Design, synthesis, and evaluation of novel indomethacin-antioxidant codrugs as gastrosparing NSAIDs

Article abstract of DOI:10.1007/s00044-011-9589-1

Indomethacin has been conjugated with different antioxidants having antiulcerogenic activity with the objective of obtaining indomethacin- antioxidant codrugs as gastrosparing NSAIDs devoid of ulcerogenic side effects. Purified synthesized codrugs have be

Full text of DOI:10.1007/s00044-011-9589-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:834–843
DOI 10.1007/s00044-011-9589-1
ORIGINAL RESEARCH
Design, synthesis, and evaluation of novel indomethacin–
antioxidant codrugs as gastrosparing NSAIDs
•
Shruti Sawraj Tilak R. Bhardawaj
•
Pritam D. Sharma
Received: 17 May 2010 / Accepted: 28 January 2011 / Published online: 17 February 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Indomethacin has been conjugated with dif-
ferent antioxidants having antiulcerogenic activity with the
objective of obtaining indomethacin–antioxidant codrugs
as gastrosparing NSAIDs devoid of ulcerogenic side
effects. Purified synthesized codrugs have been character-
ized by m.p., TLC, elemental analyses, FTIR, NMR, MS.
The synthesized derivatives have been screened for their
antiinflammatory, analgesic, and antiulcer activity. The
codrugs showed retention of antiinflammatory activity with
reduced ulcerogenic side effects. These results indicated
that indomethacin–antioxidant codrugs have the potential
to be developed as gastrosparing NSAIDs.
involved in the biosynthesis of prostaglandin H2 (PGH2)
(Vane, 1971; Hla and Neilson, 1992). It is now well known
that COX exists in two isoforms, namely COX-I and COX-
II, which are regulated differently (Xie et al., 1991). COX-I
is constitutively expressed in stomach to provide cytopro-
tection in the GIT. COX-II is inducible and plays a major
role in prostaglandin biosynthesis in inflammatory cells.
Since most of the NSAIDs used clinically inhibit both
isoforms, long term use of these agents results in gastric
ulcer and there is enough evidence that inhibition of COX-I
rather than that of COX-II underlies gastric ulcer formation
(McCarthy, 1989; Warner et al., 1999; Wolfe et al., 1999).
As a result, a number of selective COX-II inhibitors,
including Celecoxib and Rofecoxib have been introduced
for clinical use with exceptional antiinflammatory proper-
ties with reduced gastric toxicity (Xie et al., 1992; Haw-
key, 1999). But initial enthusiasm for selective COX-II
inhibitors as safer NSAIDs has faded due to emergence of
serious cardiovascular side effects on long term use and
need for design and development of safer agents still
remain (Dogne et al., 2005; Schnitzer, 2001).
Keywords NSAIDs ꢀ Indomethacin ꢀ Antioxidant ꢀ
Codrug ꢀ Ulcerogenicity
Introduction
Nonsteroidal antiinflammatory drugs (NSAIDs) are the
most widely used drugs, with prescription as well as over
the counter formulations being available in most countries.
Since the introduction of NSAIDs in the market, enormous
literature has been published regarding their side effects.
Although these agents affect renal and cardiovascular
systems, the most common, widely studied, reported, and
reviewed side effects are related to gastrointestinal tract
(GIT) (Gibson, 1988; Vane and Botting, 1998). The phar-
macological activity of NSAIDs is related to their ability to
inhibit the activity of the enzyme cyclooxygenases (COXs)
Recently, it has been well known that local generation of
various reactive oxygen species (ROS) plays a significant
role in the formation of gastric ulceration associated with
NSAID therapy (Bandyopadhyay et al., 1999; Hassan et al.,
1998). These observations indicate that antioxidants may be
used to prevent NSAIDs induced gastric ulcers. During the
past few decades, a large number of naturally occurring
compounds have been identified as antioxidants, which are
viewed as promising therapeutic agents for treating free
radical mediated diseases including NSAID induced peptic
ulcers. Large number of herbs and spices are recognized as
source of natural antioxidants and studies have confirmed
their efficacy for the treatment of gastrointestinal ulcers
(Nakatani, 2000). Based on these observations, it has been
S. Sawraj ꢀ T. R. Bhardawaj ꢀ P. D. Sharma (&)
University Institute of Pharmaceutical Sciences,
Panjab University, Chandigarh 160014, India
e-mail: pritamdevsharma@hotmail.com
123

Med Chem Res (2012) 21:834–843
835
suggested that coadminstration of antioxidants and NSA-
IDS in formulated dosage form may possibly decrease the
risk of NSAIDs induced gastrointestinal side effects
(Jimenez and Alcaraz, 1988; Repetto and Llesuy, 2002).
However, there are potential advantages in giving such
coadministered drugs having complementary pharmaco-
logical activities in the form of a single chemical entity.
Such agents are named as mutual prodrugs/codrugs which
are designed with improved physicochemical properties and
release the parent drug at the site of action (Singh and
Sharma, 1994; Bhosle et al., 2006; Leppanen et al., 2002).
Indomethacin (1) is one of the most potent NSAIDs.
However, its use is restricted due to high incidences of
ulcerogenic side effects. In the present study, this potential
NSAID has been selected. In literature various indometh-
acin-conjugates has been reported as ulcer protective
agents (Sawraj et al., 2010; Zhang et al., 2005; Doulgkeris
et al., 2006). This work aims to synthesize indomethacin–
antioxidant ester codrugs to get safer NSAIDs, devoid of
ulcerogenic side effects while retaining the antiinflamma-
tory and analgesic activity.
CHO
HO
HO
HO
HO
OCH
OCH
OCH
3
3
3
(2a)
(2b)
(2c)
(2d)
O
HO
O
O
O
HO
HO
(2f)
(2e)
(2g)
Fig. 1 Structures of naturally occurring antioxidants; guaiacol (2a),
eugenol (2b), thymol (2c), vanillin (2d), sesamol (2e), umbelliferone
(2f), and menthol (2g)
OH
H3CO
Ar / R-OH
(2a-g)
O
+
N
O
(1)
Cl
(i)
Result and discussion
OAr/R
H3CO
Chemistry
O
N
For the preparation of indomethacin–antioxidant codrugs
(3a–g), various natural antioxidants were identified for
conjugation including, guaiacol (2a), eugenol (2b), thymol
(2c), vanillin (2d), sesamol (2e), umbelliferone (2f), and
menthol (2g) (Fig. 1). These agents have been an important
part of human diet and therefore their safety profile is well
known (Cotelle, 2001; Martin et al., 1998). Sequence of
steps involved in the synthesis of various indomethacin–
antioxidant codrugs (3a–g) by conjugation of indomethacin
(1) and various antioxidant (2a–g) are shown in Scheme 1.
For this purpose, indomethacin (1) was dissolved in chlo-
roform followed by the addition of DCC and stirred at
room temperature for 1 h. To this solution, the corre-
sponding antioxidant was added along with DMAP and
reaction mixture was stirred at room temperature for 24 h.
After the processing the reaction mixture, desired product
was obtained. All the codrugs (3a–g) were prepared fol-
lowing this general procedure. These compounds were
purified by recrystallization and obtained in reasonable
yield (43–58%). Their structures were confirmed by the use
of elemental analysis and spectral studies (Table 1).
O
Cl
(3a-g)
Scheme 1 Sequence of steps involved in the synthesis of indometh-
acin–antioxidant codrugs. Reagent and conditions: (i) CH2Cl2, DCC,
DMAP, room temperature, 24 h
signals at d 2.43 for CH3 protons (s, indomethacin ring), d
3.73 for OCH3 protons (s, guaiacol), 3.83 for OCH3 protons
(s, indomethacin ring), d 3.92 for CH2 (s, indomethacin).
Aromatic protons of indomethacin and guaiacol are over-
lapped and appeared between d 6.89–7.25. Two distinct
ABq were observed at 7.44–7.47 (J = 9 Hz) and 7.65–7.68
(J = 9 Hz), respectively, representing para coupling of the
indomethacin molecule. In 13C NMR, signals appeared at d
168.39 and 169.02 for COO (ester linkage) and C=O
(indomethacin nucleus). The mass spectrum of the com-
pound 3a showed molecular ion peak at m/z 463.98 (M?).
The IR spectrum of the derivative 3b showed the
absorption peaks at 3081 cm-1 characteristic of C–H
stretching. The peaks at 1752.7, 1668.7 cm-1 showed
presence of C=O (ester linkage) and C=O (indomethacin),
respectively. The 1H NMR spectrum of 3b showed the sig-
nals at d 2.46 for CH3 protons (s, indomethacin ring), d 3.74
for OCH3 protons (s, eugenol), 3.86 for OCH3 protons (s,
indomethacin ring), d 3.95 for CH2 (s, indomethacin). The
The IR spectrum of the derivative 3a showed the
absorption peaks at 3028.1 cm-1 characteristic of C–H
stretching. The peaks at 1761.1, 1684.5 cm-1 showed
presence of C=O (ester linkage) and C=O (indomethacin),
respectively. The 1H NMR spectrum of 3a showed the
123

836
Med Chem Res (2012) 21:834–843
Table 1 Physical properties
Name
Yield M.p. Spectral and elemental data
(%)
(°C)
56
203 IR (KBr): 3028.1 (aromatic C–H st), 2928.3 (aliphatic C–H st),
2851.2 (C–H st of aromatic OCH3), 1761.1 (C=O st, ester), 1684.5
(C=O st, indomethacin), 1500.6 (benzene ring C=C st), 1258.4
(asymm C–O–C st), 1138.5 (C–C(=O)–O st), 1042.2 (symm C–O–
C st) cm-1
OCH3
O
H3CO
O
N
O
1H NMR (CDCl3): d 2.43 (s, 3H, CH3, indomethacin), 3.73 (s, 3H,
OCH3, guaiacol), 3.83 (s, 3H, OCH3, indomethacin), 3.92 (s, 2H,
CH2COO), 6.67–6.70 (dd, H, J = 9.0 Hz and 2.5 Hz Ar–H,
indomethacin), 6.89–6.94 (m, 3H, Ar–H, indomethacin, guaiacol),
6.98–7.01 (dd, 1H, J = 1.7 Hz and 7.9 Hz, Ar–H, guaiacol),
7.08–7.09 (d, 1H, J = 2.52 Hz, Ar–H, indomethacin), 7.15–7.25
(m, 1H, Ar–H, guaiacol), 7.46–7.47 (Abq, 2H, J = 9 Hz, Ar–H,
indomethacin), 7.65–7.68 (Abq, 2H, J = 9 Hz, Ar–H,
indomethacin)
Cl
2-Methoxyphenyl[1-(4-chlorobenzoyl)-5-methoxy-2-
methyl-1H-indol-3-yl]acetate (3a)
C26H22ClNO5
13C NMR (CDCl3): d 13.49 (Ar–CH3), 30.08 (Ar–CH2COO), 55.77
(OCH3), 101.61–133.99 (Ar-carbons), 136.22 ([N–C(CH3)=C),
139.3 (CH2COOCAr), 139.90 (ArC–Cl), 151.08 (COCH3,
indomethacin), 156.11 (COCH3, guaiacol), 168.39 (CH2COO),
169.02 ([NCO–Ar)
LC–MS: m/z 463.98 [M]?
Calculated for C26H22ClNO5: C, 67.31; H, 4.78; Cl, 7.64; N, 3.02.
Found: C, 67.71; H, 4.39; N, 3.37%
50
147.5 IR (KBr): 3081 (aromatic C–H st), 2933.4 (aliphatic C–H st), 2839.2
(C–H st of aromatic OCH3), 1752.7 (C=O st, ester), 1668.7 (C=O
st, indomethacin), 1506.5 (benzene ring C=C st), 1272.2 (asymm
C–O–C st), 1149.2 (C–C(=O)–O st), 1034.3 (symm C–O–C
st) cm-1
OCH3
O
H3CO
O
N
1H NMR (CDCl3): d 2.46 (s, 3H, CH3, indomethacin), 3.38–3.39 (d,
2H, J = 6.6 Hz, –CH2–, eugenol), 3.74 (s, 3H, OCH3, eugenol),
3.86 (s, 3H, OCH3, indomethacin), 3.95 (s, 2H, CH2COO),
5.09–5.13 (m, 2H, =CH2, eugenol), 5.93–6.00 (m, 1H, –CH=,
eugenol), 6.70–6.79 (m, 3H, Ar–H, indomethacin, eugenol),
6.93–6.96 (dd, 2H, J = 2.34 Hz and 9.24 Hz, Ar–H,
indomethacin, eugenol), 7.120–7.126 (d, 1H, J = 2.2 Hz, Ar–H,
indomethacin), 7.48–7.50 (Abq, 2H, J = 9 Hz, Ar–H,
indomethacin), 7.69–7.71 (Abq, 2H, J = 9 Hz, Ar–H,
indomethacin)
O
Cl
4-Allyl-2-methoxyphenyl[1-(4-chlorobenzoyl)-5-
methoxy-2-methyl-1H-indol-3-yl]acetate (3b)
C29H26ClNO5
13C NMR (CDCl3): d 13.48 (Ar–CH3), 30.07 (Ar–CH2COO), 40.10
(CH2–CH=CH2), 55.74 (OCH3), 114.93 (CH=CH2),
101.58–138.12 (Ar-carbons), 134.2 (CH=CH2), 136.16 ([N–
C(CH3)=C), 139.13 (CH2COOCAr), 139.26 (ArC–Cl), 150.82
(COCH3, eugenol), 156.09 (COCH3, indomethacin), 168.35
(CH2COO), 169.12 ([NCO–Ar)
LC–MS: m/z 503.93 [M]?
Calculated for C29H26ClNO5: C, 69.11; H, 5.20; N, 2.78. Found: C,
69.44; H, 5.15; N, 2.65%
123

Med Chem Res (2012) 21:834–843
837
Table 1 continued
Name
Yield M.p. Spectral and elemental data
(%)
(°C)
40.5 118.5 IR (KBr): 3042.2 (aromatic C–H st), 2927.6 (aliphatic C–H st), 1754.8
(C=O st, ester), 1680.3 (C=O st, indomethacin), 1504.7 (benzene ring
C=C st), 1287.1 (asymm C–O–C st), 1134.7 (C–C(=O)–O st) cm-1
1031.5 (symm C–O–C st) cm-1, 952.2 (C–H bend)
,
O
H3CO
1H NMR (CDCl3): d 1.01–1.02 (d, 6H, J = 6.9 Hz, CH(CH3)2), 2.27 (s,
3H, CH3, indomethacin), 2.45 (s, 3H, Ar–CH3, thymol), 2.73–2.77
(sept, 1H, J = 6.9, CH(CH3)2), 3.89 (s, 3H, OCH3, indomethacin), 3.91
(s, 2H, CH2COO), 6.67–6.70 (dd, 1H, J = 2.5 Hz and 9 Hz, Ar–
H indomethacin), 6.7913–6.7936 (d, 1H, J = 0.92 Hz, Ar–H, thymol),
6.89–6.91 (d, 1H, J = 9 Hz, Ar–H, indomethacin), 6.96–6.98 (d, 1H,
J = 7.92 Hz, Ar–H, thymol), 7.063–7.069 (d, 1H, J = 2.48 Hz, Ar–H,
indomethacin), 7.11–7.13 (d, 1H, J = 7.88 Hz, Ar–H, thymol),
7.42–7.46 (Abq, 2H, J = 9 Hz, Ar–H, indomethacin), 7.62–7.66 (Abq,
2H, J = 9 Hz, Ar–H, indomethacin)
O
N
O
Cl
2-Isopropyl-5-methylphenyl[1-(4-chlorobenzoyl)-5-
methoxy-2-methyl-1H-indol-3-yl]acetate (3c)
C29H28ClNO4
13C NMR (CDCl3): d 13.38 (–NC(–CH3))=C), 20.80 (Ar–CH3, thymol),
22.85 (CH(CH3)2), 27.01 (CH(CH3)2), 30.55 (CH2COO), 55.67
(OCH3), 101.18–147.86 (Ar–carbons), 136.56 ([N–C(CH3)=C), 139.6
(ArC–Cl), 147.86 (CH2COOCAr), 156.16 (COCH3), 168.24
(CH2COO), 169.50 ([NCO–Ar)
LC–MS: m/z 489.06 [M]?
Calculated for C29H28ClNO4: C, 70.22; H, 6.91; N, 2.82. Found: C,
70.34; H, 6.91; N, 2.85%
42.5 128 IR (KBr): 3064.9 (aromatic C–H st), 2933.4 (aliphatic C–H st), 2843.5
(C–H st of aromatic OCH3), 1752.9 (C=O st, ester), 1685.7 (C=O st
aldehyde), 1683.6 (C=O st, indomethacin), 1503.5 (benzene ring C=C
st), 1273.7 (asymm C–O–C st), 1138.8 (C–C(=O)–O st), 1032.9 (symm
C–O–C st) cm-1, 908.4 (C–H bend) cm-1
OCH3
O
H3CO
O
N
1H NMR (CDCl3): d 2.45 (s, 3H, CH3, indomethacin), 3.79 (s, 3H,
OCH3, vanillin), 3.83 (s, 3H, OCH3, indomethacin), 3.95 (s, 2H,
CH2COO), 6.68–6.70 (dd, H, J = 9.0 Hz and 2.5 Hz, Ar–H),
6.88–6.90 (d, 1H, J = 9.0 Hz, Ar–H), 7.06–7.07 (d, 1H, J = 2.4 Hz,
Ar–H), 7.17–7.19 (d, 1H, J = 9.0 Hz, Ar–H, vanillin), 7.432–7.436 (d,
2H, J = 2.0 Hz, Ar–H), 7.45–7.47 (Abq, 2H, J = 9 Hz, Ar–H,
indomethacin), 7.65–7.69 (Abq, 2H, J = 9 Hz, Ar–H, indomethacin),
9.92 (s, 1H, CHO)
CHO
O
Cl
(4-Formyl-2-metho xyphenyl[1-(4-chlorobenzoyl)-5-
methoxy-2-methyl-1H-indol-3-yl]acetate (3d)
C27H22ClNO6
13C NMR (CDCl3): d 13.40 (Ar–CH3), 30.00 (Ar–CH2COO), 55.75
(OCH3), 55.97 (OCH3), 37.99 (Ar–CH2), 56.15 (OCH3),
101.61–139.37 (Ar-carbons), 136.36 ([N–C(CH3)=C), 139.37 (ArC–
Cl), 144.99 (CH2COOCAr), 151.88 (COCH3, indomethacin), 156.09
(COCH3, vanillin), 168.32 (CH2COO), 169.50 ([NCO–Ar), 191.00
(CHO)
LC–MS: m/z 491.91 [M]?
Calculated for C27H22ClNO6: C, 65.92; H, 4.51; N, 2.85. Found: C,
65.04; H, 4.84; N, 2.92%
123

838
Med Chem Res (2012) 21:834–843
Table 1 continued
Name
Yield M.p. Spectral and elemental data
(%)
(°C)
48.4 77.5 IR (KBr): 3025.8 (aromatic C–H st), 2924.9 (aliphatic C–H st),
2852.9 (C–H st of aromatic OCH3), 1759.8 (C=O st, ester),
1683.1 (C=O st, indomethacin), 1482.6 (benzene ring C=C st),
1258.4 (asymm C–O–C st), 1133.4 (C–C(=O)–O st), 1029.7
(symm C–O–C st), 927.6 (C–H bend) cm-1
O
O
O
H3CO
O
N
1H NMR (CDCl3): 2.43 (s, 3H, CH3, indomethacin), 3.83 (s, 3H,
OCH3, indomethacin), 3.86 (s, 2H, CH2COO), 5.95 (s, 2H,
OCH2), 6.47–6.49 (dd, 1H, J = 8.4 Hz and 2.4 Hz, Ar–H,
sesamol), 6.562–6.568 (d, 1H, J = 2.28 Hz, Ar–H, sesamol),
6.67–6.70 (dd, 1H, J = 9.0 Hz and 2.5 Hz, Ar–H, indomethacin),
6.72–6.74 (d, 1H, J = 8.4 Hz, Ar–H, sesamol), 6.88–6.90 (d, 1H,
J = 9.0 Hz, Ar–H, indomethacin), 7.03–7.04 (d, 1H, J = 2.4 Hz,
Ar–H, indomethacin), 7.45–7.48 (Abq, 2H, J = 9 Hz, Ar–H,
indomethacin), 7.66–7.68 (Abq, 2H, J = 9 Hz, Ar–H,
indomethacin)
O
Cl
3,4-(Methylenedioxy)phenyl[1-(4-chlorobenzoyl)-5-
methoxy-2-methyl-1H-indol-3-yl]acetate (3e)
C26H20ClNO5
13C NMR (CDCl3): d 13.49 (Ar–CH3), 30.51 (Ar–CH2COO), 55.77
(OCH3), 101.20 (OCH2O), 101.78–133.86 (Ar-carbons), 136.24
([N–C(CH3)=C), 139.39 (ArC–Cl), 145.47 (CH2COOCAr),
148.02 (ArCOCH2O), 156.16 (COCH3), 168.35 (CH2COO),
169.68 ([NCO–Ar)
LC–MS: m/z 477.92 [M]?
Calculated for C26H20ClNO5: C, 65.34; H, 4.22; N, 2.93. Found: C,
65.97; H, 4.31; N, 2.85%
42.5 224.5 IR (KBr): 3036.1 (aromatic C–H st), 2927.9 (aliphatic C–H st),
1776.7 (C=O st aldehyde), 1758.9 (C=O st, ester), 1687.6 (C=O
st, indomethacin), 15037.5 (benzene ring C=C st), 1241.4 (asymm
C–O–C st), 1186.1 (C–C(=O)–O st), 1049.6 (symm C–O–C
st) cm-1, 928.4 (C–H bend) cm-1
O
O
O
H3CO
O
N
1H NMR (CDCl3): d 2.46 (s, 3H, CH3, indomethacin), 3.83 (s, 3H,
OCH3, indomethacin), 3.94 (s, 2H, CH2COO), 6.36–6.39 (d, 1H,
J = 9.5 Hz, H of lactone ring, umbelliferone), 6.68–6.71 (dd, H,
J = 9.0 Hz and 2.5 Hz, Ar–H, indomethacin), 6.86–6.89 (d, 1H,
J = 9.0 Hz, Ar–H, indomethacin), 6.99–7.00 (d, 1H, J = 2.4 Hz,
Ar–H, indomethacin), 7.02–7.03 (m, 1H, Ar–H, umbelliferone),
7.09–7.1 (d, 1H, J = 2.2 Hz, Ar–H, umbelliferone), 7.45–7.47
(m, 3H, Ar–H, indomethacin, umbelliferone), 7.65–7.69 (m, 3H,
Ar–H, indomethacin, H of lactone ring, umbelliferone)
O
Cl
22-Oxo-2H-chromen-7-yl[1-(4-chlorobenzoyl)-5-methoxy-
2-methyl-1H-indol-3-yl]acetate (3f)
C28H20ClNO6
13C NMR (CDCl3): d 13.47 (Ar–CH3), 30.56 (Ar–CH2COO), 55.79
(OCH3), 101.19–133.73 (Ar-carbons), 110.32 (Ar–CH=CH–),
136.45 ([N–C(CH3)=C), 139.50 (ArC–Cl), 142.82 (Ar–CH=
CH–), 153.14 (ArCOCO), 154.66 (CH2COOCAr), 156.09
(COCH3), 160.27 (C=O, umbelliferone), 168.32 (CH2COO),
168.68 ([NCO–Ar)
LC–MS: m/z 501.97 [M]?
Calculated for C28H20ClNO6: C, 67.00; H, 4.02; N, 2.79. Found: C,
67.09; H, 4.02; N, 2.93%
123

Med Chem Res (2012) 21:834–843
839
Table 1 continued
Name
Yield M.p. Spectral and elemental data
(%)
(°C)
42.5 82
IR (KBr): 2957.9 (aliphatic C–H st), 1730.1 (C=O st, ester), 1683.6 (C=O
st, indomethacin), 1602.5 (Ar C=C st); 1226.0 (asymm C–O–C st),
1171.6 (C–C(=O)–O st) cm-1, 1038.6 (symm C–O–C st) cm-1, 926.7
O
(C–H bend) cm-1
;
H3CO
1H NMR (CDCl3): d 0.67–0.69 (d, 3H, J = 7.0 Hz, CH3, menthol),
081–0.89 (d, 3H, CH3), 0.91–0.96 (d, 4H, CH3, CH), 0.99–1.05 (m, 2H,
CH), 1.33–1.35 (m, 1H, CH), 1.46–1.48 (m, 1H, CH), 1.63–1.74 (m, 3H,
CH), 1.99–2.02 (dd, 1H, CH), 2.38 (s, 3H, CH3, indomethacin), 3.65 (s,
2H, CH2COO), 3.83 (s, 3H, OCH3, indomethacin), 4.46–4.79 (m, 1H,
CH), 6.66–6.69 (dd, H, J = 9.0 Hz and 2.5 Hz, Ar–H), 6.88–6.91 (d, 1H,
J = 9.0 Hz, Ar–H), 6.97–6.98 (d, 1H, J = 2.4 Hz, Ar–H), 7.45–7.47
(Abq, 2H, J = 9 Hz, Ar–H, indomethacin), 7.64–7.66 (Abq, 2H,
J = 9 Hz, Ar–H, indomethacin)
O
N
O
Cl
2-Isopropyl-5-methylcyclohexyl[1-(4-
chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl]acetate (3g)
13C NMR (CDCl3): 13.45 (Ar–CH3), 30.46 (Ar–CH2COO), 55.89 (OCH3),
101.19–133.35 (Ar-carbons), 110.46 (Ar–CH=CH–), 136.51 ([N–
C(CH3)=C), 139.25 (ArC–Cl), 142.72 (Ar–CH=CH–), 153.47
(ArCOCO), 154.22 (CH2COOCAr), 157.07 (COCH3), 168.32
(CH2COO), 168.68 ([NCO–Ar)
C29H34ClNO4
LC–MS: m/z 496.0 [M]?
Calculated for C29H34ClNO4: C, 70.22; H, 6.91; N, 2.82. Found: C, 70.34;
H, 6.91; N, 2.82
Spectral and elemental data of indomethacin–antioxidant codrugs (3a–g)
signal appeared at d 3.38–3.39 for methylene (CH2–
CH=CH2) proton as doublet, at d 5.93–6.0 for methine
(CH2–CH=CH2) proton as multiplet and at 5.09–5.13 for
terminal methylene (CH2–CH=CH2) protons as multiplet of
eugenol moiety. The signals for the aromatic protons
appeared in the range of d 6.70–7.12, overlapped with Ar–
H signals of indomethacin moiety as multiplets. Two distinct
ABq were observed at 7.48–7.50 (J = 9 Hz) and 7.69–7.71
(J = 9 Hz), respectively, representing para coupling of the
indomethacin molecule. In 13C NMR, signals appeared at d
168.35 and 169.12 for COO (ester linkage) and C=O
(indomethacin nucleus). The mass spectrum of the com-
pound 3b showed molecular ion peak at m/z 503.93 (M?).
The IR spectrum of the derivative 3c showed the
absorption peaks at 3042.2 cm-1 characteristic of C–H
stretching. The peaks at 1754.8, 1680.3 cm-1 showed pres-
ence of C=O (ester linkage) and C=O (indomethacin),
respectively. The 1H NMR spectrum of 3c showed the sig-
nals at d 2.27 for CH3 protons (s, indomethacin ring), 2.45 for
CH3 protons (s, thymol), 3.89 for OCH3 protons (s, indo-
methacin ring), d 3.91 for CH2 (s, indomethacin). The signal
for gem dimethyl group of thymol appeared at d 1.01–1.02 as
doublet (J = 6.9 Hz). Methine (–CH) proton signal
appeared as septet at d 2.73–2.77 (J = 6.9 Hz) due to
splitting by gem dimethyl group. Aromatic protons appeared
in the range of d 6.79–7.13 as multiplet. Two distinct ABq
were observed at 7.42–7.46 (J = 9 Hz) and 7.62–7.66
(J = 9 Hz), respectively, representing para coupling of the
indomethacin molecule. In 13C NMR, signals appeared at d
168.24 and 169.50 for COO (ester linkage) and C=O (indo-
methacin nucleus). The mass spectrum of the compound 3c
showed molecular ion peak at m/z 489.06 (M?).
The IR spectrum of the derivative 3d showed the
absorption peaks at 3064.9 cm-1 characteristic of C–H
stretching. The peaks at 1752.9, 1685.7, and 1683.6 cm-1
showed presence of C=O (ester linkage), (C=O st alde-
hyde), and C=O (indomethacin), respectively. The 1H
NMR spectrum of 3d showed the signals at d 2.45 for CH3
protons (s, indomethacin ring), 3.79 (s, 3H, OCH3, vanil-
lin), 3.83 for OCH3 protons (s, indomethacin ring), d 3.95
for CH2 (s, indomethacin). Aromatic protons appeared in
the range of d 6.68–7.43 as multiplet. Two distinct ABq
were observed at 7.45–7.47 (J = 9 Hz) and 7.65–7.69
(J = 9 Hz), respectively, representing para coupling of the
indomethacin molecule. The signal for –CHO proton
appeared at d 9.92 as singlet. In 13C NMR, signals
appeared at d 168.32, 169.50, and 191 for COO (ester
linkage), C=O (indomethacin nucleus) and CHO (aldehyde,
vanillin). The mass spectrum of the compound 3d showed
molecular ion peak at m/z 491.91 (M?).
The IR spectrum of the derivative 3e showed the
absorption peaks at 3025.8 cm-1 characteristic of C–H
stretching. The peaks at 1759.8 and 1683.1 cm-1 showed
presence of C=O (ester linkage) and C=O (indomethacin),
respectively. The 1H NMR spectrum of 3e showed the sig-
nals at d 2.43 for CH3 protons (s, indomethacin ring), 3.83 for
OCH3 protons (s, indomethacin ring), 3.86 for CH2 (s,
indomethacin), and 5.95 for methylenedioxy (–OCH2O) of
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840
Med Chem Res (2012) 21:834–843
sesamol as singlet. Aromatic protons appeared in the range of
d 6.47–7.04 as multiplets. Two distinct ABq were observed
at 7.45–7.48 (J = 9 Hz) and 7.66–7.68 (J = 9 Hz),
respectively, representing para coupling of the indomethacin
molecule. In 13C NMR, signals appeared at d 168.35 and
169.68 for COO (ester linkage) and C=O (indomethacin
nucleus). The mass spectrum of the compound 3e showed
molecular ion peak at m/z 477.92 (M?).
Carrageenan (1% w/v) was used to produce paw edema.
Edema is presented as percentage increase in right hind
paw, in comparison to the uninjected left hind paw. Per-
centage change in paw volume was calculated and
expressed as the amount of inflammation. For antiinflam-
matory activity, the test compounds (3a–g) were adminis-
tered orally at molar equivalent doses of indomethacin
(12 mg/kg, p.o.). All codrugs at molar equivalent doses
showed significantly increased antiinflammatory activity as
compared to that produced by indomethacin (Table 2).
This increased activity may be due to the combined effect
of improved physicochemical properties of conjugates and
contribution by their corresponding promoieties (indo-
methacin and antioxidants). Furthermore, equimolar phys-
ical mixtures of indomethacin and promoieties in
equimolar proportion were also studied for the antiin-
flammatory activity. These physical mixtures showed
comparable results to the parent indomethacin, but lower
than their corresponding conjugates (Table 2).
The IRspectrumof the derivative3f showedthe absorption
peaks at 3036.1 cm-1 characteristic of C–H stretching. The
peaksat1776.7, 1758.9, and1687.6 cm-1 showedpresenceof
C=O st (aldehyde), C=O (ester linkage), and C=O (indo-
methacin), respectively. The 1H NMR spectrum of 3f showed
the signals at d 2.46 for CH3 protons (s, indomethacin ring),
3.83for OCH3 protons (s, indomethacin ring), 3.94for CH2 (s,
indomethacin). Lactoneringprotonsofumbelliferoneshowed
doublet at d 6.36 and 7.65 (J = 9.6 Hz), respectively, indi-
cating the cis configuration. Aromatic protons appeared in the
range of d 6.68–7.47 as multiplets. In 13C NMR, signals
appeared at d 160.27, 168.32, and 168.68 for C=O (umbel-
liferone), COO (ester linkage), and C=O (indomethacin
nucleus). The mass spectrum of the compound 3f showed
molecular ion peak at m/z 501.97 (M?).
Analgesic activity
For the analgesic activity, abdominal writhing assay was
performed (Koster et al., 1959). Writhing was induced by
intraperitoneal (i.p.) injection of freshly prepared acetic
acid solution (1%, 10 ml/kg, i.p.) in mice. The number of
writhes (constriction of abdomen, turning of trunk, and
extension of hind limbs) due to acetic acid was expressed
as a nociceptive response. Vehicle treated control mice
were given 1% acetic acid and writhing response was noted
for 20 min. Indomethacin (10 mg/kg, p.o.) as well as
synthesized conjugates at equimolar doses significantly
reduced the writhing response (Table 2). The results
showed that these derivatives (3a–g) possess analgesic
activity comparable to the parent drug (Table 2).
The IR spectrum of the derivative 3g showed the
absorption peaks at 2957.9 cm-1 characteristic of C–H
stretching. The peaks at 1730.1 and 1683.6 cm-1 showed
presence of C=O (ester linkage) and C=O (indomethacin),
respectively. The 1H NMR spectrum of 3g showed the sig-
nals at d 2.38 for CH3 protons (s, indomethacin ring), 3.83 for
OCH3 protons (s, indomethacin ring), 3.65 for CH2 (s,
indomethacin). The signals for methyl group of menthol
appears at d 0.67–0.69 as doublet with J = 7.0 Hz. The
methine proton appears as multiplet along with gem dime-
thyl protons at d 0.81–0.96 as multiplet. The cyclic methy-
lene and methine protons appeared as multiplets in the range
of d 0.99–2.02. Aromatic protons appeared in the range of d
6.66–6.98 as multiplets. Two distinct ABq were observed at
7.45–7.47 (J = 9 Hz) and 7.64–7.66 (J = 9 Hz), respec-
tively, representing para coupling of the indomethacin
molecule. In 13C NMR, signals appeared at d 168.32 and
168.68 for COO (ester linkage) and C=O (indomethacin
nucleus). The mass spectrum of the compound 3g showed
molecular ion peak at m/z 496 (M?).
Antiulcer activity
The codrugs (3a–g) were screened for their ulcerogenicity in
rats, using parent drug induced acute gastric ulcerations
(Cioli et al., 1979). The animals were fasted for 24 h, divided
into different groups containing six animals in each group.
Control group was treated with an equal volume of 0.5%
carboxy methyl cellulose (CMC) vehicle. Animals were
killed 8 h after the treatment. The stomach was removed,
opened along the greater curvature, washed with saline, and
observed for ulcers. For the acute gastric damage evaluation,
the parent drug indomethacin was used to produce gastric
ulcers. For this purpose, indomethacin (48 mg/kg, p.o.) was
administered which produced a significant increase in ulcer
index (5.54 0.09) as compared to the control group
(0.2 0.06). All conjugates (3a–g) showed significantly
reduced gastric damage (Table 2). The reduction in ulcer
Pharmacological evaluation
The parent drug, indomethacin has been used as reference
substance.
Antiinflammatory activity
Antiinflammatory activity was determined by using carra-
geenan induced rat paw edema model (Winter et al., 1962).
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Med Chem Res (2012) 21:834–843
841
Table 2 Antiinflammatory, analgesic, and antiulcer activity of indomethacin, indomethacin–antioxidant codrugs, and indomethacin ? anti-
oxidant physical mixtures
Compound
Antiinflammatory activity
Analgesic activity
Antiulcer activity
Dose (mg/kg, p.o.) % Increase in paw volume mean SEM Dose
% Inhibition Dose
Ulcer index
(mg/kg, p.o.) mean SEM (mg/kg, p.o.) mean SEM
2 h
4 h
Control
0.5% CMC
64.73 0.72
81.58 0.63
0.5% CMC
–
0.5% CMC 0.23 0.09
Indomethacin (1) 12.0
48.35 0.56*
46.89 0.65*,#
48.1 0.76*
57.39 0.60*
48.21 0.88*,#
54.61 0.57*
45.39 0.83*,#
52.71 1.06*,#
54.61 0.95*
59.13 1.01*,#
49.87 0.77*,#
57.51 0.62*
44.52 0.59*,#
51.36 0.69*,#
50.27 1.39*,#
56.92 1.07*,#
55.54 1.47*,#
58.85 0.79*,#
10.0
12.9
–
74.87 0.88 48
78.66 0.6# 62.2
5.54 0.84
3a
15.6
1.15 0.58*,#
4.17 0.19*,#
0.93 0.28#
4.08 0.34*,#
1.37 0.63*,#
4.77 0.46*
1.23 0.36*,#
4.26 0.76*,#
0.79 0.47#
3.92 0.14*,#
1.28 0.44*,#
4.35 0.49*,#
1.41 0.19*,#
4.96 0.51*
1 ? 2a
3b
12 ? 4.2
16.9
–
48 ? 16.6
44.33 0.97*,#
47.68 0.67*
50.25 0.65*,#
50.99 0.17*
47.13 0.96*,#
49.27 0.78*
43.17 0.81*,#
47.01 0.49*
48.06 0.61*,#
49.66 0.98*
52.01 0.89*,#
52.25 1.27*
14.08
–
84.75 0.83# 67.6
1 ? 2b
3c
12 ? 5.5
16.4
–
48 ? 22
13.69
–
73.19 0.91# 65.7
1 ? 2c
3d
12 ? 5.1
16.5
–
48 ? 20
13.75
–
81.46 1.49# 66
1 ? 2d
3e
12 ? 5.1
16.0
–
48 ? 20
13.96
–
85.98 0.79# 67.1
1 ? 2e
3f
12 ? 4.7
16.8
–
48 ? 19
13.35
–
75.23 0.54# 64.1
1 ? 2f
3g
12 ? 5.5
16.6
–
48 ? 22
13.9
–
73.94 1.78# 66.5
1 ? 2g
12 ? 5.3
–
48 ? 21
#
* P \ 0.05 as compared to control, P \ 0.05 as compared to indomethacin
index by the physical mixture of indomethacin and antioxi-
dant was negligible as compare to their conjugates (Table 2).
This may be due to the polar nature of the antioxidants
resulting in their poor bioavailability, whereas reduction in
ulcer index by the conjugates was significant which may be
due to the improved physicochemical properties and con-
tribution by the antioxidant promoiety after the cleavage of
the codrug.
damage in all these cases may be attributed to the com-
bined effect of antioxidant activity of the compounds as
well as improved physicochemical properties of the co-
drugs. Furthermore, indomethacin with antioxidants phys-
ical mixture did not effectively reduce the risk of GI side
effects in comparison to their corresponding conjugates.
These results suggest that there is a potential advantage in
giving such drugs having complementary pharmacological
activities, in the form of single chemical entity, i.e., co-
drugs which are designed with improved physicochemical
properties.
The results listed in Table 2 showed that these indo-
methacin–antioxidant codrugs (3a–g) lack gastrointestinal
ulcerogenic side effects with retention of antiinflammatory
and analgesic activity.
Experimental protocols
Conclusion
Chemistry
In our attempt to combine antiinflammatory and antioxi-
dant activities, it has been possible to synthesize indo-
methacin–antioxidant codrugs as safer NSAIDs using
different naturally occurring phytophenols as antioxidant
promoieties. Further, these agents were found to possess
encouraging results with retention of antiinflammatory and
analgesic activity with significant reduction in ulcerogenic
side effects of the parent NSAID. The indomethacin–
guaiacol (3a), indomethacin–eugenol (3b), indomethacin–
vanillin (3d), indomethacin–sesamol (3e), conjugates
showed maximum antiulcer activity. The absence of gastric
Melting points (mp) were determined on a Veego melting
point apparatus and are uncorrected. For TLC, glass plates
coated with silica gel (E. Merck) were used. The TLC
plates were activated at 110°C for 30 min and visualized
by exposure to iodine vapors. Glass columns of appropriate
sizes were used. Silica gel (60–120 mesh, BDH) was used
as adsorbent. IR spectra were recorded on Perkin Elmer
882 spectrometer using potassium bromide pellets. 1H
NMR and 13C NMR spectra were recorded with Bruker AC
300 F, 400 MHz spectrometer using CDCl3 or DMSO-d6
123

842
Med Chem Res (2012) 21:834–843
as solvents and tetramethylsilane as internal standard. Mass
spectra were obtained with Vg-11-250J 70s mass spec-
trometer at 70 eV using electron ionization (EI) sources.
The synthetic reactions were monitored by TLC. The
test drugs were administered on molar equivalent basis of
indomethacin.
Antiinflammatory activity
1
structures of all new compounds were confirmed by H
NMR, 13C NMR, IR data, elemental analysis, and mass
spectrometer; homogeneity was confirmed by TLC. Solu-
tions were routinely dried over anhydrous sodium sulfate
prior to evaporation. Guaicaol, eugenol, vanillin, menthol,
thymol, sesamol, and umbelliferone were purchased from
Sigma-Aldrich. All other reagents and solvents were of AR
grade.
Antiinflammatory activity was determined by using carra-
geenan induced rat paw edema model. Rats were divided
into different groups and the indomethacin–antioxidant
codrugs were administered to each group. Acute edema
was induced in left hind paw of rats by injecting freshly
prepared solution of carrageenan (Type IV, 0.1 ml, 1%)
under plantar region of left hind paw. In the right paw,
saline (1 ml, 0.9%) was injected, which served as control
for comparison. The increase in paw volume was measured
by using plethysmometer (water displacement, UGO BA-
SILE, Italy) at 2 and 4 h after carrageenan challenge.
Percentage change in paw volume was calculated and
expressed as the amount of inflammation.
General procedure for the synthesis of indomethacin–
antioxidant codrugs (3a–g)
Indomethacin (3.57 g, 0.01 M) was dissolved in 25 ml of
chloroform followed by the addition of DCC (2.06 g,
0.01 M). The reaction mixture was stirred at room tem-
perature for 1 h. To this solution, antioxidant (0.01 M) and
DMAP (40 mg) were added and stirred for 24 h at room
temperature. The precipitated dicyclohexyl urea was fil-
tered off and the solvent of the filtrate was removed under
reduced pressure. To the residue obtained, cold ether
(20 ml) was added. The ethereal solution was filtered and
the filtrate was washed with acetic acid (1%, 3 9 50 cml),
HCl (5%, 3 9 50 cml), NaHCO3 (5%, 3 9 50 ml), and
finally with water (3 9 50 ml). The organic layer was
dried over anhydrous sodium sulfate, filtered and solvent
was removed under reduced pressure to obtain crude
product, which was recrystallized from petroleum ether and
ethyl acetate to obtain pure compounds. This general
procedure was used starting with different antioxidants
(2a–g) to prepare various indomethacin–antioxidant co-
drugs (3a–g). The final products were obtained as solids
and recrystallized from petroleum ether and ethyl acetate
(Table 1).
Analgesic activity
Analgesic activity was determined by using abdominal
writhing assay. Mice were divided into different groups
containing six animals in each group. Writhing response
was elicited by intraperitoneal (i.p.) injection of freshly
prepared acetic acid solution (1%, 10 ml/kg, i.p.). The
number of writhes due to acetic acid was expressed as
antinociceptive response. The number of writhes per ani-
mal was counted during a 20 min period. Writhings were
counted 3 min after the injection of acetic acid solution.
% Inhibition ¼ ð1 ꢁ Nt=NcÞ ꢂ 100
where Nc number of writhes in control group and Nt
number of writhes in drug treated group
Antiulcer activity
The fasted animals (rats) were divided into different groups
containing six animals in each group. Animals were treated
with indomethacin (48 mg/kg, p.o.), equimolar doses of
indomethacin–antioxidant codrugs and their physical mix-
ture. Animals were killed 8 h after the treatment. The
stomach was removed, opened along greater curvature,
washed with saline, and observed for the ulcers. The ulcers
were scored as: 0 normal colored stomach, 0.5 red color-
ation, 1.0 spot ulcers, 1.5 hemorrhagic streaks, 2.0 ulcers
[3 but \5, 3.0 ulcers [5.
Pharmacology
Animals
Sprague-Dawley (sd) rats (weighing 150–200 g) of both
sex and LACCA mice (male, 25.35 g) procured from
central animal house, Panjab University, Chandigarh, India
were used. The animals were housed in plastic cages (five
rats/cage) under standard laboratory conditions and main-
tained on rat chow and water.
Statistical analysis
Unless otherwise stated, the following conditions were
employed in all experiments. The test compounds were
suspended in 0.5% carboxymethylcellulose (CMC) and
administered per orally (p.o.). Control animals were given
the corresponding amount of vehicle (0.5%, CMC). The
Statistical analysis was carried out on in vivo studies data.
The ulcer index data was subjected to student t test
(unpaired), analysis of variance (ANOVA) test, followed
123

Med Chem Res (2012) 21:834–843
843
Leppanen J, Huuskonen J, Nevalainen T, Gynther J, Taipale H,
Jarvinen T (2002) Design and synthesis of a novel L-dopa
entacapone codrug. J Med Chem 45:1379–1382
Martin MJ, La-casa C, Alarcon-de-la-Lastra C, Cabeza J, Villegas J,
Motilva Y (1998) Anti-oxidant mechanisms involved in gastro-
protective effects of quercetin. Z Naturforsch C 53:82–88
Mc Carthy MD (1989) Nonsteroidal antiinflammatory drug-induced
ulcers: management by traditional therapies. Gastroenterology
96:662–674
by Dunnett’s test for determining the levels of significance
in antioxidant studies. P values \0.05 were considered
statistically significant.
Acknowledgments The financial support as Senior Research Fel-
low (SS) from University Grant Commission (UGC) and the research
facilities provided by University Institute of Pharmaceutical Sciences,
Panjab University, India are gratefully acknowledged.
Nakatani M (2000) Phenolic antioxidants from herbs and spices.
Biofactors 13:141
Repetto MG, Llesuy SF (2002) Antioxidant properties of natural
compounds used in popular medicine for gastric ulcers. Br J Med
Biol Res 35:523–534
References
Bandyopadhyay U, Das D, Banerjee RK (1999) Reactive oxygen
species—oxidative damage and pathogenesis. Curr Sci
77:658–665
Sawraj S, Bhardawaj TR, Sharma PD (2010) Design, synthesis and
evaluation of novel indomethacin-flavonoid mutual prodrugs as
safer NSAIDs. Med Chem Res. doi:10.1007/s00044-010-9363-9
Schnitzer TJ (2001) COX-2-specific inhibitors: are they safe? J Am
Med 110:46–49
Singh G, Sharma PD (1994) Mutual prodrugs—a recent trend in
prodrug design. Indian J Pharm Sci 56:69–79
Vane JR (1971) Inhibition of prostaglandin synthesis as a mechanism
of action for aspirin-like drugs. Nat New Biol 231:232–235
Vane JR, Botting RM (1998) Anti-inflammatory drugs and their
mechanism of action. Inflamm Res 47:S78–S87
Warner TD, Giuliano F, Vojnovic I, Bukada A, Mitchell JA, Vane JR
(1999) Nonsteroidal drug selectivities for cyclooxygenase-1
rather than cyclooxygenase-2 are associated with human gastro-
intestinal toxicity—a full in vitro analysis. Proc Natl Acad Sci
USA 96:7563–7568
Winter CA, Risley GA, Nuss GW (1962) Carrageenan induced edema
in hind paw of the rat as an assay for antiinflammatory drugs.
Proc Soc Exp Biol Med 3:544–547
Bhosle D, Bharambe S, Garrola N, Dhaneshwar SS (2006) Mutual
prodrug concept: fundamentals and applications. Indian
Pharma Sci 68:286–294
J
Cioli V, Putzolu S, Rossi V, Barcellona SP, Corradino C (1979) The
role of direct tissue contact in the production of gastro-intestinal
ulcer by antiinflammatory drugs in rats. Toxicol Appl Pharmacol
50:283–289
Cotelle N (2001) Role of flavonoids in oxidative stress. Curr Top Med
Chem 1:569–590
Dogne JM, Supuran CT, Pratico D (2005) Adverse cardiovascular
effects of coxibs. J Med Chem 48:2251–2257
Doulgkeris CM, Galanakis D, Kourounakis AP, Tsiakitzis KC,
Gavalas AM, Eleftheriou PT, Victoratos P, Rekka EA, Kouro-
unakis PN (2006) Synthesis and pharmacochemical study of
novel polyfunctional molecules combining antiinflammatory,
antioxidant, and hypocholesterolemic properties. Bioorg Med
Chem Lett 16:825–829
Wolfe MM, Lichtenstein DR, Singh G (1999) Gastrointestinal
toxicity of non steroidal antiinflammatory drugs. N Engl J Med
340:1888–1899
Gibson T (1988) Nonsteroidal antiinflammatory drugs—another look.
Br J Rheumatol 27:87–90
Hassan A, Martin E, Puig-Parellada P (1998) Role of antioxidants in
gastric mucosal damage induced by indomethacin in rats.
Methods Find Exp Clin Pharmacol 20:849–854
Hawkey CJ (1999) COX-2 inhibitors. Lancet 353:307–314
Hla T, Neilson K (1992) Human cyclooxygenase-2 cDNA. Proc Natl
Acad Sci USA 89:7384–7388
Jimenez MJ, Alcaraz MJ (1988) Flavonoids as antiinflammatory
agents. Fitoterapia 59:25–38
Koster R, Anderson M, De Beer EJ (1959) Acetic acid for analgesic
screening. Fed Proc 18:412
Xie W, Chipman JG, Robertson DL, Erikson RL, Simmons DL
(1991) Expression of a mitogen-responsive gene encoding
prostaglandin synthase is regulated by mRNA splicing. Proc
Natl Acad Sci USA 88:2692–2696
Xie W, Robertson DL, Simmons DL (1992) Mitogen-inducible
prostaglandin G/H synthase: a new target to be a potential source
of inhibitors of NF-jB activation. Drug Dev Res 25:249–265
Zhang Y, Chen P, Guan H, Li Y (2005) Synthesis and pharmaco-
logical evaluation of novel conjugates of indomethacin with
antioxidant activity. Chinese J Chem 23:1523–1529
123