GLYCOSAMINOGLYCANES SELECTIVELY O-ACYLES, THEIR PREPARATION AND PHARMACEUTICAL COMPOSITIONS THE CONTAINER

The invention relates to selectively glycosaminoglycans 0-acylated, their method of preparation as well as pharmaceutical compositions containing them and their use in therapy.

For the term "glycosaminoglycan" refer to substances made of uronic acid chains (D-glucuronic acid or L-iduronic acid) and amino sugars, the latter being of. glucosamines or galactosamines.

Glycosaminoglycans The of natural origin consist of blends more or less homogeneous chaining of disaccharide units formed by a uronic subunit (glucuronic acid or iduronic acid) and by a sub-unit amino sugar (glucosamine or galactosamine) joined by a link or 1-M 1-"-3.

In the glycosaminoglycans, the hydroxyl groups are variously substituted with functional groups, in particular the sulfate groups, amino groups are substituted by osamine the sulfate groups and/or acetyl.

Glycosaminoglycans The considered herein include 6 product types: heparin, hëparane sulfate, chondroitin sulfate At, C chondroitin sulfate, chondroitin sulfate B, more precisely called dermatan sulfate, and hyaluronic acid. They are characterized by two base structures the disaccharide (A) and (B) following:

The glycosaminoglycans having the base structure (A) above are termed of glycosaminoglycans (or GAGs) type and comprise hëparinique heparins and heparan sulfates and glycosaminoglycans having the base structure (B) above are termed GAGs type chondroitin sulfate and chondroitin sulfates comprise the A and C and dermatan sulfate.

hyaluronic acid to the base structure (B) wherein the glucosamine galactosamine is replaced by a.

As mentioned above, in both the type that GAGs of heparin in the GAGs type chondroitin sulfate, a more or less high percentage of the hydroxyl groups of the n disaccharide units is present in the form of esters of sulfuric acid.

Therefore, the heparin can be represented by the base structure

(A) above, wherein n may vary from 1 to 80, R being in a majority of the n units a group S0 " and, for the rest of the cases, an acetyl group, the OH groups in position 6 of the glucosamine and in position 2 of the uronic acid are in the majority of the sulfated species, while the OH group in position 3 of the sulfated glucosamine is that in a minority species; the OH in position 3 of the uronic acid is substantially non-sulfated, said uronic acid being in the majority of species an iduronic acid.

The products having the base structure (A) also include heparin N- dësulfatée N-acetylated which can be prepared as described in K. Inoue Nagasawa and Y., Glucides Methods in Chemistry, Academie press, 1980, Vol. 8, pp. 291-294. Said heparin is represented by the formula (A) wherein R is a group acëtyle.

The heparin also contains species having the structure (A), with variable n 3 to 15 and the same profile chemical. The species isolatable by Fractionator according to known methods (for example US 4,692,435) and are named low molecular-weight heparins

Heparins b.p.m. having the molecular weight distribution and the biological properties substantially identical to those obtained by fractionating b.p.m. heparins have been prepared by fragmenting heparin of the following methods:

optionally sulfated on primary Vhydroxyle in position 6;

-method of enzymatic depolymerization (for example EP 244,235 and 244,236) or alkaline (for example EP 40,144) which intersects the molecule by driving the sub-units to provide a termination uronic a-S însaturé uronic acid

( b.p.m.)

-method of oxidative depolymerization (for example US 4,281,108 and EP 121067) which intersects the molecule by oxidation while maintaining the terminations "natural".

Heparins b.p.m. Other which have a molecular weight distribution different from that of natural heparin and particular biological properties can be prepared by oxidation of the natural periodic heparin followed by depolymerization by S-elimination or acid hydrolysis. Periodic oxidation cutting the non-sulfated uronic acid units between the carbon atoms in position 2 and 3. It is known that such motifs are present in the binding site antithrombin III (AT III), plasma inhibitor of various serine proteases of the coagulation cascade, and whose activity is strongly enhanced by heparin acting as co-factor. Periodic oxidation thus provides products lacking attachment sites to the AT III, and thus essentially free of anticoagulant activity.

Heparins b.p.m. of this type can be obtained in particular by implementing the method comprising the steps of:

-the controlled oxidation of heparin carried out by reacting heparin, in aqueous solution ê a final concentration of 0.5 to 5 % (p/v) with a salt of periodic acid to a final concentration of 0.5 to 1 % (p/v), at a pH between 4.5 and 6.5, preferably at a pH of 5, at a temperature of 0 to 10° C, for about 15 hours to 48, with the exclusion of light;

-the depolymerization of heparin chains obtained, by addition of a strong base such as soda, at a pH greater than about 11, in particular between 11 and 12, preferably 11.2 to 11.6, preferably about 11.5;

-reducing dépolymërisation fragments with a reducing agent and, optionally after removal of the unreacted reducing agent,

-retrieving heparin fragments reduced, by precipitation using a solvent in which they are insoluble;

-isolating fragments searched by fractionation using

alcohol, in the presence of an inorganic salt, an aqueous solution obtained by re-solution in water the precipitate previously isolated and recovering the precipitate formed.

Heparins b.p.m. These have the formula (C) below:

wherein n may vary from 6 to 15, R is in at least about 90% of the n units S0 a group ' and, for the rest of the cases, an acetyl group, the OH groups in position 3 of the glucosamine can be sulfated, and the OH groups in position 6 being sulfated glucosamine species in 70%. Furthermore, in an amount of 1 to 2 pattern of the heparin chains at least b. p.m., iduronic acid is replaced by a pattern uronioue acid (D-glucuronic or L-iduronic) non-sulphated opened between the carbon atoms in position 2 and 3, of the following formula:

Glycosaminoglycans hëparinique Other type consist of GAGs of formula (A) or (C), in which the carboxylic function carried by the uronic acid is estërifiëe , for example by condensation of an alcohol in the presence of a condensing agent, carbodiimide, as described in the Patent FR no. 2,159 724, or by alkylation of carboxyl using a halogënure alkyl in the presence of a weak base.

Also, -like glycosaminoglycans consist of chondroitin sulfate glycosaminoglycans of formula (B) in which the carboxyl functions uronic acids are esterified, for example by alkylation of carboxyl using an alkyl halide in the presence of a weak base.

Obtaining such esters for the hyaluronic acid is described ., in EP no. 215,453, by treating the hyaluronic acid in form of quaternary ammonium salt with alcohol in the presence of a catalyst, or by an etherification agent.

The heparan sulfate is represented by the formula (A) above, wherein n can varfer of 1 to 89, R is in the majority of species and an acetyl group in a minority species SOg. Furthermore, in the great majority of species, the OH groups in position 6 of glucosamine are sulfated, the uronic acid being in the majority of species glucuroniaue acid.

C A The chondroitin sulfates and are represented by the formula

(B) above wherein n may vary from 1 to 80 and, respectively, the groups GH 4-and 6-OH of glucosamine are sulfated, the uronic acid being in the majority of species glucuronic acid.

The dermatan sulfate has a structure substantially identical to that of the chondroitin sulfate A, but the subunit uronic acid is a iduronic acid in the majority of species.

Fragments dermatan sulfate, of formula (B) above, wherein n may vary from 1 to 20, can be obtained by periodic oxidation followed by a reduction in sodium borohydride and acid hydrolysis, as described in Fransson Carlstedt L.A. and I., Carbohydr. Res., 36__s (1974), 349-358.

The glycosaminoglycans have many biological activities, including their activities against coagulation factors, which may be exerted via various plasma proteins. For heparin, it is also known in the literature as heparin or derivatives thereof with or without anticoagulant activity may have a regulating activity on the proliferation of vascular smooth muscle cells (and Guyton al ., Carnauba. Res., 46, (1980), 625-634) or inhibitory activity I ' heparanase, enzyme involved in the mechanisms of metastatic dissemination (EP no. 254,067 Request).

In the following text, the term GAG is used to designate a glycosaminoglycan, thereof having either a natural structure such. that obtained by extraction, hemi-synthesis or synthesis, or a chemically modified structure on the amino or carboxyl functional groups, prior to the acylation reaction giving the GAGs selectively 0-acylated.

Despite their pharmacological activities of large interest, the natural GAGs has a drawback that the relatively short half-life, causing repeated administrations. This disadvantage is partly overcome-by the use, in the prevention and treatment of venous thrombosis, derivatives of low molecular weight heparin, subcutaneously, thereby lowering the frequency of administration to one injection per day.

However, it is a very interesting to provide derivatives having a delay action, which would further decrease the frequency of administering these products by increasing their duration of action.

It may also be of large interest for delay derivatives non-anticoagulant such as heparin N-desulfated N-acetylated whose activity as an inhibitor of hëparanase , enzyme involved in phenomena of metastatic dissemination, is biased higher.

The literature described derivatives of glycosaminoglycans modified to improve their pharmacokinetic, especially esters of the carboxylic function uronic acids or hydroxyl functions es.

Was used in particular the total or partial esterification of the carboxyl groups of heparin, processed as quaternary ammonium salt, in inert solvent, with an alcohol or a halogenated derivative, optionally in the presence of a condensing agent (FR η Br.⁰ 2 159,724 and EP no. 44,228). Hyaluronic acid derivatives obtained by esterification with alcohol in the presence of a catalyst or by reaction with an etherification agent have further been described (EP no. 216,453 Br.).

Furthermore, various means have been described in the literature to esterify the GAGs on the hydroxyl functionality is primary and secondary.

The publication Adc. J. Res., 25B , (1947), 472-476, acetylated derivative of the '. heparin corresponding to one mole of acetyl group to four saccharide units. The product is prepared by action of the ketene on heparin in acetone. Although in the publication it is specified that the product obtained is a derivative 0-acetylated, it is well known that the ketene in the presence of carboxylic groups provides anhydrides, (cf. J. March, Advanced Organic Chemistry; Reactions, Mechanisms and Structure, and J. Willy Sounds Eds ., 1985, ρρ.686-687:

" Kith ketenes , carooxylic give acid and acetic anhydride year. nydrides is prepared industrially in this manner:

"Ch"= = c = 0 + ch, - C00 h >-ch, -c = 0

4 3,3 i

0coch3")

Therefore, the publication described in fact a heparin in which 0-acetyl groups are associated with mixed anhydrides COG between the group ' of the uronic acid and the acetyl group from the ketene, which may explain their instability in water.

FR 2,100 735 The patent partial esters are hydrolysable of heparin and a non-toxic organic acid, in particular 4- chlorophénoxyisobutyrique , acid ' 4-chlorophenoxyacetic acid, cholic acid, nicotinic acid, N-oxy-nicotinic, pyridyl acetic acid, N-oxy- pyridylacétique or linoleic acid. The preparation method described in above, that ' is characterized by the reaction of a quaternary salt of heparin with the acid activated by a carbodiimide, not only provides the derivative 0-acylated, in a large quantity but also a secondary product stable as an ester derivative 0- acylisourëe. Furthermore, such a type of reaction is likely to promote intramolecular anhydrides and reactions between the acid functions and the hydroxyl functions of heparin.

JP 74/048533 The patent describes esters 0-chondroitin sulfate with aromatic acids, optionally substituted heterocyclic or arylaliphatic having prolonged. The preparation method described herein, which is characterized by the. reaction of the acid chondroîtinsulfurique with an acid chloride, provides a secondary n-acylation reaction.

The patent W0 83/00150 generally described prodrugs of drugs prepared using the various functions of the GAGs, which 1 'hydroxyl and, specifically, a 0-ester of the chondro' îtinsulfate with penicillin V. The preparation of the product is performed via carbodiimide and therefore comprises side reactions mentioned above.

EP 0-esters 46,828 provides heparin with unsaturated acids, in particular acrylic or metacrylic acid which, grafted to biomedical materials, provide long-term antithrombotic activity. The grafting by covalent bonding at links a-8 insaturêes of these esters with the surface of said materials which come into contact with blood. According to the document, the esters 0-alpha, bëta -unsaturated are prepared by reacting 1' heparin with chloride or anhydride of a carboxylic acid alpha, bëta - insaturë. Furthermore, the specification that indicates the optional use chlorides or anhydrides of alpha acids, beta- insaturës as reagents does not specify which types d ' 0-esters of heparin are thus obtained.

EP 256,880 describes the esterification of heparin or low-molecular-weight heparin by the action of acid chlorides in formamide and pyridine to give derivatives increased transmembrane permeability. Or, the method leads to derivatives that undergo partial dësulfatation and are also 0mais N-acetylated, and wherein the ratio sulfate/carboxyl is altered.

It is now found that by reacting a salt-soluble organic GAG, such as a tertiary or quaternary ammonium salt, with the anhydride of a carboxylic acid in a polar aprotic organic solvent, there is obtained a selective acylation of the free hydroxyl, without impairing the carboxylic functional groups or amino GAG implemented. Furthermore, the rate of acylation is easily scalable and can be raised without causing of impairment on the remainder of the molecule.

In particular, it has been found that no N-acyl derivative are formed and that, when small amounts of anhydrides are present, they can be easily transformed back to free acid by the action of a weak base.

It has finally found that the 0-and GAGs obtained have pharmacological activity extra-long.

Therefore, the present invention provides, according to one aspect, selectively glycosaminoglycans 0- acylës , of the following formula I:

A-G-u-B (i)

r.

wherein:

-G represents a group of formula (a):

or a group (a¹ ) of formula:

or a group of formula (b):

ü represents a group of formula (c):

or a group {di of formula:

or the residue of the group of the group (c) or (d) after oxidation followed by a periodic B-elimination or acid hydrolysis;

A is Rj, Rj a group-(c), or a group-Rj (d), (e) or a group of formula:

the residue or ' the group (cJ or group (d) after oxidation followed by a periodic B-elimination or acid hydrolysis;

B is 0-Rj, a group (has)- QRj , a group (a ' J-ORj, a group (b) ^-OR, (f) a group of formula:

or represents a group (a), a group (a¹ ), or a group (b) which is retained a residue of (c) or (d} as present after oxidation followed by a periodic S-elimination or acid hydrolysis;

α β unsaturated carboxylic-selected from:

. an alkanoyl group of 1 to 18 carbon atoms;

. alkanoyl of 2 to 3 carbon atoms substituted by

-a cycloalkyl group of 3 to 7 carbon atoms,

-a phenyl group optionally substituted by one or more alkyl radicals of 1 to 14 carbon atoms, halogen atoms or groups or OCH ^ ^ N0,

a radical hydrocarbonë -aliphatic unsaturated 4 to 16 carbon atoms;

. a benzoyl group optionally substituted by one or more alkyl radicals of 1 to 4 carbon atoms, halogen atoms or

or a group of formula (g):

represents H, ^50 or acyl, acyl is the residue of an acid groups or ^ ^ Ν0 OCH;

. a cycloalkyl group (3-7 C-) carbonyl;

R2 represents-SOj and/or an acetyl radical, with the proviso that the proportion of N-acetyl glucosamine is at most equal to that of heparin when R ^ is an acetyl radical;

-Rj is hydrogêne or an alkyl radical of 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms, or substituted alkyl group of 7 to 12 carbon atoms, or an alkali metal cation;

-n is an integer from 1 to 80.

Rj being acyl in a proportion of at least 0.1 to 3 acyl groups per disaccharide unit, preferably 0.5 to 1 acyl group, and pharmaceutically acceptable salts thereof.

In a preferred embodiment, the selected glycosaminoglycans are-like glycosaminoglycans héoarinique , i.e. heparin, Vhéparane and sulfate derivatives thereof obtained or by splitting from heparin sulfate or hëparane , either by hemi-synthesis or synthesis.

The glycosaminoglycans of this type, selectively 0- acylës according to the present invention have the following formula II:

-A has the abovementioned meanings in the formula (I);

Rj is H and-/ or S0j and/or an acyl group as defined in formula (I);

R2 represents-SOj and/or an acetyl radical, with the proviso that the proportion of N-acetyl glucosamine is at most equal to that of heparin when R ^ is an acetyl radical;

-Rg is hydrogêne , an alkyl radical of 1 I 10 carbon atoms, preferably 1 to 4 carbon atoms, a substituted alkyl radical of 7 to 12 carbon atoms, or an alkali metal cation;

-B is (has) ^-OR or (f), (a) and (f) are as defined in formula (II, or 0R ^, or a group (a) which is retained a residue of (c) or (d) as present after oxidation followed by a periodic B-elimination or acid hydrolysis;

-n is an integer from 1 to 80.

Advantageous compounds of the invention are compounds of the formula (II), where R ^ is a hydrogen atom or an alkali metal cation.

Another group of beneficial compounds corresponds to compounds of formula (II), wherein is an alkyl radical of 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms, or substituted alkyl group of 7 to 12 carbon atoms.

In a preferred aspect of the invention, used are mixtures of heparin fragments having a molecular weight less than 10,000 daltons, especially those having an average molecular weight between 2.000 and 7.000 daltons, or those having an average molecular weight of about 4.500 daltons, or those having an average molecular weight of about 2.500 daltons.

Can be advantageously used to provide a method for the depolymerization nitrous acid, as described dpar example in EP 37,319. Glycosaminoglycans selectively The 0- acylës of the invention are then represented by the following formula III:

wherein:

-A represents, or R ^-^ R or (c)-(d), as defined in formula (I);

-Rj is H, SO3 and/or and/or an acyl group as defined in formula (I);

-R2 is-SO ^, and/or an acetyl radical, with the proviso that the proportion of N- acëtyl glucosamine is at most equal to that of heparin when R ^ is an acetyl radical;

-R3 represents a hydrogen atom, and/or an alkyl radical of 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms, or substituted alkyl group of 7 to 12 carbon atoms, or an alkali metal cation;

-n is an integer from 3 to 12.

Preferred compounds of the present invention correspond to the. compounds of formula (III) in which R is a radical âlcanoyle of 4 to 10 carbon atoms.

To obtain mixtures of fragments of heparin having a molecular weight less than 10,000 daltons, it is also possible to use a method of enzymatic dépolymërisation , for example as described in the patents EP 244,235 and 244,236, or alkaline depolymerization, for example as described in EP 40,144.

In this case, selectively glycosaminoglycans 0-acylated according to the invention are represented by the following formula IV:

R-₂ represents SO ", and/or an acetyl radical, with the proviso that the proportion of N-acetyl glucosamine is at most equal to that of heparin when represents an acetyl radical;

-Rj represents hydrogen, and/or an alkyl radical of 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms, or substituted alkyl group of 7 to 12 carbon atoms, or an alkali metal cation;

-B is (has)-ORj, as defined in formula (I), or OR ^;

-n is an integer from 2 to 20.

Among the compounds of formula (IŸ), advantageous compounds are those in which a radical Rj is alkanoyl of 4 to 10 carbon atoms.

In another advantageous aspect of the invention, can be used a mixture of homogeneous heparin fragments with regard to their molecular weight, a heparin fragment obtained by synthesis, homogeneous in terms of its molecular weight that 'its fonctionnalisât !' is.

4ntéressant In another aspect of the invention, can be chosen is glycosaminoglycan heparin derivatives free of binding site antithrombin III (AT III), is that the heparin chains have been fractionated to remove the oligosaccharide chains comprising the binding site AT T III using for example to affinity chromatography on Sepharose-AT III resin or ion exchange chromatography, as described in E. And Recognize al ., Thromb. Res ., 25, (1982), pp. 443-458, is that these sites have been destroyed by example by periodate dépolymërisation followed by a S-elimination or acid hydrolysis.

Such compounds may respond to the following formula V:

wherein:

Rj-A represents-(c), R, -(d) or the residue of (c) or (d) after oxidation followed by a periodic S-elimination or acid hydrolysis;

-B is (has) ^-OR, or OR ^, or a group (a) which is retained a residue of (c) or (d) as present after oxidation followed by a periodic B-elimination or acid hydrolysis;

-Rj is as defined in formula (I);

Represents SO-R ^ ^, or an acetyl radical, the proportion of SO " is about 90 % ;

^-R represents a hydrogen atom or an alkali metal cation;

-n is an integer from 1 to 80.

Particularly advantageous compounds can be obtained from compounds prepared by the method using a periodic oxidation followed by alkaline B- ëlimination , reduction and fractionation as described above. These compounds have the following formula VI:

(VI)

-A represents R ^, Rj-(c), Rj-(d), or the residue of (c), (d) or after oxidation followed by a periodic B-elimination;

or, at a rate of one pattern for two chains at least, a uronic acid (D-glucuronic or L-iduronic) non-sulphated opened between the carbon atoms in positions 2 and 3, of formula:

-B is (has) ^-OR, or 0R ^, or a group (a) which is retained a residue of (c) or (d) as present after oxidation followed by a periodic B-elimination;

-R is as defined in formula (I);

S0-R is ', or an acetyl radical, the proportion of being 2,3 SOZ ^ of at least about 90: =;

-R represents a hydrogen atom or an alkali metal cation;

-n is an integer from 7 to 15.

Advantageous compounds of formulas (V) and (VI) are those in which R ^ is a radical alkanoyl of 4 to 10 carbon atoms.

Among the compounds of structure-free heparin binding site 1¹ AT III, and thus devoid of anticoagulant activity, a family of beneficial compounds corresponds to N-desulfated heparin derivatives N- acétylëe selectively 0-acylated having the following formula VII:

(VII) wherein:

-A is Rj, Rj-(c), or R-, -(d!, as defined in formula (I);

Rj represents a radical-alkanoyl of 2 to 18 carbon atoms;

-Rg represents a hydrogen atom or an alkali metal cation;

-B is (has)- ORp as defined in formula (I), or ^ 0R;

-n is an integer from 1 to 80.

In another advantageous aspect of the invention, selected the glycosaminoglycans are chondroitin sulfate, in other words the chondroïtin 4et 6-sulfate, dermatan sulfate, and fragments thereof.

The glycosaminoglycans of this type, selectively 0-acylated according to the invention are represented by the following formula VIII:

-Rj is H, ^ and/or 50, and/or an acyl group as defined in formula (I);

-represents a hydrogen atom, and/or a radicalk alkyl of 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms, or substituted alkyl group of 7 to 12 carbon atoms, or an alkali metal cation;

-B represents (bJ -OR ^ or (g), as defined in formula φ, or ORj, or a group (b) which is retained the residue of (c) or

(d) as present after periodic oxidation followed by a fl- ëlimination or acid hydrolysis-,

-n is an integer from 1 to 80.

Among the compounds of formula (VIII), a family advantageous corresponds to compounds in which represents a hydrogen atom or an alkali metal cation.

Other beneficial compounds of formula (VIII) are those in which R 3 represents an alkyl radical of 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms, or substituted alkyl group of. 7 to 12 carbon atoms.

Another advantageous family of compounds of formula (VIIO corresponds to compounds wherein Rj is a radical alkanoyl of 4 to 10 carbon atoms.

The invention also relates to a process for the preparation of selectively glycosaminoglycans 0-acylated.

The method of the present invention is characterized in that the glycosaminoglycan is converted into a soluble salt in an organic medium and in that comprises treating the salt with an acylating agent capable of selectively acylating the primary and secondary hydroxyl groups of the glycosaminoglycan, without impairing the functional groups, or ^ COOÎ present on the glycosaiminoglycan before the acylation reaction, the reaction is performed in a polar aprotic solvent in the presence of a catalyst and in the presence of a base which can detect the acid which is released during the acylation, at a temperature of 0° to 100 °C C.

Advantageously, the method of the invention is carried out according to the following steps:

(1) a glycosaminoglycan following formula IX:

° (IX) B

G-° represents a group (a)⁰ of formula:

or a group (a')° of formula:

or a group (b) ° of formula:

or a group (d]^c of formula:

or the residue of the group ° (c) or (d) the group ° after oxidation followed by a periodic B-elimination or acid hydrolysis;

A⁰ rj is °, a group-Rj ° (c), a group R ^ °-(dj °, or ° (e) a group of formula:

or the residue of the group (c} ° or Group ° (d) after oxidation followed by a periodic S-elimination or acid hydrolysis;

°-B OR is^⁰ , a group (has)- 0Rj °^C , a group (a')^c -0R ^ °, a group (b)-^ 0R ° °, or ° (f) a group of formula:

or a group (g)^c of formula:

or represents the groups (a)^c , (a')^c , or (b)⁼ which remains attached a residue of ° (c) or (d)^c as present after oxidation followed by a periodic B-elimination or acid hydrolysis;

R-,⁰ represents H or SOj;

-Rj is SOj, or an acetyl radical, with the proviso that the proportion of N- acëtyl glucosamine is at most equal to that. of the heparin when R ^ is an acetyl radical;

-Rj represents a hydrogen atom, or an alkyl radical of 1 to 10 carbon atoms, or an alkali metal cation;

-n is an integer from 1 to 80.

a salt said glycosaminoglycan ' soluble in a polar aprotic organic solvent;

(2) comprises treating the salt by an anhydride of formula:

Long Long-0-

wherein Long is as defined in formula (I), in said polar aprotic organic solvent, in the presence of pyridine or cataïytiques amounts of a di alkyl aminopyridine and of a proton acceptor;

(3) is precipitated the product thus obtained by the action of a solution of sodium acetate in ethanol; and

(4) the glycosaminoglycan is isolated selectively 0-acylated by dissolving in water and of the resulting precipitate by dialysis, and is optionally converted to the sodium salt of the glycosaminoglycan selectively. 0-acylated other thus obtained into a pharmaceutically acceptable salt.

In one advantageous aspect of the method of the invention, the glycosaminoglycan used in step (1) is selected from the group consisting of heparin, a mixture of heparin fragments having a molecular weight less than 10,000 daltons, a mixture of fragments of heparin having an average molecular weight between 2.000 and 7.000 daltons, a mixture of heparin fragments having an average molecular weight of about 4.500 daltons, a mixture of heparin fragments having an average molecular weight of about 2.500 daltons, ur, homogeneous mixture of heparin fragments with regard to their molecular weight, a heparin fragment obtained by synthesis, homogeneous in terms of its molecular weight and as regards the functionalization.

In another advantageous aspect of the method of the invention, the glycosaminoglycan used in step (1) is heparin, a fraction or a heparin fragment free of binding site antithrombin III.

The glycosaminoglycan used in step (1) of the method may advantageously be selected from the group consisting of dermatan sulfate and fragments thereof, or the 6-sulfate chondroïtin 4et and fragments thereof.

•Advantageously, the salt of glycosaminoglycan used in step (1) the method of the invention is a. tertiary amine salt, in dead end binding a sorting butyl ammonium salt, or a quaternary ammonium salt, in particular a tetrabutylammonium salt.

In another advantageous aspect of the method of the invention, the anhydride in step (2) is the anhydride of an acid alcanoîque containing 4 to 10 carbon atoms.

The polar aprotic solvent which is conducted in the step (2) of the method of the invention may advantageously be selected from the group consisting of dimethylformamide, hexamethylphosphoric triamide, pyridine, or a mixture of these solvents by themselves or with dichloromethane, and the catalyst from the group consisting of amines such as pyridine, dimethylaminopyridine and the.

The base for neutralising the acidity can be pyridine (which can serve both solvent, catalyst and base), or the friend triëthylamine tri butyl does.

Advantageously, the temperature and the duration of the reaction 'can vary, according to the rate of desired acylation, respectively of 0° to 100 °C C, in particular 0' C to 50° C, and advantageously at room temperature, for 1 to 24 hours for example.

The compounds of the invention, selectively 0- acylës , have-in yivo prolonged activity. For example, when these compounds are-like compounds having the heparin binding site AT III, and can thus exert antithrombotic activity, the activity is substantially prolonged in time with respect to, the same compound has not been 0-a selective acylation.

When these derivatives selectively acylated heparin 0-do not possess the binding site 1' AT III and are therefore practically devoid of anticoagulant activity, they can have in vivo activities with a delay inherent in non-anticoagulant heparin derivatives, particularly such as the inhibition of smooth muscle cells.

The invention therefore also relates to pharmaceutical compositions characterized in that they contain as active substance an effective amount of at least one glycosaminoglycan 0-acylated according to the invention, in association with a pharmaceutically acceptable vehicle.

Advantageously, the glycosaminoglycan selectively O ^ acylated is in the form of a salt acceptable dpharmaceutiquement , .such as a sodium salt, magnesium or calcium.

In an advantageous embodiment of the invention, the pharmaceutical compositions are characterized in that the pharmaceutical carrier is suitable for oral administration, and that they are in the form of enteric-coated capsules, tablets or tablets, pills, or in the form of drink solutions, advantageously containing of 50 mg to 5 g per unit prtse , preferably 100 to 1000 mg for capsules, tablets or pills, and 10 to 150 mg for the orally administered solutes, for example from one to three times, per day; or in that these compositions are in the form of an injectable solution, or sterile stërilisable , for oral, intravenous, intramuscular or subcutaneous, these solutions advantageously containing 50 to 200 mg/ml of glycosaminoglycan 0-acylated selectively when for the subcutaneous injection or 20 to 200 mg/ml of glycosaminoglycan selectively 0- acylë when for intravenous injection or by infusion '.

Doses These intervals have only a value indicative, the doses administered to, in cnaaue case, be evaluated by the clinician, account of the condition of the patient and reactivity is personal to drugs.

The invention also features as biological reagent selectively glycosaminoglycans 0-acylated of the invention, which can be used as references or standards in comparison studies for studies of structure/activity relationships in different physiological systems wherein the glycosaminoglycans may be involved.

The invention will be better understood with the aid of application examples which follow, and which have no limiting character.

from the salt of tetrabutyl annionium heparin

a) tëtrabutylammonium Preparation of heparin salt :

The sodium salt of heparin (10 g) dissolved in water (500 ml) is percolated through a cation-exchange resin column (Dowex 50 W x 4, as H⁺ ). The solution obtained is neutralized by Vhydroxyde of tëtrabutylammonium. After freeze-drying is obtained tetrabutylammonium salt of heparin (19.55 g).

b) Acétylation :

1,05 hëparinate g of tetrabutylammonium is dissolved into anhydrous dimëthylformamide (5 ml). C After cooling to 0 °, is added dropwise acetic anhydride (1 ml; 10.46 mmol) then triethylamine (1.45 ml; 10.46 mmol) and dimethylaminopyridine (64 mg; 0.5 mmol). The mixture is dropped 24 hours at room temperature. After adding water (5 ml), the solution is dialyzed for 72 hours against distilled water. The tëtrabutyl ammonium salt is converted into sodium salt by passage over a Dowex 50 resin, H⁺ c to 0 °, followed by neutralization per soda IN. After freeze-drying, is obtained 0.49 g of sodium hëparinate selectively 0- acétylë having the following features:

Ratio sulfate/ carboxÿle: 2.28 meq/g

(starting material: 2.20 meq/g) Title APTT: 91 iu/mg

Spectre 1¾ NMR (methanol 51.6 PPM, internal standard!

-signal to 23.4 PPM: ^ ^-CH CH CO-O-

to 24.5 PPM-signal (low): CH ^ of CR ^-CO-NH (identical to the starting material)

The NMR spectrum indicates the presence of about two disaccharide acetyl groups per unit.

from the sorting butyl ammonium salt of heparin

The sodium salt of heparin (10, g) is dissolved in water (500 ml) then percolated through a cation-exchange resin column (Dowex 50 W x 4, as H⁺ ). The solution is neutralized by adding a solution of tri friend does butyl to 10% in ethanol.

After being washed with ether and freeze-drying, is obtained sorting butyl ammonium salt of heparin (14.77 gl.

b) Acétylation :

A a solution cooled to 0 °C salt of the above (4 g) in anhydrous dimethylformamide (50 ml), is added dimethyl aminopyridine • (250 mg), acetic anhydride (3.9 ml} and tributylamine (9.7 ml). After 24 hours at room temperature, water (1.5 ml) is added. The mixture is then poured into a solution of ethanol saturated with sodium acetate. After washing by ethanol, the precipitate is dissolved in water, and then dialyzed against bicarbonate in 5 % in water, and then against water. Is obtained, after lyophilization, sodium Vhéparinate 0-acetylated (1.81 g).

The IR spectrum has a high band ester 1730 cm ' *.

After saoonification , the product has:

-a sulfate/carboxyl ratio of mea/2.38 g

(starting material: 2.40 meq/g)

-a title APTT of:85 iu/mg

The tetrabutylammonium salt of heparin (0.58 g) is dissolved in a mixture (1/1; v/v) pyridine (10 ml) and dimethylformamide.

Acetic anhydride (0.5 ml) is added, and the mixture is dropped at room temperature. After 24 hours, an aqueous solution is added sodium acetate (1 M, 15 ml) and the mixture is poured in ethanol (80 ml) cooled to 0 °C. After centrifugation the precipitate is redissolved in water (10 ml), and ethanol (160 ml) is added followed by aqueous sodium acetate (1 M, 10 ml). The precipitate is then recovered by the water and freeze-dried, giving 0-acetylated heparin (0.22 g).

A a solution, cooled to 0° C, hëparinate of tetra-butyl-

butyl ammonium (1.85 g) obtained as described in the example 1, in dimethylformamide (10 ml), is added dropwise propionic anhydride (2.7 ml), then triethylamine (2.9 ml) and dimethyl nopyri of the friend dines (128 mg). H At time 1, 2 h, 4 h, 8 h h and 24, a portion of the reaction mixture is removed, diluted with an equal volume of water and dialyzed for 24 hours in distilled water. After passing on a resin Dowex 50 H⁺ and neutralization by soda, the products obtained are freeze-dried and have the following features:

The proton NMR spectrum products has been recorded in the water with the 3, 3, 3- triméthylsilylpropionate (ISP) as internal standard. Signals to λ It has ^ 1, and 1 PPM / ^ 2, 4 PPM characteristics respectively ^ and residues CH CH, , CH CH ^ ^-CO-O and signals between etrv / ^ 3/5 PPM characteristics of the protons of the osidic seuelette.

The comparing the intensity of signals of the propionyl-skeleton between the product processed during time 1 and subjected to the 24 hours reaction illustrates the influence of the reaction time: in effect, a decrease in the signals is observed protons skeletal, indicating an increase in the degree of substitution.

The spectrum carbon (51.6 PPM methanol, internal standard) signals to / ^ 10, 8 and-> ^ r 30 PPM, characteristics of the radicals CHg CH2 propionates and esters.

A) Use of the tetrabutylammonium salt of heparin :

A a solution of the tetrabutylammonium salt of heparin (0.55 g) obtained as described in the example 1, in dimethylformamide (5 ml) is added, to 0^e C butyric anhydride and of the dimethyl aminopyridine. After 24 hours at room temperature, water (5 ml) is added, and then the reaction mixture is dialyzed for 72 hours against distilled water. After exchange on Dowex 50 H⁺ , soda and neutralization by lyophilization, heparin is obtained 0- butyrylée as sodium salt (0.32 g).

Ratio sulfate/carboxyl: 2.15 meq/g

(starting material: 2.20 meq/g)

Title APTT: 59 iu/mg

The proton NMR spectrum (TSP, internal standard) shows the presence of signals to 0.9 PPM; and 2.4 PPM 1.6 PPM, characteristics of the groups-CH CH ^ ^ CH ^-^-^-CH CH C0-254.0ethanolamine5.0and signals between 3 and 6 PPM, characteristics of the osidic backbone.

The analysis of the NMR spectrum indicates the presence of a chain by about butyryl disaccharide unit.

A a solution, cooled to 0^e C, héparinate of tributylamine (4 g) obtained as described in the example 2 in dimethylformamide (50 ml), is added dimethylaminopyridine (0.25 g), butyric anhydride (6.7 ml) and tributylamine (9.7 ml). The reaction mixture is left at room temperature for 24 hours. Water is added (1.5 ml) and then, after 30 minutes, a saturated solution of sodium acetate in ethanol (250 ml). The precipitate is then washed three times with ethanol, then dialyzed against a solution to 5 % bicarbonate, and against water. Is obtained, after lyophilization, the sodium salt of heparin 0- butyrylée (2.1 g).

Title APTT: 29 iu/mg

Saponification of the esters After by soda 0.5 M for 2 hours at 0° C, the product obtained has a ratio of 2.36 carboxyl sulfate/meq/g (2.40 meq/g in the starting material).

A) Use of the tetrabutylammonium salt of heparin :

A a solution of the tetrabutylammonium salt of heparin (0.55 g) obtained as described in the example 1, in dimethylformamide (5 ml), is added to 0 °C caproîque anhydride (1 ml; 6 mmol), triethylamine (0.84 ml, 6 mmoles) and dimethyl aminopyridine. After 24 hours at room temperature, water (5 ml) is added, and then the reaction mixture is dialyzed for 72 hours against a solution of bicarbonate eroism 5, then against distilled water. After exchange Dowex 50 H on..⁺ , soda and neutralization by lyophilization, is obtained- Vhéparine 0- hexanoylëe as sodium salt (0.30 g).

Title APTT: 25 iu/mg

The proton NMR spectrum (TSP, internal standard) shows signals to 0.8; 1.2 ; 1.5; 2.3 PPM, CH characteristics of the ^ ^ ^-CH CH

-(ch₂ )₈ -co-O-

A a solution, cooled to 0° C, of sorting hêparinate butyl ammonium (4 g) obtained as described in the example 2 in the dimethylformamide (50 ml), is added diméthylamincpyridine (0.25 g), tri butyl friend does '(9.7 ml) and anhydride hexano' machine (10.6 ml). After 24 hours at 20° C, water is added (1.5 ml), followed by a saturated solution of sodium acetate in ethanol. After being washed with ethanol, dialysis and lyophization, heparin is obtained O-hexanoyl (2.5 g).

A a solution, cooled to 0° C, of sorting hêparinate butyl ammonium (4 g) obtained as described in the example 2 in dimethylformamide (50 ml), is added dimethylaminopyridine (0.25 g), and anhydride octanoîque (12.1 ml) of tributylamine (9.7 ml). After 24 hours at room temperature, water (1.5 ml) is added followed by, after 30 minutes, a saturated solution of sodium acetate in ethanol.

After dialysis against a solution to 20 % ethanol, then against distilled water and ultrafiltration, the product is subjected to a cation exchange by passage over Dowex 50 H⁺ , followed by neutralization by soda. After lyophilization, heparin is obtained 0- octanoylëe (2.5 g).

A) Use of the tetrabutylammonium salt of heparin :

A a solution of the tetrabutylammonium salt of heparin (0.55 g) obtained as described in the example 1 in the dimëthylformamide (5 ml), is added to 0 °C Capric anhydride (1 ml; 6 mmol), of the triêthylamine (0.84 ml, 6 mmoles) and dimethyl aminopyridine. After 24 hours at room temperature, water (5 ml) is added, and then the reaction mixture is dialyzed for 72 hours against a solution of bicarbonate 5%, then against distilled water. After exchange on Dowex 50 H⁺ , soda and neutralization by lyophilization, heparin is obtained 0-octanoyl as sodium salt (0.30 g).

The proton NMR spectrum (TSP, internal standard) shows signals to 0.8; 1.2 ; 1.5 and 2.4 PPM, and characteristics of the ^ ^ CH CH CH₃ -(CH₂ )₈ -C0-0-

A a solution, cooled to 0° C, of sorting héparinate butyl ammonium (4 g) obtained as described in the example 2 in the dimëthylformamide (50 ml), is added dimethylaminopyridine (0.25 g), anhydride dëcanoîque (13.3 g dissolved in 20 ml of dimëthylformamide), and tributylamine (9.7 ml). After 24 hours at room temperature, water (1.5 ml) is added, followed by a saturated solution of sodium acetate in ethanol. The precipitate is dissolved in dimethyl sulfoxide then dialyzed against water, sodium bicarbonate and water. After lyophilization, heparin is obtained 0- décanoylëe (2.33 g).

tëtrabutylammonium The salt of heparin, obtained as described in the example 2, by is benzoylated benzoic anhydride under the conditions described above for the preparation of heparin 0- dëcanoylée.

The carbon NMR spectrum of the resulting product has signals to 131,-132 and 136 PPM characteristics of the groups is benzoyl.

The product is devoid of anticoagulant activity in vitro .

EXAMPLE 10 : D * Preparation heparin low pass molecular

(Mean MW daltons ~ 4500 rpm), PM interval *-"* 1.800-8.000 daltons) O- acëtylëe (IC 1945)

The low molecular weight heparin has been obtained by depolymerization partial nitrous and alcoholic as fractionation.

described in EP 181,252 and is hereinafter CY 216.

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

The salt (1.7 g) is vacuum dried for three hours at 50 °C then dissolved in anhydrous dimethylformamide (10 ml). C After cooling to 0 °, acetic anhydride (1.7 ml) is added dropwise, tracking and triëthylamine (2.4 ml) of dimethylaminopyridine (102 mg). After 20 hours of reaction, the product is chromatographed on a column of Sephadex G-25 ëluée by water. Is obtained, after conversion into sodium salt and freeze-drying, the 0- acëtylé CY 216 (0.89 g).

Carbon Its NMR spectrum (51.6 PPM mëthanol , internal standard) presents a signal to characteristic 23 PPM acetates.

The signal of the ^ CH CH-CO-NH-, to ^ / 24, 5 PPM, is identical with that of the starting material.

The ratio sulfate/carboxyl is 2.09 meq/g

(starting material: 2.05 meq/g)

-Title APTT: 18 iu/mg

-Title anti-Xa:u/205 mg (Yin and The assay of al ., J. Lab. CLin.

Med ., 1973, 81_ , 298-310)

(Mean MW ^ "* 2.50D daltons, PM interval * *"^ 1500-8. OOP daltons) O-acetylated (IC 1946)

The low molecular weight heparin has been obtained by partial nitrous depolymerization according to the process described in EP 37,319 and is hereinafter CY 222.

The CY 222 of the sodium salt is converted into sorting butyl ammonium salt by passage through a column of Dowex 50 resin H⁺ , followed by neutralization by tri butyl friend does.

The obtained salt after lyophilization (1.5 g) is dissolved in dimethylformamide (5 ml) and then, after cooling to 0° C, acetic anhydride (1.35 ml) is added dropwise, tracking and friend does triéthy1 (2 ml) of dimethylaminopyridine (85 mg). After 18 hours of reaction, water is added (20 ml) and the mixture is dialysed during 3 days against distilled water. After conversion to sodium salt and freeze-drying, is obtained Q- acëtylé CY 222 (0.86 g)-

The product has a ratio of 1.98 carboxyl sulfate/meq/g.. (starting material: 1.97 meq/g).

The spectrum of carbon NMR (methanol 51.6 PPM, internal standard) of the product contains a signal to 23 PPM characteristic of the 0-acetyl. Comparison of the signal intensities of the N-acetyl (to--^ 24.5 PPM) between the starting material and the product of arrival demonstrates that the acetylation has been selective.

-Title APTT: 8 iu/mg

-Title anti-Xa:u/191 mg (Yin and The assay of al ., J. Lab. CLin.

Med ., 1973, 81_ , 298-310)

1/ Chains-Timeout heparin using periodic acid :

10 g of injectable heparin pig mucus, as sodium salt, titrating 157 iu/mg in the assay and 155 Codex u/mg in the assay of anti-factor Xa Yin al. and are placed in solution in 250 ml of deionized water to 4 °C. The pH of the solution is adjusted to 5.0 by concentrated hydrochloric acid. 10 g of sodium mëtaperiodate (NalO ^, PM: 213.89) in solution in 250 ml of deionized water to 4 °C are added under moderate agitation. The pH of the assembly is adjusted to 5.0 by concentrated hydrochloric acid. The solution is dropped 24 hours, in the dark, cold-chamber to + 4 °C.

2/ Periodate Withdrawal of residual :

Storage the reaction solution in three casings 40 NOJAX dialysis^R , (porosity of 3 to 4.000 Da) and under a 15 hours dialysis against deionized water current.

3/ Dépolymérisation in a basic medium :

At 780 ml of solution obtained after dialysis, , are added 16 ml sodium N 10, and the assembly is stirred 3 hours at room temperature (of the order of 18-21 °C).

4/ Sub :

500 mg of sodium borohydride NaBH ^, PM: 37.83) are then added and the solution is again stirred 4 hours at room temperature. Its is then brought to pH 4 using concentrated hydrochloric acid. After 15 minutes of stirring, the pH is adjusted to 7 using concentrated caustic soda.

At 820 ml of solution thus obtained are added 16.4 g of NaCl and 1270 ml of ethanol. The assembly is left to stand 3 hours, then centrifuged to 2.500 t/minute for 20 minutes. The precipitate is collected, resuspended in 200 ml of pure ethanol, ground to D

TUltra -Turrax and finally recovered by filtration on buchner

sintered. It is then dried under vacuum at 40 °C for 5 hours.

Is recovered and 8.9 g of product.

5/ Alcoholic Splitting :

These 8.9 g are dissolved in about 120 ml of deionized water at room temperature. 1.78 g of NaCl is added and the pH of the solution is lowered to 3.5 with hydrochloric acid. The volume of the solution is adjusted to 178 ml using deionized water. 151 ml of pure ethanol are added with agitation. Agitation is held 15 minutes after completion of the addition, and then the assembly is allowed to rest 10 hours, at room temperature.

The formed precipitate is collected by centrifugation 20 minutes to 2.500 t/minute. It is resuspended in 150 ml of pure ethanol, ground to 1' Ultra-Turrax, sintered buchner recovered by filtration, washed by 300 ml of pure ethanol and dried under vacuum at 40 °C for 24 hours.

Is obtained in this manner as a white powder 5 grams of the IC product 1772, having the following features:

It is substantially free of N-acetyl glucosamine (absence of signal to 24.5 PPM over the spectrum of the carbon).

-Title Codex: 11 iu/mg

-Title APTT: 9 iu/mg

-Title anti-Xa:u/12 mg

The product obtained in step A/is converted to tetrabutylammonium salt by passage over a Dowex 50 resin H⁺ , followed by neutralization of tetrabutylammonium hydroxide. From 9.5 g sodium salt, 18 is obtained g of tetrabutylammonium salt.

A a solution of 6 g of the resulting salt, in the dimëthylformamide (55 ml) is added, after cooling to 0° C, acetic anhydride (6.2 ml; 65.6 mmol) then the triëtnylamine (9 ml; 65.5 mmol) and dimethyl aminopyridine (403 mg; 3.3 mmoles). After 24 hours, a saturated solution of SOdium acetate in ethanol (250 ml) is added. After centrifugation and washing the precipitate with ethanol, the solid is desalted on Sephadex G-25, and then subjected to ion exchange by passage over Dowex 50 H⁺ , followed by neutralization by soda. After freeze-drying, the product is produced IC 1924 (3 g).

The product has the following characteristics:

-sulfate/carboxyl ratio: 2.28 meq/g.

The spectrum of carbon NMR (methanol 51.6 PPM, internal standard) clearly shows that the product has been 0-acylated. C The signal characteristic of the £N- acëtyl -glucosamine, about 56 PPM, is as in the starting material, absent from the spectrum.

6 g of the tetrabutylammonium salt of ICs 1772, obtained as described in the example 12, are butyrylés by butyric anhydride under the conditions described for the acetylation. The product is obtained IC I925 (2.96 g) having the following features:

-sulfate/carboxyl ratio: 2.31 meq/g.

13

The C-NMR spectrum (methanol 51.6 PPM, internal standard) contains the feature signals of the butyl 15.6, 20.4 and 38.4 PPM.

for antithrombin III O- hexanoylë (IC 1926)

6 g of the tetrabutylammonium salt of ICs 1772, obtained as described in the example 12, are processed by the anhydride hexanoîque the same way as the acetylation. The product is obtained IC 1926 (3 g) which has the following features:

-sulfate/carboxyl ratio: 2.20 mea/g

The NMR spectrum carbon (51.6 PPM mêthanol , internal standard) has the characteristics of the signals ^ and CH CH_? the hexyl group to 15.5, 23.9, 26.2, 32.7 and 36.1 PPM.

The proton NMR spectrum indicates the presence of about one per unit disaccharide hexyl group.

EXAMPLE 15 : Dermatan sulfate preparation 0- acëtylë (IC 1947)

A a solution, cooled to 0^e C, tetrabutyl ammonium salt dermatan sulfate (0.91 g), obtained in the same conditions as those described for obtaining héparinate tétraDutyl ammoni of um in the example 1, in the dimethylformamide (20 ml), is added, dropwise, acetic anhydride (1.35 ml; 14.2 mmol) and triethylamine (1.97 ml; 14.2 mmol) and dimethylaminopyridine (0.7 mmol). After 24 hours at room temperature, water is added 40 ml), then dialysis for 72 hours. The sodium salt is obtained by passage through a Dowex 50 column H⁺ , to 0° C, followed by neutralization by soda. Is obtained, after freeze-drying, a beige powder (0.52 g).

0- acëtylé The dermatan sulfate has the following characteristics:

-sulfate/carboxyl ratio: 0.99 meq/g

(starting material: T, 05 meq/g)

1^

" c-NMR spectrum (51.6 PPM mêthanol , internal standard)

signal to 25.2 PPM CH.₃ cH₃ -C0-NH-(identical to the

starting material)

signal to 23.0 PPM CH.₃ cH ^-CO-O

N-desulfated and N- acéiylëe (IC 1948)

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

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

After lyophilization and drying, the salt is dissolved in dimethylformamide (10 ml) and then, after cooling to 0^e C, butyric anhydride (2 ml), tributylamine and dimethylaminopyridine (2 ml) of the (65 mg) are added. After 24 hours, water (5 ml) is added, and the mixture is dialyzed against a solution to 5 % sodium bicarbonate, and against water. After passing on Dowex 50 H⁺ , followed by neutralization by soda, is obtained the sodium salt of heparin N-acylated O- butyrylée.

The spectrum of carbon NMR (methanol 51.6 PPM, standard, internal) presents a signal to 24.6 PPM h characteristic of the '-acetyl and signals to 16, 20 and 38 PPM butyric characteristics of the esters.

Experience can be repeated with a partially N-desulfated heparin N-acetylated and lead to a product partially N-desulfated N- acétylë and 0- butyrylé.

EXAMPLE 17 : Dermatan sulfate preparation peracetylated (IC 1950)

The tetrabutylammonium salt dermatan sulfate (0.8 g) dissolved in dimethylformamide (20 ml) is acëtylé by addition of dimethylaminopyridine (76 mg), acetic anhydride (1.2 ml) and triethylamine (1.7 ml). The mixture is heated at 80 °C for 1 hour.

After returning to room temperature, water (0.45 ml) is added, 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 50 column H⁺ , followed by neutralization by soda. Is obtained, after lyophilization, dermatan sulfate peracetylated (0.51 g).

The product has a sulfate/carboxyl 1.07 meq/g (starting material: 1.05 meq/g) and contains about three disaccharide acetyl groups per unit.

EXAMPLE 18 : Preparation 0- acétylëe heparin benzyl ester (IC 1949)

A a solution of héparinate of tëtrabutylammonium (1 g) in dimethylformamide (10 ml), is added benzyl bromide (0.17 ml). After 24 hours at room temperature, is added tëtrabutylammonium acetate (220 mg). After 24 hours, is acetylation of the benzyl ester formed. For this purpose, of the dimethylaminopyridine is introduced (57 mg), followed by triethylamine (1.3 ml) and acetic anhydride (0.9 ml).

After returning to room temperature, the reaction mixture is stirred for 24 hours. Water is then added, and then the product is precipitated by a saturated solution of sodium acetate in ethanol. After dialysis against distilled water, passage on Dowex 50 H⁺ , soda and neutralization by lyophilization, is obtained the sodium salt of heparin benzyl ester 0-acetylated (0.57 g).

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

The spectrum carbon (51.6 PPM methanol, internal standard) shows signals to 23.3 PPM (0-acetyl) and 131.6 PPM (benzyl).

The signal of the ^ ^-CH CH CO-NH, to ^ 24, 5 PPM, is identical with that of the starting material.

The salt of tëtrabutylammonium dermatan sulfate (1 g) is dissolved in anhydrous dimethylformamide (15 ml). To this solution, cooled to 0° C, is added benzyl bromide (0.25 ml), and then allowing during 24 hours at room temperature.

tëtrabutylammonium In acetate (0.32 g) is then added, and then, after 24 hours at room temperature, is acetylation.

Acetic anhydride (1.5 ml) is introduced, followed by triethylamine (2.2 ml) and of dimethylaminopyridine (96 mg). After 24 hours, of Veau (0.6 ml} is added, and then the product is precipitated by adding a ëthanolique saturated solution of sodium acetate.

The product is dialyzed center of the sodium chloride to 10 %, then water.

Is obtained, after lyophilization, the benzyl ester dermatan sulfate 0- acétylë (0.63 g).

The method of the present invention has been compared to methods of FR 2,100 735 Application and EP 256,880. In particular, the characteristics of the acetic ester of heparin prepared according to the method of the invention (IC 1938, example 1) were compared to those of the acetic ester of heparin obtained by applying operating conditions described in the Patent FR 2,100 735 (product A) and acetic ester of heparin obtained according to the procedures of examples 3 and 4 of the Patent EP 256,880 (B products and C).

In a solution of tetrabutylammonium hëparinate (1 g) dissolved in anhydrous dimëthylformamide (10 ml), is introduced dichlorohexylcarbodiimide (4.2 g) dissolved in dimethylformamide (15 ml), then acetic acid (1.16 ml) dissolved in dimëthylformamide (25 ml) is added dropwise in 45 minutes to + 4 °C.

After 24 hours at room temperature, the reaction mixture is filtered and concentrated under vacuum. The residue is resuspended in ether. After filtration and washing, the precipitate is dialyzed against distilled water. The sodium salt is obtained by passage over a resin column Dowex H⁺ , then neutralizing by soda. 0,493 is obtained g of product A.

The preparation has also been carried out for 48 hours at • + 4 °C.

(b) C B Production of the products and :

C B and The products have been prepared by acetylation of heparin in a mixture of pyridine and formamide in acëtyle , using 2 ml of acetyl chloride and 40 ml B for the product for the product C, under the conditions described in the examples 3 and 4 of patent EP 256,880. The acetic ester is then relayed in the dialyzed against water and sodium chloride.

(c) Results :' The characteristics of the products are data in Table 1. The characteristics of the starting heparin used in each test are given as a reference.

Table 1:

* Titles APTT Yin and Wessler titre are measured and in vitro .

The results show that, although the titles APTT and YW are lower for all the products to those of the starting heparin, and this more strongly for the products A,. C B and, only the IC product 1938 maintains a sulfate/carboxyl ratio nearly identical to that of the starting heparin. This is a result of the fact that the method of the invention allows selectively acylating the hydroxyl functionality is without impairing the functional groups of the heparin, confirmed through chemical analysis products.

The products IC 1938, A, B and C have been analysed by carbon NMR (methanol 51.6 PPM, internal standard). The spectra, and that that of the starting heparin are given as follows:

Figure 1: starting Heparin

Figure 2:IC 1938

Figure 3:A product after 24 hours of reaction

Figure 4:A product after 48 hours of reaction

Figure 5:B product

Figure 6:C product

The results are the following:

1. IC 1938 The product present in the spectrum of the carbon (Fig. 2):

-a signal corresponding to the to 23.4 PPM CH CH CO ^-^-0

-a signal corresponding to the to 24.4 PPM CH-j CH ^-CO-NH, identical to the signal present in the starting heparin.

These signals show that the carboxyl and amino groups have been met and reliable selective acetylation at the hydroxyl groups.

2. The analysis of the product A carbon NMR spectroscopy shows that the product obtained by majority, both after 24 hours that after 48 hours (Fig. 3 and 4), is a derivative isourea heparin, whose carboxyl groups are substituted by a group

due to the use of the dicyclohexylcarbodiimide. Indeed, strong signals is observed corresponding to the carbon atoms in the group above to 28, 34, 54 and 156 PPM, while the signal corresponding to the CH CH ^ ^-CO -0 to 23 PPM is very low.

The method thus cannot realize selective O-acylation and driven an alteration carboxyl functions, that reflects increasing the ratio sulfate/carboxyl.

3. The product B present in the spectrum of the carbon (Fig. 3):

-a signal corresponding to the to 23.1 PPM CH CH CO ^-^-0

-a signal corresponding to the to 24.8 PPM CH CH ^ ^-CO-NH, significantly more intense than those of the starting heparin and IC 1938.

These signals show that not only is observed a O-acetylation, but high N-acetylation. The method used, non-selective, causes a partial N-desulfation, followed by acetylation amines, which also results in a decrease in the ratio/carboxyl sulfate.

4. C The product present in the spectrum of the carbon:

-a signal to corresponding to the 22 PPM CH₃ cH₃ -CO--0

-a signal to 24 PPM CH_ corresponding to the₃ cH₃ -CO-NH,.

significantly more intense than those of the starting heparin and IC 1938.

B As for the product, is observed both a 0et N- acëtylation. N-desulfation, larger than for the product B, causes a significant decrease in the ratio/carboxyl sulfate.

A/Active anticoagulant in vitro :

1. Evaluation of the title in vitro with respect to a range gauge:

The measurement was made using the assay "thromboplastin time kaolin sensitized" (Diagnostica Stago , Assnières , -France), on human plasma. The results are given in Table 2:

Table 2:

IC = heparin 1940 O-' butyrylëe prepared as described in example 5 the IC = heparin 1941 O- hexanoylëe prepared as described in example 6 the IC = heparin 1943 O- dëcanoylëe prepared as described in the example 8 The results show that the products selectively 0- acylës of the invention have a title in vitro lower than that of heparin, anticoagulant activity decreases when acylating the length of the chain increases.

2. Measure of anticoagulant activity in vitro

products of the invention on human blood:

The assays are performed to two doses of the products of the invention, 2 ml gjnl / ^ ^ Ig 4 and 5 ml/ml on human blood. A test sample is also performed for each product by replacing the product to be tested by the solute isotonic NaCl. After 30 minutes of incubation at room temperature, the blood is centrifuged for 20 minutes at 3000 rpm. The plasma platelet depleted is decanted to provide the following tests:

-TCK (Cêphaline Kaolin Time) using the kit " time

cephalin kaolin sensitized " (Diagnostica Stago , Marcel Saupin Stadium,

France)

©- Heptest (Hemachem , St Louis, USA).

The results are given in Table 3. Each result corresponds to the average of three tests.

Table 3:

The results show in both methods an elongation coagulation time for the products and IC IC 1940 1941. Said lengthening is proportional to the dose.

However, in such methods in vitro , 1943 iC the product has shown that a very low action on the lengthening of the coagulation time.

3. Measure of anticoagulant activity in vitro products of the invention on human blood by thromboélastographie :

The products of the invention, at doses of 2/ml judicata judicata and 4/ml are in the presence of 5 ml of human blood as described in the previous test, a reference tube being carried out for each product by replacing the product to be tested by the solute isotonic NaCl.

After the 30 minute incubation at room temperature, a trace thromboélastographique is carried out using a thromboélastographe Hellige on 0.25 ml blood recalcifié by 0.1 ml of CaCl ^ 0,058 m.

The results are given in Table 4.

Table 4:

r * = reaction time

k = clotting time corresponding to an amplitude of the trace

of 20 mm

amx = maximum amplitude

IPT = index potential thrombodynamique

The results show that IC IC and 1940 1941 increase the r + k, a decrease in the amx and 1¹ IPT, which corresponds to a increased hypocoagulation. The hypocoagulation increases both as a function of the dose used and the acyl chain length ante.

IC 1943, in this test, does not cause detectable change in the parameters.

B/anticoagulant Active in vivo :

1. Measure of anticoagulant activity in vivo products of the invention in the rabbit (i.v. path):

The tests have been performed on male rabbits neo-Zealand. A blood sample is performed at the median of the ear artery prior to the injection of the product.

Is then injected in the marginal vein of the ear a solution of the product to be tested of 25 mg of product in 5 ml of isotonic solution of NaCl. _

The testing of the TCK and I ' HEPTEStU are formed on the blood collector before the injection of the products, and samples 6 h., h. 24, 48 and 96 h. h. after injection.

The results are given in Figures 7 and 8. They exhibit a net elongation in the time of the anticoagulant activity, the length increasing with acylating the length of the chain.

Indeed, anticoagulant activity of the product IC 1940 est net still more than 6 hours after injection, while it is still net 24 hours after the injection for the IC product 1941 and that it extends to 96 hours for the product IC 1943.

2. Measure of anticoagulant activity in vivo products of the invention by thromboélastographie :

The products to be tested are injected by i.v. channel as described in the previous test (25 mg in 5 ml of isotonic solution NaCl). A trace thromboélastographique is carried out using a theomboélastographe Hellige on 0.25 ml of blood samples taken before injection and 6 h., 24 h., 48 h. and 96' h. after injection, samples are performed when the anticoagulant activity persists beyond 96 hours.

The-results are given in Tables 5, 6 and 7.

Table 5: product tested: 1940 IC

The results obtained show that for all the products, there is an increase in r + k and a decrease in the IPT to the 6ëme time, indicating an elongation of 1' hypocoagulabilitë. This extends to at least 24 hours after the injection for the IC product 1941 and is still measurable 96 hours after the injection for the IC product 1943.

There is thus that all the products have high anticoagulant activity in vivo , that this activity is extended in time and that IC 1943 which has been little or no active in assays in vitro is highly active and highly delay in the in vivo assays.