Substituted aminoalcohol ester analogs of indomethacin with reduced toxic effects

Article abstract of DOI:10.1007/s00044-007-9013-z

Synthesis and evaluation of five different N,N-disubstituted aminoethanol ester derivatives of indomethacin bearing structural resemblance to the aminoethanol ester class of anticholinergics are reported herein. The anticholinergic activity was incorporated into the intact esters to overcome the gastric toxicity of indomethacin, not only by blocking the acidic functionality but also by decreasing gastric secretions and motility. These derivatives exhibited in vitro stability in buffers of pH 2.0 and 7.4 for 6 h and were readily hydrolyzed by human plasma esterases to liberate the parent drug. All the derivatives were significantly less irritating to the gastric mucosa than the parent drug. Though only two esters showed antiinflammatory activity similar to that of the parent drug at equivalent dose levels, all the esters were equipotent to indomethacin in the mouse acetic acid-induced writhing assay for analgesic action. The present evaluation indicates that the combined pharmacological properties of these ester derivatives may prove useful for design and development of novel gastric sparing antiinflammatory molecules with potentially important therapeutic applications. Birkhaeuser 2007.

Full text of DOI:10.1007/s00044-007-9013-z

Med Chem Res (2007) 16:101–111
DOI 10.1007/s00044-007-9013-z
ORIGINAL RESEARCH
Substituted aminoalcohol ester analogs of indomethacin
with reduced toxic effects
Parmeshwari K. Halen Æ Kewal K. Chagti Æ Rajani Giridhar Æ Mange Ram Yadav
Received: 13 January 2007 / Accepted: 3 May 2007 / Published online: 21 July 2007
¨
ꢀ Birkhauser Boston 2007
Abstract Synthesis and evaluation of five different N,N-disubstituted aminoeth-
anol ester derivatives of indomethacin bearing structural resemblance to the
aminoethanol ester class of anticholinergics are reported herein. The anticholinergic
activity was incorporated into the intact esters to overcome the gastric toxicity of
indomethacin, not only by blocking the acidic functionality but also by decreasing
gastric secretions and motility. These derivatives exhibited in vitro stability in
buffers of pH 2.0 and 7.4 for 6 h and were readily hydrolyzed by human plasma
esterases to liberate the parent drug. All the derivatives were significantly less
irritating to the gastric mucosa than the parent drug. Though only two esters showed
antiinflammatory activity similar to that of the parent drug at equivalent dose levels,
all the esters were equipotent to indomethacin in the mouse acetic acid–induced
writhing assay for analgesic action. The present evaluation indicates that the
combined pharmacological properties of these ester derivatives may prove useful
for design and development of novel gastric sparing antiinflammatory molecules
with potentially important therapeutic applications.
Keywords Antiinflammatory Á Indomethacin Á Ulceration
Introduction
The market for antiinflammatory drugs, squarely focused on selective cyclooxy-
genase-2 (cox-2) inhibitors during the past few years, no longer remains dominated
by these drugs, following the alarming increase in their renal and cardiovascular
adverse effects (Paul et al., 2004; Scheen, 2004). With the withdrawal of some cox-
2 inhibitors, the demand for safer nonsteroidal antiinflammatory drugs (NSAIDs)
P. K. Halen Á K. K. Chagti Á R. Giridhar Á M. R. Yadav (&)
Pharmacy Department, Faculty of Technology and Engineering, Kalabhavan, The M.S. University
of Baroda, Vadodara 390001, India
e-mail: mryadav11@yahoo.co.in

102
Med Chem Res (2007) 16:101–111
still remains unmet. Chemical modification of the existing time-tested and clinically
used drugs to overcome their side effects would be an economical and time-saving
approach compared to new drug discovery, which is quite an expensive process.
The undesired gastrointestinal (GI) irritation principally limits the clinical utility
of most of the conventional acidic NSAIDs. The main causes of NSAID-induced
gastropathy are reduced mucosal cytoprotective prostaglandin (PG) levels,
increased gastric acidity, and increased gastric motility. The increased gastric
motility leads to a reduced mucosal blood flow, hypoxia, and destruction of the
mucous bicarbonate barrier, which prevents back diffusion of pepsin and hydrogen
ions from the lumen into the mucosal layer (Takeuchi, 1989). Moreover,
microcirculation of gastroduodenal mucosa supplies energy and oxygen to mucosal
cells, removes hydrogen ions and waste products, and transports bicarbonate to the
surface of the gastric epithelium. Thus, the mucosal blood flow plays a very crucial
role in supporting the defense mechanism of gastric mucosa (Akira and Tadashi,
1992). Local GI irritation via a direct contact effect due to the acidic carboxyl
functionality is common to all nonselective cyclooxygenase inhibitor NSAIDs
(Wallace and Cirino, 1994). To diminish the GI toxicity, many ester and amide
prodrugs of NSAIDs have been reported in the literature (Ueda et al., 1991; Bonina
et al., 2002). The inactive prodrugs temporarily block the acidic carboxyl group
until their absorption and release the active parent drugs into the blood after
absorption.
We thought of modifying the structure of NSAIDs to N,N-disubstituted
aminoalcohol esters so that it would lead to the formation of anticholinergic
molecules in the intact form, with a greatly reduced acidic character during their GI
transit before absorption. The expected advantage of incorporation of anticholin-
ergic activity was that the intact ester in stomach would inhibit gastric secretions
and motility by its local anticholinergic action. Thus, contrary to the prior prodrug
approach that relied on overcoming local GI irritation by simply blocking the acidic
group of NSAIDs, the specially designed NSAID aminoalcohol esters would afford
gastric protection by (1) masking the acidic carboxyl group temporarily until
absorption, (2) inhibiting gastric acid secretion, and (3) decreasing the gastric
motility to maintain the optimal mucosal blood flow, an important gastric mucosal
defense factor (Akira and Tadashi, 1992). Hence, N,N-disubstituted aminoalcohol
esters were specifically designed to possess a terminal tertiary ‘‘nitrogen’’ atom
with an ethylene bridge between this terminal nitrogen and the carbonyl group of
bulky ester, which are also the structural features of aminoalcohol ester class of
anticholinergics (Latin and Fifer, 1981).
Previous studies from our laboratory have revealed that esterification of the
carboxylic acid moiety, with different aminoalcohols, in NSAIDs such as
flurbiprofen (Halen et al., 2006), naproxen (Halen et al., 2007), and diclofenac
(Yadav et al., 2005) generates potent dual acting anticholinergic-antiinflammatory
moieties with significantly reduced oral gastric toxicity. The promising results from
these candidates prompted us to apply this potential approach to indomethacin (1), a
potent indoleacetic acid class of antiinflammatory drug. We report here the
synthesis and evaluation of potential esters of indomethacin that combine
anticholinergic and antiinflammatory actions. Moreover, a methodology for

Med Chem Res (2007) 16:101–111
103
esterification of carboxyl compounds possessing other sensitive groups is discussed,
ensuring a much broader synthetic utility.
Results and Discussion
Synthesis
Mahfouz et al. (1999) have reported that indomethacin esters can be obtained via
the acid chloride formation followed by reaction with the respective aminoal-
cohols. However, our efforts to obtain the esters of indomethacin via the acid
chloride yielded the unexpected esters of p-chlorobenzoic acid. The esters 2 and
3 so obtained, as per Scheme 1 (method A), were characterized as the
hydrochloride salt forms. The infrared (IR) spectra showed strong ester (C=O)
stretching bands at 1727–1739 cmÀ1 in their IR spectra and characteristic proton
magnetic resonance (PMR) signals at d 7.43–7.46 (d, 2H) for ring protons ortho
to chloro and 7.97–8.01 (d, 2H) for ring protons ortho to keto ester moiety. From
this observation, we conclude that in presence of thionyl chloride the parent acid
undergoes degradation at the indole-amide linkage to liberate the p-chlorobenzoyl
group, which results in the various undesired esters via the acid chloride.
Interestingly, it was observed that even under basic conditions (in the presence of
anhydrous potassium carbonate i.e., without being converted to acid chloride)
indomethacin (1) reacted with the aminoalcohols to yield the undesired esters of
p-chlorobenzoic acid. After failure to obtain the desired aminoalcohol esters by
the reported acid chloride method, an easy access to the desired esters was
finally made possible by using a two-step process (method B), that is (1)
preparation of the N,N-disubstituted aminoethylchloride followed by (2) the
reaction of indomethacin with the resulting chloro derivatives. The appropriate
N,N-disubstituted aminoalcohol was reacted with thionyl chloride smoothly at
room temperature to give the desired chloro derivative in good yields. The
second step, i.e., reaction of resulting chloro derivative dissolved in chloroform,
with indomethacin dissolved in aqueous bicarbonate solution, was carried out
successfully in presence of a phase transfer catalyst. The simple work up
involving the washing of the organic phase with ice-cold water, drying, and
solvent removal yielded the oily esters 4–8, which were found to be unstable on
storage when converted to the hydrochloride salts. The basic esters were
therefore characterized, preserved, and used as such for further studies. Features
such as mild reaction conditions and simple workup with good yields broadens
the scope of the present synthetic strategy for esterification of carboxylic
compounds with groups sensitive to thionyl chloride. The present procedure
offers a superior and cleaner method than the reported ones, involving use of
dicyclohexylcarbodiimide as coupling agent (Holmberg and Hansen, 1979;
Bonina et al., 1991; Abordo et al., 1998). All the esters were characterized by
their spectral data.

104
Med Chem Res (2007) 16:101–111
Method A
CH2COOH
Me
MeO
3
5
2
COOR
.HCl
Cl
N
a, b, c
O
6
2, 3
Cl
1
CH2COOR
Me
Method B
MeO
N
a, d
R-OH
O
Cl
4-8
R
R
CH2-CH2-N
2, 4
3, 5
7
8
CH2-CH2- N
N
CH2-CH2-
CH -CH -
N
O
2
2
N
CH2-CH2-
6
Scheme 1 Reagents and reaction conditions: (a) SOCl2; (b) R-OH, K2CO3; (c) dry HCl;
(d) H2O,NaHCO3. 1, Phase transfer catalyst
Hydrolysis studies
Chemical and enzymatic hydrolyses studies of the ester derivatives 4–8 were
performed in aqueous buffer solutions (pH 2.0 and pH 7.4) and in human serum
(80%), respectively. The corresponding half-lives in buffer solution and percent
release of parent drug on enzymatic hydrolysis are given in Table 1. At pH 2.0, no
noticeable hydrolysis was observed for all the derivatives until a period of 6 h. The
observed acid stability of the ester derivatives fulfills the requirement for oral
delivery system of NSAIDs, whereby the masking group should be acid stable to
prevent the direct contact effects on the gastric mucosa. Apart from this, in the
acidic pH of stomach, the polar protonated form (at the basic nitrogen) of these
esters would resist absorption into the gastric cells owing to their decreased
lipophilicity. This would prevent the intracellular entrapment of these compounds,
and hence the local inhibition of the cytoprotective prostaglandin synthesis. It is
well accepted that an easy absorption of the lipophilic form of NSAIDs at the gastric
pH into the mucosal cells, followed by the entrapment due to the alkaline
intracellular pH, is responsible for a more intense localized anti-PG action (Wallace
and Cirino, 1994).

Med Chem Res (2007) 16:101–111
105
Table 1 Chemical and enzymatic hydrolysis of ester derivatives 4–8
Compound
t (h) pH 7.4 buffer
½
% Release of indomethacin (in 80% human serum)
½ h
1 h
2 h
4
5
6
7
8
34
39
99
66
52
35.5
54.4
40.6
50.1
27.8
47.8
59.3
42.1
59.1
36.9
49.6
70.9
53.3
65.0
45.4
At pH 7.4, sufficiently long half-lives were observed for all the ester derivatives
(Table 1). These data ensure the stability of the candidates throughout their GI
transit followed by their absorption in the intact form to enter the circulation. The
enzymatic susceptibility of all the compounds toward the human plasma esterases
ensures a successful release of the parent acid after entering the circulation.
Compound 6, bearing a cyclic substituent at the N-terminus, exhibited the slowest
rate of chemical hydrolysis and low percent release of parent acid whereas
compound 5, with an open chain diethyl linkage, showed an exactly opposite
behavioral pattern toward chemical and enzymatic hydrolysis. Compound 5
exhibited not only a fast chemical hydrolysis but also the highest susceptibility
toward serum esterases. Replacing the pyrrolidino substituent in 6 with the
piperidino substituent (7) increases the susceptibility toward both types of
hydrolysis; however, the rates are lower when compared to the diethylaminoethyl
derivative 5. Further, the higher percent release of parent acid in serum by
compound 7 (piperidino derivative) in comparison to compound 8 shows that
replacement of a –CH2– group with O, i.e., changing the polarity of the N-terminal
substituent affects the binding of the esters to the esterase site. Hence, moving from
an open chain to a cyclic substituent at the N-terminal of these esters has a
substantial influence on their hydrolysis behavior.
The combined results of chemical and enzymatic hydrolyses studies reveal these
esters to survive the GI conditions and enter the circulation as intact compounds,
thereby successfully overcoming the local GI irritation. Besides this, all the esters
have appeared to possess the chemical requirements (good aqueous stability and
high enzymatic conversion) to be regarded as derivatives useful for oral
administration. Further, the aminoethylalcohol moiety released on metabolism of
the ester drug would be free of systemic toxicity and side effects owing to their
rapid elimination as highly water soluble molecules.
Biological evaluation
All ester derivatives 4–8 exhibited anticholinergic activity with pA2 values
(Table 2) ranging from 4.92 to 5.19, though the activity was much weaker than that
of the standard atropine sulfate (pA2 = 8.20). The right parallel shift observed for the
dose–response curve in the presence of the esters reveals them to be competitive

106
Med Chem Res (2007) 16:101–111
reversible inhibitors of acetylcholine at the smooth muscle receptors. Compounds 4
and 6 exhibited antiinflammatory activity comparable to that of the parent acid, and
compounds 5, 7, and 8 were less active than indomethacin at equimolar doses. A
significant reduction was observed in the ulcerogenic potential of these compounds
(4–8) when compared to their respective parent drugs (Table 2). Considering the
stability of these derivatives in aqueous buffer solutions and the rapid enzymatic
hydrolysis, it can be concluded that the observed ulcerogenicity may be due to
conversion of the esters to indomethacin after absorption from the GI tract, which is
responsible for the systemic inhibition of the prostaglandin synthesis. In acetic acid–
induced writhing syndrome for evaluation of the peripheral analgesic activity,
interesting results were observed. Except for 5, all the other derivatives were
equipotent to the parent drug in their analgesic action. In brief, in the series 4–8,
compound 4 was found to be the best among all the derivatives of indomethacin,
with a significant reduction in gastric irritation while retaining the antiinflammatory
and analgesic activity.
In conclusion, the present study indicates the tested esters represent potentially
useful indomethacin derivatives for oral administration because they (1) are stable
in aqueous solutions, (2) show anticholinergic activity in the gut, (3) are readily
hydrolyzed in human plasma, (4) retain the antiinflammatory and analgesic activity
of the parent acid, and (4) notably inhibit the GI irritation induced by indomethacin.
There are reports on indomethacin esters and amides being cox-2 selective
inhibitors (Kalgutkar et al., 2005; Khanna et al., 2006); hence, in addition, we
expect that these esters, until they remain intact in the circulation, might afford
gastric protection by having selectivity for cox-2 enzyme. These derivatives cannot
be called prodrugs because unlike prodrugs they have a biological action
(anticholinergic) before being hydrolyzed for release of the parent moiety. We
believe that the present approach is a very rare example of drug design wherein a
compound possesses a beneficial pharmacological action before entering the
circulation and elicits the desired biological action on metabolism.
Table 2 Anticholinergic, antiinflammatory, analgesic activities and ulcerogenicity of indomethacin (1)
and its derivatives 4–8
Compound
pA2 Edema volume (% inhibition) Ulcer index
No. of writhings, % inhibition
Control
—
0.920 0.06 (0.00)
0.502 0.04 (45.43)
3.0
0.000
36.60 1.44 (0.00)
Indomethacin
0.547 0.116* 21.20 3.14 (42.08)
(Dose, mg/kg)
25.0
15.0
4
5
6
7
8
5.19 0.490 0.05 (46.74)
4.49 0.602 0.12 (34.56*)
4.92 0.576 0.03 (37.50)
5.04 0.768 0.06 (16.57*)
5.16 0.708 0.10 (23.09*)
0.149 0.044* 22.00 2.19 (39.89)
0.317 .058* 28.17 1.58 (23.04*)
0.341 0.046* 23.83 1.75 (37.16)
0.134 0.042* 23.80 2.52 (34.97)
0.259 0.031* 23.60 1.50 (35.52)
*p < 0.05 versus indomethacin. Results are expressed as means SEM

Med Chem Res (2007) 16:101–111
107
Experimental
Lambda (type IV) carrageenan and the aminoalcohols were purchased from Sigma-
Aldrich (St. Louis, MO). Distilled water was used in the preparation of the buffer
solutions. Anhydrous sodium sulfate was used as the drying agent. Melting points
were taken in open capillaries and are uncorrected. The IR spectra were recorded on
a Shimadzu-8300 FT-IR using a KBr disc. Absorbance was measured on a
1
Shimadzu UV-1601 spectrophotometer. H-NMR spectra were recorded on a 300
MHz instrument for solutions in CDCl3.
Synthesis of N,N-disubstituted aminoethyl esters of indomethacin (4–8)
Method A. Indomethacin (1; 1.0 g, 2.79 mmol) was dissolved in dichloromethane
by refluxing and then treated dropwise with thionyl chloride (0.4 ml, 5.37 mmol).
The reaction mixture was stirred for 2 h at room temperature followed by the
addition of the N,N-dimethylaminoethyl alcohol (1.5 ml, 0.015 mmol) and
anhydrous potassium carbonate (1.0 g) with further stirring for 2 h at room
temperature. The reaction mixture was diluted with chloroform, filtered, and the
organic layer was washed with ice-cold water, dried, and the solvent removed to
obtain an oily residue. The oily material was dissolved in dry isopropyl ether, and
dry hydrogen chloride was passed to give a solid precipitate. The salt so obtained
was filtered, crystallized, and identified as 2. The above route was tried with the
N,N-diethylaminoethyl alcohol and the compound so obtained was characterized to
be the aminoalcohol ester 3 of p-chlorobenzoic acid.
2-Dimethylaminoethyl 4-chlorobenzoate hydrochloride (2). % Yield 57, mp
1
178–181ꢁC, IR (cm–1): 1731 (ester C=O stretching), H-NMR: d (ppm) (CDCl3):
2.94 (s, 6H, N(CH3)2),3.47–3.50 (t, 2H, CH2N), 4.82–4.85 (t, 2H, O–CH2), 7.43–
7.46 (d, 2H, Ar–3–CH and Ar–5–CH), 8.02–8.05 (d, 2H, Ar–2–CH and Ar–6–CH),
C11H15Cl2NO: Requires C, 53.2; H, 6.1; N, 5.6. Found C, 53.1; H, 6.3; N, 5.4%.
2-Diethylaminoethyl 4-chlorobenzoate hydrochloride (3). % Yield 55, mp
1
167–169ꢁC, IR (cm–1): 1727 (ester C=O stretching), H-NMR: d (ppm) (CDCl3):
2.94 (s, 6H, N(CH3)2), 3.47–3.50 (t, 2H, CH2N), 4.82–4.85 (t, 2H, O–CH2), 7.43–
7.46 (d, 2H, Ar–3–CH and Ar–5–CH), 8.02–8.05 (d, 2H, Ar–2–CH and Ar–6–CH),
C13H19Cl2NO : Requires C, 56.5; H, 6.9; N, 5.1. Found C, 56.1; H, 6.7; N, 5.3%.
Method B. N,N-Dimethylaminoethanol (1.0 ml, 0.01 mol) was reacted with
thionyl chloride (1.5 ml, 0.02 mol) in dry chloroform (20 ml) under stirring at room
temperature and refluxed for 15 min on a water bath. The reaction mixture was then
cooled and ice-cold water (20 ml) was added to it. Sodium bicarbonate (3.5 g),
potassium iodide (300 mg), indomethacin (1.5 g, 4.18 mmol), and tetraethylam-
monium bromide (PTC, phase transfer catalyst; 1.5 g) were added to the above
solution and the reaction mixture was stirred for 15–18 h. The reaction mixture was
monitored via thin-layer chromatography (TLC) for the absence of indomethacin in
the organic layer, diluted further with chloroform (200 ml), and washed several
times with ice-cold water. The chloroform layer was dried and removed completely
to afford the oily product (4). The esters were preserved as oily bases without
conversion into the corresponding hydrochloride salts, which were found to be

108
Med Chem Res (2007) 16:101–111
unstable. Compounds 5–8 (yield 60–73%) were prepared by this route (Scheme 1)
and were characterized by their spectral data, purified by column chromatography,
and purity checked by TLC. Different solvent systems were tried for crystallization
but the semisolid oily ester bases could not be crystalized.
2-Dimethylaminoethyl 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indole-
3-acetate (4). %Yield 62, UV (MeOH): kmax 227 nm (log e 4.40), IR (cm–1):
1
1733 (ester C=O stretching), 1683 amide C=O, H NMR: d (ppm) (CDCl3): 2.31
(s, 6H, N(CH3)2), 2.40 (s, 3H, ArCH3), 2.63–2.69 (t, 3H, CH2N), 3.65 (s, 2H,
Ar–CH2), 3.80 (s, 3H, Ar–OCH3) , 4.24–4.26 (t, 2H, –OCH2), 6.64–7.67(m,7H,
ArH).
2-Diethylaminoethyl 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H -indole-3-
acetate (5). % Yield 67, UV (MeOH): kmax 227 nm (log e 4.48), IR (cm–1): 1732
1
(ester C=O stretching), 1683(amide C=O stretching), H-NMR: d (ppm) (CDCl3):
1.11 (t, 6H, N(CH2CH3)2), 2.35 (s, 3H, Ar–CH3), 2.73–2.85 (q, 4H, N(CH2CH3)2),
2.98 (t, 2H, –CH2N), 3.64 (s, 2H, Ar–CH2), 3.78 (s, 3H, Ar–OCH3), 4.21–4.26
(m, 2H, OCH2), 6.63–7.66 (m,7H, ArH).
1-[2-(1-Pyrrolidino)]ethyl 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-in-
dole-3-acetate (6). % Yield 65, UV (MeOH): kmax 227 nm (log e 4.39), IR (cm–1):
1
1733 (ester C=O stretching), 1683 (amide C=O stretching), H-NMR: d (ppm)
(CDCl3): 1.90–1.95 (m, 4H, (CH2)2), 2.33 (s, 3H, Ar–CH3), 2.71–3.16 (m, 6H,
CH2N(CH2)2), 3.60 (s, 2H, Ar–CH2), 3.81(s, 3H, Ar–OCH3), 4.52–4.58 (t, 2H,
OCH2), 6.68–7.97(m,7H, ArH).
1-[2-(1-Piperidino)]ethyl
1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-in-
dole-3-acetate (7). % Yield 73, UV (MeOH): kmax 227 nm (log e 4.29), IR (cm–1):
1
1732 (ester C=O stretching), 1683 (amide C=O stretching), H-NMR: d (ppm)
(CDCl3): 1.25–1.68 (m, 6H, (CH2)3), 2.34 (s, 3H, Ar–CH3), 2.55–2.93 (m, 6H,
CH2N(CH2)2), 3.60 (s,2H, Ar–CH2), 3.79 (s, 3H, Ar–OCH3), 4.48–4.58 (t, 2H,
OCH2), 6.65–7.97 (m,7H, ArH).
1-[2-(4-Morpholino)]ethyl 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-in-
dole-3-acetate (8). % Yield 60, UV (MeOH): kmax 227 nm (log e 4.40), IR (cm–1):
1
1732 (ester C=O stretching), 1683 (amide C=O stretching), H-NMR: d (ppm)
(CDCl3): 2.40(s, 3H, Ar–CH3), 2.43–2.75(m, 6H, CH2N(CH2)2), 3.57–3.77 (m, 4H,
CH2OCH2), 3.68 (s, 2H, Ar–CH2), 3.83 (s, 3H, Ar–OCH3), 4.24–4.27 (t, 2H, OCH2),
6.64–7.67 (m, 7H, ArH).
Hydrolysis studies
Kinetics of hydrolysis of 4–8 in aqueous solutions. Reactions were initiated by
maintaining a solution of the ester 4–8 (1 mg/ml), in pH 7.4 buffer and in pH 2.0
buffer at 37 1ꢁC. At definite time intervals until a period of 6 h, samples (1.0 ml)
were withdrawn and transferred to a separating funnel containing buffer (pH 2.0,
9 ml). This acidified solution was extracted into solvent ether (3 · 5 ml). The pooled
organic extract was removed completely, methanol (10 ml) was added to the
residue, and absorbance was measured at 227 nm (k max) against a blank obtained by
similar treatment. The rate constants, k, for hydrolysis of prodrugs were determined

Med Chem Res (2007) 16:101–111
109
by linear regression of log of residual ester versus time plots. Triplicate samples
were analyzed and t1/2 was calculated (Table 1) using the equation t1/2 = 0.693/k.
Kinetics of hydrolysis of 4–8 in pooled human serum. Pooled human serum
(4.0 ml) was placed in a stoppered conical flask and maintained at 37 1ꢁC in a
water bath. To this the derivative (4) solution (1.0 ml, 5 mg/ml in pH 7.4 phosphate
buffer) was added, and at appropriate time intervals aliquots (0.5 ml) were
withdrawn and transferred to a separating funnel containing trichloroacetic acid
(1 ml, 10% wt/vol). Buffer solution (8.5 ml, pH 2.0) was added and this protein-
precipitated solution was extracted with solvent ether (3 · 5 ml). Further treatment
was as described under hydrolysis in aqueous solution. The percent release of the
parent drug was calculated. Three determinations were performed to give average
percent release of the parent drug (Table 1).
Biological evaluation
Indomethacin was administered orally as a suspension in carboxymethylcellulose
(1% wt/vol). The derivatives 4–8 were administered orally at an equivalent dose in
polyethylene glycol 400 (5% vol/vol) in distilled water. The protocol for the animal
experiments performed was approved by the IAEC (Institutional Animal Ethics
Committee) as registered under CPCSEA (Committee for the Purpose of Control
and Supervision of Experiments on Animals), Government of India.
Anticholinergic activity. The anticholinergic activity was determined on isolated
rat ileum (Winter et al., 1962; Halen et al., 2006a). Rats weighing 150–200 g were
fasted overnight and housed singly; the abdomen was dissected and the ileum was
placed in Tyrode solution at 37 1ꢁC with aeration. A 1.0–1.5 cm length of the
tissue was mounted under tension (1g). The tissue was stabilized with washings of
fresh Tyrode every 10 min and the dose–response curve (DRC) was recorded to
obtain the maximum response for acetylcholine (ACh). The tissue was allowed to be
in contact with the Tyrode containing the derivative 1(a–e) or atropine sulfate for
half an hour and the DRC was repeated again for ACh. The percentage response was
calculated to find the EC50 in the presence and absence of the antagonist (derivative
or atropine). The pA2 value was calculated via the following formula:
pA2 = Àlog[M] + log(x À 1) where, log[M] = molar conc. of antagonist and
x = EC50 found in presence of antagonist/EC50 found in the absence of antagonist.
Antiinflammatory activity. The antiinflammatory activity for the derivatives was
determined via carrageenan-induced rat hind paw edema assay as described
by Halen et al. (2006a) and Winter et al. (1962). Sprague–Dawley rats of either sex
(n = 6, 150–200 g) were used. The carrageenan-induced rat hind paw edema assay
was performed. The animals were fasted for 24 h with water ad libitum. Each animal
was injected s.c. (0.1 ml, 1% wt/vol) with carrageenan suspension into the
subplantar region of the left hind paw after 1 h of drug administration. The paw
volume was measured immediately after injection and after 3 h using a
plethysmometer (Ugo Basil). The control group received no drug. Indomethacin
(1) was given at a dose of 3 mg/kg body weight and the derivatives were
administered at dose equivalent to 3 mg/kg of indomethacin. Results (Table 2) are
expressed as percentage inhibition of edema formation, calculated via the formula,

110
Med Chem Res (2007) 16:101–111
% inhibition of paw edema = (1 À Eddrug/Edcontrol) · 100, where Eddrug and Edcontrol
are the edema volumes in drug-treated and control groups respectively.
Ulcerogenicity. Sprague–Dawley rats (n = 6, 150–200 g) of either sex were used
to determine the ulcer indices (Parmar and Desai, 1993; Halen et al., 2006a). The
rats were fasted for 36 h with water ad libitum before administration of drug
solutions and for 4 h post-dosing. The control group received no drug. Indomethacin
(25 mg/kg) was given orally and derivatives (4–8) were given at a dose equivalent to
25 mg/kg of indomethacin. The animals were killed, their stomach was dissected
out, cut along the greater curvature, washed with normal saline, and the gastric
mucosa was observed for the lesions via a 2 · 2 binocular magnifier and the results
were expressed as ulcer index using the formula, Ulcer index = 10 (Au/Am), where,
Am = total mucosal area, Au = Al + Ac + Ap, Al = area of linear lesions (l · b),
Ac = area of circular lesions, and Ap = total no. of piteaches/5.
Analgesic activity. Analgesic activity of the derivatives (4–8) was assessed by
the acetic acid–induced writhing assay in the mouse as described by Koster and
Anderson (1959).The animals were divided via randomization in groups (each of six
mice, 18–22 g) and were orally administered indomethacin (1; 15 mg/kg) or its ester
derivatives 4–8 at dose equivalent to 15 mg/kg of indomethacin. After 1 h of drug
administration, the writhing syndrome was elicited by the intraperitoneal injection
of (10 ml/kg of body weight of 0.6% vol/vol in 0.9% saline) acetic acid and the
number of writhes for each mouse was counted after 5 min of injection for a period
of 20 min. The average number of writhes was determined in each group and the
degree of analgesia was expressed as percentage inhibition calculated according to
the formula: % inhibition of writhing = (1 À T/S) · 100, where S and T are the
number of writhes in the control and drug-treated group, respectively.
Acknowledgments The authors thank AICTE (All India Council of Technical Education), New Delhi
for providing financial support through a research grant (TAPTEC Major Research Project).
References
Abordo EA, Bowden K, Huntington AP, Powell SL (1998) Prodrugs. Part 3. 2-Formylphenyl esters of
indomethacin, ketoprofen and ibuprofen and 6-substituted 2-formyl and 2-acylphenyl esters of
aspirin. Il Farmaco 53:95
Akira Y, Tadashi F (1992) Possible mechanisms of gastroduodenal mucosal damage in volunteers treated
with Non-steroidal antiinflammatory drugs: the usefulness of prodrugs. J Rheumatol 19:1075
Bonina FP, Montenegro L, De Capraris P, Bousquet E, Tirendi S (1991) 1-Alkylazacyclo alkan-2-one
esters have prodrugs of indomethacin for improved delivery through human skin. Int J Pharm 77:21
Bonina FP, Pugila C, Santagati NA, Saija A, Tomino A, Tita B (2002) Oligoethylene ester derivatives of
ketoprofen, naproxen and diclofenac as oral prodrugs: a pharmacological evaluation. Pharmazie
57:552
Halen PK, Chagti KK, Giridhar R, Yadav MR (2006) Synthesis and pharmacological evaluation of some
dual-acting aminoalcohol ester derivatives of Flurbiprofen and 2-[1,1’- Biphenyl-4-yl]acetic Acid: A
potential approach to reduce local gastrointestinal toxicity. Chem Biodiversity 3:1238
Halen PK, Raval MK, Chagti KK, Giridhar R, Yadav MR (2007) Synthesis and evaluation of some
gastrointestinal sparing anti-inflammatory aminoethyl ester derivatives of naphthalene-based
NSAIDs. Arch Pharm Chem Life Sci 340:88
Holmberg K, Hansen B (1979) Ester synthesis with dicyclohexylcarbodiimide improved by acid catalysts.
Acta Chem Scand B 33:410

Med Chem Res (2007) 16:101–111
111
Kalgutkar AS, Crews BC, Slaeh S, Prudhomme D, Marnett LJ (2005) Indolyl esters and amides related to
indomethacin are selective COX-2 inhibitors. Biol Med Chem 13:6810
Khanna S, Madam M, Vangoori A, Banerjee R, Thaimattam R, Jafar Sadik Basha SK, Mullangi R,
Casturi SR, Pal M (2006) Evaluation of glycolamide esters of indomethacin as potential
cyclooxygenase-2 (COX-2) inhibitors. Biol Med Chem 14:4820
Koster R, Anderson M (1959) Acetic acid for Analgesic Screening. J Fed Proc 18:412
Lattin DL, Fifer EK (1981) Drugs affecting cholinergic neurotransmission. In: Williams DA (ed) Foye’s
principles of medicinal chemistry, 5th edn. Williams & Wilkins, Philadelphia, p 269
Mahfouz NM, Omar FA, Aboul-Fadl T (1999) Cyclic amide derivatives as potential prodrugs II: N-
Hydroxymethyl succinimde-/isatin esters of some NSAIDs as prodrugs with an improved
therapeutic index. Eur J Med Chem 34:551
Parmar NS, Desai J (1993) A review of the current methodology for the evaluation of gastric and
duodenal anti-ulcer agents. K Indian J Pharmacol 25:120
Paul AD, Shah E, Richard MM, Peter J (2004) Lessons from the withdrawal of Rofecoxib. Br Med J
329:867
Scheen AJ (2004) Withdrawal of Rofecoxib (Vioxx): What about cardiovascular safety of cycloxygenase
selective non-steroidal anti-inflammatory drugs? Rev Med Liege 59:5650
Takeuchi T (1989) Bilateral adrenalectomy worsens gastric mucosal lesions induced by indomethacin in
the rat. Role of enhanced gastric motility. Clin Rep 8:167
Ueda I, Ishii K, Arai H, Ikeda S, Hitomi Y, Hatanaka M (1991) Design, synthesis and anti- inflammatory
activity of new indomethacin ester 2-[N-{3-(3-piperidinomethyl)phenoxy) propyl carbamoylmeth-
ylthio]ethyl 1-(p-chlorobenzoyl)-5-methoxy-2-methyl-indole-3-acetate. Chem Pharm Bull 39:697
Van Rossum JM, Hurkmans JH, Walters CJ (1965) Cumulative dose response curves. J Arch Int
Pharmacodyn Ther 143:299
Wallace JL, Cirino G (1994) The development of gastrointestinal-sparing nonsteroidal anti- inflammatory
drugs. Trends Pharmacol Sci 15:405
Winter CA, Risley EA, Nuss GW (1962) Carrageenan-induced edema in hind paw of the rat as an assay.
Proc Soc Exp Biol Med 111:544
Yadav MR, Halen PK, Chagti KK, Hemalata B, Giridhar R (2005) A novel approach towards therapeutic
optimization of diclofenac. Ars Pharm 46:263