Design, Synthesis and Pharmacological Screening of Novel Antihypertensive Agents Using Hybrid Approach

Article abstract of DOI:10.1016/j.bmc.2008.10.032

Eight derivatives of general formula 2-(2-(4-(3-((5-substituted methylene)-4-oxo-2-(phenylimino)thiazolidin-3-yl)-2-hydroxypropylamino)b enzoyl)hydrazinyl)-2-oxoethyl nitrate were synthesized and tested for electrocardiographic, antiarrhythmic, vasorelaxing and antihypertensive activity as well as for in-vitro nitric oxide (NO) releasing ability. Compound 8b 2-(2-(4-(3-(5-benzyliden-4-oxo-2-(phenylimino)thiazolidin-3-yl)-2-hydrox ypropylamino)benzoyl)hydrazinyl)-2-oxoethyl nitrate, was the most potent in this series. The pharmacological results suggested that the antiarrhythmic effects of these compounds were related to their adrenolytic properties which are believed to be due to the presence of the 5-(substituted)methylen-2-(phenylimino)thiazolidin-4-one moiety with less bulky, electron donating substituent on the phenyl ring at 5th position of the thiazolidin-4-one. In conclusion, most of the synthesized compounds were significantly potent as antiarrhythmic and antihypertensive; this might be due to the presence of different pharmacopores which might act at different locations with different mode of action. Further insights of the same can be obtained by doing investigation at receptor level. The potency of compounds 8a-8h were promising enough to continue further experiments.

Full text of DOI:10.1016/j.bmc.2008.10.032

Bioorganic & Medicinal Chemistry 17 (2009) 390–400
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, Synthesis and Pharmacological Screening of Novel Antihypertensive
Agents Using Hybrid Approach
*
Shashikant V. Bhandari , Kailash G. Bothara, Ajit A. Patil, Trupti S. Chitre, Aniket P. Sarkate,
Suraj T. Gore, Sudarshan C. Dangre, Chetan V. Khachane
Dept. of Pharm. Chem. (PG wing), A.I.S.S.M.S. College of Pharmacy, Near RTO, Kennedy Road, Pune 411 001, Maharashtra, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Eight derivatives of general formula 2-(2-(4-(3-((5-substituted methylene)-4-oxo-2-(phenylimino)thiaz-
olidin-3-yl)-2-hydroxypropylamino)benzoyl)hydrazinyl)-2-oxoethyl nitrate were synthesized and tested
for electrocardiographic, antiarrhythmic, vasorelaxing and antihypertensive activity as well as for in-vitro
nitric oxide (NO) releasing ability. Compound 8b 2-(2-(4-(3-(5-benzyliden-4-oxo-2-(phenylimino)thiaz-
olidin-3-yl)-2-hydroxypropylamino)benzoyl)hydrazinyl)-2-oxoethyl nitrate, was the most potent in this
series. The pharmacological results suggested that the antiarrhythmic effects of these compounds were
related to their adrenolytic properties which are believed to be due to the presence of the 5-(substi-
tuted)methylen-2-(phenylimino)thiazolidin-4-one moiety with less bulky, electron donating substituent
on the phenyl ring at 5th position of the thiazolidin-4-one. In conclusion, most of the synthesized com-
pounds were significantly potent as antiarrhythmic and antihypertensive; this might be due to the pres-
ence of different pharmacopores which might act at different locations with different mode of action.
Further insights of the same can be obtained by doing investigation at receptor level. The potency of com-
pounds 8a–8h were promising enough to continue further experiments.
Received 19 July 2008
Revised 11 October 2008
Accepted 14 October 2008
Available online 17 October 2008
Keywords:
Hybrid
Antiarrhythmic
Antihypertensive
Vasorelaxation
NO donor
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
in one molecule, so called hybrid molecules. These hybrid mole-
cules often consist of different pharmacophoric groups which are
Most of currently used antihypertensive agents can not be used
as a single drug therapy because of several toxicities, side effects
associated with these drugs. Multiple dose therapies make the re-
gime complicated and less successful. One of the most important
goals in organic and medicinal chemistry is the design and synthe-
sis of molecules that have value as therapeutic agents. In this re-
gard, heterocyclic compounds have proven to be versatile
support structures that offer a high degree of structural diversity.1
A problem often faced in the treatment of certain diseases is the
complex and heterogeneous pathogenesis. A variety of mediators
can be involved in the pathophysiological process leading to a dis-
ease, in many instances; treatment with a single drug cannot ade-
quately control the illness. Sometimes, the use of a therapeutic
agent alone in the treatment of a disease may be limited by side ef-
fects caused by its own action. Then combinations of drugs with
different pharmaco-therapeutic effects are feasible. Combination
drug therapy can be applied either to overcome the side effects
of the single drug or to add beneficial effects. The principle of com-
bination drug therapy can be achieved by either using concomitant
administration of two or more single active drugs or by designing
drugs in which more than one active pharmacophore are combined
linked to each other via spacers.2
In the long-term treatment of hypertension, there is no single
antihypertensive drug which is able to normalize the elevated
blood pressure in all hypertensive patients. b-Adrenoceptors are
known to play an important role in the regulation of the autonomic
nervous system and b-blockers have been shown to be useful in
the pharmacotherapy of serious and widespread cardiovascular
diseases.3,4
Nitric oxide (NO) donors have been used for many years in the
treatment of various clinical conditions, particularly coronary arte-
rial diseases.5 Though vasodilatation reduces blood pressure by
directly relaxing peripheral vascular smooth muscles, the reduc-
tion in blood pressure leads to a reflux increase in sympathetic
tone, followed by increase in heart rate, cardiac output, and plasma
rennin activity, which attenuate antihypertensive effect. It was
reported that these undesirable effects of vasodilators could be
eliminated by simultaneous treatment with b-blockers and the
possible increase in peripheral vascular resistance induced by
b-blockers was eliminated by concomitant use of vasodilators. In
order to improve patient compliance, we have developed hybrid
compounds containing three different pharmacophores viz. propa-
nolamine possessing the characteristics of a typical b-antagonist,
thiazolidinone component responsible antiarrythmic activity and
of a ‘slow NO donor’, by adding NO-donor side chain. These new
* Corresponding author. Tel.: +91 20 26058204; fax: +91 20 26058208.
E-mail address: drugdesign1@gmail.com (S.V. Bhandari).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.032

S. V. Bhandari et al. / Bioorg. Med. Chem. 17 (2009) 390–400
391
compounds, displayed vasorelaxing effects, due to the release of
NO. Therefore, hybrid molecules with vasodilating and nitric oxide
(NO), which is constitutively produced by endothelial nitric oxide
synthase and acts upon the guanylate cyclase–cGMP pathway, is
required for the maintenance of blood-vessel homeostasis, blood
pressure, and organ perfusion.6
O
N
S
COCl
C
O
H
Ph-NH2
NH4SCN
C
N
Dry Acetone
+
N
C
Δ 10 min
-NH4Cl
Dry Acetone
Δ for 1 h
H
S
Ammonium
thiocyanate
Benzoyl
chloride
benzoyl
isothiocyanate
N
-(phenylcarbamothioyl)
benzamide
NaOH
H2O
S
R
CHO
COOH
C2H5OH (95%)
N
HN
C NH2
N
C2H5OH (95%)
S
S
Fused CH3COONa
N
H
+
N
CH
R
Fused CH3COONa,
Reflux 3.5-7h
H
ClCH2COOC2H5
O
O
Ethylchloroacetate
Benzoic
acid
(2)
(1)
(3a-3h)
Cl
C2H5OH,
Caustic soda,
Reflux 1-3 h.
O
N
Epichlorhydrine
N
S
S
N
HO
HN
CH
R
i) SOCl2,
ii) NH2NH2, H2O
N
HO
HN
CH
R
NH2
N
HOOC
O
S
O
C2H5OH,
reflux 9-10 h
p-Aminobenzoic acid
N
CH
R
NaHCO3, C2H5OH
Reflux 15-17 h
O
(6a-6h)
O
(5a-5h)
CONHNH2
(4a-4h)
COOH
Triethylamine,
Reflux 9-10 h
ClCOCH2Cl,
Dry Benzene,
N
N
S
S
N
HO
HN
CH
R
N
HO
HN
CH
R
AgNO ,
CH3CN
3
O
O
Stirr 2.5-3 h at RT
CONHNHCOCH2Cl
CONHNHCOCH2ONO2
(7a-7h)
(8a-8h)
Compound code
8a
-R
Compound code
8e
-R
CH3
NO2
OCH3
OCH3
OCH3
8b
8f
8c
8d
8g
8h
OH
F
HO
Scheme 1. Synthetic route for title compounds 8a–8h.

392
S. V. Bhandari et al. / Bioorg. Med. Chem. 17 (2009) 390–400
A research group in the People’s Republic of China noted in
literature,15 using ammonium thiocyanate and benzoyl chloride.
This process was optimized in the presence of dry acetone. The
acetone is dried for at least 48 h over anhydrous calcium sulfate
and distilled just before it is used. 2-Phenyliminothiazolidin-4-
one (2) was prepared by heating of phenyl thiourea and ethyl-
chloroacetate in the presence of fused sodium acetate in ethanol
(95%).16 The reaction of (2) with substituted aromatic aldehydes
in presence of fused sodium acetate in ethanol gave correspond-
ing 5-(substituted) methylene-2-(phenylimino)thiazolidin-4-one
(3a–3h) in very good yields. The aim of the next step was to
introduce reactive 2,3-epoxypropyl moiety at the 3rd position
of thiazolidin-4-one. These compounds (3a–3h) were reacted
with epichlorhydrine.17,18 The syrupy reside, the hydrochloric
salt of the compound (4a–4h) was used immediately in the next
step. In the following step, the epoxy bridge was cleaved with
primary amine: p-aminobenzoic acid to obtain compounds (5a–
5h). The reaction of (5a–5h) with thionyl chloride followed by
hydrazine hydrate yielded the corresponding hydrazides (6a–
6h). The reaction of these hydrazides with the chloroacetyl chlo-
ride, gave the corresponding haloalkyl derivatives (7a–7h).19 The
terminal chloro function was then converted to the target nitrate
derivatives (8a–8h)19 with AgNO3 in acetonitrile. The structures
of various synthesized compounds were assigned on the basis
of different chromatographic, spectral studies and qualitative
and quantitative organic analytical studies. The physical data,
FTIR, 1H NMR and Mass spectral data for all the synthesized
compounds are reported in experimental protocols.
1979, while examining the antimalarial properties of derivatives
of Febrifugine, that one compound, changrolin, was effective as
an arrhythmic agent during clinical trials. Stout and his research
group studied the structure of changrolin for its dissimilarity with
currently marketed antiarrhythmics7–11; there are also other stud-
ies about the biological structure activity relationships.7,12,13 And
so, we take the 2-arylimino-5-arylidene-4-thiazolidinones rings
as a structural requirement to show cardiovascular effects and
we change the pyrrolidin-one14 ring to substituted thiazolidinone
ring. In this paper, we report, design synthesis and antihyperten-
sive screening of new 2-arylimino-5-aryliden-4-thiazolidinone
derivatives with significant cardiovascular actions.
These types of cardiovascular agents, as single entities, can be
applied as antiarrhythmics, vasodilators and antihypertensive
drugs. Pharmacological assays of compounds containing multiple
pharmacophores designed by a so called hybrid approach have
been proven to be successful as antihypertensive agents, indicating
correctness of rationale based on which these NCEs were designed.
2. Results and discussions
2.1. Chemistry
The synthetic route used to synthesize title compounds
is outlined in Scheme 1. Phenyl thiourea (1), the starting mate-
rial, was prepared according to the method reported in the
Table 1
Effects of an intraperitoneal injection of the investigated compounds (40 mg/kg ip) on heart rate and ECG intervals in anaesthetized Wistar rats
Compound code
Parameters
Time of observation (min)
30
0
15
45
60
8a
Beats per minute
P–Q (ms)
QRS (ms)
331.13 9.110
54.8 1.393
20.4 1.030
70.4 1.030
318.61 9.744
57 1.265
21.4 0.927
71.4 0.927
295.46 9.337*
57.8 1.393
22.4 1.030
72.4 1.030
302.16 8.015
56.2 1.020
21.8 0.860
71.8 0.860
315.86 7.085
55.6 1.364
20.4 0.927
70.4 0.927
Q–T (ms)
8b
8c
8d
8e
8f
Beats per minute
P–Q (ms)
QRS (ms)
305.51 15.394
52 1.581
18.2 0.583
80.6 1.208
292.18 14.572
52.2 1.158
19.4 0.510
81.8 1.356
269.11 13.662
53.6 1.503
21.2 0.583*
63.2 1.241
272.68 7.678*
56 1.304
276.83 12.563
54.2 1.319
20.6 0.812
84.6 1.327
258.51 5.552**
56.2 1.158
22.4 0.748
77.2 1.393
291.80 12.511
55.4 1.288
21.6 0.872**
86.2 1.241*
Q–T (ms)
Beats per minute
P–Q (ms)
QRS (ms)
307.48 11.291
53.2 1.463
19.6 0.812
73.6 1.435
290.03 8.505
55.2 1.020
20.4 0.748
74.8 1.068
278.31 5.403
54.8 1.158
20.6 0.678
74.4 1.288
21.4 0.748
76 0.949
Q–T (ms)
Beats per minute
P–Q (ms)
QRS (ms)
305.08 6.285
50.2 1.772
20 0.894
298.24 6.613
51.4 1.691
21.2 1.158
73.2q1.772
291.85 5.820
54.2 1.828
22.4 0.925
74.4 1.860
279.28 5.841*
54.6 1.503
23.4 0.812*
75.4 1.806
294.57 5.583
52.8 1.463
20.8 0.663
72.8 0.860
Q–T (ms)
72 1.949
Beats per minute
P–Q (ms)
QRS (ms)
311.36 12.127
49.2 1.562
22.6 0.927
72.2 1.281
303.44 11.524
49.6 1.435
23.2 0.800
72.8 1.158
291.92 9.614
51.2 1.715
23.6 0.927
73.4 1.166
279.98 8.305
52.0 1.517
24.0 0.707
74.8 0.970
277.12 7.480*
57.6 1.288
23.2 0.583*
82.2 0.970
297.59 8.50
50.2 1.393
22.8 0.735
72.2 0.860
Q–T (ms)
Beats per minute
P–Q (ms)
QRS (ms)
310.70 9.077
53.4 1.720
20.2 0.860
79.2 0.860
301.97 9.291
55 1.140
21.2 0.735
79.6 0.927
293.35 8.420
56.2 1.356
22 0.548
291.20 8.371
54.8 1.393
22.8 0.583*
83.8 0.800**
Q–T (ms)
80.8 1.020
8g
8h
Beats per minute
P–Q (ms)
QRS (ms)
306.87 6.011
50.6 1.503
22 1.049
300.28 5.861
51.7 1.691
22.8 1.020
67.8 1.020
289.83 5.914
52.2 1.463
23.6 0.927
68.6 0.727
294.11 5.496
52.4 1.503
23.2 0.735
68.2 0.735
300.39 5.931
52.2 1.772
22 0.548
Q–T (ms)
67 1.049
66.4 0.812
Beats per minute
P–Q (ms)
QRS (ms)
314.28 10.707
51.6 2.293
20.4 0.814
73.2 1.655
307.46 10.493
52.4 2.135
21. 0.633
295.42 9.166
53.6 2.088
22.4 0.510
75.8 1.594
302.23 9.196
53.6 2.462
21.4 0.510
74.8 1.655
308.77 8.876
52.4 2.293
20.4 0.980
73.4 1.806
Q–T (ms)
74.6 1.435
The data are the means of five experiments SEM. Statistical analyses were performed using a one-way ANOVA followed by Dunnett’s test.
*
P < 0.05.
P < 0.01.
**
*** P < 0.001.

S. V. Bhandari et al. / Bioorg. Med. Chem. 17 (2009) 390–400
393
The FTIR spectra of the title compounds (8a–8h) showed char-
acteristic functional groups like –OH, –C@N stretching vibrations,
amide C@O stretching, specific band for –NO2 vibrations etc. In
the proton NMR spectral data, all protons were seen according to
the expected chemical shift and integration values. The signals
belonging to benzylidene group were observed at aromatic region,
while the signals belonging to –NHNH2 disappeared indicating
functionalization of hydrazide to haloalkyl derivatives with chloro-
acetyl chloride.
2.2. Pharmacology
In the present study, the following pharmacological tests
were carried out to assess the effect on normal electrocardio-
gram (ECG), antiarrhythmic, hypotensive and nitric oxide releas-
ing assay of synthesized compounds. The effect on ECG intervals
and the heart rate were determined for all compounds at a dose
of 40 mg/kg ip. Electrocardiographic experiments showed that
compounds 8b, 8c and 8f at a dose of 40 mg/kg, that is, signifi-
cantly decreased the number of cardiac beats per minute, pro-
longed P–Q intervals, QRS complex (8b) and Q–T intervals (8c,
8f), (Table 1).
All compounds were tested for antiarrhythmic activity using
BaCl2 induced arrhythmia. For experimental animals, intravenous
injections of high dose of BaCl2 (32 mg/kg) caused sinus brady-
cardia and a rapid ventricular extrasystoles, ventricular tachycar-
dia and ventricular fibrillation (100%), which led to the death of
all animals within 3–5 min. Among synthesized compounds only
8b, 8c and 8d were more effective in BaCl2 induced arrhythmia
test. Compounds 8f, 8g and 8h exhibited weak antiarrhythmic
properties after ip administration, slightly reducing number of
barium chloride-induced rhythm disturbances and mortality.
The lowest activity was demonstrated by compound 8a, which
was not active at dose of 40 mg/kg ip in 100% of experimental
animals following their death within 4–5 min after BaCl2
administration.
Atenolol 8a 8b 8c 8d
100
*
**
**
**
**
80
60
40
20
0
**
**
**
**
**
**
**
**
**
**
**
0
15
30
Time (in minutes)
45
60
Figure 1. Hypotensive activity of compounds 8a, 8b, 8c and 8d tested after ip
administration in anaesthetized normotensive rats.
The title compounds (8a–8h) (40 mg/kg, ip) produced blood
pressure lowering (Fig. 1 and Fig. 2) and heart rate decreasing ef-
fects in thiopental-anesthetized normotensive Wistar rats. Blood
pressure lowering effect of compounds 8a, 8b, 8e, 8h and 8g
reached their maximum about 30 min after dosing and only com-
pounds 8c, 8d and 8f lasted for 45 min (Table 2). Atenolol at a dose
of 10 mg/kg, produced significant reduction in blood pressure at
30 min after dosing.
In isolated Wistar rat aorta rings, compounds 8a–8h compet-
itively inhibited norepinephrine-induced contraction effects,
causing a shift to the right of the norepinephrine concentration
response curves. EC50 (g/mL) values were calculated from the
cumulative concentration (dose) response curves. In order to
prove the involvement of nitric oxide in the relaxation process,
nitric oxide releasing properties of synthesized compounds were
Atenolol 8e 8f 8g 8h
100
**
**
****
**
**
**
**
**
80
60
40
20
0
**
**
**
**
**
**
0
15
30
45
60
Time (in minutes)
assessed in phosphate buffer, pH 7.4, in the presence of L-cys-
teine, relative to nitric oxide released from standard sodium ni-
trite solution (Table 3).
Figure 2. Hypotensive activity of compounds 8e, 8f, 8g and 8h tested after ip
administration in anaesthetized normotensive rats.
Table 2
Responses (MAP) versus time (min), following the administration of the compounds (40 mg/kg ip)
Compound code
Mean arterial pressure (MAP) in mm Hg
0 min
15 min
30 min
45 min
60 min
Atenolol
8a
8b
8c
8d
8e
8f
8g
8h
92.06 1.923
94.78 1.301
92.30 1.048
91.39 1.844
93.80 0.720
95.32 1.744
92.99 2.062
91.09 1.699
95.65 1.926
79.01 1.19
63.44 1.404
85.08 0.815**
62.21 0.738**
68.96 1.163**
72.44 0.605**
79.16 0.993**
73.93 1.342**
78.92 0.502**
77.54 0.933**
65.24 1.100
85.87 1.086**
67.54 0.537**
63.34 1.063**
64.32 0.619**
72.21 0.907**
62.69 1.117**
80.53 0.788**
80.16 0.920**
69.93 0.99
87.32 1.413**
78.67 0.471**
79.52 1.182**
82.91 1.132**
91.10 1.192Ns
82.81 2.052**
84.89 1.477**
84.86 1.618**
90.29 1.026*
71.29 0.645**
77.31 1.781**
76.24 0.202**
88.93 1.091**
81.25 0.979**
85.23 0.885**
90.58 0.805**
The data are the means of five experiments SEM. Statistical analyses were performed using a one-way ANOVA followed by Dunnett’s test.
*
P < 0.05.
P < 0.01.
**
*** P < 0.001.
Ns
P > 0.05.

394
S. V. Bhandari et al. / Bioorg. Med. Chem. 17 (2009) 390–400
Table 3
NMR) spectrophotometer using TMS as an internal reference
(Chemical shift represented in (d ppm). Mass spectra were re-
corded on GC–MS QP5050A System (benchtop quadruple mass
spectrophotometer). Microanalyses were performed for C, H, and
N using instrument FLASHEA 1112 series. Purity of the compounds
was checked on TLC plates using silica gel G as stationary phase
and iodine vapors as visualizing agent.
EC50 and nitric oxide releasing properties of the compounds (8a–8h)
Compound code
EC50
% NO releasea
8a
8b
8c
8d
8e
8f
141.25
34.68
32.36
45.71
28.18
60.26
48.98
34.67
0.21
0.33
0.30
0.29
0.27
0.32
0.23
0.24
4.1. Synthesis of phenyl thiourea (1)
8g
8h
The phenyl thiourea was prepared as per reported method.
Yield: 55.79% (solid); mp 152–154 °C. FTIR (KBr) cmꢀ1: 3325
(N–H); 2970 (C–H); 1221 (C@S).
a
Percentage of NO released (n = 2) relative to a theoretical maximum 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.
4.2. Synthesis of 2-phenyliminothiazolidin-4-one (2)
3. Conclusions
Phenyl thiourea 16 g (0.08 mole) was dissolved in 32.89 mL
ethanol (95%). The resulting solution was refluxed with fused so-
dium acetate 8.63 g (0.105 mole) and ethylchloroacetate 12.94 g
(11.30 mL, 0.106 mole) for 3.5 h. The mixture was then poured
into 250 mL of water. The solution deposits the crystalline prod-
uct; the product was filtered and dried. The compound was
recrystallized using ethanol (95%). Yield: 70.79% (solid); mp
172–174 °C. FTIR (KBr) cmꢀ1: 3410 (N–H); 2970 (C–H); 1742
(C@O); 1610 (C@N). 1H NMR (300 MHz, CDCl3, d ppm): 3.21 (s,
2H, CH2); 6.98–7.34 (m, 5 H, phenyl); 11.82 (br s, 1H, NH). Mass
spectra of compound exhibited molecular ion peak at m/z 192
(M+), other important fragments were observed at 193 (M+1),
194 (M+2).
The present screening of hypotensive and vasorelaxing activi-
ties of title compounds (8a–8h) has led to the identification of sev-
eral potent cardiovascular agents. The results from this study
indicate that 8b, 8c, 8d and 8f at given concentration are effective
hypotensive agents and their inhibitory effect on the contractile re-
sponse caused by norepinephrine in rat thoracic aorta may be due
to the inhibition of Ca2+ influx through both voltage-dependent
and receptor-operated Ca2+ channels. Since these compounds have
higher molecular weight, due to high lipophility, these compounds
may be easily partitioned into the cell membrane of vascular
smooth muscles where the binding events occur.
Compound 8b was the most potent in this series. The pharma-
cological results suggested that the antiarrhythmic effects of these
compounds were related to their adrenolytic properties and those
properties were due to the presence of the 5-(substituted) methy-
len-2-(phenylimino)thiazolidin-4-one moiety with less bulky,
electron donating substituent on the phenyl ring at 5th position
of the thiazolidin-4-one. The mode of action and activity of com-
pounds 8a–8h were promising enough to continue further
experiments.
From the detailed analysis of pharmacological activities of syn-
thesized compounds, we concluded that the rationale based on
which we have designed hybrid molecules, containing multiple
pharmacophore has proven to be promising at least in these preli-
minary in vivo pharmacological screening models.
4.3. General procedure for the preparation of compounds (3a–
3h)
Mixture of aldehyde (0.02 mole), 2-phenyliminothiazolidin-4-
one (0.02 mole) and fused sodium acetate (0.04 mole) was taken
in 15 mL of absolute ethanol. The resultant mixture was refluxed
for 3.5–7 h. The hot mixture was then poured into 50 mL of water
and kept overnight. The precipitate obtained was filtered and
washed with water (30–40 mL sufficient to remove unreacted
aldehyde) and then dried. The product was recrystallized using
dimethyl formamide.
4.3.1. 5-(3-Nitrobenzyliden)-2-(phenylimino)thiazolidin-4-one
(3a)
Yield: 90.77% (solid); mp 269–271 °C. FTIR (KBr) cmꢀ1: 3421
(N–H); 3031 (Ar C–H); 1738 (C@O); 1607 (C@N); 1498 (C–NO2).
1H NMR (CDCl3, d ppm): 6.92–7.36 (m, 5H, phenyl); 7.62 (s, 1H,
C@CH); 7.73–8.29 (m, 4H, 3-nitrobenzylidene); 8.9 (br s, 1H, NH).
Mass spectra of compound exhibited molecular ion peak at m/z
325 (M+), 326 (M+1), 327 (M+2).
3.1. Scope for further research
The most potent compounds will be subjected to following
studies:
1. Acute and chronic toxicity studies
2. Mechanisms of release of NO and in turn vasodilatory activity
3. Pharmacokinetic profile
4. Mechanism of action
5. Possibility of metabolic activation and if yes, mechanisms of
degradation of the compounds etc.
4.3.2. 5-Benzyliden-2-(phenylimino)thiazolidin-4-one (3b)
Yield: 82.14% (solid); mp 219–221 °C. FTIR (KBr) cmꢀ1: 3442
(N–H); 3018 (Ar C–H); 1716 (C@O); 1620 (C@N). 1H NMR (CDCl3,
d ppm): 6.99–7.60 (m, 10H, phenyl and benzylidene); 7.82 (s, 1H,
C@CH); 8.2 (br s, 1H, NH). Mass spectra of compound exhibited
molecular ion peak at m/z 280 (M+), 281 (M+1), 282 (M+2).
Latter if results are found to be promising then further decision
of starting clinical studies will be taken.
4.3.3. 5-(4-hydroxybenzyliden)-2-(phenylimino)thiazolidin-4-
one (3c)
4. Experimental
Yield: 72.64% (solid); mp 219–221 °C. FTIR (KBr) cmꢀ1: 3551
(O–H); 3467 (N–H); 3042 (Ar C–H); 1742 (C@O); 1641 (C@N). 1H
NMR (CDCl3, d ppm): 6.81 (d, 2H, benzylidene); 6.93–7.29 (m,
5H, phenyl); 7.44 (d, 2H, benzylidene); 7.68 (s, 1H, C@CH); 8.34
(br s, 1H, NH); 9.92 (s, 1H, OH). Mass spectra of compound exhib-
ited molecular ion peak at m/z 296 (M+), 297 (M+1), 298 (M+2).
Melting points were determined by open capillary tubes and are
uncorrected. FTIR spectra of the powdered compounds were re-
corded using KBr on a Jasco FTIR V 430+ spectrometer using Diffuse
Reflectance Attachment and are reported in cmꢀ1 and 1H NMR
spectra were recorded on a Varian Mercury YH300 (300 MHz FT

S. V. Bhandari et al. / Bioorg. Med. Chem. 17 (2009) 390–400
395
4.3.4. 5-(2-Hydroxybenzylidene)-2-(phenylimino)thiazolidin-4-
one (3d)
4.5. General procedure for the preparation of compounds 5a–5h
Yield: 79.39% (solid); mp 221–223 °C. FTIR (KBr) cmꢀ1: 3565
(O–H); 3442 (N–H); 3029 (Ar C–H); 1737 (C@O); 1599 (C@N). 1H
NMR (CDCl3, d ppm): 6.71–7.31 (m, 9H, phenyl and benzylidene);
7.69 (s, 1H, C@CH); 8.40 (br s, 1H, NH); 9.85 (s, 1H, OH). Mass spec-
tra of compound exhibited molecular ion peak at m/z 296 (M+), 297
(M+1), 298 (M+2).
Compound 4a–4h (0.008 mole) and 1.11 g (0.0081 mol) of p-
aminobenzoic acid was dissolved in ethanol (30 mL). To the above
solution, sodium bicarbonate 0.68 g (0.0081 mole) was added. The
reaction mixture was refluxed for 15–17 h; then concentrated to
remove excess of alcohol; cold water was added with vigorous stir-
ring. The precipitated product obtained was filtered, washed with
water and recrystallized from ethanol to yield respective deriva-
tives 5a–5h.
4.3.5. 5-(4-Methylbenzyliden)-2-(phenylimino)thiazolidin-4-
one (3e)
Yield: 73.13% (solid); mp 207–209 °C. FTIR (KBr) cmꢀ1: 3461
(N–H); 3038 (Ar C–H); 2954 (aliphatic C–H) 1741 (C@O); 1600
(C@N); 1236 (C–O). 1H NMR (CDCl3, d ppm): 2.38 (s, 3H, CH3);
6.82–7.27 (m, 5H, phenyl); 7.36 (d, 2H, benzylidene); 7.52 (d, 2H,
benzylidene); 7.79 (s, 1H, C@CH); 8.12 (br s, 1H, NH). Mass spectra
of compound exhibited molecular ion peak at m/z 294 (M+), 295
(M+1), 296 (M+2).
4.5.1. 4-(2-Hydroxy-3-(5-(3-nitrobenzyliden)-4-oxo-2-(phenyli-
mino)thiazolidin-3-yl)propylamino)benzoic acid (5a)
Yield: 57.52% (solid); mp 47–49 °C. FTIR (KBr) cmꢀ1: 3572 (O–
H); 3398 (N–H); 3044 (Ar C–H); 1657 (C@O); 1619 (C@N); 1510
(C–NO2). 1H NMR (CDCl3, d ppm): 3.18 (d, 4H, CH2 of alkyl chain
at N3); 4.09 (m, 1H, CH); 4.69 (s, 1H, OH); 6.78–7.61 (m, 13H, aro-
matic); 7.99 (s, 1H, C@CH); 8.01 (s, 1H, NH); 12.49 (s, 1H, acid OH).
Mass spectra of compound exhibited molecular ion peak at m/z
518 (M+), 519 (M+1), 520 (M+2).
4.3.6. 2-(Phenylimino)-5-(3,4,5-trimethoxybenzyliden)thiazoli-
din-4-one (3f)
Yield: 75.68% (solid); mp 123–124 °C. FTIR (KBr) cmꢀ1: 3472
(N–H); 3026 (Ar C–H); 2925 (aliphatic C–H) 1705 (C@O); 1571
(C@N); 1232 (aralkyl ether C–O–C). 1H NMR (CDCl3, d ppm): 3.49
(s, 9H, CH3); 6.76 (s, 2H, benzylidene); 6.90–7.31 (m, 5H, phenyl);
7.89 (s, 1H, C@CH); 8.82 (br s, 1H, NH). Mass spectra of compound
exhibited molecular ion peak at m/z 370 (M+), 371 (M+1), 372
(M+2).
4.5.2. 4-(3-(5-Benzyliden-4-oxo-2-(phenylimino)thiazolidin-3-
yl)-2-hydroxypropylamino)benzoic acid (5b)
Yield: 63.02% (solid); mp 68–70 °C. FTIR (KBr) cmꢀ1: 3560 (O–
H); 3420 (N–H); 3041 (Ar C–H); 1731 (C@O); 1608 (C@N). 1H
NMR (CDCl3, d ppm): 3.09 (d, 4H, CH2); 3.98 (m, 1H, CH); 4.78 (s,
1H, OH); 6.92–7.39 (m, 14H, aromatic); 7.50 (s, 1H, C@CH); 8.19
(s, 1H, NH); 12.65 (s, 1H, Acid OH). Mass spectra of compound
exhibited molecular ion peak at m/z 473 (M+), 474 (M+1), 475
(M+2).
4.3.7. 5-(4-Fluorobenzyliden)-2-(phenylimino)thiazolidin-4-
one (3g)
Yield: 75.50% (solid); mp 229–231 °C. FTIR (KBr) cmꢀ1: 3468
(N–H); 3051 (Ar C–H); 1721 (C@O); 1601 (C@N); 1035 (C–F). 1H
NMR (CDCl3, d ppm): 6.81–7.24 (m, 5H, phenyl); 7.56 (d, 2H, ben-
zylidene); 7.40 (d, 2H, benzylidene); 7.62 (s, 1H, C@CH); 8.38 (br s,
1H, NH). Mass spectra of compound exhibited molecular ion peak
at m/z 298 (M+), 299 (M+1), 300 (M+2).
4.5.3. 4-(2-Hydroxy-3-(5-(4-hydroxybenzyliden)-4-oxo-2-(phe-
nylimino)thiazolidin-3-yl)propylamino)benzoic acid (5c)
Yield: 63.54% (solid); mp 62–64 °C. FTIR (KBr) cmꢀ1: 3568 (O–
H); 3435 (N–H); 3058 (Ar C–H); 1753 (C@O); 1649 (C@N). 1H
NMR (CDCl3, d ppm): 3.32 (d, 4H, CH2); 4.09 (m, 1H, CH); 4.86 (s,
1H, aliphatic OH); 6.55–7.69 (m, 13H, aromatic); 7.81 (s, 1H,
C@CH); 8.04 (s, 1H, NH); 8.92 (s, 1H, phenol OH); 12.71 (s, 1H, Acid
OH). Mass spectra of compound exhibited molecular ion peak at
m/z 489 (M+), 490 (M+1), 491 (M+2).
4.3.8. 5-(3-Phenylallyliden)-2-(phenylimino)thiazolidin-4-one
(3h)
Yield: 86.60% (solid); mp 230–232 °C. FTIR (KBr) cmꢀ1: 3466
(N–H); 3042 (Ar C–H); 1698 (C@O); 1598 (conjugated –C@CH)
1551 (C@N). 1H NMR (CDCl3, d ppm): 6.58 (d, 1H, C@CH–Ar);
6.79 (t, 1H, C–CH@C); 7.26 (d, 1H, @CH–C); 6.99–7.57 (m, 10H,
phenyl and benzylidene); 8.10 (br s, 1H, NH). Mass spectra of com-
pound exhibited molecular ion peak at m/z 306 (M+), 307 (M+1),
308 (M+2).
4.5.4. 4-(2-Hydroxy-3-(5-(2-hydroxybenzyliden)-4-oxo-2-(phe-
nylimino)thiazolidin-3-yl)propylamino)benzoic acid (5d)
Yield: 82.07% (solid); mp 61–63 °C. FTIR (KBr) cmꢀ1: 3547 (O–
H); 3468 (N–H); 3042 (Ar C–H); 1760 (C@O); 1611 (C@N). 1H
NMR (CDCl3, d ppm): 3.19 (d, 4H, CH2); 4.13 (m, 1H, CH); 4.59 (s,
1H, aliphatic OH); 6.62–7.82 (m, 13H, aromatic); 7.70 (s, 1H,
C@CH); 8.0 (s, 1H, NH); 8.81 (s, 1H, phenol OH); 12.78 (s, 1H, Acid
OH). Mass spectra of compound exhibited molecular ion peak at
m/z 489 (M+), 490 (M+1), 491 (M+2).
4.4. General procedure for the preparation of compounds 4a–4h
A solution of 0.64 mL (0.0081 mole, 0.75 g) of epichlorhydrin
in 10 mL of ethanol was heated to 65–70 °C. This solution was
then added in a dropwise fashion over a period of 1–2 h in the
solution of (0.0081 mole) compounds 3a–3h in ethanol (20 mL)
maintaining the reaction temperature at around 70 °C. The solu-
tion was then heated for 1–3 h to gentle reflux (75–80 °C) and
cooled to 60 °C. The reflux time was monitored using TLC. Caus-
tic soda solution (10% alcoholic) was added until a distinctly
alkaline to thymol phthaleine paper. (Around 2 mL of 10% caustic
soda solution was required.) The reaction mixture was concen-
trated to 40% of volume. The precipitated sodium chloride was
filtered off and the filtrate was evaporated to dryness at 100 °C
in vacuo. The residual syrup like residue, the hydrochloride salt
of the respective compounds 4a–4h could not be isolated, so it
was used immediately in the next step in the form of suspension
in ethanol.
4.5.5. 4-(2-Hydroxy-3-(5-(4-methylbenzyliden)-4-oxo-2-(phen-
ylimino)thiazolidin-3-yl)propylamino)benzoic acid (5e)
Yield: 51.52% (solid); mp 110–112 °C. FTIR (KBr) cmꢀ1: 3569
(O–H); 3428 (N–H); 3060 (Ar C–H); 2946 (alkyl C–H); 1757
(C@O); 1592 (C@N). 1H NMR (CDCl3, d ppm): 2.14 (s, 3H, CH3);
2.98 (d, 4H, CH2); 3.99 (m, 1H, CH); 4.72 (s, 1H, OH); 7.06–7.89
(m, 13H, aromatic); 7.88 (s, 1H, C@CH); 8.23 (s, 1H, NH); 12.89
(s, 1H, Acid OH). Mass spectra of compound exhibited molecular
ion peak at m/z 487 (M+), 488 (M+1), 489 (M+2).
4.5.6. 4-(2-Hydroxy-3-(4-oxo-2-(phenylimino)-5-(3,4,5-trimet-
hoxybenzyliden)thiazolidin-3-yl)propylamino)benzoic acid (5f)
Yield: 61.89% (solid); mp 92–94 °C. FTIR (KBr) cmꢀ1: 3601 (O–
H); 3424 (N–H); 3049 (Ar C–H); 2961 (alkyl C–H); 1748 (C@O);
1639 (C@N); 1239 (aralkyl ether C–O–C). 1H NMR (CDCl3, d

396
S. V. Bhandari et al. / Bioorg. Med. Chem. 17 (2009) 390–400
ppm): 3.24 (d, 4H, CH2); 3.75 (s, 9H, OCH3); 4.12 (m, 1H, CH); 4.93
(s, 1H, OH); 6.81–7.73 (m, 11H, aromatic); 7.91 (s, 1H, C@CH); 8.0
(s, 1H, NH); 12.69 (s, 1H, Acid OH). Mass spectra of compound
exhibited molecular ion peak at m/z 563 (M+), 564 (M+1), 565
(M+2).
7.78 (s, 1H, C@CH); 8.12 (s, 1H, NH); 8.81 (s, 1H, Ar–OH); 9.41
(br s, 1H, CONH). Mass spectra of compound exhibited molecular
ion peak at m/z 503 (M+), 504 (M+1), 505 (M+2).
4.6.4. 4-(2-Hydroxy-3-(5-(2-hydroxybenzyliden)-4-oxo-2-
(phenylimino)thiazolidin-3-yl)propylamino)benzohydrazide
(6d)
4.5.7. 4-(3-(5-(4-Fluorobenzyliden)-4-oxo-2-(phenylimino)thi-
azolidin-3-yl)-2-hydroxypropylamino)benzoic acid (5g)
Yield: 67.84% (solid); mp 76–78 °C. FTIR (KBr) cmꢀ1: 3588 (O–
H); 3437 (N–H); 3051 (Ar C–H); 1760 (C@O); 1655 (C@N); 1044
(C–F). 1H NMR (CDCl3, d ppm): 3.07 (d, 4H, CH2); 4.0 (m, 1H,
CH); 4.83 (s, 1H, OH); 6.69–7.85 (m, 13H, aromatic); 7.78 (s, 1H,
C@CH); 8.31 (s, 1H, NH); 12.92 (s, 1H, Acid OH). Mass spectra of
compound exhibited molecular ion peak at m/z 491 (M+), 492
(M+1), 493 (M+2).
Yield: 53.06% (solid); mp 96–98 °C. FTIR (KBr) cmꢀ1: 3633 (O–
H); 3432 (N–H); 3040 (Ar C–H); 1662 (C@O); 1619 (C@N). 1H
NMR (CDCl3, d ppm): 3.07 (d, 4H, CH2); 4.04 (m, 1H, CH); 4.18 (s,
2H, NH2); 4.64 (s, 1H, OH); 6.60–7.44 (m, 13 H, aromatic); 7.80
(s, 1H, C@CH); 8.07 (s, 1H, NH); 8.90 (s, 1H, Ar–OH); 9.50 (br s,
1H, CONH). Mass spectra of compound exhibited molecular ion
peak at m/z 503 (M+), 504 (M+1), 505 (M+2).
4.6.5. 4-(2-Hydroxy-3-(5-(4-methylbenzyliden)-4-oxo-2-
(phenylimino)thiazolidin-3-yl)propylamino)benzohydrazide
(6e)
4.5.8. 4-(2-Hydroxy-3-(-4-oxo-5-(3-phenylallyliden)-2-(pheny-
limino)thiazolidin-3-yl)propylamino)benzoic acid (5h)
Yield: 66.91% (solid); mp 54–56 °C. FTIR (KBr) cmꢀ1: 3555 (O–
H); 3438 (N–H); 3029 (Ar C–H); 1648 (C@O); 1590 (conjugated –
C@CH); 1549 (C@N). 1H NMR (CDCl3, d ppm): 2.98 (d, 4H, CH2);
3.91 (m, 1H, CH); 4.62 (s, 1H, OH); 6.47 (d, 1H, C@CH–Ar); 6.62
(t, 1H, C–CH@C); 7.01 (d, 1H, @CH–C); 7.09–7.80 (m, 14H, aro-
matic); 8.11 (s, 1H, NH); 12.49 (s, 1H, Acid OH). Mass spectra of
compound exhibited molecular ion peak at m/z 499 (M+), 500
(M+1), 501 (M+2).
Yield: 69.44% (solid); mp 234–236 °C. FTIR (KBr) cmꢀ1: 3581
(O–H); 3410 (N–H); 3048 (Ar C–H); 2946 (C–H); 1659 (C@O);
1610 (C@N). 1H NMR (CDCl3, d ppm): 2.38 (s, 3H, Ar–CH3); 2.96
(d, 4H, CH2); 3.97 (m, 1H, CH); 4.40 (s, 2H, NH2); 4.78 (s, 1H,
OH); 6.89–7.54 (m, 13 H, aromatic); 7.84 (s, 1H, C@CH); 7.99
(s, 1H, NH); 9.55 (br s, 1H, CONH). Mass spectra of compound
exhibited molecular ion peak at m/z 501 (M+), 502 (M+1), 503
(M+2).
4.6. General procedure for the preparation of compounds 6a–
6h
4.6.6. 4-(2-Hydroxy-3-(4-oxo-2-(phenylimino)-5-(3,4,5-trimeth-
oxybenzyliden)thiazolidin-3-yl)propylamino)benzohydrazide
(6f)
A mixture of 5a–5h (1 mole) and thionyl chloride (1.5 mole)
was refluxed for 2–4 h. Excess of thionyl chloride was removed un-
der reduced pressure. The residue was refluxed with hydrazine hy-
drate (2 mole) (99%) in sufficient alcohol (3–6 mL) for 5–6 h. Add
0.4 g sodium bicarbonate just before 4–5 h of reflux. The mixture
was left to cool and evaporated under reduced pressure. The resi-
due obtained was filtered, washed with water, dried and recrystal-
lized from ethanol.
Yield: 73.78% (solid); mp 143–145 °C. FTIR (KBr) cmꢀ1: 3598
(O–H); 3430 (N–H); 3051 (Ar C–H); 2958 (alkyl C–H); 1741
(C@O); 1627 (C@N); 1241 (aralkyl ether C–O–C). 1H NMR (CDCl3,
d ppm): 3.29 (d, 4H, CH2); 3.91 (s, 9H, OCH3); 4.08 (m, 1H, CH);
4.33 (s, 2H, NH2); 4.61 (s, 1H, OH); 6.68–7.59 (m, 11H, aromatic);
8.01 (s, 1H, C@CH); 7.90 (s, 1H, NH); 9.61 (br s, 1H, CONH). Mass
spectra of compound exhibited molecular ion peak at m/z 577
(M+), 578 (M+1), 579 (M+2).
4.6.1. 4-(2-Hydroxy-3-(5-(3-nitrobenzyliden)-4-oxo-2-(phenyli-
mino)thiazolidin-3-yl)propylamino)benzohydrazide (6a)
Yield: 62.86% (solid); mp 239–241 °C. FTIR (KBr) cmꢀ1: 3545
(O–H); 3421 (N–H); 3029 (Ar C–H); 1660 (C@O); 1610 (C@N);
1500 (C–NO2). 1H NMR (CDCl3, d ppm): 2.97 (d, 4H, CH2); 3.91
(m, 1H, CH); 4.29 (s, 2H, NH2); 4.46 (s, 1H, OH); 6.82–7.81 (m, 13
H, aromatic); 7.86 (s, 1H, C@CH); 7.99 (s, 1H, NH); 9.42 (br s, 1H,
CONH). Mass spectra of compound exhibited molecular ion peak
at m/z 532 (M+), 533 (M+1), 534 (M+2).
4.6.7. 4-(3-(5-(4-Fluorobenzyliden)-4-oxo-2-(phenylimino)-
thiazolidin-3-yl)-2-hydroxypropylamino)benzohydrazide (6g)
Yield: 56.10% (solid); mp 85–87 °C. FTIR (KBr) cm-1: 3546 (O–
H); 3394 (N–H); 3048 (Ar C–H); 1758 (C@O); 1621 (C@N); 1061
(C–F). 1H NMR (CDCl3, d ppm): 2.98 (d, 4H, CH2); 4.12 (m, 1H,
CH); 4.18 (s, 2H, NH2); 4.70 (s, 1H, OH); 6.52–7.69 (m, 13H, aro-
matic); 8.14 (s, 1H, C@CH); 8.21 (s, 1H, NH); 9.52 (br s, 1H, CONH).
Mass spectra of compound exhibited molecular ion peak at m/z 505
(M+), 506 (M+1), 507 (M+2).
4.6.2. 4-(3-(5-Benzyliden-4-oxo-2-(phenylimino)thiazolidin-3-
yl)-2-hydroxypropylamino)benzohydrazide (6b)
4.6.8. 4-(2-Hydroxy-3-(4-oxo-5-(3-phenylallyliden)-2-(phenyli-
mino)thiazolidin-3-yl)propylamino)benzohydrazide (6h)
Yield: 53.10% (solid); mp 176–178 °C. FTIR (KBr) cmꢀ1: 3542
(O–H); 3398 (N–H); 3031 (Ar C–H); 1637 (C@O); 1584 (conjugated
–C@CH); 1571 (C@N). 1H NMR (CDCl3, d ppm): 3.0 (d, 4H, CH2);
4.02 (m, 1H, CH); 4.09 (s, 2H, NH2); 4.41 (s, 1H, OH); 6.39 (d, 1H,
C@CH–Ar); 6.59 (t, 1H, C–CH@C); 6.81 (d, 1H, @CH–C); 6.90–7.68
(m, 14H, aromatic); 8.11 (s, 1H, NH); 9.66 (br s, 1H, CONH). Mass
spectra of compound exhibited molecular ion peak at m/z 513
(M+), 514 (M+1), 515 (M+2).
Yield: 68.78% (solid); mp 169–171 °C. FTIR (KBr) cmꢀ1: 3561
(O–H); 3437 (N–H); 3039 (Ar C–H); 1666 (C@O); 1600 (C@N). 1H
NMR (CDCl3, d ppm): 2.93 (d, 4H, CH2); 4.01 (m, 1H, CH); 4.31 (s,
2H, NH2); 4.61 (s, 1H, OH); 6.71–7.70 (m, 14 H, aromatic); 7.73
(s, 1H, C@CH); 8.09 (s, 1H, NH); 9.60 (br s, 1H, CONH). Mass spectra
of compound exhibited molecular ion peak at m/z 487 (M+), 488
(M+1), 489 (M+2).
4.6.3. 4-(2-Hydroxy-3-(5-(4-hydroxybenzylidene)-4-oxo-2-
(phenylimino)thiazolidin-3-yl)propylamino)benzohydrazide
(6c)
4.7. General procedure for the preparation of compounds 7a–
7h
Yield: 69.78% (solid); mp 111–113 °C. FTIR (KBr) cmꢀ1: 3642
(O–H); 3429 (N–H); 3028 (Ar C–H); 1657 (C@O); 1624 (C@N). 1H
NMR (CDCl3, d ppm): 3.10 (d, 4H, CH2); 3.98 (m, 1H, CH); 4.09
(s, 2H, NH2); 4.52 (s, 1H, OH); 6.54–7.47 (m, 13 H, aromatic);
A mixture of 6a–6h (1 mole) was refluxed with chloroacetyl
chloride (1.2 mole) in dry benzene (5–10 mL) in the presence of tri-
ethylamine (0.31 mL) for 9–11 h. The solution was evaporated to

S. V. Bhandari et al. / Bioorg. Med. Chem. 17 (2009) 390–400
397
dryness. The residue obtained was filtered, washed with water and
recrystallized from ethanol.
1612 (C@N); 1237 (aralkyl ether C–O–C); 658 (C–Cl). 1H NMR
(CDCl3, d ppm): 3.20 (d, 4H, CH2); 3.82 (s, 9H, OCH3); 3.92 (m,
1H, CH); 4.32 (s, 2H, COCH2); 4.78 (s, 1H, OH); 6.68–7.51 (m,
11H, aromatic); 8.14 (s, 1H, C@CH); 8.01 (s, 1H, NH); 10.81 (br s,
2H, CONH). Mass spectra of compound exhibited molecular ion
peak at m/z 653 (M+), 654 (M+1), 655 (M+2).
0
4.7.1. N-(2-Chloroacetyl)-4-(2-hydroxy-3-(5-(3-nitrobenzylid-
en)-4-oxo-2-(phenylimino)thiazolidin-3-yl)propylamino)ben-
zohydrazide (7a)
Yield: 53.51% (solid); mp 69–71 °C. FTIR (KBr) cmꢀ1: 3565 (O–
H); 3347 (N–H); 3038 (Ar C–H); 1659 (C@O); 1629 (C@N); 1489
(C–NO2); 689 (C–Cl). 1H NMR (CDCl3, d ppm): 3.18 (d, 4H, CH2);
4.08 (m, 1H, CH); 4.30 (s, 2H, COCH2); 4.71 (s, 1H, OH); 6.98–
7.79 (m, 13H, aromatic); 7.92 (s, 1H, C@CH); 7.99 (s, 1H, NH);
10.13 (br s, 2H, CONH). Mass spectra of compound exhibited
molecular ion peak at m/z 608 (M+), 609 (M+1), 610 (M+2).
4.7.7. N0-(2-Chloroacetyl)-4-(3-(5-(4-fluorobenzyliden)-4-oxo-
2-(phenylimino)thiazolidin-3-yl)-2-hydroxypropylamino)ben-
zohydrazide (7g)
Yield: 70.87% (solid); mp 171–173 °C. FTIR (KBr) cmꢀ1: 3512
(O–H); 3382 (N–H); 3034 (Ar C–H); 1689 (C@O); 1610 (C@N);
1098 (C–F); 639 (C–Cl). 1H NMR (CDCl3, d ppm): 3.02 (d, 4H,
CH2); 4.07 (m, 1H, CH); 4.51 (s, 2H, COCH2); 4.62 (s, 1H, OH);
6.70–7.62 (m, 13H, aromatic); 8.08 (s, 1H, C@CH); 8.18 (s, 1H,
NH); 10.59 (br s, 2H, CONH). Mass spectra of compound exhib-
ited molecular ion peak at m/z 581 (M+), 582 (M+1), 583 (M+2).
4.7.2. 4-(3-(5-Benzyliden-4-oxo-2-(phenylimino)thiazolidin-3-
yl)-2-hydroxy propylamino)-N0-(2-chloroacetyl)benzohydrazide
(7b)
Yield: 71.84% (solid); mp 185–187 °C. FTIR (KBr) cmꢀ1: 3547
(O–H); 3412 (N–H); 3041 (Ar C–H); 1649 (C@O); 1619 (C@N);
659 (C–Cl). 1H NMR (CDCl3, d ppm): 3.08 (d, 4H, CH2); 3.94 (m,
1H, CH); 4.28 (s, 2H, COCH2); 4.47 (s, 1H, OH); 6.79–7.58 (m, 14
H, aromatic); 7.82 (s, 1H, C@CH); 8.10 (s, 1H, NH); 10.64 (br s,
2H, CONH). Mass spectra of compound exhibited molecular ion
peak at m/z 563 (M+), 564 (M+1), 565 (M+2).
4.7.8. N0-(2-Chloroacetyl)-4-(2-hydroxy-3-(4-oxo-5-(3-phenyl-
allyliden)-2-(phenylimino)thiazolidin-3-yl)propylamino)ben-
zohydrazide (7h)
Yield: 72.46% (solid); mp 93–95 °C. FTIR (KBr) cmꢀ1: 3551 (O–
H); 3408 (N–H); 3042 (Ar C–H); 1664 (C@O); 1579 (conjugated –
C@CH); 1597 (C@N); 658 (C–Cl). 1H NMR (CDCl3, d ppm): 3.10 (d,
4H, CH2); 4.21 (m, 1H, CH); 4.52 (s, 1H, OH); 4.60 (s, 2H, COCH2);
6.42 (d, 1H, C@CH–Ar); 6.56 (t, 1H, C–CH@C); 6.75 (d, 1H, @CH–
C); 6.83–7.54 (m, 14H, aromatic); 8.04 (s, 1H, NH); 10.76 (br s,
2H, CONH). Mass spectra of compound exhibited molecular ion
peak at m/z 589 (M+), 590 (M+1), 591 (M+2).
4.7.3. N0-(2-Chloroacetyl)-4-(2-hydroxy-3-(5-(4-hydroxybenzyl-
iden)-4-oxo-2-(phenylimino)thiazolidin-3-yl)propylamino)ben-
zohydrazide (7c)
Yield: 62.43% (solid); mp 142–144 °C. FTIR (KBr) cmꢀ1: 3637
(O–H); 3417 (N–H); 3025 (Ar C–H); 1643 (C@O); 1612 (C@N);
643 (C–Cl). 1H NMR (CDCl3, d ppm): 3.29 (d, 4H, CH2); 4.10
(m, 1H, CH); 4.37 (s, 2H, COCH2); 4.61 (s, 1H, OH); 6.70–7.51
(m, 13 H, aromatic); 7.83 (s, 1H, C@CH); 8.0 (s, 1H, NH); 8.62
(s, 1H, Ar–OH); 10.31 (br s, 2H, CONH). Mass spectra of com-
pound exhibited molecular ion peak at m/z 579 (M+), 580
(M+1), 581 (M+2).
4.8. General procedure for the preparation of compounds 8a–
8h
A solution of the appropriate chloroalkyl derivative 7a–7h
(1.8 mole) in dry acetonitrile (2–4 mL) was treated portion wise
with a solution of AgNO3 (2 mole) in dry acetonitrile (5–10 mL)
and the whole mixture was stirred at room temperature for 2.5–
3.5 h. The mixture was then filtered, evaporated to dryness, and
the residue was crystallized from absolute ethanol.
4.7.4. N0-(2-Chloroacetyl)-4-(2-hydroxy-3-(5-(2-hydroxybenzyl-
iden)-4-oxo-2-(phenylimino)thiazolidin-3-yl)propylamino)-
benzohydrazide (7d)
Yield: 71.01% (solid); mp 177–179 °C. FTIR (KBr) cmꢀ1: 3647
(O–H); 3421 (N–H); 3037 (Ar C–H); 1656 (C@O); 1631 (C@N);
661 (C–Cl). 1H NMR (CDCl3, d ppm): 3.09 (d, 4H, CH2); 4.09
(m, 1H, CH); 4.49 (s, 2H, COCH2); 4.72 (s, 1H, OH); 6.64–7.59
(m, 13 H, aromatic); 7.79 (s, 1H, C@CH); 8.17 (s, 1H, NH); 8.71
(s, 1H, Ar–OH); 10.62 (br s, 2H, CONH). Mass spectra of com-
pound exhibited molecular ion peak at m/z 579 (M+), 580
(M+1), 581 (M+2).
4.8.1. 2-(2-(4-(2-Hydroxy-3-(5-(3-nitrobenzyliden)-4-oxo-2-
(phenylimino)thiazolidin-3-yl)propylamino)benzoyl)hydrazi-
nyl)-2-oxoethyl nitrate (8a)
Yield: 77.11% (solid); mp 189–191 °C. FTIR (KBr) cmꢀ1: 3598
(O–H); 3457 (N–H); 3031 (Ar C–H); 2923 (aliphatic C–H); 1683
(C@O); 1597 (C@N); 1551 (Ar–NO2); 1490 (C–NO2). 1H NMR
(CDCl3, d ppm): 3.19 (d, 4H, CH2); 3.89 (m, 1H, CH); 4.44 (s, 1H,
OH); 4.59 (s, 2H, COCH2); 6.97 (s, 1H, C@CH); 7.01–7.74 (m, 13
H, aromatic); 8.13 (s, 1H, NH); 10.54 (br s, 2H, CONH). Mass spectra
of compound exhibited molecular ion peak at m/z 635 (M+), 636
(M+1), 637 (M+2). Anal. Calcd for C28H25N7O9S: C, 52.91; H, 3.96;
N, 15.43. Found: C, 52.97; H, 3.94; N, 15.39.
4.7.5. N0-(2-Chloroacetyl)-4-(2-hydroxy-3-(5-(4-methylbenzy-
lidene)-4-oxo-2-(phenylimino)thiazolidin-3-yl)propylamino)-
benzohydrazide (7e)
Yield: 79.13% (solid); mp 98–100 °C. FTIR (KBr) cmꢀ1: 3564 (O–
H); 3394 (N–H); 3029 (Ar C–H); 2954 (alkyl C–H); 1661 (C@O);
1634 (C@N); 647 (C–Cl). 1H NMR (CDCl3, d ppm): 2.29 (s, 3H, Ar–
CH3); 3.18 (d, 4H, CH2); 4.11 (m, 1H, CH); 4.42 (s, 2H, COCH2);
4.74 (s, 1H, OH); 6.73–7.59 (m, 13 H, aromatic); 7.90 (s, 1H, C@CH);
8.23 (s, 1H, NH); 10.64 (br s, 2H, CONH). Mass spectra of compound
exhibited molecular ion peak at m/z 577 (M+), 578 (M+1), 579
(M+2).
4.8.2. 2-(2-(4-(3-(5-Benzyliden-4-oxo-2-(phenylimino)thiazoli-
din-3-yl)-2-hydroxypropylamino)benzoyl)hydrazinyl)-2-oxoethyl
nitrate (8b)
Yield: 87.30% (solid); mp 253–255 °C. FTIR (KBr) cmꢀ1: 3572
(O–H); 3451 (N–H); 3026 (Ar C–H); 2934 (aliphatic C–H); 1657
(C@O); 1598 (C@N); 1500 (C–NO2). 1H NMR (CDCl3, d ppm): 3.21
(d, 4H, CH2); 3.81 (m, 1H, CH); 4.34 (s, 1H, OH); 4.51 (s, 2H,
COCH2); 6.98–7.41 (m, 14 H, aromatic); 7.58 (s, 1H, C@CH); 7.89
(s, 1H, NH); 10.14 (br s, 2H, CONH). Mass spectra of compound
exhibited molecular ion peak at m/z 590 (M+), 591 (M+1), 592
(M+2). Anal. Calcd for C28H26N6O7S: C, 56.94; H, 4.44; N, 14.23.
Found: C, 57.04; H, 4.47; N, 14.28.
4.7.6. N0-(2-Chloroacetyl)-4-(2-hydroxy-3-(4-oxo-2-(phenylimi-
no)-5-(3,4,5-trimethoxybenzyliden)thiazolidin-3-yl)propylami-
no)benzohydrazide (7f)
Yield: 75.20% (solid); mp 77–79 °C. FTIR (KBr) cmꢀ1: 3572 (O–
H); 3431 (N–H); 3046 (Ar C–H); 2938 (alkyl C–H); 1710 (C@O);

398
S. V. Bhandari et al. / Bioorg. Med. Chem. 17 (2009) 390–400
4.8.3. 2-(2-(4-(2-Hydroxy-3-(5-(4-hydroxybenzyliden)-4-oxo-2-
(phenylimino)thiazolidin-3-yl)propylamino)benzoyl)hydrazinyl)-
2-oxoethyl nitrate (8c)
4.8.8. 2-(2-(4-(2-Hydroxy-3-(4-oxo-5-(3-phenylallyliden)-2-
(phenylimino)thiazolidin-3-yl)propylamino)benzoyl)hydrazi-
nyl)-2-oxoethyl nitrate (8h)
Yield: 86.67% (solid); mp 247–249 °C. FTIR (KBr) cmꢀ1: 3592
(O–H); 3462 (N–H); 3014 (Ar C–H); 2947 (aliphatic C–H); 1650
(C@O); 1596 (C@N); 1504 (C–NO2). 1H NMR (CDCl3, d ppm): 2.93
(d, 4H, CH2); 3.99 (m, 1H, CH); 4.38 (s, 1H, OH); 4.57 (s, 2H,
COCH2); 6.58–7.43 (m, 13 H, aromatic); 7.72 (s, 1H, C@CH); 7.98
(s, 1H, NH); 8.45 (s, 1H, Ar–OH); 10.57 (br s, 2H, CONH). Mass spec-
tra of compound exhibited molecular ion peak at m/z 606 (M+), 607
(M+1), 608 (M+2). Anal. Calcd for C28H26N6O8S: C, 55.44; H, 4.32;
N, 13.85. Found: C, 55.36; H, 4.28; N, 13.80.
Yield: 67.02% (solid); mp 165–167 °C. FTIR (KBr) cmꢀ1: 3541
(O–H); 3466 (N–H); 3042 (Ar C–H); 2943 (aliphatic C–H); 1680
(C@O); 1598 (conjugated –C@CH); 1551 (C@N); 1519 (C–NO2).
1H NMR (CDCl3, d ppm): 3.23 (d, 4H, CH2); 3.89 (m, 1H, CH); 4.31
(s, 1H, OH); 4.65 (s, 2H, COCH2); 6.68 (d, 1H, C@CH–Ar); 6.96 (t,
1H, C–CH@C); 7.01 (d, 1H, @CH–C); 7.28–7.9 (m, 14H, aromatic);
7.99 (s, 1H, NH); 10.54 (br s, 2H, CONH). Mass spectra of compound
exhibited molecular ion peak at m/z 616 (M+), 617 (M+1), 618
(M+2). Anal. Calcd for C30H28N6O7S: C, 58.43; H, 4.58; N, 13.63.
Found: C, 58.58; H, 4.53; N, 13.69.
4.8.4. 2-(2-(4-(2-Hydroxy-3-(5-(2-hydroxybenzyliden)-4-oxo-2-
(phenylimino)thiazolidin-3-yl)propylamino)benzoyl)hydrazinyl)-
2-oxoethyl nitrate (8d)
5. Pharmacology
5.1. Animals
Yield: 80% (solid); mp 201–203 °C. FTIR (KBr) cmꢀ1: 3613 (O–
H); 3446 (N–H); 3043 (Ar C–H); 2921 (aliphatic C–H); 1650
(C@O); 1584 (C@N); 1506 (C–NO2). 1H NMR (CDCl3, d ppm): 2.99
(d, 4H, CH2); 4.04 (m, 1H, CH); 4.48 (s, 2H, COCH2); 4.58 (s, 1H,
OH); 6.61–7.17 (m, 13 H, aromatic); 7.96 (s, 1H, C@CH); 8.08 (s,
1H, NH); 8.39 (s, 1H, Ar–OH); 10.79 (br s, 2H, CONH). Mass spectra
of compound exhibited molecular ion peak at m/z 606 (M+), 607
(M+1), 608 (M+2). Anal. Calcd for C28H26N6O8S: C, 55.44; H, 4.32;
N, 13.85. Found: C, 55.38; H, 4.30; N, 13.82.
Wistar rats weighing in the range 250–300 g were obtained
from National center for cell science (NCCS), Pune, India. All the
animals were housed in standard cages, under standard ambient
conditions, temperature (25 2 °C), relative humidity of 50 5%.
A 12:12 h light:dark cycle was maintained. All the animals were al-
lowed to have free access to water and standard palletized labora-
tory. All the experimental procedures and protocols used in these
studies 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),
Government of India.20
4.8.5. 2-(2-(4-(2-Hydroxy-3-(5-(4-methylbenzyliden)-4-oxo-2-
(phenylimino)thiazolidin-3-yl)propylamino)benzoyl)-
hydrazinyl)-2-oxoethyl nitrate (8e)
Yield: 50.48% (solid); mp 193–195 °C. FTIR (KBr) cmꢀ1: 3550
(O–H); 3444 (N–H); 3010 (Ar C–H); 2957 (aliphatic C–H); 1654
(C@O); 1577 (C@N); 1519 (C–NO2). 1H NMR (CDCl3, d ppm): 2.19
(s, 3H, Ar–CH3); 2.89 (d, 4H, CH2); 4.0 (m, 1H, CH); 4.54 (s, 2H,
COCH2); 4.62 (s, 1H, OH); 6.94–7.51 (m, 13 H, aromatic); 7.88 (s,
1H, C@CH); 8.1 (s, 1H, NH); 10.7 (br s, 2H, CONH). Mass spectra
of compound exhibited molecular ion peak at m/z 604 (M+), 605
(M+1), 606 (M+2). Anal. Calcd for C29H28N6O7S: C, 57.61; H, 4.67;
N, 13.90. Found: C, 57.82; H, 4.72; N, 13.95.
5.2. Preparation of test compounds
After suspending the test compounds in 0.5% aqueous solution
of methyl cellulose, test samples were administered to test animals
intraperitoneally.
5.3. In vivo pharmacological screening
5.3.1. Influence on blood pressure21,22
4.8.6. 2-(2-(4-(2-Hydroxy-3-(4-oxo-2-(phenylimino)-5-(3,4,5-
trimethoxybenzyliden)thiazolidin-3-yl)propylamino)benzoyl)-
hydrazinyl)-2-oxoethyl nitrate (8f)
Influence on blood pressure was analyzed in adult normoten-
sive Wistar rats (250–300 g) under thiopental anesthesia (60 mg/
kg ip). The right carotid artery were exposed and cannulated with
polyethylene tube filled with small volumes of heparin solution
(20 IU/mL, in physiological saline) for the measurement of the
mean arterial pressure of the rats. The heparin solution was in-
jected in the arterial catheter at 30 min intervals, to avoid possible
blood coagulation. The catheter (PE-50) was connected to the
blood pressure transducer and the transducer was connected with
the Four Channel Data Acquisition System (BIOPAC System, Inc.,
MP35). One hour after the surgical protocol, mean arterial pressure
values were measured at 0, 15, 30, 45 and 60 min after administra-
tion of standard and test compounds. Test compounds were
administered intraperitoneally (40 mg/kg) and standard (Atenolol
10 mg/kg, ip)3 thereafter, blood pressure measurement was contin-
ued for 60 min.
Yield: 86.08% (solid); mp 219–221 °C. FTIR (KBr) cmꢀ1: 3541
(O–H); 3461 (N–H); 3046 (Ar C–H); 2925 (aliphatic C–H); 1685
(C@O); 1571 (C@N); 1521 (C–NO2); 1236 (aralkyl ether C–O–C).
1H NMR (CDCl3, d ppm): 3.14 (d, 4H, CH2); 3.7 (s, 9H, OCH3); 4.0
(m, 1H, CH); 4.28 (s, 1H, OH); 4.41 (s, 2H, COCH2); 6.32–7.85 (m,
11H, aromatic); 8.0 (s, 1H, C@CH); 8.1 (s, 1H, NH); 10.69 (br s,
2H, CONH). Mass spectra of compound exhibited molecular ion
peak at m/z 680 (M+), 681 (M+1), 682 (M+2). Anal. Calcd for
C31H32N6O10S: C, 54.70; H, 4.74; N, 12.35. Found: C, 54.55; H,
4.69; N, 12.31.
4.8.7. 2-(2-(4-(3-(5-(4-Fluorobenzyliden)-4-oxo-2-(phenylimino)
thiazolidin-3-yl)-2-hydroxypropylamino)benzoyl)hydrazinyl)-2-
oxoethyl nitrate (8g)
Yield: 89.27% (solid); mp 211–213 °C. FTIR (KBr) cmꢀ1: 3537
(O–H); 3464 (N–H); 3021 (Ar C–H); 2943 (aliphatic C–H); 1634
(C@O); 1594 (C@N); 1505 (C–NO2); 1035 (C–F). 1H NMR (CDCl3,
d ppm): 3.18 (d, 4H, CH2); 3.87 (m, 1H, CH); 4.3 (s, 2H, COCH2);
4.53 (s, 1H, OH); 6.81–7.90 (m, 13H, aromatic); 7.97 (s, 1H, NH);
8.12 (s, 1H, C@CH); 10.75 (br s, 2H, CONH). Mass spectra of com-
pound exhibited molecular ion peak at m/z 608 (M+), 609 (M+1),
610 (M+2). Anal. Calcd for C28H25FN6O7S: C, 55.26; H, 4.14; N,
13.81. Found: C, 55.38; H, 4.19; N, 13.86.
5.3.2. Influence on normal electrocardiogram14,21
Recording of the electrocardiogram is an essential tool in the
evaluation of antiarrhythmic drugs. Similar to the heart rate, the
electrocardiogram is different between various species. Electrocar-
diographic investigations were carried out with the application of
subcutaneous needle electrodes to the flexor aspect of limbs of
rat and connected with the SS2L electrode transducer which was
connected with the Four Channel Data Acquisition System (BIOPAC

S. V. Bhandari et al. / Bioorg. Med. Chem. 17 (2009) 390–400
399
System, Inc, MP 35) with the respective ECG transducer. Physiolog-
ical ECG recordings were taken from Wistar rats (250–300 g)
anaesthetized with thiopental (60 mg/kg ip) and, afterwards, com-
pounds under investigation were injected. Test compounds were
administered intraperitoneally (40 mg/kg). ECG parameters were
collected at 0, 15, 30, 45 and 60 min of administration of
compounds.
(20 lg/mL) or KCl (30 mM) and when the contraction reached a
stable plateau, the test compounds in concentration (0.1 mg/mL)
were added cumulatively.
5.4.2. Detection of nitrite19,25
A solution of the appropriate compound (20 lL) in dimethyl-
sulfoxide (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
5.3.3. Influence on heart rate14,21
MeOH, containing 5ꢁ of 10ꢀ4 M
L-cysteine. The final concentration
Heart rate recording were taken from Wistar rats (250–300 g)
with the application of subcutaneous needle electrodes to the flex-
or aspect of limbs of rat and connected with the SS2L electrode
transducer which was connected with the Four Channel Data
Acquisition System (BIOPAC System, Inc., MP 35) with the
respective ECG transducer. Rats were anaesthetized with thiopen-
tal sodium (60 mg/kg ip) and, afterwards, test compounds were
injected. Test compounds were administered intraperitoneally
(40 mg/kg). Heart rate (in beats per min) was recorded at 0, 15,
30, 45 and 60 min of administration of compounds.
of drug was 10ꢀ4 M. After 1 h at 37 °C, 1 mL of the reaction mixture
was treated with 250 lL of Griess reagent [sulfanilamide (4 g), N-
naphthylethylenediamine dihydrochloride (0.2 g), 85% phosphoric
acid (10 mL) in distilled water (final volume: 100 mL)]. After
10 min at room temperature, 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.
5.5. Statistical analyses
5.3.4. Antiarrhythmic activity14,21
The synthesized title compounds (8a–8h) were examined
in vivo for prophylactic antiarrhythmic action in animal models
of heart rhythm disturbances caused by intravenous injection of
barium chloride. Protection against rhythm disturbances caused
by injection of arrhythmogen (barium chloride) depicts the ability
of a compound to act as a potential antiarrhythmic agent. Barium
chloride takes effect by decrease of the outward potassium
currents.
The data are expressed as means SEM. The statistical signifi-
cance was calculated using a one-way ANOVA test. Differences
were considered significant when p < 0.05. All statistical calcula-
tions were performed using evaluation version of Graph PadÒ
Prism 3.0 (USA) statistical software.26
Acknowledgments
The authors are thankful to The Principal, AISSMS College of
Pharmacy, Pune, for providing infrastructure facilities. The authors
are also thankful to Head, Department of Pharmacology, for his
continuous support and guidance throughout the project. The
authors also wish to thank National Center for Cell Science (NCCS),
Pune, for providing animals for pharmacological screening and Shi-
madzu Analytical Centre and Dept. of Chemistry, University of
Pune, National Chemical Laboratories (NCL), Pune, for providing
facilities for spectral studies of compounds.
5.3.4.1. Barium chloride-induced arrhythmia. The arrhythmia
was evoked in rats anaesthetized with thiopental (60 mg/kg ip) by
iv injection of barium chloride (32 mg/kg, in a volume of 1 mL/kg).
Test compounds were administered intraperitoneally at the
constant dose of 40 mg/kg, 60 min prior to arrhythmogen adminis-
tration. Overdose of BaCl2 induced progressively increasing distur-
bances of cardiac rhythm, associated with premature ventricular
beats, ventricular tachycardia and ventricular fibrillation, leading
to death within 2–5 min. Attenuation or lack of disturbances and
the decreased mortality after BaCl2 administration were accepted
as a criterion of the antiarrhythmic effect of the compound.
References and notes
1. Nieto, R. M.; Coelho, A. Chem. Pharm. Bull. 2003, 51, 1025.
2. Christiaans, J. A. M.; Timmerman, H. Eur. J. Pharm. Sci. 1996, 4, 1.
3. Nandakumar, N.; Singh, R.; Bansal, S. K.; Bodhankar, S. L.; Jindal, D. P.; Coumar,
M. S.; Bhardwaj, S. H. Indian J. Pharmacol. 2004, 36, 381.
4. Brizzi, V.; Francioli, M.; Brufani, M.; Filocamo, L.; Bruni, G.; Massarelli, P. IL
Farmaco 1999, 54, 713.
5. Bing, R. J.; Yamamoto, T.; Kim, H.; Grubbs, R. H. Biochem. Biophys. Res. Commun.
2000, 275, 350.
6. Nguyen, J.; Velazquez, C. A.; Knaus, E. E. Bioorg. Med. Chem. 2005, 13, 1725.
7. Martinez, L.; Abrego, V.; Balboa, M. A., et al Eur. J. Med. Chem. 2008, 43,
486.
8. Stout, D. M.; Matier, W. L.; Barcelon-Yang, C.; Reynolds, R. D.; Brown, B. S. J.
Med. Chem. 1983, 26, 808.
9. Stout, D. M.; Matier, W. L.; Barcelon-Yang, C.; Reynolds, R. D.; Brown, B. S. J.
Med. Chem. 1984, 27, 1347.
10. Stout, D. M.; Matier, W. L.; Barcelon-Yang, C.; Reynolds, R. D.; Brown, B. S. J.
Med. Chem. 1985, 28, 295.
11. Glowka, M. L.; Codding, P. W. J. Med. Chem. 1991, 34, 2678.
12. Zeng, Y. L.; Wang, Y. F.; Li, J.; Xu, J. M.; Wang, M.; Qiu, H. Eur. J. Pharmacol. 1990,
183, 1849.
5.4. In vitro pharmacological screening
5.4.1. Vasorelaxing activity23,24
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 dislo-
cation under light ether anesthesia and bled. The aortae were
immediately excised, freed of extraneous tissues and prepared as
multiple-ring preparations. Then the vessels were suspended, un-
der a preload of 2 g, in 10 mL organ baths, containing Tyrode solu-
tion (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 mix-
ture of O2 (95%) and CO2 (5%). Changes in tension were recorded by
means of an isometric transducer (BIOPAC System, Inc., MP 35).
After an equilibration period of 60 min, the endothelial integrity
13. Dai, D. J. Mol. Cell. Cardiol. 1990, 22, S74.
14. Malawska, B.; Kulig, K.; Filipek, B., et al Eur. J. Med. Chem. 2002, 37, 183.
15. Frank, R. L.; Smith, P. V. Org. Synth. 1955, 3, 735.
16. Rout, M. K.; Mahapatra, G. N. Tetrahedron 1955, 77, 2427.
17. Stallmann, O. U.S. Patent 1,977,253, 1934.
18. Furniss, B.; Hannaford, A.; Smith, P.; Tatchell, A. In Vogel’s Textbook of Practical
Organic Chemistry; ELBS Publication, 1994, pp 90, 908–973.
19. Abadi, A. H.; Hegazy, G. H.; El-Zaher, A. A. Bioorg. Med. Chem. 2005, 13, 5759.
20. CPCSEA guidelines for laboratory animal facility. Indian J. Pharmacol. 2003, 35,
257. www.cpcsea.com.
was confirmed by acetylcholine (ACh) (55
lM) induced relaxation
of Norepinephrine (NE 20 g/mL) precontracted tissues. A relaxa-
l
tion P70% of the NE-induced contraction was considered repre-
sentative of an acceptable presence of the endothelial layer,
while the organs, showing a relaxation <70%, were not used in
the experimental procedures. Thirty to 40 minutes after the confir-
mation of the endothelial integrity, the aortic preparations were
21. Dylag, T.; Zygmunt, M.; Maciag, D.; Handzlik, J.; Bednarski, M.; Filipek, B.; Kiec´-
Kononowicz, K. Eur. J. Med. Chem. 2004, 39, 1013.
contracted by treatment with
a single concentration of NE

400
S. V. Bhandari et al. / Bioorg. Med. Chem. 17 (2009) 390–400
ꢀ
22. Ferrarini, P.; Mori, C.; Calderone, V.; Calzolari, L.; Nieri, P.; Saccomanni, G.;
Martinotti, E. Eur. J. Med. Chem. 1999, 34, 505.
25. Nitric
Oxide
(NO2ꢀ=NO3
Assay.
Catalog
Number
DE1500B.
info@RnDSystems.com.
23. Calderone, V.; Martinotti, E.; Scatizzi, R.; Pellegrini, A.; Breschi, M. J. Pharmacol.
Toxicol. Methods 1996, 35, 13l.
26. Graph Pad Software Inc. 11452, E-1, Camino Real, #215, San Diego, CA 92130,
USA. www.graphpad.com.
24. Ferrarini, P.; Mori, C.; Calderone, V.; Calzolari, L.; Nieri, P.; Saccomanni, G.;
Martinotti, E. Eur. J. Med. Chem. 1998, 33, 383.