Novel Arylpiperazine Derivatives of Salicylamide with α1-Adrenolytic Properties Showed Antiarrhythmic and Hypotensive Properties in Rats

Cardiovascular diseases remain one of the leading causes of death worldwide. Unfortunately, the available pharmacotherapeutic options have limited effectiveness. Therefore, developing new drug candidates remains very important. We selected six novel arylpiperazine alkyl derivatives of salicylamide to investigate their cardiovascular effects. Having in mind the beneficial role of α1-adrenergic receptors in restoring sinus rhythm and regulating blood pressure, first, using radioligand binding assays, we evaluated the affinity of the tested compounds for α-adrenergic receptors. Our experiments revealed their high to moderate affinity for α1- but not α2-adrenoceptors. Next, we aimed to determine the antiarrhythmic potential of novel derivatives in rat models of arrhythmia induced by adrenaline, calcium chloride, or aconitine. All compounds showed potent prophylactic antiarrhythmic activity in the adrenaline-induced arrhythmia model and no effects in calcium chloride- or aconitine-induced arrhythmias. Moreover, the tested compounds demonstrated therapeutic antiarrhythmic activity, restoring a normal sinus rhythm immediately after the administration of the arrhythmogen adrenaline. Notably, none of the tested derivatives affected the normal electrocardiogram (ECG) parameters in rodents, which excludes their proarrhythmic potential. Finally, all tested compounds decreased blood pressure in normotensive rats and reversed the pressor response to methoxamine, suggesting that their hypotensive mechanism of action is connected with the blockade of α1-adrenoceptors. Our results confirm the antiarrhythmic and hypotensive activities of novel arylpiperazine derivatives and encourage their further investigation as model structures for potential drugs.


Introduction
Various factors in our daily lives, including an unhealthy diet, stress, obesity, tobacco, or even a history of infections, may negatively affect our heart and blood vessel function, leading to the development of cardiovascular diseases. Globally, sudden cardiac death due to heart attack and stroke continues to be one of the most common causes of 2. Results 2.1. The Tested Compounds Showed High to Moderate Affinity for α 1

-Adrenergic Receptors
We investigated the affinity of arylpiperazine derivatives for α-adrenergic receptors. The radioligand binding assays revealed that all tested compounds possessed high to moderate affinity for α 1 -adrenergic receptors but no affinity for α 2 -adrenergic receptors ( Table 1). Table 1. The affinity of the tested compounds for α 1 -and α 2 -adrenergic receptors.

Treatment
Adrenergic Receptors-pK i α Data are represented as pK i , that is −logK i, and expressed as means ± SEM from three independent experiments performed in duplicates. Inhibition constants (K i ) were calculated according to the equation of Cheng and Prusoff [49]. Radioligand binding was performed using rat cortex tissue. The affinity values were determined using a [ 3 H]-prazosin or b [ 3 H]-clonidine. n.a. (no affinity)-compound did not bind to the receptor at the concentration 10 −5 M.

The Tested Compounds Showed Prophylactic Antiarrhythmic Activity in the Arrhythmia Model Induced by Adrenaline, but Not by Calcium Chloride or Aconitine in Rats
In the next step of our experiments, we investigated the prophylactic antiarrhythmic properties of the studied compounds in different animal models of arrhythmia. As all tested derivatives possessed high affinity for α 1 -adrenergic receptors, we used adrenaline (20 µg/kg) as an arrhythmogen to induce heart rhythm irregularities in rats. Considering the mechanisms of action of classic antiarrhythmic drugs, we also used calcium chloride (140 mg/kg), acting via cardiac calcium channels, and aconitine (20 µg/kg) acting via sodium channels, to evoke arrhythmia ( Table 2).
All the tested compounds, administered at a range of doses 0.02-1 mg/kg before adrenaline, reduced the number of heart rhythm disturbances, such as extrasystoles, conduction blocks, and bradycardia, protecting animals from death. ED 50 values for each compound are presented in Table 3.
None of the studied compounds was active in calcium chloride-and aconitine-induced models of arrhythmia and did not reduce the number of heart rhythm irregularities in rats.

The Tested Compounds Showed Therapeutic Antiarrhythmic Activity in the Adrenaline-Induced Arrhythmia Model in Rats
The prophylactic antiarrhythmic activity of the studied derivatives in adrenalineinduced models of arrhythmia set the ground for the investigation of their potential therapeutic antiarrhythmic activity in the same arrhythmia model.
All the tested compounds (except for JJGW12) significantly reduced the number of adrenaline-induced extrasystoles by 58-86% compared to the control group (F(6,30) = 10.61, p < 0.0001) (Figure 1). Table 2. The effect of arylpiperazine derivatives on the cardiac events occurrence in adrenaline-, calcium chloride-, and aconitine-induced arrhythmia models in rats. The tested compounds were administered intravenously (iv) 15 min before the experiment. The control group received no treatment except the administration of the arrhythmogen. The electrocardiogram (ECG) recordings were performed for 15 min after the iv injection of adrenaline (20 µg/kg), calcium chloride (140 mg/kg), or aconitine (20 µg/kg), i.e., during the first 2 min, at the 5th, 10th, and 15th min. The criterion of antiarrhythmic activity was the decrease in or complete absence of heart rhythm disturbances in the ECG recording compared with the control group. Results are presented as the number of animals in which specific cardiac events (fibrillations, extrasystoles, bradycardia, blocks, or mortality) occurred; n = 5-6 rats. The tested compounds were administered intravenously (iv) 15 min before the experiment. The control group received no treatment except the administration of arrhythmogen. The electrocardiogram (ECG) recordings were performed for 15 min after the iv injection of adrenaline (20 µg/kg) during the first 2 min, at the 5th, 10th, and 15th min. The ED 50 values with confidence limits were calculated according to the methods described by Litchfield and Wilcoxon [50]. Each value was obtained from three experimental groups; n = 5-6 rats.
The prophylactic antiarrhythmic activity of the studied derivatives in adrenaline-induced models of arrhythmia set the ground for the investigation of their potential therapeutic antiarrhythmic activity in the same arrhythmia model.
All the tested compounds (except for JJGW12) significantly reduced the number of adrenaline-induced extrasystoles by 58-86% compared to the control group (F(6,30) = 10.61, p < 0.0001) (Figure 1). Figure 1. The therapeutic antiarrhythmic activity of the studied compounds in the adrenaline-induced arrhythmia model in rats. After an intravenous (iv) injection of adrenaline (20 μg/kg), the tested compounds were immediately administered iv. The control group received no additional treatment. The electrocardiogram (ECG) was recorded for the first 2 min and then at the 5th, 10th, and 15th min of the experiment. The criterion of antiarrhythmic activity was the decrease in or complete absence of extrasystoles in the ECG recording compared with the control group. The results are presented as means ± SD for active or maximum tested doses (1 mg/kg for JJGW01, JJGW07, and JJGW11, and 5 mg/kg for JJGW02, JJGW03, and JJGW12). Statistical analysis: one-way ANOVA (Dunnett's post hoc); * p < 0.05, ** p < 0.01, *** p < 0.001; n = 5-6 rats.
All the tested compounds (except for JJGW12) also reduced the occurrence of bradycardia and atrioventricular blocks in adrenaline-treated rats (Table 4). The tested compounds were administered intravenously (iv) immediately after the injection of adrenaline (20 μg/kg). The control group received no treatment except the administration of arrhythmogen. The electrocardiogram (ECG) recordings were performed for 15 min. Results are Figure 1. The therapeutic antiarrhythmic activity of the studied compounds in the adrenaline-induced arrhythmia model in rats. After an intravenous (iv) injection of adrenaline (20 µg/kg), the tested compounds were immediately administered iv. The control group received no additional treatment. The electrocardiogram (ECG) was recorded for the first 2 min and then at the 5th, 10th, and 15th min of the experiment. The criterion of antiarrhythmic activity was the decrease in or complete absence of extrasystoles in the ECG recording compared with the control group. The results are presented as means ± SD for active or maximum tested doses (1 mg/kg for JJGW01, JJGW07, and JJGW11, and 5 mg/kg for JJGW02, JJGW03, and JJGW12). Statistical analysis: one-way ANOVA (Dunnett's post hoc); * p < 0.05, ** p < 0.01, *** p < 0.001; n = 5-6 rats.
All the tested compounds (except for JJGW12) also reduced the occurrence of bradycardia and atrioventricular blocks in adrenaline-treated rats (Table 4). The tested compounds were administered intravenously (iv) immediately after the injection of adrenaline (20 µg/kg). The control group received no treatment except the administration of arrhythmogen. The electrocardiogram (ECG) recordings were performed for 15 min. Results are presented as the number of animals in which specific cardiac events (bradycardia, conduction blocks, mortality) occurred; n = 5-6 rats.
2.4. The Tested Compounds Did Not Influence the ECG Parameters in Rats (Except for JJGW02 and JJGW11, Which Decreased the Heart Rate Significantly) In order to exclude potential proarrhythmic effects of the tested derivatives, we investigated their influence on normal ECG in rats.

The Tested Compounds Decreased Blood Pressure in the Normotensive Rats
Considering the high affinity of the tested compounds towards α 1 -adrenergic receptors and the role of these receptors in blood pressure regulation, we evaluated the influence of the studied derivatives on the blood pressure of the normotensive rats.
The injection of JJGW12 at a dose of 5 mg/kg decreased the systolic blood pressure in the rats in the 50th min since administration by 13% (122. 5 Table 6).

The Tested Compounds Reversed the Pressor Effect of Methoxamine in Rats
In order to establish that the hypotensive activity of the studied compounds was a result of their α 1 -adrenolytic properties, we investigated their pressor response to methoxamine, an agonist of α 1 -receptors.

Discussion
In the course of our research aiming to develop the potential candidates for the antiarrhythmic and hypotensive drugs, we have discovered that the group of novel arylpiperazine alkyl derivatives of salicylamide showed prophylactic and therapeutic antiarrhythmic properties in rodents. Furthermore, the studied compounds protected animals from death and reduced heart rhythm disturbances in the adrenaline-induced arrhythmia model. Importantly, none of the tested derivatives exposed torsadogenic potential, characteristic of many antiarrhythmic drugs, proving their beneficial safety profile. Additionally, due to antagonistic effects toward α1-adrenoceptors, all the compounds decreased systolic and diastolic blood pressure in the normotensive rats. The effect of arylpiperazine derivatives on the blood pressure response to methoxamine in rats. Pressor response to methoxamine (150 µg/kg) was estimated before and 5 min after the intravenous (iv) administration of the studied compounds at the lowest hypotensive doses (JJGW01 1mg/kg, JJGW02 2.5 mg/kg, JJGW03 5 mg/kg, JJGW07 5 mg/kg, JJGW11 2.5 mg/kg, JJGW12 5 mg/kg). The results are presented as means ± SD. Statistical analysis: paired t-test; ** p < 0.01, *** p < 0.001, **** p < 0.0001; n = 6 rats.

Discussion
In the course of our research aiming to develop the potential candidates for the antiarrhythmic and hypotensive drugs, we have discovered that the group of novel arylpiperazine alkyl derivatives of salicylamide showed prophylactic and therapeutic antiarrhythmic properties in rodents. Furthermore, the studied compounds protected animals from death and reduced heart rhythm disturbances in the adrenaline-induced arrhythmia model. Im-portantly, none of the tested derivatives exposed torsadogenic potential, characteristic of many antiarrhythmic drugs, proving their beneficial safety profile. Additionally, due to antagonistic effects toward α 1 -adrenoceptors, all the compounds decreased systolic and diastolic blood pressure in the normotensive rats.
Adrenergic receptors regulate arterial pressure, blood flow, and cardiac function, presenting the limiting stage of the cardiac response to adrenergic stimulation [52]. Dunaway et al. demonstrated that activation of cardiac α-adrenoceptors reduced cardiac output, ejection fraction, and stroke volume, negatively affecting cardiac function [53]. Correspondingly, Heusch G discovered that the blockade of coronary vascular α-adrenergic receptors might benefit coronary blood flow and myocardial function [54]. Therefore, novel drug candidates targeting these receptors are being developed [42,[55][56][57]. Many compounds with arylpiperazine fragments showed a high affinity for α-adrenergic receptors [58][59][60]. Thus, as the first step in our studies, a series of selected compounds were submitted to radioligand binding assays to assess their affinity for α 1 -and α 2 -adrenergic receptors. Our results showed a substantial affinity for α 1 -adrenoceptors (pKi = 7.41-8.40) and no significant interaction with α 2 -adrenergic receptors. Among the tested derivatives, JJGW01 demonstrated the highest affinity for α 1 -adrenergic receptors. Its affinity was even higher than that of phentolamine, the reference compound.
Emotional stress and intense exercise stimulate the sympathetic nervous system, activate cardiac adrenoceptors, and may trigger arrhythmia [61]. A number of studies suggest the role of α 1 -adrenoceptors in antiarrhythmic effects. Knowing that the tested arylpiperazine alkyl derivatives target α 1 -adrenergic receptors, in the following step, we investigated their prophylactic antiarrhythmic activity in the rat adrenaline-induced model of arrhythmia. When adrenaline, used as an arrhythmogen, was bound to the adrenergic receptors, a cascade of cardiac events, such as extrasystoles, conduction blocks, and bradycardia, was triggered. The pretreatment with all the studied compounds reduced the number of post-adrenaline heart rhythm disturbances, indicating their prophylactic antiarrhythmic activity. Significantly, the calculated ED 50 value for the most active compound, i.e., JJGW07, was 9-fold lower than that of carvedilol, a drug with proven antiarrhythmic properties [62]. The obtained results encouraged further studies on the therapeutic antiarrhythmic activity of the tested compounds in the same rat arrhythmia model. In this experiment, the studied derivatives were coadministered with adrenaline simultaneously to evaluate if they could effectively stop heart rhythm disturbances. Our studies revealed that all the compounds (except for JJGW12) reduced the number of post-adrenaline extrasystoles, conduction blocks, bradycardia, and rodent mortality. As previously mentioned, JJGW07 showed the most robust therapeutic antiarrhythmic activity and outperformed other derivatives. Together, these findings indicate that the studied arylpiperazine alkyl (except for JJGW12) could not only prevent but also treat attacks of arrhythmia induced by adrenaline. All the derivatives showed high to moderate affinity for α 1 -adrenergic receptors. Hence, most likely, the observed pharmacological effects are due to interaction with α 1 -adrenergic receptors or other-not-yet tested targets (e.g., ion channels). Our results suggest that arylpiperazine alkyl derivatives of salicylamide could effectively treat arrhythmia caused by catecholamines. However, more studies are necessary.
As the next step, we investigated the prophylactic antiarrhythmic potential of the studied compounds in rodent models of arrhythmia induced by the administration of either calcium chloride or aconitine. Calcium chloride changes intracellular Ca 2+ levels, whereas aconitine modifies Na + concentration, causing arrhythmias manifested by extrasystoles, fibrillations, blocks, bradycardia, and increased animal mortality. None of the tested compounds showed activity in these arrhythmia models, so we can assume that Ca 2+ and Na + channels do not play a significant role in their antiarrhythmic activity.
Increasing evidence suggests that sympathetic nervous system activity is associated with ventricular tachyarrhythmias and sudden death [63]. The potential to induce lifethreatening arrhythmias, i.e., torsade de pointes, is directly linked with the prolongation of the QT interval in the ECG recordings [51,64]. Therefore, next, the proarrhythmic properties of the tested derivatives were investigated by analyzing their effects on the normal ECG in rats. Notably, none of the studied compounds at the highest tested dose, i.e., 5 mg/kg, affected the QT interval and exposed the proarrhythmic potential. Therefore, if arylpiperazine alkyl derivatives showed no torsadogenic potential at the higher doses, we may assume that they do not possess proarrhythmic properties at lower doses. However, JJGW02 and JJGW11 affected the heart rate and showed negative chronotropic effects, generating a risk of developing bradycardia, but at doses 11-fold and 26-fold higher than the calculated ED 50 value in the adrenaline-induced arrhythmia model.
For approximately 50 years, drugs targeting α 1 -adrenergic receptors have been used to regulate blood pressure [65]. α 1 -adrenolytics counteract sympathetic dominance, which is considered a pathogenic factor in hypertension [66]. Therefore, as the next step in our study of novel arylpiperazine alkyl derivatives of salicylamide, we evaluated their effect on systolic and diastolic blood pressure in the normotensive rats. All the tested compounds showed significant hypotensive activity after a single intravenous administration, with JJGW01 acting the strongest. We discovered that JJGW01 decreased blood pressure at a dose 10-fold higher than the median antiarrhythmic dose in the adrenaline-induced arrhythmia. On the other hand, JJGW07, which showed the most potent antiarrhythmic effect, lowered blood pressure at a dose 125-fold higher than the ED 50 value for antiarrhythmic effect. Thus, compounds such as JJGW01 could be a promising therapeutic option for patients suffering from heart rhythm disturbances accompanied by high blood pressure, whereas JJGW07 could be beneficial for patients with arrhythmia alone.
As the last step, we evaluated the effect of the studied compounds on the vasopressor response to methoxamine, an agonist of α 1 -adrenergic receptors [67]. The inhibition of the vasopressor response to the mentioned α 1 -adrenomimetic by the pretreatment with the test derivatives proves that their mechanism of hypotensive action is a result of α 1adrenolytic properties. In our study, all studied compounds diminished the hypertensive effect induced by methoxamine, with JJGW03 acting the strongest. Therefore, we can assume that the hypotensive effect of the tested arylpiperazine alkyl derivatives was due to their α 1 -adrenolytic properties.
Our study has some limitations. First, since β-adrenoceptors play an essential role in cardiac function, in future studies using radioligand binding assays, we need to assess the affinity of the tested compounds for β-adrenergic receptors, to reveal their mechanism of antiarrhythmic and hypotensive activity fully. Even though none of the studied derivatives showed effectiveness in the calcium chloride-and aconitine-induced models of arrhythmia, we cannot entirely eliminate the role of Na + and Ca 2+ in their mechanism of antiarrhythmic action, so it requires further investigation and evaluation of their effects on voltage-gated sodium and calcium channels in radioligand binding studies. Finally, the intrinsic activity toward α 1 -adrenergic receptors needs evaluation using in vitro functional assays.

Animals
All experiments were performed on male Wistar rats, weighing 200-250 g, purchased from an accredited animal facility at the Faculty of Pharmacy, Jagiellonian University Medical College, Krakow, Poland. The animals were housed in groups of 3 rats in plastic cages (42.7 cm × 26.7 cm), in a room with controlled temperature (22 ± 2 • C), appropriate humidity (40-60%), and 12 h light/dark cycle. The standard pellet food and filtered tap water were permanently available. The animals were assigned randomly to either control or treatment groups, and each group consisted of 5-6 Wistar rats. All the injections were administered in 1 mL/kg volume by the trained experimenter blind to the treatments. The animals were used only once in each test and were immediately euthanized after each procedure. The procedures involving animals and their care were conducted according to current European Community and Polish legislation on animal experimentation.

Radioligand Binding Assay
The α 1 -and α 2 -adrenoceptor radioligand binding assays were performed on the rat cerebral cortex using previously described methods [70]. [ 3 H]-prazosin (19.5 Ci/mmol, α 1 -adrenoceptor) and [ 3 H]-clonidine (70.5 Ci/mmol, α 2 -adrenoceptor) were utilized as specific ligands. The brains were homogenized using the ULTRA-TURRAX homogenizer in 10 mL of an ice-cold 50 mM Tris-HCl buffer (pH 7.6). The homogenates were centrifuged at 20,000× g for 20 min (0-4 • C). Subsequently, the cell pellet was resuspended in the Tris-HCl buffer and centrifuged again. Radioligand binding assays were carried out in plates (MultiScreen/Millipore). The final incubation mixture, with a volume of 300 µL, consisted of 240 µL of the tissue suspension, 30 µL of the radioligand solution, and 30 µL of the buffer containing 7-8 concentrations of the studied compounds. In order to measure the unspecific binding, 10 µM phentolamine (for [ 3 H]-prazosin) or 10 µM clonidine (for [ 3 H]-clonidine) were utilized. The incubation was completed by rapid filtration through Whatman GF/C filters 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 performed in duplicates, and the inhibitory constants (Ki) were calculated.

Prophylactic Antiarrhythmic Activity in Adrenaline-, Aconitine-, and Calcium Chloride-Induced Arrhythmia
All the experiments were performed according to the method described by Szekeres and Papp [71]. The animals were anesthetized with thiopental (75 mg/kg ip). Depending on the model, either adrenaline, aconitine, or calcium chloride was used as an arrhythmogen. Adrenaline and aconitine were administered iv at a dose of 20 µg/kg, whereas calcium chloride was administered at a dose of 140 mg/kg to induce heart rhythm disturbances. The tested compounds were injected iv 15 min before the arrhythmogen. Aspel ASCARD apparatus (standard II lead, with the tape speed 50 mm/s and voltage calibration 1 mV = 1 cm) was used for ECG measurements. The ECG was recorded during the first 2 min and in the 5th, 10th, and 15th min after the arrhythmogen injection ( Figure 3A). The lack or decreased amount of extrasystoles, atrioventricular blocks, bradycardia, and fibrillation in the ECG recordings compared to the control group was the criterion of the antiarrhythmic activity. The ED 50 was calculated using the method of Litchfield and Wilcoxon [50]. All the tested antiarrhythmic activity in rat models of arrhythmia, as well as the influence on electrocardiogram (ECG)_ parameters (Panel C). Adrenaline (20 μg/kg), calcium chloride (140 mg/kg), or aconitine (20 μg/kg) were used as arrhythmogens. All the compounds were administered intravenously (iv). The ECG observation was during the first 2 min and in the 5th, 10th, and 15th min.

Therapeutic Antiarrhythmic Activity in Adrenaline-Induced Arrhythmia
The experiments were carried out according to the method described by Szekeres and Papp [71]. The heart rhythm disturbances, such as extrasystoles, conduction blocks, and bradycardia, were induced by iv injection of adrenaline (20 μg/kg) to anesthetized rats (thiopental, 75 mg/kg, ip). The studied compounds were administered iv immediately after the injection of adrenaline, at a dose of 1 mg/kg or 5 mg/kg, depending on the prophylactic activity of the studied compounds and their calculated ED50 values (if ED50 < 0.3, the dose 1 mg/kg was used-JJGW01, JJGW07, and JJGW11; if ED50 > 0.3, the dose 5 mg/kg was used-JJGW02, JJGW03, and JJGW12). The Aspel ASCARD apparatus (standard II lead, with a tape of speed 50 mm/s and voltage calibration of 1mV = 1 cm) was used for the ECG measurements. The ECG was recorded during the first 2 min and in the 5th, 10th, and 15th min after the adrenaline injection ( Figure 3B). The lack or decreased amount of extrasystoles, atrioventricular blocks, and bradycardia in the ECG recordings compared to the control group was the criterion of antiarrhythmic activity [72].

The Effect on a Normal Electrocardiogram in Rats
The procedure was performed to exclude the negative effects of the tested compounds on the normal ECG, according to the method described earlier [62]. The Aspel ASCARD apparatus (standard II lead, with the tape speed of 50 mm/s and voltage calibration of 1 mV = 1cm) was used for the ECG measurements. Firstly, animals were anesthetized with thiopental (75 mg/kg ip). The ECG recordings were created prior and in the 5th, 10th, and 15th min after iv administration of the tested compounds ( Figure 3C). The influence on PQ, QTc interval, QRS complex, and heart rate was evaluated. The Bazzett's formula: QTc = QT/√RR was used to calculate QTc [51]. All the studied compounds were administered at a dose of 5 mg/kg. antiarrhythmic activity in rat models of arrhythmia, as well as the influence on electrocardiogram (ECG)_ parameters (Panel C). Adrenaline (20 µg/kg), calcium chloride (140 mg/kg), or aconitine (20 µg/kg) were used as arrhythmogens. All the compounds were administered intravenously (iv). The ECG observation was during the first 2 min and in the 5th, 10th, and 15th min.

Therapeutic Antiarrhythmic Activity in Adrenaline-Induced Arrhythmia
The experiments were carried out according to the method described by Szekeres and Papp [71]. The heart rhythm disturbances, such as extrasystoles, conduction blocks, and bradycardia, were induced by iv injection of adrenaline (20 µg/kg) to anesthetized rats (thiopental, 75 mg/kg, ip). The studied compounds were administered iv immediately after the injection of adrenaline, at a dose of 1 mg/kg or 5 mg/kg, depending on the prophylactic activity of the studied compounds and their calculated ED 50 values (if ED 50 < 0.3, the dose 1 mg/kg was used-JJGW01, JJGW07, and JJGW11; if ED 50 > 0.3, the dose 5 mg/kg was used-JJGW02, JJGW03, and JJGW12). The Aspel ASCARD apparatus (standard II lead, with a tape of speed 50 mm/s and voltage calibration of 1mV = 1 cm) was used for the ECG measurements. The ECG was recorded during the first 2 min and in the 5th, 10th, and 15th min after the adrenaline injection ( Figure 3B). The lack or decreased amount of extrasystoles, atrioventricular blocks, and bradycardia in the ECG recordings compared to the control group was the criterion of antiarrhythmic activity [72].

The Effect on a Normal Electrocardiogram in Rats
The procedure was performed to exclude the negative effects of the tested compounds on the normal ECG, according to the method described earlier [62]. The Aspel ASCARD apparatus (standard II lead, with the tape speed of 50 mm/s and voltage calibration of 1 mV = 1 cm) was used for the ECG measurements. Firstly, animals were anesthetized with thiopental (75 mg/kg ip). The ECG recordings were created prior and in the 5th, 10th, and 15th min after iv administration of the tested compounds ( Figure 3C). The influence on PQ, QT c interval, QRS complex, and heart rate was evaluated. The Bazzett's formula: QT c = QT/ √ RR was used to calculate QT c [51]. All the studied compounds were administered at a dose of 5 mg/kg.

ECG Waveform Analysis
Automated ECG waveform analysis was evaluated as described earlier [55], using Eleven Maze software version 0.1 from Eleven Products Sp. z o.o. (Krakow, Poland), which uses artificial intelligence neural network-based models (e.g., [73]) and statistical and mixture modeling features of ECG signals [74]. The software, under expert supervision, recognized and classified the heart rhythm irregularities. It also calculated PQ, QRS, QTc, and rate parameters.

The Influence on Blood Pressure in the Normotensive Rats
The experiment was performed according to the method described earlier [75]. Normotensive Wistar rats were anesthetized with thiopental (75 mg/kg ip). The right carotid artery was cannulated with a polyethylene tube filled with heparin solution, using a Datamax apparatus (Columbus Instruments, Columbus, OH, USA) [71]. After 15 min of the stabilization period, the studied compounds were administered iv, and their effect on systolic and diastolic blood pressure was evaluated ( Figure 4A). The tested derivatives were administered at a dose of 5 mg/kg, and their dose was gradually decreased until the hypotensive activity disappeared.

ECG Waveform Analysis
Automated ECG waveform analysis was evaluated as described earlier [55], using Eleven Maze software version 0.1 from Eleven Products Sp. z o.o. (Krakow, Poland), which uses artificial intelligence neural network-based models (e.g., [73]) and statistical and mixture modeling features of ECG signals [74]. The software, under expert supervision, recognized and classified the heart rhythm irregularities. It also calculated PQ, QRS, QTc, and rate parameters.

The Influence on Blood Pressure in the Normotensive Rats
The experiment was performed according to the method described earlier [75]. Normotensive Wistar rats were anesthetized with thiopental (75 mg/kg ip). The right carotid artery was cannulated with a polyethylene tube filled with heparin solution, using a Datamax apparatus (Columbus Instruments, USA) [71]. After 15 min of the stabilization period, the studied compounds were administered iv, and their effect on systolic and diastolic blood pressure was evaluated ( Figure 4A). The tested derivatives were administered at a dose of 5 mg/kg, and their dose was gradually decreased until the hypotensive activity disappeared. . The experimental setup for studying the influence of the tested compounds on blood pressure (Panel A), and the pressor response to methoxamine (Panel B) in normotensive rats. All the compounds were administered intravenously (iv). ME-methoxamine (150 μg/kg), BP-blood pressure.

The Influence on Blood Vasopressor Response in Rats
The experiment was performed to establish the effect of the studied derivatives on the pressor response to methoxamine (150 μg/kg, iv), according to the methods described previously [75]. Normotensive Wistar rats were anesthetized with thiopental (75 mg/kg ip). The right carotid artery was cannulated with a polyethylene tube filled with heparin solution using a Datamax apparatus (Columbus Instruments, Columbus, OH, USA). After a 15-min stabilization period, the pressor response to methoxamine before (control) and 5 min after the administration of the tested compounds was measured ( Figure 4B). The tested compounds were administered at the lowest hypotensive dose.

Statistic Analysis
The results are presented as either means ± SD (or SEM in case of radioligand studies) or as a number of animals in which specific cardiac events (extrasystoles, fibrillations, bradycardias, or mortality) occurred. In our analysis, we used a paired t-test, one-way ANOVA followed by Dunnet's post hoc, and also one-way or two-way repeated measures ANOVA followed by Dunnet's or Bonferroni's post hoc. p < 0.05 was considered . The experimental setup for studying the influence of the tested compounds on blood pressure (Panel A), and the pressor response to methoxamine (Panel B) in normotensive rats. All the compounds were administered intravenously (iv). ME-methoxamine (150 µg/kg), BP-blood pressure.

The Influence on Blood Vasopressor Response in Rats
The experiment was performed to establish the effect of the studied derivatives on the pressor response to methoxamine (150 µg/kg, iv), according to the methods described previously [75]. Normotensive Wistar rats were anesthetized with thiopental (75 mg/kg ip). The right carotid artery was cannulated with a polyethylene tube filled with heparin solution using a Datamax apparatus (Columbus Instruments, Columbus, OH, USA). After a 15-min stabilization period, the pressor response to methoxamine before (control) and 5 min after the administration of the tested compounds was measured ( Figure 4B). The tested compounds were administered at the lowest hypotensive dose.

Statistic Analysis
The results are presented as either means ± SD (or SEM in case of radioligand studies) or as a number of animals in which specific cardiac events (extrasystoles, fibrillations, bradycardias, or mortality) occurred. In our analysis, we used a paired t-test, one-way ANOVA followed by Dunnet's post hoc, and also one-way or two-way repeated measures ANOVA followed by Dunnet's or Bonferroni's post hoc. p < 0.05 was considered significant. All data were statistically evaluated with Prism 9.0 software (GraphPad Software, San Diego, CA, USA).

Conclusions
Our study demonstrated that selected novel arylpiperazine alkyl derivatives could be an attractive therapeutic option for patients with heart rhythm disturbances accompanied by hypertension. However, further studies are necessary to fully determine the mechanisms involved in the cardiovascular effects of the studied compounds, as well as their safety profiles.