AU620632B2 - Selectively o-acylated glycosaminoglycans, their preparation and pharmaceutical compositions containing them - 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

0632 COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COW-Lim SPN INAME ADDRESS OF APPLICANT: Sanofi avenue George V Paris 75008 France NAME(S) OF INVENTOR(S): Maurice PETITOU o Jean-Claude LORMEAU Jean CHOAY 8 0 ADDRESS FOR SERVICE: DAVIES COLLISON Patent Attorneysj l 1 Little Collins Street, Melbourne, 3000.

COMPLETE SPECIFICATION FOR THE INVENTION ENTITLED: a 6 64 Selectively 0-acylated glycosaminoglycans, their preparation and pharmaceutical compositkins containing them The following statement is a full description of this invention, including the best method of performing it known to me/us:-

I

I.

-la- The invention relates to selectively 0-acylated glycosaminoglycans, a procedure for their preparation as well as the pharmaceutical compositions containing them and their use in therapy.

By the term "glycosaminoglycan" is meant substances constituted of repeating sequences of uronic acids (D-glucuronic acid or L-iduronic acid) and amino sugars, these latter being glucosamines or galactosamines.

The glycosaminoglycans of natural origin are constituted by more or less homogeneous mixtures of repeating sequences of disaccharide units formed from a uronic acid sub-unit (glucuronic acid or iduronic acid) and from an osamine sub-unit (glucosamine or galactosamine) linked by a 1-4 or 1!3 linkage.

o oo In the glycosaminoglycans, the hydroxyl groups are variously o substituted by functional groups, in particular sulfate groups, the 0o0 e 15 amine groups of the osamines being substituted by sulfate and/or acetyl groups.

.0 The glycosaminoglycans considered here comprise 6 types 00 0 of product: heparin, heparan sulfate, chondroitin sulfate A, chondroitin sulfate C, chondroitin sulfate B, more precisely designated as dermatan sulfate, and hyaluronic acid. They are 00,0 characterized by the following two basic disaccharide structures D and 25 300 0 O o 0*OH 0o- (A) OH NHR in which R is a SO 3 group and/or an acetyl group and the sign indicates that the carboxyl group can be below the plane of the ring (iduronic unit) or above the plane of the ring (glucuronic unit), and ii i

I-I

i

COO

CH2OH O

NHCOCH

3 The glycosaminoglycans having the basic structure above *o are designated glycosaminoglycans (or GAGs) of the heparin type and 0 0 include the heparins and the heparan sulfates and the o°o 15 glycosaminoglycans having the basic structure above are designated o e0 ,0,O GAGs of the chondroitin sulfate type and include the chondroitin o 0o .o oo sulfates A and C and dermatan sulfate.

Hyaluronic acid has the basic structure in which galactosamine is replaced by glucosamine.

As mentioned above, in both the GAGs of the heparin type o 1 and in the GAGs of the chondroitin sulfate type, a more or less high 00 0 o0 0 percentage of the hydroxyl groups of the n disaccharide units is present in the form of esters of sulfuric acid.

Thus, heparin may be represented by the basic structure above in which n may vary from 1 to 80, R being a SO 3 group in the majority of the n units and, in the remainder of the cases, an acetyl group, the majority of the OH groups in position 6 of the 0o o glucosamine and in position 2 of the uronic acid are sulfated, whereas the OH group in position 3 of the glucosamine is sulfated only in a minority of cases; the OH group in position 3 of the uronic acid is practically not sulfated, the said uronic acid being an iduronic acid in the majority of species.

The compounds having the basic structure also include N-desulfated N-acetylated heparin which may be prepared as described in Nagasawa K. and Inoue Methods in Carbohydrate Chemistry, Academic Press, 1980, Vol. 8, pp. 291-294. This heparin is represented 3 by the formula in which R is an acetyl group.

Heparin also contains species having the structure with n varying from 3 to 15 and the same chemical profile. The said species can be isolated by fractionation according to known methods (for example US 4,692,435) and are named heparins of low molecular weight L.m.w. heparins having a molecular distribution and biological properties essentially identical with those of the l.m.w.

heparins obtained by fractionation were prepared by fragmentation of heparin according to the following methods: nitrous acid method of depolymerization and reduction (for example EP 37 319 or US 4,686,288) which cleaves the molecule by attacking the glucosamine N-sulfate sub-units to give a 0 9q o terminal: o+ o00 CH 2

OH

o oa be I 0 0 9

CH

2

OH

00 9 0 09 °9 possibly sulfated at the.primary hydroxyl at position 6; enzymatic (for example EP 244 235 and 244 236) or alkaline (for 25 example EP 40 144) method of depolymerization which cleaves the molecule by attacking the uronic acid sub-units to give an 1-B unsaturated uronic acid terminal: coo 0

COO

0

OH

possibly 2-sulfated; _1 w i( a an O o a o ODL) o 0040 o 0o o e o 04 o 44 o a 6 44 QO a 4 Q 4 64 000 0 <S4& 0 o 9 0 0 S4 oxidative method of depolymerization (for example US 4,281,108 and EP 121067) which cleaves the molecule by oxidation while preserving the "natural" terminals.

Other l.m.w. heparins which have a molecular distribution different from that of natural heparin and distinctive biological properties can be prepared by periodic oxidation of natural heparin followed by depolymerization by means of B-elimination or acid hydrolysis. The periodic oxidation cleaves the non-sulfated uronic acid residues between the carbons at positions 2 and 3. It is known that such residues are present in the binding site for antithrombin III (AT III), a plasma inhibitor of various serine-proteases of coagulation, and the activity of which is strongly enhanced by heparin acting as co-factor. Periodic oxidation thus makes it possible to obtain products lacking the binding sites for AT III, and thus 15 essentially lacking anticoagulant activity.

L.m.w. heparins of this type can be obtained in particular by using the procedure comprising the following steps: limited oxidation of the heparin carried out by reacting the heparin, in aqueous solution at a final concentration of 0.5 to (wt/v) with a salt of periodic acid at a final concentration of to 1% (wt/v) at a pH situated between 4.5 and 6.5, and preferably at a pH of 5, at a temperature of 0 to 10 0 C for about 15 to 48 hours, protected from light; the depolymerization of the heparin chains obtained by addition of a strong base such as sodium hydroxide at a pH higher than about 11, in particular included between 11 and 12, advantageously 11.2 to 11.6, and preferably about 11.5; the reduction of. the depolymerization fragments with the aid of a reducing agent and, after removal if necessary of any unreacted reducing agent, the recovery of the fragments of reduced heparin by precipitation by means of a solvent in which they are insoluble; the isolation of the desired fragments by fractionation by means of alcohol in the presence of a mineral salt from an aqueous solution obtained by redissolving in water the precipitate previously isolated and by recovering the precipitate formed.

i- These l.m.w. heparins correspond to the formula below: CH2OH 0 -0 OHO

AOH

0 C OSO- NHR n in which n may vary from 6 to 15, R is a SO 3 group in about at least of the n units and, in the remainder of the cases, an acetyl ;group, the OH groups in position 3 of the glucosamine may be sulfated, i and the OH groups in position 6 of the glucosamine being sulfated s in 70% of the species. Furthermore, to the extent of 1 residue per 2 chains at least of this l.m.w. heparin, the iduronic acid is replaced by a non-sulfated uronic acid residue (D-glucuronic or Liduronic acid) open between the carbons in position 2 and 3, of the following formula: C00- 0t Such l.m.w. heparins can also be fractionated by gel filtration according to known methods so as to produce mixtures of fragments homogeneous with respect to molecular mass and lacking the AT III binding site, S, Other glycosaminoglycans of the heparin type are constituted by GAGs of formula or in which the carbuxyl function borne by the uronic acid is esterified, for example by condensation with an alcohol in the presence of a condensation reagent of the carbodiimide type as described in the patent FR No. 2 159 724, or also by alkylation of the carboxyl by means of an alkyl halide in the presence of a weak base.

l 6 Similarly, glycosaminoglycans of the chondroitin sulfate type are constituted by glycosaminoglycans of formula in which the carboxyl functions of the uronic acids are esterified, for example by alkylation of the carboxyl with the aid of an alkyl halide in the presence of a weak base.

The preparation of such esters in the case of hyaluronic acid is described in the patent EP No. 215 453, by treatment of hyaluronic acid in the form of its quaternary ammonium salt by an alcohol in the presence of a catalyst or an ether-forming reagent.

The heparan sulfate is represented by the formula above, in which n may vary from 1 to 80, R represents an acetyl group in the majority of the species and SO 3 in a minority of the species.

Furthermore, in the large majority of the species, the OH groups at position 6 of the glucosamine are sulfated, the uronic acid being S 15 glucuronic acid in the majority of the species.

fit,' The chondroitin sulfates A and C are represented by the formula above in which n may vary from 1 to 80 and the 4-OH and 6-OH groups of the glucosamine are respectively sulfated, the uronic acid being a glucuronic acid in the majority of the species.

Dermatan sulfate has a structure substantially identical with that of chondroitin sulfate A, but the uronic acid sub-unit jis an iduronic acid in the majority of the species.

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

The glycosaminoglycans possess numerous biological Sactivities, among which may be cited their activities towards coagulation factors, which can be exerted through the intermediary of various plasma proteins. As regards heparin, it is also known from the literature that heparin or some of its derivatives which may or may not possess an anticoagulant activity may have a regulatory activity on the proliferation of the smooth muscle cells of the vascular wall (Guyton et al., Circ. Res., 46, (1980), 625-634) or 7 also an inhibitory activity on heparanase, an enzyme implicated in the mechanisms of metastatic dissemination (Application EP No. 254 067). Moreover, the glycosaminoglycans form part of the larger family of sulfed poiysaccharides, soane of which having shown varying degrees of anti-viral activity (BABA et al., Antimicrob. Agents Chen ther., 32 (1988) V742-/745)and, in particular, on anti-HIV activity (human iunodeficency virus) (BABA et al., Proc. Natl. Acad.

Sci., 85 (1988) 6132-6136).

In the remainder of the text, the term GAG will be used to designate a glycosaminoglycan, the latter having either a natural structure such as that obtained by extraction, semi-synthesis or synthesis, or a structure chemically modifie at the carboxyl and amino functional groups prior to the acylation reaction giving rie S to the selectively O-acylated GAGs.

In spite of their pharmacological activities of great value, f the natural GAGs possess the disadvantage of having a relatively short half-life, making repeated administrations necessary. This it disadvantage has in part been remedied by the use for the prevention Sand treatment of venous thromboses of heparin derivatives of low molecular mass by the subcutaneous route, thus diminishing the frequency of administration to one injection per day.

Nonetheless, it would be of great interest to have available derivatives possessing a sustained-release action which would make it possible to diminish further the frequency of administration of these compounds by increasing their duration of action.

It might also be of great interest to have available nonanticoagulant sustained-release derivatives such as those of N-desulfated N-acetylated heparin, the activity of which as inhibitor of heparanase, an enzyme implicated in the phenomena of metastatic dissemination, is pointed out above.

The literature describes many derivatives of glycosaminoglycans modified so as to improve their pharmaco-kinetics, in particular esters of the carboxyl function of the uronic acids or of the hydroxyl functions.

Use has been made in particular of partial or total esterification of the carboxyl functions of heparin, treated in the L form of its quaternary ammonium salt in an inert solvent by an alcohol or a halogenated derivative in the presence of a condensation reagent i 8 if necessary (French patent No. 2 159 724 and EP No. 44 228).

Derivatives of hyaluronic acid obtained by esterification using an alcohol in the presence of a catalyst or by reaction with an etherforming reagent have also been described (European patent No, 216 h 5 453).

Moreover, different agents have been described in the literature with a view to esterifying the primary and secondary hydroxyl functions of the GAGs.

The publication Can. J. Res., 25B, (1947), 472-476, describes an acetylated derivative of heparin corresponding to one mole of acetyl group per four 'saccharide units. The product is prepared by the action of ketene on heparin in acetone. Although it is specified in this publication that the product obtained is an 0-acetylated derivative, it is well known that ketene gives anhydrides in the presence of carboxyl groups (cf. J. March, Advanced Organic Chemistry; Reactions, Mechanisms and Structure, J. Wiley and Sons Eds., 1985, pp. 686-687- "With ketenes, carboxylic acids S, give anhydrides and acetic anhydride is prepared industrially in this manner: 0 O "CH =C=0 CH--COOH-CH3- 0 S0-

COCH

3 Consequently, this publication in fact describes a heparin in which o.o. 0-acetyl groups are associated with mixed anhydrides between the a a COO- group of the uronic acid and the acetyl group derived from ketene, and this may explain their instability in water.

The patent FR 2 100 735 describes hydrolysable partial esters of heparin and a non-toxic organic acid, in particular S4-chlorophenoxyisobutyric acid, 4-chlorophenoxyacetic acid, cholic 30 acid, nicotinic acid, N-oxynicotinic acid, pyridylacetic acid, N-oxypyridylacetic acid or linoleic acid. The method of preparation described in the above patent, which is characterized by the reaction of a quaternary salt of heparin with the acid activated by a carbodiimide, gives not only the 0-acylated derivate but also a considerable amount of a by-product stable in the form of a O-acylisourea ester derivative. Furthermore, this type of reaction i. m_ h -L L I I 'r I

IY

I

is likely to promote the reactions between the ac j heparin.

formation of anhydrides and intramolecular :id functions and the hydroxyl functions of ii 4 (t 4* 4 4 4* 4 4 4* 4 44 *4 The patent JP 74/048533 describes 0-esters of chondroitin sulfate with possibly substituted aromatic, arylaliphatic or heterocyclic acids endowed with sustained activity. The method of preparation described in this document, which is characterized by the reaction of chondroitin sulfuric acid with an acid chloride, gives rise to an N-acylation as side reaction.

The patent WO 83/00150 provides a general description of prodrugs of medicines prepared by using the different functions of the GAGs, including the hydroxyl and, specifically, an 0-ester of chondroitin sulfate with penicillin V. The preparation of this compound is carried out by means of carbodiimide and thus entails the side reactions mentioned above.

The patent EP 46 828 describes O-esters of heparin with unsaturated acids, in particular with acrylic acid or metacrylic acid which, grafted on to biomedical materials, confer on them an antithrombotic activity of long duration. This grafting is carried 20 out through covalent linkage at the level of the K-B unsaturated bonds of these esters with the surface of these said materials which enter into contact with the blood. According to this document, the alpha, beta-unsaturated 0-esters are prepared by reaction of heparin with the chloride or anhydride of an alpha, beta-unsaturated carboxylic acid. Furthermore, the description which points to the indiscriminate use of chlorides or anhydrides of the alpha, betaunsaturated acids as reagents, does not specify which type of 0-esters of heparin are thus obtained.

The patent EP 256 880 describes the esterification of heparin or heparin of low molecular weight by the action of acid chlorides in formamide and pyridine in order to give rise to derivatives with enhanced transmembrane permeability. Now, this method leads to the derivatives which undergo a partial desulfatation and are 0- but also N-acetylated, and in which the sulfate/carboxyl ratio is modified.

T 1

NHCOCH

3 /2 j I The patent FR 3 066M describes the acetylation of N-monomethyl heparinamide as a result of the action of acetic anhydride-in formamide and pyridine. The starting material is a heparin derivative, the carboxyl functions of which have been replaced by amide functions.

The procedure used for the acetylation does not make use of a salt soluble in an organic medium. Consequently, the acetylation reaction cannot be precisely controlled and leads to only a low acetylation yield. Furthermore, if the procedure described is applied to heparin in which the carboxyl functions have not been blocked, considerable amounts of mixed anhydride are formed between the carboxyl groups of heparin and acetic anhydride, these undesirable by-products not being removed from the mixture.

The patent JP 5128602 describes the acylation of heparin as a result of the action of an acid anhydride in formamide. In the procedure used, the heparin is not present in the form of a salt soluble in an organic medium, and this makes .it impossible to control the acylation 4 reaction precisely and leads to a low degree of acetylation. Further- Snore, the procedure described gives rise to the presence in the medium of mixed anhydrides as by-products of the reaction.

It was thus of interest to have available more Epecific products, which can be prepared in a highly reproducible manner.

i t 4 4 i I44

I

L~.

./3 ii ::II o o.

a 00 a0 a 00 0) aD a a a 0o 0 I 04 O 0C

II

It has now been found that by causing to react in organic medium a soluble salt of a GAG 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 modifying the carboxyl or amine functional groups of the GAG used. The use of the GAG in the form of a salt soluble in an organic medium makes it possible to advantageously control the acylation reaction with high precision. Furthnnrrn, the extent of acylation can easily he nodulated and can be incre.:iid without it leading to the inpairment of the rest of the molecle. In particular, it is possible to attain an extent of acylation of 0.1 to 3,:and in particular of 0.5 to 2, acyl groups per disaccharide unit.

It has also been found that no N-acylated derivative is formed and that,when anhydrides form at carboxyl functions, the use of a 15 weak base enables them to be readily reconverted into the free acid.

Advantageously, the selective acylation of the GAGs according to the procedure of the invention makes possible a modulation of their biological activities, which in some cases are greatly enhanced.

Finally, it has been found that the 0-acylated GAGs thus obtained have a pharmacological activity of longer duration.

Thus, the subject of the present invention, according to one of its features, is selectively 0-acylated glycosaminoglycans, corresponding to the following formula I: G U- B (I) n in which: G represents a group of formula:

NHR

2 I 12 or a group of formula:

-OR

1 W) 0 N HCC

H

or a group of formula:

OR

1 R 1 0 0 *00

NHCOCH

3 0 06 U represents a group of formula: COOR 3 (C)0

ORI

OR

1 or a group of formula: 4 a

I

-13or the residue of group or of group after periodic oxidation followed by a p-elimination or acid hydrolysis; or, in a proportion of one residue for at least two I chains, a non-sulfated uronic acid (D-glucuronic acid or L-iduronic acid) open between the carbon atoms in positions 2 and 3, of formula: 0

I

CH OR CH OR 2 2 13- A represents a group R, a group or a group after or a group of formula:

COOR

3

OR

t

OR

1 cr the residue of the group or the group after periodic oxidation followed by a p-elimination or acid hydrolysis, B represents a group O-R 1 a group (a)-OR 1 a group (a')-OR 1 a group (b)-OR 1 a group of formula:

OR

M 0

OR

o

R

or a group of formula: S91112,PHHSPE.020,sanofiet,13 I 25 erioic oxdatin folowedby a -eliinatosn o acid hyrlysis,- i ,L m or represents a group a group or a group (b) to which remains attached a residue of or such as is present after periodic oxidation followed by a elimination or acid hydrolysis; 04 911122,P HHSPE.020,sanoEIlet, 13 in which R is a SO 3 group and/or an acetyl group ana ne bxsI y indicates that the carboxyl group can be below the plane of the ring (iduronic unit) or above the plane of the ring (glucuronic unit), and i.

!i' 14 R represents H and/or SO3 and/or an acyl group, acyl being the residue of a non a:-B unsaturated, mono- or dicarboxylic acid selected from among: an alkanoyl group of 1 to 18 carbon atoms; an alkanoyl group of 2 to 3 carbon atoms substituted bh a cycloalkyl group of 3 to 7 carbon atoms, a phenyl group possibly substituted by one or more alkyl radicals of 1 to 14 carbon atoms, halogen atoms or NO 2 or OCH 3 groups, an unsaturated aliphatic hydrocarbon radical of 4 to 16 carbon atoms; a benzoyl group possibly substituted by one or more alkyl radicals of 1 to 4 carbon atoms, halogen atoms or NO 2 or OCH 3 groups; a cycloalkyl (3-7 C) carbonyl group;

R

2 represents SO3 and/or an acetyl radical, provided that the proportion of N-acetyl glucosamine is not more than that in heparin 15 when R 1 represents an acetyl radical;

R

3 represents a hydrogen atom or an alkyl radical of 1 to 10 carbon o"a atoms, and preferably 1 to 4 carbon atoms, or a phenyl alkyl radical of 7 to 12 carbon atoms, or an alkali or alkaline earth metal cation; n is an integer *a4yn- from 1 to

R

1 being acyl in a proportion of at least 0.1 to 3 acyl groups, and preferably 0.5 to 2 acyl groups, per disaccharide unit, and 0o o their pharmaceutically acceptable salts.

In a preferred embodiment of the invention, the S° glycosaminoglycans selected are glycosaminoglycans of the heparin type, namely heparin, the derivatives of heparin obtained either by jo fractionation or by semi-synthesis or synthesis, heparan sulfate and its derivatives obtained by fractionation, semi-synthesis or synthesis.

The glycosaminoglycans of this type, selectively O-acylated according to the present invention correspond to the following formula II:

L

The compounds having the basic structure also include N-desulfated N-acetylated heparin which may be prepared as described in Nagasawa K. and Inoue Methods in Carbohydrate Chemistry, Academic Press, 1980, Vol. 8, pp. 291-294. This heparin is represented Iu i tl c lm r

T

j

(II)

A -I G o a s 90 0 0 Ol o 00 o 0 00 9 0 00 04. 0 0 0o o 090 0. 0 o 0o o 00 09 9 006 0 00 D 0 0 00 in which: A has the meaning given above in formula

R

I represents H and/or SO 3 and/or an acyl group such as that defined in formula

R

2 represents SO 3 and/or an acetyl radical, provided that the proportion of N-acetyl glucosamine does not exceed that in heparin when R 1 represents an acetyl radical;

R

3 represents a hydrogen atom, an alkyl radical of 1 to 10 carbon atoms, and preferably 1 to 4 carbon atoms, a phenyl alkyl radical of 7 to 12 carbon atans, or an alkali or alkaline earth metal cation; B represents (a)-OR 1 or and being such as defined in formula or OR 1 or a group to which remains attached a residue of or of such as is present after periodic oxidation followed by a B-elimination or acid hydrolysis; n is an integer vYaryia from 1 to A preferred family IIa of compounds of the invention are comapounds of formula II F r COOR r-OR f

(II)

0' -i possibly 2-sulfated; -i 16- C C iCC' S44 C 4 i cC C in which: A has the meaning given above in formula

R

1 represents H and/or SO3 and/or an acyl group such as that defined in formula

R

2 represents S0 3 and/or an acetyl radical, provided that the proportion of N-acetyl glucosamine does not exceed that in heparin when R 1 represents an acetyl radical;

R

3 represents a hydrogen atom, an alkyl radical of 1 to 10 carbon atoms, and preferably 1 to 4 carbon atoms, a phenyl alkyl radical, or an alkali or alkaline earth metal cation; B represents (a)-OR 1 or and being such as defined in formula or OR 1 or a group to which remains attached a residue of or of (d) such as is present after periodic oxidation followed by a p-elimination or acid hydrolysis; n is an integer from 1 to 80, provided that, when A represents R 1 a group R 1 or a group R 1 and that B represents O-R 1 n is an integer varying from 1 to 16.

Another preferred family IIb of compounds of the invention corresponds to compounds of formula II: 2.

NHR

2

OR

1 1 .dJA 4 cl:1C-) 91 112PHHSPE.020,sanofi.Ie,16 F 1 I I of alcohol in the presence of a mineral salt from an aqueous solution obtained by redissolving in water the precipitate previously isolated and by recovering the precipitate formed.

-i -16ain which A and B have the meanings given above in formula I;

R

1 represents H and/or SO 3 and/or an acyl group, acyl being the residue of a non-a-p unsaturated, mono- or dicarboxylic acid selected from among: S an alkanoyl group of 4 to 18 carbon atoms; an alkanoyl group of 2 to 3 carbon atoms substituted by a cycloalkyl group of 3 to 7 carbon atoms, a phenyl group possibly substituted by one or more alkyl radicals of 1 to 14 carbon atoms, halogen atoms or NO 2 or OCH 3 groups, i 1 t r (11(1 i. 4 t44 4 4

II

I I I I

((II

(I

1 (4c I (i 911122,PHHSPE.020,sanofi.Iet,16 also Dy alKylation ot the carboxyl by means of an alkyl halide in the presence of a weak base.

17 an unsatu:ated aliphatic hydrocarbon radical of 4 to 16 carbon atoms; a benzoyl group possibly substituted by one or more alkyl radicals.

of 1 to 4 carbon atoms, halogen atoms or NO 2 or OCH 3 groups; a cycloalkyl (3-7 C) carbonyl group;

R

2 represents SO 3 and/or an acetyl radical, provided that the proportion of N-acetyl glucoswmine is not more than that in heparin when R represents an acetyl radical;

R

3 represents a hydrogen atom or 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, or an alkali or alkaline earth metal cation; n is an integer varying from 1 to R being acyl in a proportion of at least 0.5 to 2 acyl greups, and Sreferably 1 acyl group, per disaccharide unit.

Advantageous compounds of the invention are compounds belonging to o Rthe families (IIa) and (IIb), in which R represents a hydrogen atom r an alkali or alkaline earth metal cation.

Other groups of advantageous compounds correspond to the compounds belonging to the families (IIa) and (lib) in which R 3 represents an alkyl radical of 1 to 10 carbon atoms, and preferably 1 to 4 carbon atoms, or a substituted alkyl radical of 7 to 12 carbon atoms.

In a preferred embodiment of the invention, use is made of mixtures of heparin fragments having a molecular mass lower than 10,000 daltons, in particular either those having a mean molecular mass varying between 2,000 and 7,000 daltons, or those having a mean t- 3 t 0 9 9 Y^ V11 L-U pLujL.LiraLion or cne smooun musc±e ce.-s 01. Lilt; vascular wall (Guyton et al., Circ. Res., 46, (1980), 625-634) or St ill 18 molecular mass of about 4,500 daltons, or even those having a mean molecular mass of about 2,500 daltons.

A depolymerization procedure using nitrous acid, as described for example in the European patent 37 319, can be advantageously used to obtain them. The selectively 0-acylated glycosaminoglycans of the invention are then resented by the following formula III:

(III)

44 4 4 4.4 4 44 9 4 9 o 44 44 49 44 4 I 44 44 44 I 4 4 4 4 15 L NHR 2

OR

1

LOR,

in which: A represents R 1 or Rl-(c) or as defined in formula

R

1 represents H and/or SO 3 and/or an acyl group such as that defined in formula

R

2 represents SO and/or an acetyl radical, provided that the proportion of N-acetyl glucosamine does not exceed that in heparin when R 1 represent an acetyl radical;

R

3 represents a hydrogen atom and/or an alkyl radical of 1 to carbon atoms, and preferably 1 to 4 carbon atoms, or a phenyl-alkyl radical of 7 to 12 carbon atmos, or an alkali or alkaline earth metal cation; n is an integer varying from 3 to 12.

Preferred compounds of the invention correspond to compounds 30 of formula (III) in which R 1 is an alkanoyl radical of 4 to 10 carbon atoms.

In order to obtain mixtures of heparin fragments having a molecular mass lower than 10,000 daltons, it is also possible to use an enzymatic method of depolymerization, for example as described in the patents EP 244 235 and 244 236, or alkaline depolymerization, for example as described in the patent EP 40 144.

44~ 4 44 4 4 44 49

I

I

i; K Use has been made in particular of partial or total esterification of the carboxyl functions of heparin, treated in the form of its quaternary ammonium salt in an inert solvent by an alcohol i or a halogenated derivative in the presence of a condensation reagent r: S( 19 In this case, the selectively 0-acylated glycosaminoglycans according to the invention are represented by the following formula

IV:

COOR

COOR

3

OR

1 COoR 3 0 0 0

.(IV)

OR

1 OR OR B IO ORo 0iy OR,

OR

1

NHR

2

OR

1 n in which: R represents H and/or SO3 and/or an acyl group such as that S' defined in formula R represents SO3 and/or an acetyl radical, provided that the 'o'o ,proportion of N-acetyl glucosamine does not exceed that in heparin owhen R 1 represents an acetyl radical;

R

3 represents a hydrogen atom and/or an alkyl radical of 1 to carbon atoms, and preferably 1 to 4 carbon atoms, or a phenyl-alkyl radical of 7 to 12 carbo, atons, or an alkali or alkaline earth metal cation; B represents (a)-0R 1 as defined in formula or OR 1 n is an integer varying from 2 to Among the compounds of formula advantageous compounds are those in which R 1 is an alkanoyl radical of 4 to 10 carbon atoms.

In another advantageous embodiment of the invention, it is possible to use a mixture of heparin fragments homogeneous with t..t respect to their molecular mass, a fragment of heparin obtained by S 30 synthesis, homogeneous both with respect to its molecular mass and its functionalization.

In another interesting embodiment of the invention, it is possible to select as glycosaminoglycan derivatives of heparin lacking the binding site for antithrombin III (AT III), either because the heparin chains have been fractionated so as to remove the oligo- -i K carbodiimide, gives not only the 0-acylated derivate but also a considerable amount of a by-product stable in the form of a O-acylisourea ester derivative. Furthermore, this type of reaction .ie~~l d~u 4: saccharide chains containing the binding site for AT III by having recourse for example to affinity chromatography on Sepharose-AT III resin or to ion exchange chromatography, as described in E. Sache et al., Thromb. Res., 25, (1982), pp. 443-458, or because these sites have been destroyed for example by depolymerization with periodate followed by a B-elimination or acid hydrolysis.

Such compounds correspond to the following formula V:

A

ft

S*(

t 1 CIa f SI a *r a a I It t I i a a a a

NHR

2 in which: A represents R 1

R

1 or the residue of or of after periodic oxidation followed by a B-elimination or acid hydrolysis; B represents (a)-OR 1 or OR 1 or a group to which remains attached a residue of or of such as that present after periodic oxidation, followed by a B-elimination or acid hydrolysis;

R

1 has the meanings given in formula

R

2 represents SO 3 or an acetyl radical, the proportion of SO3 being about

R

3 represents a hydrogen atom or an alkali or alkaline earth metal cation; n is an integer *Ajyfg. from 1 to Particularly advantageous compounds can be obtained starting from compounds prepared by the procedure making use of periodic oxidation followed by B-elimination under alkaline conditions, reduction and fractionation as described above. These compounds correspond to the following formula VI:

N,

are 0- but also N-acetylated, and in which the sulfate/carboxyl ratio is modified.

i-

A--

(VI)

NHR

2 in which: A represents R 1

R

1

R

1 or t;e residue of or of (d) after periodic oxidation followed by a B-elimination; U represents: 0oso *i 44 t i So 3 or, in a proportion of one residue for at least two chains, a nonsulfated uronic acid (D-glucuronic or L-iduronic) open between the carbon atoms at positions 2 and 3, of formula:

COOR

3 0 CH OR CH OR 2 1 2 1 B represents (a)-OR 1 or OR 1 or a group to which remains attached a residue of or such as that present after periodic oxidation, followed by a B-elimination;

R

1 has the meanings given in the formula

R

2 represents SO3, and/or an acetyl radical, the proportion Sbeing about at least

R

3 represents a hydrogen atom or an alkali or alkaline earth metal cation; of

K

11

II

N

n is an integer varying from 2 to i8.

Advantageous compounds corresponding to the formula VI are those in which n is an integer vsrying from 7 to 15 for the major species which constitute them.

Advantageous compounds corresponding to the formulae V and VI, and in particular those corresponding to formula VI in which n is an integer varying from 7 to 15 for the major species, are those in which R is an alkanoyl radical of 2 to 10 carbovk atoms, advantageously from 4 to 3.0 carbon atoms and preferably 4 or 6 carbon atoms.

Other preferred compounds corresponding to the formula VI are mixtures of compounds homogeneous with respect to their molecular mass, obtained by gel filtration, and in which n is an integer vcrying from 2 to 12.

Among the co-pounds of heparin structure lacking the binding 15 site for AT III, and t;.js lacking anticoagulent activity, a family of advantageous compounds corresponds to selectively 0-acylated derivatives of N-acetylated N-desulfated heparin corresponding to the following formula VII: 0a 'at 0ov '1 0a Q a a 'a o a Q 00 a o 00S 0 o 'a L o o 'aB oo i'a e o 1 0i' a 'aU 'al A

(VII)

-0

NHCOCH

3 in which: 30 A represents RI, R 1 or R 1 such as defined in formula

R

1 represents an alkanoyl radical of 2 to 18 carbon atoms;

R

3 represents a hydrogen atom or an alkali or alkaline earth metal cation; B represents (a)-OR 1 as defined in formula or OR 1 n is an integer varying from 1 to NHR 2 23 In another advantvsgeous embodiment ot the invention, the glycosaminoglycans selected are of the chondroitin sulfate type, iiamely chondroitins 4- and 6-sulfates, dermatan sulfate and their fragments.

The glycosaminoglycans of this type, selectively 0-acylated according to the invention, are represented by the following formula :I I 7 t 24

OR

1

COOR

R0 0B 0

NHCOCH

3 OR n in which -A has the meanings of formula

-R

1 represents H and/or SO 3 and/or an acyl group such as defined in formula carbon atoms, and preferably 1 to 4 carbon atoms, or a phenyl alkyl radical of 7 to 12 carbon atom, or an alkali or alkaline earth metal cation; -B represents (b)-OR 1 or such as defined in formula or 1 *4

OR

1 or a group to which remains attached the residue of (c) or of such as that present after periodic oxidation followed by a B-eli."niniation or acid hydrolysis; -n is an integer varying from 1 to Among the compounds of formula (VIII) an advantageous family corresponds to the compounds in which R 3 represents a hydrogen atom or an alkali or alkaline earth metal cation; Other advantageous compounds of formula (VIII) are those in which R 3 represents an alkyl radical of 1 to 10 carbon atoms, and -preferably from 1 to 4 carbon atoms, or a phenyl alkyl radical of 7 to 12 carbon atoms.

3044 cores o the avngeumi y of ismpandakaos offrua(II crepnsto tecompounds inwhich R sa laolradical o 4 to 10 carbon atoms.

Another subject of the invention is a procedure for the preparation of the selectively 0-acylated glj, ninoglycans.

911122,PHHSPE.020,sanofi.le 13 The procedure of the present invention is characterized in that the glycosaminoglycan is converted into a salt soluble in an organic medium and in that this salt is treated with an acylation reagent capable of acylating selectively the primary and secondary hydroxyl groups of this glycosaminoglycan without affecting the functional NHR 2 or COOR 3 groups present in this glycosaminoglycan before the acylation reaction, the reaction being carried out in a polar aprotic solvent in the presence of a catalyst or in the presence of a base capable of binding the acid released during the acylation at a temperature of 00C to 1000C.

The product is then precipitated by means of a solvent miscible with water such as ethanol to which may be added an adjuvant factlitating precipitation, such as a mineral salt. The 0-acylated glycosaminoglycan is isolated by dissolution of the precipitate in water and advantageously dialysed in the presence of a weak base.

In an advantageous manner, the procedure of the invention Sis implemented in the following steps: S' a glycosaminoglycan of the following formula IX: G- B° (IX) 2 0 n in which: G° represents a group (a)O of formula: OR 1 a) 0 0 O

OR:

NHR

2 or a group of formula: 1 30

OR

(a

NHCOCH

3 or a group of formula: (b)e F

U

0 represents a group of formula:

COOR

3 C)a -o0 00 000 0 0100 o 00 o 00 0 000 0 o 00 0 04 00 0 O 00 or a group of formula: (d) 0 4 4 4 r or the residue of the group or the group after periodic oxidation followed by a B-elimination or acid hydrolysis; A* represents a group R, a group Ri 0 0 a group R 1 0 0 or a group of formula:t 4 r 4 s i -1 1 27 or the residue of the group or of the group after periodic oxidation, followed by a B-elimination or acid hydrolysis; B° represents a group OR 1 0 a group (a)°-OR 1 a group (a')-OR 1 a group or a group of formula: 0or a group of formula: (g)O RI O, S« I 1 44 15 4 4 4 t t t 4 44« I i t t* €4 t 4 9 t 4 i

Q

or represents the groups (a) 0 attached a residue of (c)O or of (d periodic oxidation, followed by a B

R

1 0 represents H or SO

R

2 represents SO 3 or an acetyl rad proportion of N-acetyl glucosamine when R 1 represents an acetyl radica

R

3 represents a hydrogen atom or an atoms, or a phenyl alkyl radic or an alkali or alkaline eartt n is an integer g from 1 to 8 is converted into a salt of the said a polar aprotic organic solvent; this salt is treated with an anhyi Acyl-O-Acy in which Acyl is such as defined in fi aprotic organic solvent in the presen i r 4 4 4 1 o, or (b)O to whicti remains )o such as that present after -elimination or acid hydrolysis; ical, provided that the does not exceed that in heparin 1; alkyl radical of 1 to 10 carbon ;al of 7 to 12 carbon atoms, metal cation; 0.

glycosaminoglycan soluble in dride of formula:

I

ormula in the said polar ce of catalytic amounts of

I

NHR

2 jn r i 000 oa o 0 0 o o 0 0 o- ser

D

pyridine or of a dialkylaminopyridine and a proton acceptor; the product thus obtained is precipitated by the action of a solution of sodium acetate in ethanol; and the selectively 0-acylated glycosaminoglycan is isolated by dissolving in water the precipitate thus obtained and by dialysis in the presence of a weak base, and, if necessary, the sodium salt of the selectively 0-acylated glycosaminoglycan thus obtained is converted into another pharmaceutically acceptable salt.

In an advantageous embodiment of the procedure of the invention, the glycosaminoglycan used in step is selected from the group constituted by heparin, a mixture of heparin fragments having a molecular mass lower than 10,000 daltons, a mixture of heparin fragments having a mean molecular mass varying between 2,000 and 7,000 daltons, a mixture of heparin fragments having a mean molecular mass of about 4,500 daltons, a mixture of heparin fragments having a mean molecular mass of about 2,500 daltons, a mixture of heparin fragments homogeneous with regard to their molecular mass, a fragment of heparin obtained by synthesis, homogeneous both with regard to its molecular mass and its functionalization.

20 In another advantageous embodiment of the procedure of the invention, the glycosaminoglycan used in step is heparin, a fraction or a fragment of heparin lacking the binding site for antithrombin III.

The glycosaminoglycan used in step of the procedure 25 of the invention may also be chosen advantageously from the group constituted by dermatan sulfate and its fragments, or the chondroitin 4- and 6-sulfates and their fragments.

In an advantageous manner, the glycosaminoglycan salt used in step of the procedure of the invention is a tertiary amine 30 salt, in particular a tributylammonium salt, or a quaternary ammonium salt, in particular a tetrabutylammonium salt.

In another advantageous embodiment of the procedure of the invention, the anhydride used in step is the anhydride of an alkanoic acid containing from 2 to 10 carbon atoms, advantageously from 4 to 10 carbon atoms and preferably 4 or 6 carbon atoms.

03 00 0 0 0 Se, o oo 0 0 000 oa o 0 a0 911122,PHHSPE020,sanof.liet,16 S29 The polar aprotic solvent in which step of the procedure of the invention is conducted may be advantageously selected from the group constituted by dimethylformamide, hexamethylphosphoric triamide, pyridine, or also a mixture of these solvents with or without dichloromethane, and the catalyst is selected from the group constituted by amines such as pyridine and dimethylaminopyridine.

The base intended to neutralize the acidity may be pyridine (which can serve simultaneously as solvent, catalyst and base), triethylamine or tributylamine.

in Advantageously, the dialysis carried out in step is performed in the presence of a weak base such as sodium bicarbonate in order to eliminate possible by-products such as anhydrides.

In an advantageous manner, the temperature and the time of the reaction may vary from 000C to 1000C, in particular from 000C to 500C, and advantageously the room temperature, for 1 to 24 hours for example, depending on the extent of acylation desired.

The selectively O-acylated compounds of the invention 000 0 possess a sustained activity in vivo. For example, when these 0° compounds are compounds of the heparin type possessing the binding o 20 site for AT III and are thus capable of exerting an antithrombotic activity, this activity is markedly prolonged in time compared with that of the same compound not having undergone a selective O-acylation.

The selectively O-acylated heparin derivatives according to the a 25 invention, which may or may not possess the AT III binding site, the o. latter derivatives being practically devoid of anticoagulant activity, also possess various biological activities, in particular: I the inhibition of the proliferation of smooth muscle cells which is of considerable interest for the prevention of stenoses in surgical operations such as angioplasty, by-passes, venous and arterial grafts, organ transplants, in particular heart transplants, the inhibition of heparanase and heparitinase, enzymes implicated in metastatic disseminations, anti-viral properties, in particular directed against retroviruses, in particular against the different HIVs (human immunodeficiency virus), which makes them of great value in the treatment of AIDS, of the_ recto ma vary fro 0°Cr to 10°,i priuarfo

II

inhibitory properties towards leucocyte elastase, increase of the levels of circulating elastase inhibitors as well as selective inhibition of the synthesis of type III collagen and fibronectin, which confers on them considerable value in the treatment of diseases in which a disequilibrium of the elastasz anti-elastase system is isplicated such as enphysema, as well as in the treatment of degenerative diseases of connectie tiss such as arteriosclerosis and diabetes.

The selectively 0-acylated glycosaminoglycans of the invention may thus constitute the active principle of medicines of considerable value in many indications.

Thus, another subject of the invention is pharmaceutical compositions characterized in that they contain as active substance an efficaceous quantity of at least one 0-acylated glycosaminoglycan according to the invention in combination with a pharmaceutically acceptable vehicle.

oTe 15 In an advantageous manner, the selectively 0-acylated Soglycosaminoglycan is in the form of a pharmaceutically acceptable salt, such as a sodium salt, magnesium salt or calcium salt.

au, an tageous embodiment of the invention, the pharmaceutical compositions are characterized in that the pharmaceutical vehicle is suitable for administration by the oral route, and in that they are available in the form of gastro-resistant capsules, tablets or lozenges, pills, or also in the form of solutions to be taken orally, advantageously containing from 50 mg to 5 g per unit dose, and preferably from 100 to 1000 mg for capsules, lozenges or pills and from 10 to 150 mg for solutions to be taken orally, for example once to three times per day; or also in that these cbmpositions are available in the form of injectable, sterile or Ssterilizable solutions for administration by the intravenous, intramuscular or subcutaneous routes, such solutions containing Sb advantageously from 50 to 200 mg/ml of selectively 0-acylated glycosaminoglycan when they are intended for injection by the subcutaneous route or from 20 to 200 mg/ml of selectively 0-acylated glycosaminoglycan when they are intended for injection by the intravenous route or by perfusion.

SLi._ 31 These dose intervals are only given for guidance, it being necessary in each case that the doses administrated be assessed by the clinician in the light of the condition of the patient and his personal reactivity towards the medicines.

The invention also relates to the selectively 0-acylated glycosaminoglycans of the invention as biological reagents which can be used as reference compounds or calibration standards in comparative studies for studies of structure/activity relationships in the different physiological systems in which the glycosaminoglycans may be implicated.

The invention will be better understood with the aid of the examples of its application which follow, and which are not meant in any way to be limiting.

1/ 4 4 t II in the patents EP 244 235 and 244 236, or alkaline depolymerization, for example as described in the patent EP 40 144.

i EXMPLE 1: Preparation of 0-acetylated heparin (CI 1938) starting from the tetrabutylammonium salt of heparin a) Preparation of the tetrabutylammonium salt of heparin: The sodium salt of heparin (10 g) dissolved in water (500 ml) is allowed to percolate through a column of a cation exchange resin (Dowex 50 W x 4, in the H form). The solution obtained is neutralized by tetrabutylammonium hydroxide. After lyophilization, the tetrabutylammonium salt of heparin is obtained (19.55 g), b) Acetylation: 1.05 g of tetrabutylammonium heparinate is dissolved in anhydrous Jimethylformamide (5 ml). After cooling to 0C, acetic anhydride (1 ml; 10.46 mmoles) is added dropwise followed by triethylamine (1.45 ml; 10.46 mmoles) and dimethylaminopyridine (64 mg; 0.5 mmole). The mixture is left to stand at room temperature for 24 hours. After addition of water (5 ml), the solution is dialysed for 72 hours against distilled water. The tetrabutylammonium salt is converted into the sodium salt by passage through Dowex 50 resin, S H at C, followed by neutralization by means of IN sodium hydroxide.

After lyophilization, 0.49 g of selectively O-acetylated sodium heparinate is obtained with the following properties: Sulfate/carboxyl ratio 2.28 meq/g (starting material 2.20 meq/g) APTT Titre 91 iu/mg 13C NMR spectrum (methanol 51.6 ppm, internal standard) signal at 23.4 ppm CH 3 of CH 3 -CO-0signal at 24.5 ppm (weak) CH 3 of CH 3

-CO-NH-

(identical with starting material) The NMR Spectrum shows the presence of about two acetyl groups per disaccharide unit.

S' EXAMPLE 2: Preparation of O-acetylated heparin (CI 1938) starting from the tributylammonium salt of heparin a) Preparation of the tributylammonium salt of heparin H at0°C folowe by eutaliztio bymean ofIN sdiu hydoxie.

Aftr lophliztio, 049 ofselctiely0-aetyate soiu lacking the binaing siLe iuL il the heparin chains have been fractionated so as to remove the oligoj :i tt

I

33 The sodium salt of heparin (10 g) is dissolved in water (500 ml), then allowed to percolate through a column of a cation exchange resin (Dowex 50 W x 4, in the H+ form). The solution is neutralized by the addition of a 10% solution of tributylamine in ethanol. After washing with ether and lyophilization, the tributylamnonium salt of heparin is obtained (14,77 g).

b) Acetylation: To a solution of the above salt (4 g) in anhydrous dimethylformamide (50 ml) cooled to 0°C are added dimethylaminopyridine(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 an ethanol solution saturated with sodium acetate.

After washing with ethanol, the precipitate is dissolved in water, then dialysed against 5% bicarbonate in water, then against water.

After lyophilization, 0-acetylated sodium heparinate is obtained (1.81 g).

-1 The infra-red spectrum shows a strong ester band at 1730 cm After saponification, the product shows: a sulfate/carboxyl ratio of 2.38 meq/g 20 (starting material: 2.40 meq/g) a APTT titre of: 85 iu/mg oe O 00 r 01 o oo 0C 0 00 0, 0 00 0 0~ 0' 'Ict O 01 0 II 01 0 1 O C( EXAMPLE 3: Preparation of 0-acetylated heparin (CI 1938) using catalysis by pyridine The tetrabutylammonium salt of heparin (0.58 g) is dissolved in a mixture v/v) of pyridine and dimethylformamide (10 ml).

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

i.

r i g 9

X

ii L. yl correspond to the following formula VI:

SI.:

34 EXAMPLE 4: Preparation of heparin 0-propionylated to various degrees (CI 1939) Propionic anhydride (2.7 ml), followed by triethyiamine (2.9 ml) and dimethylaminopyridine (128 mg) are added dropwise to a solution of tetrabutylammonium heparinate (1.85 g) obtained as described in example 1 in dimethylformamide (10 ml), cooled to 0 0

C.

A sample of the reaction mixture is taken at times 1 h, 2 h, 4 h, 8 h and 24 h, diluted with an equal volume of water and dialysed for 24 hours in against distilled water. After passage through Dowex 50 resin, H and neutralization by means of sodium hydroxide, the products obtained are lyophilized and have the following properties: Reaction Sulfate/Carboxyl AP1T time time Mass meq/g (iu/mg).

24 h 496 mg 2.31 8 h 138 mg 2.35 88 4 h 122 mg 2.33 94 2 h 120 mg 2.42 109 1 h 120 mg 2.33 120 (starting material) 2.40 150 The proton NMR spectrum of the products is recorded in water with 3,3,3-trimethylsilylpropionate (TSP) as internal standard.

It gives signals at ppm and 2,4 ppm characteristic respectively of the CH 3 and CH 2 residues of CH 3

-CH

2 -CO-0 and signals between -v3 and ppm characteristic of the protons of the carbohydrate skeleton.

30 The comparison of the intensity of the signals for propionyl and the skeleton between the product treated for 1 hour and that undergoing 24 hours of reaction illustrates the effect of the duration of the reaction: in fact, a decrease is observed of the proton signals of the skeleton, which indicates an increase in the extent of substitution.

CH2 radicals of the propionate esters.

51EXAMPLE 5: Preparation of 0-butyrylated heparin (CI 1940) A) Utilization of the tetrabutylammonium salt of heparin: 3 5

I

B utyric anhydride and dimethylaminopyridpm, ine are added to shows signals at w 10.8 and it.-30 ppm, characteristic of the CH3 and CHL radicals of the propionate esters.

EXAMPLE 5: Preparation of 0-butyrylated heparin (CI 1940) A) Utilization of the tetrabutylammonium salt of heparin: Butyric anhydride and dimethylaminopyridine are added to a solution of the tetrabutylammonium salt of heparin (0.55 g), obtained as described in example 1, in dimethylformamide (5 ml), cooled to OC. After 24 hours at room temperature, water (5 ml) is added, then the reaction mixture is dialysed for 72 hours against distilled water. After ion exchange on Dowex 50 H neutralization with sodium hydroxide and lyophilization, 0-butyrylated heparin is obtained in the form of its sodium salt (0.32 g).

Sulfate/carboxyl ratio 2.15 meq/g (Starting material 2.20 meq/g) S' APTT Titre 59 iu/mg The proton MNR spectrum (TSP, internal standard) shows the presence of signals at 0.9 ppm; 1.6 ppm and 2.4 ppm, characteristic of the CH 3

-CH

2 groups of CH 3

-CH

2

-CH

2 -CO-O- and signals between 3 and 6 ppm, characteristic of the carbohydrate skeleton.

Analysis of the NMR spectrum indicates the presence of about one butyryl chain per disaccharide unit.

B) Utilization of the tributylammonium salt of heparin: Dimethylaminopyridine (0.25 butyric anhydride (6.7 ml) and tributylamine (9.7 ml) are added to a solution of tributylammonium heparinate (4 g) obtained as described in example 2 in dimethylformamide (50 ml) cooled to 0°C. The reaction mixture is left to stand at room temperature for 24 hours. Water is added (1.5 ml) followed, after 30 minutes, by a saturated solution of sodium acetate in ethahol (250 ml). The precipitate is then washed three times with ethanol, then dialysed against a 5% solution of bicarbonate, then against water. After lyophilization, the sodium salt of 0-butyrylated heparin is obtained (2.1 g).

n is an integer varying from 1 to 36 APTT titre: 29 iu/mg After saponification of the esters by means of 0.5 M sodium hydroxide for 2 hours at 0OC, the product obtained ext 'bits a sulfate/ csrboxyl ratio of 2.36 meq/g'(2.40 meq/g in the starting material).

EXAMPLE 6: Preparati' of 0-hexanoylated heparin (CI 1941) A) Utilization of the tetrabutylammonium salt of heparin: Caproic anhydride (1 ml; 6 mmoles), triethylamine (0.84 ml, 6 mmoles) and dimethylaminopyridine are added to a solution of the tetrabutylammonium salt of heparin (0.55 g) obtained as described in example 1 in dimethylformamide (5 ml), at 0 0 C. After 24 hours at room temperature, water (5 ml) is added, then the reaction mixture is dialysed for 72 hours against a 5% bicarbonate solution, then against distilled water. After ion excha .ge on Dowex 50 H 15 neutralization with sodium hydroxide and lyophilization, til 0-hexanoylated heparin is obtained in the form of its sodium salt (0.30 g).

APTT Titre 25 iu/mg S The proton 13 C NMR spectrum (TSP, internal standard) shows 20 signals at 0,8; 1.2; 1.5; 2.3 ppm, characteristic of the CH 3

-CH

2 of CH 3

-(CH

2 8

-CO-O-

B) Utilization of the tributylammonium p'lt of heparin: Dimethylaminopyridine (0.25 tributylamine (9.7 ml) and hexanoic anhydride (10.6 ml) are added to a solution of tributylammonium heparinate (4 g) obtained as described in example 2 in dimethylformaide (50 ml), cooled to 0 0 C. After 24 hours at 20 0

C,

water is added (1.5 ml), followed by a saturated solution of sodium acetate in ethanol. After washing with ethanol, dialysis and lyophilization. 0-hexanoylated heparin is obtained (2.5 g).

30 EXAMPLE 7: Preparation of 0-octanoylated heparin (CI 1942) Dimethylaminopyridine (0.25 octanoic anhydride (12.1 ml) and tributylamine (9.7 ml) are added to a solution of tributT-'ammonium heparinate (4 g) obtained as described in example 2 in dituethylformamide (50 ml), cooled to O*C. After 24 hours at room temperature, water (1.5 ml) is added followed, after 30 minutes, wr- I iL i_ C i 37 by a saturated solution of sodium acetate in ethanol. After dialysis against a 20% solution of ethanol, then against distilled water and ultrafiltration, the product is subjected to a cation exchange by passage through Dowex 50 followed by neutralization with sodium hydroxide. After lyophilization, 0-octanoylated heparin is obtained g).

EXAMPLE 8: Preparation of 0-decanoylated heparin (CI 1943) A) Utilization of the tetrabutylammonium salt of heparin: Capric anhydride (1 ml; 6 mmoles), triethylamine (0.84 ml, 6 mmoles) and dimethylaminopyridine are added to a solution of the tetrabutylammonium salt of heparin (0.55 g) obtained as described in example 1 in dimethylformamide (5 ml), cooled to 00C. After 24 hours at room temperature, water (5 ml) is added, then the reaction mixture is dialysed for 72 hours against a 5% bicarbonate solution, °oS" then against distilled water. After ion exchange on Dowex 50 H+ neutralization by hydroxide and lyophilization, 0-decanoylated 1 oo heparin is obtained In the form of its sodium salt (0.30 g).

,o o The proton NMR spectrum (TSP, internal standard) shows signals at 0.8; 1.2; 1.5 and 2.4 ppm, characteristic of the CH 3 and

CH

2 of CH 3

-(CH

2 8 B) Utilization of the tributylammonium salt of heparin: Dimethylaminopyridine (0.25 decanoic anhydride (13.3 g dissolved in 20 ml of dimethylfcrmamide) and tributylamine (9.7 ml) are added to a solution of tributylammonium heparinate 4 g) obtained as described in example 2 in dimethylformamide (50 ml), cooled to 0 C. After 24 hours at room temperature, water (1.5 ml) is added, followed by a saturated solution of sodium acetate in s ethanol. The precipitate is dissolved in dimethylsulfoxide, then 30 dialysed against water, sodium bicarbonate and again against water, r, After lyophilization, O-decanoylated heparin is obtained (2.38 g).

L f JD preparation ot the seiectiveiy u)-acyia~ea giy MIIIUK.LYUUII-'!s t( 38 EXAMPLE 9 PREPARAT1ON OF O-OLEOYLATED HEPARIN (CI 2013) The tributylamonium salt of' heparin (3 g) and N,N-dimethylaninopyridine (244 mg) are dissolved in anhydrous dimethylformamide (50 nil) After cooling to 00 C, oleic anhydride (21 synthesized according to Plusquellec et al., letrahodron, 1988, 4-4, 2471-2476, and dissolved in dichloromethane (20 mil) is added dropwise, followed by tributylainine (9.5 mil). After a reaction time of 24 hours and cooling to 00 C, 5 sodium bicarbonate (10 nil) is introduced, followed one hour later by a saturated alcoholic solution of sodium acetate. After washing with Lbsolute ethanol and dissolution in a dimethyl sulfoxide-water (4/1; v/v, 750 ml), the solution obtained is dialysed against 10 aqueous ethanol for 2 days, then against water for 3 days. The sodium salt of the oleoylated heparin is isolated after lyophilization and precipitation with ether of the lyophilizate redissolved in DMF (2.77 Oleic acid content :1.44 pmole/mg (determination according to Duconabe W. et al., Biochemical Journal, 1963, 88, 7).

EXAMPLE 10: Preparation of O-benzoylated heparin (CI 1946" The tetrabutylammonium salt of heperin, obtiined as 4 described in example 2, is benzoylated by benzoic anhydride under the conditions described .previously for the preparation of O-decancy2.ated heparin (Example 8).

13 The C NMR spectrum of the product obtained. shows signals at 131, 132 and 136 ppm, characteristic of benzoyl groups.

The product lacks anticoagulant activity in vitro.

EXAMPLE 11 :PREPARATION OF 0-3-CYCLOPENTYLPROPIONYLATED HEPARIN (CI 2014) 3-Cyclopentylpropionic anhydride is prepared accor"ding to the method of Plusquellec et Tetrahedron, 1988, 44, 2471-2476. The latter is obtained after addition of a solution of the acid (23 ml, 150' mmoles) in dichloromethane (400 mil) to a mixture containing 30 tetrabutylammonium bromide (15 mmoles), 20 sodium hydroxide (60 ml) and dichloromethane (80 ml), stirred and cooled to -100 C, followed by decantation, washings with 5 sodium bicau'bonat( I water, and cu-nzatration. to an oil (96%) Characteristic frequencies in the 1800, 1745, 1040 cm 1 ft. *44 4 4 54 4 I £4 4 44

NHCOCH,

39 The tribuylammonium salt of heparin (4 g) and N,N-dimethylaminopyridine (253 mg) are dissolved in anhydrous dimethylformamide ml). After cooling to 00 C, 3-cyclopentylpropionic anhydride (11 g) is added dropwise, followed by tributylamine (9.8 ml). After a reaction time of 24 hours at room temperature, followed by cooling to 00 C, water (1 ml) is added, followed one hour later by a saturated alcoholic solution of sodium acetate. After washing with absolute ethanol, the precipitate is dialysed against 5 sodium bicarbonate for 24 hours, then against water for 3 days. After lyophilization, 0-3-cyclopentylpropionylated heparin is obtained in the form of its sodium salt (2.64 g).

13 The C NMR spectrum in D20 (methanol 51.6 ppm, internal standard) shows characteristic signals at 27.4 ppm; 33.1 ppm; 34.6 ppm; 35.9 ppm and 41.7 ppm for the O-cyclopentylpropionyl group.

SSulfate/carboxyl ratio 2.2.

SI i 15 EXAPLE 12: Preparation of an 0-acetylated heparin of low molecular mass (mean MW e-4,500 daltons, MW interval -1,800-8,000 daltons (CI 1945) l| This heparin of low molecular mass was obtained by partial nitrous acid depolymerization and alcoholic fractionation as described I' in the patent EP 181 252 and is designated hereafter as CY 216.

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

The salt thus obtained (1.7 g) is dried in a vacuum for 1 three hours at 50'C, then dissolved in anhydrous dim.thylformamide ml). After cooling to OC, acetic anhydride (1.7 ml) is added dropwise, followed by triethylamine (2.4 ml) and dimethylamino- Spyridine (102 mg). After 20 hours of reaction, the product is t 30 chromatographed on a column of Sephadex G-25 e+ ed by water. After conversion to the sodium salt and lyophiliza-i, 0-acetylated CY 216 is obtained (0.89 g).

Its 13C NMR spectrum (methanol 51.6 ppm, internal standard) shows a signal at 23 ppm characteristic of acetates.

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

The sulfate/carboxyl ratio is 2.09 meq/g (Starting material 2.05 meq/g) APTT titre 18 iu/mg Anti-Xa titre 205 u/mg (Determination of Yin et al., J. Lab. Clin. Med., 1973, 81, 298-310) B) Utilization of the tributylammonium salt of CY 216 The sodium aalt of CY 216 is converted into the tributylammonium salt by passage through a column of Dowex 50 H resin, followed by neutralization by means of tributylamine, as eescribed for heparin.

The tributylammonium salt of CY 216 is obtained after washing with ether, lyophilization and drying in a vacuum oven.

The above salt (4 g) and N,N-dimethylaminopyridine (288 mg) are 1 5 dissolved in dimethylformamide. After cooling to 00 C, acetic anhydride (4.4 ml) is added dropwise, followed by tributylamine oo (11.2 ml). After 24 hours at room temperature and cooling to 00 C, water (1.7 ml) is addedfollowed one hour later by a saturated alcoholic solution of sodium acetate. The precipitate is washed with S ethanol, dissolved in apyrogenic water, dialysed for 36 hours against bicarbonate, then against water for 3 days. After lyophilization, the acetylated CY 216 is obtained in the form of its sodium salt (1.4 g).

13 SThe 1C NMR spectrum in D 2 0 (methanol 51.6 ppm as internal 1 2 standard) shows the signal characteristic for the acetyl group at 23 ppm.

Sulface/carboxyl ratio 2.07.

EXAMPLE 13 PREPARATION OF AN O-BUTYRYLATED HEPARIN OF LOW MOLECULAR MASS (MEAN MW- 4,500 DALTONS, MW INTERVAL 800-8,000 DALTONS) (CI 1957).

The tributylammonium salt of CY 216 (4 g) obtained as described in Example 12 and N,N-dimethylaminopyridine (288 mg) are dissolved in anhydrous dimethylformamide (40 ml); after cooling to 00 C butyric anhydride (7.68 ml) is added dropwise, followed by tributylamine (11.2 ml).

After a reaction time of 24 hours at room temperature, then cooling to 00 C, water (1.7 ml) is added, followed one hour later by a S a l i this salt is treated with an anhydride of formula: ~L 1,44 Acyl-O-Acyl[ S in which Acyl is such as defined in-formula in the said polar aprotic organic solvent in the presence of cataslytic amounts of 41K satuate alcholc slutin o sodum cette. fte wasingwit mabtrated lch2li isolt d of tefrmo t sodium actt.sfe an witer lyophilization (2.14 z).

C3 NMR spectrum in D 0 (methanol 51.6 ppm, internal standard)I shows the signals characteristic of the 0-butyryl group at 15.6 ppm; 20.5 ppm and 39.4 ppm.

Sulfate/carboxyl ratio :2.08.

EXAMPLE 14 :PREPARATION OF AN 0-1-*LXANOYLATED HEPARIN OF LOW MOEUA MASS (MEAN F3 4, 500 DALTONS, MW INEVL-,0-,0 DALTONS) (Cl 1958) The tributylammonium, salt of CY 216 (4 g) obtained as described in Example 12 and N, N-d inethy 1aminopyri dineC (288 mg) are dissolved in anhydrous dime thy lformami de (40 ml). After cooling to 00 C, caproic anhydride (10.8 ml) is added dropwise, followed by tributylamine (11.2 ml).

After a reaction time of 24 hours at roora temperature, then cooling to 00 C, water (1.7 ml) is added, followed one hour later by a saturated alcoholic eolution of sodium acetate. After washing with absolute ethanol, dissolution in apyrogenic water, dialysis against 5 sodium bicarbonate for 36 hours, then against water for 3 days, the 0-caproylated CY 216 is isolated in the form of its sodium salt after lyophilization (2.5 g).

13 The C lIMR spectrum in D 0 (methanol 51.6 ppm, internal standard) shows the characteristic signals of the caproyl group at 15.9 ppm; 24.2 ppm; 26.4 ppm; 33.1 ppm and 36.4 ppm.

Sulfate /o arboxyl ratio :2.08.

EXAMPLE 15 :PREPARATION OF AN '0-OCTANOYLATED HEPARIN OF LOW MOLECULAR M4ASS (MEAN MW.-4,500 -DALTONS, MW INTERVAL-% 1 ,800-8,0 DALTONS) (CI 19!*)C tributylammonium salt of CY 216 (4 g) obtained as described in Example 12 and N,N-dimethylaminopyridine (288 mg) are dissolved in anhydrous dimethylformamide (40 ml). After cooling to 00 C, caprylic anhydride (14 ml) is added dropwise, followed by tributylamine (11.2 ml).

t *C ae 17~1 sadd olwdoehu ae yAfter a reaction time of 24 hours at room temperature, then coolingY an alkanolc acid containing from 2 to 10 carbon atoms, advantageously from 4 to 10 carbon atoms and preferably 4 or 6 carbon atoms.

IIL

42 saturated alcoholic solution of sodium acetate. After washing with absolute ethanol, dissolution in apyrogenic water, dialysis against sodium bicarbonate for 36 hours, then against water for 3 days, O-octanoylated CY 216 is obtained in the form of its sodium salt after lyophilization (1.76 g).

The 1 3 C NMR spectrum in d 6 DMSO (methanol 51.6 ppm, internal standard) shows signals characteristic of the capryl group at 16.8 ppm; 25.0 ppm; 27.3 ppm; 30.9 ppm; 31.4 ppm; 34.1 ppm and 36.4 ppm.

Sulfate/carboxyl ratio 2.07 io EXAMPLE 16 PREPARATION OF AN O-DECANOYLATED HEPARIN OF LOW MOLECULAR MASS (MEAN MW -4,500 DALTONS, MW INTERVAL '',800-8,000 DALTONS) (CI 1960) The tributylammonium salt of CY 216 (4 g) obtained as described in Example 12 and N,N-dimethylaniinopyridine (288 mg) are dissolved in 15 dimethylformamide (40 ml). After cooling to 00 C, a solution of decanoic anhydride (15.3 ml) in anhydrous dimethylformamide (10 ml) is added dropwise, followed by tributylamine (11.2 ml). After a o reaction time of 24 hours at room temperature, then cooling to 00 C, water (1.7 ml) is added, followed one hour later by a saturated 0 0 20 alcoholic solution of sodium acetate. After washing with absolute ethanol and resuspension in apyrogenic water, the mixture is dialysed against 5 sodium bicarbonate for 2 days, against 10 sodium chloride for 2 days, then against water for 5 days. The 0-decanoylated CY 216 o is isolated in the form of its sodium salt after lyophilization (2.76 g).

l 13 5 The C NMR spectrum in D 2 0 (methanol 51.6 ppm, internal standard) shows signals characteristic for the decanoyl group at 16.4 ppm; 22.3 ppm; 25.1 ppm; 29.2 ppm; 31.9 ppm; 34.4 ppm and 0000. 36.5 ppm.

o0 30 Sulfate/carboxyl ratio 2.13.

i I tuii-vi1ra. y)LuiJjcJL ycu-bjcujgs- J.ISXLLIcUu ayUJi.iL rLUi .1 uo in particular against the different HIVs (human immunodeficiency virus), which makes them of great value in the treatment of AIDS, Si.

43 EXAMPLE 17: Preparation of an 0-acetylated heparin of low molecular mass (mean MW ,-2,500 daltons, MW interval ^1,500-8,000 daltons) (CI 1946) This heparin of low molecular mass was obtained by partial nitrous acid depolymerization according to the procedure described in the patent EP 37 319 and is designated hereafter as CY 222.

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

The salt obtained after lyophilization (1.5 g) is dissolved in dimethylformamide (5 ml) then, after the solution has been cooled to 0 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 is added (20 ml), then the mixture is o 15 dialysed for 3 days against distilled water. After conversion into o the sodium salt and lyophilization, 0-acetylated CY 222 is obtained (0.86 g).

o 0 The product has a sulfate/carboxyl ratio of 1.98 meq/g uo° (starting material: 1.97 meq/g).

13 o 20 The C NMR spectrum (methanol 51.6 ppm, internal standard) of the product contains a signal at 23 ppm characteristic of O-ace'tl.

Comparison of the starting material and the final product with respect to the intensities of the signals of the N-acetyl (at 24.5 ppm) demonstrates that acetylation was selective.

o 25 APTT titre 8 iu/mg Anti-Xa titre 191 u/mg (Determination of Yin et al., Lab. Clin. Med., 1973, 81, 298-310) 30 EXAMPLE 18: A/ Preparation of a heparin fragment lacking affinity o o for antithrombin III (CI 1772) 1/ Cleavage of the heparin chains by means of periodic acid: g of injectable heparin of porcine mucus, in the form of its sodium salt, titrating at 157 iu/mg in the Codex determination and 155 u/mg in the anti-Xa factor determination of Yin et al. are glycosaminoglycan when they are intended for injection by the intravenous route or by perfusion.

r

I

-j dissolved in 250 ml of demineralized water at 4 0

C.

The pH of the solution is adjusted to 5.0 by means of concentrated hydrochloric acid. 10 g of sodium metaperiodate (NaIO 4 MW: 213.89) dissolved in 250 ml of demineralized water at 4°C are added with moderate stirring. The pH of the whole mixture is adjusted to by means of concentrated hydrochloric acid. The solution is left to stand for 24 hours in the dark in the cold room at 4°C.

2/ Removal of residual periodate: The reaction solution is then distributed between three dialysis tubes NOJAX 4 0 R (porosity 3 to 4,000 Da) and it is subjected to dialysis for 15 hours against running demineralized water.

3/ Depolymerization in basic medium: 16 ml of 10 N sodium hydroxide are added to 780 ml of the solution obtained after dialysis and the solution thus obtained S 15 is stirred for 3 hours at room temperature (of the order of 18-21oC).

Reduction: 500 mg of sodium borohydride (NaBH MW: 37.83) are then added and the solution is stirred for a further 4 hours at room temperature. Its pH is then brought to 4 with the aid of concentrated S 20 hydrochloric acid. After 15 minutes stirring, the pH is adjusted to 7 by means of concentrated sodium hydroxide.

To 820 ml of solution thus obtained are added 16.4 g of NaC1 followed by 1270 ml of ethanol. The mixture is left to stand for 3 hours, then centrifuged at 2,500 rev/minute for 20 minutes.

The precipitate is recovered, resuspended in 200 ml of pure ethanol, ground with an Ultra-Turrax R and finally recovered by filtration on a fritted Buchner funnel. It is then dried under vacuum at 40 0

C

for 5 hours.

8.9 of product are thus recovered.

5/ Alcoholic fractionation SThese 8.9 g are dissolved in about 120 ml of demineralized water at room temperature. 1.78 g of NaC1 are added and the pH of the solution is lowered to 3.5 with the aid of hydrochloric acid. The volume of the solution is adjusted to 178 ml by means of demineralized water. 151 ml of pure ethanol are added with stirring. The stirring is maintained for 15 minutes after the *4

Z

t/ wn-- end of the addition, then the mixture is left to stand for 10 hours at room temperature.

The precipitate formed is recovered by means of centrifugation for 20 minutes at 2,500 rev/minute. It is resuspended

R

in 150 ml of pure ethanol, ground with an Ultoa-Turrax recovered by filtration on a fritted Buchner funnel, washed with 300 ml of pure ethanol and finally dried under vacuum at 40 0 C for 24 hours.

grams of product CI 1772 are thus recovered in the form of a white powder having the following properties:

SO

3 3.55 meq/g COO- 1.54 meq/g S C 5.09 meq/g S/C 2.31 meq/g It is practically devoid of N-acetyl glucosamine (absence of signal at '-24.5 ppm in the 13C spectru4 Codex titre 11 iu/mg APTT titre 9 iu/mg Anti-Xa titre 12 u/mg

I

Si 20 C L B/ Preparation of an 0-acetylated heparin fragment devoid of affinity for antithrombin III (CI 1924) The product obtained in step A/ is converted into the tetrabutylammonium salt by passage through a Dowex 50 H+ resin, followed by neutralization with the aid of tetrabutylammonium hydroxide.

18 g of the tetrabutylammonium salt are obtained starting from 9.5 g of the sodium salt.

Acetic anhydride (6.2 ml; 65.6 mmoles), followed by triethylamine (9 ml; 65.5 mmoles) and dimethylaminopyridine (403 mg; 3.3 mmoles) are added to a solution of 6 g of the salt thus obtained in dimethylformamide (55 ml), cooled to 0 0 C. After 24 hours, a saturated solution of sodium acetate in ethanol is added (250 ml).

After centrifugation and washing of the precipitate with ethanol, the solid is desalted on Sephadex G-25, then subjected to ion exchange by passage through Dowex 50 H followed by neutralization by means of sodium hydroxide. After lyophilization, the product CI 1924 is obtained (3 g).

a) rreparation or tne urlDutyIamIroiilumli sUat uL nkICILL..

i 46 This product has the following properties: sulfate/carboxyl ratio: 2.28 meq/g.

13 The C NMR spectrum (methanol 51.6 ppm, internal standard) shows clearly that the product was 0-acylated. The signal characteristic of C 2 of the N-acetyl-glucosamine at about 56 ppm is absent from the spectrum as it is from that of the starting material.

EXAMPLE 19: Preparation of an 0-butyrylated heparin fragment devoid of affinity for antithrombin III (CI 1925) 6 g of the tetrabutylammonium salt of CI 1772, obtained as described in example 12, are butyrylated by butyric anhydride under the conditions used for acetylation. The product CI 1925 (2.96 g) is obtained which has the following properties: ,'15 sulfate/carboxy. ratio 2.31 meq/g.

The C-NMR spectrum (methanol 51.6 ppm, internal standard) shows the signals characteristic of the butyl group at 15.6, 20.4 S and 38.4 ppm.

EXAMPLE 20 Preparation of an O-hexanoylated heparin fragment devoid I of affinity for antithrombin III (CI 1926) 6 g of the tetrabutylammonium salt of CI 1772, obtained as described in example 12, are treated with hexanoic anhydride in the same manner as for acetylation. The product CI 1926 (3 g) is obtained which has the following properties: sulfate/carboxyl ratio: 2.20 meq/g SThe 13 C NMR spectrum (methanol 51.6 ppm, internal standard) 0, shows the signals characteristic of CH 3 and CH2 of the hexyl group 3 at 15.5, 23.9, 26.2, 32.7 and 36.1 ppm.

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

ji (0.22 g).

4j- EXAMPLE 21 PREPARATION OF MIXTURE OF FRAGMENTS DEVOID OF AFFINITY FOR ANTITHROMBIN III, HOMOGENEOUS WITH RESPECT TO THEIR MOLECULAR MASS AND 0-BUTYRYLATED The mixture of fragments lacking anticoagulant activity described in Example 12 is fractionated into its different constituents by gel filtration. Fractions homogeneous with respect to their molecular mass are thus obtained, the masses of which are 7700, 6500, 5800, 5300, 4980, 4400, 3900, 3400, 2600, 1860 and 1210.

Example of esterification of a fraction The fraction of mass 2600 (0.20 g) is converted into its tributylammonium salt, then lyophilized and dried (0.34 This product is then dissolved in DMF (2 ml), the solution is cooled to 00 C and then dimethylaminopyridine (18 mg), butyric anhydride (0.49 ml) and tributylamine (0.70 ml) are added successively. After 24 hours at S room temperature, sodium bicarbonate (1 ml of a 5 solution) is added then, hour's later, the mixture is chromatographed on a column of Sephadex G25 (1 litre) eluted by 0.2 M sodium chloride. The product is lyophilized after desalting to give a light beige powder (0.24 g).

The other fractions are treated in the same way, giving the corresponding products, IR analysis of which revealed the presence of -1 an ester band at 1734 cm The extent of sulfation of the products (sulfate to carboxyl ratio) is unchanged after acylation.

t Determination of the extent of acylation This is determined by means of gas chromatography after butanolysis of the products using a butane-sulfuric acid mixture, followed by extraction of the butyl esters with chloroform and removal of excess butanol by washing with water.

EXAMPLE 22: Preparation of 0-acetylated dermatan sulfate (CI 1947) To a solution of the tetrabutylammonium salt of dermatan S sulfate (0.91 obtained under the same conditions as those of the skeleton, which indicates an increase in the extent of substitution.

i 48 described for the preparation of tetrabutylammonium heparinate in example 1, in dimethylformamide (20 ml) and cooled to 0 0 C are added dropwise acetic acid (1.35 ml; 14.2 mmoles) followed by triethylamine (1.97 ml; 14.2 mmoles) and dimethylaminopyridine (0.7 mmole). After 24 hours at room temperature, water (40 ml) is added, and dialysis is performed for 72 hours. The aodium salt is obtained by passage through a column of Dowex 50 H at 0°C, followed by neutralization with sodium hydroxide. After lyophilization, a beige powder is obtained (0.52 g).

The 0-acetylated dermatan sulfate has the following properties: sulfate/carboxyl ratio 0.99 meq/g (starting material 1.05 meq/g) 13 C-NMR spectrum (methanol 51.6 ppm, internal standard) .signal at 25.2 ppm CH 3 of CH 3 -CO-NH- (identical with the starting material) signal at 23.0 ppm CH 3 of CH 3

-CO-O

EXAMPLE 23 PREPARATION OF 0-SUCCINYLATED DERMATAN SULFATE (CI 2020) The tetrabutylammonium salt of dermatan sulfate (1 g) and N,N-dimethylaminopyridine (110 mg) are dissolved in anhydrous dimethylformamide. After addition of succinic anhydride (396 mg) and tributylamine (0.94 ml), the mixture is incubated under anhydrous conditions at 600 C for 2 hours. After cooling and the addition of water (2 ml), precipitation is carried out with a cold saturated 2 alcoholic solution of sodium acetate. The precipitate is washed with ethanol, dissolved in apyrogenic water, then dialysed against 5 sodium bicarbonate for 36 hours and then against water for 3 days.

After lyophilization, the 0-suc.inylated dermatan sulfate is obtained in the form of its sodium salt (0.83 mg).

30 The infra-red spectrum (KBr) shows the characteristic frequencies (wave numbers) of the carboxylic acid group at 1730 cm and 1420 cm S* 13 The C NMR spectrum in D 2 0 (methanol 51.6 ppm, internal standard) shows the signals characteristic of the succinyl group at 33, 34.5 and 183.6 ppm, and the signal characteristic of the methyl of the i" S acetamido group at 25.3 ppm, identical with that of the starting material.

Sulfate/carboxyl ratio 0.51.

i i I i: i ri| bicarbonate, then against water. After lyophilization, the sodium salt of 0-butyrylated heparin is obtained (2.1 g).

Vol. VIII, pp. 291-294).

The tributylammonium salt (1 g) is obtained by passage through a Dowex 50 H resin, followed by neutralization by means S49 of a solution of tributylamine in ethanol. n after prior After lyophilization and drying, this salt is dissolved in dimethyformamide (10 mN-desulfated) then, after being cooled to goC, butyric anhydride (2 tributylamine (2 mgasawa and Inoue (dimethylaminopyridine Vol. VIII, pp. 291-294).

mg) are added. After 24 hours, water (5 m) is added, then the mixture is dialysed against a 5% solution of sodium bicarbonate, then against water. After passage through Dowex 50 H utfollowed by acetylated -butyrylated heparin is obtained.

The 1C KR spectrum (methanol 51.6 ppm, internal standard) shows a signal at 24.6 ppm characteristic of N-acety and signals at 16, 20 and 38 ppm characteristic of butyryl esters.

S The experiment may be repeate d with partially N-desulfated N-acetylated heparibyan and le ads to a partially N-desulfated N-acetylated and 0-butyrylated product.

EXAMPLE 25 Preparation of peracetylated dermatan sulfate (CI 1950) The tetrabutylammonium salt of dermatan sulfate (0.8 g) 25 dissolved in dimethylformamide (20 mi) is acetylated by addition of dimethylaminopyridine (76 mg), acetic anhydride (1.2 ml) and triethylamine (1.7 ml). The mixture is hea'ted at 800 for 1 hour.

After being allowed to cool to room temperature, water mixtur(0.45 ml) is added, followed by a 0.3 5% solution of sodium acetate 30 in ethanol (100 ml). After centrifugation, the precipitate is dissolved in water, then dialysed against distilled water. The sodium salt is obtained by ion exchange on a column of Dowex 50 H, followed by neutralization by means of sodium hydroxide. After syophilization, peracetylated -udermatan sulfate is obtained (0.51 g shows a signal at 24.6 ppm characteristic of N-acetyl and signals I tThe experiment may be repeated with partially N-desulfated N-acetylated heparin and leads to a partially N-desulfated N-acetylated and 0-butyrylated product.

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

After being allowed to cool to room temperature, water (0.45 ml) is added, followed by a 0.3 M solution of sodium acetate S 2 isin ethanol (I00 ml). After centrifugation, the precipitate iso Sdissolved in water then dialysed against distilled water. The sodium salt is obtained by ion exchange on a column of Dowex 50 H followed by neutralization by means of sodium hydroxide. After lyophilization, peracetylated dermatan sulfate is obtained (0.51 g).

i.i 2 in dimethylformamide (50 ml), cooled to 0 0 G. After 24 hours at room temperature, water (1.5 ml) is added followed, after 30 minutes, This product has a sulfate/carbo):yl ratio 1.07 meq/g (starting mat~'ril: 1.05 meq/g) and contains about three acetyl groups per disaccha~ride unit.

EXAMJPLE 26,t Preparation of 0-acetylated heparin benzyl ester (CI 1949).

Be.nzyl bromide 17 m3J is added to a solution of tetra bu tylammon ium heparinate (1 g) Ain dimethylformamide (10 ml).

After 24 hours at room temperature, tetrabutylamnoniun acetate (220 mg) is added. Af cer 24 hours, acetylatiom of the benzyl ester formed is carried out. For that purpose, dimethylaminopyridine (57 rtg) is added, followed by triethylamine (1.3 ml) and acetic anhydride(0.9 ml).

After being allowed to cool to room emperature, the recinmxuei tirdfr2 or.Wte ste dete the product is precipitated by a saturated solution of sodium acetate 4 41 in ethanol. After dialysis against distilled water, passage through 6~t~ Dowex 50 neutrilization by sodium hydroxide and lyophilization, the sodium salt of 0-acetylated heparin benz, I ester is obtained (0,57 g).

The product has a sulfate/carboxyl ratio of 3.6 meq/g (nonbenzylatedi starting material: 2.20 meq/g).

The CNI4R spectrum (methanol 51.6 ppm, internal standard) shows~ signals at 23.3 ppm (0-acetyl) and at 131.6 ppm (benzyl).

The signal for the CH 3 of 01H 3 -CO-NH at m,./24.5 ppm is identical with that of the starting material.

EXAMPLE 27: Preparation o2f 0 -acetylated dermatan suli~i:e benzyl ester (CI 1953) 'Me~ tetrabu.yammonium salt of dermatan sulfate (1 g) is dissolved in anhydrous limethylformamide (15 ml). IBenzyl bromide (0.25 ml) is added to this solution cooled to 0 0 C, :--hen the mixture is left at room temperature for 24 hours.

Tetfcqbutylamnonium acetate (0.32 g) is then added then, after 24 hours at room temperature, the acetylation is carried out.

Acetic anhydride (1.5 ml) is added, followzi by 51 triethylamine (2.2 ml) and dimethylaminopyridine (96 mg). After 24 hours, water (0.6 ml) is added, then the product is precipitated by addition of a saturated ethanolic solution of sodium acetate.

The product is dialysed against 10% sodium chloride, then against water.

After lyophilization, the benzyl ester of 0-acetylated dermatan sulfate is obtained (0.63 g).

EXAMPLE 28 PREPARATION OF O-BUTYRYLATED DERMATAN SULFATE (CI 2018) The tributylammonium salt of dermatan sulfate (2 prepared under the same conditions as those described for the preparation of tributylamnium heparin in Exameple 2, and N,N-dimethylaminopyridine (220 mg) are dissolved in anhydrous dimethylformamide (25 ml).

After coollng to 00 C, butyric anhydride (5.9 ml) is added dropwise, followed by tributylamine (8.6 ml). After incubation for 24 hours and cooling to 00 C water (1 ml) is added; precipitation is brought about water for 3 days. After lyophilization, the sodium salt of 0-butyrylated dermataM sulfate is obtained (1.3 g).

13 The C NMR spectrum in D 2 0 (methanol 51.6 ppm, internal stadard) shows the 3ignals characteristic of the butyryl group at 15.6, 20.5 and 38.4 ppm, and the signal characteristic of the methyl of the acetamido group at 25.2 ppm, identi t with that of the starting material.

Sulfate/carboxyl ratio 1.05.

EXAMPLE 29 PREPARATION OF 0-HEXANOYL DRMATAN SULFATE (CI 2019) The tributylammonium salt of dermatan sulfate (2 g) and N,N-dinethylaminopyridine (220 mg) are dissolved in anhydrous dimethylformamide (25 ml). After cooling to 00 C, hexanoic anhydride (9.4 ml) is added dropwise, followed by tributylamine (8.6 ml).

After 24 hours at room temperature and cooling to Oo C water (1 ml) is added and precipitation is brought about with a cold saturated alcoholic olution of sodium acetate. The precipitate is washed with absolute ethanol, dissolved in apyrogenic water and dialysed agans 5 oimbcroaefr3 orte gis concentration. to an oil (96 1 Characteristic frequencies in the 1800, 1745, 1040 cm 52 for 3 days. After lyophilization, the sodium ialt of 0-hexanoylated dermatan sulfate is obtained (1 g).

The 13C NMR spectrum in D 2 0 (methanol 51.6 ppm, internal standard) shows the signals characteristic of the 0-hexanoylated group at 15.9, 24.2, 26.5, 33.1 and 36.4 ppm and the signal characteristic of the methyl of the acetamido group at 25.2 ppm, identical with that of the starling material.

Sulfate/carboxyl ratio 1.02.

EXAMPLE 30 Demonstration of selective 0-acylation by the procedure according to the invention and comparison with other acylation procedures The procedure of the present invention was compared with the procedures described in Patents FR 2 100 735 and EP 256 880.

In particular, the properties of the acetic ester of heparin prepared according to the procedure of the invention (CI 1938, example 1) were compared with those of the acetic ester of heparin obtained by applying the operating conditions described in Patent FR 2 100 735 (product A) and with the acetic esters of heparin obtained according to the procedures of examples 3 and 4 of Patent EP 256 880 (products B and C).

Preparation of the product A: To a solution of tetrabutylammonium heparinate (1 g) dissolved in anhydrous dimethylformamide (10 ml), are addsd dicyclohexylcarbodiimide (4.2 g) dissolved in dimethylformamide (15 ml), then acetic acid (1.16 ml) dissolved in dimethylformamide (25 ml) is added dropwise during 45 minutes at +4 0

C.

After 24 hours at room temperature, the reaction mixture is filtered and concentrated in a vacuum. The residue is resuspended in ether. After filtration and washing, the precipitate is dia>lyed 30 against distilled water. The sodium salt is obtained by passa6, through a column of Dowex H+ resin, followed by neutralization by means of sodium hydrixide. 0.493 g of product A is obtained.

This preparation has also been carried out for 48 hours at L Its 1C NMR spectrum (methanol 51.6 ppm, internal standard) shows a signal at 23 ppm characteristic of acetates.

j i; i

I

i i:;i nn i Preparation of the products B and C: The products B and C were prepared by acetylation of heparin in a mixture of formamide and pyridine by acetyl chloride, by using 2 ml of acetyl chloride for product B and 40 ml for product C under the conditions described in the examples 3 and 4 of the patent EP 256 880. The acetic ester is then taken up in water and dialysed against sodium chloride.

Results: The properties of the products are given in Table 1. The properties of the starting heparin used in each experiment are given for reference.

'S

C (t C CC (4r~ 4 1e 1, 4 1 _i i After a reaction time of 24 hours at room temperature, then cooling to 0° C, water (1.7 ml) is added, followed one hour later by a *In i b ii t Table 1: t Product Sulfate/carboxyl APTT titer YW titer ratio iu/mg u/mg (meq/g) CI 1938 2.28 91 Starting Hep. 2.20 160 160 A 3.40 42 4 Starting Hep. 2,40 160 160 B 2.10 57 39 C 1.15 2 0.6 Starting Hep. 2.40 160 160 4I 4 t 04 *APTT and Yin and Wessler titres are measured in vitro.

The results show that, although the APTT and YW titres are lower for all of the products than those of the starting heparin, 20 and more markedly so for the products A, B and C, only the product CI 1938 conserves a sulfate/carboxyl ratio almost identical with that of the starting heparin. This results from the fact that the procedure of the invention makes it possible to acylate the hydroxyl functions selectively without modifying the functional groups of heparin, and this confirmed by chemical analysis of the products.

The products CI 1938, A, B and C were analysed by means of 13C N4R (methanol 51.6 ppm, internal standard). The spectra of these products as well of that of the starting heparin are given as follows: ~44L 30 4 44 Figure Figure Figure Figure Figure Figure Starting hbparin CI 1938 Product A after 24 hours of reaction Product A after 48 hours of reaction Product B Product C 1 44 anhydrous dimethylformamide (40 ml). After cooling to 0° C, caprylic anhydride (14 ml) is added dropwise, followed by tributylamine (11.2 ml).

After a reaction time of 24 hours at room temperature, then cooling to 00 C, water (1.7 mi) is added, followed one hour later by a The results are as follows: 1. The product CI 1938 shows in its 13 C NMR spectrum (Fig. 2): a signal at 23.4 ppm corresponding to the CH 3 of CH 3

-CO-O

a signal at 24.4 ppm corresponding to the CH 3 of CH 3

-CO-NH,

identical to the signal present in the starting heparin.

These signals show that the amine and carboxyl groups have been left untouched and that there has indeed been selective acetylation of the hydroxyl groups.

2. The analysis of the product A by means of 1 C NMR spectroscopy shows that the major product obtained after both 24 and 48 hours (Fig. 3 and is an isourea derivative of heparin, the carboxyl functions of which are substituted by a group: NH- N I as a result of dicyclohexylcarbodiimide being used. In fact, strong So signals corresponding to the carbon atoms of the above group at 28, o o SO 34, 54 and 156 ppm are observed, whereas the signal corresponding to the CH 3 of CH 3 -CO-O at 23 ppm is very weak.

0 20 The procedure thus makes it possible to obtain a selective 0-acylation and leads to a modification of the carboxyl functions which is reflected in an increase of the sulfate/carboxyl ratio.

3. The product B shows in its 13C NMR spectrum (Fig. 3): a signal at 23.1 ppm corresponding to the CH 3 of CH 3 -CO-0 25 a signal at 24.8 ppm corresponding to the CH 3 of CH 3

-CO-NH,

markedly more intense than those of the starting heparin and of CI 1938.

o °These signals show that not only an O-acetylation but also considerable N-acetylation is observed. The acylation procedure'used is non-sel' ive and leads to partial N-desulfatation, followed by 9 0 acetylation of the amines, which also leads to a diminution of the o ou sulfate/carboxyl ratio.

13, 4. The product C shows in its '3C NMR spectrum: a signal at 22 ppm corresponding to the CH 3 of CH 3 -CO-0 a signal at 24 ppm corresponding to the CH 3 of CH 3

-CO-NH,

markedly more intense than those of the starting heparin and CI 1938.

As for the product B, both 0- and N-acetylation are observed.

The N-desulfation, more substantial that in the case of the product B, leads to a marked diminution of the sulfate/carboxyl ratio.

PHARMACOLOGICAL ACTIVITY OF THE PRODUCTS OF THE INVENTION A/ Anticoagulant activity in vitro: 1.Evaluation of the in vitro titre in comparison with a calibration series: This measurement was made with the aid of the "kaolinactivated partial thromboplastin time" test (Diagnostica, Stago, Asnidres, France) on human plasma. The results are given in Table 2: Table 2: Product Titer (iu/mg)cI 1941 28 ci 1943 7 Soo 20 Heparin 160 4o 1 CI 1940 0-butyrylated heparin prepared as described in example CI 1941 0-hexanoylated heparin prepared as described in example 6 25 CI 1943 0-decanoylated heparin prepared as described in example 8 .0 The results obtained show that the selectively 0-acylated products of the invention have an in vitro titre lower than that of heparin, their anticoagulant activity diminishing as the length of the acylating chain increases.

2. Measurement of the anticoagulant activity in vitro 4 4 of the products of the invention on human blood: 44 0 The assays were carried out at two doses of the products of the invention, 2 pg/ml and 4 pg/ml on 5 ml of human blood. A control assay was also carried out for each product by replacing the product to be tested by an isotonic solution of NaCl. After incubation for I o of its sodium salt, titrating at 157 iu/mg in the Codex determination and 155 u/mg in the anti-Xa factor determination of Yin et al. are if-cl 1 -r ~I 57 minutes at room temperature, the blood is centrifuged for 20 minutes at 3000 revs/min. The platelet-free plasma is decanted so that the following tests can carried out: TCK (kaolin cephalin time) with the aid of the "sensiblilised kaolin cephaline time" (Diagnostica Stago, Asnieres, France);

R

Heptest (Hemachem, St. Louis, USA).

The results are given in Table 3. Each result corresponds to the mean of three assays.

'Table 3: Product Dose (Yg/m)1 PTT HEPTEST (S) Control 48.00 21.50 2.70 1.73 CI 1940 2 ;08.75 61.00 45.54 t 8.52 CI 1940 4 428.00 101.00 103.43 28.36 Control 45.00 23.66 5,00 t 2.08 CI 1941 2 53.00 40.33 t 5.19 t 7.63 CI 1941 4 84.66 61.33 15.01 11.01 Control 49.66 23.00 8.50 t 4.35 CI 1943 2 50.00 23.00 t 9,54 1 4,58 CI 1943 4 56.00 24.00 S10.00 5,29 The results show a prolongation of the coagulation time in both methods in the case of the products CI 1940 and CI 1941. This prolongation is proportional to the dose.

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

i j:

E'

L I cIiu LHi pn or une solution is lowered to 3.5 with the aid ot hydrochloric acid. The volume of the solution is adjusted to 178 ml by means of demineralized water. 151 ml of pure ethanol are added with stirring. The stirring is maintained for 15 minutes after the

I

58 3. Measurement of the anticoagulant activity in vitro of the products of the invention by thromboelastography on human blood: The products of the invention at doses of 2 ig/ml and 4 pg/ml are placed in the presence of 5 mg of human blood as described in the previous assay, a control tube being introduced for each product by replacing the product to be tested by an isotonic solution of NaCl.

After incubation for 30 minutes at room temperature, a thromboelastographic tracing was recorded with the aid of a Hellige thromboelastograph on CaCI 2 0.25 ml of blood recalcified by 0.1 ml of 0.058 M The results are given in Table 4.

Table 4: '1 5 tt t4

I

l t tg|l 3Ot Dose j m Product r k J r k amx a PT Control 13.00 8.25 21,25 I 48.00 11.25 t 0.81 1.25 t 1,50 2.44 t 2.36 CI 1940 2 33.50 22.25 55.75 35.50 2,57 2.88 t 4.03 t 5.85 3.69 0.77 CI 1940 4 78,75 44.00 122.75 27.00 0.82 17.34 8.68 13.02 16 t 0.27 Control 10.50 4.50 15.00 57.00 37.00 Contr 0.70 2.12 2.82 8.48 28.28 CI (941 2 14.00 10.33 24.33 51.66 21.33 3.00 6.50 6.00 10.96 19.85 Ci 1941 4 20.33 15.00 35.33 40.33 4.66 t 3.51 4.58 t 5.03 t Z.51 t 2.08 Control 13.33 9.00 22.33 47.00 10.33 S2,,30 2.64 4.93 4.58 t 4.04 CI 1943 2 12,66 8.00 20.66 47.00 12.00 2.08 t 2.64 t 4.72 5.29 5.29 CI 1943 4 12.66 9.83 22.50 45.66 9,33 t 2.08 3.01 5.07 5.77 t 4.04 i

I

IC

(I

I)

I t r reaction time k coagulation time corresponding to a tracing amplitude of 20 mm amx maximal amplitude ITP index of thrombodynamic potential The results show that CI 1940 and CI 1941 lead to an increase of r k, a diminution of am and of ITP, and this corresponds to enhanced hypocoagulation. This hypocoagulation increases both as a function of the dose used and of the length of the acylating chain.

CI 1943 does not cause any appreciable change of the parameters in this test.

B Anticoagulant activity in vi-o: 1. Measurement of the anticoagulant activity in vivo of the products of the invention in the rabbit route): The assays were carried out on New Zealand male rabbits.

A blood sample is taken from the median artery of the ear before injection of the product.

A solution of 25 mg of the product to be tested in 5 ml 20 of an isotonic solution of NaCi is then injected into the marginal vein of the ear.

The TCK test and the HEPTEST R are carried out on a blood sample taken before the injection of the products and on blood samples taken at 6 24h., 48 h. and 96 h. after the injection.

The results are given in the figures 7 and 8. They show a distinct prolongation in time of the anticoagulant activity, this prolongation increasing with the length of the acylating chain.

In fact, the anticoagulant activity of the product CI 1940 is still marked more than 6 hours after the injection, whereas it is still marked 24 hours after injection in the case of the product CI 1941 and is prolonged for up to 96 hours in the case of product CI 1943.

2. Measurement of the anticoagulant activity in vivo of the products of the invention by thromboelastography: The products to be tested are injected by the i.v. route as described in the previous assay (25 mg in 5 ml of isotonic NaC1 I L t 1; j it solution). A thromboelastographic tracing is recorded with the aid of a Hellige thromboelastograph on 0.25 ml samples of blood taken before the injection and at 6 24 48 h. and 96 h. after the injection, additional samples being taken when the anticoagulant activity persists for more than 96 hours.

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

Table 5: Product tested: CI 1940 2I r k r+k k amx IPT Before 13 6 19 67 33 injection 6 h. after 91 60 151 56 2.1 Injection 24 h after 24 h after 12 5 17 67 Injection 96 h. after3 11 70 77 Injection 8 3 II 70 77 Table 6: Product tested: CI 1941 r k r k i amx IPT Before je on 13 5 I 18 67 6 h. after 135 31 166 45 2.6 Injection 24 h. after 19 12 31 58 11.5 Injection 48 h. after 22 10 32 72 Injection 61 Table 7: Product tested: CI 1943 0 00 oo o 0000 Q 00 6 0a 0 0 r k r k amx IPT Before 12 4 16 69 Injection 6 h. after 40 27 67 Injection 24 h. after 13 7 20 72 36 Injection 48 hafter 53 34 87 54.5 4.7 Injection 72 h. after Injection 112 185 297 38 I 96 h. after Injection 82 45 127 62 3.6 168 h. after Injection 19 9 28 68 23 192 h.after 46 Injection17 22 70 00 a 60 o 0 0*0 o o a0 0 6 Q 6 o 0 The results obtained show that a considerable increase in r k and a diminution of ITP at the 6th hour is observed for all of the products, which points to a prolongation of hypocoagulability.

This latter extends up to at least 24 hours after the injection in the case of the product CI 1941 and is still measurable 96 hours after the injection in the case of the product CI 1943.

Thus it can be seen that all of the products have a very considerable anticoagulant activity in vivo, that this activity is prolonged in time and that CI 1943, which had little or no activity in the assays in vitro, choose to have a very high and markedly sustained activity in the in vivo assays.

w 62 3. The anticoagulant activity of the products CI 1945, CI 1957 and CI 1958, prepared according to Examples 12, 13 and 14, respectively, was also tested in vivo in the rabbit.

The results show prolonged pharmacokinetics by the intravenous and subcutaneous routes for the products CI 1957 and CI 1958.

C/ ACTIVITY IN AN IN VITRO MODEL OF INHIBITION OF THE VIRUSES HIV-1 AND 2 i"-e products prepared according to the invention were tested in a model nf inhibition of the viruses HIV-I and HIV-2 in vitro as described in R. Pauwels et al., J. Virol. Methods, 1987, 16, 171-185.

All of the products tested proved to be active at doses totally lacking cytotoxicity. In particular, it was observed that the products CI 1925 and CI 1926 were more active than the starting material.

D/ ACTIVITY IN AN IN VITRO MODEL OF INHIBITION OF THE FORMATION OF

SYNCYTIA

The syncytia are giant, multi-nucleated cells, formed by the fusion of healthy T4 lymphocytes with infected T4 lymphocytes.

The products prepared according to the invention were tested in a model of co-culture of MOLT4 cells (healthy T4 lymphocytes) and 4 0I0 HUT-78/HTLV III B cells (human line of infected T4 lymphocytes).

All of the products tested proved to be active in the model, at doses totally lacking cytotoxicity. In particular, the products CI 1924, CI 1925 and C1 1926 were found to be morc active than the starting material.

E! ACTIVITY IN AN IN VITRO MODEL OF INHIBITION OF VARIOUS ENVELOPED 125 VIRUSES The activity of the products prepared according to the invention with respect to the inhibition of various enveloped DNA and RNA viruses, in particular the foliowing viruses HSV 1 and 2 (Herpes simplex virus 1 and 2, DNA viruses) VSV (vesicular stomatitis virus) RNA virus Sindbis virus All of the products tested were shown to be active in the If 63 particular, the products CI 1924, CI 1925 and CI 1926 exhibited a markedly enhanced activity towards the VSV and the Sindbis virus, compared with the starting material.

F/ ACTIVITY IN AN IN VIVO MODEL OF INHIBITION OF THE PROLIFERATION OF SMOOTH MUSCLE CELLS j The products prepared according to the invention were tested '1 in a model of inhibition of the proliferation of smooth muscle cells in vivo in the rat (balloon catheter model as describid in A.W.

Clowes and M.M.Clowes, Labor. Investig., 1985, 52 612-616.

The products tested were shown to be active. In particular, the products CI 1924, CI 1925 and CI 1926 showed enhanced activity compared with the starting mater-ial.

Claims (24)

1. Selectively 0-acylated glycosaminoglycans corresponding to the following formula I: in which: G represents a group of formula:p OR 1 0 0 4OR 1 0 150 NHR 2 or a grou~p of formula: 0 00 040 0 0 NHCOCH 3 or a group of formula: OR 4 430 Rio 0 400 0 NHCOCH 3 353 P ^i U represents a group of formula: (c) o or a group of formula: (d) S(C e i I i~ It l I tt4414 or the residue of the group or of the group after periodic oxidation, followed by a p-elimination or acid hydrolysis or, in a proportion of one residue for at least two chains, a non-sulfated uronic acid (D-glucuronic acid or L-iduronic acid) open between the carbon atoms in positions 2 and 3, of formula: COOR 3 CH2OR CH2OR 1 A represents a group Ri, a group R 1 a group R 1 or a group of formula: -4 COOR 3 0 (e) 0OR 1 OR 1 or the residue of the group or of the group after periodic I) oxidation, followed by a 1-elimination or acid hydrolysis: -B represents a group O-R 1 ,P a group (a)-0RJ, a group (a')-ORJ a c roup (b)-0R 1 a group of formula: fi t 4() 66 OR 1 OR 0 1 O OR or a group of formula: 0 R O 0 1 -OR S o 1 °i o or represents a group a group or a group to which remains attached a residue of or of such as that present S° 20 after periodic oxidation, followed by a B-elimination or acid 0 o oo hydrolysis; R 1 represents H, SO 3 or acyl, acyl being the residue of a non- c-3 unsaturated, mono- or dicarboxylic acid selected from among an alkanoyl group of 1 to 18 carbon atoms; 25 an alkanoyl group of 2 to 3 carbon atoms substituted by o a cycloalkyl group of 3 to 7 carbon atoms, a phenyl group possibly substituted by one or more alkyl radicals °o of 1 to 14 carbon atoms, halogen atoms or NO 2 or OCH 3 groups, o an unsaturated aliphatic hydrocarbon radical of 4 to 16 carbon atoms; 00 a benzoyl group possibly substituted by one or more alkyl radicals 0 o o o of 1 to 4 carbon atoms, halogen atoms or NO 2 or OCH 3 groups; a cycloalkyl (3-7 C) carbonyl group; R 2 represents SO 3 and/or an acetyl radical, provided that the proportion of N-acetyl glucosamine does not exceed that in heparin when R 1 represents an acetyl radical; R 3 represents a hydrogen atom or an alkyl radical of 1 to 10 carbon /ti 1. 67 atoms, and preferably 1 to 4 carbon atoms, or a pheny~alkyl radical of 7 to 12 carbon atoms, or an alkali or alkaline earth metal cation; n is an integer yey~itg from 1 to R being acyl in a proportion of at least 0.1 to 3 acyl groups, and preferably from 0.5 to 2 acyl group, per disaccharide unit and their pharmaceutically acceptable salts.

2. Selectively O-acylated glycosaminoglycans according to Claim 1, characterized in that they correspond to the following f o r m u l a I I OR] COOR 3 0 (II) A O OR OR 1 B NHR 2 OR 1 n in which: A has the meanings of Claim 1; R 1 represents H and/or S03 and/or an acyl group such as that defined in Claim 1; R 2 represents SO 3 and/or an acetyl radical, provided that the proportion of N-acetyl glucosamine does not exceed that in heparin when R 1 represents an acetyl radical; R 3 represents a hydrogen atom, an alkyl radical of 1 to 10 carbon atoms, and preferably from 1 to 4 carbon atoms, a phenyl-alkyl radical of 7 to 12 carbon atcas, or an alkali or alkaline earth metal cation; B represents (a)-OR 1 or and being such as detined in Claim 1, or OR 1 or a group to which remains attached a residue of or of such as that present after periodic oxidation, followed by a B-elimination or acid hydrolysis; n is an integer Aaauyig from 1 to 80, provided that when A represents R1, a group R1-(c) or a group R1-(d) and when B represents L ll 1 iM /i v -68 0-Rl 1 a group R 1 0-(a or a group R 1 n is an integer f rom 1 to 16.

3. Selectively 0-acylated glycosaminoglycans according to Claim 2, characterised in that they to the following formula II: (II) o 9A 9 0 0000 o 99 o 9 9 99, 9 9 '9 15 o '9 9 99 00 o 9 A 0 -0 N\L k NHR 2 OR 1 n' in which: B, R 1 ,9 R 2 and n have the meanings of Claim 2; R R 3 represents a hydrogen atom or an alkali or alkaline earth aietal caticon;

4. Selectively 0-acylated glycosaminoglycans accrding to Claim 2, characterized in that they correspond to the following formula II: 9 20 0 9 C I Coo, 9 4 0 o 09 -o0 in which: A, B, R 1 ,I R 2 and n have the meanings of Claim 2; R 3 represents an alkyl radical of 1 to 10 carbon atoms, and 'preferably from 1 to 4 carbon atoms, or a phenyl-alkyl radical of 7 to 12 carbon atoms. I 69 Selectively 0-acylated glycosaminoglycans according to Claim 1 characterized in that they correspond to the following formula II ORCOOR 3 0 (II) OR OR S1 1 -Ro x on NHR 2 OR n in which A has the meanings of Claim 1, R represents H and/or SO 3 and/or an acyl group, acyl being the residue of a non o-B3 unsaturated, mono- or dicarboxylic a iJ selected 0 U0 o, from among: o an alkanoyl group of 1 to 18 carbon atoms; o0 an alkanoyl group of 2 to 3 carbon atoms substituted by 0 00 o «C a cycloalkyl group of 3 to 7 carbon atoms, 2 0 a phenyl group possibly substituted by one or more alkyl radicals of 1 to 14 carbon atoms, halogen atoms or NO 2 or OCH 3 groups, an unsaturated aliphatic hydrocarbon radical of 4 to 16 carbon atoms; a benzoyl group possibly substituted by one or more alkyl radicals of 1 to 4 carbon atoms, halogen atoms or NO 2 or OCH 3 groups; a cycloalkyl (3-7 C) carbonyl group; R 2 represents SO 3 and/or an acetyl radical, provided chat the proportion of N-acetyl glucosamine is not more than that in heparin when R 1 represents an acetyl radical; 0 R 3 represents a hydrogen atom or 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, or an alkali or alkaline earth metal catio.n n is an integer w aig from 1 to p, R1 being acyl in a proportion of at least 0.5 to 2 acyl groups, and S 5 preferably 1 acyl group, per disaccharide unit.- 0

8. Selectively 0-acylated g lycosami nogly cans according Claim characterized in that they correspond to the following formula II (11) A t :4154 0~4 a. 240* in which B, Rif R 2 and n have the meanings of Claim R R 3 represents a hydrogen atom or an alkali or alkaline earth metal cation. 7. Selectively 0-acylated glycosaininoglycans according to Claim characterized in that they correspond to the following formula II (11) -o0 A0 NHRf 2 in which la, Rip R 2 and n have the meanings of" Claim 5 R R 3 represents an alkyl radical of 1 to 10 carbon atoms, and preferably from 1 to 4 carbon atoms, or a phenyl-alkyl radical of 7 to 12 carbon atoms. I F I, 71 8 Selectively 0-acylated glycosaminoglycans according to any one of the Claims 1 to 4, characterized in that the glycosaminoglycan is selected from the group constituted by a mixture of heparin fragments having a molecular mass lower than 10,000 daltons, a mixture of heparin fragments having a mean molecular mass varying between 2,000 and 7,000 daltons, a mixture of heparin fragments having a mean molecular mass of about 4,500 daltons, a mixture of heparin fragments having a mean molecular mass of about 2,500 daltons, a mixture of heparin fragments homogeneous with regard to their molecular mass, a heparin fragment obtained by synthesis, and homogeneous with respect to beth its molecular mass and its I functionalization; the acyl group being in a proportion of at least 0.1 to 3, and preferably 0.5 to 2 acyl group per disaccharide unit. i 9. Selectively 0-acylated glycosaminoglycans according to any one of the Claims 5 to 7, characterized in that the glycosamino- glycan is heparin, the acyl group being in a proportion of at least to 2 groups, mnd preferably 1 acyl group, per disaccharide unit. Selectively 0-acylated glycosaminoglycans according to any one of the Claims 1 to 8, characterized in that they correspond to the following formula III: R CiR" 3 rOR 1 1 0 -0 0 A Ro (III) NHR 2 OR, 1 n 1 in which: A represents R1 or Ri-(c) or as defined in Claim 1; R 1 represents H and/or SO 3 and/or an acyl group as defined in Claim 1; R 2 represents SO 3 and/or an acetyl radical, provided that the proportion of N-acetyl glucosamine does not exceed that in heparin when R 1 represents an acetyl radical; R 3 represents a hydrogen atom and/or an alkyl radical of 1 to carbon atoms, and preferably from 1 to 4 carbon atoms, or a phenyl-alkyl radical of 7 to 12 carbon atoms, or an alkali or alkaline earth I 72 Metal cation; n is an integer e*axya-g-from 3 to 12. 11 Selectively -acylated glycosaminoglycans according to any one of the Claims 1 to 8, characterized in that they correspond to the following formula IV: COOR3 JO COOR 3 0 o (IV) 1 OR OR 1 NHR 2 OR 1 n in which: R 1 represents H and/or SO 3 and/or an acyl group as defined in Claim 1; R 2 represents SO 3 and/or an acetyl radical, provided that the proportion of N-acetyl glucosamine does not exceed that in heparin when R 1 represents an acetyl radical; R 3 represents a hydrogen atom and/or an alkyl radical of 1 to carbon atoms, and preferably from 1 to 4 carbon atoms, or a phenyl-alkyl radical of 7 to 12 carbon atone, or an alkali or alkaline earth metal cation; B represents (a)-0R 1 as defined in Claim 1, or OR 1 n is an integer -a&riy from 2 to 12 Selectively 0-acylated glycosaminoglycans according to any one of the Claims 1 to 4, characterized in that the glycosaminoglycan selected is heparin, a fraction or fragment of heparin lacking the binding site for antithrombin III.

13. Selectively O-acylated glycosaminoglycans according to any one of the Claims 1, 2, 5 and 12 characterized in that they correspond to the following formula V: !,;rPN A 1 0. NHR 2 oso in which: A represents R 1 or R R1-(d) or the residue of or of (d) after periodic oxidation, followed by a B-elimination or acid hydrolysis; B represents (a)-OR 1 or OR 1 or a group to which remains attached a residue of or of such as that present after periodic oxidation, followed by a B-elimination or acid hydrolysis; R 1 has the meanings given in Claim 1; R 2 represents SO 3 or an acetyl radical, the proportion of SO3 being about R 3 represents a hydrogen atom or an alkali or alkaline earth metal cation; n is an integer arying-from 1 to

14. Selectively 0-acylated glycosaminoglycans according 25 to any one of the Claims 1, 2, 5 and 12 characterized in that they correspond to the following formula VI: I s 4 f 4 4 '4 I~ A 4 44 144 4 4 B (VI) NHR 2 I A4 -74- in which: A represents R 1 R 1 R 1 or the residue of or of after periodic oxidation, followed by a p-elimination; U represents: COOR 3 7OR oso Sor, in a proportion of one residue for at least two Schains, a non-sulfated uronic acid (D-glucuronic acid or L-iduronic acid) open between the carbon atoms in 15 positions 2 and 3, of formula: COOR 3 0O 0 C112 R C OR S- B represents (a)-OR 1 or OR 1 or a group to which remains attached a residue of or of (d) such as that present after periodic oxidation, followed by a p-elimination; R 1 has the meanings given in Claim 1; R 2 represents SO03 and/or an acetyle radical, the proportion of S0 3 being about at least R 3 represents a hydrogen atom or an alkali or alkaline earth metal cation; i 30 n is an integer from 2 to 18. Selectively O-acylated glycosaminoglycans according to any one of the Claims 1, 2, 5 and 12, characterised in that they correspond to the formula VI as defined in claim 14 in which: n is an integer between 7 and 15 for the major species, R 1 is an alkanoyl radical of 2 to 10 carbon atoms, ALi 4 911125,PHHSPE.020,sanoflet,74 4 7T L.i 74a advantageously from 4 to 10 carbon atomn, nd preferably 4 to 6 carbon atoms.

16. Selectively 0-acylated glycosaminoglycans according to the Claims 14 and 15, characterized in that they are constituted of a mixture of fragments homogenous with respect to their molecular mass and in which n is an integer from 2 to 12.

17. Selectively O-acylated glycosaminoglycans according to any one of the Claims 1, 2, 5 and 12 characterized in that they r 1 I i j: "i I I c 1 91112ZPH HSPE.020,sanofle 75 L \i~E4~1/ correspond to the following formula VII: -OR 1 COOR 3 0\ -N,(VII) A O 0 OR 1 B( NHCOCH OR n in which: A represents R I R1-(c) or R 1 as defined in Claim 1; R 1 represents an alkanoyl radical of 2 to 18 carbon atoms: R 3 represents a hydrogen atcm or an alkali or alkaline earth metal cation; B represents (a)-OR 1 as defined in Claim or OR 1 n is an integer a-i-a from 1 to

18. Selectively O-acylated glycosaminoglycans according to Claim 1, characterized in that they correspond to the following formula VIII: SOR 1 COOR3 0 (v .0 OR 1 (VIII) A NHCOCH 3 OR, in which: A has the meanings of Claim 1; R 1 represents H and/or SO and/or an acyl group as defined in Claim 1; R 3 represents a hydrogen atom, an alkyl radical of 1 to I p~LL 4 t. 76 carbon atoms, and preferably from 1 to 4 carbon atoms, or a phenyl-alkyl radical of 7 to 12 carbon atoms, or an alkali or alkaline earth metal cation; B represents (b)-OR or as defined in formula or OR 1 or a group to which remains attached a residue of or of (d) such as that present after periodic oxidation, followed by a B-elimination or acid hydrolysis; n is an integer ~etryifg from 1 to

19. Selectively 0-acylated glycosaminoglycans according to Claim 18, characterized in that they correspond to the following formula VIII: l r CO OR OR tO O OR 0 0 OR V I I I) 20 A NHCOCH 3 OR 1 #4 in which: A, B, R 1 and n have the meanings given in Claim 18; R 3 represents a hydrogen atom or an alkali or alkaline earth netal cation' Selectively O-acylated glycosaminoglycans according to Claim 18, characterized in that they correspond to the following formula VIII: cooR OR 1 OOR 3 SRiO 0 B .0 OR (VIII) 1 A NHCOCH 3 ORI n ^w 77 in which: A, B,R 1 and n have the meanings given in Claim 18; R represents an alkyl radical of 1 to 10 carbon atoms and preferably from 1 to 4 carbon atoms, or a phenyl alkyl radical of 7 to 12 carbon atoms.

21. Selectively O-aylated glycosaminoglycans according to any one of the Claims 1, i 1 19 and 20 characterized in that the glycosaminoglycan is selected from the group constituted by dormatan sulfate and its fragments, or the chondroitin 4- and 6-sulfates and their fragments, the acyl group being in a proportion of at least 0.1 to 3 acyl groups, and preferably from 0.5 to 2 acyl group, per disaccharide unit. 22 Selectively 0-acylated glycosaminoglycans according to any one of the Claims 1 to 21 in which R1 is an alkanoyl radical 4 to 10 carbon atoms.

23. -seeefeX ffor the preparation of selectively 0-acylated r glycosaminoglycans according to any one of the Claims 1 to 22, characterized in that I a glycosaminoglycan of the following formula IX: 4 G-U B° (IX) in which: GO represents a group (a)O of formula: OR' NHR 2 i a or a group of formula: 1 i- rE t A OR,* NHCOCH 3 or a group of formula: g 4 4 1 A U' represents a group of formula: COOR 3 or a group (d)l of formula: 4 4 444 040000 4 0 00 4 0 0 0 44 -0 ORI 444 If 79 or the residue of the group (c)O or of the group (d)O after periodic oxidation, followed by a B-elimination or acid hydrolysis; A° represents a group R 1 0 a group R 1 0 a group R° 1 or a group (e)O of formula: or the residue of the group (c)o or of the group (d)O after periodic oxidation, followed by a B-elimination or acid hydrolysis; B° represents a group ORl a group (a)°-OR 1 o, a group 1 a group (b)o-OR 1 a group (f)o of formula: S4 4'20 4 1 or a group of formula: t I 4 1 446 4444CC os o a o 4 (g) 0 or represents the groups (a) 0 or (b)O to which remains attached a residue of or of such as that present after periodic oxidation, followed by a B-elimination or acid hydrolysis; R 1 represents H or S0 3 I i I: n is an integer from 1 to 80, proviaea nar wnen a Srepresents a group RI-(c) or a group R and when B represents i i L j ii "Y d AA f t I Ij I iIr I R 2 represents SO 3 and/or an acetyl radical, provided that the proportion of N-acetyl glucosamine does not exceed that in heparin when R 1 represents an acetyl radical; R 3 represents a hydrogen atom, or an alkyl radical of 1 to 10 carbon atoms or a phenyl-alkyl radical of 7 to 12 carbon atoms or an alkali or alkaline earth metal cation; n is an integer from 1 to is converted into a salt of the said glycosaminoglycan soluble in a polar aprotic organic solvent; this salt is treated with an anhydride of formula; Acyl-0-Acyl in which Acyl is as defined in Claim 1, in the said polar aprotic organic solvent in the presence of catalytic amounts of pyridine or a dialkylaminopyridine and a proton acceptor; the product thus obtained is precipitated by the action of a solution of sodium acetate in ethanol; and the selectively O-acylated glycosaminoglycan is isolated by dissolving the precipitate thus obtained in water, followed by dialysis, and if required, the sodium salt of the selectively O-acylated glycosaminoglycan thus obtained is converted into another pharmaceutically acceptable salt.

24. Process according to Claim 23, characterized in that t-_e glycosaminoglycan used in step is selected from a group constituted by heparin, a mixture of heparin fragments having a molecular mass lower than 10,000 daltons, a mixture of heparin fragments having a mean molecular mass varying between 2,000 and 7,000 daltons, a mixture of heparin fragments having a mean molecular mass of about 4,500 daltons, a mixture of heparin fragments having a mean molecular mass of about 2500 daltons, a mixture of heparin fragments homogenous with respect to their molecular mass, a fragment of heparin obtained by S911125,PHHSPE.020,sanofilet,80 i I SI s i i- 80a synthesis, and homogeneous with respect to both its molecular mass and its functionalization. Process according to Claim 23, characterized in that the glycosaminoglycan used in step is heparin, a fraction or a fragment of heparin lacking the binding site for antithrombin III.

26. Process according to Claim 23, characterized in that the glycosaminoglycan used in step is selected from the group constituted by dermatan sulfate and its fragments, or the chondroitin 4 q 4f 1 I 4 I t i 4(4 4 tltIyl t( 1 II 911125,PHHSPE.020,sanolet81 :1 n is an integer *earig from 1 to L- R 1 being acyl in a proportion of at least 0.5 to 2 acyl groups, and co 5 preferably 1 acyl group, per disaccharide unit.. P -81 4- and 6-sulfates and their fragments.

27. Process according to any one of the Claims 23 to 26, characterized in that the salt of the glycosaminoglycan of step is a tertiary amine salt, in particular a salt of tributylamine, or a quaternary ammonium salt, in particular a tetrabutylammonium salt.

28. Process according to any one of the Claims 23 to 27, characterized in that the anhydride used in step is the anhydride of an alkanoic acid containing 2 to carbon atoms, advantageously from 4 to 10 carbon atoms and preferably 4 or 6 carbon atoms.

29. Process according to any one of the Claims 23 to 28, characterized in that the step is conducted in a polar aprotic solvent selected from the group constituted by dimethylformaide, hexamethylphosphoric triamide, pyridine or a mixture of these solvents with or without dichloromethane. Process according to any one of the Claims 23 to 29, characterized in that a base chosen from the group constituted by pyridine, triethylamine, tributylamine is used as proton acceptor in step 2.

31. Process according to any one of the Claims 23 to 30, characterized in that the dialysis in step is performed in the presence of a weak base.

32. Process according to any one of Claims 23 to 31, characterized in that step 2 is carried out at a temperature varying between 0 0 C and 100 0 C, and more particularly between 50 0 C and 100 0 C. 30 33. Pharmaceutical composition characterized in t 4 that it contains as active substance an efficacious amount of at least one selectively O-acylated glycosaminoglycan according to any one of the Claims 1 to 22 in combination with a pharmaceutical vehicle.

34. Pharmaceutical composition according to Claim 32, characterized in that the selectively O-acylated 91 5PP. _so e 911125,PHHSPE.020,sanofi.let,!| r 'i L S- va L 1. i caroorn atoms, ana preferably from 1 to 4 carbon atoms, or a phenyl-alkyl radical of 7 to 12 carbon atoms. Ib i i -r 81a glycosaminoglycan is present in the form of a pharmaceutically acceptable salt such as a sodium salt, a magnesium salt or a calcium salt. Pharmaceutical composition according to the Claims 33 and 34, characterized in that the pharmaceutical vehicle is suitable for administration by the oral route, and is available in the form of gastro- resistant capsules, tablets or lozenges, pills or also in the form of solutions to be taken orally, containing advantageously J e 4 44I d j i 1 j, ii i: 91U25z,PHHPE.02,sanfl~fet,82 r K:: R 3 represents a hydrogen atom and/or an alkyl radical of 1 to carbon atoms, and preferably from 1 to 4 carbon atoms, or a phenyl-alkyl radical of 7 to 12 carbon atoms, or an alkali or alkaline earth solutions to be taken orally. mg to 5 g, and preferably 100 to 1000 mg per dose in the case of capsules, lozenges or pills and from 10 to 150 mg in the cane of solutions to be taken orally. 36 Pharmaceutical composition according to the Claims 33 and 34, characterized in that it is available in the form of a sterile, or sterilizable injectable solution for administration by the intravenous, intramuscular or subcutaneous routes, these solutions advantageously containing from 50 to 200 mg/ml of selectively O-acylated glycosaminoglycan when it is intended for injection by the subcutaneous route, or from 20 to 200 mg/ml of selectively 0-acylated glycosaminoglycan when it is intended for injection by the intravenous route or by perfusion.

37. Pharmaceutical composition according to any one of the Claims 33 to 36 for the treatment or prevention of diseases due to enveloped viruses. 15.. 5 0' ,38. Pharmaceutical composition according to Claim 36 for the treatment or prevention of diseases due to retroviruses such as AIDS. 4 '4 44 4 I y **K L- 83

39. A method for the treatment and/or prophylaxis of viral diseases which comprises administering to a patient in need of such treatment or prophylaxis a therapeutically effective amount of compound of formula O-acylated glycosaminoglycans, methods for their manufacture or pharmaceutical compositions or methods of treatment involving them, substantially as hereinbefore described with reference to the Examples and/or drawings.

41. The steps, features, compositions and c nds disclosed herein or referred to or atied in the specification and/or cla' this application, individually lectively, and any and all combinations of ywo or more of said steps or features. I0 #1 *r a i to If (i Ir I DATED this TWENTY FIRST day of JULY 1989 Sanofi by DAVIES COLLISON Patent Attorneys for the applicant(s) 1r 0 #4 I i