Prodrug compound of levosimendan, preparation method and use thereof

Abstract

The invention relates to a prodrug compound of levosimendan as shown by the formula below, and solvates, hydrates, N-oxides, stereoisomers, and pharmaceutically acceptable salts thereof, wherein each of Ra and Rb is selected from hydrogen or C1-C6 alkyl; Rp is selected from a basic group containing N atom or an acidic group containing carboxyl group, phosphate group, sulfate group, and sulfonate group. The prodrug compound overcomes the defects of levosimendan such as poor water solubility, and has an ideal pharmacokinetic profile and excellent prospects for medicament development. ##STR00001##

Claims

1. A prodrug compound of levosimendan, characterized in that, the compound is a compound as shown by formula I, and solvates, hydrates, N-oxides, stereoisomers, and pharmaceutically acceptable salts thereof; ##STR00042## wherein each of Ra and Rb is selected from hydrogen atom or C1-C6 alkyl; and Rp is selected from a basic group containing N atom or an acidic group containing carboxyl group, phosphate group, sulfate group or sulfonate group.

2. The prodrug compound according to claim 1, characterized in that, each of Ra and Rb is hydrogen atom.

3. The prodrug compound according to claim 2, characterized in that, the basic group containing N atom is selected from 4-(morpholin-1-ylmethyl)phenyl, 4-(4-methylpiperazin-1-ylmethyl)phenyl, N,N-dimethylaminomethyl, pyridin-3-yl, 4-(piperidin-1-yl)-piperidin-1-yl, aminomethyl, pyridin-4-ylmethylaminoformylmethyl, or pyrrolidin-1-ylmethyl.

4. The prodrug compound according to claim 2, characterized in that, the acidic group is selected from carboxylmethyl, carboxylethyl, carboxylmethoxy, phosphoryloxymethyl, sulfonyloxymethyl, sulfonylmethyl, phosphoryloxyethyl, sulfonyloxyethyl, or sulfonylethyl.

5. The prodrug compound according to claim 3, characterized in that, the basic group containing N atom is selected from 4-(morpholin-1-ylmethyl)phenyl, 4-(4-methylpiperazin-1ylmethyl)phenyl, and N,N-dimethylaminomethyl, or pyridin-3-yl.

6. The prodrug compound according to claim 4, characterized in that, the acidic group is selected from carboxylmethyl, carboxyl ethyl, phosphoryloxymethyl, sulfonyloxymethyl, or sulfonylmethyl.

7. The prodrug compound according to claim 5, characterized in that, the basic group containing N atom is selected from 4-(morpholin-1-ylmethyl)phenyl or 4-(4-methylpiperazin-1-ylmethyl)phenyl.

8. Use of the prodrug compound according to any one of claims 1 to 7 in the manufacture of a medicament for treating heart failure diseases.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 .sup.1H-NMR spectrum of compound TCX1001-031;

(2) FIG. 2 ESI-MS spectrum of compound TCX1001-031;

(3) FIG. 3 Liquid chromatogram of compound TCX1001-031;

(4) FIG. 4 Change profile of the content of compound TCX1001-031 itself in the pharmacokinetic study;

(5) FIG. 5 Change profile of the content of released levosimendan in the pharmacokinetic study of compound TCX1001-031;

(6) FIG. 6 Metabolic conversion rate of compound TCX1001-031 in the pharmacokinetic study.

DESCRIPTION OF EMBODIMENTS

(7) The embodiments of the invention and the beneficial effects produced will be described in detail below through specific examples, which are intended to help readers better understand the essences and characteristics of the invention, and are not intended to limit the scope of present invention.

(8) The structures of the compounds are characterized by H nuclear magnetic resonance (.sup.1HNMR) and/or mass spectrometry (MS). .sup.1HNMR is determined with Bruker Spectrometer AVIII HD NMR spectrometer (400 MHz), and the chemical shifts (δ) are given in 10.sup.−6 (ppm). The internal standard is tetramethylsilane (TMS). Chemical shift: δ, s: singlet; d: doublet; t: triplet; q: quartet; m: multiplet. Mass spectra is determined with Agilent Accurate-Mass Q-TOF LC/MS mass spectrometer.

(9) Unless otherwise specified, benzyl carbazate is purchased from Shanghai Haohong Biomedical Technology Co., Ltd.; paraformaldehyde is purchased from Shanghai Macklin Biochemical Co., Ltd., diethyl phosphate is purchased from Shanghai Macklin Biochemical Co., Ltd.; 2,2-diethoxyethyl diethyl phosphate is purchased from Alfa Aesar (China) Chemical Co., Ltd.; tert-butoxycarbonylhydrazine is purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd.; (R)-6-((4-aminophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one is homemade in Beijing Chenguang Tongchuang Pharmaceutical Research Institute Co., Ltd. TLC refers to thin layer chromatography; TSTU refers to O-(N-succinimidyl)-N N N′N′-tetramethyltetrafluoroborate urea; DCM refers to dichloromethane; MtOH refers to methanol; DMF refers to dimethylformamide. The specifications of petroleum ether used are in the boiling range of 60-90° C.

(10) The synthesis route for N-((4-(1-(hydroxymethyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)biscyanomethylene)hydrazine (5) as the common intermediate is as follows:

(11) ##STR00020## ##STR00021##

(12) I. Preparation of tert-butyl (R)-(4-((4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)carbamate

(13) ##STR00022##

(14) (R)-6-((4-aminophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (101 g, 0.5 mol), di-tert-butyl dicarbonate (163.5 g, 0.75 mol), triethylamine (76 g, 0.75 mol), and tetrahydrofuran (1 L) were added to a 2 L single-necked flask, and stirred at room temperature for 3 days, until no remaining raw materials detectable by TLC detection. The reaction was filtered, and subjected to rotary evaporation under reduced pressure to remove the solvent. Under stirring, petroleum ether (300 mL) was added to the residue, stirred at room temperature for 20 minutes, filtered, and dried, to obtain 84 g of the title compound as a light brown solid (yield: 54%). .sup.1H-NMR (400 MHz, DMSO-d.sub.6) δ: 10.86 (s, 1H), 9.55 (s, 1H), 7.66-7.70 (m, 2H), 7.50-7.53 (m, 2H), 3.35-3.38 (m, 1H), 2.63-2.69 (m, 1H), 2.18-2.23 (m, 1H), 1.48 (s, 9H), 1.05 (d, 3H, J=7.2 Hz); ESI-MS (m/z): Calcd. For C.sub.16H.sub.21N.sub.3O.sub.3+H: 304.16; Found: 304.17.

(15) II. Preparation of tert-butyl (R)-(4-(1-(hydroxymethyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)carbamate

(16) ##STR00023##

(17) Tert-butyl (R)-(4-((4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)carbamate (100 g, 0.33 mol) and methanol (2.5 L) were added to a 3 L three-necked flask, paraformaldehyde (128.2 g, 4.27 mol) and potassium carbonate (95.3 g, 0.69 mol) were added under stirred, and stirred at room temperature overnight. The reaction was completed under TLC monitoring. The solvent was removed by rotary evaporation under reduced pressure. The concentrate was purified by column chromatography (petroleum ether:ethyl acetate, v:v=1:1), to obtain 60.0 g of the title compound as a white solid (yield: 55%). .sup.1H-NMR (400 MHz, DMSO-d.sub.6) δ: 9.56 (s, 1H), 7.74 (d, 2H, J=8.5 Hz), 7.53 (d, 2H, J=8.5 Hz), 6.13-6.17 (m, 1H), 5.10-5.15 (m, 1H), 4.95-5.00 (m, 1H), 3.35-3.41 (m, 1H), 2.67-2.73 (m, 1H), 2.28-2.33 (m, 1H), 1.48 (s, 9H), 1.06 (d, 3H, J=7.2 Hz); ESI-MS (m/z): Calcd. For C.sub.17H.sub.23N.sub.3O.sub.4+H: 334.17; Found: 334.18.

(18) III. Preparation of (R)-6-((4-aminophenyl)-2-(hydroxymethyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one hydrochloride

(19) ##STR00024##

(20) Tert-butyl (R)-(4-((4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)carbamate (20 g, 0.06 mol) was added to a 500 mL three-necked flask, and 50 mL of ethyl acetate was added. After stirring at room temperature for 5 minutes, a solution of hydrogen chloride in ethyl acetate (100 mL, 20 wt %) was dropwise added. After dropwise addition, it was stirred at room temperature for 2 hours, and No raw materials remained detectable by TLC detection. The solvent was removed by rotary evaporation under reduced pressure, to obtain 23 g of yellow solid. The crude product was directly subjected to the next reaction.

(21) IV. Preparation of (R)-N-((4-(1-(hydroxymethyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)biscyanomethylene)hydrazine

(22) ##STR00025##

(23) 23 g of compound (R)-6-((4-aminophenyl)-2-(hydroxymethyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one hydrochloride obtained in step 3 was transferred into a 2 L flask with 2.6% aqueous solution of hydrochloric acid (500 mL) and cooled in an ice-water bath. At 5˜10° C., a solution formed by dissolving 7.05 g of sodium nitrite (0.1 mol) into 50 mL of water was added to the above reaction solution. To 50 mL of water, malononitrile (6.2 g, 0.094 mol) was dissolved, and added to the above reaction solution at 5˜10° C. After addition, the ice-water bath was removed. After stirring at room temperature for 2 hours, a 20% aqueous solution of sodium acetate was slowly added, to adjust pH value to 5˜6. A yellow suspension was obtained, filtered, washed with water, and dried, to obtain 11.2 g of the title compound as a yellow solid with a two-step yield of 60%. .sup.1H-NMR (400 MHz, DMSO-d.sub.6) δ: 13.1 (br, s, 1H), 7.88-7.92 (m, 2H), 7.52-7.55 (m, 2H), 5.13 (d, 1H, J=10 Hz), 5.00 (d, 1H, J=10 Hz), 3.37-3.43 (m, 1H), 2.71-2.77 (m, 1H), 2.31-2.35 (m, 1H), 1.08 (d, 3H, J=7.2 Hz); ESI-MS (m/z): Calcd. For C.sub.15H.sub.14N.sub.6O.sub.2+H: 311.12; Found: 311.13.

Example 1: Preparation of (R)-(3-((4-(2-(dicyanomethylene)hydrazino)phenyl)-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl)methyl nicotinate (TCX-1001-029)

(24) ##STR00026##

(25) The compound (R)-N-((4-(1-(hydroxymethyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)biscyanomethylene)hydrazine (0.26 g, 0.84 mmol), TSTU (0.3 g, 1 mmol), and nicotinic acid (0.11 g, 0.92 mmol) were added to a 50 mL single-necked flask, 5 mL of dichloromethane was added, then triethylamine (0.3 g, 3 mmol) was added under stirring, and stirred at room temperature for 5 hours. After no remaining raw materials were detectable by TLC detection, the solvent was removed by rotary evaporation under reduced pressure. The resulting concentrate was purified by Pre-HPLC (eluent: DCM/MeOH, v:v=50/1), to obtain 0.12 g of the title compound as a yellow solid (yield: 34%). .sup.1H-NMR (400 MHz, DMSO-d.sub.6) δ: 13.1 (br, s, 1H), 9.08 (s, 1H), 8.83-8.85 (m, 1H), 8.27-8.30 (m, 1H), 7.90-7.92 (m, 2H), 7.54-7.60 (m, 3H), 5.98-6.08 (m, 2H), 3.50-3.54 (m, 1H), 2.91-2.97 (m, 1H), 2.51-2.53 (m, 1H), 1.13 (d, 3H, J=7.2 Hz); ESI-MS (m/z): Calcd. For C.sub.21H.sub.17N.sub.7O.sub.3+H: 416.14; Found: 416.15.

Example 2: Preparation of (R)-(3-((4-(2-(dicyanomethylene)hydrazino)phenyl)-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl)methyl glycinate (TCX-1001-032)

(26) ##STR00027##

(27) The compound (R)-N-((4-(1-(hydroxymethyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)biscyanomethylene)hydrazine (0.2 g, 0.64 mmol), glycine (0.12 g, 1.6 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (0.31 g, 1.6 mmol), 4-dimethylpyridine (0.08 g, 0.64 mmol), and 5 mL of N,N-dimethylacetamide were successively added to a 50 mL single-necked flask, and stirred at room temperature overnight. The solvent was removed by rotary evaporation under reduced pressure, and the concentrate was purified by column chromatography (eluent: DCM/MeOH, v:v=30/1), to obtain 70 mg of the title compound as a yellow solid (yield: 30%). .sup.1H-NMR (400 MHz, DMSO-d.sub.6) δ: 13.2 (br, s, 1H), 7.75-7.81 (m, 2H), 7.36-7.41 (m, 2H), 5.95-6.05 (m, 2H), 3.48-3.54 (m, 1H), 3.17-3.23 (m, 2H), 2.95-3.02 (m, 1H), 2.67-2.69 (m, 1H), 1.14 (d, 3H, J=7.2 Hz); ESI-MS (m/z): Calcd. For C.sub.19H.sub.21N.sub.7O.sub.3+H: 368.14; Found: 368.15.

Example 3: Preparation of (R)-(3-((4-(2-(dicyanomethylene)hydrazino)phenyl)-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl)methyl 2-pyrrolidin-1-yl-acetate (TCX-1001-039)

(28) ##STR00028##

(29) The compound (R)-N-((4-(1-(hydroxymethyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)biscyanomethylene)hydrazine (300 mg, 0.97 mmol), 2-(pyrrolidin-1-yl)acetic acid hydrochloride (321 mg, 1.94 mmol), EDCI (465 mg, 2.43 mmol), and DMAP (118 mg, 0.97 mmol) were dissolved in DMA (10 mL), and stirred at room temperature for 18 hours. To the system, ethyl acetate (10 mL) and water (10 mL) were added, stirred, and separated. The organic phase was washed with water (3×10 mL), and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation under reduced pressure, and the crude product was purified by Pre-HPLC, to obtain 210 mg of the title compound as a yellow solid product (yield: 51.3%). .sup.1H-NMR (400 MHz, DMSO-d.sub.6) δ: 13.0 (br, s, 1H), 7.80-7.74 (m, 2H), 7.38-7.33 (m, 2H), 6.05-5.95 (m, 2H), 3.52-3.46 (m, 1H), 3.35(s, 2H), 3.02-2.95 (m, 1H), 2.68-2.66 (m, 1H), 2.61-2.58 (m, 2H), 1.72-1.70 (m, 2H), 1.13 (d, 3H, J=7.2 Hz); ESI-MS (m/z): Calcd. For C.sub.20H.sub.20N.sub.7O.sub.3+H: 407.16; Found: 407.217.

Example 4: Preparation of (R)-(3-((4-(2-(dicyanomethylene)hydrazino)phenyl)-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl)methyl N,N-dimethylglycinate (TCX-1001-030)

(30) ##STR00029##

(31) The compound (R)-N-((4-(1-(hydroxymethyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)biscyanomethylene)hydrazine (0.2 g, 0.64 mmol), N,N-dimethylglycinate (0.16 g, 1.6 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (0.31 g, 1.6 mmol), 4-dimethylpyridine (0.08 g, 0.64 mmol), and 5 mL of N,N-dimethylacetamide were successively added to a 50 mL single-necked flask, and stirred at room temperature overnight. The solvent was removed by rotary evaporation under reduced pressure, and the concentrate was purified by column chromatography (eluent: DCM/MeOH, v:v=30/1), to obtain 0.06 g of the title compound as a yellow solid (yield: 24%). .sup.1H-NMR (400 MHz, DMSO-d.sub.6) δ: 13.0 (br, s, 1H), 7.74-7.80 (m, 2H), 7.33-7.38 (m, 2H), 5.95-6.05 (m, 2H), 3.46-3.52 (m, 1H), 3.19-3.25 (m, 2H), 2.95-3.02 (m, 1H), 2.75-2.85 (m, 6H), 2.66-2.68 (m, 1H), 1.13 (d, 3H, J=7.2 Hz); ESI-MS (m/z): Calcd. For C.sub.19H.sub.21N.sub.7O.sub.3+H: 396.17; Found: 396.18.

Example 5: Preparation of (R)-(3-((4-(2-(dicyanomethylene)hydrazino)phenyl)-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl)methyl 4-(morpholin-1-ylmethyl)benzoate (TCX-1001-031)

(32) ##STR00030##

(33) The compound (R)-N-((4-(1-(hydroxymethyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)biscyanomethylene)hydrazine (0.2 g, 0.64 mmol) was dissolved in 2 mL of pyridine, then 4-chloromethylbenzoyl chloride (0.22 g, 1.16 mmol) was added, and stirred at room temperature for 2 hours. The reaction solution was diluted with 30 mL of ethyl acetate. Then the organic phase was successively washed with distilled water and saturated brine, dried over anhydrous sodium sulfate, and spin-dried. The resulting concentrate was dissolved in DMF (3 mL), sodium iodide (0.05 g) was added, followed by morpholine (0.13 g, 1.5 mmol), and stirred at room temperature overnight. The solvent was removed by rotary evaporation under reduced pressure, and the resulting concentrate was purified by column chromatography (eluent: DCM/MeOH, v:v=70/1), to obtain 0.11 g of the title compound as a yellow solid (yield: 33%). .sup.1H-NMR (400 MHz, DMSO-d.sub.6) δ: 13.1 (br, s, 1H), 7.95-8.03 (m, 2H), 7.77-7.83 (m, 2H), 7.53-7.63 (m, 2H), 7.38-7.42 (m, 2H), 5.92-6.06 (m, 2H), 3.56-4.51 (m, 6H), 3.35-3.53 (m, 2H), 2.71-3.15 (m, 4H), 2.41-2.46 (m, 1H), 1.13 (d, 3H, J=7.2 Hz); ESI-MS (m/z): Calcd. For C.sub.27H.sub.27N.sub.7O.sub.4+H: 514.21; Found: 514.22.

Example 6: Preparation of (R)-(3-((4-(2-(dicyanomethylene)hydrazino)phenyl)-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl)methyl 4-((4-methylpiperazin-1-yl)methyl)benzoate (TCX-1001-033)

(34) ##STR00031##

(35) The compound (R)-N-((4-(1-(hydroxymethyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)biscyanomethylene)hydrazine (0.2 g, 0.64 mmol) was dissolved in 2 mL of pyridine, then 4-chloromethylbenzoyl chloride (0.22 g, 1.16 mmol) was added, and stirred at room temperature for 2 hours. The reaction solution was diluted with 30 mL of ethyl acetate, then successively washed with distilled water and saturated brine. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. The concentrate was dissolved in DMF (3 mL), sodium iodide (0.05 g) was added, followed by N-methylpiperazine (0.15 g, 1.5 mmol), and stirred at room temperature overnight. The solvent was removed by rotary evaporation under reduced pressure, and the concentrate was purified by column chromatography (eluent: DCM/MeOH, v:v=30/1), to obtain 0.05 g of the title compound as a yellow solid (yield: 15.0%). .sup.1H-NMR (400 MHz, DMSO-d.sub.6) δ: 13.2 (br, s, 1H), 7.95-7.88 (m, 2H), 7.83-7.78 (m, 2H), 7.59-7.54 (m, 2H), 7.33-7.29 (m, 2H), 6.06-5.90 (m, 2H), 3.70-3.58 (m, 2H), 3.52-3.48 (m, 1H), 3.43-3.27 (m, 5H), 3.04-2.98 (m, 1H), 2.93-2.80 (m, 3H), 2.69 (s, 3H), 2.47-2.41 (m, 1H), 1.13 (d, 3H, J=7.2 Hz); ESI-MS (m/z): Calcd. For C.sub.27H.sub.27N.sub.7O.sub.4+H: 527.12; Found: 526.80.

Example 7: Preparation of (R)-(3-((4-(2-(dicyanomethylene)hydrazino)phenyl)-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl)methyl 4-(piperidin-1-yl)piperidin-1-ylformate (TCX-1001-037)

(36) ##STR00032##

(37) 10 mL of anhydrous tetrahydrofuran, 1.24 g of (R)-N-((4-(1-(hydroxymethyl)-4-methyl oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)biscyanomethylene)hydrazine, 0.71 g of N,N′-carbonyldiimidazole, and 0.67 g of 4-piperidylpiperidine were successively added to a 50 mL single-necked flask, stirred at room temperature for 48 hours, and the reaction was completed by TLC monitoring (developer: dichloromethane/methanol=10/1). The solvent was removed by rotary evaporation under reduced pressure, and the concentrate was purified by column chromatography (eluent: DCM/MeOH, v:v=30/1), to obtain 0.50 g of the title compound as a yellow solid (yield: 24.8%). .sup.1H-NMR (400 MHz, DMSO-d.sub.6) δ: 12.35 (s, 1H), 7.76-7.80 (d, 2H), 7.40-7.43 (d, 2H), 5.94-6.02 (m, 2H), 4.25 (m, 2H), 3.43-3.49 (d, 1H), 2.70-2.76 (d, 3H), 2.60-2.65 (d, 1H), 2.5 (t, 4H), 2.3-2.45 (m, 1H), 1.8 (m, 2H), 1.51-1.65 (m, 4H), 1.3-1.5 (m, 4H), 1.08-1.14 (d, 3H); ESI-MS (m/z): Calcd. For C.sub.26H.sub.32N.sub.8O.sub.3+H: 505.6; Found: 505.61.

Example 8: Preparation of (R)-(3-((4-(2-(dicyanomethylene)hydrazino)phenyl)-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl)methyl 2-carboxyl acetate (TCX-1001-041)

(38) ##STR00033##

(39) Malonic acid (0.21 g, 2.06 mmol), triethylamine (0.36 mL, 2.6 mmol), DMAP (13 mg, 0.1 mmol), and isopropenyl chloroformate (0.08 mL, 0.73 mmol) were successively added to 10 mL of dichloromethane. After cooling to 0° C. in an ice-water bath, a solution of (R)-N-((4-(1-(hydroxymethyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)biscyanomethylene) hydrazine (0.16 g, 0.52 mmol) in dichloromethane (10 mL) was added. The reaction solution was stirred at 0° C. for 2 hours, then poured into 3 mL of 10% aqueous acetic acid solution, and stirred at room temperature for 0.5 hours. The reaction solution was successively washed with distilled water and saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation under reduced pressure, and the concentrate was purified by column chromatography (dichloromethane/methanol/formic acid, v:v:v=20/1/0.05), to obtain 80 mg of the title compound as a yellow solid (yield: 39.0%). .sup.1H-NMR (400 MHz, DMSO-d.sub.6) δ: 14.1 (br, s, 1H), 13.1 (br, s, 1H), 7.90-7.92 (m, 2H), 7.54-7.60 (m, 2H), 5.98-6.08 (m, 2H), 3.55-3.60 (m, 1H), 3.4 (s, 2H), 2.91-2.97 (m, 1H), 2.51-2.53 (m, 1H), 1.13 (d, 3H, J=7.2 Hz); ESI-MS (m/z): Calcd. For C.sub.21H.sub.17N.sub.7O.sub.3+H: 397.12; Found: 397.15.

Example 9: Preparation of (R)-(3-((4-(2-(dicyanomethylene)hydrazino)phenyl)-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl)methyl 3-carboxylpropionate (TCX-1001-034)

(40) ##STR00034##

(41) Succinic acid (0.488 g, 4.12 mmol), triethylamine (0.72 mL, 5.2 mmol), 4-dimethylaminopyridine (DMAP) (26 mg, 0.2 mmol), and isopropenyl chloroformate (0.16 mL, 1.46 mmol) were dissolved in 10 mL of dichloromethane. After cooling to 0° C. in an ice-water bath, a solution of (R)-N-((4-(1-(hydroxymethyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)biscyanomethylene) hydrazine (0.32 g, 1.04 mmol) in dichloromethane (10 mL) was added. The reaction solution was stirred at 0° C. for 2 hours, then poured into 3 mL of 10% aqueous acetic acid solution, and stirred at room temperature for 0.5 hours. The reaction solution was successively washed with distilled water and saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation under reduced pressure, and the concentrate was purified by column chromatography (dichloromethane/methanol/formic acid, v:v:v=20/1/0.05), to obtain 0.14 g of the title compound as a yellow solid (yield: 33%). .sup.1H-NMR (400 MHz, DMSO-d.sub.6) δ: 14.3 (br, s, 1H), 13.4 (br, s, 1H), 7.91-7.93 (m, 2H), 7.55-7.61 (m, 2H), 5.99-6.10 (m, 2H), 3.51-3.54 (m, 1H), 2.91-2.97 (m, 1H), 2.75-2.51 (m, 5H), 1.13 (d, 3H, J=7.2 Hz); ESI-MS m/z: Calcd. For C.sub.21H.sub.17N.sub.7O.sub.3+H: 411.14; Found: 411.15.

Example 10: Preparation of (R)-(3-((4-(2-(dicyanomethylene)hydrazino)phenyl) methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl)methyl carboxylmethyloxyformate (TCX-1001-035)

(42) ##STR00035##

(43) (R)-N-((4-(1-(hydroxymethyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)biscyanomethylene)hydrazine (0.16 g, 0.52 mmol), N,N'-carbonyldiimidazole (CDI) (95 mg, 0.57 mmol), and 2-hydroxyacetic acid (40 mg, 0.57 mmol) were dissolved in 5 mL of THF, stirred at room temperature for 48 hours, and the reaction was completed by TLC monitoring. The solvent was removed by rotary evaporation under reduced pressure, and the concentrate was purified by column chromatography (dichloromethane/methanol/formic acid, v:v:v=20/1/0.05), to obtain 60 mg of the title compound as a yellow solid (yield: 28%). .sup.1H-NMR (400 MHz, DMSO-d.sub.6) δ: 14.1 (br, s, 1H), 13.1 (br, s, 1H), 7.90-7.92 (m, 2H), 7.56-7.61 (m, 2H), 5.97-6.08 (m, 2H), 5.2 (s, 2H), 3.50-3.54 (m, 1H), 2.91-2.97 (m, 1H), 2.52-2.54 (m, 1H), 1.14 (d, 3H, J=7.2 Hz); ESI-MS (m/z): Calcd. For C.sub.21H.sub.17N.sub.7O.sub.3+H: 413.14; Found: 413.15.

Example 11: Preparation of (R)-(3-((4-(2-(dicyanomethylene)hydrazino)phenyl)-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl)methyl phosphoryloxyacetate (TCX-1001-040)

(44) ##STR00036##

(45) (R)-N-((4-(1-(hydroxymethyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)biscyanomethylene)hydrazine (0.26 g, 0.84 mmol), TSTU (0.3 g, 1 mmol), and (phosphoryloxy)acetic acid (0.16 g, 1 mmol) were added to a 50 mL single-necked flask, 5 mL of dichloromethane was added, then triethylamine (0.3 g, 3 mmol) was added under stirring, and stirred at room temperature for 15 hours. No raw materials remained detectable by TLC detection. The solvent was removed by rotary evaporation under reduced pressure, and the concentrate was separated by Pre-HPLC, to obtain 0.11 g of the title compound, as a yellow solid (yield: 29%). .sup.1H-NMR (400 MHz, DMSO-d.sub.6) δ: 13.1 (br, s, 1H), 7.90-7.92 (m, 2H), 7.54-7.60 (m, 2H), 5.98-6.08 (m, 2H), 5.7 (br, s, 2H), 4.3 (s, 2H), 3.50-3.54 (m, 1H), 2.91-2.97 (m, 1H), 2.51-2.53 (m, 1H), 1.13 (d, 3H, J=7.2 Hz); ESI-MS (m/z): Calcd. For C.sub.21H.sub.17N.sub.7O.sub.3+H: 449.0; Found: 449.10.

Example 12: Preparation of (R)-(3-((4-(2-(dicyanomethylene)hydrazino)phenyl)-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl)methyl sulfonyloxyacetate (TCX-1001-038)

(46) ##STR00037##

(47) (R)-N-((4-(1-(hydroxymethyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)biscyanomethylene)hydrazine (0.52 g, 1.68 mmol), TSTU (0.6 g, 2 mmol), and (sulfonyloxy)acetic acid (0.32 g, 2 mmol) were added to a 50 mL single-necked flask, 15 mL of dichloromethane was added, followed by triethylamine (0.6 g, 6 mmol), and stirred at room temperature for 15 hours. No raw materials remained detectable by TLC monitoring. The solvent was removed by rotary evaporation under reduced pressure, and the concentrate was separated by Pre-HPLC, to obtain 0.15 g of the title compound as a yellow solid (yield: 20%). .sup.1H-NMR (400 MHz, DMSO-d.sub.6) δ: 15.1 (br, s, 1H), 13.1 (br, s, 1H), 7.90-7.92 (m, 2H), 7.54-7.60 (m, 2H), 5.98-6.08 (m, 2H), 4.2 (s, 2H), 3.52-3.56 (m, 1H), 2.91-2.97 (m, 1H), 2.52-2.54 (m, 1H), 1.13 (d, 3H, J=7.2 Hz); ESI-MS (m/z): Calcd. For C.sub.21H.sub.17N.sub.7O.sub.3+H: 449.08; Found: 449.10.

Example 13: Preparation of (R)-(3-((4-(2-(dicyanomethylene)hydrazino)phenyl) methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl)methyl 3-oxo-3-((pyridin ylmethyl)amino)propionate (TCX-1001 -036)

(48) ##STR00038##

(49) (1) Preparation of ethyl 3-oxo-3-((pyridin-4-ylmethyl)amino)propionate

(50) ##STR00039##

(51) Ethyl chloroformylacetate (5.8 mL, 46.23 mmol) and 4-pyridinylmethylamine (5 g, 46.23 mmol) were dissolved in 200 mL of THF. The reaction solution was cooled to 0° C., then triethylamine (9.5 mL, 69.35 mmol) was slowly dropwsie added. After dropwise addition, the reaction solution was stirred at room temperature for 2 hours, and the reaction was completed by TLC monitoring. The solvent was removed by rotary evaporation under reduced pressure, the concentrate was dissolved in 200 mL of ethyl acetate, washed with distilled water and saturated brine successively, and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation under reduced pressure, and the concentrate was purified by column chromatography (dichloromethane/methanol, v:v=50/1), to obtain 5 g of the target product ethyl 3-oxo-3-((pyridin-4-ylmethyl)amino)propionate as a brown solid (yield: 48%). .sup.1H-NMR (400 MHz, CDCl.sub.3) δ: 8.57-8.55 (m, 1H), 8.13-8.10 (m, 2H), 7.35-7.30 (m, 2H), 4.62-4.60 (m, 2H), 4.26-4.18 (m, 2H), 3.4 (s, 2H), 1.32-1.37 (t, 3H, J=7.2 Hz); ESI-MS (m/z): Calcd. For C.sub.21H.sub.17N.sub.7O.sub.3+H: 223.10; Found: 222.90.

(52) (2) Preparation of 3-oxo-3-((pyridin-4-ylmethyl)amino)propanoic acid

(53) ##STR00040##

(54) The compound ethyl 3-oxo-3-((pyridin-4-ylmethyl)amino)propionate (2.2 g, 10 mmol) was dissolved in a mixed solvent of 10 mL methanol, 10 mL THF, and 5 mL water, and lithium hydroxide monohydrate (0.84 g, 20 mmol) was added. The reaction solution was stirred at room temperature for 16 hours, and the reaction was completed by TLC monitoring. The solvent was removed by rotary evaporation under reduced pressure. The system was diluted with 10 mL of water, adjusted with 3N HCl aqueous solution to adjust to pH 5˜6, then extracted with ethyl acetate (20 mL×3), and the organic phases were combined. The organic phase was successively washed with distilled water and saturated brine, and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation under reduced pressure, and the concentrate was purified by column chromatography (dichloromethane/methanol/formic acid, v:v:v=20/1/0.05), to obtain 1.5 g of target product 3-oxo-3-((pyridin-4-ylmethyl)amino)propionic acid (yield: 77%). .sup.1H-NMR (400 MHz, DMSO-d.sub.6) δ: 13.1 (br, s, 1H), 8.67-8.65 (m, 1H), 8.18-8.16(m, 2H), 7.40-7.35 (m, 2H), 4.62-4.60 (m, 2H), 3.4 (s, 2H); ESI-MS (m/z): Calcd. For C.sub.21H.sub.17N.sub.7O.sub.3+H: 195.07; Found: 195.10.

(55) (3) Preparation of (R)-(3-((4-(2-(dicyanomethylene)hydrazino)phenyl)-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl)methyl 3-oxo-3-((pyridin-4-ylmethyl)amino)propionate (TCX-1001-0361)

(56) ##STR00041##

(57) The compound (R)-N-((4-(1-(hydroxymethyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)biscyanomethylene)hydrazine (0.26 g, 0.84 mmol), TSTU (0.3 g, 1 mmol), and 3-oxo-3-((pyridin-4-ylmethyl)amino)propanoic acid (0.18 g, 0.92 mmol) were added to a 50 mL single-necked flask, 5 mL of dichloromethane was added, then triethylamine (0.3 g, 3 mmol) was added under stirring, and stirred at room temperature for 5 hours. After no remaining raw materials were detectable by TLC detection, the solvent was removed by rotary evaporation under reduced pressure. The resultant was purified by column chromatography (eluent: DCM/MeOH, v:v=50/1), to obtain 0.11 g of the title compound as a yellow solid (yield: 27%). .sup.1H-NMR (400 MHz, DMSO-d.sub.6) δ: 13.2 (br, s, 1H), 8.71-8.68 (m, 1H), 8.32-8.29 (m, 2H), 7.95-7.93 (m, 2H), 7.65-7.62 (m, 2H), 7.45-7.40 (m, 2H), 6.08-5.98 (m, 2H), 4.62-4.60 (m, 2H), 3.54-3.50 (m, 1H), 3.4 (s, 2H), 2.98-2.92 (m, 1H), 2.53-2.51 (m, 1H), 1.15 (d, 3H, J=7.2 Hz); ESI-MS (m/z): Calcd. For C.sub.21H.sub.17N.sub.7O.sub.3+H: 487.14; Found: 487.15.

(58) The present invention solves the problem of poor solubility of levosimendan in most solvents (including water). When preparing a liquid formulation of levosimendan, the defects of having to use an organic solvent and adding a toxic co-solvent at the same time are avoided.

(59) Meanwhile, due to the solubility limitation of levosimendan itself, it is difficult to make oral formulations. The novel compounds designed and prepared in the invention solve this defect and can be dispersed in water, that is, they can be dispersed in gastric juice or intestinal juice in the form of molecules or ions, which is beneficial to the oral absorption in organisms, and can solve the problem that it is difficult to prepare levosimendan into oral formulations.

Example 14: Sample Stability Test

(60) After the compound samples prepared in Examples 1-13 above were placed (in naked form) for 10 days under conditions of strong light (4500Lx±500Lx) irradiation, high temperature (60° C.±2° C.) and high humidity (90%±5%), there were no obvious changes in appearance and related substances. Thus, the compound samples prepared in Examples 1-13 are stable through preliminary investigation.

Example 15: Sample Characterization Experiment

(61) The chromatographic conditions for detection were as follows: octadecylsilane-bonded silica gel was used as the filler; phosphate buffer (1.56 g of sodium dihydrogen phosphate was weighted, 1000 ml of water was added for dissolution, and pH was adjusted to 3.5 with phosphoric acid)-methanol (30:70) was used as the mobile phase; the flow rate was 1.0 mL per minute; the column temperature was 30° C.; the detection wavelength was 254 nm; and the injection volume was 10 μL.

(62) Test solution: an appropriate amount of the product (TCX-H1001-031) was accurately weighted, and dissolved in methanol to make a solution containing 0.5 mg of sample per 1 ml.

(63) The test solution was accurately weighted, and injected into a liquid chromatograph, to record the chromatogram.

(64) FIGS. 1-3 are .sup.1H-NMR spectrum, ESI-MS spectrum, and liquid chromatogram of compound TCX-H1001-031 prepared in Example 5, respectively.

Example 16: Pharmacokinetic Study for Compound

(65) FIGS. 4-6 are the change profile of the content of compound TCX1001-031 itself in plasma after administration in the pharmacokinetic study, the change profile of the content of released levosimendan, and the pharmacokinetic metabolic conversion rate, respectively. It can be seen from the figures that TCX-1001-031 can be completely metabolized within 1 minute, that is, its content drops to 0 within 1 minute; while the content of the original drug levosimendan released by the metabolism begins to increase rapidly, and then gradually increases, and becomes completely stable with the prodrug metabolism, indicating that the metabolic conversion rate of TCX-1001-031 into the raw material within 10 minutes is about 40%, and it is a high-quality prodrug molecule.

(66) Table 3 shows the results of in vivo administration of some specific compounds of present invention, indicating that they are completely metabolized within 1-10 minutes respectively, and the content of levosimendan increases accordingly, and it is stable with the completion of the prodrug metabolism. The conversion rates of these compounds to levosimendan in plasma within 10 minutes are approximately 30-45%.

(67) TABLE-US-00003 TABLE 3 Metabolic conversion rate Compound Metabolic conversion rate  1 (TCX1001-029) 39.4%  2 (TCX1001-031) 40.0%  3 (TCX1001-032) 35.1%  4 (TCX1001-030) 38.9%  5 (TCX1001-037) 35.6%  6 (TCX1001-033) 44.6%  7 (TCX1001-036) 36.9%  8 (TCX1001-039) 35.1%  9 (TCX1001-041) 35.6% 10 (TCX1001-034) 34.9% 11 (TCX1001-035) 37.8% 12 (TCX1001-040) 37.6% 13 (TCX1001-038) 36.4%

(68) From the results of the pharmacokinetic study, among the metabolic conversion rates of 13 prodrug molecules in plasma within 10 minutes, the metabolic conversion rates of Compounds 1, 2, 4, and 6 are the highest. According to current data, the above-mentioned 13 compound molecules all have the potential to serve as prodrugs.

(69) The Examples only represent several embodiments of the invention, and the descriptions thereof are more specific and detailed, but should not be construed as a limitation to the scope of the invention. It should be pointed out that for those skilled in the art, without departing from the concept of the invention, some alterations and modifications can also be made, which all fall into the scope of the invention. Thus, the scope of the invention is defined by the appended claims.