FR2533219A1 - New disaccharides composed of units having a D-glucosamine and L-iduronic acid structure and method for preparing them - Google Patents

Abstract

New disaccharides composed of units having a D-glucosamine and L-iduronic acid structure and method for preparing them. The invention relates to disaccharides of formula: in which R represents an alkyl or acyl radical, A a hydrogen atom, an alkyl radical or an organic or inorganic cation, and R1 to R4 a hydrogen atom or a group that protects the -OH radical.

Description

"New disaccharides with patterns. D-clucosamine structure and L-iduronic acid and process for their preparation.tt
The invention relates to novel disaccharides formed with patterns having a D-glucosamine structure and L-iduronic acid and to a process for their preparation.

dans laquelle -R représente un radical aliphatique ou aromatique, notamment alcoyle comportant i à 4 atomes de carbone, en particulier un radical méthyle, ---A représente un atome d'hydrogène, un radical alcoyle comportant de 1 à 4 atomes de carbone, en particulier, un radical méthyle, ou un cation métallique, en particulier un cation alcalin, plus spécialement de sodium, ou un cation organique tel qu'un dérivé de base organique azotée,
- Y représente un anion, en particulier, un groupe sulfate, éventuellement sous forme de sel avec un cation organique ou minéral, et,dans ce dernier cas, en particulier un cation alcalin, - Z représente un groupe fonctionnel azoté, en particulier, un groupe azide,ou un groupe amine éventuellement substitué, tel qu'un groupe de type -NHY dans lequel Y présente la signification donnée ci-dessus, et -R1 à R4 représentent un atome d'hydrogène, ou un groupe protecteur de radical -OH.It relates more particularly to disaccharides in which the D-glucosamine motif is a motif
N-sulfate 6-o sulfate-D-glucosamine
These compounds correspond to the following formula (I)

in which -R represents an aliphatic or aromatic radical, in particular an alkyl radical containing i to 4 carbon atoms, in particular a methyl radical, --- A represents a hydrogen atom, an alkyl radical comprising from 1 to 4 carbon atoms, in particular, a methyl radical, or a metal cation, in particular an alkaline cation, more particularly of sodium, or an organic cation such as a derivative of nitrogenous organic base,
- Y represents an anion, in particular, a sulphate group, optionally in the form of a salt with an organic or inorganic cation, and, in the latter case, in particular an alkali cation, - Z represents a nitrogen functional group, in particular, a azide group, or an optionally substituted amine group, such as a group of type -NHY in which Y has the meaning given above, and -R1 to R4 represent a hydrogen atom, or a radical protecting group -OH .

 In a preferred group of disaccharides of the invention R1 to R4 represent hydrogen atoms.

 Preferred disaccharides of this group contain in position 2 of the glucosamine motif a radical -NHSO3, this radical advantageously being in the form of a salt, in particular with sodium.

 Other disaccharides additionally contain, as a substituent -OY a group -OSO3, also advantageously in the form of seltz, in particular with sodium.

Advantageously, the disaccharides of the invention constitute precursors or correspond to patterns constituting the heparin or heparan sulfate chain. These compounds are precursors or are also found in fragments or fractions having, in particular, a specific anti-Xa activity (Yin
Wessler) higher than that of heparin and overall anticoagulant activity, measured by titer
USP, lower.

 It will be recalled that the titles Yin and Wessler and USP are defined in particular in patent application FR No. 78 31857 of November 6, 1978 in the name of the Applicant.

 The products of the invention are therefore particularly advantageous as synthetic intermediates for obtaining products endowed with activity in specific coagulation tests for certain factors and more particularly for factor Xa.

 They are also advantageously usable as reference products for the study of this type of structure.

 The invention also relates to a process for the preparation of the compounds defined above.

 According to this process, a D-glucosamine derivative is reacted with a Liduronic acid derivative in which all the positions are blocked except the positions 1 and 4 respectively which must intervene for the establishment of the glycosidic bond.

 The halides of the glucosamine derivative being easily accessible, condensation is advantageously based on a reaction between a glucosamine halide and an alcohol function of the iduronic acid derivative.

 The other positions of these compounds are blocked by blocking groups which are not involved in the condensation reaction, which are compatible with each other and which can be eliminated either at once or in sequence to introduce the desired functional groups or to release certain -OH groups.

 For the positions intended to be occupied by -oY groups, acetyl groups are used as blocking groups in the starting materials.

 From these acetyl groups, it is easy to liberate the —OH radicals and then to subject them to a sulfation reaction. As for the positions which will be occupied by free —OH groups, they are advantageously substituted by inert radicals during the sulfation reaction such as benzyl radicals.

dans lesquelles Ac représente un groupe acétyle, Bn un groupe benzyle, A représente un radical alcoyle et
R présente les significations données ci-dessus, les conditions mises en oeuvre, en particulier de concentration en réactifs, de durée et de température étant choisies pour réaliser la condensation désirée.In addition, it proves to be advantageous to use as substituent Z, a precursor group of amine group, inert with respect to the salification reactions used; such as an azido group
According to a preferred embodiment of the invention, the monosaccharides of formula-II are reacted and
III following

in which Ac represents an acetyl group, Bn a benzyl group, A represents an alkyl radical and
R has the meanings given above, the conditions used, in particular the concentration of reactants, the duration and the temperature being chosen to achieve the desired condensation.

 The resulting disaccharide is then treated in order to first substitute position 6 of the glucosamine motif using an appropriate agent. Thus when, for example, it is desired to introduce a sulfate group in position 6, the disaccharide obtained at the end of the condensation reaction is first subjected to a saponification treatment, then to the action of an agent. sulfation such as a trimethylamine / SO3 complex.

 During a subsequent step, the disaccharide is subjected to a hydrogenation treatment in order to remove the benzyl protecting groups and to transform the azide group into an amino group which will be easily substituted, in particular sulfated, in the next step.

 The resulting disaccharide is then subjected to the action of a base in order to saponify the carboxyl group in position 6 of the iduronic acid unit and to form salts with the groups in positions 2 and 6 of the glucosamine unit.

 The iduronic acid derivative of formula (III) is advantageously obtained from the corresponding D-glucose derivative by epimerization of the group -CH20H in position 6, oxidation and esterification.

 According to a preferred embodiment of the invention for carrying out the epimerization, a Dglucose derivative is subjected to a tosqlation reaction, in particular with tosyl chloride, then to acid iodination of an agent such as sodium iodide, followed by an acetylation reaction of the OH group in position 4, using acetic anhydride, then with the elimination of hydroiodic acid which creates a double bond in position 6, and finally to a hydroboration reaction, which leads to the production of a -CH 2 OH group in position 6 in the epimer form and to the release of the -OH group in position 4.

 The specific oxidation reaction of the -CH2OH group in position 6 to the -COOH group advantageously comprises the action of trityl chloride, then the benzoylation of position 4, followed by hydrolysis of the trityl group and the oxidation of the primary function thus released.

 To obtain the corresponding esterified derivative used in the condensation reaction defined above, the acid obtained after release of the -OH group in position 4 is subjected to the action of diazomethane.

 The intermediate products used in this process are new and, as such, fall within the scope of the invention. These products have the advantage of constituting precursors of basic sequences of heparin or heparan sulfate.

 Other characteristics and advantages of the invention will appear in the examples which follow.

 The formulas of the compounds in question in these examples are represented in FIGS. 1 and 2 in which the numbers indicated correspond to those used in the examples to designate the same compounds. The symbols used in these figures have the following meanings -: Ac: acetyl, Me: methyl, Ts: tosyle.

EXAMPLE 1.

Synthesis of the monosaccharide 12.

 This synthesis is carried out according to steps 1 to 7 below.

 Stage 1: synthesis of monosaccharide 2.

This monosaccharide is prepared from compound 1 obtained according to the technique of NL Holder and B. Fraser
Reid, Canadian Journal of Chemistry, 51 (1973) page 3357.

To a solution of compound 1 (1 g, 12.67 mM) in dichloromethane (20 ml), tosyl chloride (0.55 g) is added, then dimethylaminopyridine (16 mg) and finally triethylamine (0.7 ml). After stirring under a stream of nitrogen, protected from moisture, for about 14 hours, the reaction is stopped by adding ice and water. After dilution of the reaction mixture with dichloromethane (50 ml), the dichloromethane phase is washed with 2 M hydrochloric acid, then a saturated solution of sodium bicarbonate, and finally with water until neutral pH. After drying and evaporation, a residue is obtained, namely derivative 2 (1.4 g, 97%) which is used as it is in the synthesis of derivative 3.

Step 2: synthesis of derivative 3.

 Monosaccharide 2 (31.8 g) and sodium iodide (39 g) are dissolved in acetonitrile (250 ml), then the solution is brought to reflux for 3 hours.

After. cooling the reaction mixture, the white precipitate formed is filtered. The filtrate is concentrated, the residue is taken up in chloroform, then the chloroform phase is washed with water until neutral pH, dried over sodium sulfate and concentrated to dryness. A syrup is obtained which is chromatographed on a column of silica gel (200 g, ether-hexane, 1/1, vXv). The iodine derivative 3 is thus obtained (24; 7 g, 71.5%). K720 = 240 - (1, chloro form)
The infrared spectrum, the NMR spectrum and the elementary analysis confirm the structure of 3.

Step 3: synthesis of derivative 4.

 To a solution of derivative 3, in anhydrous pyridine (200 ml), acetic anhydride (43 ml) is added. After approximately 14 hours with stirring, the reaction is terminated. The reaction mixture is concentrated to dryness, then the residue is purified on a column of silica gel, under pressure, in a solvent ethyl acetatehexane (1/6, v / v). The pure fractions are grouped.

Product 4 is thus obtained (16.4 g, 70%). This product is in the form of a syrup. z / 20 = + 4.50 (1.3, chloroform). The elementary analysis as well as the analysis of the infrared spectrum confirm the structure.

 Stage 4: synthesis of 5.

 To a solution of derivative 4 (4 g) in pyridine (100 ml), cooled to OOC, silver fluoride (kif, 6.9 g) is added. After two and a half hours, the reaction mixture is poured into a mixture containing chloroform and ether (1/4, v / v, 1 1). The suspension obtained is passed through a pleated filter. The filtrate is concentrated to dryness, then the residue is taken up in chloroform (500 ml). The chloroform phase is washed with potassium acid sulfate in 10% solution in water, then with water until neutral pH. After drying over sodium sulfate and concentration to dryness, a residue is obtained (2.7 g), which is chromatographed on a silica column (200 g) (eluent: ethyl acetate-hexane, 1/4, v / v) .The fractions containing product 5 are combined and after evaporating the solvents, a crystalline product is obtained (1.62 g, 54%).

PF: 81-820C, z / D25 = - 20 (1, chloroform).

Analysis of the infrared spectrum, elementary analysis and analysis of the nuclear magnetic resonance spectrum confirm the structure of compound 5.

 Step 5: synthesis of the derivative 6.

 Product 5 (2 g) is dissolved in methanol (20 ml) and chloroform (20 ml). To this solution is added sodium methanolate (2 M, 2 ml). After 1.5 hours, the deacetylation reaction is complete. The reaction mixture is diluted with chloroform. The hydrochloric phase is washed with water until neutral pH, dried, then evaporated to dryness. A residue, compound 5 ′, is thus obtained (1.8 g, 100%). It is immediately dissolved in tetrahydrofuran (50 ml), then boron hydride (BH3, 1M) in tetrahydrofuran; (10 ml) is then added. After one hour of reaction, the excess boron hydride is destroyed by the addition of ethanol. At the end of the evolution of gas, the reaction mixture is diluted by the addition of tetrahydrofuran (100 ml). 3M sodium hydroxide (12 ml) is then added, followed by hydrogen peroxide (120 volumes, 8 ml). After 2 hours of heating at 500C, the reaction is stopped. The solution is poured into chloroform (500 ml), then the organic phase thus obtained is washed with water, 2 M hydrochloric acid, then finally with water until neutral pH. A very milky chloroform phase is thus obtained, which becomes clear during drying over sodium sulfate. After filtration, the chloroform is evaporated and the residue obtained is chromatographed on silica (200 g chloroformmethanol, 30/1, v / v).

 The derivative of idose 6 is thus obtained (1.05 g, 55%). This product is in the form of a syrup.

 i D20 = + 85.50 (1, chloroform).

The elementary analysis as well as the NMR analysis confirm the expected structure.

 Extase 6: synthesis of derivative 9.

 This synthesis is carried out from derivative 6 in a single step (intermediates 7 and 8 are not isolated). To a solution of derivative 6 (2.25 g, 6 mM) in dichloromethane (50 ml), dimethylaminopyridine (60 mg; 0.24 -mM) of triethylamine (1.7 ml, 12 mM) is successively added. ) and trityl chloride (2.5 g; 9 mM). After about 14 hours, the reaction is complete. The derivative 7 is thus obtained in solution. Then dimethylaminopyridine (150 mg), triethylamine (1.7 ml) and benzoyl chloride (1.05 ml) are added to the reaction mixture. After 6 days, the dichloromethane is eliminated by passing a stream of nitrogen and replaced by dimethylformamide (40 ml). The reaction mixture is heated to 700C overnight. Benzoyl chloride (1 ml) and triethylamine (1.7 ml) are then added again, then the heating is maintained at 700C for 2 days. The dimethylformamide is then evaporated, then the residue is taken up in chloroform, the chloroform phase is washed with water, with a saturated sodium bicarbonate solution, then with a 2 M hydrochloric acid solution and finally with water until neutral pH. After drying, the chloFoforme is evaporated, which makes it possible to obtain compound 8.

 This is immediately subjected to a reaction to remove the trityl group in order to obtain derivative 9.

The residue containing derivative 8 is dissolved in 25 ml of chloroform and to this solution is added 10 ml of a solute of paratoluenesulfonic acid monohydrate in methanol (1 M). After 4 hours of reaction at room temperature, the reaction is terminated. The reaction mixture is then diluted with chloroform, washed with water, dried and then evaporated to dryness. The residue obtained is chromatographed on silica gel (200 g, ether-hexane, 3/1, v / v).

Derivative 9 is thus obtained in the pure state (1.5 g; 52%).

This derivative is in the form of a syrup. ru720 = - 80 (1, chloroform).

Analysis of the infrared spectrum and the NMR spectrum confirm the structure of the expected product.

 step 7: synthesis of compound 12.

 This synthesis is carried out directly from derivative 9 without isolating intermediates 10 and 11.

To the solution of compound 9 (1.2 g) in acetone (20 ml), a solution (2.9 ml) of chromium oxide (Cr03, 1.17 g) in 3.5 M sulfuric acid (5 ml). After 30 minutes of stirring at OOC, the temperature is brought back to ambient. The reaction evolves for 3 hours. The reaction mixture is then poured into a settling tank containing ice water (100 ml). The product formed is extracted with chloroform (3 x 50 ml). The chloroform phase is washed with water to neutral pH, then dried over sodium sulfate, filtered and concentrated to dryness.

The residue obtained (compound lo) is dissolved in methanol (130 ml). 3M solution of sodium hydroxide (17 ml) is added to this solution and the mixture is then stirred for approximately 14 hours. After acidification with sulfuric acid, the compound 11 is extracted with ether, then immediately methylated with diazomethane, according to the conventional method to give the compound 12.

 After evaporation of the ether, compound 12 is obtained pure by means of chromatography on silica gel (50 g; ether-hexane; 4/1, v / v). The pure fractions containing derivative 12 are combined and the solvents are removed. Derivative 12 of iduronic acid is thus obtained (587 mg, 59 or more than derivative 9).

This product is in the form of a syrup. Z / 25 = +980 (2.65, chloroform).

NMR analysis, infrared analysis and elementary analysis confirm the expected structure.

EXAMPLE 2.

Synthesis of the disaccharide 14.

 This synthesis is carried out using monosaccharide 12 prepared as above and monosaccharide 13 prepared according to the technique of H. Paulsen and W. Stenzel, chemische Berichte 111 (1978) 2234-2247.

 To a solution of compound 12 (200 mg, 0.5 mM) in dichloromethane (10 ml), compound 13 (0.450 g) of sym-collidine (150 μl) and silver triflate (260) is successively added. mg).

 The reaction mixture is kept at OOC under a stream of nitrogen and with stirring protected from moisture and light for 3 hours.

 It is then diluted with dichloromethane (100 ml) then the solids are removed by filtration on pleated filters. The solution obtained is washed with a saturated sodium bicarbonate solution with water and with 2 M sulfuric acid, then again with water until neutral pH.

 After drying over sodium sulfate and evaporation of the dichloromethane, the residue obtained is chromatographed on silica gel (50 g; chloroform / ethyl acetate; 15/1; v / v).

 Pure derivative 14 is thus obtained (327 mg, 82%).

The product is in the form of a syrup. [α] D20 = +57 (1, chloroform).

The NMR analysis as well as the elementary analysis confirm the structure and the anomerism of the disaccharide 14.

EXAMPLE 3.

Disaccharide synthesis 15.

 Disaccharide 14 (260 mg) is dissolved in methanol (5 ml) and 1 M sodium hydroxide (1 ml) is added dropwise. At the end of the reaction, the reaction mixture is introduced at the top of a column of DOWEX 50 resin in HT form (5 ml). The effluent is concentrated to dryness, taken up in methanol, and the free acid product, obtained at the end of the saponification of derivative 14, is methylated by addition of diazomethane. Derivative 15 is thus obtained which is purified by means of a column of silica gel (20 g, ether / hexane; 8/1; v / v).

The yield of compound 15 is 92 mg. This product is directly involved in the synthesis of derivative 16.

EXAMPLE 4.

Preparation of the disaccharide 16.

 The product obtained above (92 mg) is dissolved in dimethylformamide (5-ml) and then trimethylamine / sulfide trioxide complex (25 mg) is added. The solution is brought to 500C for approximately 14 hours.

After evaporation to dryness, the residue is taken up in chloroform, then the chloroform phase is washed with water, dried and concentrated to dryness. The solid obtained is purified on a column of silica gel (15 g eluent: methanol / chloroform; 1/4 v / v). After evaporation of the pure fractions, the sulfated disaccharide 16 is obtained (58 mg; 55.6%).

EXAMPLE 5.

Disaccharide synthesis 17.

 The 16 'disaccharide (58 mg) is dissolved in a methanol-water mixture (15 ml + 2 ml). Catalyst (Pd / C 5%; 60 mg) is then added and this suspension is then stirred under a hydrogen atmosphere for 48 hours. At this stage, the total disappearance of the benzyl groups carried by the derivative 16 is noted, as is the reduction of the azide group of the derivative 16 into an amino group.

The catalyst is removed by filtration, then the reaction mixture is concentrated to dryness.

 Disaccharide 17 is thus obtained which will be treated directly to obtain product 18.

EXAMPLE 6.

Disaccharide synthesis 19.

 Disaccharide 17 is dissolved in water (6 ml).

To this solution is added trimethylamine / sulfide trioxide complex (25 mg), while maintaining the pH at 9.5 by addition of sodium hydroxide (0.1N). After 45 hours of reaction, 1N sodium hydroxide is added to bring the pH to 12. Then, it is maintained at this value for 1 hour. The solution is then neutralized with 1N hydrochloric acid, then passed through a column of DOWEX 50 (5 ml) in Na + form. The eluate from this column is introduced on a column G1 x 2 (15 ml-, 1.6 x 8 cm). The column is eluted with a sodium chloride gradient from 0 to 3 M.

The fractions containing disaccharide 19 in the form of sodium salts are combined, concentrated, then the product is desalted by passage over a column of Sephadex.
G25 (50 ml) eluted with water. Disaccharide 19 is thus obtained (27 mg, 68%). After lyophilization, the product is in the form of a white powder.

 </ 20 = + 95.50 (1.3; water).

Carbon 13 NMR analysis confirms the expected structure for product 19.

Claims (1)

 CLAIM  Disaccharides formed from structural patterns D-glucosamine and L-iduronic acid, characterized in that they correspond to the formula

 in which -R represents an aliphatic or aromatic radical, in particular an alkyl radical containing 1 to 4 carbon atoms, in particular a methyl radical, AA represents a hydrogen atom, an alkyl radical comprising 1 to 4 carbon atoms, in particular , a methyl radical, or a metal cation, in particular an alkaline cation, more especially of sodium, or a crganic cation such as a derivative of nitrogenous organic base,  - X represents an anion, in particular, a sulfate group, optionally in the form of a salt with an organic or inorganic cation, and, in the latter case, in particular an alkaline cation, - Z represents a nitrogen functional group, in particular, an azide group, or an optionally substituted amine group, such as a group of type -NHY in which Y has the meaning given above, and - R1 to R4 represent a hydrogen atom, or a radical protecting group - OH.