HK1050535B - Novel oligosaccharides, preparation method and pharmaceutical compositions containing same - Google Patents

Description

New oligosaccharides, process for their preparation and pharmaceutical compositions containing them

Technical Field

The present invention relates to oligosaccharides of the formula:

their mixtures, diastereoisomers, processes for their preparation and pharmaceutical compositions containing them.

Background

Sulfated disaccharides having a 1, 6-anhydro structure at the reducing end have been described by H.P.WESSEL in J.carbohydrate Chemistry, 11(8), 1039-1052 (1992); they are not mentioned as having any pharmacological activity.

Sulfated trisaccharides containing 1, 6-anhydro units have also been described as intermediates for the preparation of higher oligosaccharides in patent EP 84999 and carbohydrate research (carbohydrate. Res.), 141, 273-282(1985) by Y.ICHIKAWA and co-workers. These trisaccharides have low anti-Xa activity.

Detailed Description

In the formula (I), n is an integer of 0 to 25, R₁、R₃、R₄And R₅Identical or different, they represent a hydrogen atom or SO₃M group, R₂And R₆Identical or different, they represent a hydrogen atom or SO₃M or COCH₃And M is sodium, calcium, magnesium or potassium.

These oligosaccharides then contain an even number of sugars.

In the formula (I), R₄Preferably a hydrogen atom.

Preferably, n is an integer from 0 to 10, in particular from 0 to 6, more in particular from 1 to 6.

The oligosaccharide of formula (I) may be prepared by reacting an alkali metal hydroxide or a quaternary ammonium hydroxide with an oligosaccharide of the formula:

wherein n is an integer of 0 to 25, R₁、R₃、R₄And R₅Identical or different, they represent a hydrogen atom or SO₃M group, R₂And R₆Identical or different, they represent a hydrogen atom or SO₃M or COCH₃And M is sodium, calcium, magnesium or potassium, or a mixture thereof.

The reaction is carried out in an aqueous medium at a temperature of 40-80 ℃ and a pH of 10-13.

As alkali metal hydroxides which can be used, sodium hydroxide, potassium hydroxide, lithium hydroxide and cesium hydroxide are cited.

As the quaternary ammonium hydroxide that can be used, tetrabutylammonium hydroxide can be cited.

The amount of alkali metal hydroxide or quaternary ammonium hydroxide should be sufficient to maintain the pH of the reaction medium stable throughout the reaction. Therefore, it is necessary to continuously add the alkali metal hydroxide or the quaternary ammonium hydroxide throughout 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 60-70 ℃.

Advantageously, the reaction pH is between 11 and 12.5.

By acidifying the reaction medium, e.g. by adding substances like Amberlite IR120^*Acidic resins such as resin (Fluka) can stop the reaction.

The oligosaccharide of formula (I) or mixture thereof prepared by the process may be isolated.

Polyacrylamide-agarose (e.g. under the trademark Ultrogel ACA 202) may be used^*Product sold (Biosepra)) type gel was optionally purified by permeation chromatography according to the following protocol to isolate the intermediate oligosaccharides of formula (II). The oligosaccharides of formula (I) wherein n is 0 or 1 can also optionally be purified using an alumina column with a water-ethanol mixture as eluent.

Enzymolysis heparan, or alkali hydrolysis heparan benzyl ester or semi-synthetic heparin benzyl ester to obtain oligosaccharide (III) mixture, and separating by gel chromatography to obtain intermediate oligosaccharide shown in formula (II) and its mixture.

Using polyacrylamide-loaded agarose (e.g. under the trade mark Ultrogel ACA 202)^*Marketed product (Biosepra)) gel-like column. Preferably, a set of polyacrylamide-sepharose columns is used. The number of columns used varies with the gel and the volume of oligosaccharide to be separated. The mixture was eluted using a solution containing phosphate and sodium chloride buffer. Preferably, the phosphate buffer is 0.02 mol/L NaH₂PO₄/Na₂HPO₄(pH7) containing 0.1 mol/l sodium chloride. The different fractions were examined by UV spectroscopy (254 nm) and by ion method (IBF). Then, thus obtainedFractions can optionally be purified, for example, by SAX (Strong anion exchange) chromatography, according to methods known to the person skilled in the art, in particular according to the methods described in K.G.Rice and R.J.Linhardt, Carbohydrate Research 190, 219-233(1989), A.Larnkjaer, S.H.Hansen and P.B.Ostergaard, Carbohydrate Research 266, 37-52(1995) and patent WO 90/01501 (example 2). These fractions are then freeze-dried and then subjected to a gel column, for example Sephadex G10^*Gel columns (Pharmacia Biochemicals) were desalted.

When purification is not carried out by SAX chromatography, these lyophilisates can optionally be purified by simple precipitation or isolated according to methods known to the person skilled in the art, in particular according to the method described in patent FR 2548672. Generally, the following protocol is followed:

the lyophilized fraction to be purified is dissolved in about 10 volumes of distilled water at 25 ℃. The desired oligosaccharides were precipitated by adding methanol or ethanol while controlling their enrichment by CLHP chromatography (high performance liquid chromatography). The addition of methanol or ethanol is determined according to the desired purity and yield of the oligosaccharide. Likewise, this operation can be carried out in several steps, one after the other, starting from the initial solution of the lyophilizate. For this purpose, the insolubilizing agent (methanol or ethanol) is added in small portions and the precipitate formed is isolated after each addition. The precipitate thus prepared was analysed by CLHP chromatography. Depending on the purity and yield required, an appropriate fraction of the precipitate may be collected.

The intermediate product represented by formula (II) in which n is 0, 1 or 2 is preferably obtained by using a mixture (III) obtained by an enzymatic depolymerization method as a starting material.

This depolymerization is carried out using heparinase I (EC 4.2.2.7) in phosphate buffer pH7 in the presence of sodium chloride and BSA (bovine serum albumin) at a temperature of 10-18 deg.C, preferably 15 deg.C, for 8-10 days, preferably 9 days. This depolymerization is stopped, for example, by heating the reaction medium to 100 ℃ for 2 minutes, and the mixture is recovered by lyophilization. Preferably 7UI heparinase I per 25 g heparin is used. Phosphate bufferThe liquor generally contains 0.05 mol/l NaH in the presence of 0.1 mol/l sodium chloride₂PO₄/Na₂HPO₄(pH 7). The BSA concentration is typically 2%.

For the intermediate products of formula (II) in which n ═ 0, 1, 2, 3 or 4, it is preferred to use as starting material the mixture (III) obtained by depolymerization of heparin benzyl ester.

Heparin benzyl esters can be prepared according to the methods described in US 5389618, EP 40144, FR 2548672. The esterification rate is preferably 50 to 100%. More preferably, the esterification rate is 70 to 90%.

The depolymerization process is carried out in an aqueous medium using 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 in a molar concentration of 0.1-0.2 mol/liter, at a temperature of 40-80 ℃ for 5-120 minutes. In a preferred embodiment, the operation is carried out at a temperature of 60 to 70 ℃ for 5 to 15 minutes using 0.15 mol/l sodium hydroxide solution. The depolymerization can be stopped by neutralization with an acid such as acetic acid. After addition of 10% sodium acetate on a weight/volume basis, the oligosaccharide mixture is precipitated by adding methanol, preferably 2 volumes per 1 volume of reaction medium, and then filtered.

According to a preferred aspect of the invention, the oligosaccharide mixture obtained after chemical depolymerization, in the form of an aqueous solution, is enriched by ultrafiltration with an ultrafiltration membrane having a suitable nominal cut-off (Pellicon-type ultrafiltration membrane made of regenerated cellulose sold by Millipore); the type of membrane varies with the enriched oligosaccharide species to be recovered. For oligosaccharides (II) where n is 0, a 1kDa nominal cut-off ultrafiltration membrane should be used, for oligosaccharides (II) where n is1, a 1kDa or 3kDa membrane should be used, for oligosaccharides (II) where n is 2, a 3kDa membrane should be used, and for oligosaccharides (II) where n is 3 or 4, a 5kDa membrane should be used. During this operation, the permeate is recovered and the retentate is discharged. Thus, the enriched product fraction may represent 50-10% of the initial oligosaccharide mixture, while retaining at least 80% of the desired oligosaccharides.

For intermediates of formula (II) wherein n-0-25, it is preferred to use as starting material a mixture (III) obtained by depolymerization of a semisynthetic benzyl ester of a sulfated polysaccharide. The semisynthetic sulfated polysaccharide benzyl ester was prepared using a semisynthetic sulfated polysaccharide from the K5 polysaccharide and following the procedures described in patents WO 94/29352 and WO 96/14425. The esterification, depolymerization and recovery conditions were the same as for heparin benzyl ester described above.

In all of the above methods, the starting heparin may be derived from porcine, ovine or bovine heparin, and may also be derived from mucus, lung or skin of the animal. Preferably, heparin from porcine, ovine mucus or bovine lung is used, more preferably from porcine mucus.

The oligosaccharides of formula (I) have anti-inflammatory properties and can therefore be used for the prevention and treatment of diseases associated with inflammatory processes involving the production of cytotoxic substances such as Nitric Oxide (NO), which are released in particular in induced forms of cytotoxic substances when neutrophils or macrophages migrate in tissues and become activated. Activation, migration, adhesion and infiltration of neutrophils occurs at the site of ischemic tissue due to arterial occlusion or spasm that floods the vessel with such tissue. These ischaemias may occur in the brain (stroke syndrome) or in the region of the myocardium (myocardial infarction) or in the lower extremities (so-called peripheral vascular ischaemia). The oligosaccharides of formula (I) can therefore also be used for the prevention and/or treatment of neurodegenerative diseases in which encephalitis plays a detrimental role which may lead to death, among which mention may be made of cerebral ischemia, myocardial ischemia (myocardial infarction), peripheral ischemia, central nervous system trauma, in particular cranial, spinal and cranial spinal trauma, macular sclerosis, neuropathic pain and non-distal neuropathy, motor neuron diseases, including amyotrophic lateral sclerosis, neurological AIDS, alzheimer's disease, parkinson's disease and Huntington's disease, and certain osteoarthritic diseases, in particular arthritic, osteoarthritis.

According to M.YAMASHITA and co-workers in Eur.J.Pharmacol, 338, 2, 151-158(1997) or J.E.SHELLITO and co-workers in am.J.Respir.CelMoBiol., 13, 1, 45-53(1995) in the induction of NO production by Lipopolysaccharide (LPS) from E.coli_x(nitrite and nitrate) tests demonstrated the anti-inflammatory activity of these products in vivo.

The male CD1 mice (Charles river, 25-35 g) were injected with 0.5 mg/kg oligosaccharide at T0 using bolus intravenous administration and 1 or 2 mg/kg oligosaccharide at T +15 min by subcutaneous administration. At T +30 min, 100 mg/kg Lipopolysaccharide (LPS) from E.coli was administered (Sigmal L3129, serotype 0127: B8). At T +3 hours, 1 or 2 mg/kg oligosaccharide was re-injected subcutaneously. Blood samples were taken at T +5 hours for 30 minutes using eye puncture and NOx (nitrite and nitrate) concentrations in the serum were determined using Griess colorimetry after nitrate was reduced to nitrite using nitrate reductase in the following manner: a12 microliter sample of serum was mixed with 88 microliter of deionized water and incubated at room temperature for 1 hour in the dark with 40 microliter of phosphate buffer (0.31M, pH7.5), 20 microliter of beta-NADPH (reduced nicotinamide adenine dinucleotide phosphate) (0.86mM), 20 microliter of FDA (flavin adenine dinucleotide) (0.11mM) and 20 microliter of nitrate reductase (2U/ml) (Boehringer Mannheim). Adding 10 microliter ZnSO₄(1M) to precipitate the proteins, and after mixing, the samples were centrifuged at 20000 g for 5 minutes. Finally, 50 microliters of the supernatant was incubated with 100 microliters of Griess reagent (1% sulfanilamide in a mixture of 0.1% phosphoric acid/naphthylethylenediamine in deionized water on a volume/volume basis) for 10 minutes at room temperature. Reading the optical density at 540 nm by microplate spectrophotometry; each point was measured twice. KNO₃And NaNO₃Used as a standard for this colorimetric method.

In this test, the oligosaccharides of the invention inhibited NO by more than 50% of the rate_xAnd (4) forming.

Among the preferred oligosaccharides of formula (I), the following oligosaccharides may be mentioned in particular, among which:

n is equal to 0, R₁And R₆Represents SO₃Na group, M is sodiumWith their diastereoisomeric mixtures,

n is equal to 1, R₁、R₂、R₃、R₅、R₆Represents SO₃Na group, R₄Represents a hydrogen atom, M is sodium, with mixtures of their diastereomers,

n is equal to 2, R₁、R₂、R₃、R₅、R₆Represents SO₃Na group, R₄Represents a hydrogen atom, M is sodium, with mixtures of their diastereomers,

n is equal to 2, R₁、R₂、R₃、R₆Represents SO₃Na group, R₅Represents a hydrogen atom or SO₃Na group, R₄Represents a hydrogen atom, M Is sodium, and mixtures of diastereomers thereof (Δ Is-IIs1, 6 anhydro derivatives).

The following examples represent the preparation of oligosaccharides and intermediates of formula (I).

Examples

In these examples, the abbreviations have the following meanings:

Δ Is: (4-deoxy-2-O-sulfo- α -L-threo-hex-enopyrano syluronic acid) - (1 → 4) -2-deoxy-2-sulfamino-6-O-sulfo- α -D-glucopyranose, tetrasodium salt or Δ UAp2S- (1 → 4) - α -D-GlcNp2S6S

Is: (2-sulfo- α -L-iduronosylturonic acid) - (1 → 4) -2-deoxy-2-sulfamino-6-O-sulfo- α -D-glucopyranose, tetrasodium salt or α -L-IdoAp2S- (1 → 4) - α -D-GlcNp2S6S

IIs: (alpha-L-iduronosylguronic acid) - (1 → 4) -2-deoxy-2-sulfamino-6-O-sulfo-alpha-D-glucopyranose, trisodium salt or alpha-L-IdoAp- (1 → 4) -alpha-D-GlcNp 2S6S

IIIs: (2-sulfo- α -L-iduronosylturonic acid) - (1 → 4) -2-deoxy-2-sulfamino- α -D-glucopyranose, trisodium salt or α -L-IdoAp2S- (1 → 4) - α -D-GlcNp2S

IdoAp: iduronyl uronic acid

GlcNp: 2-deoxy-2-aminopyranyl glucose

Δ Uap: 4-deoxy-alpha-L-threo-hex-enopyrano syluronic acid

S: sulfates of sulfuric acid

Preparation of the mixture of the formula (II)

Example a-preparation of an oligosaccharide of formula (II) wherein n-0, 1 and 2 by enzymatic depolymerization and isolation

25 g heparin dissolved in 250 ml phosphate buffer, the buffer containing 0.05 mol/L NaH₂PO₄/Na₂HPO₄(pH7), 0.2 mol/l sodium chloride and 2% BSA (bovine serum albumin). To this mixture was added 7UI heparinase I (EC 4.2.2.2.7) and the resulting solution was cooled to 15 ℃ and then kept at this temperature throughout the depolymerization reaction. The progress of the reaction was followed by sampling in aliquots in portions and analysis by gel permeation chromatography. After 9 days, the enzymatic reaction can be stopped by heating the reaction medium to 100 ℃ for 2 minutes. The cooled mixture was then lyophilized. This gives an oligosaccharide mixture of the formula (III).

The oligosaccharide mixture of formula (III) obtained is then chromatographed as follows: the column packed with polyacrylamide-agarose gel of known trade name U1trogel ACA202 was used for chromatographic separation, followed by a buffer solution containing phosphate at pH7 (0.02 mol/l NaH)₂PO₄/Na₂HPO₄) And 0.1 mol/l sodium chloride. Detection was carried out by UV spectroscopy (254 nm) and by the ion method (IBF). These products can optionally be purified by SAX (strong anion exchange) chromatography or by fractional precipitation according to the method described in patent FR 2548672. The recovered product fraction was lyophilized and then used as sephadex G10^*Desalting with a gel (Pharmacia Biochemicals) packed column.

In this way, 3 g of Is disaccharide, 1100 mg of a hexasaccharide mixture typically containing 55% of derivatives of Δ Is-Is, 35% of Δ Is-IIs and 10% of Δ Is-IIIs was obtained. This latter mixture can be purified according to methods well known to those skilled in the art, separated into individual components, or used as such for conversion into the 1, 6 anhydro derivative of formula (I). It Is noteworthy that the Δ Is-IIIs hexasaccharide did not lead to the compound of formula (I) during this transformation.

Example B-preparation of oligosaccharide of formula (II) wherein n ═ 0, 1, 2, 3, or 4 by depolymerization and isolation of heparin benzyl ester

a-preparation of heparin benzyl ester

Heparin benzyl ester was prepared according to example 3 of patent US 5389618.

b-depolymerization

100 g heparin benzyl ester was dissolved in 1.9 l demineralized water. The mixture was warmed to 60 ℃ with stirring. After a homogeneous solution was obtained, about 35 ml of 23% sodium hydroxide solution was added only once. After 10 minutes of reaction, the solution was cooled again and then neutralized with 80 ml of about 2N acetic acid solution. To this solution was added 10% sodium acetate on a weight/volume basis. The oligosaccharide mixture was precipitated by adding about 2 volumes of methanol. The precipitate was isolated by filtration, washed twice with methanol and dried at 50 ℃ under reduced pressure. After drying 73.8 g of oligosaccharide (II) mixture are obtained.

c-enrichment of oligosaccharides wherein n ═ 1

30 grams of the oligosaccharide mixture previously obtained was dissolved in about 35 volumes of water. This solution was ultrafiltered using a 3kDa membrane (Pellicon). While 600 ml of permeate was withdrawn, the retentate was diluted with 500 ml of water. This operation was continued until another 450 ml of permeate was withdrawn. The two permeates were combined and then concentrated to dryness under reduced pressure. 6.1 g of a yellowish white solid are obtained. The solid was analyzed by gel permeation chromatography and, as a result, it contained about 30% of oligosaccharide represented by formula (II) wherein n-1.

d-separation of the ultrafiltered oligosaccharide mixture

The enriched mixture was separated on a polyacrylamide-agarose column loaded with the known trade name Ultrogel ACA202 (using 4 columns in series of 10 cm diameter and 50 cm height). 5 g of the enriched mixture obtained by ultrafiltration were dissolved in 25 ml of water and then eluted with 0.2 mol/l sodium chloride solution at a rate of 5 ml/min. A plurality of 25 ml fractions were recovered at the bottom of the column. The product was detected by UV spectroscopy (254 nm) and by ion method (IBF). The product fractions where n-1 were combined, lyophilized, and desalted using sephadex G10 gel-packed columns. After lyophilization, 1 g of a derivative (R) Is obtained which typically contains 70% of a.DELTA.is-Is of the formula II₁、R₂、R₃、R₅、R6＝SO₃Na; (ii) a R4 ═ H and M ═ Na). Derivatives of Δ Is-Is may optionally be purified by SAX (strong anion exchange) chromatography or according to the method described in patent FR 2548672, by fractional precipitation according to preferred characteristics.

Example 1

To the reactor maintained at 66 ℃ was added 5 ml of 0.0063 mol/l sodium hydroxide solution. The pH of the solution was measured at this time and taken as the target value (pH 11.35). 30 mg of an oligosaccharide of the formula (II) in which n is equal to 0 and R is added in one portion with stirring₁And R6 ═ SO₃A Na group; m ═ Na. The pH can then be adjusted by continuous addition of 0.5 mol/l sodium hydroxide solution and maintained at pH 11.35. After 10 hours, the sodium hydroxide solution was stopped and the reaction mixture was cooled to 25 ℃. The pH of the solution was then adjusted to 6-7 by the addition of Amberlite IR120 resin. The mixture was filtered through a Whatman GF/B membrane and then concentrated to dryness under reduced pressure (2.7 kPa) at a temperature of about 25 ℃. The product was dissolved in 0.5 ml of distilled water and lyophilized. This gave 29 mg of a diastereoisomeric mixture of oligosaccharides of the formula (I) in which n is equal to 0 and R is₁And R₆＝SO₃A Na group; m ═ Na [ (4-deoxy-2-O-sulfo- α -L-threo-hex-4-enopyrano syluronic acid) - (1 → 4) -1, 6-anhydro-2-deoxy-2-sulfamino-beta-D-mannopyranose, trisodium salt]]: at D₂Mass spectrum in O, 400MHz, T298K, δ, in ppm: 3.15(1H, s, H2), 3.75(2H, m, H6 and H3), 3.88(1H, m, H4), 4.20(1H, d, J ═ 8Hz, H6), 4.22(1H, t, J ═ 5Hz, H3 '), 4.58(1H, m, H2'), 4.75(1H, m, H5), 5.53(1H, s, H1), 5.60(1H, dd, J ═ 6 and 1Hz, H1 '), 6.03(1H, d, J ═ 5Hz, H4'); (4-deoxy-2-O-sulfo- α -L-threo-hex-4-enopyrano syluronic acid) - (1 → 4) -1, 6-anhydro-2-deoxy-2-sulfamino- β -D-glucopyranose, trisodium salt): mass spectrum in D2O, 400MHz, T298K, δ, in ppm: 3.34(1H, dd, J ═ 7 and 2Hz, H2), 3.72(1H, t, J ═ 8Hz, H6), 3.90(1H, m, H3), 4.03(1H, s, H4), 4.20(1H, d, J ═ 8Hz, H6), 4.23(1H, t, J ═ 5Hz, H3 '), 4.58(1H, m, H2'), 4.78(1H, m, H5), 5.50(1H, s, H1), 5.60(1H, dd, J ═ 6 and 1Hz, H1 '), 6.03(1H, d, J ═ 5Hz, H4')]。

Example 2

To the reactor maintained at 62 ℃ was added 33.3 ml of 0.0063 mol/l sodium hydroxide solution.

The pH of the solution was measured at this time and taken as the target value (pH 11.15). 200 mg of an oligosaccharide of the formula (II) in which n is equal to 1 and R is added in one portion with stirring₁、R₂、R₃、R₅And R₆Represents SO₃A Na group; r₄Represents a hydrogen atom and M is sodium. The pH can then be adjusted by continuous addition of 0.5 mol/l sodium hydroxide solution and maintained at pH 11.15. After 12 hours, the addition of sodium hydroxide was stopped and the reaction mixture was cooled to 25 ℃. The pH of the solution was then adjusted to 6-7 by addition of Amberlite IR120 resin. The mixture was filtered through a Whatman GF/B membrane and then concentrated to dryness under reduced pressure (2.7 kPa) at a temperature of about 25 ℃. The product was dissolved in 3 ml of distilled water and lyophilized. This gives 230 mg of an oligosaccharide of the formula (I) in which n is equal to 1 and R₁、R₂、R₃、R₅And R₆Represents SO₃A Na group; r₄Represents a hydrogen atom and M is sodium, in diastereoisomerism(4-deoxy-2-O-sulfo-alpha-L-threo-hex-4-enopyrano syluronic acid) - (1 → 4) -2-deoxy-2-sulfamino-6-O-sulfo-alpha-D-glucopyranosyl uronic acid- (1 → 4) -2-O-sulfo-alpha-L-iduopyranosyl uronic acid- (1 → 4) -1, 6-anhydro-2-deoxy-2-sulfamino-beta-D-mannopyranose, heptasodium salt]]: at D₂Mass spectrum in O, 400MHz, T298K, δ, in ppm: 3.15(1H, s, H2), 3.25(1H, m, H2 "), 3.60(1H, m, H3"), 3.70-4.70(14H, clustered, H3/H4/H6, H2 '/H3'/H4 '/H5', H4 "/H5"/H6 ", H2 '"/H3' "), 4.75(1H, m, H5), 5.20-5.40(2H, m, H1 'and H1"), 5.45(1H, m, H1' "), 5.56(1H, m, H1), 5.94(1H, d, J ═ 5Hz, H4); (4-deoxy-2-O-sulfo- α -L-threo-hex-4-enopyrano syluronic acid) - (1 → 4) -2-deoxy-2-sulfamino-6-O-sulfo- α -D-glucopyranosyl- (1 → 4) -2-O-sulfo- α -L-iduopyranosyl uronic acid- (1 → 4) -1, 6-anhydro-2-deoxy-2-sulfamino- β -D-glucopyranosyl, heptasodium salt)]: at D₂Mass spectrum in O, 400MHz, T298K, δ, in ppm: 3.25(1H, m, H2 "), 3.42(1H, dd, J ═ 4 and 1Hz, H2), 3.60(1H, m, H3"), 3.70-4.70(14H, clustered, H3/H4/H6, H2 '/H3'/H4 '/H5', H4 "/H5"/H6 ", H2 '"/H3' "), 4.75(1H, m, H5), 5.20-5.40(2H, m, H1 'and H1"), 5.45(1H, m, H1' "), 5.52(1H, m, H1), 5.94(1H, d, J ═ 5Hz, H4)]。

Example 3

To the reactor maintained at 62 ℃ was added 16.7 ml of 0.0063 mol/l sodium hydroxide solution. The pH of the solution was measured at this time and taken as the target value (pH 11.7). 100 mg of an oligosaccharide of formula (II) wherein n is equal to 2 and R is added in one portion with stirring₁、R₂、R₃、R₅And R₆Represents SO₃A Na group; r₄Represents a hydrogen atom and M is sodium. The pH can then be adjusted by continuous addition of 0.5 mol/l sodium hydroxide solution and maintained at pH 11.7. After 10 hours, the addition of sodium hydroxide was stopped and the reaction mixture was cooled to 25 ℃. The pH of the solution was adjusted to 6-7 by the addition of Amberlite IR120 resin. The mixture was filtered through Whatman GF/B membranes and thenConcentrated to dryness at a temperature of about 25 ℃ under reduced pressure (2.7 kPa). The product was dissolved in 3 ml of distilled water and lyophilized. This gives 108 mg of an oligosaccharide of the formula (I) in which n is equal to 2 and R₁、R₂、R₃、R₅And R₆Represents SO₃A Na group; r₄Represents a hydrogen atom and M is sodium, in the form of a mixture of diastereomers. The sugar consisting of the hexasaccharide is labeled a-F, a being the 1, 6-anhydro residue and F being the unsaturated uronic acid residue. [ (4-deoxy-2-O-sulfo- α -L-threo-hex-4-enopyrano syluronic acid) - (1 → 4) -2-deoxy-2-sulfamino-6-O-sulfo- α -D-glucopyranosyl- (1 → 4) -2-O-sulfo- α -L-iduronosylropyranosyl uronic acid- (1 → 4) -2-deoxy-2-sulfamino-6-O-sulfo- α -D-glucopyranosyl- (1 → 4) -2-O-sulfo- α -L-iduronyl uronic acid- (1 → 4) -1, 6-anhydro-2-deoxy-2-sulfamino-beta-D-mannopyranose, undecyl sodium salt]]: at D₂Mass spectrum in O, 600MHz, T298K, δ, in ppm: 3.15(1H, s, H2(A)), 3.25(2H, m, H2(C + E)), 3.60(2H, m, H3(C + E)), 3.65-4.50(19H, clustered, H2(B + D)/H3(A + B + D + F)/H4(A + B + C + D + E)/H5(C + E)/H6(A + C + E)), 4.60(1H, s, H2(F)), 4.80(3H, m, H5(A + B + D)), 5.18(1H, s, H1(D)), 5.30(1H, s, H1(B)), 5.34(1H, D, H1(C)), 5.36(1H, D, H1 (E5.46 (1H, s 1)); 5.57(1H, s, H1 (A)); 5.95(1H, d, J ═ 5Hz, H4 (F)); (4-deoxy-2-O-sulfo- α -L-threo-hex-4-enopyrano syluronic acid) - (1 → 4) -2-deoxy-2-sulfamino-6-O-sulfo- α -D-glucopyranosyl- (1 → 4) -2-O-sulfo- α -L-iduopyranosyl uronic acid- (1 → 4) -1, 6-anhydro-2-deoxy-2-sulfamino-beta-D-glucopyranose, undeca-sodium salt)]: mass spectrum in D2O, 600MHz, T298K, δ, in ppm: 3.25(2H, m, H2(C + E)), 3.42(1H, m, H2(A)), 3.60(2H, m, H3(C + E)), 3.65-4.50(19H, clustered, H2(B + D)/H3(A + B + D + F)/H4(A + B + C + D + E)/H5(C + E)/H6(A + C + E)), 4.60(1H, s, H2(F)), 4.80(3H, m, H5(A + B + D)), 5.18(1H, s, H1(D)), 5.31(1H, s, H1(B)), 5.34(1H, D, H1(C)), 5.36(1H, D, H1 (E5.46 (1H, s 1)); 5.52(1H, s,H1(A))；5.95(1H，d，J＝5Hz，H4(F))。

example 4

To the reactor maintained at 62 ℃ was added 4 ml of 0.0316 mol/l sodium hydroxide solution. The pH of the solution was measured at this time and taken as the target value (pH 11.8). 100.8 mg of an oligosaccharide mixture of formula (II) wherein n Is equal to 2 and containing 55% of delta Is-Is (R) were added in one portion under stirring₁、R₂、R₃、R₅And R₆Represents SO₃A Na group; r₄Representing a hydrogen atom and M representing Na), 35% of. DELTA.is-Is-IIs (R)₁、R₂、R₃And R₆Represents SO₃A Na group; r₅Represents SO₃Na group or hydrogen atom, R₄Represents a hydrogen atom and M represents sodium), and 10% of. DELTA.is-Is-IIIs (R)₁、R₂、R₃、R₅And R₆Represents SO₃A Na group; r₄SO representing a hydrogen atom and M being sodium, C6 carbon₃The Na function is replaced by hydrogen). The pH can then be adjusted by continuous addition of 0.5 mol/l sodium hydroxide solution and maintained at pH 11.8. After 11 hours, the addition of sodium hydroxide was stopped and the reaction mixture was cooled to 25 ℃. The pH of the solution was then adjusted to 6-7 by the addition of Amberlite IR120 resin. The mixture was filtered through a Whatman GF/B membrane and then concentrated to dryness under reduced pressure (2.7 kPa) at a temperature of about 25 ℃. The product was dissolved in 1.5 ml of distilled water and lyophilized. This gives 110 mg of an oligosaccharide mixture of the formula (I) in which n Is equal to 2, in particular containing the 1, 6-anhydro derivative (. DELTA.is-Is (R)₁、R₂、R₃、R₅And R₆Represents SO₃A Na group; r₄Representing a hydrogen atom and M being sodium) and a.DELTA.is-Is-IIs 1, 6-anhydro derivative (R)₁、R₂、R₃And R₆Represents SO₃A Na group; r₅Represents SO₃Na group or hydrogen atom, R₄Represents a hydrogen atom and M is sodium). Ion-pair CLHP (high performance liquid chromatography) analysis allows to follow the conversion to the derivatives of formula (I). In this case, the CLHP assay showed that derivatives of. DELTA.is-Is, and. DELTA.is-Is-IIsThe transformation has occurred.

Example 5

To the reactor maintained at 66 ℃ was added 8.6 ml of 0.025 mol/l lithium hydroxide solution. The pH of the solution was measured at this time and taken as the target value (pH 11.68). 50 mg of an oligosaccharide of the formula (II) in which n is equal to 0 and R is added in one portion with stirring₁And R₆Represents SO₃The Na group and M are sodium. The pH can then be adjusted by continuous addition of 0.466 mol/l lithium hydroxide solution and maintained at pH 11.68. After 8 hours, the addition of lithium hydroxide was stopped and the reaction mixture was cooled to 25 ℃. Ion-pair CLHP (high performance liquid chromatography) analysis can be followed up to where n equals 0, R₁And R₆Represents SO₃Conversion of a derivative of formula (I) wherein the Na group and M are sodium or lithium. In this case, the CLHP assay indicated that the conversion reached 100%. The yield using the external standard was 81.2%.

Example 6

To the reactor maintained at 66 ℃ was added 8.3 ml of 0.0063 mol/l potassium hydroxide solution. The pH of the solution was measured at this time and taken as the target value (pH 11.1). 50 mg of an oligosaccharide of the formula (II) in which n is equal to 0 and R is added in one portion with stirring₁And R₆Represents SO₃The Na group and M are sodium. The pH can then be adjusted by continuous addition of 0.515 mol/l potassium hydroxide solution and maintained at pH 11.1. After 24 hours, the addition of potassium hydroxide was stopped and the reaction mixture was cooled to 25 ℃. Ion-pair CLHP (high performance liquid chromatography) analysis can be followed up to where n equals 0, R₁And R₆Represents SO₃Conversion of a derivative of formula (I) wherein the Na group and M are sodium or potassium. In this case, the CLHP assay indicated that the conversion reached 100%. The yield using the external standard was 75.6%.

Example 7

To the reactor maintained at 66 ℃ was added 8.3 ml of 0.0063 mol/l cesium hydroxide solution. The pH of the solution was measured at this time and taken as the target value (pH 10.75). 50 mg of compound of the formula (II) are added in one portion with stirringOligosaccharide wherein n is equal to 0, R₁And R₆Represents SO₃The Na group and M are sodium. The pH can then be adjusted by the continuous addition of 0.476 mol/l cesium hydroxide solution and maintained at pH 10.75. After 20 hours, the addition of cesium hydroxide was stopped and the reaction mixture was cooled to 25 ℃. Ion-pair CLHP (high performance liquid chromatography) analysis can be followed up to where n equals 0, R₁And R₆Represents SO₃Conversion of a derivative of formula (I) wherein the Na group and M are sodium or cesium. In this case, CLIP measurements indicated that the conversion reached 90.3%. The yield using the external standard was 73%.

Example 8

To the reactor maintained at 66 ℃ was added 8.3 ml of 0.0063 mol/l tetrabutylammonium hydroxide solution. The pH of the solution was measured at this time and taken as the target value (pH 10.95). 50 mg of an oligosaccharide of the formula (II) in which n is equal to 0 and R is added in one portion with stirring₁And R₆Represents SO₃The Na group and M are sodium. The pH can then be adjusted by the continuous addition of 0.521 mol/l tetrabutylammonium hydroxide solution and maintained at pH 10.95. After 16 hours, the addition of cesium hydroxide was stopped, the reaction mixture was cooled to 25 ℃ and the analysis by ion-pair CLHP (high performance liquid chromatography) was followed up to a value in which n is equal to 0, R₁And R₆Represents SO₃Conversion of a derivative of formula (I) wherein the Na group and M are sodium or tetrabutylammonium. In this case, CLHP measurement indicated that the conversion reached 96.7%. The yield using the external standard was 65%.

The medicament of the invention contains at least one oligosaccharide or a mixture of oligosaccharides of formula (I) as active ingredient, in the form of a composition, wherein the active ingredient in combination with any other pharmaceutically compatible compound which may be inert or physiologically active, can be administered by intravenous, subcutaneous, oral, rectal, topical or pulmonary (inhalation) 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 can be performed in a variety of ways, such as sterile filtration by adding a sterilizing agent to the composition, or by irradiation. These compositions may also be prepared as sterile solid compositions which may be dissolved in sterile water or any other injectable sterile medium at the time of use.

As solid compositions for oral administration, tablets, pills, powders (gelatin capsules, sachets) or granules may be used. In these compositions, the active ingredient is mixed with one or more inert diluents such as starch, cellulose, sugar, lactose or silica under a stream of argon. These compositions may also contain substances other than diluents, for example one or more lubricants, like magnesium stearate or talc, agents promoting absorption in the oral cavity, colouring agents, coating agents (dragees) or varnishes.

As liquid compositions for oral administration, pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing inert diluents such as water, ethanol, glycerol, vegetable or paraffin oils may be used. These compositions may also contain other substances in addition to the diluents, for example wetting agents, sweeteners, thickeners, flavorants or stabilizers.

Compositions for rectal administration are suppositories or rectal capsules containing, in addition to the active product, excipients such as coconut oil, semisynthetic glycerides or polyethylene glycols.

Topical compositions may be, for example, creams, lotions, eye washes, mouthwashes, nasal drops or aerosols.

The dosage depends on the desired effect, the time of treatment and the route of administration; these doses are generally 0.5-10 mg per kg of subcutaneous drug per day, i.e. 3-60 mg per day for an adult weighing 60 kg.

Generally, the physician will determine the appropriate dosage depending on the age, weight and all other factors inherent in the subject to be treated.

The invention also relates to the prevention or treatment of diseases associated with inflammatory processes that lead to the production of cytotoxic substances (e.g. nitric oxide, NO). The oligosaccharides of formula (I) can therefore be used for the prevention and/or treatment of neurodegenerative diseases in which encephalitis plays a detrimental role that may lead to death, among which mention may be made of central nervous system ischemia, cerebral ischemia, retinal and cochlear ischemia, myocardial ischemia (myocardial infarction), peripheral ischemia, central nervous system trauma, in particular cranial, spinal and cranial spinal trauma, retinal and cochlear trauma, macular sclerosis, neuropathic pain and peripheral neuropathy, motor neuron diseases, including amyotrophic lateral sclerosis, psychogenic aids, alzheimer's disease, parkinson's disease and Huntington's disease, and certain osteoarthritic diseases, in particular arthritic osteoarthritis.

Claims (31)

1. A purified oligosaccharide of formula (I):

wherein n is 0 or an integer of 1 to 2, R₁、R₂、R₃、R₅And R₆Which may be the same or different, represent a hydrogen atom or SO₃An M group; r₄Represents a hydrogen atom and M is sodium, calcium, magnesium or potassium.

2. The purified oligosaccharide of formula (I) of claim 1, wherein n is 0.

3. The purified oligosaccharide of formula (I) of claim 1, wherein n is 1.

4. The purified oligosaccharide of formula (I) of claim 1, wherein n is 2.

5. The purified oligosaccharide of formula (I) of claim 1, wherein n is 0 and R is₁And R₆＝SO₃Na。

6. The purified oligosaccharide of formula (I) of claim 1, wherein n is1 and R is₁、R₂、R₃、R₅And R₆＝SO₃Na。

7. The purified oligosaccharide of formula (I) of claim 1, wherein n is 2 and R is₁，R₂，R₃，R₅And R₆＝SO₃Na。

8. The purified oligosaccharide of formula (I) of claim 1, wherein n is 2 and R is₁，R₂、R₃And R₆＝SO₃Na and R₅H or SO₃Na。

9. A composition comprising a purified oligosaccharide of formula (I) or a diastereomer thereof, wherein n is 0 or an integer from 1 to 2, R₁、R₂、R₃、R₅And R₆Which may be the same or different, represent a hydrogen atom or SO₃An M group; r₄Represents a hydrogen atom and M is sodium, calcium, magnesium or potassium

10. A composition comprising a purified oligosaccharide of formula (I) or diastereoisomer thereof according to claim 9, wherein n is 0.

11. A composition comprising a purified oligosaccharide of formula (I) or diastereoisomer thereof according to claim 9, wherein n is 1.

12. A composition comprising a purified oligosaccharide of formula (I) or diastereoisomer thereof according to claim 9, wherein n is 2.

13. The composition comprising purified oligosaccharides of formula (I) or diastereomers thereof according to claim 9, wherein n is 0 and R is₁And R₆＝SO₃Na。

14. The composition comprising purified oligosaccharides of formula (I) or diastereomers thereof according to claim 9, wherein n is1 and R is₁，R₂，R₃，R₅And R₆＝SO₃Na。

15. The composition comprising purified oligosaccharides of formula (I) or diastereomers thereof according to claim 9, wherein n is 2 and R is₁，R₂，R₃，R₅And R₆＝SO₃Na。

16. The composition comprising purified oligosaccharides of formula (I) or diastereomers thereof according to claim 9, wherein n is 2 and R is₁，R₂，R₃And R₆＝SO₃Na and R₅H or SO₃Na。

17. A process for the preparation of oligosaccharides of formula (I) or mixtures thereof, wherein n is 0 or an integer from 1 to 2, R₁、R₂、R₃、R₅And R₆Which may be the same or different, may be substituted forEpi hydrogen atom or SO₃An M group; r₄Represents a hydrogen atom and M is sodium, calcium, magnesium or potassium, which process comprises reacting an oligosaccharide of formula (II) or a mixture thereof with an alkali metal hydroxide or a quaternary ammonium hydroxide, wherein the reaction is carried out in an aqueous medium at a temperature of from 40 to 80 ℃ and a pH of from 10 to 13,

in the formula (II), n, R₁，R₂，R₃，R₄，R₅And R₆As defined above.

18. The process of claim 17, wherein the reaction is carried out using a 1-5% aqueous solution of an alkali metal hydroxide or quaternary ammonium hydroxide.

19. The process of claim 17, wherein the reaction is carried out at a temperature of 60-70 ℃.

20. The process of claim 17, wherein the pH of the reaction is 11-12.5.

21. The method of claim 17, wherein the alkali metal or quaternary ammonium hydroxide is sodium hydroxide, potassium hydroxide, lithium hydroxide, cesium hydroxide, or tetrabutylammonium hydroxide.

22. The method of claim 17, wherein the oligosaccharide of formula (I) or mixture thereof is isolated.

23. An oligosaccharide of formula (I) or a mixture thereof, wherein n is 0 or an integer from 1 to 2, R₁、R₂、R₃、R₅And R₆Which may be identical or different, represent a hydrogen atom orSO₃An M group; r₄Represents a hydrogen atom and M is sodium, calcium, magnesium or potassium, which is obtainable by a process comprising reacting an oligosaccharide of the formula (II) wherein n, R are hydrogen and M is sodium, calcium, magnesium or potassium, with an alkali metal hydroxide or a quaternary ammonium hydroxide, wherein the reaction is carried out in an aqueous medium at a temperature of from 40 to 80 ℃ and a pH of from 10 to 13₁，R₂，R₃，R₄，R₅And R₆As defined above

24. A pharmaceutical composition comprising as an active ingredient at least one purified oligosaccharide according to claim 1.

25. A pharmaceutical composition comprising as an active ingredient at least one purified oligosaccharide according to any one of claims 2-8.

26. A pharmaceutical composition comprising as an active ingredient at least one composition according to claim 9.

27. A pharmaceutical composition comprising as active ingredient at least one composition according to any one of claims 10 to 16.

28. Use of a purified oligosaccharide of formula (I) according to claim 1 in the manufacture of a medicament for the prevention or treatment of a disease associated with an inflammatory process leading to nitric oxide production.

29. The use according to claim 28, wherein the disease is cerebral ischemia, myocardial ischemia, peripheral vascular ischemia, osteoarthritis, central nervous system trauma, macular sclerosis, neuropathic pain and peripheral neuropathy, motor neuron disease, neurological AIDS, Alzheimer's disease, Parkinson's disease and Huntington's disease.

30. The use of claim 29, wherein the central nervous system trauma is cranial, spinal, and cranial spinal trauma.

31. The use of claim 29, wherein the motor neuron disease is amyotrophic lateral sclerosis.