WO2013044793A1

WO2013044793A1 - High-purity heparin benzyl ester salt, preparation method therefor and application thereof

Info

Publication number: WO2013044793A1
Application number: PCT/CN2012/081981
Authority: WO
Inventors: 徐美英
Original assignee: Xu Meiying
Priority date: 2011-09-26
Filing date: 2012-09-26
Publication date: 2013-04-04

Abstract

The present invention relates to a high-purity heparin benzyl ester salt, and a preparation method therefor and an application thereof. Specifically, in the present invention, the preparation method of the heparin benzyl ester salt comprises: a heparin benzyl ester quaternary ammonium salt, or a material containing a heparin benzyl ester quaternary ammonium salt, or a heparin benzyl ester salt containing a quaternary ammonium salt reacting with an alkali metal or an aqueous solution of an alkaline-earth metal salt; adding the product of the reaction into a solvent in which the heparin benzyl ester metal salt or the alkaline-earth metal salt is insoluble, and obtaining the heparin benzyl ester salt after precipitation and separation. The heparin benzyl ester salt is featured by high purity, a small amount of residual quaternary ammonium salt, and a simple preparation method. The heparin benzyl ester salt is easily applied in industrial production, can be used for preparing high-purity heparin with a low molecular weight, and has a high yield.

Description

High-purity heparin benzyl ester salt and preparation method and application thereof

Technical field

The present invention relates to a high purity heparin benzyl ester salt, its preparation and its use in the preparation of low molecular weight heparin. Background technique

Heparin is a mixture of sulfated polysaccharides derived from animals (pig or bovine) and is widely used due to its excellent antithrombotic properties, such as preparation of antithrombotic drugs, drugs for treating cardiovascular diseases, and treatment of cerebrovascular diseases. Drugs, etc.

However, natural heparin has many drawbacks that limit the use of heparin. One major drawback is that excessive heparin administration can cause spontaneous bleeding, so clotting time should be measured before each injection. In addition, heparin can cause allergic reactions, thrombocytopenia, osteoporosis and spontaneous fractures over time.

Heparin sodium has a high risk of spontaneous bleeding, and clotting time must be monitored during clinical use, which is very inconvenient for both doctors and patients. At the same time, adverse reactions such as thrombocytopenia and osteoporosis also make doctors have concerns in their use. The use of low molecular weight heparin does not require monitoring of clotting time and greatly reduces the above-mentioned adverse effects of long-term use of heparin.

At present, the main methods for preparing low molecular weight heparin include nitrite degradation method, hydrogen peroxide degradation method, heparinase degradation method and alkali degradation method of heparin benzyl ester. Among them, the alkaline degradation method of heparin benzyl ester is to form heparin and quaternary ammonium salt, and then esterify heparin quaternary ammonium salt with benzyl chloride in dichloromethane to form heparin benzyl ester quaternary ammonium salt, into the solution The sodium acetate methanol solution is added, and the heparin benzyl ester sodium salt is obtained by filtration. According to the esterification rate, the corresponding degradation conditions are selected, and the sodium salt is degraded in an alkaline solution to obtain low molecular weight heparin. Thus, how to accurately determine the esterification rate is critical in the overall preparation process. In addition, since low molecular weight heparin is a mixture, process control impurities become very important. Therefore, a high-purity heparin benzyl ester alkali metal or alkaline earth metal salt is a prerequisite for obtaining high-quality low molecular weight heparin.

However, the above preparation method has the following disadvantages: (1) Heparin benzyl ester sodium salt has low purity: When sodium acetate methanol solution is reacted with heparin benzyl ester quaternary ammonium salt to form heparin benzyl ester sodium salt, heparin is a macromolecular long chain, heparin The solubility of the benzyl ester sodium salt in methanol is very low. With the addition of the sodium acetate methanol solution, the sodium salt of heparin containing the quaternary ammonium salt which is not in reaction is precipitated at the same time, which results in incomplete reaction. Experiments have shown that even after a long period of stirring or repeated reaction with sodium acetate methanol solution, it is suspended, and a considerable portion of the quaternary ammonium salt cannot be completely reacted, thereby remaining on the heparin benzyl ester sodium salt chain. (2) The esterification rate of heparin benzyl ester sodium salt is difficult to accurately determine: Since the reaction is in a non-solution state, both the reactants and the product are precipitated, and the degree of each reaction is not easy to reproduce, quaternary ammonium salt, benzyl chloride There is a difference between the residue and the sodium acetate residue, which brings uncertainty to the determination of the esterification rate, which affects the selection of subsequent degradation conditions, which in turn affects the quality of the product and may even affect the success rate of production. (3) Safety hazards of the final product: The quaternary ammonium salt formed during the reaction and the excess of benzyl chloride and sodium acetate are not easily washed away by methanol and will remain in the product. New impurities are also produced in subsequent degradation, posing a safety hazard to the product. (4) Difficult to industrialize: The precipitate obtained during the preparation of sodium salt of heparin benzyl ester is viscous, difficult to filter and purify, and requires a large amount of methanol washing and precipitation, which is difficult to industrialize and costly.

It is also mentioned in the literature that after the heparin benzyl ester quaternary ammonium salt is formed, the reaction solvent tetrahydrofuran is directly distilled off, and then the methanol is suspended and precipitated, and the heparin benzyl ester quaternary ammonium salt is obtained by filtration, and then added to an aqueous solution of sodium acetate to form Heparin benzyl ester sodium salt. This preparation method requires distillation of the reaction solvent, and experiments have shown that the heparin benzyl ester quaternary ammonium salt is easily soluble in methanol. Therefore, the addition of methanol after evaporation of the reaction solvent does not result in precipitation, and the difficulty in industrialization and cost are greatly increased.

Therefore, there is an urgent need in the art to develop a high-purity heparin benzyl ester salt, as well as a simple, feasible, industrially suitable preparation method and its use in the preparation of low molecular weight heparin. Summary of the invention

SUMMARY OF THE INVENTION The object of the present invention is to provide a high-purity heparin benzyl ester salt, as well as a simple and feasible preparation method suitable for industrialization and its use in the preparation of low molecular weight heparin. In a first aspect of the invention, there is provided a process for the preparation of a high purity heparin benzyl ester alkali metal salt or an alkaline earth metal salt, comprising the steps of:

(a) mixing (i) a heparin benzyl ester quaternary ammonium salt or a heparin benzyl ester quaternary ammonium salt-containing material or (ii) a quaternary ammonium salt-containing heparin benzyl ester salt with an aqueous solution of an alkali metal or alkaline earth metal salt, Thereby producing a heparin benzyl ester alkali metal salt or an alkaline earth metal salt;

(b) In the reaction mixture formed in the step (a), an insoluble solvent for the alkali metal salt of heparin benzyl ester or an alkaline earth metal salt is added, thereby precipitating and isolating the alkali metal salt or alkaline earth metal salt of the heparin benzyl ester.

In another preferred embodiment, the alkali metal salt is sodium chloride, sodium acetate, potassium chloride or sodium sulfate; and the alkaline earth metal salt is calcium chloride or magnesium chloride.

It is preferably sodium acetate, more preferably sodium chloride.

In another preferred embodiment, the aqueous solution of the alkali metal salt or alkaline earth metal salt has a concentration of from 0.1 w/v% to 95 w/v%; preferably from 3 w/v% to 50 w/v%; more preferably 5w/v%~25w/v%.

In another preferred embodiment, the weight ratio of the alkali metal salt or alkaline earth metal salt to the heparin benzyl ester quaternary ammonium salt is 0〜1〜10: 1, preferably 0. 2~5: 1, more preferably 0. 5~1 : 1; the alkali metal or alkaline earth metal salt and the heparin containing the quaternary ammonium salt 5〜1: 1。 The weight ratio of the benzyl ester salt is 0.1 to 10: 1, preferably 0. 2~5: 1, more preferably 0. 5~1: 1.

In another preferred embodiment, the solvent of the aqueous solution is water or an aqueous solution containing methanol, ethanol, acetone or a combination thereof.

In another preferred embodiment, the insoluble solvent is selected from the group consisting of alcohols, ketones, or combinations thereof.

More preferably, the alcohol is methanol, ethanol, or a combination thereof; the ketone is acetone. According to a second aspect of the present invention, there is provided a high-purity heparin benzyl alkali metal salt or an alkaline earth metal salt, wherein the content of the impurity quaternary ammonium salt is < 5 wt%.

In another preferred embodiment, the content of the impurity quaternary ammonium salt is < 1%, preferably, the content of the impurity quaternary ammonium salt is less than 0.5%, and more preferably the content of the impurity quaternary ammonium salt is < 0.1^^%.

In another preferred embodiment, the heparin benzyl ester metal salt comprises a heparin benzyl ester sodium salt, a potassium salt, a calcium salt or a magnesium salt form.

In another preferred embodiment, the heparin benzyl ester metal salt is heparin benzyl ester sodium salt.

In another preferred embodiment, the heparin benzyl ester alkali metal salt or alkaline earth metal salt is prepared by the method described in the first aspect. In a third aspect of the invention, a method of preparing a heparin-derived polysaccharide mixture is provided, comprising the steps of:

(a) mixing (i) a heparin benzyl ester quaternary ammonium salt or a heparin benzyl ester quaternary ammonium salt-containing material or (ii) a quaternary ammonium salt-containing heparin benzyl ester salt with an aqueous solution of an alkali metal or alkaline earth metal salt, Thereby reacting to form a heparin benzyl ester alkali metal salt or an alkaline earth metal salt;

(b) in the reaction mixture formed in the step (a), adding an insoluble solvent for the heparin benzyl ester alkali metal salt or alkaline earth metal salt, thereby precipitating and separating the heparin benzyl ester alkali metal or alkaline earth metal salt;

(c) Alkali degradation of a heparin benzyl ester alkali metal salt or an alkaline earth metal salt to obtain the heparin-derived polysaccharide mixture.

It is preferably sodium acetate, more preferably sodium chloride.

In another preferred embodiment, the aqueous solution concentration of the alkali metal salt or alkaline earth metal salt is 0.1 w/v% 〜 95 w/v%, preferably 3 w/v% to 50 w/v%, more preferably 5 w/v% to 25 w/v%.

2〜5: 1, More preferably, the weight ratio of the alkali metal salt or the alkaline earth metal salt to the heparin benzyl ester quaternary ammonium salt is 0.1 to 10: 1, preferably 0. 2~5: 1, more preferably 2〜5: The weight ratio of the alkali metal salt or the alkaline earth metal salt to the quaternary ammonium salt containing heparin benzyl ester salt is 0.1 to 10: 1, preferably 0. 2~5: 1, more preferably 0. 5~1: 1.

More preferably, the alcohol is methanol, ethanol, or a combination thereof; the ketone is acetone. According to a fourth aspect of the invention, a heparin-derived polysaccharide mixture is provided, which has the following features: It is prepared by the method described in the third aspect;

The weight average molecular weight is not more than 8000 Daltons;

The activity ratio of anticoagulant factor Xa to anticoagulant factor Ila is not less than 1.5;

At its non-reducing end, it contains a 4-pyranuronic acid structure.

In another preferred embodiment, the heparin-derived polysaccharide mixture has the following characteristics:

It is prepared by the method described in the third aspect;

The weight average molecular weight is 3800-5000 Daltons;

The molecular weight is less than 2000 Daltons, and the content is 12 to 20% of the total weight;

The molecular weight is between 2,000 and 8,000 Daltons, and the content is 68% to 82% of the total weight;

The activity ratio of anticoagulant factor Xa to anticoagulant factor Ila was 3.3-5.3;

At its non-reducing end, it contains a 4-pyranuronic acid structure, and the reducing end contains 15 to 25% of a 1,6-anhydrocyclic ring structure.

In another preferred embodiment, the heparin-derived polysaccharide mixture prepared by the method of the third aspect has the following characteristics:

The weight average molecular weight is 1500-5000 Daltons;

The activity ratio of anticoagulant factor Xa to anticoagulant factor Ila is not less than 10;

At its non-reducing end, it contains a 4-pyranuronic acid structure.

In another preferred embodiment, the polysaccharide mixture is in the form of an alkali metal salt or an alkaline earth metal salt, of which sodium is preferred. Salt, potassium, calcium and magnesium salts. According to a fifth aspect of the invention, there is provided a pharmaceutical composition comprising the heparin-derived polysaccharide mixture of the fourth aspect as an active ingredient.

In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In a sixth aspect of the invention, there is provided the use of a high purity heparin benzyl alkali metal salt or an alkaline earth metal salt for the preparation of a high purity heparin derived polysaccharide mixture.

In another preferred embodiment, the preparation process is as described in the third aspect. The heparin benzyl ester salt provided by the invention has high purity, especially low quaternary ammonium salt residue, simple preparation method, easy industrial production, and can be used for preparing high-purity low molecular weight heparin with high yield. detailed description

The present inventors have extensively and intensively studied, by accidentally discovering the preparation method of (i) heparin benzyl ester quaternary ammonium salt or heparin benzyl ester quaternary ammonium salt or (ii) containing season by improving the preparation method of heparin benzyl ester salt. The heparin benzyl ester salt of the ammonium salt is directly reacted with an aqueous solution of an alkali metal salt or an alkaline earth metal salt, and the heparin benzyl ester having high purity and low residual of quaternary ammonium salt, benzyl chloride and sodium acetate can be efficiently, simply and rapidly prepared. An alkali metal salt or an alkaline earth metal salt. The present invention has been completed on the basis of this. The heparin benzyl ester salt of the present invention has the following main advantages: (1) high purity: the residual amount of the quaternary ammonium salt is usually less than 3% by weight, the residual benzyl chloride is usually less than 0. lwt%, the residual amount of sodium acetate is usually less than 0. Lwt%. (2) The esterification rate is easy to measure and control, which is beneficial to the application in the preparation of low molecular weight heparin. (3) It is easily soluble in water, which is beneficial to its application in the preparation of low molecular weight heparin, that is, it is easier to select suitable degradation conditions in the preparation of low molecular weight heparin. (4) When preparing low molecular weight heparin, there are no impurities introduced by quaternary ammonium salts, benzyl chloride and sodium acetate, and the pigment content is low and easy to remove, the quality is good, and the yield is high.

As used herein, the term "heparin" refers to a polysaccharide having a molecular weight in the range of from 2000 to 30,000 Daltons.

The heparin benzyl ester quaternary ammonium salt in the term "heparin-containing benzyl ester quaternary ammonium salt-containing raw material" accounts for 20-99. 9wt, preferably 30-99wt% of the heparin salt (heparin benzyl ester quaternary ammonium salt heparin quaternary ammonium salt, etc.) in the raw material. More preferably 40_95wt%.

The term "heparin benzyl ester quaternary ammonium salt" is an esterification reaction of heparin quaternary ammonium salt with benzyl halide in an organic solvent. The resulting reaction product. Typically, the reaction product is not dequaternized.

The term "heparin benzyl ester salt containing a quaternary ammonium salt" is a reaction product of esterification of a heparin quaternary ammonium salt with a benzyl halide in an organic solvent, and the reaction product is an alcohol such as methanol, such as methanol. The quaternary ammonium salt is treated with a solution such as 8w/ _v %-20w/ _v %. Generally, the quaternary ammonium salt treatment does not completely remove the quaternary ammonium salt, so the treated product still contains a certain amount of quaternary ammonium salt.

Usually, the benzyl halide is a benzyl halide, preferably benzyl chloride or benzyl bromide, more preferably benzyl chloride.

Preferably, the organic solvent is dichloromethane.

Heparin may be heparin derived from bovine or porcine, namely porcine heparin or bovine heparin and its derivatives. In the present invention, an aqueous solvent or an aqueous solvent may be used, and the resulting solution is generally referred to as an aqueous solution. In other words, the aqueous solution of an alkali metal salt or an alkaline earth metal salt means that the solute is an alkali metal or an alkaline earth metal, and the solvent is (a) water or (b) a mixed solvent of water and an organic solvent (such as methanol, ethanol, etc.). (For example, a mixed solvent having an alcohol content of less than 90% (V/V) can be regarded as a solvent of an aqueous solution).

A preferred aqueous mixed solvent is water to an organic solvent (such as methanol, ethanol, etc.) in a volume ratio of 10-100:0. 01-90 (preferably 20-100: 0. 1-80, more preferably 50- 100: 1-50) mixed solvent. Heparin benzyl ester alkali metal salt or alkaline earth metal salt

The heparin benzyl ester alkali metal salt or alkaline earth metal salt of the present invention can be produced by the following method: (i) a heparin benzyl ester quaternary ammonium salt or a heparin benzyl ester quaternary ammonium salt-containing material or (ii) a quaternary ammonium salt containing heparin benzyl The ester salt is reacted with an aqueous solution of an alkali metal or alkaline earth metal salt to obtain an alkali metal salt or an alkaline earth metal salt of heparin benzyl ester, and an insoluble solvent for a metal salt of heparin benzyl ester or an alkaline earth metal salt is added to the reaction system, thereby The heparin benzyl ester alkali metal salt or alkaline earth metal salt of the present invention is isolated by precipitation. In a preferred embodiment of the invention, the preparation method comprises the steps of:

(a) reacting a heparin benzyl ester quaternary ammonium salt with an aqueous solution of an alkali metal or alkaline earth metal salt to obtain a heparin benzyl ester alkali metal or alkaline earth metal salt suspension;

(b) An insoluble solvent for a heparin benzyl ester alkali metal salt or an alkaline earth metal salt is added to the above system, whereby the heparin benzyl ester alkali metal salt or alkaline earth metal salt of the present invention is precipitated and isolated.

In a preferred embodiment of the invention, the preparation method comprises the steps of:

(a) a raw material containing a heparin benzyl ester quaternary ammonium salt, reacted with an aqueous solution of an alkali metal or alkaline earth metal salt to obtain heparin a benzyl ester alkali metal or alkaline earth metal salt suspension;

(b) An insoluble solvent for a heparin benzyl ester metal salt or an alkaline earth metal salt is added to the above system to precipitate a heparin benzyl ester alkali metal salt or an alkaline earth metal salt according to the present invention.

(a) reacting a heparin benzyl ester salt containing a quaternary ammonium salt with an aqueous solution of an alkali metal or alkaline earth metal salt to obtain a heparin benzyl ester alkali metal or alkaline earth metal salt suspension;

The insoluble solvent is selected from the group consisting of alcohols, ketones, or combinations thereof.

More preferably, the alcohol is methanol, ethanol, or a combination thereof; the ketone is acetone.

In the present invention, as the alkali metal salt or alkaline earth metal salt, preferred are sodium, potassium, calcium and magnesium salts.

The heparin benzyl ester salts of the present invention have higher purity and easier quality control than heparin benzyl ester salts prepared by the prior art. In other words, heparin benzyl ester salt is highly pure, while impurities such as quaternary ammonium salts, benzyl chloride and sodium acetate are very low. For example, the heparin benzyl ester salt of the present invention has a quaternary ammonium salt impurity content of 5 wt%, preferably 3 wt%, more preferably 1 wt%, even 0.5 wt%; optimally 0.1 wt% o

These heparin benzyl ester salts may be in the form of an alkali metal salt or an alkaline earth metal salt, of which a sodium salt, a potassium salt, a calcium salt and a magnesium salt are preferred. Heparin-derived polysaccharide mixture

According to the esterification rate of the high-purity heparin benzyl ester salt, it can be degraded by the corresponding conditions to obtain a high-purity heparin-derived polysaccharide mixture such as low molecular weight heparin such as enoxaparin sodium. Among these heparin-derived polysaccharide mixtures, there is an unsaturated 4,5-glucuronic acid 2-0-sulfate structural unit at one end thereof. Pharmaceutical composition and method of administration

The heparin-derived polysaccharide mixture of the present invention can be used as an antithrombotic agent for preventing venous thromboembolic disease (prevention of venous thrombosis), particularly thrombosis associated with orthopedic or general surgery. Treatment of established deep vein thrombosis with or without pulmonary embolism. Treatment of unstable angina and non-Q-wave myocardial infarction, used for hemodialysis in vitro, to prevent thrombosis.

Furthermore, the heparin-derived polysaccharide mixture of the present invention can also be used for the treatment or adjuvant treatment of cardiovascular diseases and cerebrovascular diseases.

Accordingly, the present invention also provides a pharmaceutical composition comprising the heparin derivative of the present invention as an active ingredient A mixture of polysaccharides and a pharmaceutically acceptable carrier. 1-10重量%。 A preferred pharmaceutical composition dosage form is an injection (including a solution). The dosage of the heparin-derived polysaccharide mixture of the present invention is 0. 01-50wt%, more preferably 0. l-30wt% o. And lyophilizates).

The pharmaceutical composition of the present invention can be prepared and administered by a conventional method. A preferred method of administration is subcutaneous or intravenous administration.

The main advantages of the invention are:

(1) The purity of heparin benzyl ester salt is high, and the residual amount of quaternary ammonium salt, benzyl chloride and acetate is low, and the weight content is usually less than 5%.

(2) The quality control of heparin benzyl ester salt is simple, especially the esterification rate of key indicators can be accurately determined and controlled.

(3) The preparation method of heparin benzyl ester salt is simple, and does not produce a viscous product. It can be simply filtered, and the heparin benzyl ester salt has high yield and is easy to be industrialized without long-time filtration or centrifugation.

The quaternary ammonium salt content <0. lwt%. The quaternary ammonium salt content is less than 0.1% by weight. The quaternary ammonium salt content is less than 0.1% by weight.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are only intended to illustrate the invention and not to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually in accordance with conventional conditions (such as those described in the European Pharmacopoeia) or in accordance with the conditions recommended by the manufacturer. Universal assay

(1) The degree of esterification of heparin ester or heparin ester salt can be determined by a conventional HPLC, that is, the degree of esterification is determined by measuring the amount of benzyl alcohol produced by saponification of the ester at 0 °C.

(2) The molecular weight and molecular weight distribution as well as the anti-Xa and anti-I la titers were carried out by the conventional low molecular weight heparin quality standard in the European Pharmacopoeia. Example 1 Preparation of Heparin Quaternary Ammonium Salt

1. 1 To a solution (1,050 ml) of benzethonium chloride (250 g), a solution of water (1000 ml) containing commercially available sodium heparin (100 g) (Note: porcine-derived heparin) was added. The mixture was allowed to stand for 30 minutes with stirring to form a heparin quaternary ammonium salt precipitate. Then, the reaction mixture was filtered at room temperature to give a filter cake, which was washed with water and dried in vacuo to give heparin benzyloxyethylamine salt (305 g). For use in Examples 2-5.

1. 2 Repeat Example 1. 1. Obtain heparin benzyloxyethylammonium salt (300 g). For use in Examples 6-9. Example 2 Preparation of Heparin Benzate Sodium Salt

Benzyl chloride (100 ml) was added to a solution of heparin benzyloxyethylammonium salt (100 g) in dichloromethane (500 ml). Will The solution was heated to 35 ° C for 25 hours. Then, 1 (1⁄2% aqueous sodium chloride solution (600 ml) was added, and after stirring for 1 hour, 1800 ml of methanol was added thereto to precipitate a precipitate, which was filtered, and the filter cake was washed with a small amount of methanol and dried to obtain white heparin benzyl ester sodium salt (37 g). ).

5重量%的酯。 The residual amount of the quaternary ammonium salt of the heparin benzyl ester sodium salt, the residual amount of the quaternary ammonium salt <0. 5wt%, the residual amount of sodium acetate <0. 05wt%, the residual amount of benzyl chloride <0. 05wt%, ester The rate of conversion is 12.5% (w/w). EXAMPLES Preparation of Heparin Benzate Sodium Salt

Benzyl chloride (100 ml) was added to a solution of heparin benzyloxyethylammonium salt (100 g) in dichloromethane (500 ml). The solution was heated to 35 ° C for 24 hours. Then, a 15% aqueous solution of sodium acetate (700 ml) was added, and the mixture was stirred for 1 hour, and 2100 ml of methanol was added thereto to precipitate a precipitate, which was filtered, and the filter cake was washed with a small amount of methanol and dried to give white heparin benzyl ester sodium salt (38 g).

The amount of residual benzyl chloride is <0. 05wt%, the residual amount of benzyl chloride is <0. lwt%, the residual amount of benzyl chloride is <0. 05wt%, as determined by the HPLC method. The esterification rate was 10.9% (w/w). EXAMPLES Preparation of Heparin Benzate Sodium Salt

Benzyl bromide (120 ml) was added to a solution of heparin benzyloxyethylammonium salt (100 g) in dichloromethane (500 ml). Heat the solution to 35 °C for 25 hours. Then, a 15% aqueous solution of sodium chloride (900 ml) was added, stirred, and 2700 ml of ethanol was added thereto to precipitate a precipitate, which was filtered, and the cake was washed with a small amount of ethanol and dried to give white heparin benzyl ester sodium salt (36 g). 3% (w/w) The esterification rate of the benzyl chloride salt is 0.001% by weight. . EXAMPLES Preparation of Heparin Benzate Sodium Salt

Benzyl chloride (150 ml) was added to a solution of heparin benzyloxyethylammonium salt (100 g) in dichloromethane (500 ml). The solution was heated to 35 ° C for 48 hours. Then, 800 ml of a 10 w/v% aqueous solution of sodium chloride (5 vol% aqueous solution of ethanol) was added, stirred, and 2400 ml of ethanol was added thereto to precipitate a precipitate, which was filtered, and the filter cake was washed with a small amount of ethanol and dried to obtain a white heparin benzyl ester. Sodium salt (42 g).

5% (w/w) The esterification rate of the benzyl chloride salt was determined by HPLC. The residual amount of the quaternary ammonium salt was <0. 5wt%, the residual amount of benzyl chloride was <0. 05wt esterification rate was 13. l% (w / w) . EXAMPLES Preparation of Heparin Benzate Sodium Salt

Benzyl chloride (100 ml) was added to a solution of heparin benzyloxyethylammonium salt (100 g) in dichloromethane (500 ml). Solution Heat to 35 ° C for 25 hours. Then 10% sodium acetate in methanol (600 ml) was added. After stirring, the precipitate was precipitated, filtered, washed with a small amount of methanol and dried to give pale yellow heparin benzyl ester sodium salt (38 g).

The dried heparin benzyl ester sodium salt is dissolved in 800 ml of water, 100 g of sodium chloride is added, 2400 ml of methanol is added with stirring, and the precipitate is filtered, washed with a small amount of methanol, and dried to obtain high purity white heparin benzyl ester sodium salt (35 g). ).重量百分比,酯。 The residual amount of benzyl chloride in the sodium salt of the heparin benzyl ester, the residual amount of the quaternary ammonium salt <0. 4wt%, the residual amount of sodium acetate <0. lwt%, the residual amount of benzyl chloride <0. lwt%, ester The rate of conversion was 12.3% (w/w). Example 7 Preparation of Heparin Benzylate Sodium Salt

Benzyl chloride (100 ml) was added to a solution of heparin benzyloxyethylammonium salt (100 g) in dichloromethane (500 ml). The solution was heated to 35 ° C for 20 hours. Then, a 10% sodium acetate methanol solution (700 ml) was added, stirred, and the precipitate was precipitated, centrifuged, and dried to obtain a pale yellow heparin benzyl ester sodium salt (38 g).

The dried crude heparin benzyl ester sodium salt was dissolved in 800 ml of water, 80 g of sodium chloride was added, 2400 ml of methanol was added thereto with stirring, and the precipitate was filtered, and dried to obtain high purity white heparin benzyl ester sodium salt (31⁄2).

Lwt%, residual amount of benzyl chloride <0. lwt%, residual amount of benzyl chloride <0. lwt%, 5% by weight of benzyl chloride. The esterification rate was 11.9% (w/w). Example 8 Preparation of Heparin Benzate Sodium Salt

Benzyl chloride (100 ml) was added to a solution of heparin benzyloxyethylammonium salt (100 g) in dichloromethane (500 ml). The solution was heated to 35 ° C for 25 hours. Then, a 10% sodium acetate ethanol solution (800 ml) was added to precipitate a precipitate, which was filtered and dried to give a pale yellow heparin benzyl ester sodium salt (37 g).

The dried crude heparin benzyl ester sodium salt was dissolved in 800 ml of water, 80 g of sodium chloride was added, 2400 ml of ethanol was added thereto with stirring, and the precipitate was separated by filtration, and dried to obtain high purity white heparin benzyl ester sodium salt (35 g).

Lwt%, residual amount of benzyl chloride <0. lwt%, residual amount of benzyl chloride <0. lwt%, 5% by weight of benzyl chloride. The esterification rate was 13.1% (w/w). Example 9 Preparation of Heparin Benzylate Sodium Salt

Benzyl chloride (100 ml) was added to a solution of heparin benzyloxyethylammonium salt (100 g) in dichloromethane (500 ml). The solution was heated to 40 ° C for 20 hours. Then, after adding 10% sodium acetate methanol solution (1000 ml), the precipitate was precipitated, filtered, and after washing with a small amount of methanol, the crude heparin benzyl ester sodium salt cake was dissolved in 800 ml of water, 80 g of sodium chloride was added, and 2400 ml of ethanol was added with stirring. The precipitate precipitated was filtered and dried to obtain high purity white heparin benzyl ester sodium salt (33 g).

The residual amount of the quaternary ammonium salt in the heparin benzyl ester sodium salt is determined by HPLC, <0. 4wt%, the residual of sodium acetate 8% (w/w)。 The residual amount of <0. lwt%, benzyl chloride residue <0. lwt%, esterification rate of 10.8% (w / w). The results of Examples 2-9 show that the preparation method of the invention is simple and reproducible, and the sodium salt of heparin benzyl ester is high in purity, all in white, and the quaternary ammonium salt, sodium acetate and benzyl chloride are low. Example 10 Application of Heparin Benzylate Salt

The sodium heparin benzyl ester salt (10 g) obtained in Example 2 was dissolved in water (250 ml), and the solution was colorlessly clarified and heated to 62 °C. Sodium hydroxide (lg) was added to the solution, and the reaction was carried out for 1.5 hours to degrade the sodium salt of heparin benzyl ester, and the reaction solution became a pale yellow clear solution. The solution was cooled to about 20 ° C and neutralized by the addition of dilute hydrochloric acid. Additional sodium chloride was added to bring the sodium chloride concentration in the reaction mixture to 12% (w/v). Methanol (750 ml) was added to the reaction mixture to precipitate a precipitate, which was filtered to give a filter cake. After washing with methanol, the cake was dissolved in 100 ml of water. 5 ± 0. 1。 The pH was adjusted to 8. 4 ± 0.1. The sulphate is used to remove excess amount of sodium borohydride (as a result of 10 _g / _{gram of} heparin benzyl ester sodium salt), and the solution is allowed to stand for 1 hour, and then the pH is adjusted to 4. 0-4. Boron hydride. The pH was adjusted to 7.0 with 2M NaOH. Sodium chloride was added to make a salt concentration of 10 w/v%, and then a solution volume of 3 volumes of methanol was added to precipitate a precipitate, which was filtered to obtain a filter cake, which was washed with methanol and dried under vacuum to obtain a high-purity low molecular weight heparin (white). , 7. 9g). 1%。 The yield was 89. 1%.

The properties of the heparin-derived polysaccharide mixture were determined as follows:

The quaternary ammonium salt in the product was <0.01% by weight, and the purity was high and the safety was good.

The resulting product is fully compliant with the quality requirements of the listed enoxaparin sodium. Example 11 Application of Heparin Benzylate Salt

Example 10 was repeated except that the heparin benzyl ester sodium salt (10 g) obtained in Example 3 was dissolved in water (250 ml), the solution was colorless and clarified, heated to 64 ° C; and sodium chloride was added to make the reaction mixture The concentration of sodium chloride in the solution is 10% (w/v). High purity low molecular weight heparin (white, 7.9 g) was obtained. 6%。 According to the calculation of heparin sodium, the yield was 91.6%.

The properties of the heparin-derived polysaccharide mixture were determined as follows: Measured value standard

Weight average molecular weight (Dalton) 4513 3800~5000 Component content of molecular weight less than 2000 Daltons 13% 12~20% Component content of molecular weight between 2000~8000 Daltons 78% 68-82%

Anticoagulant factor Xa activity (IU/mg) 109. 7 90-125 IU/mg Anticoagulant factor I la activity (IU/mg) 25. 1 20-35 IU/mg Anticoagulant factor Xa activity/anticoagulant factor I la activity ratio 4. 4 3. 3~5· 3

1, 6-dehydration ring content 19. 3% 15~25% By HPLC, the product has a quaternary ammonium salt <0. lwt%, high purity and good safety.

The resulting product is fully compliant with the quality requirements of the listed enoxaparin sodium. Example 12 Application of Heparin Benzylate Salt

The heparin benzyl ester sodium salt (10 g) obtained in Example 4 was dissolved in water (250 ml), and the solution was colorlessly clarified and heated to 62 °C. Sodium hydroxide (lg) was added to the solution, and the reaction was carried out for 1.5 hours to degrade the heparin benzyl ester sodium salt, and the reaction solution became a pale yellow clear solution. The solution was cooled to about 20 ° C, and hydrochloric acid was added for neutralization. Additional sodium chloride was added to bring the sodium chloride concentration in the reaction mixture to 15% (w/v). Methanol (750 ml) was added to the reaction mixture to precipitate a precipitate, which was filtered to give a filter cake. After washing with methanol, the cake was dissolved in 100 ml of water. 5 ml of hydrogen peroxide was added to the solution, and after reacting for 1 hour, sodium chloride was added to make a salt concentration of 10 w/v%, and a volume of 3 times by volume of methanol was added to precipitate a precipitate, which was filtered to obtain a filter cake, which was washed with methanol and vacuumed. It was dried to obtain high-purity low molecular weight heparin (white, 7.9 g). 7%。 According to the calculation of heparin sodium, the yield was 86.7%.

The quaternary ammonium salt in the product was <0.05% by weight, and the purity was high and the safety was good.

The resulting product is fully compliant with the quality requirements of the listed enoxaparin sodium. Example 13 Application of Heparin Benzylate Salt

The heparin benzyl ester sodium salt (10 g) obtained in Example 5 was dissolved in water (250 ml), and the solution was colorlessly clarified and heated to 65 °C. Sodium hydroxide (1. lg) was added to the solution, and the reaction was carried out for 1.5 hours to degrade the heparin benzyl ester sodium salt, and the reaction solution became a pale yellow clear solution. The solution was cooled to about 20 ° C and hydrochloric acid was added for neutralization. Add chlorination Sodium causes the sodium chloride concentration in the reaction mixture to be 10% (w/v). Methanol (750 ml) was added to the reaction mixture to precipitate a precipitate, which was filtered to give a filter cake. After washing with methanol, the filter cake was dissolved in 100 ml of water. 5 ml of hydrogen peroxide was added to the solution, and after reacting for 1 hour, sodium chloride was added to make a salt concentration of 10 w/v%, and a volume of 3 times by volume of methanol was added to precipitate a precipitate, which was filtered to obtain a filter cake, which was washed with methanol and vacuumed. It was dried to obtain high-purity low molecular weight heparin (white, 6.6 g). 5%。 According to the calculation of heparin sodium, the yield was 84.5%.

By HPLC, the quaternary ammonium salt in the product is <0. lwt%, high purity and good safety. Example 14 Application of Heparin Benzylate Salt

The heparin benzyl ester sodium salt (10 g) obtained in Example 6 was dissolved in water (250 ml) and heated to 62 °C. Sodium hydroxide (lg) was added to the solution, and the reaction was carried out for 1.5 hours to degrade the heparin benzyl ester sodium salt. The reaction mixture was then cooled to about 20 ° C and neutralized by the addition of dilute hydrochloric acid. Additional sodium chloride was added to bring the sodium chloride concentration in the reaction mixture to 10% (w/v). Methanol (750 ml) was added to the reaction mixture to precipitate a precipitate, which was filtered to give a filter cake. After washing with methanol, the cake was dissolved in 100 ml of water. 5 ± 0. 1。 The pH was adjusted to 8. 4 ± 0.1. The sulphate is used to remove excess amount of sodium borohydride (as a result of 10 _g / _{gram of} heparin benzyl ester sodium salt), and the solution is allowed to stand for 1 hour, and then the pH is adjusted to 4. 0-4. Boron hydride. The pH was adjusted to 7.0 with 2M NaOH. Sodium chloride was added to make a salt concentration of 10 w/v%, and then a solution volume of 3 volumes of methanol was added to precipitate a precipitate, which was filtered to obtain a filter cake, which was washed with methanol and dried under vacuum to obtain a high-purity low molecular weight heparin (white). , 7. 9g). 0%。 The yield was 83.0%.

Measurement value Standard weight average molecular weight (Dalton) 4518 3800~5000 Component content of molecular weight less than 2000 Daltons 16% 12~20% Component content of molecular weight between 2000~8000 Daltons 78% 68-82%

Anticoagulant factor Xa activity (IU/mg) 101. 7 90-125IU/mg Anticoagulant factor I la activity (IU/mg) 21. 6 20-35IU/mg Anticoagulant factor Xa activity/anticoagulant factor I la activity ratio 4. 7 3. 3~5· 3

1, 6-dehydration ring content 21. 3% 15~25% The quaternary ammonium salt in the product was <0.01% by weight, and the purity was high and the safety was good.

Therefore, the heparin-derived polysaccharide mixture prepared by the preparation method of the present invention fully meets the quality requirements of the listed enoxaparin sodium. At the same time, the preparation method has high yield, no need for cumbersome and long-time filtration treatment steps and a large amount of methanol rinsing filter cake, which is beneficial to reducing cost and energy consumption, and greatly enhancing the competitiveness of the product. Example 15 Application of Heparin Benzylate Salt

The heparin benzyl ester sodium salt (10 g) obtained in Example 7 was dissolved in water (250 ml) and heated to 62 °C. Sodium hydroxide (lg) was added to the solution, and the reaction was carried out for 1.5 hours to degrade the heparin benzyl ester sodium salt. The reaction mixture was then cooled to about 20 ° C and neutralized by the addition of hydrochloric acid. Additional sodium chloride was added to bring the sodium chloride concentration in the reaction mixture to 10% (w/v). Methanol (750 ml) was added to the reaction mixture to precipitate a precipitate, which was filtered to give a filter cake. After washing with methanol, the cake was dissolved in 100 ml of water. 5 ± 0. 1。 The pH was adjusted to 8. 4 ± 0.1. The sulphate is used to remove excess amount of sodium borohydride (as a result of 10 _g / _{gram of} heparin benzyl ester sodium salt), and the solution is allowed to stand for 1 hour, and then the pH is adjusted to 4. 0-4. Boron hydride. The pH was adjusted to 7.0 with 2M NaOH. Sodium chloride was added to make a salt concentration of 12 w/v%, and then a solution volume of 3 volumes of methanol was added to precipitate a precipitate, which was filtered to obtain a filter cake, which was washed with methanol and dried under vacuum to obtain a high-purity low molecular weight heparin (white). , 7. 9g). Calculated by heparin sodium, the yield was 80.6%.

The quaternary ammonium salt in the product was <0.01% by weight, and the purity was high and the safety was good. Example 16 Application of Heparin Benzylate Salt

The heparin benzyl ester sodium salt (10 g) obtained in Example 8 was dissolved in water (250 ml) and heated to 60 °C. Sodium hydroxide (lg) was added to the solution, and the reaction was carried out for 1.5 hours to degrade the heparin benzyl ester sodium salt. The reaction mixture was then cooled to about 20 ° C and neutralized by the addition of hydrochloric acid. Additional sodium chloride was added to bring the sodium chloride concentration in the reaction mixture to 10% (w/v). Methanol (750 ml) was added to the reaction mixture to precipitate a precipitate, which was filtered to give a filter cake. After washing with methanol, the cake was dissolved in 100 ml of water. 5 ± 0. 1。 The pH was adjusted to 8. 4 ± 0.1. Sodium borohydride (10 _g / k _g heparin) The benzyl hydride was added to the solution, and the mixture was allowed to stand for 1 hour, and then the pH was adjusted to 4. 0-4. 4 to eliminate excessive borohydride. The pH was adjusted to 7.0 with 2M Na0H. Sodium chloride was added to make a salt concentration of 10 w/v%, and then a solution volume of 3 volumes of ethanol was added to precipitate a precipitate, which was filtered to obtain a filter cake, which was washed with methanol and dried under vacuum to obtain a high-purity low molecular weight heparin (white). , 8. lg). 1%。 The yield was 85.1%.

The low molecular weight heparin has a quaternary ammonium salt of 0.01% by weight, which has high purity and good safety. Example 17 Application of Heparin Benzylate Salt

The heparin benzyl ester sodium salt (10 g) obtained in Example 9 was dissolved in water (250 ml) and heated to 64 °C. Sodium hydroxide (1. lg) was added to the solution for 1.5 hours to degrade the heparin benzyl ester sodium salt. The reaction mixture was then cooled to about 20 ° C and hydrochloric acid was added for neutralization. Additional sodium chloride was added to bring the sodium chloride concentration in the reaction mixture to 10% (w/v). Methanol (750 ml) was added to the reaction mixture to precipitate a precipitate, which was filtered to give a filter cake. After washing with methanol, the filter cake was dissolved in 100 ml of water. To the solution was added 5 ml of hydrogen peroxide, and after reacting for 1 hour, sodium chloride was added to make a salt concentration of 10 w/v%, and a solution of 3 volumes of methanol was added to precipitate a precipitate, which was filtered to obtain a filter cake, washed with methanol, and dried under vacuum. Thereby, high-purity low molecular weight heparin (white, 8. 1) is obtained. 2%。 The yield was 80.2%.

The quaternary ammonium salt of the low molecular weight heparin was 0.01% by weight, and the purity was high and the safety was good. Example 18 Application of Heparin Benzylate Salt

The sodium heparin benzyl ester salt (10 g) obtained in Example 8 was dissolved in water (250 ml), and the solution was colorlessly clarified and heated to 65 °C. Sodium hydroxide (1. lg) was added to the solution for 1.5 hours to degrade the heparin benzyl ester sodium salt, and the reaction solution became a pale yellow clear solution. The solution was cooled to about 20 ° C, and hydrochloric acid was added for neutralization. Further sodium chloride was added to bring the sodium chloride concentration in the reaction mixture to 10% (w/v). Methanol (750 ml) was added to the reaction mixture to precipitate a precipitate, which was filtered to give a filter cake. After washing with methanol, the filter cake was dissolved in 100 ml of water. 5 ml of hydrogen peroxide was added to the solution, and after reacting for 1 hour, sodium chloride was added to make a salt concentration of 10 w/v%, and a volume of 3 times by volume of methanol was added to precipitate a precipitate, which was filtered to obtain a filter cake, which was washed with methanol. It was dried under vacuum to obtain a high-purity low molecular weight heparin (white, 6.9 g). 5%。 According to the calculation of heparin sodium, the yield was 72.5%.

The quaternary ammonium salt in the product was <0.01% by weight, and the purity was high and the safety was good. Examples 10-18 show that the heparin benzyl ester salt obtained by the invention is an intermediate, and the prepared low molecular weight heparin has controllable quality, high yield, less impurities, high purity, good product safety, and fully meets the listed Enoch. Quality requirements for heparin sodium. At the same time, the preparation method has high yield, no need for cumbersome and long-time filtration treatment steps and a large amount of methanol rinsing filter cake, which is beneficial to reducing cost and energy consumption, and greatly enhancing the competitiveness of the product. Examples 5, 13, 16 and 18 show that, depending on the esterification rate, different molecular degradation factors can be used to obtain low molecular weight heparin with specific molecular weight distribution and potency characteristics. Comparative Example 1 Preparation of heparin benzyl ester sodium salt

Benzyl chloride (100 ml) was added to a solution of heparin benzyloxyethylammonium salt (100 g) in dichloromethane (500 ml). The solution was heated to 35 ° C for 25 hours. Then 15 w/v% sodium acetate in methanol (600 ml) was added. The precipitate was precipitated, filtered under suction for a long time, and the cake was washed with 5 L of methanol and dried to give pale yellow heparin benzyl ester sodium salt (34 g).

The content of benzyloxyethylammonium salt in the heparin benzyl ester sodium salt is 16 wt% determined by HPLC method, sodium acetate The content is 13% by weight.重量百分比。 The residual amount of benzyl chloride is 1. lwt%. It is difficult to determine the esterification rate due to the residual of benzethonium salt, benzyl chloride and sodium acetate. Comparative Example 2 Preparation of heparin benzyl ester sodium salt

Benzyl chloride (100 ml) was added to a solution of heparin benzyloxyethylammonium salt (100 g) in dichloromethane (500 ml). The solution was heated to 35 ° C for 25 hours. Then 10 w/v% sodium acetate in methanol (600 ml) was added. The precipitate was precipitated and centrifuged. The precipitate was suspended in a 10 w/v% sodium acetate methanol solution (600 ml), stirred for 2 hours, and centrifuged. After the suspension was repeatedly suspended and centrifuged twice, the precipitate was suspended in 600 ml of methanol, stirred for 2 hours, filtered, and the filter cake was repeatedly washed with methanol and dried to give pale yellow heparin benzyl ester sodium salt (31). g).

5重量%。 The residual amount of benzyl chloride is 0. 5wt%. The residual amount of benzyl chloride is 0. 5wt%. It is difficult to determine the esterification rate due to the residual of benzyloxyethylammonium salt, benzyl chloride and sodium acetate. Comparative Example 3 Preparation of heparin benzyl ester sodium salt

Benzyl chloride (100 ml) was added to a solution of heparin benzyloxyethylammonium salt (100 g) in dichloromethane (500 ml). The solution was heated to 35 ° C for 25 hours. Then 10% sodium acetate in ethanol (600 ml) was added. The precipitate was precipitated and centrifuged. The precipitate was suspended in a 10% sodium acetate ethanol solution (600 ml), stirred for 2 hours, and centrifuged. After the suspension was repeatedly suspended and centrifuged twice, the precipitate was suspended in 600 ml of ethanol, stirred for 2 hours, filtered, and the filter cake was washed with 5 L of ethanol and dried to give heparin benzyl ester sodium salt (30 g).

The residual amount of the quaternary ammonium salt was 7 wt%, the residual amount of sodium acetate was 7 wt%, and the residual amount of benzyl chloride was 1. l wt% as determined by HPLC. It is difficult to determine the esterification rate due to the residual of quaternary ammonium salt, sodium acetate and benzyl chloride. Comparative Example 4 Preparation of heparin benzyl ester sodium salt

Benzyl bromide (100 ml) was added to a solution of heparin benzyloxyethylammonium salt (100 g) in tetrahydrofuran (500 ml). The solution was heated to 35 ° C for 25 hours. The tetrahydrofuran solvent was then distilled off. Methanol was added and a precipitate was obtained. It is indicated that heparin benzyl ester benzyloxyethylammonium salt is soluble in methanol, and heparin oxime ester sodium salt cannot be obtained by a known patent method. Comparative Example 5 Application of heparin benzyl ester salt

The heparin benzyl ester sodium salt (10 g) obtained in Comparative Example 1 was dissolved in water (250 ml) to give a pale yellow mixture, which was heated to 62 °C. Sodium hydroxide (lg) was added to the solution, and the reaction was carried out for 1.5 hours, thereby lowering the sodium salt of heparin benzyl ester. Solution, the reaction solution turned into a yellow turbid liquid. The solution was then cooled to about 20 ° C and neutralized by the addition of hydrochloric acid. Additional sodium chloride was added to bring the sodium chloride concentration in the reaction mixture to 10% (w/v). Methanol (750 ml) was added to the reaction mixture to precipitate a precipitate, which was filtered to give a filter cake. After washing with methanol, the filter cake was dissolved in 100 ml of water. The pH was adjusted to 8.4 ± 0.1 by using 1M NaOH. The sulphate was used to remove the excess amount of sodium borohydride (as a percentage of 10 _g / _{gram of} heparin benzyl ester sodium salt), and the solution was allowed to stand for 1 hour, and then the pH was adjusted to 4. 0-4. Boron hydride. The pH was adjusted to 7.0 with 2M NaOH. Sodium chloride was added to make a salt concentration of 10 w/v%, and then a solution volume of 3 volumes of methanol was added to precipitate a precipitate, which was filtered to obtain a filter cake, which was washed with methanol and dried under vacuum to obtain a low molecular weight heparin (white-like). , 5. 7g). 1%。 The yield was 59.1%.

By comparison, the color is deeper than the European Pharmacopoeia's color requirements for enoxaparin sodium, and the molecular weight distribution and other indicators do not meet the requirements of the European Pharmacopoeia for enoxaparin sodium.

The benzyl chlorohydrin content of the final product is 0. 5wt%, the benzyl alcohol content introduced by the benzyl chloride is about 0. 3wt, does not meet the requirements of the injection grade enoxaparin sodium, and brings product safety. Hidden dangers. Comparative Example 6 Application of heparin benzyl ester salt

The heparin benzyl ester sodium salt (10 g) obtained in Comparative Example 2 was dissolved in water (250 ml) to give a pale yellow mixture, which was heated to 60 °C. Sodium hydroxide (lg) was added to the solution, and the reaction was carried out for 1.5 hours to degrade the heparin benzyl ester sodium salt, and the reaction liquid became a yellow turbid liquid. The solution was cooled to about 20 ° C and hydrochloric acid was added for neutralization. Additional sodium chloride was added to bring the sodium chloride concentration in the reaction mixture to 10% (w/v). Methanol (750 ml) was added to the reaction mixture to precipitate a precipitate, which was filtered to give a filter cake. After washing with methanol, the cake was dissolved in 100 ml of water. The pH was adjusted to 8.4 ± 0.1 by using 1M NaOH. The sulphate is used to remove the excess amount of the sodium borohydride (as a result of the 10 _g / _{gram of the} heparin benzyl ester sodium salt), the solution is allowed to stand for 1 hour, and then the pH is adjusted to 4. 0-4. Boron hydride. The pH was adjusted to 7.0 with 2M NaOH. Sodium chloride was added to make a salt concentration of 10 w/v%, and then a solution volume of 3 volumes of methanol was added to precipitate a precipitate, which was filtered to obtain a filter cake, which was washed with methanol and dried under vacuum to obtain a low molecular weight heparin (white-like, 7%。 The yield was 68.1%. The properties of the heparin-derived polysaccharide mixture were determined as follows:

In addition, the content of benzethonium chloride in the final product is 0. 7wt%, the content of the benzyl alcohol introduced by the benzyl chloride is about 0. 3wt, does not meet the requirements of the injection grade enoxaparin sodium, brings product safety Hidden dangers. Comparative Example 7 Application of heparin benzyl ester salt

The heparin benzyl ester sodium salt (10 g) obtained in Comparative Example 3 was dissolved in water (250 ml) to give a pale yellow mixture, which was then warmed to 65 °C. Sodium hydroxide (1.2 g) was added to the solution, and the reaction was carried out for 1.8 hours to degrade the heparin benzyl ester sodium salt, and the reaction liquid became a yellow turbid liquid. The solution was cooled to about 20 ° C and hydrochloric acid was added for neutralization. Additional sodium chloride was added to bring the sodium chloride concentration in the reaction mixture to 10% (w/v). Methanol (750 ml) was added to the reaction mixture to precipitate a precipitate, which was filtered to give a filter cake. After washing with methanol, the filter cake was dissolved in 100 ml of water. The pH was adjusted to 8.4 ± 0.1 by using 1M NaOH. The sulphate was used to remove the excess amount of sodium borohydride (as a percentage of 10 _g / _{gram of} heparin benzyl ester sodium salt), and the solution was allowed to stand for 1 hour, and then the pH was adjusted to 4. 0-4. Boron hydride. The pH was adjusted to 7.0 with 2M Na0H. Then, a solution volume of 3 times by volume of methanol was added to precipitate a precipitate, which was filtered to obtain a cake, which was washed with methanol and dried in vacuo to give a low molecular weight heparin (white, 7.3 g). 8%。 The yield was 66.8%.

By comparison, the color is deeper than the European Pharmacopoeia's color requirements for enoxaparin sodium, and molecular weight distribution and other indicators. Does not meet the requirements of the European Pharmacopoeia for enoxaparin sodium.

In addition, the content of benzethonium chloride in the final product is 0. 6wt%, the content of the benzyl alcohol introduced by the benzyl chloride is about 0. 3wt, does not meet the requirements of the injection grade enoxaparin sodium, brings product safety Hidden dangers. Comparative Examples 1 to 3 Compared with Examples 2 to 9, it is indicated that even if the amount of alcohol is increased by rinsing the filter cake during filtration or by repeatedly suspending the precipitate with sodium acetate methanol solution, a large amount of the intermediate heparin benzyl ester sodium salt remains. The quaternary ammonium salt, benzyl chloride and sodium acetate will introduce impurities into the final product, and it is difficult to precisely control the esterification rate of heparin benzyl ester sodium salt.

Comparative Examples 5 to 7 compared with Examples 10 to 18, indicating that if the quality of the sodium salt of heparin benzyl ester is different, particularly the purity and the accuracy of the esterification rate are different, it is difficult to control the degree of degradation of the sodium salt of heparin benzyl ester. The molecular weight distribution, color and impurity residue of the prepared low molecular weight heparin are difficult to meet the requirements. Example 19 Pharmaceutical Composition

Preparation of injection (equivalent to 50mg heparin-derived polysaccharide mixture / branch)

50 g of the heparin-derived polysaccharide mixture prepared in Example 10 was dissolved in 400 ml of water for injection, and the volume was adjusted to 500 ml, sterilely filtered, and filled into 1000 pieces, which were applied after passing the quality test. Example 20 Pharmaceutical Composition

Preparation of injection (equivalent to 25mg heparin-derived polysaccharide mixture / branch)

25 g of the heparin-derived polysaccharide mixture prepared in Example 17 was dissolved in 400 ml of water for injection, and the volume was adjusted to 500 ml, sterilely filtered, and filled into 1000 pieces, which were applied after passing the quality test. All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the In addition, it is to be understood that various modifications and changes may be made by those skilled in the art after the above-described teachings of the present invention, which are also within the scope of the appended claims.

Claims

Rights request

A method for preparing a high-purity heparin benzyl ester alkali metal salt or an alkaline earth metal salt, comprising the steps of:

The method according to claim 1, wherein the alkali metal salt is sodium chloride, sodium acetate, potassium chloride or sodium sulfate; and the alkaline earth metal salt is calcium chloride or magnesium chloride.

Lw/v% 〜95w/v%。 The aqueous solution of the alkali metal salt or the alkaline earth metal salt is 0. lw / v% ~ 95w / v%.

1〜10: 1; The alkali metal, the weight ratio of the alkali metal salt or the alkaline earth metal salt to the heparin benzyl ester quaternary ammonium salt is 0. 1~10: 1; 1〜10: 1。 The weight ratio of the salt or alkaline earth metal salt and the quaternary ammonium salt of the quaternary ammonium salt is 0. 1~10: 1.

The preparation method according to claim 1, wherein the solvent of the aqueous solution is water or an aqueous solution containing methanol, ethanol, acetone or a combination thereof.

6. The method according to claim 1, wherein the insoluble solvent is selected from the group consisting of alcohols, ketones, or a combination thereof.

A high-purity heparin benzyl alkali metal salt or alkaline earth metal salt characterized in that the content of the impurity quaternary ammonium salt is < 5 wt%.

8. A method of preparing a heparin-derived polysaccharide mixture, comprising the steps of:

(b) in the reaction mixture formed in the step (a), adding an insoluble solvent for the heparin benzyl ester alkali metal salt or alkaline earth metal salt, thereby precipitating and separating the heparin benzyl ester alkali metal salt or alkaline earth metal salt;

(c) Alkaline degradation of a heparin benzyl ester alkali metal salt or an alkaline earth metal salt to obtain the heparin-derived polysaccharide mixture.

9. A heparin-derived polysaccharide mixture characterized by having the following characteristics:

It is prepared by the method of claim 8;

The weight average molecular weight is not more than 8000 Daltons;

The activity ratio of anticoagulant factor Xa to anticoagulant factor I la is not less than 1. 5;

At its non-reducing end, it contains a 4-pyranuronic acid structure.

A pharmaceutical composition comprising, as an active ingredient, the heparin-derived polysaccharide mixture of claim 9 and a pharmaceutically acceptable carrier.