FI67865B - REFRIGERATOR FOR SUSPENSION AV EN COMPOSITION AV SULFATGRUPPER INNEHAOLLANDE POLYSACKARIDER - Google Patents

Description

67865

Method for preparing a mixture of polysaccharides containing sulfate groups

The present invention relates to a process for the preparation of a mixture of novel sulphate-containing polysaccharides having a lower average molecular weight than heparin, which can be used as anticoagulant and antithrombotic agents in the prevention and treatment of thrombosis and as hypolipemic agents.

Heparin is a mixture of sulfate-containing mucopolysaccharides of animal origin, which is widely used due to its anticoagulant and antithrombotic properties, especially for the prevention of postoperative venous thrombi, and its hypolipemic properties. It is known to activate the natural anticoagulant in the blood, antithrombin III, in the coagulation process. Activation of this protein inhibits the action of two proteases, X-activated factor (factor Xa) on the one hand and thrombin on the other.

The in vitro anticoagulant effect of heparin is usually measured according to official pharmacopoeial methods, in particular the American, English or French pharmacopoeia, by comparison with an international reference subject. But today it is possible to measure its specific effects in vitro, on factor Xa on the one hand and on thrombin on the other (cf., for example, A. Teien et al., Thrombosis Research, 11, 107, 1977).

Commercially available heparins are mixtures of polysaccharides with an average molecular weight of more than 10,000 67865 daltons and a molecular weight distribution of approximately 4,000 to 45,000 daltons and have the following in vitro effects: - Anticoagulant activity as measured by the French Pharmacopoeia, eighth edition, heparin Ereparin to the third international reference model (i.e. Codex anticoagulant effect): 140-200 IU / mg.

- The anti-Xa effect as measured by the above-mentioned Teien et al. according to the method, compared to the third international reference model: 160-180 k.y./mg.

- A.P.T.T. effect (Activated Partial Thromboplastin Time) as measured by Teien et al. by the method previously cited, and compared to the third international reference model: 150-170 k.y./mg.

Heparin is definitely administered parenterally (practically subcutaneously) and has a relatively short duration of action, due to its two main drawbacks: it must be administered three times a day and post-operative bleeding occurs relatively frequently.

It is known (cf. Johnson et al., Thrombos. Haemostas. Stuttg., 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;

Lasker, Adv. Exp. Med. Biol. 52, 1975, 119-130) that can be obtained by fractionation of heparin, for example by filtering over Sephadex gel fractions with an average molecular weight lower than heparin and a lower molecular weight distribution than heparin. On the one hand, as shown by in vitro and in vivo experiments, such fractions are relatively more potent against X-activated factor 67865 than against thrombin (i.e. they have a ratio of anti-Xa effect ..,......

antitr5Sbriii ^ iIk5tS5 clearly greater than 1) 3a on the other hand they are more easily absorbed into the bloodstream by subcutaneous injection than heparin itself, resulting in a stronger plasma effect and a longer duration of action than heparin.

It is also known (cf. Lasker and Chiu, cited above; U.S. Patent 3,766,167; Periin et al., Carbohydrate Research, 18, 1971, 185-194) that it is possible to prepare heparin by enzymatic hydrolysis of depolymerized substances having an average molecular weight. low (practically 5300-4500 daltons, determined by ultracentrifugation) and having an anticoagulant effect, as measured by USP No. XVII method is about 70 k.y./mg. These depolymerized materials can be fractionated by filtration over Sephadex gel into fractions having average molecular weights as determined by ultracentrifugation of 3200-5900 daltons and anti-coagulation effects of 45-95 k.y./mg (U.S.P. method). These depolymerized agents and fractions derived therefrom are as effective when administered orally as when administered parenterally.

It is also known (cf. Lasker, cited above) that it can be prepared by depolymerizing heparin with ascorbic acid and hydrogen peroxide with low average molecular weight substances. Depolymerization with ascorbic acid and hydrogen peroxide, after alcohol fractionation, gives fractions having an average molecular weight of 4000-7100 daltons and an anti-coagulant effect of 12-100 k.y./mg (U.S.P. method).

Finally, it is still known (cf. British patent application 2,002,406 A) to reconstitute, by sulphation, heparin depolymerization products which do not have anticoagulant activity, oligosaccharides having an average molecular weight of 2600-5500 daltons as determined by the Somoqy method. At 20 ° C has a temperature of + 40 ° to + 50 °, and an anticoagulant effect of less than 50 IU / mg (USP method), which would be as effective when administered orally as when administered parenterally in the prevention of thrombosis.

The invention relates to a process for the preparation of a mixture of polysaccharides containing sulphate groups, which polysaccharides have the general structure of heparin constituent polysaccharides and have the following typical physical properties in the form of the sodium salt:% sulfur content: 9-13.5% - nitrogen content: 1.8-2.5% - uronic acid content in%: 20-30% - average molecular weight: 2000 to 10 000 daltons - specific rotation at water-soluble at 20 ° C:: + 25 ° to + 55 °, the polysaccharides of which in particular correspond to the following formula: R CH2 ~ R1

) r ~ o _. / I O

/ Oh. h - o 1-GOUi 0— 'H \) H, OH (I) w 1 J- Tr 1 I il HR' K NH-R2 where R represents a hydrogen atom or a carboxyl group, in the form of an acid or a salt, R 'represents OH- a group or a sulfate group, in the form of an acid or a salt, R1 represents an OH group or a sulfate group, in the form of an acid or a salt, R2 represents a sulfonate group, in the form of an acid or a salt, or an acetyl group, -O- represents an oxygen bridge, chain rings G represent glucose typical chain rings of amines present in the structure of heparin, rings U denote uronic acid type chain rings (D-glucuronic acid, 1, -iduronic acid, sulfated L-iduronic acid) present in the structure of heparin, and n is an integer from 3 to 20, and pol is the acidic groups are in free form or in salt form.

S 67865

The process according to the invention is characterized in that the heparin ester obtained by partial or complete esterification of the carboxylic acid groups of heparin is reacted under reaction conditions with a soluble inorganic or organic base at a temperature of 20 to 80 ° C in either an aqueous medium if the ester is soluble in water, and isolating the depolymerization product thus obtained, or in an ester-inert solvent, and isolating the depolymerization product thus obtained as an alkali salt, which is then hydrolyzed with at least 1N aqueous sodium hydroxide solution at a temperature of 0-5 ° C.

In the process according to the invention, the concentration of the base in the aqueous medium is 0.1 to 0.6, using alkali hydroxide, alkali carbonate, triethylamine, triethylenediamine, quinuclidinine, diaza-1,5-bicyclo / 4.3.0 / noneene-5 or diaza-1 as the base. , 5-bicyclo [5.4.4] undecene-5.

The sulfur nitrogen contents and circulating capacity given above have been determined according to the methods of the French Pharmacopoeia, eighth edition, Special Study of Heparin.

The uronic acid content was determined by N. Blumenkrantz et al. according to the method (Analytical Biochemistry, 54, 484, 1973). The average molecular weight was determined by gel-gel permeation chromatography on polyacrylamide-agarose based on reference models of heparins of known average molecular weight according to the method of Andersson et al. method (Thrombosis Research, 9575, 1976).

The heparin esters used as starting materials in the preparation of the mixtures of polysaccharides according to the invention can be non-selective or selective esters. By non-selective esters in the context of the present invention are meant those esters of heparin in which the carboxyl groups of the rings of D-glucuronic acid, non-sulphated L-iduronic acid and sulphated L-iduronic acid have been blocked without discrimination. By selective 6,678,65 esters are meant within the scope of the present invention those esters of heparin which are partially or completely esterified, either the carboxyl groups of the D-glucuronic acid rings alone, or only the carboxyl groups of the D-glucuronic acid and non-sulfated L-iduronic acid rings, or carboxyl groups of rings of exclusively non-sulfated L-iduronic acid and sulfated L-iduronic acid, or finally carboxyl groups of rings of sulfated L-iduronic acid alone.

Suitable esters of heparin which can be used as starting materials in the processes according to the invention include, in particular, the esters of heparin mentioned in French Patent 2,150,724 and British Patent 1,501,095, as well as methyl, ethyl, ethoxycarbonylmethyl, cyanomethyl, benzyl and substituted especially chloro-4-benzyl, nitro-4-benzyl ester). Preferably, the benzyl or substituted benzyl ester of heparin is used as the starting material. The heparin esters used as starting materials in the methods of the invention may be derived from heparin of any origin (heparin from bovine lung, porcine mucosa, etc.).

Non-selective methyl, ethyl, ethoxycarbonylmethyl, cyanomethyl, benzyl, and substituted benzyl esters of heparin can be obtained, for example, by reacting a neutral quaternary ammonium salt of heparin or an amine salt of heparin with a halogenated derivative of the formula Hal-C II) in which Hal represents a chlorine, bromine or iodine atom and R represents a hydrogen atom or a methyl, ethoxycarbonyl, cyano, femyl or substituted phenyl group. This reaction takes place in solution or suspension in an inert solvent such as dimethylformamide, methylene chloride, dimethyl sulfoxide, tetrahydrofuran or acetone at a temperature between -20 ° C and + 60 ° C.

7 67865

Methyl, ethyl, ethoxycarbonylmethyl, cyanomethyl, benzyl and substituted benzyl esters of heparin with esterified, partially or completely, either exclusively carboxyl groups of D-glucuronic acid rings, or exclusively D-glucuronic acid and non-sulphated The carboxyl groups of L-iduronic acid rings are obtained by reacting a halogen derivative of formula II as defined above with an acidic quaternary ammonium salt of heparin in salt form except for sulphate groups, either exclusively carboxyl groups of D-glucuronic acid rings and non-D-glucuronic acid rings. -carboxylic groups of -uronic acid rings, and the other carboxylic groups are in the form of an acid. This reaction is carried out under the same conditions as the reaction of the halogen derivative of formula II with the neutral quaternary ammonium salt of heparin.

Acidic quaternary ammonium salts of heparin having in the form of a salt not only sulphate groups but only carboxyl groups of D-glucuronic acid rings are obtained by allowing the quaternary ammonium salt to act in aqueous heparin at pH 3-4.

Acidic quaternary ammonium salts of heparin in salt form, with the exception of sulphate groups, exclusively carboxyl groups of D-glucuronic acid and non-sulphated L-iduronic acid rings, are obtained by allowing a quaternary ammonium salt to act on heparin in aqueous solution at a sufficiently low pH form only the sulphate groups (practically at pH 2-2.5) and then selectively neutralize the carboxyl groups of the D-glucuronic acid and non-sulphated L-xduronic acid rings of the substance thus obtained by adding a calculated amount of quaternary ammonium hydroxide in dimethylformamide medium. This amount is calculated from the neutralization curve of a certain amount of said substance in a dimethylformamide medium.

Methyl, ethyl, ethoxycarbonylmethyl, cyanomethyl, benzyl and substituted benzyl esters of heparin with esterified, partially or completely, either the carboxyl groups of the sulphated L-iduronic acid rings alone or the non-sulphated L-iduronic acid and the non-sulphated L-iduronic acid alone carboxyl groups of rings, prepared by administering an alcohol of the formula HO-Cl 2 -R III, wherein R is a hydrogen atom or methyl, ethoxycarbonyl, cyano, phenyl or substituted phenyl, in heparin in an aqueous solution containing a condensation agent which is of the water-soluble carbodiimide type, such as ethyl 1- (dimethylamino-3-propyl) -3-carbodiimide, and the pH of the medium is adjusted to 3.5-4.5 in the first case and to 2-3 in the second case. In particular, methanol and ethanol can be used as the alcohol of the formula III, in which case selective methyl and ethyl esters of heparin are obtained, respectively.

By the process of the invention using the reaction of an inorganic or organic base with an ester of heparin obtained by β-elimination, partial and controlled depolymerization of heparin can be achieved without changing its general structure.

The mixtures of sulphate-containing polysaccharides according to the invention have anticoagulant and antithrombotic properties and a hypolipemic effect. Mixtures with a sufficiently low average molecular weight (practically less than or equal to 7,000 daltons) have a stronger antithrombotic effect than the anticoagulant effect. The compositions according to the invention are of very low toxicity. For example, the compound of Example 9 is atoxic at a dose of 300 mg / kg in rats and mice when administered intravenously and its toxicity is identical to that of heparin when administered subcutaneously.

Mixtures of sulphate-containing polysaccharides according to the invention, in which the acidic groups of the polysaccharides are in the form of pharmaceutically safe salts, in particular in the form of sodium, calcium or magnesium salts, can be used as anticoagulants and antithrombotic agents for the prevention and treatment of thrombosis. They can also be used to treat hyperlipemia. They can be advantageously used to replace heparin for such purposes. When administered subcutaneously, they have a longer duration of action than heparin, which makes it possible to reduce the number of injections. In addition, they have fewer side effects (causing hemorrhages) than heparin.

They can be administered as a medicament with a pharmaceutically acceptable solvent intravenously, subcutaneously, by pulmonary (inhalation), rectally, and orally low molecular weight mixtures (especially category III mixtures) orally. Doses depend on the route of administration and the desired effect (antithrombotic or hypolipemic effect).

Although the present invention relates to all mixtures as defined above, it relates in particular to those mixtures which, in the form of the sodium salt, have the typical additional physical properties shown in Table A, Categories I, II and III below.

10 67865

Table A

Category- Category- Category-

ria I ria II ria III

Average molecular weight 8,000 to 3,000 to 2,000 (in daltons) 10,000 8,000 to 7,000

Anticoagulant effect 130-160 80-140 10-80 in vitro Codex

Anti-Xa effect in 130-180 120-250 80-250 in vitro Α.Ρ, Τ.Τ — effect in 100-150 80-120 10-80 in vitro

Ratio: anti-Xa effect in vitro A.P.T.T. effect in vitro ^ ^ 2 10

The anticoagulant effect reported in Table A Codex has been determined by the method of the French Pharmacopoeia, 8th edition, Special Study of Heparin. The anti-Xa and A.P.T.T. reported in Table A as well as in Tables B and C below. effects have been determined by Teien et al. according to the method (cited above) following the procedures described below: a) Measurement of the anti-Xa effect: This effect is determined from interstitial bovine plasma using the chromogenic substrate S 2222 / chromogenic peptide of the structure (N-benzoyl) -Ile- Glu-Gly-Arg- (p-nitro) anilide / i and by comparison with a third international reference model.

To 100 [mu] l of test substance or reference substance in aqueous buffer Tris / EDTA pH 8.4, corresponding to 0.02-0.08 [mu] l of substance or reference substance, is added 100 [mu] l of bovine plasma containing citrate, previously diluted 1 1 67865 in a ratio of 2/5 using the above buffer tris / EDTA. Incubate for 3 min at 37 ° C and add 100 μl of an aqueous solution of bovine factor Xa corresponding to the 7 X Kat of factor Xa. Incubate for 30 ml and add 200 .mu.l of an aqueous solution of S2222 corresponding to 0.2 .mu.l of S2222. Incubate for 3 min and add 300 ul of acetic acid and read the optical density of the solution at 405 nm compared to distilled water.

When plotting the optical density as a function of the concentration of the test substance on the one hand and the concentration of the reference substance on the other, two lines are obtained, one corresponding to the test substance and the other corresponding to the reference substance. The effect of the test substance, expressed in international units per mg, is given by the formula: 173 The slope of the line corresponding to the test substance from the reference countries The slope of the corresponding line 173 corresponds to the value of the effect of the third international reference model. The above ratio of the slopes of the two lines is equal to the inverse ratio of the concentrations of test substance and reference substance corresponding to the same optical density.

b) A.P. Measuring the T.T. effect:

The test substance and the third international reference substance are dissolved in 0.15 M aqueous sodium chloride solution, then diluted with interstitial bovine plasma containing citrate to give test substance (or reference substance) concentrations of 0-4 μg / ml.

To 100 μl of the solution thus prepared is added 100 μl of reagent "automated APTT Precibio" (reagent based on rabbit brain phospholipids and micronized silica) and incubated for 5 min at 37 ° C and 12 μl of 100 μl of calcium chloride is added. M aqueous solution. Measure the time to coagulation using a Bio-Meri§ux fibrometer.

When plotting the logarithm of the coagulation time as a function of the concentration of the test substance on the one hand and the concentration of the reference model on the other hand, two lines are obtained. The effect of the test substance, expressed in international units per mg, is given by the formula: the slope of the line corresponding to the test substance in the reference countries The slope of the corresponding line 173 corresponds to the value of the effect of the third international reference model.

All effects in Tables A, B and C are reported in International Units per mg (k.y./mg) compared to the third international reference model.

The following examples illustrate the invention without limiting it.

The neutral benzethonium salt of heparin used as a starting material in Examples 1-3, 10, 12-15, i.e. benzethonium heparinate, is derived from heparin obtained from porcine intestines and has the following typical physical properties: - average molecular weight: 16,000 daltons 13 67865 specific rotation in aqueous solution at 20 ° C: ^ _ / D = + 41 - anticoagulant efficiency Codex = 157 IU / mg.

The neutral benzethonium salt of heparin, i.e. benzethonium heparinate, used as a starting material in Examples 4-9 and 11, is derived from heparin obtained from bovine intestines and has the following typical physical properties: average molecular weight = 11,400 daltons / ~ 7d ° = + 37 ° - anticoagulation efficiency IU / mg of.

The neutral benzethonium salt of heparin used as a starting material in Example 16, i.e. benzethonium heparinate, is derived from heparin derived from porcine mucosa and has an average molecular weight of 16,000 daltons, a specific rotation in aqueous solution at 20 ° C = + 44 ° and an anticoagulation efficiency of Codex of 180 k.y.

The sodium salt of heparin used as a starting material in Examples 17-19 corresponds to the above-mentioned heparin obtained from porcine mucosa.

Example 1

To a solution of 30 g of benzethonium heparinate in 600 ml of dimethylformamide is added 30 g of (chloro-4) -benzyl chloride. The solvent is allowed to contact for 60 h at ambient temperature (about 20 ° C) and then 600 ml of 10% methanolic sodium acetate solution are added. The precipitate formed is filtered off, washed with methanol and dried in vacuo. This gives 10.75 g of the chloro-4-benzyl ester of heparin in the form of the sodium salt.

The ester obtained is contacted with stirring with 269 ml of 14 67865 aqueous 0.4 N aqueous sodium hydroxide solution. After 2 h, neutralize by adding 0.4 N aqueous hydrochloric acid and precipitate by adding two volumes of methanol (i.e. twice the amount of the aqueous phase). Separate by filtration 8.46 g of depolymerized heparin in the form of the sodium salt.

Example 2

To a solution of 30 g of benzethonium heparinate in 600 ml of dimethylformamide is added 30 g of benzyl chloride. The solvent is allowed to come into contact for 60 h at ambient temperature, then precipitated by adding 600 ml of 10% methanolic sodium acetate solution. The precipitate is filtered off, washed with methanol and dried in vacuo. In this way, 11.4 g of benzyl ester of heparin are obtained in the form of the sodium salt.

Contact with 2 g of the ester obtained above with 75 ml of 0.4 N aqueous sodium hydroxide solution at 20-25 ° C is stirred for 2 h. It is then neutralized by adding 0.4 N aqueous hydrochloric acid and precipitated by adding 2 volumes of methanol. 2.23 g of depolymerized heparin in the form of the sodium salt are thus isolated.

Example 3

To a solution of 10 g of benzethonium heparinate in 250 ml of dichloromethane is added 10 g of benzyl chloride.

The solvent is allowed to stand for 24 hours at ambient temperature, then the solvent is evaporated off under vacuum. The residue is dissolved in 150 ml of dimethylformamide and precipitated by adding 150 ml of a 10% solution of sodium acetate in methanol. Separate by filtration 3.67 g of the benzyl ester of heparin in the form of the sodium salt.

15 67865 2 g of the ester obtained above are treated with 50 ml of 0.1 N aqueous sodium hydroxide solution at 60 ° C for 2 h with stirring. After cooling, neutralize by adding 0.1 N aqueous hydrochloric acid, then precipitate by adding 2 volumes of methanol. This gives 1.54 g of depolymerized heparin in the form of the sodium salt.

Example 4

To a solution of 5 g of benzethonium heparinate in 125 ml of dichloromethane is added 5 g of ethyl chloroacetate and the solvent is allowed to remain in contact for 3 days at ambient temperature. The solvent is evaporated off under vacuum, the residue is taken up in 75 ml of dimethylformamide and precipitated by adding 75 ml of 10% methanolic sodium acetate solution. The precipitate, which has been filtered off, is washed with methanol and then dried in vacuo. 1.72 g of carbethoxymethyl ester of heparin are thus obtained in the form of the sodium salt.

1.7 g of the ester obtained above are treated with 43 ml of 0.1 N aqueous sodium hydroxide solution at 60 ° C with stirring for 2 h. After cooling, neutralize by adding 0.1 N aqueous hydrochloric acid and precipitate by adding 2 volumes of methanol. Separate by filtration 1.33 g of depolymerized heparin in the form of the sodium salt.

Example 5

To a solution of 10 g of benzethonium heparinate in 250 ml of dichloromethane is added 10 g of (chloro-4) -benzyl chloride, which is dissolved by stirring. The solution is left to stand at ambient temperature for 24 h and then the solvent is evaporated off under vacuum. The residue is taken up in 150 ml of dimethylformamide and precipitated by adding 150 ml of 10% methanolic sodium acetate solution. Filter, wash the precipitate with methanol and dry in vacuo, then isolate 3.84 g of 16,67865 heparin chloro-4-benzyl ester in the form of the sodium salt.

Treat 2 g of the ester obtained above with 50 ml of a 0.1 N aqueous solution of sodium hydroxide at 60 ° C with stirring for 2 h. After cooling and neutralization with 0.1 N aqueous hydrochloric acid, precipitate by adding two volumes of methanol. The precipitate is filtered off, washed with methanol and dried in vacuo. This gives 1.38 g of depolymerized heparin in the form of the sodium salt.

Example 6

To a solution of 5 g of benzethonium heparinate in 125 ml of dichloromethane is added 5 g of (nitro-4) -benzyl chloride, which is dissolved with stirring. The solution is then left to stand at room temperature for 3 days, then the solvent is evaporated off under vacuum and the residue is dissolved in 75 ml of dimethylformamide. The ester formed is precipitated by adding 75 ml of 10% methanolic sodium acetate solution. The precipitate is collected by filtration, washed with methanol and dried in vacuo. This gives 1.89 g of the nitro-4-benzyl ester of heparin in the form of the sodium salt.

Treat 1.85 g of the above ester with 46 measures of 0.1 N aqueous sodium hydroxide solution at 60 ° C for 2 h with stirring. Cool and then neutralize by adding 0.1 N aqueous hydrochloric acid, then precipitate by adding 2 volumes of methanol. The precipitate is isolated by filtration, washed with methanol and dried in vacuo. This gives 1.13 g of depolymerized heparin in the form of the sodium salt.

Example 7

To a solution of 30 g of benzethonium heparinate in 500 ml of dichloromethane is added 30 g of benzyl chloride, which dissolves with stirring. The solution is then left to stand at room temperature for 24 h, then the solvent is evaporated off under vacuum and the residue 400 is taken up in any ether. The insoluble matter is separated by filtration. This gives 30 g of the benzyl ester of heparin in the form of the benzethonium salt. This ester is dissolved in 200 any dichloromethane containing 8 ml of diaza-1,5-bicyclo [4.3.0] nonene-5. Maintain under reflux for 3.5 h, then evaporate the solvent in vacuo. The residue is dissolved in 450 any dimethylformamide and an equal volume of 10% methanolic sodium acetate is added. The precipitate is collected by filtration and washed with methanol. It is then treated at 0 ° C for one hour with 1 N aqueous sodium hydroxide solution. Neutralize and precipitate by adding two 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

To a solution of 10 g of benzethonium heparinate in 250 ml of dichloromethane is added 10 g of (chloro-4) -benzyl chloride, which dissolves with stirring. The solution is left to stand at ambient temperature for 24 h, then the solvent is evaporated off under vacuum. The residue is taken up in 200 ml of ether and the precipitate formed is isolated by filtration. This gives 10 g of the chloro-4-benzyl ester of heparin in the form of the sodium salt.

Dissolve 5 g of this substance in 100 ml of dichloromethane containing 1.5 ml of diaza-1,5-bicyclo [4.3.1] 7-nonene-5 and keep under reflux for 4 h. The solvent is then evaporated off under vacuum, the residue is taken up again in 30 ml of dimethylformamide and 100 ml of 10% strength sodium acetate solution are added. The precipitate formed is isolated by filtration, washed with methanol and then treated with 24 ml of 1N aqueous sodium hydroxide solution for 1 h at 0 ° C. Neutralize by adding 1 N aqueous hydrochloric acid and precipitate by adding two volumes of methanol. The precipitate is filtered off. Wash with methanol and dry in vacuo to give 1 g of depolymerized heparin as the sodium salt.

Example 9

To a solution of 30 g of benzethonium heparinate in 600 ml of dimethylformamide is added 30 g of benzyl chloride. The solvent is left in contact for 60 h at ambient temperature, then the ester formed is precipitated by adding 1200 ml of 10% methanolic sodium acetate solution. The precipitate is isolated by filtration, washed with methanol and dried in vacuo. 11.4 g of benzyl ester of heparin are thus obtained in the form of the sodium salt.

10 g of the above ester are treated with 250 ml of 0.1 N aqueous sodium hydroxide solution at 60 ° C for 2 h with stirring. Cool and then neutralize with 0.1 aqueous hydrochloric acid and precipitate by adding two volumes of methanol. The precipitate is filtered off, washed with methanol and dried in vacuo. This gives 6.65 g of depolymerized heparin in the form of the sodium salt.

Example 10

To a solution of 120 g of benzethonium heparinate in 2.4 l of dimethylformamide is added 120 g of (chloro-4) -benzyl chloride, which dissolves with stirring. The solution is then left to stand at ambient temperature for 60 h, then 2.4 l of a 10% solution of sodium acetate in methanol are added. The precipitate formed is filtered off, washed with methanol and dried in vacuo. 46 g of chloro-4-benzyl ester of heparin are thus obtained in the form of the sodium salt.

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

Example 11

To a solution of 30 g of benzethonium heparinate in 750 ml of dichloromethane is added 30 g of methyl iodide, which dissolves with stirring. Leave the solution at ambient temperature for 48 h, then evaporate the solvent in vacuo. The residue is taken up in 450 ml of dimethylformamide and precipitated by adding 450 ml of 10% methanolic sodium acetate solution. Filter, wash the precipitate with methanol and dry in vacuo. 10.5 g of heparin methyl ester are thus isolated in the form of the sodium salt.

Treat 2 g of the above ester with 50 ml of 0.1 N aqueous sodium hydroxide solution at 60 ° C with stirring for 2 h. After cooling, the pH of the solution is adjusted to approximately 4.5 by stirring with an exchange resin having carboxylic acid ions in the H + form. The resin is then filtered off and washed with water. The combined aqueous phases are neutralized by adding dilute aqueous sodium hydroxide solution, and then lyophilized. This gives 2 g of depolymerized heparin in the form of the sodium salt.

Example 12 3 g of the heparin chloro-4-benzyl ester obtained in Example 10 are treated with 120 ml of a 10% aqueous solution of sodium carbonate at 60 [deg.] C. for 2 h with stirring. After cooling, neutralize with 0.4 N aqueous hydrochloric acid and mix by adding two volumes of methanol. Isolate by filtration 1.57 g of depolymerized heparin in the form of sodium syrup.

Example 13

To a solution of 30 g of benzethonium heparinate in 600 ml of dimethylformamide is added 30 g of ethyl chloroacetate. The solvent is allowed to come into contact for 60 h at ambient temperature, then 600 ml of a 10% solution of sodium acetate in methanol are added. The precipitate formed is filtered off, washed with methanol and dried in vacuo. This gives 10.78 g of the carbethoxymethyl ester of heparin 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 60 ° C. After 5 h, neutralize by adding aqueous hydrochloric acid, then precipitate by adding 2 volumes of methanol. Isolate by filtration 2.5 g of depolymerized heparin in the form of the sodium salt.

Example 14

To a solution of 5 g of benzethonium heparinate in 125 ml of dichloromethane is added 5 g of chloroacetonitrile which dissolves with stirring. The solution is left for 48 h at ambient temperature, then the solvent is evaporated off under vacuum. The residue is dissolved in 75 ml of dimethylformamide and precipitated by adding 75 ml of 10% methanolic sodium acetate solution. The precipitate is filtered off, washed with methanol and dried in vacuo. 1.63 g of the cyanomethyl ester of heparin in the form of the sodium salt are thus obtained.

: .7.

6 7 8 6 5

The resulting ester is treated with 40 g of 0.1 N aqueous sodium hydroxide solution at 60 ° C with stirring for 2 h. After cooling, neutralize by adding 0.1 N aqueous hydrochloric acid, then mix by adding two volumes of methanol. Isolate by filtration 1.33 g of depolymerized heparin in the form of the sodium salt.

Example 15

Dissolve with stirring 3 g of the chloro-4-benzyl ester of heparin obtained in Example 10, 120 in any 10% disodium carbonate solution. Stir for 2 h at 20 ° C to 25 ° C and adjust the pH of the solution to 6 by adding an aqueous solution of hydrochloric acid N, then add twice the volume of methanol to the volume of the aqueous solution. The precipitate formed is filtered off to give 2.1 g of chloro-4-benzyl ester of heparin.

Treat with stirring for 2 h 2 g of the above ester with 50 g of a 0.1 N aqueous solution of sodium hydroxide at 60 ° C. After cooling, neutralize by adding 0.1 N aqueous hydrochloric acid, then mix by adding two volumes of methanol. This gives 1.4 g of depolymerized heparin in the form of the sodium salt.

In Examples 1-10 and 12-15 above, before precipitating the formed product by adding two volumes of methanol, the NaCl concentration of the aqueous phase is adjusted to 10% by adding sodium chloride.

Example 16

To a solution of 10 g of benzethonium heparinate in 250 ml of dichloromethane is added 10 g of (chloro-4) -benzyl chloride, which is dissolved with stirring. The solution is left at ambient temperature for 24 h, then the ester formed is precipitated by adding a 10% methanolic solution of sodium acetate. The precipitate is filtered off, washed with methanol. 3.72 g of chloro-4-benzyl ester of heparin are thus obtained in the form of the sodium salt.

Treat at 60 ° C for 5 h a solution of 0.500 g of the above ester in 10 ml of formamide with 0.5 ml of diaza-1,5-bicyclo [4.3.0] nonene-5 th. After cooling, precipitate by adding 70 ml of acetone. 0.364 g of material is thus obtained, which is treated at 0 [deg.] C. for 2 h with 6 ml of aqueous sodium hydroxide solution. The aqueous phase is neutralized by adding an aqueous solution of hydrochloric acid N and its NaCl concentration is adjusted to 10% by adding sodium chloride. Precipitate by adding two volumes of methanol. 0.263 g of depolymerized heparin in the form of the sodium salt is thus isolated.

Example 17

To a solution of 10 g of heparin (sodium salt) in 40 ml of water is added 2.5 ml of acetic acid, then slowly and with stirring 150 ml of a 10% aqueous solution of benzethonium chloride. The precipitate that forms is taken off by centrifugation, washed with water and dried in vacuo. 19.67 g of acidic benzetonium heparinate are thus obtained.

11 g of the above substance are treated with 11 g of (chloro-4-) benzyl chloride dissolved in 110 ml of dimethylformamide for 48 h at ambient temperature. Add 220 ml of 10% methanolic sodium acetate solution. The precipitate formed is filtered off and washed with methanol. 4.70 g of heparin (chloro-4-) benzyl ester are thus obtained in the form of the sodium salt.

4 g of the above substance are dissolved 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. This gives (chloro-4) -benzyl ester of heparin in the form of the benzethonium salt.

23 67865 1 g of the above ester in the form of the benzethonium salt is dissolved in 20 g of any dimethylformamide and treated with 1 g of diaza-1,5-bicyclo [4.3.1] nonene-51 at 60 [deg.] C. for 5 h. After cooling, precipitate by adding 5 ml of 10% methanolic sodium acetate solution. This gives 0.346 g of material which is treated at 0 ° C for 2 h with 5.8 g of aqueous sodium hydroxide solution. The aqueous phase is neutralized by adding aqueous N hydrochloric acid and its NaCl concentration is adjusted to 10% by adding sodium chloride. Precipitate by adding 2 volumes of methanol. Filter and wash the precipitate with methanol to give 0.253 g of depolymerized heparin in the form of the sodium salt.

Example 18

To a solution of 10 g of heparin (sodium salt) in 40 ml of water is added 2.5 ml of formic acid and then, slowly and with stirring, 150 ml of a 10% aqueous solution of benzethonium chloride. The precipitate is collected by centrifugation, washed with water and dried in vacuo. 20.5 g of acidic benzethonium heparinate are thus obtained. The above substance is dissolved in 2.95 g of 60 ml of dimethylformamide, then 5.9 ml of a 0.1 N solution of tetrabutylammonium hydroxide in n-propanol / methanol are added.

After adding 2.95 g of (chloro-4) -benzyl chloride, the solution is left at room temperature for 5 days. Add 74 ml of 10% methanolic sodium acetate solution. Isolate by filtration 1.32 g of heparin (chloro-4) -benzyl ester 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 are added slowly with stirring. The precipitate formed is collected by centrifugation, washed with water and dried in vacuo. This gives the chloro-4-benzyl ester of heparin in the form of the benzethonium salt.

1 g of the above ester in the form of the benzethonium salt is dissolved in 20 ml of dichloromethane and 1 ml of diaza-1,5-bicyclo [4.3, ()] - nonene-5 is added. Heat under reflux for 5 h, then evaporate the solvent in vacuo, take up the residue in any dimethylformamide and add 20 ml of 10% sodium acetate in methanol. The precipitate formed is isolated by filtration and washed with methanol.

This gives 0.405 g of the substance which is treated at 0 ° C for 2 h with 6 ml of aqueous N sodium hydroxide solution. The aqueous phase is neutralized by adding aqueous N hydrochloric acid and its NaCl concentration is adjusted to 10% by adding sodium chloride. Precipitate by adding two volumes of methanol. Filter and wash the precipitate with methanol to give 0.355 g of depolymerized heparin in the form of the sodium salt.

Example 19

Dissolve 0.600 g of heparin (sodium salt) 7 in any water and adjust the pH of the solution to 3.5 by adding N aqueous hydrochloric acid. 0.300 g of ethyl 1- (dimethylamino-3-propyl) -3-carbodiimide is then added and, after dissolution, the solution is left at ambient temperature for 1 h. Add 2.5 ml of an aqueous solution of sodium chloride containing 280 g / l, then 15 ml of methanol. The precipitate formed is isolated by filtration, washed with methanol and dried in vacuo. This gives 0.527 g of heparin methyl ester in the form of a sodium salt with a degree of esterification of carboxylic acids of 50%.

0.300 g of the above ester is dissolved in 7.5 ml of 0.1 N aqueous sodium hydroxide solution and heated at 60 ° C for 2 h. After cooling, neutralize by adding 0.1 N aqueous hydrochloric acid, adjust the NaCl concentration in the medium to 10% by adding sodium chloride and mix by adding two volumes of methanol. This gives 0.200 g of depolymerized heparin in the form of the sodium salt.

The following Tables B and C show the typical physical properties of the substances prepared in Examples 1-19 (depolymerized heparins in the form of the sodium salt). The percentages of sulfur, nitrogen and uronic acids, the specific rotations of aqueous solutions at 20 ° C, the average molecular weights and the effects given in this table have been determined using the methods mentioned above. The molecular weight dispersion column contains the approximate extremes of the molecular weights of the polysaccharides as components of the mixtures, determined by gel-gel permeation chromatography on polyacrylamide-agarose. The viscosity column shows the viscosities at 25 ° C with aqueous solutions containing 10% of the substance. The UV absorption column shows the absorbances of 1 cm thick layers with solutions containing 1% of the substance in 0.01 N Helium, and the absorbance is measured as the maximum absorbance in the range 220-232 nm.

Table C

Pre- Average Absorption Anticoagulant effect marker molecular weight Anti- APTT Codex (in daltons) UV Xa in in vitro ________vitro vitro___ 16 4200 6 140 63 60 17 5500 7.96 165 80 70 18 4400 6.3 110 60 40 19 5000 10.9 150 45 50 26 67865

Table B

Pre- Uron- _ ___ Mean- Molecular-markic acids% S% N / a_ / specific weights ki% molecular dispersion lipo weight (in dalto- _ _____ _ _ _) __ 1 23.0 11.5 2.1 + 44 ° 7300 1600-12 000 2 26.0 11.6 2.0 + 44 ° 6500 1800-13 000 3 25.4 12.0 2.2 + 44 ° 6400 3000-11 000 4 23.9 10.9 2.1 + 35 ° 5500 2200-13 000 5 22.8 12.2 2.2 + 39 ° 5000 2000-13 000 6 24.5 10.8 2.3 + 33 ° 4200 1000-10 000 7 25, 2 11.3 2.0 + 33 ° 4100 1700-10 000 8 23.7 11.1 2.2 + 35 ° 4500 1000-10 000 9 26.0 11.8 2.2 + 38 ° 4000 2000-10 000 10 25.2 10.9 2.1 + 41 ° 3800 1000-9000 11 23.8 11.0 2.3 + 27 ° 2800 1000-8000 12 23.9 11.5 2.0 + 40 ° 4000 2000 -9000 13 24.3 11.8 2.2 + 45 ° 9000 3000-20 000 14 25.6 12.0 2.1 + 44 ° 8500 3000-20 000 15 25.3 11.8 2.3 +45 ° 4000 200 0-20 000 67865

Table B (continued)

Pre- Viscosity- Absorp- Anti-- Anti- APTT- significance- thio gulimis- Xa-action effect ky / 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 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 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 13 2.60 5.9 135 140 130 14 2.50 6.1 138 130 130 15 1.65 10, 64 110 170 86

Example 20

The substance of Example 1, on the one hand, and commercially available heparin, on the other, have been administered subcutaneously at various stages to five healthy volunteers at a dose of 5,000 k.y. Codex. Blood samples at 1, 3, 5, and 7 h after drug administration have been measured for anticoagulant effect in plasma by anti-Xa and A.P.T.T. experiments as defined above. The results obtained are expressed in international units per ml / plasma compared to a calibration curve plotted against reference plasma experiments in which known amounts of reference heparin have been added to the plasma (third international reference).

'• I'; 'V

28 67865

The average results obtained are summarized in the following Table D:

Table D

Time drug Anti-Xa effect APTT effect keen administration- Pre-sale Pre-sale and sample obtainable mark available tea intake substance heparin 1 substance heparin between 1 h 0.20 0.04 0.04 0.05 3 h 0.27 0.08 0.05 0.04 5 h 0.28 0.06 0.03 0.02 7 h 0.22 0.05 0.02 0.01

It is found that the agent of Example 1 has a much stronger anti-Xa effect than commercial heparin.

f.:’·· *

Claims (4)

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