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.

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) and the N-dodecyl pyridinium group (compounds 11 and 12); these activities were comparable to that of amlodipine. 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 vasodilating 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].

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.

2.2.2 Assessment of total antioxidant activity (AOA)

In this study, the phosphomolybdenum complex method was used for the evaluation of the total antioxidant activity of the synthesised 1,4-DHP derivatives. 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, respectively. 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 phosphomolybdenum complex test system.

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.

2.2.3.2 Influence on Ca²⁺-induced mitochondrial pore transition (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 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 10 μM, did not induce MPT, while in the presence of calcium, this compound at concentrations 1, 5 and 10 μM significantly potentiated calcium toxic effects. For instance, at concentration 10 μM, the potentiating effect 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 μM), indicating that compound 12 has an ability to potentiate MPT induction. In 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 mitochondria safety. Under certain pathological conditions (in our case, the calcium ion overload), it may potentiate calcium entry into mitochondria.

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 × 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 Alltima C18 column, 5 μm, 4.6 × 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 chromatography 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 (δ: 7.26) or DMSO-d₆ (δ: 2.50) for ¹H NMR spectra and CDCl₃ (δ: 77.16) or DMSO-d₆ (δ: 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) operating in the ESI positive or negative ion mode on a Xbridge C18 column (5 μm, 2.1 mm × 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 determined on an Elemental Combustion System ECS 4010 (Costech Instruments) or on an EA 1106 (Carlo Erba Instruments).

4.1.2 Synthesis

Compounds 1, 3, 13, 15 and 17 were synthesised according to the literature. Briefly, the 2′,6′-dimethyl-4-phenyl-1,4-dihydropyridine-3,5-dicarboxylic acid diethyl ester (1) [26,40] and 2′,6′-dimethyl-1′,4′-dihydro-[3,4′]bipyridinyl-3′,5′-dicarboxylic acid diethyl ester (3) [27] were obtained from the corresponding aldehyde, ethyl acetoacetate and ammonia in the classical Hantzsch synthesis; 3′,5′-bisethoxycarbonyl-1-hexadecyl-2′,6′-dimethyl-1′,4′-dihydro-[3,4′]bipyridinyl-1-ium bromide (13) and 3′,5′-bisethoxycarbonyl-1-ethoxycarbonylmethyl-2′,6′-dimethyl-1′,4′-dihydro-[3,4′]bipyridinyl-1-ium bromide (15) from 2′,6′-dimethyl-1′,4′-dihydro-[3,4′]bipyridinyl-3′,5′-dicarboxylic acid diethyl ester (3) and hexadecyl bromide [28] or ethyl bromoacetate [29], according to procedures described from Makarova et al., respectively; 1-carboxymethyl-2′,6′-dimethyl-3′,5′-bisetoxycarbonyl-1′,4′-dihydro-[3,4′]bipyridinyl-1-ium (17) from equimolar amounts of 3′,5′-bisethoxycarbonyl-1-ethoxycarbonylmethyl-2′,6′-dimethyl-1′,4′-dihydro-[3,4′]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.

4.2.1 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 European Collection of Animal Cell Cultures (ECACC, UK). The cells were grown at 37 °C in a humidified atmosphere 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.

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 for 72 h. Changes in intracellular [Ca²⁺]_i concentration 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 0.8 to 100 μM. 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⁺]_i concentration, whereas A7r5 cells 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 used as the positive control. IC₅₀ values as mean ± SD were calculated using GraphPad Prism 3.0 software.

4.2.2 Evaluation of the total antioxidant activity

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 × 10⁻³ M just before use.

The reagent solution contains 0.6 M sulphuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate 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 °C 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.

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 procedures were performed in accordance with the guidelines of Directive86/609/EEC. “European Convention for the Protection 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 centrifugation according to the conventional methods. The experimental procedures were performed in accordance to those described previously [15].

Briefly, after washing, the pellet was gently re-suspended in the washing medium at a protein concentration of about 50 mg/mL. The protein content was determined by the biuret method, using bovine serum albumin as a standard.

1,4-DHP derivatives were dissolved in DMSO; concentration did no exceed 0.1% in the medium.

4.2.4 Measurement of respiratory activities

Oxygen consumption was monitored polarographically with a Clark-type electrode at 30 °C 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 diphosphate (ADP, 150 μM) and state-4 respiration (consumption of oxygen after ADP phosphorylation). FCCP (p-trifluoromethoxyphenylhydrazone)-stimulated respiration was initiated by the addition of 1 μM 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 μM at 30 °C, 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 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 (pH 7.4), 1 mM of KH₂PO₄, and 10 μM of EGTA, supplemented with 2 μM of rotenone, as previously described [42,43]. Mitochondria (1 mg/mL), previously incubated at 30 °C 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.

4.2.6 Lipid peroxidation

Lipid peroxidation was determined by measuring thiobarbituric acid reactive substances (TBARs), using the routine thiobarbituric acid assay. Aliquots of mitochondrial 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 °C 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 × 105 mol⁻¹ cm⁻¹ and expressed as nmol TBARs/mg of protein.

4.2.7 Statistical analysis

The mitochondrial experiments were performed using three independent experiments with different mitochondrial preparations. The values are expressed as means ± SE. Means were compared using one-way ANOVA for multiple-comparisons, followed by Tukey's test. Statistical significance was set at P < 0.05.

4.2.8 Subchronic toxicity in mice

The test was performed in male ICR mice weighing 20 ± 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 accordance 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).