Use of 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium salts in the treatment of cardiovascular disease

Abstract

Salts of 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium, method of preparation thereof and use in the treatment of cardiovascular disease.

Claims

1. 3-Carboxy-N-ethyl-N,N-dimethylpropan-1-aminium dihydrogen phosphate ##STR00004##

2. A process for preparing the 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium dihydrogen phosphate according to claim 1, comprising: a. adding N,N-dimethylethylamine to ethyl 4-bromobutanoate in appropriate solvent to obtain 4-ethoxy-N-ethyl-N,N-dimethyl-4-oxo-1-butanaminium bromide; b. passing 4-ethoxy-N-ethyl-N,N-dimethyl-4-oxo-1-butanaminium bromide through ion exchange resin column to obtain 4-[ethyl(dimethyl)ammonio] butanoate; c. adding phosphoric acid in appropriate solvent to obtain the 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium dihydrogen phosphate.

3. The process according to claim 2, wherein in step a) the appropriate solvent is acetonitrile or acetone.

4. A method for treating a cardiovascular disease in a subject in need thereof, comprising administration of an effective amount of the 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium salt dihydrogen phosphate of claim 1, wherein the cardiovascular disease is ischemic heart disease or myocardial infarction.

5. The method according to claim 4, wherein the cardiovascular disease is ischemic heart disease.

6. The method according to claim 4, wherein the cardiovascular disease is myocardial infarction.

Description

DESCRIPTION OF EMBODIMENTS

(1) The present invention will be described in more detail by referring to the following non-limiting examples.

Preparation of 4-ethoxy-N-ethyl-N,N-dimethyl-4-oxo-1-butanaminium bromide (2)

(2) Procedure A

(3) To a solution of ethyl 4-bromobutanoate (1) (20.0 g, 102.5 mmol) in acetonitrile (70 ml) N,N-dimethylethylamine (15 ml, 139 mmol) was added and stirred at ambient temperature for 3 days. The reaction mixture was evaporated, the residue was triturated with acetone (50 ml), filtered, washed with ether, and dried to afford 26.051 g (94.8%) of the 4-ethoxy-N-ethyl-N,N-dimethyl-4-oxo-1-butanaminium bromide. LCMS (ESI.sup.+, m/z): [M-Br.sup.].sup.+188, purity 98.9%.

(4) .sup.1H NMR (CDCl.sub.3, HMDSO) : 1.26 (t, J=7.2 Hz, 3H); 1.44 (t, J=7.4 Hz, 3H); 2.00-2.11 (m, 2H); 2.52 (t, J=6.6 Hz, 2H); 3.40 (s, 6H); 3.64-3.73 (m, 2H); 3.69 (q, J=7.4 Hz, 2H); 4.14 (q, J=7.2 Hz, 2H).

(5) Procedure B

(6) To a solution of ethyl 4-bromobutanoate (1) (19.5 g, 100 mmol) in acetone (70 ml) N,N-dimethylethylamine (15 ml, 139 mmol) was added and stirred at ambient temperature for 3 days. The reaction mixture was filtered; the solid material was washed with an acetone, ether, and dried to afford 24.19 g (90.2%) of the title compound 2. The filtrate was evaporated; the residue (2.147 g) was triturated with ether and dried to give an extra batch (0.962 g, 3.6%) of the product 2 of the same quality as the main portion. The evaporation of the ether washings allowed recovering 0.956 g (4.9 mmol, 4.9%) of the starting material 1. 4-ethoxy-N-ethyl-N,N-dimethyl-4-oxo-1-butanaminium bromide: LCMS (ESI.sup.+, m/z): [M-Br.sup.].sup.+188, purity 98.4%.

(7) .sup.1H NMR (CDCl.sub.3, HMDSO) : 1.26 (t, J=7.2 Hz, 3H); 1.44 (t, J=7.4 Hz, 3H); 2.00-2.11 (m, 2H); 2.52 (t, J=6.6 Hz, 2H); 3.40 (s, 6H); 3.64-3.73 (m, 2H); 3.69 (q, J=7.4 Hz, 2H); 4.14 (q, J=7.2 Hz, 2H).

Preparation of 4-[ethyl(dimethyl)ammonio]butanoate (3)

(8) A solution of 4-ethoxy-N-ethyl-N,N-dimethyl-4-oxo-1-butanaminium bromide (2) (12.00 g, 44.7 mmol) in water (10 ml) was passed through Amberlite IRA-410 (OH) ion exchange resin column (250 ml) eluting slowly (ca. 10 drops/min) with ethanol (TLC control). The eluate was evaporated and the residue (12 g) was dissolved in water (50 ml). To this solution DOWEX 50WX8 ion exchange resin (5 g) was added and stirred at ambient temperature for 0.5 h. The reaction mixture was filtered through celite (1 cm) and the eluate was evaporated. The residue was azeotropically dried with isopropanol, acetonitrile, and acetone. The obtained solid was triturated with acetone (10 ml) and the mixture was kept at 0 C. for 2 h. The precipitate was filtered and dried in vacuo over P.sub.2O.sub.5 to give 4.65 g (65%) of the 4-[ethyl(dimethyl)ammonio]butanoate (3).

(9) (DMSO-d.sub.6, HMDSO) : 1.24 (t, J=7.3 Hz, 3H); 1.66-1.76 (m, 2H); 1.81 (t, J=6.4 Hz, 2H); 2.95 (s, 6H); 3.16-3.23 (m, 2H); 3.29 (q, J=7.3 Hz, 2H). LCMS (ESI.sup.+, m/z): 160 [M+H].sup.+.

(10) Anal. Calc. for C.sub.8H.sub.17NO.sub.2.1.55 H.sub.2O: C, 51.34; H, 10.82; N, 7.48.

(11) Found: C, 51.36, H, 11.40, N, 7.34.

Preparation of 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium 2-(acetyloxy)benzoate (4 a)

(12) 3-Carboxy-N-ethyl-N,N-dimethylpropan-1-aminium 2-(acetyloxy)benzoate was prepared in a form of a water mixture. Thus, ca. 90% 4-[ethyl-(dimethyl)ammonio]butanoate (3) (2.20 g, 12.44 mmol) and 2-(acetyloxy)-benzoic acid (2.266 g, 12.57 mmol) were placed in a volumetric flask and diluted with water up to 100 ml. The content of the mixture dissolves by heating and precipitates by lowering of the temperature. According to .sup.1H-NMR, the precipitated solid material consists of almost pure 2-(acetyloxy)-benzoic acid.

Preparation of 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium (2E)-3-carboxyacrylate (4 b)

(13) To a solution of 4-[ethyl(dimethyl)ammonio]butanoate (3) (2.0 g, 12.56 mmol) in anh. ethanol (10 ml) a hot (60 C.) solution of (E)-butenedioic acid (1.46 g, 12.56 mmol) in ethanol (50 ml) was added. The reaction mixture was allowed to stand at ambient temperature for 2 h, the precipitated crystals were filtered and dried over P.sub.2O.sub.5 to give 2.98 g (85%) of the 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium (2E)-3-carboxyacrylate. M.p. 122-123 C.

(14) .sup.1H-NMR (D.sub.2O, DSS) : 1.36 (tt, J=1.9, 7.3 Hz, 3H); 2.06 (m, 2H); 2.49 (t, J=7.1 Hz, 2H); 3.06 (s, 6H); 3.31 (m, 2H); 3.40 (q, J=7.3 Hz, 2H); 6.75 (s, 1.9H, CHCH).

(15) LCMS ESI.sup.+ (m/z): 160 [M+H].sup.+. Titration assays: water content (Fisher) 0.13%, betaine content (HClO.sub.4) 93.0%, (E)-butenedioic acid content 46.1%.

(16) Anal. Calc. for C.sub.8H.sub.17NO.sub.2.1.2 C.sub.4H.sub.4O.sub.4 (46.7%): C, 51.50, H, 7.36, N, 4.69.

(17) Found: C, 51.52, H, 7.35, N, 4.61.

Preparation of 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium 3-carboxypropanoate (4 c)

(18) 3-Carboxy-N-ethyl-N,N-dimethylpropan-1-aminium 3-carboxypropanoate was prepared in a form of a water solution. Thus, ca. 90% 4-[ethyl-(dimethyl)ammonio]butanoate (3) (2.20 g, 12.44 mmol) and succinic acid (1.49 g, 12.62 mmol) were placed in a volumetric flask and dissolved and diluted with water up to 100 ml.

Preparation of 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium 2,6-dioxo-1,2,3,6-tetrahydropyrimidine-4-carboxylate (4 d)

(19) To a solution of 4-[ethyl(dimethyl)ammonio]butanoate (3) (2.0 g, 12.56 mmol) in isopropanol (100 ml) 2,6-dioxo-1,2,3,6-tetrahydropyrimidine-4-carboxylic acid monohydrate (2.187 g, 12.56 mmol) was added and the reaction mixture was heated to reflux until all the carboxylic acid dissolved. The reaction mixture was allowed to cool to ambient temperature, the precipitated crystals were filtered, washed with isopropanol (5 ml) and diethyl ether (20 ml), and dried over P.sub.2O.sub.5 to give 3.238 g (97.4%) of the 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium 2,6-dioxo-1,2,3,6-tetrahydropyrimidine-4-carboxylate. M.p. 150.7 C.

(20) .sup.1H-NMR (D.sub.2O, DSS) : 1.36 (tt, J=2.0, 7.3 Hz, 3H); 2.05 (m, 2H); 2.47 (t, J=7.0 Hz, 2H); 3.07 (s, 6H); 3.31 (m, 2H); 3.41 (q, J=7.3 Hz, 2H); 6.20 (s, 1H, CCH).

(21) LCMS ESI.sup.+ (m/z): 160 [M+H].sup.+.

(22) Anal. Calc. for C.sub.8H.sub.17NO.sub.2.C.sub.5H.sub.4N.sub.2O.sub.4 (49.5%): C, 49.52, H, 6.71, N, 13.33.

(23) Found: C, 49.59, H, 6.69, N, 13.26.

Preparation of 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium dihydrogen phosphate (4 e)

(24) To a solution of 4-[ethyl(dimethyl)ammonio]butanoate (3) (6.4 g, 40 mmol) in water (10 ml) a solution of 85% aq. H.sub.3PO.sub.4 (4.73 g, 40 mmol) in acetone (10 ml) was added and the resulting solution was stirred at ambient temperature for 10 min. The reaction mixture was evaporated and azeotropically dried several times with acetone by rotary evaporator at 45 C. The obtained white crystalline substance was dried over P.sub.2O.sub.5 to give 9.82 g (95%) of the 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium dihydrogen phosphate. M.p. 110-135 C.

(25) .sup.1H-NMR (D.sub.2O, DSS) : 1.36 (tt, J=1.8, 7.3 Hz, 3H); 2.06 (m, 2H); 2.50 (t, J=7.0 Hz, 2H); 3.06 (s, 6H); 3.32 (m, 2H); 3.41 (q, J=7.3 Hz, 2H). LCMS ESI.sup.+ (m/z): 160 [M+H].sup.+. Titration assays: water content (Fisher) 0.356%, betaine content (HClO.sub.4)95.682%.

(26) Anal. Calc. for C.sub.8H.sub.17NO.sub.2.0.052 H.sub.2O (0.356%).Math.1.07 H.sub.3PO.sub.4 (39.6%): C, 36.26; H, 7.73; N, 5.29.

(27) Found: C, 36.20, H, 7.72, N, 5.11.

(28) The purity of the obtained 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium dihydrogen phosphate was increased by crystallization from methanol. Thus, the 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium dihydrogen phosphate (6.9 g) was crystallized from methanol (40 ml) to afford 5.326 g (77%) of the purified 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium dihydrogen phosphate with m.p. 139 C.

(29) Calc. for C.sub.8H.sub.17NO.sub.2.H.sub.3PO.sub.4 (38.1%): C, 37.36; H, 7.84; N, 5.45.

(30) Found: C, 37.52, H, 7.85, N, 5.39.

(31) Cardioprotective Activity

(32) Fifty male, 10 weeks old Wistar rats weighing 200-250 g were housed under standard conditions (21-23 C., 12 h light-dark cycle) with unlimited access to food (R3 diet, Lactamin AB, Sweden) and water.

(33) Rats were adapted to local conditions for two weeks before the start of treatment. Meldonium dihydrate at a dose of 20 mg/kg, gamma-butyrobetaine at a dose of 20 mg/kg and 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium salts at dose of 20 mg/kg were administered p.o. daily for 8 weeks. Control rats received water.

(34) Isolated Rat Heart Infarction Study

(35) The isolated rat heart experiment was performed essentially as described earlier (Liepinsh et al., J. Cardiovasc. Pharmacol. 2006; 48(6):314-9). Twenty-four hours after the last drug administration hearts were excised and retrogradely perfused via the aorta at a constant pressure with oxygenated Krebs-Henseleit buffer at 37 C. The heart rate, left ventricle end-diastolic pressure and left ventricle developed pressure were continuously recorded. Coronary flow was measured using an ultrasound flow detector (HSE) and the PowerLab 8/30 system from ADInstruments. The hearts were perfused for 20 min to stabilize the hemodynamic functions and then occlusion was performed for 60 min by constricting threads through a plastic tube. Successful occlusion was confirmed by a coronary flow decrease of about 40 percent. Reperfusion was achieved by releasing the threads. At the end of the 150-min reperfusion period, the risk zone was delineated with 0.1% methylene blue. The hearts were then sectioned transversely from the apex to the base in five slices 2 mm in thickness and incubated in 1% triphenyltetrazolium chloride in phosphate buffer (pH 7.4, 37 C.) for 10 min to stain viable tissue red and necrotic tissue white. Computerized planemetric analysis of Sony A900 photographs was performed using Image-Pro Plus 6.3 software to determine the area at risk and area of necrosis expressed as a % of the left ventricle. The obtained values were then used to calculate the infarct size (IS) as a % of risk area according to the formula:
Infarct Size=Area of Necrosis/Area at Risk100%.

(36) Effects in Isolated Rat Heart Infarction Model

(37) The anti-infarction effect of examined substances was investigated in an isolated rat heart infarction model. During occlusion of left coronary artery, the coronary flow in all experimental groups was decreased for 40% (from 11 ml/min to 7 ml/min). Moreover, the drop of developed left ventricular pressure for 50% was observed. The heart rate during the occlusion period did not change significantly. In reperfusion stage, coronary flow, developed left ventricular pressure, dp/dt values were recovered till about 80% of control level. There were no significant differences between control and treatment groups.

(38) Effects of Meldonium dihydrate (20 mg/kg), gamma-butyrobetaine (20 mg/kg) and 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium salts (20 mg/kg) after 2 weeks of treatment on infarct size in the isolated rat heart infarction experiment are presented in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6

(39) TABLE-US-00001 TABLE 1 Infarct size, % of control Control 100.0 5.9 Meldonium dihydrate 20 mg/kg 117.9 7.9 Gamma-butyrobetaine 20 mg/kg 87.6 11.4 3-Carboxy-N-ethyl-N,N- .sup.61.6 6.7*.sup.,#,$ dimethylpropan-1-aminium 2- (acetyloxy)benzoate 20 mg/kg

(40) Effects of Meldonium dihydrate, gamma-butyrobetaine and 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium 2-(acetyloxy)benzoate on infarct size

(41) TABLE-US-00002 TABLE 2 Infarct size, % of control Control 100.0 5.9 Meldonium dihydrate 20 mg/kg 117.9 7.9 Gamma-butyrobetaine 20 mg/kg 87.6 11.4 3-carboxy-N-ethyl-N,N- .sup.46.5 7.0*.sup.,#,$ dimethylpropan-1-aminium (2E)-3- carboxyacrylate 20 mg/kg

(42) Effects of Meldonium dihydrate, gamma-butyrobetaine and 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium (2E)-3-carboxyacrylate on infarct size

(43) TABLE-US-00003 TABLE 3 Infarct size, % of control Control 100.0 5.9 Meldonium dihydrate 20 mg/kg 117.9 7.9 Gamma-butyrobetaine 20 mg/kg 87.6 11.4 3-carboxy-N-ethyl-N,N- .sup.60.6 6.7*.sup.,#,$ dimethylpropan-1-aminium 2,6- dioxo-1,2,3,6-tetrahydropyrimidine- 4-carboxylate 20 mg/kg

(44) Effects of Meldonium dihydrate, gamma-butyrobetaine and 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium 2,6-dioxo-1,2,3,6-tetrahydropyrimidine-4-carboxylate on infarct size

(45) TABLE-US-00004 TABLE 4 Infarct size, % of control Control 100.0 5.9 Meldonium dihydrate 20 mg/kg 117.9 7.9 Gamma-butyrobetaine 20 mg/kg 87.6 11.4 3-carboxy-N-ethyl-N,N- .sup.56.1 4.4*.sup.,#,$ dimethylpropan-1-aminium dihydrogen phosphate 20 mg/kg

(46) Effects of Meldonium dihydrate, gamma-butyrobetaine and 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium dihydrogen phosphate on infarct size

(47) TABLE-US-00005 TABLE 5 Infarct size, % of control Control 100.0 5.9 Meldonium dihydrate 20 mg/kg 117.9 7.9 Gamma-butyrobetaine 20 mg/kg 87.6 11.4 3-carboxy-N-ethyl-N,N- .sup.62.9 4.7*.sup.,#,$ dimethylpropan-1-aminium 3- carboxypropanoate 20 mg/kg

(48) Effects of Meldonium dihydrate, gamma-butyrobetaine and 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium 3-carboxypropanoate on infarct size

(49) Each values in mentioned Tables from 1-5 represents the means.e.m. of 9-10 animals.

(50) *p<0.05 compared with control group; #p<0.05 compared with Gamma-butyrobetaine group, $p<0.05 compared with Meldonium dihydrate group

(51) As it is presented in Tables 1-5, Meldonium dihydrate treatment at a dose of 20 mg/kg had no therapeutical effect; gamma-butyrobetaine has decreased infarct size by 12.4%.

(52) 3-Carboxy-N-ethyl-N,N-dimethylpropan-1-aminium 2-(acetyloxy)benzoate at dose of 20 mg/kg decreased infarction size by 38.4%.

(53) Carboxy-N-ethyl-N,N-dimethylpropan-1-aminium (2E)-3-carboxyacrylate at dose of 20 mg/kg decreased infarction size by 53.5%.

(54) 3-Carboxy-N-ethyl-N,N-dimethylpropan-1-aminium 2,6-dioxo-1,2,3,6-tetrahydropyrimidine-4-carboxylate at dose of 20 mg/kg decreased infarction size by 39.4%.

(55) 3-Carboxy-N-ethyl-N,N-dimethylpropan-1-aminium dihydrogen phosphate at dose of 20 mg/kg decreased infarction size by 43.9%.

(56) 3-Carboxy-N-ethyl-N,N-dimethylpropan-1-aminium 3-carboxypropanoate at dose of 20 mg/kg decreased infarction size by 37.1%.