RU2670767C9 - Method for producing low molecular weight heparin - Google Patents

Abstract

FIELD: chemical-pharmaceutical industry.SUBSTANCE: invention relates to method for producing low molecular weight heparin, which can be used in the chemical and pharmaceutical industry. Method includes the steps of: (a) shaping the protection of sulfo groups by the interaction of high molecular weight heparin with benzethonium chloride to form heparinate benzethonium, (b) esterification of the resulting salt by benzylation in an aprotic solvent, (c) isolating an incomplete heparin benzyl ester with removal of the benzethonium protection of the sulfo groups with a saturated solution of sodium acetate in alcohol, (d) cleavage of the macarolecule of heparin by alkaline depolymerization and (e) formation of terminal 1,6-anhydrogroups β-elimination when interacting with a strong reducing agent, and differs in that that at stage (a) the washing of heparinate benzethonium from an excess of unreacted benzethonium chloride is performed by repeated fractional washing with purified water using ultrasound of an operating frequency of 30–40 kHz radiation power of 200–400 W, and at the stage (c) the isolation of the heparin benzyl ester is carried out in two successive operations: the separation of the benzyl ester benzyl ester from solution by precipitation with a methanolic solution of sodium acetate, followed by removal of the benzethonium protection of sulfo groups with a saturated methanol solution of sodium acetate.EFFECT: new effective method for obtaining low molecular weight heparin with improved purity and yield while reducing water consumption and reducing waste is proposed.4 cl, 4 ex, 8 dwg, 3 tbl

Description

FIELD OF THE INVENTION

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

Brief Description of the Drawings

In FIG. 1 shows the structure of a fragment of a high molecular weight heparin molecule.

In FIG. 2 shows a scheme for the production of NMH by oxidative depolymerization.

In FIG. 3 shows the structure of dalteparin.

In FIG. 4 shows the scheme of enzymatic depolymerization in the synthesis of tinza-parine.

In FIG. 5 shows the structure of a fragment of the NMG macromolecule (enoxaparin) obtained by alkaline hydrolysis.

In FIG. 6 shows a diagram of the reactions occurring upon receipt of benzethonium heparin.

In FIG. 7 is a diagram of the reactions of benzylation of benzetonium heparin benzylation (GB) and deprotection of sulfo groups.

In FIG. 8 is a diagram of the reactions occurring during epimerization and cyclization.

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, in acute myocardial infarction, as well as for maintaining 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 (Fig. 1) with a molecular weight of 3,000 to 30,000 Da. The pharmacokinetics and pharmacodynamics of the drug, as well as the ability of heparin to interact with blood proteins and body cells, depend on the length of the heparin molecule and the magnitude of its charge [1]. Along with the undoubted advantages, UFH preparations have serious disadvantages, such as the possibility of uncontrolled bleeding, allergic reactions, osteoporosis, the need for repeated injections, etc. [2].

Fractionation of natural heparin by known methods (gel filtration, dialysis, etc.) gives a low yield of the target fraction with a low molecular weight and high anticoagulant activity. In this respect, the so-called controlled depolymerization of heparin [3] is more effective under the action of inorganic or organic substances, enzymes, or radiation. Currently implemented methods of controlled depolymerization can be reduced to processes of hydrolysis, oxidative, radical or enzymatic degradation.

Oxidative depolymerization of UFH consists in the treatment of its aqueous solutions with various oxidizing agents, of which sodium nitrite is most often used [4]. As a result of the action of nitrous acid released on UFH, the glycosidic bonds of the main chain break [5] to form hydroxyl and aldehyde groups which are reduced, for example, with sodium borohydride [6] as shown schematically in FIG. 2. Thus, for example, dalteparin is obtained, which has an average molecular weight of about 6.0 kDa and anhydromannose terminal fragments (Fig. 3).

Enzymatic depolymerization is carried out under the influence, for example, of microbial heparinases (heparin lyases), which specifically destroy α-glycosidic bonds between N-sulfated D-glucosamine-6-sulfate and iduronic acid 2-O-sulfate. Thus, NMH tinzaparin with an average molecular weight of about 4.5 kDa and terminal 2-N, 6-O-disulfo-D-glucosamine and 4,5-unsaturated uronic acid fragments is obtained [7] (Fig. 4). Currently, methods are known. enzymatic depolymerization of UFH using other heparinases [8]. The advantages of enzymatic depolymerization include mild synthesis conditions and high selectivity of the process.

Hydrolytic depolymerization is a specific method of directed destruction of UFH, which involves the preparation of derivatives of UFH with their subsequent alkaline hydrolysis. Typically, this kind of process involves the preliminary protection of UFH sulfogroups by reaction with benzetonium chloride and the subsequent conversion of carboxyl groups to ester groups due to the esterification of the resulting product with benzyl chloride. The resulting benzethonium heparinate benzyl ester is precipitated, for example, with ethanol [9] followed by deprotection of the SO ₃ ^- groups, and the resulting partial ester is subjected to alkaline hydrolysis [5]. In this case, NMH molecules are formed with 2-O-sulfo-4-enopyranosuronic and 2-N, 6-O-disulfo-D-glucosamine end fragments (Fig. 5). Thus, ultra-low molecular weight semuloparin is obtained, as well as LMWHs such as bemiparin and enoxaparin. Additionally, depolymerization of this kind can be stimulated by microwave irradiation.

The derivative of UFH is obtained in a similar way - by reaction with benzetonium chloride and subsequent esterification with benzyl chloride. The resulting product is subjected to hydrolytic depolymerization in organic solvents such as formamide, dimethylformamide or methylene chloride, and strong bases of the phosphazene family are used as a catalyst. It is believed that the process in a non-aqueous medium takes place under relatively mild conditions, which reduces the proportion of adverse reactions and preserves the biological activity of heparin.

Closest to the technical nature of the claimed invention is a production method disclosed in the description and claims of the patent [9], which includes the steps of producing the benzethonium salt of heparin, benzylation of this salt in a non-aqueous solvent, the alcohol deposition of a partial benzyl ester of the benzethonium salt of heparin and alkaline depolymerization of this product, characterized in that the benzethonium salt of unfractionated heparin is obtained in a 0.05-0.5 M aqueous solution of sodium chloride at a temperature of 50-60 ° C, pH in the range of 8.2 -8.8 and a mass ratio of heparin / benzethonium chloride 1 / (2.35-2.70), benzylation of the benzethonium salt of heparin is carried out for 2-3 hours in a bipolar aprotic solvent with benzyl chloride in the ratio of heparin / benzyl chloride 1 / (0, 2-1.0), which is preliminarily subjected to activation in an aprotic solvent for 15-20 minutes, the precipitation of heparin benzyl ester is carried out by Spiro ethyl alcohol, previously saturated with anhydrous sodium acetate, followed by deprotection from sulfo groups, carrying out β-elimination benzyl ester with a degree of esterification of the heparin 9-13% 1 ± 0.5 N NaOH alkali at a temperature of 55 ± 5 ° C, the processing time of 40-60 minutes and a weight ratio of benzyl ester reactants / alkali 1 / (0.5-2).

The benzylation step is carried out in a mixture of aprotic solvents. The isolation of the benzyl ester of the benzethonium salt is carried out by ethanol precipitation in the presence of sodium acetate co-precipitator, the sulfo groups are deprotected by the action of a saturated ethanol solution of the same reagent. The process of washing benzethonium salt from impurities is carried out by treatment with distilled water.

Among the disadvantages of this method should be noted:

- a large flow of water for washing benzetonium heparin from an excess of benzetonium chloride, the residual amount of which in the subsequent stages of the synthesis of NMH significantly complicates the process and leads to significant losses of the target product, since the reaction products are sticky pasty substances;

- the process of precipitation of benzyl ester of benzethonium salt and the subsequent procedure for removing the protection of heparin sulfogroups by the same reagent without isolation of the intermediate product does not lead to a structured product and increases the total process time;

- the selection of the finished product with 96% ethanol is complicated by the high hygroscopicity of the powder.

The aim of the present invention is to overcome the disadvantages of the closest analogue, in particular - reducing the content of impurities in the finished product, increasing the yield of intermediates at certain stages of production, reducing the consumption of washing water. The advantages of the proposed technical solution are the reduction of the complexity of the method and the cost of manufacturing the finished NMG.

Disclosure of the invention.

The purpose of the present invention is achieved using a method of producing low molecular weight heparin, comprising the steps of:

(a) formation of sulfo group protection by the interaction of high molecular weight heparin with benzetonium chloride to form benzetonium hepinate (GB),

(b) esterifying the obtained salt with benzylation in an aprotic solvent,

(c) isolating heparin benzyl ester by precipitation and deprotecting the sulfo groups with a saturated solution of sodium acetate in methyl alcohol,

(g) alkaline depolymerization of a heparin macromolecule; and

(e) the formation of terminal 1,6-anhydro groups by β-elimination by reaction with a strong reducing agent,

in which, at stage (a), benzethonium heparin is washed from excess unreacted benzetonium chloride by repeated fractional washing with water purified using ultrasound at an operating frequency of 30-40 kHz, a radiation power of 200-400 W, and at stage (c), heparin benzyl ester is isolated precipitation and deprotection of sulfo groups is carried out as two sequential operations, and the alcohol used is methanol.

Preferably, multiple fractional washing using ultrasound is carried out with purified water at a mass ratio of GB / water equal to 1 / (50-60) and a temperature of 40-60 ° C with a duration of each cycle of ultrasonic exposure of 10-35 minutes.

It is also preferable to isolate the benzethonium heparin incomplete benzyl ester from the solution by precipitation of 1.5-2.5 volumes of an 8-10% methanol solution of sodium acetate at a temperature of from -2 to 4 ° C. with stirring for 4-6 hours.

It is further preferred that the benzethonium protection of the sulfo groups is removed after separation of the precipitate by reaction with a 4-6-fold mass amount of a saturated methanol solution of anhydrous sodium acetate at a temperature of 18-24 ° C. with stirring for 8-10 hours.

In addition, preferably, the alkaline depolymerization step is carried out in the presence of an inert perlite carrier in a weight ratio of heparin / perlite benzyl ester 1 / (0.25-0.5).

As a result of extensive research, the inventors unexpectedly found that when washing benzetonium chloride with ultrasound, its frequency and power are critical to the quality of the resulting intermediate. At an operating frequency above 40 kHz and a radiation power of more than 400 W, there is a danger of unpredictable degradation not only in the polymer chain, but also in individual units of monosaccharides, which leads to a loss of activity of the finished product.

The nature of the alcohol solvent is also of great importance for achieving the technical result of the invention. In the closest analogue [9], 96% ethanol is used to isolate intermediates from working solutions at various stages of the process. In this case, an oil-like, sticky and viscous hygroscopic substance that is impossible to extract from technological equipment without significant losses is initially released. To obtain a powder, it is necessary to grind this substance with a precipitant repeatedly until an amorphous powder of satisfactory quality is obtained.

In the proposed method, at the stage of producing benzethonium salt benzyl ester, the process of isolating a benzylation product from the solution with the non-removed benzethonium protection of sulfo groups by methanol precipitation is carried out as a separate operation, and a small amount of sodium acetate is used as a precipitant.

In accordance with the invention, the process of removing the benzethonium protection is isolated in a separate stage. The chemistry of this reaction consists in the interaction of the benzethonium compound with sodium acetate. In analogue [9], the processes of isolation and deprotection are carried out by repeated washing with ethanol saturated with sodium acetate (its maximum concentration is 2.33% by weight). This requires three to four times the treatment of the product with this solution with an intermediate allocation of substance, because in one portion of the reagent solution is not enough.

The basic mole of heparin contains three sulfo groups, and, accordingly, to remove the benzethonium protection from the macromolecule, three equivalents of sodium acetate must be consumed. In methanol, the solubility of sodium acetate is 16.22% by mass, which is seven times more than in ethanol. Therefore, the consumption of precipitant is much smaller. In addition, a significantly better sludge quality is unexpectedly achieved.

Thus, the technical result of the invention is an improved method for producing low molecular weight heparin with improved purity and overall yield of the substance with a significant reduction in the consumption of washing water and the resulting effluent.

The implementation of the invention

Further, the possibility of carrying out the invention with the achievement of a technical result will be shown in non-limiting examples.

Example 1. Synthesis of benzetonium heparin

The transformation scheme is shown in FIG. 6. In a 250-ml glass two-necked flask placed in a water bath on the platform of a thermostatically controlled magnetic stirrer, pour 50 ml of purified water and load with stirring 8.4 g of benzethonium chloride (Hyamine 1622, Lonza Group Ltd.). After complete dissolution, the pH is adjusted in the range of 6.0-6.7.

In another glass, a solution of 3.2 g of mucosal heparin in 30 ml of a 0.2 M NaCl solution is prepared at room temperature. After complete dissolution of the heparin, the pH is adjusted in the range of 8.5-8.7.

The temperature of the water bath is raised to 60 ± 1 ° C and, using a dropping funnel, the heparin solution is poured into the reactor with stirring, avoiding foaming of the contents of the flask, for 30-40 minutes. After adding the entire amount of the solution, the reaction is continued for another 50-60 minutes. The precipitate formed is hot separated by filtration on a Buchner funnel or centrifuged. The liquid phase is discarded, having previously measured its volume and optical density to quantify the content of impurities.

The precipitate is transferred to a glass with a capacity of 100 ml, 50 ml of purified water is added and placed in an ultrasonic bath (ultrasound type, sapphire). Extraction of excess unreacted reagents (washing) is carried out at a temperature of 40-60 ° C and exposure to ultrasound with an operating frequency of 30-40 kHz and a radiation power of 200-400 W with a duration of each cycle of ultrasonic exposure of 10-35 minutes. The degree of washing and the content of impurities in the wash water is determined by measuring the optical density of the solution (D ₂₇₀ ) spectrophotometrically (Cary 60 UV-Vis, Agilent) at a wavelength of 270 nm according to the calibration graph. Washing is repeated until the optical density of the washing water is less than 1.5. The results of the experiment with the use of ultrasound (US) are shown in Table 1. As can be seen from these data, a satisfactory result is achieved for 7 washing cycles, which consumes 430 ml of purified water. The washed product is benzetonium heparin (GB), freeze-dried (Iney-6 freeze dryer, FSBII IBP RAS). The GB yield is 8.23 g with a moisture content of 5.3%. The consumption of purified water per 1 g of powder is 52.2 ml.

* including wash water 350 ml.

** including with wash water 3.035 g.

In parallel, washing is carried out under the same conditions without the use of ultrasound (table 2). Moreover, to achieve the desired result, it is necessary to carry out 14-15 washing cycles, and the purified water consumption is 700 ml. The GB powder yield is 7.9 g with a moisture content of 5.1%. The consumption of purified water per 1 g of product is 88.6 ml.

* including wash water 700 ml.

** including 3.424 g.

As can be seen from the above data, the use of washing using ultrasonic treatment leads to a significant intensification of the process, reducing the consumption of purified water for washing GB, the amount of effluent and labor costs for the process by half.

Example 2. The synthesis of partial benzyl ester of heparin

The synthesis scheme is shown in FIG. 7.

2.1. Synthesis of benzethonium heparin benzyl ester

In a three-necked flask with a capacity of 1 l, equipped with a mechanical stirrer and a water lock, placed in a thermostatic water bath at a temperature of 37 ± 1 ° С, 250 ml of N, N-dimethylformamide (DMF) were poured and 45.0 g of benzetonium heparinate obtained by the method described in example 1. After complete dissolution of the GB in a flask with a solution using a dropping funnel add 40 ml of benzyl chloride. The reaction is continued at a temperature of 37 ± 1 ° C and stirring for 8 hours after which the contents of the flask are cooled to room temperature. Isolation of benzethonium heparinate benzyl ester from solution is carried out by precipitation of 525 ml of a 9% methanol solution of sodium acetate at a temperature of 2-4 ° C with stirring for 4-6 hours. The precipitate is separated on a Buchner funnel and wet used at the stage of removal of the benzethonium protection.

The yield of wet benzethonium heparin benzyl ester is 24.4 g with a heparin benzyl ester content of 16.8 g.

2.2. Deprotection of benzogonium hepinate benzyl ester sulfo groups

A 500 ml conical flat-bottomed flask placed on a magnetic stirrer platform was charged with 24.0 g of wet benzethonium hepinate benzyl ester obtained in Example 2.1, and 100 ml of a saturated methanol solution of anhydrous sodium acetate were added. The anchor of the magnetic stirrer is placed in the flask and closed with a ground stopper. The reaction is carried out at room temperature with stirring for 8-10 hours. After the end of the process, the precipitate is separated on a Buchner funnel, and dried in a vacuum oven at a temperature not exceeding 40 ° C.

The yield of incomplete benzyl ester of heparin is 16.2 g, which is 96% of theoretical.

Example 3. Hydrolysis of heparin sodium benzyl ester

The synthesis scheme is shown in FIG. 8. In a 250 ml beaker placed in a water bath on the platform of a thermostatically controlled magnetic stirrer, 75 ml of purified water is poured and 16.0 g of heparin benzyl ester obtained as described in Example 2 are loaded with stirring. The temperature is set to 60 ± 1 ° C and mix until the powder is completely dissolved. Then, the solution is adjusted to pH 11.0-11.1 with a freshly prepared 1 M NaOH solution and 8 ml of 1 M sodium hydroxide solution are added. Then, with the help of a dropping funnel, an additional 8 ml of the same solution is dosed for 30-45 minutes. The reaction is continued for 45 minutes, maintaining the pH not lower than 11.0.

At the end of the exposure, the reaction mixture is cooled to room temperature, the pH is adjusted to 6.0 ± 0.2 by the addition of hydrochloric acid (1: 3), and the solution is filtered. 1.6 g of anhydrous sodium acetate are added to the clear yellowish filtrate and, if necessary, the pH is adjusted to 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 and the product precipitated with chilled methanol (250-300 ml). The solution is placed in the refrigerator and left to mature the precipitate for 3-4 hours. The supernatant was decanted, and the precipitate formed was further washed with 50-60 ml of methanol. The precipitate is separated on a Buchner funnel and dried in a vacuum oven at a temperature not exceeding 40 ° C. The yield of hydrolyzed heparin is 11.3 g, which is 84.3% of theory.

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 11.0 g of the product obtained in Example 3 are added with stirring. After complete dissolution of the powder, the pH is adjusted to 8.4 ± 0.2 and 0.20 g of borohydride is added. sodium. The reaction is continued for 50-60 minutes At the end of the reaction, the pH was adjusted to 4.0 ± 0.2 by the addition of hydrochloric acid (1: 3) with stirring for 10 minutes, after which the pH was adjusted to 6.0 ± 0.2 by adding a 1 M sodium hydroxide solution. 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 glass with a capacity of 200 ml and the product is precipitated with chilled methanol (150-200 ml). The solution was refrigerated and left to mature for 3 hours. The supernatant was decanted and the precipitate formed was washed further with 30 ml of methanol. The precipitate is separated on a Buchner funnel and dried in vacuum at a temperature not exceeding 40 ° C. The mass of the finished product is 9.7 g.

The resulting substance is characterized by the indicators shown in table 3.

Literature

1. Hirsh J. et al. Clinicalof Chest Physicians Evidence-Based Parenteral Anticoagulants: American College Practice Guidelines (8th Edition) / Chest, 2008, V. 133, pp. 141S-159S.

2. Makarov B.A. et al. The use of heparins in clinical practice / breast cancer, 1998, No. 3, p. four.

3. Kudryavtsev V.N. / High Energy Chemistry, 1993, T. 27, No. 1, S. 41.

4. Copyright certificate SU 1570651 A3, publ. 06/07/1990.

5.Linhardt R.J. et. al. Production and chemical processing of low molecular weight heparins / Seminars in Thrombosis and Hemostasi, 1999, V. 25, N. 3, pp. 5-16; US patent 6384021 B1, publ. 05/07/2002.

6. Linhardt R.J. et. al. Differential anticoagulant activity of heparin fragments prepared using microbial heparinase / Journal of Biological Chemistry, 1982, V. 257, N. 13, pp. 7310-7313.

7. Patent RU 2295538 C2, publ. 03/20/2007.

8. Patent RU 2396282 C1, publ. 08/10/2010.

9. Patent RU 2512768 C1, publ. 04/10/2014.

Claims (9)

1. The method of obtaining low molecular weight heparin, comprising the steps of: (a) formation of sulfo group protection by the interaction of high molecular weight heparin with benzetonium chloride to form benzetonium hepinate (GB), (b) esterifying the obtained salt with benzylation in an aprotic solvent, (C) the allocation of an incomplete complex benzyl ester of heparin with the removal of the benzetonium protection of sulfo groups with a saturated solution of sodium acetate in alcohol, (d) cleavage of the heparin macromolecule by alkaline depolymerization and (e) the formation of terminal 1,6-anhydro groups by β-elimination when interacting with a strong reducing agent, characterized in that, in step (a), benzethonium heparin is washed from excess unreacted benzetonium chloride by repeated fractional washing with purified water using ultrasound with an operating frequency of 30-40 kHz, with a radiation power of 200-400 W, and at stage (c) the isolation of heparin benzyl ester is carried out in two successive operations: the isolation of benzethonium heparin benzyl ester from solution by precipitation of anolnym sodium acetate, followed by removing the protecting benzetonievoy sulfo a saturated methanolic solution of sodium acetate. 2. The method according to p. 1, characterized in that multiple fractional washing using ultrasound is carried out with purified water at a mass ratio of GB / water equal to 1 / (4-6) and a temperature of 40-60 ° C with a duration of each cycle of ultrasonic exposure of 10-35 minutes. 3. The method according to claim 1, characterized in that the isolation of benzethonium heparin benzyl ester from the solution is carried out by precipitation of 1.5-2.5 volumes of an 8-10% methanolic solution of sodium acetate at a temperature of from -2 to 4 ° C with stirring 4-6 hours. 4. The method according to p. 1, characterized in that the removal of the benzethonium protection of the sulfo groups is carried out after separation of the precipitate by interaction with a 4-6-fold mass quantity of a saturated methanol solution of anhydrous sodium acetate at a temperature of 18-24 ° C with stirring for 8-10 hours.

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