Synthesis and evaluation of anti-inflammatory, analgesic, ulcerogenicity and nitric oxide-releasing studies of novel ibuprofen analogs as nonulcerogenic derivatives

Article abstract of DOI:10.1007/s00044-010-9389-z

Since the last 41 years, Ibuprofen has been one of the most widely used Non-Steroidal Anti-Inflammatory Drug (NSAID) due to its anti-inflammatory actions. As all the NSAIDs are suffering from the deadlier GI toxicities, Ibuprofen also is no exception to these toxicities. The free -COOH group is thought to be responsible for the Gastrointestinal (GI) tract toxicity associated with all the traditional NSAIDs. Therefore, the main aim of this study was to develop new chemical entities as potential anti-inflammatory agents with less GI toxicities. In this article, synthesis of a series of Hybrid molecules containing important pharmacophore of Ibuprofen and substituted diaryl rings on 5-membered heterocycle similar to coxibs and Nitric oxidereleasing moiety are described. All the synthesized compounds were tested in vivo for their anti-inflammatory, analgesic, ulcerogenic properties, and histopathological studies and in vitro for their nitric oxide-releasing properties. Out of the six synthesized compounds, four compounds showed significant anti-inflammatory and analgesic activity which was compared with standard. All the synthesized compounds exhibited significant nitric oxide-releasing and reduced GI ulcerogenic activity. Springer Science+Business Media, LLC 2010.

Full text of DOI:10.1007/s00044-010-9389-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:795–808
DOI 10.1007/s00044-010-9389-z
ORIGINAL RESEARCH
Synthesis and evaluation of anti-inflammatory, analgesic,
ulcerogenicity and nitric oxide-releasing studies of novel
ibuprofen analogs as nonulcerogenic derivatives
•
Aniket P. Sarkate Deepak K. Lokwani
•
•
Ajit A. Patil Shashikant V. Bhandari
•
Kailash G. Bothara
Received: 25 April 2009 / Accepted: 7 July 2010 / Published online: 22 July 2010
Ó Springer Science+Business Media, LLC 2010
Abstract Sincethelast41 years, Ibuprofenhasbeenoneof
the most widely used Non-Steroidal Anti-Inflammatory
Drug (NSAID) due to its anti-inflammatory actions. As all
the NSAIDs are suffering from the deadlier GI toxicities,
Ibuprofen also is no exception to these toxicities. The free
–COOH group is thought to be responsible for the Gastro-
intestinal (GI) tract toxicity associated with all the traditional
NSAIDs. Therefore, the main aim of this study was to
develop new chemical entitiesaspotentialanti-inflammatory
agents with less GI toxicities. In this article, synthesis of a
series of Hybrid molecules containing important pharma-
cophore of Ibuprofen and substituted diaryl rings on
5-membered heterocycle similar to coxibs and Nitric oxide-
releasing moiety are described. All the synthesized com-
pounds were tested in vivo for their anti-inflammatory,
analgesic, ulcerogenic properties, and histopathological
studies and in vitro for their nitric oxide-releasing properties.
Out of the six synthesized compounds, four compounds
showed significant anti-inflammatory and analgesic activity
which was compared with standard. All the synthesized
compounds exhibited significant nitric oxide-releasing and
reduced GI ulcerogenic activity.
Introduction
Non-steroidal Anti-Inflammatory Drugs (NSAIDs) are
among the most commonly prescribed drugs to reduce
pain, inflammation, and fever. These drugs reduce pain and
edema by suppressing the formation of prostaglandins, by
inhibiting the activity of the enzyme Cyclooxygenase
(COX-1 and COX-2). Selective COX-2 inhibitors elicit
less or no GI damage and bleeding compared with con-
ventional NSAIDs, although the magnitude of this reduc-
´
tion continues to be debated in the literature (Szabo et al.,
2008; Biava et al., 2010). As widely reported in the lay-
press, the selective COX-2 inhibitors also cause significant
adverse effects in the renal and cardiovascular systems,
possibly more serious than those caused by conventional
NSAIDs. However, their use is limited by their significant
side effects upon the stomach and the kidney. Their side
effects as well as therapeutic actions are related to their
ability to inhibit cyclooxygenase enzymes involved in the
first step of the arachidonic acid cascade (Insel, 1996;
Wolfe et al., 1999).
Recent strategies adopted to minimize the side effects of
NSAIDs include the use of the dual LOX/COX inhibitors,
the use of selective COX-2 inhibitors, and the use of hybrid
molecules made up of non-selective or selective COX
inhibitors together with a nitric oxide-releasing functional
group (Bias et al., 2004; Doggrell, 2005; Velazquez et al.,
2005).
Keywords Ibuprofen ꢀ Nitric oxide ꢀ Anti-inflammatory ꢀ
Analgesic ꢀ Ulcerogenicity ꢀ Vasodilator
Recent data revealed serious cardiovascular side effects
associated with selective COX-2 inhibitors (Bias et al., 2004;
Dogne et al., 2005). In addition, such drugs only minimize
the development of new gastric ulcers but do not cure the
existing ones (Hunt et al., 2002). The strategy involving the
use of hybrid molecules made up of non-selective COX
inhibitors together with a nitric oxide-releasing moiety,
A. P. Sarkate (&) ꢀ D. K. Lokwani ꢀ A. A. Patil ꢀ
S. V. Bhandari ꢀ K. G. Bothara
Department of Pharmaceutical Chemistry,
AISSMS College of Pharmacy, Near RTO,
Kennedy Road, Pune 411001, Maharashtra, India
e-mail: aniketpharma1@gmail.com
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Med Chem Res (2011) 20:795–808
constitutes one of the most promising approaches, because
nitric oxide supports several endogenous GI defense mech-
anisms, including increase in mucus, bicarbonate secretions,
increase in mucosal blood flow and inhibition of the acti-
resulted in an increased anti-inflammatory activity with
reduced GI-ulcerogenicity (Bhandari et al., 2010; Bhandari
et al., 2009). In our attempt to continue to discover new,
safer, and potent agents for the treatment of inflammatory
diseases, we have synthesized hybrid compounds containing
pharmacophore of Ibuprofen, 1,2-diaryl triazole ring, the
pharmacophore similar to coxibs and nitric oxide-releasing
group to accentuate potency and reduce GI toxicities asso-
ciated with the traditional NSAIDs. The compounds
designed so were found to possess much significant anal-
gesic, anti-inflammatory, vasodilatory profile with signifi-
cant reduction in ulcerogenic toxicities.
´
vation of proinflammatory cells (Wey et al., 2007; Muscara
and Wallace, 2006; Chegaev et al., 2007). Moreover,
because of the beneficial cardiovascular effects (vasodila-
tion) of Nitric Oxide, such drugs are expected to be devoid of
the cardiovascular adverse effects associated with the use of
´
selective COX-2 inhibitors (Doggrell, 2005; Muscara and
Wallace, 2006).
Among those nitric oxide-NSAIDs that came into clin-
ical trials are Nitroaspirin, Nitronaproxene, Nitroketopro-
fen, Nitroibuprofen, etc. Among the nitric oxide donors
adopted to prove the validity of this principle are furoxans,
oximes, hydrazides, and organic nitrates. (Mann, 2003;
Keeble and Moore, 2002; Rehse and Brehme, 2000).
Synthetic approaches based on chemical modification of
NSAIDs have been taken with the aim of improving safety
profile and in turn therapeutic window of the resultant
NSAIDs. Our previous studies had described the synthesis of
hybrid molecules with nitric oxide-releasing group that
Results and discussion
Chemistry
The synthetic route, utilized to synthesize title compounds, is
outlined in Schemes 1, 2 and 3. The compound, 2,5-dioxo-
pyrrolidin-1-yl-2-(4-isobutylphenyl)propanoate (1), was pre-
pared according to the method reported in the literature
a
Scheme 1 Synthesis of Compounds 4a–4ca. Reagents and Condi-
acid, ethanol, reflux 4 h; (v) 4-chlorophthalic acid, ethanol, reflux 4 h
and (vi) 2-(2-chlorophenyl)acetic acid, ethanol, reflux 4 h
tions: (i) DCC, THF, overnight at 5°C; (ii) THF, thiosemicarbazide,
reflux 12 h; (iii) 10% aq. KOH, dil. HCl, reflux; (iv) 4-chlorobenzoic
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Med Chem Res (2011) 20:795–808
797
(Birsen et al., 2000) using Ibuprofen. The 2-(2-(4-isobutyl-
phenyl)propanoyl)hydrazincarbothioamide (2) was prepared
by reacting (1) with thiosemicarbazide in tetrahydrofuran.
Ring formation yielding 5-(1-(4-isobutylphenyl) ethyl)-2H-
1,2,4-triazole-3(4H)-thione (3) was carried out by reacting (2)
with 10% KOH. The different acid derivatives (4a–4c) were
prepared by reacting (3) with different acids. The reaction of
(4a–4c) with thionyl chloride followed by hydrazine hydrate
yielded corresponding hydrazides (5a–5c). The reaction of
these hydrazides with the substituted chloro acetyl chloride
gave the corresponding haloalkyl derivatives (6a–6f). The
terminal chloro function is then replaced with nitric oxide by
reacting (6a–6f) with silver nitrate to get nitrate derivatives
(7a–7f) using acetonitrile as solvent. The structures of various
synthesized compounds were assigned on the basis of results
of different chromatographic and spectral studies. The phys-
ical data, FTIR, 1H-NMR, Mass spectral data, and elemental
analysis data for all the synthesized compounds are given in
experimental protocols.
The FTIR spectra of the title compounds (7a–7f)
exhibited very similar features and showed the expected
bands for the characteristic groups which are present in the
compounds such as C=N stretching vibrations, amide C=O
stretching, and another specific band for –NO2 vibrations.
a
Scheme 2 Synthesis of Compounds 7a, 7b, 7e, and 7fa. Reagents
reflux 10 h; (iii) chloro-propionyl chloride, dry benzene, triethyl-
amine, reflux 10 h; and (iv) AgNO3, acetonitrile, stirring, RT, 3 h
and conditions: (i) Thionyl chloride, hydrazine hydrate, ethanol,
reflux 6 h; (ii) chloro-acetyl chloride, dry benzene, triethylamine,
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a
Scheme 3 Synthesis of Compounds 7c and 7da. Reagents and
conditions: (i) Thionyl chloride, hydrazine hydrate, ethanol, reflux
6 h; (ii) chloro-acetyl chloride, dry benzene, triethylamine, reflux
10 h; (iii) chloro-propionyl chloride, dry benzene, triethylamine,
reflux 10 h; and (iv) AgNO3, acetonitrile, stirring, RT, 3 h
1
In the H-NMR spectral data, all the protons were seen
model, and equimolar doses are equivalent to Ibuprofen.
Sub-planter injection of 0.1 ml of 1% Carrageenan in rat
paw increased the paw volume (edema) in all the animals
of various groups. The onset of action was evident during
1 h in various test groups. The significant (P \ 0.01)
reduction of rat paw edema was observed for most of the
test compounds, at 3 h compared to control group and
Ibuprofen (Table 1). Compounds with significant anti-
inflammatory profile were subjected to GI-ulcerogenicity
potential studies at 12 times the therapeutic doses with
additional physical (cold) stress. A thorough examination
of the results of histopathological studies indicated the
absence of the disruption of morphology of the gastric
epithelial cells and the absence of ulcers/erosion in test
(test compd.-treated) group animals which were compared
to reference standard, Diclofenac acid, and control group
animals. The results of the ulcerogenicity studies are pre-
sented in Table 3, and the results of Histopathological
studies are depicted in Fig. 1a–f. Out of the synthesized six
NCEs, compounds 7a, 7c, 7d, and 7f (61.25–62.82%)
exhibited very significant anti-inflammatory activity com-
pared to standard drug Ibuprofen (62.82% at 3 h). Thus, the
compounds with nitrate-containing moiety at ortho or para
position of phenyl ring show higher anti-inflammatory
activity whereas the bi-substitution of nitrate-containing
moiety at both ortho and para positions of phenyl in
compounds (7b and 7e) shows decreased anti-inflammatory
activity. Results indicated that the length of the chain by
according to the expected chemical shift and integration
values. The aromatic protons appeared as multiplet peaks
within the range of 7.07–7.6 d ppm, and the synthesized
derivatives had shown the peak at 6.9 d for –NH of triazole
ring that confirms the formation of triazole ring.
Pharmacology
The synthesized new chemical entities (NCEs) were sub-
jected to the evaluation of anti-inflammatory activity,
analgesic activity, and acute ulcerogenicity studies as well
as investigated for their nitric oxide-releasing properties.
Ibuprofen and Diclofenac were used as reference standards.
The experiments were performed using albino rats of
Wistar strain of either sex, weighing in the range of
140–160 g. The animals were maintained at 25 2°C,
50 5% relative humidity, and 12-h light/dark cycle. All
the animals were fasted for 24 h before the experiments,
and water was provided ad libitum. The test compounds
were suspended in 1% aqueous carboxy methyl cellulose
(CMC) solution and administered orally to experimental
animals for all the studies.
Anti-inflammatory activity
Anti-inflammatory activity of the synthesized compounds
was evaluated by carrageenan-induced rat paw edema
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799
Table 1 Results of anti-inflammatory activity of synthesized compounds against carrageenan-induced rat paw edema model in rats
Compound code/
dose (mg/kg, p.o)
Change in paw volume in (ml) after drug treatment ( SEM)
Anti-inflammatory activity (% inhibition)
1 h
2 h
3 h
1 h
2 h
3 h
Control
1.65 0.017
1.77 0.02
1.91 0.03
–
–
–
Ibuprofen (20)
7a (49.70)
7b (63.98)
7c (49.70)
7d (48.34)
7e (66.69)
7f (51.06)
0.76 0.072**
0.75 0.074**
1.04 0.042**
0.79 0.009**
0.78 0.02**
1.19 0.06**
0.80 0.03**
0.74 0.009**
0.73 0.009**
1.06 0.03**
0.76 0.01**
0.77 0.01**
1.24 0.09**
0.77 0.02**
0.71 0.02**
0.71 0.05**
1.11 0.07**
0.73 0.01**
0.74 0.01**
1.30 0.09**
0.73 0.01**
53.93
54.54
38.78
52.12
52.72
27.87
51.51
58.19
58.75
40.11
57.06
56.49
29.94
56.49
62.82
62.82
41.88
61.78
61.25
31.93
61.78
Data analyzed by one way ANOVA followed by Dunnett’s ‘t’ test, (n = 6), * P \ 0.05, ** P \ 0.01 significant from control
ns not significant
Fig. 1 Haematoxylin and Eosin Immunohistochemical staining of
gastric ulcers after ulcer induction in rats. As illustrated in Fig 1,
specimen a shows intact mucous membrane in treated control rat
showing granular tissues composed of macrophages, fibroblasts, and
endothelial cells forming microvessels. Congestion of mucosal blood
vessels was observed in Diclofenac-treated group, specimen (b). No
damage was seen to mucosa of rat treated with test compound, 7a,
specimen (c), 7c, specimen (d), 7d, specimen (e), and 7f, specimen
(f) these specimens c–f were identical to that of the control, specimen
(a). Original magnification 2009
which nitrate-containing moiety attached to phenyl ring did
not affect the anti-inflammatory activity of the compounds.
Based on the findings of these preclinical results, further
studies need to be carried out to investigate the other
specifications, such as in vitro assays, chronic ulcerogene-
city studies, toxicological studies, and mechanism by which
these drugs exhibit potential analgesic, anti-inflammatory
activities.
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Analgesic activity
Table 3 Ulcerogenic effects of synthesized compounds in compari-
son with diclofenac acid
The analgesic activity of the compounds was studied by
using acetic acid-induced writhing test in mice. The anal-
gesic activity was evaluated at equimolar doses equivalent
to 15 mg/kg (Ibuprofen) body weight. The important
analgesic profile of the compounds was measured by the
classical acetic acid-induced writhing model. From the
results, it was noticed that all the compounds possess sig-
nificant analgesic activity (Table 2). The analgesic effects
of compounds 7c (64.75%) and 7d (63.57%) were found to
be better than that of Ibuprofen (62.43%). Similar to anti-
inflammatory activity, the presence of nitrate-containing
moiety at ortho or para position of phenyl ring in com-
pounds shows higher analgesic activity as compared to the
bi-substitution of this moiety at both the ortho and para
positions of phenyl in compounds 7b and 7e. It was
exhibited that the presence of nitrate-containing moiety at
para position of phenyl ring (Compounds 7c and 7d) show
higher analgesic activity as compared to substitution at
ortho position of phenyl ring (Compounds 7a and 7f).
Compound
code
Dose
(mg/kg, p.o)
Ratio of
ulcerated
animals
Ulcer index
(mean SE)
Diclofenac
24
6/6
Nil
Nil
Nil
Nil
2.3 0.3
7a
7c
7d
7f
59.64
59.64
57.96
61.2
–
–
–
–
oral doses. The results are shown in Table 3. Thus, it was
indicated that presence of nitrate-containing moiety will
decrease the adverse effect of compounds (NSAIDS.).
Histopathological studies
The stomach specimen of Diclofenac acid-treated rats was
characterized by complete disruption of protective mucosal
layer (Fig. 1b). Histopathological analysis also showed
characteristic features of ulceration in diclofenac acid-
treated group of animals. The tissue of Diclofenac-acid
treated rats showed that some epithelial cells in the ulcer
margin were proliferated and migrated over and into the
ulcer crater. This indicated the complete disruption of
gastric epithelial layer. Scanning of stomach specimens
using electron microscope revealed that in the rats no
injury was observed in stomach mucosa when treated with
synthesized derivatives (7a, 7c, 7d, and 7f). As illustrated
in Fig. 1, specimen c, d, e, and f are seen identical to that of
the control (Fig. 1 Specimen a).
Acute ulcerogenecity studies
The compounds, which exhibited significant anti-inflam-
matory and analgesic activity comparable to that of Ibu-
profen, were subjected to acute ulcerogenecity studies.
Therefore ulcerogenic effect of the compounds 7a, 7c, 7d,
and 7f with best overall profile in animal efficacy model
was evaluated for gastric ulcerogenic potential in rat stress
model at 12 times the therapeutic doses of Diclofenac acid
(Table 3). When compared with Diclofenac acid, all the
four compounds did not cause any gastric ulceration and
disruption of gastric epithelial cells at the above mentioned
Nitric oxide-release study
In isolated Wistar rat aorta rings, compounds 7a–7f com-
petitively inhibited norepinephrine-induced contraction
effects, causing a shift to the right of the norepinephrine
concentration response curves. EC50 (lg/ml) values were
calculated from the cumulative concentration response
curves. In order to prove the involvement of nitric oxide in
the relaxation process, nitric oxide-releasing properties of
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
(Table 4).
Table 2 Results of analgesic activity of synthesized compounds
against acetic acid-induced writhing test in mice
Compound no.
Dose
(mg/kg, p.o)
No. of Writhes
in 5–15 min
% Inhibition
after treatment
(Mean SE)
Control
Acetic acid
(1% v/v)
28.83 0.65**
–
Ibuprofen
15
10.83 0.60**
11.83 0.94**
13.66 0.33**
10.16 0.47**
10.5 0.42**
14.83 0.60**
12.66 0.33**
62.43
58.96
52.61
64.75
63.57
48.56
56.08
7a
7b
7c
7d
7e
7f
37.28
47.98
37.28
35.85
49.46
37.87
Conclusions
Six hybrid derivatives were synthesized and screened for
analgesic, anti-inflammatory, ulcerogenic potential, and
nitric oxide-releasing studies. Most of the compounds (4 out
Data analyzed by one way ANOVA followed by Dunnett’s ‘t’ test,
(n = 6), ** P \ 0.01 significant from control
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801
represented in d ppm). Mass spectra were recorded on
GC-MS QP5050ASystem (benchtop quadrupole mass
spectrophotometer). Elemental analysis was recorded on
FLASHEA112 series at Shimadzu Analytical Centre and
NMR Facility, the Department of Chemistry, University of
Pune. Purity of the compounds was checked on TLC plates
using silica gel G as stationary phase and iodine vapors as
visualizing agent. The 2-(4-isobutylphenyl)propanoic acid
(Ibuprofen) was procured from Inland Pharmaceuticals Ltd.,
Pune, India.
Table 4 EC50 values and nitric oxide-releasing properties of the
compounds (7a–7f)
Compound code
EC50
% NO releasea
7a
7b
7c
7d
7e
7f
a
55.26
38.68
30.52
42.78
34.88
58.56
0.35
0.42
0.39
0.48
0.57
0.32
Percentage of NO released (n = 2) relative to a theoretical maxi-
mum release of 1 mol NO/mol of test compound; determined by
Griess reagent in the presence of 5 mM L-cysteine, at pH 7.4
Synthesis of 2,5-dioxopyrrolidin-1-yl2-
(4-isobutylphenyl)propanoate (1)
of 6) exhibited significant analgesic and anti-inflammatory
activity. Compounds (7c) and (7d) showed strong analgesic
activity in the acetic acid-induced writhing tests. Among all
the synthesized compounds, compound (7a) exhibited most
prominent and consistent anti-inflammatory activity. From
the detailed analysis of the results of histopathological
studies, we conclude that the synthesized compounds have
not only retained but showed enhanced anti-inflammatory
profile, which are devoid of the deadlier gastrointestinal
toxicities, but also all the synthesized derivatives exhibited
significant vasorelaxant activity that is devoid of CVS
toxicities associated with coxib families of COX-2 inhibi-
tors. Therefore, it can be concluded that the rational, based
on which these NCEs were designed, has been proven to be
superior compared to the currently used NSAIDs. Outcome
of this research study is very promising. The only concern to
be addressed now is the pharmacokinetic profile of the
designed NCEs, since molecular weight of all the designed
compounds is beyond 500.
Compound (1) was synthesized as per the previously
reported procedure (Birsen et al., 2000). Yield: 80%; and
M.P: 82–84°C. FTIR spectra of the compound showed
bands at 3053 (Ar C–H); 2933 (Aliphatic C–H); and 1676
(C=O).
Synthesis of 2-(2-(4-isobutylphenyl)propanoyl)
hydrazinecarbothioamide (2)
The product (2) was obtained as per the previously reported
procedure (Birsen et al., 2000). Yield: 70%; and M.P:
92–94°C. FTIR spectra of the compound showed bands at
3465 (N–H); 3043 (Ar C–H); 2941 (Aliphatic C–H); and
1680 (C=O).
Synthesis of 5-(1-(4-isobutylphenyl) ethyl)-2H-1,2,4-
triazole-3(4H)-thione (3)
The product (3) was obtained as per the previously reported
procedure (Birsen et al., 2000). Yield: 74%; and M.P:
128–130°C. FTIR spectra of the compound showed bands
at 3455 (N–H); 3061 (Ar C–H); 2941 (Aliphatic C–H); and
1546 (C=N).
The most potent derivatives are in the process for
chronic toxicity and pharmacokinetic studies; based on
those results, further action plan will be finalized.
Experimental
Synthesis of 4-(3-(1-(4-isobutylphenyl)ethyl)-5-thioxo-1H-
1,2,4-triazol-4(5H)-yl) benzoic acid (4a) (McCall et al.,
1986)
Synthetic studies
All the compounds were synthesized using the reported lit-
erature procedures, and synthetic procedures were set and
optimized as and where required. All the chemicals were
procured from Merck, Sigma-Aldrich, Mumbai. Melting
points were determined by open capillary tubes and were
uncorrected. FTIR spectra of the powdered compounds were
recorded using KBr on a Jasco FTIR V 430 ? spectrometer
with Diffuse Reflectance Attachment and are reported in
Equimolar amounts of intermediate (3) (1 g, 0.003 M) and
4-chlorobenzoic acid (0.46 g 0.003 M) were refluxed in
ethanol (10 ml) for 4 h. After completion of 3 h from the
starting of reflux, NaHCO3 (0.252 g, 0.003 M) was added
in reaction mixture to increase the rate of forward reaction.
After 4-h duration, the reaction mixture was cooled, etha-
nol was evaporated to yield product (4a). Yield: 76%; and
M.P: 152–154°C. FTIR spectra of the compound showed
bands at 3541 (O–H); 3463 (N–H); 3043 (Ar C–H); 2943
1
cm-1 and H NMR spectra were recorded on a Varian
1
Mercury YH300 (300 MHz FT NMR) spectrophotometer
using TMS as an internal reference (Chemical shift
(Aliphatic C–H); 1685 (C=O); and 1546 (C=N). H NMR
chemical shifts at (CDCl3, d ppm): 0.89 (d, 6H, –(CH3)2);
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Med Chem Res (2011) 20:795–808
1.82–1.84 (m, 1H, –CH–); 2.43 (d, 2H, –CH2–); 7.13–7.17
(m, 4H, 2,3,5,6 ArH); 3.0 (q, 1H, –CH–); 1.2 (d, 3H,
–CH3); 6.90 (s, 1H, triazole NH); 6.20–7.81 (m, 4H, 2,3,5,6
ArH); and 12.79 (s, 1H, –OH). Mass spectra of the com-
pound exhibited molecular ion peak at m/z 381(M?); other
important fragments were observed at 382 (M? ? 1), 338,
and 248.
residue was refluxed with excess hydrazine hydrate
(0.11 ml, 5 mmol) in ethanol (6 ml) for 6 h. The mixture
was left to cool and then evaporated under reduced pres-
sure. The residue obtained was filtered, washed with water,
dried, and crystallized from absolute alcohol. Yield: 68%;
and M.P. 174–176°C. FTIR spectra of the compound
showed bands at 3431 (N–H); 3042 (Ar C–H); 2938 (Ali-
phatic C–H); 1680 (C=O); and 1555 (C=N). 1H NMR
chemical shifts at (CDCl3, d ppm): 0.91 (d, 6H, –(CH3)2);
1.84–1.86 (m, 1H, –CH–); 2.43 (d, 2H, –CH2–); 7.13–7.17
(m, 4H, 2,3,5,6 ArH); 2.9 (q, 1H, –CH–); 1.2 (d, 3H,
–CH3); 6.84 (s, 1H, triazole NH); 6.17–7.53 (m, 4H, 2,3,4,5
ArH); 9.48 (s, 1H, –NH–); and 4.35 (s, 2H, –NH2). Mass
spectra of the compound exhibited molecular ion peak at
m/z 395 (M?); other important fragments were observed at
396 (M? ? 1), 397 (M? ? 2), 352, 262, and 380.
Synthesis of 4-(3-(1-(4-isobutylphenyl) ethyl)-5-thioxo-1H-
1,2,4-triazol-4(5H)-yl)phthalic acid (4b)
The product 4b was obtained as per the procedure reported
in ‘‘Synthesis of 4-(3-(1-(4-isobutylphenyl)ethyl)-5-thioxo-
1H-1,2,4-triazol-4(5H)-yl) benzoic acid (4a) (McCall et al.,
1986)’’ section. Yield: 70%; and M.P: 142–144°C. FTIR
spectra of the compound showed bands at 3532 (O–H);
3433 (N–H); 3053 (Ar C–H); 2921 (Aliphatic C–H); 1685
(C=O); and 1549 (C=N). 1H NMR chemical shifts at
(CDCl3, d ppm): 0.92 (d, 6H, –(CH3)2); 1.78–1.80 (m, 1H,
–CH–); 2.45 (d, 2H, –CH2–); 7.13– 7.17 (m, 4H, 2,3,5,6
ArH); 3.2 (q, 1H, –CH–); 1.2 (d, 3H, –CH3); 6.88 (s, 1H,
triazole NH); 6.54–8.09 (m, 3H, 2,5,6 ArH); and 12.34 (s,
2H, –(OH)2). Mass spectra of compound exhibited molec-
ular ion peak at m/z 425 (M?); other important fragments
were observed at 426 (M? ? 1), 427 (M? ? 2), 382, 292.
Synthesis of 4-(3-(1-(4-isobutylphenyl) ethyl)-5-thioxo-1H-
1,2,4-triazol-4(5H)-yl) phthalohydrazide (5b)
The product (5b) was obtained as per the previously
reported procedure (Abadi and Gehan, 2005) and reported
in ‘‘Synthesis of 4-(3-(1-(4-isobutylphenyl)ethyl)-5-thioxo-
1H-1,2,4-triazol-4(5H)-yl)benzohydrazide (5a)’’ section.
Yield: 88%; and M.P: 188–190°C. FTIR spectra of the
compound showed bands at 3445 (N–H); 3032 (Ar C–H);
1
Synthesis of 2-(2-(3-(1-(4-isobutylphenyl) ethyl)-5-thioxo-
1H-1,2,4-triazol-4(5H)-yl) phenyl) acetic acid (4c)
2933 (Aliphatic C–H); 1682 (C=O); and 1559 (C=N). H
NMR chemical shifts at (CDCl3, d ppm): 0.87 (d, 6H,
–(CH3)2); 1.82–1.84 (m, 1H, –CH–); 2.47 (d, 2H, –CH2–);
7.13–7.17 (m, 4H, 2,3,5,6 ArH); 3.0 (q, 1H, –CH–); 1.3 (d,
3H, –CH3); 6.89 (s, 1H, triazole NH); 6.42–7.88 (m, 3H, 3,
5,6 ArH); 9.56 (s, 2H, –(NH)2–); and 4.33 (s, 4H, (–NH2)2).
Mass spectra of the compound exhibited molecular ion
peak at m/z 453 (M?); other important fragments were
observed at 454 (M? ? 1), 455 (M? ? 2), 410, and 438.
The product 4c was obtained as per procedure reported in
‘‘Synthesis of 4-(3-(1-(4-isobutylphenyl)ethyl)-5-thioxo-
1H-1,2,4-triazol-4(5H)-yl) benzoic acid (4a) (McCall et al.,
1986)’’ section. Yield: 73%, and M.P: 148–150°C. FTIR
spectra of the compound showed bands at 3549 (O–H);
3445 (N–H); 3043 (Ar C–H); 2932 (Aliphatic C–H); 1678
(C=O); and 1539 (C=N). 1H NMR chemical shifts at
(CDCl3, d ppm): 0.88 (d, 6H, –(CH3)2); 1.80–1.82 (m, 1H,
–CH–); 2.43 (d, 2H, –CH2–); 7.13–7.17 (m, 4H, 2,3,5,6
ArH); 2.9 (q, 1H, –CH–); 1.1 (d, 3H, –CH3); 6.89 (s, 1H,
triazole NH); 6.34–6.82 (m, 4H, 2,3,4,5 ArH); 3.49 (s, 2H,
–CH2–); and 12.28 (s, 1H, –OH). Mass spectra of com-
pound exhibited molecular ion peak at m/z 395 (M?); other
important fragments were observed at 396 (M? ? 1), 397
(M? ? 2), 352, 262, and 380.
Synthesis of 2-(2-(3-(1-(4-isobutylphenyl) ethyl)-5-thioxo-
1H-1,2,4-triazol-4(5H)-yl) phenyl)acetohydrazide (5c)
The product (5c) was obtained as per the previously
reported procedure (Abadi and Gehan, 2005) and is
reported in ‘‘Synthesis of 4-(3-(1-(4-isobutylphenyl)ethyl)-
5-thioxo-1H-1,2,4-triazol-4(5H)-yl)benzohydrazide (5a)’’
Yield: 79%; and M.P: 178–180°C. FTIR spectra of the
compound showed bands at 3455 (N–H); 3043 (Ar C–H);
1
Synthesis of 4-(3-(1-(4-isobutylphenyl)ethyl)-5-thioxo-1H-
1,2,4-triazol-4(5H)-yl)benzohydrazide (5a)
2939 (Aliphatic C–H); 1678 (C=O); and 1549 (C=N). H
NMR chemical shifts at (CDCl3, d ppm): 0.88 (d, 6H,
–(CH3)2); 1.78–1.80 (m, 1H, –CH–); 2.43 (d, 2H, –CH2–);
7.13–7.17 (m, 4H, 2,3,5,6 ArH); 3.3 (q, 1H, –CH–); 1.2 (d,
3H, –CH3); 6.92 (s, 1H, triazole NH); 6.34–6.82 (m, 4H,
3,4,5,6 ArH); 3.44 (s, 2H, –CH2–); 9.08 (s, 1H, –NH–); and
4.35 (s, 2 H, –NH2). Mass spectra of the compound
exhibited molecular ion peak at m/z 409 (M?); other
The product (5a) was obtained as per the previously
reported procedure (Abadi and Gehan, 2005). A mixture of
(1 g, 3 mmol) of 4a and excess of thionyl chloride
(0.36 ml, 5 mmol) was refluxed for 4 h, excess thionyl
chloride was neutralized by using formic acid, and the
123

Med Chem Res (2011) 20:795–808
803
important fragments were observed at 410 (M? ? 1), 411
(M? ? 2), 366, and 394.
reported in ‘‘Synthesis of 2-chloro-N0-(2-(2-(3-(1-(4-iso-
butylphenyl)ethyl)-5-thioxo-1H-1,2,4-triazol-4(5H)-yl)
phenyl)acetyl)acetohydrazide (6a)’’ section. Yield: 75%;
and M.P: 210–212°C. FTIR spectra of the compound
showed bands at 3459 (N–H); 3042 (Ar C–H); 2934 (Ali-
phatic C–H); 1680 (C=O); 1547 (C=N); and 681 (C–Cl).
1H NMR chemical shifts at (CDCl3, d ppm): 0.87 (d, 6H,
–(CH3)2); 1.82–1.84 (m, 1H, –CH–); 2.43 (d, 2H, –CH2–);
7.13–7.17 (m, 4H, 2,3,5,6 ArH); 3.0 (q, 1H, –CH–); 1.3 (d,
3H, –CH3); 6.89 (s, 1H, triazole NH); 6.17–7.53 (m, 4H,
2,3,5,6 ArH); 10.75 (s, 1H, –NH–); 10.08 (s, 1H, –NH–);
2.46 (t, 2 H, –CH2–); and 3.66 (t, 2 H, –CH2–). Mass
spectra of the compound exhibited molecular ion peak at
m/z 485 (M?); other important fragments were observed at
486 (M? ? 1), 487 (M? ? 2), 442, and 470.
Synthesis of 2-chloro-N0-(2-(2-(3-(1-(4-
isobutylphenyl)ethyl)-5-thioxo-1H-1,2,4-triazol-4(5H)-
yl)phenyl)acetyl)acetohydrazide (6a)
The product (6a) was obtained as per the previously reported
procedure (Abadi and Gehan, 2005). A mixture of (1 g,
0.0024 M) of intermediate 5c was refluxed with 0.19 ml of
chloro-acetyl chloride in dry benzene (15 ml) and in the
presence of triethylamine (0.1 ml) for 10 h. The solution
was evaporated to dryness, the residue was washed with
acetone, the precipitate was filtered, washed with water, and
crystallized from absolute ethanol. Yield: 72%; and M.P:
208–210°C. FTIR spectra of the compound showed bands at
3455 (N–H); 3044 (Ar C–H); 2936 (Aliphatic C–H); 1684
Synthesis of N0-(2-chloroacetyl)-4-(3-(1-(4-isobutylphenyl)
ethyl)-5-thioxo-1H-1,2,4-triazol-4(5H)-yl)benzohydrazide
(6d)
1
(C=O); 1541 (C=N); and 685 (C–Cl). H NMR chemical
shifts at (CDCl3, d ppm): 0.89 (d, 6H, –(CH3)2); 1.82–1.84
(m, 1H, –CH–); 2.44 (d, 2H, –CH2–); 7.13–7.17 (m, 4H,
2,3,5,6 ArH); 3.0 (q, 1H, –CH–); 1.4 (d, 3H, –CH3); 6.83 (s,
1H, triazole NH); 6.34–6.82 (m, 4H, 3,4,5,6 ArH); 3.46 (s,
2H, –CH2–); 10.08 (s, 2H, –(NH)2–); and 4.27 (s, 2 H,
–CH2–). Mass spectra of the compound exhibited molecular
ion peak at m/z 485 (M?); other important fragments were
observed at 486 (M? ? 1), 487 (M? ? 2), 442, and 470.
The product (6d) was obtained as per the previously
reported procedure (Abadi and Gehan, 2005) and is
reported in ‘‘Synthesis of 2-chloro-N0-(2-(2-(3-(1-(4-iso-
butylphenyl)ethyl)-5-thioxo-1H-1,2,4-triazol-4(5H)-yl)
phenyl)acetyl)acetohydrazide (6a)’’ section. Yield: 82%;
and M.P: 228–230°C. FTIR spectra of the compound
showed bands at 3452 (N–H); 3043 (Ar C–H); 2926 (Ali-
phatic C–H); 1675 (C=O); 1554 (C=N); and 683 (C–Cl).
1H NMR chemical shifts at (CDCl3, d ppm): 0.84 (d, 6H,
–(CH3)2); 1.83–1.85 (m, 1H, –CH–); 2.41 (d, 2H, –CH2–);
7.13–7.17 (m, 4H, 2,3,5,6 ArH); 3.2 (q, 1H, –CH–); 1.1 (d,
3H, –CH3); 6.82 (s, 1H, triazole NH); 6.17–7.53 (m, 4H,
2,3,5,6 ArH); 10.71 (s, 1H, –NH–); 10.05 (s, 1H, –NH–);
and 4.27 (S, 2 H, –CH2–). Mass spectra of the compound
exhibited molecular ion peak at m/z 471 (M?); other
important fragments were observed at 472 (M? ? 1), 473
(M? ? 2), 428, and 456.
Synthesis of N01, N02-bis(2-chloroacetyl)-4-(3-(1-(4-
isobutylphenyl)ethyl)-5-thioxo-1H-1,2,4-triazol-4(5H)-
yl)phthalohydrazide (6b)
The product (6b) was obtained as per the previously reported
procedure (Abadi and Gehan, 2005) and is reported in
‘‘Synthesis of 2-chloro-N0-(2-(2-(3-(1-(4-isobutylphenyl)
ethyl)-5-thioxo-1H-1,2,4-triazol-4(5H)-yl)phenyl)acetyl)
acetohydrazide (6a)’’ section. Yield: 80%; and M.P:
222–224°C. FTIR spectra of the compound showed bands at
3465 (N–H); 3034 (Ar C–H); 2939 (Aliphatic C–H); 1687
1
(C=O); 1551 (C=N); and 689 (C–Cl). H NMR chemical
Synthesis of N01, N02-bis(3-chloropropanoyl)-4-(3-
(1-(4-butylphenyl) ethyl)-5-thioxo1H-1,2,4-triazol-
4(5H)-yl)phthalohydrazide (6e)
shifts at (CDCl3, d ppm): 0.84 (d, 6H, –(CH3)2); 1.85–1.87
(m, 1H, –CH–); 2.39 (d, 2H, –CH2–); 7.13–7.17 (m, 4H,
2,3,5,6 ArH); 2.9 (q, 1H, –CH–); 1.2 (d, 3H, –CH3); 6.87 (s,
1H, triazole NH); 6.42–7.88 (m, 3H, 2,5,6 ArH); 10.74 (s,
2H, –(NH)2–); 10.05 (s, 2H, –(NH)2–); and 4.25 (s, 4 H,
–(CH2)2–). Mass spectra of the compound exhibited molec-
ularion peakatm/z 605 (M?);other important fragments were
observed at 606 (M? ? 1), 607 (M? ? 2), 562, and 590.
The product (6e) was obtained as per the previously
reported procedure (Abadi and Gehan, 2005) and is
reported in ‘‘Synthesis of 2-chloro-N0-(2-(2-(3-(1-(4-iso-
butylphenyl)ethyl)-5-thioxo-1H-1,2,4-triazol-4(5H)-yl)
phenyl)acetyl)acetohydrazide (6a)’’ section. Yield: 78%;
and M.P: 214–216°C. FTIR spectra of the compound
showed bands at 3441 (N–H); 3046 (Ar C–H); 2939 (Ali-
phatic C–H); 1681 (C=O); 1552 (C=N); and 687 (C–Cl).
1H NMR chemical shifts at (CDCl3, d ppm): 0.87 (d, 6H,
–(CH3)2); 1.82–1.84 (m, 1H, –CH–); 2.43 (d, 2H, –CH2–);
7.13–7.17 (m, 4H, 2,3,5,6 ArH); 3.0 (q, 1H, –CH–); 1.2 (d,
N0-(3-chloropropanoyl)-4-(3-(1-(4-isobutylphenyl)ethyl)-5-
thioxo-1H-1,2,4-triazol-4(5H)-yl)benzohydrazide (6c)
The product (6c) was obtained as per the previously
reported procedure (Abadi and Gehan, 2005) and is
123

804
Med Chem Res (2011) 20:795–808
Synthesis of 2-(2-(4-(3-(1-(4-isobutylphenyl)ethyl)-
3H, –CH3); 6.86 (s, 1H, triazole NH); 6.42–7.88 (m, 3H, 2,
5, 6 ArH); 10.75 (s, 2H, –(NH)2–); 10.08 (s, 2H, –(NH)2–);
2.46 (t, 4 H, –(CH)2–); and 3.66 (t, 4 H, –(CH)2–). Mass
spectra of the compound exhibited molecular ion peak at
m/z 633 (M?); other important fragments were observed at
634 (M? ? 1), 635 (M? ? 2), 590, and 618.
5-thioxo-1H-1,2,4-triazol-4(5H)-yl)-2-(2-(2-
nitroacetyl)hydrazinecarbonyl) benzoyl) hydrazinyl)-bis-
2-oxoethyl nitrate (7b)
The product (7b) was obtained as per the previously
reported procedure (Abadi and Gehan, 2005) and is reported
in ‘‘Synthesis of 2-(2-(2-(2-(3-(1-(4-isobutylphenyl)ethyl)-
5-thioxo-1H-1,2,4-triazol-4(5H)-yl)phenyl)acetyl)hydrazi-
nyl)-2-oxoethyl nitrate (7a)’’ section. Yield: 70%; and M.P:
244–246°C. FTIR spectra of the compound showed bands at
3453 (N–H); 3045 (Ar C–H); 2949 (Aliphatic C–H); 1689
(C=O); 1561 (C=N); and 1519 (C–NO2). 1H NMR chemical
shifts at (CDCl3, d ppm): 1.2 (d, 9H, –(CH3)3); 2.02–2.09
(m, 1H, –CH–); 2.4 (d, 2H, –CH2–); 7.18–7.3 (m, 4H,
2,3,5,6 ArH); 3.02 (q, 1H, –CH–); 6.9 (s, 1H, triazole NH);
7.4–7.52 (m, 3H, 2, 5, 6 ArH); 8.23 (s, 4 H, –(NH)4–); and
4.4 (s, 4H, –(CH2)2–). Mass spectra of the compound
exhibited molecular ion peak at m/z 659 (M?); other
important fragments were observed at 644, 602, 498, 324,
238, and 152. Elemental analysis was observed as C
(47.36%), H (4.49%), and N (19.03%).
Synthesis of 3-chloro-N0-(2-(2-(3-(1-(4-isobutylphenyl)
ethyl)-5-thioxo-1H-1,2,4-triazol-4(5H)-
yl)phenyl)acetyl)propanehydrazide (6f)
The product (6f) was obtained as per the previously
reported procedure (Abadi and Gehan, 2005) and is
reported in ‘‘Synthesis of 2-chloro-N0-(2-(2-(3-(1-(4-iso-
butylphenyl)ethyl)-5-thioxo-1H-1,2,4-triazol-4(5H)-yl)
phenyl)acetyl)acetohydrazide (6a)’’ section. Yield: 74%;
and M.P: 204–206°C. FTIR spectra of the compound
showed bands at 3459 (N–H); 3051 (Ar C–H); 2946 (Ali-
phatic C–H); 1676 (C=O); 1551 (C=N); and 688 (C–Cl).
1H NMR chemical shifts at (CDCl3, d ppm): 0.89 (d, 6H,
–(CH3)2); 1.85–1.87 (m, 1H, –CH–); 2.46 (d, 2H, –CH2–);
7.13–7.17 (m, 4H, 2,3,5,6 ArH); 3.3 (q, 1H, –CH–); 1.1 (d,
3H, –CH3); 6.88 (s, 1H, triazole NH); 6.34–6.82 (m, 4H, 3,
4, 5, 6 ArH); 3.44 (s, 2 H, –CH2); 10.08 (s, 2H, –(NH)2–);
2.46 (t, 2H, –CH2–); and 3.66 (t, 2 H, –CH2–). Mass spectra
of compound exhibited molecular ion peak at m/z 499
(M?); other important fragments were observed at 500
(M? ? 1), 501 (M? ? 2), 456, and 484.
Synthesis of 3-(2-(4-(3-(1-(4-isobutylphenyl) ethyl)-
5-thioxo-1H-1,2,4-triazol-4(5H)-yl)benzoyl)hydrazinyl)-
3-oxopropyl nitrate (7c)
The product (7c) was obtained as per reported procedure
(Abadi and Gehan, 2005) and is reported in ‘‘Synthesis of
2-(2-(2-(2-(3-(1-(4-isobutylphenyl)ethyl)-5-thioxo-1H-
1,2,4-triazol-4(5H)-yl)phenyl)acetyl)hydrazinyl)-2-oxoeth-
yl nitrate (7a)’’ section. Yield: 80%; and M.P: 232–234°C.
FTIR spectra of the compound showed bands at 3475 (N–
H); 3053 (Ar C–H); 2933 (Aliphatic C–H); 1676 (C=O);
Synthesis of 2-(2-(2-(2-(3-(1-(4-isobutylphenyl)ethyl)-
5-thioxo-1H-1,2,4-triazol-4(5H)-
yl)phenyl)acetyl)hydrazinyl)-2-oxoethyl nitrate (7a)
The product (7a) was obtained as per the previously reported
procedure (Abadi and Gehan, 2005). A solution of the
intermediate 6a (0.85 g) in dry acetonitrile (2 ml) was
treated portionwise with a solution of AgNO3 (0.34 g) in dry
acetonitrile (5 ml), and the whole mixture was stirred at
room temperature for 3 h. The mixture was then filtered,
evaporated to dryness, and the residue was recrystallized
from absolute ethanol. Yield: 76%; and M.P: 236–238°C.
FTIR spectra of the compound showed bands at 3468 (N–H);
3043 (Ar C–H); 2951 (Aliphatic C–H); 1686 (C=O); 1549
1
1546 (C=N); and 1515 (C–NO2). H NMR chemical shifts
at (CDCl3, d ppm): 1.2 (d, 9H, –(CH3)3); 2.23–2.38 (m, 1H,
–CH–); 2.58 (d, 2H, –CH2–); 7.3–7.41 (m, 4H, 2,3,5,6
ArH); 3.1 (q, 1H, –CH–); 6.88 (s, 1H, triazole NH);
7.6–7.78 (m, 4H, 2, 3, 5, 6 ArH); 8.22 (s, 2 H, –(NH)2–);
2.58 (t, 2H, –CH2–); and 3.94 (t, 2H, –CH2–). Mass spectra
of the compound exhibited molecular ion peak at m/z 512
(M?); other important fragments were observed at 469,
379, 252, 133, and 90. Elemental analysis was observed as
C (56.10%), H (5.46%), and N (16.47%).
1
(C=N); and 1518 (C–NO2). H NMR chemical shifts at
(CDCl3, d ppm): 1.2 (d, 9H, –(CH3)3); 2.02–2.08 (m, 1H,
–CH–); 2.4 (d, 2H, –CH2–); 7.07–7.1 (m, 4H, 2,3,5,6 ArH);
3.02 (q, 1H, –CH–); 6.9 (s, 1H, triazole NH); 7.21–7.4 (m,
4H, 2, 3, 4, 5 ArH); 3.5 (s, 2 H, –CH2); 8.18 (s, 2H, –(NH)2–);
and 4.6 (s, 2H, –CH2–). Mass spectra of the compound
exhibited molecular ion peak at m/z 512 (M?), other
important fragments were observed at 497, 455, 379, 252,
134, and 104. Elemental analysis was observed as C
(56.33%), H (5.47%), and N (16.45%).
Synthesis of 2-(2-(4-(3-(1-(4-isobutylphenyl)ethyl)-
5-thioxo-1H-1,2,4-triazol-4(5H)-yl)benzoyl)hydrazinyl)-
2-oxoethyl nitrate (7d)
The product (7d) was obtained as per the previously reported
procedure (Abadi and Gehan, 2005) and iss reported in
‘‘Synthesis of 2-(2-(2-(2-(3-(1-(4-isobutylphenyl)ethyl)-
5-thioxo-1H-1,2,4-triazol-4(5H)-yl)phenyl)acetyl)
123

Med Chem Res (2011) 20:795–808
805
hydrazinyl)-2-oxoethyl nitrate (7a)’’ section. Yield: 83%;
and M.P: 254–256°C. FTIR spectra of the compound showed
bands at 3466 (N–H); 3042 (Ar C–H); 2943 (Aliphatic C–H);
7.14–7.21 (m, 4H, 3, 4, 5, 6 ArH); 3.5 (s, 2 H, –CH2–); 8.17
(s, 2H, –(NH)2–); 2.65 (t, 2H, –CH2–); and 3.82 (t, 2H,
–CH2–). Mass spectra of the compound exhibited molecular
ion peak at m/z 526 (M?); other important fragments were
observed at 483, 365, 190, 148, and 118. Elemental analysis
was observed as C (56.08%), H (5.61%), and N (16.34%).
1
1680 (C=O); 1551 (C=N); and 1523 (C–NO2). H NMR
chemical shifts at (CDCl3, d ppm): 0.84 (d, 9H, –(CH3)3);
2.01–2.3 (m, 1H, –CH–); 2.49 (d, 2H, –CH2–); 7.43–7.6 (m,
4H, 2,3,5,6 ArH); 3.22 (q, 1H, –CH–); 6.9 (s, 1H, triazole
NH); 6.63–6.77 (m, 4H, 2, 3, 5, and 6 ArH); 8.3 (s, 2 H,
–(NH)2–); and 4.3 (s, 2H, –CH2–). Mass spectra of the
compound exhibited molecular ion peak at m/z 498 (M?);
other important fragments were observed at 365, 337, 162,
119, and 76. Elemental analysis was observed as C (55.52%),
H (5.20%), and N (16.79%).
Pharmacology
Animals
Swiss albino mice of either sex weighing 20–25 g and
Wistar rats weighing in the range 140–160 g were obtained
from National Centre for Cell Sciences (NCCS), Pune,
India. All the animals were housed under standard ambient
conditions of temperature (25 2°C) and relative humid-
ity of 50 5%. A 12/12-h light/dark cycle was main-
tained. All the animals were allowed to have free access to
water and standard palletized laboratory animal diet 24 h
before pharmacological studies. All the experimental pro-
cedures and protocols used in this study were reviewed and
approved by the Institutional Animal Ethical Committee
(IAEC) of College, Pune, constituted in accordance with
the guidelines of the Committee for the Purpose of Control
and Supervision of Experiments on Animals (CPCSEA/
257, CPCSEA/300), Government of India.
Synthesis of 3-(2-(4-(3-(1-(4-isobutylphenyl)ethyl)-5-
thioxo-1H-1,2,4-triazol-4(5H)-yl)-2-(2-(2-nitroacetyl)
hydrazinecarbonyl) benzoyl) hydrazinyl)-bis-3-oxopropyl
nitrate (7e)
The product (7e) was obtained as per the previously
reported procedure (Abadi and Gehan, 2005) and is reported
in ‘‘Synthesis of 2-(2-(2-(2-(3-(1-(4-isobutylphenyl)ethyl)-
5-thioxo-1H-1,2,4-triazol-4(5H)-yl)phenyl)acetyl)hydrazi-
nyl)-2-oxoethyl nitrate (7a)’’ section. Yield: 65%; and M.P:
258–260°C. FTIR spectra of the compound showed bands at
3456 (N–H); 3031 (Ar C–H); 2935 (Aliphatic C–H); 1675
(C=O); 1541 (C=N); and 1519 (C–NO2). 1H NMR chemical
shifts at (CDCl3, d ppm): 1.24 (d, 9H, –(CH3)3); 2.17–2.22
(m, 1H, –CH–); 2.5 (d, 2H, –CH2–); 7.4–7.58 (m, 4H,
2,3,5,6 ArH); 3.18 (q, 1H, –CH–); 6.5 (s, 1H, triazole NH);
7.75–7.88 (m, 3H, 2, 5, 6 ArH); 8.18 (s, 4 H, –(NH)4–); 2.72
(t, 4H, –(CH2)2–); and 3.9 (t, 4H, –(CH2)2–). Mass spectra
of the compound exhibited molecular ion peak at m/z 687
(M?); other important fragments were observed at 644, 526,
342, 266, 180, and 124. Elemental analysis was observed as
C (49.04%), H (4.72%), and N (18.40%).
Preparation of test compounds
After suspending the test compounds in 1.0% aqueous
solution of sodium carboxymethyl cellulose (CMC), the
test samples were administered to the test animals orally.
The positive and negative control group animals received
the same experimental handling as those of the test groups
except that the drug-treated control group animals received
only appropriate volumes of vehicle and of the reference
drug, Ibuprofen.
Synthesis of 3-(2-(2-(2-(3-(1-(4-isobutylphenyl) ethyl)-
5-thioxo-1H-1,2,4-triazol-4(5H)-
yl)phenyl)acetyl)hydrazinyl)-3-oxopropyl nitrate (7f)
Anti-inflammatory activity
The product (7f) was obtained as per the previously reported
procedure (Abadi and Gehan, 2005) and is reported in
‘‘Synthesis of 2-(2-(2-(2-(3-(1-(4-isobutylphenyl)ethyl)-
5-thioxo-1H-1,2,4-triazol-4(5H)-yl)phenyl)acetyl)hydrazi-
nyl)-2-oxoethyl nitrate (7a)’’ section. Yield: 72%; and M.P;
236–238°C. FTIR spectra of the compound showed bands at
3446 (N–H); 3032 (Ar C–H); 2945 (Aliphatic C–H); 1685
Anti-inflammatory activity was evaluated using the well-
known Carrageenan-induced rat paw edema model of
Winter et al., 1962, using eight groups of six animals each.
A freshly prepared aqueous suspension of Carrageenan
(1.0% w/v, 0.1 ml) was injected in the subplanter 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 compounds, 1 h before the Carrageenan treatment. The
volume was measured before and after carrageenan treat-
ment at 60-min intervals with the help of digital plethys-
mometer (Panlab LE 7500, USA).
1
(C=O); 1545 (C=N); 1527 (C–NO2). H NMR chemical
shifts at (CDCl3, d ppm): 1.2 (d, 9H, –(CH3)3); 1.98–2.17
(m, 1H, –CH–); 2.39 (d, 2H, –CH2–); 7.21–7.4 (m, 4H,
2,3,5,6 ArH); 3.08 (q, 1H, –CH–); 6.84 (s, 1H, triazole NH);
123

806
Med Chem Res (2011) 20:795–808
Analgesic activity
In vitro and in vivo screening of nitric oxide
The analgesic activity was evaluated using the acetic acid
induced writhing method (Koster et al., 1959), using groups
of six animals each. A solution of acetic acid (1% v/v) in
distilled water was prepared and injected intraperitoneally
in a volume of 0.1 ml. One group was kept as control, and
the animals of the other group were pretreated with the test
compounds, 1 h before the acetic acid (1% v/v) treatment.
The writhing episodes were recorded for 15 min; stretching
movements consisting of arching of the back, elongation of
body, and extension of hind limbs were counted.
Vasorelaxing activity (Calderone et al., 1996; Ferrarini
et al., 1998)
In order to determine a possible vasodilator mechanism of
action, the compounds were tested on isolated aortae of
male normotensive Wistar rats (250–350 g). The rats were
sacrificed by cervical dislocation under light ether anes-
thesia and bled. The aortae were immediately excised,
freed of extraneous tissues, and prepared as multiple-ring
preparations. Then the vessels were suspended, under a
preload of 2 g, in 10-ml organ baths, containing Tyrode
solution (composition of saline in mM: NaCl 136.8; KCl
2.95; CaCl2 1.80; MgSO4 7H2O 1.05; NaH2PO4 0.41;
NaHCO3 11.9; Glucose 5.5), thermostated at 37°C and
continuously bubbled with a mixture of O2 (95%) and CO2
(5%). Changes in tension were recorded by means of an
isometric transducer (BIOPAC System, Inc., MP 35,
USA).
Acute ulcerogenicity studies
Acute ulcerogenicity screening was done according to
method reported by Cioli et al. (1979). The mucosal
damage was examined by means of an electron micro-
scope. For each stomach specimen, the mucosal damage
was assessed according to the following scoring system.
After an equilibration period of 60 min, the endothelial
integrity was confirmed by Acetylcholine (ACh) (55 lM)-
induced relaxation of Norepineohrine (NE. 20 lg/ml)
precontracted tissues. A relaxation C70% of the NE-
induced contraction was considered representative of an
acceptable presence of the endothelial layer, while the
organs, showing a relaxation \70%, were not used in the
experimental procedures. At 30–40 min after the confir-
mation of the endothelial integrity, the aortic preparations
were contracted by treatment with a single concentration of
NE (20 lg/ml) or KCl (30 mM), and when the contraction
reached a stable plateau, the test compounds in concen-
tration (0.1 mg/ml) were added cumulatively.
Score description
0.0 Normal (no injury, bleeding, and latent injury);
0.5 Latent injury or widespread bleeding ([2 mm);
1.0 Slight injury (2–3 dotted lines);
2.0 Severe injury (continuous lined injury or 5–6 dotted
injuries);
3.0 Very severe injury (several continuous lined
injuries);
4.0 Widespread lined injury or widened injury.
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 ulcer
score SEM, data analyzed by one way ANOVA fol-
lowed by Dunnett’s ‘t’ test to determine the significance of
the difference between the standard group and the rats
treated with the test compounds. The differences in results
were considered significant when P was found to be\0.01.
Detection of nitrite (Abadi and Gehan, 2005)
A solution of the appropriate compound (20 ll) in
dimethylsulfoxide (DMSO) was added to 2 ml of 1:1 v/v
mixture either of 50 mM phosphate buffer (pH 7.4) or of an
HCl solution (pH 1) with MeOH, containing 5 9 of 10-4
M L-cysteine. The final concentration of the test compound
was 10-4 M. After 1 h at 37°C, 1 ml of the reaction
mixture was treated with 250 ll of Griess reagent [sulfa-
nilamide (4 g), N-naphthylethylenediamine dihydrochlo-
ride (0.2 g), and 85% phosphoric acid (10 ml) in distilled
water (final volume: 100 ml)]. After 10 min at room tem-
perature, the absorbance was measured at 540 nm. Sodium
nitrite standard solutions (10–80 lmol/ml) were used to
construct the calibration curve. The results were expressed
as the percentage of NO released (n = 2) relative to a
theoretical maximum release of 1 mol NO/mol of test
compound.
Histopathological studies (Motilva et al., 2004; Omar
et al., 1996; Palaska et al., 2002)
For the histopathological study, rats were sacrificed 4 h after
the cold stress and their stomach specimens were removed
and put into 10% formalin solution. 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 white
solid paraffin. Morphological examination was performed
with Haematoxylin and Eosin staining to analyze histolog-
ical changes and examined under electron microscope.
123

Med Chem Res (2011) 20:795–808
Statistical analysis
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Data obtained for each set of anti-inflammatory model were
expressed as the mean of change in paw volume SD and
analyzed by one-way ANOVA followed by Dunnett’s test.
Data from acetic acid-induced writhing model were
expressed as the mean of number of writhes SEM and
analyzed by one way ANOVA followed by Dunnett’s ‘t’
test. Level of significance was set to P \ 0.05. All the
statistical calculations were performed using evaluation
version of Graph Pad_ Prism 3.0 (USA) statistical software.
Acknowledgments The authors are thankful to The Principal,
AISSMS College of Pharmacy, Pune, for providing laboratory facil-
ities. The authors are also thankful to Prof. N. S. Vyawahare, Head,
the Department of Pharmacology, for his continuous support and
guidance throughout the project. The authors also wish to thank the
National Centre for Cell Sciences (NCCS), Pune, Shimadzu Analyt-
ical Centre, and the Department of Chemistry, University of Pune, for
providing facilities for spectral studies of compounds. The authors are
also thankful to Inland Pharmaceuticals Ltd. Pune, for providing pure
Ibuprofen, and Ipca Pharmaceuticals Ltd. Pune, for providing Dic-
lofenac sodium. The authors are also thankful to Satavs pathology
laboratory, Pune, for providing facilities for the histopathological
studies.
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