Synthesis and anti-inflammatory, analgesic, ulcerogenic and lipid peroxidation activities of some new 2-[(2,6-dichloroanilino) phenyl]acetic acid derivatives

Article abstract of DOI:10.1016/j.ejmech.2004.02.008

The synthesis of a group of 1,3,4-oxadiazoles, 1,2,4-triazoles, 1,3,4-thiadiazoles and 1,2,4-triazine derived from 2-[(2,6-dichloroanilino) phenyl] acetic acid is described. The structures of new compounds are supported by IR, ¹H-NMR and Mass spectral data. These compounds were tested in vivo for their anti-inflammatory activity. The compounds, which showed activity comparable to the standard drug diclofenac, were screened for their analgesic, ulcerogenic and lipid peroxidation activities. Ten new compounds, out of 28 showed very good anti-inflammatory activity in the carrageenin induced rat paw edema test, with significant analgesic activity in the acetic acid induced writhing test together with negligible ulcerogenic action. The compounds, which showed less ulcerogenic action, also showed reduced malondialdehyde content (MDA), which is one of the byproduct of lipid peroxidation. 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:10.1016/j.ejmech.2004.02.008

European Journal of Medicinal Chemistry 39 (2004) 535–545
www.elsevier.com/locate/ejmech
Short communication
Synthesis and anti-inflammatory, analgesic, ulcerogenic
and lipid peroxidation activities of some new
2-[(2,6-dichloroanilino) phenyl]acetic acid derivatives
Mohd Amir *, Kumar Shikha
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University, Hamdard Nagar, New Delhi 110062, India
Received 31 October 2003; received in revised form 12 February 2004; accepted 26 February 2004
Available online 09 April 2004
Abstract
The synthesis of a group of 1,3,4-oxadiazoles, 1,2,4-triazoles, 1,3,4-thiadiazoles and 1,2,4-triazine derived from 2-[(2,6-dichloroanilino)
phenyl] acetic acid is described. The structures of new compounds are supported by IR, 1H-NMR and Mass spectral data. These compounds
were tested in vivo for their anti-inflammatory activity. The compounds, which showed activity comparable to the standard drug diclofenac,
were screened for their analgesic, ulcerogenic and lipid peroxidation activities. Ten new compounds, out of 28 showed very good
anti-inflammatory activity in the carrageenin induced rat paw edema test, with significant analgesic activity in the acetic acid induced writhing
test together with negligible ulcerogenic action. The compounds, which showed less ulcerogenic action, also showed reduced malondialde-
hyde content (MDA), which is one of the byproduct of lipid peroxidation. 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.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Diclofenac; 1,3,4-Oxadiazole; 1,3,4-Thiadiazole; 1,2,4-Triazole; 1,2,4-Triazine; Anti-inflammatory activity; Analgesic activity; Ulcerogenic
activity; Lipid peroxidation
1. Introduction
appreciable GI irritation, bleeding and ulceration [14]. The
incidence of clinically significant GI side effects due to
NSAIDs is high (30%) and causes some patients to abandon
NSAID therapy [4]. GI damage from NSAID is generally
attributed to two factors. Local irritation by the carboxylic
acid moiety, common to most NSAIDs (topical effect) and
decreased tissue prostaglandin production, which under-
mines the physiological role of cytoprotective prostaglandins
in maintaining GI health and homeostasis [5,15].
Synthetic approaches based upon NSAIDs chemical
modification have been taken with the aim of improving
NSAID safety profile. Studies by others described the deriva-
tization of the carboxylate function [16–18] of representative
NSAID resulted in an increased anti-inflammatory activity
with reduced ulcerogenic effect. Furthermore, it has been
reported in literature that certain compounds bearing 1,3,4-
oxadiazole/thiadiazole and 1,2,4-triazole nucleus possess
significant anti-inflammatory activity [19–23]. In our attempt
to discover new and useful agents for treatment of inflamma-
tory diseases, we have replaced the carboxylic acid group of
diclofenac with additional heterocycles, which have been
Non-steroidal anti-inflammatory drugs (NSAIDs) are
widely used for the treatment of pain, fever and inflamma-
tion, particularly arthritis [1–3]. Among the most popular
NSAIDs, diclofenac has been approved in 120 countries
since its introduction 25 years ago and ranked 30th among
the top 200 drugs with respect to new prescriptions [4].
The pharmacological activity of NSAIDs is related to the
suppression of prostaglandin biosynthesis from arachidonic
acid by inhibiting the enzyme cyclooxygenases (COXs)
[5,6]. Recently, it was discovered that COX exists in two
isoforms, COX-1 and COX-2, which are regulated differ-
ently [7–9]. COX-1 provides cytoprotection in the gas-
trointestinal (GI) tract whereas inducible COX-2 mediates
inflammation [10–12]. Since most of the NSAIDs in the
market show greater selectivity for COX-1 than COX-2 [13],
chronic use of NSAIDs, including diclofenac may elicit
* Corresponding author.
E-mail address: mamir_s2003@yahoo.co.in (M. Amir).
© 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.02.008

536
M. Amir, K. Shikha / European Journal of Medicinal Chemistry 39 (2004) 535–545
found to possess an interesting profile of anti-inflammatory
activity with significant reduction in their ulcerogenic effect.
The heterocycles reported here are 1,3,4-oxadiazoles, 1,2,4-
triazoles, 1,3,4-thiadiazoles and 1,2,4-triazine.
whereas when the 2-amino group was replaced by
2-alkyl/aryl amino group there was sharp increase in the
activity. The compound 8a having n-butyl amino group
showed the greatest activity (84.61%). It was observed that
oxadiazole derivatives having p-methyl, o-methoxy and
p-fluoro phenyl amino group at second position showed
better activity (83.65%, 82.69% and 80.76%) than the stan-
dard drug. Rest of the oxadiazole derivatives showed moder-
ate activity.
The anti-inflammatory activity of 1,2,4-triazole deriva-
tives was found between 63.46% and 82.69%. The highest
activity (82.69%) was found in the triazole derivative 9e
having p-methyl phenyl group at fourth position. When this
group was replaced by n-butyl group 9a, the activity was
found to be decreased (79.80%), whereas in case of oxadia-
zole derivative 8a, the compound having n-butyl group
showed greatest activity. It was observed that triazole deriva-
tive having o-methoxy phenyl 9g and p-fluoro phenyl group
9d, also showed good activity viz. 81.90% and 79.80%,
respectively. Other derivatives showed moderate activity.
The 1,3,4-thiadiazole derivatives of diclofenac showed
anti-inflammatory activity from 79.04% to 82.85%. The
maximum activity (82.85%) was shown by thiadiazole de-
rivative 10d having p-fluoro phenyl amino group at second
position. When this group was replaced by n-butyl amino
group the activity was found to be decreased but equivalent to
the standard drug (80.76%). Furthermore, thiadiazole deriva-
tives having p-methyl phenyl amino 10e, o-methoxy phenyl
amino 10g and p-chloro phenyl amino groups 10c at second
position showed good activity, viz. 80.00%, 80.76% and
79.04%, respectively.
2. Chemistry
The acid hydrazide 2 was prepared by esterification of
2-[(2,6-dichloroanilino) phenyl]acetic acid followed by
treatment with hydrazine hydrate in absolute ethanol. The
reaction of hydrazide 2 with carbon disulphide in alkaline
medium afforded, after acidic treatment, 5-[2-(2,6-dichlo-
roanilino)benzyl]2-mercapto-1,3,4-oxadiazole 3. Various
2-aryl substituted-1,3,4-oxadiazole 5a–d were prepared by
treatment of hydrazide with appropriate aromatic acids in
presence of phosphorus oxychloride. 3-[2-(2,6-Dichlo-
roanilino)benzyl]5-oxo-1,2,5,6-tetrahydro-1,2,4-triazine
6
was prepared by condensation of hydrazide with chloroac-
etamide (Fig. 1).
Furthermore, hydrazide 2 on treatment with various aryl
isothiocyanate
gave
N1-[2-(2,6-dichloroanilino)
phenylacetyl]N4-alkyl/arylthiosemicarbazides 7a–g. The thi-
osemicarbazides were oxidatively cyclised to 2-arylamino-5-
substituted-1,3,4-oxadiazoles 8a–g by elimination of H2S
using iodine and potassium iodide in ethanolic sodium hy-
droxide. The thiosemicarbazides 7a–g on heating with 4 N-
NaOH in ethanol underwent smooth cyclisation through de-
hydration to afford 5-substituted-4-aryl-3-mercapto-4H-
1,2,4-triazoles 9a–g. 2-Arylamino-5-substituted–1,3,4-
thiadiazoles 10a–g were obtained by cyclisation of 7a–g by
treating with cold concentrated sulphuric acid.
When the hydrazide of diclofenac was treated with
2-chloroacetamide, 1,2,4-triazine derivative was obtained,
which also showed anti-inflammatory activity equivalent to
diclofenac (80.76%).
The structures of various compounds synthesized were
assigned on the basis of elemental analysis and spectral data.
The IR, 1H-NMR and Mass spectral data of compounds are
given in experimental protocols. Physical data for the com-
pounds are given in Table 1.
3.2. Analgesic activity
1,3,4-Oxadiazole derivatives 8a, 8d, 8e and 8g showed
analgesic activity ranging from 78.57% to 81.86%, better
than the standard drug diclofenac (70.32%). p-Fluoro phenyl
amino group, present at the second position on oxadiazole
ring 8d, showed the maximum activity (81.86%). When this
group was replaced by n-butyl 8a and o-methoxy phenyl 8g,
the activity was found to be slightly decreased (81.31%).
When these groups were replaced by p-methyl phenyl amino
group 8e, the activity was further decreased (78.57%). The
result shows that an electron-withdrawing group increases
the analgesic activity of the compound.
When 1,3,4-oxadiazole nucleus was replaced by 1,2,4-
triazole nucleus, the analgesic activity was found to be de-
creased. Two triazole derivatives 9e and 9g screened, showed
72.52% and 73.62% analgesic activity, respectively. 1,3,4-
Thiadiazole derivatives 10a, 10d and 10g were also screened
and showed 77.47%, 76.92% and 72.52% analgesic activity,
respectively. The compound having p-fluoro phenyl amino
3. Pharmacological results and discussion
3.1. Anti-inflammatory activity
The anti-inflammatory activity of the synthesized com-
pounds 3, 4, 5b, 5d, 6, 8a, 8b, 8d, 8e, 8f, 8g, 9a, 9b, 9d, 9e,
9g, 10a, 10c, 10d, 10e, 10g was evaluated by carrageenan
induced paw edema method of Winter et al. [27]. The com-
pounds were tested at 10 mg/kg oral dose and were compared
with the standard drug diclofenac at the same oral dose. The
tested compounds showed anti-inflammatory activity rang-
ing from 44.05% to 84.61% (Table 2), whereas the standard
drug diclofenac showed 80.76% inhibition after 4 h. The
anti-inflammatory activity of 1,3,4-oxadiazole derivatives is
in the range from 44.05% to 84.61%. When the 2-amino
group of the oxadiazole nucleus 4 was replaced by a
2-mercapto group 3, the activity was found to be decreased,

M. Amir, K. Shikha / European Journal of Medicinal Chemistry 39 (2004) 535–545
537
COOH
NH
Cl
Cl
Absolute ethanol
Conc.H2SO4
O
OC2H5
NH
Cl
Cl
(1)
NH2NH2.H2O
N
N
O
N
N
NHNH2
NH2
SH
O
Cl
O
Cl
CS2/KOH
NH
NH
CNBr
NH
Cl
Cl
Cl
Cl
R'-COOH
POCl3
2-Chloroacetamide
(2)
(4)
Cl
(3)
O
N
R-NCS
O
R'
N
H
N
H
NH
(6)
N
N
Cl
NH
Cl
Cl
S
H
N
R
N
H
N
H
O
(5a-d)
NH
Cl
Cl
H2SO4
KI / I2
NaOH
(7a-g)
4N NaOH
N
N
R
N
N
R
O
Cl
S
NH
NH
Cl
Cl
Cl
N
N
SH
N
R
Cl
(8a-g)
(10a-g)
NH
Cl
(9a-g)
Fig. 1. Synthetic pathways to oxadiazole (3, 4, 5a-d, 8a-g), triazine (6), triazole (9a-g) and thiadiazole (10a-g) derivatives of diclofenac.
10d and n-butyl amino group 10a at second position of
thiadiazole derivative showed slight difference in their activ-
ity. When these groups were replaced by o-methoxy phenyl
amino group 10g there was decrease in the activity.
Cyclisation of carboxyl group of diclofenac into a 1,2,4-
triazine nucleus showed 79.12% analgesic activity, which
was better than 1,2,4-triazole and 1,3,4-thiadiazole deriva-
tives.

538
M. Amir, K. Shikha / European Journal of Medicinal Chemistry 39 (2004) 535–545
Table 1
Physical data of 2-[(2,6-dichloroanilino)phenyl]acetic acid derivatives
N
N
N
N
S
H
R
SH
R
N
X
N
R
N
H
N
H
NH
NH
O
NH
Cl
Cl
Cl
Cl
Cl
Cl
7a-g
9a-g
X= O (3,4,5,8); S (10)
Compound
R
Yield (%)
MP (°C)
240
Molecular formula
Molecular weight
3
4
5a
–SH
79
74
68
C
C
C
15H11ON3SCl2
15H12ON4Cl2
21H14ON3Cl3
352.24
335.20
430.72
–NH2
>360
190
Cl
5b
5c
5d
70
76
71
120
70
C
C
C
21H13ON3Cl4
22H15O2N3Cl4
21H16ON4Cl2
465.17
495.20
411.29
Cl
Cl
Cl
H2CO
Cl
295
NH2
7a
7b
–CH2–CH2–CH2–CH3
74
82
190
178
C
C
19H22ON4SCl2
21H24ON4SCl2
425.30
451.42
7c
7d
7e
7f
80
78
68
65
174
140
176
146
C
C
C
C
21H17ON4SCl3
21H17ON4SCl2F
22H20ON4SCl2
22H20ON4SCl2
479.82
463.36
459.40
459.40
Cl
F
CH3
H3C
7g
72
150
C
22H20O2N4SCl2
475.40
H3CO
8a
8b
–NH–CH2–CH2–CH2–CH3
52
40
280
220
C
C
19H20ON4Cl2
21H22ON4Cl2
391.30
417.34
NH
(continued on next page)

M. Amir, K. Shikha / European Journal of Medicinal Chemistry 39 (2004) 535–545
539
Table 1
(continued)
Compound
R
Yield (%)
65
MP (°C)
>360
Molecular formula
Molecular weight
445.74
8c
C
C
C
C
21H15ON4Cl3
21H15ON4Cl2F
22H18ON4Cl2
22H18ON4Cl2
NH
NH
NH
Cl
8d
8e
8f
47
33
55
80
429.28
425.32
425.32
F
300
320
CH3
H3C
NH
8g
46
260
C
22H18O2N4Cl2
441.32
H3CO
NH
9a
9b
–CH2–CH2–CH2–CH3
84
78
168
220
C
C
19H20N4SCl2
21H22N4SCl2
407.37
433.41
9c
9d
9e
9f
80
86
79
80
290
188
222
240
C
C
C
C
21H15N4SCl3
21H15N4SCl2F
22H18N4SCl2
22H18N4SCl2
461.80
445.35
441.39
441.39
Cl
F
CH3
H3C
9g
75
246
C
22H18ON4SCl2
457.38
H3CO
10a
10b
–NH–CH2–CH2–CH2–CH3
28
32
Semisolid
Semisolid
C
C
19H20N4SCl2
21H22N4SCl2
407.37
433.41
NH
10c
10d
44
56
120
124
C
C
21H15N4SCl3
461.80
445.35
NH
NH
Cl
F
21H15N4SCl2F
(continued on next page)

540
M. Amir, K. Shikha / European Journal of Medicinal Chemistry 39 (2004) 535–545
Table 1
(continued)
Compound
R
Yield (%)
48
MP (°C)
Molecular formula
Molecular weight
441.39
10e
Semisolid
C
22H18N4SCl2
NH
CH3
10f
33
37
Semisolid
60
C
22H18N4SCl2
441.39
457.38
H3C
NH
10g
C
22H18ON4SCl2
H3CO
NH
Satisfactory analysis for C,H,N was obtained for all the compounds within 0.4% of the theoretical values.
3.3. Acute ulcerogenesis
minimum ulcerogenic activity (0.500
0.00 and
0.517 0.11, respectively). Moreover their anti-
The compounds, which showed anti-inflammatory activ-
ity comparable to that of the standard drug diclofenac and
also showed high analgesic activity, were screened for their
ulcerogenic activity.
inflammatory activity was found to be high viz. 83.65% and
82.69%, respectively. The other two oxadiazole derivatives
8a and 8d also showed reduction in the ulcerogenic activity
(0.666 0.10 and 0.583 0.08, respectively) in comparison
to the standard drug.
The tested compounds showed significant reduction in
ulcerogenic activity ranging from 0.417
0.08 to
The triazole derivatives 9e and 9g also showed reduction
in ulcerogenic activity (0.583 0.08 and 0.517 0.11). It was
interesting to note that the compound 8g and 9g having
oxadiazole and triazole nucleus showed the same severity
index (0.517 0.11). 1,3,4-Thiadiazole derivative showed
minimum ulcerogenic activity, when compared to oxadiazole
1.50 0.00, whereas the standard drug diclofenac showed
high severity index of 2.416 0.20. The ulcerogenic activity
of 1,3,4-oxadiazole derivatives 8a, 8d, 8e and 8g ranges from
0.50 0.00 to 0.666 0.10. Compound 8e and 8g having
p-methyl phenyl and o-methoxy phenyl amino group showed
Table 2
Biological data of diclofenac derivatives
Compound
Anti-inflammatory activity Analgesic activity
Ulcerogenic activity
nmol MDA content S.E.M.)/
(% inhibition S.E.M.)
–
(% inhibition S.E.M.)
(severity index S.E.M.)
100 mg tissue
Control
Diclofenac
3
4
5b
5d
6
8a
8b
8d
8e
8f
8g
9a
9b
9d
9e
–
0.000 0.00
3.371 0.01
80.76 3.71 a
44.05 3.76 c
69.23 3.71 a
77.14 2.20 b
61.76 5.61 c
80.76 2.43 a
84.61 1.00 a
68.26 2.31 a
80.76 1.96 a
83.65 1.84 a
54.28 5.76 a
82.69 1.65 a
79.80 1.84 a
63.46 2.38 a
79.80 1.28 a
82.69 1.65 a
81.90 1.27 b
80.76 1.98 a
79.04 1.64 b
82.85 1.65 b
80.00 2.22 a
80.76 1.18 b
70.32 1.31 b
2.416 0.20
9.155 0.14
–
–
–
–
–
–
–
–
–
–
–
–
79.12 0.58 b
1.500 0.00 a
5.928 0.06 b
81.31 0.85 b
0.666 0.10 b
6.110 0.06 b
–
–
–
81.86 0.92 b
0.583 0.08 b
5.715 0.14 b
78.57 0.85 b
0.500 0.00 b
5.833 0.19 b
–
–
–
81.31 0.85 b
0.516 0.11 b
5.742 0.17 b
–
–
–
–
–
–
–
–
–
72.52 1.00 b
73.62 1.16 b
77.47 0.85 b
–
0.583 0.08 b
0.517 0.11 b
0.417 0.08 b
–
5.876 0.13 b
5.854 0.14 b
5.788 0.10 b
–
9g
10a
10c
10d
10e
10g
76.92 0.58 b
–
72.52 1.00 b
0.500 0.00 b
–
0.500 0.00 b
5.808 0.02 b
–
5.904 0.14 b
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. diclofenac. Data were analyzed by Student’s t-test for n = 6.
aP < 0.001; b P < 0.0001; c P <0.01.

M. Amir, K. Shikha / European Journal of Medicinal Chemistry 39 (2004) 535–545
541
and triazole derivatives. The compound 10a having n-butyl
amino group showed the minimum severity index
cyclohexyl amino and o-methyl phenyl amino group de-
creases the anti-inflammatory activity. It was further noted
that the presence of o-methoxy and p-methyl phenyl group at
fourth position of triazole nucleus increases the anti-
inflammatory activity than the standard drug, whereas pres-
ence of n-butyl group and p-fluoro phenyl group showed
anti-inflammatory activity slightly less than the standard
drug.
These compounds tested for ulcerogenic activity showed
significant decrease in the activity than the standard drug. It
was noted that thiadiazole derivatives showed maximum
reduction in the ulcerogenic activity followed by triazoles
and oxadiazoles. It was further concluded that presence of
o-methoxy phenyl amino and p-methyl phenyl amino at the
second position of oxadiazole and thiadiazole nucleus
showed maximum anti-inflammatory activity, minimum ul-
cerogenic activity along with minimum lipid peroxidation.
(0.417
0.83). The other two compounds 10d and 10g
having p-fluoro phenyl amino and o-methoxy phenyl amino
group showed the same severity index (0.500 0.00).
Cyclisation of carboxyl group of diclofenac into 1,2,4-
triazine nucleus also showed reduction in ulcerogenic activ-
ity (1.500 0.00). Thus it was concluded that cyclisation of
carboxylic group of diclofenac into oxadiazole, triazole, thia-
diazole and triazine nucleus resulted in the significant de-
crease of ulcerogenic activity while retaining their high anti-
inflammatory properties.
3.4. Lipid peroxidation
It has been reported in literature that compounds show-
ing less ulcerogenic activity also showed reduced malondi-
aldehyde (MDA) content, a byproduct of lipid peroxidation
5. Experimental protocols
[24,25]. 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 peroxida-
tion.
The lipid peroxidation is measured as nmol of
MDA/100 mg of tissue. The diclofenac (standard drug)
5.1. Chemistry
Melting points were measured in open capillary tubes and
are uncorrected. IR (KBr) spectra were recorded on a Perkin
Elmer 157 infracord spectrometer (mmax in cm–1) and H-
1
NMR spectra on a Bruker DRX-300 (300 MHz FT NMR)
spectrophotometer using TMS as internal reference (Chemi-
cal shift d in 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 visu-
alising agent. 2-[(2,6-Dichloroanilino)phenyl]acetic acid
(Diclofenac) was procured from Pharmax Corporation Pvt.,
New Delhi, India.
showed the maximum lipid peroxidation (9.155
0.14),
whereas the control group showed 3.370 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 com-
pounds have inhibited the induction of gastric mucosal le-
sions and the results further suggested that their protective
effect might be related to the inhibition of lipid peroxidation
in the gastric mucosa.
5.1.1. Ethyl-[2-(2,6-dichloroanilino)phenyl]acetate (1)
It was prepared by the procedure given in literature [26].
4. Conclusion
5.1.2. [2-(2,6-Dichloroanilino)phenyl]acetic acid
hydrazide (2)
Various oxadiazoles, triazoles, thiadiazoles and triazine
derivatives of 2-[(2,6-dichloroanilino)phenyl]acetic acid
were prepared with the objective of developing better anti-
inflammatory molecules with minimum ulcerogenic activ-
ity. It was interesting to note that six cyclised compounds
8a, 8e, 8g, 9e, 9g and 10d were found to have anti-
inflammatory activity greater than the standard drug (di-
clofenac, 80.76%) at 10 mg/kg p.o. Furthermore, four com-
pounds 6, 8d, 10a and 10g exhibited anti-inflammatory
activity equivalent to the standard drug against carrageenin
induced paw edema test in rats. When these compounds
were subjected to the analgesic activity, against acetic acid
induced writhing test in mice, showed increased activity
than their reference drug.
Compound 1 (0.01 mol) and hydrazine hydrate (0.02 mol)
were refluxed in absolute ethanol (50 ml) for 20 h. The
mixture was concentrated, cooled and poured in ice cold
water. The solid thus separated out was filtered, dried and
recrystallized from ethanol, yield: 72%; m.p. 134–136 °C. IR
spectra of the compound showed bands at 3325 (N–H); 2970
(C–H); 1638 (C=O). 1H-NMR (CDCl3, d ppm): 3.59 (s, 2H,
CH2CO); 4.14 (s, 2H, NH2); 6.82–6.98 (m, 4H, 3,4,5,6ArH);
7.21–7.27 (m, 3H, dichloro ArH); 7.54 (s, 1H, NH); 8.27 (s,
1H, CONH). Mass spectra of compound exhibited molecular
ion peak at m/z 309 (M+), other important fragments were
observed at 310 (M+ + 1), 311 (M+ + 2), 313 (M+ + 4), 278,
250, 214.
The presence of o-methoxy phenyl amino, p-methyl phe-
nyl amino, p-fluoro phenyl amino and n-butyl amino group at
second position in the oxadiazole and thiadiazole nucleus
increases the anti-inflammatory activity whereas presence of
5.1.3. 5-[2-(2,6-Dichloroanilino)benzyl]2-mercapto-1,3,4-
oxadiazole (3)
A mixture of 2 (0.005 mol), KOH (0.005 mol) and carbon-
disulphide (5 ml) in ethanol (50 ml) was refluxed on a steam

542
M. Amir, K. Shikha / European Journal of Medicinal Chemistry 39 (2004) 535–545
bath for 12 h. The solution was then concentrated, cooled and
acidified with dilute HCl. The solid mass that separated out
was filtered, washed with ethanol, dried and recrystallized
from ethanol (Table 1). IR spectra of the compound showed
(C–N). 1H-NMR (CDCl3, d ppm): 2.81 (s, 2H, CH2 of triazi-
none); 3.96 (s, 2H, CH2); 6.39–6.46 (bs, 2H, 2NH); 6.98–
7.50 (m, 7H, ArH); 8.02 (s, 1H, NH).
1
5.1.7. General procedure for the preparation
bands at 3354 (N–H); 1525 (C–N); 1182 (C=S). H-NMR
of
arylthiosemicarbazides (7a–g)
N1-[2-(2,6-dichloroanilino)phenyl
acetyl]N4-alkyl/
(CDCl3, d ppm): 4.13 (s, 2H, CH2); 7.25–7.30 (m, 4H,
3,4,5,6 ArH); 7.51–7.53 (m, 3H, dichloro ArH); 7.90 (s, 1H,
NH); 10.51 (s, 1H, SH).
A mixture of 2 (0.10 mol), alkyl/aryl isothiocyanate
(0.10 mol) and ethanol (50 ml) was refluxed on steam bath
for 8 h. It was then concentrated, cooled and kept overnight in
refrigerator. The solid thus separated out, was filtered,
washed with ethanol, dried and recrystallized from ethanol
(Table 1). IR spectra of the compounds 7a–g showed bands at
3300–3280 (N–H); 2960–3000 (CH); 1680–1670 (C=O);
1217–1190 (C=S). Mass spectra of compound 7a exhibited
molecular ion peak at m/z 424 (M+), other important frag-
ments were 425 (M+ + 1), 426 (M+ + 2), 428 (M+ + 4), 278,
214 and 148. 1H-NMR of 7b (CDCl3, d ppm): 0.99–1.88 (m,
11H, C6H11); 3.72 (s, 2H, CH2–CO); 6.40–6.45 (d, 1H,
cyclohexyl-NH); 6.82–7.04 (m, 4H, 3,4,5,6-Ar–H); 7.25–
7.28 (m, 3H, dichloro Ar–H); 7.61 (s, 1H, NH); 8.57 (s, 1H,
CSNH); 9.64 (s, 1H, CONH).
5.1.4.
5-[2-(2,6-Dichloroanilino)benzyl]2-amino-1,3,4-
oxadiazole (4)
To an ethanolic solution of 2 (0.001 mol), cyanogen bro-
mide (0.001 mol) was added. The reaction mixture was
warmed at 55–60 °C for 90 min. The resulting solution was
cooled and neutralized with sodium bicarbonate solution.
The solid thus separated out was filtered, washed with water,
dried and recrystallized from methanol (Table 1). IR spectra
of the compound showed bands at 3447 (NH2); 2975 (C–H);
1
1431 (C–N). H-NMR (DMSO-d6, d ppm): 4.15 (s, 2H,
CH2); 6.20 (s, 2H, NH2); 6.73–7.20 (m, 4H, 3,4,5,6 ArH);
7.55–7.67 (m, 3H, dichloro ArH); 7.82 (s, 1H, NH). Mass
spectra of compound exhibited molecular ion peak at m/z 335
(M+), other important fragments were observed at 337 (M+ +
2), 339 (M+ + 4), 289, 237.
5.1.8. General procedure for the preparation of 5-[2-(2,6-
dichloroanilino)benzyl]2-alkyl/arylamino-
1,3,4-oxadiazoles (8a–g)
5.1.5. General procedure for the preparation of 5-[2-(2,6-
dichloroanilino)benzyl]2-aryl-1,3,4-oxadiazoles (5a–d)
Compound 2 (0.001 mol) and appropriate aromatic acid
(0.001 mol) was dissolved in phosphorus oxychloride and
refluxed for 20 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 recrystallized
from ethanol (Table 1). IR spectra of the compound 5a–d
showed bands at 3450–3430 (N–H); 2970–2950 (CH2);
1490–1450 (C–N). 1H-NMR of 5b (CDCl3, d ppm): 3.71 (s,
2H, CH2); 6.65 (s, 1H, NH); 7.10–7.23 (m, 4H, 3,4,5,6ArH);
7.28–7.43 (m, 6H, dichloro ArH). 1H-NMR of 5c (CDCl3, d
ppm): 3.80 (s, 2H, CH2); 4.54 (s, 2H, OCH2); 6.67 (s, 1H,
NH); 7.06–7.20 (m, 4H, 3,4,5,6 Ar–H); 7.32–7.52 (m, 6H,
dichloro ArH). Mass spectra of compound 5c exhibited mo-
lecular ion peak at m/z 495 (M+), other important fragments
were observed at 497 (M+ + 2), 499 (M+ + 4), 295 and 246.
1H-NMR of 5d (CDCl3, d ppm): 2.88 (s, 2H, CH2); 6.67–
6.94 (m, 4H, 3,4,5,6Ar–H); 7.03 (s, 2H, NH2); 7.16–7.39 (m,
4H, 2,3,5,6 Ar–H); 7.44–7.78 (m, 4H, dichloro ArH and
1NH).
A suspension of 7a–g (0.002 mol) in ethanol (50 ml) was
dissolved in aqueous sodium hydroxide (5 N, 1 ml) with
cooling and stirring, resulting in a clear solution. To this,
iodine in potassium iodide solution (5%) was added gradu-
ally with stirring till the colour of iodine persisted at room
temperature. The reaction mixture was then refluxed for 1 h
on steam bath. It was then cooled and poured over crushed
ice. The solid mass that separated out was filtered, dried and
recrystallized from ethanol (Table 1). IR spectra of the com-
pound 8a–g showed bands at 3440–3420 (N–H); 2985–2965
1
(CH2); 1441–1425 (C–N). H-NMR of 8a (DMSO-d6, d
ppm): 0.82–0.84 (t, 3H, CH3), 1.14–1.17 (m, 2H, CH3CH2);
1.52–1.58 (m, 2H, CH3CH2CH2); 3.94–3.99 (m, 2H,
NHCH2); 4.21 (s, 2H, CH2); 6.20–6.25 (t, 1H, NHCH2);
6.58–7.24 (m, 4H, 3,4,5,6 ArH); 7.42–7.55 (m, 3H, dichloro
ArH); 8.51 (s, 1H, NH). Mass spectra of compound 8a
exhibited molecular ion peak at m/z 391 (M+), other impor-
tant fragments were at 393 (M+ + 2), 395 (M+ + 4), 376,
289 and 176. 1H-NMR of 8b (DMSO-d6, d ppm): 1.13–1.69
(complex m, 11H, cyclohexyl H); 3.94 (s, 2H, CH2); 6.86–
6.90 (m, 4H, 3,4,5,6-ArH); 7.10–7.29 (m, 3H, dichloroArH);
7.39 (s, 1H, cyclohexyl NH); 7.41 (s, 1H, NH). 1H-NMR of
8d (CDCl3, d ppm): 4.40 (s, 2H, CH2); 7.40–7.56 (complex
m, 12H, 11ArH and 1NH); 8.61 (s, 1H, NH). 1H-NMR of 8e
(DMSO-d6, d ppm): 2.39 (s, 3H, CH3); 3.39 (s, 2H, CH2);
6.59–7.51 (complex m, 12H, 11ArH and 1NH); 8.47 (s, 1H,
NH). 1H-NMR of 8f (DMSO-d6, d ppm): 2.27 (s, 3H, CH3);
3.36 (s, 2H, CH2); 6.59–7.54 (complex m, 12H, 11ArH and
1NH); 8.50 (s, 1H, NH). 1H-NMR of 8g (DMSO-d6, d ppm):
3.69 (s, 3H, OCH3); 4.01 (s, 2H, CH2); 6.58–7.45 (complex
m, 12H, 11ArH and 1NH); 8.90 (s, 1H, NH).
5.1.6. 3-[2-(2,6-Dichloroanilino)benzyl]5-oxo-1, 2,5,6-
tetra-hydro-1, 2,4-triazine (6)
To compound 2 (0.01 mol) was added chloroacetamide
(0.01 mol) and dimethyl formamide (80 ml) and the reaction
mixture was refluxed for 25 h. It was then concentrated and
cooled, whereupon the solid separated out, which was fil-
tered, washed with ethanol and recrystallized from
DMF/water, yield: 54%; m.p. 176 °C. IR spectra of the
compound showed bands at 3385 (N–H); 1640 (C=O); 1474

M. Amir, K. Shikha / European Journal of Medicinal Chemistry 39 (2004) 535–545
543
1
5.1.9. General procedure for the preparation of 5-[2-(2,6-
dichloro-anilino)benzyl]4-alkyl/aryl-3-mercapto-4H-1,2,4-
triazoles (9a–g)
1NH), 8.30 (s, 1H, NH). H-NMR of 10d (CDCl3, d ppm);
3.80 (s, 2H, CH2), 6.98–7.61 (complex m, 12H, 11ArH and
1NH), 8.32 (s, 1H, NH). 1H-NMR of 10e (DMSO d6, d ppm);
2.21 (s, 3H, CH3); 4.16 (s, 2H, CH2), 6.86–7.41 (complex m,
A suspension of 7a–g (0.002 mol) in ethanol (25 ml) was
dissolved in aqueous sodium hydroxide (4 N, 2 ml) and
gently refluxed for 2 h. The resulting solution was concen-
trated, cooled and filtered. The filtrate was adjusted to pH
5–6 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 com-
pounds 9a–g showed bands at 3375–3360 (N–H); 2960–
2926 (CH2); 1450–1435 (C–N); 1220–1199 (C=S). 1H-NMR
of 9a (CDCl3, d ppm): 0.90–0.97 (t, 3H, CH3); 1.34–1.46 (m,
2H, CH3CH2); 1.61–1.72 (m, 2H, CH3CH2CH2); 3.98–4.03
(t, 2H, NCH2); 4.14 (s, 2H, C6H4–CH2); 6.93–7.03 (m, 4H,
3,4,5,6 ArH); 7.13–7.19 (m, 3H, dichloro ArH); 7.26 (s, 1H,
1
12H, 11ArH and 1NH), 7.55 (s, 1H, NH). H-NMR of 10g
(DMSO d6, d ppm); 3.82 (s, 2H, CH2), 3.86 (s, 3H, OCH3),
6.35–7.81 (complex m, 12H, 11Ar–H and 1NH), 8.28 (s, 1H,
NH). Mass spectra of compound 10g exhibited molecular ion
peak at m/z 456 (M+), other important fragments were found
at 458 (M+ + 2), 460 (M+ + 4), 424, 350 and 107.
5.2. Pharmacology
The compounds 3, 4, 5b, 5d, 6, 8a, 8b, 8d, 8e, 8g, 9a, 9d,
9e, 9f, 9g, 10a, 10c, 10d, 10e and 10g were evaluated for
their anti-inflammatory activity. Diclofenac, the parent com-
pound was used as a reference drug. The experiments were
performed on albino rats ofWistar strain of either sex, weigh-
ing 180–200 g. The animals were maintained at 25 2 °C,
50 5% relative humidity, 12 h light/dark cycle. Food and
water were freely available upto the time of experiments. The
test compounds were dissolved in 1% carboxy methyl cellu-
lose (CMC) solution.
1
NH); 11.46 (s, 1H, SH). H-NMR of 9b (CDCl3, d ppm):
0.85–1.85 (m, 11H, C6H11); 4.21 (s, 2H, CH2); 6.96–7.04 (m,
4H, 3,4,5,6 Ar–H); 7.13–7.35 (m, 4H, dichloro ArH and
1
1NH); 10.53 (s, 1H, SH). H-NMR of 9c (CDCl3, d ppm):
3.97 (s, 2H, CH2); 6.44–6.82 (m, 4H, 3,4,5,6 ArH); 6.98–
7.11 (m, 4H, p-Cl phenyl ArH); 7.20–7.34 (m, 3H, dichloro
ArH); 7.55 (s, 1H, NH); 11.51 (s, 1H, SH). Mass spectra of
compound 9c exhibited molecular ion peak at m/z 460 (M+),
other important fragments were found at 462 (M+ + 2), 464
(M+ + 4), 364, 251 and 129. 1H-NMR of 9d (CDCl3, d ppm):
3.95 (s, 2H, CH2); 6.46–6.85 (m, 4H, 3,4,5,6 ArH); 6.90–
7.12 (m, 4H, p-F phenyl ArH); 7.22–7.38 (m, 3H, dichloro
5.2.1. Anti-inflammatory activity
This activity was performed by the following procedure of
Winter et al. [27] on groups of six animals each. A freshly
prepared suspension of carrageenin (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 1 h before the carrageenin treatment. The
volume was measured before and after 4 h of carrageenin
treatment with the help of pleythysmometer. The percent
anti-inflammatory activity was calculated according to the
formula given below:
1
ArH); 7.56 (s, 1H, NH); 11.51 (s, 1H, SH). H-NMR of 9e
(CDCl3, d ppm): 1.95 (s, 3H, CH3); 3.82 (s, 2H, CH2);
6.27–6.83 (m, 4H, ArH); 6.91–7.13 (m, 4H, 3,4,5,6 ArH);
7.30–7.43 (m, 3H, dichloroArH); 8.21 (s, 1H, NH); 10.81 (s,
1H, SH). 1H-NMR of 9f (CDCl3, d ppm): 1.81 (s, 3H, CH3);
3.82 (s, 2H, CH2); 6.24–6.81 (m, 4H, ArH); 6.93–7.18 (m,
4H, 3,4,5,6 ArH); 7.31–7.45 (m, 3H, dichloro ArH); 8.08 (s,
1
1H, NH); 10.82 (s, 1H, SH). H-NMR of 9g (DMSO-d6, d
ppm): 3.69 (s, 2H, CH2); 3.84 (s, 3H, OCH3); 6.54–6.71 (m,
4H, ArH); 6.95–7.20 (m, 4H, 3,4,5,6 Ar-H); 7.43–7.51 (m,
4H, dichloro ArH and 1NH); 10.83 (s, 1H, SH).
% Anti-inflammatory activity = (Vc – Vt/Vc) × 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., Student’s t-test was
applied to determine the significance of the difference be-
tween the control group and rats treated with the test com-
pounds. The difference in results was considered significant
when P < 0.01.
5.1.10. General procedure for the preparation
of
5-[2-(2,6-dichloroanilino)benzyl]2-alkyl/arylamino-
1,3,4-thiadiazoles (10a–g)
The thiosemicarbazide 7a–g (0.002 mol) was added
gradually with stirring to ice cold concentrated sulphuric acid
(10 ml) and the reaction mixture was further stirred for 4 h in
an ice bath. It was then poured over crushed ice and solid thus
separated was filtered, washed with water and recrystallized
5.2.2. Analgesic activity
1
from methanol (Table 1). H-NMR of 10a (DMSO-d6, d
The acetic acid induced writhing test [28] 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 individually in the test cage, before acetic
acid injection and habituated for 30 min. Screening of anal-
gesic activity was performed after p.o. administration of test
ppm): 1.16–1.21 (t, 3H, CH3), 1.23–1.28 (m, 2H, CH3CH2);
1.39–1.48 (m, 2H, CH3CH2CH2); 3.26–3.35 (m, 2H,
NHCH2); 4.15 (s, 2H, CH2); 6.86–6.90 (t, 1H, NH–CH2);
7.10–7.25 (m, 4H, 3,4,5,6 ArH); 7.39–7.41 (m, 3H, dichloro
ArH); 8.23 (s, 1H, NH). H-NMR of 10c (CDCl3, d ppm);
3.79 (s, 2H, CH2), 6.95–7.57 (complex m, 12H, 11ArH and
1

544
M. Amir, K. Shikha / European Journal of Medicinal Chemistry 39 (2004) 535–545
drugs at the dose of 10 mg/kg. The compounds, which exhib-
ited good anti-inflammatory activity comparable to that of
diclofenac, 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.
Diclofenac 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 consisting
of arching of the back, elongation of body and extension of
hind limbs were counted for 5–15 min of acetic acid injec-
tion. The analgesic activity was expressed in terms of %
inhibition. % Analgesic activity = (n – n′/n) × 100 where
n = mean number of writhes of control group and n′ = mean
number of writhes of test group.
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 spectrophotom-
eter. The results were expressed as nmol MDA/100 mg tis-
sue, using extinction coefficient 1.56 × 105 cm–1 M–1.
Data are expressed as mean S.E.M., Student’s t-test was
applied to determine the significance of the difference be-
tween the standard group and rats treated with the test com-
pounds. The difference in results was considered significant
when P < 0.01.
Data are expressed as mean S.E.M., Student’s t-test was
applied to determine the significance of the difference be-
tween the control group and rats treated with the test com-
pounds. The difference in results was considered significant
when P < 0.01.
Acknowledgements
5.2.3. Acute ulcerogenesis
The authors are thankful to head of department, pharma-
ceutical chemistry for providing laboratory facilities and
Central Drug Research Institute (CDRI) 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 pharmacological studies. One of
the author (KS) is grateful to the University Grant Commis-
sion (UGC) New Delhi, for providing financial assistance.
Acute ulcerogenesis test was done according to Cioli et al.
[29]. Albino rats have been divided into different groups
consisting of six animals in each group. Ulcerogenic activity
was evaluated after p.o. administration of test compounds or
diclofenac at the dose of 30 mg/kg. Control rats received p.o.
administration of vehicle (suspension of 1% methyl cellu-
lose). Food but not water was removed 24 h before adminis-
tration of the test compounds. After the drug treatment, the
rats were fed normal diet for 17 h and then sacrificed. The
stomach was removed and opened along the greater curva-
ture, washed with distilled water and cleaned gently by dip-
ping in saline. The mucosal damage was examined by means
of a magnifying glass. For each stomach, the mucosal dam-
age was assessed according to the following scoring system:
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