RU2725545C1 - Method of producing low-molecular heparin - Google Patents

Abstract

FIELD: chemistry.SUBSTANCE: invention relates to the technology of producing low-molecular heparin by alkaline depolymerisation of benzyl ester of high-molecular heparin sodium. Disclosed method involves preparing a benzethonium salt of heparin by reacting high-molecular heparin with benzethonium chloride to form benzethonium heparinate. Then benzotonium heparinate is washed from excess unreacted benzethonium chloride by multiple fractional extraction of the latter with purified water with intermediate filtration with water on a Buchner funnel with a paper filter or without intermediate product separation by filtration on a funnel using a laboratory device for washing off powdered products of chemical reactions from impurities. Esterification of the obtained benzethonium heparate by benzylation in an aprotic solvent and preparing the heparin benzyl ester in two steps: obtaining incomplete heparin benzyl ester by reacting with 16–17 wt% sodium acetate in methanol and depositing the product with a saturated solution of sodium acetate in methanol. Heparin macromolecule is decomposed by alkaline depolymerisation and formation of reducing and non-reducing end groups of hydrolysed heparin fragments by reaction with a strong reducing agent.EFFECT: invention is aimed at quality improvement due to reduction of content of impurities in the finished product, increase of output and reduction of labor intensity of production in comparison with analogue.4 cl, 5 dwg, 3 tbl, 4 ex

Description

FIELD OF THE INVENTION

The invention relates to a technology for the production of a pharmaceutical substance of low molecular weight heparin (NMH) by alkaline depolymerization of benzyl ether of commercial high molecular weight sodium heparin.

State of the art

Heparin is an organ preparation obtained from the lungs or mucose, the mucous membrane of cattle or pigs. It is a direct anticoagulant and is used for the preparation of dosage forms used for the prevention and treatment of thromboembolic diseases, thrombosis during operations on the heart and blood vessels, with acute myocardial infarction, and also to maintain the liquid state of the blood in cardiopulmonary bypass and hemodialysis machines.

Commercial unfractionated heparin (UFH) is a mixture of sulfated polysaccharides of various structures with a molecular weight of 3,000 to 30,000 Da (Fig. 1). The pharmacokinetics and pharmacodynamics of the drug, as well as the ability of heparin to interact with blood proteins and body cells, depends on the length of the heparin molecule and the magnitude of its charge [Kedik S.A. et al. Synthesis methods and properties of low molecular weight heparins / Biopharmaceutical Journal, 2018, V. 10, No. 2, p. 10-22].

Along with the undoubted advantages, UFH preparations have serious disadvantages, such as: the risk of hemorrhagic bleeding, allergic reactions, exacerbation of osteoporosis, the need for repeated injections, etc. [Lukashin B.P. et al. Heparin and heparinoids: sources, structure and biological effects / Bulletin of the Russian Military Medical Academy, 2007, No. 4 (20), p. 141-147]. Such shortcomings are deprived of preparations developed in recent years on the basis of substances of low molecular weight heparins (LMWH), among which preparations on the basis of the substances dalteparin (fragmentin), enoxaparin (clexane) and calcium nadroparin (fraksiparin) are most popular, the main characteristics of which are given below.

The closest in technical essence to the claimed invention is a method for producing low molecular weight heparin, disclosed in the document [patent RU2670767 C9, published 10.25.2018], including, in particular:

- at the stage of obtaining benzetonium heparin, repeated washing of benzetonium heparin is carried out from excess benzethonium chloride, which is accompanied by repeated filtration of the target product on a Buchner funnel with a paper filter;

- the stage of synthesis of heparin benzyl ester is carried out in two successive stages of precipitation of benzetonium heparin benzyl ester (BEGB) with filtration on a Buchner funnel and obtaining an incomplete heparin benzyl ester by reacting BEGB with a saturated solution of sodium acetate in methanol.

Among the disadvantages of this method, it should be noted that repeated washing of benzetonium heparin with intermediate filtration and reloading of wet sediment for the next operation leads to a significant loss of the target product. The same should be noted when carrying out the benzelation stage of benzetonium heparin.

Another significant disadvantage of this method is that the product obtained at the stage of benzelation contains a large amount of impurities of benzetonium acetate and sodium acetate, which greatly complicates the subsequent processes and leads to the loss of the target product.

Disclosure of the invention

The technical task of the invention was to overcome the disadvantages of the closest analogue.

The technical result consists in reducing the content of impurities in the finished product, increasing the yield of intermediates at certain stages of production and reducing the complexity of production.

The technical problem is solved and the technical result is achieved by the method of obtaining low molecular weight heparin, including the stage, in accordance with which:

(a) get the benzethonium salt of heparin by the interaction of high molecular weight heparin with benzetonium chloride to form benzetonium heparin,

(b) washing the benzethonium heparin from the excess of unreacted benzethonium chloride by repeated fractional extraction of the latter with water purified with intermediate filtration using a Buchner funnel with a paper filter or without intermediate isolation of the product by filtration on a funnel using a laboratory device for washing powder of chemical products from impurities,

(C) carry out the esterification of the obtained benzethonium heparin benzylation in an aprotic solvent,

(g) receive heparin benzyl ester in two stages: obtaining a partial heparin benzyl ester by interaction with 16-17 weight. % solution of sodium acetate in methanol and precipitation of the product with a saturated solution of sodium acetate in methanol,

(e) degradation of the heparin macromolecule by alkaline depolymerization and

(e) form reducing and non-reducing terminal groups of hydrolyzed heparin fragments by interaction with a strong reducing agent.

In one embodiment of the method, at the stage of producing a partial heparin benzyl ester by benzylation of benzetonium heparin followed by interaction with a saturated solution of sodium acetate in methanol, the benzetonium heparin benzyl ester is intermediately precipitated.

In yet another embodiment of the method, the resulting partial heparin benzyl ester is purified from impurities of sodium acetate and benzethonium chloride by extraction with methanol in a Soxhlet extractor.

And in yet another embodiment of the method, the reduction reaction of the end groups of hydrolyzed heparin fragments is carried out by reaction with a strong reducing agent in the presence of an active adsorbent and a strong electrolyte.

Brief Description of the Drawings

In FIG. 1 shows the chemical structure of a fragment of a heparin macromolecule.

In FIG. 2 shows a reaction scheme for the production of benzethonium heparin (establishment of sulfo group protection).

In FIG. 3 shows a diagram of a benzethonium heparin benzelation reaction.

In FIG. 4 shows a reaction scheme for removing the benzetonium protection of sulfo groups.

In FIG. 5 shows a reaction scheme for alkaline hydrolysis.

The implementation of the invention

The following examples demonstrate the feasibility of the invention to achieve a technical result. These examples are illustrative and in no way should limit the claimed scope of claims.

Example 1. Obtaining benzethonium heparin (GB). The reaction scheme is shown in FIG. 2.

In a beaker with a capacity of 250 ml placed on an adjustable heating plate with a magnetic stirrer drive, 65 ± 0.5 ml of 0.2 M sodium chloride solution is poured and 5.0 g of commercial high molecular weight heparin is added (MM _cf = 15000 Da). The temperature is increased to 35-40 ° C and dissolve within 35-45 minutes. After complete dissolution of heparin establish pH = 8.5 ± 0.1.

In another glass, a solution of 9.15 g of benzetonium chloride in 60 ± 0.5 ml of water purified at room temperature with stirring is prepared, avoiding foaming of the solution. After complete dissolution, establish pH = 5.8 ± 0.5. The solution is placed in a dropping funnel. The temperature in a glass with a heparin solution is increased to 60 ± 1 ° C and a solution of benzetonium chloride is added dropwise with stirring for 30-45 minutes. The reaction is continued for another 50-60 minutes.

The resulting suspension is quantitatively transferred to a 150 ml Schott funnel placed in an ultrasonic bath of a laboratory device for washing powder products of chemical reactions from impurities (LUUZ) [patent RU 186728 U1, published January 30, 2019], and sounding is carried out at a temperature of 50-55 ° C for 15-20 minutes. After the end of the process, the solution is aspirated into a receiving flask and the benzetonium chloride content is determined in it according to the calibration schedule. For subsequent washes, a device with a precipitate is poured into a Schott funnel with 50 ml of purified water and the process is repeated. Washing is continued until the residual content of the washed benzetonium chloride in the wash water is less than 7 mg / ml (Table 2).

As a result of washing, 20.9 g of a wet product are obtained, which is discharged into a Petri dish and dried in a freeze dryer (Iney-6, FGBUN IBP RAS). The yield of benzetonium heparinate (GB) with a moisture content of 7.0% is 15.5 g.

Example 2

2.1. Benzethonium Heparinate Benzylation

The reaction scheme is shown in FIG. 3.

In a two-necked flask with a capacity of 500 ml, placed in a water bath on the platform of an adjustable heating tile with a magnetic stirrer drive, 75 ml of N, N-dimethylformamide are poured and 15.5 g of benzetonium heparin obtained in the previous stage are loaded therein (in example 1). The throat is drowned with a ground stopper. Set the temperature to 37 ± 1 ° C and mix the contents until the GB is completely dissolved. In another throat, a dropping funnel is installed, into which 86 ml of benzyl chloride (BH) is poured and after complete dissolution of the GB, BH is added dropwise for 30-45 minutes. The reaction is continued at room temperature for 12 hours. After completion of the reaction, the contents of the reactor are cooled to room temperature and the benzethonium protection of the sulfo groups is removed. For this, two volumes of saturated 16.5 weight are added dropwise to the resulting clear solution with stirring. % solution of sodium acetate in methanol (16.5 g of sodium acetate / 100 g of methanol) with vigorous stirring. The resulting suspension was stirred for 2 hours and the precipitate was filtered off on a Buchner funnel. The wet precipitate is transferred to the stage of purification from benzetonium acetate and excess sodium acetate. The amount of sediment containing incomplete benzyl ester of high molecular weight heparin and an impurity of benetonium acetate and sodium acetate is 21 ± 3 g.

2.2. Purification of heparin benzyl ester from impurities.

The scheme for producing benzethonium heparin is shown in FIG. 4.

Partial benzyl ether is purified from excess sodium acetate and benzetonium acetate by extraction of impurities in a Soxhlet extractor. Sediment from the previous stage is placed in the cartridge of the apparatus, and 250 ml of dry methanol are poured into the cube of the extractor, heating is turned on and benzetonium acetate and sodium acetate are extracted, periodically sampling the extract to determine the content of sodium acetate in the extract. The extraction process is completed with a residual sodium acetate content of less than 0.7 mg / ml in the extract.

Purified from impurities, the powder is dried in vacuum at a pressure of 0.09 ± 0.05 MPa and a temperature of 30 ± 3 ° C. The yield of dry incomplete heparin benzyl ester is 6.27 g.

Example 3. The hydrolysis of a partial benzyl ester of heparin.

The hydrolysis reaction scheme is shown in FIG. five.

In a glass with a capacity of 250 ml, placed on the platform of a magnetic stirrer, pour 100 ml of purified water and load 6.27 g of incomplete benzyl ester of heparin therein. The anchor of a magnetic stirrer is placed in a glass and the powder is dissolved with stirring at a temperature of 35 ± 5 ° C. After the powder is completely dissolved, the temperature of the solution is increased to 60 ± 2 ° C and pH = 11-11.5 is set with a freshly prepared 0.5 M NaOH solution. 3 g of prepared filter perlite (GOST 30566) and 350 mg of NaCl are added to the solution, and 18-20 ml of a freshly prepared 1 M NaOH solution are added dropwise with a dropping funnel, maintaining the pH at 11-11.5. The hydrolysis reaction is continued for 45 minutes, after which pH = 6 ± 0.2 of 10% HCl is adjusted. The resulting suspension is filtered on a Buchner funnel with a white ribbon filter. The transparent filtrate is cooled in a refrigerator to a temperature below 10 ° C and precipitated with two volumes of cold methanol. The resulting suspension is left to ripen for 5-10 hours. The precipitate is filtered off on a Buchner funnel with a white ribbon filter and dried in vacuum at 40 ° C to constant weight. The product yield is 4.8 g.

Example 4. Borohydrogenation of raw low molecular weight sodium heparin.

50 ml of purified water is poured into a 100 ml beaker equipped with a stirrer and 4.8 g of the product obtained in Example 3 are added with stirring. After complete dissolution of the powder, pH = 7.6 ± 0.2 is added and 0.80 g is added. sodium borohydride. The reaction is continued for 50-60 minutes At the end of the reaction, pH = 4.0 ± 0.2 was added by adding 10 weight. % hydrochloric acid with stirring for 10 min, after which the addition of a 1M sodium hydroxide solution set the pH = 6.0 ± 0.2. The resulting solution is filtered through a membrane with a pore size of 0.45 μm. The filtrate is cooled to a temperature of 0-4 ° C for 30-60 minutes. The cooled filtrate is placed in a 200 ml beaker and the product is precipitated with chilled methanol (150-200 ml). The solution was placed in a refrigerator and left to mature the precipitate for 3 hours. The supernatant was decanted and the precipitate formed was washed twice with 30 ml of ethanol to wash it from methanol impurities. The precipitate is separated on a Buchner funnel and dried in vacuum at a temperature of 35 ± 2 ° C to constant weight. The product yield is 4.6 g.

Table 3 shows the results of the analysis of the obtained sample, carried out according to the methods described in the Global Fund XIV 2018 [XIV edition of the State Pharmacopoeia of the Russian Federation, T.1 (2018)].

Thus, the method proposed in the present invention allows to increase the yield of the finished product (up to 15-20%) by reducing losses in the separation and purification of intermediate products from impurities, as well as to improve quality and significantly reduce the complexity of the process.

Claims (10)

1. The method of obtaining low molecular weight heparin, characterized in that it includes the following stages, in accordance with which: (a) get the benzethonium salt of heparin by the interaction of high molecular weight heparin with benzetonium chloride to form benzetonium heparin, (b) washing the benzethonium heparin from the excess of unreacted benzethonium chloride by repeated fractional extraction of the latter with water purified with intermediate filtration using a Buchner funnel with a paper filter or without intermediate isolation of the product by filtration on a funnel using a laboratory device for washing powder of chemical products from impurities, (C) carry out the esterification of the obtained benzethonium heparin benzylation in an aprotic solvent, (d) get heparin benzyl ester in two stages: obtaining a partial heparin benzyl ester by reaction with a 16-17 wt.% solution of sodium acetate in methanol and precipitating the product with a saturated solution of sodium acetate in methanol, (e) degradation of the heparin macromolecule by alkaline depolymerization and (e) form reducing and non-reducing terminal groups of hydrolyzed heparin fragments by interaction with a strong reducing agent. 2. The method according to p. 1, characterized in that at the stage of obtaining an incomplete benzyl ester of heparin by benzylation of benzetonium hepinate, followed by interaction with a saturated solution of sodium acetate in methanol, without intermediate precipitation of benzethonium heparin benzyl ester. 3. The method according to p. 1, characterized in that the resulting partial heparin benzyl ester is purified from impurities of sodium acetate and benzetonium chloride by extraction with methanol in a Soxhlet extractor. 4. The method according to p. 1, characterized in that the reaction of the restoration of the end groups of hydrolyzed fragments of heparin is carried out by interaction with a strong reducing agent in the presence of an active adsorbent and a strong electrolyte.

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