RU2579120C1 - Method of producing creatine amides - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to pharmaceutical chemistry, in particular to a process for preparing creatine amides of general formula shown below, which exhibit anti-ischemic activity. In formula R denotes amino acid residue of amino acid; Y denotes OR¹, NR²R³, where R¹ is H, CH₂Ph, alkyl C₁-C₃, R², R³ - H, alkyl C₁-C₄; X - low-molecular C₁-C₄ organic acid or mineral acid, or water. Method involves treating creatine para-toluenesulphonic acid in dimethyl formamide, followed by reacting obtained complex with derivatives of amino acids in presence of a condensing agent and a base. Condensing agent used is formic acid monoester chloride, wherein condensing agent and base are added simultaneously. Then an alkali metal acetate is added to obtained mixture, followed by distilling not less than 80 % of dimethyl formamide, extracting active ingredient with a aprotic polar solvent containing at least one ingredient from a group including acetone, tetrahydrofuran, methyl ethyl ketone and acetonitrile. Further, solvent was distilled off, residue is dissolved in a polar aprotic solvent containing at least one ingredient from a group consisting of water, ethanol and isopropyl alcohol, acetic and formic acid, p-toluenesulphonic acid is removed by anion-exchange chromatography on a sorbent. Solvent is removed in presence of low molecular weight C₁-C₄ organic acid or inorganic acid and alcohol at not more than pH 4.5, then treated with a solvent containing at least one ingredient from A group consisting of acetone, tetrahydrofuran, methyl ethyl ketone, ethyl acetate and acetonitrile. Active ingredient is extracted with a solvent containing at least one ingredient from a group consisting of ethyl and methyl alcohols are distilled off at least 70 % of solvent and treated with a solvent containing at least one ingredient from a group consisting of acetone, tetrahydrofuran, methyl ethyl ketone, acetonitrile, and acetate.

EFFECT: disclosed method increases output and purity of end product.

22 cl, 1 tbl, 25 ex

NH=C(NH₂)-N(CH₃)-CH₂-CO-RY*X

Description

The invention relates to the field of pharmaceutical chemistry, and in particular to methods for producing biologically active substances, in particular, exhibiting anti-ischemic activity of creatine amides of the general formula NH = C (NH ₂ ) -N (CH ₃ ) -CH ₂ -CO-RY * X, where R is the amino acid residue of an amino acid; Y is OR ¹ , NR ² R ³ , where R ¹ is H, CH ₂ Ph, C ₁ -C ₃ alkyl, R ² , R ³ = H, C ₁ -C ₄ alkyl; X is a low molecular weight C ₁ -C ₄ organic acid, or mineral acid or water.

Ischemic damage to the brain and myocardium leads to the development of pathophysiological changes in tissues, accompanied by metabolic disturbances, impaired tissue tissue metabolism [Reichelt K.L. Acta Anesthesiol. Scand. 1978], the development of acidosis, the destruction of astrocytes [Hertz L.J. Cereb. Blood Flow Metab. 2004; 24: 1241-8], compression of the capillaries of the brain [Ames A.I. et al. 1968; 52: 437-53].

A large group of drugs is known - thrombolytics, antiplatelet agents, nootropics, etc., which increase the resistance of tissues, in particular the brain, to ischemia (Goodman E. Gilman's. The Pharmacological Basis of Therapeutics, 11 ed, McGraw-Hill, Medical Publ . Division, New York 2006; RU 1746886, 1991; WO 96/08527, 1996).

The disadvantages of most known drugs are a narrow spectrum of action, a significant number of side effects and contraindications, low neuroprotective activity, limited use in acute and chronic cerebral ischemia and acute myocardial infarction [The Cochrane Library, Issue 4, 2002. Oxford].

Therefore, the development of drugs for the treatment and prevention of ischemia is an important problem.

A promising direction for the prevention and treatment of ischemia is the use of creatine amides and their derivatives (RU 2354645, 2009; US 8,350,077 B2).

Creatine amides are neuroprotective in brain ischemia [Burov S.V et all. J. Pept. Sci. 2011. V. 17. N.9. P. 620-626], as well as antithrombotic and antiplatelet activity, contributing to the restoration of blood flow [O. Veselkina. and other Bulletin of the blood service of Russia, No. 3, p. 31-37, 2010], however, the technology for their preparation does not allow to obtain the target product with high yield. In addition, the range of amides obtained is limited and needs to be expanded. In this regard, it remains an important task to develop more effective methods for the synthesis of creatine amides.

A known method of producing creatine amides and aliphatic, aromatic or heteroaromatic amino acids or their derivatives, which consists in the initial production of intermediates, namely sarcosine amides and aliphatic, aromatic or heteroaromatic amino acids or their derivatives, further guanidylation of intermediates in polar organic solvents at a temperature of not more than 50 ° C and production of creatine amides [RU 2354645, 2009].

The disadvantages of this method are the low yield of the target product, the need to use expensive guanidylating agents — benzotriazole-1-carboxamidine tosylate, N, N′-dibenzyloxycarbonyl-1-H-benzotriazole-1-carboximidine in the synthesis, which generally leads to the high cost of creatine amides.

Closest to the claimed invention, is a method for producing creatine amides and amino acids or their derivatives [RU 2428414, 2011] in the form of salts, which consists in processing creatine or creatine monohydrate with para-toluenesulfonic acid in an organic solvent, subsequent processing of the reaction mixture with ester or amide derivatives of amino acids, in the presence of a sequentially introduced condensing agent and base.

The disadvantage of this method is the low yield of creatine amides, not exceeding 32% and a limited range of drugs. In addition, this method, using para-toluenesulfonic acid (p-TSC) or toluenesulfonates in the synthesis process, does not ensure their complete removal in the process of isolation and purification of the target product. At the same time, p-TSC is not a pharmacologically acceptable acid, and when it enters the body, it can form products with toxicity.

The technical problem solved by the authors was to create a method for the synthesis of creatine amides of the general formula NH = C (NH ₂ ) -N (CH ₃ ) -CH ₂ -CO-RY * X, where R is the amino acid residue of the amino acid; Y is OR ¹ , NR ² R ³ , where R ¹ is H, CH ₂ Ph, C ₁ -C ₃ alkyl; R ² , R ³ = H, alkyl C ₁ -C ₄ ; X - low molecular weight C ₁ -C ₄ organic acid or mineral acid or water, providing a higher yield of the target product and its purity in accordance with modern pharmacopoeial requirements for new pharmaceutical substances.

The technical result was achieved by treating creatine with para-toluenesulfonic acid in dimethylformamide, followed by the interaction of the obtained complex with amino acid derivatives, in the presence of a condensing agent and a base, with formic acid monoester as a condensing agent and N-methylmorpholine or triethylamine as the base. In this case, the condensing agent and the base are introduced into the reaction mixture, at the same time, unlike the analogue, where the condensing agent is first introduced, and then, after it, the base is introduced. Next, alkali metal acetate is added to the reaction mixture, at least 80% of dimethylformamide is distilled off, the active principle is extracted with a polar aprotic solvent containing at least one ingredient from the group consisting of acetone, tetrahydrofuran, methyl ethyl ketone and acetonitrile, and at least 80% of the solvent is distilled off. The active principle is extracted from the residue with a polar aprotic solvent containing at least one ingredient from the group consisting of acetone, tetrahydrofuran, methyl ethyl ketone and acetonitrile, the solvent is distilled off, the residue is dissolved in a polar proton solvent containing at least one ingredient from the group of water , ethyl and isopropyl alcohol, formic and acetic acids. Para-toluenesulfonic acid is removed by chromatography on an anion-exchange sorbent, the solvent is distilled off in the presence of a low molecular weight C ₁ -C ₄ organic acid or mineral acid and alcohol at a pH of not more than 4.5. The residue is treated with a solvent containing at least one ingredient from the group of acetone, tetrahydrofuran, methyl ethyl ketone, acetonitrile and ethyl acetate, the active principle is extracted with a solvent containing at least one ingredient from the group of ethyl and methyl alcohols, then at least 70% solvent. The distillation residue is treated with a solvent containing at least one ingredient from the group consisting of acetone, tetrahydrofuran, methyl ethyl ketone, acetonitrile and ethyl acetate.

It was found that the simultaneous introduction of a condensing agent, such as acid chloride of a monoester of formic acid, and a base, such as N-methylmorpholine, triethylamine, can significantly increase the yield of creatine amides in the synthesis stage, and alkaline acetate added to the mother liquor obtained after the synthesis stage metals, allows to achieve a more complete removal of chloride ions generated in the condensation reaction of the complex of creamide with p-TSC with amino acid derivatives, which leads to optimization of the chromatographic process fusion on an anion-exchange sorbent and provides a high yield and the required purity of the target product - the content of the main substance in the synthesized creatine amides according to RP HPLC was not less than 98%, the content of chloride ions was trace (at the level of measurement error), and the content of p-TSC was not exceeded several r.r.m.

During the synthesis, to prevent the ability of amino acid molecules (and creatine) to react with each other, one of one of the functional groups is protected to make it inert [S. Chandrudu, R. Simerska, * and I.T. Chemical Methods for Peptide and Protein Production. Molecules 2013, 18, 4373-4388; doi: 10.3390]. Get a complex of creatine with para-toluenesulfonic acid, which allows you to protect the guanidine group of creatine, and amino acid derivatives containing substituents on the carboxyl group of the molecule. As such derivatives of amino acids, etheric, amide or nitro derivatives of proteinogenic α-amino acids, ether or amide derivatives of β-alanine are used. The term "proteinogenic α-amino acids" is understood in accordance with the international nomenclature (NOMENCLATURE AND SYMBOLISM FOR AMINO ACIDS AND PEPTIDES. Pure & Appi. Chem., Vol. 56, No. 5, pp. 595-624, 1984). In particular, acyclic or aromatic α-amino acids, O-ethyl tyrosine, O-isopropyl tyrosine, proline or histidine and the like can be used as proteinogenic α-amino acids.

Anhydrous creatine or creatine monohydrate can be used as creatine in the synthesis.

As the acid chloride of the formic acid monoester, isobutyl chloroformate or ethyl chloroformate and their analogs can be used, N-methylmorpholine, triethylamine and the like as the base. Anhydrous sodium or potassium acetate, hydrates of sodium or potassium acetate can be used as alkali metal acetates.

As an anion exchange sorbent, it is optimal to use resins based on styrene copolymers or based on acrylic copolymers containing tertiary or quaternary ammonium groups, for example, Dowex 1x8, Amberlite IRA-67. As a rule, an aqueous solution of formic or acetic acids or a water-alcohol solvent is used as an eluent.

As a low molecular weight C ₁ -C ₄ organic acid, methanesulfonic, acetic, fumaric or succinic acid and the like can be used, and acids such as phosphoric, hydrochloric and the like can be used as a mineral acid.

Monitoring the progress of the reaction, as well as evaluating the yield and purity of creatine amides, was carried out using the RP-HPLC method on an Alliance chromatograph (Waters), Zorbax Eclipse C18 column, 3.5 μm, 3 * 100 mm (Agilent Technologies), linear gradient elution from 100% phase A (a mixture of a buffer solution containing 0.01 M sodium octanesulfonate and 0.02 M sodium dihydrogen phosphate (pH = 3.0) with acetonitrile in a ratio of 95: 5) to 100% phase B in 30 minutes (a mixture of buffer solution, containing 0.01 M sodium octanesulfonate and 0.02 M sodium dihydrogen phosphate (pH = 3.0) with acetonitrile in a ratio of 70:30), or linear gradient mode from 100% phase A (0.05 M orthophosphoric acid in a mixture of water: acetonitrile in a ratio of 95: 5) to 100% phase B 0.05 M orthophosphoric acid in a mixture of water: acetonitrile in a ratio of 50:50) for 30 min; detection - at 210 nm. The content of p-TSC in creatine amides was determined by RP HPLC on an Alliance chromatograph (Waters), Symmetry C18 column (Waters, USA), mobile phase — 0.1% trifluoroacetic acid solution in a mixture of water: acetonitrile (97: 3), detection at 220 nm. Sulphate ash in creatine amides was determined according to EP 8.2, the content of chloride ions was determined by argentometric titration.

The structure of the obtained compounds was confirmed by NMR spectroscopy ¹ H and ¹³ C.

The essence and advantages of the proposed method are illustrated by the following examples.

Example 1. Obtaining creatylglycine ethyl acetate

1.1. Obtaining creatylglycine ethyl acetate acetate by the present method.

Stage A. Obtaining creatylglycine tosylate ethyl ester.

Creatine monohydrate (5.69 g, 38 mmol), p-TSC (15.23 g, 80 mmol) are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, and DMF (30 ml), stirred for 10 min, a solution of glycine ethyl ester hydrochloride (5.32 g, 38 mmol) in 15 ml of DMF was added. The reaction mixture is stirred at room temperature for 5 minutes, cooled (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) and N- are added simultaneously over 30 minutes. methylmorpholine (8.48 g, 83.9 mmol), stirred at a temperature of (-) 10 ° C for 1.5 hours. The precipitate of N-methylmorpholine salts is filtered off from the cooled reaction mixture, washed on the filter with DMF cooled to 0 ° C. (2 × 5 ml). Wash liquids and mother liquor are combined. In the combined solution, the yield of tosylate ethyl ester creatin glycine and chloride ion content are determined. The yield of tosylate ethyl ester creatyl glycine is 72% mol., The total amount of chloride ions is 29.3 mMEq. Sodium acetate trihydrate (3.99 g, 29.3 mmol) was added to the solution, stirred for 1 h, 50 ml (91%) of DMF were distilled off under reduced pressure and a temperature of 45 ° C. 40 ml of acetone are added to the residue cooled to room temperature, the suspension is stirred for 20 minutes, 4 g of celite are added, and stirred for 20 minutes. 10 g of celite are placed on the filter, washed with 40 ml of acetone, the washing liquid is disposed of. The suspension is filtered through celite, the filter cake washed with 25 ml of acetone. Wash liquids and mother liquor are combined. The amount of chloride ions in the solution decreases to 5.6 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 45 ° C until the end of the run. The residue was dissolved in 20 ml of water to obtain a solution of tosylate ethyl ester creatyl glycine.

Stage B. Obtaining creatyl glycine ethyl acetate.

Removing p-TSC and replacing it with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, 49 × 210 mm column (General Electric, USA) filled with a Dowex 1x8 sorbent in acetate form). A solution of tosylate ethyl ester creatyl glycine is applied to the column, eluting with a 0.05 M acetic acid solution at a rate of 0.3 cm / min. Detection is carried out at a wavelength of 220 nm. Fractions were collected, analyzed by HPLC, and combined on the basis of the analysis results to obtain creatyl glycine ethyl acetate with a total content of unidentified impurities of not more than 1%, creatinine - not more than 1%, creatine - not more than 1%. Acetic acid was added to the combined fractions to a pH of 4.3, the solvent was distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. 100 ml of isopropyl alcohol was added to the residue, the solvent was distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 50 ml of acetone were added, stirred for 3 hours. The precipitate was filtered off, washed with acetone (3 × 25 ml), dried in vacuum at 40 ° C, dissolved in 125 ml of absolute ethanol with stirring for 20 minutes, the insoluble residue was separated by filtration, washed on the filter with absolute ethanol (2 × 20 ml), the washing liquids were combined with the mother liquor. 130 ml (78%) of ethanol are distilled off from the combined solution under reduced pressure and a temperature of 40 ° C, 50 ml of ethyl acetate are added, the mixture is kept for 2 hours at room temperature, the precipitate is filtered off, washed with ethyl acetate (2 × 50 ml), and dried under vacuum at temperature 40 ° С.

The yield of creatyl glycine ethyl acetate C ₈ H ₁₆ N ₄ O ₃ * C ₂ H ₄ O ₂ was 3.67 g (35%). The content of the main substance is 98.6%, extraneous impurities: creatinine - 1.0%, creatine - 0.5%, the maximum single unidentified impurity - 0.1%, the amount of unidentified impurities - 0.3%; sulfate ash - 0.02%, chloride ions - traces, p-TSC - 3 ppm. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.26 (3H, t, 7.17 Hz, CH ₂ CH ₃ ), 1.90 (3H, s, CH ₃ COOH), 3.07 (3H, s, NCH ₃ ), 4.06 (2H, s, N (CH ₃ ) CH ₂ CO); 4.20-4.25 (4H, m, NHCH ₂ CO and CH ₂ CH ₃ ). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 15.96, 25.98, 39.72, 44.09, 55.30, 65.34, 160.55, 172.69, 174 , 24, 184.09.

1.2. Obtaining creatylglycine ethyl ester of acetate according to the method described in the prototype [RU 2428414, 2011].

Stage A. Obtaining creatylglycine tosylate ethyl ester.

Creatine monohydrate (5.69 g, 38 mmol), p-TSC (15.23 g, 80 mmol) are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, and DMF (30 ml), stirred for 10 min, a solution of glycine ethyl ester hydrochloride (5.32 g, 38 mmol) in 15 ml of DMF was added. The reaction mixture was stirred at room temperature for 5 minutes, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) was added sequentially, then, over 30 minutes, N-methylmorpholine (8.48 g, 83.9 mmol) was added dropwise, stirred at (-) 10 ° C for 1 h, the temperature was brought to room temperature. The precipitate was filtered off from the reaction mixture. In the mother liquor, the content of creatiglycine tosylate ethyl ester, chloride ions is determined. The output of creatyl glycine ethyl tosylate is 51% mol., The total amount of chloride ions is 35.1 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 50 ° C until the end of the run. The residue is dissolved in 160 ml of chloroform, left for 10 hours at a temperature of (-) 10 ° C. The solution is filtered and extracted with water 3 times in 100 ml. The aqueous extracts are combined, 2 times extracted with chloroform, the solvent is removed from the combined aqueous extract to a volume of 50 ml under reduced pressure and a temperature of 40 ° C. A solution of creatyl glycine ethyl tosylate is obtained. Stage B. Obtaining creatyl glycine ethyl acetate.

Removing p-TSC and replacing it with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, 49 × 210 mm column (General Electric, USA) filled with a Dowex 1x8 sorbent in acetate form). A solution of tosylate ethyl ester creatyl glycine is applied to the column, eluted with water at a rate of 0.3 cm / min, controlling the pH of the eluate. Fractions with pH = 7 (water pH 5) were collected, analyzed by HPLC, combined, acetic acid was added, the solvent was distilled off from the resulting solution under reduced pressure and a temperature of 40 ° C until the end of the run. The residue is crystallized from 20 ml of acetonitrile at a temperature of (-) 10 ° C for 20 hours. The precipitate of creatylglycine ethyl acetate is filtered off, washed with cold acetonitrile, diethyl ether and dried.

The yield of creatyl glycine ethyl acetate C ₈ H ₁₆ N ₄ O ₃ * C ₂ H ₄ O ₂ is 2.83 g (27%). The content of the main substance is 97%, impurities: creatinine - 1.5%, creatine - 0.5%; the maximum single unidentified impurity is 0.5%, the sum of unidentified impurities is 0.9%, sulfate ash is 0.03%, chloride ions are 0.5 mMEq / g, p-TSC is 150 ppm. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.26 (3H, t, 7.17 Hz, CH ₂ CH ₃ ), 1.91 (3H, s, CH ₃ COOH), 3.07 (3H, s, NCH ₃ ), 4.06 (2H, s, N (CH ₃ ) CH ₂ CO); 4.20-4.25 (4H, m, NHCH ₂ CO and CH ₂ CH ₃ ). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 15.95, 25.99, 39.72, 44.11, 55.30, 65.34, 160.55, 172.71, 174 , 24, 184.09.

Example 2. Obtaining creatylglycine isopropyl ether acetate

2.1 Obtaining creatylglycine isopropyl ether acetate according to the claimed method.

Step A. Preparation of Tosylate Isopropyl Ester Creatyl Glycine

Anhydrous creatine (5 g, 38 mmol), p-TSC (15.23 g, 80 mmol) and DMF are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, (30 ml), stirred for 10 minutes, a solution of glycine isopropyl ether hydrochloride (5.84 g, 38 mmol) in 15 ml of DMF was added. The mixture was stirred at room temperature for 5 minutes, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) and N-methylmorpholine ( 8.48 g, 83.9 mmol), stirred at a temperature of (-) 10 ° C for 1.5 hours. The precipitate was filtered off from the cooled reaction mixture, washed on the filter with DMF (2 × 5 ml) cooled to 0 ° C. . Wash liquids and mother liquor are combined. In a solution, the yield of tosylate isopropyl ether creatin glycine and the chloride ion content are determined. The yield of tosylate isopropyl ester creatyl glycine is 79% mol., The total amount of chloride ions is 29.0 mMEq. Sodium acetate trihydrate (3.95 g, 29.0 mmol) was added to the solution, stirred for 1 h, 50 ml (91%) of DMF were distilled off under reduced pressure and a temperature of 45 ° C. 40 ml of acetone are added to the residue at room temperature, the suspension is stirred for 20 minutes, 4 g of celite are added, and stirred for 20 minutes. 5 g of celite are placed on the filter, washed with 40 ml of acetone, the washing liquid is disposed of. The suspension is filtered through celite, the precipitate is washed with 25 ml of acetone. Wash liquids and mother liquor are combined. The content of chloride ions in the solution is 5.7 mMEq. From the combined solution, the solvent is distilled off under reduced pressure and a temperature of 45 ° C until the end of the run. The residue is dissolved in 20 ml of a 0.05 M solution of acetic acid, and a solution of tosylate isopropyl ether is obtained.

Stage B. Obtaining creatylglycine isopropyl ether acetate.

Removing p-TSC and replacing it with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, 49 × 210 mm column (General Electric, USA) filled with a Dowex 1x8 sorbent in acetate form). A solution of tosylate isopropyl ester creatyl glycine is applied to the column, eluting with a 0.05 M acetic acid solution at a rate of 0.3 cm / min. Detection is carried out at a wavelength of 220 nm. Fractions were collected, analyzed by HPLC, combined on the basis of the analysis results to obtain isopropyl acetate acetate creatyl glycine with a total content of unidentified impurities of not more than 1%, creatinine - not more than 1%, creatine - not more than 1%, acetic acid was added to pH 4.5 . From the combined fractions, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. 100 ml of isopropyl alcohol is added to the residue, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 50 ml of acetone are added, stirred for 3 hours, the precipitate is separated by filtration, washed with acetone (3 × 25 ml), dried in vacuum at a temperature of 40 ° C. The precipitate was dissolved in 125 ml of absolute ethanol with stirring for 20 minutes, the insoluble residue was separated by filtration, washed on the filter with absolute ethanol (2 × 20 ml), and the washings were combined with the mother liquor. 130 ml (78%) of ethanol are distilled off from the solution under reduced pressure and a temperature of 40 ° C, 50 ml of ethyl acetate are added, the mixture is kept for 2 hours at room temperature. The precipitate was separated by filtration, washed with ethyl acetate (2 × 50 ml), dried in vacuum at a temperature of 40 ° C.

The yield of creatylglycine isopropyl ether acetate C ₉ H ₁₈ N ₄ O ₃ * C ₂ H ₄ O ₂ is 5.08 g (46%). The content of the main substance is 98.7%, sulfate ash - 0.01%, chloride ions - traces, p-TSC - 4 ppm, impurities: creatinine - 1.0%, creatine - 0.4%, the maximum single unidentified impurity - 0.1%, the amount of unidentified impurities - 0.3% ,. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.26 (6H, d, 6.22 Hz, CH (CH ₃ ) ₂ ), 1.90 s (3H, s, CH ₃ COOH), 3.07 s (3H, s, NCH ₃ ), 4.02 (2H, s, N (CH ₃ ) CH ₂ CO), 4.23 (2H, s, NHCH ₂ CO), 5.05 (1H, m, CH (CH ₃ ) ₂ ) . ¹³ C NMR spectrum (D ₂ O), δ, ppm: 23.49, 25.96, 39.72, 44.36, 55.29, 73.65, 160.54, 172.63, 173 69, 184.06.

2.2 Obtaining creatylglycine isopropyl ether acetate according to the method described in the prototype [RU 2428414, 2011].

Step A. Preparation of Tosylate Isopropyl Ester Creatyl Glycine

Creatine monohydrate (5.69 g, 38 mmol), p-TSC (15.23 g, 80 mmol) are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, and DMF (30 ml), stirred for 10 min, a solution of glycine isopropyl ether hydrochloride (5.84 g, 38 mmol) in 15 ml of DMF was added. The reaction mixture was stirred at room temperature for 10 minutes, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) was added sequentially, then, over 30 minutes, N-methylmorpholine (8.48 g, 83.9 mmol) was added dropwise. The reaction mixture was stirred at a temperature of (-) 10 ° C for 1 h, the temperature was brought to room temperature. The precipitate is filtered off. In the mother liquor, the content of creatine glycine isopropyl ester of tosylate, chloride ions is determined. The yield of tosylate isopropyl ester creatyl glycine is 41% mol., The total amount of chloride ions is 36.3 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 50 ° C until the end of the run. The residue is dissolved in 160 ml of chloroform, left for 10 hours at a temperature of (-) 10 ° C. The solution is filtered off, extracted with water 3 times in 100 ml. The aqueous extracts are combined, extracted 2 times with chloroform, the solvent is distilled off from the combined aqueous extract under reduced pressure and a temperature of 40 ° C to a volume of 50 ml. A solution of tosylate isopropyl ester creatyl glycine is obtained.

Stage B. Obtaining creatylglycine isopropyl ether acetate.

Removing p-TSC and replacing it with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, 30 × 250 mm column filled with Dowex 1x8 sorbent in acetate form). A solution of tosylate isopropyl ester creatyl glycine is applied to the column, eluted with water at a rate of 0.3 cm / min, controlling the pH of the eluate. Fractions with pH = 7 (water pH 5) are collected, analyzed by HPLC, combined, acetic acid is added, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. The residue is crystallized from 35 ml of acetonitrile at a temperature of (-) 10 ° C for 20 hours. The precipitate of creatylglycine isopropyl ether acetate is filtered off, washed with cold acetonitrile, diethyl ether and dried.

The yield of creatylglycine isopropyl ether acetate C ₉ H ₁₈ N ₄ O ₃ * C ₂ H ₄ O ₂ is 2.33 g (21%). The content of the main substance is 97.4%, impurities: creatinine - 1.5%, creatine - 0.4%, the maximum single unidentified impurity - 0.5%, the sum of unidentified impurities - 0.7%; sulfate ash - 0.03%, chloride ions - 0.4 mMEq / g, p-TSA - 150 ppm. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.25 (6H, d, 6.22 Hz, CH (CH ₃ ) ₂ ), 1.90 s (3H, s, CH ₃ COOH), 3.07 s (3H, s, NCH ₃ ), 4.00 (2H, s, N (CH ₃ ) CH ₂ CO), 4.23 (2H, s, NHCH ₂ CO), 5.05 (1H, m, CH (CH ₃ ) ₂ ) . ¹³ C NMR spectrum (D ₂ O), δ, ppm: 23.49, 25.96, 39.72, 44.38, 55.29, 73.63, 160.55, 172.63, 173 69, 184.06.

Example 3. Obtaining creatylglycine benzyl ester acetate and creatylglycine hydrate

3.1 Obtaining creatylglycine benzyl ether acetate and creatylglycine hydrate according to the present method.

Stage A. Obtaining creatylglycine tosylate benzyl ester.

Anhydrous creatine (5 g, 38 mmol), p-TSC (15.23 g, 80 mmol) and DMF are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, (30 ml), a solution of glycine tosylate benzyl ester (12.82 g, 38 mmol) in 15 ml of DMF. The mixture is stirred for 5 min, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° С, isobutyl chloroformate (6.25 g, 45.8 mmol) and N-methylmorpholine (8, 48 g, 83.9 mmol), stirred at a temperature of (-) 10 ° C for 1.5 hours. The precipitate was filtered off from the cooled reaction mixture, washed on the filter with DMF (2 × 5 ml) cooled to 0 ° C. Wash liquids and mother liquor are combined. In the combined solution, the content of creatinglycine tosylate benzyl ester is determined. The output of creatyl glycine benzyl ester of tosylate 60% mol. Sodium acetate trihydrate (2.10 g, 15.5 mmol) was added to the solution, stirred for 1 h, 50 ml (91%) of DMF were distilled off under reduced pressure and a temperature of 45 ° C. 40 ml of tetrahydrofuran are added to the residue at room temperature, stirred for 20 minutes, 4 g of celite are added, and stirred for 20 minutes. 10 g of celite are placed on the filter, washed with 40 ml of tetrahydrofuran, and the washing liquid is disposed of. The suspension is filtered through celite, the precipitate is washed with 25 ml of tetrahydrofuran. Wash liquids and mother liquor are combined. The amount of chloride ions in the solution is 2.6 mMEq. From the combined solution, the solvent is distilled off under reduced pressure and a temperature of 45 ° C until the end of the run. 75 ml of double-distilled water are added to the residue, 3 × 100 ml of methylene chloride are extracted. The organic extracts are combined, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, the residue is dissolved in 20 ml of a mixture of 0.05 M acetic acid: isopropyl alcohol (50:50). A tosylate benzyl ester creatinglycine solution is obtained.

Stage B. Obtaining creatylglycine benzyl ether acetate.

Removal of p-TSC and its replacement with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, column 49 × 230 mm in size, filled with a Dowex 1x8 sorbent in acetate form). A solution of tosylate benzyl ester creatiglycine was applied to the column, eluted with a mixture of 0.05 M acetic acid: isopropyl alcohol (75:25) at a rate of 0.3 cm / min. Detection is carried out at a wavelength of 220 nm. Fractions were collected, analyzed by HPLC, combined on the basis of the analysis results to obtain creatiglycine benzyl ester acetate with a total content of unidentified impurities of not more than 1%, creatinine - not more than 1%, creatine - not more than 1%, acetic acid was added to pH 4.4 . From the combined fractions, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run.

100 ml of isopropyl alcohol is added to the residue, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 50 ml of acetone are added, stirred for 3 hours, the precipitate is separated by filtration, washed with acetone (3 × 25 ml), dried in vacuum at a temperature of 40 ° C, dissolved in 125 ml of absolute ethanol with stirring for 20 minutes, the insoluble residue was separated by filtration, washed on the filter with absolute ethanol (2 × 20 ml), the washing liquids were combined with the mother liquor. 125 ml (75%) of ethanol are distilled off from the solution under reduced pressure and a temperature of 40 ° C, 50 ml of ethyl acetate are added, the mixture is kept for 2 hours at room temperature. The precipitate was separated by filtration, washed with ethyl acetate (2 × 50 ml), dried in vacuum at a temperature of 40 ° C.

The yield of creatylglycine benzyl ether acetate C ₁₃ H ₁₈ N ₄ O ₃ * C ₂ H ₄ O ₂ 5.40 g (42%). The content of the main substance is 97.9%, sulfate ash - 0.01%, chloride ions - traces, p-TSC - 3 ppm, impurities: creatinine - 1.0%, creatine - 0.3%, the maximum single unidentified impurity - 0.1%, the amount of unidentified impurities - 0.3%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.92 (3H, s, CH ₃ COOH), 2.99 (3H, s, NCH ₃ ), 4.10 (2H, s, N (CH ₃ ) CH ₂ CO), 4 19 (2H, s, NHCH ₂ CO); 5.22 (2H, s, CH ₂ Ph); 7.45 (5H, m, CH ₂ Ph). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 25.97, 39.62, 44.11, 55.26, 70.44, 131.08, 131.53, 137.80, 160 48, 172.63, 173.86, 184.09.

Step C. Preparation of Creatyl Glycine Hydrate

5.40 g of creatylglycine benzyl ester acetate obtained in stage B is dissolved in 100 ml of methanol and hydrogenated with palladium-carbon Pd / C for 3 hours. The complete removal of the benzyl group is monitored by TLC in the acetonitrile: water: acetic acid system. 6: 1: 1. The catalyst is filtered off and washed with 10 ml of a mixture of water: methanol 10:90. The mother liquor and washings are combined, the solvent is distilled off from the combined solution under reduced pressure and a temperature of 40 ° C. The product is obtained in the form of a white powder. The yield of creatylglycine hydrate C ₆ H ₁₂ N ₄ O ₃ * H ₂ O 2.67 g (37%). The content of the main substance is 98.2%, extraneous impurities: creatinine - 0.8%, creatine - 0.3%, the maximum single unidentified impurity - 0.1%, the amount of unidentified impurities - 0.2%; sulfate ash - 0.01%, chloride ions - traces, p-TSC - 2 ppm. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 3.06 (3H, s, NCH ₃ ), 3.81 (2H, s, N (CH ₃ ) CH ₂ CO), 4.20 (2H, s, NHCH ₂ CO). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 39.75, 45.94, 55.47, 160.60, 171.94, 179.22.

3.2 Obtaining creatin glycine benzyl ester acetate and creatyl glycine hydrate according to the method described in the prototype [RU 2428414, 2011].

Stage A. Obtaining creatylglycine tosylate benzyl ester.

Anhydrous creatine (5 g, 38 mmol), p-TSC (15.23 g, 80 mmol) and DMF are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube, mounted on a magnetic stirrer. (30 ml), stirred for 10 min, a solution of glycine tosylate benzyl ester glycine (12.82 g, 38 mmol) in 15 ml of DMF was added. The mixture was stirred at room temperature for 5 minutes, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) was added sequentially, then, over 30 minutes, dropwise add N-methylmorpholine (8.48 g, 83.9 mmol), stir at (-) 10 ° C for 1 h, bring the temperature to room temperature. The precipitate is filtered off. In the mother liquor, the content of creatinglicin benzyl ester of tosylate and chloride ions is determined. The yield of tosylate benzyl ester creatiglycine is 42% mol., The total amount of chloride ions is 18.0 mMeq. The solvent is distilled off from the solution under reduced pressure and a temperature of 50 ° C until the end of the run. The residue is dissolved in 300 ml of n-butyl alcohol, washed 4 times with 5% NaHCO ₃ solution, 2 times with water. The solvent is distilled off from the organic phase under reduced pressure and a temperature of 40 ° C, 60 ml of a mixture of water: isopropyl alcohol (50:50) are added to the residue, stirred, a solution of creatinglicin benzyl ester of tosylate is obtained.

Stage B. Obtaining creatylglycine benzyl ether acetate.

Removing p-TSC and replacing it with a pharmaceutically acceptable acid is carried out by RP chromatography (Buchi Sepacore chromatographic system, 22.5 × 150 mm column filled with YMC * Gel ODS sorbent). A solution of tosylate benzyl ester creatiglycine is applied to the column, eluting in a linear gradient mode from 0.05 M acetic acid to 20% isopropyl alcohol in 0.05 M acetic acid at a speed of 0.3 cm / min. Fractions are collected, analyzed by HPLC, combined based on the results of the analysis, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. The residue is crystallized from 35 ml of acetonitrile, separated by filtration and dried.

Yield of creatyl glycine benzyl ester acetate C ₁₃ H ₁₈ N ₄ O ₃ * C ₂ H ₄ O ₂ 2.83 g (22%). The content of the main substance is 97.5%, extraneous impurities: creatinine - 1.3%, creatine - 0.5%, the maximum single unidentified impurity - 0.2%, the amount of unidentified impurities - 0.5%; sulfate ash - 0.01%, chloride ions - 0.1 mMEq / g, p-TSC - 5 ppm. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.92 (3H, s, CH ₃ COOH), 2.99 (3H, s, NCH ₃ ), 4.11 (2H, s, N (CH ₃ ) CH ₂ CO), 4 19 (2H, s, NHCH ₂ CO); 5.22 (2H, s, CH ₂ Ph); 7.43 (5H, m, CH ₂ Ph). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 25.95, 39.62, 44.11, 55.26, 70.44, 131.10, 131.53, 137.80, 160 48, 172.62, 173.86, 184.09.

Step C. Preparation of Creatyl Glycine Hydrate

2.83 g of creatyl glycine benzyl ester acetate obtained in stage B is dissolved in 70 ml of methanol and hydrogenated over Pd / C for 3 hours. The complete removal of the benzyl group is monitored by TLC in acetonitrile: water: acetic acid 6: 1: one. The catalyst is filtered off and washed with 10 ml of a mixture of water: methanol 10:90. The mother liquor and washings are combined, the solvent is distilled off from the solution under reduced pressure and a temperature of 40 ° C until the end of the run. The product is obtained in the form of a white powder.

The yield of creatylglycine hydrate C ₆ H ₁₂ N ₄ O ₃ * H ₂ O 1.61 g (22%). The content of the main substance is 97.5%, extraneous impurities: creatinine - 1.3%, creatine - 0.5%, the maximum single unidentified impurity - 0.2%, the amount of unidentified impurities - 0.5%; sulfate ash - 0.01%, chloride ions - 0.1 mMEq / g, p-TSC - 5 ppm. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 3.07 (3H, s, NCH ₃ ), 3.81 (2H, s, N (CH ₃ ) CH ₂ CO), 4.21 (2H, s, NHCH ₂ CO). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 39.75, 45.94, 55.49, 160.60, 171.93, 179.22.

Example 4. Obtaining creatyl-α-alanine isopropyl ether acetate

4.1 Obtaining creatyl-α-alanine isopropyl ether acetate according to the present method.

Stage A. Obtaining creatyl-α-alanine isopropyl ester of tosylate.

Anhydrous creatine (5 g, 38 mmol), p-TSC (15.23 g, 80 mmol) and DMF are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, (30 ml), stirred for 10 min, a solution of α-alanine isopropyl ether hydrochloride (6.37 g, 38 mmol) in 15 ml of DMF was added. The mixture is stirred at room temperature for 5 minutes, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) is added dropwise at the same time over 30 minutes. and triethylamine (8.48 g, 83.9 mmol), stirred at a temperature of (-) 10 ° C for 1.5 hours. The precipitate was filtered off from the cooled reaction mixture, washed on the filter with DMF cooled to 0 ° C (2 × 5 ml). The washing liquids and the mother liquor are combined; the content of creatyl-α-alanine tosylate isopropyl ether and chloride ions is determined in the solution. The yield of creatyl-α-alanine tosylate isopropyl ether is 44% mol., The amount of chloride ions is 28.5 mMeq. Sodium acetate trihydrate (3.88 g, 28.5 mmol) was added to the solution, stirred for 1 h, 50 ml (91%) of DMF were distilled off under reduced pressure and a temperature of 45 ° C. 40 ml of acetone are added to the residue at room temperature, stirred for 20 minutes, 4 g of celite are added, and stirred for 20 minutes. 10 g of celite are placed on the filter, washed with 40 ml of acetone, the washing liquid is disposed of. The suspension is filtered through celite, the precipitate is washed with 25 ml of acetone. Wash liquids and mother liquor are combined. The amount of chloride ions is 5.9 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 45 ° C until the end of the run. The residue is dissolved in 20 ml of a 0.05 M solution of acetic acid. A solution of creatyl-α-alanine tosylate isopropyl ether is obtained.

Stage B. Obtaining creatyl-α-alanine isopropyl ether acetate.

Removing p-TSC and replacing it with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, 49 × 210 mm column (General Electric, USA) filled with a Dowex 1x8 sorbent in acetate form). A solution of creatyl-α-alanine tosylate isopropyl ether is applied to the column, eluting with a 0.05 M solution of acetic acid at a speed of 0.6 cm / min. Detection is carried out at a wavelength of 220 nm. Fractions were collected, analyzed by HPLC, and pooled on the basis of the results of analysis to obtain creatyl-α-alanine isopropyl ether acetate with a total content of unidentified impurities of not more than 1%, creatinine - not more than 1%, creatine - not more than 1%. A 5 M solution of acetic acid (16 ml, 16 mmol) was added to the combined fractions to a pH of 4.5, the solvent was distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. 100 ml of isopropyl alcohol is added to the residue, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 50 ml of acetone are added, stirred for 3 hours, the precipitate is separated by filtration, washed with acetone (3 × 25 ml), dried in vacuum at a temperature of 40 ° C. The precipitate was dissolved in 125 ml of absolute ethanol, the insoluble residue was separated by filtration, washed on the filter with absolute ethanol (2 × 20 ml), and the washing liquids were combined with the mother liquor. 130 ml (79%) of ethanol are distilled off from the solution under reduced pressure and a temperature of 40 ° C, 50 ml of ethyl acetate are added, and they are kept at room temperature for 2 hours. The precipitate was separated by filtration, washed with ethyl acetate (2 × 50 ml), dried in vacuum at a temperature of 40 ° C.

The yield of creatyl-α-alanine isopropyl ether acetate C ₁₀ H ₂₀ N ₄ O ₃ * C ₂ H ₄ O ₂ 4.32 g (37%). The content of the main substance is 98.2%, extraneous impurities: creatinine - 0.8%, creatine - 0.3%, the maximum single unidentified impurity - 0.1%, the amount of unidentified impurities - 0.2%; sulfate ash - 0.01%, chloride ions - traces, p-TSC - 4 ppm. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.25 (6H, m, OCH (CH ₃ ) ₂ ), 1.42 (3H, d, 6.98 Hz, CH ₃ HCO), 1.90 (3H, s, CH ₃ COOH), 3.05 (3H, s, NCH ₃ ), 4.20 (2H, s, N (CH ₃ ) CH ₂ CO), 4.37 (1H, m, CH ₃ CHCO), 5.02 ( 1H, m, OCH (CH ₃ ) ₂ ). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 18.48, 23.42, 25.96, 39.69, 51.92, 55.06, 73.54, 160.49, 171 69, 176.75, 183.97.

4.2 Obtaining creatyl-α-alanine isopropyl ether acetate according to the method described in the prototype [RU 2428414, 2011].

Stage A. Obtaining creatyl-α-alanine isopropyl ester of tosylate.

Creatine monohydrate (5.69 g, 38 mmol), p-TSC (15.23 g, 80 mmol) are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, and DMF (30 ml), stirred for 10 min, a solution of α-alanine isopropyl ether hydrochloride (6.37 g, 38 mmol) in 15 ml of DMF was added. The mixture was stirred at room temperature for 10 minutes, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) was added sequentially, then, over 30 minutes, triethylamine (8.48 g, 83.9 mmol) was added dropwise, stirred at a temperature of (-) 10 ° C for 1 h, the temperature was brought to room temperature. The precipitate of triethylamine salts is filtered off from the reaction mixture. The content of creatyl-α-alanine tosylate isopropyl ether and chloride ions is determined in the mother liquor. The yield of creatyl-α-alanine tosylate isopropyl ether is 36% mol., The total amount of chloride ions is 33.5 mMeq. The solvent is distilled off from the solution under reduced pressure and a temperature of 50 ° C until the end of the run. The residue is dissolved in 160 ml of chloroform, left for 10 hours at a temperature of (-) 10 ° C. The solution is filtered off, extracted with water 3 times in 100 ml. The aqueous extracts are combined, extracted 2 times with chloroform, the solvent is distilled off from the aqueous extract under reduced pressure and a temperature of 40 ° C to a volume of 50 ml. A solution of creatyl-α-alanine tosylate isopropyl ether is obtained.

Stage B. Obtaining creatyl-α-alanine isopropyl ether acetate.

Removing p-TSC and replacing it with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, 49 × 210 mm column (General Electric, USA) filled with a Dowex 1x8 sorbent in acetate form). A solution of creatyl-α-alanine tosylate isopropyl ether is applied to the column, elute with water at a rate of 0.3 cm / min, controlling the pH of the eluate. Fractions with pH = 7 (water pH 5) are collected, analyzed by HPLC, combined, 5 ml of acetic acid are added, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. The residue is crystallized from 40 ml of acetonitrile at a temperature of (-) 10 ° C for 20 hours. The precipitate of creatylglycine isopropyl ether acetate is filtered off, washed with cold acetonitrile, diethyl ether and dried.

The yield of creatyl-α-alanine isopropyl ether acetate C ₁₀ H ₂₀ N ₄ O ₃ * C ₂ H ₄ O ₂ 2.64 g (23%). The content of the main substance is 97.7%, extraneous impurities: creatinine - 1.3%, creatine - 0.5%, the maximum single unidentified impurity - 0.2%, the amount of unidentified impurities - 0.3%; sulfate ash - 0.03%, chloride ions - 0.5 mMEq / g, p-TSA - 152 ppm. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.27 (6H, m, OCH (CH ₃ ) ₂ ), 1.42 (3H, d, 6.99 Hz, CH ₃ CHCO), 1.90 (3H, s, CH ₃ COOH), 3.06 (3H, s, NCH ₃ ), 4.20 (2H, s, N (CH ₃ ) CH ₂ CO), 4.38 (1H, m, CH ₃ CHCO), 5.02 ( 1H, m, OCH (CH ₃ ) ₂ ). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 18.48, 23.42, 25.95, 39.69, 51.92, 55.06, 73.54, 160.47, 171 69, 176.75, 183.97.

Example 5. Obtaining creatyl-β-alanine isopropyl ether succinate

5.1 Obtaining creatyl-β-alanine isopropyl ether succinate according to the present method.

Stage A. Obtaining creatyl-β-alanine tosylate isopropyl ether.

Anhydrous creatine (5 g, 38 mmol), p-TSC (15.23 g, 80 mmol) and DMF are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, (30 ml), stirred for 10 min, a solution of β-alanine isopropyl ether hydrochloride (6.37 g, 38 mmol) in 15 ml of DMF was added. The mixture is stirred at room temperature for 5 minutes, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) is added dropwise at the same time over 30 minutes. and N-methylmorpholine (8.48 g, 83.9 mmol), stirred at a temperature of (-) 10 ° С for 1.5 h. The precipitate was filtered off from the cooled reaction mixture, washed on the filter with DMF cooled to 0 ° С ( 2 × 5 ml). Wash liquids and the mother liquor are combined, the content of creatyl-β-alanine tosylate isopropyl ether and chloride ions is determined in the solution. The yield of creatyl-β-alanine tosylate isopropyl ether is 67% mol., The total amount of chloride ions is 29.5 mMEq. Anhydrous potassium acetate (2.90 g, 29.5 mmol) was added to the solution, stirred for 1 h, 50 ml (91%) of DMF were evaporated under reduced pressure and a temperature of 45 ° C. 40 ml of acetone are added to the residue at room temperature, stirred for 20 minutes, 4 g of celite are added, and stirred for 20 minutes. 5 g of celite are placed on the filter, washed with 40 ml of acetone, and the washing liquid is disposed of. The suspension is filtered through celite, the precipitate is washed with 25 ml of acetone. Wash liquids and mother liquor are combined. The content of chloride ions in the solution is 5.9 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 45 ° C until the end of the run, the residue is dissolved in 20 ml of a 0.05 M solution of acetic acid. A solution of creatyl-β-alanine tosylate isopropyl ether is obtained.

Stage B. Obtaining creatyl-β-alanine isopropyl ether succinate.

Removal of p-TSA and its replacement with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, column 49 × 200 mm (General Electric, USA) filled with a Dowex 1x8 sorbent in acetate form). A solution of creatyl-β-alanine tosylate isopropyl ether is applied to the column, eluting with a 0.05 M solution of acetic acid at a rate of 0.3 cm / min. Detection is carried out at a wavelength of 220 nm. Fractions were collected, analyzed by HPLC, and pooled on the basis of the results of analysis to obtain creatyl-β-alanine isopropyl ether acetate with a total content of unidentified impurities of not more than 1%, creatinine - not more than 1%, creatine - not more than 1%. Succinic acid (1.89 g, 16 mmol) was added to the combined fractions, stirred for 20 minutes until complete dissolution (solution pH 4.4), the solvent was distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. 100 ml of isopropyl alcohol was added to the residue, the solvent was distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 50 ml of a mixture of acetone: acetonitrile 90:10 were added, stirred for 3 h, the precipitate was separated by filtration, washed with acetone (3 × 25 ml), dried in vacuum at a temperature of 40 ° C. The precipitate was dissolved in 125 ml of absolute ethanol with stirring for 20 minutes, the insoluble residue was separated by filtration, washed on the filter with absolute ethanol (2 × 20 ml), and the washings were combined with the mother liquor. 130 ml (78%) of ethanol are distilled off from the solution under reduced pressure and a temperature of 40 ° C, 50 ml of acetonitrile are added, the mixture is kept for 2 hours at room temperature. The precipitate was separated by filtration, washed with acetonitrile (2 × 50 ml), dried in vacuum at a temperature of 40 ° C.

The yield of creatyl-β-alanine isopropyl ester of succinate C ₁₀ H ₂₀ N ₄ O ₃ * C ₄ H ₆ O ₄ 4.58 g (33%). The content of the main substance is 98.3%, extraneous impurities: creatinine - 0.9%, creatine - 0.4%, the maximum single unidentified impurity - 0.1%, the sum of unidentified impurities - 0.2%; sulfate ash - 0.03%, p-TSA - 3 ppm, chloride ions - traces. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.24 (6H, d, 6.27 Hz, OCH (CH ₃ ) ₂ ), 2.56 (4H, s, (CH ₂ COOH) ₂ ), 2.59 (2H, t , 6.44 Hz, CH ₂ CH ₂ CO), 3.03 (3H, s, NCH ₃ ), 3.51 (2H, t, 6.44 Hz, CH ₂ CH ₂ CO), 4.11 (2H , s, N (CH ₃ ) CH ₂ CO), 4.99 (1H, m, OCH (CH ₃ ) ₂ ). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 23.53, 25.96, 36.69, 38.01, 39.63, 55.40, 72.58, 160.48, 171 69, 176.34, 181.85.

5.2 Obtaining creatyl-β-alanine isopropyl ether succinate according to the method described in the prototype [RU 2428414, 2011].

Stage A. Obtaining creatyl-β-alanine tosylate isopropyl ether.

Creatine monohydrate (5.69 g, 38 mmol), p-TSC (15.23 g, 80 mmol) are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, and DMF (30 ml), stirred for 10 min, a solution of β-alanine isopropyl ether hydrochloride (6.37 g, 38 mmol) in 15 ml of DMF is added. The mixture is stirred for 10 minutes, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) is added sequentially, then added dropwise over 30 minutes N-methylmorpholine (8.48 g, 83.9 mmol), stirred at a temperature of (-) 10 ° C for 1 h, brought the temperature to room temperature. The precipitate is filtered off. The content of creatyl-β-alanine tosylate isopropyl ether and chloride ions is determined in the mother liquor. The yield of creatyl-β-alanine tosylate isopropyl ether is 43% mol., The amount of chloride ions is 36.3 mMEq. The solvent is distilled off from the mother liquor under reduced pressure at a temperature of 50 ° С, the residue is dissolved in 160 ml of chloroform, left for 10 hours at a temperature of (-) 10 ° С. The solution is filtered off, extracted with water 3 times in 100 ml. The aqueous extracts are combined, extracted 2 times with chloroform, the solvent is distilled off from the aqueous extract under reduced pressure and a temperature of 40 ° C to a volume of 50 ml. A solution of creatyl-β-alanine tosylate isopropyl ether is obtained.

Stage B. Obtaining creatyl-β-alanine isopropyl ether succinate.

Removing p-TSC acid and replacing it with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, column 49 × 200 mm (General Electric, USA) filled with a Dowex 1x8 sorbent in acetate form). A solution of creatyl-β-alanine tosylate isopropyl ether is applied to the column, elute with water at a rate of 0.3 cm / min, controlling the pH of the eluate. Fractions with pH = 7 (water pH 5) are collected, analyzed by HPLC, combined, succinic acid (1.89 g, 16 mmol) is added, stirred until complete dissolution, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. The residue is crystallized from 40 ml of acetonitrile at a temperature of (-) 10 ° C for 20 hours. The precipitate of creatyl-β-alanine isopropyl ether of succinate is filtered off, washed with cold acetonitrile, diethyl ether and dried.

The yield of creatyl-β-alanine isopropyl ester of succinate C ₁₀ H ₂₀ N ₄ O ₃ * C ₄ H ₆ O ₄ 3.38 g (24%). The content of the main substance is 97.4%, sulfate ash - 0.04%, chloride ions - 0.6 mMEq / g, p-TSA - 140 ppm, impurities: creatinine - 1.4%, creatine - 0.7% , the maximum unit unidentified impurity is 0.6%, the sum of unidentified impurities is 0.9%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.24 (6H, d, 6.27 Hz, OCH (CH ₃ ) ₂ ), 2.57 (4H, s, (CH ₂ COOH) ₂ ), 2.59 (2H, t , 6.44 Hz, CH ₂ CH ₂ CO), 3.03 (3H, s, NCH ₃ ), 3.51 (2H, t, 6.44 Hz, CH ₂ CH ₂ CO), 4.11 (2H , s, N (CH ₃ ) CH ₂ CO), 4.98 (1H, m, OCH (CH ₃ ) ₂ ). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 23.53, 25.96, 36.69, 38.02, 39.63, 55.40, 72.58, 160.48, 171 68, 176.34, 181.85.

Example 6. Obtaining creatylleucine isopropylamide acetate

6.1. Preparation of isopropylamide acetate creatyl leucine according to the claimed method.

Stage A. Obtaining tosylate isopropylamide creatyl leucine.

Anhydrous creatine (5 g, 38 mmol), p-TSC (15.23 g, 80 mmol) and DMF are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, (30 ml), stirred for 10 minutes, a solution of leucine isopropylamide hydrochloride (7.93 g, 38 mmol) in 15 ml of DMF was added. The mixture is stirred at room temperature for 5 minutes, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) is added dropwise at the same time over 30 minutes. and N-methylmorpholine (8.48 g, 83.9 mmol), stirred at a temperature of (-) 10 ° C for 1.5 hours. The precipitate was filtered off from the reaction mixture, washed on the filter with DMF cooled to 0 ° C (2 × 5 ml). Wash liquids and mother liquor are combined. The solution determines the content of creatyl leucine isopropylamide tosylate and chloride ions. The yield of tosylate isopropylamide creatyl leucine is 52% mol., The amount of chloride ions is 29.3 mMEq. Sodium acetate trihydrate (3.99 g, 29.3 mmol) was added to the solution, stirred for 1 h, 50 ml (90.9%) of DMF were distilled off under reduced pressure and a temperature of 45 ° C. 40 ml of acetone was added to the residue at room temperature, the suspension was stirred for 20 minutes, 4 g of celite was added, and stirred for 20 minutes. 10 g of celite are placed on the filter, washed with 40 ml of acetone, the washing liquid is disposed of. The suspension is filtered through celite, the filter cake washed with 25 ml of acetone. Wash liquids and mother liquor are combined. The amount of chloride ions in the combined solution is 5.9 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 45 ° C until the end of the run, the residue is dissolved in 20 ml of a 0.05 M solution of acetic acid. A solution of tosylate isopropylamide creatyl leucine is obtained.

Stage B. Obtaining creatyl leucine isopropylamide acetate.

Removal of p-TSC and its replacement with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatography system, 49 x 200 mm column (General Electric, USA) filled with a Dowex 1x8 sorbent in acetate form). A solution of tosylate isopropylamide creatyl leucine is applied to the column, eluting with a 0.05 M acetic acid solution at a rate of 0.3 cm / min. Detection is carried out at a wavelength of 220 nm. Fractions were collected, analyzed by HPLC, and combined on the basis of the analysis results to obtain isopropylamide acetate creatyl leucine with a total content of unidentified impurities of not more than 1%, creatinine - not more than 1%, creatine - not more than 1%, acetic acid was added to pH 4.4. From the combined fractions, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 100 ml of isopropyl alcohol is added, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 50 ml of acetone are added, the mixture is stirred for 3 hours, the precipitate is separated by filtration, washed with acetone (3 × 25 ml), dried in vacuum at a temperature of 40 ° C. The precipitate was dissolved in 125 ml of absolute ethanol with stirring for 20 minutes, the insoluble residue was separated by filtration, washed on the filter with absolute ethanol (2 × 20 ml), and the washings were combined with the mother liquor. 130 ml (79%) of ethanol are distilled off from the combined solution under reduced pressure and a temperature of 40 ° C, 50 ml of ethyl acetate are added, and the mixture is kept for 2 hours at room temperature. The precipitate was separated by filtration, washed with ethyl acetate (2 × 50 ml), dried in vacuum at a temperature of 40 ° C.

The yield of isopropylamide acetate creatyl leucine C ₁₃ H ₂₇ N ₅ O ₂ * C ₂ H ₄ O ₂ 3.72 g (28%). The main substance content is 98.4%, sulfate ash - 0.04%, chloride ions - traces, p-TSC - 2 ppm, impurities: creatinine - 1.0%, creatine - 0.2%, maximum single unidentified impurity - 0.1%, the amount of unidentified impurities - 0.3%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 0.92 (6H, m, CH ₂ CH (CH ₃ ) ₂ ), 1.13 (6H, m, OCH (CH ₃ ) ₂ ), 1.50-1.70 (3H, m, CH ₂ CH (CH ₃ ) ₂ ), 1.91 (3H, s, CH ₃ COOH), 3.03 (3H, s, NCH ₃ ), 3.92 (1H, m, OCH (CH ₃ ) ₂ ), 4.20 (2H, s, N (CH ₃ ) CH ₂ CO), 4.26 (1H, m, NHCHCO). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 23.55, 23.90, 24.64, 25.85, 26.94, 39.70, 42.68, 44.49, 55 02, 55.59, 160.44, 171.66, 175.77, 183.75.

6.2 Obtaining creatylleucine isopropylamide acetate according to the method described in the prototype [RU 2428414, 2011].

Stage A. Obtaining tosylate isopropylamide creatyl leucine.

Creatine monohydrate (5.69 g, 38 mmol), p-TSC (15.23 g, 80 mmol) are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, and DMF (30 ml), stirred for 10 min, a solution of leucine isopropylamide hydrochloride (7.93 g, 38 mmol) in 15 ml of DMF was added. The mixture is stirred for 10 minutes, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) is added sequentially, then, dropwise, is added over 30 minutes N-methylmorpholine (8.48 g, 83.9 mmol). The mixture is stirred at a temperature of (-) 10 ° C for 1 h, the temperature is brought to room temperature. The precipitate was filtered off from the reaction mixture. In the mother liquor, the content of tosylate isopropylamide creatyl leucine is determined. The yield of tosylate isopropylamide creatyl leucine is 40% mol., The amount of chloride ions is 37.0 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 50 ° C until the end of the run. The residue is dissolved in 160 ml of chloroform, left for 10 hours at a temperature of (-) 10 ° C. The solution is filtered off, extracted with water 3 times in 100 ml, 2 times extracted with chloroform. From the combined extract, the solvent is distilled off under reduced pressure and a temperature of 40 ° C to a volume of 50 ml. A solution of tosylate isopropylamide creatyl leucine is obtained.

Stage B. Obtaining creatyl leucine isopropylamide acetate.

Removal of p-TSA and its replacement with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, column 49 × 200 mm (General Electric, USA) filled with a Dowex 1x8 sorbent in acetate form). A solution of tosylate isopropylamide creatyl leucine is applied to the column, eluted with water at a rate of 0.3 cm / min, controlling the pH of the eluate. Fractions with pH = 7 (water pH 5) are collected, analyzed by HPLC, combined, 5 ml of acetic acid are added, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. The residue is crystallized from 35 ml of acetonitrile at a temperature of (-) 10 ° C for 20 hours. The precipitate of creatyl leucine isopropylamide acetate is filtered off, washed with cold acetonitrile, diethyl ether and dried.

The yield of creatyl leucine isopropylamide acetate C ₁₃ H ₂₇ N ₅ O ₂ * C ₂ H ₄ O ₂ is 2.77 g (21%). The content of the main substance is 97.7%, sulfate ash - 0.05%, chloride ions - 0.4 mMEq / g, p-TSA - 160 ppm, impurities: creatinine - 1.4%, creatine - 0.4% , the maximum unit unidentified impurity is 0.5%, the sum of unidentified impurities is 0.8%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 0.92 (6H, m, CH ₂ CH (CH ₃ ) ₂ ), 1.13 (6H, m, OCH (CH ₃ ) ₂ ), 1.50-1.70 (3H, m, CH ₂ CH (CH ₃ ) ₂ ), 1.91 (3H, s, CH ₃ COOH), 3.03 (3H, s, NCH ₃ ), 3.92 (1H, m, OCH (CH ₃ ) ₂ ), 4.20 (2H, s, N (CH ₃ ) CH ₂ CO), 4.26 (1H, m, NHCHCO). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 23.55, 23.90, 24.64, 25.85, 26.94, 39.70, 42.68, 44.49, 55 02, 55.59, 160.44, 171.66, 175.77, 183.75.

Example 7. Obtaining creatyl leucine benzyl ester acetate and creatyl leucine hemihydrate

7.1 Preparation of creatyl leucine benzyl ester acetate and creatilleucine hemihydrate according to the claimed method.

Stage A. Obtaining creatyl leucine benzyl ester of tosylate.

Anhydrous creatine (5 g, 38 mmol), p-TSC (15.23 g, 80 mmol) and DMF are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, (30 ml), stirred for 10 minutes, a solution of leucine tosylate benzyl ester (14.95 g, 38 mmol) in 15 ml of DMF was added. The mixture is stirred at room temperature for 5 minutes, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) is added dropwise at the same time over 30 minutes. and N-methylmorpholine (8.48 g, 83.9 mmol), stirred at a temperature of (-) 10 ° С for 1.5 h. The precipitate was filtered off from the cooled reaction mixture, washed on the filter with DMF cooled to 0 ° С ( 2 × 5 ml). Wash liquids and mother liquor are combined. The solution determines the content of creatyl leucine benzyl ester of tosylate and chloride ions. The yield of tosylate benzyl ester creatyl leucine is 60% mol, the amount of chloride ions is 15.1 mMeq. Sodium acetate trihydrate (2.05 g, 15.1 mmol) was added to the solution, stirred for 1 h, 50 ml (91%) of DMF were distilled off under reduced pressure and a temperature of 45 ° C. 40 ml of methyl ethyl ketone are added to the residue at room temperature, stirred for 20 minutes, 4 g of celite are added, and stirred for 20 minutes. 10 g of celite are placed on the filter, washed with 40 ml of methyl ethyl ketone, the washing liquid is disposed of. The suspension is filtered through celite, the filter cake washed with 25 ml of methyl ethyl ketone. Wash liquids and mother liquor are combined. The amount of chloride ions in the solution is 2.6 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 45 ° C until the end of the run. 75 ml of double-distilled water are added to the residue, 3 × 100 ml of methylene chloride are extracted. The organic extracts are combined, the solvent is removed from the combined organic extract under reduced pressure and a temperature of 40 ° C until the end of the run, the residue is dissolved in 20 ml of a mixture of 0.05 M acetic acid: isopropyl alcohol (50:50). A solution of creatyl leucine benzyl ester of tosylate is obtained.

Stage B. Obtaining creatylleucine benzyl ether acetate.

Removal of p-TSA and its replacement with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, column 49 × 200 mm (General Electric, USA) filled with a Dowex 1x8 sorbent in acetate form). A solution of tosylate benzyl ester creatyl leucine is applied to the column, eluting with a mixture of 0.05 M acetic acid: isopropyl alcohol (75:25) at a rate of 0.3 cm / min. Detection is carried out at a wavelength of 220 nm. Fractions were collected, analyzed by HPLC, the fractions were combined based on the analysis results to obtain creatyl leucine benzyl ester acetate with a total content of unidentified impurities of not more than 1%, creatinine - not more than 1%, creatine - not more than 1%, acetic acid was added to pH 4, four. The solvent is distilled off from the combined fractions under reduced pressure and a temperature of 40 ° C. 100 ml of isopropyl alcohol was added to the residue, the solvent was distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 50 ml of acetone were added, stirred for 3 hours. The precipitate was filtered off, washed with acetone (3 × 25 ml), dried in vacuum at 40 ° C, dissolved in 125 ml of absolute ethanol with stirring for 20 minutes, the insoluble residue was separated by filtration, washed on the filter with absolute ethanol (2 × 20 ml), the washing liquids were combined with the mother liquor. 130 ml (79%) of ethanol are distilled off from the solution under reduced pressure and a temperature of 40 ° C, 50 ml of ethyl acetate are added, and they are kept at room temperature for 2 hours. The precipitate was separated by filtration, washed with ethyl acetate (2 × 50 ml), dried in vacuum at a temperature of 40 ° C.

The yield of creatyl leucine benzyl ether acetate C ₁₇ H ₂₆ N ₄ O ₃ * C ₂ H ₄ O ₂ is 6.00 g (40%). The content of the main substance is 97.7%, sulfate ash - 0.02%, chloride ions - traces, p-TSC - 3 ppm, impurities: creatinine - 1.2%, creatine - 0.6%, the maximum single unidentified impurity - 0.1%, the amount of unidentified impurities - 0.2%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 0.96 (6H, m, CH ₂ CH (CH ₃ ) ₂ ), 1.48-1.67 (3H, m, CH ₂ CH (CH ₃ ) ₂ ), 2.00 ( 3H, s, CH ₃ COOH), 3.04 (3H, s, NCH ₃ ), 4.24 (2H, s, N (CH ₃ ) CH ₂ CO), 4.57 (1H, m, NHCHCO), 5.27 (2H, m, CH ₂ Ph); 7.48 (5H, m, CH ₂ Ph). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 22.99, 24.31, 25.63, 26.69, 39.27, 41.42, 54.13, 54.75, 70 01, 130.66, 131.11, 137.52, 160.13, 171.60, 176.32, 183.72.

Stage C. Obtaining creatyl leucine hemihydrate.

6.0 g of creatyl leucine benzyl acetate ester obtained in step B is dissolved in 120 ml of methanol and hydrogenated over Pd / C for 3 hours. The complete removal of the benzyl group is monitored by TLC in the acetonitrile: water: acetic acid 6: 1 system: one. The catalyst is filtered off, washed with 10 ml of a mixture of water: methanol 10:90. The mother liquor and washings are combined, the solvent is distilled off from the solution under reduced pressure and a temperature of 40 ° C until the end of the run. The product is obtained in the form of a white powder.

The yield of creatilleucine hemihydrate C ₁₀ H ₂₀ N ₄ O ₃ * 0.5H ₂ O 2.65 g (33%). The content of the main substance is 98.0%, sulfate ash - 0.02%, chloride ions - traces, p-TSC - 3 ppm, impurities: creatinine - 1.0%, creatine - 0.5%, maximum single unidentified impurity - 0.1%, the amount of unidentified impurities - 0.2%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 0.91 (6H, m, CH ₂ CH (CH ₃ ) ₂ ), 1.61 (3H, m, CH ₂ CH (CH ₃ ) ₂ ), 3.04 (3H, s, NCH ₃ ), 4.12-4.26 (3H, m, N (CH ₃ ) CH ₂ CO and NHCHCO). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 23.32, 25.12, 27.35, 39.76, 43.21, 55.34, 56.79, 160.53, 171 , 32, 182.50.

7.2 Obtaining creatyl leucine benzyl ester of acetate and creatyl leucine hemihydrate according to the method described in the prototype [RU 2428414, 2011].

Stage A. Obtaining creatyl leucine benzyl ester of tosylate.

Anhydrous creatine (5 g, 38 mmol), p-TSC (15.23 g, 80 mmol) and DMF are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube, mounted on a magnetic stirrer. (30 ml), stirred for 10 min, a solution of leucine benzyl ester of tosylate (14.95 g, 38 mmol) in 15 ml of DMF was added, stirred at room temperature for 5 min, cooled with a bath (ice-salt mixture) to a temperature (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) is subsequently added, then, over 30 mi , Dropwise added N-methylmorpholine (8.48 g, 83.9 mmol). The mixture is stirred at a temperature of (-) 10 ° C for 1 h, the temperature is brought to room temperature. The precipitate is filtered off. In the mother liquor, the content of creatyl leucine benzyl ester of tosylate and chloride ions is determined. The output of creatyl leucine benzyl ester of tosylate is 48% mol., The amount of chloride ions is 17.0 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 50 ° C until the end of the run. The residue is dissolved in 300 ml of n-butyl alcohol, washed 4 times with 5% NaHCO ₃ solution, 2 times with water. The solvent is distilled off from the organic phase under reduced pressure and a temperature of 40 ° C, the residue is dissolved in 60 ml of a mixture of water: isopropyl alcohol (50:50), and a solution of creatyl leucine benzyl ester of tosylate is obtained.

Stage B. Obtaining creatylleucine benzyl ether acetate.

Removal of p-TSC and its replacement with a pharmaceutically acceptable acid is carried out by reverse phase (RP) chromatography (Buchi Sepacore chromatographic system, 22.5 × 150 mm column filled with YMC * Gel ODS sorbent in 0.05 M acetic acid) . A solution of tosylate benzyl ester creatyl leucine is applied to the column, eluting in a linear gradient mode from 0.05 M acetic acid to 20% isopropyl alcohol in 0.05 M acetic acid at a speed of 0.3 cm / min. Fractions are collected, analyzed by the method of qualitative reactions and HPLC, combined based on the results of the analysis. From the combined fractions, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. The residue is crystallized from 40 ml of acetonitrile, separated by filtration and dried.

Yield of creatyl leucine benzyl ester acetate C ₁₇ H ₂₆ N ₄ O ₃ * C ₂ H ₄ O ₂ 3.30 g (22%). The content of the main substance is 97.3%, sulfate ash - 0.02%, chloride ions - 0.1 mMEq / g, p-TSA - 5 ppm, impurities: creatinine - 1.4%, creatine - 0.6% , the maximum unit unidentified impurity is 0.2%, the sum of unidentified impurities is 0.3%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 0.96 (6H, m, CH ₂ CH (CH ₃ ) ₂ ), 1.48-1.67 (3H, m, CH ₂ CH (CH ₃ ) ₂ ), 2.00 ( 3H, s, CH ₃ COOH), 3.04 (3H, s, NCH ₃ ), 4.24 (2H, s, N (CH ₃ ) CH ₂ CO), 4.57 (1H, m, NHCHCO), 5.27 (2H, m, CH ₂ Ph); 7.48 (5H, m, CH ₂ Ph). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 22.99, 24.31, 25.63, 26.69, 39.27, 41.42, 54.13, 54.75, 70 01, 130.66, 131.11, 137.52, 160.13, 171.60, 176.32, 183.72.

Stage C Obtaining creatyl leucine hemihydrate.

3.3 g of creatyl leucine benzyl ester acetate obtained in stage B is dissolved in 90 ml of methanol and hydrogenated with Pd / C for 3 hours. The complete removal of the benzyl group is monitored by TLC in the acetonitrile: water: acetic acid 6: 1 system: one. The catalyst is filtered off and washed with 10 ml of a mixture of water: methanol 10:90. The mother liquor and washings are combined, the solvent is distilled off from the solution under reduced pressure and a temperature of 40 ° C until the end of the run. The product is obtained in the form of a white powder.

The yield of creatilleucine hemihydrate C ₁₀ H ₂₀ N ₄ O ₃ * 0.5H ₂ O 2.72 g (21%). The content of the main substance is 97.4%, impurities: creatinine - 1.4%, creatine - 0.6%, the maximum single unidentified impurity - 0.1%, the amount of unidentified impurities - 0.2%; sulfate ash - 0.02%, chloride ions - 0.1 mMEq / g, p-TSA - 5 ppm. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 0.91 (6H, m, CH ₂ CH (CH ₃ ) ₂ ), 1.61 (3H, m, CH ₂ CH (CH ₃ ) ₂ ), 3.04 (3H, s, NCH ₃ ), 4.12-4.26 (3H, m, N (CH ₃ ) CH ₂ CO and NHCHCO). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 23.32, 25.12, 27.35, 39.76, 43.21, 55.34, 56.79, 160.53, 171 , 32, 182.50.

Example 8. Obtaining creatylphenylalanine ethyl acetate

8.1 Obtaining creatylphenylalanine ethyl acetate acetate by the present method.

Stage A. Obtaining creatylphenylalanine tosylate ethyl ester.

Anhydrous creatine (5 g, 38 mmol), p-TSC (15.23 g, 80 mmol) and DMF are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, (30 ml), stirred for 10 min, a solution of phenylalanine ethyl ester hydrochloride (8.73 g, 38 mmol) in 15 ml of DMF was added, stirred at room temperature for 5 min, cooled with a bath (ice-salt mixture) to a temperature (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) and N-methylmorpholine (8.48 g, 83.9 mmol), stirred at a temperature of (-) 10 ° C for 1.5 hours. The precipitate was filtered off, washed on the filter with DMF cooled to 0 ° C. (2 × 5 ml). Wash liquids and mother liquor are combined. The solution determines the content of creatylphenylalanine tosylate ethyl ester and chloride ions. The output of creatylphenylalanine tosylate ethyl ester is 70% mol., The amount of chloride ions is 27.6 mMeq. Potassium acetate (2.69 g, 27.6 mmol) was added to the solution, stirred for 1 h, 50 ml (91%) of DMF were distilled off under reduced pressure and at a temperature of 45 ° C. 40 ml of acetonitrile was added to the residue at room temperature, the suspension was stirred for 20 minutes, 4 g of celite was added, and stirred for 20 minutes. 10 g of celite are placed on the filter, washed with 40 ml of acetonitrile, the washing liquid is disposed of. The suspension is filtered through celite, the filter cake washed with 25 ml of acetonitrile. Wash liquids and mother liquor are combined. The amount of chloride ions in the solution is 5.9 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 45 ° C until the end of the run. 75 ml of distilled water are added to the residue, 3 × 100 ml of methylene chloride are extracted. The organic extracts are combined, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, the residue is dissolved in 20 ml of a mixture of 0.05 M acetic acid: isopropyl alcohol (50:50). A solution of creatylphenylalanine tosylate ethyl ester is obtained.

Stage B. Obtaining creatinylphenylalanine ethyl acetate.

Removal of p-TSA and its replacement with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, column 49 × 200 mm (General Electric, USA) filled with a Dowex 1x8 sorbent in acetate form). A solution of tosylate ethyl ester creatylphenylalanine is applied to the column, eluting with a mixture of 0.05 M acetic acid: isopropyl alcohol (75:25) at a rate of 0.3 cm / min. Detection is carried out at a wavelength of 280 nm. Fractions were collected, analyzed by HPLC, combined on the basis of the analysis to obtain creatinyl phenylalanine ethyl acetate ethyl acetate with a total content of unidentified impurities of not more than 1%, creatinine - not more than 1%, creatine - not more than 1%, acetic acid was added to pH 4.4 . From the combined fractions, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. 100 ml of isopropyl alcohol was added to the residue, the solvent was distilled off under reduced pressure and a temperature of 40 ° С until the end of the run, 50 ml of acetone: acetonitrile mixture (80:20) were added, stirred for 3 h, the precipitate was filtered off and washed with acetone (3 × 25 ml ), dried in vacuum at a temperature of 40 ° C, dissolved in 125 ml of absolute ethanol, the insoluble residue was separated by filtration, washed on the filter with absolute ethanol (2 × 20 ml), the washing liquids were combined with the mother liquor, 130 ml (78%) of the solvent were distilled off under reduced pressure and at a temperature of 40 ° C, 50 ml of ethyl acetate are added, the mixture is incubated for 2 hours at room temperature. The precipitate was separated by filtration, washed with ethyl acetate (2 × 50 ml), dried in vacuum at a temperature of 40 ° C.

Yield of creatylphenylalanine ethyl acetate C ₁₅ H ₂₂ N ₄ O ₃ * C ₂ H ₄ O ₂ 5.44 g (38%). The content of the main substance is 98.8%, sulfate ash - 0.02%, chloride ions - traces, p-TSA - 3 ppm, impurities: creatinine - 0.7%, creatine - 0.2%, maximum single unidentified impurity - 0.1%, the amount of unidentified impurities - 0.2%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.25 (3H, t, 7.15 Hz, OCH ₂ CH ₃ ), 1.94 (3H, s, CH ₃ COOH), 2.90 (3H, s, NCH ₃ ), 3.03 (1H, m, PhCH ₂ ), 3.27 (1H, m, PhCH ₂ ), 4.09 (2H, m, N (CH ₃ ) CH ₂ CO), 4.20 (2H, q, 7.15 Hz, OCH ₂ CH ₃ ), 4.78 (1H, m, PhCH ₂ CH), 7.34 (5H, m, Ph). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 15.85, 25.91, 39.24, 39.52, 54.98, 56.88, 65.37, 129.82, 131 35, 131.78, 138.94, 160.27, 171.44, 175.46, 183.89.

8.2 Obtaining creatinylphenylalanine ethyl acetate acetate according to the method described in the prototype [RU 2428414, 2011].

Stage A. Obtaining creatylphenylalanine tosylate ethyl ester.

Anhydrous creatine (5 g, 38 mmol), p-TSC (15.23 g, 80 mmol) and DMF are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, (30 ml), stirred for 10 min, a solution of phenylalanine ethyl ester hydrochloride (8.73 g, 38 mmol) in 15 ml of DMF was added. The mixture was stirred at room temperature for 5 minutes, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) was added sequentially, then, over 30 minutes, dropwise add N-methylmorpholine (8.48 g, 83.9 mmol), stir at (-) 10 ° C for 1 h, bring the temperature to room temperature. The precipitate is filtered off. In the mother liquor, the content of creatylphenylalanine ethyl tosylate and chloride ions is determined. The yield of tosylate ethyl ester creatylphenylalanine is 57% mol., The amount of chloride ions is 37.4 mMeq. The solvent is distilled off from the solution under reduced pressure and a temperature of 50 ° C until the end of the run. The residue is dissolved in 300 ml of n-butyl alcohol, washed 4 times with 5% NaHCO ₃ solution, 2 times with water. The solvent is distilled off from the organic phase under reduced pressure and a temperature of 40 ° C until the end of the run, the residue is dissolved in 60 ml of a mixture of water: isopropyl alcohol (75:25). A solution of creatylphenylalanine tosylate ethyl ester is obtained.

Stage B. Obtaining creatinylphenylalanine ethyl acetate.

Removing p-TSC acid and replacing it with a pharmaceutically acceptable acid is carried out by RP chromatography (Sepaore Buchi chromatographic system, 22.5 × 150 mm column filled with YMC * Gel ODS sorbent in 0.05 M acetic acid). A solution of tosylate ethyl ester creatylphenylalanine is applied to the column, eluting in a linear gradient mode from 0.05 M acetic acid to 20% isopropyl alcohol in 0.05 M acetic acid at a speed of 0.4 cm / min. Fractions are collected, analyzed by HPLC, combined based on the results of the analysis, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. The residue is crystallized from 40 ml of acetonitrile, separated by filtration and dried.

The yield of creatylphenylalanine ethyl acetate acetate C ₁₅ H ₂₂ N ₄ O ₃ * C ₂ H ₄ O ₂ 3.76 g (27%). The content of the main substance is 97.7%, extraneous impurities: creatinine - 1.2%, creatine - 0.7%, the maximum single unidentified impurity - 0.2%, the amount of unidentified impurities - 0.5%; sulfate ash - 0.04%, chloride ions - 0.1 mMEq / g, p-TSA - 6 ppm. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.25 (3H, t, 7.15 Hz, OCH ₂ CH ₃ ), 1.94 (3H, s, CH ₃ COOH), 2.91 (3H, s, NCH ₃ ), 3.03 (1H, m, PhCH ₂ ), 3.27 (1H, m, PhCH ₂ ), 4.09 (2H, m, N (CH ₃ ) CH ₂ CO), 4.21 (2H, q, 7.15 Hz, OCH ₂ CH ₃ ), 4.78 (1H, m, PhCH ₂ CH), 7.34 (5H, m, Ph). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 15.85, 25.91, 39.24, 39.51, 54.98, 56.88, 65.37, 129.82, 131 35, 131.78, 138.94, 160.27, 171.44, 175.46, 183.89.

Example 9. Obtaining creatinyl-ω-nitroarginine benzyl ester acetate and creatylarginine acetate

9.1 Obtaining creatinyl-ω-nitroarginine benzyl ester acetate and creatylarginine acetate by the present method.

Stage A. Obtaining creatinyl-ω-nitroarginine benzyl ester of tosylate.

Anhydrous creatine (5 g, 38 mmol), p-TSC (15.23 g, 80 mmol) and DMF are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube, mounted on a magnetic stirrer. (30 ml), stirred for 10 min, a solution of ω-nitroarginine of tosylate benzyl ester (18.30 g, 38 mmol) in 15 ml of DMF was added. The mixture is stirred at room temperature for 5 minutes, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) is added dropwise at the same time over 30 minutes. and N-methylmorpholine (8.48 g, 83.9 mmol), stirred at a temperature of (-) 10 ° C for 1.5 hours. The precipitate was filtered off, washed on the filter with DMF cooled to 0 ° C (2 × 5 ml) ) Wash liquids and mother liquor are combined. The solution determines the content of creatyl-ω-nitroarginine benzyl ester of tosylate and chloride ions. The yield of creatyl-ω-nitroarginine of tosylate benzyl ester is 76% mol., The amount of chloride ions is 16.5 mMEq. Sodium acetate trihydrate (2.24 g, 16.5 mmol) was added to the solution, stirred for 1 h, 53 ml (92%) of DMF were distilled off under reduced pressure and a temperature of 45 ° C. 40 ml of acetone was added to the residue at room temperature, the suspension was stirred for 20 minutes, 4 g of celite was added, and stirred for 20 minutes. 10 g of celite are placed on the filter, washed with 40 ml of acetone, the washing liquid is disposed of. The suspension is filtered through celite, the precipitate is washed with 25 ml of acetone. Wash liquids and mother liquor are combined. The amount of chloride ions in the solution is 5.8 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 45 ° C. 75 ml of distilled water are added to the residue, 3 × 100 ml of methylene chloride are extracted. The organic extracts are combined, washed with a saturated solution of sodium chloride, the solvent is removed from the combined organic extract under reduced pressure and a temperature of 40 ° C until the end of the run, the residue is dissolved in 20 ml of a mixture of 0.05 M acetic acid: isopropyl alcohol (50:50). A solution of creatyl-ω-nitroarginine of tosylate benzyl ester is obtained.

Stage B. Obtaining creatyl-ω-nitroarginine benzyl ether acetate.

Removal of p-TSA and its replacement with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, column 49 × 200 mm (General Electric, USA) filled with a Dowex 1x8 sorbent in acetate form). A solution of creatyl-ω-nitroarginine of tosylate benzyl ester is applied to the column, eluting with a mixture of 0.05 M acetic acid: isopropyl alcohol (75:25) at a speed of 0.55 cm / min. Detection is carried out at a wavelength of 220 nm. Fractions were collected, analyzed by HPLC, combined to obtain creatyl-ω-nitroarginine benzyl ester of acetate with a total content of unidentified impurities of not more than 1%, creatinine - not more than 1%, creatine - not more than 1%, acetic acid was added to pH 4.5 . From the combined fractions, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. 100 ml of isopropyl alcohol is added to the residue, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 50 ml of acetone are added, stirred for 3 hours, the precipitate is separated by filtration, washed with acetone (3 × 25 ml), dried in vacuum at a temperature of 40 ° C, dissolved in 125 ml of absolute ethanol, the insoluble residue was separated by filtration, washed with absolute ethanol (2 × 20 ml), the washing liquids were combined with the mother liquor. 130 ml (78.7%) of ethanol are distilled off from the solution under reduced pressure and a temperature of 40 ° C, 50 ml of ethyl acetate are added, and they are kept at room temperature for 2 hours. The precipitate was separated by filtration, washed with ethyl acetate (2 × 50 ml), dried in vacuum at a temperature of 40 ° C.

The yield of creatyl-ω-nitroarginine benzyl ester of acetate C ₁₇ H ₂₆ N ₈ O ₅ * C ₂ H ₄ O ₂ 7.33 g (40%). The content of the main substance is 98.3%, sulfate ash - 0.02%, chloride ions - traces, p-TSA - 5 ppm, impurities: creatinine - 1.0%, creatine - 0.5%, maximum single unidentified impurity - 0.2%, the amount of unidentified impurities - 0.4% ;. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.61 (2H, m, CH ₂ ), 1.74-1.89 (2H, m, CH ₂ ), 1.93 (3H, s, CH ₃ COOH), 2.99 (3H, s, NCH ₃ ), 3.25 (2H, m, CH ₂ NHC (NH) NHNO ₂ ), 4.20 (2H, m, N (CH ₃ ) CH ₂ CO), 4.50 (1H m, NHCHCO), 5.22 (2H, m, CH ₂ Ph), 7.43 (5H, m, CH ₂ Ph). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 25.9, 30.3, 39.7, 43.0, 55.1, 55.5, 70.5, 131.2, 131 5, 137.8, 160.5, 161.5, 172.1, 175.7, 184.0.

Stage C. Obtaining creatylarginine acetate.

7.33 g of creatyl-ω-nitroarginine benzyl ester acetate obtained in stage B is dissolved in 130 ml of methanol and hydrogenated with Pd / C for 3 hours. The complete removal of the benzyl group is monitored by TLC in the acetonitrile: water: acetic acid system. 6: 1: 1. The catalyst is filtered off, washed with 10 ml of methanol. The mother liquor and washings are combined, the solvent is distilled off from the combined solution under reduced pressure and a temperature of 40 ° C. The product is obtained in the form of a white powder.

The yield of creatylarginine acetate C ₁₀ H ₂₁ N ₇ O ₃ * C ₂ H ₄ O ₂ 4.96 g (28%). The content of the main substance is 98.5%, sulfate ash - 0.02%, chloride ions - traces, p-TSA - 4 ppm, impurities: creatinine - 1.0%, creatine - 0.5%, maximum single unidentified impurity - 0.1%, the amount of unidentified impurities - 0.2%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.60-1.93 (4H, m, CH ₂ CH ₂ ), 1.93 (3H, s, CH ₃ COOH), 3.04 (3H, s, NCH ₃ ), 3 20 (2H, m, NHCH ₂ ); 4.14-4.26 (3H, m, N (CH ₃ ) CH ₂ CO and NHCHCO). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 23.28, 25.10, 29.11, 37.57, 41.10, 53.12, 55.45, 157.25, 158 33, 169.22, 178.90, 181.10.

9.2 Obtaining creatinyl-ω-nitroarginine benzyl ester acetate and creatylarginine acetate according to the method described in the prototype [RU 2428414, 2011].

Stage A. Obtaining creatinyl-ω-nitroarginine benzyl ester of tosylate.

Anhydrous creatine (5 g, 38 mmol), p-TSC (15.23 g, 80 mmol) and DMF are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube, mounted on a magnetic stirrer. (30 ml), stirred for 10 min, a solution of ω-nitroarginine of tosylate benzyl ester (18.30 g, 38 mmol) in 15 ml of DMF was added. The mixture was stirred at room temperature for 5 minutes, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (5.25 g, 45.8 mmol) was added sequentially, then dropwise over 30 minutes N-methylmorpholine (8.48 g, 83.9 mmol) was added. The reaction mixture was stirred at a temperature of (-) 10 ° C for 1 h, the temperature was brought to room temperature. The precipitate is filtered off. In the mother liquor, the content of creatyl-ω-nitroarginine of tosylate benzyl ester and chloride ions is determined. The yield of creatyl-ω-nitroarginine of tosylate benzyl ester is 60% mol., The amount of chloride ions is 20.0 mMeq. The solvent is distilled off from the solution under reduced pressure and a temperature of 50 ° C until the end of the run. The residue is dissolved in 300 ml of n-butyl alcohol, washed 4 times with 5% NaHCO ₃ solution, 2 times with water. The solvent is distilled off from the organic phase under reduced pressure and a temperature of 40 ° C, the residue is dissolved in 60 ml of a mixture of water with isopropyl alcohol (50:50). A solution of creatyl-ω-nitroarginine of tosylate benzyl ester is obtained.

Stage B. Obtaining creatyl-ω-nitroarginine benzyl ether acetate.

Removing p-TSC and replacing it with a pharmaceutically acceptable acid is carried out by RP chromatography (Sepaore Buchi chromatographic system, 22.5 × 150 mm column filled with YMC * Gel ODS sorbent in 0.05 M acetic acid). A solution of creatyl-ω-nitroarginine of tosylate benzyl ester is applied to the column, eluting in a linear gradient mode from a mixture of 0.05 M acetic acid with isopropyl alcohol (80:20) to a mixture of 0.05 M acetic acid with isopropyl alcohol (50:50) at a speed of 0.3 cm / min. Fractions are collected, analyzed by the method of qualitative reactions and HPLC, combined based on the results of the analysis, acetic acid is added to a pH of 4.4. From the combined fractions, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. The residue is crystallized from 30 ml of acetonitrile, separated by filtration and dried.

The yield of creatyl-ω-nitroarginine benzyl ester acetate C ₁₇ H ₂₆ N ₈ O ₅ * C ₂ H ₄ O ₂ 3.67 (20%). The content of the main substance is 97.4%, sulfate ash - 0.04%, chloride ions - 0.1 mMEq / g, p-TSA - 7 ppm; impurities: creatinine - 1.2%, creatine - 0.5%, the maximum single unidentified impurity is 0.2%, the sum of unidentified impurities is 0.4%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.61 (2H, m, CH ₂ ), 1.74-1.89 (2H, m, CH ₂ ), 1.92 (3H, s, CH ₃ COOH), 2.99 (3H, s, NCH ₃ ), 3.24 (2H, m, CH ₂ NHC (NH) NHNO ₂ ), 4.20 (2H, m, N (CH ₃ ) CH ₂ CO), 4.50 (1H m, NHCHCO), 5.22 (2H, m, CH ₂ Ph), 7.43 (5H, m, CH ₂ Ph). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 25.9, 30.3, 39.7, 43.1, 55.1, 55.6, 70.5, 131.2, 131 5, 137.8, 160.5, 161.6, 172.1, 175.7, 184.0.

Stage C. Obtaining creatylarginine acetate.

3.67 g of the obtained creatyl-ω-nitroarginine benzyl ester of the acetate obtained in step B are dissolved in 80 ml of methanol and hydrogenated over Pd / C for 3 hours. The complete removal of the benzyl group is monitored by TLC in the acetonitrile: water: acetic system acid 6: 1: 1. The catalyst is filtered off and washed with 10 ml of methanol. The mother liquor and washings are combined, the solvent is distilled off from the combined solution under reduced pressure and a temperature of 40 ° C. The product is obtained in the form of a white powder.

The yield of creatylarginine acetate C ₁₀ H ₂₁ N ₇ O ₃ * C ₂ H ₄ O ₂ 1.93 g (18%). The content of the main substance is 97.8%, sulfate ash 0.04%, chloride ions - 0.1 mMEq / g, p-TSC - 7 ppm, impurities: creatinine - 1.2%, creatine - 0.5%, the maximum unit unidentified impurity is 0.1%; the sum of unidentified impurities is 0.3% ;. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.60-1.93 (4H, m, CH ₂ CH ₂ ), 1.93 (3H, s, CH ₃ COOH), 3.03 (3H, s, NCH ₃ ), 3 21 (2H, m, NHCH ₂ ), 4.14-4.26 (3H, m, N (CH ₃ ) CH ₂ CO and NHCHCO). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 23.28, 25.10, 29.11, 37.57, 41.10, 53.12, 55.44, 157.25, 158 33, 169.22, 178.92, 181.10.

Example 10. Obtaining creatyl-O-isopropyl tyrosine methyl ester acetate

10.1 Obtaining creatyl-O-isopropyl tyrosine methyl ester acetate according to the present method.

Stage A. Obtaining creatyl-O-isopropyl tyrosine methyl tosylate.

Anhydrous creatine (5 g, 38 mmol), p-TSC (15.23 g, 80 mmol) and DMF are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube, mounted on a magnetic stirrer. (30 ml), stirred for 10 min, a solution of O-isopropyl tyrosine methyl ester hydrochloride (10.40 g, 38 mmol) in 15 ml of DMF was added. The reaction mixture was stirred at room temperature for 5 minutes, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) and N were added simultaneously over 30 minutes. -methylmorpholine (8.48 g, 83.9 mmol), stirred at a temperature of (-) 10 ° C for 1.0 h. The precipitate was filtered off from the cooled reaction mixture, washed on the filter with DMF cooled to 0 ° C (2 × 5 ml). Wash liquids and mother liquor are combined. The solution determines the yield of the content of creatyl-O-isopropyl tyrosine tosylate methyl ester, the amount of chloride ions. The yield of creatyl-O-isopropyl tyrosine tosylate methyl ester is 65% mol., The amount of chloride ions is 28.0 mMeq. Potassium acetate (2.75 g, 28.0 mmol) is added to the solution, stirred for 1 h, the total chloride ion content decreases to 3.1 mMEq. 50 ml (91%) of DMF are distilled off under reduced pressure and a temperature of 45 ° C. 40 ml of tetrahydrofuran is added to the residue at room temperature, the suspension is stirred for 20 minutes, 4 g of celite are added, and stirred for 20 minutes. 10 g of celite are placed on the filter, washed with 40 ml of tetrahydrofuran, and the washing liquid is disposed of. The suspension is filtered through celite, the filter cake washed with 25 ml of tetrahydrofuran. Wash liquids and mother liquor are combined. The content of chloride ions in the solution is 5.6 mmol. The solvent is distilled off from the solution under reduced pressure and a temperature of 45 ° C. 75 ml of water are added to the residue, 3 × 100 ml of methylene chloride are extracted. The organic extracts are combined, washed with a saturated solution of sodium chloride, the solvent is removed from the combined organic extract under reduced pressure and a temperature of 40 ° C until the end of the run, the residue is dissolved in 20 ml of a mixture of 0.05 M acetic acid with ethyl alcohol (60:40). A solution of creatyl-O-isopropyl tyrosine tosylate methyl ester is obtained.

Stage B. Obtaining creatyl-O-isopropyl tyrosine methyl ester acetate.

Removal of p-TSA and its replacement with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, column 49 × 200 mm (General Electric, USA) filled with a Dowex 1x8 sorbent in acetate form). A solution of creatyl-O-isopropyl tyrosine tosylate methyl ester is applied to the column, eluting with a mixture of 0.05 M acetic acid: ethyl alcohol (75:25) at a rate of 0.3 cm / min. Detection is carried out at a wavelength of 280 nm. Fractions were collected, analyzed by HPLC, fractions were combined based on the results of analysis to obtain creatyl-O-isopropyl tyrosine methyl acetate ester with a total content of unidentified impurities of not more than 1%, creatinine - not more than 1%, creatine - not more than 1%, acetic acid was added to pH 4.4. From the combined fractions, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. 100 ml of n-butyl alcohol are added to the residue, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 50 ml of acetone are added, stirred for 3 hours, the precipitate is filtered off, washed with acetone (3 × 25 ml), and dried under vacuum at temperature 40 ° C, dissolved in 125 ml of absolute ethanol with stirring for 20 minutes, the insoluble residue was separated by filtration, washed on the filter with absolute ethanol (2 × 20 ml), the washing liquids were combined with the mother liquor, 130 ml were distilled off (78.7% ) ethanol under reduced pressure enii and a temperature of 40 ° C, 50 ml of ethyl acetate, the mixture was maintained for 2 hours at room temperature. The precipitate was separated by filtration, washed with ethyl acetate (2 × 50 ml), dried in vacuum at a temperature of 40 ° C.

Yield of creatyl-O-isopropyl tyrosine methyl ester acetate C ₁₇ H ₂₆ N ₄ O ₄ * C ₂ H ₄ O ₂ 7.03 g (37%). The content of the main substance is 98%, sulfate ash - 0.02%, chloride ions - traces, p-TSA - 4 ppm, impurities: creatinine - 1.0%, creatine - 0.4%, maximum single unidentified impurity - 0 , 2%, the amount of unidentified impurities is 0.3%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.22 (6H, d, 6.11 Hz, CH (CH ₃ ) ₂ ), 1.83 (3H, s, CH ₃ COOH), 2.78 (3H, s, NCH ₃ ), 2.79-2.88 (1H, m, CH ₂ PhOiPr), 3.12-3.17 m (1H, m, CH ₂ PhOiPr), 3.68 (3H, s, OCH ₃ ), 3 90-4.07 (2H, m, N (CH ₃ ) CH ₂ CO), 4.50-4.60 (1H, m, OCH (CH ₃ ) ₂ ), 4.65-4.67 (1H m, NHCHCO), 6.87 (2H, d, 8.65 Hz, Ph), 7.11 (2H, d, 8.61 Hz, Ph). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 21.08, 23.26, 35.84, 36.82, 52.42, 53.00, 54.16, 71.81, 116 68, 129.38, 130.48, 155.84, 157.75, 168.85, 173.34, 181.37.

10.2 Obtaining creatyl-O-isopropyl tyrosine methyl ester acetate according to the method described in the prototype [RU 2428414, 2011].

Stage A. Obtaining creatyl-O-isopropyl tyrosine methyl tosylate.

Anhydrous creatine (5 g, 38 mmol), p-TSC (15.23 g, 80 mmol) and DMF are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube, mounted on a magnetic stirrer. (30 ml), stirred for 10 min, a solution of O-isopropyl tyrosine methyl ester hydrochloride (10.40 g, 38 mmol) in 15 ml of DMF was added. The mixture was stirred at room temperature for 5 minutes, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) was added sequentially, then, over 30 minutes, dropwise add N-methylmorpholine (8.48 g, 83.9 mmol), stir at (-) 10 ° C for 1 h, bring the temperature to room temperature. The precipitate was filtered off from the reaction mixture. The solution determines the content of creatyl-O-isopropyl tyrosine methyl ester of tosylate, chloride ions. The yield of creatyl-O-isopropyl tyrosine tosylate methyl ester is 45% mol., The amount of chloride ions is 37.1 mMeq. The solvent is distilled off from the solution under reduced pressure and a temperature of 50 ° C until the end of the run. The residue is dissolved in 300 ml of n-butyl alcohol, washed 4 times with 5% NaHCO ₃ solution, 2 times with water. The solvent is distilled off from the organic phase under reduced pressure and a temperature of 40 ° C until the end of the run, the residue is dissolved in 20 ml of a mixture of 0.05 M acetic acid with ethyl alcohol (60:40). A solution of creatyl-O-isopropyl tyrosine tosylate methyl ester is obtained.

Stage B. Obtaining creatyl-O-isopropyl tyrosine methyl ester acetate.

Removing p-TSC and replacing it with a pharmaceutically acceptable acid is carried out by RP chromatography (Sepaore Buchi chromatographic system, 22.5 × 150 mm column filled with YMC * Gel ODS sorbent in 0.05 M acetic acid). A solution of creatyl-O-isopropyl tyrosine tosylate methyl ester is applied to the column, eluting in a linear gradient mode from 0.05 M acetic acid to 30% isopropyl alcohol in 0.05 M acetic acid at a speed of 0.3 cm / min. Fractions are collected, analyzed by HPLC, combined based on the results of the analysis, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. The residue is crystallized from 40 ml of acetonitrile, separated by filtration and dried.

Yield of creatyl-O-isopropyl tyrosine methyl ester acetate C ₁₇ H ₂₆ N ₄ O ₄ * C ₂ H ₄ O ₂ 3.33 g (21%). The content of the main substance is 97.5%, extraneous impurities: creatinine - 1.4%, creatine - 0.6%, the maximum single unidentified impurity - 0.2%, the amount of unidentified impurities - 0.3%; sulfate ash - 0.05%, chloride ions - 0.1 mMEq / g, p-TSA - 5 ppm. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.22 (6H, d, 6.11 Hz, CH (CH ₃ ) ₂ ), 1.83 (3H, s, CH ₃ COOH), 2.79 (3H, s, NCH ₃ ), 2.79-2.88 (1H, m, CH ₂ PhOiPr), 3.12-3.17 m (1H, m, CH ₂ PhOiPr), 3.67 (3H, s, OCH ₃ ), 3 90-4.07 (2H, m, N (CH ₃ ) CH ₂ CO), 4.50-4.60 (1H, m, OCH (CH ₃ ) ₂ ), 4.65-4.67 (1H m, NHCHCO), 6.87 (2H, d, 8.65 Hz, Ph), 7.11 (2H, d, 8.61 Hz, Ph). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 21.08, 23.25, 35.84, 36.82, 52.42, 53.01, 54.16, 71.81, 116 68, 129.38, 130.48, 155.84, 157.75, 168.84, 173.34, 181.37.

Example 11. Obtaining creatyl-O-ethyl tyrosine isobutylamide acetate

11.1 Obtaining creatyl-O-ethyl tyrosine isobutylamide acetate by the present method.

Stage A. Obtaining creatyl-O-ethyl tyrosine isobutylamide tosylate.

Anhydrous creatine (5 g, 38 mmol), p-TSC (15.23 g, 80 mmol) and DMF are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, (30 ml), stirred for 10 min, a solution of O-ethyl tyrosine isobutylamide hydrochloride (12.04 g, 38 mmol) in 15 ml of DMF was added, stirred at room temperature for 5 min, cooled with a bath (ice-salt mixture) to temperature (-) 15 ° С, isobutyl chloroformate (6.25 g) is added dropwise at the same time over 30 minutes 45.8 mmol) and N-methylmorpholine (8.48 g, 83.9 mmol). The reaction mixture was stirred at a temperature of (-) 10 ° C for 1.5 hours. The precipitate was filtered off from the cooled reaction mixture, washed on a filter with DMF (2 × 5 ml) cooled to 0 ° C. Wash liquids and the mother liquor are combined, the content of creatyl-O-ethyl tyrosine tosylate isobutylamide and chloride ions is determined. The yield of creatyl-O-ethyl tyrosine tosylate isobutylamide is 62% mol., The amount of chloride ions is 26.5 mMeq. Sodium acetate trihydrate (3.61 g, 26.5 mmol) was added to the solution, stirred for 1 h, 50 ml (91%) of DMF were distilled off under reduced pressure, 40 ml of acetone was added at room temperature, stirred for 20 minutes, 4 g of celite are added, stirred for 20 minutes. 10 g of celite are placed on the filter, washed with 40 ml of acetone, the washing liquid is disposed of. The suspension is filtered through celite, the filter cake washed with 25 ml of acetone. Wash liquids and mother liquor are combined. The amount of chloride ions in the solution is 5.8 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 45 ° C. 75 ml of distilled water are added to the residue, 3 × 100 ml of methylene chloride are extracted. The organic extracts are combined, washed with a saturated solution of sodium chloride, the solvent is distilled off from the organic extract under reduced pressure and a temperature of 40 ° C, the residue is dissolved in 20 ml of a mixture of 0.05 M acetic acid: isopropyl alcohol (50:50). A solution of creatyl-O-ethyl tyrosine tosylate isobutylamide is obtained.

Stage B. Obtaining creatyl-O-ethyl tyrosine isobutylamide acetate.

Removal of p-TSA and its replacement with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, column 49 × 200 mm (General Electric, USA) filled with a Dowex 1x8 sorbent in acetate form). A solution of creatyl-O-ethyl tyrosine tosylate isobutylamide was applied to the column, eluted with a mixture of 0.05 M acetic acid: isopropyl alcohol (80:20) at a rate of 0.3 cm / min. Detection is carried out at a wavelength of 220 nm. Fractions were collected, analyzed by HPLC, combined on the basis of the analysis results to obtain creatyl-O-ethyl tyrosine isobutylamide acetate with a total content of unidentified impurities of not more than 1%, creatinine - not more than 1%, creatine - not more than 1%, acetic acid was added to pH 4.4. From the combined fractions, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 100 ml of isopropyl alcohol is added to the residue, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 50 ml of acetone are added, the mixture is stirred for 3 hours, the precipitate is separated by filtration, washed with acetone (3 × 25 ml), dried in vacuum at 40 ° C, dissolved in 125 ml of absolute ethanol with stirring for 20 minutes, the insoluble residue was filtered off, washed on the filter with ethanol (2 × 20 ml), washing liquids are combined with the mother liquor, 130 ml (79%) of ethanol are distilled off under reduced pressure and a temperature of 40 ° C, 50 ml of ethyl acetate are added, and they are kept for 2 hours at room temperature. The precipitate was separated by filtration, washed with ethyl acetate (2 × 50 ml), dried in vacuum at a temperature of 40 ° C.

Yield of creatyl-O-ethyl tyrosine isobutylamide acetate C ₁₉ H ₃₁ N ₅ O ₃ * C ₂ H ₄ O ₂ 6.29 g (38%). The content of the main substance is 98.4%, sulfate ash - 0.02%, chloride ions - traces, p-TSC - 3 ppm, impurities: creatinine - 0.9%, creatine - 0.9%, the maximum single unidentified impurity - 0.1%, the amount of unidentified impurities - 0.2%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 0.63 (6H, m, CH (CH ₃ ) ₂ ), 1.28 (3H, t, 7.02 Hz, CH ₂ CH ₃ ), 1.42-1.53 (1H m, CH (CH ₃ ) ₂ ), 1.83 (3H, s, CH ₃ COOH), 2.70-2.75 (1H, m, CH ₂ PhOEt), 2.85 (3H, s, NCH ₃ ), 2.97-2.99 (3H, m, CH ₂ PhOEt and CH ₂ CH (CH ₃ ) ₂ ), 3.98-4.11 (4H, m, CH ₂ CH ₃ and N (CH ₃ ) CH ₂ CO), 4.44 (1H, m, NHCHCO), 6.87 (2H, d, 8.69 Hz, Ph), 7.13 (2H, d, 8.67 Hz, Ph). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 13.90, 19.15, 23.23, 27.73, 36.48, 36.92, 46.75, 52.39, 55 72, 64.45, 115.00, 128.84, 130.44, 157.05, 157.75, 168.79, 172.54, 181.37.

11.2 Obtaining creatyl-O-ethyl tyrosine isobutylamide acetate according to the method described in the prototype [RU 2428414, 2011].

Anhydrous creatine (5 g, 38 mmol), p-TSC (15.23 g, 80 mmol) and DMF are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube, mounted on a magnetic stirrer. (30 ml), stirred, a solution of O-ethyl tyrosine isobutylamide hydrochloride (12.04 g, 38 mmol) in 15 ml of DMF was added, stirred at room temperature for 5 min, cooled with a bath (ice-salt mixture) to a temperature (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) is added sequentially, then, over 30 minutes, beaches, N-methylmorpholine (8.48 g, 83.9 mmol) is added. The reaction mixture was stirred at a temperature of (-) 10 ° C for 1 h, the temperature was brought to room temperature. The precipitate is filtered off. In the mother liquor, the content of creatyl-O-ethyl tyrosine tosylate isobutylamide and chloride ions is determined. The yield of creatyl-O-ethyl tyrosine tosylate isobutylamide is 51% mol., The amount of chloride ions is 39.0 mMeq. The solvent is distilled off from the solution under reduced pressure and a temperature of 50 ° C until the end of the run. The residue is dissolved in 300 ml of n-butyl alcohol, washed 4 times with 5% NaHCO ₃ solution, 2 times with water. The organic phase is evaporated under reduced pressure and a temperature of 40 ° C until the end of the run. The residue was dissolved in 20 ml of a mixture of 0.05 M acetic acid: isopropyl alcohol (50:50). A solution of creatyl-O-ethyl tyrosine tosylate isobutylamide is obtained.

Stage B. Obtaining creatyl-O-ethyl tyrosine isobutylamide acetate.

Removing p-TSC and replacing it with a pharmaceutically acceptable acid is carried out by RP chromatography (Sepaore Buchi chromatographic system, 22.5 × 150 mm column filled with YMC * Gel ODS sorbent in 0.05 M acetic acid). A solution of creatyl-O-ethyl tyrosine tosylate isobutylamide is applied to the column, eluting in a linear gradient mode from 0.05 M acetic acid to 30% isopropyl alcohol in 0.05 M acetic acid at a speed of 0.4 cm / min. Fractions are collected, analyzed by HPLC, combined based on the results of the analysis, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. The residue is crystallized from 45 ml of acetonitrile, separated by filtration and dried.

Yield of creatyl-O-ethyl tyrosine isobutylamide acetate C ₁₉ H ₃₁ N ₅ O ₃ * C ₂ H ₄ O ₂ 4.21 g (25%). The content of the main substance is 97.0%, sulfate ash - 0.04%, chloride ions - 0.15 mMEq / g, p-TSC - 7 ppm; impurities: creatinine - 1.4%, creatine - 1.0%, the maximum single unidentified impurity is 0.3%, the sum of unidentified impurities is 0.4%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 0.63 (6H, m, CH (CH ₃ ) ₂ ), 1.28 (3H, t, 7.02 Hz, CH ₂ CH ₃ ), 1.42-1.53 (1H m, CH (CH ₃ ) ₂ ), 1.84 (3H, s, CH ₃ COOH), 2.70-2.75 (1H, m, CH ₂ PhOEt), 2.83 (3H, s, NCH ₃ ), 2.97-2.99 (3H, m, CH ₂ PhOEt and CH ₂ CH (CH ₃ ) ₂ ), 3.98-4.11 (4H, m, CH ₂ CH ₃ and N (CH ₃ ) CH ₂ CO), 4.44 (1H, m, NHCHCO), 6.87 (2H, d, 8.69 Hz, Ph), 7.13 (2H, d, 8.67 Hz, Ph). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 13.91, 19.15, 23.23, 27.73, 36.48, 36.92, 46.75, 52.39, 55 72, 64.45, 115.01, 128.84, 130.44, 157.04, 157.75, 168.79, 172.54, 181.37.

Example 12. Obtaining creatyl glycine ethyl acetate.

12.1 Obtaining creatylglycine ethyl acetate acetate by the present method.

Stage A. Obtaining creatylglycine tosylate ethyl ester.

Creatine monohydrate (5.69 g, 38 mmol), p-TSC (15.23 g, 80 mmol) are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, and DMF (30 ml), stirred for 10 min, a solution of glycine ethyl ester hydrochloride (5.32 g, 38 mmol) in 15 ml of DMF was added, stirred at room temperature for 5 min, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° С, ethyl chloroformate (4.97 g, 45.8 mmo) is added dropwise at the same time over 30 minutes v) and N-methylmorpholine (8.48 g, 83.9 mmol). The reaction mixture was stirred at a temperature of (-) 10 ° C for 1.5 hours. The precipitate was filtered off, washed on a filter with DMF (2 × 5 ml) cooled to 0 ° C. Wash liquids and mother liquor are combined. The solution determines the content of creatinglycine tosylate ethyl ester and chloride ions. The yield of tosylate ethyl ester creatyl glycine is 67% mol., The amount of chloride ions is 29.2 mMEq. Sodium acetate trihydrate (3.97 g, 29.2 mmol) was added to the solution, stirred for 1 h, 50 ml (91%) of DMF were distilled off under reduced pressure and a temperature of 45 ° C. 40 ml of acetone are added to the residue at room temperature, stirred for 20 minutes, 4 g of celite are added, and stirred for 20 minutes. 10 g of celite are placed on the filter, washed with 40 ml of acetone, the eluate is disposed of. The suspension is filtered through celite, the filter cake washed with 25 ml of acetone. Wash liquids and mother liquor are combined. The amount of chloride ions is 5.6 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 45 ° C until the end of the run, the residue is dissolved in 20 ml of a 0.05 M solution of acetic acid. A solution of creatyl glycine ethyl tosylate is obtained.

Stage B. Obtaining creatyl glycine ethyl acetate.

Removal of p-TSC and its replacement with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatography system, 49 x 200 mm column (General Electric, USA) filled with a Dowex 1x8 sorbent in acetate form). A solution of tosylate ethyl ester creatyl glycine is applied to the column, eluting with a 0.035 M solution of acetic acid at a rate of 0.3 cm / min. Detection is carried out at a wavelength of 220 nm. Fractions were collected, analyzed by HPLC, combined on the basis of the results of analysis to obtain ethyl acetate ethyl acetate with a total content of unidentified impurities of not more than 1%, creatinine not more than 1%, creatine not more than 1%, acetic acid was added to pH 4.4 . From the combined fractions, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. 100 ml of isopropyl alcohol is added to the residue, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 50 ml of acetone are added, stirred for 3 hours, the precipitate is separated by filtration, washed with acetone (3 × 25 ml), dried in vacuum at a temperature of 40 ° C, dissolved in 125 ml of absolute ethanol with stirring for 20 minutes, the insoluble residue was separated by filtration, washed on the filter with absolute ethanol (2 × 20 ml), the washing liquids were combined with the mother liquor. 130 ml (78%) of ethanol are distilled off from the combined solution under reduced pressure and a temperature of 40 ° C, 50 ml of a mixture of ethyl acetate: tetrahydrofuran 80:20 are added, the mixture is kept for 2 hours at room temperature. The precipitate was separated by filtration, washed with ethyl acetate (2 × 50 ml), dried in vacuum at a temperature of 40 ° C.

Yield of creatyl glycine ethyl acetate C ₈ H ₁₆ N ₄ O ₃ * C ₂ H ₄ O ₂ 3.94 g (38%). The content of the main substance is 98.4%, sulfate ash - 0.02%, chloride ions - traces, p-TSC - 3 ppm, impurities: creatinine - 0.9%, creatine - 0.5%, the maximum single unidentified impurity - 0.1%, the amount of unidentified impurities - 0.3%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.26 (3H, t, 7.17 Hz, CH ₂ CH ₃ ), 1.90 (3H, s, CH ₃ COOH), 3.07 (3H, s, NCH ₃ ), 4.07 (2H, s, N (CH ₃ ) CH ₂ CO); 4.20-4.25 (4H, m, NHCH ₂ CO and CH ₂ CH ₃ ). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 15.96, 25.99, 39.72, 44.09, 55.30, 65.34, 160.55, 172.69, 174 , 24, 184.09.

12.2 Obtaining creatylglycine ethyl ester of acetate according to the method described in the prototype [RU 2428414, 2011].

Stage A. Obtaining creatylglycine tosylate ethyl ester.

Creatine monohydrate (5.69 g, 38 mmol), p-TSC (15.23 g, 80 mmol) are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, and DMF (30 ml), stirred for 10 min, a solution of glycine ethyl ester hydrochloride (5.32 g, 38 mmol) in 15 ml of DMF was added. The reaction mixture was stirred at room temperature for 5 minutes, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, ethyl chloroformate (4.97 g, 45.8 mmol) was added sequentially, then, over 30 minutes, N-methylmorpholine (8.48 g, 83.9 mmol) is added, the mixture is stirred at a temperature of (-) 10 ° C for 1 h, the temperature is brought to room temperature. The precipitate was filtered off from the reaction mixture. In the mother liquor, the content of tosylate ethyl ester of tosylate and chloride ions is determined. The yield of tosylate ethyl ester creatyl glycine is 55% mole., The amount of chloride ions is 36.0 mMEq. The solvent is distilled off from the solution under reduced pressure at a temperature of 50 ° C until the end of the run. The residue is dissolved in 160 ml of chloroform, left for 10 hours at a temperature of (-) 10 ° C. The solution is filtered off, extracted with water 3 times in 100 ml. The aqueous extracts are combined, 2 times extracted with chloroform. From the combined aqueous extract, the solvent is distilled off under reduced pressure and a temperature of 40 ° C to a volume of 50 ml. A solution of creatyl glycine ethyl tosylate is obtained.

Stage B. Obtaining creatyl glycine ethyl acetate.

Removal of p-TSA and its replacement with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, column 49 × 200 mm (General Electric, USA) filled with a Dowex 1x8 sorbent in acetate form). A solution of tosylate ethyl ester creatyl glycine is applied to the column, eluted with water at a rate of 0.3 cm / min, controlling the pH of the eluate. Fractions with pH = 7 (water pH 5) are collected, analyzed by HPLC, combined, acetic acid is added, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. The residue is crystallized from 20 ml of acetonitrile at a temperature of (-) 10 ° C for 20 hours. The precipitate of creatylglycine ethyl acetate is filtered off, washed with cold acetonitrile, diethyl ether and dried.

Yield of creatyl glycine ethyl acetate C ₈ H ₁₆ N ₄ O ₃ * C ₂ H ₄ O ₂ 2.30 g (22%). The basic substance content is 97%, p-TSA - 150 ppm, sulfate ash - 0.03%, chloride ions - 0.5 mMEq / g, impurities: creatinine - 1.5%, creatine - 0.5%, maximum single unidentified impurity - 0.5%, the sum of unidentified impurities - 0.9%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.26 (3H, t, 7.17 Hz, CH ₂ CH ₃ ), 1.91 (3H, s, CH ₃ COOH), 3.07 (3H, s, NCH ₃ ), 4.07 (2H, s, N (CH ₃ ) CH ₂ CO); 4.20-4.25 (4H, m, NHCH ₂ CO and CH ₂ CH ₃ ). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 15.95, 25.99, 39.72, 44.11, 55.30, 65.32, 160.55, 172.71, 174 , 24, 184.09.

Example 13. Obtaining creatyltryptophan methyl ester acetate

13.1 Obtaining creatyltryptophan methyl ester acetate according to the present method.

Step A. Preparation of Tosylate Creatyltryptophan Methyl Ester

Anhydrous creatine (5 g, 38 mmol), p-TSC (15.23 g, 80 mmol) and DMF are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, (30 ml), stirred for 10 min, a solution of tryptophan methyl ester hydrochloride (9.82 g, 38 mmol) in 15 ml of DMF was added. The mixture was stirred at room temperature for 5 minutes, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) and N-methylmorpholine were added simultaneously over 30 minutes. (8.48 g, 83.9 mmol), stirred at a temperature of (-) 10 ° C for 1.5 hours. The precipitate was filtered off, washed on the filter with DMF (2 × 5 ml) cooled to 0 ° C. Wash liquids and mother liquor are combined. The solution determines the content of creatyltryptophan methyl ester of tosylate and chloride ions. The yield of tosylate creatyltryptophan methyl ester was 67% mol., The amount of chloride ions 29.5 mMEq. Potassium acetate (2.92 g, 29.7 mmol) was added to the combined solution, stirred for 1 h, 44 ml (80%) of DMF were distilled off under reduced pressure and a temperature of 45 ° C. 40 ml of methyl ethyl ketone are added to the residue at room temperature, the suspension is stirred for 20 minutes, 4 g of celite are added, and stirred for 20 minutes. 10 g of celite are placed on the filter, washed with 40 ml of methyl ethyl ketone, and the washing liquid is disposed of. The suspension is filtered through celite, the filter cake washed with 25 ml of methyl ethyl ketone. Wash liquids and mother liquor are combined. The content of chloride ions in the solution is 5.9 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 45 ° C until the end of the run. The residue is dissolved in 200 ml of methylene chloride, the solution is washed 4 times with 100 ml of water. The solvent is distilled off from the organic phase under reduced pressure and a temperature of 40 ° C until the end of the run, the residue is dissolved in 20 ml of a mixture of 0.05 M acetic acid: isopropyl alcohol (50:50). A solution of creatyltryptophan methyl tosylate is obtained.

Stage B. Obtaining creatyltryptophan methyl ester acetate.

Removal of p-TSC and its replacement with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, column 49 × 230 mm in size, filled with a Dowex 1x8 sorbent in acetate form). A solution of tosylate methyl ester tryptophan is applied to the column, eluting with a mixture of 0.05 M acetic acid: isopropyl alcohol (80:20) at a speed of 0.45 cm / min. Detection is carried out at a wavelength of 220 nm. Fractions were collected, analyzed by HPLC, combined on the basis of the analysis results to obtain creatyl tryptophan methyl acetate ester with a total content of unidentified impurities of not more than 1%, creatinine - not more than 1%, creatine - not more than 1%, acetic acid was added to pH 4.4 . From the combined fractions, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. 100 ml of n-butyl alcohol are added to the residue, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 50 ml of ethyl acetate are added, the mixture is stirred for 3 hours. The precipitate is filtered off, washed with ethyl acetate (3 × 25 ml), and dried under vacuum at at a temperature of 40 ° C, dissolved in 125 ml of absolute ethanol with stirring for 20 min, the insoluble residue was separated by filtration, washed with absolute ethanol (2 × 20 ml), the washing liquids were combined with the mother liquor. 116 ml (70%) of ethanol are distilled off from the solution under reduced pressure and a temperature of 40 ° C, 50 ml of ethyl acetate are added, the mixture is kept for 2 hours at room temperature. The precipitate was separated by filtration, washed with ethyl acetate (2 × 50 ml), dried in vacuum at a temperature of 40 ° C.

The yield of creatyltryptophan methyl ester acetate C ₁₆ H ₂₁ N ₅ O ₃ * C ₂ H ₄ O ₂ 7.8 g (52%). The content of the main substance is 98.0%, sulfate ash - 0.02%, chloride ions - traces, p-TSA - 4 ppm, impurities: creatinine - 1.0%, creatine - 0.8%, the maximum single unidentified impurity - 0.1%, the amount of unidentified impurities - 0.2%. ¹ H NMR Spectrum (DMSO-d ⁶ ), δ, ppm (J, Hz): 1.67 (3H, s, C H ₃ COOH), 2.74 (3H, s, OS H ₃ ) 3.05-3.20 (2H, m, Ind-C H ₂ ) , 3.59 (3H, s, NC H ₃ ), 4.02 (2H, m, N (CH ₃ ) C H ₂ CO), 4.57 (1H, t, 5.87 Hz, COC H NH

CO), 6.99 (1H, m, ArH), 7.07 (1H, m, ArH), 7.19 (1H, s, ArH), 7.34 (1H, d, 8.31 Hz, ArH ), 7.50 (1H, d, 7.82 Hz, ArH). ¹³ C NMR spectrum (DMSO-d ⁶ ), δ, ppm: 1.18, 19.26, 19.32, 28.43, 29.12, 29.28, 75.69, 80.30, 80.40, 105.24, 107.69, 141.40, 145.79, 150.31, 152.43, 174.74

13.2 Obtaining creatyltryptophan methyl ester acetate according to the method described in the prototype [RU 2428414, 2011].

Step A. Preparation of Tosylate Creatyltryptophan Methyl Ester

Anhydrous creatine (5 g, 38 mmol), p-TSC (15.23 g, 80 mmol) and DMF are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube, mounted on a magnetic stirrer. (30 ml), stirred for 10 min, a solution of tryptophan methyl ester hydrochloride 9.82 g (38 mmol) in 15 ml of DMF was added, stirred at room temperature for 5 min, cooled with a bath (ice-salt mixture) to a temperature ( -) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) is added sequentially, then, over 30 minutes, N-methylmorpholine (8.48 g, 83.9 mmol) was added dropwise. The mixture is stirred at a temperature of (-) 10 ° C for 1 h, the temperature is brought to room temperature. The precipitate was filtered off from the reaction mixture. The solution determines the content of creatyltryptophan methyl ester of tosylate and chloride ions. The yield of tosylate creatyltryptophan methyl ester is 50% mol., The amount of chloride ions is 37.8 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 50 ° C until the end of the run. The residue is dissolved in 300 ml of n-butyl alcohol, washed 4 times with 5% NaHCO ₃ solution, 2 times with water. The solvent is distilled off from the organic phase under reduced pressure and a temperature of 40 ° C until the end of the run, the residue is dissolved in 20 ml of a 0.05 M acetic acid: isopropyl alcohol mixture (50:50). A solution of creatyltryptophan methyl tosylate is obtained.

Stage B. Obtaining creatyltryptophan methyl ester acetate.

Removing p-TSC and replacing it with a pharmaceutically acceptable acid is carried out by RP chromatography (Sepaore Buchi chromatographic system, 22.5 × 150 mm column filled with YMC * Gel ODS sorbent in 0.05 M acetic acid). A solution of creatyltryptophan methyl tosylate is applied to the column, eluting in a linear gradient mode from 0.05 M acetic acid to 20% isopropyl alcohol in 0.05 M acetic acid at a rate of 0.3 cm / min. Fractions are collected, analyzed by HPLC, combined based on the results of the analysis, acetic acid is added to pH 4.5, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. The residue is crystallized from 40 ml of acetonitrile, separated by filtration and dried.

Yield of creatyltryptophan methyl ester acetate C ₁₆ H ₂₁ N ₅ O ₃ * C ₂ H ₄ O ₂ 5.1 g (34%). The content of the main substance is 97.3%, sulfate ash - 0.02%, chloride ions - 0.1 mMEq / g, p-TSC - 7 ppm, impurities: creatinine - 1.4%, creatine - 0.8% , the maximum unit unidentified impurity is 0.2%, the sum of unidentified impurities is 0.3%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.67 (3H, s, C H ₃ COOH), 2.74 (3H, s, OS H ₃ ) 3.05-3.20 (2H, m, Ind-C H ₂ ) , 3.59 (3H, s, NC H ₃ ), 4.02 (2H, m, N (CH ₃ ) C H ₂ CO), 4.57 (1H, t, 5.87 Hz, COC H NHCO), 6 99 (1H, m, ArH), 7.07 (1H, m, ArH), 7.19 (1H, s, ArH), 7.34 (1H, d, 8.31 Hz, ArH), 7, 50 (1H, d, 7.82 Hz, ArH). ¹³ C NMR spectrum (DMSO-d ⁶ ), δ, ppm: 1.18, 19.26, 19.32, 28.43, 29.12, 29.28, 75.69, 80.30, 80.40, 105.24, 107.69, 141.40, 145.79, 150.31, 152.43, 174.74

Example 14. Obtaining creatyl leucine diethylamide acetate

14.1 Obtaining creatyl leucine diethylamide acetate by the present method.

Stage A. Obtaining creatylleucine diethylamide tosylate.

Anhydrous creatine (5 g, 38 mmol), p-TSC (15.23 g, 80 mmol) and DMF are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, (30 ml), stirred for 10 min, a solution of leucine diethylamide hydrochloride 8.47 g (38 mmol) in 15 ml of DMF was added, stirred at room temperature for 5 min, cooled with a bath (ice-salt mixture) to a temperature (- ) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) is added dropwise at the same time over 30 minutes and N-methylmorpholine (8.48 g, 83.9 mmol) was stirred at a temperature of (-) 10 ° C for 1.5 hours. The precipitate was filtered off, washed on the filter with DMF (2 × 5 ml) cooled to 0 ° C. . Wash liquids and mother liquor are combined. The solution determines the content of creatyl leucine diethylamide tosylate and chloride ions. The yield of tosylate diethylamide creatyl leucine is 60% mol., The amount of chloride ions is 27.5 mMEq. Potassium acetate (2.70 g, 27.5 mmol) was added to the solution, stirred for 1 h, 50 ml (91%) of DMF were distilled off under reduced pressure and a temperature of 45 ° C. 40 ml of acetonitrile is added to the residue at room temperature, stirred for 20 minutes, 4 g of celite are added, and stirred for 20 minutes. 10 g of celite are placed on the filter, washed with 40 ml of acetonitrile, the washing liquid is disposed of. The suspension is filtered through celite, the precipitate is washed with 25 ml of acetonitrile. Wash liquids and mother liquor are combined. The content of chloride ions in the solution is 5.7 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 45 ° C. The residue was dissolved in 20 ml of a mixture of 0.05 M acetic acid: isopropyl alcohol (50:50). A solution of creatyl leucine diethylamide tosylate is obtained.

Stage B. Obtaining creatyl leucine diethylamide acetate.

Removal of p-TSA and its replacement with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, column 49 × 200 mm (General Electric, USA) filled with a Dowex 1x8 sorbent in acetate form). A solution of tosylate diethylamide creatyl leucine was applied to the column, eluted with a mixture of 0.05 M acetic acid: isopropyl alcohol (80:20) at a rate of 0.3 cm / min. Detection is carried out at a wavelength of 220 nm. Fractions were collected, analyzed by HPLC, the fractions were combined based on the results of HPLC analysis to obtain creatilleucine diethylamide acetate with a total content of unidentified impurities of not more than 1%, creatinine - not more than 1%, creatine - not more than 1%, acetic acid was added to pH 4, four. From the combined fractions, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. 100 ml of isopropyl alcohol was added to the residue, the solvent was distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 50 ml of ethyl acetate were added, stirred for 3 hours, the precipitate was filtered off, washed with ethyl acetate (3 × 25 ml), dried in vacuum at 40 ° C, dissolved in 125 ml of absolute ethanol with stirring for 20 minutes, the insoluble residue was separated by filtration, washed on the filter with absolute ethanol (2 × 20 ml), the washing liquids were combined with the mother liquor. 130 ml (79%) of ethanol are distilled off from the solution under reduced pressure and a temperature of 40 ° C, 50 ml of ethyl acetate are added, and they are kept at room temperature for 2 hours. The precipitate was separated by filtration, washed with ethyl acetate (2 × 50 ml), dried in vacuum at a temperature of 40 ° C.

Yield of creatyl leucine diethylamide acetate C ₁₄ H ₂₉ N ₅ O ₂ * C ₂ H ₄ O ₂ 5.39 g (40%). The content of the main substance is 98.0%, sulfate ash - 0.04%, chloride ions - traces, p-TSC - 4 ppm, impurities: creatinine -1.0%, creatine - 0.7%, the maximum single unidentified impurity - 0.1%, the amount of unidentified impurities - 0.2%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 0.85 (3H, d, 3.02 Hz, CH (CH ₃ ) ₂ ), 0.87 (3H, d, 2.52 Hz, CH (CH ₃ ) ₂ ), 1, 06 (3H, t, 7.05 Hz, CH ₂ CH ₃ ), 1.25 (3H, t, 7.05 Hz, CH ₂ CH ₃ ), 1.43 (1H, m, CH (CH ₃ ) ₂ ), 1.65 (2H, m, CH ₂ CH (CH ₃ ) ₂ ), 1.88 (3H, s, CH ₃ COOH), 2.96 (3H, s, NCH ₃ ), 3.07-3 58 (4H, m, CH ₂ CH ₃ ), 4.18 (2H, m, N (CH ₃ ) CH ₂ CO), 4.70 (1H, m, COCHNHCO, under the signal of residual water). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 11.96, 13.23, 20.30, 22.48, 24.48, 37, 08, 41.28, 42.63, 49 01, 52.36, 157.77, 168.97, 173.05, 181.25. ¹ H NMR Spectrum (DMSO-d ⁶ ), δ, ppm (J, Hz): 0.89 (3H, d, 4.16 Hz, CH (CH ₃ ) ₂ ), 0.907 (3H, d, 4.40 Hz, CH (CH ₃ ) ₂ ), 1.01 ( 3H, t, 7.09 Hz, CH ₂ CH ₃ ), 1.18 (3H, t, 7.09 Hz, CH ₂ CH ₃ ), 1.32 (1H, m, CH (CH ₃ ) ₂ ), 1.66 (2H, m, CH ₂ CH (CH ₃ ) ₂ ), 1.69 (3H, s, CH ₃ COOH), 2.85 (3H, s, NCH ₃ ), 3.07-3.50 (4H, m, CH ₂ CH ₃ ), 4.03 (2H, m, N (CH ₃ ) CH ₂ CO), 4.74 (1H, m, COCHNHCO), 7.0-9.5 (5H, COCHNHCO and NH ₂ (NH) C * AcOH).

14.2 Obtaining creatyl leucine diethylamide acetate according to the method described in the prototype [RU 2428414, 2011].

Stage A. Obtaining creatylleucine diethylamide tosylate.

Anhydrous creatine (5 g, 38 mmol), p-TSC (15.23 g, 80 mmol) and DMF are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube, mounted on a magnetic stirrer. (30 ml), the suspension is stirred for 10 min, a solution of leucine diethylamide hydrochloride 8.47 g (38 mmol) in 15 ml of DMF is added, stirred at room temperature for 5 min, cooled with a bath (ice-salt mixture) to a temperature ( -) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) is added sequentially, then After 30 minutes, N-methylmorpholine (8.48 g, 83.9 mmol) was added dropwise. The reaction mixture was stirred at a temperature of (-) 10 ° C for 1 h, the temperature was brought to room temperature. The precipitate was filtered off from the reaction mixture. In the mother liquor, the content of creatyl leucine tosylate diethylamide and chloride ions is determined. The yield of tosylate diethylamide creatyl leucine is 41% mol., The amount of chloride ions is 38.2 mMeq. The solvent is distilled off from the solution under reduced pressure and a temperature of 50 ° C until the end of the run, the residue is dissolved in 300 ml of n-butyl alcohol, washed 4 times with 5% NaHCO ₃ solution, 2 times with water. The solvent is distilled off from the organic phase under reduced pressure and a temperature of 40 ° C until the end of the run. The residue was dissolved in 20 ml of a mixture of 0.05 M acetic acid: isopropyl alcohol (50:50). A solution of creatylleucine tosylate diethylamide is obtained.

Stage B. Obtaining creatyl leucine diethylamide acetate.

Removing p-TSC and replacing it with a pharmaceutically acceptable acid is carried out by RP chromatography (Sepaore Buchi chromatographic system, 22.5 × 150 mm column filled with YMC * Gel ODS sorbent in 0.05 M acetic acid). A solution of tosylate diethylamide creatyl leucine is applied to the column, eluting in a linear gradient mode from 0.05 M acetic acid to 20% isopropyl alcohol in 0.05 M acetic acid at a speed of 0.35 cm / min. Fractions are collected, analyzed by HPLC, combined based on the results of the analysis, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. The residue is crystallized from 20 ml of acetonitrile, separated by filtration and dried.

The yield of creatyl leucine diethylamide acetate C ₁₄ H ₂₉ N ₅ O ₂ * C ₂ H ₄ O ₂ 3.31 g (24%). The content of the main substance is 97.2%, sulfate ash - 0.02%, chloride ions - 0.15 mMEq / g, p-TSA - 8 ppm, impurities: creatinine - 1.1%, creatine - 0.8% , the maximum unit unidentified impurity is 0.2%, the sum of unidentified impurities is 0.3%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 0.85 (3H, d, 3.02 Hz, CH (CH ₃ ) ₂ ), 0.87 (3H, d, 2.52 Hz, CH (CH ₃ ) ₂ ), 1, 06 (3H, t, 7.05 Hz, CH ₂ CH ₃ ), 1.25 (3H, t, 7.05 Hz, CH ₂ CH ₃ ), 1.43 (1H, m, CH (CH ₃ ) ₂ ), 1.65 (2H, m, CH ₂ CH (CH ₃ ) ₂ ), 1.88 (3H, s, CH ₃ COOH), 2.96 (3H, s, NCH ₃ ), 3.07-3 58 (4H, m, CH ₂ CH ₃ ), 4.18 (2H, m, N (CH ₃ ) CH ₂ CO), 4.70 (1H, m, COCHNHCO, under the signal of residual water). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 11.96, 13.23, 20.30, 22.48, 24.48, 37.08, 41.28, 42.63, 49 01, 52.36, 157.77, 168.97, 173.05, 181.25. ¹ H NMR Spectrum (DMSO-d ⁶ ), δ, ppm (J, Hz): 0.89 (3H, d, 4.16 Hz, CH (CH ₃ ) ₂ ), 0.907 (3H, d, 4.40 Hz, CH (CH ₃ ) ₂ ), 1.01 ( 3H, t, 7.09 Hz, CH ₂ CH ₃ ), 1.18 (3H, t, 7.09 Hz, CH ₂ CH ₃ ), 1.32 (1H, m, CH (CH ₃ ) ₂ ), 1.66 (2H, m, CH ₂ CH (CH ₃ ) ₂ ), 1.69 (3H, s, CH ₃ COOH), 2.85 (3H, s, NCH ₃ ), 3.07-3.50 (4H, m, CH ₂ CH ₃ ), 4.03 (2H, m, N (CH ₃ ) CH ₂ CO), 4.74 (1H, m, COCHNHCO), 7.0-9.5 (5H, COCHNHCO and NH ₂ (NH) C * AcOH).

Example 15. Obtaining creatylproline n-butylamide acetate

15.1 Obtaining creatylproline n-butylamide acetate by the present method.

Stage A. Obtaining creatylproline n-butylamide tosylate

Creatine monohydrate (5.7 g, 38 mmol), p-TSC (15.23 g, 80 mmol) are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube, mounted on a magnetic stirrer, and DMF (30 ml), stirred for 10 min, a solution of proline n-butylamide (6.49 g, 38 mmol) in 15 ml of DMF was added. The mixture was stirred at room temperature for 5 minutes, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) and N-methylmorpholine were added simultaneously over 30 minutes. (8.48 g, 83.9 mmol), stirred at a temperature of (-) 10 ° C for 1.5 hours. The precipitate was filtered off from the cooled reaction mixture, washed on a DMF filter (2 × 5 ml), cooled to 0 ° C. Wash liquids and mother liquor are combined. The content of creatylproline n-butylamide tosylate and chloride ions is determined in the solution. The yield of creatylproline n-butylamide tosylate is 52% mol., The amount of chloride ions is 28.0 mMEq. Sodium acetate trihydrate (3.8 g, 28.0 mmol) was added to the solution, stirred for 1 h, 53 ml (93%) of DMF were distilled off under reduced pressure and a temperature of 45 ° C. 40 ml of acetone was added to the residue at room temperature, the suspension was stirred for 20 minutes, 4 g of celite was added, and stirred for 20 minutes. 8 g of celite are placed on the filter, washed with 40 ml of acetone, the washing liquid is disposed of. The suspension is filtered through celite, the filter cake washed with 25 ml of acetone. Wash liquids and mother liquor are combined. The amount of chloride ions in the solution is 3.8 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 45 ° C until the end of the run. The residue is dissolved in 20 ml of a 0.05 M solution of acetic acid. A solution of creatylproline n-butylamide tosylate is obtained.

Stage B. Obtaining creatylproline n-butylamide acetate.

Removing p-TSC and replacing it with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, column 49 × 210 mm in size (General Electric, USA) filled with a Dowex 1x8 sorbent in acetate form). A solution of creatylproline n-butylamide tosylate is applied to the column, eluting with a 0.05 M acetic acid solution at a rate of 0.7 cm / min. Detection is carried out at a wavelength of 220 nm. Fractions were collected, analyzed by HPLC, and pooled on the basis of the results of analysis to obtain isotropyl ether acetate acetate with a total content of unidentified impurities of not more than 1%, creatinine not more than 1%, creatine not more than 1%, acetic acid was added to pH 4.3 . From the combined fractions, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. 100 ml of isopropyl alcohol is added to the residue, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 50 ml of acetone are added, stirred for 3 hours, the precipitate is separated by filtration, washed with acetone (3 × 25 ml), dried in vacuum at a temperature of 40 ° C until the end of the run, dissolved in 125 ml of absolute ethanol with stirring for 20 minutes, the insoluble residue was separated by filtration, washed with absolute ethanol (2 × 20 ml), the washing liquids were combined with the mother liquor. 134 ml (79%) of ethanol are distilled off from the solution under reduced pressure and a temperature of 40 ° C, 50 ml of ethyl acetate are added, the mixture is kept for 2 hours at room temperature. The precipitate was separated by filtration, washed with ethyl acetate (2 × 50 ml), dried in vacuum at a temperature of 40 ° C.

Yield of creatylproline n-butylamide acetate C ₁₃ H ₂₅ N ₅ O ₂ * C ₂ H ₄ O ₂ 3.78 g (29%). The content of the main substance is 97.1%, sulfate ash - 0.03%, chloride ions - traces, p-TSC - 3 ppm, impurities: creatine - 0.5%, creatinine - 1.0%, the maximum single unidentified impurity - 0.2%, the amount of unidentified impurities - 0.7%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 0.88 (3H, m, CH ₂ CH ₂ CH ₂ CH ₃ ), 1.30 (2H, m, CH ₂ CH ₂ CH ₂ CH ₃ ), 1.47 (2H, m, CH ₂ CH ₂ CH ₂ CH ₃ ), 1.90 (3H, s, CH ₃ COOH), 1.91 (1H, m, Pro), 2.01 (2H, m, Pro), 2.26 (1H m, Pro), 3.04 (3H, s, NCH ₃ ), 3.19 (2H, m, CH ₂ CH ₂ CH ₂ CH ₃ ), 3.58 (2H, m, Pro), 4.36 (3H, m, Pro and N (CH ₃ ) CH ₂ CO). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 15.62, 21.97, 25.98, 27.05, 32.25, 33.11, 39.91, 41.71, 49 45, 55.12, 63.70, 160.34, 170.08, 176.54, 183.97.

15.2 Obtaining creatylproline n-butylamide acetate according to the method described in the prototype [RU 2428414, 2011].

Stage A. Obtaining creatylproline n-butylamide tosylate.

Creatine monohydrate (5.69 g, 38 mmol), p-TSC (15.23 g, 80 mmol) are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, and DMF (30 ml), stirred for 10 min, a solution of proline n-butylamide (6.49 g, 38 mmol) in 15 ml of DMF was added. The mixture was stirred at room temperature for 5 minutes, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) was added sequentially, then, over 30 minutes, dropwise add N-methylmorpholine (8.48 g, 83.9 mmol), stir at (-) 10 ° C for 1 h, bring the temperature to room temperature. The precipitate was filtered off from the reaction mixture. In the mother liquor, the content of creatylproline n-butylamide tosylate is determined. The yield of creatylproline n-butylamide tosylate is 2.7% mole. The reaction is practically not going on.

Example 16. Obtaining creatylhistidine methyl ester diacetate

16.1 Obtaining creatylhistidine methyl ester of diacetate according to the claimed method

Step A. Preparation of Tosylate Creatyl Histidine Methyl Ester

Anhydrous creatine (5 g, 38 mmol), p-TSC (7.62 g, 40 mmol) and DMF are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, (30 ml), stirred for 10 minutes, a suspension of histidine methyl ester dihydrochloride (9.22 g, 38 mmol) in 15 ml of DMF was added, and stirred at room temperature for 5 minutes. The mixture is cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° С, isobutyl chloroformate (6.25 g, 45.8 mmol) and N-methylmorpholine (12.33 g) are added dropwise at the same time over 30 minutes. , 121.9 mmol), stirred at a temperature of (-) 10 ° C for 1.5 hours. The precipitate was filtered off from the cooled reaction mixture, washed with DMF (2 × 5 ml) cooled to 0 ° C. Wash liquids and mother liquor are combined. The solution determines the content of creatyl histidine methyl ester of tosylate, chloride ions. The yield of tosylate methyl ester creatyl histidine is 33% mol., The amount of chloride ions is 29.9 mMEq. Sodium acetate trihydrate (3.98 g, 29.9 mmol) was added to the solution, stirred for 1 h, 53 ml (92%) of DMF were distilled off under reduced pressure and a temperature of 45 ° C. 40 ml of acetone are added to the residue, stirred for 20 minutes at room temperature, 4 g of celite are added, and stirred for 20 minutes. 7 g of celite are placed on the filter, washed with 40 ml of acetone, the washing liquid is disposed of. The suspension is filtered through celite, the precipitate is washed with 25 ml of acetone. Wash liquids and mother liquor are combined. The amount of chloride ions in the solution is 4.9 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 45 ° C until the end of the run. The residue was dissolved in 20 ml of 0.05 M acetic acid. A solution of creatyl histidine methyl tosylate is obtained.

Stage B. Obtaining creatylhistidine methyl ester diacetate.

Removal of p-TSC and its replacement with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, column 49 × 230 mm in size, filled with a Dowex 1x8 sorbent in acetate form). A solution of tosylate methyl ester creatyl histidine is applied to the column, eluting with a 0.035 M solution of acetic acid at a rate of 0.3 cm / min. Detection is carried out at a wavelength of 220 nm. Fractions were collected, analyzed by HPLC, combined on the basis of the analysis to obtain creatyl histidine methyl ester diacetate with a total content of unidentified impurities of not more than 1%, creatinine - not more than 1%, creatine - not more than 1%, acetic acid was added to pH 4.3 . From the combined fractions, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. 100 ml of n-butyl alcohol is added to the residue, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 50 ml of acetone are added, stirred for 3 hours, the precipitate is filtered off, washed with acetone (3 × 25 ml), dried under vacuum at temperature 40 ° С. The precipitate was dissolved in 125 ml of absolute ethanol, the insoluble residue was separated by filtration, absolute ethanol (2 × 20 ml), the washings were combined with the mother liquor. 127 ml (78%) of ethanol are distilled off from the solution under reduced pressure and a temperature of 40 ° C, 50 ml of ethyl acetate are added, the mixture is kept for 2 hours at room temperature. The precipitate was separated by filtration, washed with ethyl acetate (2 × 50 ml), dried in vacuum at a temperature of 40 ° C.

Yield of creatyl histidine methyl ester of diacetate C ₁₁ H ₁₈ N ₆ O ₃ * 2C ₂ H ₄ O ₂ 3.03 g (23%). The content of the main substance is 98.1%, sulfate ash - 0.03%, chloride ions - traces, p-TSC - 4 ppm, impurities: creatinine - 1.0%, creatine - 0.4; maximum single unidentified impurity - 0 , 1%, the sum of unidentified impurities - 0.4%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 2.14 (3H, s, CH ₃ CO ₂ H), 2.89 (3H, s, NCH ₃ ), 3.00-3.20 (2H, m, imidazole-C H ₂ CHNHCO ₂ CH ₃ ), 3.69 (3H, s, imidazole-CH ₂ CHNHCO ₂ C H ₃ ), 4.00-4.15 (2H, m, NHCH ₂ CO), 8.43 (1H, s, HCO ₂ H) 4.70 (1H, m, imidazole-CH ₂ C H NHCO ₂ CH ₃ , under the signal of residual water), 6.98 (1H, s, H at position 2 of the imidazole ring), 7.82 (1H , c, H at position 5 of the imidazole ring). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 28.85, 31.79, 38.47, 54.16, 54.72, 55.44, 118.40, 136.61, 137 44, 158.67, 170.69, 174.21, 176.13.

16.2 Obtaining creatylhistidine methyl ester of diacetate according to the method described in the prototype [RU 2428414, 2011].

Stage A. Obtaining creatylhistidine methyl tosylate.

Anhydrous creatine (5.0 g, 38 mmol), p-TSC (15.23 g, 80 mmol) are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, and DMF (30 ml), stirred for 10 min, a suspension of histidine methyl ester dihydrochloride (9.22 g, 38 mmol) in 15 ml of DMF was added, stirred at room temperature for 5 min. The mixture was cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° С, isobutyl chloroformate (6.25 g, 45.8 mmol) was added sequentially, then N-methylmorpholine was added dropwise over 30 minutes (8, 48 g, 83.9 mmol). The reaction mixture was stirred at a temperature of (-) 10 ° C for 1 h, the temperature was brought to room temperature. The precipitate was filtered off from the reaction mixture. The content of creatyl histidine tosylate methyl ester is determined in the mother liquor. The yield of tosylate methyl ester creatyl histidine is 8% mole.

Due to the low yield of creatine amide tosylate, no further procedure was performed.

Example 17. Obtaining creatylglutamine dimethyl ether acetate

17.1 Obtaining creatylglutamine dimethyl ether acetate according to the present method.

Stage A. Obtaining creatylglutamine tosylate dimethyl ether.

Anhydrous creatine (5 g, 38 mmol), p-TSC (7.62 g, 40 mmol) and DMF are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, (30 ml), stirred for 10 min, a suspension of glutamic acid dimethyl ester hydrochloride (8.04 g, 38 mmol) in 15 ml of DMF was added, stirred at room temperature for 5 min, cooled with a bath (ice-salt mixture) to temperature (-) 15 ° С, isobutyl chloroformate is added simultaneously, over 30 minutes (6.2 5 g, 45.8 mmol) and N-methylmorpholine (8.48 g, 83.9 mmol). The mixture is stirred at a temperature of (-) 10 ° C for 1.5 hours. The precipitate is filtered off, washed on the filter with DMF (2 × 5 ml) cooled to 0 ° C. Wash liquids and mother liquor are combined. The content of creatylglutamine tosylate dimethyl ether and chloride ions is determined in the solution. The output of creatylglutamine tosylate dimethyl ether is 44% mol., The amount of chloride ions is 28.5 mMEq. Sodium acetate trihydrate (3.72 g, 28.5 mmol) was added to the solution, stirred for 1 h, 50 ml (91%) of DMF were distilled off under reduced pressure and a temperature of 45 ° C. 40 ml of acetone was added to the residue at room temperature, the suspension was stirred for 20 minutes, 4 g of celite was added, and stirred for 20 minutes. 7 g of celite are placed on the filter, washed with 40 ml of acetone, the washing liquid is disposed of. The suspension is filtered through celite, the precipitate is washed with 25 ml of acetone. Wash liquids and mother liquor are combined. The amount of chloride ions in the solution is 5.0 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 45 ° C, the residue is dissolved in 20 ml of 0.05 M acetic acid. A solution of creatylglutamine tosylate dimethyl ether is obtained.

Stage B. Obtaining creatylgiltamine dimethyl ether acetate.

Removing p-TSC and replacing it with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, 49 × 210 mm column (General Electric, USA) filled with a Dowex 1x8 sorbent in acetate form). A solution of creatylglutamine tosylate dimethyl ether is applied to the column, eluting with a 0.05 M solution of acetic acid at a rate of 0.3 cm / min. Detection is carried out at a wavelength of 220 nm. Fractions were collected, analyzed by HPLC, combined on the basis of the analysis to obtain creatylglutamine dimethyl ether acetate with a total content of unidentified impurities of not more than 1%, creatinine not more than 1%, creatine not more than 1%, acetic acid was added to pH 4.3 . From the combined fractions, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. 100 ml of n-butyl alcohol are added to the residue, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 50 ml of acetone are added, stirred for 3 hours, the precipitate is filtered off, washed with acetone (3 × 25 ml), and dried under vacuum at temperature 40 ° С. The precipitate was dissolved in 125 ml of absolute ethanol with stirring for 20 minutes, the insoluble residue was separated by filtration, washed on the filter with absolute ethanol (2 × 20 ml), and the washings were combined with the mother liquor. 125 ml of ethanol (75%) are distilled off from the solution under reduced pressure and a temperature of 40 ° C, 50 ml of ethyl acetate are added, the mixture is kept for 2 hours at room temperature. The precipitate was separated by filtration, washed with ethyl acetate (2 × 50 ml), dried in vacuum at a temperature of 40 ° C.

The yield of creatylglutamine dimethyl ether acetate C ₁₁ H ₂₀ N ₄ O ₅ * C ₂ H ₄ O ₂ 3.62 g (33%). The content of the main substance is 98.1%, sulfate ash - 0.03%, chloride ions - traces, p-TSA - 4 ppm, impurities: creatinine - 1.0%, creatine - 0.7%, the maximum single unidentified impurity - 0.1%, the amount of unidentified impurities - 0.5%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.84 (3H, s, CH ₃ CO ₂ H), 1.90-2.25 (2H, m, CH ₃ O ₂ CCH ₂ C H ₂ CHNHCO ₂ CH ₃ ), 2, 98 (3H, s, NCH ₃ ), 2.44 (2H, t, 6.85 Hz, CH ₃ O ₂ CC H ₂ CH ₂ CHNHCO ₂ CH ₃ ), 3.63 (3H, s, C H ₃ O ₂ CCH ₂ CH ₂ CHNHCO ₂ CH ₃ ), 4.15 (2H, m, NHCH ₂ CO), 3.69 (3H, s, CH ₃ O ₂ CCH ₂ CH ₂ CHNHCO ₂ C H ₃ ), 4.45 (1H, m, 3.63 (3H, s, CH ₃ O ₂ CCH ₂ CH ₂ C H NHCO ₂ CH ₃ ). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 26.05, 28.47, 32.81, 39.98, 55.10, 55.27, 55.40, 56.01, 160.79, 172.38, 176.38, 178.52, 184.06. 17.2 Preparation creatylglutamine dimethyl ether acetate according to the method described in the prototype [RU 2428414, 2011].

Stage A. Obtaining creatylgiltamine dimethyl tosylate ether.

Anhydrous creatine (5 g, 38 mmol), p-TSC (15.23 g, 80 mmol) and DMF are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, (30 ml), stirred for 10 min, a solution of glutamic acid dimethyl ester hydrochloride (8.04 g, 38 mmol) in 15 ml of DMF was added. The mixture was stirred at room temperature for 5 minutes, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) was added sequentially, then, over 30 minutes, dropwise, N-methylmorpholine (8.48 g, 83.9 mmol), stirred at a temperature of (-) 10 ° C for 1 h, brought to room temperature. The precipitate was filtered off from the reaction mixture. The content of creatylglutamine tosylate dimethyl ether and chloride ions is determined in the mother liquor. The output of creatylglutamine tosylate dimethyl ether is 29% mol., The amount of chloride ions is 39.9 mMEq. The solvent is distilled off from the mother liquor under reduced pressure and a temperature of 50 ° C until the end of the run. The residue is dissolved in 160 ml of chloroform, left for 10 hours at a temperature of (-) 10 ° C, the solution is filtered off, extracted with water 3 times in 100 ml. The aqueous extracts are combined, 2 times extracted with chloroform. The aqueous extract is evaporated under reduced pressure and a temperature of 40 ° C to a volume of 50 ml. A solution of creatylglutamine tosylate dimethyl ether is obtained.

Stage B. Obtaining creatylgiltamine dimethyl ether acetate.

Removal of p-TSA and its replacement with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatography system, column 49 × 210 mm (General Electric, USA, filled with a Dowex 1x8 sorbent in acetate form). on a column, elute with water at a speed of 0.3 cm / min, controlling the pH of the eluate. before The residue is crystallized from 50 ml of acetonitrile at a temperature of (-) 10 ° C for 20 hours. The precipitate of creatylglutamine dimethyl ether acetate is filtered off, washed with cold acetonitrile, diethyl ether and dried.

The yield of creatylgglutamine dimethyl ether acetate C ₁₁ H ₂₀ N ₄ O ₅ * C ₂ H ₄ O ₂ 2.39 g (22%). The content of the main substance is 96.4%, sulfate ash - 0.05%, chloride ions - 0.5 mmol / g, p-TSA - 156 ppm, impurities: creatinine - 1.7%, creatine - 0.9% , the maximum unit unidentified impurity is 0.4%, the sum of unidentified impurities is 0.8%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 2.99 (3H, s, NCH ₃ ), 1.85 (3H, s, CH ₃ CO ₂ H), 1.90-2.25 (2H, m, CH ₃ O ₂ CCH ₂ C H ₂ CHNHCO ₂ CH ₃ ), 2.44 (2H, t, 6.85 Hz, (CH ₃ O ₂ CC H ₂ CH ₂ CHNHCO ₂ CH ₃ ), 3.63 (3H, s, C H ₃ O ₂ CCH ₂ CH ₂ CHNHCO ₂ CH ₃ ), 3.69 (3H, s, CH ₃ O ₂ CCH ₂ CH ₂ CHNHCO ₂ C H ₃ ), 4.15 (2H, m, NHCH ₂ CO), 4, 45 (1H, m, 3.63 (3H, s, CH ₃ O ₂ CCH ₂ CH ₂ C H NHCO ₂ CH ₃ ). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 26.06 , 28.47, 32.81, 39.98, 55.10, 55.26, 55.40, 56.01, 160.79, 172.37, 176.38, 178.52, 184.06.

Example 18. Obtaining creatyl glycine ethyl ester fumarate

18.1. Obtaining creatiglycine ethyl ester of the fumarate according to the claimed method.

Stage A. Obtaining creatylglycine tosylate ethyl ester.

Creatine monohydrate (5.69 g, 38 mmol), p-TSC (15.23 g, 80 mmol) are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, and DMF (30 ml), stirred for 10 min, a solution of glycine ethyl ester hydrochloride (5.32 g, 38 mmol) in 15 ml of DMF was added. The mixture is stirred at room temperature for 5 minutes, cooled (ice-salt mixture) to a temperature of (-) 15 ° С, isobutyl chloroformate (6.25 g, 45.8 mmol) and N-methylmorpholine are added simultaneously over 30 minutes. (8.48 g, 83.9 mmol), stirred at a temperature of (-) 10 ° C for 1.5 hours. The precipitate was filtered off, washed with DMF (2 × 5 ml) cooled to 0 ° C. The washing liquids and the mother liquor are combined, the content of tosylate ethyl ester of tosylate and chloride ions is determined. The yield of tosylate ethyl ester creatyl glycine is 71% mol., The amount of chloride ions is 29.4 mMEq. Sodium acetate trihydrate (4 g, 29.4 mmol) was added to the solution, the suspension was stirred for 1 h, 50 ml of DMF (91%) was distilled off under reduced pressure and a temperature of 45 ° C. 40 ml of acetone are added to the residue at room temperature, stirred for 20 minutes, 4 g of celite are added, and stirred for 20 minutes. 8 g of celite are placed on the filter, washed with 40 ml of acetone, the washing liquid is disposed of. The suspension is filtered through celite, the precipitate is washed with 25 ml of acetone. Wash liquids and mother liquor are combined. The content of chloride ions in the solution is 5.6 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 45 ° C until the end of the run, the residue is dissolved in 20 ml of a 0.05 M solution of acetic acid, and a solution of tosylate ethyl glycine is obtained.

Stage B. Obtaining creatylglycine ethyl ester fumarate.

Removal of p-TSC and its replacement with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, 49 × 210 mm column (General Electric, USA, filled with a Dowex 1x8 sorbent in acetate form). A solution of tosylate ethyl ester is applied per column, elute with a 0.05 M solution of acetic acid at a speed of 0.8 cm / min, Detection is carried out at a wavelength of 220 nm, fractions are taken, analyzed by HPLC, combined based on the results of the analysis to obtain creatiglycine et acetate acetate with a total content of unidentified impurities of not more than 1%, creatinine - not more than 1%, creatine - not more than 1%. Fumaric acid (1.86 g, 16 mmol) was added to the combined fractions, stirred for 20 minutes until complete dissolution ( pH 4.0), the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. 100 ml of isopropyl alcohol is added to the residue, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 50 ml of acetone are added, stirred for 3 hours the precipitate is separated by filtration, romyvayut (3 × 25 ml) with acetone, dried in vacuo at 40 ° C. 125 ml of absolute ethanol was added to the precipitate, stirred for 20 minutes, the insoluble residue was filtered off, washed (2 × 20 ml) with absolute ethanol on the filter, and the washing liquids were combined with the mother liquor. 130 ml (78%) of ethanol are distilled off from the solution under reduced pressure and a temperature of 35 ° C, 50 ml of ethyl acetate are added, and they are kept at room temperature for 2 hours. The precipitate was separated by filtration, washed (2 × 20 ml) with ethyl acetate, dried in vacuum at a temperature of 40 ° C.

Yield of creatyl glycine ethyl ester of the fumarate C ₈ H ₁₆ N ₄ O ₃ * C ₄ H ₄ O ₄ 4.99 g (40%). The main substance content is 98.3%, sulfate ash - 0.02%, chloride ions - traces, p-TSC - 2 ppm, impurities: creatinine - 1.0%, creatine - 0.2%, maximum single unidentified impurity - 0.1%, the amount of unidentified impurities - 0.2%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.16 (3H, t, 7.17 Hz, CH ₂ CH ₃ ), 2.96 (3H, s, NCH ₃ ), 3.96 (2H, s, N (CH ₃ ) CH ₂ CO), 4.20-4.25 (4H, m, NHCH ₂ CO and CH ₂ CH ₃ ), 6.85 (2H, s, HO ₂ CCH = CHCO ₂ H). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 13.23, 37.00, 41.37, 52.59, 62.64, 134.71, 157.82, 170.00, 171 52, 171.69.

18.2. Obtaining creatiglycine ethyl ester of fumarate according to the method described in the prototype [RU 2428414, 2011].

Stage A. Obtaining creatylglycine tosylate ethyl ester.

Creatine monohydrate (5.69 g, 38 mmol), p-TSC (15.23 g, 80 mmol) are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, and DMF (30 ml), stirred for 10 min, a solution of glycine ethyl ester hydrochloride (5.32 g, 38 mmol) in 15 ml of DMF was added, stirred at room temperature for 5 min, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) is added sequentially, then over 30 minutes, dropwise added N-methylmorpholine (8.48 g, 83.9 mmol). The mixture is stirred at a temperature of (-) 10 ° C for 1 h, the temperature is brought to room temperature. The precipitate was filtered off from the reaction mixture. In the mother liquor, the content of creatiglycine of tosylate ethyl ester and chloride ions is determined. The yield of tosylate ethyl ester creatyl glycine is 50% mol., The amount of chloride ions is 35.2 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 50 ° C until the end of the run. The residue is dissolved in 160 ml of chloroform, left for 10 hours at a temperature of (-) 10 ° C. The solution is filtered off, extracted with water 3 times in 100 ml. The aqueous extracts are combined, extracted 2 times with chloroform, the solvent is distilled off from the aqueous extract under reduced pressure and a temperature of 40 ° C to a volume of 50 ml. A solution of creatyl glycine ethyl tosylate is obtained.

Stage B. Obtaining creatylglycine ethyl ester fumarate.

Removal of p-TSC and its replacement with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, column 49 × 210 mm (General Electric, USA, filled with a Dowex 1x8 sorbent in acetate form). per column, elute with water at a speed of 0.7 cm / min, controlling the pH of the eluate Fractions with pH = 7 (pH 5 of water) are collected, analyzed by HPLC, combined, fumaric acid (1.86 g, 16 mmol) is added, mixed until completely dissolved, the solvent is distilled off b under reduced pressure and a temperature of 40 ° C until the end of the run, the residue is crystallized from 80 ml of acetonitrile at a temperature of (-) 10 ° C for 20 hours.

Yield of creatyl glycine ethyl ester of the fumarate C ₈ H ₁₆ N ₄ O ₃ * C ₄ H ₄ O ₄ 3.01 g (24%). The content of the main substance is 97.7%, sulfate ash - 0.04%, chloride ions - 0.45 mMEq / g, p-TSA - 140 ppm, impurities: creatinine - 1.0%, creatine - 0.4% ; the maximum single unidentified impurity is 0.5%, the sum of unidentified impurities is 0.9%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.15 (3H, t, 7.17 Hz, CH ₂ CH ₃ ), 2.96 (3H, s, NCH ₃ ), 3.96 (2H, s, N (CH ₃ ) CH ₂ CO), 4.20-4.25 (4H, m, NHCH ₂ CO and CH ₂ CH ₃ ), 6.85 (2H, s, HO ₂ CCH = CHCO ₂ H). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 13.24, 37.00, 41.37, 52.59, 62.64, 134.71, 157.82, 170.00, 171 52, 171.69.

Example 19. Obtaining Creatyl Glycine Ethyl Succinate

19.1. Obtaining creatiglycine ethyl ester of succinate by the present method.

Stage A. Obtaining creatylglycine tosylate ethyl ester.

Creatine monohydrate (5.69 g, 38 mmol), p-TSC (15.23 g, 80 mmol) are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, and DMF (30 ml), stirred for 10 min, a solution of glycine ethyl ester hydrochloride (5.32 g, 38 mmol) in 15 ml of DMF was added, stirred for 5 min, cooled (ice-salt mixture) to a temperature (- ) 15 ° С, isobutyl chloroformate (6.25 g, 45.8 mmol) and N-methylmorpholine (8.48 g, 83.9 mmol) are added simultaneously, over 30 min. stirred at a temperature of (-) 10 ° C for 1.5 hours. The precipitate is filtered off from the cooled reaction mixture, washed on the filter with DMF (2 × 5 ml) cooled to 0 ° C. Wash liquids and mother liquor are combined. The solution determines the content of creatinglycine tosylate ethyl ester and chloride ions. The yield of tosylate ethyl ester creatyl glycine is 73% mol., The amount of chloride ions is 29 mMEq. Sodium acetate trihydrate (3.94 g, 29 mmol) was added to the solution, stirred for 1 h, 50 ml (91%) of DMF were distilled off under reduced pressure and a temperature of 45 ° C. 40 ml of acetone are added to the residue at room temperature, the suspension is stirred for 20 minutes, 4 g of celite are added, and stirred for 20 minutes. 8 g of celite are placed on the filter, washed with 40 ml of acetone, the washing liquid is disposed of. The suspension is filtered through celite, the filter cake washed with 25 ml of acetone. Wash liquids and mother liquor are combined. The content of chloride ions in the solution is 2.6 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 45 ° C until the end of the run. The residue is dissolved in 20 ml of a 0.05 M acetic acid solution, and a solution of tosylate ethyl ester creatyl glycine is obtained.

Stage B. Obtaining Creatyl Glycine Ethyl Succinate.

Removal of p-TSC and its replacement with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, 49 × 210 mm column (General Electric, USA, filled with a Dowex 1x8 sorbent in acetate form). A solution of tosylate ethyl ester is applied per column, elute with a 0.05 M solution of acetic acid at a speed of 0.5 cm / min, Detection is carried out at a wavelength of 220 nm, fractions are taken, analyzed by HPLC, combined based on the results of analysis to obtain creatiglycine et acetate acetate with a total content of unidentified impurities of not more than 1%, creatinine - not more than 1%, creatine - not more than 1%. Succinic acid (1.89 g, 16 mmol) was added to the combined fractions, stirred for 20 minutes until complete dissolution ( pH 4.3), the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. 100 ml of isopropyl alcohol is added to the residue, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 50 ml of methyl ethyl ketone are added, stirred for 3 hours filter cake tion, washed 3 times with 25 ml of methyl ethyl ketone and dried in vacuo at 40 ° C. 20 ml of absolute methanol are added to the precipitate, stirred for 20 minutes, 40 ml of absolute ethanol are added, i.e. dissolve the creatyl glycine ethyl ester succinate in a mixture of methyl and ethyl alcohol (20:40), the insoluble residue is separated by filtration, washed with absolute ethanol on the filter (2 × 20 ml), the washing liquids are combined with the mother liquor. 80 ml (80%) of the solvent are distilled off from the solution under reduced pressure and a temperature of 40 ° C, 30 ml of acetone are added, the mixture is kept for 2 hours at room temperature. The precipitate was separated by filtration, washed (2 × 20 ml) with acetone, dried in vacuum at a temperature of 40 ° C.

Yield of creatyl glycine ethyl succinate ethyl ester C ₈ H ₁₆ N ₄ O ₃ * C ₄ H ₆ O ₄ 5.34 g (38%). The main substance content is 98%, sulfate ash - 0.02%, chloride ions - traces, p-TSC - 3 ppm, impurities: creatinine - 1.0%, creatine - 0.4%, maximum single unidentified impurity - 0 , 1%, the amount of unidentified impurities is 0.3%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.27 (3H, t, 7.17 Hz, CH ₂ CH ₃ ), 2.56 (4H, s, (CH ₂ COOH) ₂ ), 3.08 (3H, s, NCH ₃ ), 4.07 (2H, s, N (CH ₃ ) CH ₂ CO), 4.20-4.25 (4H, m, NHCH ₂ CO and CH ₂ CH ₃ ). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 15.90, 33.56, 39.67, 44.09, 55.325, 65.29, 160.50, 172.58, 174.17 , 181.85.

19.2. Obtaining creatiglycine ethyl ester of succinate according to the method described in the prototype [RU 2428414, 2011].

Stage A. Obtaining creatylglycine tosylate ethyl ester.

Creatine monohydrate (5.69 g, 38 mmol), p-TSC (15.23 g, 80 mmol) are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, and DMF (30 ml), stirred for 10 min, a solution of glycine ethyl ester hydrochloride (5.32 g, 38 mmol) in 15 ml of DMF was added, stirred at room temperature for 5 min, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) is added sequentially, then, over 30 minutes, N-methylmorpholine (8.48 g, 83.9 mmol) was added dropwise. The mixture is stirred at a temperature of (-) 10 ° C for 1 h, the temperature is brought to room temperature. The precipitate was filtered off from the reaction mixture. In the mother liquor, the content of tosylate ethyl ester of tosylate and chloride ions is determined. The yield of tosylate ethyl ester creatyl glycine is 51% mol., The amount of chloride ions is 35.1 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 50 ° C until the end of the run. The residue is dissolved in 160 ml of chloroform, left for 10 hours at a temperature of (-) 10 ° C. The solution is filtered off, extracted with water 3 times in 100 ml. The aqueous extracts are combined, extracted 2 times with chloroform, the solvent is distilled off from the aqueous extract under reduced pressure and a temperature of 40 ° C to a volume of 50 ml. A solution of creatyl glycine ethyl tosylate is obtained.

Stage B. Obtaining Creatyl Glycine Ethyl Succinate.

Removing p-TSC and replacing it with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, 30 × 250 mm column filled with Dowex 1x8 sorbent in acetate form). A solution of tosylate ethyl ester creatyl glycine is applied to the column, eluted with water at a rate of 0.3 cm / min, controlling the pH of the eluate. Fractions with pH = 7 (water pH 5) are collected, analyzed by HPLC, combined, succinic acid (1.89 g, 16 mmol) is added, stirred until complete dissolution, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. The residue is crystallized from 85 ml of acetonitrile containing 0.5% water at a temperature of (-) 10 ° C for 20 hours. The precipitate of creatyl glycine ethyl ester succinate is filtered off, washed with cold acetonitrile, diethyl ether and dried.

Yield of creatyl glycine ethyl ester of succinate C ₈ H ₁₆ N ₄ O ₃ * C ₄ H ₆ O ₄ 3.36 g (26%). The content of the main substance is 97.5%, sulfate ash - 0.02%, chloride ions - 0.4 mMEq / g, p-TSA - 145 ppm, impurities: creatinine - 1.8%, creatine - 0.4% ; the maximum single unidentified impurity is 0.4%, the sum of unidentified impurities is 0.8%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.27 (3H, t, 7.17 Hz, CH ₂ CH ₃ ), 2.56 (4H, s, (CH ₂ COOH) ₂ ), 3.07 (3H, s, NCH ₃ ), 4.07 (2H, s, N (CH ₃ ) CH ₂ CO), 4.20-4.25 (4H, m, NHCH ₂ CO and CH ₂ CH ₃ ). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 15.90, 33.56, 39.66, 44.09, 55.325, 65.29, 160.51, 172.58, 174.17, 181 , 85.

Example 20. Obtaining Creatylglycine Ethyl Dihydrogen Phosphate

20.1 Obtaining creatylglycine ethyl dihydrogen phosphate according to the present method.

Step A. Preparation of Tosylate Ethyl Creatyl Glycine

Creatine monohydrate (5.69 g, 38 mmol), p-TSC (15.23 g, 80 mmol) are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, and DMF (30 ml), stirred for 10 min at room temperature, a solution of glycine ethyl ester hydrochloride (5.32 g, 38 mmol) in 15 ml of DMF was added, stirred at room temperature for 5 min, cooled (ice - salt) to a temperature of (-) 15 ° С, isobutyl chloroform is added simultaneously, over 30 minutes at (6.25 g, 45.8 mmol) and N-methylmorpholine (8.48 g, 83.9 mmol) are stirred at (-) 10 ° C for 1.5 hours. The precipitate is filtered off, washed on a filter chilled to a temperature of 0 ° C DMF (2 × 5 ml). Wash liquids and mother liquor are combined. The solution determines the content of creatinglycine tosylate ethyl ester and chloride ions. The yield of tosylate ethyl ester creatyl glycine is 70% mol., The amount of chloride ions is 29.7 mMEq. Sodium acetate trihydrate (4.04 g, 29.7 mmol) was added to the solution, stirred for 1 h, 50 ml (91%) of DMF were distilled off under reduced pressure and a temperature of 45 ° C. 40 ml of acetone are added to the residue, stirred for 20 minutes, 4 g of celite are added, the suspension is stirred for 20 minutes. 10 g of celite are placed on the filter, washed with 40 ml of acetone, the washing liquid is disposed of. The suspension is filtered through celite, the precipitate is washed with 25 ml of acetone. Wash liquids and mother liquor are combined. The content of chloride ions in the solution is 5.8 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 45 ° C until the end of the run. The residue is dissolved in 20 ml of a 0.05 M acetic acid solution, and a solution of tosylate ethyl ester creatyl glycine is obtained.

Stage B. Obtaining creatylglycine dihydrogen phosphate ethyl ester.

Removal of p-TSC and its replacement with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, 49 × 210 mm column (General Electric, USA, filled with a Dowex 1x8 sorbent in acetate form). A solution of tosylate ethyl ester is applied per column, elute with a 0.05 M solution of acetic acid, the elution speed is 0.55 cm / min Detection is carried out at a wavelength of 220 nm, fractions are collected, analyzed by HPLC, combined on the basis of analysis results to obtain creatine lglycine ethyl acetate with a total content of unidentified impurities of not more than 1%, creatinine - not more than 1%, creatine - not more than 1% 85% orthophosphoric acid (1.84 g, 16 mmol) was added to the combined fractions to a pH of 4.0 the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 100 ml of isopropyl alcohol is added to the residue, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 50 ml of tetrahydrofuran are added, stirred for 3 hours, the precipitate is separated by filtration, washed 3 times 25 ml of tetrahydrofuran, dried in vacuum at a temperature of 40 ° C. 40 ml of methanol are added to the precipitate, stirred for 20 minutes, the insoluble residue is separated by filtration, washed with methanol on a filter (2 × 5 ml), the washing liquids are combined with the mother liquor. 40 ml (80%) of methanol are distilled off from the combined mother liquor under reduced pressure and a temperature of 40 ° C, 30 ml of methyl ethyl ketone are added, and the mixture is kept for 2 hours at room temperature. The precipitate was separated by filtration, washed on a filter (2 × 20 ml) with methyl ethyl ketone, and dried in vacuum at a temperature of 40 ° C.

The yield of creatyl glycine ethyl ester dihydrogen phosphate C ₈ H ₁₆ N ₄ O ₃ * H ₃ PO ₄ 4.40 g (35%). The content of the main substance is 98%, sulfate ash - 0.03%, chloride ions - traces, p-TSC - 2 ppm, impurities: creatinine - 1.0%, creatine - 0.3%, maximum single unidentified impurity - 0 , 1%, the amount of unidentified impurities - 0.4% ;. NMR spectrum ^l H (D ₂ O), δ, ppm (J, Hz): 1.29 (3H, t, 7.17 Hz, CH ₂ CH ₃ ), 3.09 (3H, s, NCH ₃ ), 4.08 (2H, s, N (CH ₃ ) CH ₂ CO), 4.20-4.25 (4H, m, NHCH ₂ CO and CH ₂ CH ₃ ). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 15.92, 39.69, 44.07, 55.27, 65.30, 160.51, 172.64, 174.20.

20.2 Obtaining creatylglycine ethyl ester of dihydrogen phosphate according to the method described in the prototype [RU 2428414, 2011].

Step A. Preparation of Tosylate Ethyl Creatyl Glycine

Creatine monohydrate (5.69 g, 38 mmol), p-TSC (15.23 g, 80 mmol) are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, and DMF (30 ml), stirred for 10 min, a solution of glycine ethyl ester hydrochloride (5.32 g, 38 mmol) in 15 ml of DMF was added, stirred at room temperature for 5 min, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) is added sequentially, then over 30 minutes, dropwise added N-methylmorpholine (8.48 g, 83.9 mmol). The mixture is stirred at a temperature of (-) 10 ° C for 1 h, the temperature is brought to room temperature. The precipitate is filtered off. In the mother liquor, the content of tosylate ethyl ester of tosylate and chloride ions is determined. The output of tosylate ethyl ester creatyl glycine is 56% mol., The amount of chloride ions is 35.5 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 50 ° C until the end of the run. The residue is dissolved in 160 ml of chloroform, left for 10 hours at a temperature of (-) 10 ° C. The solution is filtered off, extracted with water 3 times in 100 ml. The aqueous extracts are combined, extracted 2 times with chloroform, the solvent is distilled off from the aqueous extract under reduced pressure and a temperature of 40 ° C to a volume of 50 ml. A solution of creatyl glycine ethyl tosylate is obtained.

Stage B. Obtaining creatylglycine dihydrogen phosphate ethyl ester.

Removal of p-TSC and its replacement with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, column 49 × 210 mm (General Electric, USA, filled with a Dowex 1x8 sorbent in acetate form). A solution of tosylate ethyl glycine is applied per column, elute with a speed of 0.3 cm / min with water, controlling the pH of the eluate Fractions with pH = 7 (pH 5 water) are collected, analyzed by HPLC, combined, 85% orthophosphoric acid (1.84 g, 16 mmol) is added. stirred, the solvent is distilled off from the solution at reduced pressure and a temperature of 40 ° C until the end of the run, the residue is crystallized from 80 ml of acetonitrile at a temperature of (-) 10 ° C for 20 hours, the precipitate of creatyl glycine ethyl dihydrogen phosphate is filtered off, washed with cold acetonitrile, diethyl ether and dried.

The yield of creatyl glycine ethyl ester dihydrogen phosphate C ₈ H ₁₆ N ₄ O ₃ * H ₃ PO ₄ 3.20 g (27%). The content of the main substance is 97.3%, sulfate ash - 0.03%, chloride ions - 0.5 mMEq / g, p-TSA - 148 ppm, impurities: creatinine - 1.3%, creatine - 0.6% ; the maximum unit unidentified impurity is 0.4%; the sum of unidentified impurities is 0.8% ;. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.29 (3H, t, 7.17 Hz, CH ₂ CH ₃ ), 3.09 (3H, s, NCH ₃ ), 4.08 (2H, s, N (CH ₃ ) CH ₂ CO), 4.20-4.25 (4H, m, NHCH ₂ CO and CH ₂ CH ₃ ). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 15.92, 39.69, 44.07, 55.27, 65.30, 160.51, 172.64, 174.20.

Example 21. Obtaining creatylglycine ethyl ester hydrochloride

21.1 Obtaining creatylglycine ethyl ester hydrochloride according to the claimed method.

Stage A. Obtaining creatylglycine tosylate ethyl ester.

Creatine monohydrate (5.69 g, 38 mmol), p-TSC (15.23 g, 80 mmol) are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, and DMF (30 ml), stirred for 10 min, a solution of glycine ethyl ester hydrochloride (5.32 g, 38 mmol) in 15 ml of DMF was added, stirred at room temperature for 5 min, cooled (ice-salt mixture) to temperature (-) 15 ° С, isobutyl chloroformate (6.25 g, 45.8 mmol) and N-methylm are added simultaneously, over 30 minutes orfolin (8.48 g, 83.9 mmol), stirred at a temperature of (-) 10 ° C for 1.5 hours. The precipitate was filtered off from the cooled mixture, washed on the filter with DMF cooled to 0 ° C (2 × 5 ml) ) Wash liquids and mother liquor are combined. The solution determines the content of creatinglycine tosylate ethyl ester and chloride ions. The yield of tosylate ethyl ester creatyl glycine is 72% mol., The amount of chloride ions is 29.2 mMEq. Sodium acetate trihydrate (3.97 g, 29.2 mmol) was added to the solution, stirred for 1 h, 53 ml (92%) of DMF were distilled off under reduced pressure and a temperature of 45 ° C. 40 ml of acetone are added to the residue at room temperature, stirred for 20 minutes, 4 g of celite are added, and stirred for 20 minutes. 10 g of celite are placed on the filter, washed with 40 ml of acetone, the washing liquid is disposed of. The suspension is filtered through celite, the filter cake washed with 25 ml of acetone. Wash liquids and mother liquor are combined. The content of chloride ions in the solution is 5.8 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 45 ° C until the end of the run. The residue is dissolved in 20 ml of a 0.05 M acetic acid solution, and a solution of tosylate ethyl ester creatyl glycine is obtained.

Stage B. Obtaining creatylglycine ethyl ester hydrochloride.

Removal of p-TSC and its replacement with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, column 49 × 210 mm (General Electric, USA, filled with a Dowex 1x8 sorbent in acetate form). A solution of tosylate ethyl glycine is applied per column, elute with 0.05 M acetic acid solution, elution speed 0.7 cm / min Detection is carried out at a wavelength of 220 nm, fractions are collected, analyzed by HPLC, and the fractions are combined based on the results of analysis to obtain I am creatyl glycine ethyl acetate with a total content of unidentified impurities of not more than 1%, creatinine - not more than 1%, creatine - not more than 1%. To the combined fractions add 1 M hydrochloric acid solution (16 ml, 16 mmol) to pH 3.6 the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 100 ml of isopropyl alcohol is added to the residue, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 50 ml of acetonitrile are added, the mixture is stirred for 3 hours, the precipitate is separated by filtration, rinse 3 times with 25 ml of acetonitrile and dried in vacuo at 40 ° C. 140 ml of absolute ethanol are added to the precipitate, stirred for 20 minutes, the insoluble residue is separated by filtration, washed with absolute ethanol on the filter (2 × 20 ml), the washing liquids are combined with the mother liquor. 128 ml (78%) of ethanol are distilled off from the solution under reduced pressure and a temperature of 40 ° C, 30 ml of tetrahydrofuran are added, the mixture is kept for 2 hours at room temperature. The precipitate was separated by filtration, washed on the filter (2 × 20 ml) with tetrahydrofuran, dried in vacuum at a temperature of 40 ° C.

Yield of creatyl glycine ethyl ester of hydrochloride C ₈ H ₁₆ N ₄ O ₃ * HCl 3.14 g (34%). The content of the main substance is 98.1%, sulfate ash - 0.03%, p-TSC - 4 ppm, impurities: creatinine - 1.0%, creatine - 0.2%, maximum single unidentified impurity - 0.1%, the amount of unidentified impurities is 0.4%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.27 (3H, t, 7.17 Hz, CH ₂ CH ₃ ), 3.09 (3H, s, NCH ₃ ), 4.08 (2H, s, N (CH ₃ ) CH ₂ CO), 4.20-4.25 (4H, m, NHCH ₂ CO and CH ₂ CH ₃ ). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 15.91, 39.70, 44.08, 55.27, 65.30, 160.51, 172.64, 174.20.

21.2 Obtaining creatylglycine ethyl ester hydrochloride according to the method described in the prototype [RU 2428414, 2011].

Step A. Preparation of Tosylate Ethyl Creatyl Glycine

Creatine monohydrate (5.69 g, 38 mmol), p-TSC (15.23 g, 80 mmol) are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, and DMF (30 ml), stirred for 10 min, a solution of glycine ethyl ester hydrochloride (5.32 g, 38 mmol) in 15 ml of DMF was added, stirred at room temperature for 5 min, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) is added sequentially, then, over 30 minutes, N-methylmorpholine (8.48 g, 83.9 mmol) was added dropwise. The mixture is stirred at a temperature of (-) 10 ° C for 1 h, the temperature is brought to room temperature. The precipitate is filtered off. In the mother liquor, the content of tosylate ethyl ester of tosylate and chloride ions is determined. The yield of tosylate ethyl ester creatyl glycine is 52% mol., The amount of chloride ions is 35.2 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 50 ° C until the end of the run. The residue is dissolved in 160 ml of chloroform, left for 10 hours at a temperature of (-) 10 ° C. The solution is filtered off, extracted with water 3 times in 100 ml. The aqueous extracts are combined, extracted 2 times with chloroform, the solvent is distilled off from the aqueous extract under reduced pressure and a temperature of 40 ° C to a volume of 50 ml. A solution of creatyl glycine ethyl tosylate is obtained.

Stage B. Obtaining creatylglycine ethyl ester hydrochloride.

Removal of p-TSC and its replacement with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Buchi Sepacore chromatographic system, column 49 × 210 mm (General Electric, USA, filled with a Dowex 1x8 sorbent in acetate form). on a column, elute with water at a speed of 0.55 cm / min, controlling the pH of the eluate. the solvent is distilled off from the solution under reduced at a pressure and a temperature of 40 ° C until the end of the run, the residue is crystallized from 55 ml of acetonitrile at a temperature of (-) 10 ° C for 20 hours.

The yield of creatyl glycine ethyl ester of hydrochloride C ₈ H ₁₆ N ₄ O ₃ * HCl 2.31 g (25%). The content of the main substance is 97.1%, sulfate ash - 0.04%, p-TSA - 152 ppm, impurities: creatinine - 1.3%, creatine - 0.7%; the maximum single unidentified impurity is 0.4%, the sum of unidentified impurities is 0.8%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.27 (3H, t, 7.17 Hz, CH ₂ CH ₃ ), 3.09 (3H, s, NCH ₃ ), 4.09 (2H, s, N (CH ₃ ) CH ₂ CO), 4.20-4.25 (4H, m, NHCH ₂ CO and CH ₂ CH ₃ ). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 15.91, 39.71, 44.08, 55.27, 65.30, 160.51, 172.64, 174.20.

Example 22. Obtaining creatylglycine ethyl ester of methanesulfonate

22.1. Obtaining creatyl glycine ethyl ester of methanesulfonate by the present method.

Stage A. Obtaining creatylglycine tosylate ethyl ester.

The process is carried out as described in Example 1.1 to Stage B.

The yield of tosylate ethyl ester creatyl glycine is 79% mol., The amount of chloride ions is 3.3 mMEq.

Stage B. Obtaining Creatyl Glycine Ethanesulfonate Ethyl Ester.

Removing p-TSA and replacing it with formic acid is carried out by ion exchange chromatography using a Buchi Sepacore chromatography system, a 49 × 210 mm column (General Electric, USA, filled with a Dowex 1x8 sorbent in formate form. A solution of tosylate ethyl glycine is applied to column, elute with a 0.025 M solution of formic acid at a speed of 0.35 cm / min Detection is carried out spectrophotometrically at a wavelength of 220 nm, fractions are collected, analyzed by HPLC, the fractions are combined based on the results of analysis for I receive creatine glycine methanesulfonate ethyl ester with a total content of unidentified impurities of not more than 1%, creatinine - not more than 1%, creatine - not more than 1%. Methanesulfonic acid (1.92 g ml, 20 mmol) is added to the combined fractions to pH 3, 5. The solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 100 ml of isopropyl alcohol is added to the residue, the solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 50 ml of acetone are added, the mixture is stirred for 3 hours, the acetone layer is decanted It is added, ethyl acetate is added, it is stirred for 2 hours, the ethyl acetate layer is decanted, the residue is dried in vacuum at 40 ° С, dissolved in 125 ml of absolute ethanol with stirring for 20 minutes, the insoluble residue is filtered off, and the filter is washed with absolute ethanol (2 × 20 ml), washings are combined with the mother liquor. The solvent was distilled off from the solution under reduced pressure and a temperature of 40 ° C, while distilling 130 ml of ethanol, 50 ml of ethyl acetate was added, the mixture was kept for 2 hours at room temperature. The precipitate was separated by filtration, washed with ethyl acetate (2 × 50 ml), dried in vacuum at a temperature of 40 ° C.

Yield of creatyl glycine ethyl ester of methanesulfonate C ₈ H ₁₆ N ₄ O ₃ * CH ₃ SO ₃ H 4.75 g (40%). The content of the main substance is 98.3%, sulfate ash 0.03%, chloride ions - traces, p-TSC - 5 ppm, impurities: creatinine - 1.0%, creatine - 0.3%, the maximum single unidentified impurity - 0.1%, the amount of unidentified impurities - 0.3%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.26 (3H, t, 7.17 Hz, CH ₂ CH ₃ ), 2.79 (3H, s, CH ₃ SO ₃ H), 3.06 (3H, s, NCH ₃ ), 4.06 (2H, s, N (CH ₃ ) CH ₂ CO), 4.20-4.25 (4H, m, NHCH ₂ CO and CH ₂ CH ₃ ). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 15.95, 39.73, 41.13, 44.08, 55.30, 65.33, 160.51, 172.65, 174 , 21. 22.2.

Obtaining creatiglycine ethyl ester of methanesulfonate according to the method described in the prototype [RU 2428414, 2011].

Stage A. Obtaining creatylglycine tosylate ethyl ester.

The process is carried out as described in Example 1.2 to Stage B.

The yield of tosylate ethyl ester creatyl glycine is 52% mol., The total amount of chloride ions is 23.0 mMEq.

Stage B. Obtaining Creatyl Glycine Ethanesulfonate Ethyl Ester.

Removal of p-TSC and its replacement with methanesulfonic acid is carried out by ion exchange chromatography, using the Buchi Sepacore chromatographic system, a column 30 × 250 mm in size, filled with a Dowex 1x8 sorbent in formate form. A solution of tosylate ethyl ester creatyl glycine is applied to the column, eluted with water at a rate of 0.3 cm / min, controlling the pH of the eluate. Fractions with pH = 7 (water pH 5) are collected, analyzed by HPLC, and combined on the basis of the results of analysis to obtain creatiglycine ethyl methanesulfonate with a total content of unidentified impurities of not more than 1%, creatinine - not more than 1%, creatine - not more than 1% . Methanesulfonic acid (1.92 g ml, 20 mmol) was added to the combined fractions. The solvent is distilled off under reduced pressure and a temperature of 40 ° C until the end of the run. 100 ml of isopropyl alcohol was added to the residue, the solvent was distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 50 ml of acetone were added, stirred for 3 hours, the acetone layer was decanted, ethyl acetate was added, stirred for 2 hours, the ethyl acetate layer was decanted, the residue was dried in vacuo at a temperature of 40 ° C, dissolved in 125 ml of absolute ethanol, the insoluble residue was separated by filtration, washed on the filter with absolute ethanol (2 × 20 ml), the washing liquids were combined with the mother liquor. The solvent was distilled off from the solution under reduced pressure and a temperature of 40 ° C, while distilling 130 ml of ethanol, 50 ml of ethyl acetate was added, the mixture was kept for 2 hours at room temperature. The precipitate was separated by filtration, washed with ethyl acetate (2 × 50 ml), dried in vacuum at a temperature of 40 ° C.

Yield of creatyl glycine ethyl methanesulfonate C ₈ H ₁₆ N ₄ O ₃ * CH ₃ SO ₃ H 2.85 g (40%). The content of the main substance is 97.9%, sulfate ash - 0.05%, chloride ions - 0.19 mMEq / g, p-TSC - 55 ppm, impurities: creatinine - 1.0%, creatine - 0.4% , the maximum unit unidentified impurity is 0.5%, the sum of unidentified impurities is 0.5% ;. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.26 (3H, t, 7.17 Hz, CH ₂ CH ₃ ), 2.79 (3H, s, CH ₃ SO ₃ H), 3.06 (3H, s, NCH ₃ ), 4.06 (2H, s, N (CH ₃ ) CH ₂ CO), 4.20-4.25 (4H, m, NHCH ₂ CO and CH ₂ CH ₃ ). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 15.95, 39.73, 41.13, 44.08, 55.30, 65.33, 160.51, 172.65, 174 , 21.

Example 23. Obtaining creatylglycine isopropyl ether acetate

23.1 Obtaining creatylglycine isopropyl ether acetate according to the present method.

Step A. Preparation of Tosylate Isopropyl Ester Creatyl Glycine

Creatine monohydrate (5.69 g, 38 mmol), p-TSC (15.23 g, 80 mmol) are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, and DMF (30 ml). The suspension is stirred for 10 minutes, a solution of glycine isopropyl ether hydrochloride (5.84 g, 38 mmol) in 15 ml of DMF is added. The mixture was stirred at room temperature for 5 minutes, then cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) was added simultaneously over 30 minutes and triethylamine (8.48 g, 83.9 mmol), the mixture was stirred at (-) 10 ° C for 1.5 h. The precipitate was filtered off, washed with DMF (2 × 5 ml) cooled to 0 ° C. Wash liquids and mother liquor are combined. The solution determines the content of the basic substance, the amount of chloride ions. The yield of tosylate isopropyl ester creatyl glycine is 75% mol., The amount of chloride ions is 30.1 mMeq. Sodium acetate trihydrate (4.1 g, 29.0 mmol) was added to the solution, stirred for 1 h, 50 ml (91%) of DMF were distilled off under reduced pressure and a temperature of 45 ° C. 40 ml of acetone are added to the residue at room temperature, stirred for 20 minutes, 4 g of celite are added, and stirred for 20 minutes. 5 g of celite are placed on the filter, washed with 20 ml of acetone, the washing liquid is disposed of. The suspension is filtered through celite, the filter cake washed with 25 ml of acetone. Wash liquids and mother liquor are combined. The content of chloride ions in the solution is 5.7 mMEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 45 ° C until the end of the run. The residue is dissolved in 20 ml of a 0.05 M solution of acetic acid, and a solution of tosylate isopropyl ether is obtained.

Stage B. Obtaining creatylglycine isopropyl ether acetate.

Removing p-TSC and replacing it with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Sepaore Buchi chromatographic system, 49 × 210 mm column (General Electric, USA, filled with Amberlite IRA-67 sorbent in acetate form). Isopropyl ether creatyl glycine solution the tosylate is applied to the column, eluted with a 0.05 M solution of acetic acid at a speed of 0.3 cm / min, Detection is carried out at a wavelength of 220 nm, fractions are collected, analyzed by HPLC, and the fractions are combined based on the analysis results to obtain isopropyl acetate acetate glycine with a total content of unidentified impurities of not more than 1%, creatinine not more than 1%, creatine not more than 1%, acetic acid is added to a pH of 4.4, the solvent is removed from the combined fractions under reduced pressure and a temperature of 40 ° C 100 ml of isopropyl alcohol are added to the residue, the solvent is removed from the suspension under reduced pressure and a temperature of 40 ° C until the end of the run, 50 ml of acetone are added, the mixture is stirred for 2 hours, the precipitate is filtered off and washed with acetone (3 × 25 ml), dried in vacuum at a temperature of 40 ° С, dissolved in 125 ml of absolute ethanol with stirring for 20 minutes, the insoluble residue was separated by filtration, washed on the filter with absolute ethanol (2 × 20 ml), the washing liquids were combined with the mother liquor. 135 ml (80%) of ethanol are distilled off from the solution under reduced pressure and a temperature of 40 ° C, 50 ml of ethyl acetate are added, the mixture is kept for 2 hours at room temperature. The precipitate was separated by filtration, washed with ethyl acetate (2 × 50 ml), dried in vacuum at a temperature of 40 ° C.

Yield of creatyl glycine isopropyl ether acetate C ₉ H ₁₈ N ₄ O ₃ * C ₂ H ₄ O ₂ 4.86 g (44%). The content of the main substance is 98.1%, extraneous impurities: creatinine - 0.7%, creatine - 0.5%, the maximum single unidentified impurity - 0.1%, the amount of unidentified impurities - 0.5%; sulfate ash - 0.04%, chloride ions - traces, p-TSK -5 ppm. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.26 (6H, d, 6.22 Hz, CH (CH ₃ ) ₂ ), 1.90 s (3H, s, CH ₃ COOH), 3.08 s (3H, s, NCH ₃ ), 4.02 (2H, s, N (CH ₃ ) CH ₂ CO), 4.24 (2H, s, NHCH ₂ CO), 5.05 (1H, m, CH (CH ₃ ) ₂ ) . ¹³ C NMR spectrum (D ₂ O), δ, ppm: 23.49, 25.96, 39.72, 44.36, 55.29, 73.64, 160.54, 172.62, 173 69, 184.06.

23.2 Obtaining creatylglycine isopropyl ether acetate according to the method described in the prototype [RU 2428414, 2011]

Step A. Preparation of Tosylate Isopropyl Ester Creatyl Glycine

Creatine monohydrate (5.69 g, 38 mmol), p-TSC (15.23 g, 80 mmol) are placed in a 100 ml three-necked flask equipped with a thermometer, two dropping funnels, an air cooler with a calcium chloride tube mounted on a magnetic stirrer, and DMF (30 ml). The mixture is stirred for 10 minutes, a solution of glycine isopropyl ether hydrochloride (5.84 g, 38 mmol) in 15 ml of DMF is added, stirred at room temperature for 10 minutes, cooled with a bath (ice-salt mixture) to a temperature of (-) 15 ° C, isobutyl chloroformate (6.25 g, 45.8 mmol) was successively added, then triethylamine (8.48 g, 83.9 mmol) was added dropwise over 30 minutes. The reaction mixture was stirred at a temperature of (-) 10 ° C for 1 h, the temperature was brought to room temperature. The precipitate is filtered off. In the mother liquor, the content of creatine glycine isopropyl ester of tosylate and with chloride ions is determined. The yield of tosylate isopropyl ester creatyl glycine is 45% mol., The amount of chloride ions is 37.1 mMEq. The solvent is distilled off from the solution under reduced pressure at a temperature of 50 ° C until the end of the run. The residue is dissolved in 160 ml of chloroform, left for 10 hours at a temperature of (-) 10 ° C. The solution is filtered off, extracted with water 3 times in 100 ml. The aqueous extracts are combined, extracted 2 times with chloroform, the solvent is distilled off from the aqueous extract under reduced pressure and a temperature of 40 ° C to a volume of 50 ml. A solution of tosylate isopropyl ester creatyl glycine is obtained.

Stage B. Obtaining creatylglycine isopropyl ether acetate.

Removing p-TSC and replacing it with a pharmaceutically acceptable acid is carried out by ion exchange chromatography (Sepaore Buchi chromatographic system, 49 × 210 mm column (General Electric, USA, filled with Amberlite IRA-67 sorbent in acetate form). Isopropyl ether creatyl glycine solution the tosylate is applied to the column, eluted with water at a speed of 0.3 cm / min, controlling the pH of the eluate. Fractions with pH = 7 (water pH 5) are collected, analyzed by HPLC, combined, acetic acid is added, the solvent is distilled off under reduced pressure and temperature 40 ° C d The residue is crystallized from 20 ml of acetonitrile at a temperature of (-) 10 ° C for 20 hours. The precipitate of creatylglycine isopropyl ether acetate is filtered off, washed with cold acetonitrile, diethyl ether and dried.

The yield of creatylglycine isopropyl ether acetate C ₉ H ₁₈ N ₄ O ₃ * C ₂ H ₄ O ₂ 2.22 g (20%). The content of the main substance is 97.3%, impurities: creatinine - 1.5%, creatine - 0.4%, the maximum single unidentified impurity - 0.2%, the amount of unidentified impurities - 0.5%; sulfate ash - 0.04%, chloride ions - 0.45 mMEq / g, p-TSA - 155 ppm. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.25 (6H, d, 6.22 Hz, CH (CH ₃ ) ₂ ), 1.90 s (3H, s, CH ₃ COOH), 3.07 s (3H, s, NCH ₃ ), 4.01 (2H, s, N (CH ₃ ) CH ₂ CO), 4.23 (2H, s, NHCH ₂ CO), 5.05 (1H, m, CH (CH ₃ ) ₂ ) . ¹³ C NMR spectrum (D ₂ O), δ, ppm: 23.49, 25.96, 39.72, 44.38, 55.29, 73.63, 160.55, 172.62, 173 69, 184.06.

Example 24. Obtaining creatylglycine ethyl acetate in accordance with the present method (industrial process)

Stage A. Obtaining creatylglycine tosylate ethyl ester.

Anhydrous creatine (944.1 g, 7.2 mol), p-TSC (2881.8 g, 15.15 mol) and DMF are placed in a 25 L reactor equipped with a thermocouple, a mechanical stirrer, a cooling system and two dropping funnels. (5.7 L). The suspension is stirred for 20 minutes at room temperature until the substances are almost completely dissolved, a solution of glycine ethyl ester hydrochloride (1005.0 g, 7.2 mol) in 3.9 L of DMF is added. The reaction mixture was stirred at room temperature for 30 minutes, cooled to (-) 15 ° C, and isobutyl chloroformate (1184.1 g, 8.67 mol) and N-methylmorpholine (1608) were added dropwise simultaneously over 1 hour. 3 g, 15.90 mol). The reaction mixture was stirred at a temperature of (-) 10 ° C for 1.5 hours. The precipitate was filtered off from the cooled reaction mixture and washed with DMF (2 × 0.75 L) cooled to 0 ° C. Wash liquids and mother liquor are combined. The target product is determined in the solution - the content of tosylate ethyl ester creatyl glycine, the content of chloride ions. The yield of tosylate ethyl ester creatyl glycine is 75% mole., The total amount of chloride ions is 4.5 MEq. Sodium acetate trihydrate (612.3 g, 4.5 mol) was added to the solution, stirred for 1 h, 29.7 L (89%) of DMF were distilled off under reduced pressure and a temperature of 45 ° C. 7.5 L of acetone is added to the residue at room temperature, stirred for 20 minutes, 300 g of celite are added, and stirred for 20 minutes. 1050 g of celite are placed on the filter, washed with 4.5 l of acetone, the washing liquid is disposed of. The suspension is filtered through celite, the filter cake is washed with 3 L of acetone. Wash liquids and mother liquor are combined. The content of chloride ions in the solution is 0.87 MEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 45 ° C. The residue was dissolved in 7.5 L of a 0.05 M solution of acetic acid. A solution of creatyl glycine ethyl tosylate is obtained.

Stage B. Obtaining creatyl glycine ethyl acetate.

Removing p-TSC and replacing it with a pharmaceutically acceptable acid is carried out by ion exchange chromatography using a Bio Rad - Skid 00 chromatographic platform, a column with a diameter of 448 mm and a Dowex 1x8 sorbent layer in the acetate form 400 mm in height. A solution of tosylate ethyl ester creatyl glycine is applied to the column, eluting with a 0.05 M solution of acetic acid at a rate of 0.2 cm / min. Detection is carried out spectrophotometrically at 220 nm and conductometric. Fractions were collected, analyzed by HPLC, combined on the basis of the results of analysis to obtain ethyl acetate ethyl acetate with a total content of unidentified impurities of not more than 1%, creatinine not more than 1%, creatine not more than 1%, acetic acid was added to pH 4.3 . The solvent is distilled off from the solution under reduced pressure and a temperature of 40 ° C until the end of the run. 3 L of isopropyl alcohol was added to the residue, the solvent was distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 5 L of acetone was added, stirred for 3 hours, the precipitate was filtered off, washed 2 times with 2 L of acetone, dried in vacuum at 40 ° C. FROM. The precipitate was dissolved in 20 L of absolute ethanol, the insoluble residue was separated by filtration, washed with absolute ethanol on the filter (1 L), the washing liquids were combined with the mother liquor. 15.4 L (73%) of ethanol is distilled off from the solution under reduced pressure and a temperature of 40 ° C, 5 L of ethyl acetate is added, the mixture is kept for 2 hours at room temperature. The precipitate was filtered off, washed with 3 L of ethyl acetate, and dried in vacuo at a temperature of 40 ° C.

Yield of creatyl glycine ethyl acetate C ₈ H ₁₆ N ₄ O ₃ * C ₂ H ₄ O ₂ 740.3 g (37%). The content of the main substance is 98.0%, sulfate ash - 0.02%, chloride ions - traces, p-TSC - 3 ppm, impurities: creatinine - 1.0%, creatine - 0.2%, the maximum single unidentified impurity - 0.3%, the amount of unidentified impurities - 0.7%. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.26 (3H, t, 7.17 Hz, CH ₂ CH ₃ ), 1.90 (3H, s, CH ₃ COOH), 3.07 (3H, s, NCH ₃ ), 4.06 (2H, s, N (CH ₃ ) CH ₂ CO); 4.20-4.25 (4H, m, NHCH ₂ CO and CH ₂ CH ₃ ). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 15.96, 25.98, 39.72, 44.09, 55.30, 65.34, 160.55, 172.69, 174 , 24, 184.09.

Example 25. Obtaining creatylglycine ethyl ester of fumarate according to the present method (industrial process)

Stage A. Obtaining creatylglycine tosylate ethyl ester.

Anhydrous creatine (944.1 g, 7.2 mol), p-TSC (2881.8 g, 15.15 mol) and DMF are placed in a 25 L reactor equipped with a thermocouple, a mechanical stirrer, a cooling system and two dropping funnels. (5.7 L). The suspension is stirred for 20 minutes until the substances are almost completely dissolved, a solution of glycine ethyl ester hydrochloride (1005.0 g, 7.2 mol) in 3.9 L of DMF is added. The reaction mixture was stirred at room temperature for 30 minutes, cooled to (-) 15 ° C, and isobutyl chloroformate (1184.1 g, 8.67 mol) and N-methylmorpholine (1608.3 g) were added simultaneously over 1 h. 15.90 mol). The reaction mixture was stirred at a temperature of (-) 10 ° C for 1.5 hours. The precipitate was filtered off from the cooled reaction mixture and washed with DMF (2 × 0.75 L) cooled to 0 ° C. Wash liquids and mother liquor are combined. The solution determines the content of creatyl glycine ethyl ester of tosylate, chloride ions. The yield of tosylate ethyl ester creatyl glycine was 74% mol., The amount of chloride ions was 4.44 MEq. Sodium acetate trihydrate (603.9 g, 4.44 mol) was added to the solution, stirred for 1 h, 29.7 L (89%) of DMF were distilled off under reduced pressure and a temperature of 45 ° С, 7.5 L was added to the residue. acetone at room temperature, stirred for 20 minutes, 300 g of celite were added, stirred for 20 minutes. 1050 g of celite are placed on the filter, washed with 4.5 l of acetone, the washing liquid is disposed of. The suspension is filtered through celite, the precipitate is washed with 3 l of acetone. Wash liquids and mother liquor are combined. The content of chloride ions in the solution is 0.84 MEq. The solvent is distilled off from the solution under reduced pressure and a temperature of 45 ° C, the residue is dissolved in 7.5 l of a 0.05 M acetic acid solution. A solution of creatyl glycine ethyl tosylate is obtained.

Stage B. Obtaining creatylglycine ethyl ester fumarate.

Removing p-TSC and replacing it with a pharmaceutically acceptable acid is carried out by ion exchange chromatography using a Bio Rad - Skid 00 chromatographic platform, a column with a diameter of 448 mm and a Dowex 1x8 sorbent layer height in the acetate form 400 mm. A solution of tosylate ethyl ester creatyl glycine is applied to the column, eluting with a 0.05 M solution of acetic acid at a rate of 0.2 cm / min. Detection is carried out spectrophotometrically at 220 nm and conductometric. Fractions were collected, analyzed by HPLC, and combined on the basis of the analysis results to obtain creatyl glycine ethyl acetate with a total content of unidentified impurities of not more than 1%, creatinine - not more than 1%, creatine - not more than 1%. Fumaric acid (288.9 g, 2.49 mol) was added to the combined fractions, stirred for 20 minutes until dissolved (pH 4.1). The solvent is distilled off from the solution under reduced pressure and a temperature of 40 ° C until the end of the run. 3 L of isopropyl alcohol was added to the residue, the solvent was distilled off under reduced pressure and a temperature of 40 ° C until the end of the run, 5 L of acetone was added, stirred for 3 hours, the precipitate was filtered off, washed 2 times with 2 L of acetone, dried in vacuum at 40 ° C. FROM. The precipitate was dissolved in 20 L of absolute ethanol, the residue was separated by filtration, washed with absolute ethanol (1 L), and the washings were combined with the mother liquor. 15.4 L (73%) of ethanol was distilled off from the solution under reduced pressure and a temperature of 40 ° C, 5 L of ethyl acetate was added, and it was kept at room temperature for 2 hours. The precipitate was separated by filtration, washed with 3 L of ethyl acetate, dried in vacuum at a temperature of 40 ° C.

The yield of creatyl glycine ethyl ester of the fumarate C ₈ H ₁₆ N ₄ O ₃ * C ₄ H ₄ O ₄ was 916.0 g (38%). The content of the main substance is 98.0%, sulfate ash - 0.03%, chloride ions - traces, p-TSA - 3 ppm, impurities: creatinine - 1.0%, creatine - 0.6%, maximum single unidentified impurity - 0.1%, the amount of unidentified impurities - 0.4% ;. ¹ H NMR spectrum (D ₂ O), δ, ppm (J, Hz): 1.16 (3H, t, 7.17 Hz, CH ₂ CH ₃ ), 2.96 (3H, s, NCH ₃ ), 3.96 (2H, s, N (CH ₃ ) CH ₂ CO), 4.20-4.25 (4H, m, NHCH ₂ CO and CH ₂ CH ₃ ), 6.85 (2H, s, HO ₂ CCH = CHCO ₂ H). ¹³ C NMR spectrum (D ₂ O), δ, ppm: 13.23, 37.00, 41.37, 52.59, 62.64, 134.71, 157.82, 170.00, 171 52, 171.69.

Summarized information about the results of the synthesis of drugs according to the claimed invention in comparison with the closest analogue are shown in table 1.

The above examples show that the inventive method for producing creatine amides can increase the yield of the target product by 7-38%, to increase the purity of the product, as well as to obtain creatine amides that cannot be synthesized by the prototype.

Claims (22)

1. The method of producing creatine amides of General formula

where R is the amino acid residue of an amino acid; Y is OR ¹ , NR ² R ³ , where R ¹ is H, CH ₂ Ph, C ₁ -C ₃ alkyl; R ² , R ³ —H, C ₁ -C ₄ alkyl; X is a low molecular weight C ₁ -C ₄ organic acid or mineral acid, or water, consisting in treating creatine with para-toluenesulfonic acid in dimethylformamide followed by interaction of the resulting complex with amino acid derivatives in the presence of a condensing agent and a base, characterized in that as a condensing agent formic acid monoester is introduced, the condensing agent and the base being introduced simultaneously, followed by the addition of alkali metal acetate to the resulting mixture s, distilling at least 80% dimethylformamide, extraction of the active principle aprotic polar solvent containing at least one ingredient from the group consisting of acetone, tetrahydrofuran, methyl ethyl ketone, and acetonitrile; distilling off the solvent, dissolving the residue in a polar protic solvent containing at least one ingredient from the group consisting of water, ethyl and isopropyl alcohol, formic and acetic acids, removing para-toluenesulfonic acid by chromatography on an anion exchange sorbent, distilling off the solvent in the presence of low molecular weight С C ₁ -C ₄ organic acid or inorganic acid and alcohol at a pH less than 4.5 by treatment with a solvent containing at least one ingredient from the group consisting of aij tone, tetrahydrofuran, methyl ethyl ketone, acetonitrile and ethyl acetate; extraction of the active principle with a solvent containing at least one ingredient from the group consisting of ethyl and methyl alcohols; distillation of at least 70% of the solvent; treatment with a solvent containing at least one ingredient from the group consisting of acetone, tetrahydrofuran, methyl ethyl ketone, acetonitrile and ethyl acetate. 2. The method according to claim 1, characterized in that the proteinogenic α-amino acid is used as the amino acid. 3. The method according to claim 1, characterized in that β-alanine is used as the amino acid. 4. The method according to claim 1, characterized in that ω-nitroarginine is used as the amino acid. 5. The method according to claim 1, characterized in that O-isopropyl tyrosine is used as the amino acid. 6. The method according to claim 1, characterized in that O-ethyl tyrosine is used as the amino acid. 7. The method according to claim 1, characterized in that methanesulfonic or acetic, or fumaric, or succinic acid is used as a low molecular weight C ₁ -C ₄ organic acid. 8. The method according to claim 1, characterized in that orthophosphoric acid or hydrochloric acid is used as a mineral acid. 9. The method according to claim 1, characterized in that anhydrous creatine is used as creatine. 10. The method according to claim 1, characterized in that as creatine use creatine monohydrate. 11. The method according to claim 1, characterized in that the derivatives of amino acids are ether, amide or nitro derivatives of proteinogenic α-amino acids. 12. The method according to claim 1, characterized in that as the derivatives of amino acids using ether or amide derivatives of β-alanine. 13. The method according to claim 1, characterized in that N-methylmorpholine is used as the base. 14. The method according to claim 1, characterized in that triethylamine is used as the base. 15. The method according to claim 1, characterized in that isobutyl chloroformate is used as the acid chloride of the formate acid monoester. 16. The method according to claim 1, characterized in that ethyl chloroformate is used as the acid chloride of the formate acid monoester. 17. The method according to claim 1, characterized in that as the alkali metal acetate, sodium acetate or potassium acetate is used. 18. The method according to claim 1, characterized in that as the anion-exchange sorbent use resins based on styrene copolymers or based on acrylic copolymers containing tertiary groups, such as Dowex 1 × 8. 19. The method according to claim 1, characterized in that as the anion exchange sorbent use resins based on styrene copolymers or based on acrylic copolymers containing quaternary ammonium groups, such as Amberlite IRA-67. 20. The method according to claim 1, characterized in that the chromatography is carried out using an aqueous solution of formic or acetic acids or an aqueous-alcoholic solvent as an eluent. 21. The method according to claim 20, characterized in that as a water-alcohol solvent using a mixture of water with isopropyl alcohol or ethyl alcohol. 22. The method according to claim 1, characterized in that isopropyl alcohol or n-butyl alcohol is used as the alcohol.