Synthesis and biological evaluation of ester derivatives of indomethacin as selective COX-2 inhibitors

Article abstract of DOI:10.1007/s00044-011-9747-5

The ester derivatives of indomethacin were prepared by condensing indomethacin with an equimolar quantity of an appropriate alcoholic compound in anhydrous dichloromethane in the presence of DCC and DMAP. Spectral studies comprising of IR, ¹H NMR, mass, and microanalysis were performed in order to confirm their structures. In vivo anti-inflammatory studies were carried out using carrageenan rat paw edema method and in vivo ulcerogenic studies by ulcer index method for the panel of synthesized compounds. Out of eleven compounds, the compound IIc displayed moderate anti-inflammatory activity with no observable ulcerogenic effect when compared to indomethacin. Furthermore, compound IIc, indomethacin and celecoxib were tested at a concentration of 20 μM against COX-1 and COX-2 enzymes by colorimetric COX inhibitor screening assay. Compound IIc was found to be active against COX-2 and COX-1 enzymes exhibiting 62.0 and 12.9% inhibition, respectively. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9747-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:2223–2228
DOI 10.1007/s00044-011-9747-5
ORIGINAL RESEARCH
Synthesis and biological evaluation of ester derivatives
of indomethacin as selective COX-2 inhibitors
•
M. Arockia Babu Rakesh Shukla
•
•
Chandishwar Nath S. G. Kaskhedikar
Received: 18 April 2010 / Accepted: 8 July 2011 / Published online: 16 July 2011
Ó Springer Science+Business Media, LLC 2011
Abstract The ester derivatives of indomethacin were
prepared by condensing indomethacin with an equimolar
quantity of an appropriate alcoholic compound in anhy-
drous dichloromethane in the presence of DCC and DMAP.
Introduction
NSAIDs are used extensively to alleviate inflammation,
pain, rheumatoid arthritis, and osteoarthritis. Long-term
regimens of NSAIDs have been greatly shortened due to
their gastrointestinal side effects (Laine, 2001). These
adverse effects are driving the research toward develop-
ment of NSAIDs with minimal, if any, gastrointestinal side
effects. Vane proposed that the mechanism-of-action of
classical NSAIDs viz., aspirin, ibuprofen, diclofenac,
mefenamic acid, indomethacin, sulindac, and picroxicam is
through their inhibition of prostaglandin biosynthesis
(Vane, 1971). We have therefore directed our efforts to
develop safer NSAIDs over conventional NSAIDs. In
addition, the structures of flurbiprofen (Bayly et al., 1999),
indomethacin (Kalgutkar et al., 2000), meclofenamic acid
(Kalgutkar et al., 2002), ketrolac (Black et al., 1996;
Bhandari et al., 2007; Mishra et al., 2008), etc. have been
modified with selective COX-2 inhibitory activity. These
modified structures were found to have similar or moderate
efficacy to that of selective COX-2 inhibitors, but with
greater gastrointestinal safety. Recently, several research
groups independently tried to modify the structure of
Indomethacin (Kalgutkar et al., 2000; Wood et al., 2001;
Khanna et al., 2006), which has led to clinical trials of
L-761, 000, as a potent selective COX-2 inhibitor (Leblanc
et al., 1996). This is encouraged us to attempt to synthesize
safer indomethacin esters via modifications of the carbox-
ylic group.
1
Spectral studies comprising of IR, H NMR, mass, and
microanalysis were performed in order to confirm their
structures. In vivo anti-inflammatory studies were carried
out using carrageenan rat paw edema method and in vivo
ulcerogenic studies by ulcer index method for the panel of
synthesized compounds. Out of eleven compounds, the
compound IIc displayed moderate anti-inflammatory
activity with no observable ulcerogenic effect when com-
pared to indomethacin. Furthermore, compound IIc, indo-
methacin and celecoxib were tested at a concentration of
20 lM against COX-1 and COX-2 enzymes by colori-
metric COX inhibitor screening assay. Compound IIc was
found to be active against COX-2 and COX-1 enzymes
exhibiting 62.0 and 12.9% inhibition, respectively.
Keywords Indomethacin esters ꢀ COX-2 inhibitors ꢀ
Colorimetric COX inhibitor screening assay
M. Arockia Babu (&)
School of Pharmacy and Health Sciences, International Medical
University, 126, Jalan 19/155B, Bukit Jalil, 57000 Kuala
Lumpur, Malaysia
e-mail: babuphd2001@yahoo.co.in
R. Shukla ꢀ C. Nath
Chemistry
Division of Pharmacology, Central Drug Research Institute,
Lucknow 226 001, India
The indomethacin derivatives were prepared according to
the synthetic route illustrated in Scheme 1. The indo-
methacin esters were prepared from condensation between
S. G. Kaskhedikar
Department of Pharmacy, Shri G. S. Institute of Technology and
Science, Indore 452003, India
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Med Chem Res (2012) 21:2223–2228
Scheme 1 Synthesis of
indomethacin esters
OR
OH
MeO
MeO
O
O
N
O
N
ROH
a
O
Cl
Cl
I
IIa-k
Reagents and conditions: (a) N,N'-Dicyclohexyl carbodiimide (DCC),
Dimethyl amino pyridine (DMAP), Dichloromethane, 5h
R=
COO-(CH2)3-CH3
CH2
CH2
IIa
IIb
COO-CH2-CH3
IIc
N
CH2 CH2
IId
CH2
N
N
CH2
IIe
CH2-CH3
IIf
CH2
CH2
IIg
NO2
O
IIi
IIh
Cl
Cl
Cl
Cl
Cl
Cl
II
IIj
k
indomethacin and hydroxyl group containing alcoholic
compounds in the presence of DCC and DMAP (Kalgutkar
et al., 2000). The synthesized compounds were evaluated
for their in vivo anti-inflammatory activity, gastrointestinal
toxicity, and COX-1 and COX-2 enzyme inhibitory activ-
ities. All the final compounds were chemically character-
ized by melting point, infrared (IR) spectra, nuclear
magnetic resonance (NMR) spectra, mass spectra, and
elemental analysis.
Experiment
Melting points were determined by the open capillary
method using a Besto melting point apparatus without
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Med Chem Res (2012) 21:2223–2228
2225
correction. Indomethacin was procured from Sun Pharma,
Gujarat (India). All other chemicals and solvents were
purchased from Lancaster and Merck, respectively, except
anhydrous methylene chloride which was purchased from
Sigma-Aldrich. Precoated TLC plates from Merck India
were used to follow the course of reactions. Developed
plates were visualized by iodine vapor. Silica gel
(60–120 mesh) was used for column chromatography and
purchased from Spectrochem Pvt. Ltd. (Mumbai). IR
spectra were recorded on a Perkin-Elmer 237 spectropho-
Table 1 Anti-inflammatory activity of indomethacin esters in car-
rageenan induced rat paw edema
Group
(N = 5)
0 h
1.5 h
3 h
%
Inhibition
Control
IIa
IIb
IIc
1.00 0.057 1.60 0.10 1.66 0.07
–
0.92 0.01 1.51 0.08 1.53 0.08 7.83
0.93 0.04 1.42 0.07 1.51 0.12 9.04
0.98 0.03 1.22 0.05 1.33 0.08b 19.88
1.01 0.03 1.42 0.06 1.54 0.09 7.23
IId
IIe
1.00 0.01 1.63 0.14 1.67 0.13
0
1
tometer using KBr pellets. H NMR spectra were recorded
IIf
1.04 0.02 1.52 0.04 1.62 0.04 2.41
1.00 0.09 1.49 0.29 1.60 0.26 3.61
0.95 0.09 1.47 0.12 1.58 0.13 4.81
in CDCl3 on a Bruker DRX300 (300 MHz FT NMR)
instrument. Mass spectra were determined on a Joel
SX-102 spectrometer. Carbon, hydrogen, and nitrogen
analysis were performed on CHN analyses Carlo Erba
1108, Heracus at the Central Drug Research Institute
(CDRI), Lucknow.
IIg
IIh
IIi
0.98 0.08 1.53 0.23 1.77 0.18
0.92 0.89 1.47 0.25 1.71 0.30
1.00 0.07 1.52 0.17 1.66 0.20
0
0
0
IIj
IIk
Indomethacin 1.03 0.01 1.27 0.06 1.43 0.06a 13.86
Each value is the mean SEM in five rats
a
Synthesis of indomethacin esters
P \ 0.05 compared to control
b
P \ 0.01 compared to control
A reaction mixture containing indomethacin (0.003 mol) in
6 ml of anhydrous dichloromethane (CH2Cl2) was treated
with DCC (2.76 mmol), DMAP (252 lmol), and appro-
priate alcohol (0.003 mol). After stirring at room temper-
ature for 5 h, the reaction mixture was filtered and the
filtrate was concentrated in a vacuum. The residue was
diluted with water (*30 ml) and extracted with ethyl
acetate (2 9 30 ml). The combined organic solution was
washed with 5% acetic acid (2 9 30 ml), 1 N sodium
hydroxide (2 9 30 ml) and water (*100 ml), dried in
sodium sulphate, filtered and the solvent was removed
under a vacuum. The product was obtained as a solid or oil.
This procedure was used to synthesize all of the com-
pounds (IIa–k). Physical data of compounds are given in
Table 1.
[1-(4-Chloro-benzoyl)-5-methoxy-2methyl 1H-indol-3-yl]
acetic acid 2-(2,4,6-trimethyl-phenyl)-ethyl ester (IIb)
1
Yield 88%; mp = 98–100°C; H NMR (CDCl3) d 2.04 (s,
9H, CH3), 2.32 (s, 3H, CH3), 2.93–2.98 (m, 2H, CH2), 3.66
(s, 2H, CH2COO), 3.82 (s, 3H, OCH3), 4.16–4.21 (m, 2H,
COOCH2), 6.66–6.69 (d, 2H, J = 8.7 Hz, Ar–H),
6.82–6.93 (m, 2H, Ar–H), 7.26 (s, 1H, Ar–H), 7.46–7.49 (d,
2H, J = 8.1 Hz, Ar–H), 7.65–7.68 (d, 2H, J = 8.1 Hz, Ar–
H); IR (KBr) 1729 cm-1 (C=O, ester); FAB–MS m/z 505
[M ? H]?; Anal. cald. For C30H30ClNO4; C, 71.49; H,
6.00; N, 2.78; Found: C, 71.50; H, 6.10; N, 2.80.
[4{2-[1-(4-Chloro-benzoyl)-5-methoxy-2-methyl 1H-indol-
3-yl] acetoxy}-benzoic acid butyl ester (IIa)
4{2-[1-(4-Chloro-benzoyl)-5-methoxy-2-methyl 1H-indol-
3-yl] acetoxy} benzoic acid ethyl ester (IIc)
Yield 90.6%; mp 68–71°C; 1H NMR (CDCl3)
d
0.94–0.99 (m, 3H, CH3), 1.43–1.50 (m, 2H, CH2),
1.69–1.76 (m, 2H, CH2), 2.45 (s, 3H, CH3), 3.84 (s, 3H,
OCH3), 3.92 (s, 2H, –CH2–COO–), 4.26–4.34 (m, 2H,
COO–CH2), 6.69–6.71 (d, 2H, J = 7.5 Hz, Ar–H),
6.72–6.90 (m, 2H, Ar–H), 7.04 (s, 1H, Ar–H), 7.13–7.16
(d, 1H, J = 8.7 Hz, Ar–H), 7.46–7.49 (d, 1H,
J = 8.1 Hz, Ar–H), 7.66–7.69 (d, 2H, J = 8.1 Hz,
Ar–H), 8.04–8.06 (d, 2H, J = 8.4 Hz, Ar–H); IR (KBr)
1738 cm-1 (C=O, ester); FAB–MS m/z 535 [M ? H]?;
Anal. cald. for C30H28ClNO6; C, 67.48; H, 5.29; N, 2.62;
Found: C, 67.50:, H, 5.27; N,2.64.
Yield 74.0%; 1H NMR(CDCl3) d 1.05–1.13 (m, 3H, CH3),
2.34 (s, 3H, CH3), 3.08 (s, 2H, –CH2COO), 3.77 (s, 3H,
–OCH3), 4.28–4.35 (m, 2H, –COOCH2–), 6.59–6.63
(d, 2H, J = 9.00 Hz, Ar–H), 7.10 (m, 2H, Ar–H),
7.34–7.41 (d, 2H, J = 8.1 Hz, Ar–H), 7.55–7.58 (d, 2H,
J = 8.1 Hz, Ar–H), 7.83–7.86 (d, 2H, J = 8.4 Hz, Ar–H),
8.07–8.10 (d, 1H, J = 8.4 Hz, Ar–H); IR (KBr) 1742 cm-1
(C=O, ester); FAB–MS m/z 507 [M ? H]?; Anal. cald. for
C28H24ClNO6; C, 66.47; H, 4.78; N, 2.77; Found: C, 66.50;
H, 4.78; N, 2.80.
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Med Chem Res (2012) 21:2223–2228
[1-(4-Chloro-benzoyl)-5-methoxy-2-methyl-1H indol-3-yl]
acetic acid-2-piperidin-1-yl ethyl ester (IId)
[1-(4-Chloro-benzoyl)-5-methoxy-2methyl-1H-indol-3-yl]-
acetic acid 4-nitro phenyl ester (IIh)
1
Yield 69.7%; mp = 130–132°C; 1H NMR(CDCl3) d
1.11–1.90 (m, 10H, Piperidyl), 2.37 (s, 3H, CH3), 3.20
(s, 2H, –CH2COO–), 3.84 (s, 3H, –OCH3), 3.98 (m, 2H,
–COOCH2–), 3.98 (m,2H, –COOCH2), 6.75–6.78 (d, 2H,
J = 8.4 Hz, Ar–H), 7.13–7.16 (2H, d, J = 8.4 Hz, Ar–H),
7.28–7.32 (m, 2H, Ar–H), 7.87 (s,1H, Ar–H); IR (KBr)
1742 cm-1 (C=O, ester); FAB-MS m/z 470 [M ? H]?;
Anal. cald. for C26H29ClN2O4; C, 66.59; H, 6.23; N, 5.97;
Found: C, 66.61; H, 6.19; N, 5.91.
Yield 78%; H NMR(CDCl3) d 2.48 (s, 3H, CH3), 3.71
(s, 2H, –CH2–COO–), 3.95 (s, 3H, OCH3), 6.69–6.72
(d, J = 9.3 Hz, 2H, Ar–H), 6.86–6.89 (d, J = 9.3 Hz, 2H,
Ar–H), 7.03 (s, 1H, Ar–H), 7.47–7.50 (m, 2H, Ar–H),
7.47–7.50 (d, J = 7.8 Hz, 2H, Ar–H), 8.24–8.27 (d, J =
8.4 Hz, 2H, Ar–H); FAB-MS m/z 479 [M ? H]?; IR (KBr)
1740 cm-1 (–C=O, ester); Anal. cald. for C25H19ClN2O6;
C, 62.70; H, 4.00; N, 5.85; Found: C, 62.80; H, 4.10; N, 5.90.
1-[1-(4-Chloro-benzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl]–acetic acid furan-2-yl methyl ester (IIi)
[1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H indol-3-yl]
acetic acid-2 piperazin-1-yl ethyl ester (IIe)
Yield 84%; 1H NMR(CDCl3) d2.35 (s, 3H, CH3), 3.69
(s, 2H, CH2), 3.80 (s, 2H, CH2-CO), 3.99 (s, 3H, OCH3),
6.36–6.39 (d, J = 8.4 Hz, 2H, Ar–H), 6.65–6.68
(d, J = 8.7 Hz, 2H, Ar–H), 6.86–6.96 (m, 2H, Ar–H),
7.38–7.48 (m, 2H, Ar–H), 7.64–7.66 (d, J = 8.4 Hz, 2H,
Ar–H); IR (KBr) 1680 cm-1 (C=O, ester); FAB-MS m/z
439 [M ? H]?; Anal. cald. for C24H20ClNO5; C, 65.83; H,
4.60; N, 3.20; Found: C, 65.90; H, 3.10; N, 3.27.
Yield 67.0%; mp = 128–130°C; 1H NMR(CDCl3) d
1.07–1.90 (m, 8H, piperazinyl, CH2), 2.00 (s, 1H, NH),
2.37 (s, 3H, CH3), 3.61–3.66 (m, 2H, CH2COO–), 3.54 (m,
2H, CH2), 3.84 (s, 3H, OCH3), 3.98–4.01 (m, 2H, –CO-
OCH2), 6.74–6.78 (d, J = 8.2 Hz, 2H, Ar–H), 7.13–7.15
(d, J = 8.7 Hz, 2H, Ar–H), 7.27–7.33 (m, 2H, Ar–H), 7.85
(s, 1H, Ar–H); IR (KBr) 1769 cm-1 (–C=O, ester); FAB-
MS m/z 471 [M ? H]?, Anal. cald. for C25H28ClN3O4; C,
63.89; H, 6.01; N, 8.94; Found: C, 63.91; H, 6.10; N, 8.96.
[1-(4-Chloro-benzoyl)-5-methoxy-2methyl 1H-indol-3-yl]
acetic acid 4-chloro-phenyl ester (IIj)
[1-(4-Chloro-benzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]
acetic acid 4-ethyl phenyl ester (IIf)
1
Yield 76%; H NMR(CDCl3) d 2.45 (s, 3H, CH3), 3.84
(s, 2H, –CH2–CO–)3.97 (s, 3H, OCH3), 6.78 (d,
J = 9.3 Hz, 2H, Ar–H), 6.89–6.92 (d, J = 9.0 Hz, 2H,
Ar–H), 7.06 (s, 1H, Ar–H), 7.10–7.32 (m, 2H, Ar–H),
7.45–7.48 (d, J = 8.1 Hz, 2H, Ar–H), 7.65–7.68 (d,
J = 8.1 Hz, 2H, Ar–H); IR (KBr) 1750 cm-1 (–C=O,
ester); FAB-MS m/z 469 [M ? H]?; Anal. cald. for
C25H19Cl2NO4; C, 64.11; H, 4.09; N, 2.99; Found: C,
64.44; H, 4.09; N, 2.89.
Yield 87.0%; mp = 128–130°C; 1H NMR(CDCl3) d
1.19–1.26 (m, 3H, CH3), 2.44 (s, 3H, CH3), 2.59–2.64 (m,
2H, –CH2CH3), 3.61–3.66 (m, 2H, CH2) 3.84 (s, 3H,
–OCH3), 6.68–6.71 (d, 2H, J = 8.1 Hz, Ar–H), 6,89–6.97
(m, 1H, Ar–H), 7.06 (1H, s, Ar–H), 7.15–7.18 (d, 2H,
J = 8.4 Hz, Ar–H), 7.36 (s, 1H, Ar–H), 7.46–7.49 (d, 2H,
J = 8.4 Hz, Ar–H), 7.66–7.70 (d, 2H, J = 8.4 Hz, Ar–H);
IR (KBr) 1752 cm-1 (C=O, ester); FAB-MS m/z 463
[M ? H]?; Anal. cald. for C27H24ClNO4; C, 70.20; H,
5.24; N, 3.03; Found: C, 70.94; H, 5.69; N, 2.99.
[1-(4-Chloro-benzoyl)-5-methoxy-2-methyl-1H- indol-3-yl]
acetic acid pentachlorophenyl ester (IIk)
Yield 58%; 1H NMR(CDCl3) d2.46 (s, 3H, CH3), 3.84
(s, 2H, –CH2CO–), 4.12 (s, 3H, OCH3), 6.68–6.71
(d, J = 9.0 Hz, 1H, Ar–H), 7.47–7.49 (d, J = 7.8 Hz, 2H,
Ar–H), 7.65–7.68 (d, J = 7.8 Hz, 2H, Ar–H), 7.65–7.68
(d, J = 7.8 Hz, 2H, Ar–H); IR (KBr) 1680 cm-1 (–C=O,
ester); FAB-MS m/z 607 [M ? H]?; Anal. cald. for
C25H15Cl6NO4; C, 49.54; H, 2.49; N, 2.31; Found: C,
49.58; H, 2.49; N, 2.39.
[1-(4-Chloro-benzoyl)-5-methoxy-2-methyl 1H-indol-3-yl]
acetic acid benzyl ester (IIg)
1
Yield 72%; H NMR(CDCl3) d 2.38 (s, 3H, CH3), 3.71
(s, 2H, CH2–COO–), 3.77 (s, 3H, –OCH3), 5.36 (s, 2H,
CH2–C6H5), 6.75–6.78 (d, J = 8.7 Hz, 1H, Ar–H), 6.70
(s, 1H, Ar–H), 7.14–7.17 (d, J = 8.7 Hz, 1H, Ar–H),
7.31–7.42 (m, 6H, Ar–H), 7.63–7.66 (d, J = 8.4 Hz, 1H,
Ar–H), 7.78 (s, 1H, Ar–H), 7.99–8.02 (d, J = 8.4 Hz, 1H,
Ar–H); IR (KBr) 1752 cm-1 (–C=O, ester); FAB-MS m/z
448 [M ? H]?, Anal. cald. for C26H22ClNO4; C, 69.72; H,
4.95; N, 3.13; Found: C, 69.89; H, 4.95; N, 3.11.
Pharmacology
Pharmacological tests were carried out on adult male
Sprague–Dawley rats (100–150 g) to evaluate the anti-
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Med Chem Res (2012) 21:2223–2228
2227
inflammatory and ulcerogenic properties of the panel of
indomethacin derivatives. Animals were obtained from the
Animal House, Central Drug Research Institute (CDRI),
Lucknow, India. The animals were housed 5 per cage and
kept on standard laboratory diet with free access to water.
component of cyclooxygenase. The peroxidase activity was
assayed colorimetrically by monitoring the appearance of
oxidized N,N,N0,N0-tetramethyl-p-phenylenediamine (TMPD)
at 590 nm. The preparation of reagents was followed the
instructions given with the assay kit (Catalog no. 760111).
Carrageenan-induced rat paw edema method
Procedure
The inhibitory activity of the compounds on carrageenan
induced rat paw edema was performed according to
method of (Winter and Porter 1957). Sprague–Dawley rats
of either sex, weighing between 100–150 g, and fasted
overnight were taken and divided into thirteen groups with
five animals in each group. These animals were humanely
restrained and 100 ll of 1% w/v carrageenan solution in
sterile normal saline (0.9% w/v NaCl) was injected into the
sub plantar site of the right hind footpad by inserting the
needle bevel down through the callus at an angle nearly
parallel to the footpad. The paw volume was measured by
dislocation of the water column in a Plethysmometer (Ugo
Basile, Italy) at 0, 1.5, and 3 h after carrageenan injection.
Indomethacin and its esters suspended in 2% gum acacia
were administrated at 10 mg/kg orally, as a standard and
test, respectively. The percentage inhibition of edema was
calculated using the formula, Percent edema inhibi-
tion = [1-(Vt/Vc)] 9 100 where, Vt and Vc are mean
edema volume in the drug treated and control groups,
respectively. The differences were compared using one-
way ANOVA followed by Dunnett’s test. P values of 0.05
were considered significance.
(1) Background wells—160 ll of Assay Buffer and 10 ll
of heme were added to 96-well plates.
(2) The 100% Initial activity wells—150 ll of Assay
Buffer, 10 ll of heme, and 10 ll of enzyme (COX-1
or COX-2) were added.
(3) Inhibitor wells—150 ll of assay buffer, 10 ll of
heme, and 10 ll of enzyme (COX-1 or COX-2) were
added.
(4) The 10 ll of inhibitors (20 lM) were added to the
inhibitor wells and 10 ll of solvent (DMSO) was
added to the 100% Initial activity wells and back-
ground wells.
(5) The plate was shaken for a few seconds and incubated
for 5 min at 25°C.
(6) The 20 ll of colorimetric substrate solution (TMPD)
was added to all the wells.
(7) The 20 ll of arachidonic acid was added to all the
wells.
(8) The plate was shaken for a few seconds and incubated
for 5 min at 25°C.
(9) The absorbance was measured at 590 nm using the
Bioteck instrument.
Ulcerogenic activity
Calculations
Eight hours after the oral administration of indomethacin
and test compounds, the rats were sacrificed under ether
anesthesia. Their stomachs were removed, opened through
greater curvature, washed under running water, and fixed in
saline solution. The degree of ulcerogenicity was deter-
mined by viewing the gastric epithelial ulceration with a
magnascope at five magnifications and rated by ulcer score.
Ulcer score was used to grade the incidence and severity of
the lesions such as (i) Shedding of epithelium—10 (ii)
Petechial and frank hemorrhages—20 (iii) One or more
ulcers—30 (iv) More than two ulcers—40 (v) Perforated
ulcers—50 (srivastava et al., 1991).
The calculations were carried out in the following manner:
(1) The average absorbance of all the samples was
determined.
(2) The absorbance of the background wells was sub-
tracted from absorbance’s of the 100% Initial activity
and the inhibitor wells
(3) Percent inhibition = (100% Initial activity-Inhibitor
wells/100% Initial activity) 9 100
Results and discussion
Colorimetric COX inhibitor screening assay
The anti-inflammatory effects were estimated for the panel
of synthesized compounds and indomethacin using the
carrageenan induced rat paw edema method. The results
are shown in Table 1. Out of eleven compounds, com-
pound IIc showed enhanced anti-inflammatory activity
(19.88%) at 10 mg/kg over indomethacin (13.86%) as a
The percentage of COX-1 and COX-2 inhibition was
measured using the Colorimetric COX (ovine) Inhibitor
Screening Assay Kit, Catalog no. 760111 supplied by
Caymen Chemicals, USA. The Colorimetric COX (ovine)
Inhibitor Screening Assay utilizes the peroxidase
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Med Chem Res (2012) 21:2223–2228
Acknowledgments The author MAB is grateful to the Director,
SGSITS, Indore and Director, CDRI, Lucknow for providing the
necessary facilities for this study.
Table 2 Ulcer index for compound IIc and indomethacin
No.
Compound
Ulcer index
1
2
Compound IIc
Indomethacin
0
5 9 10 = 50
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No.
Compound
COX-2 inhibitory
activity (%)
COX-1 inhibitory
activity (%)
1
2
3
a
Compound IIca
Indomethacina
Celecoxiba
62.0
76.0
95.3
12.9
71.0
2.0
The mean of the duplicate values for each sample was taken
reference drug. Other compounds, such as IIa, IIb, and IId
showed moderate activity (7.83, 9.04, and 7.23%). Com-
pounds IIe, IIi, IIj, and IIk inhibited the edema in
carrageenan-induced rat- paw edema model at 0 and 1.5 h.
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Compound IIc was therefore subjected for further
evaluation to check its ulcerogenic property. It was
observed from the present study that compound IIc did not
promote observable ulceration in the stomachs of rats.
However, the reference compound indomethacin promoted
ulcers in the stomachs of rats. A total of five areas of
shedding of epithelium were observed in rats administrated
with indomethacin. The results are given in Table 2. Fur-
thermore, it was analyzed that the ulceration can be
reduced via masking the free carboxylic group into an ester
derivative.
Further, colorimetric COX inhibitor screening assay was
employed to characterize compound IIc together with
reference compounds. Compound IIc, indomethacin, and
celecoxib were tested at a concentration of 20 lM in the
assay. Compound IIc was found to be active against
COX-2 and COX-1 enzymes exhibiting 62.0 and 12.9%
inhibition, respectively (Table 3). However, indomethacin
has produced 76 and 71% inhibition against COX-2 and
COX-1 enzymes, respectively. It can thus be concluded
that compound IIc displays more selective inhibition
against COX-2 enzyme than COX-1 enzyme.
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