RU2670767C1 - 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

The technical field to which the invention relates.

The invention relates to the technology of pharmaceutical substance of low molecular weight heparin (LMWH) alkaline depolymerization of benzyl ester of commercial high molecular weight heparin sodium.

Brief Description of the Drawings

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

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

FIG. 3 shows the structure of dalteparin.

FIG. 4 shows the scheme of enzymatic depolymerization in the synthesis of tinse-parin.

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

FIG. 6 shows a diagram of the reactions occurring in the preparation of heparinate benzene.

FIG. 7 shows the scheme of benzylation reactions of heparinate benzethonium (GB) and removal of the protection of sulfo groups.

FIG. 8 given the scheme of reactions occurring during epimerization and cyclization.

The level of technology

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, thrombus formation during operations on the heart and blood vessels, in acute myocardial infarction, as well as to maintain the liquid state of the blood in cardiopulmonary bypass and hemodialysis.

Commercial unfractionated heparin (UFH) is a mixture of sulfated polysaccharides of various structures (Fig. 1) with a molecular weight of from 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. The so-called controlled depolymerization of heparin [3] under the action of inorganic or organic substances, enzymes or radiation is more effective in this respect. Currently implemented methods of controlled depolymerization can be reduced to the 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 release of nitrous acid on UFH, the breaking of glycosidic bonds of the main chain occurs [5] with the formation of hydroxyl and aldehyde groups which are reduced, for example, with sodium borohydride [6] as schematically shown in FIG. 2. In this way, for example, dalteparin is obtained, which has an average molecular weight of about 6.0 kDa and anhydro terminal terminal fragments (Fig. 3).

The enzymatic depolymerization is carried out under the influence of, for example, microbial heparinases (heparin lyase), specifically destroying the α-glycosidic bonds between N-sulfated D-glucosamine-6-sulfate and 2-O-sulfate of iduronic acid. In this way, LMWH tinzaparin is obtained with an average molecular weight of about 4.5 kDa and terminal 2-N, 6-O-disulfo-D-glucosamine and 4,5-unsaturated acid-acid fragments [7] (Fig. 4) At present, methods are known enzymatic depolymerization of UFH with the help of 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 targeted destruction of UFH, which involves the preparation of derivatives of UFH with their subsequent alkaline hydrolysis. Typically, a process of this kind involves the preliminary protection of the sulfo groups of UFH by reaction with benzene chloride and the subsequent conversion of carboxyl groups to ester due to the esterification of the resulting product with benzyl chloride. The resulting benzyl ester of heparinate benzethonium is precipitated, for example, with ethanol [9], followed by removal of protection from the SO ₃ ^- groups, and the resulting partial ester is subjected to alkaline hydrolysis [5]. At the same time, NMH molecules are formed with 2-O-sulfo-4-enopyranosuronic and 2-N, 6-O-disulfo-D-glucosamine terminal fragments (Fig. 5). In this way, ultra low molecular weight semloparin is obtained, as well as such LMWHs as bemiparin and enoxaparin. Additionally, depolymerization of this kind can be stimulated by microwave irradiation.

Derivative NFG is obtained in a similar way - by reaction with benzene 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 in relatively mild conditions, thereby reducing the proportion of adverse reactions and preserving the biological activity of heparin.

The closest to the technical nature of the claimed invention is a method of obtaining, disclosed in the description and claims to the patent [9], including the stage of obtaining benzethonium salt of heparin, benzylation of this salt in a non-aqueous solvent, alcohol precipitation of partial benzyl ether of benzethonium salt of heparin and alkaline depolymerization product, characterized in that the benzethonium salt of unfractionated heparin receive 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 medium with benzyl chloride in a ratio of heparin / benzyl chloride 1 / (0, 2-1.0), which is preliminarily subjected to activation in an aprotic solvent for 15–20 minutes, precipitation of heparin benzyl ester is carried out by the Spiro method with ethyl alcohol, previously saturated with anhydrous sodium acetate, followed by removal of the protection from the sulfo groups, 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. Isolation of the benzyl ester of the benzethonium salt is carried out by precipitation with ethanol in the presence of a sodium acetate co-precipitator, and the removal of the sulfo group protection is carried out with a saturated ethanolic 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:

- high water consumption for washing heparinate benzethonium from excess benzethonium chloride, the residual amount of which at subsequent stages of the synthesis of LMWH significantly complicates the process and leads to significant losses of the target product, since the reaction products are sticky pasty substances;

- carrying out the processes of precipitating the benzyl ether of the benzethonium salt and the subsequent procedure of removing the protection of heparin sulfo groups with the same reagent without isolating the intermediate product does not result in a structured product and increases the total duration of the process;

- isolation 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 shortcomings of the closest analogue, in particular, a decrease in the content of impurities in the finished product, an increase in the yield of intermediates at certain stages of production, a reduction in the consumption of wash water. The advantages of the proposed technical solution are the reduction of the complexity of the method and the cost of producing the finished LMWH.

Disclosure of the invention.

The purpose of the present invention is achieved by using the method of obtaining low molecular weight heparin, which includes stages:

(a) the formation of protection of sulfo groups by the interaction of high molecular weight heparin with benzethonium chloride to form benzethonium heparinate (GB),

(b) the esterification of the resulting salt by benzylation in an aprotic solvent,

(c) isolating heparin benzyl ester by precipitating and removing the sulfo group protection with a saturated solution of sodium acetate in methyl alcohol,

(d) alkaline depolymerization of the heparin macromolecule and

(e) the formation of terminal 1,6-anhydrogroups by β-elimination when interacting with a strong reducing agent,

wherein in stage (a) hepatinate benzethonium is washed from excess unreacted benzethonium chloride by repeated fractional washing with purified water using ultrasound operating frequency 30-40 kHz, radiation power 200-400 W and at stage (c) separation of heparin benzyl ester sedimentation and removal of the protection of sulfo groups are carried out as two successive operations, and the alcohol used is methanol.

Preferably, multiple fractional washing with the use of 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 for a duration of each ultrasound cycle of 10-35 minutes.

It is also preferable to separate the partial benzyl ester of heparinate benzene from the solution by precipitation with 1.5-2.5 volumes of 8-10% methanolic sodium acetate solution at a temperature of -2 to 4 ° C with stirring for 4-6 hours.

Next, it is preferable to remove the benzethonium protection of the sulfo groups after separation of the precipitate by reacting with a 4-6-fold mass amount of a saturated methanolic solution of anhydrous sodium acetate at a temperature of 18-24 ° C with stirring for 8-10 hours.

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

As a result of extensive research conducted, the inventors unexpectedly found that when washing benzethonium chloride using ultrasound, its frequency and power are crucial to the quality of the semi-product obtained. With an operating frequency higher than 40 kHz and a radiation power of more than 400 W, there is a danger of unpredictable destruction not only in the polymer chain, but also in individual links of monosaccharides, which leads to a loss of activity of the finished product.

The nature of the alcohol solvent is also of great importance to achieve the technical result of the invention. In the closest analogue [9], 96% ethanol is used to isolate intermediates from working solutions at different stages of the process. At the same time, an oil-like, sticky and viscous hygroscopic substance is initially released, which cannot be extracted from the process equipment without significant losses. To obtain a powder, it is necessary to repeatedly grind this substance with a precipitant to obtain a satisfactory quality amorphous powder.

In the proposed method, at the stage of obtaining benzyl ester of benzethonium salt, the process of separating the benzylation product from the solution with unremoved benzethonium protection of the sulfo groups by precipitation with methanol is carried out as a separate operation, with a small amount of sodium acetate being used as co-precipitant.

In accordance with the invention, the process of removing benzethonium protection is isolated in a separate stage. The chemistry of this reaction is the interaction of the benzethonium compound with sodium acetate. In the analogue of [9], the processes of isolation and removal of protection 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 release of the substance, since in one portion of the reagent solution is not enough.

The basis of heparin contains three sulfo groups, and accordingly, three equivalents of sodium acetate must be consumed to remove the benzethonium protection from the macromolecule. In methanol, the solubility of sodium acetate is 16.22% by weight, which is seven times greater than in ethanol. Therefore, the precipitator consumption is much smaller. In addition, significantly better sediment quality is unexpectedly achieved.

Thus, the technical result of the present 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 flow of wash water and the resulting effluent.

The implementation of the invention

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

Example 1. Synthesis of heparinate benzene

The transformation scheme is shown in FIG. 6. In a 250 ml glass two-neck flask placed in a water bath on a magnetic thermostatted agitator platform, 50 ml of purified water is poured and 8.4 g of benzethonium chloride (Hyamine 1622, Lonza Group Ltd.) are loaded with stirring. After complete dissolution, the pH is adjusted to 6.0-6.7.

In another glass, a solution of 3.2 g of mucosal heparin in 30 ml of 0.2 M NaCl solution is prepared at room temperature. After complete dissolution of 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 into the reactor with stirring, avoiding foaming of the contents of the flask, the heparin solution is poured in for 30-40 minutes. After adding the total amount of solution, the reaction is continued for another 50-60 minutes. The resulting precipitate is hot separated by filtration on a Buchner funnel or centrifuged. The liquid phase is discarded, pre-measuring its volume and optical density for the quantitative determination 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 (type UZV, 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 for the duration of each ultrasound cycle of exposure to 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 a calibration graph. Washing is repeated until the optical density of the wash 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 in already 7 washing cycles, which consumes 430 ml of purified water. The washed product - heparinate benzethonium (GB) is freeze dried (lyophilic dryer Iney-6, FSBUN IBP RAS). The output of GB 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 flushing water 350 ml.

** including with wash water 3.035 g.

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

* including wash water 700 ml.

** including with wash water 3,424 g.

As can be seen from the above data, the use of washing with ultrasonic treatment leads to a significant intensification of the process, reducing the consumption of purified water for washing GB, the amount of wastewater and labor costs for carrying out the process twice.

Example 2. Synthesis of incomplete benzyl ester of heparin

The synthesis scheme is shown in FIG. 7

2.1. Synthesis of benzyl ester heparinate benzethonium

In a three-neck flask with a capacity of 1 l, equipped with a mechanical stirrer and hydrolock, placed in a thermostatically controlled water bath at 37 ± 1 ° C, pour 250 ml of N, N-dimethylformamide (DMF) and add with stirring 45.0 g of heparinate benzene prepared using method described in example 1. After complete dissolution of 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. Separation of benzyl ester of heparinate benzethonium from the solution is carried out by precipitation of 525 ml of 9% methanolic sodium acetate solution at a temperature of 2-4 ° C with stirring for 4-6 hours. The precipitate is separated on a Buchner funnel and wet is used at the stage of removal of benzethonium protection.

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

2.2. Removing the protection of the sulfo groups of the benzyl ester of heparinate benzene

In a conical flat-bottomed flask with a capacity of 500 ml, placed on a magnetic stirrer platform, 24.0 g of the benzyl heparinate benzethonium benzyl ester prepared according to Example 2.1 was charged and 100 ml of a saturated methanolic solution of anhydrous sodium acetate was poured. An 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 process is completed, the precipitate is separated on a Buchner funnel, and dried in a vacuum oven at a temperature not higher than 40 ° C.

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

Example 3. Hydrolysis of benzyl ester of heparin sodium

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

At the end of the exposure, cool the reaction mass to room temperature, adjust the pH to 6.0 ± 0.2 by adding hydrochloric acid (1: 3), and filter the solution. 1.6 g of anhydrous sodium acetate are added to the clear yellowish filtrate and the pH is adjusted to 6.0 ± 0.2, if necessary. 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 is precipitated with chilled methanol (250-300 ml). The solution is placed in a fridge and left to mature for 3-4 hours. The supernatant is decanted, and the precipitate formed is 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 higher than 40 ° C. The yield of hydrolyzed heparin is 11.3 g, which is 84.3% of the theoretical.

Example 4. Borgohydriana raw low molecular weight sodium heparin

In a glass with a capacity of 100 ml, equipped with a stirrer, 50 ml of purified water are poured and 11.0 g of the product obtained in Example 3 is added with stirring. After the powder is completely dissolved, the pH is set 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 is adjusted to 4.0 ± 0.2 by adding hydrochloric acid (1: 3) with stirring for 10 minutes, after which by adding 1 M sodium hydroxide solution the pH is set to 6.0 ± 0.2. The resulting solution is filtered through a 0.45 µm membrane. 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 cooled methanol (150-200 ml). The solution is placed in the fridge and left to mature for 3 hours. The supernatant is decanted, and the precipitate formed is further washed with 30 ml of methanol. The precipitate is separated on a Buchner funnel and dried in vacuum at a temperature not higher than 40 ° C. The mass of the finished product is 9.7 g.

The resulting substance is characterized by the indicators given 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. Use of Heparins in Clinical Practice / Breast Cancer, 1998, No. 3, p. four.

3. Kudryavtsev V.N. / Chemistry of High Energies, 1993, Vol. 27, No. 1, p. 41.

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

5.Linhardt R.J. et. al. 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, which includes stages: (a) the formation of protection of sulfo groups by the interaction of high molecular weight heparin with benzethonium chloride to form benzethonium heparinate (GB), (b) the 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 by β-elimination when interacting with a strong reducing agent, characterized in that in step (a) washing benzethonium heparinate from excess unreacted benzethonium chloride is performed by repeated fractional washing with purified water using ultrasound of an operating frequency of 30-40 kHz, radiation power 200-400 W, and at the stage (c) separation of the heparin benzyl ester is carried out in two successive operations: extraction of the benzethonium heparinate benzethonium ester from the solution by precipitation 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 with the use of 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 for the duration of each cycle of ultrasonic exposure 10-35 minutes 3. The method according to p. 1, characterized in that the separation of benzyl ester of heparinate benzethonium from solution is produced by precipitation of 1.5-2.5 volumes of 8-10% methanolic solution of sodium acetate at a temperature of from -2 to 4 ° C with stirring during 4-6 hours. 4. A method according to claim 1, characterized in that the removal of the benzethonium protection of sulfo groups is carried out after separation of the precipitate by reacting with 4-6 times the mass of a saturated methanolic solution of anhydrous sodium acetate at a temperature of 18-24 ° C with stirring for 8-10 hours.