Synthesis and investigation of anti-inflammatory activity of novel nitric oxide donating hybrid drugs

Article abstract of DOI:10.1007/s00044-012-0345-y

A small library of nitric oxide donating groups, 4-acetamidophenyl-2-[{2- (nitrooxy)ethyl}(phenyl) amino]benzoate (5a-e) possessing a variety of substituents (-H, -NO₂, -CH₃, -acetamidophenyl, -SO ₂NH₂) attached to the fourth position of phenyl ring were synthesized and evaluated for anti-inflammatory, analgesic and ulcerogenic potential. Structure-activity relationship data showed that the 2-phenylaminobenzoic acid skeleton is required for selective COX-2 inhibition. Among all compounds 4-acetamidophenyl-2-[{2-(nitrooxy)ethyl}(phenyl)amino] benzoate (5a) has shown potent anti-inflammatory activity while 4-acetamidophenyl-2-[{4-{(4-acetamidophenoxy)carbonyl} phenyl}{2-(nitrooxy) ethyl}amino]benzoate (5d) has shown potent analgesic activity compared to standard drug diclofenac.

Full text of DOI:10.1007/s00044-012-0345-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:3510–3517
DOI 10.1007/s00044-012-0345-y
ORIGINAL RESEARCH
Synthesis and investigation of anti-inflammatory activity
of novel nitric oxide donating hybrid drugs
•
Shailesh L. Chandak Amol S. Bansode
•
•
•
Prashant R. Murumkar Monika G. Shinde
Kailash G. Bothara
Received: 5 July 2012 / Accepted: 7 November 2012 / Published online: 25 November 2012
Ó Springer Science+Business Media New York 2012
Abstract A small library of nitric oxide donating groups,
4-acetamidophenyl-2-[{2-(nitrooxy)ethyl}(phenyl) amino]
benzoate (5a–e) possessing a variety of substituents (–H,
–NO2, –CH3, –acetamidophenyl, –SO2NH2) attached to the
fourth position of phenyl ring were synthesized and evalu-
ated for anti-inflammatory, analgesic and ulcerogenic
potential. Structure–activity relationship data showed that
the 2-phenylaminobenzoic acid skeleton is required for
selective COX-2 inhibition. Among all compounds 4-acet-
amidophenyl-2-[{2-(nitrooxy)ethyl}(phenyl)amino]benzo-
ate (5a) has shown potent anti-inflammatory activity while
4-acetamidophenyl-2-[{4-{(4-acetamidophenoxy)carbonyl}
phenyl}{2-(nitrooxy)ethyl}amino]benzoate (5d) has shown
potent analgesic activity compared to standard drug
diclofenac.
mechanism by which NSAIDs exert their anti-inflammatory
activity is through inhibition of cyclooxygenase derived
prostaglandin synthesis, which is also responsible for the
gastrointestinal (Cryer, 2005; James and Hawkey, 2003;
Lazzaroni and Bianchi, 2004; Naesdal and Brown, 2006;
Schneider et al., 2006), renal (Mounier et al., 2006;
Zadrazil, 2006; Adebayo and Bjarnason, 2006) and hepatic
(Vela’zquez et al., 2007) side effects in patients undergoing
long term treatment. Therefore, the challenge exists for the
pharmaceutical industry to develop effective anti-inflam-
matory agents with enhanced safety profile. The most
common side effects associated with NSAID therapy are
upper GI irritation, ulceration, dyspepsia, bleeding, and in
some cases death also (Wallace and Miller, 2000).
Three different strategies have emerged to improve the
safety profile of NSAIDs: (a) the development of selective
cyclooxygenase-2 (COX-2) inhibitors; (b) the co-adminis-
tration of a proton pump inhibitor with the NSAID; and
(c) the linkage of a nitrate-based nitric oxide (NO)-releasing
moiety to classical NSAIDs (NO–NSAIDs) (Takeuchi et al.,
1998). Each strategy presents a different set of advantages
and limitations. Scientific literature shows that serious car-
diovascular side effects are associated with selective COX-2
inhibitors (Doggrell, 2005; Dogne et al., 2005). In addition,
such drugs only minimise the development of new
gastric ulcers but do not affect the existing ones (Hunt et al.,
2002). Due to serious adverse effects several selective
coxibs (rofecoxib, valdecoxib) were withdrawn from the
market (Brown et al., 1993; Holm et al., 1998; Wallace et al.,
1999).
Keywords NO–NSAIDs ꢀ Anti-inflammatory ꢀ
Analgesic ꢀ Ulcerogenicity ꢀ Fenamate ꢀ Paracetamol ꢀ
Nitric oxide
Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are one of
the most useful clinical therapies for the treatment of pain,
fever and inflammation throughout the world (Singh and
Triadafilopoulos, 1999). In 2006, NSAIDs market in US
alone was valued at $3.2 billion and it was projected to
reach $4.6 billion by 2013 (Botting, 2006). The major
A potential strategy to overcome side effects associated
with NSAIDs is to design NO–NSAIDs which are capable
of generating the radical biomediator and gastro-protective
agent NO (Iwata and Leung, 1995). The rationale behind
developing this class of drugs is that, NO by maintaining
S. L. Chandak ꢀ A. S. Bansode ꢀ P. R. Murumkar ꢀ
M. G. Shinde ꢀ K. G. Bothara (&)
Department of Pharmaceutical Chemistry, Sinhgad Institute
of Pharmacy, Narhe, Pune 411 041, Maharashtra, India
e-mail: kgbothara@gmail.com
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Med Chem Res (2013) 22:3510–3517
3511
gastric mucosal blood flow and preventing leucocyte
adherence to the vascular endothelium of the splanchnic
circulation may counteract the detrimental effect of COX-1
suppression so that mucosal injury does not occur (Wallace
1993). The general structural features of NO–NSAIDs
enable a large number of variations within the linking
spacer and the NO-donating moiety. Owing to the ease of
formation of these nitrate esters, several derivatives could
be prepared for a given spacer. A significant amount of
work on NO–NSAIDs and other related compounds has
been reported (Wallace and Del Soldato 2003). These
so-called ‘‘NO–NSAIDs’’ (also known as COX inhibiting
nitric oxide donors, CINODS) have been claimed to have
comparable or superior anti-inflammatory and analgesic
activities in acute and chronic inflammation model in rat,
while sparing the gastrointestinal tract and kidney of injury.
Interestingly, NO–NSAIDs have been shown to accelerate
the healing of pre-existing gastric ulcers (Elliott et al.,
1995) and restore renal function and structure in rats when
subjected to renal ablation (Fujihara, 1998; Halen et al.,
2009).
As a part of our ongoing research programme to
develop anti-inflammatory agents with a greater safety
profile, we now report the synthesis, in vivo anti-inflam-
matory activity, analgesic activity, ulcerogenicity studies,
histopathological examination, and in vitro NO releasing
ability of hybrid NSAIDs having fenamate, acetamino-
phen moieties to which NO is covalently attached via
ethyl linker.
Chemistry
A group of 4-acetamidophenyl-2-[{2-(nitrooxyethyl)phenyl}
amino]benzoate (5a–e), the target compound possessing a
variety of substituents (–H, –NO2, –CH3, –acetaminophen,
–SO2NH2) attached on phenyl ring of amino benzoate were
synthesized using reaction sequence illustrated in Scheme 1.
Briefly, the first step is Ullmann synthesis involves the con-
densation of 2-iodobenzoic acid with different substituted
aniline (1a–e) in the presence of copper powder and anhy-
drous potassium carbonate to give N-(substituted phenyl)
Scheme 1 Scheme for
synthesis of compounds (5a–e)
NHCOCH3
O
using substituted aniline and
2-iodobenzoic acid
NH2
COOH
+
O
S
O
COOH
+
iii
i
NH
Cl
Cl
NH
ii
-HI
-SO2;-2HCl
I
R
R
1a-e
3a-e
R
2a-e
Paracetamol moiety
-HBr
iv
O
N
Fenamate moiety
O
O
N
NHCOCH3
Me
v
O
N
H
O
ONO2
-AgCl
Cl
N
i
t
r
i
R
c
O
x
R
i
d
5a-e
e
R
e
l
e
a
s
i
n
4a-e
g
g
r
o
u
p
i=Cupric Oxide;Cu;Anhydrous K2CO3;
Ethanol;Reflux;110oC;24-36 h.
ii=Thionyl chloride;Reflux 4h.
iii=Acetaminophen;Reflux 30 min.
iv=1-Bromo-2-chloroethane; Reflux 20-30 h.
v=AgNO3;CH3CN;Stirr Room Temp.; 3-3.5 h
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Med Chem Res (2013) 22:3510–3517
anthranillic acids 2a (60.75 %), 2b (59.23 %), 2c (30.70 %),
2d (81.08 %) and 2e (81.29 %). These intermediates were
then oxidised with thionyl chloride which gave acyl chloride
derivatives, and then acetaminophen was refluxed with the
resulting molecules to obtain esterified derivatives (3a–e).
These derivatives were further reacted with 1-bromo-2-chlo-
roethanetoformalkyl halidederivatives(4a–e) and finally the
NO moiety was covalently appended to these deriva-tives
using silver nitrate cyanomethane. These synthesized com-
pounds are the hybrids of fenamate, acetaminophen and NO.
compounds. However, sulphonamide substituted compound
(5e) showed the lowest activity.
Analgesic activity
The analgesic activities of the compounds were studied
using acetic acid induced writhing test in mice. All the
synthesized derivatives were screened for analgesic activ-
ity. From the results of acetic acid induced writhing test, it
was noticed that compounds 5a, 5b, 5c and 5d possess
significant analgesic activity (Table 2). The compounds 5d
(61.34 %) and 5b (56.06 %) and 5a were found to exhibit
significant analgesic activity compared to standard drug
diclofenac (71.81 %). It can be observed from the biolog-
ical result that compound containing acetamidophenoxy-
carbonyl group (5d) is showing comparable activity with
the diclofenac whereas sulphonamide containing com-
pound (5e) showed the least analgesic activity. Less bulky
substituted compounds (5a and 5b) also showed significant
analgesic activity but less than compound (5d).
Results and discussion
Anti-inflammatory activity
Anti-inflammatory activity of the synthesized compounds
was evaluated by carrageenan induced rat paw oedema
model, at equimolar doses, to that of 20 mg/kg p.o. of
diclofenac. Subplantar injection of 0.1 ml, 1 % carra-
geenan produced increase in paw volume (oedema) in all
the animals of various groups. The onset of action was
evident from 1 h in various test groups. The significant
reduction (p \ 0.01) of rat paw oedema was observed by
most of the test compounds at 3 and 4 h compared to
control group (Table 1).
Ulcerogenicity study
Ulcerogenic effect of compounds 5a–c with the best
overall profile in animal efficacy model was evaluated for
gastric ulcerogenic potential in rats (Table 2). When
compared with diclofenac, compounds did not cause any
gastric ulceration at same doses equivalent to 20 mg/kg
diclofenac. Thus, compounds (5a–c) did not show any
ulcerogenic effect due to the presence of NO releasing
moiety in the structure.
4-Acetamidophenyl-2-[{2-(nitrooxy)ethyl}(phenyl)amino]
benzoate (5a) showed higher activity compared to its
4-substituted derivatives (5b–e) and has slightly higher activity
compared to diclofenac after 2 h whereas significant activity
was observed at 3 and 4 h (p\0.001). The compounds 5d and
5e show non-significant activity compared to diclofenac. Thus,
it was observed from the biological activity that the compounds
having less bulkier groups like hydrogen (5a) as well as methyl
(5c) attached to the fourth position of phenyl ring showed
higher activity as compared to more bulkier groups like nitro
(5b) and acetamidophenoxycarbonyl (5d) containing
Histopathological studies
Scanning of stomach specimens using electron microscopy
revealed that in rats treated with compounds 5a–c there
Table 1 Results of anti-inflammatory activity of compounds (5a–e) against carrageenan induced rat paw oedema model in rats
Change in paw volume in (ml) after drug treatment ( SEM) Anti-inflammatory activity (% Inhibition)
Compound
1 h
2 h
3 h
4 h
1 h
2 h
3 h
4 h
Control
0.28 0.0374 0.76 0.1122
1.8 0.0836 1.92 0.1881
–
–
–
–
Diclofenac (20 mg/kg) 0.14 0.0509 0.38 0.0489 0.48 0.0374 0.48 0.0374 44.99*
42.08*
55.19**
11.95ns
17.99ns
18.67ns
11.49ns
73.26***
65.82***
23.32*
24.37*
13.70ns
8.46ns
73.42***
59.61***
32.11**
36.75***
12.13ns
9.41ns
5a
5b
5c
5d
5e
0.18 0.0374 0.32 0.0583 0.60 0.0836 0.74 0.0509 26.67ns
0.24 0.0509 0.76 0.0509 1.36 0.0927 1.26 0.0509
6.67ns
0.18 0.0374 0.60 0.0707 1.34 0.0678 1.16 0.0678 29.99ns
0.20 0.0316 0.66 0.0509 1.54 0.0509 1.84 0.0812 11.66ns
0.26 0.04
0.74 0.0509 1.68 0.0860 1.90 0.0707
8.33ns
N = 5
ns non-significant
*
**
p \ 0.05, p \ 0.01,
***
p \ 0.001
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3513
Table 2 Results of analgesic activity against acetic acid induced writhing tests in mice, ulcer index test and NO releasing properties of
compounds (5a–e)
Compound
Dose
(mg/kg, p.o.)
No. of writhes in
25 min after treatment
(mean SEM)
%
Inhibition
Ratio of
ulcerated
animals
Ulcer index
(mean SE)
% NO
release
Control
Acetic acid (1 % v/v)
29.00 1.000
8.200 0.8602
15.40 1.208
13.80 1.356
21.80 0.8602
11.20 0.8602
26.20 1.655
–
–
–
–
Diclofenac (20 mg/kg)
20
14
13
14
10
12
71.81***
46.368**
56.06**
24.63*
61.344**
8.482ns
5/5
–
2.3 0.3
–
5a
5b
5c
5d
5e
–
–
–
–
–
10.71
6.98
7.31
2.46
5.88
–
–
–
–
N = 5
ns non-significant
*
**
p \ 0.05, p \ 0.01,
***
p \ 0.001
was no injury observed in stomach mucosa. As illustrated
in Fig. 1, specimen (c), (d) and (e) is identical to that of the
control (a). The stomach specimens of diclofenac sodium
treated rats were characterised by complete damage of the
protective mucosal layer and congestion of mucosal blood
vessels (b).
For example, diclofenac (having 2-phenylaminoacetic acid
nucleus) is well known for selective COX-2 inhibitory activity.
Selective COX-2 inhibitors exhibit ulcerogenic gastrointestinal
side effects. The synthesized derivatives having NO releasing
group overcomes the problem of gastrointestinal side effects.
Detection of nitrite
Experimental
The nitric oxide releasing properties of the synthesized
compounds were assessed in phosphate buffer, pH 7.4, in
the presence of L-cysteine, relative to nitric oxide released
from standard sodium nitrite solution. The nitrate con-
taining molecules 5a–e were found to release NO
(Table 2). Thus, it is evident from this study that com-
pounds having NO releasing moiety did not show any
ulcerogenic effect. The % NO release for the compound
5a–5c is higher as compared to 5d and 5e.
Chemistry
All reagents and anhydrous solvents were generally used as
it is received from the commercial supplier. Reactions were
routinely performed in oven-dried glassware. Melting
points were determined with Veego VMP-D digital melting
point apparatus and are uncorrected. FTIR spectra of the
powdered compounds were recorded using KBr on a Jasco
FTIR 4100 series spectrophotometer and are reported in
cm-1. 1H NMR spectra were recorded on Varian 300 MHz
in CDCl3 unless otherwise specified and chemical shifts are
reported relative to tetramethylsilane as an internal stan-
dard. Low resolution mass spectra were obtained using
atmospheric pressure-turbo ion spray ionisation. Pre-
parative TLC was performed using 3 9 6 cm precoated
silica gel G plates (Whatman). Spots were visualised under
UV chamber at 254 nm light or by using iodine vapours.
Conclusion
A group of 4-acetamidophenyl-2-[{2-(nitrooxy)ethyl}(phenyl)
amino]benzoate (5a–e) possessing a variety of substituents
(–H, –NO2, –CH3, –acetamidophenyl, –SO2NH2) attached
to the fourth position of phenyl ring were synthesized and
evaluated for anti-inflammatory, analgesic and ulcerogenic
potential. Compound 5a, 5b and 5c showed significant anti-
inflammatory, analgesic and non-ulcerogenic activity. The
compound 5d showed potent analgesic activity. As for as % NO
releaseis concerned, the potent analgesic compound 5d showed
poor % NO release (2.46) as compared to other compounds.
The structures of all the synthesized derivatives are having
similarity with the pharmacophore, 2-phenylaminobenzoic
acid skeleton which is required for selective COX-2 inhibition.
General procedure for synthesis of 2-(phenylamino)
benzoic acid derivatives (2a–e)
A mixture of 2-iodobenzoic acid 2.5 g (0.01 mol) and
aniline (1a, 0.02 mol) [q = 1.021, 1.86 ml, 1.9 g] or nitro-
aniline (1b, 0.02 mol) or 4-methylphenylamine (1c,
0.02 mol) or PABA (1d, 0.02 mol) or sulphanilamide (1e,
0.02 mol) dissolved in ethanol, cupric oxide (0.1 g),
123

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Med Chem Res (2013) 22:3510–3517
Fig. 1 Haematoxylin and eosin
immunohistochemical staining
of stomach
General procedure for synthesis of 4-acetamidophenyl-
anhydrous potassium carbonate (0.08 g) and copper (0.2 g)
was heated under reflux for 24–36 h. The resulting com-
pound was suspended in water, filtered and finally precip-
itated with the help of dilute hydrochloric acid. The
precipitate was dried and recrystallised from ethanol
(95 %) to get compound (2a–e) (Joshi et al., 2007). The
physical characterisation of the synthesized compounds is
mentioned in Table 3.
2-(phenylamino)benzoate derivatives (3a–e)
A mixture of intermediate 2a (1.18 g, 0.005 mol) or 2b
(1.54 g, 0.005 mol) or 2c (0.70 g, 0.003 mol) or 2d (2.10 g,
0.008 mol) or 2e (2.39 g, 0.008 mol) and excess of thionyl
chloride (for 2a, 2b—0.71 g, 0.006 mol; for 2c—0.46 g,
0.003 mol; for 2d—2.38 g, 0.009 mol; for 2e—1.14 g,
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Med Chem Res (2013) 22:3510–3517
3515
Table 3 Characterisation of 2-(phenylamino)benzoic acid deriva-
tives (2a–e)
Table 5 Characterisation of 4-acetamidophenyl 2-[(2-chloroethyl)
(phenyl)amino]benzoate derivatives (4a–e)
Compound m.p. (°C)
id
Rf
Yield
%
Compound m.p. (°C)
ID
Rf
Yield
%
valuea
valuea
2a
2b
2c
179–180
120–123
0.56
0.66
0.53
60.75
59.23
30.70
4a
No decomposition/any change in
colour up to 310
85–87
0.52
29.07
4b
4c
4d
4e
a
0.85
0.40
0.62
0.74
56.89
40.35
51.61
32.27
No decomposition/any change in
colour up to 310
60–61
232
2d
2e
a
255–256
150
0.60
0.58
81.08
81.29
230–232
solvent system used: 2a, c–e: toluene:methanol (3.5:1.5), 2b:
n-Hexane: ethyl acetate:methanol (2:2:1)
solvent system used: 4a–e: n-Hexane: chloroform:methanol (4:3:1)
General procedure for synthesis of 4-acetamidophenyl-
2-[{2-(nitrooxy)ethyl}(phenyl)amino]benzoate
derivatives (5a-e)
0.009 mol) was refluxed for 4 h. Excess of thionyl chloride
was neutralised using formic acid. The residue obtained
was then refluxed with paracetamol (for 2a, 2b—0.75 g,
0.005 mol; for 2c—0.45 g, 0.003 mol; 2d—2.41 g,
0.016 mol; 2e—1.20 g, 0.008 mol) for 30 min in water
bath to get compound (3a–e) (Furniss et al., 2005). The
physical characterisation of the synthesized compounds is
mentioned in Table 4.
A solution of intermediate 4a (0.40 g, 0.0009 mol) or 4b
(0.95 g, 0.0019 mol) or 4c (0.40 g, 0.0009 mol) or 4d (0.16
g, 0.0002 mol) or 4e (1.11 g, 0.002 mol) in dry acetonitrile
(1-2 ml) was treated in portion with a solution of AgNO3
(4a—0.17 g, 0.001 mol; 4b-0.41 g, 0.0024 mol; 4c—0.17
g, 0.001 mol; 4d—0.055 g, 0.0003 mol; 4e—0.46 g,
0.0027 mol) in dry acetonitrile (4–5 ml) and stirred at
room temperature for 3 h, evaporated to dryness and the
residue was recrystallised from absolute ethanol to get
compound (5a–e) (Abadi et al., 2005).
General procedure for synthesis of 4-acetamidophenyl-
2-[(2-chloroethyl)(phenyl)amino]benzoate derivatives
(4a–e)
Compounds 4a–e were prepared by refluxing intermediates
3a (1.19 g, 0.003 mol) or 3b (1.44 g, 0.003 mol) or 3c (0.4
g, 0.001 mol) or 3d (0.28 g, 0.0005 mol) or 3e (3.00 g,
0.007 mol) with 1-bromo-2-chloroethane (3a—0.715 g,
0.005 mol; 3b—0.858 g, 0.006 mol; 3c—0.286 g, 0.002
mol; 3d—0.10 g, 0.007 mol; 3e—2.00 g, 0.014 mol) for
20–30 h. The solid product thus obtained was treated with
excess of NaOH solution, the organic layer was separated,
dried and distilled (Furniss et al., 2005). The physical
characterisation of the synthesized compounds is men-
tioned in Table 5.
Synthesis of 4-acetamidophenyl-2-[{2-
(nitrooxy)ethyl}(phenyl)amino]benzoate (5a)
Yield 29.07 %; m.p. No decomposition or any change in
colour up to 310°C; FTIR (KBr) cm-1: 1731 (C=O, ester),
3302 (N–H, amide), 1500, 1456 (C=C, Ar), 3060 (C–H,
Ar), 1671 (C=O, amide), 1600, 1502 (C–C, Ar), 3010 (C–H,
Me), 1476 (C–H, bending), 1235 (C–O Stretch) 1609, 1254
1
(N=O, NO3); H NMR (CDCl3, d ppm) : 2.46 –CH3 (s,
3H), 3.3 –CH2– (m, 2H), 3.76 –CH2– (m, 2H), 6.7
–NH– (S, 1H), 7.07–7.6 Ar attached to amide (d, 4H),
8.03–8.06 Ar attached to ester (d, 1H), 7.81–7.87 Ar
attached to ester (t, 2H), 7.39–7.42 Ar attached to ester (d,
1H), 7.2 Ar attached to tertiary amine (m, 5H).
Table 4 Characterisation of 4-acetamidophenyl-2-(phenylamino)
benzoate derivatives (3a–e)
Synthesis of 4-acetamidophenyl-2-[{2-
Rf valuea
Yield %
(nitrooxy)ethyl}(4-nitrophenyl)amino]benzoate (5b)
Compound id
m.p. (°C)
3a
3b
3c
3d
3e
a
196–199
103–105
99–102
0.64
0.69
0.64
0.48
0.59
61.98
61.8
Yield 56.89 %; m.p. 85–87°C; FTIR (KBr) cm-1: 1735
(C=O, ester), 3275 (N–H, amide), 1510, 1480 (C=C, Ar),
1560,1360 (NO2), 3052 (C–H, Ar), 1682 (C=O, amide),
3302 (N–H, amide), 2982 (C–H, Me), 1682 (C=O, amide),
1600, 1502 (C–C, Ar), 1476 (C–H, bending), 1130(C–O
Stretch), 1633,1261 (N=O, NO3); 1H NMR (CDCl3,
44.54
6.71
140–142
180–182
86.45
solvent system used: 3a–e: toluene:methanol (4.5:0.5)
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Med Chem Res (2013) 22:3510–3517
d ppm): 2.5 –CH3 (s, 3H), 7.56 –NH in amide (s, 1H), 3.34
–CH2– (m, 2H), 3.76 –CH2– (m, 2H).
paw of each rat. One group was kept as control and the
animals of the other group were pretreated with the test
drugs, 1 h before the carrageenan treatment. The volume was
measured before and after carrageenan treatment at the 1 h
interval with the help of digital plethysmometer v1.5.
Synthesis of 4-acetamidophenyl-2-[{2-
(nitrooxy)ethyl}(4-methyl phenyl)amino]benzoate (5c)
Yield 40.35 %; m.p. 60–61°C; FTIR (KBr) cm-1: : 1720
(C=O, ester), 3282 (N–H, amide), 1600, 1572 (C=C, Ar),
3034 (C–H, Ar), 3302 (N–H, amide), 2950 (C–H, Me),
1243 (C–O, ester), 1673 (C=O, amide), 1512 (C–C, Ar),
Analgesic activity
The analgesic activity was evaluated using the acetic acid
induced writhing method (Koster et al., 1959).
1
3020 (C–H, Ar Me), 1270 (N=O, NO3); H NMR (CDCl3,
d ppm): 2.4 –CH3 in amide (s, 3H), 7.23 Ar attached to
amide (m, 4H), 6.5–6.6 Ar attached to 3°N (m, 4H),
8.11–8.14 Ar attached to ester (d, 2H), 7.52–7.54 Ar
attached to ester (t, 2H), 3.6 –CH2– (m, 2H), 3.76 –CH2
– (m, 2H), 2.07 –CH3 in toluene (s, 3H).
Ulcerogenicity studies (El-Sayed et al., 2010)
Acute ulcerogenicity of test compounds 5a–e was deter-
mined using adult albino rats of both sexes weighing
between 120 and 150 g. Animals were divided into groups
each of five animals. Animals were divided into groups
each of five animals. Rats were fasted 20 h before drug
administration. The tested compounds and diclofenac
sodium were given orally in a dose of 20 mg/kg suspended
in 1 % CMC while one group received vehicle (1 %
CMC). Rats were fasted for 2 h, allowed to feed for 2 h
and then fasted for another 20 h. Rats were given another
two doses on the second and third day. On the fourth day,
rats were sacrificed, the stomach removed, opened along
with the greater curvature and rinsed with 0.9 % saline.
The number of mucosal damage (red spots) was counted
and their severity (ulcerogenic severity) was graded from 0
to 4 according to the following score assignment:
Synthesis of 4-acetamidophenyl-2-[{4-{(4-
acetamidophenoxy)carbonyl}phenyl}{2-
(nitrooxy)ethyl}amino] benzoate (5d)
Yield 51.61 %; m.p. 232°C; FTIR (KBr) cm-1: 1735
(C=O, ester), 3369 (N–H, amide), 1572, 1500 (C=C, Ar),
1243 (C–O, ester), 3076 (C–H, Ar), 2994 (C–H, Me), 1670
(C=O, amide), 1501 (C–C, Ar), 1257 (C–O, ester), 1270
(N=O, NO3); 1H NMR (CDCl3, d ppm) : 1.57 –CH3
attached to amide (s, 3H), 2.36 –CH2– (m, 2H), 3.76
–CH2– (m, 2H), 6.9 –NH in amide (s, 1H), 7.5 Ar attached
to amide (d, 2H), 7.19–7.22 Ar attached to amide (d, 2H),
7.91–7.94 Ar attached to ester (d, 1H), 7.13–7.16 Ar
attached to ester (t, 2H).
Score: Normal (No injury) 0, Latent small red spot 1,
Wide red spot 2, Slight injury 3, Severe injury 4.
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. Data were analysed by one-way ANOVA followed
by Dunnett’s test to determine the significance of the dif-
ference between the standard group and rats treated with
the test compounds.
Synthesis of 4-acetamidophenyl-2-[{2-
(nitrooxy)ethyl}(4-sulphamoylphenyl)amino]benzoate
(5e)
Yield 32.27 %; m.p. 230–232°C; FTIR (KBr) cm-1: 1730
(C=O, ester), 3219 (N–H, amide), 3004 (C–H, Me), 3031
(C–H, Ar), 1637 (C=O, amide), 1505 (C–C, Ar), 1278
1
(N=O, NO3), 1187 (C–O, ester), 1323 (S=O); H NMR
Histopathological studies
(CDCl3, d ppm): 2.29 –NH2 in SO2NH2 (s, 2H), 2.56 –CH3
in amide (s, 3H), 7.27 –NH in amide (s, 1H), 3.49 –CH2
– (m, 2H), 3.76 –CH2– (m, 2H).
For the histopathological study, rats were sacrificed and
their stomach specimens were removed and put into 10 %
formalin solution. The samples were then sent to Dhande
laboratory, Pune for further examination. A longitudinal
section of stomach along the greater curvature, which
included the ulcer base and both sides of the ulcer margin
was taken and fixed in 10 % formalin for 24 h at 4 °C and
embedded in paraffin. Morphological examination was
performed with haematoxylin and eosin staining for histo-
logical changes and examined under electron microscope.
Anti-inflammatory activity
Anti-inflammatory activity was evaluated using the well-
known carrageenan induced rat paw oedema model (Winter
et al., 1962) using groups of six animals each. A freshly
prepared aqueous suspension of carrageenan (1.0 % w/v,
0.1 ml) was injected in the subplantar region of right hind
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