The experiments were carried out using male normotensive Wistar rats [Krf: (WI) WU], weighing 200–250 g. Animals were kept in plastic cages (three rats per cage) at constant room temperature of 22 ± 2°C, with 12:12 h light/dark cycle. Rats had free access to food (standard pellet diet) and water. Each experimental and control groups consisted of four to six animals. The animals were killed by cervical dislocation immediately after the experiment. All injections were given in a volume of 1 ml/kg. All experimental procedures were approved by the Local Ethics Committee for Experiments on Animals of the Jagiellonian University in Krakow, Poland (approval numbers 110/2014 and 246/2015) and cared for in accordance with the Guide to the Care and Use of Experimental Animals.

Six studied compounds (Figure 1): 1-[(2,6-dimethylphenoxy)ethoxyethyl]-4-(2-methoxyphenyl)piperazine hydrochloride (HBK14), 1-[(2-chloro-6-methylphenoxy)ethoxyethyl]-4-(2-methoxyphenyl)piperazine hydrochloride (HBK15), 1N-[3-(2-chloro-5-methylphenoxy)propyl]-4N-(2-methoxyphenyl)piperazine hydrochloride (HBK16), 1N-[3-(2,5-dimethylphenoxy)propyl]-4N-(2-methoxyphenyl)piperazine hydrochloride (HBK17), and 1N-[3-(2,4,6-trimethylphenoxy)propyl]-4N-(2-methoxyphenyl)piperazine hydrochloride (HBK18), 1N-[3-(2,3,5-trimethylphenoxy)propyl]-4N-(2-methoxyphenyl)piperazine hydrochloride (HBK-19) were synthesized in the Department of Bioorganic Chemistry, Chair of Organic Chemistry, Faculty of Pharmacy, Jagiellonian University (Waszkielewicz et al., 2015). The investigated compounds were dissolved in saline and administered intraperitoneally (i.p.) or intravenously (i.v.). Thiopental (Rotexmedica, Germany) was dissolved in saline and administered i.p. Chloroethylclonidine (Sigma, Germany), noradrenaline (Sigma, Germany), johimbine (Tocris, United Kingdom), propranolol (Fluka, USA), phentolamine (Sigma, Germany) were dissolved in saline or dimethyl sulfoxide (DMSO, Sigma, Germany) and used in radioligand or biofunctional studies. Adrenaline (Polfa S.A., Warsaw), carvedilol (Sigma, Germany), methoxamine (Sigma, China), calcium chloride (Fluka, Germany), and barium chloride (Sigma, Germany) were dissolved in saline and administered i.v. Heparin (Polfa S.A., Warsaw) was used as anticoagulant during experiments. The control groups received 0.9% NaCl solution. Other chemicals used were obtained from POCh (Polish Chemical Reagents, Poland).

The α₁- and β₁-adrenoceptor radioligand binding assay was performed on rat cerebral cortex. [³H]-prazosin (19.5 Ci/mmol, α₁-adrenoceptor) and [³H]-CGP-12177 (48 Ci/mmol, β₁-adrenergic receptor) were used. The brains were homogenized in 20 volumes of an ice-cold 50 mM Tris-HCl buffer (pH 7.6) and were centrifuged at 20,000 × g for 20 min (0−4°C). The cell pellet was resuspended in the Tris–HCl buffer and centrifuged again. Radioligand binding assays were performed in plates (MultiScreen/Millipore). The final incubation mixture (final volume 300 μl) consisted of 240 μl of the membrane suspension, 30 μl of [³H]-prazosin (0.2 nM), [³H]-CGP-12177 (0.2 nM) solution and 30 μl of the buffer containing seven to eight concentrations (10⁻¹¹−10⁻⁴ M) of the tested compounds. In order to measure the unspecific binding, 10 μM phentolamine (for [³H]-prazosin) and 1 μM of propranolol (for [³H]-CGP-12177) were applied. The incubation was terminated by rapid filtration through glass fiber filters (Whatman GF/C) using a vacuum manifold (Millipore). The filters were then washed twice with the assay buffer and placed in scintillation vials with a liquid scintillation cocktail. Radioactivity was measured in a WALLAC 1409 DSA liquid scintillation counter (Perkin Elmer, USA). All the assays were made in duplicate and the inhibitory constants (K_i) were estimated.

Rats were anesthetized with thiopental (75 mg/kg i.p.) and the middle part of the ventral caudal artery was removed, cleaned of surrounded tissues and uncovered of endothelium by gentle rubbing in the Krebs-Henseleit solution. The isolated artery, cut into ~4 mm long rings, was then horizontally put up between two stainless steel hooks (diameter 0.15 mm). One hook was fastened to the bottom of the chamber and the other to an isometric FDT10-A force displacement transducer (DMT Model 750TOBS, Denmark), linked with Powerlab 4/26 analyzer (ADInstruments), and processed by LabChart 7 software. The isolated rings were incubated in the Krebs-Henseleit solution (20 ml) at the temperature 37°C and pH 7.4 with constant oxygenation (O₂/CO₂, 19:1). The initial optimal tissues tension was set at 0.75 g. Chloroethylclonidine (3 μM) as the preferential α_1B-adrenoreceptor alkylating agent was used during incubation and after 30 min it was completely washed off. Through 100 min of equilibration tissues were stimulated with noradrenaline (1 μM) four times with washing out until the contractile response become constant. Two cumulative concentration-response curves to noradrenaline, at an interval of 60 min, were established on each arterial ring both in the presence and absence of antagonist. The incubation with antagonists went on for 30 min. The experiments were carried out in the constant presence of yohimbine (0.1 μM) and propranolol (1 μM) to block α₂- and β-adrenoceptors, respectively in order to minimize the involvement of these adrenoceptors in the response to noradrenaline.

The influence on α_1B-adrenoceptors was evaluated using the isolated mouse spleen. The spleen was removed from male mice right after killing the anesthetized animal by cervical dislocation. The isolated tissue was incubated in 20 ml cup filled with the Krebs-Henseleit solution at the temperature 37°C and pH 7.4 with constant oxygenation (O₂/CO₂, 19:1). The initial optimal tissues tension was set at 1.0 g. Through the 100 min of equilibration tissues were stimulated with noradrenaline (0.1−10.0 μM) three times with washing out until the contractile response become constant. Two cumulative concentration-response curves to noradrenaline, at an interval of 60 min, were established on each tissue both in the presence and absence of antagonist. The incubation with antagonists went on for 30 min. The experiments were carried out in the constant presence of propranolol (1 μM) to block β-adrenoceptors, and minimize the involvement of these adrenoceptors in the response to noradrenaline.

Finally, the influence on α_1D-adrenoreceptors was investigated using the isolated rat aorta. Rats anesthetized with thiopental and killed by cervical dislocation had aorta removed, denuded of endothelium and incubated in the Krebs-Henseleit solution at the temperature 37°C and pH 7.4 with constant oxygenation (O₂/CO₂, 19:1). The aorta rings were maintained at optimal tension of 2.0 g. During 3 h of equilibration tissues were stimulated with noradrenaline three times (0.3 μM). Two cumulative concentration-response curves to noradrenaline, at an interval of 60 min, were established on each tissue both in the presence and absence of antagonist. The incubation with antagonists went on for 30 min. The experiments were carried out in the constant presence of yohimbine (0.1 μM) and propranolol (1 μM) to block α₂- and β-adrenoceptors, respectively.

The procedures were performed according to the method described by Szekeres and Papp (1975). The heart rate disturbances were evoked by the intravenous administration of adrenaline (20 μg/kg), barium chloride (32 and 10 mg/kg) or calcium chloride (140 and 25 mg/kg) solution into the caudal vein in anesthetized rats (thiopental, 75 mg/kg). The studied compounds were administered i.p. 45 min before the injection of adrenaline, calcium chloride, or barium chloride. The electrocardiogram (ECG) was recorded during the first 2 min as well as in the 5th, 10th, and 15th min after the arrhythmogen injection. The criterion of antiarrhythmic activity was the lack of extrasystoles and inhibition of cardiac arrhythmia in comparison with the control group in adrenaline-induced arrhythmia or the progressive disappearance of the arrhythmia and reinstatement of the sinus rhythm in barium chloride- and calcium chloride-induced arrhythmias. The ED₅₀ (a dose producing a 50% inhibition of ventricular contractions) with 95% confidence limits was determined by computer log-probit analysis according to Litchfield and Wilcoxon (1949) and Szekeres and Papp (1975). The compounds were administered at the dose 10 mg/kg. We gradually decreased the dose by half until the disappearance of antiarrhythmic activity.

The experiment was performed according to the method described by Szekeres and Papp (1968). The arrhythmia was evoked by the intravenous administration of adrenaline (20 μg/kg) into the caudal vein in anesthetized rats (thiopental, 75 mg/kg). The tested compounds were injected i.v. at the peak of arrhythmia directly after adrenaline injection, at the ED₈₄ (a dose producing a 84% inhibition of premature ventricular contractions established in prophylactic adrenaline-induced arrhythmia). The range of doses was 0.325−0.504 mg/kg. The ECG was recorded constantly for 5 min as well as in the 10th and 15th min of the experiment. The criterion of antiarrhythmic activity was the reduction of premature ventricular contractions in comparison with the control group (Sapa et al., 2011).

The experiment was carried out to exclude the influence of tested compounds on normal ECG. The ECG was recorded (ASPEL ASCARD B5 apparatus, standard lead II and paper speed of 50 mm/s) prior and also 5, 10, 15, 20, 30, 40, 50, and 60 min after the i.p. administration of tested compounds. The influence on QRS complex, PQ interval, heart rate (RR), and QTc interval was determined. The QTc was calculated according to the Bazzett's formula: QTc = QT/√RR (De Clerck et al., 2002). The compounds were administered at the ED₈₄ (a dose producing a 84% inhibition of premature ventricular contractions established in prophylactic adrenaline-induced arrhythmia).

Normotensive rats were anesthetized with thiopental (75 mg/kg ip). The right carotid artery was cannulated with a polyethylene tube filled with heparin solution to allow pressure measurements, using a Datamax apparatus (Columbus Instruments, USA; Kubacka et al., 2013b). The tested compounds were administered i.p. after 15 min of stabilization period. The compounds were administered at the dose 10 mg/kg. We gradually decreased the dose by half until the disappearance of antiarrhythmic activity.

To verify if the hypotensive activity was a result of α-adrenolytic properties, we studied the influence of tested compounds on the pressor response to methoxamine (150 μg/kg) The experiment was carried out for all active compounds, which were administered (i.v.) to the caudal vein at the lowest hypotensive dose (Kubacka et al., 2013b). Pressor response to methoxamine injected i.v. was measured before (control) and 5 min after the tested compounds. We administered the tested compounds at the lowest possible doses, not to lose selectivity for α₁-adrenoceptors.

This experiment was performed according to the method described by Yue et al. (1992). The rat brain homogenate containing 10 mg tissue/ml was prepared in 0.9% saline. The rates of membrane lipid peroxidation were measured by the formation of thiobarbituric acid reactive substances (TBARS). Rat brain homogenates (1 ml) were incubated at 37°C for 5 min with 10 μl of a tested compound or vehicle. Lipid peroxidation was initiated by the addition of 50 μl of 0.5 mM FeCl₂ and 50 μl of 2.0 mM ascorbic acid. After 30 min of incubation, the reaction was stopped by adding 0.1 ml of 0.2% butylated hydroxytoluene (BHT). Thiobarbituric acid reagent was then added and the mixture was heated for 15 min in a boiling water bath. Carvedilol was used as reference compound. The compounds were tested at a concentration of 10⁻³ M. The TBARs were measured at 532 nm.

In radioligand binding studies, the obtained data were fitted to a one-site curve-fitting equation with Prism 6.0 (GraphPad Software), and inhibition constants (K_i) values were estimated from the Cheng−Prusoff equation (Cheng and Prusoff, 1973): Ki=IC501+LOKD

L_O–labeled ligand concentration, K_D–dissociation constant of labeled ligand

In functional bioassays the concentration–response curves were analyzed using GraphPad Prism 6.0 software (GraphPad Software Inc., San Diego, CA, USA) as previously described by Kubacka et al. (2013b). Data are means ± S.E.M. of at least 4 separate experiments. To establish Hill slopes for the agonist concentration–response curves and calculate EC₅₀ values, curves were fitted to all the data by non-linear regression. The EC₅₀ value in the presence and absence of antagonists was used to ascertain the concentration ratio (CR). Schild analysis was performed. If the slope was not significantly different from unity, the relative antagonistic potencies (pA₂: −log of the concentration of an antagonist that doubles the concentration of the agonist needed to elicit the original submaximal response obtained in the absence of antagonist) were determined by plotting the log (CR-1) against the −log of antagonist concentration (Arunlakshana and Schild, 1959).

In case of in vivo experiments the results are presented as the means ± S.E.M. Statistically significant differences between groups were calculated using one-way analysis of variance (ANOVA) with repeated measurements followed by Dunnett's or Bonferroni's test post-hoc or Student's t-test. The criterion for significance was set at p < 0.05.

Antioxidant activity was expressed as the percentage reduction of the sample absorbance during the reaction at wavelength 532 nm.

The log-probit method described by Litchfield and Wilcoxon (1949) was used to determine median effective doses (ED₅₀—doses producing 50% inhibition of premature ventricular contractions) and doses producing 84% of the maximal effect (ED₈₄) for compounds in arrhythmia models.

All studied compounds possessed high affinity for α₁-adrenoceptors but none of them bound to β₁-adrenoceptors (Table 1).

The tested compounds antagonized noradrenaline evoked contraction in isolated rat aorta and tail artery, as well as mouse spleen, and concentration-dependently, shifted the noradrenaline response to the right, without affecting the maximum response. The obtained pA₂ values with Schild slopes not significantly different from unity indicated a competitive antagonism at α_1A-, α_1B-, and α_1D-adrenoceptors (Tables 2A,B).

HBK-14, HBK-15, HBK-16, HBK-17, and HBK-18 showed stronger antagonistic properties at α_1D- than α_1A- or α_1B-adrenoceptors. HBK-19 antagonized α_1A-adrenoceptors stronger than two other subtypes. All compounds antagonized α_1A-adrenoceptors stronger than α_1B-subtype. Among the studied compounds the strongest antagonist of α_1A-adrenoceptor was HBK-19, α_1B-adrenoceptor—HBK-18, and α_1D-adrenoceptor—HBK-16.

Table 3 shows the influence of tested compounds on normal ECG in rats.

HBK-14 and HBK-15 administered at the dose 6.154 and 20.218 mg/kg, respectively did not influence the ECG parameters throughout the experiment. HBK-16 at the dose 0.363 mg/kg did not influence PQ interval, QRS complex or QTc interval but it significantly reduced heart rate by 11−13%, since the 40th min of the observation. Similarly, HBK-17 at the dose 0.504 mg/kg significantly decreased heart rate by 9−12%, 20 min after administration, without affecting PQ interval, QRS complex or QTc interval. HBK-18 at the dose 0.325 mg/kg significantly reduced the heart rate by 13−23%, since the 15th min of the observation and did not affect PQ interval, QRS complex or QTc interval. HBK-19 at the dose 0.444 mg/kg between the 15th and 30th min after administration reduced heart rate by 21−22% without the influence on PQ interval, QRS complex, or QTc interval.

In anesthetized rats i.v. injection of adrenaline (20 μg/kg) caused atrioventricular disturbances, ventricular and supraventricular extrasystoles in 100% of the animals, which led to the death of ~70% of the animals. The studied compounds administered 45 min (i.p.) prior to adrenaline, decreased the number of extrasystoles and mortality (Figures 2, 3, Table 4). Table 5 shows the ED₅₀ values (doses producing 50% inhibition of premature ventricular contractions).

The injection (i.v.) of barium chloride (32 mg/kg) caused rapid ventricular extrasystoles in all animals (100%), which led to the death within 3−5 min. Lower dose of barium chloride (10 mg/kg) caused ventricular extrasystoles in around 60% of rats and did not lead to the death of animals. We did not observe a reproducible negative effect on heart rhythm when barium chloride was used at the dose 10 mg/kg. None of the tested compounds administered (i.p.) 45 min before barium chloride were active in barium chloride-induced model of arrhythmia (data not shown).

In anesthetized rats injection (i.v.) of calcium chloride (140 mg/kg) caused rapid ventricular fibrillation in all animals (100%), which led to death within 3−5 min. The intravenous administration of calcium chloride (25 mg/kg) caused ventricular fibrillation in all animals, but did not lead to the death of rats. None of the tested compounds administered (i.p.) 45 min prior to calcium chloride were active in the above model of arrhythmia (data not shown).

All tested compounds administered i.v. at the peak of adrenaline-induced arrhythmia (20 μg/kg) reduced the number of premature ventricular contractions (Figure 4).

Table 6 presents the lowest dose of each tested compound, which significantly lowered systolic and diastolic blood pressure in rats.

HBK-14 at the dose 0.625 mg/kg, 10 min after injection, significantly reduced systolic blood pressure by 5−10%, and diastolic blood pressure by 9−14% since the 20th min of the observation. HBK-15 at the dose 5.0 mg/kg significantly reduced systolic blood pressure by 13−19%, and diastolic blood pressure by 13−20%, 5 min after administration. HBK-16 at the dose 0.1 mg/kg, 20 min after administration, significantly reduced systolic blood pressure by 8−11% and diastolic blood pressure by 6−10%. HBK-17 at the dose 0.1 mg/kg significantly reduced systolic blood pressure by 8−11% and diastolic blood pressure by 10−16%, 20 min after administration. HBK-18 at the dose 0.01 mg/kg, from the 20th min of the observation, significantly reduced systolic and diastolic blood pressure by 23−28 and 16−18%, respectively. HBK-19 at the dose 0.625 mg/kg since the 5th min after i.p. administration, significantly reduced systolic blood pressure by 5−17%, whereas diastolic blood pressure by 9−15% since the 10th min of the observation.

In the control group the increase of blood pressure after methoxamine (150 μg/kg) was ranging from 62.7 ± 10.4 to 94.2 ± 2.7 mmHg. Figure 5 shows that all tested compounds at the lowest hypotensive doses, significantly antagonized the pressor response to methoxamine.

Carvedilol, HBK-16, HBK-17, HBK-18, and HBK-19 inhibited lipid peroxidation (Table 7). HBK-14 and HBK-15 were inactive in this test.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.