NUMER 6.Vp:Corelventura

SYNTHESIS AND CARDIOVASCULAR ACTIVITY OF NEW 8-ALKYLAMINO-1,3-DIMETHYL-7-(2-HYDROXY-3-PIPERAZI- NOPROPYL)-3,7-DIHYDRO-1H-PURINE-2,6-DIONES

Gra¿yna Ch³oñ-Rzepa1, Maciej Paw³owski1,#, Ma³gorzata Zygmunt2, Barbara Filipek2, Dorota Maci¹g2 Department of Pharmaceutical Chemistry, Department of Pharmacodynamics, Laboratory of Pharmacological Screening, Collegium Medicum, Jagiellonian University, Medyczna 9, PL 30-688 Kraków, Poland

Synthesis and cardiovascular activity of new 8-alkylamino-1,3-dimethyl- -7-(2-hydroxy-3-piperazinopropyl)-3,7-dihydro-1H-purine-2,6-diones. G. CH£OÑ- -RZEPA, M. PAW£OWSKI, M. ZYGMUNT, B. FILIPEK, D. MACI¥G. Pol. J. Pharmacol., 2004, 56, 755–766.

7-{2-Hydroxy-3-[4-(2-phenoxyethyl)-piperazinyl-1-yl]-propyl}-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione dihydrochloride (2), and several of its 8-alkylamino substituted derivatives (11–17) were synthesized and tested for electrocardiographic, antiarrhythmic and hypotensive activity. Also their a - and a -adrenoreceptor affinities were determined. It was found that compound 2, and its analogue 15 with 8-(2-morpholin-4-yl-ethylamino) substituent displayed a strong prophylactic antiarrhythmic activity in experimentally induced arrhythmia (LD#/ED# = 54.9 and 55.0, respectively). The hypotensive activity was observed for 8-benzylamino (11) or 8-(pyridin-2-yl- methylamino) (12) analogues. All the new derivatives (11–17) and 2 showed a weak affinity for a -(KE = 0.225–1.400 mM) and a -(KE = 0.152–4.299 mM) receptors.

Key words: purine-2,6-diones, antiarrhythmics, hipotensive agents, a - and a -receptor ligands

correspondence;e-mail: [email protected] G. Ch³oñ-Rzepa, M. Paw³owskI, M. Zygmunt, B. Filipek, D. Maci¹g

INTRODUCTION arrhythmic and hypotensive activity as well as for a1- and a2-adrenoreceptor binding affinities. Theophylline (1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione) and a number of its salts and 7-sub- MATERIAL and METHODS stituted derivatives exhibit multidirectional pharmacological properties. Their activities such as CNS stimulation, peripherial smooth muscle re- Chemical part laxation (broncho- and vasodilatation) heart rate in- crease result from the antagonism at adenosine re- Melting points (m.p.) were determined with ceptors and non-selective inhibition of the cyclic a Büchi SMP-20 apparatus and are uncorrected. nucleotide phosphodiesterases [23]. These com- UV spectra were recorded on a Perkin Elmer Lambda 12 UV-VIS spectrometer in5×105 mol/l pounds are commonly used in the treatment of sev- 1 eral diseases such as asthma (theophylline, amino- methanolic solutions. H-NMR spectra (Mercury Varian BB 300 MHz NMR spectrometer) were de- phylline, etophylline, proxyphylline, diprophyl- termined in CDCl solution, with TMS as an inter- line), cardiac failure, and peripheral circulation 3 nal standard. Chemical shifts (d) are reported in disorders (xanthinol nicotinate). It was also found ppm, coupling constants (J) are given in Hz. Mass that pyrimidine-8-on[2,1-f] theophylline-9-alkylcarb- spectra (MS) were taken with a AMD-604 mass oxylic acids derivatives possess hypnotic, sedative spectrometer (EI mode, 70 eV). Thin-layer chro- and tranquillizing properties [4]. matography (TLC) was performed on Merck pre- In the course of our search for new biologically coated silica gel 60 F aluminium plates, using: active compounds belonging to the group of 7,8- 254 S : benzene/acetone/methanol (1:1:1, v/v/v); S : disubstituted theophylline derivatives, we have 1 2 methanol/25% NH3 (100:5, v/v) as mobile phases. found that 7-(2-hydroxypropyl)-8-benzylaminotheo- The spots were visualized under UV light (l = 254 phyllines exhibited hypotensive effects and re- nm) or by iodine solution (0.05 M in 10% HCl). duced heart rate, as well as stimulated respiratory Elemental analyses (C, H, N) were carried out with activity [12]. Pharmacological investigation of Elementar Vario EL III apparatus and were within 7-(2-hydroxy-3-alkylamino)-propyl derivatives of ± 0.4% of the theoretical values. 8-benzylaminotheophylline and 7-(2-hydroxy-3-al- The 1,3-dimethyl-7-oxiranylmethyl-3,7-dihy- kylamino)-propyl derivatives of 8-(2-furfurylami- dro-1H-purine-2,6-dione (1) [24] and 8-bromo- no)-theophylline, revealed also hypotensive and 7-(3-chloro-2-hydroxypropyl)-1,3-dimethyl-3,7-di- bronchodilatatory effects [5, 14, 15]. It is known hydro-1H-purine-2,6-dione (3) [3] were used as that aryl and arylalkyl substituted piperazines show starting materials for the synthesis of new com- a -adrenolytic, hypotensive and vasodilatatory ac- pounds 2 and 11–17. tivity [6, 16, 18, 20]. The described pharmacologi- Compound 2 was synthesized by aminolysis cal effects prompted us to synthesize new theophyl- of 1 with 1-(2-phenoxyethyl)-piperazine [13]. The line derivatives with 1-(2-phenoxyethyl)-piper- 8-alkylamino derivatives of 7-{2-hydroxy-3-[4-(2- azine moiety attached to theophylline nucleus as phenoxyethyl)-piperazin-1-yl]-propyl}-3,7-dihyd- potential agents acting on the cardiovascular sys- ro-1H-purine-2,6-dione (11–16) were obtained by tem. two step procedure (Scheme 1). Compound 3 in the In this paper, we report the synthesis as well as reaction with the appropriate aryl-(heteroaryl-, the results of pharmacological evaluation (in vivo morpholino- or diethylamino)-alkylamine gave in- and in vitro tests) of 7-{2-hydroxy-3-[4-(2-phen- termediate products, previously described 4 and 7 oxyethyl)-piperazin-1-yl]-propyl}-1,3-dimethyl-3,7- [14], and the new ones 5, 6, 8 and 9. In the second dihydro-1H-purine-2,6-dione dihydrochloride (2) step, compounds 4–9 heated with 1-(2-phe- and its 8-alkylamino analogues (11–16). In order to noxyethyl)-piperazine in anhydrous ethanol (for establish the role of the arylalkyl substituent at compounds 11, 12, 14, 16, 17) or toluene (for com- 8 position, the unsubstituted 8-amino analogue (17) pounds 13, 15) in the presence of anhydrous was also obtained. Compound 2 and the newly syn- K2CO3 yielded final products 11–16 (Scheme 1). thesized 11–17 in a form of water soluble hydro- The reaction of 3 with 25% NH3 in dioxane yielded chlorides were tested for electrocardiographic, anti- the intermediate product 10, which treated with

756 Pol. J. Pharmacol., 2004, 56, 755–766 CARDIOVASCULAR ACTIVITY OF NEW 8-ALKYLAMINO-PURINE-2,6-DIONES

Cl O OH H C N 3 N Br O N N 3 CH3

H2N-(CH2)n-R 25% NH3 methanol dioxane

Cl Cl O O OH OH H C N H C N 3 N 3 N NH NH (CH2)n R 2 O N N O N N 4-9 10 CH CH3 3

O O HN N HN N

K CO , ethanol K2CO3, ethanol or toluene 2 3

O O N N N N O O H C OH H C 3 N 3 N OH N 17 N 11-16 NH NH (CH )n R 2 2 N O N N O N CH CH 3 3

No 4, 11 5, 12 6, 13 7, 14 8, 15 9, 16

C H R N O N 2 5 O C H N N 2 5 n1 1 2 1 2 2 Scheme 1. Synthesis of compounds 4–17

1-(2-phenoxyethyl)-piperazine yielded the final General procedure for preparation of the 8-al- 8-aminoderivative 17 (Scheme 1). kylamino derivatives of 7-(3-chloro-2-hydroxy Compounds 11–17 were isolated and examined propyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6- 1 as free bases for their H-NMR, MS and UV spec- dione (5-6, 8 and 9) tra as well as by elemental analyses. For pharmacological studies the free bases 11–17 were con- verted into water soluble hydrochloride salts. The A solution of 3 (3.52 g, 0.01 mol) and appropri- salts were recrystallized and their composition was ate amine (0.02 mol) in methanol (40 ml) was established on the basis of an elemental analysis. heated under reflux for 5 h. Then the reaction mix- Yields and physicochemical properties of the ob- ture was cooled down. The precipitated solid prod- tained compounds (free bases and their salts) are uct was filtered off and purified by recrystallization presented in Table 1. from suitable solvent (Tab. 1).

ISSN 1230-6002 757 G. Ch³oñ-Rzepa, M. Paw³owskI, M. Zygmunt, B. Filipek, D. Maci¹g

Table 1. Physicochemical data of new compounds 5, 6 and 8–17

Comp. M.p. (°C) Yield (%) (base) Molecular formula (molecular RB (solvent system) Cryst. solvent weight)* Base Salt

5 171–172 52 C16H19N6O3Cl 0.88 (S1) methanol (378.8)

6 204–206 87 C17H21N6O3Cl 0.89 (S1) 2-methoxyethanol (392.8)

8 153–155 69 C16H25N6O4Cl 0.82 (S1) methanol (400.9)

9 147–149 71 C16H27N6O3Cl 0.43 (S2) methanol (386.9)

10 232–233 51 C10H14N5O3Cl 0.43 (S1) ethanol (287.7)

11 95–97 242–244 48 C29H37N7O4 ×2HCl×H2O 0.82 (S1) ethanol (638.6)

12 165–166 249–251 85 C28H36N8O4 × 3 HCl 0.58 (S1) ethanol (658.0)

13 140–142 180–185 75 C29H38N8O4 × 3 HCl 0.55 (S1) methanol (672.0)

14 118–120 231–233 55 C27H35N7O5 ×2HCl×H2O 0.80 (S1) methanol (628.5)

15 134–136 215–217 54 C28H42N8O5 × 3 HCl 0.15 (S1) acetone (680.0)

16 105–107 215–218 56 C28H44N8O4 ×3HCl×H2O 0.23 (S2) acetone (666.1)

17 154–156 262–264 56 C22H31N7O4 × 2 HCl × 1/2 H2O 0.33 (S1) ethanol (539.4)

* calculated from elemental analysis

7-(3-Chloro-2-hydroxy-propyl)-1,3-dimethyl-8- CH3), 3.44 (s, 3H, N3-CH3), 3.51–3.70 (m, 2H, [(pyridin-2-yl-methyl)-amino]-3,7-dihydro-1H- NHCH2CH2), 3.77–3.90 (m, 2H, CH2Cl), 4.09–4.39 purine-2,6-dione (5) (m, 3H, N7-CH2,CHOH), 6.17 (t, 1H, NH), 7.14–7.18 (m, 1H, 5’pyridine), 7.23–7.25 (d, 1H, l e 1 UV: max (log ): 294 nm (4.26). H NMR: 3.34 3’pyridine), 7.60–7.69 (m, 1H, 4’pyridine), (s, 3H, N1-CH3), 3.49 (s, 3H, N3-CH3), 3.57–3.77 8.41–8.43 (d, 1H, 6’pyridine). (m, 2H, CH2Cl), 4.22–4.46 (m, 3H, N7-CH2, Analysis for C17H21N6O3Cl (392.8): C, H, N. CHOH), 4.73–4.76 (m, 2H, NHCH2) 6.48 (t, J = 5.2, 1H, NH), 7.18–7.25 (m, 1H, 5’pyridine), 7-(3-Chloro-2-hydroxy-propyl)-1,3-dimethyl-8-(2- 7.31–7.33 (d, 1H, 3’pyridine), 7.65–7.71 (t, 1H, morpholin-4-yl-ethylamino)-3,7-dihydro-1H-pu- 4’pyridine), 8.49–8.51 (d, 1H, 6’pyridine). rine-2,6-dione (8) Analysis for C16H19N6O3Cl (378.8): C, H, N. 1 UV: lmax (log e): 295 nm (4.29). HNMR: 7-(3-Chloro-2-hydroxy-propyl)-1,3-dimethyl-8- (2- 2.47–2.50 (t, 4H, CH2NCH2), 2.58–2.62 (t, 2H, pyridin-2-yl-ethylamino)-3,7-dihydro-1H-purine- NHCH2CH2), 3.34 (s, 3H, N1-CH3), 3.51–3.58 (m, -2,6-dione (6) 2H, NHCH2CH2), 3.54 (s, 3H, N3-CH3), 3.60–3.65 (m, 2H, CH2Cl), 3.67–3.69 (t, 4H, CH2OCH2), 1 UV: lmax (log e): 295 nm (4.12). H NMR: 3.11 4.14–4.34 (m, 3H, N7-CH2,CHOH), 5.76 (t, J = (t, J = 5.77, 2H, NHCH2CH2), 3.35 (s, 3H, N1- 4.6 Hz, 1H, NH).

758 Pol. J. Pharmacol., 2004, 56, 755–766 CARDIOVASCULAR ACTIVITY OF NEW 8-ALKYLAMINO-PURINE-2,6-DIONES

Analysis for C16H25N6O4Cl (400.9): C, H, N. 8-Benzylamino-7-{2-hydroxy-3-[4-(2-phenoxyethyl)-piperazin-1-yl]-propyl}-1,3-dimethyl-3,7- 7-(3-Chloro-2-hydroxy-propyl)-8-(2-diethylami- dihydro-1H-purine-2,6-dione (11) no-ethylamino)-1,3-dimethyl-3,7-dihydro-1H-pu- 1 rine-2,6-dione (9) UV: lmax (log e): 296 nm (3.29). HNMR: 1 2.30–2.46 (m, 10H, CH2N(CH2)4N), 2.64 (t, J = UV: lmax (log e): 296 nm (4.224). H NMR (d, 5.7, 2H, CH2CH2O), 3.34 (s, 3H, N1-CH3), 3.54 (s, ppm): 0.99–1.03 (t, J = 7.15, 6H, (CH2CH3)2), 3H, N3-CH3), 3.99–4.18 (m, 2H, N7-CH2), 4.03 (t, 2.52–2.59 (q, J = 7.15, 4H, (CH2CH3)2), 2.63–2.68 J = 5.7, CH2CH2O), 4.39–4.45 (m, 1H, CHOH), (t, J = 6.5, 2H, NHCH2CH2), 3.36 (s, 3H, N1-CH3), 4.57–4.80 (m, 2H, NHCH2), 6.86–6.92 (m, 3H, 3.46–3.55 (m, 2H, NHCH2CH2), 3.56 (s, 3H, N3- arom.), 6.95 (t, J = 6.3, 1H, NH), 7.24–7.42 (m, 7H, CH3), 3.58–3.64 (m, 2H, CH2Cl), 4.10–4.33 (m, arom.). 3H, N7-CH2,CHOH), 5.84 (t, 1H, NH). Analysis for C29H37N7O4 (547.6): C, H, N. Analysis for C16H27N6O3Cl (386.9): C, H, N. 7-{2-Hydroxy-3-[4-(2-phenoxyethyl)-piperazin-1- 8-Amino-7-(3-chloro-2-hydroxypropyl)-1,3-di- -yl]-propyl}-1,3-dimethyl-8-[pyridin-2-ylmethyl) methyl-3,7-dihydro-1H-purine-2,6-dione (10) -amino]-3,7-dihydro-1H-purine-2,6-dione (12) A mixture of 3 (3.52 g, 0.01 mol), 4 ml (0.06 1 UV: lmax (log e): 292 nm (4.11). H NMR: mol) of 25% NH3 and dioxane (12 ml) was stirred 2.37–2.54 (m, 10H, CH2N(CH2)4N), 2.73 (t, J = at room temperature for 48 h in a closed flask. The 5.7, 2H, CH2CH2O), 3.37 (s, 3H, N1-CH3), 3.52 (s, precipitated solid product was filtered off, washed 3H, N3-CH3), 4.05 (t, J = 5.7, 2H, CH2CH2O), with ethanol and purified by recrystallization from 4.09–4.20 (m, 2H, N7-CH2), 4.40–4.48 (m, 1H, anhydrous ethanol. CHOH), 4.74–4.81 (m, 2H, NHCH ), 6.86–6.98 l e 1 2 UV: max (log ): 288 nm (4.06). H NMR: 3.14 (m, 3H, arom.), 7.17–7.39 (m, 4H, 3’-pyridine, 5’- (s, 3H, N1-CH3), 3.29 (s, 3H, N3-CH3), 3.47–3.63 pyridine, 2H, arom. Ph), 7.62–7.73 (t, 1H, 4’- (m, 2H, CH2Cl), 3.94–4.14 (m, 3H, N7-CH2, pyridine), 8.52–8.53 (d, J = 4.9, 1H, 6’-pyridine). CHOH), 6.78 (s, 2H, NH2). Analysis for C28H36N8O4 (548.6): C, H, N. Analysis for C10H14N5O3Cl (287.7): C, H, N. 7-{2-Hydroxy-3-[4-(2-phenoxyethyl)-piperazin-1- General procedure for the synthesis of 8-al- -yl]-propyl}-1,3-dimethyl-8-(2-pyridin-2-yl-ethyl- kylamino derivatives of 7-{2-hydroxy-3-[4-(2- amino)-3,7-dihydro-1H-purine-2,6-dione (13) phenoxyethyl)-piperazin-1-yl]-propyl}-3,7-dihy- 1 dro-1H-purine-2,6-dione (11-17) H NMR: 2.28–2.52 (m, 10H, CH2N(CH2)4N), 2.80 (t, J = 5.7, 2H, CH2CH2O), 3.12 (t, J = 6.6, A mixture of the appropriate 8-alkylamino de- 2H, NH-CH2CH2), 3.35 (s, 3H, N1-CH3), 3.52 (s, rivative of 7-(3-chloro-2-hydroxypropyl)-1,3-di- 3H, N3-CH3), 3.74–3.90 (m, 2H, N7-CH2), methyl-3,7-purine-2,6-dione (4–10) (0.01 mol), 3.91–4.11 (m, 2H, NHCH2), 4.09 (t, J = 5.7, 2H, 1-(2-phenoxyethyl)-piperazine (2.06 g, 0.01 mol), CH2CH2O), 4.25–4.31 (m, 1H, CHOH), 6.87–6.97 anhydrous K2CO3 (2.76 g, 0.02 mol) and 30 ml of (m, 3H, arom. Ph), 7.01 (t, J = 5.8, 1H, NH), anhydrous ethanol (11, 12, 14, 16, 17) or toluene 7.10–7.17 (m, 2H, arom. Ph), 7.24–7.30 (m, 2H, (13, 15) was heated under reflux for 8 h. Then, the 3’,5’-pyridine). 7.59 (t, J = 5.77, 1H, 4’-pyridine). inorganic salt was filtered off and the filtrate was 8.52–8.53 (d, J = 4.9, 1H, 6’-pyridine). cooled and refrigerated overnight. The precipitated Analysis for C29H38N8O4 (562.7): C, H, N. solid product was filtered off, purified by recrystallization from an appropriate solvent. 8-[(Furan-2-yl-methyl)-amino]-7-{2-hydroxy-3- [4-(2-phenoxyethyl)-piperazin-1-yl]-propyl}-1,3- Preparation of HCl salts dimethyl-3,7-dihydro-1H-purine-2,6-dione (14) 1 Free bases of 11–17 were suspended in an anhy- UV: lmax (log e): 293 nm (4.06). HNMR: drous ethanol and saturated with HCl gas, then 2.36–2.53 (m, 10H, CH2N(CH2)4N), 2.78 (t, J = cooled down to –10°C. The precipitated solid salt 5.7, 2H, CH2CH2O), 3.37 (s, 3H, N1-CH3), 3.57 (s, was filtered off and then recrystallized from etha- 3H, N3-CH3), 3.95–4.17 (m, 2H, N7-CH2), 4.06 (t, nol (Tab. 1). J = 5.7, 2H, CH2CH2O), 4.36–4.44 (m, 1H,

ISSN 1230-6002 759 G. Ch³oñ-Rzepa, M. Paw³owskI, M. Zygmunt, B. Filipek, D. Maci¹g

CHOH), 4.45–4.65 (m, 2H, NH-CH2), 6.29–7.36 3.56 (s, 3H, N3-CH3), 3.70–3.73 (t, J = 4.56, 4H, (m, 8H, arom.), 7.45 (t, J = 5.7 1H, NH). MS (m/z): CH2OCH2), 4.07 (m, 4H, CH2CH2O, NHCH2), 537(26) [M+], 444(26), 430(7), 318(12), 245(45), 4.29–4.35 (m, 1H, CHOH), 6.85–6.98 (m, 4H 219(100), 208(3), 195(2), 107(3), 81(72). arom., NH), 7.24–7.28 (m, 2H, arom.). MS (m/z): + Analysis for C27H35N7O5 (537.6): C, H, N. 570(21) [M ], 540(33), 470(100), 463(1), 351(8), 245(9), 219(15), 204(7), 107(1), 100(27), 93(1). 7-{2-Hydroxy-3-[4-(2-phenoxyethyl)-piperazin- Analysis for C28H42N8O5 (570.7): C, H, N. 1-yl]-propyl}-1,3-dimethyl-8-(2-morpholin-4-yl- ethylamino)-3,7-dihydro-1H-purine-2,6-dione (15) 8-(2-Diethylamino-ethylamino)-7-{2-hydroxy-3-[4- (2-phenoxyethyl)-piperazin-1-yl]-propyl}-1,3- Yield (base): 54%, m.p. 134–136°C (acetone). 1 dimethyl-3,7-dihydro-1H-purine-2,6-dione (16) Rf = 0.15 (S1). UV: lmax (log e): 294 nm (4.33). H 1 NMR: 2.35–2.76 (m, 10H, CH2N(CH2)4N), 2.78 (t, UV: lmax (log e): 296 nm (4.67). HNMR: 2H, CH2CH2O), 2.84 (t, J = 5.6, 2H, NHCH2CH2), 1.00–1.05 (t, J = 6.8, 6H, N(CH2CH3)2), 2.30–2.67 3.36 (s, 3H, N1-CH3), 3.48–3.56 m, 2H, N7-CH2); (m, 16H, CH2N(CH2)4N, CH2N(CH2)2), 2.80 (t, J =

Table. 2. Effects of an iv injection of the investigated compounds on heart rate and ECG intervals in anesthetized male Wistar rats (60 mg of thiopental/kg ip) Compound Dose (mg/kg) Parameters Time of observation (min) 01515 2 38.4 Beats/min 435 ± 15 465 ± 15 510±17 480±24 P–Q (ms) 40 ± 0 27±2*** 25±3*** 20±0*** QRS (ms) 20±0 20±0 20±0 20±0 Q–T (ms) 70.0 ± 0 42±2 40±0 40±0*** 11 15 Beats/min 400.6 ± 18.6 348.7 ± 18,1 355.6 ± 25.5 388.9 ± 30.8 P–Q (ms) 49.3 ± 1.5 52.3 ± 1.7 52.2 ± 2.0 52.0 ± 2.7 QRS (ms) 18.3 ± 0.4 19.0 ± 0.4 18.5 ± 0.6 18.7 ± 0.6 Q–T (ms) 72.3 ± 3.6 79.5 ± 3.6 77.3 ± 2.8 72.3 ± 3.9 12 41.0 Beats/min 394.4 ± 10.2 306.1 ± 6 **** 449±12*** 406.9 ± 14.5 P–Q (ms) 48.6 ± 0.8 61.6 ± 2 **** 41.5 ± 0.5 *** 52.5 ± 1.2 QRS (ms) 18.3 ± 0.2 20.5 ± 0.8 *** 17.4 ± 0.2 18.3 ± 0.4 Q–T (ms) 67.6 ± 2.0 94.1 ± 4 **** 60.0 ± 0.7* 68.0 ± 2.5 13 17.4 Beats/min 430.5 ± 13.6 406.7 ± 13.8 440.3 ± 23.8 445.0 ± 22.6 P–Q (ms) 46.7 ± 2.1 50.0 ± 2.6 45.0 ± 2.2 44.8 ± 3.7 QRS (ms) 19.0 ± 0.4 19.3 ± 0.4 19.0 ± 0.4 19.0 ± 0.4 Q–T (ms) 70.0 ± 3.6 77.5 ± 1.7 67.5 ± 2.5 66.2 ± 3.3 14 8.6 Beats/min 361.0 ± 14 342.0 ± 9.0 312 ± 20.3 * 290.7 ± 9 *** * P–Q (ms) 50.0 ± 0 52.7 ± 1.4 53.3 ± 2.9 54.3 ± 1.7 QRS (ms) 19.3 ± 0.7 19.5 ± 0.5 20.5 ± 0.3 20.3 ± 0.3 * Q–T (ms) 76.6 ± 3.3 83.3 ± 0.9 85.6 ± 0.7* 87.0 ± 1.5 15 39.6 Beats/min 377.7 ± 19.4 464±20*** 464±20*** 453±17** P–Q (ms) 50.7 ± 1.5 43.0 ± 1.2 45.0 ± 2 * 47.5 ± 1.4 QRS (ms) 20.2 ± 0.2 17.7 ± 0.2 18.5 ± 0.8 19.0 ± 1 Q–T (ms) 62.5 ± 1.4 56.2 ± 2.4 55.0 ± 2.9 54.5 ± 1.6 * 16 13.1 Beats/min 378.0 ± 21.3 342.5 ± 18.2 350.8 ± 19.1 358.4 ± 22.3 P–Q (ms) 55.0 ± 6.9 61.7 ± 8.3 59.5 ± 7.2 60.1 ± 5.2 QRS (ms) 18.2 ± 1.3 19.5 ± 0.9 18.9 ± 1.3 19.3 ± 0.9 Q–T (ms) 76.0 ± 3.2 82.0 ± 6.3 78.4 ± 2.9 80.0 ± 7.5 17 10 Beats/min 365.2 ± 8.6 335.0 ± 13.9 347.6 ± 17.1 335.2 ± 11.4 P–Q (ms) 50.1 ± 5.2 55.7 ± 3.1 53.4 ± 4.8 55.6 ± 2.5 QRS (ms) 19.0 ± 1.6 20.0 ± 0.9 19.8 ± 1.3 20.5 ± 0.9 Q–T (ms) 80.7 ± 4.3 85.0 ± 5.6 83.3 ± 2.2 85.2 ± 3.8

The data are the means of 6 experiments ± SEM. Statistical analyses were performed using a one-way ANOVA test; * p < 0.05, ** p < 0.02, *** p < 0.01, **** p < 0.001

760 Pol. J. Pharmacol., 2004, 56, 755–766 CARDIOVASCULAR ACTIVITY OF NEW 8-ALKYLAMINO-PURINE-2,6-DIONES

5.7, 2H, CH2CH2O), 3.35 (s, 3H, N1-CH3), Non-working heart perfusion 3.44–3.62 (m, 2H, NHCH ), 3.51 (s, 3H, N3-CH ), 2 3 Hearts from thiopental-anesthetized (45–60 mg/kg, 3.99–4.18 (m, 2H, N7-CH ), 4.09 (t, J = 5.7, 2H, 2 ip) rats were perfused according to the Langendorff CH CH O), 4.27–4.31 (m, 1H, CHOH), 6.82–6.95 2 2 technique [8] at constant pressure of 70 cm H O (m, 4H, arom., NH), 7.23–7.29 (m, 2H, arom.). MS 2 (6.87 kPa) with Chenoweth-Koelle solution con- (m/z): 556(2) [M+], 470(100), 463(1), 449 (0.5), tinuously gased with 95% O plus 5% CO of the 367(14), 245(4), 219(8), 208(1), 204(4) 100(6), 2 2 following composition (mmol/l): NaCl (120.0), 86(39), 72(1), 58(4). KCl (5.6), MgCl (2.2), NaHCO (19.0), CaCl Analysis for C H N O (556.7): C, H, N. 2 3 2 28 44 8 4 (2.4), and glucose (10.0). The effect of the tested compounds, applied at 8-Amino-7-{2-hydroxy-3-[4-(2-phenoxyethyl)-pi- concentrations from 109 to 105 M, on coronary perazin-1-yl]-propyl}-1,3-dimethyl-3,7-dihydro- flow (cardiac effluent), electrocardiogram (ob- 1H-purine-2,6-dione (17) tained by two stainless steel electrodes, one in- 1 serted into the muscle of the ventricular wall and UV: lmax (log e): 290 nm (4.39). H NMR: 2.31–2.59 (m, 10H, CH N(CH ) N), 2.81 (t, J = another attached to the metal aortic cannula) were 2 2 4 assessed 15–20 min after initial stabilization. 5.7, 2H, CH2CH2O), 3.35 (s, 3H, N1-CH3), 3.59 (s, 3H, N3-CH3), 4.06 (t, J = 5.7, 2H, CH2CH2O), The effect on normal electrocardiogram (ECG) 4.09–4.20 (m, 2H, N7-CH2), 4.40–4.48 (m, 1H, Electrocardiographic investigations were car- CHOH), 5.91(s, 2H, NH2), 6.86–6.98 (m, 3H, arom.); 7.22–7.51 (m, 2H, arom.). MS (m/z): ried out using Multicard 30 apparatus, standard 457(21) [M+], 364(36), 350(24), 294(16), 281(30), lead II and paper speed of 50 mm/s. The tested 245(50), 219(100), 205(20), 195(16), 98(21). compounds were administered intravenously (iv)at a dose of 1/5 LD . The ECG was recorded just be- Analysis for C22H31N7O4 (457.5): C, H, N. 50 fore and 1, 5, 15 min after administration of the compounds. Pharmacological part Adrenaline-induced arrhythmia according to Szekeres and Papp [22] The arrhythmia was evoked in rats anesthetized Materials with thiopental (60 mg/kg, ip)byiv injection of m Compounds. Barium chloride (POCh, Poland), adrenaline (20 g/kg). The tested compounds were [3H]clonidine (Amersham), epinephrine (adrena- administered iv 15 min before adrenaline. The cri- linum hydrochloricum, Polfa, Poland), [3H]prazo- terion of antiarrhythmic activity was the lack of sin (Amersham), heparin sodium (Polfa, Poland), premature beats and inhibition of cardiac arrhyth- thiopental sodium (Biochemie, GmbH, Vienna, mia in comparison with the control group. Austria). Barium chloride-induced arrhythmia according Animals. The experiments were carried out on to Szekeres and Papp [22] male albino Swiss mice (18–25 g) and male Wistar rats (180–250 g). Animals were housed in plastic Barium chloride solution was injected into the cages in room at a constant temperature of 20 caudal vein of rat (32 mg/kg, in a volume of ± 2°C with natural light-dark cycles. The animals 1 ml/kg). The tested compounds were given iv had free access to standard pellet diet and water, 15 min before the arrhythmogen. and were used after a minimum of 3 days of accli- The criterion of antiarrhythmic activity was mation to the housing conditions. Control and ex- a gradual disappearance of the arrhythmia and res- perimental groups consisted of 8–10 animals each. toration of the sinus rhythm. Statistical analysis. The data are expressed as Influence on the blood pressure the means ± SEM. The statistical significance was calculated using a one-way ANOVA or Student’s Male Wistar normotensive rats were anesthe- t-test. Differences were considered significant tized with thiopental (50–75 mg/kg) by intraperito- when p < 0.05. neal (ip) injection. The right carotid artery was can-

ISSN 1230-6002 761 G. Ch³oñ-Rzepa, M. Paw³owskI, M. Zygmunt, B. Filipek, D. Maci¹g nulated with polyethylene tubing filled with hepa- The effect on normal electrocardiogram in vivo rin in saline to facilitate pressure measurements The effect on ECG intervals and heart rate were using a Datamax apparatus (Columbus Instru- determined for all compounds at a dose of 1/5 LD ments). The studied compounds were injected at a 50 iv (Tab. 2). Electrocardiographic experiments sho- dose of 1/10 LD iv into the cadual vein, after a 5 50 wed that compounds 2 and 15 administered iv in- min stabilization period, in a volume equivalent to creased the heart rate by 6.8–17.2% and 19.9–22.8%, 1 ml/kg. respectively, diminished P-Q (32–50% and 11.2 a-Adrenoceptor binding assay –15.2%) and Q-T (42% and 11.9–12.8%) intervals and did not change (2) or slightly diminished (15) The experiment was carried out on the rat cerebral 3 the QRS complex. Compound 14 significantly de- cortex [10, 19]. [ H]prazosin (22 Ci/mmol, a1-nor- 3 creased the heart rate by 13.5–19.4%, prolonged adrenergic receptor) and [ H]clonidine (25.5 Ci/mmol, P-Q and Q-T intervals by 8.6% and 11.7–13.5%, a2-noradrenergic receptor) were used as ligands. respectively. Compound 12 had different effect on The incubation mixture (final volume of 550 ml) the ECG: within 1 min it significantly reduced consisted of 450 ml of membrane suspension, 50 ml 3 3 heart rate by 22.4% and induced prolongation of ofa[H]prazosin (0.6 nM) or [ H]clonidine (2 nM) the P-Q and Q-T intervals and QRS complex by solution and 50 ml of buffer containing seven or 26.7%, 39.2% and 12%, respectively. After 5 min, eight concentrations (1 nM to 100 mM) of the in- it increased the number of cardiac beats per minute vestigated compounds. For measuring unspecific by 13.8%. The examined compounds (11, 13, 16 binding, phentolamine (in the case of [3H]prazosin) 3 and 17) did not significantly affect the normal ECG and clonidine (in the case of [ H]clonidine) at a fi- in anesthetized rats. nal concentration of 10 mM were present. Antiarrhythmic activity Acute toxicity according to Litchfield and Wil- coxon [9] All compounds were tested in two arrhythmia models using adrenaline- and barium chloride- The compounds dissolved in 0.9% saline were induced arrhythmia. In anesthetized rats, iv injec- injected into the caudal vein (10 ml/kg). Each dose tions of adrenaline (20 mg/kg) caused sinus brady- was given to 6 animals. The LD50 values were cal- cardia (100%), atrioventricular disturbances, ven- culated according to the method of Litchfield and tricular and supraventricular extrasystoles (100%) Wilcoxon [9] after 24 h observation period. which led to death of approximately 50% of animals within 10–5 min. The tested compounds administered 15 min prior to adrenaline injection, de- RESULTS

Non-working heart perfusion Table 3. Prophylactic antiarrhythmic activity in anesthetized rats As shown in Table 2, compounds 2 and 15, given at doses of 109–105 M increased the heart Compounds Adrenaline-induced arrhythmia rate by 16.2–38.1%, diminished P-Q (by ED50 iv (mg/kg) IT LD50/ED50 12.4–37.5%) and Q-T (by 16.1–25.0%) intervals 2 3.5 (2.9–4.1) 54.9 and did not change or slightly diminished the QRS complex. Compounds 11, 14, 16 and 17, adminis- 11 21.2 (16.5–27.8) 3.5 tered at the same doses, decreased the number of 12 16.5 (9.6–25.1) 12.1 cardiac beats per minute by 5.7–52.3%, produced a 13 9.2 (6.3–17.5) 9.5 prolongation of the P-Q (6.4–72.9%) and Q-T 14 1.08 (0.64–1.76) 40.2 (6.7–61.5%) intervals and QRS complex (1.7–34.1 15 3.6 (2.6–5.0) 55.0 %), whereas compounds 12, 13 administered at the 16 7.1 (6.9–8.6) 9.2 doses of 109–3×106 M reduced the heart rate by 2.3–41.1%, prolonged the P-Q (10.9–41.7%) and 17 1.25 (0.74–1.89) 40.2 Q-T (2.3–49.4%) intervals and QRS complex Propranolol* 1.05 (0.64–1.73) 37.0 (1.9–16.5%). The investigated compounds 2 and Each value was obtained from 3 experimental groups. Each 11–17 reduced the coronary flow by 2.0–30.9%. group consisted of 6 animals. * Ref. data [12]

762 Pol. J. Pharmacol., 2004, 56, 755–766 CARDIOVASCULAR ACTIVITY OF NEW 8-ALKYLAMINO-PURINE-2,6-DIONES creased the number of premature ventricular and and 17 exhibited important antiarrhythmic effects supraventricular beats and reduced mortality. with ED50 values ranging between 1.08–3.6 mg/kg The ED50 values (a dose producing a 50% inhi- iv. Compounds 11–13 and 16 also inhibited bition of premature ventricular beats) and therapeu- adrenaline-induced arrhythmia in the anesthetized tic index of prophylactic antiarrhythmic activity in rats (ED50 = 7.1–21.2 mg/kg iv), but their therapeu- the adrenaline-induced arrhythmia are presented in tic index was not as favorable as in case of com- Table 3. These compounds administered 15 min be- pounds 2, 14, 15 and 17. fore adrenaline prevented and/or reduced in a sta- Rapid iv injection of high dose of barium chlo- tistically significant manner the number of prema- ride (32 mg/kg) caused in rats sinus bradycardia ture ventricular beats. Four compounds 2, 14, 15 and rapid ventricular extrasystoles, ventricular

Table 4. Hypotensive activity of tested compounds 2 and 11–17 in anesthetized normotensive rats Compound Dose Blood Time of observation (min) (mg/kg) pressure (mm Hg ± SEM) Before 1 5 10 30 60 2 19.2 systolic 152.0 136.1 144.0 137.6 129.0 118.2 ± 7.2 ± 5.2 ± 9.1 ± 6.5 ± 9.2 * ± 7.8 ** diastolic 134.6 123.4 129.5 124.6 115.1 104.0 ± 8.5 ± 7.5 ± 7.3 ± 8.3 ± 8.6 * ± 6.2 ** 11 7.5 systolic 136.0 85.4 104.0 105.4 113.2 111.8 ± 5.4 ± 4.3 **** ± 7.3 **** ± 6.8 **** ± 7.3 *** ± 2.8 *** diastolic 112.8 50.4 79.6 84.6 93.6 88.4 ± 6.8 ± 3.5 **** ± 8.2 **** ± 6.3 **** ± 4.5 * ± 12.8 *** 12 20.5 systolic 138.2 68.6 86.8 102.2 105.2 115.2 ± 6.5 ± 15.2 **** ± 19.0 **** ± 15.9 *** ± 18.8 *** ± 20.5 diastolic 112.0 33.8 64.4 9.2 80.6 86.8 ± 30.5 ± 19.3 **** ± 23.8 *** ± 20.0 ** ± 21.5 * ± 22.1 13 8.7 systolic 136.2 92.2 101.0 108.2 113.7 124.0 ± 10.2 ± 2.8 *** ± 6.3 ** ± 9.2 * ± 10.3 ± 9.2 diastolic 119.0 65.2 83.0 91.5 96.0 108.2 ± 11.2 ± 3.1 *** ± 9.0 * ± 19.3 ± 10.3 ± 11.3 14 4.3 systolic 148.0 98.3 132.4 131.0 129.7 121.0 ± 4.5 ± 22.8 *** ± 11.2 ± 9.4 ± 6.8 ± 5.1 diastolic 131.0 77.7 118.0 117.3 116.3 106.0 ± 4.8 ± 24.0 *** ± 11.1 ± 9.3 ± 6.2 ± 5.7 15 19.8 systolic 152.0 93.3 143.3 139.3 136.7 135.3 ± 12.5 ± 4.2 **** ± 9.5 ± 6.6 ± 2.3 ± 0.3 diastolic 138.3 81.7 127.3 125.7 125.7 125.7 ± 12.5 ± 6.4 **** ± 9.5 ± 6.5 ± 2.9 ± 0.6 16 6.5 systolic 128.0 85.8 96.7 109.5 116.5 118.6 ± 4.3 ± 11.2 *** ± 5.8 ** ± 7.7 ± 7.0 ± 9.3 diastolic 105.2 62.5 75.3 97.2 91.6 83.7 ± 6.5 ± 8.5 *** ± 9.0 ** ± 4.8 ± 5.2 ± 6.4 17 5.0 systolic 120.3 92.1 101.3 103.4 113.3 113.0 ± 7.1 ± 8.6 * ± 5.8 ± 7.3 ± 9.5 ± 5.7 diastolic 102.0 77.7 93.9 95.5 94.7 95.4 ± 4.3 ± 6.2 * ± 8.9 ± 9.6 ± 8.0 ± 8.2

The data were the means of 5–6 experiments ± SEM. Statistical analyses were performed using a one-way ANOVA test: * p < 0.05, ** p < 0.02, *** p < 0.01, **** p < 0.001

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Table 5. Affinity for different a-adrenoceptor subtypes in the Table 6. Acute toxicity of the investigated compounds rat cerebral cortex Compound Route LD# mg/kg ! ! Compound [ H]Prazosin (a rec.) [ H]Clonidine (a rec.) KE (mM) ± SEM KE(mM) ± SEM 2 iv 192.0 (176.9–208.3) 11 iv 75.0 (59.0–95.2) 2 1.400 ± 0.100 0.400 ± 0.033 11 0.462 ± 0.015 0.152 ± 0.007 12 iv 205.0 (184.0–228.0) 12 0.876 ± 0.041 0.575 ± 0.059 13 iv 87.6 (69.0–102.3) 13 0.366 ± 0.035 4.299 ± 0.300 14 iv 43.4 (32.6–57.0) 14 0.570 ± 0.100 0.524 ± 0.075 15 iv 198.0 (179.6–216.2) 15 0.880 ± 0.100 0.980 ± 0.090 16 iv 65.6 (52.0–87.4) 16 0.225 ± 0.011 0.372 ± 0.023 17 iv 50.3 (42.3–65.7) 17 1.300 ± 0.140 0.790 ± 0.170 aminophylline iv 190 *

The data are median lethal doses with 5% confidence limits in tachycardia and ventricular fibrillation (100%), parentheses. * Ref. data [22] which led to death of all animals within 3–5 min. The tested compounds, administered 15 min be- 0.152 to 4.299 mM to cortical a1- and a2-adreno- fore barium chloride, at doses of 1/5 LD50 slightly ceptors, respectively. The shape of the curves sug- protected the animals against fatal consequence of gested competitive binding. The results are summa- barium chloride administration. Compounds 2, 11 rized in Table 5. and 15 diminished the occurrence of ventricular fibrillation in 17–34% of animals and also de- Acute toxicity creased (by approximately 34–42%) the mortality. The toxicity of compounds 2, 12 and 15 was comparable to aminophylline, while the toxicity of Influence on the blood pressure compounds 11, 13, 14, 16 and 17 was about 2–4 Hypotensive activity of compounds 2 and times higher than that of aminophylline. The sum- 11–17 was determined after their iv administration marized data are presented in Table 6. to normotensive anesthetized rats. The results are presented in Table 4. The studied compounds were DISCUSSION injected at a dose of 1/10 LD50. The highest hypotensive activity was observed for compounds 11 The effects of the newly synthesized com- and 12. The compounds significantly decreased the pounds on heart rate frequencies and the ECG in- systolic (50.4–16.6% and 37.2–16.7%) and dia- tervals (PQ, QRS, QT) were experimentally stud- stolic (69.8–22.5% and 55.3–17.0%) blood pres- ied on isolated rat heart and on anesthetized rats. sure in normotensive rats and this hypotensive ef- The conducted preliminary studies showed that fect lasted throughout the whole observation pe- these compounds changed the ECG in vitro and in riod. Compound 2, given at a dose of 1/10 LD50 iv, vivo. Compounds 2 and 15 increased the frequency produced a significant decrease in systolic of isolated rat heart, shortened the duration of P-Q, (15.1–22.2%) and diastolic (14.5–22.7%) blood Q-T intervals or QRS complex (15), whereas other pressure. The significant hypotensive effect was compounds (11–14 and 16–17) decreased the heart observed 30–60 min after administration of the rate, lengthened the duration of P-Q, Q-T intervals compound. Compounds 13–17 injected iv signifi- and the QRS complex, and diminished the coronary cantly decreased the systolic (23.4–38.6%) and dia- outflow. Compounds 2 and 15, given iv, also in- stolic (23.8–45.2%) pressure, but a significant ef- creased the heart rate and diminished the P-Q, Q-T fect lasted 1–10 min (Tab. 4). intervals and the QRS complex (15). Our results in- dicate that these two compounds possess activity a-Adrenoceptor binding assay similar to methylxanthines [16]. On the other hand, All the tested compounds inhibited [3H]prazo- compound 12, after iv administration, initially di- sin binding with Ki ranging from 0.225 to 1.4 mM minished heart rate and lengthened the duration of 3 and [ H]clonidine binding with Ki ranging from P-Q, Q-T intervals and QRS complex and later in-

764 Pol. J. Pharmacol., 2004, 56, 755–766 CARDIOVASCULAR ACTIVITY OF NEW 8-ALKYLAMINO-PURINE-2,6-DIONES creased the number of cardiac beats per minute and groups were the most active. Compounds 11 and 12 produced a slight prolongation of P-Q and Q-T in- caused a significant hypotensive effect throughout tervals. These features substantially differ from the whole observation period, whereas compound methylxanthines. 2 reduced the arterial blood pressure in the second The antiarrhythmic effects of the novel com- phase of observation. pounds were examined on rats, using two models The results of the binding assays on a1- and of arrhythmia (adrenaline-induced and barium a2-adrenoceptors presented in Table 5 demonstrate chloride-induced arrhythmia). that these derivatives (2, 11, 12) display low affin- Compound 2 and its seven analogues (11–17), ity at a1- and a2-adrenoceptors, thus, their hypo- given iv 15 min before arrhythmogen, prevented or tensive effect is not related to their a1-adrenolytic attenuated the symptoms of adrenaline-induced ar- properties. rhythmia. The data reported in Table 3 suggest that The acute toxicity of compounds 2, 12 and 15 compounds 2 and 15 showed more beneficial thera- was similar to aminophylline [21], whereas other peutic index than propranolol. The other tested tested compounds were more toxic. The results compounds possessed therapeutic index similar to suggest that the introduction of the pyridin-2-yl- (14, 17) or worse (12, 13, 16, 11) than that of pro- methylamino (12) or 2-morpholin-4-yl-ethylamino pranolol. (15) group in the 8 position of 2 does not change Compounds 2, 11 and 15 also delayed the onset the toxicity. of this arrhythmia, reduced the incidence of ven- In summary, some of the newly synthesized de- tricular tachycardia and ventricular fibrillation, rivatives of 1,3-dimethyl-7-(2-hydroxy-3-piperazino- prolonged the survival time and prevented or di- propyl)-3,7-dihydropurine-2,6-dione showed anti- minished mortality caused by BaCl2. Other com- arrhythmic effect on adrenaline (2, 14, 15, 17)- or pounds did not change the occurrence of distur- barium chloride (2, 11, 15)-induced arrhythmia and bances in barium chloride-induced model of ar- significant hypotensive activity (2, 11, 12). Further rhythmia. experiments are needed to elucidate the exact Adrenaline is an agonist of b-adrenergic recep- mechanism of the hypotensive and antiarrhythmic tors, which are located also in heart muscle tissue. action of these active compounds. The stimulation of b-adrenoceptors increases the 2+ magnitude of ICa-L (Ca current), If (inward current + + REFERENCES carried by Na and K ), ICl (induced Cl current), + IKur (ultra-rapid delayed rectifying K current), 1. Boyett MR, Harrison SM, Janvier NC, McMorn SO, + IKATP (ATP-independent K current), Ist and these Owen JM, Shui Z: A list of vertebrate cardiac ionic actions are mediated by cAMP-dependent protein currents. Nomenclature, properties, function and clo- ned equivalents. Cardiovasc Res, 1996, 32, 455–481. kinase [1, 2], whereas barium ions decrease the 2. Carmeliet E: Cardiac ionic currents and acute ische- outward potassium currents by blocking the ultra- + mia: from channels to arrhythmias. Physiol Rev, 1999, rapid delayed rectifying K channels (IKur) and in- 79, 917–1017. + + wardly rectifying K channels (Kir), pacemaker K 3. Eckstein M: A search for new drugs in the group of + channels (IK1), Ach-induced K current (IK,ACh) [1, xanthine derivatives (Polish). Diss Pharm, 1962, 14, 2, 7]. 401–409. 4. Based on our results, we hypothesize that the Fhid O, Paw³owski M, Filipek B, Horodyñska R, Maci¹g D: Central nervous system activity of new antiarrhythmic effects of the tested compounds are pyrimidine-8-on[2,1-f]-theophylline-9-alkylcarboxylic probably related to the arrest of the intflow of Ca2+ ++ acids derivatives. Pol J Pharmacol, 2002, 54, 245–254. into the cardiac cells via voltage-dependent Ca 5. Gorczyca M, Paw³owski M, Mrozikiewicz A, and IKur channels. The mode of antiarrhythmic ac- Koz³owska T, Wasik A: Chemical and pharmacologi- tion of these compounds was promising enough to cal properties of some 7-b-hydroxy-g-piperazinopropyl- continue further experiments. -8-benzylamino-theophyllines. Pol J Pharmacol Pharm, Hypotensive activity of the investigated com- 1986, 38, 85–90. 6. Hayao S, Havera HJ, Strycker WG, Leipzig TJ, pounds was determined after their iv administration Rodrigues R: New antihypertensive aminoalkyltetra- to normotensive anesthetized rats. Compound 2 zoles. J Med Chem, 1967, 10, 400–402. and their 8-substituted derivatives with ben- 7. Ino M, Fishbein M, Mandel W, Chen PS, Karagueuz- zylamino (11) or pyridin-2-yl-methylamino (12) ian HS: Cellular mechanisms of ventricular bipolar

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