阅读说明：本技术 一种阿加曲班杂质g的制备方法 (Preparation method of argatroban impurity G ) 是由 宋更申 陈艾夫 刘兆国 于 2020-12-25 设计创作，主要内容包括：本发明属于医药化合物的合成领域,具体涉及一种阿加曲班杂质G的制备方法,包括阿加曲班盐在精氨酸酶和锰离子的催化作用下进行水解得到阿加曲班杂质G盐的步骤。本发明所述合成方法成功制备得到了纯度＞98％的阿加曲班杂质G,且路线合理,操作方法简单易行,收率高,纯度高。(The invention belongs to the field of synthesis of medical compounds, and particularly relates to a preparation method of argatroban impurity G. The synthesis method successfully prepares the argatroban impurity G with the purity of more than 98 percent, and has the advantages of reasonable route, simple and easy operation method, high yield and high purity.)

1. A preparation method of argatroban impurity G is characterized by comprising the step of hydrolyzing argatroban salt under the catalytic action of arginase and manganese ions to obtain argatroban impurity G salt.

2. The method according to claim 1, wherein the arginase is an immobilized arginase.

3. The process according to claim 1, wherein the argatroban salt is argatroban hydrochloride.

4. The preparation method according to claims 1 to 3, wherein the argatroban salt is prepared by adding an acid to the argatroban solution until the pH of the system is 7 to 8.

5. The method according to claim 2, wherein the mass ratio of argatroban to arginase in the argatroban salt is 1: 0.1 to 0.25.

6. The production method according to claim 1 or 2, wherein the mass ratio of argatroban to the manganese ion in the argatroban salt is 1: 0.01-0.02.

7. A process according to any one of claims 1 to 5, wherein the hydrolysis reaction is carried out at a temperature of from 20 ℃ to 40 ℃, preferably from 35 ℃ to 40 ℃.

8. The method according to any one of claims 1 to 5, wherein the end point of the reaction is monitored by thin layer chromatography.

9. The preparation method according to claim 8, wherein a developing solvent used in the thin layer chromatography is a mixed solution of dichloromethane and methanol at a volume ratio of 1: 4.8-5.2.

10. The method according to any one of claims 1 to 9, further comprising a step of desalting the argatroban impurity G salt by adding ammonium carbonate or ammonium bicarbonate to the argatroban impurity G salt to produce argatroban impurity G.

Technical Field

The invention belongs to the field of synthesis of medicinal compounds, and particularly relates to a preparation method of argatroban impurity G.

Background

Argatroban is a novel antithrombin active substance, is an intravenous injection, is already on the market in many countries, and has good clinical application prospect. Research shows that argatroban is easy to produce decomposition product argatroban impurity G [ 2R,4R) -4-methyl-1- [ N- [ (3-methyl-1, 2,3, 4-tetrahydro-8-quinolyl) sulfonyl ] -L-ornityl ] -2-piperidinecarboxylic acid ] in the synthesis and storage process, and the structural formula is as follows:

the molecular formula is as follows: c₂₂H₃₄N₄O₅S

Molecular weight: 466.60

CAS number: 188659-43-0

The argatroban impurity G is a degradation impurity and a process impurity, and the impurity G needs to be qualitatively and quantitatively detected in order to ensure the product quality, so that a reference substance of the impurity needs to be obtained.

The patent (CN201410254533.2) discloses the synthesis of argatroban impurity G, the method is to use strong alkali to destroy and hydrolyze argatroban at high temperature, guanidino in the molecular structure of argatroban is hydrolyzed into terminal primary amino, the used strong alkali is barium hydroxide, sodium hydroxide, calcium hydroxide and potassium hydroxide aqueous solution, and the temperature is 60-100 ℃. Experiments prove that the hydrolysis degree is low and impurities are more under the condition, and the argatroban structure is provided with a plurality of hydrolyzable groups (amide, guanidyl and sulfonamide, and carbamide intermediate state is generated by hydrolysis of guanidyl), so that the separation difficulty is high, the product yield and the purification efficiency are extremely low, and the difficulty in impurity preparation is increased.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a preparation method of argatroban impurity G, which comprises the step of hydrolyzing argatroban salt under the catalytic action of arginase and manganese ions.

The invention discovers that argatroban has poor solubility in water, has good solubility after being formed into argatroban salt, can efficiently generate hydrolysis reaction in the presence of arginase and manganese ions to generate argatroban impurity G salt, has few side reactions of the hydrolysis reaction, generates few impurities, has simple purification and separation steps at the later stage, and can obtain a large amount of argatroban impurity G with high purity after treatment.

Preferably, the arginase is immobilized arginase. The immobilized arginase is convenient to be recycled after filtration, and is more suitable for industrial large-scale production.

Preferably, the argatroban salt is argatroban hydrochloride. In the general production process, argatroban hydrochloride is generated by adding hydrochloric acid into argatroban for reaction. The work-up of argatroban hydrochloride is easier than other salts, which are relatively difficult, such as sulphate.

Preferably, the argatroban salt is prepared by adding acid into the argatroban solution until the pH of the system is 7-8.

Preferably, the mass ratio of argatroban to arginase in the argatroban salt is 1: 0.1 to 0.25. Arginase may more desirably exert a catalytic action at the above-mentioned amount.

Preferably, the mass ratio of argatroban to manganese ions in the argatroban salt is 1: 0.01 to 0.02. The manganese ion can more ideally play a role in catalysis at the dosage

Preferably, the temperature of the hydrolysis reaction is 20-40 ℃.

Further preferably, the temperature of the hydrolysis reaction is 35-40 ℃.

More preferably, the temperature of the hydrolysis reaction is 34-36 DEG C

Preferably, the end point of the reaction is monitored by thin layer chromatography. Specifically, the reaction solution is developed by thin layer chromatography, and if no color reaction occurs, the reaction is finished.

Preferably, the developing solvent adopted in the thin-layer chromatography developing process is a mixed solution of dichloromethane and methanol in a volume ratio of 1: 4.8-5.2.

Preferably, the method further comprises the step of desalting the argatroban impurity G salt by adding ammonium carbonate or ammonium bicarbonate to prepare the argatroban impurity G.

As a preferred mode of operation, the argatroban impurity G salt obtained by the reaction is subjected to desalting treatment by a method comprising the steps of:

1) concentrating the reaction solution obtained after hydrolysis reaction to dry, redissolving with water, decolorizing with activated carbon, filtering to remove activated carbon, and adding anhydrous ethanol into the filtrate until Argatroban impurity G salt is separated out;

2) and dissolving the argatroban impurity G salt with water, adding ammonium carbonate until no bubbles are generated, adding ethyl acetate to extract the argatroban impurity G in the solution, and volatilizing the ethyl acetate to obtain the argatroban impurity G.

The method comprises the following steps:

hydrolyzing the argatroban hydrochloride under the catalytic action of arginase and manganese ions to obtain argatroban impurity G salt; the mass ratio of argatroban to arginase is 1: 0.48-0.52, wherein the mass ratio of argatroban to manganese ions is 1: 0.014-0.016, and the reaction temperature is 34-36 ℃.

The invention has the following beneficial effects:

the synthesis method successfully prepares the high-purity argatroban impurity G, the purity of the argatroban impurity G is more than 98% under ideal conditions, the synthesis route is reasonable, the operation method is simple and easy to implement, the yield is high, and the purity is high.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

The embodiment provides a preparation method of argatroban impurity G, which comprises the following specific steps:

1) adding 20g argatroban into a 1L three-mouth reaction bottle, adding 3.5ml hydrochloric acid (12mol/L) and 100ml water, and stirring at room temperature until the solution is clear to obtain an argatroban hydrochloride aqueous solution; adding 4g of immobilized arginase and 0.31g of manganese chloride tetrahydrate into the reaction solution, heating in a water bath at 35 ℃, stirring at the stirring speed of 150rpm, and reacting for 12 hours;

sample thin layer chromatography to monitor the reaction, developing solvent dichloromethane: the methanol is 1:5, and the reaction is finished until the argatroban raw material point does not develop color any more;

2) filtering the reaction solution, and concentrating the filtrate at 60 ℃ and-0.09 MPa under reduced pressure to dryness to obtain a white solid; adding 40G of purified water, stirring to dissolve a concentrated product, adding 2G of activated carbon, decoloring for 30 minutes, filtering, dropwise adding 80G of absolute ethyl alcohol into the filtrate, separating out white powder, cooling to 0 ℃ for crystallization for 2 hours, and filtering to obtain argatroban impurity G hydrochloride;

3) dissolving argatroban impurity G hydrochloride with 40G water, adding 4G ammonium carbonate to generate a large amount of bubbles, stirring until no bubbles are generated, measuring pH value to be neutral, adding 40ml ethyl acetate for 3 times of extraction, combining organic phases, washing the organic phases with 60ml saturated saline water, drying the organic phases for 30 minutes by taking 20G anhydrous sodium sulfate, filtering, concentrating the filtrate to dryness, addingPulping with 100ml petroleum ether, filtering, and air drying at 50 deg.C to obtain white powder Argatroban impurity G, weighing 13.15G, yield 71.66%, and purity (HPLC) 98.65%. And (3) product detection: MS (M/z), theoretical 466.2266, found 467.2279[ M + H ]]⁺。

Example 2

The embodiment provides a preparation method of argatroban impurity G, which comprises the following specific steps:

1) adding 20g of argatroban into a 1L three-mouth reaction bottle, adding 7ml of hydrochloric acid (12mol/L) and 100ml of water, and stirring at room temperature until the solution is clear to obtain an argatroban hydrochloride aqueous solution; adding 2g of immobilized arginase and 0.31g of manganese chloride tetrahydrate into the reaction solution, heating in a water bath at 35 ℃, stirring at the stirring speed of 150rpm, and reacting for 18 hours;

sample thin layer chromatography to monitor the reaction, developing solvent dichloromethane: the methanol is 1:5, and the reaction is finished until the argatroban raw material point does not develop color any more;

2) filtering the reaction solution, and concentrating the filtrate at 60 ℃ and-0.09 MPa under reduced pressure until the filtrate is dry to obtain a white solid; adding 40G of purified water, stirring to dissolve a concentrated product, adding 2G of activated carbon, decoloring for 30 minutes, filtering, dropwise adding 80G of absolute ethyl alcohol into the filtrate, separating out white powder, cooling to 0 ℃ for crystallization for 2 hours, and filtering to obtain argatroban impurity G hydrochloride;

3) the argatroban impurity G hydrochloride is dissolved by 40G of water, 4G of ammonium carbonate is added to generate a large amount of bubbles, the mixture is stirred until no bubbles are generated, the pH value is measured to be neutral, 40ml of ethyl acetate is added for 3 times of extraction, the organic phases are combined, 60ml of saturated saline solution is used for washing the organic phases, 20G of anhydrous sodium sulfate is dried for 30 minutes, the filtration is carried out, the filtrate is concentrated to be dry, 100ml of petroleum ether is added for pulping, the filtration is carried out, the forced air drying is carried out at the temperature of 50 ℃, and the argatroban impurity G which is white-like powder is obtained, the weight is 8.46G, the yield is 46.10. And (3) product detection: MS (M/z), theoretical 466.2258, found 467.2279[ M + H ]]⁺。

Example 3

The embodiment provides a preparation method of argatroban impurity G, which comprises the following specific steps:

1) adding 20g argatroban into a 1L three-mouth reaction bottle, adding 3.5ml hydrochloric acid (12mol/L) and 100ml water, and stirring at room temperature until the solution is clear to obtain an argatroban hydrochloride aqueous solution; adding 4g of immobilized arginase and 0.25g of manganese chloride tetrahydrate into the reaction solution, heating in a water bath at 40 ℃, stirring at the stirring speed of 150rpm, and reacting for 12 hours; sample thin layer chromatography to monitor the reaction, developing solvent dichloromethane: the methanol is 1:5, the argatroban raw material point disappears, and the reaction is ended;

2) filtering the reaction solution, and concentrating the filtrate at 60 ℃ and-0.09 MPa under reduced pressure until the filtrate is dry to obtain a white solid; adding 40G of purified water, stirring to dissolve a concentrated product, adding 2G of activated carbon, decoloring for 30 minutes, filtering, dropwise adding 80G of absolute ethyl alcohol into the filtrate, separating out white powder, cooling to 0 ℃ for crystallization for 2 hours, and filtering to obtain argatroban impurity G hydrochloride;

3) the argatroban impurity G hydrochloride is dissolved by 40G of water, 4G of ammonium carbonate is added to generate a large amount of bubbles, the mixture is stirred until no bubbles are generated, the pH value is measured to be neutral, 40ml of ethyl acetate is added for 3 times of extraction, the organic phases are combined, 60ml of saturated saline solution is used for washing the organic phases, 20G of anhydrous sodium sulfate is dried for 30 minutes, the filtration is carried out, the filtrate is concentrated to be dry, 100ml of petroleum ether is added for pulping, the filtration is carried out, and the air blowing drying at 50 ℃ is carried out to obtain the argatroban impurity G which is white-like powder, 11.84G of argatroban impurity G is weighed, the yield is. And (3) product detection: MS (M/z), theoretical 466.2257, found 467.2279[ M + H ]]⁺。

Example 4

The embodiment provides a preparation method of argatroban impurity G, which comprises the following specific steps:

1) adding 20g of argatroban into a 1L three-mouth reaction bottle, adding 6ml of hydrochloric acid (12mol/L) and 100ml of water, and stirring at room temperature until the solution is clear to obtain an argatroban hydrochloride aqueous solution; adding 3g of immobilized arginase and 0.40g of manganese chloride tetrahydrate into the reaction solution, heating in a water bath at 20 ℃, stirring at the stirring speed of 150rpm, and reacting for 8 hours; sample thin layer chromatography to monitor the reaction, developing solvent dichloromethane: the methanol is 1:5, the argatroban raw material point disappears, and the reaction is ended;

2) filtering the reaction solution, and concentrating the filtrate at 60 ℃ and-0.09 MPa under reduced pressure until the filtrate is dry to obtain a white solid; adding 40G of purified water, stirring to dissolve a concentrated product, adding 2G of activated carbon, decoloring for 30 minutes, filtering, dropwise adding 80G of absolute ethyl alcohol into the filtrate, separating out white powder, cooling to 0 ℃ for crystallization for 2 hours, and filtering to obtain argatroban impurity G hydrochloride;

3) the argatroban impurity G hydrochloride is dissolved by 40G of water, 4G of ammonium carbonate is added to generate a large amount of bubbles, the mixture is stirred until no bubbles are generated, the pH value is measured to be neutral, 40ml of ethyl acetate is added for 3 times of extraction, organic phases are combined, 60ml of saturated saline solution is used for washing, 20G of anhydrous sodium sulfate is dried for 30 minutes, the mixture is filtered, filtrate is concentrated to be dry, 100ml of petroleum ether is added for pulping, the filtration is carried out, air blowing drying is carried out at 50 ℃, and the argatroban impurity G which is white-like powder is obtained, 10.43G of argatroban impurity G is weighed, the yield is 56.84%, and the purity. And (3) product detection: MS (M/z), theoretical 466.2270, found 467.2279[ M + H ]]⁺。

Comparative example

CN105273045A discloses a method for obtaining a related substance B (namely, an impurity G) from argatroban through strong alkali hydrolysis in 1 step. In the third, fourth and fifth examples, calcium hydroxide, barium hydroxide, potassium hydroxide and sodium hydroxide aqueous solution are refluxed to destroy argatroban, and argatroban impurity G is obtained. In the embodiment, the yield of the argatroban impurity G is 9.8-14.5% and the purity is 88.7-93.6% based on argatroban.

The comparison shows that the yield and the purity of the synthesis method of the argatroban impurity G are obviously higher than those of a comparative example.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.