyme is induced by stimuli such as mitogens, oncogenes, growth
factors, hormones and disorders of water-electrolyte homeostasis that triggers inflammatory
responses [5-7].Whereas, COX-3 does not show any prostaglandin involved COX activity [8].
Prolonged use of classical NSAID’s causes side-effects such as gastric bleeding, ulceration, renal
dysfunction and hepatotoxicity (Nimesulide and Lumiracoxib). These side effects are due to non-
selective inhibition of COX-2 versus COX-1 [9-12].
Development of selective COX-2 inhibitors is a challenge to drug discovery because of
the close structural similarity between both the COX isoforms [13].They both have hydrophobic
tunnel through which the substrate enter the active site of enzyme. The presence of Ile523 in
COX-1 makes it`s binding shorter in comparison to the COX-2 which has Val523 at same place.
This Val523 in COX-2 produces a conformational change in COX-2, thereby forming an
additional hydrophobic secondary internal pocket protruding off the primary binding site which
is absent in COX-1[14]. The selective COX-2 inhibitors (coxibs) [ celecoxib , valdecoxib and
rofecoxib] are developed for the treatment of inflammation have shown to produce lower
gastrointestinal (GI) side effects as compared to traditional NSAID’s. However, prolonged use of
few coxibs alter the COX biochemical pathway results in increased incidences of high blood
pressure and myocardial infarction, due to which valdecoxib and rofecoxib are withdrawn from
the market [15-16]. Therefore, it is essential to come out with the scaffolds which can selectively
and potentially inhibit COX-2 and have an anti-inflammatory effect with fewer side-effects and
improved gastric safety profile [17-19].
Benzamides and benzoxazole derivatives are reported as potential inhibitory candidate
for many biological processes such as anti-inflammatory [20-25], anti-bacterial [25-28],
antifungal [28-29], anti-convulsant [30] and analgesic activity [26, 31-32]. This data also
suggested that benzamides and benzoxazole moiety can be a potent template for COX-2
3

inhibitory activity, with GI safety margins [23, 25, 33-35]. Whereas, 3,4,5-trimethoxybenzyl
moiety also found to plays an important role in anti-inflammatory activity [26].
Taking account of this literature study, previously we reported di-substituted
benzoxazole derivatives as anti-inflammatory molecules [36]. Encouraged by the findings of the
previous work, we extended the work and this present work aimed to synthesize benzoxazole
derivatives where, we combined 3,4,5-trimethoxybenzyl moiety with benzoxazole, benzamides
and reported the synthesis of N-(2-(3,4,5-Trimethoxybenzyl)benzoxazole-5-yl)benzamide
derivatives and their biological evaluation by in vitro COX-1/COX-2 enzyme inhibition assay,
in vivo anti-inflammatory and ulcerogenic activity.
Additionally, to validate our results, an in silico study was conducted to understand the
binding mechanism of newly synthesized compounds on resolved structure of COX-2 from
RCSB protein data bank. ADME parameters were also analyzed to evaluate their suitability as an
active drug candidate. Computer aided drug designing plays an imperative role in designing
selective and potent inhibitors as well as vaccines. Among them, 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 [37-39].
2. Results and discussion
2.1 Chemistry
The synthetic route for formation of title compounds is outlined in scheme 1. 2,4-Diaminophenol
dihydrochloride were reacted with 3,4,5-trimethoxyphenylacetic acid in presence of
polyphosphoric acid to obtain 2-(3,4,5-trimethoxybenzyl)benzoxazol-5-amine (1).The second
reaction was accompanied by reaction of different substituted acids with
dicyclohexylcarbodiimide to form their respective anhydride (2a). These prepared anhydrides
were reacted with benzoxazole amine in presence of Zn dust to accelerate the formation of title
compounds in moderate to good yield. The structure was proven by IR spectral bands of –
NHCO- ranges from 1651 – 1692cm-1 and –NH stretching in the range of 3230 – 3335 cm-1
indicated the synthesis of the compound. The impression of IR absorption band at 1100 – 1160
cm-1in case of compound 3a - 3c and 3m indicated the presence of Ar-Cl group at ortho, meta
and para position (3a - 3c, 3m). The presence of Ar – OH group in compound 3k, displayed
4

peak at 3520 cm-1. The presence of Ar - NO2 group in compound 3l – 3m displayed symmetric
and asymmetric stretches in the range of 1372 – 1386cm-1 and 1528 – 1532 cm-1.
1H NMR spectra showed significant signal of methyl/ethyl, methoxy/dimethoxy and
aromatic protons of benzoxazole ring. Coupling constants (J) are given in Hz and spin multiples
are given as s (singlet), d (doublet), dd (double doublet), t (triplet), m (multiplet) and br (broad).
Synthesized compounds showed singlet around 3.41 - 4.02 δ ppm revealed the presence of -
OCH3 group and singlet around 2.70 - 4.16 δ ppm indicates the presence of linker (-CH2-) group
attach to trimethoxyphenyl group. Compound 3i - 3j and 3e - 3g showed additional singlet due to
the presence of dimethoxy group and methyl group around 3.79 - 3.84 δ ppm and 2.35 - 2.37 δ
ppm respectively. While, in case of compound 3h peak around 1.50 - 4.17 δ ppm corresponds to
presence of ethyl group. The compound 3k showed singlet at 5.92 δ ppm due to the existence of
13
hydroxyl group. The elemental analysis data were within ± 0.4% of the theoretical values. C
NMR spectra of prepared analogues were recorded in CDCl3 and the spectral signals were in
accordance with the proposed structure.
O
O
OH
O
N
70 -80o C
6 -7 h
O
C
H2
C
H2
C
O
+
O
OH
H2N
. 2HCl
H2N
NH2
O
O
(1)
O
Room Temp
3 -4 h
O
O
O
N C N
R C OH
+
+
R C O C R
N C N
H
H
(2a-2n)
O
O
O
O
N
O
H2
C
H2
C
O
O
O
Room Temp
4 h
O
O
+
R C O C R
N
R
N
H2N
H
O
(3a-3n)
5

R= 3a = phenyl; 3b = 4-chlorophenyl; 3c = 3-chlorophenyl; 3d = 2-chlorophenyl; 3e = 4-methylphenyl; 3f = 3-
methylphenyl; 3g = 2-methylphenyl; 3h = 4-ethylphenyl; 3i= 3,5-dimethoxyphenyl; 3j = 3,4-dimethoxyphenyl; 3k =
4-hydroxyphenyl; 3l= 4-nitrophenyl; 3m= 2-chloro-4-nitrophenyl; 3n = 4-methoxyphenyl.
Scheme 1: Synthetic scheme of title compounds (3a-3n). Reagent and conditions: (a) PPA; (b) Dichloromethane;
(c) Zn dust, Glacial acetic acid, and Dichloromethane.
2.2. Biological Screening
2.2.1. In vitro COX-1 and COX-2 inhibitory activities
The inhibitory activity of synthesized compounds 3a-3n was evaluated against ovine COX-1
and human recombinant COX-2 using enzyme immunoassay (EIA) kit. IC50 (µM) values were
determined, the concentration of compound that leads to 50% inhibition of COX enzyme, for an
average of two determinants (Table 1) (Fig. 1). The IC50 values of celecoxib for COX-1 and
COX-2 observed as 6.20 µM and 0.15 µM, respectively. The result of the in vitro COX-1 and
COX-2 inhibitory studies revealed that all the compounds potentially inhibited COX-2 (IC50 =
0.14 - 11.24 µM range) over COX-1 (IC50 = 3.26 – 14.08 µM range). Further, the selectivity
index (SI) found to be in the range of 1.25 - 31.42. The results showed that the compounds 3b
(COX-1:COX-2::4.40:0.14), 3d (COX-1:COX-2::6.10:0.28), 3e (COX-1:COX-2::11.80:0.69),
3h (COX-1:COX-2::7.41:0.98), 3l (COX-1:COX-2::3.26:0.19) and 3m (COX-1:COX-
2::5.76:0.23) were found to be more potent inhibitor of COX-2 in comparison to COX-1 among
all the synthesized compounds. Compound 3b found to be most potent inhibitor of the series
with IC50= 0.14 µM (1.07 fold higher) as compared to celecoxib (IC50=0.15 µM). These six
most active COX-2 inhibitors (3b, 3d, 3e, 3h, 3l and 3m), IC50 < 1 µM, were further evaluated
for in vivo anti-inflammatory activity.
Table 1
IC50 of the synthesized compounds by in vitro COX-1 and COX-2 enzymatic assay and COX-2 selectivity index (SI)
data.
IC50 (µM)a
Compound
SIb
COX-1
8.85
COX-2
1.37
6.45
31.42
5.00
3a
3b
3c
3d
3e
4.40
0.14
6.81
1.36
6.10
0.28
21.78
17.10
11.80
0.69
6

10.84
9.43
4.66
2.29
0.48
7.79
2.12
11.24
0.19
0.23
2.92
0.15
1.08
2.32
4.11
3f
3g
7.41
15.43
1.73
3h
13.55
10.30
14.08
3.26
3i
4.85
3j
3k
1.25
17.15
25.04
2.11
3l
5.76
3m
6.18
3n
6.20
41.33
Celecoxib
1.42
1.31
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).
100
90
80
70
60
50
40
30
20
10
0
0.001µM
0.01µM
0.1µM
1µM
10µM
3b
3d
3e
3h
3l
3m
Compound
Fig 1. In vitro percentage inhibition of COX-2 of most potent compounds (3b, 3d, 3e, 3h, 3l, 3m) at different
concentrations.
2.2.2. In-vivo anti-inflammatory activity
7

Anti-inflammatory activity of the screened compounds (3b, 3d, 3e, 3h, 3l and 3m) was
evaluated in Wistar albino rat by the carrageenan-induced rat paw edema as per the method of
Winter et al. The biological data of selected compounds is shown in Table 2, which clearly
implies that the synthesized compounds exhibited varying degree of anti-inflammatory activity
ranging from 59.09% to 79.54%. Among all the tested compounds, N-(2-(3,4,5-
trimethoxybenzyl)benzoxazol-5-yl)-4-chlorobenzamide (3b) showed the highest anti-
inflammatory activity with 79.54% inhibition than standard drug ibuprofen (65.90%).The other
compounds namely N-(2-(3,4,5-trimethoxybenzyl)benzoxazol-5-yl)-4-nitrobenzamide (3l), N-
(2-(3,4,5-trimethoxybenzyl)benzoxazol-5-yl)-2-chloro-4-nitrobenzamide (3m) and N-(2-(3,4,5-
trimethoxybenzyl)benzoxazol-5-yl)-2-chlorobenzamide (3d) also showed appreciable activity
with 75%, 72.72% and 68.18% inhibition respectively. Whereas, compounds N-(2-(3,4,5-
trimethoxybenzyl)benzoxazol-5-yl)-4-ethylbenzamide
(3h)
and
N-(2-(3,4,5-
trimethoxybenzyl)benzoxazol-5-yl)-4-methylbenzamide (3e) exhibited weak anti-inflammatory
activity with percent inhibition of 61.36 and 59.09 than standard drug.
Table 2
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
0.74
0.68
0.67
0.64
0.71
0.65
0.7
0.84
0.80
0.85
0.81
0.76
0.77
1.14
0.80
0.098± 0.004
0.144±0.029
0.180±0.014
0.174±0.016
0.110±0.010
0.120±0.012
0.440±0.048
0.150±0.022
79.54
68.18
59.09
61.36
75.00
72.72
-
3b
3d
3e
3h
3l
3m
Control
0.65
65.90
Ibuprofen
- = not applicable
a) Values are determined after 3 h and are expressed as Mean ± SEM
8

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
The acute ulcerogenic activity was done for the compounds which exhibited significant anti-
inflammatory activity (% inhibition > standard drug).The four tested compound showed
severity index in the range of 0.70 to 1.80, while standard drug ibuprofen exhibited high severity
index of 2.20 as shown in (Table 3) (Supplementary Fig S1) . Compound N-(2-(3,4,5-
trimethoxybenzyl)benzoxazol-5-yl)-4-chlorobenzamide (3b) showed least severity index of 0.70
followed by N-(2-(3,4,5-trimethoxybenzyl)benzoxazol-5-yl)-4-nitrobenzamide (3l) with a
severity index of 1.20. Compounds namely N-(2-(3, 4, 5-trimethoxybenzyl) benzoxazol-5-yl)-2-
chloro-4-nitrobenzamide
(3m)
and
N-(2-(3,4,5-trimethoxybenzyl)benzoxazol-5-yl)-2-
chlorobenzamide (3d) showed severity index of 1.30 and 1.80 respectively. All the compounds
showed better GI safety profiles than ibuprofen. Compound 3b found to be 3.14 times less
severe on GI than ibuprofen. Thus, it could be proposed that reaction of 2-(3,4,5-
trimethoxybenyl)benzoxazol-5-amine
and
respective
anhydride
to
N-(2-(3,4,5-
trimethoxybenzyl)benzoxazol-5-yl)substituted benzamide (3b, 3d, 3l and 3m) results in
reduction of gastric toxicity.
Table 3
In vivo ulcerogenic activity of the most active synthesized compounds in rat model.
Ulcerogenic activity (a)
Compound
(severity index) (b), (c)
(Mean ± SD)
3b (60 mg/kg)
3d (60 mg/kg)
0.70 ± 0.447
1.80 ± 0.570
1.20 ± 0.447
1.30 ± 0.273
2.20 ± 0.44
-
3l (60 mg/kg)
3m (60 mg/kg)
Ibuprofen (60 mg/kg)
Control (normal saline)
a) Number of animals in each group = 5.
9

b)
Severity index = Mean score of treated group – Mean score of control group.
c) p< 0.05 (significant difference).
2.2.3.1. Histopathological studies
Histopathological study is the examination of biological tissue in order to observe the appearance
of diseased cell in microscopic detail. Examining lesions produced after the drug intake was
performed to explore the ulcerogenic effect of the compounds on stomach in comparison to
standard ibuprofen. The stomach specimen of ibuprofen treated rat showed spot ulcers, increase
in inflammatory cells, redness and perforation of gastric mucosal layer (Fig. 2e).
Histopathological analysis also showed intact lining mucosa with redness and mild increase in
inflammation in superficial stomach mucosa in 3b treated group of animal (Fig. 2a). The tissue
of 3l treated rat showed congested blood vessels with spot ulcers (Fig. 2c), while, 3m in addition
to congested vessels also showed hemorrhagic streaks (Fig. 2d). Scanning of stomach specimens
using electron microscope revealed that the rats treated with 3d, showed hemorrhagic streak and
spot ulcers in superficial stomach mucosa as compared to standard ibuprofen.
10

Fig 2. Histological section of the mucosa of stomach of rats treated with compound a) 3b, b) 3d, c) 3l, d) 3m, e)
ibuprofen, f) control. Black filled arrow indicates hemorrhagic streaks, white arrow indicates spot ulcer and black
line arrow indicates redness and increase in inflammation.
2.2.4. Acute toxicity study
A dose of 2000 mg/kg and 200 mg/kg resulted in death of animals under test and dose of 20
mg/kg was found to be safe as no mortality and behavioral changes was observed.
2.3. In silico studies
2.3.1. Molecular Docking
The molecular 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 are conserved with COX-2 active site. Also, π-π interaction with Tyr355 is
conserved in all molecules (Table 4). In addition to, π-cation interaction with Arg120 was also
observed in the series. Compounds 3b, 3d, 3l and 3m 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 (Fig. 3). Apart from above
mentioned residues, some other residues (Tyr385- 3i, 3l, Met522- 3k, 3l and Ser530- 3m) also
found to be involved in hydrogen bond interaction and among them compounds 3l and 3m found
to be amongst the 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).
In addition to 3,4,5-trimethoxyphenyl, substituent established π-cation interaction with Arg120.
Docking score showed that the presence of electron withdrawing chloro group at ortho and para
position of phenyl ring (3b, 3d), nitro group at para position (3l)
and chloro-nitro di substitution at ortho and para position on phenyl ring (3m) leads to increase
in activity. Whereas, substitution of electron donating methyl and ethyl group at para position
(3e, 3h) produces moderate activity. Activity is found to be decreased with unsubstitued phenyl
ring (3a) and electron withdrawing group substituent at meta position (3c). Also, electron
11

donating group other than methyl or ethyl (3i - 3k, 3n) leads to decrease in activity of
compounds. In addition to, effect of substitution of methyl group also found to be position
specific. Methyl group other than para position, at meta position (3f) of phenyl ring produced
detrimental effect on the activity profile of compounds. 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 (Fig 3f). The non-selective inhibitor Ibuprofen showed no H-bond with
COX-2 but salt bridge with Arg120 (Fig 3e). The substituted benzoxazole derivatives showed
characteristic features to make H-bonds, π-cation and π-stacking with COX-2 (Fig 3, Fig 4).
2.3.2. ADME study
Finally, the absorption, distribution, metabolism and excretion (ADME) parameters for all
synthesized ligands were calculated using Qikprop 4.0 (Table 5). Assessment of ADME
property is imperative because they exclude weak or toxic molecule at an early stage of drug
discovery and development process. The desirable ADME properties of these compounds make
them promising candidates as COX-2 inhibitors.
12

Fig 3. Docked pose of compound (a) 3b, (b) 3d, (c) 3l, (d) 3m, (e) ibuprofen, (f) celecoxib with COX-2. Blue line
indicates hydrogen bond, yellow dashed line indicates hydrophobic interaction and pink dashed line indicates π - π
stacking, green dashed line indicates π -cation and red line indicate salt bridge.
Table 4
13

Glide score (kcal/mol), type of interactions and interacting residues of the COX-2 protein with synthesized
compounds.
Glide
Type of Interactions
Score
(kcal/
mol)
Hydrogen bonds
π –Interactions
Comp
Atom
of
Dist
(Å)
Dist
(Å)
Amino
acids
Amino
acids
Type
Ring/Group
Ligand
N
N
N
N
N
N
Arg120
Arg120
Arg120
Arg120
Arg120
Arg120
2.6
2.2
2.1
1.7
2.8
2.4
π-stacking
π-cation
π-stacking
π-cation
π-stacking
π-cation
π-stacking
π-stacking
π-cation
π-stacking
π-cation
π-stacking
π-cation
π-stacking
π-cation
Benzoxazole
Tyr355
5.3
3.8
4.9
3.5
5.4
3.7
5.0
4.9
3.6
5.1
3.9
5.2
3.8
5.3
3.7
5.2
3.7
5.4
3.7
-9.6
-10.5
-9.4
3a
3b
3c
3,4,5-trimethoxyphenyl Arg120
Benzoxazole Tyr355
3,4,5-trimethoxyphenyl Arg120
Benzoxazole Tyr355
3,4,5-trimethoxyphenyl Arg120
Benzoxazole
Benzoxazole
Tyr355
Tyr355
N
Arg120
2.1
-10.8
3d
N
N
N
N
N
N
N
N
N
N
N
OCH3
N
N
N
N
OH
N
N
Arg120
Arg120
Arg120
Arg120
Arg120
Arg120
Arg120
Arg120
Arg120
Arg120
Arg120
Tyr385
Arg120
Arg120
Arg120
Arg120
Met522
Arg120
Arg120
Tyr385
Met522
Arg120
Arg120
Ser530
Arg120
1.6
2.5
2.1
2.5
2.1
2.8
2.4
2.4
2.0
2.6
2.2
2.8
2.5
2.1
2.2
2.8
2.6
2.2
1.6
2.1
2.2
2.2
1.6
2.5
2.9
3,4,5-trimethoxyphenyl Arg120
Benzoxazole Tyr355
3,4,5-trimethoxyphenyl Arg120
Benzoxazole Tyr355
3,4,5-trimethoxyphenyl Arg120
Benzoxazole Tyr355
3,4,5-trimethoxyphenyl Arg120
Benzoxazole Tyr355
3,4,5-trimethoxyphenyl Arg120
Benzoxazole Tyr355
3,4,5-trimethoxyphenyl Arg120
-10
-9.3
-9.5
-10.2
3e
3f
3g
3h
π-stacking
π-cation
π-stacking
π-cation
-8.8
-9.7
-9.6
3i
3j
π-stacking
π-cation
π-stacking
π-stacking
π-cation
Benzoxazole
3,4,5-trimethoxyphenyl Arg120
Benzoxazole
Benzoxazole
3,4,5-trimethoxyphenyl Arg120
Benzoxazole Tyr355
Tyr355
5.2
3.8
5.3
5.4
3.7
5.1
Tyr355
Tyr355
3k
π-stacking
-10.5
-10.6
3l
NO2
NO2
N
N
NO2
N
π-cation
3,4,5-trimethoxyphenyl Arg120
3.6
π-stacking
π-stacking
π-cation
Benzoxazole
Benzoxazole
3,4,5-trimethoxyphenyl Arg120
Benzoxazole
Benzoxazole
3,4,5-trimethoxyphenyl Arg120
Tyr355
Tyr355
5.1
4.9
3.7
5.3
5.2
3.7
3m
3n
π-stacking
Tyr355
Tyr355
-9.7
-7.4
N
Arg120
-
2.5
π-cation
Ibuprof
en
Salt bridge
COOH
Arg120
3.5
NH2
NH2
SO2
SO2
Gln192
Ser353
Arg513
Phe518
3.6
2.1
2.8
2.7
Celecox
ib
-
-
-
-
-
-
-
-
-
-
-
-
-10.5
14

Fig 4. 2D sketch of N-(2-(3,4,5-trimethoxybenzyl)-benzoxazole-5-yl) benzamide derivatives. The region making
different type of interactions is encircled in different colors
15

Table 5. Predicted absorption, distribution, metabolism and excretion (ADME) properties of
synthesized compounds.
d
#
o
r
#
m
e
n
acc QPl
pt ogP
HB o/w
QP
% Human Rule
t
mol_
MW
SAS
A
QPPCa QPlogB
Comp
CNS
FOSA
FISA
o
Log
oral
of
v
co
B
t
r
Khsa absorption
five
F
G
a
b
H
B
418.4 747.4
452.8 770.8
452.8 767.0
452.8 762.3
432.4 775.4
432.4 775.3
432.4 766.5
290.5
292.9
290.5
290.5
378.7
378.6
364.2
62.1
63.7
63.7
56.1
63.8
63.7
52.3
6.7
6.7
6.7
6.7
6.7
6.7
6.7
4.7 2549.28
5.2 2461.40
5.2 2462.71
5.1 2906.82
5.0 2459.47
5.0 2460.19
5.0 3158.80
-0.54
-0.39
-0.39
-0.33
-0.57
-0.57
-0.43
5
5
5
5
6
6
6
0.55
0.671
0.66
100.00
100.00
100.00
100.00
100.00
100.00
100.00
0
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
3a
3b
3c
3d
3e
3f
0
0.649
0.703
0.703
0.681
0
0
0
0
3g
3h
3i
0
478.5 821.7
478.5 824.3
434.4 755.4
463.4 780.5
497.8 806.1
448.4 782.7
471.5
466.8
290.5
286.0
286.0
385.7
61.6
63.8
8.2
8.2
7.5
7.7
7.7
7.5
4.9 2576.03
4.9 2458.28
-0.68
-0.71
-1.22
-1.71
-1.50
-0.63
7
7
6
6
6
6
0.546
0.562
0.398
0.484
0.593
0.552
100.00
100.00
100.00
94.69
0
0
0
0
0
0
0
0
0
-1
-1
-2
-2
-2
0
1
1
2
1
1
1
3j
118.4
160.3
150.6
63.6
3.9
4.0
4.5
745.52
298.59
368.91
3k
3l
0
0
0
100.00
3m
3n
4.8 2469.09
100.00
92.78
Ibuprofe
n
0
-1
206.2
477.0
278.8
91.23
1
2.0
3.5
342.27
-0.48
2
0.06
0
Parameters
#rtvFG
Number of reactive functional groups. The presence of these groups can lead to false positives in
HTS assays and to decomposition, reactivity, or toxicity problems in vivo (0-2)
Predicted central nervous system activity -2 (inactive) to 2(active)
Molecular weight of the molecule(130.0 -725.0)
CNS
mol_MW
16

SASA
Total solvent accessible surface area (SASA) in square angstroms using a probe with a 1.4 Å
radius (300.0 – 1000.0). ). It is the surface area of a biomolecule that is accessible to a solvent and
is used in calculating the transfer free energy required to move a biomolecule from aqueous
solvent to a lipid environment.
FOSA
Hydrophobic component of the SASA (saturated carbon and attached hydrogen) (0.00 - 750.0)
Hydrophilic component of the SASA (SASA on N, O, and H on heteroatoms). (7.0 – 330.0)
Estimated number of hydrogen bonds that would be donated by the solute to water molecules in
an aqueous solution (0.0 – 6.0)
FISA
donorHB
accptHB
Estimated number of hydrogen bonds that would be accepted by the solute from water molecules
in an aqueous solution (2.0 – 20.0)
QPlogPo/w
QPPCaco
Predicted octanol/water partition coefficient (-2.0 – 6.5)
Predicted apparent Caco-2 cell permeability in nm/sec. . Caco-2 cells are a model for the gut-
blood barrier (<25 poor >500 great)
QPlogBB
Predicted brain/blood partition coefficient (range or recommended value for 95% of known drug -
3 – 1.2)
#metab
Number of likely metabolic reactions (1 – 8)
QPlogKhsa
% Human
Prediction of binding to human serum albumin. (-1.5 – 15)
Predicted human oral absorption on 0 to 100% scale (25% - 80%). The prediction is based
on a quantitative multiple linear regression model. This property usually correlates well with
human oral absorption, as both measures the same property.
oral absorption
Rule of five
Indicates the number of violation of Lipinski’s rule of five. Compounds that satisfy these rules are
considered drug-like (maximum is 4).
3. Conclusion
A series of N-(2-(3,4,5-Trimethoxybenzyl)-benzoxazole-5-yl) benzamide derivatives (3a- 3n)
were synthesized and evaluated for their in vitro COX- I/II , in vivo anti-inflammatory and
ulcerogenic activity. The structures of the synthesized analogues were confirmed by analytical
1
techniques such as AT-FTIR, H-NMR, 13C-NMR and mass spectrometry. The results of
docking studies found to be concordant with the experimental data and the emerged SAR showed
that the benzoxazole ring plays an important role in interacting with the COX-2 enzyme. Also,
electron withdrawing substitutions at ortho and para positions to phenyl ring have favorable
effects on activity of compounds while substituting the electron donating methyl and ethyl group
at para position also produces significant activity. Among the prepared analogues compounds
3b, 3d, 3l and 3m showed significant anti-inflammatory and ulcerogenic activity. While, the
17

histopathological examination revealed that these screened compounds reduce greatly the
incidence of gastric ulceration. The compound 3b emerged as the most effective compound of
the series with improved safety profile.
4. Experimental
4.1. Chemistry
The chemical reagents and solvents used were procured from commercial vendors and used
without purification. The melting points of the synthesized analogs were determined on LAB-
India MR-VIS visual melting point apparatus and are uncorrected. The Infrared (IR) spectra were
1
recorded on Bruker Optics Spectrophotometer and were expressed in wavenumber (cm-1). H
NMR and 13C NMR were recorded in CDCl3 on Bruker, Avance DPX-300 spectrometer at
300MHz and 75 MHz respectively using tetramethylsilane (TMS) as an internal standard.
Chemical shifts (δ) were determined in parts per million (ppm) and J values are given in Hz.
Mass spectral data of analogs were recorded on API 200 LCMS/MS Applied Biosystem
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 on thin layer
chromatography (TLC) on pre-coated plates (Merck, Germany) and the 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-(3,4,5-Trimethoxybenyl)benzoxazol-5-amine (1)
2-(3,4,5-Trimethoxybenzyl)benzoxazol-5-amine (1) was prepared by heating 2,4-diaminophenol
dihydrochloride (0.01 mol) with 3,4,5-trimethoxyphenylacetic acid (0.01 mol) in presence of
cyclizing agent PPA (0.07 mol). The mixture was heated at 70 - 80 °C for 6 - 7 h. After the
completion of reaction (monitored by TLC), mixture was cooled to room temperature and 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 (2a-2n)
18

To 50 mL of methylene chloride (DCM), substituted acid derivative (0.02 mol) and
dicyclohexylcarbodiimide (DCC) (0.01mol) was added and stirred at room temperature for 3-4
h. After then the solvent was separated (2a-2n), the reaction mixture was filtered to remove
precipitate of dicyclohexylurea and the filtrate was collected, washed three times with 10%
sodium bicarbonate and three time with water to make it free from alkali and dried over
anhydrous magnesium sulfate. The dried extract was filtered, and the solvent was removed
under reduced pressure. The residue was dissolved in ethanol and water was added until
turbidity appeared. It was then kept in freezer overnight. The oily precipitate obtained was
solidified and collected (2a-2n) [40-42].
4.4. General procedure for synthesis of N-(2-(3,4,5-Trimethoxybenzyl)-benzoxazol-5-yl)
substituted benzamide (3a-3n)
A mixture of 1 (0.012mol), respective anhydride 2a-2n (0.01 mol), zinc dust (0.010gm),
glacial acetic acid (0.01 mol) and DCM (15ml) were placed in 100 ml flask. Contents were
refluxed for 4 h with constant stirring at room temperature. It was then poured in ice cold water
and the precipitated product was collected and recrystallized [41-42].
4.4.1. N-(2-(3,4,5-Trimethoxybenzyl)-benzoxazol-5-yl)benzamide (3a)
Yield: 67%. M.P.: 122-124°C. ATR-FTIR (cm-1): 3282.5 (NH), 2926.80 (-CH), 1684.50 (-
1
CONH-), 1628.26, 1476 (Aromatic C=C), 1212.20 (C-N), 1110 (C-O), 733.6 (oop). H NMR
(300 MHz, CDCl3): δ 3.41 (s, 9H, 3× -OCH3), 3.88 (s, 2H, -CH2), 6.50 (s, 2H, Hb’, Hf’), 6.61 (d,
2H, J= 6Hz, Hb, Hf), 6.85 (t, 1H, J= 6Hz, Hd), 7.02 (t, 2H, J= 3Hz, Hc, He), 7.36 - 7.52 (dd, 3H,
J1= 9Hz, J2= 9 Hz, H7, H4 and H6 of benzoxazole), 8.01 (s, 1H, -NH) .13C NMR (75 MHz,
CDCl3): δ 32.76, 55.94, 56.07, 56.60, 106.10, 106.66, 110.96, 127.56, 128.96, 128.99, 130.60,
132.28, 134.33, 136.60, 141.62, 145.67, 150.44, 160.08, 164.67. MS (m/z): 419 [M +1] +. Anal
calc for C24H22N2O5; C, 69.40; H, 5.99; N, 6.70; Found C, 69.43; H, 6.01; N, 6.73.
4.4.2. N-(2-(3,4,5-Trimethoxybenzyl)-benzoxazol-5-yl)-4-chlorobenzamide (3b)
Yield: 70%. M.P.: 132-133°C. ATR-FTIR (cm-1): 3276.2 (NH), 2925.8 (-CH), 1674.20 (-
CONH-), 1620.32, 1458 (Aromatic C=C), 1216.2 (C-N), 1150.21 (Ar-Cl), 1100.20 (C-O), 734.4
(oop).1H NMR (300 MHz, CDCl3): δ 3.65 (s, 9H, 3× -OCH3), 3.89 (s, 2H, -CH2), 6.71 (s, 2H,
19

Hb’, Hf’), 7.06 (t, 2H, J= 12Hz,Hb, Hf), 7.41(t, 2H, J= 6Hz, Hc, He), 7.51 (d, 1H, J= 6Hz, H7 of
benzoxazole), 7.67 (d, 2H, J= 6Hz, H4, H6 of benzoxazole), 8.04 (s, 1H, -NH).13C NMR (75
MHz, CDCl3): δ 32.29, 56.48, 56.71, 57.18, 106.46, 106.69, 110.99, 112.41, 128.29, 128.68,
128.79, 131.70, 132.31, 135.67, 137.78, 141.69, 145.62, 150.74, 160.91, 164.86. MS (m/z): 453
+
[M +1] . Anal calc for C24H21ClN2O5; C, 63.65; H, 4.67; N, 6.19; Found C, 63.68; H, 4.65; N,
6.20.
4.4.3. N-(2-(3,4,5-Trimethoxybenzyl)-benzoxazol-5-yl)-3-chlorobenzamide (3c)
Yield: 62%. M.P.: 126-128°C. ATR-FTIR (cm-1): 3265.20 (NH), 2892.2 (-CH), 1670.14 (-
CONH-), 1616.25, 1450 (Aromatic C=C), 1220.12(C-N), 1100.56 (Ar-Cl), 1108.01 (C-O),
730.23 (oop).1H NMR (300 MHz, CDCl3): δ 3.82 (s, 9H, 3× -OCH3), 4.08 (s, 2H, -CH2), 6.42 (s,
2H, Hb’, Hf’), 6.49 (d, 1H, J= 6Hz, Hd), 6.60 (s, 1H, Hb), 6.88 (d, 1H, J= 15Hz, Hd), 6.98 (t,
1H, J= 12Hz, He), 7.43 (d, 1H, J= 6Hz, H7 of benzoxazole), 7.99 (d, 2H, J= 6Hz, H4 , H6 of
benzoxazole), 8.08 (s, 1H, -NH). 13C NMR (75 MHz, CDCl3): δ 32.23, 56.19, 59.58, 60.86,
106.55, 106.58, 110.76, 112.35, 125.50, 128.21, 130.16, 131.54, 133.51, 134.60, 135.61, 141.72,
+
145.67, 150.57, 160.96, 164.75. MS (m/z): 453 [M +1] . Anal calc for C24H21ClN2O5; C, 63.65;
H, 4.67; N, 6.19; Found C, 63.63; H, 4.69; N, 6.21.
4.4.4. N-(2-(3,4,5-Trimethoxybenzyl)-benzoxazol-5-yl)-2-chlorobenzamide (3d)
Yield: 68%. M.P.: 119-121 °C. ATR-FTIR (cm-1): 3230.14 (NH), 2886.41 (-CH), 1673.42 (-
CONH-), 1620.51, 1456 (Aromatic C=C), 1219.28 (C-N), 1112.20 (Ar-Cl), 1108.06 (C-O),
738.34 (oop).1H NMR (300 MHz, CDCl3): δ 3.80 (s, 11H, 3×-OCH3 merged with –CH2), 6.17 (s,
2H, Hb’, Hf’), 6.52 (d, 1H, J= 3Hz, Hc), 6.86 (t, 2H, J= 15Hz, Hd, He), 7.28 (d, 1H, J= 6Hz,
Hc), 7.61 (d, 1H, J = 3Hz, H7 of benzoxazole), 7.91 (d, 2H, J= 6Hz, H4, H6 of benzoxazole),
8.23 (s, 1H, -NH). 13C NMR (75 MHz, CDCl3): δ 32.24, 56.23,56.26, 56.44, 60.81, 106.26,
106.35, 106.51, 110.88, 112.47, 127.00, 128.92, 129.47, 130.69, 132.39, 133.65, 135.11, 141.79,
+
145.57, 150.76, 150.88, 160.65, 164.82. MS (m/z): 453 [M +1] . Anal calc for C24H21ClN2O5;
C, 63.65; H, 4.67; N, 6.19; Found C, 63.64; H, 4.66; N, 6.17.
4.4.5. N-(2-(3,4,5-Trimethoxybenzyl)-benzoxazol-5-yl)-4-methylbenzamide (3e)
Yield: 61%. M.P.: 138-140 °C. ATR-FTIR (cm-1): 3322.62(NH), 2993.41 (-CH stretching),
1682.06 (-CONH-), 1635.12, 1456.32 (Aromatic C=C), 1460.82 (-CH bend), 1110.14 (C-O),
780.16 (oop).1H NMR (300 MHz, CDCl3): δ 2.37 (s, 3H, -CH3), 3.71 (s, 9H, 3× -OCH3), 4.08 (s,
20

2H, -CH2), 6.60 (s, 2H, Hb’, Hf’), 7.20 - 7.29 (br, 3H, Hc, He and H7 of benzoxazole), 7.45 (d,
1H, J= 9Hz, H4 of benzoxazole), 7.60 (d, 1H, J= 6Hz, H6 of benzoxazole), 7.80 ( d, 2H, J= 9
Hz, Hb, Hf), 7.99 (s, 1H, -NH).13C NMR (75 MHz, CDCl3): δ 24.46, 32.36, 55.94, 56.07, 56.60,
106.10, 106.66, 110.96, 127.56, 128.96, 128.99, 130.60, 132.28, 134.33, 136.60, 141.62, 145.67,
+
150.44, 160.08, 164.67. MS (m/z): 433 [M +1] . Anal calc for C25H24N2O5; C, 69.43; H, 5.59;
N, 6.48; Found C, 69.45; H, 5.62; N, 6.47.
4.4.6. N-(2-(3,4,5-Trimethoxybenzyl)-benzoxazol-5-yl)-3-methylbenzamide (3f)
Yield: 54%. M.P.: 151-153 °C. ATR-FTIR (cm-1): 3318.42(NH), 2853.41 (-CH stretching),
1681.14 (-CONH-), 1621.02, 1476.40 (Aromatic C=C), 1464.80 (-CH bend), 1112.54 (C-O),
810.10 (oop).1H NMR (300 MHz, CDCl3): δ 2.38 (s, 3H, -CH3), 3.78 (s, 9H, 3× -OCH3), 3.99 (s,
2H, -CH2), 6.50 (s, 2H, Hb’, Hf’), 6.74 (d, 3H, J= 9Hz, Hb, Hc, He), 7.06 (d, 2H, J= 21Hz, Hf
and H7 of benzoxazole), 7.33 (s, 1H, H4 of benzoxazole), 7.65 (d, 1H, J= 15Hz, He, Hf and H7
13
of benzoxazole), 7.94 (s, 1H, -NH). C NMR (75 MHz, CDCl3): δ 23.99, 32.09, 56.29, 56.32,
56.66, 106.28, 106.43, 106.59, 110.97, 112.42, 124.56, 127.82, 128.84, 130.62, 132.59, 134.11,
135.17, 136.20, 138.54, 141.71, 145.60, 150.71, 150.74, 160.61, 164.86.MS (m/z): 433 [M +1] +.
Anal calc for C25H24N2O5; C, 69.43; H, 5.59; N, 6.48; Found C, 69.41; H, 5.60; N, 6.50.
4.4.7. N-(2-(3,4,5-Trimethoxybenzyl)-benzoxazol-5-yl)-2-methylbenzamide (3g)
Yield: 54%. M.P.: 151-153 °C. ATR-FTIR (cm-1): 3313.18 (NH), 2850.15 (-CH stretching),
1680.19 (-CONH-), 1619.25, 1471.62 (Aromatic C=C), 1465.54(-CH bend), 1100.56 (C-O),
790.06 (oop).1H NMR (300 MHz, CDCl3): δ 2.35 (s, 3H, -CH3), 3.51 (s, 3H, -OCH3), 3.59 (s,
6H, 2× -OCH3), 3.98 (s, 2H, -CH2), 6.02 (s, 2H, Hb’, Hf’), 7.05 (d, 2H, J = 12Hz, Hc, H7 of
benzoxazole),7.49 (t, 1H, J = 6Hz, He), 7.65 (t, 1H, J = 15 Hz, Hd), 7.82 (d, 2H, H4 and H6 of
benzoxazole), 7.88 (d, 1H, Hf), 8.01 (s, 1H, -NH).13C NMR (75 MHz, CDCl3): δ 17.14, 32.09,
56.27, 56.51, 57.10, 106.25, 106.48, 110.99, 112.40, 125.94, 127.42, 129.29, 130.61, 132.16,
+
135.12, 136.26, 141.81, 150.70, 160.63, 164.84. MS (m/z): 433 [M +1] . Anal calc for
C25H24N2O5; C, 69.43; H, 5.59; N, 6.48; Found C, 69.44; H, 5.62; N, 6.46.
4.4.8. N-(2-(3,4,5-Trimethoxybenzyl)-benzoxazol-5-yl)-4-ethylbenzamide (3h)
Yield: 70%. M.P.: 144-147 °C. ATR-FTIR (cm-1): 3310.10 (NH), 2885.20 (-CH stretching),
1678.26 (-CONH-), 1658.26, 1470.02 (C=C), 1460.62 (-CH bend), 1109.15 (C-O), 785.68
21

(oop).1H NMR (300 MHz, CDCl3): δ 1.47 (t, 3H, J= 12Hz, -CH3), 2.57-2.70 (q, 2H, -CH2*-
CH3), 3.76 (s, 9H, 3× -OCH3), 4.17 (s, 2H, -CH2), 6.42 (s, 2H, Hb’, Hf’), 7.12-7.21 (q, 3H,Hc
merge with He, H7 of benzoxazole), 7.41 (d, 2H, J = 9Hz, H4and H6 of benzoxazole), 7.75 (d,
2H, J = 9Hz, Hb, Hf), 7.96 (s, 1H, -NH).13C NMR (75 MHz, CDCl3): δ 14.56, 32.21, 56.16,
57.21, 60.82, 106.21, 106.49, 110.94, 112.40, 126.99, 127.93, 128.01, 128.24, 130.32, 134.27,
+
136.67, 141.70, 145.62, 150.44, 160.59, 164.84. MS (m/z): 447 [M +1] .Anal calc for
C26H26N2O5; C, 69.94; H, 5.87; N, 6.27; Found C, 69.95; H, 5.85; N, 6.29.
4.4.9. N-(2-(3,4,5-Trimethoxybenzyl)-benzoxazol-5-yl)3,5-dimethoxybenzamide (3i)
Yield: 66%. M.P.: 124-127 °C.ATR-FTIR (cm-1): 3335.24 (NH), 2945.15 (-CH stretching),
1654.42 (-CONH-), 1620.45, 1465.40 (C=C), 1475.58 (-CH bend), 1108.26 (C-O), 810.40
(oop).1H NMR (300 MHz, CDCl3): δ 3.79 (s, 6H, 2× -OCH3), 3.82 (s, 9H, 3× -OCH3), 4.16 (s,
2H, -CH2), 6.20 (s, 3H, Hb, Hf), 6.67 (s/d, 3H, Hb, Hf, H7 of benzoxazole), 7.60 (d, 2H, J =
6Hz, H4, H6 of benzoxazole), 7.85 (s, 1H, -NH).13C NMR (75 MHz,CDCl3): δ 32.13, 55.58,
55.60 , 57.91, 106.12, 107.71, 110.40, 112.09, 115.41, 120.82, 127.58, 130.62, 135.70, 136.25,
141.40, 145.63, 149.94, 150.72, 160.91, 164.84.(+)ESI-MS(m/z): 479 [M+1]+. Anal calcd for
C26H26N2O7; C, 65.26; H, 5.48; N, 5.85; found C, 65.27; H, 5.47; N, 5.60.
4.4.10. N-(2-(3,4,5-Trimethoxybenzyl)-benzoxazol-5-yl)-3,4-dimethoxybenzamide (3j)
Yield: 60%. M.P.: 133-136 °C.ATR-FTIR (cm-1): 3320.15 (NH), 2956.42 (-CH stretching),
1672.20 (-CONH-), 1630.18, 1468.57 (C=C), 1476.25 (-CH bend), 1112.23 (C-O), 790.20
(oop).1H NMR (300 MHz, CDCl3): δ 3.71(s, 6H, 2× -OCH3), 3.94 (s, 9H, 3× -OCH3), 4.12 (s,
2H, -CH2), 6.41 (s, 2H, Hb’, Hf’), 6.55-6.60 (dd, 2H, J1= 6Hz, J2= 6Hz, He, Hf), 7.14 (d, 1H, J
= 3Hz, H7 of benzoxazole),7.52 (s, 2H, Hb, H4 of benzoxazole), 7.64 (d, 1H, J= 6Hz, H6 of
benzoxazole),7.83 (s, 1H, -NH).13C NMR (75 MHz,CDCl3): δ 32.34, 56.00, 56.05, 56.15,
106.14, 106.51, 110.33, 1132.35, 115.47, 120.65, 127.46, 130.81, 136.26, 141.78, 145.67,
149.92, 150.45, 153.57, 160.96, 164.82. (+)ESI-MS(m/z): 479 [M+1]+. Anal calcd for
C26H26N2O7; C, 65.26; H, 5.48; N, 5.85; found C, 65.25; H, 5.47; N, 5.83.
4.4.11. N-(2-(3,4,5-Trimethoxybenzyl)-benzoxazol-5-yl)-4-hydroxybenzamide (3k)
Yield: 57%. M.P.: 125-127 °C.ATR-FTIR (cm-1): 3520.20 (-OH), 3316.18 (NH), 2868.46 (-CH
stretching), 1651.14 (-CONH-), 1618.18, 1460.12 (C=C), 1475.17 (-CH bend), 1100.14 (C-O),
720.58 (oop). 1H NMR (300 MHz, CDCl3): δ 3.60 (s, 3H, -OCH3), 3.84 (s, 6H,2× -OCH3), 4.14
22

(s, 2H, -CH2), 5.92 (s, 1H, -OH), 6.57 (s, 2H, Hb’, Hf’), 6.81- 6.96 (dd, 2H, J1= 6Hz, J2= 3Hz,
Hc, He), 7.48-7.64 (dd, 3H, J1= 9Hz, J2= 6Hz, Hb, Hf, H7 of benzoxazole), 7.86 (s, 1H, H7 of
benzoxazole), 8.08-8.30 (s/d, 2H, H6 merge with –NH). 13C NM R (75 MHz, CDCl3): δ 32.25,
56.17, 56.30, 56.32, 106.22, 106.45, 112.52, 116.30, 126.84, 129.29, 130.68, 135.18, 136.25,
141.62, 145.60, 150.77, 150.79, 160.63, 161.90, 164.80. (+)ESI-MS (m/z): 435 [M+1] +. Anal
calcd for C24H22N2O6; C, 66.35; H, 5.10; N, 6.45; found C, 66.33; H, 5.13; N, 6.46.
4.4.12. N-(2-(3,4,5-Trimethoxybenzyl)-benzoxazol-5-yl)-4-nitrobenzamide (3l)
Yield: 62%. M.P.: 142-144 °C. ATR-FTIR (cm-1): 3282.4 (NH), 2936.4 (-CH stretching), 1660.4
(-CONH-), 1628.5, 1525.5 (C=C), 1528.16, 1386.4 (Ar-NO2), 1484.8 (-CH bend), 1012.20 (C-
O), 725.20 (oop).1H NMR (300 MHz, CDCl3): δ 3.71 (s, 3H, -OCH3), 3.85 (s, 6H, 2 ×-OCH3),
3.93 (s, 2H, -CH2), 6.16 (s, 2H, Hb’, Hf’), 7.09 (s, 1H, H7 of benzoxazole), 7.49 (d, 2H, J= 9Hz,
H4, H6 of benzoxazole), 7.59 (d, 1H, J= 9Hz, Hb), 7.72 (d, 1H, J= 9Hz, Hf), 7.85 (s, 1H,-NH),
8.08(d, 2H, J = 9Hz, He, Hc). 13C NM R (75 MHz, CDCl3): δ 32.13, 56.28, 56.65, 56.96, 106.24,
106.48, 110.87, 112.41, 121.30, 128.31, 128.44, 130.63, 135.13, 136.24, 140.36, 141.78, 145.66,
150.71, 151.93, 160.62, 164.83.(+)ESI-MS(m/z): 464 [M+H]+. Anal calcd for C24H21N3O7; C,
62.20; H, 4.57; N, 9.07; found C, 62.21; H, 4.59; N, 9.06.
4.4.13. N-(2-(3,4,5-Trimethoxybenzyl)-benzoxazol-5-yl)-2-chloro-4-nitrobenzamide (3m)
Yield: 60%. M.P.: 159-161 °C. ATR-FTIR (cm-1): 3310.2 (NH), 2908.6 (-CH stretching), 1659.4
(-CONH-), 1630.4, 1518.4 (C=C), 1532.4, 1372.15 (Ar-NO2), 1475.4 (-CH bend), 1160.18 (Ar-
1
Cl), 1115.40 (C-O), 810.2 (oop). H NMR (300 MHz, CDCl3): δ 3.93 (s, 3H, -OCH3), 3.95 (s,
6H, 2× -OCH3), 4.08 (s, 2H, -CH2), 6.08 (s, 2H, Hb’, Hf’), 7.00 (d, 1H, H7 of benzoxazole), 7.63
(d, 2H, H4, H6 of benzoxazole), 7.74 (s, 1H, -NH), 7.86 (d, 1H, Hf), 8.06(s, 1H, Hc), 8.23 (d,
1H, He). 13C NMR (75 MHz, CDCl3): δ 32.00, 56.19, 56.46, 56.74, 106.36, 106.53, 106.62,
110.88, 112.42, 119.67, 124.56, 129.80, 130.61, 133.24, 135.12, 136.78, 138.81, 141.69, 145.62,
150.7 153.21, 160.63, 164.86.(+)ESI-MS(m/z): 498 [M+H]+. Anal calcd for C24H20ClN3O7; C,
57.90; H, 4.05; N, 8.44; found C, 57.91; H, 4.03; N, 8.45.
4.4.14. N-(2-(3,4,5-Trimethoxybenzyl)-benzoxazol-5-yl)-4-methoxybenzamide (3n)
Yield: 49%. M.P.: 180-182 °C. ATR-FTIR (cm-1): 3308.10 (NH), 2896.30 (-CH stretching),
1692.70 (-CONH-), 1630.40, 1474.68 (C=C), 1476.18 (-CH bend), 1100.20 (C-O), 790.40
(oop).1H NMR (300 MHz, CDCl3): δ 3.96 (s, 3H, -OCH3), 4.02 (s, 9H, 3 × -OCH3), 4.17 (s, 2H,
-CH2), 6.50 (s, 2H, Hb’, Hf’), 6.87-6.99 (m, 4H, Hc, He, Hb and Hf), 7.46 (d, 1H, J = 9Hz, H7
23

of benzoxazole), 7.57 (d, 2H, J = 9Hz, H4, H6 of benzoxazole), 7.88 (s, 1H, -NH). 13C NMR (75
MHz,CDCl3): δ 32.14, 55.61, 55.83, 56.11, 106.16, 106.25, 106.48, 110.91, 112.40, 114.40,
114.41, 126.62, 128.58, 130.60, 135.14, 136.26, 141.72, 145.62, 150.61, 160.72, 164.12,
164.81.(+)ESI-MS (m/z): 449 [M+H] +. Anal calcd for C25H24N2O6; C, 66.95; H, 5.39; N, 6.25;
found C, 66.96; H, 5.41; N, 6.26.
4. 5. Biological Screening
4.5.1. In vitro COX-1 and COX-2 inhibitory activities
The inhibitory activity of synthesized compounds 3a-3n against both cyclooxygenase
COX-1 and COX-2 was determined by the commercial available COX inhibitory screening assay
kit (catalog no. 560131, Cayman Chemicals Inc., Ann Arbor, MI, USA). The kit consisted of
ovine COX-1 and human recombinant COX-2 which exploits enzyme immunoassay to measure
directly PGF2α by stannous chloride reduction of COX derived PGH2. According to the
manufacturer’s protocol, 10 µl of test compounds were incubated for 10 min at 37 °C with assay
buffer, 10 µl of heme and either ovine COX-1 or human recombinant COX-2 enzyme solution
(10 µM). Arachidonic acid (10 µl) was then added and incubated for 2 min at 37 °C. HCl (1M,
10 µl) was further added to stop the enzyme catalysis. Stannous chloride was added to convert
the obtained PGH2to PGF2α. Prostanoids were finally quantified by EIA and their amount was
determined through interpolation from a standard curve. The percent inhibition of target enzyme
by test compounds was calculated by comparing PGF2α produced in a compound treated sample
with that of compound-free, control sample [43]. The efficiencies of the test compounds that
causes 50% inhibition of COX-2 was calculated as IC50 from the log concentration vs. %
inhibition curve using five different concentrations of the inhibitors.
4.6. 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
24

Pharmaceutical Sciences and Research, New Delhi. All results are expressed in Mean ± SEM
and the p- values < 0.01 were statistically significant.
4.6.1. Anti-inflammatory activity
The synthesized benzoxazole derivatives were evaluated for their anti-inflammatory
activity on either sex of Wistar albino rat by adopting carrageenan induced rat paw edema
method [41, 44-45]. The animals were randomly divided into groups comprising of five rats in
each group. Test compounds were administered orally at a dose of 20 mg/kg b.wt. and the
volume of paw was determined using plethysmometer (Ugo-Basyl, Italy). Control group
received equivalent volume of normal saline and the standard drug ibuprofen (20 mg/kg b.wt.)
was administered orally to the reference group and the injection of 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
Where,
Vs= paw volume in sample treated group
Vc= paw volume in control group.
4.6.2. Acute ulcerogenic activity
Acute ulcerogenesis test of the prepared analogs was performed according to the reported
procedure [41,46].Ulcerogenic potential was evaluated 6 h post administration of compound to
rats at a dose of 60 mg/kg. The dose quantity was made three times of the administered dose for
anti-inflammatory studies (20 mg/kg). The control group received only normal saline. The rats
were sacrificed; stomach was removed, inflated 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.
4.6.2.1. Histopathological studies [47-48]
For the histopathological study, a longitudinal section of stomach specimen were fixed into 10%
formalin solution for 24 h at room temperature and embedded in white solid paraffin.
25

Morphological examination was performed with haematoxylin and eosin staining to analyze
histological changes and examined under electron microscope.
4.6.3. Acute toxicity study
Acute oral toxicity test was carried out according the Organization for Economic Co-operation
and Development (OECD) guideline for testing of chemicals number 423. Female Wistar rats
were used in this procedure. They were randomly divided into groups containing three rats each.
First group served as a control and was given equivalent volume of normal saline. The selected
analogue was taken at a dose of 2000, 200 and 20 mg/kg body weight and was given orally to
the remaining groups. All the animals were observed continuously for first 30 min, periodically
during the first 24 h and daily thereafter 14 days for behavioral changes and/or death.
4.7. In silico studies
4.7.1. Software and workstation
The in silico studies were carried out on Fujitsu linux workstation (Xeon quad-core E3-
1220 processor). Docking and ADME studies were performed using LigPrep 3.0, Impact 6.3,
Glide 6.3 and QikProp 4.0 modules of Maestro 9.8 (Schrödinger, LLC, New York, NY, 2014-2)
[49]. The protein and ligand interactions were studied using Protein-ligand profiler server [50].
4.7.2. Molecular docking
The recently resolved crystal structure of human COX-2 (PDBID: 5F19) was used for molecular
docking study. Since, this structure is reported as aspirin-acetylated human COX-2structure
therefore, before carrying out docking, the acetylated Ser530 of this structure was mutated to
Ser530 [51].This protein structure preparation, ligand preparation and grid generation were
performed using Protein Preparation Wizard (Impact 6.3, Schrodinger), Ligprep as previously
described [52-54].
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
26

protein minimization. The glide grid 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 analysed for docking score H-bonding, salt bridge, π-π and π-cation interactions.
4.7.3. ADME study
The ADME properties play important role for deciding the fate of a new molecule in drug
development process and so are vital for rational drug design [55-59]. The lack of optimum
ADME properties of a molecules leads to its 508 rejection in the advanced stages of drug
development process. All the newly synthesized small molecules were 509 analyzed for ADME
parameters by QikProp 4.0. The module provides the insight into critical properties of a 510
molecules which decide its druggable behavior like molecular weight, molecular volume, no. of
H-bond donors and 511 acceptors, polar surface area, predicted octanol/water partition
coefficient and violations related to Lipinski's ‘Rule 512 of 5' [60].
Acknowledgements
The authors are thankful to Delhi Institute of Pharmaceutical Sciences and Research, New Delhi
for providing infrastructure and other necessary facilities for research work. One of the author,
Avneet Kaur is grateful 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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