Synthesis and Evaluation of Reducing Capacity and Calcium Channel Blocking Activity of Novel 3,5-Dipropargylcarbonyl-Substituted 1,4-Dihydropyridines

Article abstract of DOI:10.1007/s10593-014-1607-z

Novel pyridinium salts based on 4-(3-pyridyl)-3,5-dipropargylcarbonyl-1,4-dihydropyridine were obtained by quaternization of pyridine moiety with different alkyl halides. The reducing capacity of the synthesized compounds was evaluated using the phosphomolybdenum complex method. The obtained results confirmed that all tested compounds possessed reducing capacity. Ca²⁺ channel antagonist and agonist activities of the compounds were additionaly assayed by changes in intracellular Ca²⁺ ion concentration in H9C2 and A7R5 cell lines. The obtained data confirmed that all synthesized 1,4-dihydropyridine derivatives have smooth muscle selective antagonist activities, and in the case of 4-phenyl derivative the activity was 4.7 times higher than that of amlodipine.

Full text of DOI:10.1007/s10593-014-1607-z

DOI 10.1007/s10593-014-1607-z
Chemistry of Heterocyclic Compounds, Vol. 50, No. 10, January, 2015 (Russian Original Vol. 50, No. 10, October, 2014)
SYNTHESIS AND EVALUATION OF REDUCING CAPACITY
AND CALCIUM CHANNEL BLOCKING ACTIVITY OF
NOVEL 3,5-DIPROPARGYLCARBONYL-SUBSTITUTED
1,4-DIHYDROPYRIDINES*
M. Rucins1,2, M. Gosteva1, I. Domracheva1, I. Kanepe-Lapsa1, S. Belyakov1, M. Plotniece1,
K. Pajuste1, B. Cekavicus1, M. Jekabsone2, A. Sobolev1, I. Shestakova1, and А. Plotniece1**
Novel pyridinium salts based on 4-(3-pyridyl)-3,5-dipropargylcarbonyl-1,4-dihydropyridine were
obtained by quaternization of pyridine moiety with different alkyl halides. The reducing capacity of the
synthesized compounds was evaluated using the phosphomolybdenum complex method. The obtained
results confirmed that all tested compounds possessed reducing capacity. Ca2+ channel antagonist and
agonist activities of the compounds were additionaly assayed by changes in intracellular Ca2+ ion
concentration in H9C2 and A7R5 cell lines. The obtained data confirmed that all synthesized
1,4-dihydropyridine derivatives have smooth muscle selective antagonist activities, and in the case of
4-phenyl derivative the activity was 4.7 times higher than that of amlodipine.
Keywords: N-alkyl pyridinium, 1,4-dihydropyridines, calcium antagonists, Hantzsch synthesis,
quaternization, reducing capacity.
The 1,4-dihydropyridine (1,4-DHP) derivatives are nitrogen-containing heterocyclic compounds
playing an important role in medicinal, synthetic, and bioorganic chemistry [1-3]. Several representatives of
1,4-DHPs such as nifedipine, nitrendipine, amlodipine, and nisoldipine belong to the class of calcium channel
modulating agents [4-6] used worldwide. Depending on the chemical structure peculiarities, dihydropyridine
derivatives also possess other activities that are not related to calcium modulating, such as antimutagenic,
antidiabetic, neuro- and radioprotective, anti-inflammatory, antianginal, and antihypertensive action, besides
being regarded as perspective gene transfection agents [7]. The observation that 1,4-DHP derivatives, such as
nicardipine [8], show activity as inhibitors of the multidrug resistance transporter has increased renewed interest
for DHPs [9-11]. 1,4-DHPs are regarded as analogs of 1,4-dihydronicotinamide and model compounds of redox
coenzymes NAD(P)-H. Depending on their structure, 1,4-DHPs possess antioxidant, radical scavenging
properties, and they quench reactive oxygen species [12]. Antiradical and antioxidant properties of
_______
*Dedicated to Academician Professor G. Duburs on the occasion of his 80th birthday.
**To whom correspondence should be addressed, e-mail: aiva@osi.lv.
1Latvian Institute of Organic Synthesis, 21 Aizkraukles St., Riga LV-1006, Latvia.
2Faculty of Medicine, University of Latvia, 19 Raina Blvd., Riga LV-1586, Latvia; е-mail: rucins@osi.lv.
_________________________________________________________________________________________
Published in Khimiya Geterotsiklicheskikh Soedinenii, No. 10, pp. 1557-1568, October, 2014. Original
article submitted August 18, 2014.
0009-3122/15/5010-1431©2015 Springer Science+Business Media New York
1431

some representative of 1,4-DHPs have been discovered, as they act as hydrogen and electron donors to oxidants,
reactive oxygen species, reactive nitrogen species, free radicals, or activated unsaturated compounds [13].
Changes in calcium level regulate important functions in the cells and in the organism.
Dihydropyridines affect calcium levels, and they are widely used for treatment of many cardiovascular diseases
[14]. Calcium channel blockers based on 1,4-DHP also selectively inhibit the influx of Ca2+ ions through
calcium channels in the smooth muscle membrane, and they have been used in the therapy of angina pectoris
and hypertension [15].
On the other hand, during the last decades the neurotropic properties of 1,4-DHP have been widely studied.
Several research groups have experimentally demonstrated the ability of 1,4-DHPs to penetrate the blood-brain
barrier [16, 17]. Moreover, the propargyl moiety is known to play an important role in providing
neuroprotective properties in antidepressants, such as selegiline and rasagiline [18]. The combination of two
active pharmacophore groups or compounds into the same molecule is one of the strategies in medicinal
chemistry for drug design and drug discovery [19].
Our group has developed various pyridinium moiety-containing 1,4-DHPs for studies of their
membranotropic effects [20]. Calcium antagonistic properties and antioxidant activity of 1,4-DHP derivatives
containing a cationic pyridinium moiety at position 4 and N-propargyl group at position 1 of the 1,4-dihydro-
pyridine cycle have also been studied by our research group giving a basic background which permits drawing
conclusions concerning structure–activity relationships [21]. However, it would be beneficial to continue studies
on the influence of propargyl substituent position in the 1,4-DHP molecule on the mentioned activities for final
conclusions.
The aim of the present work was to obtain novel pyridinium salts based on 4-pyridyl-substituted 3,5-di-
propargylcarbonyl-1,4-dihydropyridines, varying the substituent at the nitrogen atom in the pyridine ring by
quaternization of the pyridine fragment of 2,6-dimethyl-3,5-dipropargylcarbonyl-4-(3-pyridyl)-1,4-dihydro-
pyridine as potential biologically active compounds. Studies of reducing capacity (RC) and calcium channel
blocking activity of the synthesized compounds were another objective of this work.
The propargyl acetoacetate was synthesized according to the procedure described by Cruciani et al. [22]
starting from propargyl alcohol and 2,2,6-trimethyl-1,3-dioxin-4-one. Hantzsch-type 1,4-DHPs 1 and 2 for our
studies were synthesized with 61 and 82% yields, respectively, via typical synthetic routes, such as the Hantzsch
synthesis [23, 24]. It involves a one-pot cyclocondensation of the corresponding aldehyde, propargyl
acetoacetate, and ammonium acetate under 24 h reflux in ethanol (Scheme 1).
Scheme 1
X
CH
CH
CH
X
O
NH4OAc
O
O
O
O
+
, EtOH, 24 h
O
O
Me
O
Me
N
Me
H
1 X = C, 2 X = N
In order to decrease the reaction time and improve the product yield, the synthesis of compound 2 with
the same reagent ratio was performed under microwave conditions. The irradiation during 30 min at 80°C gave
the 1,4-DHP derivative 2 in 52% isolated yield.
1,4-DHP derivative 1 has been described in the literature by Liepinsh et al. [24]. For this compound,
only 13C NMR data and melting point (mp 143-144°C, recrystallized from ethanol) were reported previously
[24]. In this case, the melting point of compound 1 was lower – 124-126°C. This could be explained by using a
different technique for melting point determination and crystallization from methanol instead of ethanol. In the
1H NMR spectra, the signal of the CH proton of the propargyl groups was observed as a triplet at 2.41 ppm with
1432

the coupling constant 2.4 and 2.5 Hz for 1,4-DHP derivatives 1 and 2, respectively, while the signals for the
CH2 group protons were detected at 4.64 and 4.63 ppm with different multiplicities: an AB system of double
doublets with coupling constants 15.6 and 2.4 Hz for compound 1 and a doublet with coupling constant 2.5 Hz
for derivative 2.
For an objective assessment of the structure of compounds 1 and 2, an X-ray crystallographic analysis
of the crystals of these compounds was undertaken. The crystal structure of compound 1 significantly differs
from that of compound 2. There are four independent molecules in the asymmetric unit in crystal of compound 1
(Fig. 1) The deviations of nitrogen atom N(1) from the C(2)–C(3)–C(5)–C(6) plane of dihydropyridine are
Fig. 1. Four independent molecules of the 1,4-DHP derivative 1 in the asymmetric unit; the atoms are
represented as thermal vibration ellipsoids of 50% probability.
Fig. 2. Molecular structure of 1,4-DHP derivative 2 with atoms represented as thermal vibration ellipsoids of
50% probability.
1433

TABLE 1. The Main Crystallographic Data and Refinement Parameters of
the Structures of 1,4-DHP Derivatives 1 and 2*
Parameters
Compound 1
Compound 2
C20H18N2O4
Empirical formula
Molecular weight
Crystal system
a, Å
C21H19NO4
349.39
350.37
Monoclinic
12.8538(1)
11.8689(1)
23.6725(3)
93.9579(4)
3602.87(6)
Pc
Monoclinic
8.6757(3)
15.4092(7)
13.9313(7)
97.882(2)
1844.8(1)
P21/a
b, Å
c, Å
, deg
V, Å3
Space group
Z
8
4
μ, mm–1
0.089
0.089
dcalc, g/cm3
1.288
1.262
2θmax for data, deg.
Reflections collected
Independent reflections
Reflections with I > 3σ(I)
Final R factor
wR2 index for all data
CCDC deposit number
56.0
56.0
26251
7652
8585 (Rint 0.032)
7032
4365 (Rint 0.036)
2782
0.071
0.053
0.201
0.238
CCDC 1013346
CCDC 1013345
_______
*Crystal structure refinement was performed using SIR97 [27] and maXus
[28] software.
different for each of the four molecules: 0.135(3), 0.149(3), 0.160(3), and 0.168(3) Å. The deviations of C(4)
atom are 0.294(4), 0.304(4), 0.334(4), and 0.361(1) Å, respectively. The phenyl rings are nearly perpendicular
to the mean planes of DHP. In the crystal structure, there are intermolecular hydrogen bonds between N−H
group and carbonyl oxygen of ester group. The lengths of these hydrogen bonds are in the range from 2.890(3)
to 2.985(3) Å.
Figure 2 shows a perspective view of the molecule of 1,4-DHP derivative 2 in a crystal. The
dihydropyridine cycle is characterized by the usual boat conformation [25, 26]. The deviations of atoms N(1)
and C(4) from the plane C(2)–C(3)–C(5)–C(6) are equal to 0.155(2) and 0.341(3) Å, respectively. The dihedral
angle between pyridine ring and the mean plane of dihydropyridine cycle is 87.9(5)°. In the crystal structure of
compound 2, there are strong intermolecular hydrogen bonds of NH···N type between nitrogen atom N(16) and
N(1)−H group; the length of this bond is 2.899(2) Å. By means of these bonds chains along crystallographic
direction (100) are formed in the crystals. Basic crystallographic data, conditions, and parameters of the X-ray
diffraction structure refinement of 1,4-DHP derivatives 1 and 2 are listed in Table 1.
As the next step of our work, the synthesis of various 1,4-DHP N-alkylpyridinium salts 3-9 was
performed by alkylation of dipropargyl 1,4-DHP ester 2 with the corresponding alkyl bromides in acetone under
reflux (Scheme 2, Table 2).
Scheme 2
R
Br
CH
N+
N
CH
CH
CH
O
O
RBr
O
O
, Me2CO
O
O
O
O
Me
N
Me
Me
N
Me
H
H
3–9
2
1434

TABLE 2. Alkylation Reaction Parameters for the Synthesis of 1,4-DHP
N-Alkylpyridinium Derivatives 3-9
Com-
Ratio
Amount of RBr,
mmol/ml
R
Reaction time, h
Yield, %
pound
1,4-DHP 2 – RBr
Et
1:8
1:3
1:3
1:3
1:3
1:2
1:4
6.88/0.52
2.58/0.28
2.58/0.36
2.58/0.45
2.58/0.54
1.72/0.42
3.44/1.04
75
100
201
77
65
36
57
64
65
73
65
3
4
5
6
7
8
9
n-Bu
C6H11
C8H19
C10H21
C12H25
C14H29
201
128
222
The typical procedure for the quaternization of pyridine derivatives has been reported by several
research groups [29]. Quaternization includes treating of pyridines with the corresponding alkyl halides in
acetone under reflux. An approach to reduce the reaction time of quaternization by the use of an excess of alkyl
bromide was also reported [30]. In our case, the solution of 2-8-fold excess of the corresponding alkyl bromide
was added to the starting dipropargyl ester 2, and the reaction mixture was refluxed in a pressure tube for
75-222 h (Table 2). However, in a pressure tube the alkylation reactions were still found to be rather slow and
proceeded with moderate yields of the products. In contrast, quaternization of the pyridine moiety of
3,5-diethoxycarbonyl-1,4-DHP with various alkyl bromides occurred in 5-26 h and with higher yields [21].
In order to attempt optimization of reaction conditions for the quaternization reaction of the pyridyl
moiety in compound 2 with tetradecyl bromide, the reaction was performed under microwave conditions using
the same reagent ratio (1:4) as in the conventional quaternization procedure. The reaction course was monitored
by HPLC every 15 min. It was found that the desired quaternized 1,4-DHP derivative 9 was formed in only 8%
yield under microwave conditions in 1 h at 70°C.
1
All the synthesized 1,4-DHP alkylpyridinium salts 3-9 were crystalline substances. In the H NMR
spectra, characteristic signals for the NCH2 group protons were observed at 4.56-4.73 ppm. On the other hand,
the shift of the proton signals of the pyridine ring into the 8.05-8.93 ppm range confirms the presence of a
positive charge on the nitrogen atom.
The structures of the synthesized new 1,4-DHP derivatives 2-9 and the already reported derivative 1
1
13
were confirmed by H, C NMR, MS, and elemental analysis data. Elemental analyses of 1,4-DHP derivatives
were in accordance with the structures, with the exception for compound 3 where elemental analysis showed
1
that this compound was crystallized with 0.1 mole of acetone. This was confirmed also by H NMR data. The
purities of the studied 1,4-DHP derivatives were at least 97% as detected by HPLC method. Molecular weights
of all studied compounds were detected by LC/MS technique and the obtained values were in good agreement
with the calculated ones.
1,4-DHP derivatives as nitrogen heterocycles play an important role in biological systems, for example, as
part of redox coenzymes NAD-H or NADP-H. Oxidative aromatization reactions of 1,4-DHP derivatives take
place in biological systems in the presence of certain enzymes, for example, Cytochrome P450 (CYP) [31].
Additionally, antiradical and antioxidant properties of DHP derivatives have also been discovered [12, 13, 32, 33].
The reducing capacity of the synthesized 1,4-DHP derivatives 1-9 was tested using the method reported
by Prieto et al. [34]. The assay is based on the reduction of Mo(VI) with the subsequent formation of a green
phosphate/Mo(V) complex, having a characteristic absorbance maximum at 695 nm, which can be detected by
UV/Vis spectroscopy. Diludine (diethyl 1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate), a well-known
1,4-DHP derivative with antioxidant and antiradical properties, was used as a positive control [35]. The
obtained data on RC of the synthesized compounds are summarized in Figure 3.
The obtained results show that all synthesized 1,4-DHP derivatives 1-9 possess reducing capacity under
the tested conditions. It should be noted that RC of 1,4-DHP derivatives 1 and 2 was higher than that of
1435

diludine. Other 1,4-DHP derivatives 3-9, containing pyridinium moiety, showed lower RC values than diludine.
We assume that RC values of the tested 1,4-DHP derivatives are mainly related to the ability of their
aromatization at the phosphomolybdenum complex system leading to full oxidation of dihydropyridine to the
corresponding pyridine derivative (according to UV/Vis spectrum data).
Ca2+ channel antagonist and agonist activities of compounds 1-9 were assayed by changes in intracellular
Ca2+ ion concentration [Ca2+]i in H9C2 cells, derived from the embryonic rat ventricle, and A7R5 cells (rat smooth
muscle cells). The model for investigation of the Ca2+ channel blocking activity of DHP derivatives is based on the
analysis of the effect of KCl on intracellular Ca2+ mobilization in H9C2 and A7R5 cells. The well-known calcium
channel inhibitor – amlodipine (3-ethyl-5-methyl 2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-6-methyl-1,4-di-
hydropyridine-3,5-dicarboxylate) was used as a positive control in this test. The results are summarized in
Table 3.
Fig. 3. RC of 1,4-DHP derivatives 1-9 and diludine (at 100 mM concentration), evaluated by the
phosphomolybdenum complex method; the results are expressed as absorbance of sample at 695 nm against
absorbance of a blank solution. All data are presented as a mean with standard deviation.
TABLE 3. Antagonist and Agonist Effects of 1,4-DHP Derivatives 1-9 on
[Ca2+]i in H9C2 and A7R5 Cells
Antagonist activity (IC50, µM)
Agonist activity (RFU*, 100 µM)
Compound
H9C2
A7R5
H9C2
A7R5
Amlodipine
–
–
–
–
–
–
–
–
–
–
0.14±0.01
0.03±0.01
0.60±0.06
>100
–
+
–
–
–
–
+
+
+
+
–
+
–
–
–
–
+
+
+
+
1
2
3
4
5
6
7
8
9
95±2
2.5±0.6
0.80±0.07
0.70±0.05
0.40±0.03
0.50±0.05
_______
+ – activity was detected.
– – no activity was detected in tested concentrations.
*Relative fluorescence units.
1436

As shown in Table 3, none of 1,4-DHP derivatives 1-9 in the tested doses showed antagonist effect in
the H9C2 cell line, some of them, however, showed such effect in A7R5 cells. Furthermore, 1,4-DHP derivative
1 demonstrated an activity 4.7 times higher than amlodipine, whereas 1,4-DHP derivatives 2 and 5-9 were about
3-8 times less active than amlodipine, and compounds 3 and 4 possessed low activity. The obtained results
indicate that the compounds target only calcium channels in vascular smooth muscle cells and do not affect
calcium channels in cardiac cells.
The calcium channel agonist effect of 1,4-DHP derivatives 1-9 was also tested on both cell lines.
Results are summarized in Table 3. The obtained time dependency data for one of the active compounds,
1,4-DHP derivative 7, are represented in Fig. 4.
The obtained data show a rapid increase of [Ca2+]i, which subsequently turns into a ''plateau''. This is a
classical ''biphase'' response confirming that in the response to the stimulus not only intracellular but also extra-
cellular Ca2+ stores are involved. A similar effect was also observed for a known 1,4-DHP agonist ВауK8644 [36].
Fig. 4. Ca2+ responses by Fluo-4 NW-loaded cells (a) A7R5, (b) H9C2 stimulated by the addition of agonist 7 in
concentrations 20 and 100 µM. The arrow indicates the time of addition of compound 7.
In summary, it has been established that all synthesized pyridinium salts based on 4-pyridyl-3,5-di-
propargylcarbonyl-substituted 1,4-dihydropyridine with different alkyl substituents at the nitrogen atom
possessed RC. Studies of Ca2+ channel antagonist and agonist activities of the compounds, assayed by changes
in intracellular [Ca2+]i concentration in H9C2 and A7R5 сells, indicate that the compounds target only calcium
channels in vascular smooth muscle cells and do not affect calcium channels in cardiac cells. It has been found
that 1,4-DHP derivative 1 possess 4.7 times higher antagonist activity than amlodipine in the A7R5 cells. This
study could serve as the basis for further design and synthesis of novel Ca2+ channel antagonist-agonist agents
on the 1,4-DHP core.
EXPERIMENTAL
1H and 13C NMR spectra were recorded on a Varian Mercury BB (400 and 100 MHz, respectively) or
on a Bruker Fourier 300 (300 and 75 MHz, respectively) spectrometer. The chemical shifts of the atoms are
reported relative to the residual signal of the solvent: DMSO-d6 (δ 2.50 ppm) or CDCl3 (δ 7.26 ppm) for
1H NMR spectra and DMSO-d6 (δ 39.5 ppm) or CDCl3 (δ 77.2 ppm) for 13C NMR spectra. 13C NMR signal
assignments were based on the spectra of similar compounds [21, 37] and theoretical calculations according to
[38]. Mass spectra were obtained on a Waters Acquity UPLC system connected to a Waters SQ Detector-2
operating in the ESI positive ion mode on a Waters Acquity UPLC® BEH C18 column (1.7 µm, 2.1 × 50 mm)
using a gradient elution with MeCN (0.01% CF3CO2H) in water (0.01% CF3CO2H) at a flow rate 0.5 ml/min
and processed with the Waters MassLynx 4.1 chromatography data system. Elemental analyses were performed
on a Costech Instruments Elemental Combustion System ECS 4010 or on a Carlo Erba Instruments EA 1106
1437

instrument. Melting points were determined on a SRS OptiMelt apparatus. The compounds were analyzed by
HPLC on a Waters Alliance 2695 system equipped with a Waters 2485 UV/Vis detector and an Alltima CN
column (5 µm, 4.6×150 mm, Grace) using gradient elution with MeCN (0.1% H3PO4) in water, at a flow rate of
1 ml/min. Peak areas were determined electronically with the Waters Empower 2 chromatography data system.
TLC was performed on Silica gel 60 F254 aluminum sheets 20×20 cm (Merck). Reducing capacity was
measured at 695 nm using a UV/Vis Camspec M501 spectrometer. Calcium agonist activity was measured using
a Thermo Ascient fluorescence spectrophotometer. Reactions under microwave conditions were performed on a
Biotage® Initiator Classic microwave synthesizer with a single-mode microwave applicator.
Fluo-4 NW Calcium Assay Kit was purchased from Invitrogen (Sweden). H9C2 and A7R5 cell lines were
obtained from European Collection of Animal Cell Cultures. All chemical reagents were purchased from Acros or
Sigma-Aldrich and used without further purification.
Bis(prop-2-ynyl) 2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3,5-dicarboxylate (1) was obtained
from propargyl acetoacetate, benzaldehyde, and ammonia via classical Hantzsch synthesis following an already
reported method [24]. Yield 0.64 g (61%), white crystals, mp 124-126°C (MeOH) (mp 143-144°C (EtOH)
[24]). 1H NMR spectrum (300 MHz, CDCl3), δ, ppm (J, Hz): 2.35 (6H, s, 2,6-CH3); 2.41 (2H, t, J = 2.4, 2C≡CH);
4.64 (4H, dd, AB system, J = 15.6, J = 2.4, 2CH2); 5.03 (1H, s, 4-CH); 5.78 (1H, s, NH); 7.10-7.16 (1H, m, H-4 Ph);
13
7.18-7.25 (2H, m, H-3,5 Ph); 7.27-7.33 (2H, m, H-2,6 Ph). C NMR spectrum (100 MHz, CDCl3), δ, ppm: 19.8
(2,6-CH3); 39.3 (C-4); 51.4 (2OCH2); 74.3 (2C≡CH); 78.2 (2C≡CH); 103.6 (C-3,5); 126.4 (C-4 Ph); 127.9 (C-3,5
Ph); 128.0 (C-2,6 Ph); 145.0 (C-2,6); 147.1 (C-1 Ph); 166.6 (2C=O). Mass spectrum, m/z (Irel, %): 350 [M+H]+
(100). Found, %: C 72.11; H 5.49; N 4.01. C21H19NO4. Calculated, %: C: 72.19; H 5.48; N 4.01.
Bis(prop-2-ynyl) 2,6-dimethyl-4-(3-pyridyl)-1,4-dihydropyridine-3,5-dicarboxylate (2) was synthe-
sized from propargyl acetoacetate (1.50 g, 11.0 mmol), pyridine-3-carboxaldehyde (0.52 ml, 0.59 g, 5.5 mmol),
and ammonium acetate (0.51 g, 6.6 mmol) in EtOH (50 ml). The reaction mixture was refluxed for 24 h and
separated by flash chromatography (petroleum ether–EtOAc, 1:1 to 0:1); the obtained residue was recrystallized
1
from MeOH. Yield 1.58 g (82%), yellow crystals, mp 180-182°C (MeOH). H NMR spectrum (300 MHz,
CDCl3), δ, ppm (J, Hz): 2.34 (6H, s, 2,6-CH3); 2.41 (2H, t, J = 2.5, 2C≡CH); 4.63 (4H, d, J = 2.5, 2CH2); 5.01
(1H, s, 4-CH); 7.10 (1H, s, NH); 7.17 (1H, ddd, J = 7.8, J = 4.8, J = 0.7, H-5 Py); 7.66 (1H, ddd, J = 7.8, J = 2.4,
J = 1.9, H-6 Py); 8.37 (1H, dd, J = 4.8, J = 1.6, H-4 Py); 8.54 (1H, d, J = 2.4, H-2 Py). 13C NMR spectrum
(100 MHz, CDCl3), δ, ppm: 19.4 (2,6-CH3); 37.6 (C-4); 51.5 (2OCH2); 74.6 (2C≡CH); 78.2 (2C≡CH); 102.5
(C-3,5); 123.4 (C-5 Py); 136.0 (C-3 Py); 143.4 (C-4 Py); 146.5 (C-2,6); 147.3 (C-6 Py); 149.3 (C-2 Py); 166.3
(2C=O). Mass spectrum, m/z (Irel, %): 351 [M+H]+ (100). Found, %: C 68.28; H 5.18; N 7.86. C20H18N2O4.
Calculated, %: C 68.56; H 5.18; N 8.00.
Synthesis of 1,4-DHP Alkylpyridinium Salts 3-9 (General Method). The corresponding alkyl halide
in 2-8-fold excess was added to a solution of 1,4-DHP derivative 2 (0.30 g, 0.86 mmol) in acetone (20 ml). The
reaction mixture was refluxed in a pressure tube for 75-222 h (Table 2), then cooled to 0°C. The resulting
precipitate was filtered off, washed with cold acetone, recrystallized from acetone, and dried in vacuo.
1-Ethyl-3-{2,6-dimethyl-3,5-bis[(prop-2-ynyloxy)carbonyl]-1,4-dihydropyridin-4-yl}-pyridinium
1
Bromide (3). Yield 0.26 g (65%), yellowish powder, mp 208-210°C (Me2CO). H NMR spectrum (400 MHz,
DMSO-d6), δ, ppm (J, Hz): 1.53 (3H, t, J = 7.1, N+CH2CH3); 2.31 (6H, s, 2,6-CH3); 3.46 (2H, t, J = 2.2,
2C≡CH); 4.57-4.73 (6H, m, 2OCH2, N+CH2); 5.00 (1H, s, 4-CH); 8.05 (1H, dd, J = 7.9, J = 5.9, H-5 Py); 8.38
(1H, d, J = 7.9, H-6 Py); 8.88 (1H, s, H-2 Py); 8.92 (1H, d, J = 5.9, H-4 Py ); 9.38 (1H, s, NH). 13C NMR
spectrum (75 MHz, DMSO-d6), δ, ppm: 16.3 (N+CH2CH3); 17.9 (2,6-CH3); 37.4 (C-4); 50.7 (2OCH2); 55.9
(N+CH2); 74.8 (2C≡CH); 78.4 (2C≡CH); 98.5 (C-3,5); 127.9 (C-5 Py); 142.0 (C-3 Py); 142.2 (C-4 Py); 143.9
(C-6 Py); 147.6 (C-2 Py); 148.4 (C-2,6); 164.6 (2C=O). Mass spectrum, m/z (Irel, %): 379 [M-Br]+ (100). Found,
%: C 57.20; H 5.25; N 5.64. C22H23N2O4Br · 0.1 (CH3)2CO. Calculated, %: C 57.58; H 5.11; N 6.02.
1-Butyl-3-{2,6-dimethyl-3,5-bis[(prop-2-ynyloxy)carbonyl]-1,4-dihydropyridin-4-yl}pyridinium
1
Bromide (4). Yield 0.15 g (36%), yellowish powder, mp 206-208°C (Me2CO). H NMR spectrum (400 MHz,
DMSO-d6), δ, ppm (J, Hz): 0.90 (3H, t, J = 7.3, CH3); 1.21-1.31 (2H, m, CH2CH3); 1.82-1.91 (2H, m,
1438

N+CH2CH2); 2.32 (6H, s, 2,6-CH3); 3.46 (2H, t, J = 2.4, 2C≡CH); 4.58-4.70 (6H, m, 2OCH2, N+CH2); 4.99 (1H,
s, 4-CH); 8.06 (1H, dd, J = 8.3, J = 6.1, H-5 Py); 8.39 (1H, dt, J = 8.3, J = 1.3, H-6 Py); 8.86 (1H, s, H-2 Py);
8.91 (1H, dt, J = 6.1, J = 1.3, H-4 Py); 9.40 (1H, s, NH). 13C NMR spectrum (75 MHz, DMSO-d6), δ, ppm: 13.3
(CH3); 18.4 (2,6-CH3); 18.7 (CH2CH3); 32.9 (N+CH2CH2); 37.8 (C-4); 51.1 (2OCH2); 60.4 (N+CH2); 77.2
(2C≡CH); 78.8 (2C≡CH); 99.0 (C-3,5); 128.3 (C-5 Py); 142.6 (C-3 Py); 142.9 (C-4 Py); 144.4 (C-6 Py); 147.9
(C-2 Py); 148.8 (C-2,6); 165.0 (2C=O). Mass spectrum, m/z (Irel, %): 407 [M-Br]+ (100). Found, %: C 59.08;
H 5.55; N 5.62. C24H27N2O4Br. Calculated, %: C 59.14; H 5.58; N 5.75.
1-Hexyl-3-{2,6-dimethyl-3,5-bis[(prop-2-ynyloxy)carbonyl]-1,4-dihydropyridin-4-yl}-pyridinium
Bromide (5). Yield 0.25 g (57%), recrystallized twice, pale-yellow powder, mp 158-160°C (Me2CO). 1H NMR
spectrum (300 MHz, DMSO-d6), δ, ppm (J, Hz): 0.85 (3H, t, J = 6.7, CH3); 1.17-1.32 (6H, m, (CH2)3CH3);
1.81-1.94 (2H, m, N+CH2CH2); 2.32 (6H, s, 2,6-CH3); 3.45 (2H, t, J = 2.4, 2C≡CH); 4.57-4.71 (6H, m, 2OCH2,
N+CH2); 5.00 (1H, s, 4-CH); 8.06 (1H, dd, J = 8.2, J = 6.0, H-5 Py); 8.38 (1H, d, J = 8.2, H-6 Py); 8.85 (1H, s,
H-2 Py); 8.92 (1H, d, J = 6.0, H-4 Py); 9.40 (1H, s, NH). 13C NMR spectrum (100 MHz, DMSO-d6), δ, ppm:
14.2 (CH3); 18.8 (2,6-CH3); 22.2 (CH2CH3); 25.4 (N+(CH2)3CH2); 30.9 (N+(CH2)2CH2); 31.4 (N+CH2CH2); 38.3
(C-4); 51.5 (2OCH2); 61.0 (N+CH2); 77.6 (2C≡CH); 79.1 (2C≡CH); 99.4 (C-3,5); 128.6 (C-5 Py); 143.0 (C-3
Py); 143.4 (C-4 Py); 144.7 (C-6 Py); 148.3 (C-2 Py); 149.1 (C-2,6); 165.4 (2C=O). Mass spectrum, m/z (Irel, %):
435 [M-Br]+ (100). Found, %: C 60.59; H 6.06; N 5.43. C26H31N2O4Br. Calculated, %: C 60.30; H 6.11; N 5.54.
3-{2,6-Dimethyl-3,5-bis[(prop-2-ynyloxy)carbonyl]-1,4-dihydropyridin-4-yl}-1-octylpyridinium
Bromide (6). Yield 0.30 g (64%), pale-yellow powder, mp 182-184°C (Me2CO). 1H NMR spectrum (300 MHz,
DMSO-d6), δ, ppm (J, Hz): 0.85 (3H, t, J = 6.7, CH3); 1.18-1.30 (10H, m, CH2)5CH3); 1.82-1.93 (2H, m,
N+CH2CH2); 2.32 (6H, s, 2,6-CH3); 3.46 (2H, t, J = 2.4, 2C≡CH); 4.56-4.71 (6H, m, 2OCH2, N+CH2); 4.99 (1H,
s, 4-CH); 8.05 (1H, dd, J = 8.1, J = 6.0, H-5 Py); 8.38 (1H, d, J = 8.1, H-6 Py); 8.86 (1H, s, H-2 Py); 8.92 (1H,
d, J = 6.0, H-4 Py); 9.40 (1H, s, NH). 13C NMR spectrum (75 MHz, DMSO-d6), δ, ppm: 13.9 (CH3); 18.3
(CH2CH3); 22.0 (2,6-CH3); 25.3 (N+(CH2)5CH2); 28.3 (N+(CH2)4CH2); 28.4 (N+(CH2)3CH2); 31.0
(N+(CH2)2CH2); 31.1 (N+CH2CH2); 37.9 (C-4); 51.1 (2OCH2); 60.6 (N+CH2); 77.2 (2C≡CH); 78.7 (2C≡CH);
99.0 (C-3,5); 128.3 (C-5 Py); 142.5 (C-3 Py); 143.0 (C-4 Py); 144.3 (C-6 Py); 147.9 (C-2 Py); 148.7 (C-2,6);
165.0 (2C=O). Mass spectrum, m/z (Irel, %): 463 [M-Br]+ (100). Found, %: C 61.87; H 6.50; N 5.11.
C28H35N2O4Br. Calculated, %: C 61.88; H 6.49; N 5.15.
1-Decyl-3-{2,6-dimethyl-3,5-bis[(prop-2-ynyloxy)carbonyl]-1,4-dihydropyridin-4-yl}pyridinium
Bromide (7). Yield 0.32 g (65%), pale-yellow powder, mp 182-184°C (Me2CO). 1H NMR spectrum (300 MHz,
DMSO-d6), δ, ppm (J, Hz): 0.85 (3H, t, J = 6.7, CH3); 1.16-1.31 (14H, m, (CH2)7CH3); 1.81-1.94 (2H, m,
N+CH2CH2); 2.32 (6H, s, 2,6-CH3); 3.45 (2H, t, J = 2.4, 2C≡CH); 4.56-4.71 (6H, m, 2OCH2, N+CH2); 4.99 (1H,
s, 4-CH); 8.06 (1H, dd, J = 8.2, J = 6.0, H-5 Py); 8.38 (1H, d, J = 8.2, H-6 Py); 8.86 (1H, s, H-2 Py); 8.92 (1H, d,
J = 6.0, H-4 Py); 9.40 (1H, s, NH). 13C NMR spectrum (100 MHz, DMSO-d6), δ, ppm: 14.4 (CH3); 18.8 (CH2CH3);
22.5 (2,6-CH3); 25.7 (N+(CH2)7CH2); 28.8 (N+(CH2)6CH2); 29.1 (N+(CH2)5CH2); 29.2 (N+(CH2)4CH2); 29.3
(N+(CH2)3CH2); 31.4 (N+(CH2)2CH2); 31.7 (N+CH2CH2); 38.3 (C-4); 51.5 (2OCH2); 61.0 (N+CH2); 77.6 (2C≡CH);
79.2 (2C≡CH); 99.4 (C-3,5); 128.7 (C-5 Py); 143.0 (C-3 Py); 143.4 (C-4 Py); 144.8 (C-6 Py); 148.3 (C-2 Py); 149.2
(C-2,6); 165.5 (2C=O). Mass spectrum, m/z (Irel, %): 491 [M-Br]+ (100). Found, %: C 62.92; H 6.82; N 4.88.
C30H39N2O4Br. Calculated, %: C 63.04; H 6.88; N 4.90.
1-Dodecyl-3-{2,6-dimethyl-3,5-bis[(prop-2-ynyloxy)carbonyl]-1,4-dihydropyridin-4-yl}-pyridinium
Bromide (8). Yield 0.38 g (73%), pale-yellow powder, mp 164-166°C (Me2CO). 1H NMR spectrum (300 MHz,
DMSO-d6), δ, ppm (J, Hz): 0.85 (3H, t, J = 6.7, CH3); 1.17-1.30 (18H, m, (CH2)9CH3); 1.80-1.93 (2H, m,
N+CH2CH2); 2.32 (6H, s, 2,6-CH3); 3.45 (2H, t, J = 2.4, 2C≡CH); 4.56-4.71 (6H, m, 2OCH2, N+CH2); 4.99 (1H,
s, 4-CH); 8.06 (1H, dd, J = 8.1, J = 6.1, H-5 Py); 8.38 (1H, d, J = 8.1, H-6 Py); 8.86 (1H, s, H-2 Py); 8.93 (1H,
d, J = 6.1, H-4 Py); 9.41 (1H, s, NH). 13C NMR spectrum (100 MHz, DMSO-d6), δ, ppm: 13.3 (CH3); 17.6
(CH2CH3); 21.4 (2,6-CH3); 24.6 (N+(CH2)9CH2); 27.7 (N+(CH2)8CH2); 28.0 (N+(CH2)7CH2); 28.1
(N+(CH2)6CH2); 28.2 (N+(CH2)5CH2); 28.3 (N+(CH2)4CH2); 28.4 (N+(CH2)3CH2); 30.3 (N+(CH2)2CH2); 30.6
(N+CH2CH2); 37.2 (C-4); 50.4 (2OCH2); 59.9 (N+CH2); 76.5 (2C≡CH); 78.0 (2C≡CH); 98.3 (C-3,5); 127.6
1439

(C-5 Py); 141.9 (C-3 Py); 142.3 (C-4 Py); 143.6 (C-6 Py); 147.2 (C-2 Py); 148.1 (C-2,6); 164.3 (2C=O). Mass
spectrum, m/z (Irel, %): 519 [M-Br]+ (100). Found, %: C 63.83; H 7.38; N 4.67. C32H43N2O4Br. Calculated, %:
C 64.10; H 7.23; N 4.67.
3-{2,6-Dimethyl-3,5-bis[(prop-2-ynyloxy)carbonyl]-1,4-dihydropyridin-4-yl}-1-tetradecylpyridinium
Bromide (9). Yield 0.35 g (65%), pale-yellow powder, mp 153-155°C (Me2CO). 1H NMR spectrum (300 MHz,
DMSO-d6), δ, ppm (J, Hz): 0.85 (3H, t, J = 6.7, CH3); 1.16-1.30 (22H, m, (CH2)11CH3); 1.80-1.94 (2H, m, N+CH2CH2);
2.32 (6H, s, 2,6-CH3); 3.45 (2H, t, J = 2.4, 2C≡CH); 4.56-4.71 (6H, m, 2OCH2, N+CH2); 4.99 (1H, s, 4-CH); 8.05 (1H,
dd, J = 8.0, J = 6.0, H-5 Py); 8.38 (1H, d, J = 8.0, H-6 Py); 8.85 (1H, s, H-2 Py); 8.92 (1H, d, J = 6.0, H-4 Py); 9.40 (1H,
s, NH). 13C NMR spectrum (75 MHz, DMSO-d6), δ, ppm: 13.9 (CH3); 18.3 (CH2CH3); 22.1 (2,6-CH3); 25.3
(N+(CH2)11CH2); 28.3 (N+(CH2)10CH2); 28.4 (N+(CH2)9CH2); 28.5 (N+(CH2)8CH2); 28.6 (N+(CH2)7CH2); 28.7
(N+(CH2)6CH2); 28.8 (N+(CH2)5CH2); 28.9 (N+(CH2)4CH2); 29.0 (N+(CH2)3CH2); 29.1 (N+(CH2)2CH2); 31.3
(N+CH2CH2); 37.8 (C-4); 51.1 (2OCH2); 59.8 (N+CH2); 77.1 (2C≡CH); 78.7 2C≡CH); 99.0 (C-3,5); 128.2 (C-5 Py);
142.5 (C-3 Py); 143.0 (C-4 Py); 144.3 (C-6 Py); 147.9 (C-2 Py); 148.7 (C-2,6); 165.0 (2C=O). Mass spectrum, m/z (Irel,
%): 547 [M-Br]+ (100). Found, %: C 64.83; H 7.78; N 4.38. C34H47N2O4Br. Calculated, %: C 65.06; H 7.55; N 4.46.
X-ray diffraction data were collected at -100°C on a Bruker-Nonius KappaCCD diffractometer using
graphite monochromated MoKα radiation (λ 0.71073 Å). The crystal structures were solved by direct methods and
refined by full-matrix least squares. The main crystallographic data and refinement parameters of the crystal
structures are listed in Table 1. For further details, see crystallographic data for these compounds deposited at the
Cambridge Crystallographic Data Center.
Evaluation of the Reducing Capacity. The RC of the synthesized 1,4-DHP derivatives 1-9 was studied
using spectrophotometric quantitation through the formation of phosphomolybdenum complex [34]. Stock
solutions (1.0 mM) of the test compounds and reference compound diludine were prepared in EtOH just before
use. The reagent solution contained sulfuric acid (0.6 M), sodium phosphate (28 mM), and ammonium
molybdate (4 mM) 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°C for 90 min.
After the samples were cooled to room temperature, the absorbance of each sample solution was measured at
695 nm against a blank sample using a UV/Vis spectrometer. A typical blank solution contained 3 ml of the
reagent solution and EtOH (0.3 ml), and was incubated under the same conditions as the rest of the samples.
Each assay was performed in triplicate.
Intracellular Ca2+ measurements. Cells were grown in Dulbecco's modified Eagle medium containing
1% non-essential amino acids and 2 mM glutamine, supplemented with 10% fetal bovine serum (Sigma) at
37°C under 5% CO2. Cells were passaged once a week using 0.25% trypsin and 0.53 mM EDTA solution and
grown in 75 mm2 plastic culture flasks until confluence and seeded in 96-well plates for experiments. Cells were
seeded into 96-well plates at a density of 30,000 cells per well and incubated for 72 h. [Ca2+]i was measured in
confluent cell monolayers with the Ca2+-sensitive fluorescent indicator Fluo-4 NW.
For investigation of Ca2+ channel antagonist activity of compounds 1-9, the cells were preincubated in the
dark for 15 min with test compounds at concentrations from 0.16 to 100 μM. KCl (50 mM for A7R5 cells and
200 mM for H9C2 cells) was applied to Fluo-4 NW-loaded cells to stimulate intracellular Ca2+ mobilization.
Amlodipine was used as the positive control.
For investigation of Ca2+ channel agonist activity, Fluo-4 NW-loaded cells were stimulated by the addition
of test compounds (concentration range from 4 to 100 µM). BayK8644 was used as the positive control. Changes in
[Ca2+]i were measured from the fluorescence emitted at 516 nm due to alternate excitation at 494 nm using a
fluorescence spectrophotometer. IC50 values for the tested compounds were calculated using GraphPad Prism 4.0
software.
This research work was supported by the European Social Fund Project No. ESF
2013/0002/1DP/1.1.1.2.0/13/APIA/VIAA/005 and the European Social Fund within the project ''Support for
Doctoral Studies at University of Latvia'' (for Martins Rucins).
1440

We are also grateful to Dr. Ilga Mutule for assistance in the work with MW equipment and B.Sc.
Katrina Veinberga for technical assistance.
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