DK168824B1 - Process for Preparing Mixtures of Sulfated Polysaccharides with a General Heparin Polysaccharide Structure - Google Patents

Description

DK 168824 B1

The present invention relates to a process for the preparation of novel mixtures of sulfated polysaccharides having a general average molecular weight heparin polysaccharide structure with heparin, having an ethylenic double bond at one chain end and with the physicochemical and in the preamble of claim 1. biological properties, the process of which is characterized by the characterizing part of claim 1. The mixtures can be used as anticoagulant and antithrombotic agents for the prevention and treatment of thrombosis and as hypolipemic agents.

Heparin is a mixture of sulfated mucopolysaccharides of animal origin that is predominantly used for its anticoagulant and antithrombotic properties, especially for the prevention of post-operative venous thrombosis, and because of its hypo-lipemic properties. It is known to act in the coagulation process by activating a natural blood clotting inhibitor, antithrombin III. The activation of this protein has the effect of inhibiting the action of two proteases, the X-activated factor (factor Xa) and the thrombin-20 bin.

The in vitro anticoagulant effect of heparin is generally measured by the official methods of the pharmacopoeia, in particular the American, English or French pharmacopoeia, by comparing it to an international standard. But it is now possible to measure its specific activities in vitro, partly in relation to factor Xa and partly in relation to thrombin (cf., e.g., A. Teien et al., Thrombosis Research, 11, 107, 1977 ).

The industrial heparins, which are mixtures of polysaccharides, whose average molecular weight by weight is greater than 30 10,000 daltons and whose molecular weight distribution ranges from 4,000 to approx. 45,000 daltons, exhibits the following activities in vitro:

Anticoagulant effect measured by the method of the French Pharmacopoeia, 8th edition, heparin monograph, by comparing DK 168824 B1 2 with the third international standard (or anticoagulant activity code): 140 - 200 i.u./mg.

Anti-Xa activity measured by the method of Teien et al. (mentioned above) when compared to the third international standard: 160 - 180 i.u./mg.

A.P.T.T. activity (activated partial thromboplastin time) measured by the method of Teien et al. (mentioned above) when compared to the third international standard: 150 - 170 i.u./mg.

10 Heparin is obligately administered parenterally (in practice by subcutaneous route) and the effect is relatively short, giving the following two important drawbacks: The need to perform three administrations per second. per day and the relatively high frequency of post-operative bleeding accidents.

15 It is known (cf. Johnson et al., Thrombos. Haemostas. Stuttgart, 1976, 35, 586-591, Lane et al., Thrombosis Research 16, 651-662, Pergamon Press Ltd., 1979, Lasker , Chiu, Annals, NY, Acad.Sci., 222, 1973, 971-977, and Lasker, Adv. Exp. Med. Biol., 52, 1975, 119-130), that by fractionating heparin, 20 f. eg. by filtration over "Sephadex" gel, fractions having an average molecular weight less than the molecular weight of heparin and having a molecular weight distribution less than that of the heparin can be obtained. Tests carried out in vitro and in vivo show that such fractions, on the one hand, are relatively more active against the X-activated factor than against thrombin (i.e., they exhibit an anti-Xa activity greater anti-thrombin activity re than 1) and, on the other hand, are more readily absorbed into the circuit from a subcutaneous injection than the heparin itself, which is why they have a higher and more prolonged plasmatic activity than heparin.

It is also known (cf. Lasker & Chiu, cited above, Lasker, cited above, U.S. Patent No. 3,766,167, Perlin et al., Carbo-DK 168824 B1 3 hydrate Research, 18, 1971, 185-194 ) by enzymatic hydrolysis of heparin depolymerization products having a low average molecular weight (in practice, 5,300 - 4,500 daltons, determined by ultracentrifugation) and an anticoagulant activity, as determined by the method of US Pharmacopoeia No. XVII, of 70 i.u./mg. These depolymerization products can be fractionated by filtration over "Sephadex" gel into fractions whose average molecular weight, determined by ultracentrifugation, is from 3,200 to 5,900 daltons and whose anticoagulant activities are from 45 to 95 iu / mg (USP method) . These depolymerization products and the resulting fractions are active, taken both orally and parenterally.

It is also known (see Lasker, mentioned above) to depolymerize heparin by ascorbic acid and hydrogen peroxide 15 to produce low average molecular weight products. Depolymerization of the ion by means of ascorbic acid and hydrogen peroxide, after fractionation in alcohol, leads to fractions having an average molecular weight of 4,000 to 7,100 daltons and an acti-coagulation activity of 12-100 i.u./mg (U.S.P. method).

Finally, in the resulphation of depolymerization products of heparin which do not have anti-coagulant activity, it is known to produce oligopolysaccharides having an average molecular weight, as determined (see British Patent Application No. 2,002 to 406A, published 21/2 1979). by the Somogy method, of 2,600 -25,500 daltons, a specific rotary ability measured in aqueous solution at 20 ° C of + 40 ° - + 50 °, and an anti-coagulation activity less than 50 iu / mg (USP method) that would be as active, taken orally as parenterally, to prevent thrombosis. However, these oligopolysaccharides exhibit a less favorable ratio of anti-Xa activity to anti-thrombin activity than the sulfated polysaccharides produced by the practice of the present invention.

The mixtures of sulfated polysaccharides prepared by the process according to the invention are mixtures of sulfated polysaccharides which have the general structure of polysaccharides constituting heparin, ie. exhibit the same elemental bonds as the polysaccharides constituting heparin, which elemental bonds are bound in the same manner as in the latter. However, unlike the polysaccharides which make up heparin, the polysaccharides of the present invention comprise an ethylenic double bond at one of the chain ends.

In the compositions of the invention, the acid groups of the sulfated polysaccharides may be in free form or in the form of a salt, especially in the form of a sodium, calcium or magnesium salt.

The mixtures prepared according to the invention exhibit in the form of the sodium salt the following properties: 15% sulfur: 9% - 13.5%% nitrogen: 1.8% - 2.5%% uronic acids: 20% - 30% Weight average molecular weight: 2,000 - 7,000 daltons Specific rotating ability in aqueous solution at 20 ° C: 20 M10: + 25 ° - + 55 °.

The sulfur content and nitrogen content as well as the rotating ability shown above have been determined by the methods of the French Pharmacopoeia, 8th edition, heparin monograph. The content of uronic acids has been determined by the method of N.

25 Blumenkrantz et al. (Analytical Biochemistry, 54, 484, 1973). The average molecular weight by weight has been determined by gel-per-meation chromatography on polyacrylamide agarose gel by comparison with the standard constituted by heparins of known average molecular weight, according to the method of Andersson et al. (Thrombosis Research 9, 575, 1976).

DK 168824 B1 5

The polysaccharides constituting the compositions of the process according to the invention correspond in particular to the following formula:, R HRH'H NH-R₂ where R is a hydrogen atom or a carboxylic group in free acid state or in the form of a salt, R 'is an OH group or a sulfate group in free acid form or in the form of a salt, R 1 is an OH group or a sulfate group in free acid form or in the form of a salt, R 2 is a sulfonic acid group in free acid form or in the form of a salt or an acetyl group, -O- is an oxygen bridge, the G-10 bonds are glucosamine-type bonds present in the heparin structure, the U-bonds are uronic acid (D-glucuronic acid, L-iduronic acid, sulfated L) bonds. -iduronic acid) present in the structure of heparin and n is an integer from 3 to 20.

In the above formula I, as mentioned above, the acid groups in the polysaccharide may be in free form or in the form of a salt, especially the sodium, calcium or magnesium salt.

The present invention relates to the preparation of the mixtures which exhibit in the form of the sodium salt the additional 20 properties shown in the following Table A.

DK 168824 B1 6 TABLE A.

Average Molecular Weight 2,000-7,000 by Weight (in Dalton)

Anticoagulant activity 10-80 5 in vitro, codex

In vitro activity, 80-250 anti-Xa

In Vitro Activity, 10-80 A.P.T.T.

Ratio of activity in vitro, anti-Xa 2-10 activity in vitro, A.P.T.T.

The anti-coagulant activity code shown in the above table has been determined by the method of the 15th French Pharmacopoeia, 8th edition, heparin monograph. The anti-Xa and A.P.T.T. activity shown in Table A as well as those shown in subsequent Tables B and C have been determined by the method of Teien et al. (already mentioned) by the following procedure: 20 a) Determination of anti-Xa activity:

This activity is determined on a platelet-free bovine plasma by the chromogenic substrate S 2222 [chromogenic peptide of structure: (N-benzoyl) Ile-Glu-Gly-Arg (p-nitro) anilide] by comparison with the third international standard.

25 100 µl citrate beef plasma, diluted from 2 to 5 with an aqueous buffer Tris / EDTA, pH 8.4, is added to 100 μΐ of a solution of the product to be tested, or of the standard, in an aqueous buffer Tris / EDTA , pH 8.4, which 100 μΐ corresponds to 0.02 - 0.08 of the product or standard. After 3 minutes incubation time at 37 ° C, 100 μΐ of an aqueous solution of bovine Xa factor, corresponding to 7 n Cat of factor Xa, is added. After 30 seconds of incubation, 200 μΐ of 5 is added to an aqueous solution of S 2222, corresponding to 0.2 μπιοί S 2222. After 3 minutes of incubation, 300 μΐ of acetic acid is added and the optical density of the solution is measured at 405 nm, compared to distilled water. .

By deducing the optical density as a function of the concentration of the product or standard, two straight lines are obtained, one concerning the product to be examined and the other concerning the standard. The activity of the product, expressed in international units per mg, is given by the formula 173 slope of the straight line about the product to be examined for inclination of the straight line with respect to. the standard where the number 173 corresponds to the value of the activity for the thirty-third international standard.

b) Determination of A.P.T.T. activity:

The product to be tested and the third international standard dissolved in a 0.15 M aqueous solution of sodium chloride are then diluted with citrate-containing bovine plasma, free of platelets, so as to obtain concentrations of the product to be tested ( or of the standard) from 0 to 4 μg / ml.

Add 100 μΐ of the "Automated APTT Precibio" reagent (rabbit brain phospholipids reagent and micronized silicon dioxide reagent) to 100 μΐ of the resulting solution. After 25 5 minutes incubation at 37 ° C, 100 μΐ of a 0.025 M is added

aqueous solution of calcium chloride. Coagulation time is measured by a Bio-Mériux fibrometer.

By plotting the logarithm of the coagulation time as a function of the concentration of the product or of the standard, two straight lines are obtained, one concerning the product to be investigated and the other concerning the standard. The activity of the product, expressed in international units per mg, is given by the formula: 173 slope of the straight line vedx. the product to be won is sought inclination of the right line regarding. the standard 5 where the number 173 corresponds to the value of the activity of the third international standard.

All activities shown in Tables A, B and C are expressed in international units per year. mg (i.u./mg), compared to the third international standard.

The mixtures prepared by the process of the invention are prepared by the action of an inorganic or organic base on a heparin ester which results from partial or total esterification of the carboxyl groups in the heparin. In this ester, the acid groups in the heparin which are not esterified (i.e., the acid sulfate groups and optionally part of the carboxylic acid groups) may be in the free state or in the form of salts, in particular an alkali metal salt such as the sodium salt, an alkaline earth metal salt such as the calcium salt, magnesium salt or quaternary long chain ammonium salt such as the benzethonium salt.

In the case where the heparin ester is soluble in water (e.g., where the non-esterified acid groups are in the form of the sodium salt), the reaction between the ester and the base can be effected in water at a temperature of 20 - 80 ° C, molar concentration of the base in the medium is preferably between 0.1 and 0.6.

Bases which can be used are bases which are soluble in water, especially sodium hydroxide, potassium hydroxide, alkali metal carbonates, triethylamine, triethylethylenediamine, quinuclidine, 1,5-diaza-bicyclo [4,3,0] -5-nonene and 1, 5-diaza-bicyclo [5.4.0] -5-undecene. Once the reaction is complete, the resulting depolymerization product, e.g. by precipitation by the addition of DK 168824 B1 9 sodium chloride and then methanol.

Heparin esters which can be used in the process of the invention may be non-selective esters or selective esters. Non-selective esters are understood to mean heparin esters in which the 5 carboxyl groups of D-glucuronic acid, unsulfated L-iduronic acid and sulfated L-iduronic acid bonds are indistinguishably esterified. Selective esters are understood to mean heparin esters in which all or part of the carboxyl groups of the D-glucuronic acid linkages or only the carboxyl groups of the D-glucuronic acid and unsulfated L-iduronic acid bonds or the carboxyl groups of the unsulfated L-iduronic acid and sulfates are esterified. L-iduronic acid bonds or only the carboxyl groups of the sulfated L-iduronic acid bonds.

Heparin esters which can be used as starting products in the methods of the invention are in particular the heparin esters described in French Patent No. 2,150,724 and British Patent No. 1,501,095, as well as methyl, ethyl, ethoxycarbonylmethyl, cyanomethyl -, benzyl and substituted benzyl (especially the 4-chlorobenzyl, 4-nitrobenzyl) esters of heparin. As the starting product, benzyl or substituted benzyl esters of heparin are preferably used. The heparin esters used as starting substances in the methods of the invention can come from heparin of any origin (bovine lung heparin, pig mucosal heparin, bovine intestinal heparin, etc.).

The non-selective methyl, ethyl, ethoxycarbonylmethyl, cyanomethyl, benzyl and substituted benzyl esters of heparin can be obtained e.g. by reaction of a neutral quaternary ammonium or amine salt of heparin with a halogenated derivative of the formula Hal - CH 2 - R (II) wherein Hal is chloro, bromo or iodo, and R is a hydrogen atom or a methyl, ethoxycarbonyl, cyano, phenyl, or substituted phenyl group. This reaction is carried out in solution or in suspension in an inert solvent such as dimethylformamide, methylene chloride, dimethylsulfoxide, tetrahydrofuran or acetone at a temperature between -20 ° C and + 60 ° C.

Methyl, ethyl, ethoxycarbonylmethyl, cyanomethyl, benzyl and substituted benzyl esters of heparin wherein only or partially the carboxyl groups of the D-glucuronic acid bonds or only the carboxyl groups of the D-glucuronic acid and unsulfated L-iduronic acid rings are esterified , is obtained by reaction of a halo gene derivative of the above formula II with an acidic quaternary ammonium salt of heparin wherein, in addition to the sulfate groups, only the salt is formed either the carboxyl groups of the D-glucuronic acid binders or only the carboxyl groups of the D-glucuronic acid and unsulfated L -iduronic acid bonds, the other carboxyl groups being in free acid form. This reaction is carried out under the same conditions as the reaction of the halogen derivative of formula II with a neutral quaternary ammonium salt of heparin.

The acidic quaternary ammonium salts of heparin, in which, in addition to the sulfate groups, only the salt-formed carboxyl groups of the D-glucuronic acid bonds are prepared, by reaction of a quaternary ammonium salt with heparin in an aqueous medium whose pH is between 3 and 4.

The acidic quaternary ammonium salts of heparin, in which, in addition to the sulfate groups, only the salt-formed carboxyl groups of the D-glucuronic acid and unsulfated L-iduronic acid bonds are obtained by reacting a quaternary ammonium salt with heparin in an aqueous medium whose pH is low enough to form the quaternary ammonium salt of heparin in which only the sulfate groups are salt-formed (in practice, the pH is from 2 to 2.5), and then selectively neutralize the carboxyl groups of D-glucuronic acid and unsulfated L-iduronic acid bonds in the resulting product at addition of a certain amount of quaternary ammonium hydroxide in a medium of dimethylformamide.

The amount of quaternary ammonium hydroxide to be added is derived from the neutralization curve of a dimethylformamide medium for a sample of the product of known weight.

DK 168824 B1 11

The methyl, ethyl, ethoxycarbonylmethyl, cyanomethyl, benzyl and the substituted benzyl esters of heparin in which all or part of the carboxyl groups of the sulfated L-iduronic acid bonds only or the carboxyl groups 5 of the unsulfated L-iduronic acid and sulfated L-iduronic acid bonds, are prepared by reaction of an alcohol of the formula HO - CH 2 - R (III) wherein R is a hydrogen atom or a methyl, ethoxycarbonyl, cyano, phenyl or substituted phenyl group, with heparin in an aqueous medium in the presence of a water-soluble condensing agent of the carbodiimide type, e.g. 1-Ethyl-3- (3-dimethyl aminopropyl) carbodiimide, setting the pH of the medium to a value in the range 3.5 - 4.5 in the first case and in the range 2 - 3 in the second case. As the alcohol of formula III which may be used, in particular, methanol and ethanol may be mentioned, in which case a selective methyl ester of heparin and a selective ethyl ester of heparin are obtained, respectively.

The methods of the invention using the action of an inorganic or organic base on a heparin ester, which are / 3 elimination processes, allow a partial or controlled depolymerization of the heparin without altering its general structure.

The mixtures of sulfated polysaccharides prepared by the process of the invention have anti-coagulant, anti-thrombotic and hypolipemic activity. Furthermore, the mixtures have little toxicity. For example, the product of Example 9 is non-toxic at a dose of 300 mg / kg when administered intravenously to rats and mice. When administered subcutaneously, its toxicity is the same as that of heparin.

The mixtures of sulfated polysaccharides prepared by the process of the invention in which the acidic groups of the polysaccharides are in the form of pharmaceutically useful salts, especially in the form of the sodium, calcium or magnesium salt, can be used as anticoagulants and anti-thrombotic agents. for DK 168824 B1 12 prevention and treatment of thrombosis. They can also be used to treat hyperlipemia. They may advantageously be used in place of heparin for such use. In fact, when administered subcutaneously, they exhibit a longer-lasting effect than hepatrine, which reduces the frequency of injections. Furthermore, they produce fewer secondary effects (bleeding effects) than heparin.

They can be administered in admixture with a pharmaceutically usable carrier, intravenously, subcutaneously, via the lungs (inhalation), recum and, for mixtures of sufficiently low average molecular weight, orally. The doses administered depend on the mode of administration and the desired effect (anti-thrombotic or hypolipemic effect).

The following Examples 5-12 and 16-19 illustrate the invention.

The neutral benzethonium salt of heparin or benzethonium heparinate used as a starting material in Examples 1-3, 10, 12-15 is derived from porcine heparin having the following properties:

Average molecular weight by weight: 16,000 daltons 20 Specific rotation in aqueous solution at 20 ° C: [a] d °: + 41 °

Anti-coagulant activity code: 157 i.u./mg.

The neutral benzethonium salt of heparin or benzethonium heparinate used as a starting product in Examples 4-9 25 and 11 is derived from heparin from the intestine with the following properties:

Average molecular weight by weight: 11,400 daltons Specific rotation in aqueous solution at 20 ° C: [a] d °: + 37 °

Anti-coagulant activity code: 128 i.u./mg.

DK 168824 B1 13

The neutral benzethonium salt of heparin or benzethonium heparinate used as the starting product of Example 16 is derived from a pig mucosal heparin having an average molecular weight of 16,000 daltons, a specific rotation in aqueous solution at 20 ° C at + 44 ° and an anti-coagulant activity code of 180 iu / mg.

The sodium salt of heparin used as the starting product in Examples 17 - 19 corresponds to the aforementioned swine mucosa-10 heparin.

EXAMPLE 1.

30 g of (4-chloro) benzyl chloride are added to a solution of 30 g of benzethonium heparinate in 600 ml of dimethylformamide. After dissolving, the solution is allowed to stand for 60 hours at ambient temperature (about 20 ° C) and then 600 ml of a 10% solution of sodium acetate in methanol is added. The precipitate formed is separated by filtration, washed with methanol and dried in vacuo. Thereby, 10.75 g of the 4-chlorobenzyl ester of heparin are obtained in the form of the sodium salt.

The resulting ester is reacted with stirring with 269 ml of a 0.4N aqueous solution of sodium hydroxide at 25 ° C. After 2 hours, neutralization is done by the addition of a 0.4N aqueous solution of hydrochloric acid and precipitation is made by the addition of 2 volumes (ie twice the volume of the aqueous phase) of methanol. By filtration, 8.46 g of depolymerized heparin in the form of the sodium salt is isolated.

EXAMPLE 2.

30 g of benzyl chloride are added to a solution of 30 g of benzethoni umheparinate in 600 ml of dimethylformamide. After dissolution, the solution is left for 60 hours at ambient temperature, and precipitation is done by adding 600 ml of a 10% solution of sodium acetate in methanol. The precipitate is isolated by filtration, washed with methanol and dried in vacuo. Thereby 11.4 g of the benzyl ester of heparin is obtained in the form of the sodium salt.

3 g of the above ester are reacted for 2 hours with stirring with 75 ml of a 0.4N aqueous solution of sodium hydroxide at 20-25 ° C. The solution is then neutralized by the addition of a 0.4N aqueous solution of hydrochloric acid and precipitation is made by the addition of 2 volumes of methanol. Thereby 2.23 g of 10 depolymerized heparin in the form of the sodium salt is isolated.

EXAMPLE 3.

10 g of benzyl chloride are added to a solution of 10 g of benzethonium heparinate in 250 ml of dichloromethane.

After dissolving, the mixture is allowed to stand for 24 hours at ambient temperature and then the solvent is evaporated in vacuo. The residue is dissolved in 150 ml of dimethylformamide and precipitation is done by adding 150 ml of a 10% solution of sodium acetate in methanol. 3.67 g benzyl ester of heparin is separated by filtration in the form of the sodium salt.

20 g of the above ester are treated for 2 hours with stirring with 50 ml of a 0.1N aqueous solution of sodium hydroxide at 60 ° C. After cooling, the solution is neutralized by the addition of a 0.1N aqueous solution of hydrochloric acid and then precipitated by the addition of 2 volumes of methanol. 1.54 g of depolymerized heparin in the form of the sodium salt is thus obtained.

EXAMPLE 4.

5 g of ethyl chloroacetate are added to a solution of 5 g of benzethonium heparinate in 125 ml of dichloromethane and after dissolution, the solution is allowed to stand for 3 days at ambient temperature. The solvent is evaporated in vacuo, the residue is taken up in 75 ml of dimethyl formamide and precipitation is made by adding 75 ml of a 10% solution of sodium acetate in methanol. The precipitate, separated by filtration, is washed in methanol and dried in vacuo. There is thus obtained 1.72 g of the carbethoxymethyl ester of the heparin in the form of the sodium salt.

1.7 g of the above ester are treated with 43 ml of a 0.1N aqueous solution of sodium hydroxide at 60 ° C with stirring for 2 hours. After cooling, the solution is neutralized by the addition of a 0.1N aqueous solution of hydrochloric acid and precipitation is effected by the addition of 2 volumes of methanol. By filtration, 1.33 g of depolymerized heparin in the form of the sodium salt is isolated.

EXAMPLE 5.

10 g (4-chloro) benzyl chloride is added to a solution of 10 g benzethonium heparinate in 250 ml dichloromethane and dissolved by stirring. The solution is allowed to stand for 24 hours at ambient temperature and then the solvent is evaporated in vacuo. The residue is taken up in 150 ml of dimethylformamide and precipitation is done by adding 150 ml of a 10% solution of sodium acetate in methanol. After filtration, washing the precipitate with methanol and drying in vacuo, 3.84 g of the (4-chloro) benzyl ester of heparin in the form of the sodium salt is isolated.

2 g of the above ester are treated with 50 ml of a 0.1N aqueous solution of sodium hydroxide at 60 ° C with stirring for 2 hours. After cooling and neutralization with a 0.1N aqueous solution of hydrochloric acid, precipitation is made by adding 2 volumes of methanol. The precipitate is isolated by filtration, washed with methanol and dried in vacuo. Thereby, 1.38 g of depolymerized heparin is obtained in the form of the sodium salt.

EXAMPLE 6.

30 g of (4-nitro) benzyl chloride are added to a solution of 5 g of benzethonium heparinate in 125 ml of dichloromethane and dissolved by stirring. The solution is left to stand for 3 days at ambient temperature, then the solvent is evaporated in vacuo and the residue is dissolved in 75 ml of dimethylformamide. The ester formed is precipitated by the addition of 75 ml of a 10% solution of sodium acetate in methanol. The precipitate is collected by filtration, washed with methanol and dried in vacuo. Thereby, 1.89 g (4-nitro) benzyl ester of heparin is obtained in the form of the sodium salt.

1.85 g of the above ester are treated with 46 ml of a 0.1N aqueous solution of sodium hydroxide at 60 ° C for 2 hours with stirring. After cooling, neutralization is done by adding a 0.1 N aqueous solution of hydrochloric acid and precipitation is done by adding 2 volumes of methanol. The precipitate is isolated by filtration, washed with methanol and dried in vacuo. Thereby 1.13 g of depolymerized heparin is obtained in the form of the sodium salt.

EXAMPLE 7.

30 g of benzyl chloride are added to a solution of 30 g of benzethonium heparinate in 500 ml of dichloromethane and dissolved by stirring. The solution is allowed to stand at ambient temperature for 24 hours, 20 and then the solvent is evaporated in vacuo and the residue is taken up in 400 ml of ether. The insoluble material is separated by filtration. Thereby, 30 g of the benzyl ester of heparin is obtained in the form of the benzethonium salt. This ester is dissolved in 200 ml of dichloromethane containing 8 ml of 1,5-diaza-bicyclo [4,3,0] -5-nonene. The solution is heated under reflux for 3 1/2 hours and then the solvent is evaporated in vacuo. The residue is dissolved in 450 ml of dimethylformamide and an equal volume of a 10% solution of sodium acetate in methanol is added. The precipitate is collected by filtration and washed in methanol. It is then treated at 0 ° C for one hour with an 1N aqueous solution of sodium hydroxide. After neutralization, precipitation is made by adding 2 volumes of methanol. The precipitate is isolated by filtration, washed with methanol and dried in vacuo. 6.6 g of depolymerized heparin are obtained in the form of the sodium salt.

EXAMPLE 8.

10 g (4-chloro) benzyl chloride is added to a solution of 10 g benzethonium heparinate in 250 ml dichloromethane and dissolved by stirring. The solution is allowed to stand at ambient temperature 5 for 24 hours and then the solvent is evaporated in vacuo. The residue is taken up in 200 ml of ether and the precipitate formed is isolated by filtration. Thereby 10 g (4-chloro) benzyl ester of heparin is obtained in the form of the benzethonium salt.

Dissolve 5 g of this product in 100 ml of dichloromethane containing 1.5 ml of 1,5-diaza-bicyclo [4,3,0] -5-nonene and reflux for 4 hours. Then, the solvent is evaporated in vacuo, the residue is taken up in 30 ml of dimethylformamide and 100 ml of a 10% solution of sodium acetate in methanol is added. The precipitate formed is isolated by filtration, washed with methanol and treated with 24 ml of 1N aqueous solution of sodium hydroxide for one hour at 0 ° C. The solution is neutralized by the addition of an 1N aqueous solution of hydrochloric acid and precipitation is made by the addition of 2 volumes of methanol. The precipitate is separated by filtration. After washing with methanol and drying in vacuo, 1 g of depolymerized heparin is obtained in the form of the sodium salt.

EXAMPLE 9.

30 g of benzyl chloride are added to a solution of 30 g of benzethonium heparinate in 600 ml of dimethylformamide. After dissolution, the reaction participants were left in contact for 60 hours at ambient temperature, and then the ester formed precipitated by the addition of 1,200 ml of a 10% solution of sodium acetate in methanol. The precipitate is isolated by filtration, washed with methanol and dried in vacuo. Thereby 11.4 g of the benzyl ester of heparin is obtained in the form of the sodium salt.

30 g of the above ester are treated with 250 ml of a 0.1N van

you solution of sodium hydroxide at 60 ° C for 2 hours with stirring. After cooling, the solution is neutralized with a 0.1N

Aqueous solution of hydrochloric acid is precipitated and precipitation is made by adding 2 volumes of methanol. The precipitate is filtered, washed with methanol and dried in vacuo. 6.65 g of depolymerized heparin is thus obtained in the form of the sodium salt.

EXAMPLE 10.

120 g (4-chloro) benzyl chloride is added to a solution of 120 g benzethonium heparinate in 2.4 liters of dimethylformamide and dissolved by stirring. The solution is then allowed to stand at ambient temperature for 60 hours, then 2, 4 liters of a 10% solution 10 of sodium acetate in methanol are added. The precipitate formed is separated by filtration, washed with methanol and dried in vacuo. Thereby 46 g (4-chloro) benzyl ester of heparin is obtained in the form of the sodium salt.

20 g of the above ester are treated with 500 ml of a 0.1 N aqueous solution of sodium hydroxide at 60 ° C for 2 hours with stirring. After cooling and neutralization, precipitation is made by adding 2 volumes of methanol. By filtration, 11.7 g of depolymerized heparin in the form of the sodium salt is isolated.

EXAMPLE 11.

20 g of methyl iodide are added to a solution of 30 g of benzethonium heparinate in 750 ml of dichloromethane and dissolved by stirring. The solution is allowed to stand for 48 hours at ambient temperature and then the solvent is evaporated in vacuo. The residue is taken up in 450 ml of dimethylformamide and precipitation is made by adding 450 ml of a 10% solution of sodium acetate in methanol. After filtration, the precipitate is washed with methanol and dried in vacuo. Thereby, 10.5 g of the methyl ester of heparin is isolated in the form of the sodium salt.

2 g of the above ester are treated with 50 ml of a 0.1N aqueous solution of sodium hydroxide at 60 ° C with stirring for 2 hours. After cooling, the pH of the solution is adjusted to approx.

By stirring with a carboxyl ion-exchange resin in H + form. The resin is then separated by filtration and washed with water. The combined aqueous phases are neutralized by the addition of a dilute aqueous solution of sodium hydroxide and then lyophilized. Thereby, 2 g of depolymerized heparin is obtained in the form of the sodium salt.

EXAMPLE 12.

3 g of the (4-chloro) benzyl ester of heparin obtained in Example 10 is treated with 120 ml of a 10% aqueous solution of disodium carbonate at 60 ° C for 2 hours with stirring. After cooling, the solution is neutralized with a 0.4N aqueous solution of hydrochloric acid and precipitated by the addition of 2 volumes of methanol. 1.57 g of depolymerized heparin is isolated by filtration in the form of the sodium salt.

EXAMPLE 13.

30 g of ethyl chloroacetate are added to a solution of 30 g of benzethonium heparinate in 600 ml of dimethylformamide. After dissolution, the solution is left to stand at ambient temperature for 60 hours, then 600 ml of a 10% solution of sodium acetate in methanol is added. The precipitate formed is separated by filtration, washed with methanol and dried in vacuo. Thereby, 10.78 g of the carbethoxymethyl ester of heparin is obtained in the form of the sodium salt.

3 g of the above ester are contacted with 100 ml of a 3% aqueous solution of triethylamine at a temperature of 60 ° C. After 5 hours, the solution is neutralized by the addition of an aqueous solution of hydrochloric acid and then precipitated by the addition of 2 volumes of methanol. 2.5 g of depolymerized heparin is isolated by filtration in the form of the sodium salt.

EXAMPLE 14.

5 g of chloroacetonitrile are added to a solution of 5 g of benzethonium heparinate in 125 ml of dichloromethane and dissolved by stirring. The solution is allowed to stand for 48 hours at ambient temperature and then the solvent is evaporated in vacuo. The residue is dissolved in 75 ml of dimethylformamide and precipitation is done by adding 75 ml of a 10% solution of sodium acetate in methanol. The precipitate is separated by filtration, washed with methanol and dried in vacuo. Thereby 1.63 g of cyanomethyl ester 10 of heparin is obtained in the form of the sodium salt.

The resulting ester is treated with 40 ml of a 0.1N aqueous solution of sodium hydroxide at 60 ° C with stirring for 2 hours. After cooling, the solution is neutralized by the addition of a 0.1 N aqueous solution of hydrochloric acid and then precipitated by the addition of 2 volumes of methanol. By filtration, 1.33 g of depolymerized heparin in the form of the sodium salt is isolated.

EXAMPLE 15.

3 g of the (4-chloro) benzyl ester of heparin obtained in Example 10 is dissolved with stirring in 120 ml of a 10% aqueous solution of disodium carbonate. After stirring for 2 hours at a temperature of 20 to 25 ° C, the pH of the solution is adjusted to 6 by the addition of an N aqueous solution of hydrochloric acid and then 1 volume of methanol equal to twice the volume of the aqueous solution is added. The precipitate formed is isolated by filtration to give 2.1 g of the (4-chloro) benzyl ester of heparin.

2 g of the above ester are treated with stirring for 2 hours with 50 ml of a 0.1N aqueous solution of sodium hydroxide at 60 ° C. After cooling, the solution is neutralized by the addition of a 30 µl aqueous hydrochloric acid solution and then precipitated by the addition of 2 volumes of methanol. Thereby 1.4 g of depolymerized heparin is obtained in the form of the sodium salt.

Prior to precipitation of the product formed by the addition of the 2 volumes of methanol in the preceding Examples 1 - 10 and 12 - 15, the concentration of NaCl in the aqueous phase was adjusted to 10% by the addition of sodium chloride.

EXAMPLE 16.

10 g (4-chloro) benzyl chloride is dissolved with stirring in a solution of 10 g benzethonium heparinate in 250 ml dichloromethane. The solution is allowed to stand for 24 hours at ambient temperature, then a 10% solution of sodium acetate in 10 methanol is added. The resulting precipitate is filtered and washed with methanol. There is thus obtained 3.72 g (4-chloro) benzyl ester of heparin in the form of the sodium salt.

A solution of 0.500 g of the above ester in 10 ml of formamide is treated at 60 ° C for 5 hours with 0.5 ml of 1,5-diaza-bicyclo-15 [4,3,0] 5-nonene. After cooling, 70 ml of acetone is added. By filtration 0.364 g of a precipitate is collected. This precipitate is treated at 0 ° C for 2 hours with 6 ml of an N aqueous solution of sodium hydroxide. The aqueous phase is neutralized by the addition of an N aqueous solution of hydrochloric acid, and the concentration of NaCl in the medium is adjusted to 10% by the addition of sodium chloride. Precipitation is effected by the addition of 2 volumes of methanol. There is thus obtained 0.263 g of depolymerized heparin in the form of the sodium salt.

EXAMPLE 17

2.5 ml of acetic acid and then, slowly and with stirring, 150 ml of a 10% aqueous solution of benzethonium chloride are added to a solution of 10 g of heparin (sodium salt) in 40 ml of water. The formed precipitate is collected by centrifugation, washed with water and dried. 19.67 g of acidic benzethonium heparinate are obtained.

Dissolve 11 g of the above product in 110 ml of dimethylformamide and add 11 g (4-chloro) benzyl chloride. The reaction part is preferably left in contact for 48 hours at ambient temperature, then 220 ml of a 10% solution of sodium acetate in methanol is added. The precipitate formed is isolated by filtration, washed with methanol and dried in vacuo. There is thus obtained 4.70 g (4-chloro) benzyl ester of heparin in the form of the sodium salt.

Dissolve 4 g of the above ester in 20 ml of water and 40 ml of a 20% aqueous solution of benzethonium chloride are slowly added with stirring. The precipitate formed is collected by centrifugation, washed with water and dried in vacuo. Thereby, the (4-chloro) benzyl ester of heparin is obtained in the form of the benzethonium salt.

1 g of the above ester (benzethonium salt) is dissolved in 20 ml of dimethylformamide and treated with 1 ml of 1,5-diaza-bicyclo [4,3,0] - 5-nonene at 60 ° C for 5 hours. After cooling, 50 ml of 15 is added to a 10% solution of sodium acetate in methanol. The precipitate formed (0.346 g) is collected and treated at 0 ° C for 2 hours with 5.8 ml of an N aqueous solution of sodium hydroxide. The aqueous phase is neutralized by the addition of an N aqueous solution of hydrochloric acid, and the concentration of NaCl in the medium is adjusted to 20 to 10% by the addition of sodium chloride. Precipitation is done by adding 2 volumes of methanol. The precipitate is filtered and washed with methanol to give 0.253 g of depolymerized heparin in the form of the sodium salt.

EXAMPLE 18.

2.5 ml of formic acid and then, slowly and with stirring, 150 ml of a 10% aqueous solution of benzethonium chloride are added to a solution of 10 g of heparin (sodium salt) in 40 ml of water. The precipitate is collected by centrifugation, washed with water and dried in vacuo. Thereby 20.5 g of acidic benzethonium heparinate are obtained.

Dissolve 2.95 g of the above product in 60 ml of dimethyl formamide, and then 5.9 ml of a 0.1 N solution of tetrabutylammonium hydroxide in a mixture of n-propanol and methanol is added.

DK 168824 B1 23

After the addition of 2.95 g (4-chloro) benzyl chloride, the solution is left to stand for 5 days at ambient temperature. 74 ml of a 10% solution of sodium acetate in methanol is added.

1.32 g (4-chloro) benzyl ester of heparin is isolated by filtration 5 in the form of the sodium salt.

The above ester is dissolved in 6.4 ml of water and 12.8 ml of a 20% aqueous solution of benzethonium chloride is slowly added with stirring. The precipitate formed is collected by centrifugation, washed with water and dried in vacuo. Thereby, the (4-chloro) benzyl ester of heparin is obtained in the form of the benzethonium salt.

Dissolve 1 g of the above ester (benzethonium salt) in 20 ml of dichloromethane and add 1 ml of 1,5-diaza-bicyclo (4,3,0) - 5-nonene. The solution is heated at reflux for 5 hours, then the solvent is evaporated in vacuo, the residue is taken up in 15 ml of dimethylformamide and 20 ml of a 10% solution of sodium acetate in methanol is added. The precipitate formed is separated by filtration and washed with methanol. There is thus obtained 0.405 g of a product which is treated at 0 ° C for 2 hours with 6 ml of an N aqueous solution of sodium hydroxide. The aqueous phase is neutralized by the addition of an N aqueous solution of hydrochloric acid and the concentration of NaCl in the medium is adjusted to 10% by the addition of sodium chloride. The precipitate is made by adding 2 volumes of methanol. The precipitate is separated by filtration and washed with methanol. Thereby, 0.355 depolymerized heparin is obtained in the form of the sodium salt.

EXAMPLE 19

0.600 g of heparin (sodium salt) is dissolved in 7 ml of water and the pH of the solution is adjusted to 3.5 by the addition of an N aqueous solution of hydrochloric acid. 0.300 g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide are added and, after dissolution, the solution is allowed to stand for 1 hour at ambient temperature. Add 2.5 ml of an aqueous solution of sodium chloride containing 280 g per ml. liter, and then 15 ml of methanol. The precipitate formed is isolated by filtration, washed with methanol and dried in vacuo. There is thus obtained in the form of the sodium salt 0.527 g of the methyl ester of heparin, whose esterification percentage of the carboxyl groups is 50%.

Dissolve 0.3 0.3 g of the above ester in 7.5 ml of a 0.1N sodium hydroxide solution and heat the solution to 60 ° C for 2 hours. After cooling, the solution is neutralized by the addition of a 0.1N aqueous solution of hydrochloric acid, and the concentration of NaCl in the medium is adjusted to 10% by the addition of 10 sodium chloride, and precipitation is effected by the addition of 2 volumes of methanol. Thereby, 0.200 g of depo lymeris is a heparin in the form of the sodium salt.

The following Tables B and C indicate the properties of the products (depolymerized heparins in the form of the sodium salt) prepared in Examples 1-19. The percentages of sulfur, nitrogen and uronic acids, the specific rotational capabilities in aqueous solution at 20 ° C, the average molecular weight by weight, and the activities listed in the tables have been determined by the methods previously described. Column 20 entitled "Molecular Weight Distribution" lists the approximate extreme values of the molecular weight of the polysaccharides which make up the mixtures as determined by gel permeation chromatography on a polyacrylamide agarose gel. The viscosity column shows the viscosities at 25 ° C of a 10% aqueous solution of the products. In the UV absorption column, the absorbances are shown at a 1 cm thickness of a 1% solution of the product in 0.01N HCl, which absorptions are measured at the wavelength of the absorption maximum occurring in the range 220-232 nm.

DK 168824 B1 TABLE B.

Average% Molecular Weight Spread of

Ex. uric acid% S% N_Γα] n ° _ (in dalton) _ molecular weight 5 1 23.0 11.5 2.1 + 44 ° 7.300 1.600-12.000 2 26.0 11.6 2.0 + 44 ° 6,500 1.800-13.000 3 25.4 12.0 2.2 + 44 ° 6.400 3.000-11.000 4 23.9 10.9 2.1 + 35 ° 5.500 2.200-13.000 5 22.8 12.2 2.2 + 39 ° 5.000 2.000-13.000 10 6 24.5 10.8 2.3 + 33 ° 4.200 1,000-10.000 7 25.2 11.3 2.0 + 33 ° 4.100 1.700-10.000 8 23.7 11.1 2.2 + 35 ° 4.500 1.000 -10,000 9 26.0 11.8 2.2 + 38 ° 4,000 2,000-10,000 10 25.2 10.9 2.1 + 41 ° 3,800 1,000- 9,000 15 11 23.8 11.0 2.3 + 27 ° 2,800 1,000- 8,000 12 23,9 11,5 2,0 + 40 ° 4,000 2,000- 9,000 13 24,3 11,8 2,2 + 45 ° 9,000 3,000-20,000 14 25,6 12,0 2,1 + 44 ° 8,500 2,000-20,000 15 25.3 11.8 2.3 + 45 ° 4,000 2,000-20,000

TABLE B

(Continued).

Anti-koagul.

Activity Anti-Xa- A.P.T.T. Code Activity Activity Viscosity Absorption, in vitro in vitro in vitro

Ex. (centipoise) UV_ (i.ii./ma) (iu / ma) (iu / mg) 1 2.37 7.5 127 180 105 2 2.25 7.8 116 148 100 3 2.08 7.9 112 163 89 30 4 1.93 8.2 83 130 52 5 1.91 9.7 75 118 43 6 1.74 12.5 60 140 33 7 1.64 15.0 50 130 30 8 1.61 16.4 54 110 33 DK 168824 B1 26 9 1.62 14.0 80 159 45 10 1.57 15.4 62 159 40 11 1.50 22.8 40 90 20 12 1.60 14.4 75 140 35 5 13 2 , 60 5.9 135 140 130 14 2.50 6.1 138 130 130 15 _1.65_10.64_110_170_86 TABLE C.

Average s- Anti-coagulant activity Molecular Weight Absorption, Anti-Xa A.P.T.T. code

Ex. (in dalton) in vitro in vitro in vitro 16 4,200 6 140 63 60 17 5,500 7,96 165 80 70 18 4,400 6,3 110 60 40 15 19_5,000_10,9_150_45_50 Example 20.

The product of Example 1, on the one hand, and an industrial heparin, on the other, has been administered at various times by subcutaneous route to 5 healthy volunteers at a dose of 20 of 5,000 i.u. Code. On blood samples taken 1 hour, 3 hours, 5 hours and 7 hours after administration, the plasma anti-coagulant effect was measured by the anti-Xa and A.P.T.T. tests mentioned above. The results obtained have been expressed in international units per year. 25 ml by plasma compared to a standard curve, recorded on the basis of experiments performed with a control plasma to which known amounts of a reference heparin (third international standard) were added.

The obtained average results are shown in Table D.

Claims (2)

A process for preparing mixtures of sulfated polysaccharides having a general average molecular weight heparin polysaccharide structure with heparin having an ethylene double bond at one chain end or salts thereof, which form the sodium salt of polysaccharides 20 having the following properties: sulfur content: 9.0 - 13.5% nitrogen content: 1.8 - 2.5% uronic acid content: 20.0 - 30.0% Specific rotation in aqueous solution at 20 ° C: 25 [a]% °: + 25 ° to + 55 ° Weight average molecular weight: 2000 - 7000 daltons anticoagulant activity in vitro codex: 10 - 80 iu / mg 30 in vitro antiXa activity: 80 - 250 iu / mg activity in vitro APTT: 10 - 80 iu / mg DK 168824 B1 28 .c -, .- ·, -! in vitro anti Xa activity. conditions we believe OpenW 2 characterized in that the carboxyl groups of heparin are esterified partially or completely, after which the formed heparin ester is reacted with a water-soluble inorganic or organic base in an aqueous medium at a temperature of 20 ° C. to 80 ° C, 5 and the resulting depolymerization product is isolated. Process according to claim 1, characterized in that the molar concentration of the base in the medium is 0.1 - 0.6.