Synthesis and pharmacological evaluation of 2(3H)-furanones and 2(3H)-pyrrolones, combining analgesic and anti-inflammatory properties with reduced gastrointestinal toxicity and lipid peroxidation

Article abstract of DOI:10.1016/j.ejmech.2008.10.030

A series of 3-arylidene-5-(4-chloro-phenyl)-2(3H)-furanones (2-13) and their nitrogen analogues 1-benzylpyrrolones (14-18) were synthesized. The compounds were evaluated for their anti-inflammatory, analgesic, ulcerogenic and lipid peroxidation actions. Some of the newly synthesized compounds showed good anti-inflammatory and analgesic activities with low GI toxicity and reduced lipid peroxidation. The biological activity was found to improve upon replacement of oxygen of furanone ring with benzylamine moiety i.e. 1-benzylpyrrolones. Similarly, compounds containing halogen group(s), compounds 15 and 17, showed higher degree of anti-inflammatory activity and their activity was comparable to that of the standard. These compounds showed interesting profile of analgesic activity in acetic acid induced writhing test (peripheral effect) and in the hot-plate test (central effect). The compounds were also tested for their ulcerogenic and lipid peroxidation action and showed superior GI safety profile along with reduction in lipid peroxidation as compared to that of the standard.

Full text of DOI:10.1016/j.ejmech.2008.10.030

European Journal of Medicinal Chemistry 44 (2009) 2636–2642
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and pharmacological evaluation of 2(3H)-furanones and
2(3H)-pyrrolones, combining analgesic and anti-inflammatory properties
with reduced gastrointestinal toxicity and lipid peroxidation
,
a
,
,
,
,4
M.M. Alam a , Asif Husain 1, S.M. Hasan a 2, Suruchi b 3, T. Anwer b
*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard, Hamdard University, Hamdard Nagar, New Delhi 110 062, India
b Department of Pharmacology, Faculty of Pharmacy, Hamdard University, New Delhi 110 062, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3-arylidene-5-(4-chloro-phenyl)-2(3H)-furanones (2–13) and their nitrogen analogues 1-
benzylpyrrolones (14–18) were synthesized. The compounds were evaluated for their anti-inflammatory,
analgesic, ulcerogenic and lipid peroxidation actions. Some of the newly synthesized compounds showed
good anti-inflammatory and analgesic activities with low GI toxicity and reduced lipid peroxidation. The
biological activity was found to improve upon replacement of oxygen of furanone ring with benzylamine
moiety i.e. 1-benzylpyrrolones. Similarly, compounds containing halogen group(s), compounds 15 and 17,
showed higher degree of anti-inflammatory activity and their activity was comparable to that of the
standard. These compounds showed interesting profile of analgesic activity in acetic acid induced
writhing test (peripheral effect) and in the hot-plate test (central effect). The compounds were also
tested for their ulcerogenic and lipid peroxidation action and showed superior GI safety profile along
with reduction in lipid peroxidation as compared to that of the standard.
Received 17 April 2008
Received in revised form
16 October 2008
Accepted 20 October 2008
Available online 5 November 2008
Keywords:
Furanone
Anti-inflammatory
Analgesic
Ulcerogenic
Lipid peroxidation
Ó 2008 Elsevier Masson SAS. All rights reserved.
1. Introduction
incorporated into a wide variety of therapeutically interesting
drug candidates having anti-inflammatory [2], cardiotonic activity
2-Furanone, also known as furan-2-one, is a heterocyclic
chemical compound classified as a lactone. It is a common
component of natural products synthesized by biochemical path-
ways in organisms, especially plants of the genus Angelica. 2-Fur-
[3], analgesic [4], anticancer [5], anti-convulsant [6], anti-micro-
bial [7] and antiviral activities [8]. There are marketed drugs
containing furanone ring, e.g., basidalin as anticancer, ascorbic
acid as an antioxidant, narthogenin and butalactin as antibiotic,
rofecoxib as a specific COX-2 inhibitor. Moreover, there are many
furanones which are patented as an anti-inflammatory agent e.g.,
3-alkyl-5-hydroxy-3,4-dihydrofuran-2-ones (United States Patent
5202350), 4-[3,6-dihydro-6-hydroxy-5-(3-phenylpropyl)-2H-py-
ran-2-yl]-5-hydroxy-2(5H)-furanone (European Patent EP042-
9287).
We have previously described the synthesis of a series of new
anti-inflammatory and analgesic agents with reduced gastric
ulceration having furanone structure [9]. A recent literature
survey revealed that many of the halogen containing heterocycles
has attracted attention due to the ability of halogen to act as polar
hydrogen or hydroxyl mimic. Substitution of hydrogen by
halogen has been a strategy in designing molecules having
potential biological activities [10,11]. Prompted by these investi-
gations and in continuation of our research work [9,12] on studies
of novel O- & N-heterocyclic compounds of pharmaceutical
interest, it was thought to be interesting to study various
compounds containing 2(3H)-furanone and 2(3H)-pyrrolone
moieties.
anone is also known as g-crotonolactone or b-angelica lactone. It is
the simplest butenolide compound and is colloquially called
‘‘butenolide’’ in the context of natural product synthesis.
Compounds derived from furanones are generated by some plants
exposed to high heat in brush fires and trigger seed germination in
plants whose reproduction depends on exposure to fire [1].
In the last few decades, the chemistry of furanones has
received considerable attention owing to their synthetic and
effective biological importance. Furanone moieties have been
* Corresponding author. Tel.: þ91 11 26059681, 26059688x5645, Mobile: þ91
9868611258; fax: þ91 11 6988874.
E-mail addresses: hamlucky@rediffmail.com (M.M. Alam), drasifhusain@
yahoo.com (A. Husain), smhasan@jamiahamdard.ac.in (S.M. Hasan), suranv@
rediffmail.com (S.M. Suruchi), tanwer_26@yahoo.com (T. Anwer).
1
Tel.: þ91 11 26059681, 26059688x5647, Mobile: þ91 9891116086; fax: þ91 11
6988874.
2
Tel.: þ91 11 26059681, 26059688x5660; fax: þ91 11 6988874.
3
Tel.: þ91 11 26059681, 26059688x5667; fax: þ91 11 6988874.
4
Tel.: þ91 11 26059681, 26059688x5614; fax: þ91 11 6988874.
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.10.030

M.M. Alam et al. / European Journal of Medicinal Chemistry 44 (2009) 2636–2642
2637
2. Chemistry
yields (on an average 75%) with reduced reaction time for
2(3H)-furanones were achieved when the reaction was performed
by one pot synthesis (method ii). Mixed melting points of the 2-
(3H)-furanones (2–13) obtained by two above-mentioned methods
have not shown any depression. The IR, 1H NMR and MS spectral
data of these compounds were also identical.
Some of the 2(3H)-furanones (2–13) synthesized above were
further converted to nitrogen derivatives i.e. 1-benzylpyrrolones
(14–18). The 3-arylidene-5-(4-chloro-phenyl)-1-benzyl-2(3H)-
pyrrolones (14–18) were synthesized by reacting appropriate
Overall eighteen compounds were synthesized. Synthesis of
twelve 2(3H)-furanones were brought about by two-step reaction
using Friedel–Craft’s acylation reaction and modified Perkin reac-
tion conditions (Scheme 1). The 3-arylidene-5-(4-chloro-phenyl)-
2(3H)-furanones 2–13 were prepared by condensing different
aromatic aldehydes with 3-(4-chloro-benzoyl)propionic acid 1 in
presence of triethylamine and acetic anhydride under anhydrous
conditions. The required 3-(4-chloro-benzoyl)-propionic acid 1 was
prepared by reacting dry chlorobenzene with succinic anhydride in
presence of anhydrous aluminum chloride following Friedel–Craft’s
acylation reaction conditions. In an attempt to prepare the 2(3H)-
furanones in higher yields, two different synthetic methods were
applied. Thus the condensation of above-mentioned 3-(4-chloro-
benzoyl)propionic acid with aromatic aldehydes was carried out
using: (a) 15 mL acetic anhydride (method i) [9] and (b) 5–8 drops
acetic anhydride; one pot synthesis i.e. fusion reaction (method ii)
by heating on a heating mantle. Substrates were mixed together in
molar ratio (3 mmol; 1:1). It was found that both methods led
directly to the formation of desired 2(3H)-furanones. Higher
2(3H)-furanone with benzylamine in dry benzene to give
benzylamide, which was then lactamized (cyclized) in 6 N HCl to
give the corresponding 1-benzylpyrrolone. Calculations of -values
g-keto-
d
using incremental parameters for the hydrogen (semicyclic double
bond) suggest (E)-configuration.
In the 1H NMR spectral data all the compounds showed two
singlets of one proton each around
assigned to the ring H and the olefinic hydrogen of the arylidene
substituent. Other peaks were observed at appropriate -values.
d 6.5 and d 7.4 which could be
b
d
The fragmentation pattern observed on electron impact mass
spectrum can be summarized as follows:
The 3-arylidene-5-(4-chloro-phenyl)-2(3H)-furanones gave
M
þ peak in reasonable intensities. The major fragment appears to
be Cl–C6H4–C^Oþ arising from the heterocyclic oxygen and
g-
O
carbon with its substituent. Subsequently it loses CO to give Cl–
C6Hþ4 . There appeared a peak at m/z 77 that corresponds to C6Hþ5 .
Occasionally the aryl ring of the arylidene moiety also appeared as
Arþ. In case of 1-benzylpyrrolones, loss of 91 mass units corre-
sponding to benzyl moiety from the molecular ion is observed
along with peaks at m/z 91, 77. Other pathway is via Cl–C^NþH
arising from C-2 and its substituent, which appears to be novel.
This also loses HCN to give Cl–C6Hþ4 . The molecular ion or other
related ions produced the appropriate isotopic abundances due to
presence of chlorine atom(s). The analytical data are mentioned in
Table 1.
i
Cl
Cl
OH
O
1
ii
Cl
R
O
Table 1
O
Physical constants of 2(3H)-furanones and 1-benzyl-2(3H)-pyrrolones.
2(3H)Furanone 2-13
Cl
iii
R
Cl
X
O
2-18
R
Compounds
X
R
Rf valuea Molecular formulab
O
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
O
O
O
O
O
O
O
O
O
O
O
O
H
0.76
0.74
0.74
0.82
0.82
0.80
0.82
0.78
0.78
0.76
0.72
C17H11O2Cl
O
HN
2-Chloro
3-Chloro
4-Chloro
2-Nitro
3-Nitro
4-Nitro
4-Fluoro
4-Methoxy
3,4-Dimethoxy
2,4-Dichloro
4-Acetoxy-3-methoxy 0.72
H
C17H10O2Cl2
C17H10O2Cl2
C17H10O2Cl2
C17H10O4NCl
C17H10O4NCl
C17H10O4NCl
C17H10O2ClF
C18H13O3Cl
C19H15O4Cl
C17H9O2Cl3
C20H15O5Cl
C24H18ONCl
C24H17ONCl2
C24H17O3N2Cl
C24H17ONClF
C25H20O3NCl
γ-Ketobenzylamide
iv
Cl
–NCH2C6H5–
0.71
0.70
0.76
0.78
R
–NCH2C6H5– 4-Chloro
–NCH2C6H5– 4-Nitro
–NCH2C6H5– 4-Fluoro
N
O
–NCH2C6H5– 4-Hydroxy-3-methoxy 0.78
a
Rf values for compounds 2–13 in solvent system (toluene:ethylacetate:formic
acid, 5:4:1) and for compounds 14–18 in solvent system (petrol:toluene:ethylace-
tate, 10:5:3).
1-Benzyl-2(3H)Pyrrolone 14-18
b
Scheme 1. (i) Anhydrous AlCl3, succinic anhydride; (ii) aryl aldehyde, Ac2O, triethyl-
amine, reflux; (iii) dry benzene, benzylamine; (iv) 6 N HCl.
The micro analysis values for C, H and N were within ꢀ 4% of the theoretical
values.

2638
M.M. Alam et al. / European Journal of Medicinal Chemistry 44 (2009) 2636–2642
also have furanone nucleus in their structure. It gives the
impression that these compounds would also possess selective
cyclooxygenase-2 inhibition. The studies are under progress in
our laboratory to acquire more information regarding their
mechanism of action or biological targets.
3. Pharmacological results and discussion
3.1. Anti-inflammatory activity
The in-vivo anti-inflammatory activity of the synthesized
compounds 2–18 was evaluated by carrageenan-induced rat paw
edema method [13]. Ibuprofen was used as a standard drug for
comparison. All the compounds were administered orally and
assayed at a dose level of 20 mg/kg of body weight. The obtained
pharmacological results revealed that replacement of oxygen atom
of furanone ring with benzylamine moiety resulted in markedly
increase in anti-inflammatory activity (Table 2).
Test compounds that exhibited good anti-inflammatory activity,
namely 2(3H)-furanones 3, 5, 9 and 12 and 1-benzylpyrrolones 15
and 17, were further evaluated for their analgesic, ulcerogenic and
lipid peroxidation actions.
3.2. Analgesic activity
Following points could be made after analyzing the anti-
inflammatory results:
The compounds 3, 5, 9, 12, 15 and 17 were evaluated for their
peripherally and centrally mediated analgesic effects using acetic
acid induced writhing method [14] and Turner hot-plate method
[15], respectively.
ꢁ Among the compounds bearing benzylamine moiety i.e. 1-
benzyl-2(3H)-pyrrolones, the maximum activity was shown by
the 3-(4-chloro-benzylidene)-5-(4-chloro-phenyl)-1-benzyl-
2(3H)-pyrrolone 15 and 3-(4-fluoro-benzylidene)-5-(4-chloro-
phenyl)-1-benzyl-2(3H)-pyrrolone 17 with 88.88% and 89.5%
inhibition respectively. The other three compounds, 3-(ben-
zylidene)-5-(4-chloro-phenyl)-1-benzyl-2(3H)-pyrrolone 14,
3-(4-nitro-benzylidene)-5-(4-chloro-phenyl)-1-benzyl-2(3H)-
pyrrolone 16 and 3-(4-hydroxy-3-methoxy-benzylidene)-5-(4-
chloro-phenyl)-1-benzyl-2(3H)-pyrrolone 18, showed 37.03,
19.75 and 41.97% inhibition, respectively.
ꢁ Among the compounds of 2(3H)-furanone series, good results
were obtained with 3-(2-chloro-benzylidene)-5-(4-chloro-
phenyl)-2(3H)-furanone 3, 3-(4-chloro-benzylidene)-5-(4-chlo
ro-phenyl)-2(3H)-furanone 5, 3-(4-fluoro-benzylidene)-5-(4-
chloro-phenyl)-2(3H)-furanone 9 and 3-(2,4-dichloro-benzy-
lidene)-5-(4-chloro-phenyl)-2(3H)-furanone 12 with inhibi-
tion range of 69.13–81.48%.
ꢁ Compound 17 having fluoro at 4-position showed higher anti-
inflammatory activity than compound 15 having chloro group
at 4-position which indicates that activity increases with an
increase in electronegativity on phenyl moiety. That effect
further increases on replacement of oxygen moiety of furanone
ring with benzylamine moiety.
ꢁ DFP [3-(2-propyloxy)-(4-methyl-sulfonylphenyl)-(5,5-dimeth
yl)-furanone], a furanone derivative shows selective cycloo-
xygenase-2 inhibition [4]. The compounds under investigation
The results of analgesic activity (Table 3) indicated that few
tested compounds showed central analgesic activity (hot-plate
test), compounds 15 and 17 showed activity comparable to that of
standard ibuprofen. Compounds 3, 5, 9 and 12 showed less central
analgesic activity. Also, Table 3 shows that, compounds 3, 9, 15 and
17 were found to have significant peripheral analgesic activity
(acetic acid induced writhing method) in the range of 50–59.03%,
which was comparable to that of standard drug ibuprofen (65.06%).
These findings present an important advantage of compound 15
having chloro group at 4-position and compound 17 having fluoro
group at 4-position of phenyl moiety as anti-inflammatory with
high analgesic activity equal to that of ibuprofen.
3.3. Acute ulcerogenesis
The compounds which were screened for analgesic activity were
further tested for their ulcerogenic activity. Compounds 3, 5, 9, 12,
15 and 17 were tested according to the method reported by Cioli
et al. [16]. The tested compounds showed low ulcerogenic activity
ranging from 0.1 ꢀ0.1 to 0.4 ꢀ 0.1 whereas the standard drug
ibuprofen showed high severity index of 0.9 ꢀ 0.1. The maximum
reduction in ulcerogenic activity (0.1 ꢀ0.1) was found in the chloro
derivatives of furanones and benzylpyrrolones. The benzylpyrro-
lone derivatives 17 and 18 showing high anti-inflammatory activity
and analgesic activity also showed reduction in ulcerogenic activity.
Table 2
Anti-inflammatory activity along with ulcerogenic and lipid peroxidation effect of the synthesized compounds 2–18.
Compounds
Paw volume ꢀ SEMa
% Inhibition ꢀ SEMb
Severity indexa
Lipid peroxidationc
After 2 h
After 3 h
After 2 h
After 3 h
Control
Ibuprofen
2
3
5
6
8
9
10
11
12
13
14
15
16
17
18
0.75 ꢀ 0.01
0.81 ꢀ 0.02
–
–
0.00 ꢀ 0.00
0.9 ꢀ 0.36*
0.9 ꢀ 0.36*
0.2 ꢀ 0.12
0.2 ꢀ 0.12
0.6 ꢀ 0.36
0.4 ꢀ 0.40
0.4 ꢀ 0.10
0.5 ꢀ 0.27
0.3 ꢀ 0.12*
0.1 ꢀ 0.10
0.3 ꢀ 0.12*
0.6 ꢀ 0.36
0.1 ꢀ 0.10
0.4 ꢀ 0.10*
0.2 ꢀ 0.12
0.5 ꢀ 0.27
0.238 ꢀ 0.002b,***
0.608 ꢀ 0.001a,***
0.522 ꢀ 0.002a,b,***
0.494 ꢀ 0.001a,b,***
0.403 ꢀ 0.002a,b,***
0.429 ꢀ 0.001a,b,***
0.549 ꢀ 0.001a,b,***
0.329 ꢀ 0.001a,b,***
0.545 ꢀ 0.001a,b,***
0.533 ꢀ 0.001a,b,***
0.388 ꢀ 0.002a,b,***
0.597 ꢀ 0.001a,b,**
0.605 ꢀ 0.001a,***
0.310 ꢀ 0.001a,b,***
0.403 ꢀ 0.002a,b,***
0.401 ꢀ 0.002a,b,***
0.558 ꢀ 0.001a,b,***
0.14 ꢀ 0.01***
0.60 ꢀ 0.02**
0.28 ꢀ 0.01***
0.45 ꢀ 0.02***
0.72 ꢀ 0.01
0.085 ꢀ 0.02***
0.518 ꢀ 0.02***
0.15 ꢀ 0.00***
0.25 ꢀ 0.01***
0.66 ꢀ 0.01**
0.518 ꢀ 0.01***
0.24 ꢀ 0.01***
0.548 ꢀ 0.02***
0.518 ꢀ 0.01***
0.221 ꢀ 0.01***
0.51 ꢀ 0.01***
0.51 ꢀ 0.01***
0.09 ꢀ 0.01***
0.65 ꢀ 0.03**
0.085 ꢀ 0.01***
0.47 ꢀ 0.03***
80.88 ꢀ 2.02
18.88 ꢀ 3.94***
62.66 ꢀ 1.50***
40 ꢀ 3.13***
4 ꢀ 1.37***
89.50 ꢀ 2.56
36.00 ꢀ 3.13***
81.48 ꢀ 0.63*
69.13 ꢀ 1.65***
18.51 ꢀ 2.29***
36.00 ꢀ 1.36***
70.37 ꢀ 1.56***
32.30 ꢀ 2.78***
36.00 ꢀ 1.69***
72.63 ꢀ 2.21***
37.03 ꢀ 2.20***
37.03 ꢀ 2.20***
88.88 ꢀ 2.06
19.75 ꢀ 4.54***
89.50 ꢀ 1.79
0.72 ꢀ 0.00
4 ꢀ 0.91***
0.44 ꢀ 0.02***
0.72 ꢀ 0.00
41.33 ꢀ 3.04***
4 ꢀ 0.76***
0.68 ꢀ 0.01*
0.35 ꢀ 0.01***
0.57 ꢀ 0.02***
0.57 ꢀ 0.02***
0.31 ꢀ 0.02***
0.66 ꢀ 0.02*
0.31 ꢀ 0.02***
0.48 ꢀ 0.02***
9.33 ꢀ 2.57***
53.33 ꢀ 2.36***
24 ꢀ 3.81***
24 ꢀ 3.81***
58.66 ꢀ 3.44***
12 ꢀ 3.07***
58.66 ꢀ 3.13***
36 ꢀ 3.83***
41.97 ꢀ 3.74***
*p < 0.05; **p < 0.01; ***p < 0.001.
a
Relative to their respective control and data were analyzed by one-way ANOVA followed by Turkey test for n ¼ 6.
Relative to the standard (Ibuprofen) and data were analyzed by one-way ANOVA followed by Turkey test for n ¼ 6.
Lipid peroxidation activity is expressed as nmoles of MDA/mg of protein.
b
c

M.M. Alam et al. / European Journal of Medicinal Chemistry 44 (2009) 2636–2642
2639
Table 3
may provide valuable therapeutic intervention for the treatment of
pain and inflammation.
Analgesic effects the some 2(3H)-furanones and 1-benzyl-2(3H)-pyrrolones.
Among the newer derivatives, two compounds 15 and 17
emerged as lead compounds. It is conceivable that these derivatives
could be further modified to develop potent and safer anti-
inflammatory and analgesic agents. Further studies to acquire more
information about quantitative structure–activity relationship
(QSAR) are in progress in our laboratory.
Compounds Dose
Central analgesic activity
Peripheral analgesic
(mg/kg) [Hot-plate test (Reaction time)] activity (Writhing test)
0 min
60 min
No. of writhes/
30 min
%
Protection
Control
Ibuprofen
1 mL
20
9.39 ꢀ 0.029 10.57 ꢀ 0.026
10.18 ꢀ 0.034 14.40 ꢀ 0.024*** 29 ꢀ 1.154***
9.87 ꢀ 0.028 12.69 ꢀ 0.030*** 39 ꢀ 1.897***
10.1 ꢀ 0.023 12.67 ꢀ 0.029*** 46 ꢀ 1.612***
65.06
53.01
44.57
50.60
49.39
57.83
59.03
3
20
5
20
5. Experimental protocols
9
20
10.30 ꢀ 0.016
12.9 ꢀ 0.026*** 41 ꢀ 1.932***
12
15
17
20
20
20
9.14 ꢀ 0.020 11.86 ꢀ 0.023*** 42 ꢀ 1.264***
8.46 ꢀ 0.029 11.55 ꢀ 0.026*** 35 ꢀ 1.238***
9.76 ꢀ 0.024 13.3 ꢀ 0.019*** 34 ꢀ 1.290***
5.1. Chemistry
Chemicals were purchased from Merck and Sigma-Aldrich as
‘synthesis grade’ and used without further purification. Melting
points (m.p.) were determined in open capillary tubes and are
given uncorrected. Elemental analyses were performed on a Per-
kin–Elmer analyzer and were in range of ꢀ 0.4% for each element
analyzed (C, H, N). The IR spectra were measured as potassium
bromide pellets using a Perkin–Elmer 1725X spectrophotometer. 1H
NMR spectra were recorded on Bruker spectropsin DPX-300 MHz
in CDCl3 with tetramethylsilane (TMS) as an internal standard;
***p < 0.001, Relative to the control and data were analyzed by one-way ANOVA
followed by Turkey test for n ¼ 6.
The other tested compounds also exhibited better GI safety profile
as compared to the standard drug ibuprofen. Results are presented
in Table 2.
3.4. Lipid peroxidation
chemical shifts (d) are reported in parts per million (ppm) down-
field from TMS. The splitting pattern abbreviations are as follows: s,
singlet; d, doublet; dd, double doublet; t, triplet; q, quartet; m,
multiplet. Mass spectroscopic analyses for compounds were per-
formed on a JEOL JMS-D 300 instrument fitted with a JMS 2000
data system at 70 eV. Spectral data are consistent with assigned
structures. The molecular ion for compounds containing chloro
group was calculated according to 35Cl isotope. Thin-layer chro-
matography was carried out to monitor the reactions using silica
gel (Merck No. 5554). Dry solvents were used throughout.
The activated inflammatory cells could be involved in the
pathogenesis of mucosal damage. Gastric mucosal cells metabolise
arachidonic acid via both the lipoxygenase and cyclooxygenase
pathways [17] and the presence of inflammatory cells infiltrate in
the gastric mucosa results in the production of large quantities of
oxygen derived free radicals that could cause cell damage and leads
ultimately to mucosal injury.
Lipid peroxidation refers to the oxidative degradation of lipids.
This process proceeds by free radical chain reaction in which free
radicals steal electrons from the lipids in cell membrane and
consequently damages the cell. It most often affects poly-
unsaturated fatty acids forming malondialdehyde (MDA).
The colorimetric reaction of thiobarbituric acid (TBA) with MDA,
a secondary product of lipid peroxidation (LPO) has been widely
adopted as a sensitive assay method for measuring LPO in animal
tissues. It is used as an index of the extent to which LPO has pro-
gressed. Since the assay procedure estimates the amount of TBA
reactive substances e.g., MDA, it is also referred to as TBARS (Thi-
obarbituric Acid Reactive Substance) test.
Therefore, an attempt was made to correlate the decrease in
ulcerogenic activity of the compounds with that of lipid perox-
idation. All the compounds screened for ulcerogenic activity were
also analyzed for lipid peroxidation using Ohkawa et al. method
[18]. The lipid peroxidation was measured as nmoles of MDA/mg of
protein. Ibuprofen exhibited high lipid peroxidation 0.608 ꢀ 0.001
whereas control group showed 0.238 ꢀ 0.002. It was found that all
the furanone and its benzyl pyrrolones derivatives showed less
ulcerogenic activity along with reduced lipid peroxidation (Table 2).
5.1.1. Preparation of 3-(4-chloro-benzoyl) propionic acid (1)
Succinic anhydride (10 g, 10 mmol) was reacted with dry chlo-
robenzene (50 mL) in presence of anhydrous aluminum chloride
(15 g, 11.25 mmol). The reaction mixture was heated on a heating
mantle for 2 h. It was purified by dissolving in sodium hydroxide
solution, filtering, followed by addition of hydrochloric acid. The
solid mass so obtained was filtered, washed with cold water, dried
and crystallized from methanol to give 65 g (62%) of the desired
compound as a off-white crystalline solid, m.p. 124 ꢂC which gave
effervescence with sodium bicarbonate solution, 1H NMR (CDCl3)
d
2.81 and 3.38 (t each, 2 ꢃ CH2), 7.45, 7.92 (d each, 2 ꢃ A2B2, p-
substituted phenyl).
5.1.2. General procedure for the synthesis of 3-arylidene-5-(4-
chloro-phenyl)-2(3H)-furanones 2–13
5.1.2.1. Method i. A solution of 3-(4-chloro-benzoyl)propionic acid
(3 mmol) and aromatic aldehyde (equimolar, 3 mmol) in acetic
anhydride (15 mL) with triethylamine (3–4 drops) was refluxed for
4 h under anhydrous conditions. After completion of reaction, the
contents were poured into crushed ice in small portions while
stirring. A colored solid mass separated out, which was filtered,
washed with water and crystallized from a mixture of methanol/
chloroform (1:1) to give 2–13.
4. Conclusion
Seventeen new 2(3H)-furanones and their nitrogen analogues
i.e. 1-benzyl-2(3H)-pyrrolones were successfully synthesized. Bio-
logical evaluation results showed that the compounds are prom-
ising anti-inflammatory and analgesic agents with lesser GI toxicity
and reduced lipid peroxidation.1-Benzyl-2(3H)-pyrrolones showed
better anti-inflammatory activity than their parent compounds i.e.
2(3H)-furanones. Similarly, compounds containing halogen
group(s) showed improved anti-inflammatory activity. These
compounds showed superior GI safety profile along with reduction
in lipid peroxidation as compared to that of ibuprofen. The fur-
anone and 1-benzylpyrrolone derivatives discovered in this study
5.1.2.2. Method ii. 3-(4-Chloro-benzoyl)propionic acid (3 mmol)
and aromatic aldehyde (equimolar, 3 mmol) in acetic anhydride (5–
8 drops) were fused together for 5 min then triethylamine (2 drops)
was added and the contents were refluxed for 15 min. A colored
solid mass formed, which was crystallized from a mixture of
methanol/chloroform (1:1) to give 2–13.
5.1.2.3. 3-(Benzylidene)-5-(4-chloro-phenyl)-2(3H)-furanone (2). Yield:
52%, m.p. 194 ꢂC. 1H NMR (CDCl3)
d 6.93 (s, 1H, furanone ring), 7.41 (m,

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M.M. Alam et al. / European Journal of Medicinal Chemistry 44 (2009) 2636–2642
1H, olefinic H), 7.62 and 7.68 (d each, 2 ꢃ A2B2, p-substituted phenyl),
7.41 (m, 5H, H-2,3,4,5,6, arylidene). 13C NMR (CDCl3)
170.6 (C-1), 127.2
p-substituted phenyl). MS: m/z 312(Mþ), 111. IR (cmꢄ1, KBr): 1755,
1597, 844. Anal. Calcd. for C18H13ClO3: C, 69.13; H, 4.19. Found: C,
68.93; H, 4.17.
d
(C-2), 118.3 (C-3), 129.3 (C-4), 98.4 (C-5), 130.0 (C-6), 132.8 (C-7,11),
120.6 (C-8,10), 124.3 (C-9), 153.7 (C-12), 119.8 (C-13,17), 131.0 (C-14,16),
156.6 (C-15). MS: m/z 282(Mþ), 139, 111, 77. IR (cmꢄ1, KBr): 1763, 1602,
836. Anal. Calcd. for C17H10ClO2: C, 72.22; H, 3.92. Found: C, 72.43; H,
3.90.
5.1.2.12. 3-(3,4-Dimethoxy-benzylidene)-5-(4-chloro-phenyl)-2(3H)-
furanone (11). Yield: 64%, m.p. 218 ꢂC. 1H NMR (CDCl3)
d 3.92 (s, 6H,
2 ꢃ OCH3), 6.85 (s, 1H, furanone ring), 6.98 (d, 1H, H-5, arylidene),
7.17 (s, 1H, H-2, arylidene), 7.39 (d, 1H, H-6, arylidene), 7.59 (s, 1H,
olefinic H), 7.45 and 7.74 (d each, 2 ꢃ A2B2, p-substituted phenyl).
MS: m/z 342(Mþ) 139, 111. IR (cmꢄ1, KBr): 1771, 1598, 837. Anal.
Calcd. for C19H15ClO4: C, 66.58; H, 4.41. Found: C, 66.80; H, 4.43.
5.1.2.4. 3-(2-Chloro-benzylidene)-5-(4-chloro-phenyl)-2(3H)-furanone
(3). Yield: 64%, m.p. 220 ꢂC. 1H NMR (CDCl3)
d 6.84 (s, 1H, furanone
ring), 7.46 (s, 1H, olefinic H), 7.44 and 7.67 (d each, 2 ꢃ A2B2,
p-substituted phenyl), 7.64 (m, 4H, H-3,4,5,6, arylidene). MS: m/z
317(Mþ), 139, 111. IR (cmꢄ1, KBr): 1768, 1605, 831. Anal. Calcd. for
C17H10Cl2O2: C, 64.38; H, 3.18. Found: C, 64.20; H, 3.19.
5.1.2.13. 3-(2,4-Dichloro-benzylidene)-5-(4-chloro-phenyl)-2(3H)-
furanone (12). Yield: 52%, m.p. 172 ꢂC. 1H NMR (CDCl3)
d 6.41 (s, 1H,
furanone ring), 7.42 (s, 1H, olefinic H), 7.3 and 7.48 (d each, 2 ꢃ A2B2,
p-substituted phenyl), 7.54 (m, 2H, H-3,5, arylidene), 7.37 (m,1H, H-
6, arylidene). MS: m/z 351(Mþ), 139, 111, 77. IR (cmꢄ1, KBr): 1776,
1611, 833. Anal. Calcd. for C17H9Cl3O2: C, 58.07; H, 2.58. Found: C,
57.26; H, 2.57.
5.1.2.5. 3-(3-Chloro-benzylidene)-5-(4-chloro-phenyl)-2(3H)-furanone
(4). Yield: 60%, m.p. 240 ꢂC. 1H NMR (CDCl3)
d 6.78 (s, 1H, furanone
ring), 7.42 (s, 1H, olefinic H), 7.52 and 7.69 (d each, 2 ꢃ A2B2,
p-substituted phenyl), 7.36 (m, 3H, H-4,5,6, arylidene), 7.62 (s, 1H,
H-2). MS: m/z 317(Mþ), 139, 111, 77. IR (cmꢄ1, KBr): 1773, 1605, 832.
Anal. Calcd. for C17H10Cl2O2: C, 64.38; H, 3.18. Found: C, 64.19; H, 3.16.
5.1.2.14. 3-(4-Acetoxy-3-methoxy-benzylidene)-5-(4-chloro-phenyl)-
2(3H)-furanone (13). Yield: 52%, m.p. 148 ꢂC. 1H NMR (CDCl3)
d 2.34
5.1.2.6. 3-(4-Chloro-benzylidene)-5-(4-chloro-phenyl)-2(3H)-furanone
(s, 3H, OCOCH3), 3.90 (s, 3H, OCH3), 6.67 (s, 1H, furanone ring), 7.32
and 7.64 (d each, 2 ꢃ A2B2, p-substituted phenyl), 7.42 (s,1H, olefinic
H), 7.36 (d, 1H, H-5, arylidene), 7.54 (m, 2H, H-2,6, arylidene). MS:
m/z 370(Mþ),139, 77. IR (cmꢄ1, KBr): 1767,1597, 829. Anal. Calcd. for
C20H15ClO5: C, 64.79; H, 4.08. Found: C, 64.58; H, 4.09.
(5). Yield: 68%, m.p. 284 ꢂC. 1H NMR (CDCl3)
d 6.87 (s, 1H, furanone
ring), 7.45 (s, 1H, olefinic H), 7.55 and 7.69 (d each, 2 ꢃ A2B2,
p-substituted phenyl), 7.41 and 7.53 (d each, 2 ꢃ A2B2, arylidene).
MS: m/z 317(Mþ),139, 77. IR (cmꢄ1, KBr): 1763,1603, 835. Anal. Calcd.
for C17H10Cl2O2: C, 64.38; H, 3.18. Found: C, 64.55; H, 3.20.
5.1.3. General procedure for the synthesis of 3-arylidene-5-(4-
chloro-phenyl)-1-benzyl-2(3H)-pyrrolones (14–18)
5.1.2.7. 3-(2-Nitro-benzylidene)-5-(4-chloro-phenyl)-2(3H)-furanone
(6). Yield: 70%, m.p. 240 ꢂC. 1H NMR (CDCl3)
d
6.60 (s, 1H, furanone
Synthesis of these compounds involved the following two steps:
ring), 7.66 (s, 1H, olefinic H), 7.54 and 7.68 (d each, 2 ꢃ A2B2,
p-substituted phenyl), 7.84 (s, 1H, H-5, arylidene), 8.20 (d, 1H, H-3,
arylidene), 8.43 (m, 2H, H-4,6, arylidene). MS: m/z 327(Mþ), 122, 111,
77. IR (cmꢄ1, KBr): 1769, 1608, 828. Anal. Calcd. for C17H10ClNO4: C,
62.30; H, 3.08; N, 4.27. Found: C, 62.48; H, 3.09; N, 4.26.
ꢁ Synthesis of
g-ketobenzylamide
Furanone (3 mmol) and benzylamine (4 mmol) were refluxed in
dry benzene for 2 h. On completion of reaction, excess benzene was
distilled off and a solid mass so obtained was washed with petro-
leum ether and dried. The compound obtained was used without
crystallization.
5.1.2.8. 3-(3-Nitro-benzylidene)-5-(4-chloro-phenyl)-2(3H)-furanone
(7). Yield: 72%, m.p. 226 ꢂC. 1H NMR (CDCl3)
d 6.67 (s, 1H, furanone
ring), 7.62 (s, 1H, olefinic H), 7.52 and 7.69 (d each, 2 ꢃ A2B2,
p-substituted phenyl), 7.64 (dd,1H, H-5, arylidene), 7.81 (d, 1H, H-6,
arylidene), 8.16 (d, 1H, H-4, arylidene), 8.23 (s, 1H, H-2, arylidene).
MS: m/z 327(Mþ), 139, 122, 77. IR (cmꢄ1, KBr): 1771, 1606, 826. Anal.
Calcd. for C17H10ClNO4: C, 62.30; H, 3.08; N, 4.27. Found: C, 62.12; H,
3.05; N, 4.29.
ꢁ Cyclization of
g-ketobenzylamide
g
-Ketobenzylamide (3 mmol) was refluxed in 6 N hydrochloric
acid (20 mL) for 1 h. The contents were then cooled and a solid
mass so obtained was collected, washed with water and crystal-
lized from methanol to give 14–18.
5.1.2.9. 3-(4-Nitro-benzylidene)-5-(4-chloro-phenyl)-2(3H)-furanone
(8). Yield: 74%, m.p. 264 ꢂC. 1H NMR (CDCl3)
d
6.82 (s, 1H, furanone
5.1.3.1. 3-Benzylidene-5-(4-chloro-phenyl)-1-benzyl-2(3H)-pyrro-
ring), 7.56 (s, 1H, olefinic H), 7.50 and 7.63 (d each, 2 ꢃ A2B2,
p-substituted phenyl), 7.59 and 8.12 (d each, 2 ꢃ A2B2, arylidene).
MS: m/z 327(Mþ), 139, 111. IR (cmꢄ1, KBr): 1765, 1608, 835. Anal.
Calcd. for C17H10ClNO4: C, 62.30; H, 3.08; N, 4.27. Found: C, 62.47; H,
3.10; N, 4.28.
lone (14). Yield: 44%, m.p. 124 ꢂC. 1H NMR (CDCl3)
d 4.82 (s, 2H,
CH2), 6.25 (s, 1H, furanone ring), 7.55 (s, 1H, olefinic H), 7.05 and
7.64 (d each, 2 ꢃ A2B2, p-substituted phenyl), 7.46 (m, 3H, H-3,4,5,
arylidene), 7.62 (m, 2H, H-2,6, arylidene), 7.37 (m, 5H, H-2,3,4,5,6,
benzyl). 13C NMR (CDCl3)
d 173.2 (C-1), 126.9 (C-2), 118.3 (C-3), 127.1
(C-4), 100.0 (C-5), 129.5 (C-6), 132.1 (C-7,11), 119.4 (C-8,10), 124.0 (C-
9), 152.5 (C-12), 124.0 (C-13,17), 131.2 (C-14,16), 155.3 (C-15), 44.6
(C-10), 133.6 (C-20), 128.5 (C-30,70), 129.1 (C-40,60), 119.8 (C-50). MS:
m/z 371(Mþ), 282, 139, 91, 77. IR (cmꢄ1, KBr): 1752, 1613, 805. Anal.
Calcd. for C24H18ClNO: C, 77.52; H, 4.88; N, 3.77. Found: C, 77.24; H,
4.87; N, 3.78.
5.1.2.10. 3-(4-Fluoro-benzylidene)-5-(4-chloro-phenyl)-2(3H)-furanone
(9). Yield: 56%, m.p. 220 ꢂC. 1H NMR (CDCl3)
d 6.87 (s, 1H, furanone
ring), 7.42 (s, 1H, olefinic H), 7.63 and 7.68 (d each, 2 ꢃ A2B2,
p-substituted phenyl), 7.14 (m, 2H, H-3,5, arylidene), 7.42 (m, 3H,
H-2,6, arylidene). MS: m/z 300(Mþ), 139, 77. IR (cmꢄ1, KBr): 1761,
1601, 833. Anal. Calcd. for C17H10ClFO2: C, 67.90; H, 3.35. Found: C,
67.68; H, 3.36.
5.1.3.2. 3-(4-Chloro-benzylidene)-5-(4-chloro-phenyl)-1-benzyl-2(3H)-
pyrrolone (15). Yield: 48%, m.p. 136 ꢂC. 1H NMR (CDCl3)
d 4.82 (s,
5.1.2.11. 3-(4-Methoxy-benzylidene)-5-(4-chloro-phenyl)-2(3H)-fur-
2H, CH2), 6.18 (s, 1H, furanone ring), 7.37 and 7.55 (d each, 2 ꢃ A2B2,
arylidene), 7.46 (s, 1H, olefinic H), 7.04 and 7.31 (d each, 2 ꢃ A2B2, p-
substituted phenyl), 7.24 (m, 5H, H-2,3,4,5,6, benzyl). MS: m/z 406
(Mþ), 315, 111, 91, 77. IR (cmꢄ1, KBr): 1749, 1593, 811. Anal. Calcd. for
anone (10). Yield: 64%, m.p. 214 ꢂC. 1H NMR (CDCl3)
d 3.82 (s, 3H,
OCH3), 6.90 (s, 1H, furanone ring), 6.93 and 7.45 (d each, 2 ꢃ A2B2,
arylidene), 7.42 (s, 1H, olefinic H), 7.50 and 7.67 (d each, 2 ꢃ A2B2,

M.M. Alam et al. / European Journal of Medicinal Chemistry 44 (2009) 2636–2642
2641
C24H17Cl2NO: C, 70.95; H, 4.22; N, 3.45. Found: C, 71.15; H, 4.20; N,
3.43.
individually in the test cage, before acetic acid injection and
habituated for 30 min. Screening of analgesic activity was per-
formed after p.o. administration of test drugs at a dose of 20 mg/
kg. The compounds, which exhibited good anti-inflammatory
activity comparable to that of ibuprofen, were screened for
analgesic activity. All compounds were dissolved in 1% CMC
solution. One group was kept as control and received p.o.
administration of 1% CMC. Ibuprofen was used as reference drug.
After 1 h of drug administration 0.10 mL of 1% acetic acid solution
was given to mice intraperitoneally. Stretching movements con-
sisting of arching of the back, elongation of body and extension of
hind limbs were counted for 5–15 min of acetic acid injection. The
analgesic activity was expressed in terms of percentage
inhibition.
5.1.3.3. 3-(4-Nitro-benzylidene)-5-(4-chloro-phenyl)-1-benzyl-2(3H)-
pyrrolone (16). Yield: 46%, m.p. 200 ꢂC. 1H NMR (CDCl3)
d 6.19 (s,
1H, furanone ring), 7.50 (s, 1H, olefinic H), 7.04 and 7.75 (d each,
2 ꢃ A2B2, p-substituted phenyl), 7.34 and 8.25 (d each, 2 ꢃ A2B2,
arylidene), 7.24 (m, 5H, H-2,3,4,5,6, benzyl). MS: m/z 416 (Mþ), 325,
139, 91, 77. IR (cmꢄ1, KBr): 1739, 1613, 795. Anal. Calcd. for
C24H17ClN2O3: C, 69.15; H, 4.11; N, 6.72. Found: C, 68.97; H, 4.09; N,
6.70.
5.1.3.4. 3-(4-Fluoro-benzylidene)-5-(4-chloro-phenyl)-1-benzyl-2(3H)-
pyrrolone (17). Yield: 48%, m.p. 146 ꢂC. 1H NMR (CDCl3)
d 4.85 (s,
2H, CH2), 6.19 (s, 1H, furanone ring), 7.49 (s, 1H, olefinic H), 7.18 and
7.31 (d each, 2 ꢃ A2B2, p-substituted phenyl), 7.11 and 7.61 (d each,
2 ꢃ A2B2, arylidene), 7.21 (m, 5H, H-2,3,4,5,6, benzyl). MS: m/z 389
(Mþ), 298, 139, 91, 77. IR (cmꢄ1, KBr): 1736, 1606, 813. Anal. Calcd.
for C24H17ClFNO: C, 73.94; H, 4.40; N, 3.59. Found: C, 73.68; H, 4.38;
N, 3.40.
% Analgesic activity ¼ fðn ꢄ n0Þ=ng ꢃ 100
where, n ¼ mean number of writhes of control group, n0 ¼ mean
number of writhes of test group.
5.3.2. Hot-plate test
The experiment was carried out as described by Turner [15]
using hot-plate apparatus, maintained at 53 ꢀ 0.5 ꢂC. Swiss Albino
mice (25–30 g) of either sex in a group of six animals were used.
The mice were divided and received the same doses of tested
compounds and ibuprofen as mentioned before. The reaction time
of the mice to the thermal stimulus was the time interval between
placing the animal on the hot plate and when it licked its hind paw
or jumped. Reaction time was measured prior to aqueous suspen-
sion of synthesized compounds and drug treatment (0 min) and 1 h
after oral administration of the same.
5.1.3.5. 3-(4-Hydroxy-3-methoxy-benzylidene)-5-(4-chloro-phenyl)-
1-benzyl-2(3H)-pyrrolone (18). Yield: 48%, m.p. 150 ꢂC. 1H NMR
(CDCl3) d 3.92 (s, 3H, OCH3), 4.84 (s, 2H, CH2), 5.8 (s, 1H, OH), 6.21 (s,
1H, furanone ring), 7.07 and 7.35 (d each, 2 ꢃ A2B2, p-substituted
phenyl), 6.96 (d,1H, H-6, arylidene), 7.11 (d,1H, H-5, arylidene), 7.21
(s, 1H, H-2, arylidene), 7.49 (s, 1H, olefinic H), 7.24 (m, 5H,
H-2,3,4,5,6, benzyl). MS: m/z 417 (Mþ), 326, 91, 77. IR (cmꢄ1, KBr):
1748, 1606, 815. Anal. Calcd. for C25H20ClNO3: C, 71.85; H, 4.82; N,
3.35. Found: C, 71.67; H, 4.80; N, 3.33.
5.2. Anti-inflammatory activity
5.4. Acute ulcerogenesis
The synthesized compounds were evaluated for their anti-
inflammatory activity using carrageenan-induced paw edema
method of Winter et al. [13]. The experiment was performed on
Albino rats of Wistar strain of either sex, weighing 180–200 g. The
animals were randomly divided into groups of six. Group I was kept
as control, and received only 0.5% carboxymethyl cellulose (CMC)
solution. Groups II was kept as standard and received ibuprofen
(20 mg/kg p.o.). Carrageenan solution (0.1% in sterile 0.9% NaCl
solution) in a volume of 0.1 mL was injected subcutaneously into the
sub-plantar region of the right hind paw of each rat, 30 min after the
administration of the test compounds and standard drugs. The paw
volume was measured by saline displacement shown on screen of
digital Plethysmometer (Ugo Basile) at 2 and 3 h after carrageenan
injection. Thus the edema volume in control group (Vc) and edema
volume in groups treated with test compounds (Vt) was measured
and the percentage inhibition of edema was calculated using the
formula: anti-inflammatoryactivity (% inhibition) ¼ (Vc–Vt)/Vc ꢃ 100.
Acute ulcerogenesis test was done according to Cioli et al. [16].
Albino rats (150–200 g) were divided into different groups con-
sisting of six animals in each group. Ulcerogenic activity evaluated
after p.o. administration of test compounds or ibuprofen at the dose
of 60 mg/kg. Control rats received p.o. administration of vehicle
(suspension of 1% methyl cellulose). Food but not water was
removed 24 h before administration of the test compounds. After
the drug treatment the rats were fed with normal diet for 17 h and
then sacrificed. The stomach was removed and opened along the
greater curvature, washed with distilled water and cleaned gently
by dipping in normal saline. The mucosal damage was examined by
means of a magnifying glass. For each stomach the mucosal damage
was assessed according to the following scoring system: 0.5:
redness, 1.0: spot ulcers, 1.5: hemorrhagic streaks, 2.0: ulcers > 3
but < 5, 3.0: ulcers > 5. The mean score of each treated group minus
the mean score of control group was regarded as severity index of
gastric mucosal damage.
5.3. Analgesic activity
Experimental models used in this study were selected to
investigate both centrally and peripherally mediated analgesic
effects of the tested compounds. For this purpose; the acetic acid
abdominal constriction method was used to elucidate the periph-
eral effect and the hot-plate test to reveal central activity of the
tested compounds.
5.5. Lipid peroxidation
Lipid peroxidation in the gastric mucosa was determined
according to the method of Ohkawa et al. [18]. After screening for
ulcerogenic activity, the gastric mucosa was scraped with two glass
slides and 10% of that tissue was homogenized at 10,000 rpm in
1.8 mL of 1.15% ice-cold KCl solution. 1 mL of suspension medium
was taken from the supernatant, 0.5 mL of 30% trichloroacetic acid
(TCA) followed by 0.5 mL of 0.8% thiobarbituric acid (TBA) reagent
were added to it. The tubes were covered with aluminum foil and
kept in a shaking water bath for 30 min at 80 ꢂC. After 30 min tubes
were taken out and kept in ice-cold water for 10 min. These were
then centrifuged at 3000 rpm for 15 min. The absorbance of
5.3.1. Writhing test
The abdominal constriction in mice (Writhing effect) was
determined by acetic acid induced writhing method [14] in Swiss
Albino mice (25–30 g) of either sex. A 1% aqueous acetic acid
solution (i.p. injection in a volume of 0.1 mL) was used as writhing
induced agent. In each group six mice were kept. Mice were kept

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M.M. Alam et al. / European Journal of Medicinal Chemistry 44 (2009) 2636–2642
0.5
0.45
0.4
charge animal house, Jamia Hamdard, New Delhi, for providing
animals for pharmacological studies.
y = 0.0734x - 0.0047
R2 = 0.9962
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0.35
0.3
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The authors are thankful to Prof. M.S.Y. Khan for valuable
suggestions. Thanks are also due to Dr. (Mrs.) Shaukat Shah, in-