Synthesis, evaluation and docking studies on 3-alkoxy-4-methanesulfonamido acetophenone derivatives as non ulcerogenic anti-inflammatory agents

Article abstract of DOI:10.1016/j.ejmech.2012.01.018

A series of 3-alkoxy-4-methanesulfonamido acetophenone derivatives were synthesized and evaluated for their anti-inflammatory activity in carrageenan-induced rat paw edema model. The synthesized compounds were also investigated for their gastric ulcerogenic potential. The compounds 4a, 4c and 4d showed comparable anti-inflammatory activity to rofecoxib and indomethacin, the standard drugs taken in both studies and were also non ulcerogenic at the test doses. In silico (docking studies) were done to investigate the hypothetical binding mode of the target compounds to the cyclooxygenase isoenzyme (COX-2). A binding model has been proposed based on the docking studies. Selected physicochemical properties were calculated for theoretical ADME profiling of the compounds and excellent compliance was shown with Lipinski's rules.

Full text of DOI:10.1016/j.ejmech.2012.01.018

European Journal of Medicinal Chemistry 49 (2012) 397e405
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, evaluation and docking studies on 3-alkoxy-4-methanesulfonamido
acetophenone derivatives as non ulcerogenic anti-inflammatory agents
*
Alka Bali , Ruchika Ohri, Pran Kishore Deb
University Institute of Pharmaceutical Sciences, UGC Center of Advanced Study, Panjab University, Chandigarh 160014, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3-alkoxy-4-methanesulfonamido acetophenone derivatives were synthesized and evaluated
for their anti-inflammatory activity in carrageenan-induced rat paw edema model. The synthesized
compounds were also investigated for their gastric ulcerogenic potential. The compounds 4a, 4c and 4d
showed comparable anti-inflammatory activity to rofecoxib and indomethacin, the standard drugs taken
in both studies and were also non ulcerogenic at the test doses. In silico (docking studies) were done to
investigate the hypothetical binding mode of the target compounds to the cyclooxygenase isoenzyme
(COX-2). A binding model has been proposed based on the docking studies. Selected physicochemical
properties were calculated for theoretical ADME profiling of the compounds and excellent compliance
was shown with Lipinski’s rules.
Received 30 June 2011
Received in revised form
7 January 2012
Accepted 10 January 2012
Available online 18 January 2012
Keywords:
Anti-inflammatory activity
Carrageenan-induced paw edema
Docking
Ó 2012 Elsevier Masson SAS. All rights reserved.
In silico
Field align
3D similarity
1. Introduction
The term non-steroidal anti-inflammatory drugs or NSAIDs
refers to a group of drugs with diverse structures of heterogeneous
Inflammation is a protective response of our body that releases
cells and mediators in order to combat foreign substances and to
prevent infections [1]. Prostaglandins, produced by mast cells are
the products of arachidonic acid metabolism which act as media-
tors and play an essential modulatory role in inflammation. Pros-
taglandins PGE2, PGI2 and PGD2 are the powerful vasodilators in
their own right and synergize with other inflammatory vasodila-
tors, accounting for the characteristic vasodilation and erythema at
the site of inflammation [2]. PGE2 is reported to act synergistically
with the primary mediators of inflammation, bradykinin and
histamine producing inflammatory pain and it is considered as
a principal prostaglandin for acute inflammation and chronic
diseases such as rheumatoid arthritis and inflammatory bowel
disease [3]. PGI2 (prostacyclin) acts as a highly potent antith-
rombotic agent by inhibiting platelet aggregation (antithrombo-
genic effect). It is also involved in the maintenance of electrolyte
balance for normal renal function in the kidneys and shows cyto-
protective effect in the gastric mucosa [4e6]. Thromboxane TXA2
found at the site of inflammation is known to have vasoconstrictive
and platelet aggregative effects [4].
chemically unrelated agents, sharing common therapeutic actions
and side effects. These drugs having analgesic, antipyretic (at low
doses) and anti-inflammatory effects (at high doses), are usually
indicated for the treatment of pain, fever and acute or chronic
inflammatory diseases such as osteoarthritis, rheumatoid arthritis,
dysmenorrhea and postoperative pain [7]. All NSAIDs are postu-
lated to disrupt the biosynthesis of the prostaglandins and
thromboxanes by inhibiting the enzyme cyclooxygenase [8,9].
However, inhibition of the gastrointestinal tract or renal prosta-
glandins results in their mechanism based toxicities manifested as
gastric bleeding, life threatening gastrointestinal ulcers and on long
term use it can lead to abnormal renal physiology with resultant
suppression of the renal functions [10,11]. Indomethacin, ketopro-
fen and piroxicam appear to have highest prevalence of gastric
adverse effects, while ibuprofen and diclofenac appear to have
lower rates of gastric side effects [12]. Hence, the therapeutic
usefulness of these potent and effective drugs gets considerably
limited on account of their undesirable side effects and efforts are
underway to design better anti-inflammatory drugs having lacking
gastric and renal side effects.
COX-2 is the inducible form of cyclooxygenase enzyme and
selective COX-2 inhibitors have been introduced in the recent years
to improve upon the profile of traditional NSAIDs (t-NSAIDs).
* Corresponding author. Tel.: þ91 172 2534104; fax: þ91 172 541142.
E-mail addresses: alka.bali@rediffmail.com, alkaa.bali@gmail.com (A. Bali).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.01.018

398
A. Bali et al. / European Journal of Medicinal Chemistry 49 (2012) 397e405
Table 1
However, additional risks associated with several of these agents
such as hepatotoxicity (nimesulide and more recently, lumiracoxib)
and cardiotoxicity (rofecoxib, valdecoxib, parecoxib) has prompted
their early withdrawal from the market in many countries [13]. This
leaves plenty of scope for research to be carried out in this area.
In this context, the present work describes the design, synthesis
and the investigation of anti-inflammatory properties and ulcero-
genic potential of new 3-alkoxy-4-methanesulfonamido aceto-
phenone derivatives based on structural modification of selective
COX-2 inhibitors nimesulide and flosulide. Further, a hypothetical
binding model has been proposed for the compounds with respect
to the target enzyme COX-2 based on in silico docking studies.
Chemical structures of the synthesized compounds.
.
Compound No.
R1
R2
3a
3b
3c
3d
4a
4b
4c
4d
H
H
H
H
n-C4H9
n-C5H11
n-C6H13
cyclohexyl
n-C4H9
n-C5H11
n-C6H13
cyclohexyl
CH3SO2ꢁ
CH3SO2ꢁ
CH3SO2ꢁ
CH3SO2ꢁ
2. Results and discussion
2.1. Design strategy and field alignment studies
Our design strategy involved modification of the structures of
known selective COX-2 inhibitors nimesulide and flosulide in order
to arrive at methanesulfonamido aryl ether class of compounds
(Fig. 1). Chemical structures of the prepared compounds are shown
in Table 1. Structure Activity Relationship (SAR) studies on the
methanesulfonamido aryl ether series of compounds (nimesulide,
flosulide, etc.) have emphasized the importance of an electron
withdrawing moiety at para position with respect to meth-
anesulfonamido moiety for their activity [14]. Amongst these para
substituted methanesulfonamido aryl ethers, the compounds with
nitro grouping are most active followed by cyano and acetyl groups
in decreasing order. Nimesulide and NS-398 contains a nitro group
and Flosulide contains an indanone carbonyl as electron with-
drawing functionalities. An acetyl group of acetophenone system
was included as the electron withdrawing moiety in our series. The
results obtained for 3D similarity are shown in Table 2. The
proposed compounds showed excellent 3D similarity to flosulide
(73e82%) and good similarity to nimesulide. Fig. 2 displays the near
perfect alignment of 4d (maximum 3D similarity) with flosulide
molecule along with very good superposition of field points. As
expected, somewhat lower values were obtained with respect to
the diaryl substituted heterocycles rofecoxib and celecoxib. In all
the cases, similarity values for the compounds possessing meth-
anesulphonamido system (4ae4d) were higher than the corre-
sponding compounds having only amino functionality (3ae3d) in
the same position.
polyphosphoric acid and acetic acid gave 6-acetyl-2-(3H)-benzox-
azolinone 1, which on alkaline hydrolysis yielded 4-amino-3-
hydroxy acetophenone 2 in very good yields. This was followed
by reaction of 2 with selected alkyl bromides employing pyridine/
potassium hydroxide system to give the corresponding 1-(4-
amino-3-alkoxyphenyl)ethanone derivatives 3ae3d. The ether
derivatives 3ae3d were, then subjected to reaction with methane
sulfonyl chloride affording the target compounds 4ae4d in good
yields (65e80%). All the reactions were standardized with respect
to various reaction conditions by monitoring their progress by thin
layer chromatography. The structures of the final products were
authenticated and their purity ascertained by various spectroscopic
techniques including UV, IR, NMR and mass spectroscopic data.
2.3. Pharmacological evaluation
2.3.1. In vivo anti-inflammatory studies
The prepared test compounds 3ae3d and 4ae4d were subjected
to in vivo anti-inflammatory studies using carrageenan-induced rat
paw edema model [16]. Rofecoxib and indomethacin were taken as
standard drugs. Doses were selected by initial titration at different
dose levels. Three dose levels were employed for the standard
drugs as well as the test compounds, i.e., rofecoxib (15; 35; 45 mg/
kg); indomethacin (5; 10; 15 mg/kg); test compounds (25; 50;
100 mg/kg). The standard drugs and the target compounds were
suspended in the vehicle (0.5%w/v solution of carboxy methylcel-
lulose CMC). Solution of carrageenan was prepared in 0.9% saline
solution (900 mg in 100 ml of distilled water). Prior permission of
the Institutional Animal Ethics Committee (IAEC), Panjab Univer-
sity, Chandigarh, India was obtained and all experiments were
conducted according to the approved protocol. All the animals were
allowed free access to food and water (ad libidum), in a constant
lightedark cycle. The general behavior of the animals was normal
during the course of the experiment. Statistical comparison of the
2.2. Synthesis of compounds
Synthetic scheme for the preparation of target compounds
4ae4d is summarized in Fig. 3. In the first step (protection step), 2-
aminophenol was refluxed with urea in presence of concentrated
hydrochloric acid at 150 ꢀC to give the cyclized product 2-(3H)-
benzoxazolinone through a previously reported procedure [15].
Acetylation of the 2-(3H)-benzoxazolinone in the presence of
Fig. 1. Chemical structure correlation of the designed compound series (A) with the structures of the representative drugs.

A. Bali et al. / European Journal of Medicinal Chemistry 49 (2012) 397e405
399
Table 2
results obtained in the test groups with control and standard
groups was carried out using one way ANOVA (p < 0.001) (Jandel
Sigmastat version 2.0) followed by TUKEY test fixing the signifi-
cance level at p < 0.05.
Three dimensional similarity of the test compounds to reference drugs.
Compound No.
Nimesulide
3D SIMILARITY
Flosulide
Rofecoxib
Celecoxib
4a
4b
4c
4d
3a
3b
3c
3d
0.699
0.660
0.649
0.713
0.629
0.623
0.619
0.682
0.752
0.782
0.728
0.818
0.745
0.730
0.737
0.727
0.662
0.663
0.658
0.690
0.592
0.604
0.613
0.618
0.694
0.672
0.677
0.710
0.614
0.618
0.631
0.636
The results obtained for the maximum percentage edema and
maximum percentage inhibition of edema in the control (carra-
geenan treatment), standard (rofecoxib and indomethacin) and test
groups (3ae3d; 4ae4d) are given in Table 3 and graphically rep-
resented in Figs. 4 and 5. The final target compounds 4a, 4c and 4d
were found to demonstrate good anti-inflammatory activity with
the maximum reduction in edema after 5 h ranging from 72.66% to
76.75% which was comparable to those obtained for the standard
drugs rofecoxib and indomethacin (81.80% and 79.74% respec-
tively). Compound 4b gave least reduction in paw edema. ED50
values were calculated for the test compounds and the standard
drugs from the results of the three dose groups. Compound 4a was
found to be the most potent amongst the series and 4b was the
least potent. ED50 values of 4a, 4c and 4d were also found to be
comparable to the standard drugs (Table 3) and suggest good effi-
cacy. In comparison, the compounds 3ae3d lacking the meth-
anesulphonamido function showed very low efficacy in the paw
edema assay producing 9.88e19.02% reduction in edema after 5 h.
These results further emphasize the importance of meth-
anesulphonyl moiety for the anti-inflammatory activity of these
compounds as reported for some other compound series [17,18].
2.3.2. Potential ulcerogenicity
The potential ulcerogenic effects of the compounds 3ae3d and
4ae4d were studied in comparison with rofecoxib and indometh-
acin (Table 3). None of the compounds showed any ulcerogenic
effect at their highest employed doses in the three level doses
studied. Only indomethacin showed red coloration, streaks and
spots which increased with increase in the employed dose levels
viz. 10, 25, 50 and 100 mg/kg.
2.4. In silico evaluation and computational studies
2.4.1. In silico (docking) studies
Docking (in silico) studies were performed on the compounds
4ae4d as well as nimesulide found active in rat paw edema assay in
order to postulate a hypothetical binding model for their interac-
tion with COX-2 isoenzyme using the X-ray crystal structure of
COX-2 (PDB ID: 1CX2). To investigate the ability of molecular
Fig. 2. Alignment of compound 4d (thin violet sticks) with flosulide (shown as green
capped sticks). Spheres, and dodecahedra depict field points for 4d and flosulide
respectively. Cyan, maroon, yellow and brown colors depict negative field, positive
field, surface field and hydrophobic field points. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Synthetic scheme for preparation of the target compounds.

400
A. Bali et al. / European Journal of Medicinal Chemistry 49 (2012) 397e405
Table 3
Results for anti-inflammatory activity and ulcerogenic potential of the tested
compounds.
c
Treatment
Max. % edemaa Max. % reduction ED50
Lesion
in edema from
control group
(mg/kg) score (mm)d
Control
Rofecoxib
100 ꢂ 2.73
0
_
2.50
0
3
18.20 ꢂ 2.70b
81.80
(30 mg/kg)
Indomethacin 20.26 ꢂ 54.23b 79.74
2.00
22.3 ꢂ 1.45
(10 mg/kg)
4a
4b
4c
4d
3a
3b
3c
3d
27.34 ꢂ 2.27b
40.40 ꢂ 5.76b
23.25 ꢂ 3.13b
72.66
59.60
76.75
2.00
8.18
2.30
2.23
e
5
4
4
3
2
2
3
3
26.35 ꢂ 12.65b 73.65
80.17 ꢂ 13.03
90.12 ꢂ 11.79
85.92 ꢂ 12.78
80.98 ꢂ 10.73
13.83
09.88
14.08
19.02
Fig. 5. Observed percentage paw edema in control, standard and test groups at 5h.
_
_
_
at a distance of 2.42782 Å. The nitro group of nimesulide formed
hydrogen bonds with eNH of imidazole ring of His90 at a distance of
2.24116 Å and also formed a hydrogen bond with Arg513 at a distance
a
Values expressed as mean ꢂ SEM at highest amongst the selected three dose
levels after 5 h.
b
c
Statistically significant vs. control (p < 0.001); one way ANOVA.
Calculated from results for anti-inflammatory activity at three graded doses.
Lesions, streaks and red spots noted at highest among the three dose levels
of 2.58613 Å. The phenyl group showed a prominent pep stacking
d
interactionwith the phenyl ring of Tyr355 at 1.92819 Å along with the
van der Waals interactions with Val523 and Met522 at distances of
3.1028 Å and 3.35674 Å respectively.
employed.
docking to reproduce an experimentally observed ligand-binding
mode, the co-crystallized ligand SC-558 (a selective COX-2 inhib-
itor) was used as reference ligand. SC-558 is a highly selective COX-
2 inhibitor belonging to the vicinal diaryl heterocyclic class. These
compounds are characterized by a central carbocyclic or hetero-
cyclic ring system bearing two vicinal aryl moieties. For optimal
activity, one aromatic ring must have a sulphonamide substituent
in the para position. The two fluorines of the trifluoromethyl group
(eCF3) of SC-558 showed strong bonding interactions with the NH2
group of Arg120 at distances of 2.86504 Å and 2.03016 Å. The same
group (eCF3) was also found to be located in the vicinity of amino
acids Leu531, Val116, Leu359, Val349 and Tyr355 which were within
a distance of 4 Å. Further, sulphonamide eNH2 was found to
hydrogen bond with the nitrogen atom of imidazole ring of His90 at
a distance of 2.50664 Å. The same eNH2 group interacted with
Ser353 and Gln192 at a distance of 2.84342 Å and 2.66487 Å. The
sulphonamide oxygen was found to hydrogen bond with His90 and
Arg513 at a distance of 2.11901 Å and 2.42782 Å respectively. The
pyrazole ring of SC-558 was found in the vicinity of Val349 and
The docking studies with compounds 4ae4d suggested that one
of the most important interaction of these compounds with COX-2
enzyme is hydrogen bonding of the sulphonamide oxygen with
Arg120 as seen with nimesulide. Fig. 6 shows the hypothetical
binding model for interactions of nimesulide and the test
compounds 4a, 4c and 4d within the COX-2 active site. A strong
bonding was observed for the compounds 4c, 4a and 4d at
distances of 2.243 Å, 2.422 Å and 2.085 Å respectively. Interestingly,
the compound with least in vivo efficacy i.e., 4b formed the weakest
bond with Arg120 at 3.2712 Å. The sulphonamide oxygen was also
showing strong hydrogen bonding with Tyr355 in compounds 4a
and 4d at distances of 2.096 Å and 1.853 Å respectively. These
interactions were also seen for compound 4c at a distance of
3.533 Å. These additional interactions were found to be insignifi-
cant in 4b. The hydrogen bonding interactions of 4d with exocyclic
eNH2 of His90 and Arg513 Å at distances of 3.373 Å and 1.97122 Å
were quite similar to interaction of nitro group in nimesulide with
the same amino acids. Incidentally, the compound with maximum
in vivo efficacy 4a showed much stronger (>nimesulide) bonding of
the carbonyl oxygen with exocyclic eNH2 of His90 at a distance of
1.7313 Å. Although, interactions with Arg513 Å were not there,
additionally, the methyl group next to the carbonyl group showed
strong interaction with Phe518 at a distance of 2.6521Å. These
interactions were not seen in case of compounds 4b and 4c but the
carbonyl group was located in the vicinity of Trp387 and Tyr385
(within a distance of 4 Å) in 4b and in the vicinity of Trp387, Tyr385
and Gly526 in 4c. Further, the alkyl chain also showed additional van
der Waals interactions with the active site in compounds 4a and 4c.
In 4a, the alkyl chain was interacting with Val523, Tyr387, Ser530 and
Tyr385 at distances of 3.16965 Å, 3.03821 Å, 2.1011 Å and 2.27149 Å
respectively. The alkyl chain of 4c was located in the vicinity of the
amino acids Arg513, Ser353, Tyr355, His90, Val523 and Gln192 and
strong interactions were seen with Ser353 and His90 at distances of
2.01664 Å and 1.87751 Å respectively. The cyclohexyl group of 4d
was also found to show moderate interactions with amino acids
Ser530, Tyr385, Ala527, Val523, Tyr387 and Leu352 within a distance of
3.8 Å. Minimum interactions of the alkyl group were seen in 4b and
Ala527
.
Further, the docking studies performed for nimesulide showed
interactions with Arg120, His90 and Arg513 similar to that of SC-558.
The oxygen atom of methane sulfonyl group of nimesulide formed
a strong hydrogen bond with eNH2 of Arg120 at a distance of
1.62049 Å. The methanesulphonamido group interacted with Val349
alkyl group was present in the vicinity of amino acids His90, Ser353
,
Gln192 and Ile517 within a distance of 4.0 Å. The final DockScore
based on overall interaction energies for the test and standard
compounds is given in Table 4 and the maximum score was ob-
tained for compounds 4a and 4d which had nearly similar values.
Fig. 4. Plot of mean score of change in paw volume at various time intervals for all
treated groups.

A. Bali et al. / European Journal of Medicinal Chemistry 49 (2012) 397e405
401
Fig. 6. Hypothetical binding model for interaction of nimesulide superimposed over SC-558 (yellow) (top left) and compounds 4a (top right), 4c (bottom right) and 4d (bottom left)
showing important H-bonding interactions with amino acid residues (Arg120, His90, Tyr355) in the COX-2 active site. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
A comparison of the docking studies with the observed phar-
macological results suggests a very good correlation as the most
potent test compound 4a was also shown to dock best in the active
the active site and the least efficacious compound (4b) showed
none at all.
site consisting of the amino acids Arg120, His90, Tyr385, Tyr387
,
2.4.2. Computation of physicochemical parameters and ADME
profiling
Tyr355, Ser530, Val523, and Phe518. These studies particularly high-
lighted the importance of two features in the target compounds for
COX-2 inhibition, firstly, the presence of the sulphonamide group
for bonding with Arg120 and Tyr355 and secondly, the presence of
carbonyl group for hydrogen bonding with His90. Both these
interactions were noted for the compounds 4a and 4d which were
also seen to demonstrate the best pharmacological profile. A
slightly lower activity was seen for 4c which may be accounted for
a strong (in fact, strongest amongst the tested compounds) inter-
action with Arg120 and Tyr355, although, no bonding was seen with
His90. The least potent compound 4b showed the weakest inter-
action of the sulphonamide group with Arg120 and Tyr355 and no
significant interaction was seen between the carbonyl group
and His90. The docking studies also suggested the importance of
alkyl group for COX-2 binding as the best compound (4a) amongst
the series also demonstrated best alkyl group interactions with
Table 5 lists the values of selected molecular parameters for
the compounds 4ae4d as well as four representative anti-
inflammatory drugs having gastrointestinal tolerability. Amongst
these, we included the prototypic drugs nimesulide and flosulide
and two drugs belonging to the ‘diaryl substituted heterocycle’
category viz rofecoxib and celecoxib. Computation was done
employing ChemBio3D Ultra version12.0 after carrying out MM2
minimization of the compound structures. Some of these param-
eters such as log P, topological polar surface area, TPSA are recog-
nized parameters for prediction of drug transport properties.
Further, steric and molecular surface descriptors, e.g., SAS, SEV and
ovality and other parameters such as Molecular Topological Index
MTI and Weiner Index WI were calculated.
ADME prediction methods were used to assess the bioavailability
of the active compounds 4ae4d and the reference drugs. Herein, we
calculated the compliance of the prepared compounds to the Lip-
inski’s ‘rule of five’ [19] which has been widely used as a filter for
substances that could likely be further developed in drug design
programs. According to this rule, poor absorption or permeation is
more likely when there are more than five H-bond donors, ten H-
bond acceptors, the molecular weight (MW) is greater than 500 and
the calculated Log P (CLogP) is greater than 5. Molecules violating
more thanone of these rulesmay haveproblems with bioavailability.
Further, TPSA, which is a measure of a molecule’s hydrogen bonding
capacity, is another key property that has been linked to drug
Table 4
DockScore of the active compounds.
Compound No.
Dock score
Rank score
4a
4b
4c
4d
55.247
23.688
39.156
54.543
92.748
66.683
1
4
3
2
Nimesulide
SC-558
Standard
Standard

402
A. Bali et al. / European Journal of Medicinal Chemistry 49 (2012) 397e405
bioavailability. Passively absorbed molecules with a TPSA >140 are
thought to have low oral bioavailability [20]. Predictions of ADME
properties for studied compounds are given in Table 5. The results
show that the synthesized compounds comply with these rules and
the standard drugs also do not show any violation. Theoretically,
these compounds should present good passive oral absorption and
differences in their bioactivity may not be attributed to this aspect.
3. Conclusion
A
series of 3-alkoxy-4-methanesulfonamido acetophenone
derivatives have been designed based on field alignment studies with
nimesulide and flosulide. The pharmacological evaluation of the
synthesized compounds has shown good anti-inflammatory effect in
compounds 4a, 4c and 4d. In silico (docking studies) were done to
investigate the hypothetical binding mode of the target compounds
to the cyclooxygenase isoenzyme (COX-2). The results from the
docking studies were found to be in good agreement with the results
from pharmacological studies and suggested the importance of sul-
phonamido and carbonyl functions for biological activity which also
explains the absence of anti-inflammatory activity in compounds
3ae3d. Further, the compounds comply with Lipinski’s rule of five
which signifies a good absorption and hence, good bioavailability so
that the observed differences in bioactivity of the compounds may be
attributable to the differences in their chemical structures.
4. Experimental protocols
Infrared spectra were recorded in KBr pellets on Perkin Elmer
RX 1 spectrophotometer. Proton NMR was recorded on Bruker
Avance-II, 400 MHz instrument. For NMR, solutions were made in
deuterated chloroform employing tetramethylsilane as internal
reference. Mass spectra were obtained with Vg-11e250 J70S
spectrometer at 70eV using electron ionization (EI source). For
mass spectra, solutions were made in HPLC grade methanol.
4.1. Synthesis of compounds
4.1.1. 6-Acetyl benzo[d]oxazol-2-(3H)-one (1)
To a solution of 5.4 g (40 mmol) benzoxazolinone in 100.0 g of
polyphosphoric acid, 3.6 ml (40 mmol) of glacial acetic acid was
added with stirring. The mixture was heated to a temperature of
90e100 ꢀC, and stirred at this temperature for 3 h. After cooling, the
reaction mixture was added to 1000 ml of cold water. The precip-
itates obtained were filtered, dried and recrystallized from 95%
alcohol to give pure 6-acetylbenzoxazolinone as light brown crys-
tals. Yield 60%. m.p. 222e223 ꢀC. UV (lMax (MeOH)) 323 nm (Vmax
50,661). FTIR (KBr, cmꢁ1): 3401, 3150, 3079, 2979, 2880, 1720, 1680,
1595, 1451, 1385, 1283, 1201, 1036, 765 and 664. 1H NMR (400 MHz;
CDCl3,
d
J): 7.51 (broad s, 1H); 7.26 (dd, 1H, J ¼ 6.5 Hz, 1.7 Hz); 7.24
(d, 1H, J ¼ 3.0 Hz); 7.12 (d, 1H, J ¼ 4.0 Hz); 2.40 (s, 3H). MS [EI, m/z
(relative intensity)]: 177 (100) [M.þ].
4.1.2. 1-(4-Amino-3-hydroxyphenyl)ethanone (2)
To 60 ml of 10% aqueous sodium hydroxide solution in 120 ml
ethanol, was added 6-acetylbenzoxazoline 5.3 g (30 mmol) and
refluxing was carried out for 6 h. After cooling, the solution was
acidified with concentrated hydrochloric acid and then, the satu-
rated solution of sodium carbonate was added slowly till the
effervescence ceased. The precipitates obtained were filtered, dried
and recrystallized from 95% ethanol. Yield 3.84 g (85%). m.p.
168e169 ꢀC. UV (lmax (MeOH)) 331 nm (Vmax 20,940). FTIR (KBr,
cmꢁ1): 3470e3372, 3130, 2922, 2852, 1680, 1508, 1460, 1404, 1260
and 755. 1H NMR (400 MHz; CDCl3,
d J): 7.40 (m, 2H); 7.00 (broad s,

A. Bali et al. / European Journal of Medicinal Chemistry 49 (2012) 397e405
403
1H); 6.90 (d, 1H, J ¼ 10.4 Hz); 5.34 (broad s, 2H); 2.17 (s, 3H). MS [EI,
m/z (relative intensity)]: 151 (100) [M.þ].
dichloromethane. The organic layer was washed with
1
M
hydrocholoric acid and then evaporated to give 3-alkoxy-4-
methanesulfonamido acetophenone. The crude product was
recrystallized from toluene.
4.1.3. General procedure for preparation of 1-(4-amino-3-
alkoxyphenyl)ethanones (3ae3d)
To 4-amino-3-hydroxy acetophenone (30 mmol) dissolved in
21.0 ml of pyridine, 1.6 g (30 mmol) of potassium hydroxide was
suspended into this solution. To the resulting viscous solution,
50 mmol of 1-bromoalkane was added and refluxing was carried
out for 7e8 h. Extraction was carried out with a mixture of 1 M
conc. hydrochloric acid solution and dichloromethane (100 ml,
10:90). Dichloromethane was evaporated under vaccum to give
the crude 4-amino-3-alkoxy acetophenone. The crude product
was recrystallized from toluene.
4.1.4.1. N-(4-acetyl-2-butoxyphenyl)methanesulfonamide (4a). Yield
73%. m.p.110e112 ꢀC. UV (lMax (MeOH)) 206 nm (Vmax 19,843). FTIR
(KBr, cmꢁ1): 3372, 3100, 2922, 2852, 1680, 1508, 1460, 1411, 1370,
1122,1256,1036 and 803. 1H NMR (400 MHz; CDCl3,
d J): 8.30 (d,1H,
J ¼ 6.7 Hz); 7.40 (broad s, 1H); 6.90 (m, 2H); 4.10 (t, 2H, J ¼ 6.6 Hz);
3.00 (s, 3H); 2.30 (s, 3H); 1.80 (quintet, 2H, J ¼ 6.3 Hz); 1.50 (sextet,
2H, J ¼ 5.0 Hz); 1.00 (t, 3H, J ¼ 7.4 Hz). 13C NMR (CDCl3)
d 199.6,
143.9, 120.9, 133.0, 127.8, 117.0, 113.7, 69.5, 43.6, 32.0, 27.2, 19.5, 14.7.
MS [EI, m/z (relative intensity)]: 285 (12.8) [M.þ], 207 (3.0) [M-
H2C]SO2], 151(12.8) [m/z 207eC4H8] , 150 (100) [m/z 207eC4H9],
139 (2.1), 138 (23.8). Anal. Calcd. for C13H19NO4S: C, 54.72; H,
6.71; N, 4.91. Found: C, 54.54; H, 6.42; N, 4.50.
4.1.3.1. 1-(4-Amino-3-butoxyphenyl)ethanone (3a). Yield 40%. m.p.
86e88 ꢀC. UV (lMax (MeOH)) 331 nm (Vmax 11,580). FTIR (KBr,
cmꢁ1): 3399, 3100, 2962, 2950, 1680, 1632, 1488, 1411, 1340, 1256,
1113, 1186, 1024 and 684. 1H NMR (400 MHz; CDCl3,
d
J): 7.40 (d, 1H,
4.1.4.2. N-(4-acetyl-2-pentoxyphenyl)methanesulfonamide(4b). Yield
63%. m.p. 113e115 ꢀC. UV (lMax (MeOH)) 271 nm (Vmax 19,349). FTIR
(KBr, cmꢁ1): 3380, 3150, 3025, 2926, 2854, 1660, 1496, 1453, 1431,
J ¼ 8.1 Hz); 7.10 (m, 2H); 4.10 (t, 2H, J ¼ 7.0 Hz); 3.60 (broad s, 2H);
2.40 (s, 3H); 1.80 (quintet, 2H, J ¼ 7.5 Hz); 1.40 (sextet, 2H,
J ¼ 7.5 Hz); 0.90 (t, 3H, J ¼ 7.0 Hz). 13C NMR (CDCl3)
d
199.8, 146.4,
1400, 1350, 1258, 1157, 1042 and 965. 1H NMR (400 MHz; CDCl3,
d J):
140.8, 125.4, 121.6, 117.6, 112.8, 68.4, 30.7, 24.5, 20.0, 14.9. MS [EI, m/
z (relative intensity)]: 207 (100) [M.þ]. Anal. Calcd. for C12H17NO2: C,
69.54; H, 8.27; N, 6.76. Found: C, 69.30; H, 8.12; N, 6.45.
7.50 (d, 1H, J ¼ 6.7 Hz); 7.30 (broad s, 1H); 6.70 (m, 2H); 4.10 (t, 2H,
J ¼ 6.5 Hz); 3.40 (s, 3H); 2.10 (s, 3H); 1.70 (quintet, 2H, J ¼ 7.3 Hz);
1.40 (m, 4H); 0.90 (t, 3H, J ¼ 7.3 Hz). 13C NMR (CDCl3)
d 199.5, 145.0,
132.3, 125.6, 120.6, 117.1, 113.7, 72.5, 43.5, 29.7, 27.8, 26.3, 22.5, 14.5.
MS [EI, m/z (relative intensity)]: 299 (45.3) [M.þ], 256 (3.9) [M -
COCH3], 221 (6.5) [MeCH2SO2], 192 (12.7) [m/z 256eSO2] , 151 (12.9)
[m/z 221eC5H10], 150 (100) [m/z 221eC5H11], 136 (25.1) [m/z
151eCH3] , 122 (4.5) [m/z 151eCO]. Anal. Calcd. for C14H21NO4S: C,
56.16; H, 7.07; N, 4.68. Found: C, 55.94; H, 6.82; N, 4.22.
4.1.3.2. 1-(4-Amino-3-pentoxyphenyl)ethanone (3b). Yield 45%.
m.p. 90e93 ꢀC. UV (lMax (MeOH)) 329 nm (Vmax 39,696). FTIR (KBr,
cmꢁ1): 3380, 3150, 3025, 2926, 2854, 1660, 1496, 1453, 1400, 1258,
1042, and 695. 1H NMR (400 MHz; CDCl3,
d J): 7.30 (d, 1H,
J ¼ 6.7 Hz); 6.60 (m, 2H); 4.20 (t, 2H, J ¼ 6.6 Hz); 3.60 (broad s, 2H);
2.20 (s, 3H); 1.80 (quintet, 2H, J ¼ 6.4 Hz); 1.40 (m, 4H); 1.00 (t, 3H,
J ¼ 6.6 Hz). 13C NMR (CDCl3)
d
200.0, 146.1, 141.8, 125.6, 121.7, 116.9,
4.1.4.3. N-(4-acetyl-2-hexoxyphenyl)methanesulfonamide (4c). Yield
76%. m.p.118e120 ꢀC. UV (lMax (MeOH)) 271 nm (Vmax 19,349). FTIR
(KBr, cmꢁ1): 3200, 3098, 2928, 2862, 1636, 1499, 1462, 1400, 1370,
114.0, 70.5, 29.3, 28.6, 26.9, 23.0, 14.9. MS [EI, m/z (relative inten-
sity)]: 221 (100) [M.þ]. Anal. Calcd. for C13H19NO2: C, 70.56; H, 8.65;
N, 6.33. Found: C, 69.94; H, 8.22; N, 6.25.
1250,1110,1052 and 969. 1H NMR (400 MHz; CDCl3,
d J): 8.30 (d, 1H,
J ¼ 8.0 Hz); 7.70 (broad s, 1H); 6.90 (m, 2H); 4.00 (t, 2H, J ¼ 6.5 Hz);
4.1.3.3. 1-(4-Amino-3-hexoxyphenyl)ethanone (3c). Yield 50%. m.p.
94e95 ꢀC. UV (lMax (MeOH)) 329 nm (Vmax 39,509). FTIR (KBr,
cmꢁ1): 3404, 3098, 2928, 2862, 1636, 1499, 1453, 1462, 1400, 1270,
2.80 (s, 3H); 2.20 (s, 3H); 1.70 (quintet, 2H, J ¼ 7.3 Hz); 1.40 (m, 6H);
0.80 (t, 3H, J ¼ 7.0 Hz). 13C NMR (CDCl3)
d 200.0, 143.8, 142.0, 127.8,
120.4, 117.5, 114.0, 72.5, 43.2, 29.5, 28.3, 26.7, 23.0, 19.9, 14.2. MS [EI,
m/z (relative intensity)]: 313 (79.6) [M.þ], 270 (53.6) [M - COCH3],
235 (38.7) [MeCH2SO2], 220 (16.8) [m/z 235eCH3], 206 (45.0) [m/z
270eSO2] , 151 (12.9), 150 (21.3) [m/z 235eC6H13], 136 (100) [m/z
220eC6H12] , 122 (5.3) [m/z 150eCO]. Anal. Calcd. for C15H23NO4S:
C, 57.48; H, 7.40; N, 4.47. Found: C, 57.24; H, 6.99; N, 4.45.
1052 and 752. 1H NMR (400 MHz; CDCl3,
d
J): 7.50 (d,1H, J ¼ 8.0 Hz);
6.80 (m, 2H); 4.30 (broad s, 2H); 4.00 (t, 2H, J ¼ 7.6 Hz); 2.40 (s, 3H);
1.70 (quintet, 2H, J ¼ 7.3 Hz); 1.40 (m, 6H); 0.90 (t, 3H, J ¼ 6.6 Hz).
13C NMR (CDCl3)
d 199.7, 145.1, 142.0, 127.7, 120.8, 116.7, 114.0, 70.5,
29.0, 28.7, 25.9, 22.7, 19.7, 15.0. MS [EI, m/z (relative intensity)]: 235
(100) [M.þ]. Anal. Calcd. for C14H21NO2: C, 71.46; H, 8.99; N, 5.95.
Found: C, 70.54; H, 8.30; N, 5.40.
4.1.4.4. N-(4-acetyl-2-cyclohexyloxyphenyl)methanesulfonamide (4d).
Yield 73%. m.p. 121e122 ꢀC. UV (lMax (MeOH)) 227 nm (Vmax 40,405).
FTIR (KBr, cmꢁ1): 3399, 3100, 2927, 2856, 1637, 1575, 1499, 1460, 1400,
4.1.3.4. 1-(4-Amino-3-cyclohexyloxyphenyl)ethanone (3d). Yield 40%.
m.p. 98e101 ꢀC. UV (lMax (MeOH)) 211 nm (Vmax 31,925). FTIR (KBr,
cmꢁ1): 3399, 3100, 2927, 2856, 1637, 1575, 1499, 1460, 1377, 1270,
1360, 1270, 1115, 1052 and 950. 1H NMR (400 MHz; CDCl3,
d J): 7.10 (d,
1H, J ¼ 8.0 Hz); 6.90 (m, 2H); 6.80 (broad s,1H); 4.30 (septet,1H); 3.00
1152, 1052 and 1050. 1H NMR (400 MHz; CDCl3,
d
J): 7.10 (d, 1H,
(s, 3H); 2.40 (s, 3H); 2.10 (m, 2H); 1.80 (m, 2H); 1.50 (m, 3H); 1.40 (m,
J ¼ 8.0 Hz); 6.90 (m, 2H); 4.40 (septet, 1H); 3.60 (broad s, 2H); 2.40
2H). 13C NMR (CDCl3)
d 198.9, 143.8, 133.4, 127.8, 121.3, 117.4, 112.8,
(s, 3H); 2.00 (m, 2H); 1.80 (m, 2H); 1.50 (m, 3H). 13C NMR (CDCl3)
78.0, 42.7, 33.8, 33.8, 26.8, 25.5, 24.5, 24.5. MS [EI, m/z (relative
intensity)]: 311 (20.2) [M.þ], 233 (38.7) [MeCH2SO2], 229 (35.7)
[M ꢁ C6H10], 151 (34.1) [m/z 233eCH2SO2] ,, 150 (100) [m/z
229eCH2SO2], 136 (18.5) [m/z 151eCH3] , 122 (4.4) [m/z 150eCO].
Anal. Calcd. for C15H21NO4S: C, 57.86; H, 6.80; N, 4.50. Found: C,
56.68; H, 6.17; N, 4.00.
d
200.1, 144.8, 142.3, 127.0, 116.5, 114.3, 120.9, 79.5, 34.2, 34.2, 26.7,
24.4, 24.4, 26.0. MS[EI, m/z (relative intensity)]:235 (100)[M.þ]. Anal.
Calcd. for C14H19NO2: C, 72.07; H, 8.21; N, 6.00. Found: C, 71.54; H,
8.02; N, 5.86.
4.1.4. General procedure for preparation of N-(4-acetyl-2-
alkoxyphenyl)methane sulfonamides (4ae4d)
4.2. Pharmacological evaluation
To a solution of 4-amino-3-butoxy acetophenone (10 mmol) in
150.0 ml of dichloromethane, 15.0 ml pyridine and 1.5 ml meth-
anesulfonylchloride were added. The mixture was stirred for 6 h
at room temperature. It was then partitioned between water and
4.2.1. Anti-inflammatory activity
The anti-inflammatory activity was evaluated using in vivo
carrageenan-induced rat paw edema model considered as the

404
A. Bali et al. / European Journal of Medicinal Chemistry 49 (2012) 397e405
most conventional one for acute inflammation. The wistar rats
(150e200 g) of either sex were divided into 19 groups of six rats
each. A mark was made on both the hind paws (right and left) just
beyond tibio-tarsal junction to ensure constant paw volume. In all
the groups, 0.1 ml of 1% w/v carrageenan solution was injected in
the plantar region of the left paw of the rats and paw volume was
noted at 0, 1, 2, 3, 4, and 5 h. In the control group, 0.5% solution of
CMC was administered. In treated groups (three dose groups per
compound), the test compounds were administered p.o. as
a suspension in CMC, 30 min after the injection of carrageenan
solution. Similarly, in the standard groups, rofecoxib and indo-
methacin were administered as a suspension in CMC p.o. 30 min
after the injection of carrageenan solution. The paw volume was
noted before treatment and after treatment at different time
intervals with the help of plethysmograph by mercury displace-
ment method. Percentage edema and percentage reduction in
edema were calculated according to the formula.
consisting of three independent folding units: an N-terminal
epidermal growth factor domain (EGF), a membrane-binding
motif and a C-terminal catalytic domain, which contains the
active cyclooxygenase and peroxidase active sites. Out of the
structures lodged in the PDB, 1CX2 was selected as it contains
a COX-2 selective inhibitor SC-558 bound to the enzyme and the
same could be employed as a validation system also.
4.3.1. Protein preparation
The crystal structure of cyclooxygenase-2 complexed with SC-
558 was taken from the Protein Data Bank (PDB Code: 1CX2) and
prepared using Schrodinger protein preparation wizard tool.
Hydrogen atoms were added to the crystal structure. As a pre-
processing step, the water molecules, bound ligand and cofactors
were removed from the template. The protein was subjected to
minimization using the CHARMm force field implemented in
Accelrys-Discovery studio-2.1. The minimization iterations were set
at 1000 steps and minimization gradient tolerance of 0.001.The
prepared structure was saved in mol2 file format.
% edema ¼ 100 ꢁ ½ð1 ꢁ Vt=VcÞ ꢃ 100ꢄ
% reduction in edema ¼ ð1 ꢁ Vt=VcÞ ꢃ 100
Vt and Vc designate edema volume in drug treated and control
4.3.2. Preparation of ligands
All ligand 2D structures were built in ISIS Draw and then
transferred to Accelrys-Discovery Studio-2.1. After selection of
ligand hydrogen atoms were added. The ligands were subjected to
minimization using the CHARMm force field and prepared by using
‘prepare ligands’ module of Accelrys-Discovery Studio-2.1. The
prepared ligands were saved as mol2.
groups
4.2.2. Evaluation of gastric ulcerogenic potential
Wistar rats of either sex weighing 150e200 g were divided into
control, standard (rofecoxib and indomethacin) and various test
compound groups (n ¼ 6). The test compounds and indomethacin
were administered orally at three dose levels. Six hours later, the
animals were sacrificed by cervical dislocation and their stomachs
were removed, inflated by injecting 7.0 ml 2% formalin, immersed in
2% v/v formalin solution for 10 min to fix the gastric wall, and then
opened along the greater curvature. The area of each lesion that had
developed in the granular mucosa was measured under a dissecting
microscopewith a square grid (10x), summed per stomach, and used
as a lesion score. The lengths of the longest diameters of the lesions
are measured and summated to give a total lesion score (in mm) for
each animal, the mean count for each group being calculated.
4.3.3. Docking
The ligand conformations were generated using LigandFit
Monte-Carlo techniques which were subsequently docked into the
active site using a shape-based initial docking. The docked poses
were minimized using CHARMm force field followed by generation
of docking scores for the same. Simulations were performed with
1000 iterations. A grid resolution was set to 0.5 Aꢀ (default), and the
ligand-accessible grid was defined such that minimum distance
between a grid point and the protein is 2.0 Aꢀ for hydrogen and 2.5
Aꢀ for heavy atoms. The grid extends from the defined active site to
a distance of 3.0 Aꢀ in all directions. For analysis of docking, surface
was created around the ligand molecule and then receptor-ligand
hydrogen bonds were measured. Dockscore, LigScore1, LigScore2,
PLP1, PLP2, Jain and PMF scores were calculated for all the confor-
mations of the ligands to evaluate the fitness score relationship
with its biological activity.
4.3. Docking studies
The computational studies were carried out using Dell
Precision workstation T3400 (Intel Core 2 Duo Processor; 4 GB
RAM, 250 GB hard disk and Nvidia Quodro FX 4500 graphics
card) using Accelrys-Discovery Studio-2.1 (license no. FOD
297AOA463E4AA2E34). The docking poses were taken by using
GLIDE (Schrodinger Inc.) [21]. The synthesized molecules were
evaluated in silico (docking) using the crystal structure of
cyclooxygenase-2 complexed with a selective inhibitor, SC-558
(PDB Code: 1CX2) [22,23] and fitness scores were calculated
with LigandFit (Accelrys-Discovery Studio-2.1) software. COX-2
enzyme contains 587 amino acids and one ferric heme group.
The X-ray structure of mouse (Musmusculus) COX-2 has been
resolved as uninhibited, as well as in the presence of four
inhibitors. There is 87% sequence similarity between COX-2
enzyme from human and the mouse origin. Further, the strict
conservation of the active sites in both the species [22] suggests
that the structure of human COX-2 should be similar to the
mouse enzyme and the mouse COX-2 may be taken as a model
for the human COX-2 enzyme. The COX-2 protein exists in situ
as a dimer but the monomer structure alone has always been
considered in molecular modeling studies assuming the inter-
actions governing the COX-2 inhibitor binding to be reproduc-
ible using one monomer. COX-2 crystallizes with each monomer
4.4. Field align studies
The three dimensional and field similarity of the compound set
with respect to standard drugs was assessed using FieldAlign2.1.1Ô
(Cresset BioMolecular Discovery Ltd., UK). The reference drugs
nimesulide, flosulide, rofecoxib and celecoxib were imported from
ChemBioDraw Ultra 12.0 in sdf (MDL mol) format. Molecules to be
aligned were imported in 2D from ChemBioDraw Ultra 12.0 as sdf
(MDL mol) files. The maximum number of conformations generated
for any molecule was limited to 200 in order to have a balance of the
quality of alignments and calculation time. Number of high
temperature dynamics for flexible rings was set at 5. Gradient cut-off
for conformer minimization was 0.5. Coarseness of the sampling of
conformational space was controlled by filtering duplicate
conformers at rms 0.5. Standard scoring function was used based on
50% shape similarityand 50% dice volume similarity to derive overall
similarity between two conformations. Statistical analysis of the
similarity results for the test compounds was done by comparison of
different drug groups by All Pairwise Multiple Comparison test
(Tukey Test) (p < 0.05) (Jandel Sigmastat version 2.0).

A. Bali et al. / European Journal of Medicinal Chemistry 49 (2012) 397e405
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