Anti-inflammatory and gastro sparing activity of some new indomethacin derivatives

Article abstract of DOI:10.1002/ardp.200400891

Indomethacin is a non-steroidal anti-inflammatory drug but its use is associated with high degree of gastric toxicity therefore it is prescribed only in severe conditions. In order to reduce the gastric toxicity of indomethacin, various oxadiazole, triazole, thiadiazole and triazine derivatives have been synthesized. Out of thirteen cyclized derivatives, eleven were screened for anti-inflammatory activity by Winter et al. method. Four compounds showed highly significant activity and were further tested for analgesic, ulcerogenic and lipid peroxidation activities. The tested compounds showed anti-inflammatory activities in the range from about 32% to 85% as compared to that of indomethacin of about 96%. The compounds showing high anti-inflammatory activity also exhibited reduction in severity index. These compounds also produced less malondialdehyde content in gastric mucosa than the standard drug indomethacin. The study showed that the compounds inhibited the induction of gastric mucosal lesions and it can be suggested from our results that their protective effects may be related to inhibition of lipid peroxidation in the gastric mucosa.

Full text of DOI:10.1002/ardp.200400891

24
DOI 10.1002/ardp.200400891
Arch. Pharm. Chem. Life Sci. 2005, 338, 24−31
Anti-inflammatory and Gastro Sparing Activity of
Some New Indomethacin Derivatives
Mohd. Amir* and Shikha Kumar
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University, Hamdard Nagar,
New Delhi, India
Indomethacin is a non-steroidal anti-inflammatory drug but its use is associated with high degree of
gastric toxicity therefore it is prescribed only in severe conditions. In order to reduce the gastric toxicity
of indomethacin, various oxadiazole, triazole, thiadiazole and triazine derivatives have been synthesi-
zed. Out of thirteen cyclized derivatives, eleven were screened for anti-inflammatory activity by Winter
et al. method. Four compounds showed highly significant activity and were further tested for analgesic,
ulcerogenic and lipid peroxidation activities. The tested compounds showed anti-inflammatory activi-
ties in the range from about 32% to 85% as compared to that of indomethacin of about 96%. The
compounds showing high anti-inflammatory activity also exhibited reduction in severity index. These
compounds also produced less malondialdehyde content in gastric mucosa than the standard drug
indomethacin. The study showed that the compounds inhibited the induction of gastric mucosal le-
sions and it can be suggested from our results that their protective effects may be related to inhibition
of lipid peroxidation in the gastric mucosa.
Keywords: Indomethacin; Anti-inflammatory; Analgesic; Ulcerogenic; Lipid peroxidation
Received: April 7, 2004; Accepted: November 11, 2004 [FP891]
Introduction
intestinal tract [13]. It has been suggested that oxygen free
radicals may also play an important role in the pathogenesis
of tissue injury [14]. Furthermore, lipid peroxidation me-
diated by oxygen free radicals is believed to be an important
cause of destruction and damage to cell membranes, be-
cause polyunsaturated fatty acids of the cellular membranes
are degraded by the lipid peroxidation with consequent dis-
ruption of membrane integrity [15]. Synthetic approaches
based upon NSAIDs chemical modification have been taken
with the aim of improving NSAID safety profile. Studies by
others described the derivatization of the carboxylate func-
tion [8, 16, 17] of representative NSAID, resulted in an in-
creased anti-inflammatory activity with reduced ulcerogenic
effect. Furthermore, certain compounds bearing 1,3,4-oxa-
diazole/thiadiazole and 1,2,4-triazole nucleus have been re-
ported with significant anti-inflammatory activity [18Ϫ22].
In our attempt to discover new and useful agents for treat-
ment of inflammatory diseases we have replaced the car-
boxylic acid group of indomethacin with additional hetero-
cycles, which have been found to possess an interesting pro-
file of anti-inflammatory activity with significant reduction
in their ulcerogenic effect along with reduced lipid peroxi-
dation. The heterocycles reported here are 1,3,4-oxadi-
azoles, 1,2,4-triazoles, 1,3,4-thiadiazoles and 1,2,4-triazine.
Non-steroidal anti-inflammatory drugs (NSAIDs) are used
as anti-inflammatory agents but their use often causes gas-
tric erosions and ulcers, which are among the most serious
clinical problems [1, 2]. NSAID induced gastric damage has
been attributed to suppression of endogenous prostaglan-
dins, direct toxic effect on gastric epithelial cells such as
apoptosis or necrosis [3]. The inhibition of prostaglandin
synthesis, which has a cytoprotective effect on gastric mu-
cosa, was thought to be the major mechanism of gastroin-
testinal problems with NSAIDs [4]. The pharmacological
activity of NSAIDs is related to the suppression of prostag-
landin biosynthesis from arachidonic acid by inhibiting the
enzyme cyclooxygenases (COXs) [5, 6]. Recently, it was dis-
covered that COX exists in two isoforms, COX-1 and COX-
2, which are regulated differently [7, 8]. COX-1 provides
cytoprotection in the gastrointestinal (GI) tract whereas in-
ducible COX-2 mediates inflammation [9, 10]. Since most
of the NSAIDs in the market show greater selectivity for
COX-1 than COX-2 [11], chronic use of NSAIDs may elicit
appreciable GI irritation, bleeding and ulceration [12].
Indomethacin,
a non-steroidal anti-inflammatory drug
(NSAID), is known to induce erosions and ulcers in gastro-
Correspondence: Mohd. Amir, Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, Hamdard University, Hamdard
Nagar, New Delhi 110062, India. Phone: ϩ91 11 26059688, Ext. 5626,
5624, Fax: ϩ91 11 26059663, e-mail: mamirϪs2003@yahoo.co.in
Chemistry
The acid hydrazide 2 was prepared by esterification
of 1-(4-chlorobenzoyl)-5-methoxy-2-methyl indole-3-acetic
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

acid followed by treatment with hydrazine hydrate in abso-
lute ethanol. The reaction of hydrazide 2 with carbon disul-
phide in alkaline medium afforded, after acidic treatment,
5-[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-indol-3-yl-
methyl]-2-mercapto-1,3,4-oxadiazole 3. Treatment of hydra-
zide with cyanogen bromide afforded 5-[1-(4-chloroben-
zoyl)-5-methoxy-2-methyl-indol-3-yl-methyl]-2-amino-
1,3,4-oxadiazole 4. Various 2-aryl-1,3,4-oxadiazoles 5-8
were prepared by treatment of hydrazide with appropriate
aromatic acids in presence of phosphorus oxychloride. 3-[1-
(4-Chlorobenzoyl)-5-methoxy-2-methyl-indol-3-yl-methyl]-
1,2,5,6-tetrahydro-1,2,4-triazin-5-one 9 was prepared by
condensation of hydrazide with chloroacetamide.
When the carboxylic group of indomethacin was converted
into 1,2,4-triazole nucleus having an n-butyl group 14 at 4th
position, significant activity was found (76.34%), whereas
compound 15 having cyclohexyl group showed a sharp de-
crease in the activity (56.60%). The cyclization of carboxylic
group of indomethacin into 1,3,4-thiadiazole nucleus also
showed significant anti-inflammatory activity. The com-
pound 16 having n-butyl amino group at 2nd position of
thiadiazole nucleus showed maximum activity (84.70%)
among all the tested compounds. The activity was decreased
when n-butyl group was replaced by cyclohexyl group 17
(70.58%). Further modification of carboxylic group into 6-
membered heterocyclic ring, 1,2,4-triazin-5-one 9 also ex-
hibited highly significant anti-inflammatory activity (84.61%).
Furthermore, hydrazide 2 on treatment with n-butyl/cyclo-
hexylisothiocyanate gave N1-[1-(4-chlorobenzoyl)-5-meth-
oxy-2-methylindol-3-yl-acetyl]N4-n-butyl/cyclohexyl thio-
semicarbazides 10Ϫ11. The thiosemicarbazides were oxida-
tively cyclized to 2-n-butyl/cyclohexylamino-5-substituted-
1,3,4-oxadiazoles 12Ϫ13 by elimination of H2S using iodine
and potassium iodide in ethanolic sodium hydroxide. The
thiosemicarbazides 10Ϫ11 on heating with 4N-NaOH in
ethanol underwent smooth cyclisation through dehydration
to afford 5-substituted-4-n-butyl/cyclohexyl-3-mercapto-
1,2,4(H)-triazoles 14Ϫ15. 2-n-Butyl/cyclohexylamino-5-
substitutedϪ1,3,4-thiadiazoles 16Ϫ17 were obtained by
cyclization of 10Ϫ11 by treating with cold concentrated sul-
furic acid (Figure 1). The structures of various compounds
synthesized were assigned on the basis of elemental analysis
as well as IR, 1H-NMR and mass spectral data. Physical
data for the compounds are given in Table 1.
Few selected compounds 4, 9, 14, 16, which showed around
80% anti-inflammatory activities were tested for their anal-
gesic activity. 1,3,4-Thiadiazole derivative of indomethacin
16 showed maximum activity (76.37%) followed by triazole
14 and triazine 9 derivatives. It was noted that the oxadi-
azole derivative 4 showing maximum anti-inflammatory
activity showed minimum analgesic activity.
The compounds, which were screened for analgesic activity,
were further screened for their ulcerogenic activity. All the
compounds were tested at 20 mg/kg oral dose. The tested
compounds showed a significant reduction in ulcerogenic
activity ranging from 0.500 0.00 to 1.583 0.08, whereas
oral administration of indomethacin at 20 mg/kg induced
heavy gastric damage (4.500
0.18). The maximum re-
duction in ulcerogenic activity (0.500 0.00) was found in
1,3,4-thiadiazole derivative 16 having n-butyl amino group
whereas the minimum reduction in severity index was found
in triazine 9 and triazole 14 derivatives (1.583 0.08). The
oxadiazole derivative 4 also showed reduction in ulcerogenic
activity (1.333 0.17) in comparison to standard reference
drug indomethacin.
Biological Studies
The anti-inflammatory activities of the synthesized com-
pounds 3Ϫ6, 9, and 12Ϫ17 were evaluated by carrageenan
induced paw edema method of Winter et al. The com-
pounds were tested at 10-mg/kg oral dose and were com-
pared with the standard drug indomethacin. The tested
compounds showed anti-inflammatory activity ranging
from 31.76 to 84.70% (Table 2), whereas standard drug in-
domethacin showed 95.57% inhibition after 4 hours. The
anti-inflammatory activity of 1,3,4-oxadiazole derivatives
was found in the range from 31.76% to 80.58%. The oxadi-
azole derivative 4 having an amino group showed the maxi-
mum activity (80.58%). When this group was replaced by
n-butyl amino group 12, there was slight decrease in the
activity (76.34%). The replacement of n-butyl amino group
by a cyclohexyl amino group 13 further reduced the activity
(64.71%). The substitution of amino group by a p-chloro-
phenyl group 5 also showed significant anti-inflammatory
activity, whereas replacement of this by 2,4-dichlorophenyl
group 6 resulted in slight reduction in the activity. The mini-
mum activity was observed in the compound 3 having a
mercapto group at 2nd position of the oxadiazole nucleus.
It has been reported in literature that compounds showing
less ulcerogenic activity also showed reduced malondial-
dehyde (MDA) content, a byproduct of lipid peroxidation
[23, 24]. Therefore, an attempt was made to correlate the
decrease in ulcerogenic activity of the compounds with that
of lipid peroxidation. All the compounds screened for ul-
cerogenic activity were also analyzed for lipid peroxidation.
The lipid peroxidation is measured as nmol of MDA/100
mg of tissue. The indomethacin (standard drug) showed the
maximum lipid peroxidation (13.162
0.38), whereas the
control group showed 3.371 0.01. It was found that all
the cyclised derivatives showing less ulcerogenic activity also
showed reduction in lipid peroxidation (Table 2). Thus these
studies showed that synthesized compounds have inhibited
the induction of gastric mucosal lesions and the results
further suggested that their protective effect might be re-
lated to the inhibition of lipid peroxidation in the gastric
mucosa.

26
Amir et al.
Arch. Pharm. Chem. Life Sci. 2005, 338, 24−31
Figure 1. Synthetic pathways to oxadiazole (3, 4, 5Ϫ8, 12Ϫ13), triazine (9), triazole (14Ϫ15) and thiadiazole (16Ϫ17)
derivatives of indomethacin.
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Table 1. Physical data of indomethacin derivatives.
Compound
R
Yield (%)
M.P. (°C)
Mol. formula
Mol. Wt.
-SH
3
4
5
73
67
60
>300
>300
C20H16N3O3SCl
C20H17N4O3Cl
C26H19N3O3Cl2
413
396
491
-NH2
300Ϫ302
6
7
64
67
>300
130
C25H18N3O3Cl3
C26H20N3O4Cl3
525
555
8
50
90
84
46
48
63
72
52
53
295
162Ϫ164
198
C26H21N4O3Cl
C24H27N4O3SCl
C26H29N4O3SCl
C24H25N4O3Cl
C26H27N4O3Cl
C24H25N4O2SCl
C26H27N4O2SCl
C24H25N4O2SCl
C26H27N4O2SCl
472
486
512
452
478
468
494
468
494
10
11
12
13
-CH2-CH2CH2-CH3
-NH-CH2-CH2-CH2-CH3
CH2-CH2-CH2-CH3
>300
>300
14
15
130
110
16
17
-NH-CH2-CH2-CH2-CH3
220Ϫ222
196Ϫ200
Satisfactory analysis for C,H,N was obtained for all the compounds within 0.4% of the theoretical values.
In conclusion, various oxadiazoles, triazoles, thiadiazoles
and triazine derivatives of indomethacin were prepared with
the objective of developing better anti-inflammatory mol-
ecules with minimum ulcerogenic activity. It was interesting
to note that 2-amino-1,3,4-oxadiazole derivative of indo-
methacin showed significant anti-inflammatory activity.
The effect of alkyl and cycloalkyl (n-butyl and cyclohexyl)
groups on oxadiazole, thiadiazole and triazole derivatives of
indomethacin were also studied and was noted that intro-
duction of n-butyl group increases the activity in all the

28
Amir et al.
Arch. Pharm. Chem. Life Sci. 2005, 338, 24−31
Table 2. Biological data of indomethacin derivatives.
Compound
Anti-inflammatory
activity
(Inhibition S.E.M)
[%]
Analgesic
activity
(Inhibition S.E.M)
[%]
Ulcerogenic
activity
(Severity index S.E.M)
[nmol MDA
content S.E.M)/
100 mg tissue]
Control
Indomethacin
Ϫ
Ϫ
0.000 0.00†
4.500 0.18
3.371 0.01†
13.162 0.38
95.57 0.64†
31.76 6.38#
80.58 2.48†
74.70 5.61†
70.58 2.63†
84.61 2.22†
76.34 1.84†
64.70 7.07‡
76.34 1.83†
56.60 3.25†
84.70 0.81†
70.58 2.63†
71.42 1.53†
3
Ϫ
Ϫ
Ϫ
4
34.06 1.00‡
1.333 0.17†
8.237 0.32†
5
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
6
9
53.29 0.71†
1.583 0.08†
9.647 0.38†
12
13
14
15
16
17
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
73.62 0.58†
1.583 0.08†
9.824 0.37†
Ϫ
Ϫ
76.37 0.58†
Ϫ
Ϫ
0.500 0.00†
Ϫ
8.606 0.23†
Anti-inflammatory and analgesic activities of the test compounds were compared w.r.t. control. Ulcerogenic and lipid peroxidation were
†
‡
#
compared w.r.t. standard drug i.e. indomethacin. P < 0.0001; P<0.001; P < 0.005; Data were analyzed by student’s t-test for n ϭ 6.
agent. 1-(4-Chlorobenzoyl)-5-methoxy-2-methylindole-3-acetic acid
derivatives, whereas introduction of cyclohexyl group de-
(indomethacin) was procured from Agarwal Pharmaceuticals, New
creases the activity. The four cyclized compounds 4, 9, 14,
Delhi, India as a gift sample. All chemicals used were LR and AR
grade and were obtained from S. D. Fine Chem. Ltd., Central Drug
and 16 having significant anti-inflammatory activity were
tested for ulcerogenic activity and showed significant re-
duction in the severity index than the standard reference
drug. From these studies, compound 16, a thiadiazole de-
rivative has emerged as the lead compound, which showed
maximum reduction in ulcerogenic activity and lipid peroxi-
dation.
House Pvt. Ltd. and Merck Limited New Delhi, India.
Chemistry
Ethyl-[1-(4-chlorobenzoyl)-5-methoxy-2-methylindole-3]-ace-
tate (1)
It was prepared by the procedure given in literature [25].
Acknowledgments
1-(4-Chlorobenzoyl)-5-methoxy-2-methylindol-3-yl acetic acid hydra-
zide (2)
The authors are thankful to head of department, pharma-
ceutical chemistry for providing laboratory facilities and
Central Drug Research Institute (C.D.R.I) for spectral
analysis of the compounds. Authors are also thankful to
Mrs. Shaukat Shah, in-charge animal house for providing
Wistar strain rats and Swiss albino mice for pharmacologi-
cal studies. One of the authors (SK) is grateful to the Uni-
versity Grants Commission (U.G.C) New Delhi, for provid-
ing financial assistance.
Compound 1 (0.01 mol) and hydrazine hydrate (0.2 mol) were re-
fluxed in absolute ethanol (50 ml) for 30 h. The mixture was concen-
trated, cooled and poured in crushed ice in small portions while
stirring and kept for 3Ϫ4h at room temperature. The solid thus
separated out was filtered, dried and crystallized from ethanol, m.p.
140Ϫ142°C, yield 52%, IR: 3310 (N-H); 2998 (C-H); 1662 (CϭO)
1
cmϪ1. H-NMR (DMSO-d6): 2.49 (s, 3H, CH3); 3.47 (s, 3H, CH2);
3.84 (s, 3H, OCH3); 4.49 (s, 2H, NH2); 7.49Ϫ7.52 (d, 3H, methoxy
ArH); 7.80Ϫ7.83 (d, 4H, p-Cl ArH); 9.83 (s, 1H, CONH).
5-[1-(4-Chlorobenzoyl)-5-methoxy-2-methylindol-3-yl)-meth-
yl]-2-mercapto-1,3,4-oxadiazole (3)
Experimental
A mixture of 2 (0.005 mol), KOH (0.005 mol) and carbondisulphide
(5 ml) in ethanol (50 ml) was refluxed on a steam bath for 12 h.
The solution was then concentrated, cooled and acidified with di-
luted HCl. The solid mass that separated out was filtered, washed
with ethanol, dried and crystallized from ethanol (Table 1). IR spec-
tra of the compound showed bands at 2926 (C-H); 1682 (CϭN);
General
Melting points were measured in open capillary tubes and are
uncorrected. IR (KBr) spectra were recorded on a Nicolet, 5PC
FT-IR spectrometer and 1H-NMR spectra on a Bruker DRX-300
(300 MHz FT NMR) spectrophotometer using TMS as internal
reference (Chemical shift in δ, ppm). Mass spectra were recorded at
Jeol SX-102 (FAB) spectrometer. Purity of the compounds was
checked on silica gel G plates using iodine vapours as visualising
1120 (CϭS) cmϪ1 1H-NMR (DMSO-d6): 2.33 (s, 3H, CH3); 3.63
.
(s, 2H, CH2); 3.84 (s, 3H, OCH3); 7.14Ϫ7.41 (m, 7H, ArH); 7.78 (s,
1H, SH).
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

5-[1-(4-Chlorobenzoyl)-5-methoxy-2-methylindol-3-yl)-meth-
yl]-2-amino-1,3,4-oxadiazole (4)
stirring, resulting of a clear solution. To this, iodine in potassium
iodide solution (5%) was added gradually with stirring till the color
of iodine persisted at room temperature. The reaction mixture was
then refluxed for 3Ϫ4 h on water bath. It was then cooled and
poured over crushed ice. The solid mass that separated out was
filtered, dried and crystallized from ethanol (Table 1). IR spectra of
the compound 12Ϫ13 showed bands at 3451-3426 (N-H); 2930-2920
To an ethanolic solution of 2 (0.001 mol), cyanogen bromide (0.001
mol) was added. The reaction mixture was stirred with heating at
55Ϫ60°C for 2h. The resulting solution was cooled and neutralized
with sodium bicarbonate solution. The solid thus separated out was
filtered, washed with water, dried and crystallized from methanol
(Table 1). IR spectra of the compound showed bands at 3447 (N-
H); 2926 (C-H); 1620 (CϭN) cmϪ1. 1H-NMR (DMSO-d6): 2.50 (s,
3H, CH3); 3.21 (s, 2H, CH2); 3.71 (s, 3H, OCH3); 3.99 (s, 2H, NH2);
7.61Ϫ7.63 (d, 3H, methoxy ArH); 7.78Ϫ7.80 (d, 4H, chloro ArH).
Mass spectra exhibited molecular ion peak at m/z 396 (Mϩ), other
peaks were obtained at m/z 353, 312, 173, 119.
(C-H); 1653Ϫ1632 (CϭN) cmϪ1
.
1H NMR (DMSO-d6) 12:
0.69Ϫ0.77 (t, 3H, CH2CH3); 1.07Ϫ1.09 (m, 2H, CH3CH2);
1.41Ϫ1.44 (m, 2H, CH3CH2CH2); 2.50 (s, 3H, CH3); 3.25 (s, 2H,
CH2); 3.41 (s, 3H, OCH3); 3.96Ϫ3.98 (m, 2H, CH2-NH); 7.52Ϫ7.59
(m, 8H, 7ArH 1NH). Mass spectra of compound 13 does not exhi-
bit molecular ion peak at m/z 478 (Mϩ), other important fragments
were found at m/z 395 (Mϩ-C6H11), 339, 338, 312, 212 and 166.
General procedure for the synthesis of 5-([1-(4-chlorobenzoyl)-5-
methoxy-2-methylindol-3-yl)-methyl]-2-aryl-1,3,4-oxadiazoles (5Ϫ8)
General procedure for the synthesis of 5-[(1-(4-chloroben-
zoyl)-5-methoxy-2-methylindol-3-yl)-methyl]-4-n-butyl/cyclo-
hexyl-3-mercapto-1, 2,4(H)-triazoles (14Ϫ15)
Compound 2 (0.001 mol) and appropriate aromatic acid (0.001 mol)
was dissolved in phosphorus oxychloride and refluxed for 20Ϫ25 h.
The reaction mixture was slowly poured over crushed ice and kept
overnight. The solid thus separated out was filtered, washed with
water, dried and crystallized from ethanol (Table 1). IR spectra of
the compound 5-8 showed bands at 2930Ϫ2910 (C-H) and 1658-
1625 (CϭN) cmϪ1. In case of 8, there was an additional N-H
A suspension of 10Ϫ11 (0.002 mol) in ethanol (25 mL) was dis-
solved in aqueous sodium hydroxide (4n, 2 mL) and gently refluxed
for 6Ϫ7 h. The resulting solution was concentrated, cooled and fil-
tered. The filtrate was adjusted to pH 5Ϫ6 with dilute acetic acid
and was kept aside for 1 h. The crystals produced were filtered,
washed with water, dried and recrystallized from ethanol (Table 1).
IR spectra of the compounds 14Ϫ15 showed bands at 2928-2924
stretching at 3424 cmϪ1 1H-NMR (CDCl3) 7: 2.19 (s, 3H, CH3);
.
3.77 (s, 2H, CH2); 3.85 (s, 3H, OCH3); 4.70 (s, 2H, OCH2);
6.70Ϫ8.18 (m, 10H, ArH).
(CH); 1625Ϫ1610 (CϭN); 1097Ϫ1090 (CϭS) cmϪ1 1H NMR
.
(DMSO-d6) 14: 0.71Ϫ0.76 (t, 3H, CH2CH3); 1.05Ϫ1.17 (m, 2H,
CH3CH2); 1.43Ϫ1.53 (m, 2H, CH3CH2CH2); 2.49 (s, 3H, CH3);
3.44 (merged singlets, 5H, CH2ϩOCH3); 3.99Ϫ4.04 (t, 2H, N-CH2);
7.61Ϫ7.64 (d, 3H, methoxy ArH); 7.69Ϫ7.71 (d, 4H, p-Cl-ArH);
8.20 (s, 1H, SH). Mass spectra of compound 14 exhibited molecular
ion peak at m/z 468 (Mϩ), other important fragments were found
3-[1-(4-Chlorobenzoyl)-5-methoxy-2-methylindol-3-yl)-meth-
yl]-1,2,5,6-tetrahydro-1,2,4-triazin-5-one (9)
To compound 2 (0.01 mol) was added chloroacetamide (0.01 mol)
and dimethyl formamide (80 mL) and the reaction mixture was re-
fluxed for 30 h. It was then concentrated and cooled, whereupon
the solid separated out, was filtered, washed with ethanol and crys-
tallized from DMF / water, m.p. 182°C, yield 23%, IR spectra of
the compound showed bands at 3387 (N-H); 2918 (C-H); 1653 (Cϭ
1
at m/z 435 and 312. H NMR (DMSO-d6) 15: 1.20Ϫ1.90 (m, 11H,
cyclohexyl); 2.50 (s, 3H, CH3); 3.40 (merged singlets, 5H, CH2
ϩ
OCH3); 7.25Ϫ7.32 (d, 3H, methoxy ArH); 7.87Ϫ7.96 (d, 4H, p-Cl-
ArH); 8.30 (s, 1H, SH).
O); 1583 (CϭN) cmϪ1
.
General procedure for the synthesis of 5-[(1-(4-chlorobenzoyl)-
5-methoxy-2-methylindol-3-yl)-methyl]-2-n-butyl/cyclohexyl-
amino-1, 3,4-thiadiazoles (16Ϫ17)
General procedure for the synthesis of N1-[(1-(4-chlorobenzoyl)-5-
methoxy-2-methylindol-3-yl)-acetyl]N4- n-butyl/cyclohexyl thiosemi-
carbazides (10Ϫ11)
The thiosemicarbazide 10Ϫ11 (0.002 mol) was added gradually with
stirring to cold concentrated sulphuric acid (10 mL) during 10 min.
The reaction mixture was further stirred for another 3Ϫ4 h in ice
bath. It was then poured over crushed ice with stirring. The solid
so separated was filtered, washed with water, dried and recrys-
tallized with methanol (Table 1). The IR spectra of compounds
16Ϫ17 showed bands at 3414Ϫ3410 (N-H); 2930Ϫ2928 (C-H);
A mixture of 2 (0.10 mol), n-butyl/cyclohexyl isothiocyanate (0.10
mol) and ethanol (50 mL) was refluxed on steam bath for 10 h. It
was then concentrated, cooled and kept overnight in refrigerator.
The solid thus separated out, was filtered, washed with ethanol,
dried and crystallized from ethanol (Table 1). IR spectra of the com-
pounds 10Ϫ11 showed bands at 3289Ϫ3280 (N-H); 2959Ϫ2948 (C-
H); 1672Ϫ1664 (CϭO); 1098Ϫ1093 (CϭS) cmϪ1
.
1H-NMR
1630Ϫ1624 (CϭN) cmϪ1 1H NMR (DMSO-d6) 16: 0.88Ϫ0.93 (t,
.
(DMSO-d6) 10: 0.83-0.88 (t, 3H, CH2CH3); 1.18Ϫ1.31 (m, 2H,
CH3CH2); 1.41Ϫ1.51 (m, 2H, CH3CH2CH2); 2.48 (s, 3H, CH3);
3.30 (s, 3H, CH2CO); 3.32 (s, 2H, OCH3); 3.40Ϫ3.45 (m, 2H, CH2-
NH); 7.55Ϫ7.58 (d, 3H, methoxy ArH); 7.90Ϫ7.93 (d, 4H, p-Cl
ArH); 8.06Ϫ8.10 (t, 1H, CH2-NH); 9.22 (s, 1H, CSNH); 10.36 (s,
IH, CONH). 1H-NMR (DMSO-d6) 11: 1.01Ϫ2.10 (m, 11H, cyclo-
hexyl); 2.50 (s, 3H, CH3); 3.30 (s, 3H, CH2CO); 3.40 (s, 2H, OCH3);
7.50Ϫ7.67 (d, 3H, methoxy ArH); 7.90Ϫ8.00 (d, 4H, p-Cl ArH);
8.20 (s, 1H, NH); 9.30 (s, 1H, CSNH); 10.40 (s, 1H, CONH). Mass
spectra of compound 11 exhibited molecular ion peak at m/z 512
(Mϩ), 429, 340, 312, 281, 172, 141, 119.
3H, CH3CH2); 1.35Ϫ1.37 (m, 2H, CH3CH2); 1.55Ϫ1.57 (m, 2H,
CH3CH2CH2); 2.50 (s, 3H, CH3); 3.40 (s, 2H, CH2); 3.45 (s, 3H,
OCH3); 4.00Ϫ4.10 (m, 2H, NH-CH2); 7.52Ϫ7.95 (t, 1H, NH-CH2);
7.76Ϫ7.79 (d, 3H, methoxy ArH); 7.92Ϫ7.95 (d, 4H, p-Cl-ArH).
Mass spectra of 16 does not exhibit molecular ion peak at m/z 468
(Mϩ), other important fragments were found at m/z 425 (Mϩ-
C3H7), 344, 266, 139.
Biological evaluation
The experiments were performed on albino rats of Wistar strain of
either sex, weighing 180Ϫ200 g. The animals were maintained at 25
2°C, 50 5% relative humidity, 12 h light/ dark cycle. Analgesic
activity was carried out on Swiss albino mice of either sex, weighing
35Ϫ45 g. Food and water were freely available upto the time of
experiments. The test compounds were dissolved in 1% carboxy
methyl cellulose (CMC) solution.
General procedure for the synthesis of 5-[(1-(4-chlorobenzoyl)-
5-methoxy-2-methylindol-3-yl)-methyl]-2-n-butyl/cyclohexyl
amino-1,3,4-oxadiazoles (12Ϫ13)
A suspension of 10Ϫ11 (0.002 mol) in ethanol (50 mL) was dis-
solved in aqueous sodium hydroxide (5n, 1 mL) with cooling and

30
Amir et al.
Arch. Pharm. Chem. Life Sci. 2005, 338, 24−31
Ant-iinflammatory activity
The mean score of each treated group minus the mean score of
control group was regarded as severity index of gastric mucosal
damage. Data are expressed as mean S.E.M., the student’s t-test
was applied to determine the significance of the difference between
the standard group and rats treated with the test compounds.
This activity was performed by the following procedure of Winter
et al. [26] on groups of six animals each. A freshly prepared suspen-
sion of carrageenan (1.0% w/v, 0.1 mL) was injected in the planter
region of right hind paw of each rat. One group was kept as control
and the animals of the other group were pretreated with the test
drugs suspended in 1.0% CMC given orally 1h before the carra-
geenan treatment. The volume was measured before and after 4h of
carrageenan treatment with the help of pleythysmometer. The per-
cent anti-inflammatory activity was calculated according to the for-
mula given below:
Lipid peroxidation
Lipid peroxidation in the gastric mucosa was determined according
to the method of Ohkawa et al. [29]. After screening for ulcerogenic
activity, the gastric mucosa was scrapped with two glass slides,
weighed (100 mg) and homogenized in 1.8 mL of 1.15% ice cold
KCl solution. The homogenate was supplemented with 0.2 mL of
8.1% sodium dodecyl sulfate (SDS), 1.5 mL of acetate buffer (pH
3.5) and 1.5 mL of 0.8% thiobarbituric acid (TBA). The mixture
was heated at 95°C for 60 min. After cooling, the reactants were
supplemented with 5 mL of the mixture of n-butanol: pyridine (15:1
v/v), shaken vigorously for 1 min and centrifuged for 10 min at 4000
rpm. The supernatant organic layer was taken out and absorbance
was measured at 532 nm on UV spectrophotometer. The
results were expressed as nmol MDA/100 mg tissue, using extinction
% Anti-inflammatory activity ϭ (Vc-Vt / Vc) ϫ 100
Where, Vt represents the mean increase in paw volume in rats
treated with test compounds and Vc represents the mean increase
in paw volume in control group of rats.
Data are expressed as mean S.E.M., the student’s t-test was ap-
plied to determine the significance of the difference between the
control group and rats treated with the test compounds.
coefficient 1.56 ϫ 105 cmϪ1 MϪ1
.
Analgesic activity
Data are expressed as mean S.E.M., the student’s t-test was ap-
plied to determine the significance of the difference between the
standard group and rats treated with the test compounds.
The acetic acid induced writhing test [27] was performed by an i.p.
injection of 1% aqueous acetic acid solution in a volume of 0.1 mL.
In each group six albino mice were kept. Mice were kept individu-
ally in the test cage, before acetic acid injection and habituated for
30 min. Screening of analgesic activity was performed after p.o. ad-
ministration of test drugs at the dose of 10 mg/kg. The compounds,
which exhibited good anti-inflammatory activity comparable to that
of indomethacin, were screened for analgesic activity. All com-
pounds were dissolved in 1% CMC solution. One group was kept
as control and received p.o. administration of 1% CMC. Indometh-
acin was used as reference drug. After 1 h of drug administration
0.10 mL of 1% acetic acid solution was given to mice intra-
peritoneally. Stretching movements consisting of arching of the
back, elongation of body and extension of hind limbs were counted
for 5Ϫ15 min of acetic acid injection. The analgesic activity was
expressed in terms of% inhibition.
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