DD285606A5 - PROCESS FOR THE PRODUCTION OF SELECTIVELY O-AZYLATED GLUCOSAMINOGLYCANES - Google Patents

Abstract

The glycosaminoglycans of the invention are selectively O-acylated in a controllable manner on their free -OH groups, the carboxylic or amino functional groups being unaltered. These compounds have a pharmacological activity of very long duration.

Description

For this 8 pages drawings 4 1 page formulas

Field of application of the invention

The invention relates to a process for the preparation of selectively O-acylated. Glucosaminoglycans and the pharmaceutical compositions containing them and their application in the Therapeutic

Characteristic of the known state of the art

The term "glucosaminoglycans" is understood to mean the substances which are formed from uronic acid chains (D-glucuronic acid or L-iduronic acid) and amino sugars, the latter being glucosamines or galactosamines The glucosaminoglycans of natural origin consist of more or less homogeneous mixtures of Linkages of disaccharide units formed by a uron subunit (glucuronic acid or iduronic acid) and by an osamine subunit (glucosamine or galactosamine) joined together through a 1-4 bond or a 1-3 bond ,

In the case of the glucosaminoglycans, the hydroxyl groups are differently substituted by functional groups, in particular by sulfate groups, and the amino groups of the osamines also by sulfate and / or acetyl groups. The glucosaminoglycans contemplated herein include six product types: heparin, heparan sulfate, chondroitin sulfate A, chondroitin sulfate C, chondr'-tin sulfate B, more specifically air dermatan sulfate, and hyaluronic acid. They are characterized by two disaccharide structures u aase of the formula (A) and the formula (B) (see formula sheet), wherein in the formula (A) R is an SOY group and / or an acetyl group and the wavy line indicates that the carboxylic acid group is either below the ring plane (iduron unit) or above it (glucuronic unit). The glucosaminoglycans having the structure of the base of formula (A) are referred to as heparin-type glycosaminoglycans (or GAGs) and include the heparins and the heparan sulfates, and the glucosaminoglycans having the structure of the base of formula (B) are called Chondroitin sulphate type GAGs comprising the choncroitin sulfates A and C and dermatan sulfate.

The hyaluronic acid has the structure of the base of the formula (B) in which the galactosamine is replaced by a glucosamine As mentioned above, the heparin-type GAJs and the chondroitin sulfate-type GAGs are included Therefore, the heparin can be represented by the structure of the base of formula (A) in which η can vary between 1 and 80, R in the majority of η units is an SGy group and in the remainder of the cases an acetyl group, the OH groups in position 6 of the glucosamine and in position 2 of the uronic acid in the majority of the species are sulfated while the OH Group in position 3 of glucosamine is sulfated only in the minority of species, wherein the OH group in position 3 of the uronic acid is virtually unsulfated and this uronic acid represents an iduronic acid in the majority of species.

The products having the structure of the base of formula (A) also include N-desulfatoortes N-acetylated heparin, which is as in Nagasawa, K. and Inoue, Y., Method in Carbohydrate Chemistry, Academic Press, 19O0, Vol.8, pp. 291-294,

can be produced. This heparin is represented by the formula (A) wherein R is an acetyl group.

The heparin also contains species having the structure of the formula (A) with a variable η of 3 to 15 and the same

own chemical profile. These species can be isolated by fractionation by known methods (e.g., US 4692435), referred to as low molecular weight heparins (b.p. m.).

Heparins (b.p.m.), which, with their molecular distribution and their biological properties substantially with the Fractionation of heparins obtained (b.p.m.) are identical, were by splitting heparin according to the following Methods made:

Method of nitrous depolymerization and reduction (for example EP 37319 or US 4686288) which breaks the molecule into action on the N-sulfated glucosamine unit in order to obtain a 2,5-anhydromannitol end group of the formula (A '), optionally sulfated at the first hydroxyl in position β;

- Method of enzymatic depolymerization (for example FP 244235 and 244236) or alkaline depolymerization (for example EP 40144), which separates the molecule by attacking the uronic subunits to obtain an α-β-unsaturated uronic acid end group of the formula (A ") which is optionally 2-sulfated:

- Method of oxidative depolymerization (for example, US 4281108 and EP121067), which separates the molecule by oxidation while preserving the "natural" end groups.

Other heparins (b.p. m.) Having a molecular distribution that is inferior to that of natural heparin and having particularly biological properties can be prepared by periodic oxidation of natural heparin followed by depolymerization by β-elimination or acid hydrolysis. The periodic oxidation separates the unsulfonated uronic acid moieties between the carbon atoms in positions 2 and 3. It is known that such parts are present in the binding position to antiothrombin III (AT III), whereby the plasmatic inhibitor of the coagulation of various serine proteeses and its activity by heparin, which acts as a cofactor, is considerably increased. The periodic oxidation therefore makes it possible to obtain products which are freed from the binding layer to AT III and dumit essentially of their anticoagulant effect.

In particular, heparins (b.p. m.) Of this type can be obtained by the use of the process comprising the following steps:

gentle oxidation of heparin, which is carried out by reacting heparin in aqueous solution with a final concentration of 0.5 to 5% (w / v) with a periodic acid salt with a final concentration of 0.5 to 1% ( W / v) at a pH between 4.5 and 6.5, preferably at a pH of 5, and at a temperature of 0 to 10 ⁰ C, while 15 to 48 hours with exclusion of light;

Depolymerization of the resulting heparin chains by addition of a stckcn base, such as sodium hydroxide, "at a pH greater than about 11, in particular between 11 and 12, advantageously from 11.2 to 11.6, preferably at about 11.5 ;

Reduction of the depolymerization fragments with the aid of a reducing agent and, optionally after removal of the unreacted reducing agent,

- the recovery of the reduced heparin fragments by precipitation by means of a solvent in which they are insoluble;

- Isolation of the fragments sought by fractionation of an aqueous solution, which was obtained by dissolving the above-isolated precipitate in water, with the aid of an alcohol in the presence of a mineral salt and then recovering the precipitate formed.

These heparins (b.p. m.) Correspond to the formula (C) in which η can vary from 6 to 15, R at least at least 90% of the

η-units represents an SOj group and in the rest of these cases an acetyl group, the OH groups in position 3 of the

Glucosamine can be sulfated and the OH groups in position 6 of gluosamine in 70% of the species are sulfated. In addition, the induronic acid is in the ratio of 1 group per at least 2 chains of this heparin (b.p. m.) Through a

sulfated uronic acid moiety (D-glucuronic acid or L-iduronic acid) substituted between the carbon atoms in

Position 2 and 3 is open and corresponds to the formula (C). Such heparins (b.p.m.) may additionally be fractionated by gel filtration in accordance with known techniques to: To obtain mixtures of fragments which are homogeneous in terms of their molecular weight and the binding layer to AT III Other heparin-type glycosaminoglycans consist of GAGs of formula (A) or (C), in which the carboxylic acid function

is carried by the uronic acid and esterified, for example by condensation of an alcohol in the presence of a

Carbodiimide type condensation agent as described in patent FR 2159724, or also by alkylation of the Carboxyls with an alkyl halide in the presence of a weak base. Similarly, chondroitin sulfate-type glucosaminoglycans consist of glucosaminoglycans of formula (B), in

where the carboxylic acid function of the uronic acids is esterified, for example by alkylation of the carboxyl by means of a

Alklyhalogenids in the presence of a weak base. The preparation of such esters for the hyaluronic acid is described in the patent EP 21545L, it is carried out by treatment

the hyaluronic acid in the form of the quaternary ammonium salt with an alcohol in the presence of a catalyst or with an etherifying agent.

The heparan sulfate is represented by the formula (A) in which η can vary from 1 to 80, R in the majority of species

represents an acetyl group and in the minority of species SO ₃ -. In addition, the OH groups are in position 6 of the

Glucosamine is not sulfated in the majority of species, and the uronic acid is expressed in the majority of species by glucuron Acid shown. The chondroitin sulfato A and C are represented by the formula (B) in which η can vary from 1 to 80, the groups

4-OH and 6-OH of gluocsamin are sulfated and the uronic acid in the majority of species is a glucuronic acid.

The dermatan sulfate has a structure that is substantially identical to that of the chondroitin sulfate, but the uranium Acid subunit in the majority of species an iduronic acid. Fragments of dermatan sulfate corresponding to the formula (B) in which η can vary between 1 and 20 can be obtained by

periodic oxidation, followed by reduction with sodium borohydride and acid hydrolysis as described in FRANSSON L.A.

and CARLSTEDT I., Carbohydr. Res., 36 (1974), 349-358.

The Glucosaminoglycarie possess numerous biological activities, under which their effectiveness against the Coagulation factors that act via mediating, different plasmatic proteins can highlight. What that As concerns heparin, it is also known from the literature that heparin or some of its derivatives which are anticoagulating Possessing effect or not, a regulator effect for lid proliferation of the cells smooth muscle of the vascular wall

(GUYTON et al., Circ. Res., 46 [1980], 625-634) or also an inhibitory activity of heparanase, an enzyme involved in the mechanisms of metastatic dissemination (EP 254067). In addition, the form

Glucosaminoglycans form part of the very large family of sulfated polysaccharides, with some being more or less

significant antiviral activity (BABA et al., Antimicrob. Agents Chemother., 32 [1938], 337-339) and, in particular, anti-HIV activity (human immunodeficiency virus), cf. BABA et al., Proc. Natl. Acad. Sei., 85 [1388] 6132-

In the following text, the term GAG will be used to designate a glucosaminoglycan which is either a

natural structure as obtained by extraction, semisynthesis or synthesis, or one attached to the carboxylic acid or amine.

Has functional groups chemically modified structure, wherein previously the acylation reaction was carried out by the

one receives the selectively O-acylated GAGs.

Despite their very interesting pharmacological activities, natural GAGs have the disadvantage of being relatively short Half-life on what appears in repeated administrations. This disadvantage was partially at the Use mitigated for the prevention and treatment of venous thrombosis, where the heparin derivatives with lower Molecules are administered by subcutaneous route and the number of dosages is therefore limited to one injection per day

remains.

Nevertheless, it is of great interest to be able to dispose of derivatives with a sustained-release effect, which is the number of Reduced administration of these products and increases their duration of action. It may also be of great interest to use non-anticoagulant derivatives such as N-desulatated-N-acetylated Heparin to be involved in their activity as an inhibitor of meparanase, one with the phenomenon of metastatic Dissemination-related enzyme, was further reminded. The literature describes numerous glucosaminoglycan derivatives which are modified in such a way that their pharmacokinetics

is improved, in particular esters of the carboxylic acid function of the uronic acids or the hydroxyl functions.

In particular, the partial or complete esterification of the carboxylic acid functions of heparin, which has been used

in the form of the quaternary ammonium salt in an inert solvent with an alcohol or a halogen derivative, optionally in the presence of a condensing agent, has been treated (FR N 2159724 and EP 44228).

Also described are hyaluronic acid derivatives obtained by esterification with the aid of an alcohol in the presence

a catalyst or by reaction with an etherifying agent (EP 216453).

In addition, various means have been reported in the literature for the esterification of GAGs at their primary or secondary sites

secondary hydroxyl functions are described.

The publication Can. J. Res., 25B (1947), 472-476 describes an acetylated heparin dorivate containing 1 mole of acetyl group per

4 saccharide units corresponds. The product is prepared by the action of ketene on heparin in acetone. While it is stated in this publication that the product obtained is an O-acetylated derivative, it is well known that ketene gives anhydride in the presence of carboxylic acid groups. (See J. March, Advancceci Organic Chemistry;

Reactions, Mechanisms and Structure, J. Wiley ans Sons Eds. 1935, pp. 686-687: "With ketene, carboxylic acids give anhydrides

and acetics anhydride is prepared industrially in this manner.

CH ₂ = C = O + CH ₃ --COOH τ> CH ₃ - COO - COCHj ")

Accordingly, this publication actually describes a heparin in which the O-acetylated mixed anhydride groups are present between the COO- group. Uron acid and the acetyl group derived from the ketene, which may explain their instability in water.

Patent FR 2100735 describes partially hydrolyzable esters of heparin and a non-toxic organic acid, in particular 4-chlorophenoxyisobutyric acid, 4-chlorophenoxyacetic acid, cholic acid, nicotinic acid, pyridylacetic acid, N-oxypyridylacetic acid or linoleic acid. The production method described in said patent, which is characterized by the reaction of a quaternary heparin salt with a carbodiimide-activated acid, not only gives the O-acetylated derivative but also a substantial amount of a stable sucrose product in the form of an O-acetylated derivative. Acylisoharnstoffester-Dorivates. In addition, such a type of reaction is suitable to promote the formation of anhydrides and intramolecular reactions between the acid functions and the hydroxyl functions of heparin. The patent JP 74/048533 describes O-esters of chondroitin sulfate with aromatic, araliphatic or heterocyclic acids which are optionally substituted and doped with a view to prolonged action. The preparation method described in this document is characterized by the reaction of chondroitin sulfuric acid with an acid chloride and results in an N-acylation as a side reaction. Patent WO 83/00150 describes in general terms precursors of medicaments prepared using various functions the GAGs, their hydroxyl, and specifically, an O-ester of chondroitin sulfate with penicillin V. The preparation of this product is via carbodiimide and therefore has the abovementioned side reactions.

EP 46828 describes O-esters of heparin with unsaturated acids, in particular acrylic acid or methacrylic acid, which, grafted onto biomedical materials, give them a long-lasting antithrombotic activity. This grafting takes place by covalent bonding in the region of the α-β-unsaturated bonds of these esters with the surface of said materials which comes into contact with the blood. According to this document, the α-β-unsaturated O-esters are prepared by reaction of heparin with the chloride or anhydride of an α-β-unsaturated carboxylic acid. In addition, this description, which indifferently indicates the use of chlorides or anhydrides of the α-β-unsaturated acids as reactants, does not specify which of the O-esters of heparin are obtained in this way.

Patent EP 256880 describes the esterification of heparin or heparin (b.p. m.) By the action of acid chlorides in formamide and pyridine to obtain derivatives having increased permembrane permeability. This method thus leads to derivatives which have been subjected to partial desulfation and are 0- but also N-acetylated and in which the ratio sulfate / carboxvl is inferior.

Patent FR 4 066M describes the acetylation of N-monomethylheparinamide by the action of acetic anhydride in formamide and pyridine. The starting product is a heparin derivative whose carboxyl functions are replaced by amide functions.

The method used for the acetylation does not use a salt soluble in the organic medium. Therefore, the acylation reaction can not be controlled accurately and only makes it possible to obtain a low acetylation yield. In addition, when applying the described method to heparin whose carboxyl functions are not blocked, a significant proportion of mixed anhydrides is obtained between the carboxyl groups of the heparin and the acetic anhydride, these unwanted by-products not being removed from the medium , Patent JP-PS 5128602 describes the acetylation of heparin by the action of an acid anhydride in formamide. In this method used, the heparin is not in the form of a salt soluble in the organic medium, whereby an exact control of the Acyliarungs reaction is possible and only a small degree of acylation can be achieved. In addition, mixed anhydrides and secondary products of the reaction in the medium are present in the process described.

Object of the invention

The aim of the invention is to provide more specific products whose biological activity is significantly higher.

Explanation of the essence of the invention

The invention has for its object to find new Glucosaminoglycane with the desired properties and processes for their preparation, which ensure good reproducibility.

It has now been found that, by reaction of a GAG salt soluble in the organic medium, such as a tertiary or quaternary ammonium salt, with the anhydride of a carboxylic acid in a polar aprotic organic solvent, selective acylation of the free hydroxyls is obtained without the carboxyl or amino Function groups of the used GAG negatively influence or attack. The GAG used in the form of a salt soluble in the organic medium advantageously makes it possible to control the acylation reaction with great accuracy. In addition, the degree of acylation is easily modifiable and can be increased without fear of degradation or attack on the rest of the molecule. Thus, it is possible to achieve an acylation degree of from 0.1 to 3 acyl groups per disaccharide unit, in particular from 0.5 to 2 acyl groups.

It has also been found that no N-acylated derivative is formed and that when anhydrides are formed in the range of carboxylic functions, they can be easily recycled to the free acid by the use of a weak base. Advantageously, selective acylation of GAGs according to the present invention allows for modification of their biological activities, which in some cases grow strongly.

Finally, it has been found that the O-acylated GAGs obtained in this way have a pharmacological activity of longer duration.

The present invention therefore has, according to one of its aspects, selectively O-acylated glucosaminoglycans which correspond to the general formula (I) (see formula sheet) in which

G represents a group of the formula (a) or a group of the formula (a ') or a group of the formula (b),

U is a group of formula (c) or a group of formula (d) or the radical of group (c) or group (d) after periodic oxidation, followed by β-elimination or acid hydrolysis,

A is a group R ₁ , a group of the formula R _r (c), a group of the formula R, - (d) or a group of the formula (e) or the radical of the group (c) or the group (d) periodic oxidation, followed by β-elimination or acid hydrolysis,

B is a group OR "a group of the formula (a) -OR _{1 (} a group of the formula (a ') -OR ₍ , a group of the formula (b) -OR ₁ , a group of the formula (f) or a group of the formula (g), or a group of the formula (a), a group of the formula (a ¹ ) or a group of the formula (b), the radicals thereof having a radical of (c) or of (d), such as it is present after periodic oxidation, followed by β-elimination or acid hydrolysis,

R _{1 represents} hydrogen, SO ₃ or acyl, acyl being the radical of a non-α-unsaturated carboxylic acid or dicarboxylic acid selected from:

An alkanoyl group having 1 to 18 carbon atoms,

An alkanoyl group having 2 to 3 carbon atoms substituted by:

a cycloalkyl group having 3 to 7 carbon atoms,

a phenyl group, optionally substituted by one or more alkyl radicals having 1 to 14 carbon atoms, by halogen atoms or the groups NC ₁ or OCH ₃ ,

an unsaturated aliphatic hydrocarbon radical having 4 to 16 carbon atoms,

A benzoyl group, optionally substituted by one or more alkyl radicals having from 1 to 4 carbon atoms, by halogen atoms or the groups NO ₂ or OCH ₃ ,

A cycloalkyl (3-7 CJ-carbonyl group,

R _{2 represents} SO ₃ - and / or an acetyl radical, with the proviso that the ratio of N-acetyl-glucosamine is at most equal to that of heparin when R _{1 is} an acetyl radical,

R _{3 represents} a hydrogen atom or an alkyl radical having 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms, or a phenyl-alkyl radical having 7 to 12 carbon atoms, or an alkali metal or alkaline earth metal cation,

- η is an integer from 1 to 80,

wherein R ₁ in a ratio of at least 0.1 bir. 3 acyl groups per disaccharide unit, preferably 0.5 to 2 groups, is acylated; and their pharmaceutically acceptable salts.

In a preferred embodiment of the invention, the selected glucosaminoglycans are those of the heparin type, namely heparin and heparin derivatives, either obtained by fractionation, by semisynthesis or synthesis, and heparan sulfate and its derivatives also obtained by fractionation, semisynthesis or synthesis.

The selectively O-acylated glucosaminoglycans of this type according to the present invention correspond to the general formula (II) in which

A has the meanings given above for the formula (I),

R _{1 is} hydrogen and / or SO ₃ - and / or an acyl group as defined in formula (I),

R 2 represents R a- and / or an acetyl radical, with the proviso that the ratio of N-acetyl-glucosamine is at most equal to that of heparin when R 1 is an acetyl radical,

R 3 is a hydrogen atom or an alkyl radical having 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms, or a substituted alkyl radical having 7 to 12 carbon atoms, or an alkali metal or alkaline earth metal cation,

- B groups of the formulas Ia) -OR ₁ or (f), (a) and (f) as defined in the formula (I) or ORi dai, or a group of the formula (a), the remainder with a residue of (c) or of (d), as it is present after periodic oxidation, followed by β-elimination or acid hydrolysis,

- η is an integer from 1 to 80.

A preferred family (IIa) of the compounds according to the invention are the compounds of the formula (II) in which

A has the meanings given above for the formula (I),

R, hydrogen and / or SO ₃ - and / or eien as defined in the formula (I) defined acyl group,

R _{1 represents} SO ₃ - and / or an acetyl radical, with the proviso that the ratio of N-acetyl-glucosamine is at most equal to that of heparin when R _{1 is} an acetyl radical,

R _{3 represents} a hydrogen atom or an alkyl radical having 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms, or a substituted alkyl radical having 7 to 12 carbon atoms, or an alkali metal or alkaline earth metal cation,

B represents groups of the formulas (B) -OR ₁ or (f), (a) and (f) as defined in the formula (I), or OR, or a group of the formula (a) whose Residue with a residue of (c) or of (d), as it is present after periodic oxidation, followed by a β-elimination or an acidic hydrolysis,

- η is an integer from 1 to 80, with the proviso that when AR _{1 is} a group of the formula R ₁ - (C) or a group of the formula R _{1 is} -Id) and B is 0-R ₁ , η is an integer from 1 to 16.

Another preferred family (Mb) of the compounds according to the invention are the compounds of the formula (II) in which

A has the meanings given above for the formula (I),

R _{1 is} hydrogen and / or SO ₃ - and / or an acyl group, acyl being the radical of a non-α-unsaturated carboxylic acid or dicarboxylic acid, selected from:

An alkanoyl group of up to 18 carbon atoms,

An alkanoyl group having 2 to 3 carbon atoms substituted by:

a cycioalkyl group having 3 to 7 carbon atoms,

a phenyl group, optionally substituted by one or more alkyl radicals having 1 to 14 carbon atoms, by halogen atoms or the groups NO ₂ or UCH ₃ ,

an unsaturated aliphatic hydrocarbon radical having 4 to 16 carbon atoms,

A benzoyl group, optionally substituted by one or more alkyl radicals having from 1 to 4 carbon atoms, by halogen atoms or the groups NO ₂ or OCH ₃ ,

A cycloalkyl (3-7C) -carbonyl group,

R _{2 represents} SO ₃ - and / or an acetyl radical, with the proviso that the ratio of N-acetyl-glucosamine is at most equal to that of heparin when R _{1 is} an acetyl radical,

IJ ₃ denotes a hydrogen atom or an alkyl radical having 1 to 10 carbon atoms, before 1 to 4 carbon atoms, or a phenyl-alkyl radical having 7 to 12 carbon atoms, or an alkali metal or alkaline earth metal cation,

- η is an integer from 1 to 80,

wherein R _{1 is} acylated in a ratio of at least 0.5 to 2 acyl groups per disaccharide unit, preferably 1 group

Advantageous compounds of the invention are those which belong to the families (Ha) and (Nb) and in whose formula (I!) R _{3 represents} a hydrogen atom or an alkali metal or alkaline earth metal cation.

Other groups of advantageous compounds correspond to the compounds belonging to the families (Ha) and (lib) and in whose formula (II) R _{3 is} an alkyl radical having 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms, or a substituted alkyl Radical with 7 to 12 carbon atoms.

In a preferred aspect of the invention, use is made of mixtures of heparin fragments having a molecular weight of below 10,000 daltons, in particular those whose average molecular weight is between 2,000 and 7,000 daltons, those having an average molecular weight of approximately 4,500 daltons or those having an average molecular weight of about 2500 daltons.

For their preparation, one can advantageously use a process for depolymerization by means of nitrous acid, as described for example in the patent EP 37319. The selectively D-acylated glucosaminoglycans of the invention are therefore represented by the general formula (III) in which

- AR ₁ or a group of the formol R ₁ -Jc) or a group of the formula R ₁ -Jd), as defined in the formula (I),

R _{1 is} hydrogen and / or SO ₃ - and / or an acyl group as defined in the formula (I),

R _{2 represents} SO ₃ - and / or an acetyl radical, with the proviso that the ratio of N-acetyl-glucosamine is at most equal to that of heparin when R _{1 is} an acetyl radical,

R ₃ denotes a hydrogen atom and / or an alkyl fiadical having 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms, or a phenylalkyl radical having 7 to 12 carbon atoms, or an alkali metal or alkaline earth metal cation,

- η is an integer from 3 to 12.

-11- 28b 605

Preferred compounds of the invention correspond to the compounds of formula (III) wherein R 1 is an alkanoyl radical of 4 to

Is 10 carbon atoms.

To obtain mixtures of heparin fragments with a molecular weight of below 10OvO alton, one can also Apply method for enzymatic depolymerization, as described for example in the patents EP 244235 and EP 244236, or for alkaline depolymerization, as described for example in the patent EP 40144. In this case, the selectively O-acylated glucosaminoglycans of the invention are represented by the formula (IV) in which

Ri is hydrogen and / or SO ₃ - and / or an acyl group, as defined in formula (I),

R _{2 represents} S y- and / or an acetyl radical, with the proviso that the ratio of N-acetyl-glucosamine is at most equal to that of heparin, if R _{t is} an acetyl radical,

R _{3 represents} a hydrogen atom and / or an alkyl radical having from 1 to 10 carbon atoms, preferably from 1 to 4 carbon atoms, or a phenyl-alkyl radical having from 7 to 12 carbon atoms, or an alkali metal or alkaline earth metal cation,

B is a group of the formula (a) -OR, as defined in the formula (I), or ORi darstel;

- η is an integer from 2 to 20.

Among the compounds of the formula (IV), those are those in which R _{1 represents} an alkanoyl radical having 4 to 10 carbon atoms.

In another advantageous aspect of the invention kanr. .nan use a mixture of heparin Frat elements, which are homogeneous in terms of their molecular weight and a synthesized heparin fragment, which is partly in terms of its molecular weight and partly, as regards its executionalization, homogeneous.

In a further inteiessanten aspect of the invention can be selected as glucosaminoglycans heparin derivatives that differ from their binding position to antithrombin II! (AT III), whether the heparin chains were fractionated so that the oligosaccharide chains containing the binding site to AT III were removed by, for example, affinity chromatography on Sepharose AT Mi resin or ionic redotografts, described in E. Sache et al., Thromb. Res., 25 (1982), pp. 442-458, or that these binding sites were destroyed, for example, by depolymerization by means of periodate, followed by β-elimination or acid hydrolysis

 Such compounds may correspond to the formula (V) in which

A is a group of the formula R 1 - (c), a group of the formula R 1 (d) or the group (c) or group (d) after periodic oxidation, followed by β-elimination or acid hydrolysis, represents,

B is a group of formula (a) -OR _t or a group of formula (a1), the radical thereof having a radical of (c) or of (d), as after periodic oxidation, followed by β-elimination or a acid hydrolysis, is present, is verbundsn,

Ri has the meanings given in formula (I),

R _{2 is} SO ₃ - or an acetyl radical, the ratio of SO _{1 being} about 90%,

R _{3 represents} a hydrogen atom or an alkali metal or alkaline earth metal cation,

- η is an integer from 1 to 80.

Particularly advantageous compounds can be obtained starting from compounds prepared by the process of periodic oxidation, followed by alkaline β-elimination, reduction and fractionation as described above. These compounds correspond to the formula (VI) in which

- AR ₁ , a group of the formula R _r (c), a group of the formula R ₁ -Id) or the radical of (c) or of (d) after periodic oxidation, followed by a β-elimination,

- U is a group of formula (Vl '), or, in the ratio of one group per two chains at least, is a uronic acid (D-glucuronic acid or L-iduronic acid) which is not sulfated and open between the carbon atoms in position 2 and 3 and the general formula (VI) is taught,

B is a group of the formula (a) -OR, or OR; your group is a group of F <"Yiel (a) whose residue is connected to a residue of (c) or of (d) as it exists after periodic oxidation followed by β-elimination;

Ri has the meanings given in formula (I),

R ₂ SO ₃ - or an acetyl radical, where the ratio of SO ₃ - is at least about 90%,

R _{3 represents} a hydrogen atom or an alkali metal or alkaline earth metal cation,

- η is an integer from 2 to 18.

Advantageous compounds corresponding to the formula (VI) are those in which η is an integer from 7 to 15 fü. the Representing a majority of the species that make up it. Advantageous compounds corresponding to the Formein (V) and (Vl) and in particular those of the Form! (Vl)

corresponding to η as an integer from 7 to 15 for the majority of species are those in which R 1 is an alkanoyl radical of 2 to 10 carbon atoms, advantageously 4 to 10, preferably 4 or 10 carbon atoms.

Other preferred compounds corresponding to formula (VI) are mixtures of compounds which are suitable with respect to their Molar mass are homogeneous and would be obtained by gel filtration. With respect to the formula (VI), η here represents an integer of Among the compounds with heparin structure, that of the binding layer to AT III and, consequently, of their anticoagulant Liberated, one family of advantageous compounds corresponds to the selectively O-acylated, N-desulfated-N-

acetylated heparin derivatives of the formula (VII) in which

A Ri, a group of the formula R _r (c) or a group of the formula R, - (d), as defined in the formula (I),

Ri represents an alkanoyl radical having 2 to 18 carbon atoms,

R _{3 represents} a hydrogen atom or an alkali metal or alkaline earth metal cation,

B is a group of the formula (a) -OR ,, as in the formula (I) definie ¹ 1, or ORi means

- η is an integer from 1 to 80.

In another advantageous aspect of the invention, the selected glucosaminoplycans are of the chondroitin sulfate type, namely the chondroitin 4- and 6-sulphates, the dermatan sulphate and their fragments.

The selectively O-acylated glucosaminoglycans of this type according to the invention are represented by the formula (VIII) in which

A has the meanings of the formula (I),

R) represents hydrogen and / or SOr- and / or an acyl group as defined in the formula (I),

R _{3 represents} a hydrogen atom or an alkyl radical having 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms, or a phenyl-alkyl radical having 7 to 12 carbon atoms, or an alkali metal or alkaline earth metal cation,

- B groups of the formulas (b) -OR ₁ or (g), as defined in the formula (I)., Or OR ₁ or a group of the formula <b), the radical with a radical of (c) or of (d) associated with periodic oxidation followed by β-elimination or acid hydrolysis;

- η is a whole number from 1 to 80.

Among the compounds of the formula (VIII), a preferred family corresponds to the compounds in which R _{3 is} a Represents hydrogen atom or an alkali metal or alkaline earth metal cation. Other advantageous compounds of the formula (VIII)

are those in which R _{3 represents} an alkyl radical of 1 to 10 carbon atoms and preferably 1 to 4 carbon atoms or a phenyl-alkyl radical of 7 to 12 carbon atoms.

Another advantageous family of the compounds of the formula VIII) corresponds to the compounds in which R _{1 is} an alkanoyl Represents radical with 4 to 10 carbon atoms. The process according to the invention for the preparation of the selectively O-acylated glucosaminoglycans is characterized in that

that the glucosaminoglycan is converted into a soluble salt in organic medium and that this salt with a

Acylating treated, which is capable of the primary and secondary hydroxyl groups of this Glucosaminoglycans too

acylate without affecting or attacking the NHR ₃ OdBrCOOR ₃ functional groups present in the glucosaminoglycan acylation reaction, the reaction taking place in a polar aprotic solvent at a temperature of from ⁰ ° C to 100 ° C in the presence of a catalyst and in the presence of a base, which is suitable for binding the acid in the Acylierungfreiwerdonde.

The product is then precipitated with the aid of a water-miscible solvent, such as ethanol, to which is added a Precipitation supportive additive may give, such as a mineral salt. The O-acylated glucosaminoglycan is dissolved by dissolution

of the precipitate in water and, advantageously, isolated by dialysis in the presence of a weak base.

In an advantageous manner, the process according to the invention is carried out according to the following steps, by

(1) a glucosaminoglycan of the general formula (IX) (see formula sheet) in which

G ^{0 represents} a group of the formula (a) ° or a group of the formula (a ') ° or a group of the formula (b) °,

- U ° a group of the formula (c) ° or a group of the formula (d) ° or the radical of the group (c) ° or the group (d) ° after periodic oxidation, followed by a β-elimination or an acid hydrolysis , means

A ° is a group R ₁ ⁰ , a group of the formula Ri ° - (c) °, a group of the formula R ₁ ¹ Md) ⁰ or a group of the formula (e) ° or the radical of the group (c) ° or of group (d) ° after periodic oxidation, followed by β-elimination or acid hydrolysis,

- B ^{0 is} a group ORi ⁰ , a group of formula (a) ° -0Ri °, a group of formula (a ') - ORi ⁰ or a group of formula (f) ° or a group of formula (g) ° or the groups (a) °, (a ') ° or (b) °, their radicals having a radical of (c) ° or of (d) °, as after periodic oxidation, followed by β-elimination or an acidic hydrolysis, are connected,

R, ° represents hydrogen or SO ₃ ,

- R ₃ SO ₇ - or an acetyl radical, with the proviso that the ratio of N-acetyl-glucosamine is at most equal to that of heparin, when R _{1 represents} an acetyl radical,

R _{3 represents} a hydrogen atom or an alkyl radical having 1 to 10 carbon atoms or a phenyl-alkyl radical having 7 to 12 carbon atoms, or an alkali metal-ortho-alkaline earth metal cation,

- η is an integer from 1 to 80,

converted into a salt of the gonucleosaccharide glucosaminoglycan which is soluble in polar aprotic organic solvents:

(2) this salt with an anhydride of the formula:

Acyl-O-acyl ₍

in which acyl is defined as in formula (I), treated in said polar aprotic organic solvent in the presence of a catalytic amount of pyridine or a dialkylaminopyridine and a proton acceptor;

(3) the product thus obtained is precipitated with the aid of a solution of sodium acetate in ethanol, and

(4) isolating the selectively O-acylated glucosaminoglycan by dissolving the precipitate thus obtained in water and by dialysis in the presence of a weak base, and optionally converting the thus-obtained sodium salt of the selectively O-acylated glucosaminoglycan to another pharmaceutically acceptable salt In an advantageous aspect of the process of the invention, the glucosaminoglycan used in step (1) is selected from heparin, a mixture of heparin fragments having a molecular weight of below 10,000 daltons, a mixture of medium molecular weight heparin fragments, which is between 2000 and 7000 daltons, a mixture of heparin fragments with an average molecular weight of about 4500 daltons, a mixture of heparin fragments with an average molecular weight of about 2500 daltons, a mixture of heparin fragments, which, in view of their Molar mass is homogeneous and a hepar obtained by synthesis in-frayment, partly in terms of its molecular weight and partly homogeneous in terms of its functionalization, formed group.

In another advantageous aspect of the process according to the invention, the glucosaminoglycan used in step (1) is heparin, a fraction or a heparin fragment which has been freed from its binding position to antithrombin IU.

The glucosaminoglycan used in the step (1) of the method of the present invention can be advantageously selected from the group formed by dermatan sulfate and its fragments or chondroitin 4- and 6-sulfate and its fragments.

In an advantageous manner, the salt used in stage (1) of the process according to the invention is Glucosaminoglycan the salt of a tertiary amine, in particular a tributylamine salt, or a quaternary ammonium salt,

in particular a tetrabutylammonium salt.

In a further advantageous aspect of the process according to the invention, the anhydride used in step (2) is the Anhydride of an alkanoic acid having 2 to 10 carbon atoms, preferably 4 to 10, preferably 4 to 6 Carbon atoms. The polar aprotic solvent in which step (2) of the process according to the invention is carried out can

be advantageously selected from that by dimethylformamide, hexamethylphosphoric triamide, pyridine or a

Mixture of these solvents with each other or with dichloromethane, formed group and the catalyst from the through Amines, such as pyridine and Oimethylaminopyridin formed group. The base intended to neutralize acidity may be pyridine (which simultaneously acts as a solvent, catalyst and base

can serve), triethylamine or tributylamine.

In an advantageous manner, the dialysis performed in step (4) takes place in the presence of a weak base such as Sodium bicarbonate to remove any by-products formed, such as anhydrides. Advantageously, the temperature and duration of the reaction may vary depending on the desired Acylierurigsgrad fluctuate, each of 0 ° C b's 100 ⁰ C, in particular from ^{0 0} C to 50 ⁰ C, and advantageously takes place Reaction at ambient temperature, for example within 1 to 24 hours. The selectively O-acylated compounds of the present invention have prolonged activity in vivo. If those Compounds are, for example, those of the heparin type which have the binding position to AT III and therefore enable s' .d,

To exert an antithrombotic activity, this effect is significantly prolonged in time, in proportion to the same

Compound that was not selectively O-acylated. The selectively O-acylated heparin derivatives according to the invention which either retain their binding position to AT III or

neither, the latter being virtually free of their anticoagulant activity, also have various biological activities, in particular:

Smooth muscle cell proliferation inhibition, which is of great interest for the prevention of restenosis in procedures such as angioplasty, by-pass surgery, venous and arterial transplantation, organ transplantation, and especially heart transplantation,

Inhibition of heparanase and heparitinase, enzymes associated with metastatic dissemination,

antiviral properties, in particular to retroviruses, especially to the various HIV (human immunodeficiency virus), which is of great interest in the treatment of AIDS,

Properties for inhibiting leukocyte elastase, increasing the circulatory level of elastase inhibitors, as well as selectively inhibiting the synthesis of type III collagen and fibronectin, which is of great interest in the treatment of diseases associated with system imbalance Elastase-related elastase, like emphysema. such as in treating degenerative diseases of the connective tissue, such as arteriosclerosis and diabetes.

The selective O-acylated glucosaminoglycans of the invention can therefore be the drug of large-sized drugs Meaning for numerous indications. The invention therefore also relates to pharmaceutical compositions which are characterized

in that it contains as active ingredient an effective amount of at least one novel, selectively O-acylated

Glucosaminoglycan in association with a pharmaceutically acceptable carrier. In an advantageous manner, the selectively O-acylated glucosaminoglycan is in the form of a pharmaceutically acceptable Salt, such as a sodium, magnesium or calcium salt. In an advantageous embodiment of the invention, the pharmaceutical compositions are characterized

in that the pharmaceutical carrier is suitable for administration by oral route and in the form of gastro-resistant capsules, tablets or tablets, pills or also in the form of drinkable solutions, advantageously 50 mg to 5 g per unit dose, preferably 100 to 1000 mg for capsules, tablets or pills and 10 to 150 mg for drinkable solutions, for example one to three times a day; or by the fact that these

Compositions in the form of sterile or sterilizable injection solutions are for administration to intravenous,

intramuscular or subcutaneous route, these solutions advantageously being 50 to 200 mg / ml selectively O-acylated

Glucosaminoglycan, when provided for injection by the subcutaneous route, or from 20 to 200mg / ml selectively O-acylated glucosaminoglycan, when intended for intravenous or perfusion injection. These dosing intervals are indicative only and the doses to be administered will be determined by the Clinician calculates, taking into account the condition of the sufferer and his personal response with regard to the Medications. The invention likewise relates as biological reactants to the selectively O-acylated glucosaminoglycans of the invention,

which may be used as reference products or standards in comparative experiments for the structure / activity relationship of different physiological systems in which the glucosaminoglycans intervene.

-14- 285 606 Export Certificate

The invention will be further elucidated with the help of the following application examples, which are not restrictive in character.

example 1 Preparation of O-acetyllertem Heparin (IC 1938), starting from Heparln tetrabutylammonium salt

a) Preparation of the heparin tetrabutylammonium salt

The dissolved in water (500 ml) heparin sodium salt (1 Og) is percolated through a cation exchange resin column (Dowex 50 Wx 4, in H ⁺ form). The resulting solution is neutralized with tetrabutylammonium hydroxide. After lyophilization, the Hepdrin Totrabutylammonium salt (19.55 g) is obtained.

b) Acotyllerung

1.05 g of tetrabutylammonium heparinate are dissolved in anhydrous dimethylformamide (5 ml). After cooling to a temperature of ⁰ ° C., acetic anhydride (1 ml, 10.46 mmol) is added dropwise, followed by triethylamine (1.45 ml, 10.46 mmol) and dimethylaminopyridine (64 mg, 0.5 mmol). Then the mixture is allowed to stand at ambient temperature for 24 hours. After adding water (5 ml), the solution is dialyzed against distilled water for 72 hours. The tetrabutylammonium salt is converted to the sodium salt by passage through a Dowex 50, H ⁺ resin at ⁰ ° C, followed by neutralization by 1 N sodium hydroxide solution. After lyophilization, 0.49 g of selectively O-acylated sodium hoparinate is obtained which has the following characteristics:

Sulfate / carboxyl ratio: 2.28meq / g

(Starting product: 2.20meq / g)

APTT titer: 91 μl / mg Spectrum ¹³ C NMR (methanol 51.6 ppm, internal standard)

Signal at 23.4 ppm: CH ₃ of CH ₃ -CO-O-

Signal at 24.5 ppm (weak): CH ₃ of CH ₃ -CO-NH- (identical to the starting product)

The NMR spectrum shows the presence of about two acetyl groups per disaccharide unit. Example 2 Preparation of O-acetyllertem Heparin (IC 1938) starting from Heparln-Trlbutylammonlum salt

a) Preparation of Heparln tributylammonium salts

The dissolved in water (500ml) heparin sodium salt (10g) is percolated through a Katlonoriaustauscherharz column (Dowex 50 Wx4, in H ⁺ form). The solution is neutralized by adding a 10% solution of tributylamine in ethanol. After washing in ether and lyophilization, the tributylammonium salt of heparin (14.77 g) is obtained.

b) acetylation

To a solution of the above salt (4g) in anhydrous dimethylformamide (SOmI) cooled to ⁰ ° C are added dimethylaminopyridine (250mg), acetic anhydride (3.9ml) and tributylamine (9.7ml). After standing at ambient temperature for 24 hours, water (1.5 ml) is added. Then the mixture is poured into a saturated solution of sodium acetate in ethanol. After washing with ethanol, the precipitate is dissolved in water and then dialyzed against bicarbonate (5% in water) and then against water. The O-acetylated sodium heparinate (1.81 g) is obtained after lyophilization. The infrared spectrum has a strong ester band at 1730 cm "'

 After saponification, the product has:

a sulphate / carboxyl ratio of 2.33meq / g (starting product 2.40meq / g),

- an APTT titer of 85 III / mg.

Example 3 Preparation of O-acetyllertem heparin (IC 1938) using pyridine catalysis The tetrabutylammonium salt of heparin (0.58 g) is mixed (1/1, v / v) with pyridine and dimethylformamide

(10ml) solved. Acetic anhydride (0.5 ml) is added and the mixture allowed to stand at ambient temperature. After 24 hours, add an aqueous solution of sodium acetate (1M, 15 ml) and then pour the mixture into ethanol cooled to a temperature of 0 ° C (80 ml). After centrifugation, the precipitate is redissolved in water (10 ml) and then ethanol (160 ml) and an aqueous solution of sodium acetate (1M, 10 ml) are added. The precipitate is then taken up in water and lyophilized to give the O-acetylated heparin (0.22 g).

Example 4 Preparation of O-propylated heparin in different stages (IC 1939) To a cooled to a temperature of 0 ° C solution of tetrabutylammonium heparinate (1.85 g), obtained as in Example 1

In dimethylformamide (10 ml) are added dropwise propionic anhydride (2.7 ml) and then triethylamine (2.9 ml) and dimethylaminopyridine (128 mg). At 1,2,4,8 and 24 hours, part of the reaction mixture is taken, diluted with the same volume of water and dialyzed in distilled water for 24 hours. After passage over Dowex 50H ⁺ resin and neutralization by sodium hydroxide, the resulting products are lyophilized and have the following characteristics:

reaction time Dimensions Sulfate / carboxyl ΑΡΤΓ meq / g (U / mg) 24 hours 490 mg 2.31 80 8h 138 mg 2.35 88 4h 122 mg 2.33 94 2h 120 mg 2.42 109 1h 120 mg 2.33 120 Output product 2.40 150

The proton NMR spectrum of the products was recorded in water with 3,3,3-trimethylsilylpropionate (TSP) as internal standard. It has signals at 1.1 ppm and 2.4 ppm, which are characteristic of the CH ₃ and CH ₂ radicals of CH ₃ -CHj-CO-O and signals between 3 and "" Dpm, which are protons of the Osid Gorüstes speak.

The comparison of the intensity of the signals of the propionyl and the skeleton of the products treated for 1 hour and those which were subjected to the reaction for 24 hours shows the influence of the reaction time: if one observes an increase in the signals of the framework protons indicates an increase in the degree of substitution.

The carbon spectrum (methanol 51.6 ppm, internal standard) shows signals at 10.8 and 30 ppm, which are characteristic of the radicals CH ₃ and CH _{2 of} the propionic esters.

Example 5 Production of O-butyrylated heparin (IC 1940)

a) Use of the heparin tetrabutylammonium salt

To a solution of the heparin tetrabutylammonium salt (0.55 g), obtained as described in Example 1, in dimethylformamide (5 ml) are added at a temperature of ^{0 0} C butyric anhydride and dimethylaminopyridine. After 24 hours at ambient temperature, water (5 ml) is added and the reaction mixture is then dialyzed against distilled water for 72 hours. After exchange on Dowex 50H ⁺ , neutralization by sodium hydroxide and lyophilization, the O-butyrylated heparin is obtained in the form of the sodium salt (0.32 g).

Sulfate / carboxyl ratio: 2.15 meq / g

(Starting product: 2.20 meq / g)

APTT titer: 59 μl / mg

The proton NMR spectrum (TSP, internal standard) shows the presence of signals at 0.9 ppm; 1.6ppm and 2.4ppm, which are characteristic of CHr-CH ₂ groups of CH ₃ -CH ₂ -CH ₂ -CO-O- and signals between 3 and 6 ppm, which speak for the Osid scaffold

 Analysis of the NMR spectrum shows the presence of about one butyryl chain per disaccharide inclusion.

b) Use of the heparin-tributylammonium salt

To a cooled to 0 ° C solution of the heparinate of tributylamine (4g) obtained as described in Example 2, in dimethylformamide (50ml) are added dime'.iiylaminopyridine (0.25g), butyric anhydride (6.7ml) and tributylamine ( 9.7 ml). Then the reaction mixture is left for 24 hours at ambient temperature. Add water (1.5 ml) and, after 30 minutes, add a saturated solution of sodium acetate in ethanol (25OmI). The precipitate is then washed three times with ethanol, then dialyzed against a 5% solution of bicarbonate and then against water. After lyophilization, the O-butyrylated sodium salt of heparin (2.1 g) is obtained. APTT titer: 29 μl / mg After saponification of the esters with 0.5 M sodium hydroxide solution within 2 hours at a temperature of ⁰ ° C., the product obtained has a sulphate / carboxyl ratio of 2.36meq / g. (Starting product 2.40 meq / g).

Example β

Preparation of O-liexanolyllic Heparin (IC 1941)

a) Use of the Heperin TetrabutvlammonlunvSalzes

To a solution of the yetrabutylammonium salt of heparin (0.55 g), obtained as described in Example 1, in dimethylformamide (3 ml), caprylic anhydride (1 ml, 6 mmol), triethylamine (0.84 ml) is added at a temperature of ⁰ ° C. 6 mMol) and dimethylaminopyridine. After standing at ambient temperature for 24 hours, water (5 ml) is added, followed by dialysing the reaction mixture for 72 hours against a 5% bicarbonate solution and against deionized water. After exchange on Dowex 50 H ⁺ , neutralization with sodium hydroxide and lyophilization gives the O-hcxanoylated heparin in the form of the sodium salt (0.30 g)

 APTT titer: 25ui / mg

The proton NMR spectrum (TSP, i internal standard) has signals at 0.8; 1.2; 1.5 and 2.3 ppm, which are characteristic of CH ₃ -CH ₂ of CH ₃ - (CH ₂ Ig-CO-O-

b) Use of the heparin tributylammonium salt

To a solution of tributylammonium heparinate (4 g) cooled to a temperature of ⁰ ° C., obtained as described in Example 2, in dimethylformamide (50 ml) are added dimethylaminopyridine (0.25 g), tributylamine (9.7 ml) and hexanoic anhydride ( 10,6ml). After standing at 20 ° C. for 24 hours, water (1.5 ml) is added, followed by a saturated solution of sodium acetate in ethanol. After washing with ethanol, dialysis and lyophilization, O-hexanoylated heparin (2.5 g) is obtained.

Example 7

Preparation of O-octenoylated Heparin (IC 1942)

To a cooled to a temperature of O ⁰ C solution of tributylammonium heparinate (4g) obtained as described in Example 2, in dimethylformamide (50ml) are added dimethylaminopyridine (0.25g), tributylamine (9.7ml) and octanoic anhydride (12 , 1 ml). After standing for 24 hours at 20 ⁰ C, water (1.5 ml) is added and after 30 minutes a saturated solution of sodium acetate in Etl.anol. After dialysis against a 20% ethanol solution and then against distilled water and after ultrafiltration, the product is subjected to cation exchange by passage over Dowex 50H ⁺ , followed by neutralization by sodium hydroxide. After lyophilization, the O-octanoylated heparin (2.5 g) is obtained.

Example 8 Preparation of O-decanoylated Heparin (IC 1943)

a) Use of Heparln Tetrabutylammonlum salt

To a solution of the tetrabutylammonium salt of heparin (0.55 g), obtained as described in Example 1, in dimethylformamide (5 ml), caprylic anhydride (1 ml, 6 mmol), triethylamine (0.84 ml) is added at a temperature of ⁰ ° C. , 6 mmol) and dimethylaminopyridine. After standing at ambient temperature for 24 hours, water (5 ml) is added followed by dialysis of the reaction mixture for 72 hours against a 5% bicarbonate solution and then against distilled water. After exchange on Dowex 50H ⁺ , neutralization with sodium hydroxide and lyophilization gives the O-decanoylated heparin in the form of the sodium salt (0.30 g).

The proton NMR spectrum (TSP, internal standard) has signals at 0.8; 1.2; 1.5 and 2.4 ppm, which are characteristic of CH ₃ and CH ₂ of CHr- (CH ₂ ) a-CO-O-.

b) Use of the Heparln tributylammonium salt

To a cooled to a temperature of 0 ° C solution of tributylammonium heparinate (4g), obtained as described in Example 2, in dimethylformamide (50ml) are added dimethylaminopyridine (0.25g), tributylamine (9.7ml) and decanoic anhydride (13 , 3g, dissolved in 20 ml of dimethylformamide). After standing at ambient temperature for 24 hours, water (1.5 ml) is added followed by a saturated solution of sodium acetate in ethanol. Dor precipitate is dissolved in dimethyl sulfoxide and then dialyzed against water, sodium bicarbonate and again against water. After lyophilization, the O-decanoylated heparin (2.38 g) is obtained. '

Example 9

Preparation of O-oleoylated Heprin (IC 2013)

The tributylammonium salt of heparin (3 g) and N, N-dimethylaminopyridine (244 mg) are dissolved in anhydrous dimethylformamide (50 ml). After cooling to a temperature of ⁰ ° C., oleic anhydride (21 g), synthesized according to Plusquellec et al., Tetrahedron, 1988, 44, 2471-2476, in solution of dichloromethane (20 ml) is added dropwise, followed by tributylamine (9, 5ml). After 24 hours of reaction and cooling to a temperature »i O ⁰ C, one carries a 5% bicarbonate solution (10 ml), and then, after one hour, a saturated alcoholic solution of sodium acetate.

After washing with absolute ethanol and dissolution in a mixture of dimethylsulfoxide-water (4/1, v / v, 750 ml), dialysis is for 2 days against 10% aqueous ethanol and then against water for 3 days. The sodium salt of O-oleoylated heparin is isolated after lyophilization and precipitation of the reconstituted in DMF (2.77 g) lyophilisate in ethyl ether.

Oleic acid content: 1.44 pmol / mg (dosage according to Ducombe W. et al., Biochemical Journal, 1963, 88, 7).

Example 10 Preparation of O-benzoylated heparin (IC 1944)

The tetrabutylammonium salt of heparin, obtained as described in Example 2, is benzoylated with benzoic anhydride under the conditions previously described in the preparation of O-decanoylated heparin (Example 8).

The carbon NMR spectrum of the product obtained has signals at 131.132 and 136 ppm, which are characteristic of the benzoyl groups.

The product is free of anticoagulant activity in vitro.

Example 11 Preparation of O-3-cyclopentyl-propionylated heparin (IC 2014)

The S-cyclopentyl-propionic anhydride is prepared according to the method of Plusquellec et al., Tetrahedron, 1988, 44, 247-2476. It is obtained after adding the acid (23 ml) in solution of dichloromethane (400 ml) to a stirred and cooled to a temperature of -10 ° C mixture containing tetrabutylammonium bromide (15 mmoles), 20% sodium hydroxide solution (SOmI) and Dichloromethane (80ml), and after decantation, wash with 5% sodium bicarbonate and water and concentrate to an oil (96%). Characteristic Infrared Frequency: 1800, 1745, 1040 cm " ^1. Dissolve the tributylammonium salt of heparin (4 g) and the N, N-dimethylaminopyridine (253 mg) in anhydrous dimethylformamide (40 mL) and allow to cool to 0 ° C 3-Cyclopentylpropionic anhydride (11 g) and then tributylamine (9.8 ml) are added dropwise After 24 hours reaction time at ambient temperature and subsequent cooling to ⁰ ° C., water (1 ml) and after 1 hour a saturated, alcoholic sodium acetate solution After washing with absolute ethanol, the precipitate is dialysed for 24 hours against a 5% sodium bicarbonate solution and then against water for 3 days After lyophilization, the O-3-cyclopentylpropionylated heparin is obtained in the form of its sodium salt (2.64 g). ,

The carbon NMR spectrum in D ₂ O (methanol to 61.6 ppm, internal standard) exhibits the characteristic signals at 27.4; 33.1; 34.8; 35.9 and 41.7 ppm of the O-cyclopentylpropionyl group

 Ratio of sulphate / carboxyl: 2,2

Example 12

Preparation of Low Molecular Weight O-Acetyllic Heparin (Mean Molecular Weight 4500 Daltons, Molecular Weight Interval of 1800-8000 Daltons) (IC 1945)

This low molecular weight heparin was obtained by partial nitroso depolymerization and alcoholic fractionation according to the patent EP181252 and is referred to below as CY 216.

a) Use of the tetrabutylammonium salt of CY 216

The sodium salt of CY 216 (1 g) is converted to the tetrabutylammonium salt by passage through a Dowex 50H ⁺ resin column, followed by neutralization by tetrabutylammonium hydroxide.

The salt thus obtained (1.7 g) is dried for three hours under vacuum at a temperature of 50 ⁰ C and then dissolved in anhydrous dimethylformamide (10 ml). After cooling to a temperature of ⁰ ° C., acetic anhydride (1.7 ml) is added dropwise, followed by triethylamine (2.4 ml) and dimethylaminopyridine (102 mg). After 20 hours of reaction, the product is chromatographed on a Sephadex column G-25 eluting with water. After conversion to the sodium salt and lyophilization, the O-acetylated CY 216 (0.89 g) is obtained. Its carbon NMR spectrum (methanol 51.6 ppm, internal standard) shows a signal at 23 ppm, which is characteristic of acetates. The signal of CH ₃ of CHa-CO-NH- at 24.5 ppm is identical to that of the starting material.

Sulfate / carboxyl ratio: 2.09 meq / g

(Starting product: 2.05meq / g)

APTT titer: 18ui / mg

anti-Xa titer: 205 u / mg

(Dosage of Yin et al., J. Lab. CNn. Med., 1973 81, 298-310)

b) Use of tributylammonium salt of CY 216

The sodium salt of CY 216 is converted to the tributylammonium salt by passage through a resin Kolonno Dowex 50H ⁺ , followed by neutralization with tributylamine, as previously described for heparin. The tributylammonium salt of CY 216 is obtained after washing with ether, lyophilization and drying in a vacuum oven.

The above-mentioned salt (4 g) and the Ν, Ν-dimethylaminopyridine (288 mg) are dissolved in dimethylformamide. After cooling to a temperature of 0 ° C, acetic anhydride (4.4 ml) and then tributylamine (11.2 ml) are added dropwise. After standing for 24 hours at ambient temperature and cooling to a temperature of ^{0 0} C, water (1.7 ml) is added and after one hour a saturated, alcoholic solution of sodium acetate.

The precipitate is washed with ethanol, dissolved in pyrogen-free water, dialyzed against 5% bicarbonate solution for 36 hours and then dialyzed against water for 3 days. After lyophilization, the acetylated CY 216 in the form of the sodium salt (1.4 g) is obtained.

The carbon NMR spectrum in D ₂ O (methanol) at 51.6 ppm as internal standard) shows at 23 ppm the signal characteristic of the acetyl group.

Sulfate / carboxyl ratio: 2.07.

Example 13

Preparation of a Low Molecular Weight O-Butyrylated Heparin (Mean Molecular Weight 4500 Daltons, Molecular Weight Interval of 1800-8000 Daltons) (IC 1957)

The tributylammonium salt of CY 216 (4g) obtained as described in Example 12 and the Ν, Ν-dimethylaminopyridine (288mg) are dissolved in anhydrous dimethylformamide (40ml). After cooling to a temperature of ⁰ ° C., butyric anhydride (7.68 ml) is added dropwise, followed by tributylamine (11.2 ml). After 24 hours of reaction at ambient temperature and then cooling to ⁰ ° C, water (1.7 ml) is added and after one hour a saturated alcohol solution of sodium acetate. After washing with ethanol, dissolving in pyrogen-free water, dialyzing against a 5% sodium bicarbonate solution for 36 hours and then against water for 3 days, the O-butyrylated CY 216 is isolated after lyophilization in the form of the sodium salt (2.14 g).

The carbon NMR spectrum in D ₂ O (methanol) at 51.6 ppm, internal standard) is 15.6; 20.5 and 39.4ppm the signals characteristic of the O-butyryl group

 Sulfate / carboxyl ratio: 2.08.

Example 14

Preparation of Low Molecular Weight O-hexanoylated Heparin (Average Molecular Weight 4500 Daltons, Molecular Weight Interval of 1800-8000 Daltons) (IC 1958)

The tributylammonium salt of CY 216 (4g) obtained as described in Example 12 and the Ν, Ν-dimethylaminopyridine (288mg) are dissolved in anhydrous dimethylformamide (40ml). After cooling to a temperature of 0 ° C is added dropwise capric anhydride (10.8ml) and then tributylamine (11.2ml). After 24 hours of reaction at ambient temperature and then cooling to ⁰ ° C, water (1.7 ml) is added and after one hour a saturated alcohol solution of sodium acetate. After washing with ethanol, dissolving in pyrogen-free water, dialyzing for 36 hours against a 5% sodium bicarbonate solution and then against water for 3 days, the O-caproylated CY 216 is isolated after lyophilization in the form of the sodium salt (2.5 g).

The carbon NMR spectrum in D ₂ O (methanol at 51.6 ppm, internal standard) is 15.9; 24.2 and 26.4ppm the characteristic of the caproyl group signals

 Sulfate / carboxyl ratio: 2.08.

Example 15

Preparation of Low Molecular Weight O-Octanoylated Heparin (Mean Molecular Weight 4500 Daltons, Molecular Weight Interval of 1800-6000 Daltons) (IC 1959)

The tributylammonium salt of CY 216 (4g) obtained as described in Example 12 and the Ν, Ν-dimethylaminopyridine (288mg) are dissolved in anhydrous dimethylformamide (40ml). After cooling to a temperature of ⁰ ° C., dropwise tricarboxylic anhydride (14 ml) is added followed by tributylamine (11.2 ml). After 24 hours of reaction at ambient temperature and subsequent cooling to OX, water (1.7 ml) is added and after one hour a saturated alcoholic solution of sodium acetate. After washing with absolute ethanol, dissolving in pyrogen-free water, dialysis against a 5% sodium bicarbonate solution for 3 hours and then against water for 3 days, the O-octanoylierto CY 216 is isolated after lyophilization in the form of the sodium salt (1.76 g).

The carbon NMR spectrum in d ⁶ DMSO (methanol at 51.6 ppm, internal standard) is 16.8; 25.0; 27.3; 30.9; 31.4; 34.1 and? 5.4ppm signals characteristic of the capryloyl group. Sulfate / carboxyl ratio: 2.07.

Example 16

Preparation of a Low Molecular Weight O-Decanoylated Haparin (Average Molecular Weight 4500 Daltons, Molecular Weight Interval of 1800-8000 Daltons) (IC 1960)

The tributylammonium salt of CY 216 (4g) obtained as described in Example 12 and the N, N-dimethylaminopyridine (288mg) are dissolved in anhydrous dimethylformamide (40ml). After cooling to a temperature of 0 ° C is added dropwise decanoic anhydride (15.3ml) in solution of anhydrous dimethylformamide (10ml) and then tributylamine (11.2ml). After 24 hours of reaction at ambient temperature and then cooling to ⁰ ° C., water (1.7 ml) is added and after one hour a saturated alcoholic solution of sodium acetate. After washing with absolute ethanol and suspending in pyrogen-free water, it is dialyzed against a 10% NaCl solution for 2 days and then against water for 5 days. The O-decanoylated CY 216 is isolated after lyophilization in the form of the sodium salt (2.76 g).

The carbon NMR spectrum in D ₂ O (methanol at 51.6 ppm, internal standard) shows at 16.4; 22.3; 25.1; 29.2; 31.9; 34.4 and 36.5ppm signals characteristic of the decanoyl group

 Sulfate / carboxyl ratio: 2.13.

Example 17

Preparation of a Low Molecular Weight O-acetylated Heparin (Mean Molecular Weight 2500 Daiton, Molecular Weight Interval of 1500-8000 Daltons) (IC 1946)

This low molecular weight heparin was obtained by partial nitroso depolymerization according to the method described in patent EP 37319 and is referred to below as CY 222.

The sodium salt of CY 222 is converted to the tributylammonium salt by passage through a Dowex 50H ⁺ resin column, followed by neutralization by tributylamine.

The salt (1.5 g) obtained by r.ech lyophilization is dissolved in dimethylformamide (5 ml). After cooling to a temperature of ⁰ ° C., acetic anhydride (1.35 ml) is added dropwise, followed by triethylamine (2 ml) and dimethylaminopyridine (85 mg). After 18 hours of reaction, water (20 ml) is added and the mixture is then dialyzed against distilled water for 3 days. After conversion to the sodium salt and lyophilization, the 0-acylated CY 222 (0.86 g) is obtained.

The product has a sulphate / carboxyl ratio of 1.98meq / g (starting product: 1.97meq / g). The carbon NMR spectrum (methanol 51.6 ppm, internal standard) of the product contains a signal at 23 ppm, which is characteristic of O-acetyl. Intensity comparison of the signals of N-acetyl (at 24.5 ppm) between the starting product and the final product demonstrates that acetylation was selective.

APTT titer: 8ui / mg

anti-Xa titer: 191 u / mg

(Dosage of Yin et al., J. Lab. CNn. Med., 1973 81, 298-310).

Example 18

a) Preparation of a Heparln Fragment that Is Free from Affinity to Antithrombrn III (IC 1772)

1. Separation of the Heparln chains by means of Perlod-Säura

10 g of injectable heparin from porcine mucus, in the form of the sodium salt, which titrates 157 μl / mg in the dosage Codex and 155 μg / mg in the dosage of anti-factor Xa from Yin et al. is dissolved in 25OmI demineralized water at a temperature of 4 ⁰ C in solution. The pH of the solution is adjusted to 5.0 by concentrated hydrochloric acid. Then add 10 g sodium metaperiodate (NaIO ₄ , MW: 213.89) in solution of 250 ml demineralized water at 4 ° C with moderate stirring. The pH of the whole is adjusted to 5.0 by concentrated hydrochloric acid. Then, the solution is kept for 24 hours in the dark and in a cooling chamber at -I 4 ⁰ C.

2. Removal of the remaining periodate

The reaction solution is then distributed to three dialysis tubing NOJAX 40 ⁿ (porosity 3 to 4000Da) and subjected to dialysis for 15 hours against demineralized tap water.

3. Depolymerization In basic medium

To 780 ml of the solution obtained after dialysis, 16 ml of 10N sodium hydroxide solution are added and the whole is stirred for 3 hours at ambient temperature (of the order of 18 to 21 ° C).

4. reduction

600mg of sodium borohydride (NsBH ₄ , MW: 37.83) are then added and the solution stirred anew for 4 hours at ambient temperature. The pH is then adjusted to 4 by addition of concentrated hydrochloric acid. After stirring for 15 minutes, the p, i value is adjusted to 7 with the aid of concentrated sodium hydroxide solution. To 820 ml of the solution thus obtained are added 16.4 g of NaCl and then 1270 ml of ethanol. The whole is allowed to stand for 3 hours and then centrifuged at 2 500 rpm for 20 minutes. The precipitate is collected, suspended in 200 ml of pure ethanol, minced with an Ultra-Türrax, and finally recovered by filtration through a Buchner frit, then dried under vacuum at a temperature of 40 ° C. for 5 hours in this way 8,9g product.

5. Alcoholic fractionation

These 8,9g are dissolved in about 120ml demineralized water at ambient temperature. Add 1.78 g of NaCl and the pH of the solution is lowered to 3.5 with hydrochloric acid. Then the volume of the solution is adjusted to 178ml with demineralized water. Then 151 ml of pure ethanol are added with stirring. After the end of the addition, stirring is continued for 15 minutes and then allowed to stand for 10 hours at ambient temperature. The resulting precipitate is recovered by centrifuging at 2500 rpm for 20 minutes. It is suspended in 150 ml of pure ethanol, comminuted with the Ultra-Turrax®, recovered by filtration through a Buchner frit, washed with 300 ml of pure ethanol and finally dried under reduced pressure at 40 ° C. for 24 hours in this way 5 g of the product IC 1772 in the form of a white powder, which has the following characteristics:

- SO ₃ ": 3.55meq / g

COO ": 1.54 meq / g

- S + C: 5.09meq / g

S / C: 2.31 meq / g

It is virtually exempt from N-acetyl-glucosamine (absence of the signal at 24.5 ppm in the carbon NMR spectrum).

Codex titer: 11 μl / mg

- APTT titer: 9 μl / mg

Anti-Xa titer: 12 μl / mg

a) Preparation of an O-acetyllic heparin fragment freed from the affinity for antithrombin III (IC 1924) The product obtained in step a) is purified by passage through a Dowe.x 50 H ⁺ resin, followed by neutralization with tetrabutylammonium Hydroxide, into the tetrabutylammonium salt. Starting from 9.5 g of sodium salt, 18 g of tetrabutylammonium salt are obtained

To a solution of 6 g of the salt thus obtained in dimethylformamide (55 ml), after cooling to a temperature of ⁰ ° C., acetic anhydride (6.2 ml, 65.6 mmol) and then triethylamine (9 ml, 65.5 mmol ) and dimethylaminopyridine (403 mg, 3.3 mmol). After standing for 24 hours, a saturated alcoholic solution of sodium acetate (250 ml) is added. After centrifuging and washing the precipitate with ethanol, the solid is desalted via Sephaden G-25 and then subjected to ion exchange by passage through Dowex 50H ⁺ , followed by neutralization with sodium hydroxide. After lyophilization, the product IC 1924 (3 g) is obtained.

This product has the following characteristics:

Sulfate / carboxyl ratio: 2.28 meq / g.

The carbon NMR spectrum (methanol 51.6 ppm, internal standard) clearly shows that the product was O-acylated. The characteristic signal of C ₂ from N-acetyl-glucosamine, at about 56ppm, is absent, as in the case of the starting material, completely in the spectrum.

Example 19

Preparation of an O-butyrylated Heparin Fragment Free from Affinitiate to Antithromb'i III (IC 1925) 6 g of the tetrabutylammonium salt of IC 1772, obtained as described in Example 18, are butyrylated with butyric anhydride under the same conditions, as described in the acetylation. The product IC 1925 (2.96 g) is obtained, which has the following characteristics:

Sulfate / carboxyl ratio: 2.31 meq / g

The ^<3 C NMR spectrum (methanol 51.6 ppm, internal standard) contains the signals characteristic of the butyl group at 15.6; 20.4 and 38.4 ppm.

Example 20

Preparation of an O-hexanoylated Heparin Fragment Released from Affinity for Antithrombin III (IC 1926) 6g of the tetrabutylammonium salt of IC 1772, obtained as described in Example 12, are treated with hexanoic anhydride in the same manner as in the acetylation , The product IC 1926 (3 g) is obtained which has the following characteristics:

Sulfate / carboxyl ratio: 2.20meq / g

The carbon NMR spectrum (methanol 51.6 ppm, internal standard) shows signals at 15.5; 23.9; 26.2; 32.7 and 36.1 ppm, which are characteristic of CH ₃ and CH _{2 of} the hexyl group. The proton NMR spectrum shows the presence of about one hexyl group per disaccharide unit.

Example 21

Preparation of an O-butyrylated mixture of fragments which release their affinity for antithrombin III and are homogeneous in terms of their molecular weight

The mixture of fragments released from the anticoagulant activity was fractionated by gel filtration into its various components as described in Example 12. In this way homogeneous fractions are obtained with respect to the molar mass, the masses of which are: 7700,6500,5800, 53, 40, 440, 380, 400, 900, 400, 6400, 1860 and 1210.

Example of the esterification of a fraction

The fraction of mass 2600 (0.20 g) is converted to the tributylammonium salt and then lyophilized and dried (0.43 g). This product is then dissolved in DMF (2 ml) and, after cooling to a temperature of 0 ° C, dimethylamirjopyridine (18 mg), butyric anhydride (0.49 ml) and tributylamine (0.7 ml) are added successively. After leaving to stand at ambient temperature for 24 hours, sodium bicarbonate (1 ml of a 5% solution) is added and, 2 seconds later, the mixture is passed through a Sephadex G-25 column (1 liter) eluting with sodium chloride (0.2 M) ) Chromatography. The product is lyophilized after desalting to give a light beige powder (0.24g). The other fractions are treated in the same manner and result in corresponding products, the IR analysis shows the presence of an ester band at 1734cm ^'1. The degree of sulfation of the products (sulfate / carboxyl ratio) is unchanged after the acylation

 Determination of AcyllerungsgrddiJ

The degree of acylation is determined by gas phase chromatography, butanolysis of the products by a butyric acid / sulfuric acid mixture, followed by extraction of the butyl esters by chloroform and removal of the excess butanol by washing with water.

Example 22 Preparation of O-acetylated dermatan sulfate (IC 1947)

To a cooled to a temperature of O ⁰ C solution of the tetrabutylammonium salt of dermatan sulfate (0.91 g) obtained under the same conditions as in the preparation of the tetrabutylammonium heparinate described in Example 1, in dimethylformamide (20ml) Acetic anhydride (1.35 mL, 14.2 mmol) was added dropwise followed by triethylamine (1.97 mL, 14.2 mmol) and dimethylaminopyridine (0.7 mmol). After standing at ambient temperature for 24 hours, water (40 ml) is added and dialyzed for 72 hours. The sodium salt is obtained by passage through a Dowex 50 H ⁺ resin column at ⁰ ° C., followed by neutralization with sodium hydroxide. After lyophilization, a beige powder (0.52 g) is obtained.

 The O-acetylated dermatan sulfate has the following characteristics:

Sulfate / carboxyl ratio: 0.99meq / g

(Starting product: 1.05meq / g)

^u C NMR spectrum (methanol 61.6 ppm, internal standard):

Signal at 25.5 ppm CH ₃ of CH ₃ -CO-NH- (identical to the starting product),

Signal at 23.0 ppm CH ₃ of CHj-CO-O-

Beisplel23

Production of O-succinylated dermatan sulfate (IC 2020) The tetrabutylammonium salt of dermatan sulfate (1 g) and the Ν, Ν-dimethylaminopyridine (110 mg) are incorporated in

dissolved anhydrous dimethylformamide. After addition of succinic anhydride (396 mg) and tributylamine (0.94 ml) is the

Mixture for 2 hours under anhydrous conditions at a temperature of 60 ° C incubated. After cooling and Addition of water (2 ml) is carried out by precipitation in a saturated with sodium acetate, ice-cold alcoholic solution. The precipitate is washed with ethanol, dissolved in pyrite-free water and then countered to 5% for 36 hours Sodium bicarbonate solution and dialyzed against water for 3 days. Obtained after lyophilization, the O-succinylated Dermatan sulfate in the form of the sodium salt (0.83 mg). The infrared spectrum (KBr) shows at 1730 and 142C cm " ¹ the frequencies characteristic of the carboxyl group

(Wave number) on.

The carbon NMR spectrum in D ₂ O (methanol 51.6ρρτ>, internal standard) indicates at 33; 34.5 and 183.6ppm for the Succinyl group characteristic signals and at 25.3 ppm the characteristic signal for methyl of the acetamido group,

which is identical to the starting product.

Sulfate / carboxyl ratio: 0.51 meq / g Example 24 Preparation of O-butyrylated heparin previously N-desulfated and N-acetylated (IC 1948)

The heparin is N-desulfated and then N-acetylated according to the technique described by Nagasawa and Inoue (Methods Carbohydr., Chem. Vol. VIII, p.291-294).

The tributylammonium salt (1 g) is obtained by passage over a Dowex 50H ⁺ resin followed by neutralization with a solution of tributylamine in ethanol.

After lyophilization and drying, this salt is dissolved in dimethylformamide (10 ml), and then, after cooling to a temperature of ⁰ ° C., butyric anhydride (2 ml) and tributylamine (2 ml) and dimethylaminopyridine (65 mg) are added. After 24 hours, water (5 ml) is added and the mixture is dialyzed against a 5% sodium bicarbonate solution and then against water. After passage over Dowex 50 H ⁺ , followed by neutralization with sodium hydroxide solution, the N-acetylated-O-butyrylated heparin sodium salt is obtained.

The carbon NMR spectrum (Methanoi 51.6 ppm, internal standard) shows a signal at 24.6 ppm which is characteristic of N-acetyl and signals at 16.20 and 38 ppm corresponding to the butyric acid esters.

The experiment can be repeated with a partially N-desulfated-N-acetylated heparin and then results in a partially N-desulfated-N-acetylated and O-butyrylated product.

Example 25 Production of Peracetylated Dermat Sulfate (IC 1950)

The tetrabutylammonium salt of the dermatan sulfate (0.8 g) dissolved in dimethylformamide (20 ml) is acetylated by addition of donkey's anhydride (1.2 ml), dimethylaminopyridine (76 mg) and triethylamine (1.7 ml). The mixture is heated for one hour at a temperature of 80 ° C.

After returning to ambient temperature add water (0.45 ml) followed by a 0.3 M solution of sodium acetate in ethanol (100 ml). After centrifugation, the precipitate is dissolved in water and then dialyzed against distilled water.

The sodium salt is obtained by exchange on a Dowex 50H ⁺ resin column, followed by neutralization with sodium hydroxide. After lyophilization, the peracetylated dermatan sulfate (0.51 g) is obtained.

This product has a sulphate / carboxyl ratio of 1.07meq / g (starting product: 1.05meq / g) and contains about three acetyl groups per disaccharide unit.

Example 26 Preparation of O-acetyllic Heparin Benzyl Ester (IC 1949)

To a solution of tetrabutylammonium heparinate (1 g) in dimethylformamide (10 ml) is added benzyl bromide (0.17 ml). After standing at ambient temperature for 24 hours, tetrabutylammonium acetate (220 mg) is added. After a further 24 hours, the acetylation of the benzyl ester formed is carried out. For this purpose, dimethylaminopyridine (57 mg) is added, followed by triethylamine (1.3 ml) and acetic anhydride (0.9 ml).

Upon return to ambient temperature, the reaction mixture is allowed to stir for 24 hours. Then water is added and then the product is precipitated by a saturated solution of sodium acetate in ethanol.

After dialysis against distilled water, passage over Dowex 50 H ⁺ , neutralization by sodium hydroxide and lyophilization, the sodium salt of the O-acetylated heparin benzyl ester (0.57 g) is obtained.

The product has a sulfate / carboxyl ratio of 3.6 meq / g (non-benzylated starting product: 2.20 meq / g).

The carbon NMR spectrum (methanol 51.6 ppm, internal standard) shows signals at 23.3 (O-acetyl) and 131.6 ppm (benzyl).

The signal of the CHa of CH ₃ -CO-NH at 24.5 ppm is identical to that of the starting material.

Example 27 Preparation of O-acetyllic DermaUn Sulfate Benzyl Ester (IC 1953)

The tetrabutylammonium salt of dermatan sulfate (1 g) is dissolved in anhydrous dimethylformamide (15 ml). Benzyl bromide (0.25 ml) is added to this solution, cooled to a temperature of ⁰ ° C., and then allowed to stand at ambient temperature for 24 hours.

Thereafter, tetrabutylammonium acetate (0.32 g) is added, and after standing at ambient temperature for 24 hours, the acetylation is carried out.

Acetic anhydride (1.5 ml) is added, followed by triethylamine (2.2 ml) and dimethylaminopyridine (96 mg). After standing for 24 hours, water (0.6 ml) is added and then the product is precipitated by adding a saturated ethanolic sodium acetate solution.

The product is dialyzed against 10% sodium chloride solution and then against water.

Men, after lyophilization, receive benzyl ester of the O-acetylated dermatan sulfate (0.63 g).

Example 28 Preparation of O-butyrylated Der. latan sulfate (IC 2018)

The tributylammonium salt of dermatan sulfate (2g) obtained under the same conditions as described in the preparation of the tributylammonium heparinate in Example 2 and the N, N-dimethylaminopyridine (220 mg) are dissolved in anhydrous dimethylformamide (25 ml) , After cooling to a temperature of ⁰ ° C., add dropwise butyric anhydride (5.9 ml) and then tributylamine (8.6 ml). After 24 hours incubation and cooling to a temperature of 0 ° C, water (1 ml) is added and then the precipitation is carried out in a saturated with sodium acetate, ice-cold alcoholic solution.

The precipitate is washed with ethanol, dissolved in pyrogen-free water and dialysed for 36 hours against a 5% sodium bicarbonate solution and against water for 3 days. After lyophilization, the sodium salt of the O-butyrylated dermatan sulfate (1.3 g) is obtained.

The carbon NMR spectrum in D ₂ O (methanol 51.6 ppm, internal standard) shows signals at 15.6; 20.5 and 38.4 ppm, which are characteristic of the Buty ryl group and a signal at 25.2 ppm, which corresponds to the methyl of the acetamido group and which is identical to the starting material.

Sulfate / carboxyl ratio: 1.05meq / g

Example 29 Preparation of O-hexanoyliomate dermatan sulfate (IC 2019)

The tributylammonium salt of Dermatan sulfate (2g) and the Ν, Ν-dimethylaminopyridine (220mg) are dissolved in anhydrous dimethylformamide (25ml), cooled to 0 ° C and added dropwise with hexanoic anhydride (9.4m). After 24 hours of standing at ambient temperature and cooling to a temperature of ⁰ ° C., water (1 ml) is added and then the precipitate is poured into an ice-cold, alcoholic solution saturated with sodium acetate washed with absolute ethanol, dissolved in pyrogen-free water and dialyzed for 36 hours against a 5% sodium bicarbonate solution and against water for 3 days to give, after lyophilization, the nitro acid salt of the O-hexanoylated dermatan sulfate (1 g).

The carbon NMR spectrum in D ₂ O (methanol 51.6 ppm, internal standard) shows signals at 15.9; 24.2; 26.5; 33.1 and 36.4 ppm, which are characteristic of the O-hexanoyl group and a signal at 25.2 ppm, which corresponds to the methyl of the acetamido group and which is identical to the starting product

 Sulfate / carboxyl ratio: 1.02 meq / g

Example 30

Explanation of Selective O-Acetylation of the Process of the Invention and Comparison with Other Acylation Procedures

The process of the present invention has been compared with the processes of patents FR 2100735 and EP 256880.

In particular, the characteristics of the acetic acid esters of heparin prepared by the method of the invention (IC 19:18, Example 1) were compared with the acetic acid esters of heparin obtained using the process conditions described in patent FR 2100735 (product A) and the acetic acid esters of heparin prepared according to the process conditions described in Examples 3 and 4 of patent EP 256880 (products B and C).

a) Preparation of the product * A

To a solution of tetrabutylammonium heparinate (1 g) in anhydrous dimethylformamide (10 ml) is added dicyclohexylcarbodiimide (4.2 g) dissolved in dimethylformamide (15 ml) and then added dropwise within 45 minutes and at a temperature of 4 ° C acetic acid (1.16ml) dissolved in dimethylformamide (25ml).

After standing at room temperature for 24 hours, the reaction mixture is filtered and concentrated under vacuum.

The residue is suspended in ether. After filtration and washing, the precipitate is dialyzed against distilled water. The sodium salt is obtained by passage through a Dowex 50H ⁺ resin column, followed by neutralization by sodium hydroxide. This gives 0.493 g of product A.

This production was also realized within 48 hours at a temperature of 4 ° C.

Production of products B and C

Products B and C were prepared by acetylating heparin in a mixture of formamide and pyridine with acetyl chloride, using 2 ml of acetyl chloride for product B and 40 ml for product C and those described in examples 3 and 4 of patent EP 256880 process conditions. The acetic acid ester is then taken up in water and dialyzed against sodium chloride. ,

c) Results

The characteristics of the products are listed in Table 1. The characteristics of the starting heparin are given as references in each experiment.

TABLE 1

product relationship APTT titre " YW titers · Sulfate / carboxyl ui / mg u / mg (MeqAj) IC 1938 2.28 91 70 Outp-Hep. 2.20 160 160 A 3.40 42 41 Outp-Hep. 2.40 160 160 B 2.10 57 39 C 1.15 <2 0.6 Outp-Hep. 2.40 160 160

The titers APTT and Yin and Wessler were measured in vitro.

The results show that, although the titers APTT and YW are lower for all products than for the parent heparin, and even more so for the products A, B and C, only the product IC 1938 retains a sulfate / carboxyl ratio, which is practical identical to that of the parent heparin. This is due to the fact that the process of the present invention enables the hydroxyl functions to be selectively acylated without affecting or attacking the functional groups of the heparin, as confirmed by the chemical analysis of the products.

The products IC 1938, A, B and C were analyzed by colic NMR (methanol 51.6 ppm, internal standard). The spectra of these products as well as those of parent heparin are as follows;

Fig. 1: starting heparin

Fig. 2: IC 1938

3: product A after 24 hours of reaction

Fig. 4: Product A after 48 hours of reaction

Fig.5: Products

Fig.6: Products

The results are the following:

1. The product IC 1938 has in the carbon NMR spectrum of Figure 2:

a signal at 23.4 ppm, which corresponds to CH ₃ of CH ₃ -CO-O,

a signal at 24.4 ppm, which corresponds to CH ₃ of CH ₃ -CO-NH and is identical to the signal of the parent heparin. These signals indicate that the amine and carboxyl groups have not been attacked and that selective acetylation has occurred in the region of the hydroxyl groups.

2. The analysis of product A in the carbon NMR spectrum shows that the predominantly obtained product, both after 24 hours and after 48 hours reaction (Figures 3 and 4), is an isourea derivative of heparin, its carboxyl functions due to the use of dicyclohexylcarbodiimide substituted by a group of formula (X) (see formula sheet). In fact, we observe significant signals that correspond to the carbon atoms of the above-mentioned group, in 28,34,54 and 156ppm, as well as the signal CH ₃ CH ₃ -CO-O corresponds, at 23ppm, which is very weak.

The process therefore does not allow to obtain a selective O-acylation and leads to a deterioration of the carboxyl functions, which is reflected by the increase in the ratio sulfate / carboxyl.

3. The product S has in the carbon NMR spectrum of Figure 2:

a signal at 23.1 ppm corresponding to CH ₃ of CH ₃ -CO-O,

a signal at 24.8 ppm which corresponds to CH ₃ of CH ₃ -CO-NH and which is significantly more intense than that of the starting heparin and the IC 1938.

These signals show that not only has O-acetylation occurred, but also strong N-acetylation. The applied non-selective acylation process entails partial N-desulfation, followed by acetylation of the amines, which also causes a reduction in sulfate / carboxyl ratio.

4. The product C has in the carbon NMR spectrum:

a signal at 22 ppm corresponding to CH ₃ of CH ₃ -CO-O,

a signal at 24 ppm corresponds to the CH ₃ of CH ₃ -CO-NH and is significantly more intense than that of the starting heparin and the IC 1938.

As with product B, both O- and N-acetylation are observed. N-desulfation, more significant than boim product B, causes a large reduction in sulfate / carboxyl ratio.

PHARMACOLOGICAL ACTIVITY. OF THE INVENTION PRODUCTS

a) Atitikoagulergic activity in vitro

1. Calculation of titer in vitro in relation to a standard:

This measurement was performed on human plasma using the Temps de cephaline Kaolin sensibilise (Diagnostica Stago, Asnieres, France) test, and the results are shown in Table 2.

TABLE 2

product Titre (ui / mg) IC 1940 113 IC 1941 28 IC1943 7 heparin 160

IC 1940 = O-butyrylated heparin prepared as described in Example 5 IC 1941 = O-hexanoynated heparin prepared as described in Example 6 IC 1943 = O-decanoylated heparin prepared as described in Example 8

The results obtained show that the selectively O-acylated products of the invention have a titer lower than that of heparin in vitro, their anticoagulant activity decreasing as the length of the acyl chain increases. 2. Measurement of the anticoagulant activity of the products according to the invention in vitro on human blood: The experiments were carried out in two dosages of the products according to the invention, 2 μg / ml and 4 μg / ml per 5 ml of human blood. For each product, a control experiment was carried out simultaneously, replacing the product to be tested by isotonic NaCl solution. After incubation for 30 minutes at ambient temperature, the blood is centrifuged for 20 minutes at 3000 rpm. The depleted plasma is decanted to allow the following tests to be performed:

- TCK (Temps de Cephaiine kaolin) using the kit "Temps de cephaline kaolin sensibilise" (Diagnostics Stago, Asnieres, Fr.),

- Heptest "(Herrischem, St Louis, USA).

The results are shown in Table 3. Each result corresponds to the average of three attempts.

TABLE 3

product Dose ^ g / ml) TCK (S) HEPTEST (S) Kontrollo 48,00 21,50 ± 2.70 ± 1.73 IC 1940 2 208.75 61,00 ± 45.54 ± 8.52 IC 1940 4 428.00 101.00 ± 103.43 ± 28.36 control 45,00 23.66 ± 5.00 ± 2.08 IC 1941 2 53,00 40,33 ± 5.19 ± 7.63 IC 1941 4 84.66 61.33 ± 15.01 ± 11.01 control 49,66 23.00 ± 8.50 ± 4.35 IC 1943 2 50,00 23.00 ± 9.54 ± 4.58 IC 1943 4 56,00 24.00 ± 10.00 ± 5.29

The results show in the two methods an extension of the coagulation time for the products IC 1940 and IC 1941.

This extension is proportional to the dose.

In contrast, in these methods, in vitro, the product IC 1943 shows only a very weak effect on the prolongation of the coagulation time.

3. Measurement of anticoagulant activity of the products of the invention in vitro by thromboelastography on human blood: The products of the invention, at doses of 2pg / ml and 4 (ig / ml are combined with 5 ml of human blood as described in the previous experiment for each product, a control was performed by replacing the product to be tested with an isotonic NaC 'solution.

After 30 minutes of incubation at ambient temperature, a thromboelastographic scan is performed using a Hellige thromboelastograph with 0.25 ml recalcified blood per 0.1 ml of 0.058M CaCl ₂ .

The results are shown in Table 4.

TABLE 4

product dose r k r + k amx IPT (Mg / ml) control 13,00 8.25 21:25 48,00 11.25 ± 0.81 ± 1.25 ± 1.50 ± 2.44 ± 2.35 IC 1940 2 33.50 22.25 55.75 35,50 2.57 ± 2.88 ± 4.03 ' ± 5.85 ± 3.69 ± 0.77 IC 1940 4 78.75 44,00 122.75 27.00 0.82 ± 17 34 ± 8.68 ± 13.02 ± 0.16 ± 0.27 control 10.50 4.50 15.00 57,00 37,00 ± 0.70 ± 2.12 ± 2.82 ± 8.48 ± 28.28 IC 1941 2 14.00 10.33 24.33 51.66 21.33 ± 3.00 ± 6.50 ± 6.00 ± 10.96 ± 19.85 IC 1941 4 20.33 15.00 35.33 40,33 4.66 ± 3.51 +4.58 ± 5.03 ± 2.51 ± 2.03 control 13.33 9.00 22.33 47,00 10.33 ± 2.30 ± 2.64 ± 4.93 ± 4.58 ± 4.04 IC 1943 2 12.66 8.00 20.66 47,00 12,00 ± 2.08 ± 2.64 ± 4.72 ± 5.29 ± 5.29 IC 1943 4 12.66 9.83 22,50 45.66 9.33 ± 2.08 ± 3.01 ± 5.07 ± 5.77 ± 4.04

r = reaction time

k = coagulation time, corresponding to an amplitude of the crack of 20 mm

amx = maximum amplitude

IPT - index of thrombodynamic potential

The results show that IC 1940 and IC 1941 result in an increase in r + k as well as a decrease in amx and IPT, corresponding to increased hypocoagulation. This hypocoagulation increases depending on the dose applied and at the same time on the length of the acyl chain.

IC 1943 causes no noticeable change in the parameters in this test.

b) Anticoagulant activity in vivo

1. Measurement of the anticoagulant activity of the products according to the invention in vivo on rabbit (i.V.):

The experiments were carried out on New Zealand male rabbits. Prior to injecting the product, a blood sample was taken from the middle artery of the ear.

Then a solution of the product to be tested of 25 mg of product in 5 ml of isotonic NaCl solution is injected into the peripheral vein of the ear.

The test of TCK and the HEPTEST are performed on the blood taken before the injection of the products and on the samples taken 6.24, 48 and 96 hours after the injection.

The results are given in FIGS. 7 and 8. They show a marked increase in the time of the anticoagulant activity, this increase increasing with the length of the acyl chain.

In fact, this anticoagulant activity of the IC product 1940 is still significantly longer than 6 hours after the injection, while it persists for 24 hours after the injection of the IC 1941 product and extends to 96 hours for the IC 1943 product.

2. Measurement of the Anicocoagulant Activity of the Products According to the Invention In vivo by Thromboelastography: The products to be investigated were injected intravenously as described in the preceding experiment (25 mg in 5 ml of isotonic NaCl solution). A thromboelastographic incision was made using a sacred thromboelastograph on 0.25 ml of blood samples taken before injection and 6,24,48 and 96 hours after injection, with additional samples taken when the anticoagulant activity was longer than 96 hours.

The results are listed in Tables 5,6 and 7.

Table 5 Test product: IC 1940

Table β Table 7 r r 13 r 12 k 6 k 5 4 r + k amx IPT Before injection Test product: IC 1941 Test product: IC 1943 13 135 40 60 31 27 19 67 33 eStd.nachlnj. 91 19 13 5 12 7 151 56 2.1 24Std.nachlnj. Before injection Before injection 12 22 53 3 10 34 17 67 40 96Std.nachlnj. 6h. nachlnj. 6 hours after Inj. 8th 112 185 11 70 77 24Std.nachlnj. 24 hours after Inj. 82 45 48 hours nachlnj. 48 hours nachlnj. 19 k 9 72 hours after Inj. 17 5 r + k amx IPT 96 hours after Inj. 18 67 40 168 hours after Inj. 166 45 2.6 192 hours after Inj. 31 68 11.5 32 72 25 r + k amx IPT 16 69 55 67 > 40 - 20 72 36 87 54.5 4.7 297 > 38 - 127 62 3.6 28 68 23 22 70 46

The results obtained show that in all products a strong increase in r + k and a decrease in the IPT at

6th hour, indicating an increase in hypocculability. This is prolonged until at least 24 hours after the injection of the product IC 1941 and is still measurable 96 hours after the injection of the product IC 1943.

It is therefore found that all products have a strong anticoagulant activity in vivo, that this activity is prolonged in time and that IC 1943, which was only slightly or not at all effective in the experiments in vitro, is very active with a strong sustained-release action proves in vivo in the experiments.

3. Anticoagulant activity of the products IC 1945, IC 1957 and IC 1958, each prepared according to Examples 12, 13 and 14 and also tested in vivo on the rabbit:

The results showed prolonged pharmacokinetics for the IC 1957 and IC 1958 products when administered intravenously and subcutaneously.

c) Activity of inhibition of HIV-1 urinary HIV-2 virus in an in vitro model

The products according to the invention were made to anr. Model of virus inhibition HIV-1 and HIV-2 tested in vitro as described in R. Pauwels et al., J. Virol. Methods, 1987, 16, 171-185.

All products tested were found to be active at doses completely free of cytotoxicity. In particular, it was found that the products IC 1925 and IC 1926 were more active than the starting product.

d) Activity of inhibiting the formation of syncytia in an in vitro model

The syncitia are giant cells with multiple nuclei, which are formed by fusion of healthy lymphocytes T4 with infected T4 lymphocytes.

The products prepared according to the invention were tested in a model of co-culture of MOLT4 cells (healthy lymphocytes T4) and of HUT-78 / HTLVIII B cells (infected human T4 lymphocytes).

All products tested were found to be active in this model at doses that are completely free of cytotoxicity. In particular, the products IC 1924, IC 1925 and IC 1926 were more active than the starting product.

e) Activity of inhibition of various enveloped viruses in an in vitro model

Activity of the products according to the invention against inhibition of various enveloped DNA or RNA viruses, in particular the following viruses:

HSV 1 and 2 (herpes simplex virus 1 and 2, DNA viruses)

- VSV (Vasicular stomatitis virus, RNA virus)

- Sindbis virus, RNA virus

All products tested were found to be active in inhibiting these viruses at doses that are cytotoxic. In particular, the products IC 1924, IC 1925 and IC 1926 exhibited greatly enhanced VSV and Sindbis virus activity relative to the starting product.

f) Inhaled cell proliferation activity of smooth muscle cells in an in vivo model

The proteins produced according to the invention were obtained in a model of inhibition of cell smooth muscle cell proliferation

in rat tested in vivo (balloon catheter model as described in A.W. Clowes and M.M. Clowes, Labor, Investig., 1985, [6], 612-616).

The tested products proved to be active. In particular, the products IC 1924, IC 1925 and IC 1926 showed a

increased activity in relation to the Auspangsprodukt.

Report sup the result of the examination on protectability and evaluation of the technical-economic effectiveness Ad 1) A search was carried out by the Institut National de la Propriete Industrielle in France. Research has been done in the patent literature, International Classes C08B as well as in Chemical Abstracts and Patent Abstracts

from Japan.

The following publications were determined: FR-M-3066 (ROUSSEL-UCLAF) Chemical Abstracts Vol.75,1971, page 100, no. 7782a Vol. 77, 1972, pages 371-372, no. 24821 j Re 2) The patent application is still based on the following publications:

Japanese Patent 5128602 Japanese Patent 74/048533 FR-N 2 159724 FR-PS 2100735 FR-PS 2159724 US-PS 4,281,108 US-PS 4692435 US-PS 4686288 EP-PS 37319 EP-PS 40144 EP-PS 44228 EP-PS 46828 EP-PS 121067 EP-PS 215453 EP-PS 216453 EP-PS 244235 EP-PS 244 236 EP-PS 256880

Nagasawa, K., and Y. Inoue, Methods in Carbohydrate Chemistry, Academic Press, 1980, Vol.8, p.291-294

L.A. Fransson et al., Carbohydr. Res., 36 (1974), 349-358

BABA et al.

Antimicrob. Agents Chomother. 32 (1988) 337-339

BABA et al.

Proc. Natl. Sci., 85 (1988) 6132-6136 Guytonetal.,

Circ. Res. 46 (1980), 620-634 Can. J. Res., 25B (1947), 472-476

Ynietal.,

J. Lab. Chim. Med. 1973, 81, 298-310

Glucosaminoglycano are already known. These are understood to mean substances formed from uronic acid chains (D-glucuronic acid or L-iduronic acid) and amino sugars, the latter being glucosamines or galactosamines.

The glucosaminoglycans of natural origin consist of more or less homogeneous mixtures of disaccharide unit linkages formed by a uron subunit (glucuronic acid or iduronic acid) and by an osamine subunit (glucosamine or galactosamine), via a 1-4 Bond or a 1-3 bond are connected together.

In the case of the glycosaminoglycans, the hydroxyl groups are differently substituted by functional groups, in particular by sulfate groups, and the amino groups of the osamines also by sulfate and / or acetyl groups.

The glucosaminoglycans include various types of products, e.g. Heparin, heparin sulfate, chondroitin sulfate A, chondroitin sulfate C, chondroitin sulfate B, referred to as dermatan sulfate, and hyaluronic acid.

It is also known that the glucosaminoglycans have numerous biological activities, in particular an activity against the coagulation factors, which act via mediating, different plasmatic proteins. The glucosaminoglycans also form part of the large family of polysaccharides, some of which have antiviral activity to a greater or lesser extent.

The natural glucosaminoglycans have, in addition to their interesting pharmacological activities, only a relatively short half-life, which becomes clear with repeated administrations.

These disadvantages have been ameliorated by numerous glucosaminoglycan derivatives modified to improve their pharmacokinetics, particularly esters of the carboxylic acid function of the uronic acids or the hydroxyl functions.

Also known are the partial or complete esterification of the carboxylic acid functions of heparin, the esterification of glycosaminoglycans at their primary or secondary hydroxyl functions, and many others.

3) The method according to the invention is used in the pharmaceutical industry in the field of drug synthesis.

Ad 4) According to the invention new more specific Glucosaminoglycane provided whose biological activity is much higher, and a process for their preparation, which ensures good reproducibility. The selectively O-acylated compounds of the present invention have prolonged activity in vivo. For example, if these compounds are heparin-type and exert antithrombotic activity, this effect is markedly prolonged over the same compound that was not O-acylated.

The selectively O-acylated heparin derivatives of the present invention also exhibit various biological activities, in particular:

Smooth muscle cell proliferation inhibition, which is of great interest for the prevention of restenosis in procedures such as angioplasty, by-pass surgery, venous and arterial transplantation, organ transplantation, especially heart transplantation,

Inhibition of heparanase and heparitinase, enzymes associated with metastatic dissemination,

antiviral properties, especially against retroviruses, especially against the various H / V (human immunodeficiency) virus, which has great interest in the treatment of AIDS,

Properties for the treatment of leukocyte elastase, for increasing the circulatory level of elastase inhibitors as well as the selective inhibition of the synthesis of type III collagen and fibronectin, suitable for the treatment of diseases such as emphysema, as well as connective tissue degenerative diseases such as arteriosclerosis and diabetes.

The selectively O-acylated glucosaminoglycans according to the invention are therefore outstandingly suitable as active substance for medicaments of great importance for numerous indications.

To 5) experimental results regarding the pharmacological properties of the compounds according to the invention are given on pages 54 to 62 of the application documents.

2 8 5 6 O 6

(A)

CH ₂ OH

0 -

(B)

I.

CH ₀ OH

CH ₂ OK

COO

/ Coo "

OSO.

CH ₂ OH

NHR

(C)

Claims (38)

1. selectively O-acylated Glucosaminoglycane the general formula (I) (see formula sheet), in the G represents a group of the formula (a) or a group of the formula (a ') or a group of the formula (b) U is a group of formula (c) or a group of formula (d) or the radical of group (c) or group (d) after periodic oxidation, followed by β-elimination or acid hydrolysis, A is a group R ₁ , a group of the formula R ₁ - (C), a group of the formula RHd) or a group of the formula (e) or the group (c) or group (d) after periodic oxidation, followed by β-elimination or acid hydrolysis, B is a group 0-R ₁ , a group of the formula (aJ-ORv is a group of the formula (a'J-OR ,, a group of the formula (b) -ORv is a group of the formula (f) or a group of the formula (g), or a group of the formula (a), a group of the formula (a ') or a group of the formula (b), the radicals thereof having a radical of (c) or of (d), as in periodic oxidation, followed by β-elimination or acid hydrolysis, are present, R _{1 represents} hydrogen, SO ₃ or acyl, acyl being the radical of a non-α-unsaturated carboxylic acid or dicarboxylic acid selected from: An alkanoyl group having 1 to 18 carbon atoms, An alkanecyl group of 2 to 3 carbon atoms substituted by: a cycloalkyl group having 3 to 7 carbon atoms, a phenyl group, optionally substituted by one or more alkyl radicals having 1 to 14 carbon atoms, by halogen atoms or the groups NO ₂ or OCH ₃ , an unsaturated aliphatic hydrocarbon radical having 4 to 16 carbon atoms, A benzoyl group, optionally substituted by one or more alkyl radicals having from 1 to 4 carbon atoms, by halogen atoms or the group NO ₂ or OCH ₃ , A cycloalkyl (3-7 O-carbonyl group, R _{2 represents} SO ₃ - and / or an acetyl radical, with the proviso that the ratio of N-acetyl-glucosamine is at most equal to that of heparin when R _{1 is} an acetyl radical, R _{3 represents} a hydrogen atom or an alkyl radical having 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms, or a phenyl-alkyl radical having 7 to 12 carbon atoms, or an alkali metal or alkaline earth metal cation, - η is an integer from 1 to 80, wherein R ₁ is acylated in a ratio of at least 0.1 to 3 acyl groups per disaccharide unit, preferably 0.5 to 2 groups; and their pharmaceutically acceptable salts. 2. SeleKiiv O-acylated Glucosaminoglycane according to claim 1, characterized in that they correspond to the general formula (II) (see formula sheet), in which - A has the meanings of claim 1, R _{1 is} hydrogen and / or SO ₃ - and / or an acyl group as defined in formula (I), R _{2 represents} SO ₃ - and / or an acetyl radical, with the proviso that the ratio of N-acetyl-glucosamine is at most equal to that of heparin when R _{1 is} an acetyl radical, R _{3 represents} a hydrogen atom or an alkyl radical having 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms, or a phenyl-alkyl radical having 7 to 12 carbon atoms, or an alkali metal or alkaline earth metal cation, - B groups of formulas Ja) -OR ₁ or (f), (a) and (f), as defined in claim 1, or represents OR ₁ , or a group of formula (a), the remainder having a Remainder of (c) or of (d) as it is after periodic oxidation, followed by β-elimination or acid hydrolysis, - η is an integer from 1 to 80, with the proviso that when AR _{1 is} a group of the formula R ₁ - (C) or a group of the formula RHd) and B 0 -R ₁ , a group of the formula ( 8J-OR ₁ or a group of formula (b) -ORi, η is an integer from 1 to 16. 3. Selective O-acylated Glucosaminoglycane according to claim 1, characterized in that they correspond to the general formula (II) (see formula sheet), in which A, B, R ₁ , R ₂ and η are as defined in claim 2, R _{3 represents} a hydrogen atom or an alkali metal or alkaline earth metal cation. 4. Selectively O-acylated Glucosaminoglycane according to claim 2, characterized in that they correspond to the general formula (II) (see formula sheet), in the A, B, R ₁ , R ₂ and η are as defined in claim 2, R 3 is an alkyl radical having 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms, or a phenyl-alkyl radical! With? represents up to 12 carbon atoms. 5. Selective O-acylated Glucosaminoglycane according to claim 1, characterized in that they correspond to the general formula (II) (see formula sheet), in which A has the meanings of claim 1, R _{1 is} hydrogen and / or SO ₃ - and / or an acyl group, acyl being the radical of a non-α-unsaturated carboxylic acid or dicarboxylic acid selected from: An alkanoyl group having 4 to 18 carbon atoms, An alkanoyl group having 2 to 3 carbon atoms substituted by: a cycloalkyl group having 3 to 7 carbon atoms, a phenyl group, optionally substituted by one or more alkyl radicals having 1 to 14 carbon atoms, by halogen atoms or the groups NO ₂ or OCH ₃ , an unsaturated aliphatic hydrocarbon radical having 4 to 16 carbon atoms, A benzoyl group, optionally substituted by one or more alkyl radicals having from 1 to 4 carbon atoms, by halogen atoms or the group NO ₂ or OCH ₃ , A cycloalkyl (3-7C) -carbonyl group, R _{2 represents} SO ₃ - and / or an acetyl radical, with the proviso that the ratio of N-acetyl-glucosamine is at most equal to that of heparin when R _{1 is} an acetyl radical, R _{3 represents} a hydrogen atom orl 'an alkyl radical having 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms, 0 a phenyl-alkyl radical having 7 to 12 carbon atoms, or an alkali metal or alkaline earth metal cation, - η is an integer from 1 to 80, wherein R ₁ is acylated in a ratio of at least 0.5 to 2 acyl groups per disaccharide unit, preferably 1 group. 6. Selectively O-acylated Glucosaminoglycane according to claim 5, characterized in that they correspond to the general formula (II) (see formula sheet), in the A, B, R ₁ , R ₂ and η are as defined in claim 5, R _{3 represents} a hydrogen atom or an alkali metal or alkaline earth metal cation. 7. Selectively O-acylated Glucosaminoglycane according to claim 5, characterized in that they correspond to the general formula (II) (see formula sheet), in the A, B, R ₁ , R ₂ and η are as defined in claim 5, R _{3 represents} an alkyl radical of 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms, or a phenyl-alkyl radical of 7 to 12 carbon atoms. 8. Selectively O-acylated glucosaminoglycans according to any one of claims 1 to 4, characterized in that the glucosaminoglycan is selected from the group consisting of: a mixture of heparin fragments having a molecular weight below 10000 daltons, a mixture of heparin With a mean molecular weight of between 2,000 and 7,000 daltons, a mixture of heparin Fragrr ^ n having an average molecular weight of about 4500 daltons, a mixture of heparin Fragrnenten with an average molecular weight of about 2500 daltons, a mixture of heparin fragments, which are homogeneous in terms of their molecular weight, a through Synthesis obtained heparin fragment, which partly in terms of its molecular weight and partly, as regards its functionalization, is homogeneous; wherein the acyl group is present in a ratio of at least 0.1 to 3 groups, preferably 0.5 to 2 groups, per disaccharide unit. 9. A selective O-acylated glucosaminoglycan according to any one of claims 5 to 7, characterized in that the glucosaminoglycan is heparin, wherein the acyl group in a ratio of at least 0.5 to 2 groups, preferably 1 group, per disaccharide unit is present. 10. Selectively O-acylated glucosaminoglycans according to any one of claims 1 to 8, characterized in that they correspond to the general formula (III) (see formula sheet), in which - AR ₁ or a group of the formula RHc) or a group of the formol R ₁ - (O ¹ ), as defined in claim 1, R _{1 is} hydrogen and / or SO ₃ - and / or an acyl group as defined in claim 1, R ₂ represents S ₃ - and / or an acetyl radical, with the proviso that the ratio of N-acetyl-glucosamine is at most equal to that of heparin when R _{1 is} an acetyl radical, R _{3 represents} a hydrogen atom and / or an alkyl radical having 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms, or a phenyl-alkyl radical having 7 to 12 carbon atoms, or an alkali metal or alkaline earth metal cation, - η is an integer from 3 to 12. 11. Selectively O-acylated glucosaminoglycans according to any one of claims 1 to 8, characterized in that they correspond to the general formol (IV) (see formula sheet), in which R _{1 is} hydrogen and / or SO 3 and / or an acyl group as defined in claim 1, R ₂ represents S ₃ - and / or an acetyl radical, with the proviso that the ratio of N-acetyl-glucosamine is at most equal to that of heparin when R _{1 is} an acetyl radical, R _{3 represents} a hydrogen atom and / or an alkyl radical having 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms, or a phenyl-alkyl radical having 7 to 12 carbon atoms, or an alkali metal or alkaline earth metal cation, B represents a group of formula (a) -ORi as defined in claim 1 or OR ₁ , - η is an integer from 2 to 20. 12. A selectively O-acetylated Glucosaminoglycane according to any one of claims 1 to 4, characterized in that the Glucosaminoglycan is selected from heparin, a fraction or a heparin fragment, which are exempt from their binding position to antithrombin III. Selective O-acylated glucosaminoglycans according to any one of claims 1, 2, 5 and 12, characterized in that they correspond to general formula (V) (see formula sheet), in which A is a group of formula R ₁ - (C), a group of formula RHd) or the group (c) or group (d) after periodic oxidation, followed by β-elimination or acid hydrolysis, B is a group of formula (8J-OR ₁ or a group of formula (a) whose radical is a radical of (c) or of (d), as after periodic oxidation, followed by β-elimination or a acid hydrolysis, is present, is connected, R _{1 has} the meanings given in claim 1, R _{2 is} SO ₃ - or an acetyl radical, the ratio of SO _{3 being} about 90%, R _{3 represents} a hydrogen atom or an alkali metal or alkaline earth metal cation, - η is an integer from 1 to 80. 14. Selective O-acylated glucosaminoglycans according to any one of claims 1, 2, 5 and 12, characterized in that they correspond to the general formula (VI) (see formula sheet), in which - AR ₁ , a group of the formula Ri- (c), a group of the formula R ₁ -Jd) or the radical of (c) or of (d) after periodic oxidation, followed by a β-elimination, - U is a group of formula (Vl '), or, in the ratio of one group per two chains at least, is a uronic acid (D-glucuronic acid or L-iduronic acid) which is not sulfated and open between the carbon atoms in position 2 and 3 is and corresponds to the general formula (Vl "), B represents a group of the formula (8J-OR ₁ or OR ₁ or a group of the formula (a) the radical of which has a radical of (c) or of (d), as after periodic oxidation, followed by a β- Elimination, present, connected, Ri has the meanings given in claim 1, R ₂ SO ₃ - or an acetyl radical, wherein the ratio of SO ₃ - is at least about 90%, R _{3 represents} a hydrogen atom or an alkali metal or alkaline earth metal cation, - η is an integer from 2 to 18. Selective O-acetylated glucosaminoglycans according to any of claims 1, 2, 5 and 12, characterized in that they correspond to the general formula (VI) (see formula sheet), in which - η is an integer from 7 to 15 for the majority of species, R _{1 represents} an alkanoyl radical having 2 to 10 carbon atoms, advantageously 4 to 10 carbon atoms, preferably 4 to 6 carbon atoms. 16. A selectively O-acylated Glucosaminoglycane according to claim 14 and 15, characterized in that they consist of a mixture of fragments which are homogeneous in Minblick on their molecular weight and in which η is an integer from 2 to 12. 17. Selective O-acylated glucosaminoglycans according to any of claims 1, 2, 5 and 12, characterized in that they correspond to the general formula (VII) (see formula sheet), in which - AR ₁ , a group of the formula Ri- (c) or a group of the formula Ri- (d), as defined in the formula (I), R _{1 is} an alkanoyl radical having 2 to 18 carbon atoms, R _{3 is} a hydrogen atom or a. Represents alkali metal or alkaline earth metal cation, B is a group of the formula Ia) -OR ₁ , as defined in the formula (I), or OR ₁ , - η is an integer from 1 to 80. 18. Selective O-acetylated Glucosaminoglycane according to claim 1, characterized in that they correspond to the general formula (VIII) (see formula sheet), in the A has the meanings of claim 1, R _{1 represents} hydrogen and / or SOA and / or an acyl group as defined in claim 1, R _{3 represents} a hydrogen atom or an alkyl radical J having 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms, or a phenyl-alkyl radical having 7 to 12 carbon atoms, or an alkali metal or alkaline earth metal cation, B represents groups of the formulas (b) -ORi or (g) as defined in the formula (I), or OR ₁ or a group of the formula (b) whose radical is substituted by a radical of (c) or of (i ) associated with periodic oxidation followed by β-elimination or acid hydrolysis, - η is an integer from 1 to 80. 19. Selective O-acylated glucosaminoglycans according to claim 18, characterized in that they correspond to the general formula (VIII) (see formula sheet), in which A, B, R ₁ and η are as defined in claim 18, R _{3 represents} a hydrogen atom or an alkali metal or alkaline earth metal cation. 20. Selective O-acetylated Glucosaminoglycane according to claim 18, characterized in that they correspond to the general formula (VIII) (see formula sheet), in which A, B, R ₁ and η are as defined in claim 18, R _{3 represents} an alkyl radical of 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms, or a phenyl-alkyl radical of 7 to 12 carbon atoms. Selective O-acylated glycosaminoglycans according to any one of claims 1, 18, 19 and 20, characterized in that the glucosaminoglycan is selected from the group formed by dermatan sulphate and its fragments or chondroitin 4- and 6-sulphates and their fragments, wherein the acyl group is present in a ratio of at least 0.1 to 3 groups, preferably from 0.5 to 2 groups, per disaccharide unit. 22. Selectively O-acylated glucosaminoglycans according to any one of claims 1 to 21, wherein R _{1 is} an alkanoyl radical having 4 to 10 carbon atoms. 23. A process for the preparation of selectively O-acylated Glucosaminoglycanen according to any one of claims 1 to 22, characterized in that one comprises (1) a Glucosaminoglycan the general formula (IX) (see formula sheet), in the G ^{0 represents} a group of the formula (a) ° or a group of the formula (a ') ° or a group of the formula (b) ° , U ° is a group of formula (c) ° or a group of formula (d) ° or the radical of group (c) ° or of group (d) ° after periodic oxidation, followed by β-elimination or acid hydrolysis , means - A ⁰ is a group R ₁ ^{is 0,} a group of formula R ₁ Mc) ^0, a group of formula R ₁ Md) is ⁰ or a group of formula (e) ° or the residue of group (c) ° or of group ( d) after periodic oxidation, followed by β-elimination or acid hydrolysis, - B ^{0 is} a group OR ₁ ⁰ , a group of formula (3JM) R ₁ ⁰ , a group of formula (3'J-OR ₁ ⁰ or a group of formula (f) ° or a group of formula (g) ° represents, or the groups (a) °, (a ') ° or (b) °, whose radicals with a radical of (c) ° or of (d) °, as they after periodic oxidation, followed by a ß- Elimination or an acidic hydrolysis are present, R ₁ ^{0 represents} hydrogen or SO ₃ , R2 represents an SO3 or an acetyl radical, with the proviso that the ratio of N-acetyl-glucosamine is at most equal to that of heparin, if Ri is an acetyl radical, R 3 represents a hydrogen atom or an alkyl radical having 1 to 10 carbon atoms or a phenylalkyl radical having 7 to 12 carbon atoms, or an alkali metal or alkaline earth metal cation, - η is an integer from 1 to 80, converted into a salt of said glucosaminoglycan which is soluble in polar aprotic organic solvents; (2) this salt with an anhydride of the formula: Acyl-O-acyl in which acyl is defined as in claim 1, in which said polar: aprotic solvent in the presence of a catalytic amount of pyridine or a dialkylamiriopyridine and a proton acceptor are treated; (3) The product thus obtained is precipitated with the aid of a solution of sodium acetate in ethanol, and (4) selectively isolating O-acylated glucosaminoglycan by dissolving the obtained precipitate in water and by dialysis, and optionally converting the thus-obtained sodium salt of the selectively O-acylated glucosaminoglycan to another pharmaceutically acceptable salt. 24. The method according to claim 23, characterized in that the glucosaminoglycan used in step (1) wild selected from the heparin, a mixture of heparin fragments having a molecular weight of below 10,000 daltons, a mixture of heparin fragments with a middle Molecular weight, which is between 2000 and 7000 daltons, a mixture of heparin fragments having an average molecular weight of about 4500 daltons, a mixture of heparin fragments with an average molecular weight of about 2500 daltons, a mixture of heparin fragments, with respect is homogeneous to its molecular weight and a group formed by synthesis heparin fragment which is partly in terms of its molecular weight and partly homogeneous in terms of its Funktionaüsierung formed. 25. The method according to claim 23, characterized in that the glucosaminoglycan used in step (1) is heparin, a fraction or a heparin fragment, which are freed from their binding position to antithrombin III. 26. The method according to claim 23, characterized in that the glucosaminoglycan used in step (1) is selected from the group formed by dermatan sulfate and its fragments or chondroitin 4- and 6-sulfate and its fragments. 27. The method according to any one of claims 23 to 26, characterized in that the glucosaminoglycan of step (1) is the salt of a tertiary amine, in particular a Tributylamin-SaIz, or a quaternary Ammoniumsa'z, in particular a tetrabutylammonium salt. 28. A method according to any one of claims 23 to 27, characterized in that the anhydride used in step (2) is the anhydride of an alkanoic acid having 2 to 10 carbon atoms, advantageously 4 to 10 carbon atoms, preferably 4 to 6 carbon atoms , 29. The method according to any one of claims 23 to 28, characterized in that the step (2) is carried out in a polar aprotic solvent selected from the group formed by dimethylformamide, hexamethylphosphoric triamide, pyridine or a mixture of these solvents with each other or with dichloromethane. 30. The method according to any one of claims 23 to 29, characterized in that is used as Deprotonenakzeptor in step (2) a base selected from the group formed by pyridine, triethylamine and tributylamine. 31. The method according to any one of claims 23 to 30, characterized in that the dialysis of step (4) is carried out in the presence of a weak base. 32. The method according to any one of claims 23 to 31, characterized in that the step (2) at a temperature between 0 ⁰ C and 100 ⁰ C, in particular between 50 ⁰ C and 100 ⁰ C, is performed. 33. Pharmaceutical composition, characterized in that it contains as active ingredient an effective amount of at least one selectively O-acylated glucosaminoglycan according to any one of claims 1 to 22, in association with a pharmaceutical carrier. 34. A pharmaceutical composition according to claim 33, characterized in that the selectively O-ac / glated Glucosaminoglycan in the form of "a pharmaceutically acceptable salt, such as a sodium, magnesium or calcium salt is present. 35. Pharmaceutical composition according to claim 33 and 34, characterized in that the pharmaceutical carrier is suitable for administration by oral route and that it is in the form of gastro-resistant capsules, tablets, tablets or pills, or in the form of drinkable solutions, wherein it advantageously comprises 50 mg to 5 g per unit dose, preferably 100 to 1000 mg for capsules, tablets or pills, and 10 to 150 mg for drinkable solutions. 36. Pharmaceutical composition according to claim 33 and 34, characterized in that it is in the form of a sterile or sterilizable injectable solution for administration by intravenous, intramuscular or subcutaneous route, these solutions advantageously from 50 to 200mg / ml of selectively O-acylated glucosaminoglycan when intended for injection by the subcutaneous route, or 20 to 200 mg / ml of selective O-acylated glucosaminoglycan when intended for injection by intravenous route or perfusion. 37. A pharmaceutical composition according to any one of claims 33 to 36 for the treatment or prevention of diseases associated with enveloped viruses. 38. A pharmaceutical composition according to claim 36 for the treatment or prevention of diseases associated with retroviruses such as AIDS.