RU2709496C1 - Method of producing avatrombopag - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to an improved method for preparing avatrombopag or maleate thereof. Compound corresponds to the following structural formula (I), has the properties of a thrombopoietin receptor agonist (aTPO-r) and can be used for treating thrombocytopenia.

(1)

Method comprises adding dropwise Br₂ to solution of 2-acetyl-4-chlorothiophene (1) in diethyl ether, extraction is carried out with ethyl ether of acetic acid (EtOAc), the residue is dissolved in ethyl alcohol, thiourea is added and subjected to boiling. Resulting residue is dissolved in a mixture of EtOAc/hexane, having ratio by volume of 1:1, dropped after cooling to room temperature, filtered precipitate of 4-(4-chlorothiophen-2-yl)thiazole-2-amine (2) is washed with a mixture of EtOAc/hexane and dried in a vacuum at room temperature, is dissolved in dioxane and N-bromosuccinimide is added to obtain 4-(4-chlorothiophen-2-yl)thiazole-4-bromo-2-amine (3), as well as a characteristic impurity of 5-bromo-4-(5-bromo-4-chlorothiophen-2-yl)thiazole-2-amine, which then reacts similarly to (3), dissolving the product in acetonitrile, adding 1-cyclohexylpiperazine and triethylamine, carrying out reaction during boiling. Precipitate of 4-(4-chlorothiophen-2-yl)-5-(4-cyclohexylpiperazin-1-yl)thiazole-2-amine (4) precipitated during cooling is filtered, washed with water, hexane and diethyl ether and dried, the obtained product is added to pyridine solution of 5-chloro-6-(4-ethoxycarbonyl)piperidin-1-yl)nicotinic acid (7), which is obtained by adding to the solution of ethyl-piperidine-4-carboxylate (5) in dimethylsulphoxide 5,6-dichloronicotinic acid (6) and triethylamine, followed by heating to temperature of 150 °C, crystallization, washing with water, hexane, drying at room temperature and dissolving (7) in pyridine. Then cooled solution of mixture (4) and (7) is added with phosphorus oxychloride (POCl₃) so that mixture temperature does not exceed 10 °C is heated to room temperature, crystallization reaction is carried out, obtained crystalline precipitate of ethyl ether of avatrombopag (8) is washed with water and hexane, the powder is dried, dissolved in a mixture of toluene and ethyl acetate, having ratio by volume of 10 to 1, purified by flash chromatography on silica gel, suspended in a mixture of distilled water and tetrahydrofuran, adding sodium hydroxide at room temperature, stirring, diluting with water, adding acetic acid to the obtained solution until pH=5–5.5, stirring at room temperature until crystallization ends and precipitation of avatrombopag (9). Obtained precipitate is filtered and washed with water, a mixture of tetrahydrofuran and water, then with water, hexane and ethyl acetate, and dried with subsequent treatment of the obtained product with maleic acid to obtain maleate of avatrombopag.

EFFECT: method enables to exclude heating during hydrolysis of ether, significantly reduce the characteristic impurity of 5-bromo-4-(5-bromo-4-chlorothiophen-2-yl)thiazole-2-amine to trace amounts and improve purification of the obtained intermediate compounds; yield of the obtained product is 94,5 %, purity is 98 % NMR.

1 cl, 3 ex

Description

The invention relates to the field of chemistry and the pharmaceutical industry and relates to a new method for the synthesis of the substance of avatrombopag in the form of a base or its pharmaceutically acceptable salt.

- состояние, характеризующееся снижением количества тромбоцитов, которое сопровождается повышенной кровоточивостью и проблемами с остановкой кровотечений. Цель лечения нарушения - сведение к минимуму риска геморрагических осложнений путем повышения числа тромбоцитов до безопасного уровня, не вызывая выраженных побочных эффектов, с использованием минимально токсичных вариантов лечения, что особенно важно при длительной терапии пациентов с рефрактерным или рецидивирующим течением заболевания. Основным цитокином, стимулирующим образование тромбоцитов, является тромбопоэтин (ТПО).

- a condition characterized by a decrease in platelet count, which is accompanied by increased bleeding and problems with stopping bleeding. The purpose of treating the disorder is to minimize the risk of hemorrhagic complications by increasing the platelet count to a safe level without causing pronounced side effects using minimally toxic treatment options, which is especially important for long-term treatment of patients with a refractory or recurrent course of the disease. The main cytokine that stimulates platelet formation is thrombopoietin (TPO).

Currently, the use of thrombopoietin receptor agonists (aTPO-p) represents a new approach to the treatment of patients with thrombocytopenia with the ineffectiveness of previous methods. One of these clinically tested agonists is avatrombopag. The chemical name of avatrombopag is: 1- [3-chloro-5 - [[[4- (4-chloro-2-thienyl) -5- (4-cyclohexyl-1-piperazinyl) -2-thiazolyl] amino] carbonyl] -2pyridinyl ] -4-piperidinecarboxylic acid.

Avatrombopag is an orally bioavailable, low molecular weight TPO receptor agonist that stimulates the proliferation and differentiation of megakaryocytes from bone marrow progenitor cells, resulting in increased platelet production. The drug does not compete with TPO for binding to the TPO receptor and has an additive effect with TPO on platelet production. However, when taking the drug, a number of side effects are manifested. Therefore, new methods of obtaining a higher purity substance are required.

A known method of obtaining compounds from international application WO03062233 (A1), publ. 2003-07-31. The document describes methods for the synthesis of derivatives of 2-acylaminothiazole, the general formula of which includes the formula of avatrombopag.

The initial is the scheme:

Further reactions depend on reactive derivatives or a condensing agent. Purification is carried out on silica gel chromatographic columns.

As the closest analogue, the method according to CN 107383000 (A), publ. 2017-11-24. According to this source, the production method includes the following steps: preparing intermediate 1 and intermediate 2 for carrying out an amidation reaction in the presence of a catalyst in an aprotic solvent; adding alkali to the solvent of intermediate A to carry out an alkaline hydrolysis reaction to obtain the desired compound. Intermediate 2 is first synthesized and then reacts with intermediate 1; and the synthetic route of the process is an amplification reaction. An operation such as column chromatography is excluded; the method is suitable for an industrial production process.

The general scheme:

The present invention is to develop synthesis of avatrombopag with a high degree of purity and product yield while minimizing decomposition products.

The problem is solved by the synthesis of Avatrombopag (1- (3-chloro-5 - ((4- (4-chlorothiophen-2-yl) -5- (4-cyclohexylpiperazin-1-yl) thiazol-2-yl) carbomoyl) pyridin-2 -yl) piperidine-4-carboxylic acid (9) according to the following scheme:

The proposed new method for producing avatrombopag consists in adding Br2 dropwise to a solution of 2-acetyl-4-chlorothiophene (1) in diethyl ether, extraction with ethyl acetate, EtOAc, the residue is dissolved in ethyl alcohol, thiourea is added, and subjected By boiling, the obtained residue was dissolved in EtOAc / hexane (1: 1), the precipitated precipitate, after cooling to room temperature, of the filtered precipitate of 4- (4-chlorothiophen-2-yl) thiazol-2-amine (2) was washed with EtOAc / hexane and dried in vacuum at room temperature, dried the cage is dissolved in dioxane, and with the addition of N-bromosuccinimide, 4- (4-chlorothiophen-2-yl) thiazol-4-bromo-2-amine (3) is obtained, it is dissolved in acetonitrile, 1-cyclohexylpiperazine and triethylamine are added, carrying out the reaction upon boiling, the precipitated upon cooling, the precipitate 4- (4-chlorothiophen-2-yl) -5- (4-cyclohexylpiperazin-1-yl) thiazol-2-amine (4) is filtered off, washed with water, hexane and diethyl ether and dried, add the resulting product to a solution of 5-chloro-6- (4- (ethoxycarbonyl) piperidin-1-yl) nicotinic acid (7), previously obtained by adding to a solution of ethyl piperidine-4-carboxylate (5) in 5,6-dichloronicotinic acid dimethyl sulfoxide (6) and triethylamine, followed by heating to 150 ° C, crystallization, washing with water, hexane, drying at room temperature and dissolution (7) in pyridine, then phosphorus oxychloride (POC) is added to the cooled solution of the mixture (4) and (7) so that the temperature of the mixture does not exceed 10 ° C, it is heated to room temperature, a crystallization reaction is carried out, the obtained crystalline precipitate of avatrombopag ethyl ester (8) washed with water and hexane, the powder is dried, dissolved in a mixture of toluene and ethyl acetate, having a volume ratio of 10 to 1, purified by flash chromatography on silica gel, suspended in a mixture of distilled water and tetrahydrofuran, sodium hydroxide is added at room temperature, stirred, diluted with water, acetic acid is added to the resulting solution until pH = 5-5.5 is established, stirred at room temperature until crystallization is complete and avatrombopag precipitate forms (9), the resulting precipitate is filtered off and washed with water , Tetragidrofuran-water mixture, then with water, hexane and ethyl acetate.

If necessary, a pharmaceutically acceptable salt of avatrombopag can be obtained further, for example, hydrochloric, nitrate, succinate, glutamate, maleate, etc. It is preferable to obtain avatrombopag maleate salt, which is sold under the trade name Doptelet.

The possibility of carrying out the invention is demonstrated by the following examples:

Example 1. Synthesis of avatrombopag

Stage 1. Synthesis of amine 2

Br ₂ (22 g, 0.138 mol) was slowly added dropwise to a solution of 2-acetyl-4-chlorothiophene 1 (20 g, 0.125 mol) in diethyl ether (150 ml) for 20 minutes at 0 ° С. The resulting reaction mixture was stirred at 0 ° C for 2 hours. At the end of the reaction (control: TLC, silica gel, toluene, R _f = 0.7), the mixture was diluted with water (300 ml), stirred for 30 minutes, extracted with ethyl acetate (EtOAc) (3 × 150 ml), washed with water (2 × 100 ml) and saturated sodium chloride solution (2 × 50 ml). The solution was dried over anhydrous sodium sulfate and evaporated. The resulting residue was dissolved in ethanol (150 ml), thiourea (9.5 g, 0.125 mol) was added and boiled for 1 hour. At the end of the reaction (control: TLC, CHCl ₃ / EtOH = 97: 3, R _f = 0.6), the mixture was evaporated. The resulting residue was dissolved in 300 ml of a mixture of EtOAc / hexane (1: 1) and boiled for 1 hour. Then the solution was cooled to room temperature, the precipitate of amine hydrobromide 2 was filtered off, washed with EtOAc / hexane (1: 1) (2 × 50 ml) and dried in vacuum (10 mmHg) at room temperature for 5 hours. A suspension of the obtained product in 500 ml of an aqueous solution of K ₂ CO ₃ (40 g, 0.3 mol) was stirred vigorously for 1 hour at room temperature. Then the precipitate was filtered off, washed with water and dried in air at room temperature for 2 days. Yellowish, crystalline, 21.9 g (81% yield, 95% purity by NMR), mp = 147-150 ° С.

The use of a mixture of hexane and ethyl acetate in washing at this stage improves the purification of the intermediate.

Stage 2. Synthesis of amine 4

To a solution of 4- (4-chlorothiophen-2-yl) thiazol-2-amine 2 (21.6 g, 0.1 mol) in dioxane (100 ml), while cooling to 10 ° C, N-bromosuccinimide (12.4 g, 0.105) was added portionwise. mol) for 1 hour (temperature control inside the reaction medium — temperature no higher than 15 ° С). At the end of the reaction (control: TLC, EtOAc / hexane = 25: 75, R _f = 0.5), the reaction mixture was poured vigorously into 2 liters of cold (10 ° C) distilled water and stirred for about 1 hour until the precipitate crystallized. Then the precipitate was filtered off, washed with water (2 × 200 ml), hexane (2 × 100 ml) and dried in air at room temperature for 2 days. Gray. Crist., ~ 30 g (yield ~ 100%, purity 96 +% by NMR), mp = 143-150 ° С. A characteristic impurity of 5-bromo-4- (5-bromo-4-chlorothiophen-2-yl) thiazol-2-amine is also formed in small amounts at this stage, which then reacts similarly to the target 5-bromo-4- (4 -chlorothiophen-2-yl) thiazol-2-amine.

To a solution of 4- (4-chlorothiophen-2-yl) thiazol-4-bromo-2-amine 3 (29.5 g, 0.1 mol) in acetonitrile (250 ml) was added 1-cyclohexylpiperazine (20.2 g, 0.12 mol) and triethylamine ( 21.5 ml, 0.15 mol) and boiled for 4 hours (control: TLC, EtOAc / hexane = 25: 75, R _f = 0.1), the reaction mixture was poured into 2 L of water and stirred for 30 minutes at room temperature. Then the precipitate was filtered off, washed with water (2 × 200 ml), hexane (2 × 100 ml) and diethyl ether (4 × 75 ml). The resulting product was dried in air at room temperature for 2 days. Gray, crystalline, 28.7 g (80% yield, 95% purity by NMR), mp = 172-174 ° С.

Stage 3. Synthesis of acid 7

To a solution of ethyl piperidine-4-carboxylate 5 (18.8 g, 0.12 mol) in dimethyl sulfoxide (100 ml) was added 5,6-dichloronicotinic acid (19.2 g, 0.1 mol) and triethylamine (25.3 g, 0.25 mol). The mixture was stirred until the reagents were completely dissolved, heated in an oil bath to a temperature of 150 ° C and kept at this temperature for 8 hours (control: TLC, CHCl ₃ / EtOH = 95: 5, R _f = 0.7). Then the reaction mixture was cooled to room temperature, poured into 2 L of water and acetic acid was added to the resulting solution until pH = 4 (approximately 0.25 mol) was established. The resulting suspension was stirred for 2 hours at room temperature until final crystallization. The resulting precipitate was filtered off, washed with water (3 × 500 ml) and hexane (3 × 300 ml). The resulting product was dried in air at room temperature for 2 days. Yellowish, crystalline, 29.7 g (95% yield, 95 + purity by NMR), mp = 131-133 ° С.

Stage 4. Synthesis of ether 8

This stage was carried out as in patent CN 107383000 A. Since the conditions proposed there are optimal. However, the invention proposed another method of purification of the final substance 8, which allows you to get a cleaner product in the next stage.

To a solution of 5-chloro-6- (4- (ethoxycarbonyl) piperidin-1-yl) nicotinic acid 7 (3.15 g, 0.01 mol) in pyridine (30 ml) was added 4- (4-chlorothiophen-2-yl) -5 - (4-cyclohexylpiperazin-1-yl) thiazol-2-amine 4 (3.8 g, 0.01 mol) and cooled to 0 ° C in an ice bath. POCl ₃ (2.25 g, 0.015 mol) was slowly added to the cooled solution with stirring so that the temperature of the mixture did not exceed 10 ° C (about 20 minutes). Then the mixture was warmed to room temperature in 2 hours and kept at room temperature for 2 hours (control: TLC, CHCl ₃ / EtOH = 95: 5, R _f = 0.7). Then the reaction mixture was poured into 500 ml of water. The resulting suspension was stirred for 2 hours at room temperature until final crystallization. The resulting bright red precipitate was filtered off, washed with water (3 × 100 ml) and hexane (2 × 50 ml). The brick-colored powder was dried in air at room temperature for 2 days.

The resulting powder was dissolved in a mixture of toluene and ethyl acetate (10 to 1) and purified by flash chromatography on silica gel using the same mixture as eluent. The fractions containing the product were evaporated on a rotary evaporator at a temperature not exceeding 40 ° C. The resulting red powder (or solid foam) was used further without further purification. 4.7 g (yield 87%, purity -96% by NMR).

Stage 5. Synthesis of avatrombopag 9

This step in the patent CN 107383000 A was carried out with heating, which leads to the formation of a by-product.

According to the invention, the solvent (tetrahydrofuran instead of alcohol) and the purification method (sequential washing with a series of different solvents) were changed. Moreover, the new process does not include heating.

Starting ether 8 (13.4 g, 0.0198 mol) was suspended in a mixture of distilled water (50 ml) and tetrahydrofuran (150 ml), after which NaOH (2.2 g, 0.055 mol) was added to the suspension. Observed complete dissolution. The mixture was stirred at room temperature for 15-20 hours, (control: TLC, CHCl ₃ / EtOH = 95: 5, R _f = 0.2). The mixture was filtered from dust and other foreign inclusions, diluted with water (200 ml) and acetic acid was added to the resulting solution until pH = 5-5.5 was established. The resulting emulsion was stirred for about 2 hours at room temperature until crystallization was complete and a precipitate formed. The resulting precipitate was filtered and washed with water (100 ml), a mixture of tetrahydrofuran-water (1 to 1 2 times 30 ml), water (4 times 100 ml), hexane (2 times 100 ml) and ethyl acetate (3 times 20 ml). The resulting cream-colored crystalline product was dried in air at room temperature for 3 days.

Cream, crystall., 2.75 g (yield 94.5%, purity 98 +% by NMR). T. pl. ~ 265 ° C with decomposition.

As a result of the formation of the characteristic impurity 5-bromo-4- (5-bromo-4-chlorothiophen-2-yl) thiazol-2-amine at stage 2, as a result of all stages of the synthesis, the final substance of avatrombopag (1- (3-chloro-5 - ((4- (4-chlorothiophen-2-yl) -5- (4-cyclohexylpiperazin-1-yl) thiazol-2-yl) carbomoyl) pyridin-2-yl) piperidin-4-carboxylic acid) contains trace amounts characteristic impurity 1- (3-chloro-5 - ((4- (5-bromo-4-chlorothiophen-2-yl) -5- (4-cyclohexylpiperazin-1-yl) thiazol-2-yl) carbomoyl) pyridin- 2-yl) piperidin-4-carboxylic acid.

Example 2. Synthesis of salt avatrombopag

The stages are similar to 1-5 in example 1.

Stage 6. Synthesis of Avatrombopag maleate 10

Stage includes dissolving a mixture of avatrombopag 9 and maleic acid in DMF, precipitation of the salt with methylene chloride.

As a result of the formation at stage 2 of a characteristic impurity of 5-bromo-4- (5-bromo-4-chlorothiophen-2-yl) thiazol-2-amine, as a result of all stages of the synthesis, the final substance of avatrombopag maleate (salts of 1- (3-chloro -5 - ((4- (4-chlorothiophen-2-yl) -5- (4-cyclohexylpiperazin-1-yl) thiazol-2-yl) carbomoyl) pyridin-2-yl) piperidine-4-carboxylic acid with maleic acid) contains trace amounts of a characteristic impurity of the salt 1- (3-chloro-5 - ((4- (5-bromo-4-chlorothiophen-2-yl) -5- (4-cyclohexylpiperazin-1-yl) thiazol-2- il) carbomoyl) pyridin-2-yl) piperidin-4-carboxylic acid with maleic acid.

These decomposition products are unique features for the proposed method and allow to distinguish the final substance of avatrombopag synthesized by the claimed method from similar substances obtained by other methods.

Example 3. Pharmacodynamic studies of the obtained substance for the treatment of mice with idiopathic thrombocytopenic purpura

Tests were performed on healthy BALB / C mice with SPF level (Specefic pathogen Free), weight 18-22 g, males.

Breakdown of animals into groups: control group, avatrombopag group.

Modeling method: blood was taken from the orbital vein of BALB / C mice followed by anticoagulation treatment with EDTA-Na2, and then the platelets were separated and washed. The final platelet suspension solution was mixed uniformly with an equal amount of Freund's complete adjuvant and Freund's incomplete adjuvant, respectively. In the first week, antigen containing Freund's complete adjuvant was subcutaneously injected into the hind paw, back and groin of the guinea pig. Injections were performed only 4 times, with 5 points for each injection and 100 μl for each point. On the 6th week, whole blood not subjected to anticoagulation was taken from guinea pigs and centrifuged for 10 minutes. at 3000 rpm./min., receiving the supernatant, which is the serum of anti-mouse platelets (GP-APS) of guinea pigs. The supernatant was placed in a water bath (56 ° C) for 30 min to inactivate complement, and then the inactivated supernatant was diluted with normal saline, then the antibody titers were tested by diffusion in agar and finally stored in a refrigerator at -20 ° C for later use.

The mice were injected with guinea pig anti-mouse platelet serum (APS) at a dose of 100 μl / 20 g of body weight by intraperitoneal injection in the 1st, 3rd, 5th, 7th, 9th, 11th and 13th experiment day.

Observation of developed subcutaneous purpura:

Class I, slight bleeding is observed at the injection sites, and hemorrhagic spots spread to other places (40% of mice);

Class II, clear bleeding is observed at injection sites, and ecchymosis and petechiae are observed at other sites (35%);

Class III, severe bleeding is observed at the injection sites, a large amount of ecchymosis and petechiae appeared on the skin, and ulceration and blackening was noted on the skin (25%).

Avatrombopag was administered at a dose of 20 mg / kg. Platelet count was measured over 2 weeks.

Platelet count (average) before modeling:

Control - 748.2 × 10 ⁹ / l

Avatrombopag group - 749.0 × 10 ⁹ / l

Platelet count on day 14:

Control - 382.4 × 10 ⁹ / L

The group of avatrombopagus is 582.8 × 10 ⁹ / L.

The platelet count in mice showed that avatrombopag can effectively limit the decrease in platelet count and can be used for thrombopenia.

The substance according to the invention has a minimal manifestation of side effects, in particular there is practically no hyperthermia and swelling.

Claims (1)

A method of producing avatrombopag or its maleate, characterized in that Br _{2 is} added dropwise to a solution of 2-acetyl-4-chlorothiophene (1) in diethyl ether, extraction is carried out with ethyl acetate, EtOAc, the residue is dissolved in ethyl alcohol, thiourea is added subjected to boiling, the resulting residue was dissolved in a mixture of EtOAc / hexane, having a volume ratio of 1: 1, precipitated after cooling to room temperature, the filtered precipitate of 4- (4-chlorothiophen-2-yl) thiazol-2-amine (2) was washed with EtOAc / hexane and dried in vacuo at temperature, dissolved in dioxane and with the addition of N-bromosuccinimide, 4- (4-chlorothiophen-2-yl) thiazol-4-bromo-2-amine (3), as well as a characteristic impurity of 5-bromo-4- (5- bromo-4-chlorothiophen-2-yl) thiazol-2-amine, which then reacts similarly to (3), dissolves the product in acetonitrile, 1-cyclohexylpiperazine and triethylamine are added, carrying out the reaction under reflux, the precipitate is 4- ( 4-chlorothiophen-2-yl) -5- (4-cyclohexylpiperazin-1-yl) thiazol-2-amine (4) is filtered off, washed with water, hexane and diethyl ether and dried, added the resulting product to a solution of 5-chloro-6- (4- (ethoxycarbonyl) piperidin-1-yl) nicotinic acid (7), previously obtained by adding to a solution of ethyl piperidin-4-carboxylate (5) in 5,6- dimethyl sulfoxide dichloronicotinic acid (6) and triethylamine, followed by heating to a temperature of 150 ° C, crystallization, washing with water, hexane, drying at room temperature and dissolving (7) in pyridine, then oxychloride is added to the cooled solution of mixture (4) and (7) phosphorus (POCl ₃₎ so that the temperature did not exceed 10 ° C, heated about room temperature, a crystallization reaction is carried out, the obtained crystalline precipitate of avatrombopag ethyl ester (8) is washed with water and hexane, the powder is dried, dissolved in a mixture of toluene and ethyl acetate, having a volume ratio of 10 to 1, purified by flash chromatography on silica gel, suspended in a mixture of distilled water and tetrahydrofuran, add sodium hydroxide at room temperature, mix, dilute with water, add acetic acid to the resulting solution until pH = 5-5.5, mix at at room temperature until crystallization and precipitation of avatrombopag are completed (9), the resulting precipitate is filtered off and washed with water, a mixture of tetrahydrofuran with water, then repeatedly with water, hexane and ethyl acetate and dried, followed by, if necessary, processing the resulting product with maleic acid to obtain avatrombopag maleate .