Design, synthesis and pharmacological screening of hybrid molecules as antihypertensives

Article abstract of DOI:10.1007/s00044-010-9374-6

The synthesis and pharmacological profile of a series of hybrid compounds bearing the dihydropyrimidine moiety and substituted phenylthioureas joined via typical b-blocker aryloxypropanolamine group are described. Seven out of 10 new compounds tested proved to be endowed with negative inotropic and chronotropic effect as compared to Adrenaline on isolated perfused frog heart. Also ECG studies that were carried out on normotensive male rats showed that the most of the compounds exhibited significant reduction in Rmax value in the first 30 min although maintaining little change in heart rate, with hypotensive effect lasting for 1 h.We concluded that the most of the synthesized novel compounds exhibited hypotensive as well as antihypertensive effects which could be attributed to blockade of Ca²⁺ entry and β-adrenoreceptor blocking activities due to introduction of aryloxypropanolamine linked to N-1 substituted dihydropyrimidine moiety. Brady cardiac effects of compounds 5a-5d, 6c-6e resulting from Ca²⁺entry and β-adrenoreceptor blocking attenuate the sympathetic activation-associated reflex tachycardia in the heart. We selected compound 5c as a promising lead for further detailed pharmacological and preclinical evaluation studies. Springer Science+Business Media, LLC 2010.

Full text of DOI:10.1007/s00044-010-9374-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:705–713
DOI 10.1007/s00044-010-9374-6
ORIGINAL RESEARCH
Design, synthesis and pharmacological screening of hybrid
molecules as antihypertensives
•
Satarupa Acharjee Kailash G. Bothara
•
•
Shashikant V. Bhandari Tapan K. Maity
Received: 30 January 2010 / Accepted: 21 May 2010 / Published online: 17 June 2010
Ó Springer Science+Business Media, LLC 2010
Abstract The synthesis and pharmacological profile of a
series of hybrid compounds bearing the dihydropyrimidine
moiety and substituted phenylthioureas joined via typical
b-blocker aryloxypropanolamine group are described.
Seven out of 10 new compounds tested proved to be
endowed with negative inotropic and chronotropic effect as
compared to Adrenaline on isolated perfused frog heart.
Also ECG studies that were carried out on normotensive
male rats showed that the most of the compounds exhibited
significant reduction in Rmax value in the first 30 min
although maintaining little change in heart rate, with hypo-
tensive effect lasting for 1 h. We concluded that the most of
the synthesized novel compounds exhibited hypotensive as
well as antihypertensive effects which could be attributed to
blockade of Ca2? entry and b-adrenoreceptor blocking
activities due to introduction of aryloxypropanolamine
linked to N-1 substituted dihydropyrimidine moiety. Brady
cardiac effects of compounds 5a–5d, 6c–6e resulting from
Ca2? entry and b-adrenoreceptor blocking attenuate the
sympathetic activation-associated reflex tachycardia in the
heart. We selected compound 5c as a promising lead for
further detailed pharmacological and preclinical evaluation
studies.
Keywords Hybrid ꢀ Antihypertensive ꢀ
Calcium channel blocker ꢀ Beta blocker
Introduction
The pharmacological treatment of diseases having complex
heterogeneous pathogenesis like hypertension could pose a
problem due to intervention with a single dose therapy
because of side effects associated with single drugs (Bisi
et al., 2003) i.e., b-blockers: bradycardia, calcium channel
blockers: reflex tachycardia. In order to overcome these
effects, a multiple drug therapeutic regime is commonly
practised by combining two or more drugs for managing
hypertension. Multiple dose therapies make the regime
complicated and less successful mainly due to patient’s
non-compliance. Recently the drug discovery trend is to
design drugs in which the single active agents are com-
bined in one molecule called hybrid molecule often con-
sisting of different pharmacophoric groups linked to each
other via spacers (Christiaans and Timmerman, 1996).
Many antihypertensives having distinct mechanisms of
action share guanidine as a common structural functional-
ity (Hussain et al., 1988) exemplified by a-1/a-2 receptor
agonist Clonidine and general vasodilator Minoxidil.
Hence, it was desired to use a bioisostere of guanidine like
thioureas as one of the moieties of our designed com-
pounds. A variety of N-(alkyl/alkenyl/aryl-N-heterocyclic)
ureas and thioureas have been reported (Vajragupta et al.,
1996); the most active compound showing 29.1% reduction
in systolic blood pressure as well as substituted phenyl-
thiourea derivatives (I) demonstrated to have potent oral
S. Acharjee (&)
Department of Pharmaceutical Chemistry, NSHM College
of Pharmaceutical Technology, Kolkata 700053, West Bengal,
India
e-mail: satarupa_a@yahoo.co.in
K. G. Bothara ꢀ S. V. Bhandari
Department of Pharmaceutical Chemistry, AISSMS College
of Pharmacy, Kennedy Road, Pune 411001, Maharashtra, India
T. K. Maity
Division of Pharmaceutical Chemistry, Department
of Pharmaceutical Technology, Jadavpur University,
Kolkata, West Bengal, India
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Med Chem Res (2011) 20:705–713
antihypertensive activity in two hypertensive rat models
1992). Although N-1 is suggested to be unsubstituted for
hydrogen bonding for receptor bound conformation (Kap-
pe, 1998), Singh et al., (2009) demonstrated the novel
synthetic strategy and minor calcium channel blocking
activity (compared to Nifedipine) for N-1 alkylated
DHPMs. In continuation of the earlier reported hybrid
approaches, we undertook designing of our novel series
of compounds [II] utilizing the aryloxypropanolamine
group as a linker between substituted phenyl thioureas
and 1,4 dihydropyrimidine 2-thiones. The contribution of
the channel and receptor blockades of these compounds
was suggested towards providing a novel balance among
them.
(Tilley et al., 1980).
S
H2N
NH
NH
Cl
1-(2-aminoethyl)-3-(2,6 dichlorophenyl)thiourea (I)
However, in the long-term treatment of hypertension,
the use of vasodilators alone does not suffice and it is the
concomitant use of b-blockers which has proved to be
useful for achieving adequate control of blood pressure
(Bisi et al., 2003). Keeping the basic aryloxypropanol-
amine nucleus intact for significant b-adrenoreceptor
antagonistic activity, a variety of aromatic ring or nitrogen
substituents can be tolerated for the design of therapeutic
agents that combine b-adrenoreceptor antagonistic activity
with other pharmacological actions including a-adrenore-
ceptor blockade (Uchida et al., 1993), calcium channel
blockade (Shibasaki et al., 1994), direct vasodilation
(Hieble, 1991), inhibition of angiotensin converting
enzyme (Allan et al., 1987) and class III antiarrhythmic
activity (Argentieri et al., 1993).
S
R
S
N
H
NH
N
R1
N
O
OH
O
H3C
O
H3C
II
The aim of this study was to elicit the synergistic effects
of both b-adrenoreceptor blockade and vasodilator effects
of calcium channel blockers and substituted thioureas
without the presence of untoward circulatory effects in
classical short-acting dihydropyridines and b-blockers used
alone. The use of these novel compounds are hence
expected to exhibit enhanced antihypertensive effect with
lesser side effects as compared to concommitant use of two
or more than two single active drugs.
In recent years, there has been a great deal of interest in
exploiting multiple functional groups in the design of novel
structures capable of performing variety of functions.
Hybrid cardiovascular agents involving heterocycles, aryl-
oxypropanolamine, dihydropyrimidine nuclei have aroused
enormous interest as they display a wide variety of bio-
logical properties such as antiarrhythmic, vaso relaxing and
anti hypertensive activity as well as in vitro nitric oxide
releasing activity (Bhandari et al., 2009) and this approach
has also been fruitful in enhancing original dihydropyridine
associated calcium channel blocking activity in vascular
smooth muscle (Liang et al., 2002). Dihyropyrimidines (aza
analogs of 1,4 dihydropyridines (Kappe, 1998) being less
cardiodepressant have been proved to have beneficial anti-
hypertensive effects when combined with b-blockers
(Honda et al., 1985; Dargie, 1986; Asano et al., 1990;
Shibasaki et al., 1997) as it attenuates the undesirable
effects of reflex tachycardia associated with vasodilatory
action of dihydropyridines (Liang et al., 2002).
Results and discussions
Chemistry
The synthetic route used to synthesize title compounds is
outlined in Scheme 1. N-Substituted Phenyl thioureas (1a–
1e), were prepared according to the method reported in the
literature (Frank and Smith, 1955), using ammonium thi-
oscyanate, benzoyl chloride and substituted anilines. This
process was optimized in the presence of dry acetone.
Acetone is dried for at least 48 h over anhydrous calcium
sulfate and distilled just before it is used. Schiff’s bases 2a–
2e were prepared by heating substituted phenylthioureas
with para-amino benzaldehyde for 3 h in the presence of
dicyclohexylcarbodimide (DCCI) in THF. Compounds 2a–
2e were reacted with epichlorhydrin in the presence of
Na2CO3, without isolating the intermediate 3a–3e; the
According to structure–activity relationships established
for 1,4 dihydropyridines (Singh et al., 2009; Rodenkircken
et al., 1979; Bossert et al., 1981) substituted-2-hetero (S
[O [N)-1,4 Dihyropyrimidines (DHPMs) mimic the
activity of 1,4-DHPs, and ortho and/or meta aromatic
substitutions of ester alkyl group were found to be major
determinants of in vitro potency (Atwal et al., 1991;
Rovnyak et al., 1992; Atwal et al., 1990; Rovnyak et al.,
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Med Chem Res (2011) 20:705–713
707
Scheme 1 Synthetic route for
title compounds (5a–6e).
Reagents and conditions: a
DCCI, THF, reflux 3–6 h;
p-hydroxy benzaldehyde;
S
a,b
c
S
R
R
d
Δ
NH
NH2
NH
N
S
(1a-1e)
c epichlorhydrin; reflux 4 h,
d Na2CO3 and e reflux 12–15 h
OH
(2a-2e)
S
H
R
R1
N
NH
N
HN
+
O
O
H3C
O
(3a-3e)
O
(4a-4b)
H3C
e
S
R
H
S
NH
N
R1
N
N
O
OH
H3C
O
O
H3C
reaction mixture was used immediately in the next step.
The epoxy bridge was cleaved with secondary amine
functionality of DHPM 4a–4b to get the designed com-
pounds 5a–5e and 6a–6e. The structures of the synthesized
activity of synthesized compounds. The effects of hybrid
moieties were evaluated on an isolated perfused heart at
various dose levels and compared with the activity of
Adrenaline under identical experimental conditions. The
pharmacological activities were compared with a b-blocker
and calcium channel blocker under identical conditions.
In the isolated frog heart investigation, compounds [5a–
5d] and [6c–6e] showed dose-dependent decrease in force
of contraction when compared to Adrenaline (1 lg/ml),
thereby showing b-adrenergic blocking activity (as isolated
frog heart contains b-receptors only). 5e, 6a and 6b could
not be screened due to very poor solubility in all acceptable
vehicles; 5a showed 25% inhibition (p \ 0.01) in force of
contraction at the concentration of 100 lg/ml, 38% at
200 lg/ml and 40% at 400 lg/ml and 800 lg/ml (Table 1)
with respect to Adrenaline (1 lg/ml). 5b showed a
1
compounds were established by IR, H NMR and mass
spectral assignments. The FTIR spectra of title compounds
[5a–5e] and [6a–6e] showed characteristic functional
groups like –OH, ꢁCꢂN stretching vibrations, –C–S
stretching, sec–NH stretching vibrations and specific bands
for –NO2 vibrations etc. In the proton NMR spectral data,
the chemical shifts are in agreement with the structures.
Biological results
In this study, pharmacological tests were carried to assess the
effect on normal electrocardiogram and antihypertensive
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Med Chem Res (2011) 20:705–713
ECG measurements showed that 5a, 5b, 5c, 5d (40 mg/kg
ip) and 6c (50 mg/kg ip) decreased the heart rate at the first
30 min of administration while it remained constant later.
6e showed a gradual decrease in the heart rate from 0 to
60 min duration (Table 2).
Table 1 Percentage inhibition on force of contraction of investigated
compounds
S. no
Log dose
Percentage inhibition (%)
5a
5b
5c
5d
6c
6d
6e
1
2
3
4
5
2
25
38
20
20
31
32
10
23
22
36
30
50
50
66
–
50
43
43
50
–
23
5
23
16
18
18
50
13
20
4
It was interesting to note that unlike frog heart experi-
ment results the title compounds (5a–5d, 6d, 6e) at dose of
40 mg/kg ip and 6c (50 mg/kg ip)showed a significant
(p \ 0.05) reduction in Rmax value after 30 min of
administration and is extremely significant (p \ 0.001)
after 45 min of administration while the reduction after 1 h
of administration is not significant which is same as indi-
cated by propanolol (10 mg/kg ip) and nifedipine (30 mg/
kg ip) (Table 3). Reduction in Rmax is an indication of
decrease in force of contractility of cardiac muscles in
normotensive situation exhibiting hypotensive action of
novel series of designed hybrid compounds.
2.3
2.6
2.9
3
25
30
–
25
–
nonlinear response. 5c and 5d showed measurable response
ranging between 30 and 66% and 50% inhibition
(p \ 0.001) in a dose range of 200–800 lg/ml, respectively
(Table 1); 6d, 6e and 6f (200–800 lg/ml) showed 23–30%
inhibition in the dose level of 0.1–0.8 lg/ml as compared
to 35–66% reduction in force of contraction shown by
Propanolol (p \ 0.001) and 13–33% as evidenced by
Nifedipine (p \ 0.001) (Table 1). Measurement of heart
rate indicated that, among the designed compounds [5a–
5d] and [6c–6e], all of them showed negative chronotropic
action compared to increased heart rate due to Adrenaline
control while the data are nonsignificant (results not
shown). 5c and 5d were found to be the most promising
having shown extremely significant (p \ 0.001) reduction
in force of contraction (30–66%, 50%) and maintaining
constant heart rate as compared to positive inotropic and
chronotropic action of adrenaline. Nifedipine at a dose of
2–16 lg/ml also produced a marked increase in heart rate.
In the rat ECG experiment, the effects of standard drugs
and test compounds (5a–5d, 6c–6e) on Rmax value (mv)
and heart rate (bpm) were studied in 0-, 30-, 45- and 60-
min intervals at a dose ranging from 40 to 50 mg/kg ip.
Conclusion
The newly synthesized compounds were screened for their
anti hypertensive activity using isolated perfused frog heart
and hypotensive effect by ECG technique using normo-
tensive male Wistar rats. Most of the compounds possessed
significant antihypertensive action when compared against
positive control indicating b-adrenergic receptor blocking
activity. Compound 5c having methoxy group at ortho
position of phenyl ring of substituted thiourea and electron
withdrawing nitro functionality at ortho position of
substituted aromatic ring at DHPM moiety, respectively,
has shown maximum inhibition of force of contractility of
(30–66%) as compared to adrenaline (p \ 0.001) and
nonsignificant negative chronotropic action. These findings
Table 2 Effects of an intraperitonial injection of investigated compounds on Heart rate in anaesthetized Wistar rats
Compound code
Dose
Heart rate (beats per minute)
S. no
0 min
30 min
45 min
60 min
1
2
Control
0.004 ml/kg
10 mg/kg
30 mg/kg
40 mg/kg
40 mg/kg
40 mg/kg
40 mg/kg
50 mg/kg
40 mg/kg
40 mg/kg
307.69 (0.035)
428.57 (0.84)
352.94 (0.48)
363.53 (0.265)
272 (0.01)
307.62 (0.439)
400 (0.359)
307.62 (0.023)
333 (0.128)
307.62 (0.053)
333 (0.107)
Propanolol
3
Nifedipine
363.63 (0.155)
266.08 (0.275)
226.62 (0.437)
272.19 (0.28)
315.87 (0.223)
399.26 (0.356)
324.05 (0.167
323.85 (0.228)
425.61 (0.159)
266.08 (0.275)
214.17 (0.127)
266.41 (0.098)
300.22 (0.072)
399.26 (0.356)
324.05 (0.167)
299.78 (0.354)
521.73 (0.223)
266.08 (0.274)
215.7 (0.298)
266.41 (0.098)
300.22 (0.072)
399.26 (0.356)
315.25 (0.29)
260.42 (0.529)
4
5a
5b
5c
5d
6c
6d
6e
5
6
352.94 ((0.01)
315.7 (0.025)
428.57 (0.075)
333.33 (0.015)
342.87 (0.435)
7
8
9
10
The data are the means of six experiments SEM; Statistical analyses were performed using a one way ANOVA
* p \ 0.05, ** p \ 0.01, *** p \ 0.001
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Med Chem Res (2011) 20:705–713
709
Table 3 Effects of an intraperitonial injection of investigated compounds on Rmax value in anaesthetized Wistar rats
Compound code
Dose
Rmax (mv)
0 min
S.no
30 min
45 min
60 min
1
2
Control
0.004 ml/kg
10 mg/kg
30 mg/kg
40 mg/kg
40 mg/kg
40 mg/kg
40 mg/kg
50 mg/kg
40 mg/kg
40 mg/kg
0.08 (0.008)
0.15 (0.007)
0.17 (0.008)
0.16 (0.01)
0.13 (0.008)
0.18 (0.008)
0.15 (0.007)
0.12 (0.01)
0.14 (0.008)
0.06 (0.005)
0.08 (0.008)
0.04 (0.009)*
0.04 (0.012)*
0.1 (0.01)*
0.08 (0.008)
0.07 (0.004)
0.13 (0.015)
0.15 (0.013)
0.13 (0.011)
0.19 (0.009)
0.13 (0.011)
0.14 (0.112)
0.12 0.01
Propanolol
0.02 (0.005)***
0.1 (0.012)***
0.05 (0.011)***
0.03 (0.009)***
0.02 (0.008)***
0.07 (0.012)***
0.05 (0.013)*
3
Nifedipine
4
5a
5b
5c
5d
6c
6d
6e
5
0.08 (0.012)**
0.09 (0.012)**
0.04 (0.007)*
0.03 (0.006)*
0.08 (0.008)*
0.03 (0.007)*
6
7
8
9
0.02 (0.008)***
0.01 (0.005)***
0.09 (0.005)
0.07 (0.009)
10
The data are the means of six experiments SEM; Statistical analyses were performed using a one way ANOVA
* p \ 0.05, ** p \ 0.01, *** p \ 0.001
were further confirmed in rat ECG as test compounds had
little effect in heart rate but change in Rmax value was
prominent after 30 and 45 min of administration in nor-
motensive rats. 5c and 6d (having p-carboxylic acid
function in phenyl thiourea moiety and o-nitro group at
substituted phenyl ring at DHPM) showed 50% and 43%
reduction in Rmax value, respectively, in the first 30 min
of administration although maintaining little change in
heart rate as compared to reflex tachycardiac effects
exhibited by traditional b-blockers and Calcium channel
blockers used alone. Hence, it can be concluded that these
compounds elicit their antihypertensive action by binding
to b-receptors and Calcium channel receptors and can be
considered as promising hits in development of hybrid
cardiovascular agents especially in situations like long-
term hypertension where a single drug is not effective in
normalizing elevated blood pressure in hypertensive
patients. However, additional studies needs to be carried
out on potent compounds regarding direct measurement of
mean arterial pressure, elucidation of mode of action of
hybrid leads, acute and chronic toxicity studies and phar-
macokinetic profile to enhance the usefulness of this class
of hybrid compounds.
were recorded on a Varian MercuryYH300 (300 MHz)
Spectrophotometer using TMS as an internal reference.
Chemical shift is represented in dppm. Mass spectra were
recorded on GC–MS QP5050A system (Benchtop qua-
druple mass spectrophotometer). Purity of compounds was
checked on TLC plates using Silica Gel G as the stationery
phase and various polarities of solvent systems, and iodine
vapor/UV as visualizing agent.
General procedure for the synthesis of N-substituted phenyl
thiourea (1a–1e)
The substituted phenyl thioureas were prepared as per
previously reported procedure (Frank and Smith, 1955).
General procedure for the synthesis of N-(Substituted
phenyl) N0-(p-hydroxy phenyl imino)thioureas (2a–2e)
N-(substituted phenyl) thiourea (0.01 M) was dissolved in
15 ml of ethanol (95%); to this solution, p-hydroxy benz-
aldehyde (0.01 M) dissolved in 10 ml of ethanol (95%)
was added, and the solution was refluxed for 2 h. in the
presence of 1.5 gm of DCCI in 5 ml of THF. After this, the
mixture was filtered and the filtrate evaporated to half its
original volume and allowed to cool. Crystals of the
product were filtered, dried and recrystalized from ethanol.
Experimental procedures
Chemistry
General procedure for synthesis of 6-methyl-4-(substituted
phenyl)-2-thioxo 1,2,3,4-tetrahydropurimidine-5-
carboxylic acid ethyl ester (Ranu et al., 2002) (4a–4b)
Melting points were determined using open capillary tubes
and are uncorrected. FTIR spectra of the powdered com-
pounds were recorded using KBr on a Jasco FTIR V
430 ? Spectrophotometer using Diffuse Reflectance
Attachment and are reported in cm-1, and 1H NMR spectra
A mixture of substituted benzaldehyde (0.01 M), ethyl
acetoacetate (0.01 M) and thiourea (0.01 M), was shaken
by hand for 2 min. The reaction mixture was then heated in
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Med Chem Res (2011) 20:705–713
a water bath at 90°C for one hour. With progress of the
reaction. a solid started to deposit and after one hour, the
flask is full of solid. The solid was taken out carefully with
a spatula or spoon in a conical flask. The yellow solid was
washed with cold water (1 ml) and then recrystallized from
rectified spirit to give a colorless solid.
(s, 1H, NH–C=S); 9.30 (s, 1H, Ar–NH–C=S). m/z 667
(M?). Anal. % (C32H33N5O7S2) C, H, N, S. Calcd. 57.90,
5.01, 10.55, 9.66. Found 57.99, 4.99, 10.50, 9.72.
N1(phenyl)N3{4[2-hydroxy-3(5-carbethoxy,6-methyl-2-
thio-4(2nitro-phenyl)1,2,3,4-tetrahydropyrimidine-1-
yl)propoxy]benzylidene}thiourea (5e)
General procedure for the synthesis of N (substituted
phenyl) N0{10,6-methyl (substituted phenyl]-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylic acid ethyl
ester}30-[400-imino phenoxy] propane-2-ol thioureas
(5a–6e)
Yield: 50%, m.p.: 185–183°C; FTIR (KBr) cm-1: 3298
(NH); 1678 (C=N); 1631 (C=C); 1553 (C=O); 1342
1
(OH); 1275(C–O); 1228(C=S), H NMR (DMSO––d6,
d
ppm):1.20 (t, 3H, –O–CH2–CH3, J = 7.2 Hz); 2.34 (s, 3H,
–CH3); 3.12 (m, 2H, N–CH2–); 3.40 (m, 2H, Ar–O–CH2–);
3.56 (m, 1H, CH–OH); 3.82 (q, 2H, –O–CH2–CH3,
J = 7 Hz); 5.10 (s, 1H, CH–OH); 5.62 (s, 1H, Ar–CH);
6.20 (s, 1H, –N=CH–Ar); 7.2–7.9 (m, 13H, Ar–H); 7.92 (s,
1H, NH–C=S); 9.20 (s, 1H, Ar–NH–C=S); m/z 633 (M?).
Anal. % (C31H31N5O6S2) C, H, N, S. Calcd. 58.76, 4.89,
11.05, 10.11. Found 58.70, 4.90, 11, 10.15.
Synthesized intermediates (2a–2e) (0.0027 M), epichlorhydrin
(0.0034 M) and 1.05 g of anhydrous sodium carbonate taken in
10 ml of acetone were refluxed for 37 h with intermittent stir-
ring. It was then shaken with water, and the organic layer was
separated. To this organic layer DHPMs (4a–4b) (0.0052 M)
was added to break the epoxide formed and refluxed for 15 h.
Keeping the condensate in a bath of crushed ice, sodium
hydroxide flakes were added until precipitation occurs. Then,
the precipitate was filtered, dried and recrystallized from
ethanol.
N1(4-carboxyphenyl)N3{4[2-hydroxy-3(5-carbethoxy-
6-methyl-2-thio-4(2nitro-phenyl)1,2,3,4-tetrahydropyrimidine-
1-yl)propoxy]benzylidene}thiourea (6b)
N1(4-methoxy phenyl)N3{4[2-hydroxy-3(5-carbethoxy,
6-methyl-2-thio-4(2nitro-henyl)1,2,3,4-tetrahydropyrimidine-
1-yl)propoxy]benzylidene}thiourea (5a)
Yield: 62%, M.P. 200–202°C, FTIR (KBr) cm-1: 3332
(NH); 1642 (C=N); 1630 (C=C); 1529 (C=O); 1348 (OH);
1253(C–O); 1228(C=S), 3081 (–COOH),1H NMR (DMSO-
d6,d ppm): 1.20 (t, 3H, –O–CH2–CH3, J = 7.2 Hz); 2.34 (s,
3H, –CH3); 3.12 (m, 2H, N–CH2–); 3.40 (m, 2H, Ar–O–
CH2–); 3.56 (m, 1H, CH–OH); 3.82 (q, 2H, –O–CH2–CH3,
J = 7 Hz); 3.56 (s, 1H, CH–OH); 5.62 (s, 1H, Ar–CH);
7.92 (s, 1H, –N=CH–Ar); 7.2–7.9 (m, 12H, Ar–H); 9.20 (s,
1H, Ar–NH–C=S),11.40 (s,1H, Ar–COOH), m/z 677 (M?)
Anal. % (C32H31N5O8S2), C, H, N, S Calcd. 56.72, 4.57,
10.33, 9.45 Found 56.70, 4.60, 10.26, 9.50.
Yield: 60%, mp: 180–83°C; mp FTIR (KBr) cm-1: 3637
(NH); 1553 (C=N); 1645 (C=C); 1678 (C=O); 1321 (OH);
1
1275(C–O); 1227(C=S), H NMR (DMSO––d6, d ppm):
1.20 (t, 3H, –O–CH2–CH3, J = 7.2 Hz); 2.36 (s, 3H,
–CH3); 3.15 (m, 2H, N–CH2–); 3.41 (m, 2H, Ar–O–CH2–);
3.58 (m, 1H, CH–OH); 3.71 (s, 3H, –OCH3); 3.90 (q, 2H,
–O–CH2–CH3, J = 7.2 Hz); 4.92 (s, 1H, CH–OH); 5.45 (s,
1H, Ar–CH); 6.17 (s, 1H, –N=CH–Ar); 7.10–7.83 (m, 12H,
Ar–H); 7.90 (s, 1H, NH–C=S); 9.17 (s, 1H, Ar–NH–C=S).
m/z 667(M?). Anal.% (C32H33N5O7S2) C, H, N, S. Calcd.
57.9, 5.01, 10.55, 9.66. Found 57.85, 4.99, 10.60, 9.70.
N1(4-chlorophenyl)N3{4[2-hydroxy-3(5-carbethoxy,
6-methyl-2-thio-4(3nitro-phenyl)1,2,3,4-tetrahydropyrimidine-
1-yl)propoxy]benzylidene}thiourea (6e)
N1(2-methoxy phenyl)N3{4[2-hydroxy-3(5-carbethoxy,
6-methyl-2-thio-4(2nitro-phenyl)1,2,3,4-tetrahydropyrimidine-1-yl)
propoxy]benzylidene}thiourea (5c)
Yield: 52%, M.P. 180–85°C; FTIR (KBr) cm-1: 3511
(NH); 1660 (C=N); 1655 (C=C); 1689 (C=O); 1341 (OH);
1
1260(C–O); 1216(C=S), H NMR (DMSO––d6, d ppm):
Yield: 57%, m.p.: 200°C; FTIR (KBr) cm-1: 3297 (NH);
1677 (C=N); 1643 (C=C); 1554 (C=O); 1345 (OH);
1.22 (t, 3H, –O–CH2–CH3,J = 7.2 Hz); 2.36 (s, 3H,
–CH3); 3.22 (m, 2H, N–CH2–); 3.50 (m, 2H, Ar–O–CH2–);
3.66 (m, 1H, CH–OH); 3.92 (q, 2H, –O–CH2–CH3,
J = 7.2 Hz); 4.82 (s, 1H, CH–OH); 5.58 (s, 1H, Ar–CH);
6.08 (s, 1H, –N=CH–Ar); 7.28–7.96 (m, 12H, Ar–H); 8.12
(s, 1H, NH–C=S); 9.34 (s, 1H, Ar–NH–C=S). m/z 668
(M?). Anal. % (C31H30ClN5O6S2) C, H, N, S. Calcd. 55.77,
4.49, 10.49, 9.45. Found 5.70, 4.51, 10.51, 9.40.
1
1275(C–O); 1227(C=S), H NMR (DMSO––d6, d ppm):
1.22 (t, 3H, –O–CH2–CH3, J = 7.0 Hz); 2.33 (s, 3H,
–CH3); 3.09 (m, 2H, N–CH2–); 3.40 (m, 2H, Ar–O–CH2–);
3.52 (m, 1H, CH–OH); 3.86 (q, 2H, –O–CH2–CH3,
J = 7.2 Hz); 5.14 (s, 1H, CH–OH); 5.62 (s, 1H, Ar–CH);
6.20 (s, 1H, –N=CH–Ar); 7.02–7.72 (m, 12H, Ar–H); 8.12
123

Med Chem Res (2011) 20:705–713
711
Biology
Animals
The experiments were carried out on male Wistar rats
weighing in the range 250–300 g. All the animals were
housed in standard cages under standard ambient conditions,
temperature (25 2°C), relative humidity of 50 5%, and
were exposed to a 12/12 h light–dark cycle. All the animals
were allowed to have free access to water and standard
pellated food which was given ad libitum. All the experi-
mental procedures and protocols used in these studies were
reviewed by 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), Gov-
ernment of India.
Fig. 1 Proposed receptor-bound dihydropyrimidine confirmation
(antagonist) Kappe, (1998)
the muscle-forced transducer, and this is connected to a
‘physiograph’. Normal heart rate, contractile amplitude was
recorded. Various concentrations of compounds to be tested
were prepared using 1% sodium carboxymethyl cellulose
and administered to the ringer flowing through the cannula,
and the effectswere recorded atdifferent dose level, (200 lg,
400 lg, 600 lg, 800 lg and 1 mg/ml). Only one compound
was tested in each preparation (Table 1). During the inter-
action studies, Propanolol, a b-adrenergic blocker (2, 4, 8,
16 lg/ml), Nifedipine, a Calcium channel blocker (2, 4, 8,
16 lg/ml) in frog ringers’ solution and adrenaline (0.1 lg,
0.2 lg, 0.4 lg, 0.8 lg and 1 mg/ml) were administered to
the ringer solution, and the recordings were noted. The data
presented in figures are the means SEM. One-way
ANOVA was used for statistical analysis. p values \0.01
were considered to be statistically significant. (Fig. 1).
Drugs and chemicals
Adrenaline (Adr), urethane was purchased from Sigma,
Mumbai. Nifedipine (Calcigard) was obtained from Torrent.
Preparation of test compounds
After suspending the test compunds in 0.5% aqueous
solution of methyl cellulose, test samples were adminis-
tered to test animals intraperitoneally.
In vivo pharmacological screening (Sujatha et al.,
2006)
ECG measurements (Malawska et al., 2002;
Dylag et al., 2004)
Cardiovascular activity
Electrocardiographic investigations were carried out with
the application of subcutaneous needle electrodes to the
flexor aspect of limbs of rat, connected with the SS2L
electrodes to the flexor aspect of limbs of rat and connected
with the SS2L electrode transducer connected with the
Four channel Data Acquisition System (BIOPAC System
Inc, MP 35) with the respective ECG Transducer. Physi-
ological ECG recordings were taken from Wistar rats
(250–300 g) anaesthatized with thiopental (60 mg/kg ip)
and afterwards, compounds under investigation were
injected. Test compounds were administered intraperito-
nially (40 mg/kg). ECG parameters were collected at 0, 30,
45 and 60 min of administration of compounds.
Common Indian adult frogs of Rana tigerina species were
used in this study. The great advantage of frog tissues is that
they can function as isolated preparations for many hours
when handled carefully and to maintain the tissue prepara-
tion, No extra supply of oxygen is needed as the frog muscles
can directly imbibe oxygen from the atmosphere. The ani-
mals were maintained as per the norms of Committee for the
purpose of Control and Supervision of Experiments on
Animal (CPCSEA), and this experiment was cleared by
CPCSEA and Institutional Animal Ethics Committee con-
stituted for the purpose. Frogs were dissected from ventral
surface to expose the heart, and pericardium was removed.
The frog heart was isolated, and perfusion was done by
Bulbring’s method as described by Burn (1952) Frog ringer
with the following composition was used as the perfusion
fluid: NaCl, 110.0; KCl, 1.90; CaCl2, 1.10; NaH2PO4, 0.06;
NaHCO3, 2.40; dextrose, 11.10 lM made up to 1000 ml with
distilled water. The apex of ventricle was attached to the
sterling heart lever, which in turn was attached by a thread to
Influence on heart rate (Malawska et al., 2002;
Dylag et al., 2004)
Heart rate recordings were taken from Wistar rats (250–
300 g) with the application of subcutaneous electrodes to
123

712
Med Chem Res (2011) 20:705–713
Bhandari SV, Bothara KG, Patil AV, Chitre TS, Sarkate AP, Gore ST,
Dangre SC, Khachane CV (2009) Design, synthesis and
pharmacological screening of novel antihypertensive agents
using hybrid approach. Bioorg Med Chem 17:390–400
Bisi A, Rampa A, Budriesi, Gobbi S, Belluti F, Ioan P, Valoti E,
Chiarini A, Valenti P (2003) Cardiovascular hybrid drugs: new
benzazepinone derivatives as bradycardiac agents endowed with
selective b1-non competitive antagonism. Bioorg Med Chem
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Bossert F, Meyer H, Wehnger E et al (1981) 4-aryl dihydropyridines,
a new class of highly active calcium antagonists. Int Ed Engl
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Burn JH (1952) Practical pharmacology. Oxford Blackwell, London,
pp 55–67
the flexor aspect of limbs of rat and connected with the
Four channel Data Acquisition System (BIOPAC System
Inc.MP35) with the respective ECG Transducer. Rats were
anaesthetized with Thiopental Sodium (60 mg/kg ip) and,
afterwards, test compounds were injected. Test compounds
were administered intraperitonially (mg/kg). Heart rate (in
beats per min) was recorded at 0, 30, 45 and 60 min of
administration of compounds.
Statistical analysis
The data are expressed as means SEM. The statistical
significance was calculated using a one-way ANOVA test.
Differences were considered significant when p \ 0.05. All
statistical calculations were performed using evaluation
version of Graph Pad Instat3.0 (USA) statistical software.
Christiaans JAM, Timmerman H (1996) Cardiovascular hybrid drugs:
combination of more than one pharmacological property in one
molecule. Eur J Pharm Sci 4:1–22
Dargie HJ (1986) Combination therapy with b-adrenoceptor blockers
and calcium antagonists. Br J Pharmacol 21:155S–160S
Dylag T, Zygmunt M, Maciag D, Hanzlik K, Bendarski M, Filipek B,
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CA 92130, USA. www.graphpad.com
Acknowledgements The authors are thankful to the Principal,
AISSMS College of Pharmacy, Pune for providing infrastructural
facilities. The authors also wish to thank Shimadzu Analytical Centre
and Dept. of Chemistry, Pune University, National Chemical Labora-
tories, Pune for providing facilities for spectral studies of compounds.
Hieble JP (1991) Adrenoceptors: molecular biology. In: Ruffolo R R
Jr (ed) Biochemistry and pharmacology, vol 7. Basel, Karger,
pp 105–110
Declaration of interest The authors report no conflicts of interest.
The authors alone are responsible for the content and writing of the
article.
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