Evaluation of glycolamide esters of indomethacin as potential cyclooxygenase-2 (COX-2) inhibitors

Article abstract of DOI:10.1016/j.bmc.2006.03.023

A number of novel indomethacin glycolamide esters were synthesized and tested for their cyclooxygenase (COX-1 and COX-2) inhibition properties in vitro. Many of these compounds proved to be selective COX-2 inhibitors, and subtle structural changes in the substituents on the glycolamide ester moiety altered the inhibitory properties as well as potencies significantly. Their in vitro data were rationalized through molecular modeling studies. Few of them displayed anti-inflammatory activity in vivo. Compound 32, [1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetic acid 2-morpholin-4-yl-2-oxo ethyl ester, was identified as a promising compound in this class and its good anti-inflammatory activity was demonstrated in the in vivo model.

Full text of DOI:10.1016/j.bmc.2006.03.023

Bioorganic & Medicinal Chemistry 14 (2006) 4820–4833
Evaluation of glycolamide esters of indomethacin as
potential cyclooxygenase-2 (COX-2) inhibitors
q
a
a
b
a
Smriti Khanna, Manjula Madan, Akhila Vangoori, Rahul Banerjee,
a
b
b
Ram Thaimattam, S. K. Jafar Sadik Basha, Mullangi Ramesh,
b
a,
*
Seshagiri Rao Casturi and Manojit Pal
a
Department of Chemistry, Discovery Research, Dr. Reddy’s Laboratories Ltd, Bollaram Road, Miyapur, Hyderabad 500049, India
b
Department of Biology, Discovery Research, Dr. Reddy’s Laboratories Ltd, Bollaram Road, Miyapur, Hyderabad 500049, India
Received 31 January 2006; revised 13 March 2006; accepted 13 March 2006
Available online 11 April 2006
Abstract—A number of novel indomethacin glycolamide esters were synthesized and tested for their cyclooxygenase (COX-1 and
COX-2) inhibition properties in vitro. Many of these compounds proved to be selective COX-2 inhibitors, and subtle structural
changes in the substituents on the glycolamide ester moiety altered the inhibitory properties as well as potencies significantly.
Their in vitro data were rationalized through molecular modeling studies. Few of them displayed anti-inflammatory activity in vivo.
Compound 32, [1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetic acid 2-morpholin-4-yl-2-oxo ethyl ester, was
identified as a promising compound in this class and its good anti-inflammatory activity was demonstrated in the in vivo model.
ꢀ
2006 Elsevier Ltd. All rights reserved.
1. Introduction
H2NO2S
H3CO2S
N
Nonsteroidal anti-inflammatory drugs (NSAIDs) are
useful for the treatment of pain and inflammation. They
reduce the pain and swelling of joints by inhibiting pros-
taglandin synthesis via blocking the cyclooxygenation of
N
O
N
CF3
O
CH3
H2N
S
H3C
O
O
O
arachidonic acid (AA) to prostaglandin G (PGG ) and
2
Valdecoxib (III)
2
Celecoxib (I)
Rofecoxib (II)
their use suffers from undesirable gastric side effects, for
1
Chart 1.
example, mucosal damage and bleeding. Since NSAIDs
2
directly target cyclooxygenases (COXs), the discovery
of second and inducible isozyme (COX-2, responsible
for inflammatory effects) in addition to constitutive iso-
zyme (COX-1, responsible for cytoprotective effects) has
opened the possibility of developing selective COX-2
inhibitors for therapeutic use as effective as NSAID
without the gastric side effects. Accordingly, three
COX-2 selective inhibitors that belong to 1,2-diarylhet-
erocyclic class have been marketed in the US, for
6
withdrawn recently because of evidence of increased
cardiovascular risk in select, high-dose patients. Cele-
coxib, however, has not shown similar adverse cardio-
7
vascular effects. While celecoxib and valdecoxib are
shown to be effective and comparable to conventional
NSAIDs in terms of analgesic, antipyretic, and anti-
inflammatory activity, these sulfonamide-containing
3
4
5
8
example, celecoxib, rofecoxib, and valdecoxib (see
structures I–III in Chart 1). Despite their initial
commercial success, rofecoxib (Vioxx) has been
inhibitors can also inhibit carbonic anhydrase II there-
by raising concerns about enzyme specificity. Thus,
development of alternative COX-2 inhibitors keeping a
slight effect on COX-1 at therapeutic dosage is desirable.
Keywords: Indomethacin glycolamide esters; COX-1 and COX-2
inhibition; Structure–activity relationship (SAR) study.
Several strategies have been reported on the modifica-
tion of the well-known nonselective agents for the design
and development of novel COX-2 inhibitors. These
include lengthening or derivatization of the carboxylic
q
DRL Publication No. 500.
Corresponding author. Tel.: +91 40 23045439; fax: +91 40 23045438/
3045007; e-mail: manojitpal@drreddys.com
*
2
0
968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.03.023

S. Khanna et al. / Bioorg. Med. Chem. 14 (2006) 4820–4833
4821
O
These recent findings and our interest in the develop-
1
O
5
OH
ment of novel COX-2 inhibitors as well as in the syn-
16
R
OH
Cl
n
MeO
thesis of indoles prompted us to design potent and
selective inhibitors of COX-2 based on indomethacin.
MeO
N
N
The crystal structures of indomethacin derivatives
bound to COX enzymes wherein the carboxylic group
of the ligand is oriented toward the narrow cross-section
of a cone-shaped solvent accessible channel suggest that
the esters of indomethacin can also be accommodated
inside the active sites. This is also evident from the
SAR studies on esters or amides of indomethacin report-
O
Cl
B
A
Br
Cl
O
R2
R
MeO
COOCHCH2N
R1
R2
9b
ed by Kalgutkar and co-workers. Therefore, our strat-
N
N
egy to develop new COX-2 inhibitors was based on
derivatization of the carboxylic side chain of indometh-
acin. In the course of these investigations, we have
synthesized and evaluated a number of novel indometh-
O
R
Cl
D
C, R = OR', NHR'
Figure 1. Structures of some indomethacin derivatives as COX-2
inhibitors.
acin derivatives possessing substituted glycolamide ester
(
1
–CO CH CONRR ) in place of acid side chain for
2
2
cyclooxygenase activities. We felt that substituted glyco-
lamide ester moiety might be worth exploring, as it is
capable of forming additional hydrogen bonds directly
or indirectly inside the solvent accessible channel. The
results indicate that indeed many of these derivatives
inhibited COX-2 selectively and subtle structural chang-
es in the substituents of the glycolamide ester can alter
the inhibitory properties as well as potencies significant-
ly. The present class of indomethacin derivatives there-
fore represents a new variety of COX-2 inhibitors.
9
a,9b
side chain of indomethacin (A–D, Fig. 1), develop-
ment of 5-methylsulfonyl derivatives of indole-2-carbox-
9c
ylic acids (structurally related to indomethacin), and
modification of the basic framework of zomepirac or
The chemical structures of these COX-
inhibitors including lumiracoxib clearly indicate that
the basic framework of diarylheterocycles is not the
exclusive prerequisite for COX-2 inhibition. This was
supported by the recent report on the development of
novel tetrahydro-2H-isoindoles as COX-2 inhibitor
where 1,2-disubstitution by two aryl groups on a central
core was missing. In an earlier effort on the develop-
ment of COX-2 inhibitor from nonselective NSAID,
Black and co-workers reported synthesis of a series of
COX-2 inhibitors based on indomethacin (B, Fig. 1).
In this approach, the size of the indomethacin nucleus
was increased by replacing the 4-chlorobenzoyl group
by a bulky group to produce a compound that would
still fit into COX-2 active site but not in COX-1 consid-
ering the hypothesis that COX-2 enzyme might have a
larger active site than COX-1. Recently Kalgutkar
and co-workers described a general, biochemical-based
strategy for the identification of COX-2 selective inhib-
9
d,e
flurbiprofen.
2
9
f
2. Chemistry
The synthesis of target compounds, that is, indometha-
cin glycolamide ester (1–33) is shown in Scheme 1. Easy
access to these compounds was made possible by using a
two-step process, that is, (a) preparation of 2-chloro N-
1
0
1
7
substituted acetamide followed by (b) the reaction of
1
8
indomethacin with the resulting amide. Thus, chloro-
acetyl chloride reacted with appropriate amine smoothly
in 1,4-dioxane at 0–25 ꢁC in the absence of a base to af-
ford the desired acetamide derivatives in good yields.
The second step, that is, reaction of the resulting 2-chlo-
ro N-substituted acetamide with indomethacin was car-
ried out successfully in the presence of catalytic amount
of N,N-dimethyl-4-aminopyridine using triethylamine as
a base. The structure of all the compounds synthesized
was confirmed by spectroscopic methods. In the IR
spectra of all compounds, carbonyl-stretching bands
belonging to ester and amide were seen at 1740 and
1
1
1
2
itors from carboxylic acid NSAIDs, for example, indo-
8
b
methacin (C, Fig. 1). It was observed that large alkyl,
aryl, aralkyl and heterocyclic esters or amides of indo-
methacin showed high potency and selectivity. Further
continuation of their study led to the identification of
indomethacin alkanolamides as enantiospecific, selective
COX-2 inhibitors. More recently, a series of N-substi-
tuted indole-2-carboxylic acid esters have been studied
as possible COX-2 selective inhibitors (D, Fig. 1).
1
3
ꢀ
1
1
1670 cm . In the H NMR spectra, methylene protons
of –COOCH CO– group appeared at d 4.5–4.8 ppm.
1
4
2
H CO
3
O
1
NR R
O
O
ClCH COCl
2
indomethacin
1
N
CH3
RR NH
1
ClCH CONR R
2
1
0
,4-dioxane
-25 C, 1h
Et N, DMAP
3
I
II
o
o
O
25 C, 24h
1-33
Cl
Scheme 1. Synthesis of indomethacin derivatives 1–33.

4822
S. Khanna et al. / Bioorg. Med. Chem. 14 (2006) 4820–4833
3
. Results and discussions
tory activity at 30 mg/kg dose in the carrageenan-in-
2
duced rat paw edema model.
0
All the new compounds synthesized were tested in vitro
initially at 10 lM for selectivity and potency against hu-
man COX-2 (expressed in sf9 insect cells using baculovi-
rus) and COX-1 (ram seminal vesicles) enzymes. On the
basis of their in vitro efficacy, selected compounds were
tested at 1.0 lM concentration as well. The IC values
for COX-1 and COX-2 were determined for selected/
promising compounds. Celecoxib and indomethacin
were used as reference compounds for the in vitro assay.
Eight compounds were tested in vivo for anti-inflamma-
Biological evaluation of the title compounds showed that
the conversion of the free acid group of non-selective
inhibitor indomethacin to the N-substituted glycolamide
1
ester (–CO CH CONRR ) gives rise to compounds pos-
2
2
sessing varying degrees of COX-2 selectivity (see Table
1). Initially we examined the effect of various aryl and het-
eroaryl groups attached to the nitrogen of the glycolamide
5
0
1
9
1
ester side chain, that is, R = H, R = aryl or heteroaryl.
Among the fluorinated aryl derivatives (1–5) compound
Table 1. In vitro and in vivo data for indomethacin derivatives
COOCH2CONRR1
Compound
In vitro % inhibition at
a
In vivo % inhibition at
30 mg/kg (rat paw edema)
b
H3CO
1
0 lM
CH3
N
COC6H4Cl-p
1
R
R
COX-1
COX-2
1
2
3
4
5
6
7
8
9
3-Fluorophenyl
2-Fluorophenyl
4-Fluorophenyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
20
0
98, 63 (1)
47
56 ± 3
14 ± 5
80 ± 2
68 ± 4
19 ± 2
52 ± 3
22 ± 2
n.d.
38
0
51
86, 56 (1)
2,4-Difluorophenyl
2,3,4-Trifluorophenyl
2-Chlorophenyl
20
0
71
100, 53 (1)
4-Chlorophenyl
0
54
51
2,6-Dichlorophenyl
3-Chloro-4-fluorophenyl
2-Bromophenyl
0
0
98, 60 (1)
24
50 ± 4
n.d.
n.d.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0
3-Bromophenyl
4-Bromophenyl
0
27
29
11
15
0
19 ± 3
11 ± 2
39 ± 3
10 ± 3
26 ± 2
22 ± 1
62 ± 3
40 ± 5
n.d.
4-Bromo-2-methylphenyl
2-Methyl-4-iodophenyl
2,3-Dimethylphenyl
3-Methoxyphenyl
3,4-Dimethoxyphenyl
Benzo[d][1,3]dioxol-5-yl
2-Nitrophenyl
33
4
39
0
42
23
15
22
19
23
24
20
32
0
55
51
44
32
3-Nitrophenyl
2-Pyrimidinyl
35
54
21 ± 2
n.d.
n.d.
1,3-Thiazol-2-yl
4-Methyl-1,3-thiazol-2-yl
Benzo[d][1,3]thiazol-2-yl
47
74
80
69
n.d.
n.d.
6-Methoxybenzo[d][1,3]thiazol-2-yl
2-Methoxycarbonyl-3-thienyl
5-Methyl-3-isoxazolyl
9
74
74
n.d.
26 ± 3
n.d.
23
0
N1-(5-carbamoyl-1-methyl-3-propyl-1H-4-pyrazolyl)-
Benzyl
4-Methoxybenzyl
86, 55 (1)
79
84
18
0
40 ± 3
17 ± 1
59 ± 4
Octyl
0
78, 48 (1)
COOCH2COR
H3CO
CH3
R=
N
COC6H4Cl-p
3
2
3
N-Morpholinyl
0, 0 (1)
0
93, 53 (1)
90, 55 (1)
97
88 ± 3
44 ± 4
3
N-Thiomorpholinyl
Indomethacin
Celecoxib
100
0
100, 98 (1)
49 ± 2
n.d., not determined.
a
Human COX-2 (expressed in sf9 insect cells using baculovirus) and COX-1 (ram seminal vesicles) enzyme. Figures in the brackets indicate
concentration in micro molar. The result is the average of at least three determinations, and the deviation from the mean is <10% of the average
value.
b
The carrageenan-induced rat paw edema assay was carried out using six animals (male Wistar rats)/group following oral dose of the test compound.

S. Khanna et al. / Bioorg. Med. Chem. 14 (2006) 4820–4833
4823
4
possessing 2,4-difluorophenyl group showed good selec-
that the acid group is almost buried and thus not ex-
posed to the solvent-accessible channel while retaining
crucial interactions with Arg120. Docking studies were
carried out in order to understand the basis for COX-
2 selectivity of compounds 6 and 32 using FlexX. The
simulated binding poses of these molecules are similar
to that of indomethacin inside the active site of the
COX-2 crystal structure. However, FlexX failed to dock
these molecules in the COX-1 active site. Hence, mole-
cules 6 and 32 were manually positioned inside the
active site of COX-1 such that the binding pose of each
resembled that of iodoindomethacin in the COX-1.
tivity in COX-2 inhibition over COX-1. However,
replacement of 2,4-difluorophenyl by a 2-chlorophenyl
group increased the selectivity and potency further
against COX-2 (compound 6). Shifting of chloro or pres-
ence of additional chloro decreased the potency (com-
pounds 7 and 8). Presence of both chloro and fluoro on
the benzene ring was found to be beneficial (compound
9
). Contrary to chloro and fluoro, other halogens such
as bromo or iodo or mild electron-donating methyl
groups in general were found to be less attractive (com-
pounds 10–15 did not inhibit COX-2 significantly). The
presence of a strong electron-donating group such as alk-
oxy (compounds 16–18) or a strong electron-withdrawing
group such as nitro (compounds 19–20) on the benzene
ring favored neither COX-2 selectivity nor potency. We
then focused on replacing the aryl groups by heteroaryl
moieties. A variety of heteroaryl moieties were examined
and all of them induced moderate togoodCOX-2 selectiv-
ity (compounds 21–28). The best compound however was
found to be 28 which possessed N1-(5-carbamoyl-1-meth-
yl-3-propyl-1H-4-pyrazolyl)- as a heterocycle in the side
chain. We also examined the effect of alkyl and alkylaryl
moieties in place of aryl/heteroaryl groups and the results
were found to be encouraging (compounds 29–31).
Remarkably, selectivity and potency were observed when
the cyclic amine such as morpholine or thiomorpholine
moiety was introduced in place of primary amine in the
glycolamide ester side chain (compounds 32–33).
Molecules 6 and 32 show hydrogen bonding interactions
with Ser530 in the active site of COX-2 (see Fig. 2 and
Table 2). In both cases, the ester group is positioned
in the solvent-accessible channel. In addition, the car-
bonyl O-atom of the ester group (of both 6 and 32) is in-
volved in bifurcated hydrogen bonding interactions with
Arg120. Furthermore, molecule 6 shows additional
hydrogen bonding interactions with Tyr355 and Lys83
involving the glycolamide oxygen and the morpholine
a
Indomethacin is a non-selective inhibitor of COX iso-
zymes. The crystal structures of COX enzymes
2
1
(
4COX.pdb and 1PGG.pdb) show that the binding
poses of indomethacin derivatives are distinct in the
two isoforms because of the structural differences in
the active sites, in particular, the amino acid residue at
position 523. Val at this position in COX-2 is replaced
with Ile in COX-1. The smaller size of Val at 523-posi-
tion in COX-2 leaves a side pocket, while Ile523 restricts
access to the side pocket in COX-1. As a result, indo-
methacin moiety is flipped by about 180ꢁ (in 1PGG.pdb)
such that the phenyl and carboxylate groups occupy
similar environments in both isoforms (4COX.pdb and
b
1
PGG.pdb). However, the orientation of the carboxyl-
ate group is significantly different in the two isoforms.
In spite of these differences, the acid group, which is ori-
ented toward the solvent-accessible channel, forms
attractive interactions with Arg120 and Tyr355 in both
isoforms. In the alternative binding pose, the orientation
of iodo analogue of indomethacin (see structure V in
Chart 2) in the active site of COX-1 (1PGF.pdb) is such
COOH
H CO
3
CH3
N
O
X
Figure 2. Stereoview of (a) 6 and (b) 32 in the active site of COX-2
(4COX.pdb). Molecules 6 and 32 are shown in pink C-atoms. The
crystal structure conformation of indomethacin (green C-atoms,
IV; Indomethacin (X = Cl)
V; Iodoindomethacin (X = I)
˚
COX.pdb) is superimposed for reference. Residues within 4 A radius
4
around the ligand are shown in orange sticks, and hydrogen atoms are
not shown for clarity. Hydrogen bonds are shown in white lines.
Chart 2.

4824
S. Khanna et al. / Bioorg. Med. Chem. 14 (2006) 4820–4833
Table 2. Hydrogen bond geometries of molecules 6 and 32 inside the COX-2 active site
O2
Cl
O
R
HN
N
H3CO
6; R =
CH3 O3
N
3
2; R =
O
O1
Cl
˚
Compound
Functional group
Amino acid residue in the active site
Distance X–Y (A) for X–Hꢂ ꢂ ꢂY
Angle X–H–Y (ꢁ)
6
C@O(1)
C@O(2)
C@O(3)
Ser530
Arg120
Arg120
2.63
2.40
3.01
160.88
166.60
125.70
32
C@O(1)
C@O(2)
C@O(2)
C@O(3)
Morpholine
Ser530
Arg120
Arg120
Tyr355
Lys83
2.70
2.70
2.40
2.53
2.65
161.61
130.13
153.58
166.83
167.25
group, respectively. However, in case of COX-1 docking
experiments it indicated that there is little room to
accommodate the ester group (1PGF.pdb) or the ester
group would have steric clashes with Tyr355 and/or
Arg120 (1PGG.pdb). In summary, molecules 6 and 32
fit comfortably inside the active site of COX-2, while
showing steric clashes in COX-1. A closer analysis indi-
cates that these differences are due to differences in the
orientation of the indomethacin/ester group inside the
COX active sites. These observations suggest that esters
of indomethacin in general bind poorly to the COX-1
active site.
coxib in terms of calculated logp value. Pharmacological
studies were carried out to assess the in vivo anti-inflam-
matory activity of some of these compounds. The anti-
inflammatory activity of these compounds was tested
in the standard rat model of inflammation and
compared with celecoxib. Encouragingly, a number of
compounds showed fair to good inhibition in carrageen-
an-induced rat paw edema assay when dosed orally at
30 mg/kg (Table 1). Compounds 1, 4, 6, 9, 18, 31, and
32 showed more than 50% inhibition that was either
comparable or better than celecoxib’s 49% inhibition
in the same assay. Maximum inhibition of 88% was
achieved with the N-morpholino analogue 32. The
ED30 and/or ED50 of compounds 1, 4, 6, 31, and 32
are shown in Table 3. N,N-disubstituted glycolamide
esters have previously been reported as potentially use-
On the basis of in vitro data, few compounds were
selected for further studies. The IC50 values for com-
pounds 1, 4, 6, 9, 28, and 31–33 are shown in Table 3
and all of them are found to be potent and selective
inhibitors of COX-2. Some of them are either compara-
ble or superior to celecoxib in terms of selectivity index
18
ful biolabile prodrug type for carboxylic acids. Indeed,
a quite rapid rate of hydrolysis of N,N-diethyl glycola-
mide ester of indomethacin was observed in 80% human
plasma. However, the ease of plasma-catalyzed hydroly-
sis was altered with the change of substituents on amide
(
e.g., SI ꢁ220 for 1 or 225 for 4 or 273 for 6 vs 219 for
celecoxib). Notably, calculated logp values for all the
eight compounds were higher than those of indometha-
cin and celecoxib except for 28 and 32 (Table 3), which
contain hydrophilic groups. Compound 32 was better
than 33 and comparable to indomethacin as well as cele-
2
2a
1
1
nitrogen atom (e.g., R and R of –CO CH CONRR
2
2
moiety) and monosubstituted or unsubstituted glycola-
mide esters were found to be more resistant than N,
N-disubstituted glycolamide esters. Moreover, the
d
Table 3. In vitro and in vivo (rat paw edema assay ) data for indomethacin derivatives
a
b
Compound
IC50 (lM)
logp
Selectivity index (COX-1/COX-2)
ED30 (mg/kg)
ED50 (mg/kg)
COX-1
COX-2
c
1
4
6
9
>180
110 ± 5
0.82 ± 0.03
0.49 ± 0.01
0.55 ± 0.03
0.83 ± 0.10
0.73 ± 0.08
0.60 ± 0.10
0.30 ± 0.05
0.44 ± 0.01
7.8 ± 0.11
5.85 ± 0.1
5.48 ± 0.1
5.57 ± 0.1
6.65 ± 0.1
3.33 ± 0.1
7.30 ± 0.51
4.14 ± 0.30
4.88 ± 0.30
4.18 ± 0.10
4.37 ± 0.1
>220
ꢁ225
>273
ꢁ36
>10
nd
nd
12 ± 0.03
nd
nd
c
>150
c
10 ± 0.25
nd
nd
>30
20 ± 2
28
31
32
33
ꢁ27
nd
c
>50
25 ± 3
>83
ꢁ83
nd
nd
8.1 ± 0.07
5.3 ± 0.11
nd
c
>20
0.067 ± 0.001
15.33 ± 0.03
>46
ꢁ0.0085
ꢁ219
nd
nd
Indomethacin
Celecoxib
2.4 ± 0.20
7.9 ± 0.14
0.07 ± 0.005
nd
nd, not determined.
a
The result is the mean value of two determinations, and the deviation from the mean is <10% of the mean value.
Calculated using SYBYL.
Precipitation of compound observed beyond this concentration.
b
c
d
ED30 and ED50 values were determined using a minimum of four dose points, six animals (male Wistar rats)/group followed by oral administration.

S. Khanna et al. / Bioorg. Med. Chem. 14 (2006) 4820–4833
4825
glycolamide esters of aspirin failed to deliver aspirin in
human plasma when morpholine was the corresponding
4. Conclusion
2
2a
amine of the amide moiety.
dence seems to hold true for the present compounds
1–33, Table 1) in rats, which is reflected by ED30 or
This structural depen-
With the aim of developing a potent but moderately
selective COX-2 inhibitor we have shown that in addi-
tion to the earlier report of converting it into simple es-
ter or amide nonselective indomethacin can also be
transformed into COX-2 inhibitors via converting the
acid moiety to N-substituted glycolamide esters. Physi-
cal properties like hydrophilicity and hydrophobicity
along with the size of the N-substituent played a key role
in modulating COX-2/COX-1 selectivity. Some of these
compounds synthesized were identified as good to mod-
erately selective COX-2 inhibitors, and their in vitro
data were rationalized through molecular modeling
studies. While the discovery of COX-2 initiated a race
to the development of the most selective inhibitors, the
question now remains to be answered is whether a high-
er degree of selectivity confers any advantages. On the
other hand, the design of a COX-2 ‘preferential’ inhibi-
tor keeping a slight effect on COX-1 at therapeutic dos-
age could theoretically limit the imbalance of
prostacyclin/thromboxane A2. Therefore, the novel
indomethacin glycolamide esters developed by us seem
to have advantages over the other COX-2 inhibitors. Be-
cause of their distinctly different chemical structures
they are not expected to show affinity for carbonic anhy-
drase isozymes like 1,2-diaryl sulfonamide class or cause
marked increase in nonenzymatic generation of isopros-
(
ED50 values of compounds 1, 4, 6, 31, and 32. Their rel-
atively higher ED30 or ED50 values than indomethacin
(
Table 3) apparently indicated that these compounds
are perhaps more resistant to enzymatic hydrolysis by
plasma in rats. However, to gain further evidence stabil-
ity studies were carried out using compounds 32 and 33
in the presence of 50 mM Tris buffer (pH 7.4) initially
where 70% of 32 and 99% of 33 remained unchanged
after 2 h incubation in buffer, respectively. In a separate
study, these compounds were incubated for varying
times with rat liver microsomes (RLM) in the presence
of NADPH. After quenching with acetonitrile, the incu-
bation mixtures were analyzed by HPLC and generation
of any product that coeluted with indomethacin was
examined. The RLM stability of these compounds (32
and 33) was found to be moderate (20–30%) and there-
fore it remained unclear that the in vivo activity of these
compounds was due to their selective COX-1/COX-2
properties and not due to hydrolysis to indomethacin.
Nevertheless, the ED50 of compound 32 was found to
be 5.3 mg/kg after oral administration, which is compa-
rable to that of celecoxib (7.9 mg/kg). Thus, compound
3
2 was identified as selective inhibitor of COX-2 with
2
4
good anti-inflammatory activity. It was however not
the most selective inhibitor in this chemical class and
therefore may have the reduced cardiovascular liability
of certain COX-2 inhibitors that showed very high selec-
tanes like diaryl methansulfones. Compound 32 was
identified as a promising candidate in this class and its
good anti-inflammatory activity was demonstrated in
the in vivo model.
2
3a
tivity for COX isozyme.
To test the ulcerogenicity of compound 32 and then
compare with that of indomethacin, we conducted
qualitative acute stomach ulceration study in swiss
albino mice (n = 5). The UD30 (30% ulcerative dose)
of compound 32 was found to be ꢁ5-fold higher than
5. Experimental
5.1. Chemical methods
Unless stated otherwise, reactions were performed under
nitrogen atmosphere. All the solvents used were com-
mercially available and distilled before use. Reactions
were monitored by thin-layer chromatography (TLC)
on silica gel plates (60 F254), visualizing with ultraviolet
light or iodine spray. Flash chromatography was per-
formed on silica gel (230–400 mesh) using distilled
petroleum ether, ethyl acetate, dichloromethane, chloro-
1
.5 mg/kg/po noted for indomethacin after 6th hour of
2
3b
oral administration.
Gastrointestinal toxicity of
several glycolamide esters of non-steroidal anti-inflam-
matory drugs including indomethacin has been investi-
gated earlier and all these esters showed markedly
2
2b,22c
reduced GI toxicity than parent drugs in rats.
Temporarily masked acidic moiety of the parent drugs
in the form of glycolamide ester that avoid the accu-
mulation of the acid within gastric mucosal cells was
thought to be the reason for their reduced GI toxici-
1
form, and methanol. H NMR spectra were determined
in CDCl solution on 200 MHz spectrometers. Proton
3
chemical shifts (d) are relative to tetramethylsilane
(TMS, d = 0.00) as internal standard and expressed in
ppm. Spin multiplicities are given as s (singlet), d (dou-
blet), t (triplet), and m (multiplet) as well as b (broad).
Coupling constants (J) are given in hertz. Infrared spec-
tra were recorded on a FT-IR spectrometer. Melting
points were determined by using melting point appara-
tus and are uncorrected. MS spectra were obtained on
a mass spectrometer. Chromatographic purity by HPLC
was determined by using area normalization method
and the condition specified in each case: column, mobile
phase (range used), flow rate, detection wavelength, and
retention times. Microanalyses were performed using a
C H N S/O analyzer. Elemental data are within ±0.4%
of the theoretical values. All yields reported are
9
b
ties. However, others and we in the present study
have shown that converting the free acid side chain
of indomethacin into simple or glycolamide esters
afforded COX-2 inhibitors with moderate to high selec-
tivity. Thus, the reduced GI toxicities shown by indo-
methacin esters are perhaps related to their COX-2
inhibiting properties rather than masked acidic carbox-
yl group. Nevertheless, the desired physicochemical
properties along with high chemical stability can be ob-
tained via glycolamide ester preparation of a carboxylic
1
8,22a
acid
and therefore, the glycolamide esters of indo-
methacin described in the present study are attractive
chemical class for the development of novel anti-in-
flammatory agents.

4826
S. Khanna et al. / Bioorg. Med. Chem. 14 (2006) 4820–4833
unoptimized. Celecoxib and Rofecoxib were prepared
3
according to the literature procedure.
1.0 mL/min, UV 235 nm, retention time 13.4 min; Ele-
mental analysis found C, 63.60; H, 4.50; N, 5.57;
C H ClFN O requires C, 63.72; H, 4.36; N, 5.50.
,4b
2
7
22
2
5
5.1.1. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
3
-yl]acetic acid (3-fluoro phenylcarbamoyl) methyl ester (1).
5.1.4. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
-yl]acetic acid (2,4-difluoro phenylcarbamoyl) methyl
ester (4).
3
O
H3CO
F
O
F
NH
CH3
O
N
O
H3CO
O
O
NH
F
Cl
CH3
N
O
1
Yield 10%; off white solid; mp 152–154 ꢁC; H NMR
200 MHz, CDCl ) d 7.60 (d, J = 8.8 Hz, 2H), 7.42 (d,
J = 8.3 Hz, 2H), 7.18–7.0 (m, 2H), 6.93 (s, 1H), 6.86
O
(
3
Cl
1
(
(
(
(
m, 1H), 6.75 (m, 3H), 6.61 (d, J = 8.3 Hz, 1H), 4.75
s, 2H), 3.84 (s, 2H), 3.79 (s, 3H), 2.45 (s, 3H); IR
Yield 56%; off white solid; mp 131–134 ꢁC; H NMR
(200 MHz, CDCl ) d 8.08 (m, 1H), 7.66 (d, J = 8.3 Hz,
2H), 7.46 (d, J = 8.8 Hz, 2H), 6.96 (s, 1H), 6.85–6.75 (m,
3H), 6.65 (d, J = 9.3 Hz, 1H), 4.74 (s, 2H), 3.84 (s, 2H),
3.71 (s, 3H), 2.45 (s, 3H); IR (KBr, cm ): 3251, 1737
3
ꢀ
1
KBr, cm ): 3314, 1722 (C@O), 1644, 1616; Mass
m/z) 509 (M , 100%); HPLC 99.7%, column: Hichrom
+
ꢀ
1
RPB (250 · 4.6) mm, mobile phase: H O/CH CN
2
3
+
(
time 11.1 min; Elemental analysis found C, 63.79; H,
30:70), flow rate 1.0 mL/min, UV 210 nm, retention
(C@O), 1689; Mass (m/z) 527 (M , 33%), 139 (100%);
HPLC 97.1%, column: ECLIPSE XDB C8 (150 · 4.6)
4
.37; N, 5.45; C H ClFN O requires C, 63.72; H,
5
mm, mobile phase: H O/CH CN (45:55), flow rate
2 3
27
22
2
4
.36; N, 5.50.
1.0 mL/min, UV 215 nm, retention time 14.64 min; Ele-
mental analysis found C, 61.60; H, 4.10; N, 5.20;
5.1.2. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
C
27
H
21ClF
N O requires C, 61.54; H, 4.02; N, 5.32.
2 2 5
3-yl]acetic acid (2-fluoro phenylcarbamoyl) methyl ester (2).
5
.1.5. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-
O
H3CO
indol-3-yl]acetic acid (2,3,4-trifluoro phenylcarbamoyl)
methyl ester (5).
O
NH
F
CH3
N
O
F
O
O
H3CO
F
O
Cl
NH
F
CH3
1
N
O
Yield 34%; off white solid; mp 133–135 ꢁC; H NMR
200 MHz, CDCl ) d 8.18 (d, J = 7.8 Hz, 1H), 7.80 (s,
(
O
3
1
7
6
(
(
(
(
H), 7.66 (d, J = 8.3 Hz, 2H), 7.46 (d, J = 8.3 Hz, 2H),
.19–7.01 (m, 3H), 6.96 (s, 1H), 6.82 (d, J = 9.3 Hz, 1H),
.65 (d, J = 9.2 Hz, 1H), 4.75 (s, 2H), 3.85 (s, 2H), 3.79
s, 3H), 2.47 (s, 3H); IR (KBr, cm ): 3308, 1721
C@O), 1651, 1619; Mass (m/z) 509 (M , 30%), 139
100%); HPLC 97.9%, column: Hichrom RPB
250 · 4.6) mm, mobile phase: H O/CH CN (30:70), flow
rate 1.0 mL/min, UV 240 nm, retention time 8.96 min;
Elemental analysis found C, 63.67; H, 4.36; N, 5.55;
C H ClFN O requires C, 63.72; H, 4.36; N, 5.50.
Cl
1
Yield 45%; white solid; mp 142–145 ꢁC; H NMR
(200 MHz, CDCl ) d 7.81 (br s, 1H), 7.68 (d, J = 8.3 Hz,
2H), 7.58 (s, 1H), 7.48 (d, J = 8.3 Hz, 2H), 6.99–6.62 (m,
H), 4.75 (s, 2H), 3.85 (s, 2H), 3.77 (s, 3H), 2.48 (s, 3H);
IR (KBr, cm ): 3344, 1745 (C@O), 1707, 1679, 1610;
Mass (m/z) 545 (M , 55%), 139 (100%); HPLC 99.3%, col-
umn: Hichrom RPB (250 · 4.6) mm, mobile phase: H O/
CH CN (30:70), flow rate 1.0 mL/min, UV 225 nm, reten-
tion time 10.3 min; Elemental analysis found C, 59.41; H,
ꢀ
1
3
+
4
ꢀ1
2
3
+
2
3
2
7
22
2
5
3.72; N, 5.10; C H ClF N O requires C, 59.51; H, 3.70;
27 20 3 2 5
N, 5.14.
5
.1.3. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
3-yl]acetic acid (4-fluoro phenylcarbamoyl) methyl ester (3).
F
5.1.6. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
3-yl]acetic acid (2-chloro phenylcarbamoyl) methyl ester (6).
O
H3CO
O
O
NH
CH3
H3CO
N
O
O
O
NH Cl
CH3
N
O
Cl
1
Yield 55%; white solid; mp 132–134 ꢁC; H NMR
200 MHz, CDCl ) d 7.61 (d, J = 8.8 Hz, 2H), 7.44 (d,
J = 8.3 Hz, 2H), 7.05–7.04 (m, 2H), 6.93–6.90 (m, 4H),
O
Cl
(
3
1
Yield 61%; white solid; mp 124–126 ꢁC; H NMR
(200 MHz, CDCl ) d 8.33 (d, J = 8.3 Hz, 1H), 8.22 (br s,
1H), 7.66 (d, J = 8.8 Hz, 2H), 7.46 (d, J = 8.3 Hz, 2H),
7.34 (d, J = 8.8 Hz, 2H), 7.12–6.65 (m, 4H), 4.78 (s, 2H),
3.87 (s, 2H), 3.80 (s, 3H), 2.45 (s, 3H); IR (KBr, cm ):
6
3
1
9
.76–6.70 (m, 2H), 4.71 (s, 2H), 3.85 (s, 2H), 3.76 (s,
3
ꢀ
1
H), 2.45 (s, 3H); IR (KBr, cm ): 3340, 1735 (C@O),
684, 1615; Mass (m/z) 509 (M , 45%), 139 (100%); HPLC
+
ꢀ
1
8.1%, column: Inertsil ODS 3 V (250 · 4.6) mm, mobile
+
phase: 0.01 M KH PO /CH CN (30:70), pH 3.5, flow rate
3253, 1739 (C@O), 1678, 1610; Mass (m/z) 525 (M ,
2
4
3

S. Khanna et al. / Bioorg. Med. Chem. 14 (2006) 4820–4833
4827
ꢀ
1
4
5%), 139 (100%); HPLC 97.8%, column: symmetry C18
(s, 2H), 3.74 (s, 3H), 2.45 (s, 3H); IR (KBr, cm ):
3286, 1721 (C@O), 1698; Mass (m/z) 545 (M+2, 30%),
544 (M+1, 30%), 543 (M , 42%), 139 (100%); HPLC
(
150 · 4.6) mm, mobile phase: H O/CH CN (30:70), flow
2
3
+
rate 1.0 mL/min, UV 210 nm, retention time 8.6 min; Ele-
mental analysis found C, 61.79; H, 4.23; N, 5.30;
C H Cl N O requires C, 61.72; H, 4.22; N, 5.33.
96.5%, column: ECLIPSE XDB-C8 (150 · 4.6) mm, mo-
bile phase: H O/CH CN (40:60), flow rate 1.0 mL/min,
2
7
22
2
2
5
2
3
UV 240 nm, retention time 14.0 min; Elemental analysis
found C, 59.61; H, 3.90; N, 5.20; C H Cl FN O re-
quires C, 59.68; H, 3.90; N, 5.16.
5
.1.7. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
2
7
21
2
2
5
3-yl]acetic acid (4-chloro phenylcarbamoyl) methyl ester (7).
Cl
O
5.1.10. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl]acetic acid (2-bromo phenylcarbamoyl) methyl ester (10).
H3CO
O
NH
O
CH3
N
O
H3CO
O
O
NH Br
CH3
Cl
N
O
1
Yield 31%; off white solid; mp 159–162 ꢁC; H NMR
200 MHz, CDCl ) d 7.6 (d, J = 8.3 Hz, 2H), 7.44 (d,
J = 8.3 Hz, 2H), 7.17 (d, J = 8.8 Hz, 2H), 7.05 (s, 1H),
O
(
Cl
3
1
Yield 52%; off white solid; mp 135–136 ꢁC; H NMR
200 MHz, CDCl ) d 8.29 (m, 1H), 7.66 (d, J = 8.3 Hz,
H), 7.48 (m, 3H), 7.34 (m, 1H), 7.01 (m, 3H), 6.85 (d,
J = 8.8 Hz, 1H), 6.67 (d, J = 8.8 Hz, 1H), 4.78 (s, 2H),
.88 (s, 2H), 3.84 (s, 3H), 2.44 (s, 3H); IR (KBr,
6
(
.9 (m, 3H), 6.75 (d, J = 9.2 Hz, 1H), 4.71 (s, 2H), 3.84
s, 2H), 3.77 (s, 3H), 2.46 (s, 3H); IR (KBr, cm ): 3334,
(
2
3
ꢀ1
+
1
734 (C@O), 1686; Mass (m/z) 525 (M , 100%); HPLC
9.2%, column: Hichrom RPB (250 · 4.6) mm, mobile
9
phase: H O/CH CN (30:70), flow rate 1.0 mL/min, UV
3
ꢀ
1
2
3
cm ): 3251, 1737 (C@O), 1675; Mass (m/z) 572
+
M+2, 15%), 570 (M , 33%), 139 (100%); HPLC
250 nm, retention time 11.6 min; Elemental analysis
found C, 60.67; H, 4.20; N, 5.40; C H Cl N O requires
C, 61.72; H, 4.22; N, 5.33.
(
2
7
22
2
2
5
96.4%, column: ECLIPSE XDB C8 (150 · 4.6) mm, mo-
bile phase: H O/CH CN (45:55), flow rate 1.0 mL/min,
2
3
UV 215 nm, retention time 18.39 min; Elemental analy-
sis found C, 56.70; H, 3.85; N, 4.99; C H BrClN O
5
requires C, 56.91; H, 3.89; N, 4.92.
5
3
.1.8. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
-yl]acetic acid (2,6-dichloro phenylcarbamoyl) methyl
2
7
22
2
ester (8).
O
5.1.11. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl]acetic acid (3-bromo phenylcarbamoyl) methyl ester (11).
H3CO
Cl
O
NH Cl
CH3
Br
N
O
O
O
O
H3CO
NH
Cl
CH3
O
1
N
Yield 35%; white solid; H NMR (200 MHz, CDCl ) d
3
7
(
.62 (d, J = 8.3 Hz, 2H), 7.45 (d, J = 8.3 Hz, 2H), 7.32
d, J = 8.8 Hz, 2H), 7.22–7.13 (m, 2H), 6.98 (d,
O
Cl
1
Yield 46%; off white solid; mp 152–154 ꢁC; H NMR
200 MHz, CDCl ) d 7.62 (d, J = 8.3 Hz, 2H), 7.44 (d,
J = 8.8 Hz, 2H), 7.26–6.75 (m, 8H), 4.72 (s, 2H), 3.85 (s,
2
1
J = 2.4 Hz, 1H), 6.77 (d, J = 8.8 Hz, 1H), 6.55 (d,
J = 9.3 Hz, 1H), 4.81 (s, 2H), 3.85 (s, 2H), 3.74 (s, 3H),
(
3
ꢀ
1
2
1
9
.45 (s, 3H); IR (KBr, cm ): 3422, 1742 (C@O), 1675,
610; Mass (m/z) 560 (M , 55%), 139 (100%); HPLC
ꢀ
1
+
H), 3.76 (s, 3H), 2.46 (s, 3H); IR (KBr, cm ): 3253,
744 (C@O), 1673; Mass (m/z) 572 (M+2, 15%), 570
8.3%, column: symmetry C18 (150 · 4.6) mm, mobile
+
M , 15%), 139 (100%); HPLC 97%, column: Hichrom
(
phase: H O/CH CN (30:70), flow rate 1.0 mL/min, UV
210 nm, retention time 6.5 min; Elemental analysis found
C, 57.80; H, 3.77; N, 5.12; C H Cl N O requires C,
2
3
RPB (250 · 4.6) mm, mobile phase: H O/CH CN
30:70), flow rate 1.0 mL/min, UV 210 nm, retention time
2
3
(
2
7
21
3
2
5
1
4
1.6 min; Elemental analysis found C, 56.71; H, 3.90; N,
.90; C H BrClN O requires C, 56.91; H, 3.89; N, 4.92.
5
7.93; H, 3.78; N, 5.00.
2
7
22
2
5
5
3
.1.9. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
-yl]acetic acid (3-chloro-4-fluoro phenylcarbamoyl) meth-
5.1.12. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl]acetic acid (4-bromo phenylcarbamoyl) methyl ester (12).
yl ester (9).
Br
F
O
O
H3CO
H3CO
Cl
O
O
NH
NH
CH3
CH3
N
N
O
O
O
O
Cl
Cl
1
1
Yield 42%; off white solid; mp 158–160 ꢁC; H NMR
200 MHz, CDCl ) d 7.62 (d, J = 8.3 Hz, 2H), 7.44 (d,
J = 8.8 Hz, 2H), 7.25–6.73 (m, 7H), 4.70 (s, 2H), 3.83
Yield 42%; off white solid; mp 160–164 ꢁC; H NMR
(
(200 MHz, CDCl ) d 7.59 (d, J = 8.8 Hz, 2H), 7.44 (d,
J = 8.3 Hz, 2H), 7.33–6.78 (m, 8H), 4.71 (s, 2H), 3.84 (s,
3
3

4828
S. Khanna et al. / Bioorg. Med. Chem. 14 (2006) 4820–4833
ꢀ
1
H), 3.77 (s, 3H), 2.45 (s, 3H); IR (KBr, cm ): 3322, 1733
2
(
J = 8.7 Hz, 1H), 6.62 (d, J = 8.8 Hz, 1H), 4.76 (s, 2H),
3.83 (s, 2H), 3.72 (s, 3H), 2.44 (s, 3H), 2.25 (s, 3H), 1.82
+
C@O), 1684; Mass (m/z) 572 (M+2, 15%), 570 (M ,
5%), 139 (100%); HPLC 96%, column: symmetry C18
150 · 4.6) mm, mobile phase: H O/CH CN (30:70), flow
rate 1.0 mL/min, UV 210 nm, retention time 8.75 min;
Elemental analysis found C, 56.78; H, 3.87; N, 4.98;
C H BrClN O requires C, 56.91; H, 3.89; N, 4.92.
ꢀ
1
1
(s, 3H); IR (KBr, cm ): 3282, 1741 (C@O), 1670; Mass
(m/z) 519 (M , 40%), 180 (100%); HPLC 98.2%, column:
+
(
2
3
Hichrom RPB (250 · 4.6) mm, mobile phase: H O/CH CN
2
3
(30:70), flow rate 1.0 mL/min, UV 210 nm, retention time
10.2 min; Elemental analysis found C, 67.34; H, 5.22; N,
2
7
22
2
5
5.44; C H ClN O requires C, 67.11; H, 5.24; N, 5.40.
29 27 2 5
5.1.13. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
3-yl]acetic acid (4-bromo-2-methyl phenylcarbamoyl) methyl
ester (13).
5.1.16. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl]acetic acid (3-methoxy phenylcarbamoyl) methyl ester (16).
Br
O
O
H3CO
OCH3
H3CO
O
O
CH3
NH
N
O
NH CH3
CH3
N
O
O
O
Cl
1
Cl
Yield 48%; off white solid; mp 142–144 ꢁC; H NMR
1
Yield 89%; off white solid; mp 168–170 ꢁC; H NMR
200 MHz, CDCl ) d 7.59 (d, J = 8.3 Hz, 2H), 7.44 (d,
J = 8.8 Hz, 2H), 7.39–7.25 (m, 4H), 6.95–6.59 (m, 3H),
.74 (s, 2H), 3.83 (s, 2H), 3.73 (s, 3H), 2.43 (s, 3H), 1.83
s, 3H); IR (KBr, cm ): 3257, 1756 (C@O), 1673; Mass
m/z) 586 (M+2, 15%), 584 (M , 45%), 139 (100%); HPLC
97%, column: ECLIPSE XDB-C8 (150 · 4.6) mm, mobile
phase: H O/CH CN (40:60), flow rate 1.5 mL/min, UV
210 nm, retention time 10.3 min; Elemental analysis
found C, 57.66; H, 4.16; N, 4.75 C H BrClN O re-
quires C, 57.60; H, 4.14; N, 4.80.
(200 MHz, CDCl ) d 7.60 (d, J = 8.8 Hz, 2H), 7.42 (d,
3
(
J = 8.3 Hz, 2H), 7.27 (m, 2H), 7.08 (m, 2H), 6.79 (m,
2H), 6.31 (d, J = 7.8 Hz, 1H), 4.71 (s, 2H), 3.84 (s, 2H),
3
ꢀ
1
3.76 (s, 6H), 2.45 (s, 3H); IR (KBr, cm ): 3276, 1740
4
(
(
ꢀ1
+
(C@O), 1672, 1609; Mass (m/z) 521 (M , 100%); HPLC
+
96.9%, column: ECLIPSE XDB C8 (250 · 4.6) mm, mo-
bile phase: H O/CH CN (40:60), flow rate 1.0 mL/min,
2
3
2
3
UV 220 nm, retention time 8.69 min; Elemental analysis
found C, 64.59; H, 4.83; N, 5.32; C H ClN O requires
C, 64.55; H, 4.84; N, 5.38.
2
8
25
2
6
2
8
24
2
5
5.1.17. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl]acetic acid (3,4-dimethoxy phenylcarbamoyl) methyl ester (17).
5.1.14. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
3-yl]acetic acid (4-iodo-2-methyl phenylcarbamoyl) methyl
ester (14).
OCH3
O
H3CO
OCH3
I
O
O
NH
CH3
H3CO
N
O
O
O
NH CH3
CH3
N
O
Cl
O
1
Yield 42%; purple colored solid; mp 168–170 ꢁC; H
Cl
NMR (200 MHz, CDCl ) d 7.58 (d, J = 8.3 Hz, 2H),
7.41 (d, J = 8.3 Hz, 2H), 7.20–6.63 (m, 7H), 6.14–6.10
(m, 1H), 4.69 (s, 2H), 3.83 (s, 9H), 3.75 (s, 2H), 2.44
(s, 3H); IR (KBr, cm ): 3233, 1719 (C@O), 1685,
1611; Mass (m/z) 551 (M+1, 100%); HPLC 99.6%, col-
umn: HICHROM RPB (250 · 4.6) mm, mobile phase:
H O/CH CN (30:70), flow rate 1.0 mL/min, UV
3
1
Yield 74%; light purple solid; mp 166-168 ꢁC; H NMR
200 MHz, CDCl ) d 7.60 (d, J = 8.3 Hz, 2H), 7.48–6.61
m, 9H), 4.75 (s, 2H), 3.84 (s, 2H), 3.75 (s, 3H), 2.45 (s,
(
(
3
1
1
3
ꢀ
1
ꢀ
+ +
1
H), 1.82 (s, 3H); IR (KBr, cm ): 3276, 1728 (C@O),
672; Mass (m/z) 630 (M , 8%), 505 (M ꢀ127, 15%),
39 (100%); HPLC 97.5%, column: ECLIPSE XDB-C8
2
3
(
150 · 4.6) mm, mobile phase: H O/CH CN (40:60), flow
210 nm, retention time 6.63 min; Elemental analysis
found C, 63.39; H, 4.93; N, 5.00; C H ClN O re-
quires C, 63.22; H, 4.94; N, 5.08.
2
3
rate 1.0 mL/min, UV 240 nm, retention time 16.5 min;
Elemental analysis found C, 53.50; H, 3.83; N, 4.40
C H ClIN O requires C, 53.31; H, 3.83; N, 4.44.
2
9
27
2
7
2
8
24
2
5
5.1.18. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl]acetic acid benzo[1,3]dioxol-5-ylcarbamoylmethyl ester (18).
5.1.15. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl]acetic acid (2,3-dimethyl phenylcarbamoyl) methyl ester (15).
O
O
O
H3CO
O
H3CO
CH3
NH CH3
O
O
NH
CH3
CH3
N
O
N
O
O
O
Cl
Cl
1
1
Yield 51%; off white solid; mp 152–155 ꢁC; H NMR
200 MHz, CDCl ) d 7.56 (d, J = 8.3 Hz, 2H), 7.42 (d,
J = 8.3 Hz, 2H), 7.09 (m, 3H), 6.98 (s, 1H), 6.78 (d,
Yield 30%; white solid; mp 155–160 ꢁC; H NMR (200 MHz,
CDCl ) d 7.58 (d, J = 8.3 Hz, 2H), 7.41 (d, J = 8.3 Hz, 2H),
7.14 (s, 1H), 7.02 (m, 2H), 6.87 (d, J = 9.3 Hz, 1H), 6.70 (d,
(
3
3

S. Khanna et al. / Bioorg. Med. Chem. 14 (2006) 4820–4833
4829
J = 11.2 Hz, 1H), 6.59 (d, J = 8.3 Hz, 1H), 6.22 (d, J = 8.3 Hz,
H), 5.91 (s, 2H), 4.67 (s, 2H), 3.81 (s, 2H), 3.75 (s, 3H), 2.43 (s,
1H), 6.88 (d, J = 9.2 Hz, 1H), 6.67 (d, J = 8.8 Hz, 1H),
5.14 (s, 2H), 3.84 (s, 5H), 2.43 (s, 3H); IR (KBr,
1
ꢀ1
ꢀ1
3
H); IR (KBr, cm ): 3278, 1741 (C@O), 1670, 1610; Mass
cm ): 3442, 1742 (C@O), 1706, 1673; Mass (m/z) 493
(M , 20%), 138 (100%); HPLC 98.7%, column: Hichrom
+
m/z) 535 (M , 40%), 372 (70), 139 (100%); HPLC 96.9%, col-
+
(
umn: symmetry C18 (150 · 4.6) mm, mobile phase: H O/
RPB (250 · 4.6) mm, mobile phase: 0.01 M KH PO /
2
2
4
CH CN (50:50), flow rate 1.2 mL/min, UV 210 nm, retention
time 23.2 min; Elemental analysis found C, 62.96; H, 4.32; N,
CH CN (60:40), flow rate 1.0 mL/min, UV 235 nm,
retention time 8.1 min; Elemental analysis found C,
60.78; H, 4.19, N, 11.50; C H ClN O requires C,
3
3
5.31; C H ClN O requires C, 62.87; H, 4.33; N, 5.24.
28 23 2 7
25 21
4
5
60.92; H, 4.29; N, 11.37.
5.1.19. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl]acetic acid (2-nitro phenylcarbamoyl) methyl ester (19).
5.1.22. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
3-yl]acetic acid thiazol-2-ylcarbamoyl methyl ester (22).
O
H3CO
O
H3CO
O
N
NH NO2
CH3
O
CH3
N
O
S
NH
N
O
O
O
Cl
1
Yield 65%; light yellow solid; H NMR (200 MHz,
Cl
CDCl ) d 11.09 (s, 1H), 8.80 (d, J = 8.3 Hz, 1H), 8.26
1
3
Yield 43%; white solid; mp 190–192 ꢁC; H NMR
200 MHz, CDCl ) d 10.2 (br s, 1H), 7.69 (d, J = 8.3 Hz,
2
(
J = 8.8 Hz, 2H), 7.26–6.63 (m, 5H), 4.81 (s, 2H), 3.95
d, J = 8.8 Hz, 1H), 7.70 (d, J = 8.3 Hz, 2H), 7.48 (d,
(
3
H), 7.46 (d, J = 8.3 Hz, 2H), 7.39 (d, J = 3.4 Hz, 1H),
.0 (d, J = 3.4 Hz, 1H), 6.97 (s, 1H), 6.85 (d, J = 8.8 Hz,
H), 6.67 (m, 1H), 4.85 (s, 2H), 3.82 (s, 5H), 2.44 (s,
ꢀ
1
(
3
(
s, 2H), 3.79 (s, 3H), 2.44 (s, 3H); IR (KBr, cm ):
338, 1743 (C@O), 1700, 1670, 1607; Mass (m/z) 536
M , 50%), 139 (100%); HPLC 98.9%, column: Hi-
7
1
3
+
ꢀ1
H); IR (KBr, cm ): 1750 (C@O), 1710, 1669; Mass
chrom RPB 250 mm, mobile phase: H O/CH CN
2
+
(m/z) 498 (M , 50%), 143 (100%); HPLC 96.0%, column:
3
(30:70), flow rate 1.0 mL/min, UV 225 nm, retention
time 11.8 min; Elemental analysis found C, 60.45; H,
symmetry C18 (150 · 4.6) mm, mobile phase: H O/
CH CN (40: 60), flow rate 1.0 mL/min, UV 260 nm, reten-
tion time 8.3 min; Elemental analysis found C, 57.91; H,
2
3
4
4
.13, N, 7.90; C H ClN O requires C, 60.51; H,
27 22 3 7
.14; N, 7.84.
4
.03, N, 8.48; C H ClN O S requires C, 57.89; H,
24 20 3 5
4
.05; N, 8.44.
5.1.20. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl]acetic acid (3-nitro phenylcarbamoyl) methyl ester (20).
5.1.23. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl]acetic acid (4-methyl thiazol-2-ylcarbamoyl) methyl ester (23).
O
H3CO
NO2
CH3
O
O
H3CO
NH
CH3
N
N
O
O
CH3
S
O
N
NH
O
Cl
O
1
Yield 48%; pale yellow solid; mp 164–166 ꢁC;
H
Cl
NMR (200 MHz, CDCl ) d 7.93 (d, J = 7.3 Hz, 1H),
3
1
Yield 16%; off white solid; mp 180–183 ꢁC; H NMR
7
7
.66 (d, J = 8.3 Hz, 2H), 7.46 (d, J = 8.8 Hz, 6H),
.05–6.71 (m, 3H), 4.76 (s, 2H), 3.87 (s, 2H), 3.73
(
(
200 MHz, CDCl ) d 7.67 (d, J = 8.8 Hz, 2H), 7.46
d, J = 8.8 Hz, 2H), 6.99 (s, 1H), 6.88 (d, J = 9.3 Hz,
3
ꢀ
1
(
s, 3H), 2.48 (s, 3H); IR (KBr, cm ): 3271, 1726
+
1
2
3
H), 6.62 (d, J = 9.3 Hz, 1H), 6.58 (m, 1H), 4.84 (s,
H), 3.91 (s, 2H), 3.84 (s, 3H), 2.44 (s, 3H), 2.33 (s,
H); IR (KBr, cm ): 3216, 1712 (C@O), 1639; Mass
(
3
C@O), 1703, 1667, 1605; Mass (m/z) 536 (M ,
5%), 139 (100%); HPLC 96.3%, column: symmetry
ꢀ
1
C18 (150 · 4.6) mm, mobile phase: H O/CH CN
2
3
+
(
m/z) 512 (M , 100%); HPLC 98.6%, column: Inertsil
(
time 5.4 min; Elemental analysis found C, 60.57; H,
30:70), flow rate 1.2 mL/min, UV 240 nm, retention
ODS 3V (150 · 4.6) mm, mobile phase: H O/CH CN
(30:70), flow rate 1.0 mL/min, UV 210 nm, retention
time 6.52 min; Elemental analysis found C, 59.45; H,
2
3
4
4
.20, N, 7.70; C H ClN O requires C, 60.51; H,
27 22 3 7
.14; N, 7.84.
4
4
.37, N, 8.18; C H ClN O S requires C, 58.65; H,
25 22 3 5
.33; N, 8.21.
5.1.21. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl]acetic acid pyrimidin-2-ylcarbamoyl methyl ester (21).
5.1.24. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl]acetic acid benzothiazol-2-ylcarbamoyl methyl ester (24).
O
H3CO
N
O
N
NH
CH3
N
O
O
O
H3CO
N
S
O
Cl
NH
1
CH3
Yield 67%; off white solid; mp 205–207 ꢁC; H NMR
200 MHz, CDCl ) d 8.54 (m, 3H), 8.38 (s, 1H), 7.68
d, J = 8.8 Hz, 2H), 7.46 (d, J = 8.8 Hz, 2H), 7.03 (s,
N
O
(
(
3
O
Cl

4830
S. Khanna et al. / Bioorg. Med. Chem. 14 (2006) 4820–4833
1
Yield 10%; off white solid; mp 188–190 ꢁC; H NMR
5.1.27. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
3-yl]acetic acid (5-methyl isoxazol-3-ylcarbamoyl) methyl
ester (27).
(
200 MHz, CDCl ) d 7.82 (d, J = 8.3 Hz, 2H), 7.71 (d,
3
J = 8.3 Hz, 2H), 7.45 (d, J = 8.3 Hz, 2H), 7.37 (d,
J = 7.8 Hz, 2H), 7.01 (s, 1H), 6.89 (d, J = 9.2 Hz, 1H),
CH3
O
6
3
.74 (d, J = 7.8 Hz, 1H), 4.89 (s, 2H), 3.89 (s, 2H),
H3CO
ꢀ
1
.84 (s, 3H), 2.45 (s, 3H); IR (KBr, cm ): 3441, 1745
O
N
O
CH3
+
(
(
(
C@O), 1714, 1690; Mass (m/z) 548 (M , 10%), 139
100%); HPLC 98.6%, column: Hichrom RPB
250 · 4.6) mm, mobile phase: H O/CH CN (25:75),
NH
N
O
O
2
3
Cl
flow rate 1.0 mL/min, UV 210 nm, retention time
1
6
.65 min; Elemental analysis found C, 61.49; H, 4.00,
N, 7.60; C H ClN O S requires C, 61.37; H, 4.05; N,
Yield 53%; off white solid; mp 175–178 ꢁC; H NMR
(200 MHz, CDCl ) d 8.32 (br s, 1H), 7.73 (d,
J = 8.3 Hz, 2H), 7.48 (d, J = 8.3 Hz, 2H), 6.96 (s, 1H),
.85 (d, J = 8.8 Hz, 1H), 6.69 (m, 1H), 6.64 (s, 1H),
4.75 (s, 2H), 3.82 (s, 5H), 2.45 (s, 3H), 2.39 (s, 3H); IR
2
8
22
3
5
3
7
.67.
6
5
.1.25. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl]acetic acid (6-methoxy benzothiazol-2-ylcarbamoyl) methyl
ester (25).
ꢀ
1
(KBr, cm ): 1741 (C@O), 1714, 1677, 1635; Mass
(m/z) 496 (M , 50%), 157 (100%); HPLC 98.6%, column:
+
Inertsil ODS 3 V (250 · 4.6) mm, mobile phase: H O/
2
OCH3
CH CN (30:70), flow rate 1.0 mL/min, UV 220 nm,
3
O
retention time 9.5 min; Elemental analysis found C,
60.47; H, 4.46, N, 8.50; C H ClN O requires C,
60.55; H, 4.47; N, 8.47.
H3CO
N
S
O
25 22
3
6
NH
CH3
N
O
O
5
.1.28. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
-yl]acetic acid (5-carbamoyl-1-methyl-3-propyl-1H-pyra-
Cl
3
zol-4-ylcarbamoyl) methyl ester (28).
1
Yield 10%; yellow solid; mp 187–189 ꢁC; H NMR
200 MHz, CDCl ) d 7.70 (d, J = 8.3 Hz, 2H), 7.58 (d,
J = 8.8 Hz, 2H), 7.45 (d, J = 8.3 Hz, 2H), 7.04 (d,
CH3
N
(
N
3
O
NH2
O
H3CO
NH
J = 9.2 Hz, 2H), 6.97 (s, 1H), 6.89 (d, J = 9.2 Hz, 1H),
4
O
CH3
O
.87 (s, 2H), 3.89 (s, 2H), 3.84 (s, 6H), 2.47 (s, 3H); IR
N
ꢀ1
(
(
KBr, cm ): 3413, 1745 (C@O), 1606; Mass (m/z) 578
M , 20%), 139 (100%); HPLC 95.4%, column:
+
O
Cl
ECLIPSE XDB (150 · 4.6) mm, mobile phase: H O/
1
2
Yield 42%; white solid; mp 168–170 ꢁC; H NMR
200 MHz, CDCl ) d 7.59 (d, J = 8.3 Hz, 2H), 7.46 (d,
J = 8.3 Hz, 2H), 7.14 (s, 1H), 6.98 (s, 1H), 6.78 (d,
CH CN (45:55), flow rate 1.0 mL/min, UV 210 nm,
retention time 13.79 min; Elemental analysis found C,
3
(
3
6
0.43; H, 4.15, N, 7.30; C H ClN O S requires C,
29 24 3 6
J = 9.2 Hz, 1H), 6.62 (d, J = 9.2 Hz, 1H), 4.75 (s, 2H),
3
6
0.26; H, 4.18; N, 7.27.
.98 (s, 3H), 3.84 (s, 2H), 3.74 (s, 3H), 2.45 (s, 3H), 2.15
m, 2H), 1.44 (m, 2H), 0.84 (m, 3H); IR (KBr, cm ):
ꢀ
1
(
3
2
5.1.26. 3-(2-{2-[1-(4-Chlorobenzoyl)-5-methoxy-2-meth-
yl-1H-indol-3-yl]acetoxy} acetylamino) thiophene-2-car-
boxylic acid methyl ester (26).
+
330, 1730 (C@O), 1677; Mass (m/z) 580 (M , 30%),
23 (100%); HPLC 96.8%, column: Hichrom RPB
250 · 4.6) mm, mobile phase: H O/CH CN (50: 50), flow
rate 1.0 mL/min, UV 220 nm, retention time 18.3 min;
Elemental analysis found C, 60.15; H, 5.20, N, 12.10;
(
2
3
O
H3CO
S
C
29
H
30ClN
O requires C, 60.05; H, 5.21; N, 12.07.
5 6
O
CH3
NH
2 3
CO CH
N
O
5.1.29. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-
indol-3-yl]acetic acid benzylcarbamoylmethyl ester (29).
O
Cl
O
H3CO
O
1
NH
Yield 82%; off white solid; mp 165–168 ꢁC; H NMR
200 MHz, CDCl ) 10.95 (s, 1H), 8.12 (d,
J = 5.4 Hz, 1H), 7.67 (d, J = 8.3 Hz, 2H), 7.48 (m,
H), 7.05 (s, 1H), 6.86 (d, J = 9.2 Hz, 1H), 6.67 (d,
J = 11.2 Hz, 1H), 4.79 (s, 2H), 3.98 (s, 2H), 3.90 (s,
CH3
N
O
(
d
3
O
Cl
3
1
Yield 23%; off white solid; mp 135–137 ꢁC; H NMR
(200 MHz, CDCl ) d 7.59 (d, J = 8.3 Hz, 2H), 7.45 (d,
J = 8.3 Hz, 2H), 7.24 (m, 4H), 7.05 (m, 1H), 6.95 (s,
1H), 6.82 (d, J = 9.2 Hz, 1H), 6.66 (d, J = 8.8 Hz, 1H),
6.01 (m, 1H), 4.64 (s, 2H), 4.25 (d, J = 5.9 Hz, 2H), 3.77
ꢀ
1
3
1
H), 3.79 (s, 3H), 2.46 (s, 3H); IR (KBr, cm ): 3325,
748 (C@O), 1701, 1674; Mass (m/z) 555 (M , 100%);
3
+
HPLC 98.8%, column: Hichrom RPB (250 · 4.6) mm,
mobile phase: H O/CH CN (25:75), flow rate 1.0 mL/
2
3
ꢀ1
min, UV 220 nm, retention time 9.9 min; Elemental
analysis found C, 58.40; H, 4.20, N, 5.00;
C H ClN O S requires C, 58.43; H, 4.18; N, 5.05.
(s, 5H), 2.35 (s, 3H); IR (KBr, cm ): 3297, 1746
+
(C@O), 1662; Mass (m/z) 505 (M , 100%); HPLC
97.9%, column: Hichrom RPB (250 · 4.6) mm, mobile
2
7
23
2
7

S. Khanna et al. / Bioorg. Med. Chem. 14 (2006) 4820–4833
4831
ꢀ
1
phase: H O/CH CN (30:70), flow rate 1.0 mL/min, UV
2
3H); IR (KBr, cm ): 3435, 1731 (C@O), 1661, 1603;
Mass (m/z) 485 (M , 50%), 139 (100%); HPLC 97.7%,
3
+
2
10 nm, retention time 7.83 min; Elemental analysis
found C, 66.49; H, 5.00, N, 5.61; C H ClN O requires
column: ECLIPSE XDB (150 · 4.6) mm, mobile phase:
H O/CH CN (45:55), flow rate 1.0 mL/min, UV 225 nm,
2
8
25
2
5
C, 66.60; H, 4.99; N, 5.55.
2
3
retention time 5.54 min; Elemental analysis found C,
61.99; H, 5.21, N, 5.68; C H ClN O requires C,
61.92; H, 5.20; N, 5.78.
5.1.30. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl]acetic acid (4-methoxy benzylcarbamoyl) methyl ester (30).
2
5
25
2
6
O
H3CO
5.1.33. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
3-yl]acetic acid 2-oxo-2-thiomorpholin-4-ylethyl ester (33).
O
OCH3
NH
CH3
N
O
O
O
S
O
H3CO
N
Cl
CH3
O
1
N
Yield 36%; white solid; mp 150–152 ꢁC; H NMR
200 MHz, CDCl ) d 7.59 (d, J = 8.8 Hz, 2H), 7.45 (d,
(
O
3
Cl
J = 8.3 Hz, 2H), 6.98 (d, J = 8.8 Hz, 2H), 6.93 (s, 1H),
.85 (d, J = 9.2 Hz, 1H), 6.77 (d, J = 8.3 Hz, 2H), 6.67
m, 1H), 5.96 (m, 1H), 4.62 (s, 2H), 4.18 (d, J = 5.9 Hz,
H), 3.78 (s, 3H), 3.77 (s, 3H), 3.75 (s, 2H), 2.34 (s, 3H);
1
Pale yellow solid; mp 164–165 ꢁC; H NMR (200 MHz,
6
(
2
CDCl ) d 7.66 (d, J = 8.3 Hz, 2H), 7.46 (d, J = 8.3 Hz,
2H), 7.02–6.65 (m, 3H), 4.73 (s, 2H), 3.84 (s, 2H), 3.82
(s, 3H), 3.59 (br s, 4H), 2.60 (br s, 4H), 2.39 (s, 3H);
3
ꢀ
1
IR (KBr, cm ): 3295, 1735 (C@O), 1664; Mass (m/z)
ꢀ1
+
35 (M , 20%), 139 (100%); HPLC 98.9%, column: sym-
IR (KBr, cm ): 3439, 1733 (C@O), 1662; Mass (m/z)
5
+
03 (M+2, 25%), 502 (M+1, 40%), 501 (M , 55%), 139
5
metry C18 (150 · 4.6) mm, mobile phase: H O/CH CN
2
3
(
(
100%); HPLC 98.5%, column: Hichrom RPB
250 · 4.6) mm, mobile phase: H O/CH CN (30:70),
(
40: 60), flow rate 1.0 mL/min, UV 225 nm, retention time
2
3
1
5
0.3 min; Elemental analysis found C, 65.19; H, 5.07, N,
.10; C H ClN O requires C, 65.11; H, 5.09; N, 5.24.
flow rate 1.0 mL/min, UV 210 nm, retention time
6.7 min; Elemental analysis found C, 59.91; H, 5.00,
N, 5.60; C H ClN O S requires C, 59.93; H, 5.03; N,
2
9
27
2
6
2
5
25
2
5
5
indol-3-yl]acetic acid octylcarbamoyl methyl ester (31).
.1.31.
[1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-
5.59.
5.2. Docking studies
O
H3CO
O
Docking studies were carried out using FlexX in both
forms of COX. The crystal structures of sheep COX-1
with iodo analogue of indomethacin (1PGG.pdb and
1PGF.pdb) and murine COX-2 with indomethacin
NH
CH3
N
O
O
Cl
(
4COX.pdb) were used in the study. The structural dif-
1
Yield 29%; white solid; mp 88–90 ꢁC; H NMR (200 MHz,
ferences in the active sites of COX-1 and COX-2 from
different species are negligible compared to those be-
tween COX-1 and COX-2 themselves. Docking studies
were carried out using FlexX. In the cases where
FlexX failed, the bioactive conformations of iodoindo-
methacin (1PGG.pdb and 1PGF.pdb) was used to
CDCl ) d 7.66(d, J = 8.8 Hz, 2H), 7.49 (d, J = 8.3 Hz, 2H),
3
6
1
3
.98 (s, 1H), 6.89 (d, J = 8.8 Hz, 1H), 6.71 (d, J = 8.8 Hz,
H), 5.64 (m, 1H), 4.57 (s, 2H), 3.83 (s, 3H), 3.78 (s, 2H),
.03 (d, J = 5.9 Hz, 2H), 2.42 (s, 3H), 1.18 (m, 12H), 0.88
25
ꢀ
1
(
t, J = 12.7 Hz, 3H); IR (KBr, cm ): 3302, 1743 (C@O),
+
657; Mass (m/z) 527 (M , 100%); HPLC 98.3%, column:
1
25
construct the 3D models using SYBYL. Hydrogen
symmetry C18 (150 · 4.6) mm, mobile phase: H O/
CH CN (30:70), flow rate 1.5 mL/min, UV 210 nm, reten-
tion time 10.9 min; Elemental analysis found C, 66.19; H,
2
atoms were added to the proteins while all the basic
and acidic amino acid residues were considered to be
charged. In addition, the acid group of ligands was
considered negatively charged. The ligand–receptor
complexes were minimized using MMFF94 force
fields, and Powell method with a convergence gradient
value of 0.001 kcal/mol, and a distance dependent
dielectric constant value of 1. Atomic charges were
computed using MMFF94 method. All amino acid
3
6
N, 5.32.
.61, N, 5.52; C H ClN O requires C, 66.09; H, 6.69;
29 35 2 5
5.1.32. [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl]acetic acid 2-morpholin-4-yl-2-oxo ethyl ester (32).
O
H3CO
˚
residues within 6 A radius around the ligand were
minimized while treating the rest of the protein as
an aggregate.
O
N
O
CH3
N
O
O
Cl
5.3. Biological assays
1
Yield 24%; off white solid; mp 152–153 ꢁC; H NMR
200 MHz, CDCl ) d 7.67 (d, J = 8.3 Hz, 2H), 7.47 (d,
J = 8.3 Hz, 2H), 7.03 (s, 1H), 6.88 (d, J = 8.8 Hz, 1H),
(
5.3.1. In vitro biochemical assays (spectrophotometric
assay of COX-1 and COX-2). Microsomal fraction of
ram seminal vesicles were used as a source of COX-1
3
6
.67 (d, J = 9.3 Hz, 1H), 4.74 (s, 2H), 3.85 (s, 3H),
.83 (s, 2H), 3.63 (br s, 4H), 3.34 (br s, 4H), 2.39 (s,
2
6
3
enzyme and microsomes from sf-9 cells infected with

4832
S. Khanna et al. / Bioorg. Med. Chem. 14 (2006) 4820–4833
baculovirus containing human COX-2 c-DNA were
2
Acknowledgments
7
used as a source of COX-2 enzyme. Enzyme activity
was measured using a chromogenic assay based on oxi-
The authors thank Dr. A. Venkateswarlu, Dr. R. Raja-
gopalan, and Prof. J. Iqbal of Dr. Reddy’s Laboratories
Limited, Hyderabad, India, and Dr. U. Saxena and
Dr. I. Khanna of Reddy US Therapeutics, Norcross,
GA, for their constant encouragement. Authors also
thank the analytical research group for excellent support
and the IPM group for literature support.
0
0
dation of N,N,N ,N -tetramethyl-p-phenylenediamine
TMPD) during the reduction of PGG to PGH as
(
2
2
2
8
per the procedure described by Copeland et al., with
the following modifications. The assay mixture
(
1
1000 lL) contained 100 mM Tris pH 8.0, 3 mM EDTA,
5 lM hematin, 150 units enzyme and 8% DMSO. The
mixture was pre-incubated at 25 ꢁC for 15 min before
initiation of enzymatic reaction in the presence of com-
pound/vehicle. The reaction was initiated by the addi-
tion of 100 lM arachidonic acid and 120 lM TMPD.
The enzyme activity was measured by estimation of
the initial velocity of TMPD oxidation over the first
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0
0501 5 January 1995.
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