Antiarrhythmic, hypotensive and α1-adrenolytic properties of new 2-methoxyphenylpiperazine derivatives of xanthone

European Journal of Pharmacology 735 (2014) 10–16

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Cardiovascular pharmacology

Antiarrhythmic, hypotensive and α1-adrenolytic properties of new 2-methoxyphenylpiperazine derivatives of xanthone Anna Rapacz a,n, Karolina Pytka a, Jacek Sapa b, Monika Kubacka a, Barbara Filipek a, Natalia Szkaradek c, Henryk Marona c a

Department of Pharmacodynamics, Faculty of Pharmacy, Jagiellonian University, Medical College, Medyczna 9, 30-688 Kraków, Poland Department of Pharmacological Screening, Chair of Pharmacodynamics, Jagiellonian University, Medical College, Medyczna 9, 30-688 Kraków, Poland c Department of Bioorganic Chemistry, Chair of Organic Chemistry, Jagiellonian University, Medical College, Medyczna 9, 30-688 Kraków, Poland b

art ic l e i nf o

a b s t r a c t

Article history: Received 29 November 2013 Received in revised form 26 March 2014 Accepted 1 April 2014 Available online 18 April 2014

The main goal of this study was to assess antiarrhythmic and hypotensive activity of new 2-methoxyphenylpiperazine derivatives of xanthone. In order to better understand mechanism of action of studied compounds, their abilities to antagonize the increase in blood pressure elicited by adrenaline, noradrenaline and methoxamine, as well as the antagonistic properties for α1-adrenoceptors on isolated rat aorta were evaluated. Therapeutic antiarrhythmic activity was investigated in an adrenaline-induced model of arrhythmia. Hypotensive activity in normotensive rats was evaluated after oral administration. Influence on blood vasopressor response and α1-adrenoceptors in rat thoracic aorta was evaluated to determine if the observed cardiovascular effects could be related to α1-adrenolytic properties. Tested compounds produced antiarrhythmic and hypotensive activity. The most active compound was MH-99 – (R,S)-4-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazine-1-yl)propoxy)-9H-xanthen-9-one hydrochloride. All studied compounds showed α1-adrenolytic properties in the in vivo and in vitro tests. The results indicate that the new valuable compounds with antiarrhythmic and hypotensive activity might be found in the group of xanthone derivatives. Further pharmacological utility of these compounds should be investigated. & 2014 Elsevier B.V. All rights reserved.

Keywords: Antiarrhythmic Hypotensive α1-adrenoceptor antagonist Xanthone derivatives Piperazine derivatives

1. Introduction Cardiovascular diseases are major contributors to morbidity and mortality in developed countries. In the past decade, arrhythmias including ventricular fibrillation account for nearly one quarter of all cardiovascular-related deaths. Drugs that are currently marketed as antiarrhythmics are not uniformly effective and frequently cause adverse effects, such as bradycardia, tiredness, dizziness or thyroid dysfunction. Nevertheless, the most important of these side effects is the potential to generate new life-threatening arrhythmias (proarrhythmic effect), (Estrada and Darbar, 2008). It has been reported, that the drug-induced arrhythmia can be recognized in up to 5% of patients receiving antiarrhythmic drugs such as amiodarone, flecainide, sotalol and quinidine. In particular, pre-existent cardiac disease, bradyarrhythmias and liver disease have been identified as clinical risk factors for drug-induced arrhythmia (Petropoulou et al., 2014). Another, well-recognized cardiovascular risk factor is hypertension. Despite the vast population of hypertensive drugs, some patients do

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Corresponding author. Tel.: þ 48 12 620 55 37. E-mail address: [email protected] (A. Rapacz).

http://dx.doi.org/10.1016/j.ejphar.2014.04.010 0014-2999/& 2014 Elsevier B.V. All rights reserved.

not reach an adequate blood pressure though they use three different antihypertensive agents. Moreover, available antihypertensive as well as antiarrhythmic drugs frequently cause side effects, such as bradycardia, tiredness, sleep disturbances, changes in mood, dry mouth, blurry vision, diarrhea or impotence (Bardage and Isacson, 2000; Oliveras and de la Sierra, 2014). Therefore new potential antiarrhythmic and hypotensive agents are still urgently required for the treatment of a wide range of cardiovascular diseases. Xanthones are a class of heterocyclic compounds, widely distributed in nature. Nowadays, xanthones and xanthone derivatives are isolated from plants or synthesized chemically. They have been shown to possess profitable effects on several cardiovascular diseases, such as hypertension, ischemic heart disease and atherosclerosis. It has been reported that xanthone derivatives possess antiarrhythmic and hypotensive (Librowski et al., 2004; Marona et al., 2009; Rapacz et al., 2011), vasorelaxant and antiplatelet (Cheng and Kang, 1997; El-Seedi et al., 2010), antithrombotic (Lin et al., 1996) and antioxidant activities (Jiang et al., 2004). In our previous studies, we demonstrated significant prophylactic antiarrhythmic activity in adrenaline-induced arrhythmia as well as high hypotensive activity after i.v. administration of several xanthone derivatives with a 2-methoxyphenylpiperazine moiety (Szkaradek et al., 2013). Three of them, designated as MH-94,

A. Rapacz et al. / European Journal of Pharmacology 735 (2014) 10–16

MH-99 and MH-105, revealed also the highest affinity for α1adrenoceptors in radioligand binding assay (Ki¼4, 18 and 50 nM, respectively). It is well known that sympathetic activation plays an important role in the pathogenesis of cardiac dysfunctions. Therefore, agents that antagonize adrenergic receptors are effective and widely used in the treatment of cardiovascular diseases. Herein, we continue our pharmacological investigations in this group of xanthone derivatives. We evaluated their therapeutic antiarrhythmic activity in adrenaline-induced arrhythmia and hypotensive activity after p.o. administration. Furthermore, in order to verify that the hypotensive activity is associated with their α1-adrenoceptor blocking properties, we evaluated their ability to antagonize the increase in blood pressure elicited by adrenaline, noradrenaline and methoxamine. Moreover, we examined their antagonistic properties for α1-adrenoceptors on isolated rat aorta. Because the studied compounds as well as urapidil (α1-adrenoceptors antagonist) contain the 2-methoxyphenylpiperazine moiety in their structure, we used it as a reference compound.

2. Materials and methods 2.1. Animals All experiments were performed on normotensive male Wistar rats weighing 180–250 g (Source: Animal House, Faculty of Pharmacy, Jagiellonian University Medical College, Kraków, Poland, stocks name KRF: WI(WU)). The animals were kept in plastic cages in a room at constant temperature of 2074 1C, under a 12/12 h light/dark cycle (light on from 7 a.m. to 7 p.m.). They had free access to food (standard laboratory pellets) and water before experiments. The control and study groups consisted of five to eight animals each. The experiments were performed between 8 a.m. and 3 p.m. The animals were killed by cervical dislocation immediately after the experiment. All the procedures were conducted according to the Animal Care and Use Committee guidelines, and approved by the Local Ethics Committee of the Jagiellonian University in Kraków (resolution no. 99/2010 and 116/2011). 2.2. Therapeutic antiarrhythmic activity in adrenaline-induced arrhythmia Therapeutic antiarrhythmic activity was determined according to the method of Szekeres and Papp (1968). The arrhythmia was evoked in rats under anesthesia with thiopental (75 mg/kg, i.p.) by intravenous (i.v.) injection of adrenaline (20 mg/kg, in volume of 1 ml/kg). The tested compounds were administered by i.v. route at the peak of arrhythmia, immediately after administration of adrenaline. The ECG was recorded continuously for 5 min. The criterion of antiarrhythmic activity was the reduction of premature beats in comparison to the control group (Sapa et al., 2011a). The ED50 values were calculated according to the method of Litchfield and Wilcoxon (1949). 2.3. Influence on blood pressure in rats Male Wistar normotensive rats were anesthetized with thiopental (75 mg/kg) by i.p. injection. The right carotid artery was cannulated with polyethylene tube filled with heparin in saline to facilitate pressure measurements using a Datamax apparatus (Columbus Instruments, USA), (Kubacka et al., 2013). The studied compounds were administrated orally using an intragastric probe in a wide range of doses, after a 15 min stabilization period, at a constant volume of 1 ml/kg.

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2.4. Influence on blood vasopressor response in rats The influence of studied compounds, given intravenously at the doses of 0.31 and/or 0.62 mg/kg on the increase in blood pressure elicited by adrenaline (2 mg/kg), noradrenaline (2 mg/kg) and methoxamine (150 mg/kg), was examined according to the previously described method (Kubacka et al., 2013). Adrenaline, noradrenaline and methoxamine were injected into caudal vein before administration of tested compounds (control group) and again 5 min after the studied compounds were given.

2.5. Influence on

α1-adrenoceptors in rat thoracic aorta

Isolated rat aorta was used to examine the effect on α1adrenoceptors according to the method previously described by Marona et al. (2011) with some minor modifications. Thoracic aorta was carefully isolated from anaesthetized rats (thiopental sodium, 75 mg/kg i.p.) and immersed in a Krebs–Henseleit solution (NaCl 119 mM, KCl 4.7 mM, CaCl2 1.9 mM, MgSO4 1.2 mM, KH2PO4 1.2 mM, NaHCO3 25 mM, glucose 11 mM, and EDTA 0.05 mM). Then the aorta was cleaned of surrounding fat and connective tissues. Prior to this, the aorta was cut into ring preparations of about 4 mm in length, and the endothelium was removed by gently rubbing the luminal surface. Subsequently, the arterial preparation was suspended using stainless steel pins in 30 ml chambers filled with a medium at 37 1C and pH 7.4 with constant oxygenation (O2/CO2, 19:1). Muscle tension changes were recorded with an isometric FDT10-A force displacement transducer (BIOPAC Systems, Inc., COMMAT Ltd., Turkey). The aortic rings were equilibrated at optimal tension of 2 g for 2 h in a medium containing yohimbine (0.1 μM) and propranolol (1 μM) in order to block α2- and β-adrenoceptors. During an equilibration period, rat aorta was exposed to 0.1 mM noradrenaline and washed every 30 min (Villalobos-Molina et al., 2002). In the last stimulation with noradrenaline, the aorta was exposed to carbachol (1 mM) to verify the functionality of endothelium. The absence of endothelium was confirmed by the lack of a relaxing response to carbachol (Furchgott and Zawadzki, 1980). Two cumulative concentration– response to phenylephrine (Phe) curves were obtained for each aortic ring in the absence and presence of antagonist by the method of Van Rossum (1963). Each arterial ring was incubated with antagonist for 30 min. In order to avoid fatigue of the arterial preparation, a 60-min recovery period was allowed between phenylephrine curves.

2.6. Data analysis Results are expressed as mean7S.E.M. The significance differences between mean values were calculated using GraphPad Prism 4.0 software (GraphPad Software Inc., San Diego, CA, USA) by one-way analysis of variance (ANOVA) with the post hoc Dunnett's multiple comparison test. The influence on the blood pressure was calculated by repeated measures ANOVA. Differences were considered statistically significant at Po0.05. Concentration–response curves were analyzed by non-linear regression using GraphPad Prism 4.0 software (GraphPad Software Inc., San Diego, CA, USA), as previously described by Kubacka et al. (2013). Data are the mean 7S.E.M. of 4–8 separate experiments. Schild analysis was performed to determine the pA2 values (Arunlakshana and Schild, 1997). The log-probit method described by Litchfield and Wilcoxon (1949) was used to establish median effective doses (ED50) for compounds in the adrenaline-induced model of arrhythmia.

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A. Rapacz et al. / European Journal of Pharmacology 735 (2014) 10–16

Table 1 Schematic structure of the studied 2-methoxyphenylpiperazine derivatives of xanthone. Compound

Structure

MH-94

O

O

N

MH-99

CH3

. HCl

CH3

. HCl

O

O

N

OH

O

N

O MH-105

O

N

O

O H3C O O

. HCl

O

O

N N

H3C

O

2.7. Drugs and compounds Three compounds, 2-methoxyphenylpiperazine derivatives of xanthone: MH-94 (4-(3-(4-(2-methoxyphenyl)piperazine-1-yl) propoxy)-9H-xanthen-9-one hydrochloride), MH-99 ((R,S)-4-(2hydroxy-3-(4-(2-methoxyphenyl)piperazine-1-yl)propoxy)-9Hxanthen-9-one hydrochloride), MH-105 ((R,S)-4-(2-acetoxy-3(4-(2-methoxyphenyl)piperazine-1-yl)propoxy)-9H-xanthen-9-one hydrochloride), (Table 1) were synthesized in the Department of Bioorganic Chemistry, Chair of Organic Chemistry, Jagiellonian University. The synthesis of the investigated compounds was described earlier (Szkaradek et al., 2013). The following drugs were used: adrenaline, noradrenaline, sodium heparin (Polfa, Poland), methoxamine, phenylephrine (Sigma-Aldrich, Germany), and thiopental sodium (Biochemie Gmbh, Austria). Buffer components were obtained from Polskie Odczynniki Chemiczne (Poland). The tested compounds were dissolved in saline and administered intravenously or orally to rats at a constant volume of 1 ml/kg.

3. Results

Fig. 1. Therapeutic antiarrhythmic activity of compounds MH-94, MH-99 and MH105 in adrenaline induced arrhythmia. Statistical analysis of the results was conducted using one-way analysis of variance (ANOVA), followed by Dunnett's post-hoc test: F(3,21) ¼6.17; P o 0.01 (MH-94); F(3,19) ¼ 10.78; Po 0.001 (MH-99); F(3,22) ¼ 6.17, Po 0.01 (MH-105). Significant difference compared to the control group: *P o0.05, **Po 0.01, ***Po 0.001.

3.1. Therapeutic antiarrhythmic activity in adrenaline-induced arrhythmia 3.2. Influence on blood pressure in rats The compounds MH-94, MH-99 and MH-105 administered intravenously at the peak of arrhythmia at the doses of 0.62– 2.5 mg/kg reduced the number of premature ventricular beats. The results were highly statistically significant: F(3,21) ¼6.17; P o0.01 (MH-94); F(3,19) ¼10.78; P o0.001 (MH-99); F(3,22) ¼ 6.17, Po 0.01 (MH-105) (Fig. 1). The ED50 values (a dose producing 50% inhibition of premature ventricular beats) were calculated according to the log-probit method described by Litchfield and Wilcoxon (1949). The ED50 value obtained for compound MH-94 was 0.65 mg/kg, for compound MH-99 0.32 mg/kg, whereas for compound MH-105 it was 0.38 mg/kg.

Prominent hypotensive activity of three tested compounds after intravenous injection in rats was reported previously (Szkaradek et al., 2013). Herein, hypotensive activity after oral administration in rats was determined. Tested compounds significantly decreased systolic and/or diastolic blood pressure in rats (Table 2). Compound MH-94 at the dose of 1.25 mg/kg, 30 min after p.o. administration, significantly reduced the systolic blood pressure by 8–17% and diastolic blood pressure by 10–24% and the effect lasted for the whole 90 min period of observation. At a lower dose (0.62 mg/kg), the compound MH-94 significantly reduced

A. Rapacz et al. / European Journal of Pharmacology 735 (2014) 10–16

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Table 2 The influence of compounds MH-94, MH-99 and MH-105 on the blood pressure in anesthetized normotensive rats after p.o. administration. Comp.

MH-94

Dose (mg/kg)

1.25 0.62 0.31

MH-99

1.25 0.62 0.31

MH-105

5

Blood pressure (mmHg)

systolic diastolic systolic diastolic systolic diastolic systolic diastolic systolic diastolic systolic diastolic systolic diastolic

Time of observation (min) 0

30

50

70

90

125.6 7 2.7 110.4 73.4 130.0 7 2.6 112.2 7 2.8 130.0 7 5.4 104.67 3.8 128.7 7 3.1 111.2 7 3.5 138.4 7 3.6 113.8 74.5 138.8 7 1.6 110.8 72.2 136.0 7 1.8 110.2 72.4

117.8 7 2.5a 104.67 2.2a 122.2 7 3.9a 104.07 4.1a 126.4 7 6.0 102.2 74.5 119.0 7 4.1a 101.5 7 4.9a 123.8 7 3.9b 104.47 5.2b 133.2 7 3.2 109.8 72.1 128.7 7 1.9 103.7 73.6

111.0 7 3.9b 93.4 7 4.2b 119.4 7 3.0a 101.6 7 2.8a 122.4 7 5.6 98.6 7 4.6 114.5 7 6.7b 93.2 7 7.8b 120.0 7 4.2b 101.8 7 4.8b 131.2 7 3.3a 108.6 72.2a 127.5 7 1.3a 101.0 7 3.9a

107.4 73.0c 89.2 7 5.2c 116.8 7 3.7a 99.6 7 4.2a 119.4 7 5.4 95.2 7 4.5 114.5 7 0.9a 94.2 7 1.2a 108.0 74.8c 90.0 7 6.4c 127.4 7 3.3b 104.27 2.6b 121.0 7 1.6c 96.2 7 1.9c

103.8 7 3.3c 84.2 75.3c 113.0 7 3.3b 95.8 73.6b 120.6 7 6.4 96.8 74.4 106.0 75.4b 85.5 75.2b 109.8 7 3.3c 92.4 74.6c 125.2 7 2.1c 101.6 7 3.5c 119.7 7 1.9c 95.0 71.8c

The values are expressed as mean of 5–6 experiments 7 S.E.M. Statistical analysis: repeated measures ANOVA test, post hoc Dunnett test. a b c

P o0.05. Po 0.01. Po 0.001.

systolic blood pressure by 9–13% and diastolic blood pressure by 10–15% that also began 30 min after administration. At the dose of 0.31 mg/kg, compound MH-94 did not present any significant influence on blood pressure. Compound MH-99 at the dose of 1.25 mg/kg, 20 min after administration, significantly reduced the systolic blood pressure by 11–18% and diastolic blood pressure by 15–23%. At a lower dose (0.62 mg/kg), the compound MH-99 10 min after administration significantly reduced systolic blood pressure by 11–21% and diastolic blood pressure by 13–19%. At the dose of 0.31 mg/kg, compound MH-99 was also active and significantly reduced systolic blood pressure by 5–10% and diastolic blood pressure by 6–8%, 40 min after administration. At the dose of 0.16 mg/kg, compound MH-99 did not present any significant influence on blood pressure (data not shown). Compound MH-105 at the dose of 5 mg/kg significantly reduced the systolic blood pressure by 5–12% and diastolic blood pressure by 13–14%, 40 min after p.o. administration. At the dose of 2.5 mg/kg, compound MH105 after p.o. administration had no effect in this test (data not shown). We previously reported that urapidil showed hypotensive activity at doses of 5–2.5 mg/kg and its effect was observed 10–20 min after p.o. administration (Kubacka et al., 2013). In this experiment two compounds: MH-94 and MH-99 revealed stronger hypotensive effect than that of urapidil, whereas compound MH-105 was less active. The results showed that in the group of xanthone derivatives there are compounds with strong hypotensive activity.

Fig. 2. Effect of MH-94 on the blood pressor response to adrenaline, noradrenaline and methoxamine. All values represent the mean 7S.E.M. in 6–8 rats. Statistical analysis of the results was conducted using one-way analysis of variance (ANOVA), followed by Dunnett's post hoc test: **P o0.01, ***Po 0.001.

3.3. Influence on blood vasopressor response in rats Adrenaline (2 mg/kg), noradrenaline (2 mg/kg) and methoxamine (150 mg/kg) were given i.v. to rats to induce vasopressor response. In the control group the increases in systolic blood pressure elicited by adrenaline were from 51.14 75.97 to 57.37 74.10, for noradrenaline from 59.28 72.31 to 69.62 76.22 and for methoxamine were from 71.127 7.81 to 72.60 75.70. Compounds MH-94 and MH-99, given i.v. at the dose 0.62 mg/kg, restrained the increase in blood pressure elicited by adrenaline, noradrenaline and methoxamine (Figs. 2 and 3). These compounds, given i.v. at the dose 0.31 mg/kg and compound MH-105 given i.v. at the dose 0.62 mg/kg restrained the increase in blood pressure elicited by adrenaline and methoxamine but did not influence the effect of noradrenaline (Fig. 4). In our previous papers we reported that urapidil at the dose of 0.62 mg/kg and also at a lower dose

Fig. 3. Effect of MH-99 on the blood pressor response to adrenaline, noradrenaline and methoxamine. All values represent the mean 7S.E.M. in 6–8 rats. Statistical analysis of the results was conducted using one-way analysis of variance (ANOVA), followed by Dunnett's post-hoc test: *Po 0.05, **P o0.01, ***P o0.001.

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of 0.31 mg/kg caused the potent inhibition of the vasopressor response to adrenaline and methoxamine, but it did not influence the effect of noradrenaline (Kubacka et al., 2013; Kulig et al., 2010).

3.4. Influence on

α1-adrenoceptors in rat thoracic aorta

The effect of compounds MH-94, MH-99 and MH-105 on the phenylephrine contraction was measured in rat aorta. The contractile response was inhibited by tested compounds in dosedependent manner without affecting the maximum response (Figs. 5–7). The pA2 values were obtained with Schild regression slope not significantly different from unity, indicating a competitive interaction of compounds with the α1-adrenoceptors present in this tissue. The strongest antagonistic activity revealed compound MH-94 with pA2 value of 8.877 0.06 (s¼1.08 70.03). Both compounds MH-99 and MH-105 revealed similar antagonistic activity. For compound MH-99 the pA2 value was 8.36 70.06 (s ¼1.16 70.02), for compound MH-105 it was 8.39 70.07 (s ¼0.90 70.02). Under the same experimental condition, urapidil revealed weaker antagonistic potency: the pA2 value was 7.33 70.04 (s¼ 0.90 70.02) (Kubacka et al., 2013).

Fig. 6. Effect of compound MH-99 on α1-adrenoceptors. Concentration–response curves to phenylephrine (Phe) in the absence ( ) or presence of increasing concentration of MH-99 (filled symbols). Results are expressed as percentage of the maximal response to Phe in the corresponding concentration–response curve. Each point represents the mean 7 S.E.M. (n ¼4–8).

Fig. 7. Effect of compound MH-105 on α1-adrenoceptors. Concentration–response curves to phenylephrine (Phe) in the absence ( ) or presence of increasing concentration of MH-105 (filled symbols). Results are expressed as percentage of the maximal response to Phe in the corresponding concentration-response curve. Each point represents the mean 7 S.E.M. (n ¼4–8).

Fig. 4. Effect of MH-105 on the blood pressor response to adrenaline, noradrenaline and methoxamine. All values represent the mean 7S.E.M. in 6–8 rats. Statistical analysis of the results was conducted using one-way analysis of variance (ANOVA), followed by Dunnett's post hoc test: *Po 0.05, **P o 0.01, ***P o 0.001.

Fig. 5. Effect of compound MH-94 on α1-adrenoceptors. Concentration–response curves to phenylephrine (Phe) in the absence ( ) or presence of increasing concentration of MH-94 (filled symbols). Results are expressed as percentage of the maximal response to Phe in the corresponding concentration–response curve. Each point represents the mean7 S.E.M. (n¼ 4–8).

4. Discussion In this paper, an extended investigation regarding cardiovascular activity of new 2-methoxyphenylpiperazine derivatives of xanthone is demonstrated. Here, we show that the studied compounds have therapeutic antiarrhythmic activity in adrenaline-induced arrhythmia, hypotensive activity after p.o. administration and α1-adrenolytic activity (in vivo and in vitro tests). In our previous study, synthesis and preliminary pharmacological activity of nine piperazine derivatives of xanthone were described (Szkaradek et al., 2013). We demonstrated that three of them: MH-94, MH-99 and MH-105 had significant prophylactic antiarrhythmic activity in adrenaline-induced arrhythmia and high hypotensive activity after i.v. administration. They did not significantly influence ECG intervals (PR, QRS and QT), as well as the heart rate after i.v. administration. What is particularly important, they did not lengthen the QT interval, which means that they have not shown proarrhythmic potential. The results from our studies demonstrate that α1-adrenoceptor blocking properties of 2-methoxyphenylpiperazine derivatives of xanthone tested in the present work could contribute to their antiarrhythmic and hypotensive activities. These compounds possess high affinity for α1-adrenoceptors in radioligand binding assay (Ki ¼4, 18 and 50 nM, correspondingly), whereas affinity for α2-adrenoceptors is much weaker (Ki¼0.69, 4.3 and 2.1 mM, respectively). They reveal also weak affinity for β1-adrenoceptors

A. Rapacz et al. / European Journal of Pharmacology 735 (2014) 10–16

(Ki¼ 3.2, 7.6 and 11.2 mM, correspondingly). It is well established that α1-adrenoceptors are involved in the control of blood pressure and prostatic function. In the recent years the role of α1-adrenoceptors in various types of arrhythmia was investigated. It was reported that some drugs with α1-adrenoceptor blocking properties, including phentolamine or prazosin, showed potent antiarrhythmic action in ischemia-induced arrhythmias in a variety of animal models (Benfey et al., 1984; Bralet et al., 1985; Gwilt et al., 1992; Tö lg et al., 1997). Tö lg et al. (1997) have demonstrated that prazosin at the concentrations of 10–5 and 10–6 M reduced the incidence of ventricular fibrillation in rat heart. Whereas Bralet et al. (1985) have reported that phentolamine and nicergoline diminished or prevented reperfusion arrhythmias, but prazosin at the concentration of 5.2  10  6 M failed to alter significantly the occurrence of arrhythmias. In our previous studies, prazosin at the concentration of 10  6 M significantly diminished the occurrence of ventricular tachycardia and ventricular fibrillation in the isolated rat heart. At the same concentration another α1-adrenolytic agent—urapidil has been found to be effective in this model of arrhythmia (Sapa et al., 2011b). Urapidil, as well as tolazoline, revealed also potent antiarrhythmic effect in adrenaline-induced arrhythmia after i.v. administration with ED50 values 1.26 and 3.4 mg/kg, respectively (Kulig et al., 2010). It is suggested that α1-adrenoceptors in the heart can be of importance in the genesis of ischemia- and reperfusion-related ventricular arrhythmias (Heusch, 1990). Furthermore, cardiac α1-adrenoceptors can alter myocardial hypertrophy, electrophysiological properties and myocardial inotropy and chronotropy (Tanoue et al., 2003; Woodcock et al., 2008). However, it is worth noting that the effects mediated via α1-adrenoceptors are species dependent. The density of α1-adrenoceptors in rat heart seems to be exceptionally high, five times greater than in the human heart (Steinfath et al., 1992). Hence, these rodent models could not be good predictors of new agents' with α1-adrenolytic activity effectiveness in arrhythmias in humans. On the other hand, recent studies indicate important role of cardiac α1-adrenoceptors in sustaining cardiac contractility in the failing human heart. In physiological state the density of human cardiac α1-adrenoceptors: α1A, α1B and α1D, is only about 10–15% of that of β-adrenoceptors, but pathological settings are involved with decrease of β1-adrenoceptors proportion, whereas the proportion of α1-adrenoceptors is maintained. As a result, the function of α1adrenoceptors under pathological conditions seems to play a crucial role in sustaining cardiac increase (Brodde et al., 2006; Woodcock et al., 2008). In the present study the compounds were tested for their therapeutic antiarrhythmic activity in the adrenaline-induced model of arrhythmia in rats. All tested compounds MH-94, MH-99 and MH-105 showed high activity in this experiment (ED50 values were 0.65, 0.32, 0.38 mg/kg, respectively). It is worth noting, that tested compounds demonstrated prominent antiarrhythmic activity in both prophylactic (given i.v. 15 min before arrhythmogen) and therapeutic (given i.v. at the peak of arrhythmia) models of adrenaline-induced arrhythmia. Agents with that property could have wider usage, not only to prevent, but also to quick inhibition of arrhythmia episode. Compounds MH-94 and MH-99 had the highest hypotensive activity after intravenous (Szkaradek et al., 2013) and oral administration. These compounds were significantly active at lower doses (0.16 mg/kg, i.v. and 0.31–0.62 mg/kg, p.o.) than those of urapidil (0.62 m/kg, i.v. and 2.5 mg/kg, p.o.) (Kubacka et al., 2013), which is a well known α1adrenolytic drug. On the other hand the active dose of compound MH-105 was the same (0.62 mg/kg) after i.v. administration and higher (5 mg/kg) after per os administration than those of this reference compound. To learn more about the mechanism of action displayed by the tested compounds, we examined if their activity is a result of

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their α1-adrenoceptor blocking properties. Therefore we studied their influence on the vasopressor responses to adrenaline, noradrenaline and methoxanine, as well as antagonistic activity for α1-adrenoceptors in isolated rat aorta. It is of interest to note that α1-adrenoceptor antagonists inhibit the increase in blood pressure elicited by adrenaline, diminish the hypertensive effect of methoxamine (specific α1-adrenoceptor agonist) and only partially inhibit or have no influence on the increase of blood pressure elicited by noradrenaline. α2-adrenoceptor antagonists restrain the vasopressor response to noradrenaline and inhibit the increase in blood pressure elicited by adrenaline with no effect to methoxamine (Kubacka et al., 2013; Kulig et al., 2010; Orallo et al., 2003). In this experiment compounds MH-94 and MH-99 given at the dose of 0.31 mg/kg significantly antagonized the action of adrenaline and methoxamine but they did not reverse the hypertension induced by noradrenaline which could indicate their higher selectivity for α1- than for α2-adrenoceptors. Compound MH-105 and urapidil at the dose of 0.62 mg/kg had the same effect. On the other hand, compounds MH-94 and MH-99 given at the dose of 0.62 mg/kg caused a prominent inhibition of the hypertension effect of adrenaline, noradrenaline and methoxamine, which means that at a higher dose they lack the selectivity for α1-adrenoceptors and the hypotensive effect of these compounds is due to the blockade of both vascular α1- and α2-adrenoceptors. The present results of pharmacological experiments for studied compounds and urapidil are in agreement with the previously described radioligand binding assay in which these compounds revealed higher affinity for α1- than α2-adrenoceptors (Marona et al., 2011; Szkaradek et al., 2013). Compound MH-99 was 236 fold more selective for α1- than α2-adrenoceptor, whereas compounds MH-94 and MH-105 were more selective for α1-adrenoceptor 173 and 42 fold, respectively. Our results from functional studies show that MH-94, MH-99 and MH-105 displaced phenylephrine to the right in isolated rat aorta with pA2 values of 8.87, 8.36 and 8.39 respectively, which means that they possess high antagonistic activity for α1-adrenoceptors. Furthermore, since the Schild slopes are not different from unity, the data indicate that the antagonism is competitive. It is worth emphasizing that all studied compounds possessed an 2-methoxyphenylpiperazine moiety which probably plays an important role in the affinity to α1-adrenoceptors. Moreover, compound MH-94 is devoid of hydroxyl group and contains 3aminepropan-1-yloxy linker instead of typical β-blocker: 3-amine2-hydroxypropan-1-yloxy linker, which contains compound MH-99. Also in compound MH-105 the hydroxyl group in 3-amine-2-hydroxypropan-1-yloxy linker is acetylated. Detailed analysis of presented in vivo and in vitro results indicates greater potency of compound MH-99 than those of MH-94 and MH-105 in vivo, which does not thoroughly correlate with the previous binding data and the present phenylephrine-induced aortic ring contractility. A possible explanation of this phenomenon is the difference in the pharmacokinetic/ pharmacodynamics correlations. Hence, estimation of pharmacokinetic variables and the relationship between pharmacodynamics effect and plasma concentration could be useful to improve known properties of tested xanthone derivatives. In conclusion, our in vitro and in vivo pharmacological screenings indicate that examined structures act via α1-adrenoceptors rather than β1/α2-adrenoceptors.

5. Conclusions The results obtained in this study indicate that some xanthone derivatives with a 2-methoxyphenylpiperazine moiety possess antiarrhythmic, hypotensive and α1-adrenolytic activity. The data suggest that the antiarrhythmic and hypotensive effects of studied compounds are related to their α1-adrenolytic properties. Further

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pharmacological studies will be aimed at identifying the other possible mechanism of cardiovascular action of these compounds (i.e. antioxidant and vasorelaxant effects or impact on nitric oxide pathway).

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