SK5272002A3 - Novel oligosaccharides, preparation method and pharmaceutical compositions containing same - Google Patents

Abstract

The invention concerns oligosaccharides of formula (I), their mixtures, their diastereomers, methods for preparing them and pharmaceutical compositions containing them.

Description

Oligosaccharides, process for their preparation and pharmaceutical compositions containing them

Technical field

The invention relates to oligosaccharides of the formula I (I)

mixtures thereof, diastereoisomers thereof, processes for their preparation and pharmaceutical compositions containing these oligosaccharides.

BACKGROUND OF THE INVENTION

Sulphated disaccharides having a reducing end

The 1,6-anhydro structure has been described by H. P. Wessel in J. Carbohydrate Chemistry, 11 (8), 1039-1052 (1992), and no pharmacological activity has been described for these compounds in this publication.

Sulphated trisaccharides containing a 1,6-anhydro repeat unit have also been described in EP 84 999 and Y. Ichikawa et al. in Carbohydr.Res.141,273-282 (1985), as intermediates for the preparation of higher oligosaccharides. These trisaccharides have poor anti-Xa activity.

In the above general formula I, n denotes an integer from 0 to 25, R ¹ , R ³ , R ⁴ and R ⁵ which are the same or different denote an integer from 0 to 25. · · · · ···

a hydrogen atom or a SO ₃ M group or a COCH ₃ group and M denotes sodium, calcium, magnesium or potassium.

These oligosaccharides thus contain an even number of carbohydrates.

In the general formula, IR ⁴ preferably denotes a hydrogen atom.

In the general formula I n preferably denotes an integer from 0 to 10, in particular from 0 to 6 and in particular from 1 to 6.

Oligosaccharides of formula (I) may be prepared by treating alkali metal (II) (II) with alkali metal or quaternary ammonium hydroxide.

wherein n denotes an integer from 0 to 25, R ¹ , R ³ , R ⁴ and R ⁵ , which are the same or different, denote a hydrogen atom or a SO 3 M group, R ² and R ⁶ , which are the same or different, denote a hydrogen atom or SO 3 M or COCH ₃ and M denotes sodium, calcium, magnesium or potassium or a mixture thereof.

This reaction is carried out in an aqueous medium at a temperature of 40 to 80 ° C and a pH of 10 to 13.

Useful alkali metal hydroxides include sodium hydroxide, potassium hydroxide, lithium hydroxide and cesium hydroxide.

A useful quaternary ammonium hydroxide is tetrabutylammonium hydroxide.

ο · ·

The amount of alkali metal hydroxide or quaternary ammonium hydroxide must be sufficient to maintain the pH of the reaction mixture throughout the duration of the reaction. It is therefore necessary to continuously add alkali metal hydroxide or quaternary ammonium hydroxide during the reaction.

Preferably, the alkali metal hydroxide or quaternary ammonium hydroxide is in the form of a 1-5% aqueous solution.

Preferably, the reaction is carried out at a temperature of 60 to 70 ° C.

Preferably, the pH of the reaction mixture is 11 to 12.5.

The reaction is quenched by acidifying the reaction mixture,. for example by adding an acidic resin, for example Amberlite IR120 ^r resin (Fluka).

Optionally, the oligosaccharides of formula I may be purified by permeation gel chromatography using a polyacraylamidagarose type gel such as the commercially available Ultrogel ACA202 ^R gel (Biosphera), according to the procedure described below, for the separation of intermediate oligosaccharides of formula II. Alternatively, the oligosaccharides of formula (I) in which n is 0 or 1 can be purified on an alumina column using a water / ethanol mixture.

Intermediate oligosaccharides of formula II and mixtures thereof may be obtained by gel separation of a mixture of oligosaccharides III obtained by enzymatic depolymerization of heparin or by basic depolymerization of benzyl ester of heparin or semisynthetic benzyl ester of heparin.

This chromatography is carried out on columns packed with a polyacrylamidagarose type gel, such as a gel which is commercially available under the trade name Ultrogel.

• ·····························

ACA202 ^R (Biosepra). Preferably, a battery of columns filled with polyacrylamidagarose gel is used. The number of columns used depends on the volume, self gel and oligosaccharides to be separated. The mixture was eluted with a solution containing phosphate buffer and sodium chloride. Preferred is a phosphate buffer containing 0.02 mol / 1 Ν3Η ₂ Ρ0 ₄ / Ν32ΗΡ04 (pH 7) and 0.1 mol / 1 NaCl. Detection of individual fractions is performed using ultraviolet (254 nm) and ionic (IBF) spectrometry. The fractions thus obtained may then optionally be purified, for example, by strong anion exchange (SAX) chromatography, according to methods known to those skilled in the art, in particular those described by KGRice and RJ Linhardt in Carbohydrate Research 190, 219-233 (1989), A. Larnkjaer, SHHansen and PBOstergaard in Carbohydrate Research, 266, 37-52 (1995) and in WO90 / 01501 (Example 2).

These fractions are then lyophilized and subsequently desalted on a gel-packed column, such as a Sephadex G10 gel column (Pharmacia Biochemicals).

If the purification is not carried out by SAX chromatography, the lyophilisates can be purified by simple or fractional precipitation by methods known to those skilled in the art, in particular as described in French patent FR 2 548 672. In general, the following procedure is used.

The lyophilized fraction to be purified is dissolved at about 25 ° C in about ten volumes of distilled water. Addition of methanol or ethanol precipitates the desired oligosaccharide, checking its purity by high performance liquid chromatography (CLHP, Chromatography Liquide Haute Performance). The addition of methanol or ethanol is determined depending on the desired purity and yield of said oligosaccharide. Likewise, this operation can be carried out in several successive steps starting from the original lyophilisate solution. For this purpose, a non-solubilizing agent (methanol or ethanol) is added in small portions and after each addition of the above mentioned.

the precipitate obtained is recovered. The precipitates thus obtained are analyzed by high performance liquid chromatography. Depending on the desired purity and the desired yield, only those fractions of the precipitate which combine the desired purity and yield are combined.

In the case of intermediates of formula II in which n = 0, 1 or 2, it is preferable to start from the mixture III obtained by enzymatic depolymerization.

This enzymatic depolymerization is carried out using heparin I (EC 4.5.5.7) in a pH 7 phosphate buffer solution, in the presence of sodium chloride and bovine serum albumin (BSA), at a temperature between 10 and 18 ° C, preferably at 15 ° C , for 8 to 10 days, preferably 9 days. This depolymerization is interrupted, for example, by heating the reaction mixture to 100 ° C for two minutes, then the mixture is freeze-dried. It is preferred to use 7 µl haparinase I per 25 g heparin. The phosphate buffer solution generally contains 0.05 mol / L NaCl / NaHPCl (pH 7) in the presence of 0.1 mol / L sodium chloride. The bovine serum albumin concentration is generally 2%.

In the case of intermediates of the formula II in which n = 0, 1, 2, 3 or 4, it is preferable to start from the mixture III obtained by depolymerization of the benzyl ester of heparin.

The heparin benzyl ester may be prepared according to the procedures described in U.S. Pat. Nos. 5,389,618, EP 40,144 and FR 2,546,772. The degree of esterification is 50 to 100%, preferably 70 to 90%.

Said depolymerization is carried out in an aqueous medium with an alkali metal hydroxide (e.g. lithium hydroxide, sodium hydroxide, potassium hydroxide or cesium hydroxide) or a quaternary ammonium hydroxide (e.g. tetrabutylammonium hydroxide), preferably at a molarity of 0.1 to 0.2 mol / l, at 40 ° C. to 80 ° C and for 5 to 120 minutes. Preferably, the reaction is carried out for 5 to 15 minutes at a temperature of 60 to 70 ° C and using a hydroxide solution. 9 9 9 Sodium with a concentration of 0.15 mol / l. The depolymerization reaction is interrupted by neutralization by addition of an acid such as acetic acid. After addition of 10% w / v. sodium acetate, the oligosaccharide mixture is precipitated by the addition of methanol, preferably 2 volumes of methanol per 1 volume of the reaction mixture, and filtered.

According to a preferred embodiment of the invention, the oligosaccharide mixture obtained after chemical depolymerization in the form of an aqueous solution is enriched by ultrafiltration on membranes with an appropriate nominal separation threshold (Pellicon type from regenerated cellulose commercially available from Millipore), the membrane type being selected depending on the type enriched. the oligosaccharide to be isolated. For oligosaccharides of formula II in which n is 0, a membrane with a nominal separation threshold of 1 kDa is used, and for oligosaccharides of formula II in which n is 1, a membrane with a separation threshold of 1 kDa or 3 kDa is used. For example, in the case of oligosaccharides of formula II in which n is 2, a membrane with a separation threshold of 3 kDa is used, and in the case of oligosaccharides of formula II in which n is equal to 3 or 4, a membrane with a separation threshold of 5 kDa is used. During this operation, the permeate is retained while the retained fraction is considered waste. Thus the fraction of the enriched product may represent 50 to 10% of the initial oligosaccharide mixture, while maintaining at least 80% of the desired oligosaccharide.

In the case of intermediates of formula II in which n is 0 to 25, it is preferable to start from the mixture III obtained by depolymerization of the benzyl ester of a semisynthetic sulphated polysaccharide. Said polysynthetic sulphated polysaccharide benzyl ester is prepared from polysynthetic sulphated polysaccharides obtained from polysaccharide K5 by the procedures described in WO94 / 29352 and WO96 / 14425. The esterification, depolymerization and isolation conditions are the same as those described above for the heparin benzyl ester.

··· ················· φ φ φ φ φ φ φ · φ φ φ

In all of the above procedures, the starting heparin may be of pig, sheep, goat or bovine origin and may come from mucus, lungs or animal skin. Preferably, pork or sheep mucus heparin or bovine lung is used, and more preferably pork mucus heparin.

The oligosaccharides of the formula I have anti-inflammatory properties and can thus be used for the prevention or treatment of diseases associated with inflammatory processes involving the production of cytotoxic substances, such as nitric oxide (NO), the inducible form of which is mainly released by neutrophils or macrophages. are activated at the tissue level. Neutrophil activation, migration, adhesion and infiltration occurs at the level of tissue zones ischemized as a result of occlusion or spasm of the vascularizing arteries of said tissue. These ischemia may occur either at the cerebral level (vascular stroke) or at the level of the myocardium (myocardial infarction) or at the level of the lower extremities (the so-called peripheral ischemia).

The oligosaccharides of the formula I can thus be used for the prevention and / or treatment of neurodegenerative diseases in which cerebral inflammation plays a deleterious and possibly death-causing role, including cerebral ischemia, cardiac ischemia (myocardial infarction), peripheral ischemia, central injury nervous system and in particular skull, spine or combined skull and spine injuries, multiple sclerosis, neuropathic pain and peripheral neuropathy, motor neuron diseases, and in particular amyotrophic lateral sclerosis, neuro-AIDS, Alzheimer's disease, Parkinson's disease and Huntington's form , especially at the joint level.

Anti-inflammatory activity of these products is demonstrated in vivo in the test output N0 _x (nitrite and nitrate) • ···· ·· · · · · · · · · • · · · · · · · · · · • • • · · Lipopolysaccharide (LPS) induced by Escherichia coli induced by the protocol described by M. Yamashita et al. in Eur. J. Pharmacol., 338.2,

151-158 (1997 or authors)

J. E. Shellito et al.

Am.J.Respir.Cel Mol.Biol., 13,1,45-53 (1995).

Male CD1 mice (Charles River) weighing between 25 and 35 g were injected intravenously with 0.5 mg / kg oligosaccharide bolus at T0, followed by 1 or 2 mg / kg oligosaccharide subcutaneous injection at T + 15 minutes. At T + 30 minutes, they are given 100 mg / kg of lipopolysaccharide (LPS) derived from Escherichia coli (Sigma L3129, serotype 0127: B8). At T + 3 hours, test animals are re-injected subcutaneously with 1 or 2 mg / kg oligosaccharide. At T + 5 hours and 30 minutes, blood samples are collected by eye puncture and the NO _x concentration (nitrite and nitrate) is determined by the Griess colorimetric method after reduction of nitrate to nitrite by nitrate reductase using the following procedure: 12 μΐ plasma samples are mixed with 88 μΐ of deionized water and the resulting mixture is incubated in the dark for one hour at room temperature in the presence of 40 μΐ of phosphate buffer (0,31 M, pH 7,5), 20 μΐ β-NADPH (reduced nicotinamide adenine dinucleotide phosphate) (0,86 mM ), 20 μΐ of FDA (0.11 mM) and 20 μΐ of nitrated reductase (2U / ml) (Boehringer Mannheim). 10 μΐ ZnSO ₄ (IM) is then added to the mixture to precipitate the proteins and after mixing the samples are centrifuged at 20,000g for 5 minutes. Finally, 50 μΐ of the supernatant is incubated for 10 minutes at

100 μΐ Griess reaction mixture of phosphoric acid and room temperature in the presence of reagent (1 ^% sulranilamide in 0,1 ° naphthylethylenediamine in deionized water (v / v)). Read optical values at 540 nm using a microplate spectrophotometer. Each value is determined twice. KNO3 and NaNO2 are used as the standard for the colorimetric method. In this test, the oligosaccharides of the invention inhibit more

• as 50s of NO _X formation.

Preferred oligosaccharides of the formula I are, in particular, oligosaccharides of the formula I in which:

n is 0,

R ¹ and R ⁶ denote the group SO ₃ Na a

M denotes sodium and a mixture of its diastereoisomers;

n is equal to 1,

R ¹ , R ² , R ³ , R ⁵ and R ⁶ denote the group SO ₃ Na,

R denotes a hydrogen atom and

M denotes sodium and a mixture of its diastereoisomers;

n is 2,

R ¹ , R ² , R ³ , R ⁵ and R ⁶ denote the group SO ₃ Na,

R ⁴ denotes a hydrogen atom and

M denotes sodium and a mixture of its diastereoisomers;

n is 2,

R ¹ , R ² , R ³ , R ° denote the group SO ₃ Na,

R ⁵ denotes a hydrogen atom or a SO ₃ Na group,

R ⁴ denotes a hydrogen atom and

M denotes sodium and a mixture of its diastereoisomers (derivative 1,6 anhydro ols-16). The following examples illustrate the preparation of oligosaccharides of formula I and the corresponding intermediates.

In these examples, the following abbreviations have the following meanings:

úls: (4-deoxy-2-C-sulfo-α-L-threo-hex-4-enopyranosyluronic acid) - (1-> 4) -2-deoxy-2-sulfoamino-6-O-sulfo-α-D-glucopyranose, ··· ··· ··· ··· ····

-Ί ........ · .... .... .... .... .... .... .... .... • • • • • • • • • • • • • • • disodium salt or AUAp2S- (1-> 4) -αD-GlcNp2S6S;

Is: (2-sulfo-α-L-idopyranosyluronic acid) - (1-> 4) -2-deoxy-2-sulfoamino-6-O-sulfo-α-D-glucopyranose, four-sodium salt or also αL-IdoAp2S- (1 -> 4) -aD-GlcNp2S6S;

lis: (α-L-idopyranosyluronic acid) - (1-> 4) -2-deoxy-2-sulfoamino-6-O-sulfo-α-D-glucopyranose, trisodium salt or also αL-IdcAp- (1- 4) -ad-GlcNp2S6S;

IIIs: (2-sulfo-.alpha.-idopyranosyluronic acid) - (1-> 4) -2-deoxy-2-sulfoamino-.alpha.-glucopyranose, trisodium salt or also .alpha.-IaoAp2S- (1-> 4) -aD-GlcNp2S ;

IdoAp: idopyranosyluronic acid;

Glc'p: 2-deoxy-2-aminoglucopyranose;

ΔΟΑρ: 4-deoxy-α-L-threo-hex-enopyranosyluronic acid;

S: s o 1 f.

DETAILED DESCRIPTION OF THE INVENTION

Example A

Preparation of oligosaccharides of the formula II in which n is equal to 0, 1 and 2 by enzymatic depolymerization and separation.

g of heparin is dissolved in 250 ml of phosphate buffer containing 0.05 mol / 1 NaH 2 PO ₄ / Na ₂ HPO 4 (pH 7), 0.2 mol / 1 NaCl and 2% bovine serum albumin. 701 heparinase I (EC 4.2.2.2.7) is added to this mixture and the resulting solution is cooled to 15 ° C and then maintained at this temperature throughout the depolymerization reaction. The progress of this reaction is monitored by taking aliquots of the reaction mixture and theirs

···· • · ···· permeation gel analysis. After nine days, the enzymatic reaction is stopped by heating the reaction mixture at 100 ° C for two minutes. The cooled reaction mixture is then lyophilized. A mixture of oligosaccharides III is thus obtained.

The mixture of oligosaccharides III obtained is then chromatographed as follows.

Chromatography is carried out on polyacrylamidagarose gel columns known as Ultrogel ACA 202 and the mixture is eluted with a phosphate buffer solution (0.02 mol / l NaH ₂ PO ₄ / Na ₂ HPO ₄ ) with pH = 7 and 0.1 mol / l chloride solution. Detected by ultraviolet (254 nm) and ionic (IBF) spectrometry. If desired, the products obtained can be purified by strong anion exchange (SAX) chromatography or fractional precipitation according to the procedure described in FR 2,548,672. The isolated product fractions are lyophilized and then desalted on a Sephadex G10 ^R gel column (Pharmacia Biochemicals).

This procedure yielded 3 g of disaccharide Is, 1100 mg of a mixture of hexasaccharide typically containing 55% of the Als-Is-IIs derivative and 10% of the Als-Is-IIIs. The latter mixture can either be purified by methods known to those skilled in the art to separate all the ingredients of the mixture or used as such to convert to the 1,6-anhydro derivative of formula I. It should be noted that during this conversion hexasaccharide Als-Is -IIIs does not provide compounds of formula I.

Example B

Preparation of oligosaccharides of the general formula II in which n is equal to 0, 1, 2, 3 or 4 by depolymerization of benzyl heparin ester and separation

a) Preparation of Heparin Benzyl Ester · · · · · hep · · · · · · · · · * · * · · · * hep · · · · · · · · · · · · · · · · · · · ·

The heparin benzyl ester is prepared as described in Example 3 of US Patent 5,389,618.

b) Depolymerization

100 g of heparin benzyl ester is dissolved in 1.9 l of demineralized water. The mixture was heated to 60 ° C with stirring. After obtaining a homogeneous solution, about 35 ml of 23% sodium hydroxide solution are added to the solution at once. After 10 minutes, the solution was cooled and neutralized with 80 mL of about 2N acetic acid solution. To this solution was added 10% (w / v) sodium acetate. The oligosaccharide mixture is precipitated by the addition of about 2 volumes of methanol. The precipitate was collected by filtration, washed twice with methanol and then dried under reduced pressure at 50 ° C. After drying, 73.8 g of a mixture of oligosaccharides II are obtained.

c) Oligosaccharide enrichment in which n = 1 g of the oligosaccharide mixture obtained as described above is dissolved in about 35 volumes of water. This solution is subjected to ultrafiltration through a 3 kDa membrane (Pellicon). When 600 ml permeate is withdrawn, this is diluted with 500 ml water. The operation is continued until a further 450 ml of additional permeate is withdrawn. The two permeate fractions are combined and then concentrated to dryness under reduced pressure. 6.1 g of a yellow-white product are obtained. Analysis of the solid product by permeation gel chromatography shows that it contains about 30% of the oligosaccharide of formula II wherein n is 1.

d) Fractionation of the mixture of oligosaccharides subjected to ultrafiltration

The enriched mixture is fractionated on columns packed with polyacrylamidagarose gel, known as

Ultrogel ACA 202 (four columns are used in series with column diameter 10 cm and column height 50 cm). 5 g of the mixture enriched in ultrafiltration are dissolved in 25 ml of water and eluted with a 0.2 mol / l sodium chloride solution at a flow rate of 5 ml / min. At the bottom of the column, 25 ml of eluate fraction were collected. Detection of the products is done by ultraviolet (254 nm) and ion (IBF) spectrometry. The product fractions for which n = 1 are isolated, lyophilized and then desalted on a Sephadex G10 gel column. After lyophilization, 1 g of tetrasaccharide containing typically 70% of an Als-Is derivative of formula II (R ¹ , R ² , R ³ ,

P. ³ , R ⁶ = SO 4 Na and M = Na). This Als-Is derivative can, if necessary, be purified by strong anion exchange (SAX) chromatography, or preferably by fractional precipitation by the procedure described in patent FR 2,548,672.

Example 1

5 ml of 0.0063 mol / L sodium hydroxide solution are added to the reactor maintained at 66 ° C. The solution value is then measured and determined as a target value (pH = 11.35). While stirring, 30 mg of an oligosaccharide of formula II in which n is equal to 0, R ¹ and R 0 denote the group SO 4 Na and M denotes sodium are added at once. The pH is then checked and maintained at pH = 11.35 by continuous addition of 0.5 M sodium hydroxide solution. After 10 hours, the addition of sodium hydroxide is discontinued and the reaction mixture is cooled to 25 ° C. The pH of the solution is then adjusted to between 6 and 7 by the addition of Amberlite IR120 resin. The mixture is filtered through a Whatman GF / B membrane and then concentrated under reduced pressure (2.7 kPa) at a temperature close to 25 ° C. The product is washed with 0.5 ml of distilled water and then lyophilized. This gives 29 mg of a mixture of diastereoisomers of the oligosaccharide of formula I in which n is 0, R 'and R ^c denote the group SC 4 Na and M denotes sodium [4-deoxy-2-O-sulfo-α-L-threo-hex- 4-enopyranosyluronic- (1-> 4) -1,6-anhydro-2-deoxy-2-sulfoamino-β-D-manopyranose, trisodium salt,

9 9 9

9 ·

999 9 proton spectrum (D, 0.440 MHz, T = 298 K, delta in ppm):

3.15 (1H, s, H 2),

3.75 (2H, m, H 6 and H 3),

3.8 (1H, m, H4),

4.20 (lH, d, J = 8 Hz, H6);

4.22 (1H, t, J = 5 Hz, H-3 '),

4.58 (lH, m, H-2 '),

4.75 (lH, m, H-5),

5.53 (1H, s, H1),

5.60 (1H, dd, J = 6 and ΙΗζ, ΗΙ '),

6.03 (1H, d, J = 5 Hz, H 4 ');

4-deoxy-2-O-sulfo-α-L-threo-hex-4-enopyranosyluronic acid - (1-> 4) -1,6-anhydro-2-deoxy-2-sulfoamino-β-D-glucopyranose, trisodium salt, proton spectrum (3? 0, 400 MHz, T = 298 K, delta in ppm):

3.34 (1H, dd, J = 7 = 2Hz, H 2),

3.72 (lH, t, J = 8 Hz, H6);

3.90 (lH, m, H 3),

4.03 (1H, s, H-4),

4.20 (lH, d, J = 8 Hz, H6);

4.23 (1 H, t, J = 5 Hz, H-3 '),

4.58 (1H, m, H2 '), ο · · · · · · · · ·

4.78 (1H, m, H5);

5.50 (1Η, s, Hl),

5.60 (1H, dd, J = 6 and 1Hz, H1 '),

6.0 (1H, d, J = 5 Hz, H 4 ')].

Example 2

To the reactor maintained at 62 ° C was added 33.3 ml of 0.0063 mol / L sodium hydroxide solution. The solution value is then measured and determined as a target value (pH = 11.15). While stirring, 200 mg of an oligosaccharide of formula TT in which n is equal to 1, R ¹ , R ² , R ³ , R ⁵ and R ⁶ denote an SO 4 Na group, R ⁴ denotes a hydrogen atom and M denotes sodium are added at once. The pH is then checked and maintained at pH = 11.15 by the continuous addition of 0.5 M sodium hydroxide solution. After 12 hours, the addition of sodium hydroxide is discontinued and the reaction mixture is cooled to 25 ° C. The pH of the solution was adjusted to between 6 and 7 by adding Amberlite IR120 resin. The mixture is filtered through a Whatman GF / B membrane and then concentrated under reduced pressure (2.7 kPa) at a temperature close to 25 ° C. The product is washed with 3 ml of distilled water and then lyophilized. 230 mg of an oligosaccharide of the formula I in which n is equal to 1, R ^@ 1, R ^{@ 2} , R ^{@ 3} , R @ ⁵ and R @ ⁶ denote a SO3Na group, R @ ⁴ denotes a hydrogen atom and M denotes sodium are obtained in the form of a mixture of diastereoisomers 4-deoxy-2-0-α-sulfο-L-threo-hex-4-enopyranosyluronic acid- (l-> 4) -2-deoxy-2-sulphoamino-6-0-sulfo-D-glucopyranosyl (l- > 4) 2-O-sulfo-α-L-idopyranosyluronic acid- (1-> 4) -1,6-anhydro-2-deoxy-2-sulfoamino-β-D-manopyranose, heptasodium salt:

proton spectrum (DjO, 400 MHz, T = 298 K, delta in ppm):

3, 15 (1H, s, H2), t

3.25 (1H, m, H2);

3.60 (1H, m, H3 ''), between 3.70 and 4.70 (14H, block, H3 / H4 / H6, H2 '/ H3' / H4 '/ H5',

H4 '' / H5 '' / H6 '', H2 '' '/ H3'),

4.75 (1H, m, H5), between 5.20 and 5.40 (2H, m, H1 'and H1' '),

5.45 (lH, m, H '' '),

5.56 (1H, m, H),

5,94 (1H, d, J = 5Hz, H4), 4-deoxy-2-O-sulfo-α-L-threo-hex-4-enopyranosyluronic acid) - (1-> 4) -2-deoxy-2- sulfoamino-α-D-glucopyranosyl- (1-> 4) -acid-2-O-sulfo-α-L-idopyranosyluronic- (1-> 4) -1,6-anhydro-2-deoxy-2-sulfoamino-β-D-glucopyranose, heptasodium salt, proton spectrum (D ₂ O, 400 MHz, T = 298 K, delta in ppm):

3.25 (lH, m, H-2 '),

3.42 (1H, dd, J = 4H and 1Hz, H2),

3.60 (1H, m, H3 ''), between 3.70 and 4.70 (14H, block, H3 / H4 / H6, H2 '/ H3' / H4 '/ H5',

H4 '' / H5 '' / H6 '', H2 '' '/ H3' ''),

4.75 (1H, m, H5), between 5.20 and 5.34 (2H, m, H1 and H1),

5.45 (1H, m, H1 ''),

5.52 (1H, m, H1)

5.94 (lH, d, J = 5Hz, H4)].

Example 3

16.7 ml of 0.0063 mol / L sodium hydroxide solution are added to the reactor maintained at 62 ° C. The solution value is then measured and determined as the target value (pH = 11.7). While stirring, added in one portion 100 mg of an oligosaccharide of formula II wherein n is 2, R ^1, R ^2, R ^3, R ⁵ and R ⁶ denote a group So3n, R ⁴ denotes a hydrogen atom and M is sodium. The pH is then controlled and maintained at pH = 11.7 by continuous addition of 0.5 M sodium hydroxide solution. After 10 hours, the addition of sodium hydroxide is discontinued and the reaction mixture is cooled to 25 ° C. The pH of the solution was adjusted to between 6 and 7 by adding Amberlite IR120 resin. The mixture is filtered through a Whatman GF / B membrane and then concentrated under reduced pressure (2.7 kPa) at a temperature close to 25 ° C. The product is washed with 3 ml of distilled water and then lyophilized. This gives 108 mg of an oligosaccharide of formula I in which n is 2, R ¹ , R ² , R ³ , R ⁵ and R ⁶ denote a SO 3 Na group, R ⁴ denotes a hydrogen atom and M denotes sodium in the form of a mixture of diastereoisomers; A to F denotes the constituent sugars of hexasaccharides, with A denoting a 1,6-anhydro radical and F denoting an unsaturated uronic acid radical [4-deoxy-2-O-sulfo-α-L-threo-hex-4-enopyranosyluronic acid - (1-> 4) -2-deoxy-2-suifoamino-6- (2-sulfo-α-D-glucopyranosyl- (1-> 4) -acid)

2-O-sulfo-α-L-idopyrancyluronic- (1-> 4) -acid-2-O-sulfo-α-L-idopyranosyluronic- (1-> 4) -1,6-anhydro-2-deoxy-2-sulfoamino-β -D-manopyranose, undecasodic salt:

proton spectrum (D ₂ O, 600 MHz, T = 298 K, delta in ppm):

3.15 (1H, s, H2 (A)),

3.25 (2H, m, H2 (C + E)), · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·· ···· · · · · · · · · · · ·

3.60 (2Η, m, Η3 (C + E)), between 3.65 and 4.50 (19H, block, H2 (B + D) / H3 (A + B + D + F) / H4 (A) + B + (C + D + E) / H5 (C + E) / H6 (A + C + E))

4.60 (1 H, s, H2 (F)),

4.80 (3H, m, H5 (A + B + D)),

5.18 (1H, s, H 1 (D)),

5.30 (1H, s, H (B)),

5.34 (1H, d, H1 (C)),

5.36 (1H, d, H (E)),

5.46 (1H, s, H 1 (F)),

5.57 (1H, s, H1 (A)),

5.95 (1H, d, J = 5Hz, H 4 (F));

4-deoxy-2-O-sulfo-α-L-threo-hex-4-enopyranosyluronic acid - (1-> 4) -2-deoxy-2-sulfoamino-6-O-sulfo-α-D-glucopyranosyl- (1- > 4) 2-O-sulfo-α-L-idopyranosyluronic acid- (1-> 4) -2-deoxy-2-sulfoamino-6-O-sulfo-α-D-glucopyranosyl- (1-> 4) -acid 2- C-Sulfo-α-L-idopyranosyluronic- (1-> 4) -1,6-anhydro-2-deoxy-2-sulfoamino-β-D-glucopyranose

proton spectrum (D ₂ O, 600 MHz, T = 298 K, delta in ppm):

3.25 (2H, m, H 2 (C + E)),

3.42 (lH, m, H 2 (A)),

3.60 (2H, m, H 3 (C + E)), between 3.65 and 4.50 (19 H, block, H2 (B + D) / H 3 (A + B + D + F) / H 4 (A) + B + (C + D + E) / H5 (C + E) / H6 (A + C + E)), · · · · · · · · ··· · ·········· · · · · ·

4.60 (1Η, p, Η2 (F)),

4.80 (3H, m, H5 (A + B + D));

5.18 (1H, s, H1 (D)),

5.31 (1H, s, H (B)),

5.34 (lH, d, H (C)),

5.36 (1H, d, H 1 (E)),

5.46 (1H, s, H 1 (F)),

5.52 (1H, s, H1 (A)),

5.95 (1H, d, J = 5Hz, H 4 (F)).

Example 4

4 ml of 0.0316 mol / L sodium hydroxide solution are added to the reactor maintained at 62 ° C. The solution value is then measured and determined as the target value (pH = 11.8). The stirring and in one 100.8 mg of an oligosaccharide of formula II wherein n is 2, comprising 55% of the ALS derivative is-is (R ^1, R ^2, R ^3, R ⁵ and R ⁶ denote a group of SC / Na, R ⁴ denotes a hydrogen atom and M denotes sodium), 35% of the Al 5 -Is-IIs derivative (R ¹ , R ² , R ³ , and R ⁶ denote a group SC 4 Na, R ⁵ denotes either a group SC 4 Na or an oxygen atom, R ⁴ denotes a hydrogen atom and M denotes sodium) and 10% of the Als-Is-IIIs derivative (R ¹ , R ² , R ³ , R ⁵ and R ⁶ denote a group

SO3N3, R ⁴ denotes a hydrogen atom and M is sodium, and the pool SOIMA C6 carbon is replaced by hydrogen). The pH is then controlled and maintained at pH = 11.8 by continuous addition of 0.5 M sodium hydroxide solution. After 11 hours, the addition of sodium hydroxide is discontinued and the reaction mixture is cooled to 25 ° C. The pH of the solution was adjusted to between 6 and 7 by adding Amberlite IR120 resin. The mixture is ·· ·· ► · · 4

Filter through a Whatman GF / B membrane and then concentrate under reduced pressure (2.7 kPa) at a temperature near 25 ° C. The product is washed with 1.5 ml of distilled water and then lyophilized. In this way 110 mg of an oligosaccharide of the formula I in which n is equal to 2, containing in particular a 1,6-anhydro-Al-Is-Is derivative (R ^{@ 1} , R ^{@ 2} , R ^{@ 3} , R @ ⁵ and R @ ⁶ denote SO3Na, R). ⁴ denotes a hydrogen atom and M denotes sodium) and a 1,6-anhydro-Al-Is-IIs derivative (R ¹ , R ² , R ³ , and R ⁶ denote a SO ₃ Na group, R ⁵ denotes either an SO ₃ Na group or a hydrogen atom , R ⁴ denotes a hydrogen atom and M denotes sodium). Analysis carried out by high-performance liquid chromatography in ion pair mode (ion-pair chromatography) allows the transformation to a derivative of the general formula I to be observed. In this case, high-performance liquid chromatography shows that transformation has occurred for the Als-Is-Is, Als-Is-IIs derivatives.

Example 5

To the reactor, maintained at 66 ° C, was added 8.6 mL of 0.025 mol / L lithium hydroxide solution. The solution value is then measured and determined as the target value (pH = 11.68). While stirring, 50 mg of an oligosaccharide of formula II in which n is equal to 0, R ¹ and R ⁶ denote the SO 3 Na group and M denotes sodium are added at once. The pH is then controlled and maintained at pH = 11.68 by continuous addition of 0.466 mol / L lithium hydroxide solution. After 8 hours, the addition of lithium hydroxide is discontinued and the reaction mixture is cooled to 25 ° C. Analysis by high-performance liquid chromatography in ion pair mode makes it possible to observe the transformation to a derivative of the formula I in which n is 0, R ¹ and R ⁶ denote SO 3 Na and M denotes sodium or lithium. In this case, high performance liquid chromatography showed that the transformation was 100%. The yield determined using an external standard is 81.2%.

Example 6 • ·

• · · · · · · · · · · · ·

8.3 ml of 0.0063 mol / L potassium hydroxide solution are added to the reactor maintained at 66 ° C. The solution value is then measured and determined as the target value (pH = 11.1). While stirring, 50 mg of an oligosaccharide of formula II in which n is equal to 0, R ¹ and R ⁶ denote the group SO 4 Na and M denotes sodium are added at once. The pH is then controlled and maintained at 11.1 by continuous addition of 0.515 mol / L potassium hydroxide solution. After 24 hours, the addition of potassium hydroxide is discontinued and the reaction mixture is cooled to 25 ° C. Analysis by high-performance liquid chromatography in ion pair mode allows the transformation to a derivative of the general formula I in which n is 0, R ¹ and R ⁶ SO ₃ Na and M denotes sodium or potassium. In this case, high performance liquid chromatography showed that the transformation was 100%. The yield determined using an external standard is 75.6%.

Example 7

8.6 ml of 0.025 mol / l cesium hydroxide solution are added to the reactor maintained at 66 ° C. The solution is then measured and determined as a target value (pH = 10.75 µm. 50 mg of an oligosaccharide of formula II in which n is equal to 0, R ¹ and R ⁶ denote SO 3 Na and M denotes sodium are added with stirring. was monitored and maintained at pH 10.75 by continuous addition of a solution of cesium hydroxide in a concentration of 0.476 mol / first 20 hours after the addition of cesium hydroxide discontinued and the reaction was cooled to ^25, C. analysis by high performance liquid chromatography mode ion pairs to track the transformation of the compound of formula I, wherein n is 0, R ¹ and R ⁶ denote a group So3n, and M is sodium or cesium. in this case, high performance liquid chromatography shows that that transformation was carried out at 90, 3% Yield ·· ·· · · · · · · ·

73% determined using an external standard.

Example 8

8.6 ml of 0.0063 mol / l tetrabutylammonium hydroxide solution are added to the reactor maintained at 66 ° C. The solution value is then measured and determined as a target value (pH = 10.95). While stirring, 50 mg of an oligosaccharide of formula II in which n is equal to 0, R ¹ and R ⁶ denote the SO 3 Na group and M denotes sodium are added at once. The pH is then controlled and maintained at 10.95 by continuous addition of 0.521 mol / L tetrabutylammonium hydroxide solution. After 16 hours, the addition of tetrabutylammonium hydroxide is discontinued and the reaction mixture is cooled to 25 ° C. Analysis by high-performance liquid chromatography in ion pair mode allows the transformation to a derivative of formula I in which n is 0, R ¹ and R ⁶ denote a group. SO3Na and M denote sodium or tetrabutylammonium. In this case, high performance liquid chromatography showed that the transformation was 96.7%. The yield determined using an external standard is 65%.

The medicaments according to the invention contain as active ingredient at least one oligosaccharide of the formula I or a mixture of oligosaccharides of the formula I in the form of a composition in which said oligosaccharide or said mixture of oligosaccharides is contained in combination with any other pharmaceutically compatible product which may be inert or physiologically active. The medicaments of the invention may be administered by intravenous, subcutaneous, oral, rectal, topical or inhalational administration.

Sterile compositions for intravenous or subcutaneous administration are generally aqueous solutions. These compositions may also contain additives, in particular wetting agents, isotonizing agents, emulsifying agents, dispersing agents and stabilizing agents. Sterilization of the compositions may be accomplished by several sterilization compositions. By methods such as sterilizing filtration, irradiation, or adding sterilizing agents to the composition. The compositions may also be prepared in the form of solid compositions which may be dissolved immediately before use in sterile water or any other sterile injectable medium.

As solid compositions for oral administration, tablets, pills, powders (gelatin capsules, capsules) or granules may be used. In these compositions, the active ingredient is mixed under a stream of argon with one or more inert diluents such as starch, cellulose, sucrose, lactose or silica. These compositions may also contain substances other than diluents, for example one or more lubricants, such as, in particular, magnesium stearate or talc, an oral absorption enhancer, a colorant, an encapsulating material (dragees), or a lacquer.

As liquid compositions for oral administration, pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing inert diluents such as, in particular, water, ethanol, glycerol, vegetable oils or paraffin oil may be used. These compositions may also contain substances other than diluents, for example wetting, sweetening, thickening, flavoring or stabilizing agents.

Compositions for rectal administration are suppositories or rectal capsules which contain, in addition to the active ingredient, excipients such as cocoa butter, semisynthetic glycerides or polyethylene glycols.

Compositions for topical administration may be, for example, creams, iotions, patches, collars, colutoria, nasal drops or aerosols.

The doses of the active compounds according to the invention depend on the desired effect, the time of treatment and the route of administration chosen; these doses generally range between 0.5 and 0.5. and 10 mg per kilogram body weight of patient per day by subcutaneous administration, i 3 to 60 mg per day for an adult patient weighing 60 kg.

Appropriate dosages will generally be determined by the attending physician depending on the age, body weight and other individual factors of the patient.

The invention also relates to a method for preventing or treating diseases associated with an inflammatory process involving the production of cytotoxic agents, including, in particular, nitric oxide (NO). The oligosaccharides of the formula I can thus be used for the prevention and / or treatment of neurodegenerative diseases in which cerebral inflammation plays a malignant role, which can lead to death and which may include central nervous system ischemia, cerebral ischemia, retina and cochlea ischemia, cardiac ischemia ( myocardial infarction), peripheral ischemia, central nervous system disorders, and in particular skull, spine and combined skull and spine injuries, retinal and cochlear injuries, multiple sclerosis, neuropathic pain and peripheral neuropathy, motor neuron diseases and sclerosis, amyotrophic lateral , Alzheimer's disease, Parkinson's disease, Huntington's chorea and some forms of osteortritis, especially at the joint level.

Claims (15)

Oligosaccharides of the formula I (I) in which n is an integer of 0 to 25, R ¹ , R ³ , R ⁴ and R ⁵ , which are the same or different, denote a hydrogen atom or a SO 3 M, R ² and R ^{6 group} , which are the same or different denote a hydrogen atom or an SO ₃ M group or a COCH ₃ group and M denotes sodium, calcium, magnesium or potassium, a mixture of these oligosaccharides and their diastereoisomers. Oligosaccharides of the formula I according to claim 1, in which R ⁴ represents a hydrogen atom, a mixture of these oligosaccharides and their diastereoisomers. Oligosaccharides of the formula I according to any one of claims 1 or 2, wherein n is an integer from 0 to 10, a mixture of these oligosaccharides and their diastereoisomers. 4. Oligosaccharides of the formula I as claimed in claim 1, wherein n is an integer from 0 to 6, a mixture of these oligosaccharides and their diastereoisomers. 5. Oligosaccharides of the formula I as claimed in one of the claims 1. Metal 1 or 2, wherein n is an integer from 1 to 6, a mixture of these oligosaccharides and their diastereoisomers. Process for the preparation of oligosaccharides of the formula I according to claim 1, characterized in that the alkali metal or quaternary ammonium hydroxide is treated with oligosaccharides of the formula II in which n is an integer from 0 to 25, R ¹ , R ³ , R ⁴ and R ^5, identical or different, denote H or SO 3 M, R ² and R ^6, identical or different, denote a hydrogen atom or a radical SO 3 M or COCH ₃ and M is sodium, calcium, magnesium or potassium, or their mixture, and then the obtained oligosaccharides or mixtures thereof are isolated. 7th The method of claim 6, n by that the the reaction is carried out in water ambient temperature at 40 ° C until 80 ’C and a pH of 10 to 13th Process according to either of Claims 6 and 7, characterized in that a 1 to 5% aqueous solution of alkali metal hydroxide or quaternary ammonium hydroxide is used. A process according to any one of claims 6 to 8, characterized in that the reaction is carried out at a temperature of 60 to 80 ° C. • about · · • · ··· · 70'C Process according to claims 6 to 9, characterized in that the reaction pH is from 11 to 12.5. Process according to claims 6 to 10, characterized in that the alkali metal hydroxide or quaternary ammonium hydroxide is sodium hydroxide, potassium hydroxide, lithium hydroxide, cesium hydroxide or tetrabutylammonium hydroxide. A pharmaceutical composition comprising as active ingredient at least one oligosaccharide according to claim 1. Pharmaceutical composition comprising as active ingredient at least one oligosaccharide according to any one of claims 1 to 5. Use of the oligosaccharides of the formula I according to any one of claims 1 to 5 for the preparation of a medicament for the prevention or treatment of diseases associated with an inflammatory process involving the production of nitric oxide (NO). Use of the oligosaccharides of the general formula I according to claim 14 for the preparation of a medicament intended for the prevention and treatment of cerebral, cardiac or vascular peripheral ischemia, osteoarthritis, central nervous system injury, skull injury, spine injury and combined skull and spine injury, scattered pain, , peripheral neuropathies, motor neoron diseases, amyotrophic lateral sclerosis, neuro-AIDS, Alzheimer's disease, Parkinson's disease and Huntington's chorea.