Synthesis and studies of calcium channel blocking and antioxidant activities of novel 4-pyridinium and/or N-propargyl substituted 1,4-dihydropyridine derivatives

Article abstract of DOI:10.1016/j.crci.2013.07.003

The novel 1,4-dihydropyridine derivatives containing the cationic pyridine moiety at the position 4, and the N-propargyl group as a substituent at position 1 of the 1,4-DHP cycle were designed, synthesised, and assessed in biological tests. Among all the

Full text of DOI:10.1016/j.crci.2013.07.003

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Full paper/Memoire
Synthesis and studies of calcium channel blocking and
antioxidant activities of novel 4-pyridinium and/or
N-propargyl substituted 1,4-dihydropyridine derivatives
Martins Rucins a, Dainis Kaldre a, Karlis Pajuste a,b, Maria A.S. Fernandes c,
Joaquim A.F. Vicente c, Linda Klimaviciusa b, Elina Jaschenko a,
Iveta Kanepe-Lapsa a, Irina Shestakova a, Mara Plotniece a,
Marina Gosteva a, Arkadij Sobolev a, Baiba Jansone b, Ruta Muceniece b,
Vija Klusa b, ,1, Aiva Plotniece a, ,2
*
*
a Latvian Institute of Organic Synthesis, Aizkraukles 21, Riga LV-1006, Latvia
b Faculty of Medicine, University of Latvia, Sarlotes 1A, Riga LV-1001, Latvia
c IMAR–CMA, Department of Life Sciences, University of Coimbra, 3001–401 Coimbra, Portugal
A R T I C L E I N F O
A B S T R A C T
Article history:
The novel 1,4-dihydropyridine derivatives containing the cationic pyridine moiety at the
position 4, and the N-propargyl group as a substituent at position 1 of the 1,4-DHP cycle
were designed, synthesised, and assessed in biological tests. Among all the novel
compounds, the 4-(N-dodecyl) pyridinium group-containing compounds 11 (without the
N-propargyl group) and 12 (with the N-propargyl group) demonstrated the highest
Received 28 March 2013
Accepted after revision 8 July 2013
Available online xxx
Keywords:
calcium antagonistic properties against neuroblastoma SH-SY5Y (IC50 about 5–14
mM)
1,4-Dihydropyridines
N-Dodecyl pyridinium
Propargyl substituent
Calcium antagonists
Antioxidant activity
Mitochondrial processes
Structure–activity relationships
and the vascular smooth muscle A7r5 cell (IC50 – 0.6–0.7 M) lines, indicating that they
m
predominantly target the L-type calcium channels. These compounds showed a slight total
antioxidant activity. At concentrations close to those of L-type calcium channel blocking
ones, compound 12 did not affect mitochondrial functioning; also, no toxicity was
obtained in vivo. The N-propargyl group as a substituent at position 1 of the 1,4-DHP cycle
did not essentially influence the compounds’ activity. The 4-(N-dodecyl) pyridinium
moiety-containing compounds can be considered as prototype molecules for further
chemical modifications and studies as cardioprotective/neuroprotective agents.
´
ß 2013 Academie des sciences. Published by Elsevier Masson SAS. All rights reserved.
1. Introduction
their calcium channel modulating properties, which are
considered as the basis for 1,4-DHPs’ cardioprotective
Derivatives of 1,4-dihydropyridine (1,4-DHP) are one
of the major categories of drugs with a wide variety of
reported pharmacological activities, particularly due to
[1,2], neuroprotective, anti-inflammatory, anti-ischemic,
anti-diabetic, and many other actions [3]. The multi-
faceted effects of 1,4-DHPs were explained by the
privileged structure of the 1,4-DHP scaffolds with appro-
priate substituents, capable of interacting with diverse
receptors and ion channels [4]. During the last decade,
interest has arisen to neurotropic effects of different 1,4-
DHPs, since experimental and clinical studies demon-
strated their ability to penetrate the blood–brain barrier
and reduce the risk of developing neurodegenerative
*
Corresponding authors.
E-mail addresses: vijaklus@latnet.lv (V. Klusa),
aiva@osi.lv (A. Plotniece).
1
Biology.
2
Chemistry.
1631-0748/$ – see front matter ß 2013 Acade´mie des sciences. Published by Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.crci.2013.07.003
Please cite this article in press as: Rucins M, et al. Synthesis and studies of calcium channel blocking and antioxidant
activities of novel 4-pyridinium and/or N-propargyl substituted 1,4-dihydropyridine derivatives. C. R. Chimie (2013),
http://dx.doi.org/10.1016/j.crci.2013.07.003

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M. Rucins et al. / C. R. Chimie xxx (2013) xxx–xxx
diseases [5,6]. For instance, about a 27% reduction in
Parkinson’s disease risk was observed in patients who had
taken centrally acting calcium channel blocking drugs [7].
These drugs (e.g., isradipine) are found to be beneficial also
for the Alzheimer’s disease treatment strategy [8–10]. 1,4-
DHP derivatives of typical calcium antagonist [11] or
atypical (neuronally non-antagonist) class [12] showed
neuroprotective properties also in ischemic stroke models.
2. Results and discussion
2.1. Chemistry
Different pharmacophore moieties were introduced
into the 1,4-DHP cycle:
ꢀ
ꢀ
at position 4, the pyridinium substituent with alkyl
moieties of different lengths at quaternised nitrogen
atom;
Search
for
memory-improving/dementia-preventing
effects of 1,4-DHP derivatives is still on the agenda in
experimental laboratories and clinics [13,14]. Taking into
account mitochondrial abnormalities as the essential
cause of neurodegeneration, particular importance
is related to 1,4-DHP mitochondria-protecting effects
[15–18].
at position 1, the propargyl moiety.
Previously, we had found a direct correlation between
the length of the alkyl chain substituent at N-quaternised
4- -pyridyl-l,4-dihydropyridines and their improved
b
The present study is devoted to the design,
synthesis and biological studies of novel 1,4-DHP deriva-
tives by adding to their structures some pharmacophore
moieties:
membranotropic effects, such as incorporation in the
liposomal membranes and bilayer fluidity [24]. Further-
more, the dodecyl substituent was shown as the optimal-
length moiety among different alkyl groups in alkyltri-
phenylphosphonium or alkylrhodamine derivatives for
their mitochondria-targeted antioxidant activity [23]. In
the present study, some representative types of com-
pounds containing a N-ethyl, N-hexyl, N-octyl, N-dodecyl
or N-hexadecyl pyridinium moiety at position 4 of the 1,4-
DHP ring (compounds 5–14) were synthesised. The
propargyl moiety was introduced in the 1,4-DHP molecule
at position 1; better mitochondria-regulating properties
were expected because this group was considered as
essential in the antidepressant rasagiline molecule [20].
Moreover, N-alkylation of benzimidazole derivatives with
propargyl halides led to more active compounds [25]. All
1,4-DHP derivatives were synthesised as N–H (N-unsub-
stituted) compounds (1, 3, 5, 7, 9, 11, 13, 15 and 17) and N-
propargyl substituted compounds (2, 4, 6, 8, 10, 12, 14, 16
and 18). Our synthetic plan is shown in Scheme 1.
ꢀ
ꢀ
a pyridine substituent at position 4 (compounds 3 and 4)
or N-alkyl pyridinium moiety at the position 4 (com-
pounds 5–18);
a N-propargyl substituent at position 1 (compounds 2, 4,
6, 8, 12, 14, 16, 18).
The propargyl moiety is known to play an important
role in providing neuro- and mitochondria-protecting
properties of propargyl group-containing anti-depres-
sants, selegiline and rasagiline [19–21]. The one and only
literature source about the propargyl group-containing
1,4-DHP derivative – propargyl nitrendipine – demon-
strated the store-operated channel blocking effect in
leukemic HL-60 cells [22]. In turn, the 4-(N-dodecyl)
pyridinium group was considered as a lipophilicity-
giving moiety for the improvement of 1,4-DHP deriva-
tive penetration through the membranes and easier
delivery to cellular targets. The dodecyl chain is found
as an optimal-length alkyl substituent of different
alkylphosphonium compounds for improving their
mitochondria-targeted antioxidative properties [23].
The aim of the present study was to find the
structure–activity relationships and to identify the
importance of structure moieties as the above-men-
tioned 1,4-DHP derivatives for the future design of novel
cytoprotective drugs. The studies were performed in
vitro to assess:
The structures and characterisation of synthesised
novel and reference compounds: N–H (N-unsubstituted)
and N-propargyl-substituted 1,4-DHP derivatives are
presented in Table 1.
The data in the literature reported that the synthesis of
the well-known 4-phenyl- (1) and 4-pyridyl- (3) sub-
stituted Hantzsch-type 1,4-DHP is usually performed via
the classical synthetic procedures; they can be obtained in
good (78–80%) yields [26,27]. In our case, the 1,4-DHP
derivatives 1 and 3 were synthesised with high yields, 92%
and 78% respectively, via typical synthetic routes – the
classical Hantzsch method [30], which involves the one-
pot cyclocondensation of the corresponding aldehyde,
ethyl acetoacetate and ammonia in ethanol under reflux
for 5–7 h. The corresponding N-propargyl-substituted 1,4-
DHP derivatives 2 and 4 were obtained by analogy with the
synthesis of other N-substituted 1,4-DHP ones [31] – via
modified Hantzsch-type cyclisation using propargyl amine
hydrochloride as a nitrogen source instead of ammonia
and pyridine as a solvent for the reaction mixture, under
reflux for 5–12 h. The one and only literature data about
the synthesis and studies of N-propargyl-substituted 1,4-
DHPs, such as N-propargyl nitrendipine (N-propargyl
substituted (RS)-ethyl methyl-2,6-dimethyl-4-(3-nitro-
phenyl)-1,4-dihydropyridine-3,5-dicarboxylate) demon-
strated the alternative synthesis of N-substituted
ꢀ
the compounds’ calcium antagonistic properties in
neuroblastoma SH-SY5Y cells and smooth muscle A7r5
cell line;
ꢀ
ꢀ
the compounds’ antioxidant activity with the phospho-
molybdenum complex method;
the importance of the amphiphilic substituent in the 1,4-
DHP ring. For that, two structurally similar compounds,
12 [the 4-(N-dodecyl) pyridinium group-containing
derivative] and 4 (without 4-(N-dodecyl) pyridinium
moiety), were tested and compared in isolated rat liver
mitochondria;
ꢀ
in addition, the safety of compound 12 – in vivo
subchronic toxicity test in mice.
Please cite this article in press as: Rucins M, et al. Synthesis and studies of calcium channel blocking and antioxidant
activities of novel 4-pyridinium and/or N-propargyl substituted 1,4-dihydropyridine derivatives. C. R. Chimie (2013),
http://dx.doi.org/10.1016/j.crci.2013.07.003

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3
Scheme 1. Synthesis of 1,4-DHP derivatives 1–18. Reactions conditions: (a) compounds 1 and 3: EtOH, reflux for 5–7 h; (b) compounds 2 and 4: pyridine,
reflux for 5–12 h; (c) alkylbromide, acetone, reflux for 5–26 h; (d) NaOH, EtOH, water, 50 8C, 3 h.
Table 1
Structures and characterisation of the studied 1,4-DHP derivatives 1–18.
X
O
O
O
O
N
R
1-18
Compound
X
R
Yield (%)
Mp (8C)
1
CH
H
92 (78[26])
19 (40a)
158–160 (153[26])
111–113
2
CH
–CH2CBCH
3
N
H
78 (80[27])
22 (47a)
189–191 (192–194[27])
154–156
4
N
–CH2CBCH
5
N+–(CH2)nCH3; n = 1; Brꢁ
N+–(CH2)nCH3; n = 1; Brꢁ
N+–(CH2)nCH3; n = 5; Brꢁ
N+–(CH2)nCH3; n = 5; Brꢁ
N+–(CH2)nCH3; n = 7; Brꢁ
N+–(CH2)nCH3; n = 7; Brꢁ
N+–(CH2)nCH3; n = 11; Brꢁ
N+–(CH2)nCH3; n = 11; Brꢁ
N+–(CH2)nCH3; n = 15; Brꢁ
N+–(CH2)nCH3; n = 15; Brꢁ
N+–CH2COOC2H5; Brꢁ
N+–CH2COOC2H5; Brꢁ
N+–CH2COOꢁ
H
86
189–191
6
–CH2CBCH
77
205 (decomp.)
220–222
7
H
51
8
–CH2CBCH
69
176–178
9
H
54
157–159
10
11
12
13
14
15
16
17
18
–CH2CBCH
56
146–148
H
68
162–164
–CH2CBCH
57
161–163
H
60 (63[28])
76
134–135 (135–136[28])
149–151
–CH2CBCH
H
87 (88[29])
78
210–212 (217 (decomp.)[29])
157–159
–CH2CBCH
H
82 (84[29])
53
128–130 (128–130[29])
105 (decomp.)
N+–CH2COOꢁ
–CH2CBCH
a
Yield of product, performing the reaction in a pressure tube as the reaction vessel.
Please cite this article in press as: Rucins M, et al. Synthesis and studies of calcium channel blocking and antioxidant
activities of novel 4-pyridinium and/or N-propargyl substituted 1,4-dihydropyridine derivatives. C. R. Chimie (2013),
http://dx.doi.org/10.1016/j.crci.2013.07.003

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Table 2
1,4-DHP derivatives via N-alkylation of the corresponding
N-unsubstituted 1,4-DHP derivative with propargyl bro-
mide in the presence of NaH as a base with only 22% yield
of product [22]. In our hands, the yields of synthesised
Calcium overload-preventing activity (IC50) of novel 1,4-DHP derivatives
in SH-SY5Y and A7r5 cells.
Compound
Ca2+ antagonism, IC50
(mM)
N-propargyl-substituted 1,4-DHP derivatives
–
2,6-
Cell
dimethyl-4-phenyl-1-prop-20-ynyl-1,4-dihydropyridine-
30,50-dicarboxylic acid diethyl ester (2) and 20,60-dimethyl-
10-prop-2-ynyl-10,40-dihydro-[3,40]bipyridinyl-30,50-dicar-
boxylic acid diethyl ester (4) – were 19% and 22%,
respectively. Hantzsch-type cyclisation was carried out
SH-SY5Y
A7r5
1
#
0.0098 ꢂ 0.0004*
2
> 100*
#
2
ꢂ 0.2*
3
1.5 ꢂ 0.2*
4
53 ꢂ 8*
4.5 ꢂ 1*
5
#
#
with
a propargyl amine hydrochloride refluxing the
6
100 ꢂ 8*
#
> 100*
reaction mixture for 12 h. Meanwhile, the yields of the
desired products 2 and 4 were increased to 40% and 47%,
7
#
8
35 ꢂ 3
53 ꢂ 8
12 ꢂ 3
#
respectively; these reactions, when performed in
a
9
#
pressure tube as the reaction vessel, were completed in
just 5 h.
10
#
11
5
ꢂ 0.8
0.7 ꢂ 0.3*
12
14 ꢂ 5
0.6 ꢂ 0.06*
The synthesis of all the pyridinium moieties containing
1,4-DHP derivatives 5–14 were performed by alkylation of
4-pyridyl-1,4-DHP derivatives 3 or 4 with the correspond-
ing alkyl bromides in acetone under reflux. The typical
procedure for the quaternisation of pyridine derivatives
has been reported by several research groups [32–34].
Reaction times and yields of the product are dependent on
the structure of the alkyl halide. An excess of the alkylation
agent was used to reduce the reaction time [29].
Compounds 15 and 16 were synthesised as synthones
for the target products 17 and 18. Pyridinium betaines 17
and 18, synthesised in 82 and 53%, yields respectively,
were obtained by treatment of the corresponding 4-(N-
ethoxycarbonylmethyl) pyridinium-1,4-DHP bromides 15
and 16 with NaOH as a base, in analogy with the procedure
described by our research group [29].
The structures of all newly synthesised derivatives 2, 4–
12, 14, 16 and 18, and of those already reported
compounds 1 [26], 3, [27], 13 [28], 15 [29] and 17 [29]
were established and confirmed by 1H NMR, 13C NMR, MS
and elemental analysis data. Molecular weights of 1,4-DHP
derivatives measured by LC/MS technique were in good
agreement with the calculated values for all the com-
pounds. Details of the syntheses and full physically
chemical characterisation for 1,4-DHPs 1–18 are given in
Section 4. The purities of the studied compounds 1–18
were at least 97% according to high-performance liquid
chromatography (HPLC) data.
13
#
#
14
#
#
15
#
#
16
#
#
17
#
#
18
#
#
Amlodipine
Nimodipine
11 ꢂ 5
0.140 ꢂ 0.012
53 ꢂ 3*
0.008 ꢂ 0.0014*
#: No Ca2+-channel blocking activity was detected. Data are presented as
a mean ꢂ SD.
*
P < 0.05 vs. amlodipine; Student’s paired t-test, with a two-tailed
distribution.
and the N-dodecyl pyridinium group (compounds 11 and
12); these activities were comparable to that of amlodi-
pine. Other compounds showed either weak calcium
antagonism (compounds 4, 8 and 9), or this activity was
not obtained at all. Calcium antagonistic properties were
found to be higher in A7r5 cells, indicating that compounds
predominantly target the L-type calcium channels in
vascular smooth muscles. The highest activity comparable
to that of nimodipine was revealed for compound 1, which
is a well-known 4-phenyl-1,4-DHP derivative with calcium
antagonistic properties [35]. In A7r5 cells, compounds 11
and 12 demonstrated at least 10-fold higher activity than
that in SH-SY5Y cells, indicating their potential vasodilat-
ing activity, whereas compounds 2, 3 and 4 were about 3–
4-fold less active than compounds 11 and 12; others lacked
activity. Compounds 5, 7 and 13–18 did not show calcium
channel blocking activity in none of the used cell lines.
Our results about compounds 1 and 3 are in good
agreement with literature data, which demonstrated their
calcium channel blocking activities [35,36].
2.2. Biology
2.2.1. Assessment of calcium channel blocking activity in SH-
SY5Y and A7r5 cell lines
The influence of novel synthesised compounds on
intracellular calcium [Ca2+]i concentration was assessed in
both the neuroblastoma SH-SY5Y cells (containing L- and
N-type Ca2+ channels) and the rat aorta smooth muscle
A7r5 cells, which express functional L-type Ca2+ channels.
The well-known calcium channel inhibitors – amlodipine
and nimodipine – were used as the positive controls in
this test.
The comparison of the Ca2+-channel blocking activity of
structurally related compounds – N–H (N-unsubstituted)
(compounds 1, 3, 5, 7, 9, 11, 13, 15, and 17) and N-
propargyl-substituted (compounds 2, 4, 6, 8, 10, 12, 14, 16,
and 18) 1,4-DHP derivatives shows either slightly
increased blocking activity only for compounds 2, 4, 6 vs
1, 3, 5, and 7 in SH-SY5Y cells, or decreased activity for
compound 2 vs compound 1 in A7r5 cells.
As it is shown in Table 2, the most potent calcium
antagonistic properties in SH-SY5Y cells demonstrated
that the 1,4-DHP derivatives contained at position 4 of the
1,4-DHP ring the N-octyl pyridinium group (compound 10)
2.2.2. Assessment of total antioxidant activity (AOA)
In this study, the phosphomolybdenum complex
method was used for the evaluation of the total
Please cite this article in press as: Rucins M, et al. Synthesis and studies of calcium channel blocking and antioxidant
activities of novel 4-pyridinium and/or N-propargyl substituted 1,4-dihydropyridine derivatives. C. R. Chimie (2013),
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2.2.3. Influence on mitochondrial functions of rat liver
isolated mitochondria
Now it is known that 1,4-DHP compounds may
penetrate the blood–brain barrier [5,6]. Moreover, they
are capable of penetrating cell membranes, even those of
organelles, reaching mitochondria and nucleus [39].
Suggesting that an amphiphilic (N-dodecyl pyridinium)
group may dramatically increase the lipophilicity of
compound 12 and enhance its mitochondria-penetrating
ability, we tested it in rat liver isolated mitochondria and
compared their action with that of compound 4, which
lacks this moiety. Both compounds also contain the N-
propargyl group. We used a concentration close to IC50
obtained in the A7r5 cell line.
2.2.3.1. Effects on mitochondrial bioenergetics. Uncoupled
respiration was initiated by the addition of 1
mM of p-
trifluoromethoxyphenylhydrazone (FCCP). The respiratory
control ratio (RCR) and ADP-to-oxygen ratio (ADP/O) were
calculated. The results, presented in Table 3, show that
Fig. 1. Total antioxidant activity of 1,4-DHP derivatives 1–18 (at
concentration 100
mM) evaluated by the phosphomolybdenum
complex method. Results are expressed as absorbance unit (at 695 nm)
of sample against absorbance of a blank solution. Data are presented as a
mean ꢂ SD. *P < 0.05 vs diludine (taken as 100%), one-way ANOVA followed
by Bonferroni’s multiple-comparison test.
compound 12 at concentration
mitochondrial bioenergetics; however, at higher concen-
tration of 10 M (about 15 times higher than IC50 in A7r5
1 mM did not affect
m
cells), it perturbed the respiration and phosphorylation
efficiency of mitochondria by affecting the permeability
(integrity) of the inner mitochondrial membrane to
cations, as reflected by a stimulation of states-2 and 4
respiration rate, with a parallel decrease of state-3 one (but
not FCCP state) and RCR and ADP/O ratio, when glutamate/
malate (Table 3) or succinate (not shown) were used as the
respiratory substrates. These data indicate that the N-
dodecyl group plays some role in the compound’s ability to
influence mitochondrial bioenergetics at higher concen-
antioxidant activity of the synthesised 1,4-DHP deriva-
tives. Diludine (diethyl-1,4-dihydro-2,6-dimethyl-3,5-
pyridinecarboxylate), a well-known 1,4-DHP derivative
with antioxidant properties [37,38], was used as a positive
control. The obtained results (Fig. 1) show that all
synthesised 1,4-DHP derivatives possessed antioxidant
properties. The highest activity (about 0.5 absorbance
units) was evidenced for the N-unsubstituted
derivatives – compounds 1 and 17, containing the 4-
phenyl or N-carboxymethyl pyridinium moiety, respec-
tively. The AOA of compounds 3, 6 and 15 was comparable
to that of diludine. Other 1,4-DHP derivatives showed a
lower AOA than diludine and even compounds that
demonstrated a high calcium channel blocking activity
(compounds 11 and 12), indicating a lack of correlation
between their antioxidant and calcium antagonistic
properties. We suggested that the AOA of the tested 1,4-
DHP derivatives is mainly related to the ability of their N–H
group to protonate/deprotonate at the phosphomolybde-
num complex test system.
trations, while compound
4 (without this structural
moiety) did not show any influence on mitochondrial
bioenergetics.
2.2.3.2. Influence on Ca2+-induced mitochondrial pore transi-
tion (MPT). The influences of compounds 12 and 4 were
also studied on Ca2+-induced MPT to evaluate
their abilities to increase the susceptibility of mitochondria
to MPT induction by measuring the increase in oxygen
consumption, which is a typical phenomenon that follows
the induction of MPT. Mitochondria can tolerate a certain
amount of Ca2+
, but ultimately, their capacity of
Table 3
Effects of compounds 12 and 4 on the respiratory parameters (states 2, 3, 4, FCCP), and respiratory indices RCR and ADP/O ratio using a glutamate/malate
respiratory substrate.
Compound
Concentration (
m
M)
Oxygen consumption (% of control)
RCR
ADP/O
State 2
State 3
State 4
State FCCP
12
0 (control)
100.0 ꢂ 0.0
110.8 ꢂ 2.9
157.5 ꢂ 25**
100.0 ꢂ 0.0
93.8 ꢂ 5.5
76.7 ꢂ 2.7**
100.0 ꢂ 0.0
110.6 ꢂ 3.4
149.2 ꢂ 18.4*
100.0 ꢂ 0.0
95.2 ꢂ 2.3
81.07 ꢂ 4.9
4.9 ꢂ 0.5
4.5 ꢂ 0.6
2.5 ꢂ 0.2**
3.2 ꢂ 0.2
3.0 ꢂ 0.2
2.4 ꢂ 0.1*
1
10
4
0 (control)
10
100.0 ꢂ 0.0
98.3 ꢂ 2.4
100.0 ꢂ 0.0
93.8 ꢂ 3.9
100.0 ꢂ 0.0
100.0 ꢂ 0.0
100.1 ꢂ 7.7
7.9 ꢂ 1.0
7.2 ꢂ 1.1
3.0 ꢂ 0.05
2.9 ꢂ 0.13
115.0 ꢂ 10.0
The values are given in percentage of control (without compounds). Control values are expressed in nmol O2 mgꢁ1 protein minꢁ1. Data are presented as a
mean ꢂ SE, one-way ANOVA for multiple-comparisons, followed by Tukey’s test. For compound 12: state 2 = 7.9 ꢂ 0.4; state 3 = 44 ꢂ 0.2; state 4 = 9.4 ꢂ 0.9;
FCCP = 70 ꢂ 8.8. ND: not determined. For compound 4: state 2 = 7.32 ꢂ 0.49; state -3 = 43.67 ꢂ 3.47; state 4 = 4.27 ꢂ 0.49; state FCCP = 54.08 ꢂ 7.7.
*
P < 0.05.
**
P < 0.01.
Please cite this article in press as: Rucins M, et al. Synthesis and studies of calcium channel blocking and antioxidant
activities of novel 4-pyridinium and/or N-propargyl substituted 1,4-dihydropyridine derivatives. C. R. Chimie (2013),
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contrast, compound
4 neither protected MPT nor
potentiated its induction (not shown).
Therefore, mitochondrial transition pore opening is not
induced by compound 12 per se, indicating the mitochon-
dria safety. Under certain pathological conditions (in our
case, the calcium ion overload), it may potentiate calcium
entry into mitochondria.
2.2.3.3. Influence on mitochondrial oxidative stress. The
effects of compounds 12 and 4 on mitochondrial oxidative
damage were assessed by detecting mitochondrial mem-
brane lipid peroxidation induced by the pro-oxidant pair
ADP/Fe2+ by evaluating the formation of thiobarbituric
acid reactive substances (TBARs). Both compounds 12 and
4 did not affect TBARs formation, indicating that they
do not possess mitochondrial antioxidant properties
(Table 4).
Fig. 2. A. Effects of compound 12 on MPT induction evaluated by
measuring succinate (Suc)-supported mitochondrial respiration (oxygen
consumption). Control assays show MPT induction by Ca2+ at
a
concentration of 100 nmol/mg protein and its inhibition by CsA
(0.75 nmol/mg protein). B. Influence of compound 12 at concentrations
1,
5 and 10 mM
(1, 5, 10, respectively) on MPT induction at Ca2+
concentration of 60 nmol/mg protein. Assay conditions: +Ca2+: presence
of calcium; – Ca2+: absence of calcium. The traces in experiments A and B
are representative of assays performed with three different mitochondrial
preparations.
2.3. Subchronic toxicity in mice for compound 12
Ca2+-loading is overwhelmed and mitochondria are
completely uncoupled due to a change in the inner
membrane permeability. This effect is prevented by
cyclosporin A (CsA), a specific inhibitor of MPT. Assays
in the absence and presence of Ca2+ (100 nmol/mg protein)
as well as assays in the presence of Ca2+ (100 nmol/mg
protein) plus CsA (0.75 nmol/mg protein) were used as the
controls for MPT induction (Fig. 2A).
When the influence of compounds on MPT induction
was tested, we used a Ca2+ concentration of 60 nmol/mg
of protein that did not induce MPT by itself. The obtained
results (Fig. 2B) showed that in the absence of calcium,
compound 12, even at the higher concentration of
To clarify whether the changes in mitochondrial
bioenergetics caused by compound 12 may play a crucial
role in cell survival, we tested this compound in vivo for
its subchronic toxicity. Compound 12 was administered
for 7 days as a single daily intraperitoneal dose of
100 mg/kg. Mice were observed during 10 days. No toxic
symptom was observed. Therefore, it can be considered
that some toxic influences found in vitro at higher
concentrations are not essential for cell-integrative in
vivo processes, and the compound can be considered as a
cell-safe agent.
3. Conclusions
10
calcium, this compound at concentrations 1, 5 and
10 M significantly potentiated calcium toxic effects. For
instance, at concentration 10 M, the potentiating effect
mM, did not induce MPT, while in the presence of
The obtained data have demonstrated that among the
tested N-quaternised 4-b-pyridyl-l,4-dihydropyridines 5–
m
m
14, the highest calcium channel blocking activity was
shown by compounds comprising the amphiphilic group –
the N-dodecyl pyridinium moiety at position 4 of the 1,4-
DHP cycle (compounds 11 and 12). This activity, in smooth
muscle cell line A7r5, was considerably higher than that in
neuroblastoma cell line SH-SY5Y, suggesting that these
compounds are predominantly targeting the L-type
calcium channels. These compounds possessed low anti-
oxidant activity if compared to 1,4-DHP derivatives 1, 3
and 17. Compound 12 at concentrations close to those of
others demonstrating L-type calcium channel blocking
activity did not affect mitochondrial functioning. This
compound can be considered as a safe agent also in vivo
(no toxicity up to 100 mg/kg). The propargyl group at
position 1 of 1,4-DHP ring in general did not influence
essentially the biological activity of the tested 1,4-DHP
derivatives assessed in the used test systems. The
compounds containing the N-dodecyl pyridinium moiety
at position 4 of the 1,4-DHP cycle can be regarded as the
prototype molecules for further chemical modifications
and studies in animal models of cardiovascular or
neurological diseases.
reached the control level when
a concentration of
100 nmol Ca2+/mg protein was used (Fig. 2A). CsA totally
blocked the potentiating effect of compound 12
(Fig. 2B, +Ca2+ + CsA + 10
mM), indicating that compound
12 has an ability to potentiate MPT induction. In
Table 4
Effect of compounds 12 and 4 on membrane lipid peroxidation of rat liver
mitochondria induced by the pro-oxidant pair ADP/Fe2+ by evaluating
TBARs formation.
Compound
Concentration
TBARs (nmol/mg
protein,10 min)
(m
M)
12
0
1
11.3 ꢂ 0.5
11.0 ꢂ 0.2
11.7 ꢂ 1.0
10.9 ꢂ 1.3
11.5 ꢂ 1.6
12.0 ꢂ 1.1
10
20
50
100
4
0
11.3 ꢂ 0.5
10.4 ꢂ 0.5
60
-ADP/Fe2+
0.44 ꢂ 0.25
The structure–activity relationships of synthesised 4-
pyridinium and/or N-propargyl substituted 1,4-DHP deri-
vatives are summarised in Fig. 3.
The data are presented as mean ꢂ SE. Means were compared using one-way
ANOVA for multiple-comparisons, followed by Tukey’s test.
Please cite this article in press as: Rucins M, et al. Synthesis and studies of calcium channel blocking and antioxidant
activities of novel 4-pyridinium and/or N-propargyl substituted 1,4-dihydropyridine derivatives. C. R. Chimie (2013),
http://dx.doi.org/10.1016/j.crci.2013.07.003

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7
Fig. 3. Structure–activity relationships of the studied 4-pyridinium and/or N-propargyl-substituted 1,4-DHP derivatives.
4. Experimental
4.1.2. Synthesis
Compounds 1, 3, 13, 15 and 17 were synthesised
according to the literature. Briefly, the 20,60-dimethyl-4-
phenyl-1,4-dihydropyridine-3,5-dicarboxylic acid diethyl
ester (1) [26,40] and 20,60-dimethyl-10,40-dihydro-
[3,40]bipyridinyl-30,50-dicarboxylic acid diethyl ester (3)
[27] were obtained from the corresponding aldehyde,
ethyl acetoacetate and ammonia in the classical Hantzsch
synthesis;
dimethyl-10,40-dihydro-[3,40]bipyridinyl-1-ium bromide
(13) and
30,50-bisethoxycarbonyl-1-ethoxycarbonyl-
methyl-20,60-dimethyl-10,40-dihydro-[3,40]bipyridinyl-1-
ium bromide (15) from 20,60-dimethyl-10,40-dihydro-
[3,40]bipyridinyl-30,50-dicarboxylic acid diethyl ester (3)
and hexadecyl bromide [28] or ethyl bromoacetate [29],
according to procedures described from Makarova et al.,
respectively; 1-carboxymethyl-20,60-dimethyl-30,50-bise-
toxycarbonyl-10,40-dihydro-[3,40]bipyridinyl-1-ium (17)
from equimolar amounts of 30,50-bisethoxycarbonyl-
1-ethoxycarbonylmethyl-20,60-dimethyl-10,40-dihydro-
[3,40]bipyridinyl-1-ium bromide (15) and metallic sodium
in absolute ethanol, according to the procedure described
by Makarova et al. [29]. 1H NMR spectra data and
elemental analysis of compounds 1, 3 13, 15 and 17 were
in agreement with those reported in the corresponding
literature.
4.1. Chemistry
4.1.1. General
All the reagents and solvents were purchased from
commercial suppliers: Acros, Sigma–Aldrich, Alfa Aesar
or Merck and used without further purification. TLC was
performed on Silica gel 60 F254 Aluminium sheets
20 ꢃ 20 cm (Merck). The purities of compounds were
determined by HPLC on a Waters Alliance 2695 system
and Waters 2489 UV–vis detector equipped with an
30,50-bisethoxycarbonyl-1-hexadecyl-20,60-
Alltima C18 column,
5
m
m, 4.6 ꢃ 150 mm, using
a
gradient elution with acetonitrile/H3PO4 (0.1%) in water,
at a flow rate of 1 mL/min. Peak areas were determined
electronically with a Waters Empower 2 chromatogra-
phy data system. 1H NMR spectra were recorded on a
Varian-Mercury BB (200 MHz or 400 MHz) spectrometer.
13C NMR spectra were recorded on a Varian-Mercury BB
(100 MHz) spectrometer using CDCl3 or DMSO-d6 as the
solvents. The chemical shifts of the atoms are reported in
parts per million (ppm) relatively to the residual signals
of the solvent: CDCl3
1H NMR spectra and CDCl3
(
d
: 7.26) or DMSO-d6
(d: 2.50) for
(
d
: 77.16) or DMSO-d6 (d:
39.52) for 13C NMR. Multiplicities are abbreviated as: s,
singlet; d, doublet; t, triplet; q, quartet; quint, quintet;
m, multiplet; dq, double quartet; dd, double doublet; dq,
double quartet; bs, broad singlet. The coupling constants
are expressed in Hz. Mass spectral data were determined
on a Waters Alliance 2695 Separation module connected
with a Micromass 3100 mass detector (Waters) operat-
ing in the ESI positive or negative ion mode on a Xbridge
4.1.2.1. 20,60-Dimethyl-4-phenyl-1,4-dihydropyridine-3,5-
dicarboxylic acid diethyl ester (1). Yield: 92%. Mp: 158–
160 8C (78%; M: 153 8C [26]. 1H NMR (400 MHz, CDCl3)
d:
1.20 (t, 6H, J = 7.4); 2.29 (s, 6H); 4.01–4.12 (m, 4H); 4.97 (s,
1H); 5.85 (bs, 1H); 7.08–7.12 (m, 1H); 7.16–7.20 (m, 2H);
7.24–7.28 (m, 2H). MS (+ESI) m/z (relative intensity) 330
([M]+, 60%). Anal. calcd for C19H23NO4: C, 69.28; H, 7.04; N,
4.25; found: C, 69.35; H, 7.04; N, 4.19.
C18 column (5
m
m, 2.1 mm ꢃ 50 mm; Waters) using a
gradient elution with acetonitrile/formic acid (0.1%) in
water, at a flow rate of 0.6 mL/min. Melting points were
determined on an OptiMelt (SRS Stanford Research
Systems) equipment. Elemental analyses were deter-
mined on an Elemental Combustion System ECS 4010
(Costech Instruments) or on an EA 1106 (Carlo Erba
Instruments).
4.1.2.2. 20,60-Dimethyl-10,40-dihydro-[3,40]bipyridinyl-30,50-
dicarboxylic acid diethyl ester (3). Yield: 78%. Mp: 189–
191 8C (80%; M: 192–194 8C [27]. 1H NMR (200 MHz,
CDCl3) d: 1.21 (t, 6H, J = 7.3); 2.33 (s, 6H); 4.08 (q, 4H,
Please cite this article in press as: Rucins M, et al. Synthesis and studies of calcium channel blocking and antioxidant
activities of novel 4-pyridinium and/or N-propargyl substituted 1,4-dihydropyridine derivatives. C. R. Chimie (2013),
http://dx.doi.org/10.1016/j.crci.2013.07.003

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M. Rucins et al. / C. R. Chimie xxx (2013) xxx–xxx
J = 7.3); 4.97 (s, 1H); 6.03 (bs, 1H); 7.12–7.18 (m, 1H); 7.57–
7.61 (m, 1H); 8.36 (d, 1H, J = 5.1); 8.52 (s, 1H). 13C NMR
residue was recrystallized from ethyl acetate, then from
ethanol, and dried in vacuo.
4.1.2.6.1. 2,6-Dimethyl-4-phenyl-1-prop-20-ynyl-1,4-dihy-
dropyridine-30,50-dicarboxylic acid diethyl ester (2). Yield:
19 or 40%. Mp: 111–113 8C. 1H NMR (200 MHz, DMSO-d6)
(CDCl3)
d
: 14.24; 19.18; 37.87 (4-C–DHP); 59.80 (3,5-
OCH2); 103.13 (3,5-C–DHP); 123.20; 135.95; 143.76;
145.05; 147.00; 149.40; 167.53 (C5O). MS (+ESI) m/z
(relative intensity) 331 ([M]+, 100%). Anal. calcd for
d
: 1.14 (t, 6H, J = 7.3); 2.51 (s, 6H); 3.50 (t, 1H, J = 2.2); 4.04
(q, 4H, J = 7.3); 4.50 (d, 2H, J = 2.2); 4.93 (s, 1H); 7.10–7.20
(m, 5H). 13C NMR (CDCl3)
: 14.22; 15.21; 36.38 (N–CH2);
C18H22N2O4: C, 65.44; H, 6.71; N, 8.48; found: C, 65.40;
H, 6.73; N, 8.56.
d
38.52 (4-C–DHP); 60.21 (3,5-OCH2); 73.82 (–CB); 79.48
(BCH); 107.97 (3,5-C–DHP); 125.94; 127.25; 128.14;
146.11; 147.83; 167.78 (C5O). MS (+ESI) m/z (relative
intensity) 368 ([M]+, 30%). Anal. calcd for C22H25NO4: C,
71.91; H, 6.86; N, 3.81; found: C, 71.59; H, 6.90; N, 3.77.
4.1.2.3. 30,50-Bisethoxycarbonyl-1-hexadecyl-20,60-dimethyl-
10,40-dihydro-[3,40]bipyridinyl-1-ium bromide (13). Yield:
60%. Mp: 134–135 8C (63%; M: 135–136 8C [28]. 1H NMR
(400 MHz, DMSO-d6) d: 0.84 (t, 3H, J = 7.0); 1.12 (t, 6H, J =
7.0); 1.18–1.26 (m, 26H); 1.85 (quint, 2H, J = 7.0); 2.30 (s,
6H); 4.00 (dq, 4H, J = 7.0 and J = 3.5); 4.64 (t, 2H, J = 7.0);
4.98 (s, 1H); 8.05 (dd, 1H, J = 8.2 and J = 6.0); 8.35 (d, 1H, J =
8.2); 8.84 (bs, 1H); 8.92 (d, 1H, J = 6.0); 9.20 (s, 1H). 13C
4.1.2.6.2.
[3,40]bipyridinyl-30,50-dicarboxylic
(4). Yield: 22 or 47%. Mp: 154–156 8C. 1H NMR
(200 MHz, CDCl3) : 1.25 (t, 6H, J = 7.3); 2.45 (t, 1H,
20,60-Dimethyl-10-prop-2-ynyl-10,40-dihydro-
acid diethyl ester
d
NMR (DMSO-d6)
d
: 14.54; 14.76; 18.95; 22.70; 25.80;
J = 2.2); 2.60 (s, 6H); 4.14 (q, 4H, J = 7.3); 4.32 (d, 2H,
J = 2.2); 5.06 (s, 1H); 7.12 (dd, 1H, J = 8.1 and J = 5.1); 7.54
(d, 1H, J = 8.1); 8,37 (dd, 1H, J = 5.1 and J = 2.2); 8.46 (d, 1H,
28.98; 29.30; 29.36; 29.46; 29.56; 29.58; 29.61; 29.63;
29.64; 29.66; 31.50; 31.89; 38.71 (4-C-DHP); 60.03 (N+-
CH2); 61.09 (3,5-OCH2); 100.14 (3,5-C-DHP); 128.68;
143.25; 143.39; 144.82; 148.20; 148.99; 166.66 (C=O).
MS (+ESI) m/z (relative intensity) 555 (79Br) ([M–Br]+,
100%). Anal. calcd for C34H55N2O4Br: C, 64.24; H, 8.72; N,
4.41; found: C, 64.01; H, 8.78; N, 4.29.
J = 2.2). 13C NMR (CDCl3)
d: 14.27; 16.18; 36.14 (N–CH2);
36.84 (4-C–DHP); 60.18 (3,5-OCH2); 73.93 (-CB); 79.00
(BCH); 106.98 (3,5-C–DHP); 123.08; 135.08; 141.37;
147.56; 148.47; 148.48; 149.30; 167.22 (C5O). MS
(+ESI) m/z (relative intensity) 369 ([M]+, 100%). Anal. calcd
for C21H24N2O4: C, 68.46; H, 6.57; N, 7.60; found: C, 68.08;
H, 6.57; N, 7.31.
4.1.2.4. 30,50-Bisethoxycarbonyl-1-ethoxycarbonylmethyl-
20,60-dimethyl-10,40-dihydro-[3,40]bipyridinyl-1-ium bromide
(15). Yield: 87%. Mp: 210–212 8C. (88%; Mp: 217 8C
4.1.2.7. General method for synthesis of compounds (5–12,
14, 16). The corresponding alkyl (ethyl, hexyl, octyl,
dodecyl or hexadecyl) bromide or ethyl bromoacetate
(9.0 mmol) was added to a solution of the corresponding
20,60-dimethyl-10,40-dihydro-[3,40]bipyridinyl-30,50-dicar-
boxylic acid diethyl ester (3) or 20,60-dimethyl-10-prop-2-
ynyl-10,40-dihydro-[3,40]bipyridinyl-30,50-dicarboxylic acid
diethyl ester (4) (3.0 mmol) in acetone (30 mL). The
mixture was refluxed for 5–26 h, cooled to 0 8C, the
resulting precipitate was filtered off, washed with cooled
acetone, recrystallized from acetone, and dried in vacuo.
(decomp.) [29]. 1H NMR (400 MHz, DMSO-d6)
d: 1.10 (t,
6H, J = 7.0); 1.23 (t, 3H, J = 7.0); 2.30 (s, 6H); 3.99 (dq, 4H,
J = 7.0 and J = 3.5); 4.22 (q, 2H, J = 7.0); 5.00 (s, 1H); 5.70 (s,
2H); 8.13 (dd, 1H, J = 7.8 and J = 5.9); 8.45 (d, 1H, J = 7.8);
8.84 (bs, 1H); 8.87 (d, 1H, J = 5.9); 9.21 (s, 1H). 13C NMR
(DMSO-d6) d: 13.67; 13.88; 18.12; 37.93 (4-C–DHP); 59.25
(3,5-OCH2); 60.05 (N+-. . .OCH2); 62.12 (N+-CH2); 99.25
(3,5-C–DHP); 127.30; 143.91; 144.31; 145.15; 147.44;
148.00; 165.78 (3,5-C5O); 166.16 (N+-. . .C=O). MS (+ESI)
m/z (relative intensity) 417 (79Br) ([M–Br]+, 100%). Anal.
calcd for C22H29N2O6Br: C, 53.13; H, 5.88; N, 5.63; found: C,
52.73; H, 5.81; N, 5.47.
4.1.2.7.1.
10,40-dihydro-[3,40]bipyridinyl-1-ium bromide (5). Yield:
86%. Mp: 189–191 8C. 1H NMR (200 MHz, CDCl3)
: 1.23
30,50-Bisethoxycarbonyl-1-ethyl-20,60-dimethyl-
d
4.1.2.5. 1-Carboxymethyl-20,60-dimethyl-30,50-bisethoxycar-
(t, 6H, J = 6.9); 1.71 (t, 3H, J = 7.3); 2.51 (s, 6H); 4.09 (q, 4H,
J = 6.9); 4.90 (q, 2H, J = 7.3); 5.10 (s, 1H); 7.93 (dd, 1H, J = 8.0
and J = 5.9); 8.37 (bs, 1H); 8.39 (d, 1H, J = 8.0); 8.85 (s, 1H);
9.10 (d, 1H, J = 5.9). 13C NMR (CDCl3)
d: 14.29; 17.31; 19.48;
bonyl-10,40-dihydro-[3,40]bipyridinyl-1-ium
(17). Yield:
82%. Mp: 128–130 8C (84%; M: 128–130 8C [29]). 1H
NMR (200 MHz, CDCl3) : 1.22 (t, 6H, J = 7.0); 2.28 (s,
d
6H); 4.06 (q, 4H, J = 7.0); 5.04 (s, 1H); 5.16 (s, 2H); 7.67 (dd,
1H, J = 8.2 and J = 5.9); 8.29 (d, 1H, J = 8.2); 8.37 (d, 1H,
J = 5.9); 8.77 (s, 1H); 9.39 (bs, 1H). MS (+ESI) m/z (relative
intensity) 389 ([M]+, 100%). Anal. calcd for C20H24N2O6: C,
56.69; H, 6.65; N, 6.60; found: C, 56.45; H, 6.60; N, 6.33.
39.02 (4-C–DHP); 57.37 (N+–CH2); 60.23 (3,5-OCH2); 99.99
(3,5-C–DHP); 127.99; 142.10; 142.33; 144.68; 148.67;
150.14; 167.15 (C5O). MS (+ESI) m/z (relative intensity)
359 (79Br) ([M–Br]+, 100%). Anal. calcd for C20H27N2O4Br: C,
54.68; H, 6.19; N, 6.38; found: C, 54.77; H, 6.17; N, 6.32.
4.1.2.7.2. 30,50-Bisethoxycarbonyl-1-ethyl-20,60-dimethyl-10-
prop-2-ynyl-10,40-dihydro-[3,40]bipyridinyl-1-ium bromide
(6). Yield: 77%. Mp: 205 8C (decomp.). 1H NMR
4.1.2.6. General method for synthesis of compounds (2 and
4). The solution of ethyl acetoacetate (14.2 g, 0.109 mol,
13.7 mL), propargylamine hydrochloride (5.0 g, 0.055 mol)
and the corresponding aldehyde (benzaldehyde or pyr-
idine-3-carboxaldehyde) (0.055 mol) in pyridine (15 mL)
was refluxed for 5–12 h, the reaction mixture was poured
in an ice/water mixture, extracted by diethyl ether
(3 ꢃ 15 mL), then the organic phase was dried over Na2SO4.
The solvent was evaporated in vacuo, after which the
(400 MHz, CDCl3)
d: 1.24 (t, 6H, J = 7.0); 1.63 (t, 3H,
J = 7.4); 2.51 (t, 1H, J = 2.4); 2.68 (s, 6H); 4.14 (q, 4H, J = 7.0);
4.67 (d, 2H, J = 2.4); 5.07 (q, 2H, J = 7.4); 5.14 (s, 1H); 7.95
(dd, 1H, J = 8.2 and J = 6.3); 8.42 (d, 1H, J = 8.2); 8.77 (s, 1H);
9.29 (d, 1H, J = 6.3). 13C NMR (CDCl3)
d: 9.63; 12.06; 12.50;
33.14 (N–CH2); 33.23 (4-C–DHP); 52.60 (N+–CH2); 55.96
(3,5-OCH2); 69.41 (–CB); 74.79 (BCH); 98.78 (3,5-C–DHP);
Please cite this article in press as: Rucins M, et al. Synthesis and studies of calcium channel blocking and antioxidant
activities of novel 4-pyridinium and/or N-propargyl substituted 1,4-dihydropyridine derivatives. C. R. Chimie (2013),
http://dx.doi.org/10.1016/j.crci.2013.07.003

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9
123.07; 137.48; 138.12; 140.01; 142.93; 146.58; 162.39
(C5O). MS (+ESI) m/z (relative intensity) 397 (79Br) ([M–
Br]+, 100%). Anal. calcd for C23H29N2O4Br: C, 57.87; H, 6.12;
N, 5.87; found: C, 57,74; H, 6,08; N, 5.74.
calcd for C29H41N2O4Br: C, 62.03; H, 7.36; N, 4.99; found: C,
61.74; H, 7.41; N, 4.77.
4.1.2.7.7. 30,50-Bisethoxycarbonyl-20,60-dimethyl-1-dodecyl-
10,40-dihydro[3,40]bipyridinyl-1-ium bromide. Yield: 68%.
Mp: 162–164 8C. 1H NMR (200 MHz, CDCl3)
d: 0.85 (t,
4.1.2.7.3.
10,40-dihydro-[3,40]bipyridinyl-1-ium bromide (7). Yield:
51%. Mp: 220–222 8C. 1H NMR (400 MHz, DMSO-d6)
30,50-Bisethoxycarbonyl-1-hexyl-20,60-dimethyl-
3H, J = 7.4); 1.21 (t, 6H, J = 7.0); 1.22–1.25 (m, 18H) overlap;
1.98 (quint, 2H, J = 7.4); 2.50 (s, 6H); 4.07 (q, 4H, J = 7.0);
4.78 (t, 2H, J = 7.4); 5.07 (s, 1H); 7.91 (dd, 1H, J = 8.2 and
J = 6.3); 8.22 (d, 1H, J = 8.2); 8.48 (bs, 1H); 8.76 (s, 1H); 9.02
d
:
0.83 (t, 3H, J = 7.0); 1.11 (t, 6H, J = 7.0); 1.16–1.26 (m, 6H);
1.85–1.90 (quint, 2H, J = 7.0); 2.30 (s, 6H); 4.09 (dq, 4H,
J = 7.0 and J = 3.9); 4.66 (t, 2H, J = 7.0); 4.78 (s, 1H); 8.06 (dd,
1H, J = 8.2 and J = 5.9); 8.35 (d, 1H, J = 8.2); 8.45 (bs, 1H);
(d, 1H, J = 6.3). 13C NMR (CDCl3)
d: 14.07; 14.36; 19.44;
22.65; 26.03; 28.98; 29.00; 29.28; 29.31; 29.44; 29.51;
29.54; 31.84 (N+–CH2–CH2); 31.92 (4-C–DHP); 39.01 (N+–
CH2); 60.21 (3,5-OCH2); 100.02 (3,5-C–DHP); 127.97;
141.81; 142.44; 144.62; 148.67; 149.91; 167.36 (C5O).
MS (+ESI) m/z (relative intensity) 499 (79Br) ([M–Br]+,
100%). Anal. calcd for C30H47N2O4Br: C, 62.34; H, 8.17; N,
4.83; found: C, 62.34; H, 8.22; N, 4.72.
8.94 (d, 1H, J = 5.9); 9.22 (s, 1H). 13C NMR (DMSO-d6)
d:
13.86; 14.24; 18.43; 21.90; 24.92; 30.60; 30.93; 38.18 (4-
C–DHP); 59.53 (3,5-OCH2); 60.56 (N+–CH2); 99.60 (3,5-C–
DHP); 128.14; 142.66; 142.93; 144.34; 147.71; 148.17;
166.15 (C5O). MS (+ESI) m/z (relative intensity) 415 (79Br)
([M–Br]+, 100%). Anal. calcd for C24H35N2O4Br: C, 58.18; H,
7.12; N, 5.65; found: C, 58.18; H, 7.14; N, 5.55.
4.1.2.7.4. 30,50-Bisethoxycarbonyl-1-hexyl-20,60-dimethyl-10-
prop-2-ynyl-10,40-dihydro-[3,40]bipyridinyl-1-ium bromide
(8). Yield: 69%. Mp: 176–178 8C. 1H NMR (400 MHz,
4.1.2.7.8. 30,50-Bisethoxycarbonyl-20,60-dimethyl-1-dodecyl-
10-prop-2-ynyl-10,40-dihydro-[3,40]bipyridinyl-1-ium
bro-
mide (12). Yield: 57%. Mp: 161–163 8C. 1H NMR
(200 MHz, CDCl3) : 0.90 (t, 3H, J = 6.6); 1.22–1.39 (m,
d
DMSO-d6)
d
: 0.83 (t, 3H, J = 6.7); 1.18 (t, 6H, J = 7.1);
22H); 1.50–1.65 (m, 2H); 1.99–2.04 (m, 2H); 2.51 (t, 1H,
J = 2.2); 2.74 (s, 6H); 4.21 (q, 4H, J = 7.3); 4.70 (d, 2H,
J = 2.2); 5.03 (t, 2H, J = 6.6); 5.20 (s, 1H); 8.01 (dd, 1H, J = 8.1
and J = 5.9); 8.33 (d, 1H, J = 8.1); 8.69 (s, 1H); 8.91 (d, 1H,
1.20–1.25 (m, 6H); 1.81–1.90 (m, 2H); 2.58 (s, 6H); 3.58 (s,
1H); 4.10 (q, 4H, J = 7.1); 4.60 (s, 2H); 4.66 (t, 2H, J = 6.7);
5.02 (s, 1H); 8.05 (dd, 1H, J = 7.8 and J = 5.1); 8.37 (d, 1H,
J = 7.8); 8.73 (s, 1H); 8.97 (d, 1H, J = 5.1). 13C NMR (DMSO-
J = 5.9). 13C NMR (CDCl3)
d: 14.07; 14.30; 16.79; 22.63;
d6)
d
: 14.02; 14.37; 16.16; 22.07; 25.16; 30.80; 31.15;
25.98; 29.05; 29.28; 29.32; 29.44; 29.46; 29.52; 29.54;
31.84 (N+–CH2–CH2); 32.00 (N–CH2); 37.88 (4-C–DHP);
60.70 (N+–CH2); 62.04 (3,5-OCH2); 74.12 (–CB); 79.54
(BCH); 103.34 (3,5-C–DHP); 127.87; 142,36; 143.24;
144.78; 147.46; 151.39; 167.13 (C5O). MS (+ESI) m/z
(relative intensity) 537 (79Br) ([M–Br]+, 100%). Anal. calcd
for C33H49N2O4Br: C, 64.17; H, 8.00; N, 4.50; found: C,
64.31; H, 8.04; N, 4.50.
36.55 (N–CH2); 37.09 (4-C–DHP); 60.36 (3,5-OCH2); 60.93
(N+–CH2); 76.69 (–CB); 80.45 (BCH); 103.80 (3,5-C–DHP);
128.46; 142.69; 143.34; 144.22; 146.42; 151.13; 163.30
(C5O). MS (+ESI) m/z (relative intensity) 453 (79Br) ([M–
Br]+, 100%). Anal. calcd for C27H37N2O4Br: C, 60.79; H, 6.99;
N, 5.25; found: C, 60.59; H, 7.01; N, 5.12.
4.1.2.7.5.
10,40-dihydro-[3,40]bipyridinyl-1-ium bromide (9). Yield:
54%. Mp: 157–159 8C. 1H NMR (400 MHz, DMSO-d6)
30,50-Bisethoxycarbonyl-20,60-dimethyl-1-octyl-
4.1.2.7.9.
30,50-Bisethoxycarbonyl-1-hexadecyl-20,60-
d:
dimethyl-10-prop-2-ynyl-10,40-dihydro-[3,40]bipyridinyl-1-
0.83 (t, 3H, J = 7.0); 1.11 (t, 6H, J = 7.0); 1.16–1.27 (m, 10H);
1.87 (quint, 2H, J = 7.0); 2.30 (s, 6H); 4.00 (dq, 4H, J = 7.0
and J = 3.5); 4.65 (t, 2H, J = 7.0); 4.98 (s, 1H); 8.05 (dd, 1H,
J = 8.2 and J = 6.0); 8.35 (d, 1H, J = 8.2); 8.85 (bs, 1H); 8.95
ium bromide (14). Yield: 76%. Mp: 149–151 8C. 1H NMR
(400 MHz, DMSO-d6) d: 0.85 (t, 3H, J = 6.6); 1.18 (t, 6H,
J = 6.8); 1.19–1.30 (m, 26H); 1.85 (quint, 2H, J = 6.9); 2.58
(s, 6H); 3.57 (s, 1H); 4.09 (dq, 4H, J = 6.6 and J = 3.0); 4.59 (s,
2H); 4.64 (t, 2H, J = 6.9); 5.02 (s, 1H); 8.05 (dd, 1H, J = 7.8
and J = 5.7); 8.36 (d, 1H, J = 7.8); 8.72 (s, 1H); 8.97 (d, 1H,
(d, 1H, J = 6.0); 9.23 (s, 1H). 13C NMR (DMSO-d6)
d: 13.44;
13.72; 17.90; 21.58; 24.75; 27.89; 27.97; 30.47; 30.63;
37.65 (4-C–DHP); 58.99 (3,5-OCH2); 60.03 (N+–CH2); 99.08
(3,5-C–DHP); 127.62; 142.15; 142.40; 143.81; 147.19;
147.94; 165.62 (C5O). MS (+ESI) m/z (relative intensity)
443 (79Br) ([M–Br]+, 100%). Anal. calcd for C26H39N2O4Br: C,
59.65; H, 7.51; N, 5.35; found: C, 59.52; H, 7.50; N, 5.24.
4.1.2.7.6. 30,50-Bisethoxycarbonyl-20,60-dimethyl-1-octyl-10-
prop-2-ynyl-10,40-dihydro-[3,40]bipyridinyl-1-ium bromide
(10). Yield: 56%. Mp: 146–148 8C. 1H NMR (400 MHz,
J = 5.7). 13C NMR (DMSO-d6)
d: 13.80; 13.96; 15.74; 21.94;
25.11; 28.25; 28.55; 28.62; 28.73; 28.82; 28.84; 28.86;
28.88; 28.89; 28.91; 30.79; 31.14; 36.12; 36.68 (4-C–DHP);
59.93 (3,5-OCH2); 60.50 (N+–CH2); 76.29 (–CB); 80.12
(BCH); 103.41 (3,5-C–DHP); 128.06; 142.23; 142.91;
143.76; 146.05; 150.41; 165.88 (C5O). MS (+ESI) m/z
(relative intensity) 593 (79Br) ([M–Br]+, 100%). Anal. calcd
for C37H57N2O4Br: C, 65.96; H, 8.53; N, 4.16; found: C,
65.62; H, 8.57; N, 4.03.
DMSO-d6) d: 0.84 (t, 3H, J = 7.0); 1.18 (t, 6H, J = 7.0); 1.19–
1.27 (m, 10H); 1.85 (quint, 2H, J = 7.0); 2.58 (s, 6H); 3.58 (t,
1H, J = 2.0); 4.05 (dq, 4H, J = 7.0 and J = 3.0); 4.95 (d, 2H,
J = 2.0); 4.65 (t, 2H, J = 7.0); 5.02 (s, 1H); 8.05 (dd, 1H, J = 8.0
and J = 6.0); 8.36 (d, 1H, J = 8.0); 8.73 (s, 1H); 8.96 (d, 1H,
4.1.2.7.10. 30,50-Bisethoxycarbonyl-1-ethoxycarbonylmethyl-
20,60-dimethyl-10-prop-2-ynyl-10,40-dihydro-[3,40]bipyridinyl-
1-ium bromide (16). Yield: 78%. Mp: 157–159 8C. 1H NMR
(400 MHz, CDCl3) d: 1.28 (t, 6H, J = 7.0); 1.29 (t, 3H, J = 7.0);
J = 6.0). 13C NMR (DMSO-d6)
d: 14.09; 14.30; 16.08; 22.22;
2.46 (t, 1H, J = 1.9); 2.71 (s, 6H); 4.14 (q, 4H, J = 7.0); 4.25 (q,
2H, J = 7.0); 4.65 (d, 2H, J = 1.9); 5.20 (s, 1H); 6.35 (s, 2H);
7.88 (dd, 1H, J = 8.2 and J = 5.5); 8.48 (d, 1H, J = 8.2); 8.77 (s,
1H); 9.09 (d, 1H, J = 5.5). 13C NMR (CDCl3)
d: 14.49; 14.73;
17.18; 38.25; 38.41 (4-C-DHP); 61.14 (3,5-OCH2); 61.64
(N+–. . .OCH2); 63.63 (N+–CH2); 74.46 (–CB); 80.25 (BCH);
25.45; 28.54; 28.60; 31.12; 31.27; 36.46 (N–CH2); 37.02
(4-C–DHP); 60.28 (3,5-OCH2); 60.85 (N+–CH2); 76.60 (–
CB); 80.36 (BCH); 103.75 (3,5-C–DHP); 128.39; 142.62;
143.22; 144.15; 146.39; 150.75; 166.22 (C5O). MS (+ESI)
m/z (relative intensity) 481 (79Br) ([M–Br]+, 100%). Anal.
Please cite this article in press as: Rucins M, et al. Synthesis and studies of calcium channel blocking and antioxidant
activities of novel 4-pyridinium and/or N-propargyl substituted 1,4-dihydropyridine derivatives. C. R. Chimie (2013),
http://dx.doi.org/10.1016/j.crci.2013.07.003

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M. Rucins et al. / C. R. Chimie xxx (2013) xxx–xxx
103.60 (3,5-C–DHP); 127.18; 144.39; 145.50; 146.07;
147.70; 152.18; 166.36 (N+–. . .C5O); 167.40 (3,5-C5O).
Anal. calcd for C25H31N2O6Br: C, 56.08; H, 5.84; N, 5.23;
found: C, 56.00; H, 5.78; N, 5.09.
used as the positive control. IC50 values as mean ꢂ SD were
calculated using GraphPad Prism 3.0 software.
4.2.2. Evaluation of the total antioxidant activity
4.1.2.7.11. 1-Carboxymethyl-20,60-dimethyl-30,50-bisethoxy-
carbonyl-10prop-2-ynyl-10,40-dihydro-[3,40]bipyridinyl-1-ium
(18). 30,50-Bisethoxycarbonyl-1-ethoxycarbonylmethyl-
20,60-dimethyl-10-prop-2-ynyl-10,40-dihydro-[3,40]bipyridi-
]bipyridinyl-1-ium bromide (16) (0.5 g, 0.93 mmol) was
dissolved in ethanol (5 mL) and a solution of NaOH
(37.2 mg, 0.93 mmol) in water (2.5 mL) was added, then,
the reaction mixture was heated to 50 8C for 3 h. The
reaction mixture was concentrated in vacuo till approxi-
mately half of solvent was evaporated off, after which the
residue was mixed with water. The resulting precipitate
was filtered off, recrystallized from water and dried in
vacuo. Yield: 53% Mp: 105 8C (decomp.). 1H NMR
The total antioxidant activity of the synthesised 1,4-
DHP derivatives was studied using a spectrophotometric
quantitation of the antioxidant capacity through the
formation of the phosphomolybdenum complex [41].
Stock solutions of the tested compounds and of reference
diludine were prepared in ethanol with concentration
1 ꢃ 10ꢁ3 M just before use.
The reagent solution contains 0.6 M sulphuric acid,
28 mM sodium phosphate, and 4 mM ammonium molyb-
date solutions in water.
An aliquot of 0.3 mL of the sample solution was
combined in an Eppendorf tube with 3 mL of the reagent
solution. The tubes were capped and incubated in a
thermal block at 95 8C for 90 min. After the samples had
been cooled to room temperature, the absorbance of the
sample solution of each was measured at 695 nm against a
blank sample. A typical blank solution contained 3 mL of
the reagent solution and the appropriate volume of ethanol
as the solvent, and was incubated under the same
conditions as the rest of the samples.
(200 MHz, CDCl3)
d: 1.28 (t, 6H, J = 7.3); 2.49 (t, 1H,
J = 2.3); 2.64 (s, 6H); 4.19 (q, 4H, J = 7.3); 4.48 (d, 2H,
J = 2.3); 5.05 (s, 2H); 5.22 (s, 1H); 7.71 (dd, 1H, J = 8.0
and J = 5.9); 8.33 (d, 1H, J = 8.0); 8.42 (s, 1H); 8.56 (d, 1H
J = 5.9). MS (+ESI) m/z (relative intensity) 427 ([M]+, 100%).
Anal. calcd for C23H26N2O6: C, 64.78; H, 6.14; N, 6.35;
found: C, 64.39; H, 6.13; N, 6.38.
4.2. Biology
4.2.3. Assays in rat liver isolated mitochondria
Male Wistar rats (250–350 g) were obtained from
Central Vivarium, Faculty of Medicine, University of
Coimbra, Coimbra, Portugal). All the experimental proce-
dures were performed in accordance with the guidelines of
Directive86/609/EEC. ‘‘European Convention for the Pro-
tection of Vertebrate Animals Used for Experimental and
Other Scientific Purposes’’ (1986) and were approved by
the National Ethics Committee. All the chemicals were
obtained from Sigma (St Louis, MO, USA).
Liver mitochondria were isolated by differential cen-
trifugation according to the conventional methods. The
experimental procedures were performed in accordance to
those described previously [15].
4.2.1. Intracellular Ca2+ measurements
Fluo-4 NW Calcium Assay Kit was purchased from
Invitrogen (Sweden), all the others from Sigma–Aldrich
(St Louis, MO, USA). A7r5 (rat aorta smooth muscle cells)
were obtained from the ATTC cell collection (USA), SH-
SY5Y (human neuroblastoma cell line) from the Eur-
opean Collection of Animal Cell Cultures (ECACC, UK).
The cells were grown at 37 8C in a humidified atmo-
sphere with 5% CO2/95% air in Dulbecco’s modified Eagle
medium (DMEM), containing 2 mM of glutamine and
supplemented with 10% fetal bovine serum. Cells were
passaged once a week using 0.25% trypsin, 0.53 mM
EDTA solution and grown in 75 mm2 plastic culture
flasks until confluence.
Briefly, after washing, the pellet was gently re-
suspended in the washing medium at a protein concen-
tration of about 50 mg/mL. The protein content was
determined by the biuret method, using bovine serum
albumin as a standard.
For the experiment, SH-SY5Y cells were plated into 96-
well plates at a density of 30,000 cells per well and
incubated for 24 h. A7r5 cells were seeded into 96-well
plates at a density of 10,000 cells per well and incubated
1,4-DHP derivatives were dissolved in DMSO; concen-
tration did no exceed 0.1% in the medium.
for 72 h. Changes in intracellular [Ca2+
] concentration
i
were studied using the Fluo-4 NW Calcium Assay Kit,
accordingly to the manufacturer’s instructions. The SH-
SY5Y and A7r5 cells were loaded with Fura-4NW for
45 min. Then, the cells were pre-incubated in the dark for
15 min with the tested compounds at concentrations from
4.2.4. Measurement of respiratory activities
Oxygen consumption was monitored polarographi-
cally with a Clark-type electrode at 30 8C in a closed
glass chamber equipped with a magnetic stirring device.
Mitochondria (1 mg/mL) were incubated in a respiratory
medium containing 130 mM of sucrose, 5 mM of HEPES
(pH 7.2), 50 mM of KCl, 2.5 mM of K2HPO4, and 2.5 mM of
MgCl2 (in the presence and absence of compounds 4 or
12) for 3 min before energisation with 10 mM glutamate/
5 mM malate. State-2 respiration (consumption of
oxygen in the presence of substrate); state-3 respiration
(consumption of oxygen in the presence of substrate
and ADP) was induced by the addition of adenosine
0.8 to 100 mM. The compounds were dissolved in DMSO.
Application of carbachol (100 nM) to Fura-4 loaded SH-
SY5Y cells was used to stimulate changes in the
intracellular [Ca2+
] concentration, whereas A7r5 cells
i
were treated with 1.5 mM CaCl2 and KCl (50 mM) for
5 min to induce an increase in the [Ca2+]i concentration.
The intracellular [Ca2+]i concentration was measured using
the fluorescence spectrophotometer’s (Thermo Ascient,
Finland) settings appropriate for an excitation at 494 nm
and an emission at 516 nm. Amlodipine, nimodipine were
diphosphate (ADP, 150
mM) and state-4 respiration
Please cite this article in press as: Rucins M, et al. Synthesis and studies of calcium channel blocking and antioxidant
activities of novel 4-pyridinium and/or N-propargyl substituted 1,4-dihydropyridine derivatives. C. R. Chimie (2013),
http://dx.doi.org/10.1016/j.crci.2013.07.003

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CRAS2C-3792; No. of Pages 12
M. Rucins et al. / C. R. Chimie xxx (2013) xxx–xxx
11
(consumption of oxygen after ADP phosphorylation).
4.2.8. Subchronic toxicity in mice
FCCP (p-trifluoromethoxyphenylhydrazone)-stimulated
The test was performed in male ICR mice weighing
20 ꢂ 2 g (obtained from the Laboratory of Experimental
Animals, Riga Stradins University, Riga, Latvia).
Compound 12 was administered as a single daily
intraperitoneal dose of 100 mg/kg for 7 days. Number of
animals n = 10. Control group (n = 10) received a saline
solution. The behaviour of the mice was observed during
10 days.
All experimental procedures were performed in accor-
dance with the EU Directive 2010/63/EU for animal
experiments and were approved by the Animal Ethics
Committee of the Food and Veterinary Service (Riga,
Latvia).
respiration was initiated by the addition of 1
mM of
FCCP. The respiratory control ratio (RCR), which is
calculated by the ratio between states 3 and 4, is an
indicator of the mitochondrial membrane integrity. The
ADP/O ratio, which is expressed by the ratio between the
amounts of ADP and the oxygen consumed during state-
3 respiration, is an index of oxidative phosphorylation
efficiency. Respiration rates were calculated assuming
that the saturation of oxygen concentration was 250
mM
at 30 8C, and the values are expressed in percentage of
control (% of control).
4.2.5. Ca2+-induced mitochondrial membrane transition pore
Ca2+–induced MPT was evaluated by measuring
Acknowledgements
changes in oxygen consumption using
a Clark-type
electrode. The reactions were conducted in a medium
containing 200 mM of sucrose, 10 mM of MOPS–Tris
This study was supported by ESF project No. 2009/
0217/1DP/1.1.1.2.0/09/APIA/VIAA/031; the EuroNa-
noMed project ‘‘CheTherDel’’; Portuguese Research Coun-
cil (FCT), Faculty of Medicine, Centre for Neuroscience and
Cell Biology (CNC) and Marine and Environmental
Research Centre (IMAR–CMA) of the University of
Coimbra, Portugal.
(pH 7.4), 1 mM of KH2PO4, and 10
mM of EGTA, supple-
mented with 2 M of rotenone, as previously described
m
[42,43]. Mitochondria (1 mg/mL), previously incubated at
30 8C for 3 min (in the absence and presence of compounds
4 or 12), were energised with 10 mM succinate, and a
single addition of Ca2+ (100 mol/mg protein) was used to
induce MPT. Control assays, in both the absence and
presence of Ca2+, and in the presence of Ca2+ plus
0.75 nmol/mg of protein cyclosporin A (CsA) and the
compound (when necessary), were also performed. To
evaluate the ability of the compounds to increase the
susceptibility of mitochondria to MPT induction, we used a
Ca2+ concentration (60 nmol/mg of protein) that did not
induce MPT by itself.
We thank colleagues from the University of Coimbra,
´
particularly Catarina R. Oliveira, Antonio J. M. Moreno and
Maria S. Santos for the assistance to perform mitochondrial
studies. We obliged also the students from the University
of Latvia: Nelda Lencberga, Marta Pavasare, Evija Sidorova,
Katrina Hauka for their participation in mitochondrial
assays at University of Coimbra, in a framework of
ERASMUS programme, as well as Elga Poppela for technical
assistance to carry out in vivo test. Also thanks to Dr.
Gunars Tirzitis for fruitful discussions, Dr. Brigita Cekavi-
cus for synthesis of reference compounds 1 and 3.
4.2.6. Lipid peroxidation
Lipid peroxidation was determined by measuring
thiobarbituric acid reactive substances (TBARs), using
the routine thiobarbituric acid assay. Aliquots of mito-
chondrial suspensions (0.5 mL each), removed 10 min after
the addition of ADP/Fe2+, were added to 0.5 mL of ice cold
40% trichloroacetic acid. Then, 2 mL of 0.67% of aqueous
thiobarbituric acid containing 0.01% of 2,6-di-t-butyl-p-
cresol was added. The mixtures were heated at 90 8C for
15 min, then cooled on ice for 10 min, and centrifuged at
850 g for 10 min. Controls, in the absence of ADP/Fe2+, were
performed under the same conditions. The supernatant
fractions were collected and lipid peroxidation was
estimated spectrophotometrically at 530 nm. As blanks,
we used control reactions performed in the absence of
mitochondria and ADP/Fe2+. The amount of TBARs formed
was calculated using a molar extinction coefficient of
1.56 ꢃ 105 molꢁ1 cmꢁ1 and expressed as nmol TBARs/mg
of protein.
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Please cite this article in press as: Rucins M, et al. Synthesis and studies of calcium channel blocking and antioxidant
activities of novel 4-pyridinium and/or N-propargyl substituted 1,4-dihydropyridine derivatives. C. R. Chimie (2013),
http://dx.doi.org/10.1016/j.crci.2013.07.003

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Please cite this article in press as: Rucins M, et al. Synthesis and studies of calcium channel blocking and antioxidant
activities of novel 4-pyridinium and/or N-propargyl substituted 1,4-dihydropyridine derivatives. C. R. Chimie (2013),
http://dx.doi.org/10.1016/j.crci.2013.07.003