Effect of 4-aryl-2-methyl-5-nitro-1,4-dihydropyridine-3-carboxylates on the guinea pig papillary muscle and isolated human vena saphena magna that is used for coronary artery bypass grafting

Article abstract of DOI:10.1016/j.ejmech.2011.07.018

Background: The goal of this study was to estimate: (i) the action of 5-nitro-substituted 1,4-dihydropyridines as well as Bay K 8644 (CAS [71145-03-4]) and CGP 28392 (CAS [89289-93-0]) on cardiac action potential duration (APD) and isometric contraction i

Full text of DOI:10.1016/j.ejmech.2011.07.018

European Journal of Medicinal Chemistry 46 (2011) 4441e4447
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Effect of 4-aryl-2-methyl-5-nitro-1,4-dihydropyridine-3-carboxylates on the
guinea pig papillary muscle and isolated human vena saphena magna that is used
for coronary artery bypass grafting
,
a
b
c
c
Vida Garaliene a , Vygantas Barsys , Antanas Macys , Brigita Vigante , Aivars Krauze
*
ꢀ
a Institute of Cardiology, Medical Academy of Lithuanian university of Health Sciences, Kaunas Sukileliu 17, Kaunas, LT 50009, Lithuania
b Department of Cardiothoracic and Vascular Surgery, Lithuanian University of Health Sciences, Eiveniu 2, Kaunas, LT 50009, Lithuania
c Latvian Institute of Organic Synthesis, Aizkraukles 21, Riga LV 1006, Latvia
a r t i c l e i n f o
a b s t r a c t
Article history:
Background: The goal of this study was to estimate: (i) the action of 5-nitro-substituted 1,4-
dihydropyridines as well as Bay K 8644 (CAS [71145-03-4]) and CGP 28392 (CAS [89289-93-0]) on
cardiac action potential duration (APD) and isometric contraction in the isolated guinea pig papillary
muscles; (ii) whether the effects of 2-propoxyethyl 4-(2-difluoromethoxyphe-nyl)-2-methyl-5-nitro-1,4-
dihydropyridine-3-carboxylate on the lengthening of cardiac APD were related to certain potassium
channels (e.g., IK1, KATP and IK); and (iii) the modulation of the contraction-relaxation effects on isolated
human vena saphena magna samples using three 5-nitro-substituted 1,4-dihydropyridine derivatives,
displaying the positive inotropic and AP duration effects.
Received 11 April 2011
Received in revised form
9 June 2011
Accepted 9 July 2011
Available online 19 July 2011
Keywords:
5-Nitro-substituted 1,4-dihydropyridine
Human vena saphena magna
Guinea pig’s papillary muscles
Methods: The experiments were conducted on isolated human vena saphena magna samples and
papillary muscles from adult guinea pigs. Isometric contractions and APs were recorded using a force
transducer and microelectrode technique, respectively.
Results:
2-Propoxyethyl
4-(2-difluoromethoxyphenyl)-2-methyl-5-nitro-1,4-dihydropyridine-3-
carboxylate significantly increased APD and isometric contractions in a concentration-dependent
manner. Its effects were suppressed by DL-sotalol. Other derivatives tested, such as Bay K 8644 and
CGP 28392, showed either negligible effects or increased the contraction force but did not influence the
APD. Compounds possessing positive inotropic properties at a concentration of 10ꢀ7 to 10ꢀ4 M signifi-
cantly relaxed the isolated vessel samples pre-contracted with phenylephrine (10ꢀ4 M). The weakest
response was shown by 2-propoxyethyl 4-(2-difluoromethoxyphenyl)-2-methyl-5-nitro-1,4-
dihydropyridine-3-carboxylate.
Conclusion: These results show that 5-nitro-substituted 1,4-dihydropyridine derivatives with positive
inotropic action significantly relaxed isolated vein samples that were pre-contracted with phenylephrine
in
a dose-dependent manner. 2-Propoxyethyl 4-(2-difluoromethoxyphenyl)-2-methyl-5-nitro-1,4-
dihydropyridine-3-carboxylate prolongs the cardiac APD, which could be determined by the rapid
component IKr of the delayed potassium current IK blocker.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
diseases will continue to explore new methods of treatment, such
as medication and surgery. Today, coronary artery bypass graft
Despite the efforts to reduce cardiovascular disease incidence,
the desired results have not been achieved. Consequently,
research geared towards the prevention and treatment of these
operations have been widely used in the clinic to improve the
quality of life of many patients with various cardiovascular
diseases. Over time, ischemic cardiovascular disease may develop
into congestive heart failure (CHF), which is characterised by
weakened contraction forces in the myocardium. During the
surgical operation and postoperative period in patients with
ischemic cardiovascular disease, the main problems are vaso-
spasms and their resultant complications, which, in patients with
CHF, are associated with the additional problem of requiring
Abbreviations: AP, action potential; APD, action potential duration; IK1, inward
rectifier potassium current; KATP, ATP-sensitive Kþ channels; IKr, rapid component
of the delayed rectifier IK current; CHF, congestive heart failure.
* Corresponding author. Tel.: þ370 37 302871; fax: þ370 37 302872.
E-mail address: vida.garaliene@gmail.com (V. Garaliene).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.07.018

4442
V. Garaliene et al. / European Journal of Medicinal Chemistry 46 (2011) 4441e4447
increased contraction force from a weak myocardium. CHF drug
therapy is based on three principles: (i) unloading of the heart
with diuretics; (ii) preload and afterload reduction with vasodi-
lators; and (iii) increasing myocardial contractility by using
positive inotropic agents. The current experimental studies dis-
playing the relaxing mechanism developed in smooth muscle of
blood vessels [1e4] encourage us to synthesise and investigate 4-
aryl-2-methyl-5-nitro-1,4-dihydropyridine-3-carboxylates,
among which the dihydropyridine derivatives with the positive
inotropic properties were discovered [5e7]. In addition to their
positive inotropic effects, their action on arterial smooth muscle
has been intensively studied as well [8e11].
butyrate [15] and 2-nitrovinylamine [16] by continuous refluxing in
a mixture of ethanol and acetic acid.
3. Pharmacological results
3.1. Effects of 1,4-dihydropyridine derivatives on cardiac AP duration
and isometric contraction in the isolated guinea pig papillary muscles
The obtained data show that the inotropic effects of the tested
dihydropyridine derivatives are not unambiguous, although their
chemical structures prima facie seemverysimilar to each other. The
analysis of the structureeactivity relationship of the tested
compounds has shown that their inotropic properties and the
influence on the APD by the nature and position of the substituent’s
at the 1,4-dihydropyridine and phenyl rings are determined. So,
when exposed to compound OSI 9961, the isometric contraction of
isolated guinea pig papillary muscles increased (by an average of
11.5%) regardless of what concentration was used; however, the
absence of the 6-methyl group (OSI 4161) promotes the negative
inotropic properties and elongation of the APD, more pronounced
at 90% repolarization (22.0 ms vs. 7.9 ms in OSI 4161 and OSI 9961,
respectively). The similar evidence manifests in the case of the
compounds Bay K 8644 and OSI 9963: the absence of the 6-methyl
group at the 1,4-dihydropyridine ring varies towards the decrease
of the positive inotropic action and lengthening of the APD,
statistically significant at the early repolarization (APD10 and
APD25) as shown in Table 1. The substitution of 2-difluormethoxy-
to 2-trifluormethyl- at the phenyl ring (OSI 4161 and OSI 9963,
respectively) has not any substantial influence in respect of the
inotropic action and AP duration. Meanwhile, such substitution in
the case of the compounds Bay K 8644 and OSI 9961 enhances only
the positive inotropic effects. The other tested compounds (OSI
9968 and OSI 9719) which chemical structure differs by the nature
and position (4-trifluoromethyl- and 2-trdifluoromethoxy-,
The goals of the present study were to investigate: (i) the effects
of 5-nitro-substituted 1-4-dihydropyridines on cardiac APD
and isometric contractions in isolated guinea pig papillary muscles;
(ii)
whether
the
influence
of
2-propoxyethyl
4-(2-
difluoromethoxyphenyl)-2-methyl-5-nitro-1,4-dihydropyridine-3-
carboxylate on the lengthening of cardiac AP duration was related to
selected potassium channels (e.g., IK1, KATP and IK) using single doses
of BaCl2 (3.1 mM), glibenclamide (20 mM) and DL-sotalol (1.0 mM),
respectively, for the blockade of the inward rectifier potassium
current IK1, IATP and the rapid component of the delayed rectifier
potassium current (IKr); and (iii) the modulation of contraction-
relaxation effects on isolated human vena saphena magna
samples, which is the vessel of choice for coronary artery bypasses
grafting, using three 5-nitro-substituted 1,4-dihydropyridine deriv-
atives displaying positive inotropic and AP duration effects.
2. Chemistry
4-Aryl-2,6-dimethyl-1,4-dihydropyridine derivatives bearing
nitro group at 5 position were synthesized before [12,13] as well as
4-aryl-2-methyl-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine
derivative [14], which exhibit pronounced inotropic effect:
F
F
F
F
O
F
O
O
O
F
O
O
O
O
N
O
O
O
N
F
F
F
N
O
O
O
O
O
O
O
O
N
H
N
H
N
H
N
H
BAY K8644
OSI-9961
CGP-28392
OSI-4161
A new type of 5-nitro-1,4-dihydropyridines, 5-nitro-4-aryl-1,4-
dihydropyridines, bearing lipophilic 2-propoxyethoxycarbonyl
substituent were prepared using a modified Hantzsch reaction of
the appropriate 2-propoxyethyl 2-(1-arylmethylidene)-3-oxo-
respectively) of the substituent’s at the phenyl ring demonstrates
the similar properties as concerns the duration of AP. As for
compounds OSI 4161 and OSI 9719, the substituent 2-
propoxyethyl- causes not only the further lengthening of the AP
but also enhanced the positive inotropic effects. For instance, the
maximum lengthening of the AP duration at a concentration of
10ꢀ4 M (18.5, 39.5, 46 and 50 ms at 10, 25, 50 and 90% repolarisa-
tion, respectively) by the action of OSI 9719 followed dose-
dependently, and it coincided with an augmentation of the
contraction force. The variation of the APD in the case of Bay K 8644
was negligible as well as in case of CGP 28392, which are known as
calcium channel agonists enhancing Ca2þ influx through voltage-
operated calcium channels [5,6]. In our experimental conditions,
the latter compound showed a positive inotropic effect; the
isometric contraction of papillary muscles increased by an average
of 46.0% regardless of the concentration used (10ꢀ7e10ꢀ4 M) (these
data are not presented in Table 1).
R
R
O
O
1
1
NO
R
2
NO
2
R
O
O
+
H N
2
O
N
H
(1a) OSI-9719: R= 2-OCHF2, R1= CH2CH2OC3H7-n
(1b) OSI-9963: R= 2-CF3, R1= CH3
(1c) OSI-9968: R= 4-CF3, R1= CH2CH2OC3H7-n

V. Garaliene et al. / European Journal of Medicinal Chemistry 46 (2011) 4441e4447
4443
Table 1
Effect of cumulative doses on cardiac action potential duration and contraction force in the isolated papillary muscles of guinea pig hearts.
Compound
Dose, M
Contraction force,
Action potential duration, ms
P/Pe ꢁ mx
%
APD10
AFD25
APD50
APD90
Pre-drug
OSI-4161 (n ¼ 4)
100.0
39.0 ꢁ 8.6
36.0 ꢁ 8.0
35.0 ꢁ 7.9
39.5 ꢁ 4.4
39.5 ꢁ 6.9
96.0 ꢁ 12.0
90.6 ꢁ 12.2
93.7 ꢁ 11.8
111.6 ꢁ 3.8
112.6 ꢁ 6.3
161.0 ꢁ 12
153.0 ꢁ 6.9
161.0 ꢁ 5.7
177.0 ꢁ 2.1
178.0 ꢁ 4.9
198.0 ꢁ 4.3
190.0 ꢁ 5.8
202.0 ꢁ 7.1
217.0 ꢁ 8.0
220.0 ꢁ 7.5
l ꢂ 10ꢀ7
l ꢂ 10ꢀ6
l ꢂ 10ꢀ5
l ꢂ 10ꢀ4
78.85 ꢁ 12.6
73.5 ꢁ 14.3
90.3 ꢁ 16.0
92.8 ꢁ 16.0
Pre-drug
OSI-9961 (n ¼ 4)
100.0
47.0 ꢁ 3.8
51.3 ꢁ 6.0
54.5 ꢁ 5.5
55.3 ꢁ 1.7
53.8 ꢁ 1.2
98.3 ꢁ 5.4
101.7 ꢁ 7.7
110.3 ꢁ 4.7
110.8 ꢁ 2.6
109.2 ꢁ 2.6
141.0 ꢁ 5.4
144.8 ꢁ 7.0
149.0 ꢁ 1.6
147.0 ꢁ 4.0
148.5 ꢁ 3.7
168.8 ꢁ 6.3
178.3 ꢁ 5.0
178.3 ꢁ 2.3
175.0 ꢁ 3.4
176.7 ꢁ 7.3
l ꢂ 10ꢀ7
l ꢂ 10ꢀ6
l ꢂ 10ꢀ5
l ꢂ 10ꢀ4
114.4 ꢁ 8.4
112.6 ꢁ 7.4
108.3 ꢁ 7.0
111.0 ꢁ 4.0
Pre-drug
Bay K 8644 (n ¼ 5)
100.0
40.0 ꢁ 6.5
44.5 ꢁ 7.0
42.0 ꢁ 7.0
41.0 ꢁ 7.5
42.7 ꢁ 7.2
88.0 ꢁ 8.0
92.7 ꢁ 6.9
91.0 ꢁ 6.9
85.1 ꢁ 10.3
89.0 ꢁ 10.4
133.4 ꢁ 7.7
135.6 ꢁ 8.5
133.8 ꢁ 8.9
129.0 ꢁ 10.0
133.8 ꢁ 10.2
218.5 ꢁ 5.5
220.0 ꢁ 9.0
218.2 ꢁ 6.5
217.0 ꢁ 9.0
223.5 ꢁ 10.0
l ꢂ 10ꢀ7
l ꢂ 10ꢀ6
l ꢂ 10ꢀ5
l ꢂ 10ꢀ4
123.0 ꢁ 9.2
155.0 ꢁ 13.2
175.0 ꢁ 22.0
176.0 ꢁ 31.0
Pre-drug
OSI-9963 (n ¼ 4)
100.0
37.8 ꢁ 2.2
40.0 ꢁ 1.5
38.5 ꢁ 1.5
44.7 ꢁ 4.8
47.0 ꢁ 1.0**
86.7 ꢁ 5.2
92.5 ꢁ 5.7
132.7 ꢁ 7.8
138.0 ꢁ 13.0
140.5 ꢁ 10.5
149.8 ꢁ 7.7
156.5 ꢁ 12.5
169.0 ꢁ 7.0
171.5 ꢁ 8.6
172.0 ꢁ 8.0
175.5 ꢁ 11.2
184.0 ꢁ 13.0
l ꢂ 10ꢀ7
l ꢂ 10ꢀ6
l ꢂ 10ꢀ5
l ꢂ 10ꢀ4
101.0 ꢁ 7.3
98.5 ꢁ 10.0
103.0 ꢁ 10.2
97.8 ꢁ 8.2
91.4 ꢁ 6.3
103.2 ꢁ 3.2*
111.3 ꢁ 6.4*
Pre-drug
OSI-9968 (n ¼ 6)
100.0
30.0 ꢁ 2.2
35.0 ꢁ 6.7
36.0 ꢁ 7.2
37.5 ꢁ 6.0
38.2 ꢁ 8.3
84.0 ꢁ 10.3
91.0 ꢁ 12.4
96.0 ꢁ 14.4
103.0 ꢁ 11.3
101.0 ꢁ 10.8
136.4 ꢁ 12.8
136.6 ꢁ 12.3
149.6 ꢁ 12.2
161.2 ꢁ 12.5
158.7 ꢁ 12.9
163.0 ꢁ 12.7
166.7 ꢁ 12.3
180.0 ꢁ 9.4
188.0 ꢁ 11.5
189.6 ꢁ 11.3
l ꢂ 10ꢀ7
l ꢂ 10ꢀ6
l ꢂ 10ꢀ5
l ꢂ 10ꢀ4
89.5 ꢁ 7.7
83.7 ꢁ 9.5
81.0 ꢁ 9.7
73.0 ꢁ 10.6
Pre-drug
OSI-9719 (n ¼ 6)
100.0
37.0 ꢁ 5.7
40.0 ꢁ 7.0
41.4 ꢁ 7.7
46.0 ꢁ 6.3
55.5 ꢁ 5.0*
107.4 ꢁ 10.0
112.7 ꢁ 10.6
118.6 ꢁ 9.8
133.0 ꢁ 5.9
146.9 ꢁ 5.7**
176.0 ꢁ 8.4
188.5 ꢁ 7.0
196.0 ꢁ 6.8
205.8 ꢁ 5.7
222.0 ꢁ 5.2***
206.0 ꢁ 10.4
221.6 ꢁ 9.4
229.0 ꢁ 7.0
238.5 ꢁ 5.9*
256.0 ꢁ 4.0**
l ꢂ 10ꢀ7
l ꢂ 10ꢀ6
l ꢂ 10ꢀ5
l ꢂ 10ꢀ4
108.0 ꢁ 3.7
120.0 ꢁ 6.0
134.0 ꢁ 8.8
190.8 ꢁ 23.7
Data are expressed as means ꢁ SE. Stimulation rate. 1 Hz; P, contraction force at a given concentration; P0, control (100%); APD10, APD25, APD50, APD90 AP duration at 10, 25, 50
and 90% repolarization; n is the number of preparations (papillary muscles); control, physiological solution containing less than 1
***P < 0.001 vs. pre-drug value.
mM DMSO; *P < 0.05; **P < 0.01;
3.2. Effect of glibenclamide, DL-sotalol and BaCl2 on the effects of the
dihydropyridine derivative OSI-9719 on action potential duration
and contraction force in the isolated papillary muscles of guinea pig
hearts
lengthening of the AP duration in guinea pig papillary muscle by
5.5 ms, 10 ms and 11.8 ms at 25, 50 and 90% repolarisation,
respectively, for OSI-9719. The common application of sotalol and
OSI-9719 at a concentration of 10ꢀ7 to 10ꢀ5 M significantly sup-
pressed the contraction force and AP duration, more evidenced at
10% and 25% repolarisation. A 10-fold higher concentration of
tested compound reduces the sotalol effects because both the
contraction forces and AP durations (APD50 and APD90) approached
the initial levels (Table 1). According to the EC50 values for the
contraction force, DL-sotalol decreases the positive inotropic action
of OSI-9719 about 20 times 1.6 ꢁ 0.98 ꢂ 10ꢀ6 M for OSI-9719 vs.
3.6 ꢁ 2.48 ꢂ 10ꢀ5 M for OSI-9719 þ sotalol, and a dose-effect curve
moves to a higher concentrations of OSI-9719 (Fig. 1). Based on the
EC50 values for APD90, sotalol conditions a lesser effect of OSI-9719
on the APD as well (2.7 ꢁ 2.52 ꢂ 10ꢀ6 M for OSI-9719 vs.
1.5 ꢁ 2.0 ꢂ 10ꢀ5 M for OSI-9719 þ sotalol).
To evaluate whether the prolongation of cardiac AP duration
does not occur during the inhibition of some potassium channels,
we used the sulphonylurea compound glibenclamide that acts as
a specific blocker of the ATP-sensitive Kþ channels (KATP) [17].
Glibenclamide (20
mM) caused a slight lengthening of the AP
duration, being more pronounced at 90% repolarisation (8 ms, 3.5%,
P > 0.05), and had no effect on the isometric contraction (Table 2).
Application of both agents together did not cause a more
significant prolongation of the AP nor augment the contraction
force compared to OSI-9719 alone. The calculated EC50 (half-
maximum effective dose induced by the tested agent) values for the
isometric contraction were 1.6 ꢁ 0.98 ꢂ 10ꢀ6 M for OSI-9719 and
6.9 ꢁ 2.7 ꢂ 10ꢀ6 M for OSI-9719 þ glibenclamide and for APD90
were 2.7 ꢁ 2.52 ꢂ 10ꢀ6 M for OSI-9719 and 3.28 ꢁ 2.16 ꢂ 10ꢀ6 M for
OSI-9719 þ glibenclamide. However, according to the dose-effect
curves shown in Fig. 1, glibenclamide caused a slight shift of the
contraction force to the lower concentrations of compound OSI-
9719.
To assess the role of the inward rectifier potassium current, IK1
,
on the action of OSI-9719, we used barium chloride, which is
reported to selectively blocks IK1 [19,20]. Our results obtained after
a 20-min exposure of guinea pig papillary muscles to 3.1 m
M Ba2þ
showed that BaCl2 caused a slight (an average of 6.6%) lengthening
of the AP duration at all the tested repolarisation levels. The
common application of Ba2þ and OSI-9719, which was used in
a concentration-dependent manner, resulted in a dose-effect curve
for the contraction force shift to the left, i.e., to a lower concen-
tration of the compound tested. In this case, the calculated EC50
values appeared to be 1.03 ꢁ 0.83 ꢂ 10ꢀ6 M and 1.3 ꢁ 0.61 ꢂ10ꢀ6 M
for the contraction force and APD90, respectively.
Sotalol is a well-known blocker of the rapid component (IKr) of
the delayed rectifier potassium channel [18], i.e., under its influ-
ence, the APD and effective refractory period increases. Our pre-
sented data (Table 2 and Fig. 1) show that DL-sotalol results in
a
reduction of the contraction force by 19% and minimal

4444
V. Garaliene et al. / European Journal of Medicinal Chemistry 46 (2011) 4441e4447
Table 2
Effect of cumulative doses of compound OSI-9719, glibenclamide, DL-sotalol and BaCI2 on cardiac AP duration and contraction force in the isolated papillary muscles of guinea
pig hearts.
Compound
Concentration, M
Contraction force,
Action potential duration, ms
P/Pe ꢁ mx
%
APD10
APD25
APD50
APD90
Pre-drug
Gilbenclamide only
þ OSI-9719
100.0
52.6 ꢁ 3.3
53.7 ꢁ 3.8
59.5 ꢁ 3.3
59.6 ꢁ 3.9
61.0 ꢁ 4.2
65.0 ꢁ 4.2*
131.0 ꢁ 3.0
197.0 ꢁ 5
222.4 ꢁ 5.0
2 ꢂ 10ꢀ5
1 ꢂ 10ꢀ7
1 ꢂ 10ꢀ6
1 ꢂ 10ꢀ5
1 ꢂ 10ꢀ4
100.0 ꢁ 3.2
103.0 ꢁ 7.4
120.0 ꢁ 4.2
163.0 ꢁ 5.2
211.0 ꢁ 6.4
136.7 ꢁ 5.5
204.8 ꢁ 8.0
209.0 ꢁ 7.5
219.0 ꢁ 6.8*
235.0 ꢁ 8.0***
247.0 ꢁ 8.5***
230.4 ꢁ 8.0
141.0 ꢁ 4.0
234.5 ꢁ 7.0
148.0 ꢁ 3.2**
155.0 ꢁ 5.2***
164.8 ꢁ 5.6***
247.0 ꢁ 5.9**
265.0 ꢁ 8.9***
276.0 ꢁ 8.8***
Pre-drug
100.0
52.0 ꢁ 3.2
54.5 ꢁ 3.6
54.2 ꢁ 2.7
49.0 ꢁ 4.5
54.6 ꢁ 2.6
55.7 ꢁ 2.3
120.5 ꢁ 2.8
12.6 ꢁ 2.8
180.8 ꢁ 6.0
190.8 ꢁ 7.7
195.0 ꢁ 7.7
192.0 ꢁ 7.0
199.0 ꢁ 3.6*
218.9 ꢁ 7.4***
209.0 ꢁ 6.0
220.8 ꢁ 7.4
224.4 ꢁ 7.4
224.0 ꢁ 7.4
227.7 ꢁ 3.4*
250.8 ꢁ 8.0***
Sotalol only
þ OSI-9719
1 ꢂ 10ꢀ6
1 ꢂ 10ꢀ7
1 ꢂ 10ꢀ6
1 ꢂ 10ꢀ5
1 ꢂ 10ꢀ4
81.0 ꢁ 3.5
85.0 ꢁ 5.0
93.6 ꢁ 10.9
100.2 ꢁ 9.0
154.8 ꢁ 18.4
130.0 ꢁ 2.2*
132.9 ꢁ 4.2*
133.6 ꢁ 1.6*
142.0 ꢁ 4.3***
Pre-drug
BaCI2 only
þ OSI-9719
100.0
54.2 ꢁ 0.6
57.7 ꢁ 3.6
129.3 ꢁ 4.4
192.7 ꢁ 8.6
203.9 ꢁ 4.9
210.0 ꢁ 8.5
224.0 ꢁ 3.8**
231.0 ꢁ 5.0**
238.0 ꢁ 7.9**
218.3 ꢁ 7.5
3.1 ꢂ 10ꢀ6
1 ꢂ 10ꢀ7
1 ꢂ 10ꢀ6
1 ꢂ 10ꢀ5
1 ꢂ 10ꢀ4
107.0 ꢁ 12.0
111.4 ꢁ 14.0
118.0 ꢁ 18.5
193.0 ꢁ 23.7
204.5 ꢁ 29.0
138.8 ꢁ 4.2
233.5 ꢁ 6.3
64.3 ꢁ 3.6*
60.9 ꢁ 2.0**
63.4 ꢁ 2.0**
61.36 ꢁ 1.7***
146.5 ꢁ 3.4**
149.7 ꢁ 2.5**
155.0 ꢁ 2.9***
158.0 ꢁ 3.8***
237.0 ꢁ 10.7
250.7 ꢁ 5.4**
260.5 ꢁ 6.5***
267.9 ꢁ 9.7***
The values are expressed as means ꢁ SE of six preparations at each concentration. Stimulation rate 1 Hz; P, contraction force at a given concentration; P0, control (100%);
APD10, APD25, APD50, APD90, the AP duration at a level of 10, 25, 50 and 90% repolarization; control, physiological solution containing less than 1 mM DMSO; *P < 0.05;
P < 0.01**; ***P < 0.001 vs. pre-drug value.
3.3. Effect of 1,4-dihidropyridine derivatives OSI-9719, Bay K 8644
and CGP 28392 on phenylephrine-induced contractions in human
vena saphena magna rings
10 samples reached the baseline in the Bay K 8644 and CGP 28392
groups, respectively. At a concentration of 10ꢀ4 M, such samples
were 11 and 6, respectively. In addition, some vein rings relaxed
below baseline by 19e49%, whereas the relaxation effect of OSI-
9719 occurred at a concentration of 10ꢀ5 M.
Phenylephrine (10ꢀ4 M) added to the physiological solution
caused a contraction of the vein rings to 1056 ꢁ 143 mg. All of the
tested agents at a concentration of 10ꢀ7 to 10ꢀ4 M significantly
relaxed the smooth muscle, more significantly in the cases of Bay K
8644 and CGP 28392, as shown in Fig. 2. The significant efficacy of
4. Discussion
In this work, we characterised the effects of six 5-nitro-
substituted 1-4-dihydropyridines and CGP 28392, three of which
have an original structure, on cardiac AP duration and isometric
contractions of guinea pig papillary muscles and on isolated human
vena saphena magna samples. The following findings were
obtained.
these derivatives has already occurred at the 0.1-mM concentrations
and under their influence the isometric contraction of the vein
samples decreased about 50%, whereas in case of OSI-9719, it was
26%. At a concentration of 10ꢀ5 M, the relaxation of 7 of 16 and 3 of
(i) All of the tested compounds, except OSI-9968, at concen-
trations from 10ꢀ7 to 10ꢀ4 M show more positive than negative
inotropic effects, which are not characterised by a majority of 1,4-
Fig. 2. Relaxation responses induced by 1,4-dihydropyridine derivatives OSI-9719 (n ¼
11: 4 female, 7 male), Bay K 8644 (n ¼ 16: 6 female, 10 male) and CGP 28392 (n ¼ 10: 2
female, 6 male) in the isolated human vena saphena magna rings precontracted by
10ꢀ4 M phenylephrine; n is the number of vein samples. Results are expressed as
means ꢁ SE. *P < 0.05 vs. Bay K 8644 and CGP 28392; *#P < 0.01 vs. CGP 28392.
Fig. 1. Influence of the 1,4-dihydropyridine derivative OSI-9719 at a concentration of
10ꢀ7 to 10ꢀ4 of glibenclamide (20
mM), DL-sotalol (1 mM), and BaCl2 (3.1 mM) on the
contraction force in isolated papillary muscles of guinea pig hearts. Data are expressed
as means ꢁ SE of six preparations for the every curve. *P < 0.05 vs. OSI-9719.

V. Garaliene et al. / European Journal of Medicinal Chemistry 46 (2011) 4441e4447
4445
dihydropyridine derivatives [21]. In addition, they also had more or
less prolonged AP durations, and in the cases of OSI-9963 and OSI-
9719, it became statistically significant (Table 1). Bay K 8644 and
CGP 28392, which are well known as calcium channel agonists that
increase Ca2þ entry through voltage-dependent calcium channels
[22e24], in our experimental conditions did not have any influence
on the AP duration but simultaneously increased the isometric
contraction of the papillary muscles. Thus, on the basis of these
data, we speculate that in the mode of action of the tested 5-nitro-
substituted 1-4-dihydropyridine derivatives (except Bay K 8644
and CGP 28392), potassium channels could take part in the repo-
larisation processes of the cardiac membrane potential [25].
(ii) It has been shown in many publications that 1,4-
dihydropyridine derivatives are characterised not only as the
blockers of the slow calcium channels but some of them also reduce
KATP-efflux from ischemic myocardial cells and thereby are protec-
tive against ischemic injury [26,27]. An analysis of our results
obtained with glibenclamide, a blocker of sarcolemmal KATP chan-
nels, shows that glibenclamide does not intensify the effects of OSI-
9719 on the repolarisation state because the common action of both
agents does not cause a further lengthening of the AP duration at any
of the tested repolarisation levels. The ratio of the calculated
EC50(OSI-9719)/EC50(OSI 9719þglibenclamide) for APD90 appears to be 1:1.32
(P > 0.05). The insignificant shift of the isometric contraction curve
tolowerconcentrations of OSI-9719 confirmthe proposition that the
sarcolemmal KATP channels are not involved in the mode of action of
the 5-nitro-substituted 1,4-dihydropyridine derivative OSI-9719.
The inward rectifier potassium current in the cardiac muscles
cells, IK1, has been suggested to play a central role in the mainte-
nance and stabilisation of membrane resting potential as well as in
repolarisation of the later phase of AP [28e31]. In the present study,
we found that in response to a concerted influence of OSI-9719 and
BaCl2, a specific blocker of IK1, an insignificant effect of these agents
was observed on the changes of AP duration. It seems that this
finding suggests a lack of involvement of IK1-dependent mecha-
nisms in the action of the tested compounds on the cardiac
membrane potential. However, according to the ratio of EC50 for
APD90 (1:0.6, P > 0.05) and for contraction force (1:0.5, P > 0.05),
BaCl2 shows a tendency to increase the sensitivity of cardiac tissue
to the action of OSI-9719.
properties relax the vein samples significantly from constriction
induced by 10ꢀ4 M phenylephrine. The relaxing effects of Bay K 8644
and CGP 28392 at concentrations of 0.1e1.0 mM were expressed
thoroughly. It is known that the mechanism underlying the positive
inotropic effects of the compounds Bay K 8644 and CGP 28392 is
closely associated with the increased Ca2þ entry into myocardial
cells via voltage-gated calcium channels and trigged Ca2þ release
from the sarcoplasmic reticulum [34e37]. Today, we know that
potassium (Kþ) channels play an important role in the regulation of
vascular tone [3,4,38]. There are three subtypes of calcium-activated
potassium channels (KCa) of large (BKCa), intermediate (IKCa) and
small (SKCa) conductance found in vascular smooth muscle cells that
represent a significant proportion of all the Kþ channels [3,39,40].
BKCa channels are preferentially expressed in vascular smooth
muscle cells, whereas IKCa and SKCa are preferentially located in
endothelial cells. The augmentation of Ca2þ in the endothelium
contributes to the activation of the endothelial KCa channels, nitric
oxide generation, endothelium-dependent hyperpolarisation and
reduction in intracellular calcium [41,42]. It seems likely that the
tested calcium agonists Bay K 8644 and CGP 28392 increased Ca2þ
entry into the endothelial and smooth muscle cells resulting in
elevated Ca2þ sparks from the sarcoplasmic reticulum via
ryanodine-sensitive calcium channels, which lead to an increase in
the potassium current through KCa located both in the endothelium
(IKCa and SKCa) and in smooth muscle (BKCa) [39,43]; the ultimate
result is the relaxation of the vein rings. Theweaker relaxing effect of
OSI-9719 compared to Bay K 8644 and CGP 28392 could be
explained by its blocking action on the other potassium channels.
5. Conclusion
The results presented in this study showed that 5-nitro-
substituted 1,4-dihydropyridine derivatives possessing positive
inotropic action significantly relaxed isolated vein samples pre-
contracted with phenylephrine in a dose-dependent manner. In
addition to the positive inotropic and relaxing properties, the other
1,4-dihydropyridine derivative, i.e., 2-propoxyethyl 4-(2-
difluoromethoxyphenyl)-2-methyl-5-nitro-1,4-dihydropyridine-3-
carboxylate, prolongs the cardiac action potential duration, which
could be determined by the rapid component IKr of the delayed
potassium current IK blocker.
The delayed rectifier potassium current, IK, is a major outward
current that is responsible for ventricular AP repolarisation
[20,28,30e32]. In most species, including the guinea pig, IK can be
separated into rapid (IKr) and slow (IKs) components by both kinetic
and pharmacological differences, i.e., they differ from one another
in terms of their sensitivity to various drugs [33]. Experimental
studies have demonstrated that the most selective inhibitor of the
rapidly activating component IKr is sotalol, a representative of class
III anti-arrhytmic agents [28]. Our results show that DL-sotalol,
which decreased the contraction force of papillary muscle by 19%
and prolonged the APD50 and APD90 by 10 and 11 ms, respectively,
suppresses the effects of OSI-9719 because under the influence of
simultaneous application of both agents, the contraction force did
not exceed the baseline level and the APD decreased compared to
OSI-9719 alone at concentrations from 10ꢀ7 to 10ꢀ5 M. The impact
of sotalol was significantly weakened at a concentration of 10ꢀ4 M.
The ratio of EC50 values for the isometric contraction (1:22.5) and
for ADP90 (1:5.5), as well as a significant shift in the “dose-effect”
curve for the contraction force towards the major concentrations of
OSI-9719 suggests that DL-sotalol and OSI-9719 have competitive
properties in regards to the rapid component (IKr) of the delayed
potassium current and Ca2þ ions participating in the generating of
cardiac muscle contraction.
6. Materials and methods
6.1. Chemistry
TLC was performed on 20 ꢂ 20 cm Silica gel TLC-PET F254 foils
(Fluka). NMR spectra were recorded with
a Varian 400-MR
(400 MHz). Chemical shifts are reported in ppm relative to hexame-
thyldisiloxane ( 0.055). Multiplicities are abbreviated as follows: s,
d
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; and br, broad.
The coupling constants are expressed in hertz. Mass spectral data
were determined on an Acquity UPLC system (Waters) connected to
a Q-TOF micro hybrid quadrupole time of flight mass spectrometer
(Micromass) operating in the ESI positive or negative ion mode on an
Acquity UPLC BEH C18 column (1.7
m
m, 2.1 mm ꢂ 50 mm) using
a gradient elution with acetonitrile/formic acid (0.1%) in water.
Melting points (mp) were determined on an OptiMelt (SRS Stanford
Research Systems). Elemental analyses were determined on an EA
1106
(Carlo
Erba
Instruments).
Methyl
4-(2-difluoro-
methoxyphenyl)-2-methyl-5-nitro-1,4-dihydropyridine-3-carbo-
xylate CAS [165289-46-3] (OSI-4161) was synthesised as previously
described [44], methyl 4-(2-difluoromethoxyphenyl)-2,6-dimethyl-
5-nitro-1,4-dihydropyridine-3-carboxylate-CAS[104616-66-2](OSI-
(iii) Data presented in this study show that 5-nitro-substituted
1,4-dihydropyridine derivatives, possessing positive inotropic
9961)
as
previously
described
[12],
methyl
4-(2-

4446
V. Garaliene et al. / European Journal of Medicinal Chemistry 46 (2011) 4441e4447
trifluoromethylphenyl)-2,6-dimethyl-5-nitro-1,4-dihydropyridine-
3-carboxylate CAS [71145-03-4] (Bay K 8644) as previously described
[13], and ethyl 4-(2-difluoromethoxyphenyl)-2-methyl-5-oxo-
1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate CAS [89289-93-
0] (CGP 28392) as previously described [14].
KCl, 4; TrisC1, l0 and glucose, 5; the reaction occurred at pH 7.3e7.4
and 37 ꢃC and was aerated with oxygen continuously. A 1.0 Hz
stimulation frequency was used.
Action potential (AP) recordings were obtained with standard
microelectrodes that were made from borosilicate glass capillaries
(GC150F-10, Harvard Part No. 30-0057, England), filled with 2.5 M
KCl and connected to the high-input impedance amplification
system. Both signals (the inotropic response and AP) were digitised
at a minimum sampling rate of 10 kHz with a 12-bit A/D converter
(Digidata 1200, Keithley Instruments, Inc., Cleveland, Ohio, USA)
and recorded with a computer. The signals were preserved there
and analysed to obtain the maximal contraction amplitude and the
6.1.1. General method for the synthesis of 4-aryl-2-methyl-5-nitro-
1,4-dihydropyridine-3-carboxylates 1 aec
A solution of 2-nitrovinylamine (0.46 g, 5.2 mM) and the
appropriate ester of 2-(1-aryl-methylidene)-3-oxobutyric acid
(5.0 mM) in
a mixture of ethanol and glacial acetic acid
(30 mL þ 5 mL) was refluxed with stirring for 48 h. The solvents
were removed under reduced pressure. The residue was dissolved
in 80 mL of chloroform and washed with water (2 ꢂ 30 mL). The
organic extract was dried over MgSO4, and the solvent was
removed under vacuum. Purification of the residue using silica gel
column chromatography (eluent: chloroform:hexane:acetone at
9:7:2) yielded a bright yellow substance, which was crystallised
from methanol.
AP duration at 10, 25, 50 and 90% repolarisation (APD10, APD25
,
APD50 and APD90, respectively). After a 1-h equilibration period, the
tested compounds affected each papillary muscle in a dose-
dependent manner.
For the investigations of the blood vessel segments, the vein
samples were obtained from 37 patients who underwent coronary
artery bypass grafting at the Department of Cardiothoracic and
Vascular Surgery at the Lithuanian University of Health Sciences.
The age of the patients was in the range of 37e89 years, and the
average age was 66.13 ꢁ 11.2 years; 33.44% were women, and
66.56% were men. All patients were informed and signed a letter of
consent, and the study was approved by the Regional Ethics
Committee of the Biomedical Research on 05/11/2010 (Licence No.
BE-2-64, Kaunas, Lithuania).
For the investigations of the blood vessel segments, the system
obtained from the Global Town Microtechnology Company (Sar-
asota, FL, USA) was used. Human vena saphena magna samples
were carefully cleaned of connective tissue, cut into 3e4 mm long
rings, hung on a vascular holder whose upper hook was attached
to an isometric force transducer (i-FOT10) and dipped in 5-mL
tissue baths filled with Tyrode’s solution that was warmed
(37 ꢃC) and continuously bubbled with 100% O2. The solution had
the following composition (in mM): NaCl, 137; KCl, 5.4; CaCl2, 1.8;
MgCl2, 0.9; Tris HCl, 10; glucose, 5; and pH ¼ 7.4. The preparations
were allowed to equilibrate for at least 45e60 min. During the
investigation period, the vessels were washed every 15 min with
fresh Tyrode’s solution. A vasoconstrictor (10ꢀ4 M phenylephrine)
or the tested agents were then added to the solution to study the
activity of the vessel examples. Cumulative concentration-
contraction curves of the tested agents (10ꢀ7 to 10ꢀ4 M) were
established.
6.1.1.1. Methyl
dihydropyridine-3-carboxylate (1a, OSI-9963). Yield 35%, mp
254 ꢃC 1H NMR (400 MHz, CDCl3):
2.30 (s, 3H, 2eCH3), 3.54 (s, 3H,
2-methyl-5-nitro-4-(2-trifluoromethylphenyl)-1,4-
d
OCH3), 5.88 (s, 1H, 4eCH), 6.08 (d, J ¼ 5.5 Hz, 1H, NH), 7.24e7.50 (m,
4H, aromatic protons), 7.83 (d, J ¼ 6.3 Hz, 1H, 6eCH). MS(þESI) m/z
(relative intensity) 343 ([M
C15H13F3N2O4: C 52.64; H 3.83; N 8.18; found: C 52.37, H 3.87, N 7.99.
þ
H]þ, 80). Anal. Calcd. for
6.1.1.2. 2-Propoxyethyl
nitro-1,4-dihydropyridine-3-carboxylate (1b, OSI-9719). Yield 32%,
mp 98e100 ꢃC 1H NMR (400 MHz, CDCl3):
4-(2-difluoromethoxyphenyl)-2-methyl-5-
d
0.86 (t, J ¼ 7.3 Hz, 3H,
CH3), 1.50e1.56 (m, 2H, CH2CH3), 2.26 (s, 3H, 2eCH3), 3.37 (t,
J ¼ 7.3 Hz, 2H, CH2), 3.59 (t, J ¼ 7.3 Hz, 2H, CH2), 4.12 (m, 2H, CH2),
5.56 (s,1H, 4eCH), 6.46 (t, J ¼ 74.0 Hz,1H, OCHF2), 6.61 (d, J ¼ 6.0 Hz,
1H, NH), 6.94e7.34 (m, 4H), 7.77 (d, J ¼ 6.2 Hz,1H, 6eCH). ESI-MS m/z
(relative intensity) 411 ([M ꢀ H]þ 100). Anal. Calcd. for C19H22F2N2O6
: C 55.34; H 5.38; N 6.79; found C 55.10; H 5.33; N 6.70.
6.1.1.3. 2-Propoxyethyl 2-methyl-5-nitro-4-(4-(trifluoromethylphenyl)-
1,4-Dihydropyridine-3-carboxylate (1c, OSI-9968). Yield 21%, mp
103e105 ꢃC 1H NMR (400 MHz, CDCl3):
d
0.86 (t, J ¼ 7.3 Hz, 3H, CH3),
1.53 (m, 2H, CH2CH3), 2.34 (s, 3H, 2eCH3), 3.30 (t, J ¼ 7.3 Hz, 2H, CH2),
3.48e3.50 (m, 2H, CH2), 4.10e4.13 (m, 2H, CH2), 5.38 (s, 1H, 4eCH),
6.12 (d, J ¼ 5.3 Hz, 1H, NH), 7.39e7.46 (m, 4H), 7.82 (d, J ¼ 6.2 Hz, 1H,
2eCH). ESI-MS m/z (relative intensity): 413 ([M ꢀ H]þ, 100). Anal.
Calcd. for C19H21F3N2O5: C 55.07; H 5.11; N 6.76; found C 55.14; H
5.30; N 6.60.
6.3. Drugs and chemicals
The 5-nitro-substituted 1-4-dihydropyridine derivatives were
synthesised at the Latvian Institute of Organic Synthesis, Riga,
Latvia. Phenylephrine dihydrochloride, DL-sotalol and glibencla-
mide were obtained from SigmaeAldrich Chemie (Taufkirchen,
Germany). The test compounds were prepared as stock solutions of
0.1 M in dimethylsulfoxide; phenylephrine dihydrochloride was
dissolved in deionised water to make a stock solution of 0.1 mM. All
stock solutions were stored in refrigerator.
6.2. Experimental pharmacological methods
The experiments were preformed on isolated human vena
saphena magna samples obtained from conventional myocardial
revascularisation operations and on papillary muscle from adult
guinea pigs.
The positive inotropic activity and transmembrane AP duration
(APD) of the presented dihydropyridine derivatives was evaluated
on the guinea-pig papillary muscles according to previously
described procedures [45]. This procedure was reviewed and
approved by the Lithuanian State Food and Veterinary Office
(Permission to use the laboratory animals in the research, 29/10/
2005, No. 0139). The animals were obtained from the Lithuanian
Veterinary Academy (Licence No. B-76, 06/06/2005).
6.4. Statistics
Data are presented as means ꢁ SE. The significance of the
difference between the means was analysed by the Student’s t-test.
A level of P < 0.05 was adopted to indicate statistical significance.
Briefly, guinea pig right ventricle papillary muscles were
mounted in an organ bath with a circulation of Tyrode’s solution of
the following composition (in mM): NaCl, 144; CaCl2, 1.8; MgCl2, 1;
Conflict of interest
The authors report no conflicts of interest with respect to this
paper.

V. Garaliene et al. / European Journal of Medicinal Chemistry 46 (2011) 4441e4447
4447
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This work has been supported by the Institute of Cardiology at
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