阅读说明：本技术 一种左西孟旦的前药化合物、制备方法及其应用 (Levosimendan prodrug compound, preparation method and application thereof ) 是由 黄少林 郭长彬 李伟 马良 杨清娟 宋鹏飞 范井旺 毛晓松 于 2020-12-08 设计创作，主要内容包括：本发明涉及一种左西孟旦的前药化合物及其溶剂合物、水合物、N-氧化物、立体异构体和药学上可接受的盐,其结构式如下所示,其中R_a和R_b分别选自氢或C1-C6烷基；Rp选自含有N原子的碱性基团或含有羧基、磷酸酯基、硫酸酯基或磺酸基的酸性基团；所述前药化合物解决了左西孟旦水溶性差等缺点,并且具有理想的药物代谢动力学曲线,具有极佳的药物应用前景。(The invention relates to a levosimendan prodrug compound and a solvate, a hydrate, an N-oxide, a stereoisomer and a pharmaceutically acceptable salt thereof, wherein the structural formula of the levosimendan prodrug compound is shown as follows, wherein R is a And R b Are independently selected from hydrogen or C1-C6 alkyl; rp is selected from basic group containing N atom or carboxyl and phosphoric acidAcid groups of ester groups, sulfate groups or sulfonic acid groups; the prodrug compound overcomes the defects of poor water solubility of levosimendan and the like, has an ideal pharmacokinetic curve and has an excellent medicament application prospect.)

1. A prodrug compound of levosimendan, which is a compound having a structure represented by the general formula I, a solvate, a hydrate, an N-oxide, a stereoisomer, and a pharmaceutically acceptable salt thereof;

wherein Ra and Rb are respectively selected from hydrogen atoms or alkyl groups of C1-C6;

rp is selected from a basic group containing N atoms or an acidic group containing carboxyl, phosphate, sulfate and sulfonic acid groups.

2. The prodrug compound of claim 1, wherein Ra and Rb are each a hydrogen atom.

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

4. The prodrug compound of claim 2 wherein the acidic group is selected from the group consisting of carboxymethyl, carboxyethyl, carboxymethoxy, phosphoryloxymethyl, sulfonyloxymethyl, sulfomethyl, phosphoryloxyethyl, sulfonyloxyethyl, and sulfoethyl.

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

6. The prodrug compound of claim 4 wherein the acidic group is selected from the group consisting of carboxymethyl, carboxyethyl, phosphoryloxymethyl, sulfonyloxymethyl, and sulfomethyl.

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

8. A process for the preparation of a prodrug compound of levosimendan according to claim 1, comprising the steps of:

(1) tert-butyl (R) - (4- ((4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) carbamate is prepared by reacting di-tert-butyl dicarbonate with (R) -6- ((4-aminophenyl) -5-methyl-4, 5-dihydropyridazin-3 (2H) -one;

(2) reacting tert-butyl (R) - (4- ((4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) carbamate with paraformaldehyde in the presence of a base to obtain tert-butyl (R) - (4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) carbamate;

(3) converting (R) - (4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) carbamic acid tert-butyl ester with de-Boc groups under acidic conditions to (R) -6- ((4-aminophenyl) -2- (hydroxymethyl) -5-methyl-4, 5-dihydropyridazin-3 (2H) -one;

(4) reacting (R) -6- ((4-aminophenyl) -2- (hydroxymethyl) -5-methyl-4, 5-dihydropyridazin-3 (2H) -one with sodium nitrite and malononitrile under an acidic condition to prepare (R) -N- ((4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) dicyanomethylene) hydrazine;

(5) and (R) -N- ((4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) dicyanomethylene) hydrazine prepared in the step (4) is respectively reacted with carboxylic acid of a corresponding substituent or an activated form thereof to obtain the prodrug compound.

9. Use of the prodrug compound of any one of claims 1 to 7 for the preparation of a medicament for the treatment of a heart failure disease.

10. The process of claim 8 wherein said intermediate of steps (2), (3) and (4) is tert-butyl (R) - (4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) carbamate, use of (R) -6- ((4-aminophenyl) -2- (hydroxymethyl) -5-methyl-4, 5-dihydropyridazin-3 (2H) -one and (R) -N- ((4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) dicyanomethylene) hydrazine for the preparation of a medicament for the treatment of heart failure disorders.

Technical Field

The invention relates to a levosimendan prodrug compound, a preparation method and application thereof, belonging to the fields of chemical synthesis and medicaments.

Technical Field

Acute heart failure (heart failure) refers to the clinical syndrome of a series of symptoms such as reduction of myocardial contractility, reduction of cardiac output, increase of pulmonary circulation pressure, increase of peripheral circulation resistance, and insufficient tissue and organ perfusion, wherein the symptoms and signs of heart failure occur or worsen rapidly. The annual fatality rate of acute heart failure in China is as high as 34 percent (the recent prognosis condition and influencing factors of the elderly patients with acute decompensation heart failure, Chinese medicine guidance, 2017, 14 (14): 52-55).

According to the basic diagnosis and treatment guidelines for acute heart failure (2019) in China, acute heart failure needs to be treated by medicines such as positive inotropic drugs, diuretics, vasodilators and the like. Commonly used inotropic drugs include calcium sensitizers (e.g., levosimendan), beta adrenergic receptor agonists (e.g., dobutamine), phosphodiesterase inhibitors (e.g., milrinone), and the like. (Primary guideline for acute Heart failure (2019), J. Zhonghua general practitioner, 2019, 10, 925-930).

Levosimendan is used clinically mainly for treating various acute heart failure diseasesSymptoms, developed by the company orleon (Orion) finland, first appeared on the market in sweden in 10 months of 2000. The chemical name of which is (R) - [ [4- (1,4,5, 6-tetrahydro-4-methyl-6-oxo-pyridazin-3-yl) phenyl]-hydrazono group]-malononitrile, of formula: c₁₄H₁₂N₆O, molecular weight: 280.28, CAS. NO.131741-08-7, having a molecular structure represented by the formula:

as a multi-target drug, the major mechanisms of action of levosimendan in the treatment of acute heart failure can be summarized in the following two points: first, levosimendan, as a calcium ion sensitizer, exerts a positive inotropic effect on cardiac myocytes by enhancing the sensitivity of cardiac fibrillar troponin C to calcium ions, without increasing the calcium ion concentration in the cardiac myocytes; secondly, levosimendan opens ATP-dependent potassium channels (K) on cardiomyocytes and vascular smooth muscle cells_ATP) Causing vascular smooth muscle cell hyperpolarization, dilating coronary and peripheral blood vessels and reducing resistance of the circulatory system. Furthermore, levosimendan has a weak phosphodiesterase-III (PDE-III) inhibitory effect, and increases cyclic adenosine monophosphate (cAMP) concentration in myocardial cells to some extent, thereby increasing intracellular calcium ion concentration, but studies have shown that this mechanism does not produce significant effects within the clinically effective therapeutic range.

Traditional heart failure treatment drugs such as dobutamine and milrinone and the like all play a positive inotropic role by increasing cAMP concentration through different mechanisms and further increasing intracellular calcium ion concentration. Levosimendan exerts a positive inotropic effect through a calcium ion sensitizing mechanism and does not increase myocardial energy expenditure because it does not act through a mechanism of intramolecular calcium ion enhancement or intermolecular calcium ion migration. Moreover, studies have shown that levosimendan has a unique cardioprotective action, which can bring long-term beneficial hemodynamic and energy-balancing effects to patients, and these characteristics make levosimendan more advantageous than traditional therapeutic drugs for heart failure. (Levosimendan in Cardiac Surgery: Evaluating the evaluation. journal of cardio and Vascular Anesthesia,2019,33, 1146-.

Since levosimendan has poor solubility and stability in water, it is important to study how to improve its water solubility. The injection is prepared from sterile absolute ethyl alcohol solution of levosimendan which is clinically adopted at present, a cosolvent is required to be added, povidone K12 is adopted as the cosolvent by original manufacturers, and polyethylene glycol PEG400 or hydroxypropyl-beta-cyclodextrin is adopted as the cosolvent by Chinese imitation pharmaceutical manufacturers. In addition, there are a number of patent documents reporting the use of a cosolvent to improve the water solubility of levosimendan, such as substituted cyclodextrins as a solubilizer (a pharmaceutical formulation for injection containing levosimendan and a process for its preparation, Chinese patent publication (publication) No. 108261398A; lyophilized formulation of levosimendan and a process for its preparation, Chinese patent publication (publication) No. 1626085A); polyvinylpyrrolidone is used as a solubilizer (Parential formulations of levosimendan, world patent WO 2017037737A 1); povidone K12PF is used as solubilizer (a pharmaceutical preparation for injection containing levosimendan and its preparation method, Chinese patent publication (publication) No. 108261398A), etc.

The problem of poor water solubility of the medicine is solved by adopting a non-aqueous solvent and a cosolvent, so that the problem is not an ideal solution, and the absolute ethyl alcohol enters a circulatory system to bring adverse effects and side effects for patients with acute heart failure. The aforementioned co-solvents, such as povidone K12, tend to cause unwanted side effects.

Therefore, there is a need for a levosimendan derivative compound which can effectively solve the problem of solubility and can be prepared in a simple manner, thereby reducing the technical defects of inconvenience in use caused by insolubility and instability of levosimendan in water.

Disclosure of Invention

In view of the above technical problems, the present inventors have provided a prodrug compound of levosimendan, which is a compound having a structure represented by the general formula I, solvate, hydrate, N-oxide, stereoisomer, and pharmaceutically acceptable salt thereof;

wherein Ra and Rb are respectively selected from hydrogen atoms or alkyl groups of C1-C6;

rp is selected from a basic group containing N atoms or an acidic group containing carboxyl, phosphate, sulfate and sulfonic acid groups.

The basic group containing an N atom is a basic group which can form a salt with an inorganic acid or an organic acid. The acidic group is an acidic group which can form a salt with an inorganic base or an organic base.

Wherein Ra and Rb are preferably each a hydrogen atom.

In the technical aspect, levosimendan is insoluble in water and extremely difficult to form a salt, so that the solubility problem cannot be solved in a salt forming mode, the prodrug molecule provided by the invention has a water-soluble substituent, and the water-soluble substituent is not added randomly and has the following characteristics:

1. the synthesiser is able to synthesise and prepare the molecule;

2. the molecules have excellent water solubility or form soluble salts with other acids, bases, organic solvent reagents;

3. has the characteristics of a prodrug molecule, namely can be rapidly metabolized in vivo to form levosimendan.

4. Has a unified mother ring structure, and has the characteristic of no toxic or side effect in vivo metabolism after corresponding substituent groups are broken.

Therefore, in order to find compound molecules satisfying these three conditions, the inventors screened the following kinds of substituents and parent ring structures, i.e., formula I, based on years of drug research experience in combination with the prior art.

Firstly, connecting-CH on the 1-N position of tetrahydropyridazinone ring in the levosimendan structural formula₂-O-structure, which in combination with other substituents, is rapidly cleavable during in vivo metabolism and converted to levosimendan with high conversion. Thirdly, the substituent must have experimental verification that no additional side effects are brought after metabolism.

Thus, for the substituent, the basic group containing an N atom is selected from 4- (morpholin-1-ylmethyl) phenyl, 4- (4-methylpiperazin-1-ylmethyl) phenyl, N-dimethylaminomethyl, pyridin-3-yl, 4- (piperidin-1-yl) -piperidin-1-yl, aminomethyl, pyridin-4-ylmethylaminoformylmethyl or pyrrolidin-1-ylmethyl.

Preferably, the acidic group is selected from carboxymethyl, carboxyethyl, carboxymethoxy, phosphoryloxymethyl, sulfonyloxymethyl, sulfomethyl, phosphoryloxyethyl, sulfonyloxyethyl or sulfoethyl.

The present inventors have not studied the prodrug direction of levosimendan and have developed a series of compounds as described above, and first, the present inventors have introduced some solubilizing groups of specific structure to all compounds having the structure of formula I as described above for the problem of poor water solubility of levosimendan.

Structurally, the substituent is introduced to the N-position 1 of the tetrahydropyridazinone ring in the levosimendan structural formula, and the introduction of the side chain has the following characteristics compared with the introduction of the side chain at other sites through earlier experiments:

1) the preparation method is simple;

2) the introduced side chain can smoothly pass through N-CH₂The inter-covalent bond is broken to form the parent drug levosimendan, which contributes to the improvement of the decomposition activity of the prodrug.

The inventors have preferred a series of compounds, including the following structural formula, Rp group name and chemical name, see table 1.

TABLE 1 structural formulae and chemical names of preferred Compounds

After the structure is formed, the water solubility of the prodrug molecule can be obviously improved. Levosimendan is not only insoluble in water, but also insoluble or poorly soluble in most organic solvents, whereas the water solubility of levosimendan prodrug compounds provided by the inventors ranges between very slightly soluble and very readily soluble.

Solubility determination

The determination is carried out according to a pentadeca-based test method of the routine in the four parts of China pharmacopoeia 2020 edition: weighing the test sample ground into fine powder, adding water with the temperature of about 25 ℃ gradually in a water bath with the temperature of 25 +/-2 ℃, shaking strongly for 30 seconds every 5 minutes, and observing the dissolution condition within 30 minutes, wherein if no visible solute particles exist, the test sample is regarded as being completely dissolved.

TABLE 2 definition of solubility

Is very easy to dissolve	It means that 1g (ml) of solute can be dissolved in less than 1ml of solvent
		Is easy to dissolve	It means that 1g (ml) of solute can be dissolved in 1-less than 10ml of solvent
Dissolution	It means that 1g (ml) of solute can be dissolved in 10-less than 30ml of solvent
		Slightly soluble	It means that 1g (ml) of solute can be dissolved in 30-less than 100ml of solvent
Slightly soluble	It means that 1g (ml) of solute can be dissolved in 100-less than 1000ml of solvent
		Minimal dissolution	It means that 1g (ml) of solute can be dissolved in 1000-less than 10000ml of solvent
Hardly soluble or insoluble	It means that 1g (ml) of solute cannot be completely dissolved in 10000ml of solvent

According to the related studies of levosimendan, levosimendan is found to be hardly salifiable (whether by adding inorganic or organic acids or bases) and to have extremely poor solubility in most solvents (including organic solvents and water). Therefore, levosimendan preparations on the market are all prepared into injections dissolved in organic solvents (anhydrous ethanol is selected as the organic solvent of the commercial products), and meanwhile, the levosimendan preparations can be prepared only by adding cosolvent (povidone K12 with high toxicity is selected as the cosolvent of the commercial products at present). In conclusion, the preparation of levosimendan by introducing an organic solvent as a solvent due to the solubility problem of levosimendan and adding a cosolvent is easy to bring toxicity or side effects, thereby causing medication safety accidents.

The invention can synthesize levosimendan prodrug molecule, belongs to the first synthesis, and is a molecule directly having solubility between extremely-soluble and extremely-soluble. And the substituent of the prodrug molecule has the characteristic of containing a basic group of an N atom or containing an acidic group of carboxyl, phosphate group, sulfate group, sulfonic group and the like, so that salts are easily formed in water, or an acid-base regulator (strong acid, strong base, weak acid, weak base, pH regulator with a stabilizing effect and the like) is added to achieve the purpose of easier dissolution. Therefore, the levosimendan prodrug molecule prepared by the invention has milestone effect in the pharmaceutical prospect.

The invention solves the problem that levosimendan has poor solubility in most solvents (including water). The liquid formulation of levosimendan avoids the disadvantages of having to use organic solvents and at the same time requiring the addition of toxic co-solvents.

Meanwhile, levosimendan cannot be dispersed in gastric juice and intestinal juice due to the solubility limit of levosimendan per se, and is difficult to prepare into an oral preparation. The novel compound designed and prepared by the invention solves the defect, can be dispersed in water, namely can be dispersed in gastric juice or intestinal juice in a molecular or ionic form, is beneficial to oral absorption of organisms, and solves the problem that levosimendan is difficult to prepare into an oral preparation.

In addition, the metabolic behavior of the compound which is designed and prepared by the inventor to be metabolized and converted into levosimendan in a human body can be utilized to prepare a controlled release preparation which is more suitable for exerting the drug effect of levosimendan.

These characteristics will make the present invention extremely valuable both clinically and commercially.

Unless otherwise indicated, these terms have the following meanings.

"alkyl" means a straight or branched chain alkyl group consisting of 1 to 6 carbons.

"pharmaceutically acceptable salt" means a pharmaceutically acceptable acid or base addition salt. The compounds of formula (I) having basic properties can be converted into their pharmaceutically acceptable acid addition salts by treatment with a suitable acid. Suitable acids include, for example, inorganic acids, such as hydrohalic acids, e.g., hydrochloric or hydrobromic acid; sulfuric acid; nitric acid; acids such as phosphoric acid; or organic acids such as acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclamic acid, salicylic acid, p-aminosalicylic acid, pamoic acid and the like. The compounds of formula (I) having acidic properties can be converted into their pharmaceutically acceptable base addition salts by treating the acid form with a suitable organic or inorganic base. Suitable base salt forms include, for example, ammonium salts, alkali and alkaline earth metal salts such as lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases such as N, N' -dibenzylethylenediamine, N-methyl-D-glucamine, diethylamine, diethanolamine, and salts with amino acids such as arginine, lysine and the like.

The invention can also be prepared into preparations which are prepared by the compound of the invention with effective dose and pharmaceutically acceptable auxiliary materials. The pharmaceutically acceptable auxiliary materials refer to auxiliary materials required for preparing any pharmaceutical dosage form suitable for human or animal, for example, when the pharmaceutically acceptable auxiliary materials are prepared into an oral solid preparation, the pharmaceutically acceptable auxiliary materials refer to diluents, adhesives, wetting agents, disintegrants, lubricants and glidants; when the injection is prepared, the pharmaceutically acceptable auxiliary materials refer to a pH regulator, a cosolvent, an antioxidant, an isotonic agent and the like.

Preferably: the basic group containing N atoms is selected from 4- (morpholine-1-ylmethyl) phenyl, 4- (4-methylpiperazin-1-ylmethyl) phenyl, N-dimethylaminomethyl or pyridin-3-yl.

Further preferably, the basic group containing an N atom is selected from 4- (morpholin-1-ylmethyl) phenyl or 4- (4-methylpiperazin-1-ylmethyl) phenyl.

The invention provides a preparation method of the compound, which comprises the following steps:

(1) tert-butyl (R) - (4- ((4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) carbamate is prepared by reacting di-tert-butyl dicarbonate with (R) -6- ((4-aminophenyl) -5-methyl-4, 5-dihydropyridazin-3 (2H) -one;

(2) reacting tert-butyl (R) - (4- ((4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) carbamate with paraformaldehyde in the presence of a base to obtain tert-butyl (R) - (4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) carbamate;

(3) converting tert-butyl (R) - (4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) carbamate, de-Boc groups, to (R) -6- ((4-aminophenyl) -2- (hydroxymethyl) -5-methyl-4, 5-dihydropyridazin-3 (2H) -one under acidic conditions;

(4) reacting (R) -6- ((4-aminophenyl) -2- (hydroxymethyl) -5-methyl-4, 5-dihydropyridazin-3 (2H) -one with sodium nitrite and malononitrile under an acidic condition to prepare (R) -N- ((4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) dicyanomethylene) hydrazine;

(5) and (R) -N- ((4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) dicyanomethylene) hydrazine prepared in the step (4) is respectively reacted with carboxylic acid of a corresponding substituent or an activated form thereof to obtain the prodrug compound.

The synthetic route is shown as the following formula:

in the above preparation method, the present invention also provides the intermediate (R) - (4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) carbamic acid tert-butyl ester in the above steps (2), (3) and (4), use of (R) -6- ((4-aminophenyl) -2- (hydroxymethyl) -5-methyl-4, 5-dihydropyridazin-3 (2H) -one and (R) -N- ((4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) dicyanomethylene) hydrazine for the preparation of a medicament for the treatment of heart failure disorders.

The invention provides a preparation method of a compound of a general formula I, which comprises the following steps:

(1) preparation of (R) - (4- ((4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) carbamic acid tert-butyl

Placing (R) -6- ((4-aminophenyl) -5-methyl-4, 5-dihydropyridazin-3 (2H) -one, di-tert-butyl dicarbonate, triethylamine and tetrahydrofuran in a container, stirring for 1-5 days at room temperature, detecting no raw material by TLC, filtering, performing reduced pressure rotary evaporation to remove the solvent, adding petroleum ether into the concentrate, pulping for 10-60 minutes at room temperature, filtering, and drying the obtained solid to obtain a light brown solid.

(2) Preparation of (R) - (4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) carbamic acid tert-butyl ester

Putting the (R) - (4- ((4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) carbamic acid tert-butyl ester obtained in the step (1) and methanol into a container, adding paraformaldehyde and potassium carbonate while stirring, stirring at room temperature until the TLC monitoring reaction is complete, carrying out reduced pressure rotary evaporation to remove the solvent, purifying the concentrate by column chromatography, and obtaining a white solid by using an eluent which is a mixed solvent of petroleum ether and ethyl acetate (v: v 1: 1).

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

And (2) placing the tert-butyl (R) - (4- ((4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) carbamate obtained in the step (2) into a container, adding ethyl acetate, stirring at room temperature for 1-10 minutes, dropwise adding an ethyl acetate solution of hydrogen chloride, stirring at room temperature after dropwise adding until no raw material remains after TLC detection, and concentrating under reduced pressure to remove the solvent to obtain a crude product of (R) -6- ((4-aminophenyl) -2- (hydroxymethyl) -5-methyl-4, 5-dihydropyridazin-3 (2H) -one hydrochloride and a yellow solid, wherein the crude product is directly subjected to the next reaction.

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

And (2) transferring the (R) -6- ((4-aminophenyl) -2- (hydroxymethyl) -5-methyl-4, 5-dihydropyridazin-3 (2H) -one hydrochloride crude product obtained in the step (3) into a container by using a hydrochloric acid aqueous solution, adding a sodium nitrite aqueous solution into the reaction solution under the control of an ice water bath at 5-10 ℃, continuing stirring for 10 minutes to 2 hours under the ice water bath after the addition is finished, adding a malononitrile aqueous solution into the reaction solution at 5-10 ℃, removing the ice water bath after the addition is finished, stirring for 1 to 5 hours at room temperature, slowly adding a sodium acetate aqueous solution, adjusting the pH value to 5-6 to obtain a yellow suspension, filtering, washing with water and drying to obtain a yellow solid.

(5) Preparation of the Compounds of the formula I

And (R) -N- ((4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) dicyanomethylene) hydrazine prepared in the step (4) is respectively esterified with carboxylic acid with corresponding substituent groups or activated forms thereof to obtain the compound with the general formula.

All compounds of the invention not only have general structures, but also can be prepared by carrying out esterification reaction on a common intermediate product (R) -N- ((4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) dicyanomethylene) hydrazine, namely, by preparing a front-end intermediate product, and then carrying out esterification reaction on subsequent substituent compounds by carboxylic acid of corresponding substituents or activated forms thereof, so that the preparation route of the preparation method is that the intermediate product is prepared firstly and then various specific compounds of the prodrug compound are synthesized by a main mode of esterification.

In the preparation method, the compound (R) -N- ((4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) dicyanomethyl) hydrazino, TSTU and carboxylic acid substituted by different Rp groups are taken in a container in the step (5), dichloromethane is added, triethylamine is added under stirring, the mixture is stirred for 1-10 hours at room temperature, no raw material is left after TLC detection, the solvent is removed by reduced pressure rotary evaporation, the obtained concentrate is purified by column chromatography, and the eluent is a mixed solvent of dichloromethane and methanol (v: v 50:1), so that the prodrug compound of levosimendan is obtained.

The invention further provides the application of the prodrug compound in preparing a medicament for treating heart failure diseases.

Has the advantages that:

the invention provides a levosimendan prodrug compound, which is prepared by carrying out corresponding substitution on 1-N of a tetrahydropyridazinone ring in a levosimendan structural formula, solves the technical defect of extremely poor water solubility of levosimendan, and has the characteristics of good stability, easier drug metabolism, convenient use and the like. The preparation does not need to adopt absolute ethyl alcohol as a solvent and other cosolvents, and is greatly convenient for clinical use. The levosimendan prodrug compound can be quickly converted into levosimendan in vivo, has high conversion rate and good drug-forming property. Based on the pharmacokinetic data obtained, it can be concluded that the compounds provided by the present invention are capable of acting as prodrugs of levosimendan. These compounds also have the advantage of pharmacokinetic control of the rate of levosimendan release in vivo. In addition, the problem that levosimendan is not suitable for oral administration can be solved.

Abbreviation interpretation:

TSTU: 2-succinimidyl-1, 1,3, 3-tetramethyluronium tetrafluoroborate;

TLC: thin layer chromatography;

¹H-NMR: nuclear magnetic resonance hydrogen spectroscopy;

TMS: tetramethylsilane

DMSO-d₆: hexadeuterio dimethyl sulfoxide;

ESI-MS: electrospray ionization-mass spectrometry;

K_ATP: ATP-dependent potassium ion channels;

PDE-III: phosphodiesterase-III;

cAMP: cyclic adenosine monophosphate;

PEG: polyethylene glycol.

DCM: dichloromethane;

MtOH: methanol;

DMF: n, N-dimethylformamide;

boc group: an amino protecting group, t-butyloxycarbonyl, is commonly used.

Drawings

Process for preparing TCX1001-031 compound of FIG. 1¹H-NMR chart;

FIG. 2 ESI-MS plot of TCX1001-031 compound;

FIG. 3 liquid chromatogram of compound TCX 1001-031;

FIG. 4 is a graph of the change in self-content in pharmacokinetic experiments for compounds TCX 1001-031;

FIG. 5 is a graph of the change in the amount of levosimendan released in the pharmacokinetic test of the compound TCX 1001-031;

FIG. 6 metabolic conversion in pharmacokinetic experiments for compound TCX 1001-031.

Detailed Description

The following detailed description is provided for the purpose of illustrating the embodiments and the advantageous effects thereof, and is not intended to limit the scope of the present disclosure.

The structure of the compound is determined by nuclear magnetic resonance hydrogen spectrum (¹HNMR) and/or Mass Spectrometry (MS).¹HNMR were determined by Bruker Spectrometer AVIII HD NMR Spectrometer (400MHz) with a chemical shift (. delta.) of 10^-6The units in (ppm) are given. Internal standard Tetramethylsilane (TMS), chemical shift: δ, s: a single peak; d: double peaks; t: a triplet; q: quartering; m: multiple peaks. Mass spectra were determined on an Agilent Accurate-Mass Q-TOF LC/MS Mass spectrometer.

Unless otherwise stated, benzyl carbazate was purchased from Shanghai Haohnhong biomedical science and technology, Inc., paraformaldehyde was purchased from Shanghai Michelin Biotechnology, Inc., diethyl phosphate was purchased from Shanghai Michelin Biotechnology, Inc., diethyl 2, 2-diethoxyethyl phosphate was purchased from Afahesa (China) chemical, Inc., tert-butoxycarbonylhydrazine was purchased from Shanghai Bigdi medicinal science, Inc., (R) -6- ((4-aminophenyl) -5-methyl-4, 5-dihydropyridazin-3 (2H) -one was purchased from Beijing Cheng Guang Co., Ltd., (TLC was thin-layer chromatography), TSTU was O- (N-succinimidyl) -N N N 'N' -tetramethyltetrafluoroborate, DCM was dichloromethane, MtOH is methanol, DMF is dimethylformamide, and the specification of petroleum ether is 60-90 ℃.

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

preparation of (R) - (4- ((4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) carbamic acid tert-butyl ester

(R) -6- ((4-aminophenyl) -5-methyl-4, 5-dihydropyridazin-3 (2H) -one (101g,0.5mol), di-tert-butyl dicarbonate (163.5g,0.75mol), triethylamine (76g,0.75mol) and tetrahydrofuran (1L) were placed in a 2L one-necked flask, and stirred at room temperature for 3 days, and TLC showed no starting material remaining.filtration, removal of the solvent by rotary evaporation under reduced pressure, petroleum ether (300mL) was added to the residue with stirring, and stirred at room temperature for 20 minutes, filtered, and dried to give the title compound 84g, a pale brown solid in 54% yield.¹H-NMR(400MHz,DMSO-d₆)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＝7.2Hz)；ESI-MS(m/z):Calcd.For C₁₆H₂₁N₃O₃+H:304.16；Found: 304.17。

Preparation of di- (R) - (4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) carbamic acid tert-butyl ester

Tert-butyl (R) - (4- ((4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) carbamate (100g, 0.33mol), and 2.5L of methanol were placed in a 3L three-necked flask, and paraformaldehyde (128.2g,4.27mol) and potassium carbonate (95.3g,0.69mol) were added with stirring, stirred at room temperature overnight, the reaction was monitored by TLC for completion, the solvent was removed by rotary evaporation under reduced pressure, and the concentrate was purified by column chromatography (petroleum ether: ethyl acetate, v: v ═ 1:1) to obtain 60.0g of the title compound as a white solid in 55% yield.¹H-NMR(400MHz,DMSO-d₆)δ:9.56(s,1H),7.74(d,2H,J＝8.5Hz),7.53 (d,2H,J＝8.5Hz),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＝7.2Hz)；ESI-MS (m/z):Calcd.For C₁₇H₂₃N₃O₄+H:334.17；Found:334.18。

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

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

Preparation of tetra (R) -N- ((4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) dicyanomethylene) hydrazine

Transferring 23g of the compound (R) -6- ((4-aminophenyl) -2- (hydroxymethyl) -5-methyl-4, 5-dihydropyridazin-3 (2H) -one hydrochloride obtained in the third step to a 2L beaker by using a 2.6% hydrochloric acid aqueous solution (500mL), cooling the mixture in an ice-water bath, adding a solution prepared by dissolving 7.05g of sodium nitrite (0.1mol) in 50mL of water at 5 to 10 ℃ to the reaction solution, continuing to stir for 30 minutes in the ice-water bath after the addition is finished, dissolving malononitrile (6.2g,0.094mol) in 50mL of water, adding the mixture to the reaction solution at 5 to 10 ℃ and removing the ice-water bath after the addition is finished, stirring the mixture at room temperature for 2 hours, slowly adding a 20% sodium acetate aqueous solution, adjusting the pH value to 5 to 6 to obtain a yellow suspension, filtering, washing with water, drying to obtain 11.2 g of the title compound, yellow solid, two-step yield 60%.¹H-NMR(400MHz,DMSO-d₆)δ:13.1(br,s,1H),7.88-7.92(m, 2H),7.52-7.55(m,2H),5.13(d,1H,J＝10Hz),5.00(d,1H,J＝10Hz),3.37-3.43(m,1H), 2.71-2.77(m,1H),2.31-2.35(m,1H),1.08(d,3H,J＝7.2Hz)；ESI-MS(m/z):Calcd.For C₁₅H₁₄N₆O₂+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)

The compound (R) -N- ((4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) dicyanomethylene) hydrazine (0.26g,0.84mmol), TSTU (0.3g,1mmol) and nicotinic acid (0.11g,0.92mmol) were put in a 50mL one-necked flask, 5mL of dichloromethane was added, triethylamine (0.3g,3mmol) was added with stirring, and the mixture was stirred at room temperature for 5 hours. TLC showed no material remaining, the solvent was removed by rotary evaporation under reduced pressure and the resulting concentrate was purified by Pre-HPLC (eluent: DCM/MeOH, v: v ═ 50/1) to give the title compound 0.12g as a yellow solid in 34% yield.¹H-NMR(400 MHz,DMSO-d₆)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＝7.2Hz)；ESI-MS(m/z):Calcd.For C₂₁H₁₇N₇O₃+H: 416.14；Found:416.15。

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

The compound (R) -N- ((4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) dicyanomethylene) hydrazine (0.2g,0.64mmol), glycine (0.12g,1.6mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (0.31g,1.6mmol), 4-lutidine (0.08g,0.64mmol) and 5mL of N, N-dimethylacetamide were sequentially added to a 50mL 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 give the title compound 70mg as a yellow solid in 30% yield.¹H-NMR (400MHz,DMSO-d₆)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＝7.2Hz)；ESI-MS(m/z):Calcd.For C₁₉H₂₁N₇O₃+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)

Compound (R) -N- ((4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) dicyanomethylene) hydrazine (300mg,0.97mmol), 2- (pyrrolidin-1-yl) acetic acid hydrochloride (321mg,1.94mmol), EDCI (465mg, 2.43mmol), DMAP (118mg,0.97mmol) was dissolved in DMA (10mL) and stirred at room temperature for 18 hours. Ethyl acetate (10mL) and water (10mL) were added to the system, and the mixture was stirred and separated. The organic phase was washed with water (3 × 10mL) 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 give the title compound 210mg as a yellow solid product in 51.3% yield.¹H-NMR(400MHz,DMSO-d₆)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＝7.2Hz)；ESI-MS(m/z): Calcd.For C₂₀H₂₀N₇O₃+H:407.16；Found:407.217。

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

The compound (R) -N- ((4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) dicyanodimethylene) hydrazine (0.2g,0.64mmol), N, N-dimethylglycine (0.16g,1.6mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (0.31g,1.6mmol), 4-lutidine (0.08g,0.64mmol), and 5mL of N, N-dimethylacetamide were sequentially added to a 50mL single-neck 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 give the title compound 0.06g as a yellow solid in 24% yield.¹H-NMR(400MHz,DMSO-d₆)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＝7.2Hz)；ESI-MS(m/z):Calcd.For C₁₉H₂₁N₇O₃+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)

The compound (R) -N- ((4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) dicyanodimethylene) hydrazine (0.2g,0.64mmol) was dissolved in 2mL of pyridine, 4-chloromethylbenzoyl chloride (0.22g,1.16 mmol) was added thereto, and the mixture was stirred at room temperature for 2 hours, and 30mL of ethyl acetate was added to dilute the reaction solution, and the organic phase was washed with distilled water and saturated brine in this order, dried over anhydrous sodium sulfate, and spin-dried, and the resulting concentrate was dissolved in DMF (3mL), and sodium iodide (0.05g) was added thereto, morpholine (0.13g, 1.5mmol) was added thereto, and the mixture was 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 give the title compound 0.11g as a yellow solid in 33% yield.¹H-NMR(400MHz,DMSO-d₆)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＝7.2Hz)；ESI-MS (m/z):Calcd.For C₂₇H₂₇N₇O₄+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)

The compound (R) -N- ((4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) dicyanomethylene) hydrazine (0.2g,0.64mmol) was dissolved in 2mL of pyridine, 4-chloromethylbenzoyl chloride (0.22g,1.16 mmol) was added thereto, the mixture was stirred at room temperature for 2 hours, and 30mL of ethyl acetate was added to dilute the reaction solution, which was then washed with distilled water and saturated brine in this order. The organic phase was dried over anhydrous sodium sulfate, the solvent was removed by rotary evaporation under reduced pressure, the concentrate was dissolved in DMF (3mL), sodium iodide (0.05g) was added, then N-methylpiperazine (0.15g,1.5mmol) was added, and the mixture was 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 give the title compound 0.05g as a yellow solid in 15.0% yield.¹H-NMR(400MHz,DMSO-d₆)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＝7.2Hz)；ESI-MS(m/z):Calcd.For C₂₇H₂₇N₇O₄+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-ylcarboxylate (TCX-1001-) -037)

10mL of anhydrous tetrahydrofuran, 1.24g of (R) -N- ((4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydro-l-o-ne)Pyridazin-3-yl) phenyl) dicyanomethylene) hydrazine, 0.71g N, N' -carbonyldiimidazole and 0.67g 4-piperidinopiperidine were put in this order into a 50mL single-necked flask, and after the reaction was stirred at room temperature for 48 hours, TLC monitoring (developing solvent: dichloromethane/methanol 10/1) was complete. 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 give the title compound 0.50g as a yellow solid in 24.8% yield.¹H-NMR(400MHz,DMSO-d₆)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 C₂₆H₃₂N₈O₃+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-carboxy-acetate (TCX-1001-041)

Malonic acid (0.21g,2.06mmol), triethylamine (0.36mL,2.6mmol), DMAP (13mg,0.1mmol), isopropenyl chloroformate (0.08mL,0.73mmol) were added successively to 10mL of dichloromethane, the temperature in an ice-water bath was lowered to 0 ℃, then a solution of (R) -N- ((4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) dicyanomethylene) hydrazine (0.16g,0.52mmol) in dichloromethane (10mL) was added, and the reaction was stirred at 0 ℃ for 2 hours, then poured into 3mL of a 10% aqueous acetic acid solution and stirred at room temperature for 0.5 hours. The reaction mixture was washed with distilled water and saturated brine in this order, 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 give the title compound 80mg as a yellow solid in 39.0% yield.¹H-NMR (400MHz,DMSO-d₆)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＝7.2Hz)；ESI-MS(m/z):Calcd.For C₂₁H₁₇N₇O₃+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-carboxypropionate (TCX-1001-034)

Succinic acid (0.488g,4.12mmol), triethylamine (0.72mL,5.2mmol), 4-dimethylaminopyridine DMAP (26mg,0.2mmol), isopropenyl chloroformate (0.16mL,1.46mmol) were dissolved in 10mL of dichloromethane, the temperature in an ice-water bath was reduced to 0 ℃, then a solution of (R) -N- ((4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) dicyanomethylene) hydrazine (0.32g,1.04mmol) in dichloromethane (10mL) was added, and the reaction was stirred at 0 ℃ for 2 hours, then poured into 3mL of 10% aqueous acetic acid and stirred at room temperature for 0.5 hours. The reaction mixture was washed with distilled water and saturated brine in this order, 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 give the title compound 0.14g as a yellow solid in 33% yield.¹H-NMR(400MHz,DMSO-d₆)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＝7.2Hz)；ESI-MS m/z:Calcd.For C₂₁H₁₇N₇O₃+H:411.14；Found:411.15。

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

Taking (R) -N- ((4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) benzeneYl) Dicyanomethylene) hydrazine (0.16g,0.52mmol), N, N' -carbonyldiimidazole CDI (95mg,0.57mmol), 2-hydroxyacetic acid ((40mg, 0.57mmol) was dissolved in 5mL THF, stirred at room temperature for 48 hours, TLC monitored reaction completion. 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 give the title compound 60mg as a yellow solid in 28% yield.¹H-NMR(400MHz,DMSO-d₆)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＝7.2Hz)；ESI-MS(m/z):Calcd.For C₂₁H₁₇N₇O₃+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 phosphonooxyacetate (TCX-1001-040)

(R) -N- ((4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) dicyanomethylene) hydrazine (0.26g,0.84mmol), TSTU (0.3g,1mmol) and (phosphoryloxy) acetic acid (0.16g,1mmol) were taken in a 50mL one-neck flask, 5mL of dichloromethane were added, triethylamine (0.3g,3mmol) was added with stirring, stirring at room temperature for 15 hours, and no starting material remained by TLC. The solvent was removed by rotary evaporation under reduced pressure and the concentrate was isolated by Pre-HPLC preparative to give the title compound 0.11g as a yellow solid in 29% yield.¹H-NMR(400MHz,DMSO-d₆)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＝7.2Hz)；ESI-MS (m/z):Calcd.For C₂₁H₁₇N₇O₃+H:449.09；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-

A50 mL single vial of (R) -N- ((4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) dicyanomethylene) hydrazine (0.52g,1.68mmol), TSTU (0.6g,2mmol) and (sulfooxy) acetic acid (0.32g,2mmol) was charged with 15mL of dichloromethane, triethylamine (0.6g,6mmol) was added with stirring, stirred at room temperature for 15 hours and monitored by TLC for no starting material remaining. The solvent was removed by rotary evaporation under reduced pressure and the concentrate was isolated by Pre-HPLC preparative to give the title compound 0.15g as a yellow solid in 20% yield.¹H-NMR(400MHz,DMSO-d₆)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＝7.2Hz)；ESI-MS (m/z):Calcd.For C₂₁H₁₇N₇O₃+H:449.08；Found:449.10。

Example 13: 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- & lt 036 & gt)

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

Ethyl chloroformylacetate (5.8mL,46.23mmol) and 4-pyridylmethylamine (5g,46.23mmol) were dissolved in 200mL of THF, the reaction was cooled to 0 deg.C, triethylamine (9.5mL,69.35mmol) was slowly added dropwise, and the reaction was stirred at room temperature for 2 hours after the addition. TLC monitored the reaction complete. Removing solvent by rotary evaporation under reduced pressure, dissolving the concentrate in 200mL ethyl acetate, washing with distilled water and saturated sodium chloride water, and mixing the organic phase with anhydrous sodium sulfateAnd (5) drying. 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 give the desired product ethyl 3-oxo-3- ((pyridin-4-ylmethyl) amino) propionate 5g as a brown solid in 48% yield.¹H-NMR(400MHz, CDCl₃)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＝7.2Hz)；ESI-MS(m/z):Calcd. For C₂₁H₁₇N₇O₃+H:223.10；Found:222.90。

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

Ethyl 3-oxo-3- ((pyridin-4-ylmethyl) amino) propionate (2.2g,10mmol) was dissolved in a mixed solvent of 10mL of methanol, 10mL of THF and 5mL of water, and lithium hydroxide monohydrate (0.84g,20mmol) was added to stir the reaction solution at room temperature for 16 hours. TLC monitored the reaction complete. The solvent was removed by rotary evaporation under reduced pressure, diluted with 10mL of water, adjusted to pH 5-6 with 3N HCl aqueous solution, extracted with ethyl acetate (20 mL. times.3), and the organic phases were combined. The organic phase was washed with distilled water and saturated brine in this order, 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 give the desired product, 3-oxo-3- ((pyridin-4-ylmethyl) amino) propionic acid, 1.5g, in 77% yield.¹H-NMR(400MHz,DMSO-d₆)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 C₂₁H₁₇N₇O₃+H:195.07；Found:195.10。

(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- & lt 036 & gt)

The compound (R) -N- ((4- (1- (hydroxymethyl) -4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) dicyanomethylene) hydrazine (0.26g,0.84mmol), TSTU (0.3g,1mmol), 3-oxo-3- ((pyridin-4-ylmethyl) amino) propanoic acid (0.18g,0.92mmol) was taken in a 50mL one-necked flask, 5mL of dichloromethane was added, triethylamine (0.3g,3mmol) was added with stirring, stirring was carried out at room temperature for 5 hours, no starting material remained by TLC detection, the solvent was removed by rotary evaporation under reduced pressure, and the resultant was purified by column chromatography (eluent: DCM/MeOH, v: 50/1) to give 0.11g of the title compound as a yellow solid in 27% yield.¹H-NMR(400MHz,DMSO-d₆)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＝ 7.2Hz)；ESI-MS(m/z):Calcd.For C₂₁H₁₇N₇O₃+H:487.14；Found:487.15。

The invention solves the problem that levosimendan has poor solubility in most solvents (including water). The liquid formulation of levosimendan avoids the disadvantages of having to use organic solvents and at the same time requiring the addition of toxic co-solvents.

Meanwhile, levosimendan itself is difficult to prepare into an oral preparation due to its solubility limit. The novel compound prepared by the invention solves the defect, can be dispersed in water, namely can be dispersed in gastric juice or intestinal juice in a molecular or ionic form, is beneficial to oral absorption of organisms, and can solve the problem that levosimendan is difficult to prepare into an oral preparation.

Example 14: sample stability test

After the compound samples prepared in examples 1-13 above were exposed (exposed) to strong light (4500Lx + -500 Lx), high temperature (60 deg.C + -2 deg.C) and high humidity (90% + -5%) for 10 days, no significant change in appearance or related substances was observed. Thus, the samples of the compounds prepared in examples 1-13 were initially tested for stability.

Example 15: sample determination test

The detection chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; phosphate buffer solution (weighing 1.56g of sodium dihydrogen phosphate, adding 1000ml of water for dissolving, adjusting pH value to 3.5 with phosphoric acid) -methanol (30: 70) is used as a mobile phase; the flow rate was 1.0mL per minute; the column temperature is 30 ℃; the detection wavelength is 254 nm; the injection volume is 10. mu.L.

Test solution: taking a proper amount of the product (TCX-H1001-031), precisely weighing, and dissolving in methanol to obtain a solution containing 0.5mg of sample per 1 ml.

Precisely measuring the test solution, injecting into a liquid chromatograph, and recording the chromatogram.

FIGS. 1 to 3 are the compounds TCX-H1001-031, respectively, prepared in example 5¹H-NMR chart, ESI-MS chart, liquid chromatogram.

Example 16: pharmacokinetic testing of Compounds

FIGS. 4-6 are graphs of the change in plasma levels of TCX-1001-031 compound, the change in the release of levosimendan, and the pharmacokinetic metabolic conversion, respectively. As can be seen from the figure, TCX-1001-031 can be completely metabolized within 1 minute, i.e., its content decreases to 0 within 1 minute; the content of the original drug levosimendan released in the metabolism begins to rise rapidly and then gradually and completely tends to be stable along with the metabolism of the prodrug, which shows that the metabolic conversion rate of TCX-1001-031 converted into the raw material is about 40 percent in 10 minutes, and the prodrug belongs to a good-quality prodrug molecule.

Table 3 shows the results of in vivo administration of certain specific compounds of the present invention, showing that they are completely metabolized within 1-10 minutes, respectively, with levosimendan levels increasing and becoming completely stable with prodrug metabolism. The conversion of these compounds to levosimendan in plasma is about 30-45% within 10 minutes.

TABLE 3 metabolic conversion

Compound (I)	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％

From the results of the drug-induced experiments, the metabolic conversion rates of the 13 prodrug molecules in plasma are within 10 minutes, wherein the metabolic conversion rates are the highest among 1, 2, 4 and 6, and the 13 compound molecules all have the potential of becoming prodrugs according to the existing data.

The examples merely represent several embodiments of the present invention, which are described in more detail and detail, but are not to be construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention, and therefore the scope of the present patent is defined by the appended claims.