HU227307B1 - Intermediates for the preparation of 3-deoxy-oligosaccharides - Google Patents

Abstract

The present invention relates to compounds of formula (IX), (X) and (XI). In the formulas, Bz is a benzoyl group, R 1 is a linear or branched C 1 -C 6 alkoxy group, an acyclic C 1-6 acyloxy group, preferably an acetoxy group, an aromatic acyloxy group, preferably a benzoyloxy group or 2 - C7-C2-alkenyloxy, preferably allyloxy, - P1 and P2 are the same or different, and represent a protecting group such as acyclic C1-C6 acyl, preferably acetyl, aromatic acyl, preferably benzoyl, 2- C 7 is a 2-alkenyl group, preferably allyl or benzyl, - P 4 is a protecting group such as C 1-6 alkyl, preferably methyl or benzyl, and - Pg is levulinyl or chloroacetyl. The compounds are important intermediates for the preparation of anti-thrombotic 3-deoxyoligosaccharides. The scope of the description is 16 pages (including 2 tabs)

Description

The present invention relates to intermediates for the preparation of 3-deoxyoligosaccharides. 3-Deoxyoligosaccharides are useful in medicine for their anticoagulant and antithrombotic activity.

Heparin is a polysaccharide of the glucose-aminoglycan family and is known for its anticoagulant activity. It is known that blood coagulation is a complex physiological phenomenon [I. Jörk and U. Lindahl, Molecular and Cellular Biochemistry (1982), Dr. W. Junk Publishers - The Netherlands]. Certain stimuli, such as contact activation and tissue factors, result in the sequential activation of several coagulation factors present in the plasma. Depending on the nature of the stimulus, the final steps are the same: activated factor X (Xa) activates factor II (also known as prothrombin), which in its activated form (factor Ha, also known as thrombin) causes partial proteolysis of soluble fibrinogens and , liberates insoluble fibrin.

Under normal physiological conditions, the activity of coagulation factors is regulated by proteins such as antithrombin III (AT III) and heparin II cofactor (HC II), which are also present in plasma. AT III inhibits a number of coagulation factors, particularly Factors Xa and Ha.

Factors Xa and Ha are thus favorable means for inducing anticoagulant and antithrombotic activity, since these two factors are involved in the last two steps of coagulation, which are independent of the trigger.

The pentasaccharide of Formula XXI shows the minimum sequence of heparin required for AT III binding. This compound (R = -SO ₃ ^_) were produced entirely by chemical synthesis approximately 10 years ago [P. Sinai, Carbohydrate Research (1984) 132, C5],

Subsequently, a number of synthetically produced oligosaccharides have been described as having antithrombotic and anticoagulant activity.

EP-0 084 999 discloses compounds containing uronic acid (glucuronic acid or iduronic acid) and glucosamine monosaccharide units which have advantageous antithrombotic properties. In addition to the hydroxy substituent, the compounds also contain N-sulfate groups and N-acetyl groups, and in some cases, the anomeric hydroxyl groups are replaced by methoxy groups.

EP 0 165 134 also describes synthetic oligosaccharides with antithrombotic activity. These compounds consist of uronic acid and glucosamine monosaccharide units and contain O-sulfate or O-phosphate groups. Compounds containing uronic acid and glucosamine having O-sulfate groups at the 3-position of the glucosamine unit are also described in EP 0 301 618. These compounds have beneficial antithrombotic and anticoagulant activity. EP 0 454 220 discloses uronic acid and glucose derivatives having O-alkyl and O-sulfate groups. These latter compounds also have antithrombotic and anticoagulant properties.

Sulfated glucose amino glucanoid derivatives in which the N-sulfate, N-acetate and hydroxyl functional groups are replaced by alkoxy, aryloxy, aralkyloxy or O-sulfate groups are also disclosed in EP 0 529 715. . These compounds have advantageous antithrombotic properties. They also inhibit the overgrowth of smooth muscle cells.

Oligosaccharides, in particular pentasaccharides, analogous to the minimal heparin sequence required for AT III binding are described in Angew. Chem. Int. Ed. Engl. (1933), 32, (3), 434-I436. pages of literature. The compounds contain glucuronic acid and glucose units in which the hydroxyl groups are replaced by O-sulfate or O-methyl groups.

Surprisingly, it has been found that when one or more hydroxyl groups or O-alkyl or O-sulfate groups are substituted at the 3-position in one or more saccharide units, oligosaccharides having a beneficial biological property are obtained. The compounds prepared from the intermediates of the present invention differ from other synthetic heparinoids described in the literature in their novel structure and strong, unexpected biological properties. The compounds of the present invention which are prepared from the intermediates of the present invention are 3-deoxyoligosaccharides which have very strong anti-Factor Xa activity and high affinity for AT III. In addition, the compounds of the present invention are readily absorbed in the gastrointestinal tract so that the products can be administered orally.

In particular, the intermediates of the present invention can be used to prepare compounds of formula (I)

X is -OSO ₃ ^_ group, or (A), (B) or a group of formula (C),

Y is a group of formula D,

R is linear or branched

C 1-6 alkyl,

R _{1; R3, R5, R7, R8, R10, R12} and _R13 are the same or different and represent a hydroxyl group, a linear or branched C1-6 alkoxy, or -OSO ₃ ^_ a radical of formula

R _2, R _4, R _{6, Rg} and R ^ are the same or different and represent a hydrogen atom, a linear or branched C1-6 alkoxy, or -OSO ₃ ^_ group of the formula, with the proviso that R ₂ At least one of the substituents R ₄ , R ₆ , R 8 and R 4 is hydrogen - in the form of their pharmaceutically acceptable salts and the corresponding acids.

Hereinafter, the monosaccharide moiety is understood to mean the glycoside moiety of Formula XXII, regardless of which substituents at the 2-, 3-, and 5-positions in the unit are attached.

In the groups B, C and D, jelenti means that the carbon atom in the substituent bonded through this bond has the configuration R and S, respectively.

The compounds of the present invention may be prepared from 3-deoxysaccharide derivatives.

Accordingly, among the monosaccharide units from which the inter2 of the present invention is derived

At least one of the compounds produced from the media is at least one of the structures 3-deoxymonosaccharide.

Thus, at least one of the substituents R ₂ , R ₄ , R ₆ , R 8 and R _{11 in} formula (I) is hydrogen.

Preferred compounds of the invention are those wherein R ₂ is hydrogen.

Also preferred are compounds of the present invention wherein X is a group of formula (B) or (C).

These compounds correspond to (Ia) and the general formulas (Ib), wherein R _h R _2, R _3, R _4, R _5, R @, R _7, R _8, Rg, R _10, R 1, R 12 and R ₁₃ is as defined for formula (I).

Particularly preferred are compounds of formula (Ia).

Particularly preferred are the compounds of formula I according to formulas Ia and Ib, wherein

- _{R3, R3, R5, R7, R8, R10} and _R13 are the same or different, are linear or branched C 1-6 alkoxy group or -OSO ₃ ^_ formula

- R ₂ , R ₄ , R ₆ and R 9 are the same or different and are hydrogen or C 1-6 -alkoxy, linear or branched,

- R-ι ι is a hydrogen atom, a linear or branched C1-6 alkoxy group or -OSO ₃ ^_ formula, with the proviso that R _2, R _4, R _{6, Rg} and R, at least one of _three a is hydrogen; and

- R ₁₂ represents a hydroxyl group or a group of formula-OSO ₃ ^_.

Particularly preferred are compounds of formula I according to formulas Ia and Ib wherein

- R ₂ and R 8 are hydrogen,

- R _3, R 1 and R 12 is -OSO ₃ ^_ n and

- R ₁₃ is a linear or branched C 1 -C 6 alkoxy group.

Also particularly preferred are compounds of formula I according to formulas Ia and Ib wherein the alkoxy group is methoxy.

The compounds of formula (IX), (X), (XI) of the present invention are important intermediates in the process for the preparation of compounds of formula (I). According to this method, a compound of the formula II is represented by the formula

- X 'is chloroacetoxy, laevulinyloxy, A, Bi or C 1-6 a group of the general formula

- P _{1 (} P ₂ , P ₃ and P ₅ are the same or different and represent a protecting group such as acyclic C 1 -C 6 acyl, preferably acetyl, aromatic acyl, preferably benzoyl, C 2 -C 7 alkenyl, preferably allyl or benzyl) .

- P ₄ and P ₆ are the same or different and are a protecting group such as C 1-6 alkyl, preferably methyl or benzyl,

- Rf, _R5 'and R _8' are identical or different, are straight or branched chain C 1-6 alkoxy, C 1-6 acyclic acyloxy group, preferably an acetoxy group, an aromatic acyloxy group such as benzoyloxy or C2-C7-alkenyloxy, preferably allyloxy,

- R ₇ 'and R ₁₀ ' are the same or different and have the meaning given for Rf or a chloroacetoxy group or a levulinyloxy group,

- R ₂ ', R 8' and R 8 'are the same or different and are hydrogen or the substituents on RJ with a compound of formula (III)

- P ₇ is a substituent given by P ₄ in formula II,

- P ₃ represents the substituents given by P _{r in} formula (II),

- R ₄ 'and R 11' are the same or different and have the meaning given to R ₂ 'in formula (II),

- R ₃ ', R ₁₂ ' and R ₁₃ 'are the same or different and have the meaning given by R ₃ ' in the formula (II) to give the compound of the formula (IV)

- X ', P-], P ₂ , P ₃ , P ₄ , R- |' and R ₂ 'is as defined for formula (II),

- P ₇ , R ₃ 'and R ₄ ' are as defined for formula (III), and

- Y 'is a group of the formula:

- P ₈ , R ₁₁ ', R ₁₂ ' and R ₁₃ 'are either hydrogenated catalytically as defined in formula (III) and then saponified and converted to the sulfate ion or first saponified and then converted to the sulfate ion and then catalytically hydrogenated or hydrogenated first is converted to the sulfate ion and then saponified to give compounds of formula (I).

The compounds of formulas IX, X and XI according to the invention play an important role in this synthesis.

In Formula IX, Bz is benzoyl.

In formula (XI)

- R 1 is a linear or branched C 1 -C 6 alkoxy group, acyclic

C 1-6 acyloxy, preferably acetoxy, aromatic acyloxy, preferably benzoyloxy or C 2-7

2-alkenyloxy, preferably allyloxy,

- P ₁ and P ₂ are the same or different and are a protecting group such as acyclic C 1-6 acyl, preferably acetyl, aromatic

An acyl group, preferably a benzoyl group, a C 2 -C 7 alkenyl group, preferably an allyl group or a benzyl group,

P ₄ is a protecting group such as C 1-6 alkyl, preferably methyl or benzyl, and

- P ₉ is levulinyl or chloroacetyl.

Compounds of formula (II) wherein X 'represents a group of formula (B1) may be prepared from an anomeric carbon activated monosaccharide, such as a compound of formula (V) wherein P ₅ , R ₅ ', R ₆ 'and R ₇ ' are as defined for B1, with a compound of Formula VI, wherein Rf, R ₂ ', P ₄ , P ₂ and P ₄ are as defined in Formula II T. Peters et al., Can. J. Chem. (1989), 62, 491-496. and GH Veeneman and JH van Boom, Tetrahedron Letters (1990) 31, 275-278. pages 5 to 9, and thus the compounds of formula VII wherein P ₅ , R ₅ ', R ₆ ' and R ₇ 'are as defined in formula V, and R f, R ₂ ', P -), P ₂ and P ₄ are as defined for formula (II).

The compound thus obtained is known in the art [R. Schmidt, Angew. Chem. Int. Ed. England (1986), 25 (3), p. 212-235], in particular with acetolysis, by treatment with benzylamine followed by treatment with trichloroacetonitrile to give the compound of the formula II, wherein X 'is the compound of the formula B1.

Many of the compounds of formula VII, especially those wherein R ₂ 'and R ₈ ' are substituents at R ₄ 'and are other than hydrogen, may be prepared by J. Basten et al., Bioorganic and Medical Chemistry Letters (1992), 2 , (No. 9), 905-910. 3, p.

Compounds of formula (VII) may be prepared by reacting a compound of formula (VI) with another activated monosaccharide, such as a compound of formula (V) wherein P ₅ , R ₅ ', R ₆ ' and R ₇ 'are as defined in formula (V). of formula.

Compounds of formula (V ') wherein R ₆ ' represents the substituents at R ₆ and are other than hydrogen are known compounds and are known from M. Petitou and CAA van Boeckel, in Chemical Synthesis of Heparin Fragments and Analogues 203-210. Springer Verlag Vienna - NY (1992).

Compounds of formula (V) wherein R ₆ 'is hydrogen may be prepared from compound (VIII) wherein Bz is benzoyl.

Compounds of formula (VIII) may be prepared from 3-deoxy-β-ribo-hexapyranose (prepared by T. V. Rajanbabu, J. Org. Chem. (1988) 53, 4522-4530. 2, which is reacted with benzoyl chloride in an alkaline organic solvent. The resulting compound is reacted with ethanethiol to give compound (VIII).

Compounds of formula (VI) wherein R ₂ 'is hydrogen may also be prepared from compound (VIII), which is then reacted with 1,6: 2,3-dianhydro-pD-mannopyranose to give A compound of Formula IX wherein Bz is benzoyl.

This compound is reacted with a strong base to give 1,6: 2,3-dianhydro-4-O- (3-deoxy-p-D-ribo-hexapyranosyl) -? - D-mannopyranose according to formula (X).

The compound of formula (X) is known in the art [G. Jaurand et al., Bioorganic and Medicinal Chemistry Letters (1992), 2 (no. 9), 897-900. side; J. Basten et al., Bioorganic and Medicinal Chemistry Letters (1992), 2 (no. 9), 901-904. side; J. Basten et al., Bioorganic and Medicinal Chemistry Letters (1992), 2 (no. 9), 905-910. side; M. Petitou and C.A. A. Boeckel, "Chemical Synthesis of Heparin Fragments and Analogues," 203-210. Page - Progress in Chemistry of Organic Natural Products, Ed. Springer Verlag Vienna - N.Y. (1992)], a compound of formula (XI)

R ', P ₁ , P ₂ and P ₄ are as defined for formula (VII), and

- P ₉ is levulinyl or chloroacetyl.

The resultant compound of formula XI is then reacted with hydrazine to give compounds of formula VI wherein R ₂ 'is hydrogen.

Compounds of formula (IX), (X) and (XI) are novel products and are the subject of the present invention.

Compounds of formula (VI) wherein R ₂ 'is a substituent at R ₄ ' and is other than hydrogen may be prepared in a similar manner to the protected glucose derivatives activated on their anomeric carbon instead of (VIII). using. The preparation of such compounds is described in M. Petitou and CAA van Boeckel, Chemical Synthesis of Heparin Fragments and Analogues, pp. 203-210. Springer Verlag Vienna - NY (1992). Said authors also disclose an analogous method for the preparation of compounds of formula IX having a protected hydroxyl group in the 3-position.

Compounds of formula (II) wherein X 'is a group of formula (C _1a )

- R ₅ ', R ₆ ', R _g ', R ₉ ', R ₁₀ ', P ₅ and P ₆ have the meanings given in the formula (C 1 -), a compound of the formula (VI) may be prepared by compound of the formula: - wherein R ₅ ', R', R ', R', R ₀ ', P ₅ and P ₆ is by means (C ₄₎ given and hints formula reaction in which the compound of formula (XIIIa) - in the formula

HU 227 307 Β1

- R ₅ ', R', R _g ', R', R ₁₀ ', P ₅ and Pg are as | defined in formula, and (C)

R 1 ', R ₂ ', P ₁ , P ₂ and P ₄ are as defined for formula (II).

The resulting compound of formula (XIIIa) is then reacted as described in the preparation of compounds of formula (VII) to give compounds of formula (II) wherein X 'is C _1a .

Compounds of formula (XIIa) wherein R ₉ 'is hydrogen may be prepared from compounds of formula (XIV):

R ₅ ', R ₈ ', R ₈ ', R ₁₀ ' and Pg are the acetolyses of formula (C 4), deprotected and then reacted with trichloroacetonitrile. Compounds of formula (XIV) can be prepared by the reaction of (X). from the compound of formula (XI) as described in the preparation of the compound of formula (XI).

Compounds of formula (XIIa) wherein R ₉ 'is a substituent at R ₁ ' other than hydrogen may be prepared in the form of 1,6: 2,3-dianhydro-4-O- (p-D-glucopyranosyl). ) -DD-mannopyranose derivative as described above. 1,6: 2,3-Dianhydro-4-O- (p-D-glucopyranosyl) -pD-mannopyranose derivatives are known compounds and are described, for example, in J. Basten et al., Bioorganic and Medicinal Chemistry Letters (1992), 2 (No. 9), 905-910. pages of literature.

Compounds of formula (II) wherein X 'is a group of formula (C _1b )

R ₅ ', R ₈ ', R ₈ ', R ₈ ', R ₁₀ ', P ₅ and P ₆ are as defined in formula (0-), may be prepared by the preparation of a compound of formula (XIIIb)

- R ₅ ', R ₈ ', R ₈ ', R ₈ ', R ₁₀ ', P ₅ and P ₆ are reacted with a compound of formula (VI) as defined in formula (C 1) wherein the compound of formula (XIIIb) compounds of the formula - in the formula

- R- |, R ₂ , Rg, Rg, Rg, R ₉ , R- | o ^ 1> ^ 2>? 4> ® ^and Pg are as defined in formula (XIIIa) obtained.

Compound of formula (XIII b) may then be treated with a (XIIIa) described the preparation of compounds of formula, to thereby give a compound (II) of the formula: wherein X 'is (C _1b) radical.

Compounds of formula (XIIIb) wherein R ₉ 'is hydrogen may be prepared from compound (X). This compound is converted to a compound of formula XV by Ichikawa et al., Carbohydrate Research, 1988, 172, 37-64. as described in the literature.

The compound thus obtained is known to G., Jaurand et al., Bioorganic and Medicinal Chemistry Letters (1992), 2, No. 9, 897-900. page J.

Basten et al., Bioorganicand Medical Chemistry Letters (1992), 2 (no. 9), 901-904. J. Basten et al., Bioorganicand Medicines Chemistry Letters (1992) 2 (no. 9), 905-910. and P. Petitou and CAA van Boeckel, "Chemical Synthesis of Heparin Fragments and Analogues," pp. 203-210. Transformation according to Organic Natural Products, Ed. Springer Verlag Vienna - NY (1992) gives compounds of Formula XVI wherein R ₅ ', R ₆ ', R ₈ ', R ₁₀ ' and P ₆ is as defined in formula (XIIb).

The resulting compounds are then subjected to acetolysis followed by treatment with benzylamine and trichloroacetonitrile to give compounds of formula XIIIb.

Compounds of formula (XIIIb) wherein R ₉ 'is other than hydrogen but otherwise have the substituents at Rf can be prepared in a known manner.

Intermediates for the preparation of formula (XIIb) compounds, those wherein R ', R', R ', R _10', Ρ ₅ and Pg are as defined in formula (C]), and R ₉ 'are the Rf which are other than hydrogen are known products and are prepared by G. Jaurand et al., Bioorganic and Medicinal Chemistry Letters (1992) 2, No. 9, 897-900. pages of literature.

Compounds of formula (II) wherein X 'is a group of formula (A) and R ₂ ' is hydrogen may be prepared from compound (X) which is converted to compound (XVII).

The resulting compound (XVII) is then treated with either sodium benzylate followed by acylation or treatment with an aralkyl halide, preferably benzyl bromide or 2-alkenyl halide, preferably allyl bromide, followed by treatment with sodium benzylate, followed by acylation prepared as the compound of formula (XVIII), wherein R f, P ₁ and P ₂ are as defined in formula (II).

The compound of formula (XVIII) thus obtained is then converted to the compound of formula (XIX) in an acidic medium wherein R R / P. and P ₂ is as defined for formula (II).

The resulting compound of formula (XIX) is then selectively silylated at the 6'-position, treated with levulinic anhydride, oxidized by Jones reaction and then esterified with an alkyl halide to remove the levulinyl group to give the resulting compound in an acidic or neutral medium. Compounds of formula (II) wherein X 'is a group of formula (A) and R ₂ ' is hydrogen.

Similarly, compounds of formula (II) wherein X 'is a group of formula (A) and R ₂ ' are the substituents at R _{1 can be} prepared. Suitable intermediates for the preparation of said compounds are described in the literature

See, for example, J. Basten et al., Bioorganic and Medicinal Chemistry Letters (1992), 2 (no. 9), 905-910. and C.A.A. van Boeckel et al., J. Carbohydrate Chem. (1985), 4, 293.

Compounds of formula (III) wherein R ₄ 'is hydrogen may be prepared from compounds of formula (XX) wherein

R ₃ ', R 1', P ₇ and P ₈ are as defined for formula (III), and

- P ₁₀ is levulinilcsoport or chloroacetyl group.

The compound of formula (XX) is subjected to acetolysis and the resulting compound is treated with benzylamine, then treated with Vilsmeier reagent, treated with alcohol in the presence of silver carbonate, and then reacted with hydrazine to give the desired compound (III). (R ₄ '= H).

Compounds of formula (III) wherein R ₄ 'is a substituent at R ₄ ' are known compounds. The preparation of such compounds is described by J. Basten et al., Bioorganic and Medicinal Chemistry Letters (1992), 2 (no. 9), 905-910. pages of literature.

By the methods known above, the compounds of the present invention can be obtained in the form of their salts. To prepare the appropriate acids, the compound in salt form is contacted with a cation exchange resin in acid form.

The compounds in acid form can then be neutralized with a base to give the desired salts.

Any inorganic or organic base which forms a pharmaceutically acceptable salt with a compound of formula (I) may be used to prepare the salts of the compounds of formula (I).

Preferably, sodium, potassium, calcium or magnesium hydroxide is used. Preferred salts of the compounds of formula I are the sodium and calcium salts.

Compounds prepared from the intermediates of the present invention have advantageous pharmacological and biochemical properties. The compounds have potent Factor Xa activity and a high affinity for AT III.

As mentioned above, in the coagulation cascade, factor Xa activates prothrombin into thrombin, which causes proteolysis of soluble fibrinogens simultaneously with the release of the main component of the blood clot, insoluble fibrin. Inhibition of factor Xa is thus a preferred means of achieving anticoagulant and antithrombotic activity.

The activity of the compounds of the present invention to produce factor Xa (anti-Xa) can be determined by Teien A.N. and Lie M., Thrombosis Research (1977), 10, 399-410. The pH of the compounds of the present invention is the same as or greater than the known synthetic heparinoids, the pH of which is 8.4.

The affinity of the compounds of formula (I) for AT III was determined spectrofluorometrically by D. Atha et al., Biochemistry (1987) 26, 6454-6461. 7, p. Based on the results of the assay, the compounds of the invention have a very high affinity for AT III.

In addition, the average antithrombotic activity of the compounds of formula I in rats was investigated in a model of venous congestion and thromboplastin induction in J. Reyers et al., Thrombosis Research (1980) 18: 669-674. 3, p. The compounds of the invention have an ED _{50 of} at least the same order of magnitude or less than that of other known synthetic heparinoids. Thus, the compounds of the present invention have particularly advantageous specific activity and anticoagulant and antithrombotic activity.

Various pharmacokinetic studies with compounds of the present invention have shown that the compounds are very well absorbed and have a long half-life. Accordingly, consideration should be given to the possibility of administering the compounds once a day in therapy.

Studies in this regard have shown that the compounds of formula (I) obtainable from the intermediates of the invention are absorbed in the gastrointestinal tract and there is no need to limit the amount administered in human medicine. These compounds are suitable for the preparation of pharmaceutical compositions which may be administered parenterally or orally.

The compounds of formula (I) are of extremely low toxicity and are suitable for pharmaceutical use.

Compounds prepared from the intermediates of the present invention are also useful in the treatment of smooth muscle cell proliferation since they have been shown to have significantly greater inhibitory effects on smooth muscle cell growth than heparin.

These compounds are also active in the formation of blood vessels and are thus useful in the treatment of infections caused by retroviruses.

In addition, the compounds of the present invention have protective and regenerative effects on nerve fibers.

These compounds are very stable and thus are particularly useful as active ingredients in pharmaceutical formulations.

The pharmaceutical compositions containing the compounds of formula (I) or their pharmaceutically acceptable salts may optionally contain one or more inert, suitable excipients.

The pharmaceutical compositions prepared as above are preferably in various forms, for example, solutions for injection or oral administration, dragees, tablets, hard gelatine capsules. Injectable solutions are preferred pharmaceutical dosage forms.

HU 227 307 Β1

The pharmaceutical compositions containing at least one compound of the formula I or a salt thereof are particularly suitable for the prevention or treatment of vascular disorders such as atherosclerosis, atherosclerosis, as well as for surgical interventions, tumor development or bacterial, viral or enzymatic inducers for the treatment of hypercoagulable states following disturbances.

The dosage may vary within wide limits, depending on the age, weight, health, nature and severity of the disease and the mode of administration of the patient. The dosage may be one or more doses per day and may be in the range of about 0.5 mg to 1000 mg per day, preferably about 1 to 100 mg per day, for example about 20 mg per day, and may be administered intramuscularly or subcutaneously or at intervals of time. administration. The daily dosage amount may be about 200 mg to 1000 mg for oral administration.

The formulations can be administered to patients with the symptoms listed above, and should be subjected to a blood test. Subcutaneous administration is preferred.

The invention is illustrated by the following examples.

Production examples

Preparation I

Ethyl 2,4,6-tri-O-benzoyl-3-deoxy-1-thio-D-ribohexopyranoside (Compound VIII)

The step

1,2,4,6-Tetra-O-benzoyl-3-deoxy \ ^J> -D-ribohexopiranóz mmol of 3-deoxy-1,2: 5,6-di-O-isopropylidene-D-ribohexofuranóznak [TV Rajanbabu , J. Org. Chem. (1988) 53, 4522-4530. The resulting solution in water / ethanol was heated at 60 ° C for 4 hours in the presence of Dowex acidic resin to give 3-deoxy-D-ribohexopyranose. The reaction mixture is evaporated to dryness and dried by evaporation in the presence of pyridine.

The remaining syrup was dissolved in 150 mL of pyridine and 356 mmol of benzoyl chloride was added. The resulting reaction mixture was stirred for 3 hours at room temperature. The reaction mixture was evaporated to dryness, diluted with dichloromethane, washed with water and crystallized from ethyl acetate. There are thus obtained 7.66 g of 1,2,4,6-tetra-O-benzoyl-3-de-o-β-D-ribo-hexopyranose.

Yield: 30%.

Melting point: 164 ° C.

[α] D 20 = + 1 ° (c = 1.33 in CH ₂ Cl ₂ ).

Step β

The material from the previous step, 4.51 mmol, was dissolved in anhydrous toluene at 20 ° C under argon, and to the resulting solution was added 9.03 mmol of ethanethiol.

To the reaction mixture was added a solution of boron trifluoride (4.51 mmol) in ethyl ether and the reaction mixture was stirred for 3 hours. The resulting reaction mixture was washed with water and evaporated to dryness, and the resulting syrup was purified on a silica gel column. This gives a mixture of the α- and β-anomers of ethyl 2,4,6-tri-O-benzoyl-3-deoxy-1-thioD-ribo-hexopyranoside. The yield was 1.64 g.

The resulting compound was used without further purification.

Yield: 70%.

//. production

Ethyl 2,4,6-tri-O-benzyl-3-deoxy-1-thio-D-ribo-hexopyranoside (compound of formula V)

The material obtained in Preparation I, 5.82 mmol, was dissolved in a mixture of methanol and dichloromethane (1: 1 v / v). To the resulting solution was added 0.90 mmol of sodium methanolate. The reaction mixture was stirred at 20 ° C for 3 hours and then neutralized with Dowex acidic resin (AG 50 WX2). The resulting reaction mixture was filtered and evaporated to dryness. The residue was dissolved in 18 ml of anhydrous dimethylformamide, and 19.7 mmol of sodium hydride and 17.0 mmol of benzoyl bromide were added at 0 ° C. After stirring for 2 hours, methanol (34.1 mmol) was added.

The solvent was evaporated and the residue was purified on a silica gel column. 2.12 g of the desired product are thus obtained in the form of a mixture of anomers.

Yield: 76%.

Example 1

1,6: 2,3-Dianhydro-4-O- (2,4,6-tri-O-benzoyl-3-deoxyoxy-D-ribo-hexopyranosyl) -? - D-mannopyranose (Compound IX) ]

G. H. Veeneman and J. H. van Boom (Tetrahedron Letters (1990) 31, 275-278). 83.53 mmol of the compound of Preparation I and 7.25 mmol of 1,6: 2,3-dianhydro-β-D-mannopyranose in 220 ml of toluene are sieved through a sieve.

In the presence of 21.3 mmol of N-iodosuccinimide, 1.7 mmol of 0.04 M trifluoromethanesulfonic acid solution is added dropwise. The reaction mixture was stirred for 2.5 hours, filtered and purified on a silica gel column to give 3.07 g of 1,6: 2,3-dianhydro-4-O- (2,4,6-tri-O-benzoyl-3-). deoxy-D-ribo-hexopyranosylXD-mannopyranose is obtained.

The product was crystallized from a mixture of ethyl acetate and hexane (90:10 v / v).

Yield: 65%.

Mp 153 ° C.

[a] ² D = -12 ° (c = 1.10 in CH ₂ Cl _2).

Example 2

1,6: 2,3-dianhydro-4-O- (3-deoxy \ ^J) -D-ribohexopyran osyl) - () - D-mannopyranose [compound (X) formula]

1,6: 2,3-dianhydro-4-O- (2,4,6-tri-O-benzoyl-3-deoxy-ϋ-πόο-ΙιβχορίΓθηοζίΟ-β-Ο-ιτίθηηορίΓθηόζΙ) is debenzoylated to give 1.6. 2,3-Dianhydro-4-O- (3-deoxy-D-ribo-hexopyranosylXD-mannopyranose is obtained.

HU 227 307 Β1

Yield: 95%.

[ «] ² d ^j = -46 ° (c = 1.02 in _CH3 OH).

Example 3

3-O-acetyl-1,6-anhydro-2-O-benzyl-4-O- (benzyl 2-O-acetyl-3-deoxy-4-O-levulinyl - [- D- ribo hexopiranoziluronát) - ( > -D-glucopyranose (compound of formula XI)

The title compound was prepared from 1,6: 2,3-dianhydro-4-O- (3-deoxy-β-ribo-hexopyranosyl) -pD-mannopyranose, the compound of Example 2 by M. Petitou and CAA van Boeckel. Synthesis of Heparin Fragments and Analogues, 203-210. page Progress in Chemistry of Organic Natural Products, Ed. Springer Verlag Vienna - N.Y. (1992). Reference is made in particular to the preparation of 1,6: 2,3-dianhydro-4-O- (3-deoxy-4,6-O-isopropylidene-β-D-ribo-hexapyranosyl) -pD-mannopyranose by reaction with sodium benzylate. and subsequent acetylation, removal of the isopropylidene group with acetic acid, silylation, reaction with levulinic anhydride, Jones oxidation, and esterification with benzyl bromide.

The various steps are listed below.

The step

Scheme 1

1.5 mmol of p-toluenesulfonic acid (camphorsulfonic acid may also be used) and 250 mmol of 2,2-dimethoxypropane under argon are added dropwise to 1,6: 2,3-dianhydro-4-O- (3-deoxy-pD-ribo-hexopyranosyl) -pD- mannopyranose in dimethylformamide (5 mmol in 35 ml solution). After stirring for 2.5 hours, triethylamine (1.8 mmol) was added. The reaction mixture was diluted with dichloromethane, washed with water, dried over anhydrous sodium sulfate, filtered and evaporated to dryness. 1,6: 2,3-Dianhydro-4-O- (3-deoxy-4,6-O-isopropylidene-β-D-ribohexopyranosyl) -? - D-mannopyranose is thus obtained. The resulting compound was used in the next step without purification.

step

Scheme 2 Sodium benzylate (1 M in benzyl alcohol) was added to the compound of Step A (5.0 mmol). After stirring for 30 minutes at 110 ° C, the reaction mixture was cooled, neutralized with Dowex acidic resin (AG 50 WX2), filtered and the benzyl alcohol evaporated in vacuo. The residue was purified on a silica gel column using toluene / acetone (3: 1 v / v) as eluent. The desired product is obtained as a syrup.

Yield: 81%.

Step C

Scheme 3

Step B (4.90 mmol) was dissolved in dichloromethane (22 mL), cooled to 0 ° C, and acetic anhydride (19.6 mmol), 4-dimethylaminopyridine (1.96 mmol) and triethylamine (9.84 mmol) were added. The resulting reaction mixture was stirred for 45 minutes at room temperature, then methanol was added and stirring was continued for another 30 minutes. The reaction mixture was diluted with dichloromethane, washed with aqueous KHSO ₄ , then with water, dried over anhydrous sodium sulfate and evaporated to dryness to give the desired product as a syrup. The resulting syrup was purified on a silica gel column using toluene / acetone (9: 1 v / v) as eluent.

Yield: 85%.

Step D.

Scheme 4

The product of the preceding step was dissolved in 4.11 mmol

2.2 ml of 1,1-dichloroethane and 123 ml of an aqueous acetic acid solution (70%) are added. The reaction mixture was stirred at 50 ° C for 35 minutes. The reaction mixture was evaporated, toluene was added and evaporated to give the desired product as a syrup. The syrup was purified on a silica gel column using cyclohexane / acetone (1: 1 v / v) as eluent. Yield: 90%.

This step

Scheme 5

3.61 mmol of the product of Step D are dissolved

3.3 ml of dichloromethane and 1.42 mmol of 4-dimethylaminopyridine, 10.82 mmol of triethylamine and 5.41 mmol of tert-butyldimethylsilyl chloride are added.

After stirring for about 1 hour at 20 ° C, 43 ml of anhydrous dichloromethane and 10.8 mmol of levulinic anhydride are added. After stirring for 2 hours, dichloromethane (150 mL) was added, washed first with aqueous KHSO ₄ , then with aqueous NaHSO ₄ , dried over anhydrous sodium sulfate, filtered and concentrated to a brown syrup. The product obtained is used in the next step.

Step F

Scheme 6

The syrup obtained in the previous step (2.51 g) was dissolved in acetone (26 ml), cooled to 0 ° C, and chromium trioxide (9.56 mmol) was added and

4.2 ml of 3.5 M sulfuric acid solution.

After stirring for 4 hours at room temperature, 250 ml of dichloromethane are added, the reaction mixture is washed with water, dried over anhydrous sodium sulfate, filtered and evaporated to a brown syrup.

Step G

3-O-acetyl-1,6-anhydro-2-O-benzyl-4-O- (benzyl 2-O-acetyl-3-deoxy-4-O-levulinyl (-) - D-ribo-hexopiranoziluronát) - [ > -D-glucopyranose (compound of formula XI)

Scheme 7

The product obtained in the preceding step (2.15 g) was dissolved in anhydrous dimethylformamide (22 ml) and argon8

7.22 mmol of potassium bicarbonate and 10.83 mmol of benzyl bromide are added under an atmosphere of 301 301. After stirring for 3 hours, methanol (0.5 mL) was added and stirring was continued for 1 hour at room temperature.

The reaction mixture was diluted with ethyl acetate, washed with water, dried over anhydrous sodium sulfate, filtered and evaporated to a brown syrup.

The average yields of steps E, F and G are 81%.

///. production

Scheme 8

3.61 mmol 3-O-acetyl-1,6-anhydro-2-O-benzyl-4-O (benzyl-2-O-acetyl-3-deoxy-4-O-levulinyl-pD-ribohexopyranosyl uronate) β-D-glucopyranose, the compound of Example 3, Step G, was dissolved in 13 mL of pyridine. The resulting solution was cooled to 0 ° C and 18.1 mmol of hydrazine (1M, 3: 2 v / v in pyridine / acetic acid) was added. The reaction mixture was stirred at room temperature for 15 minutes, then concentrated, and dichloromethane was added. wash with aqueous KHSO ₄ , water, then aqueous NaHCO ₃ , and then again with water. The reaction mixture was dried over sodium sulfate, filtered and evaporated to dryness to give a brown syrup. The resulting 3- O -acetyl-1,6-anhydro-2-O-benzyl-4-O- (benzyl-2-O-acetyl-3-deoxy-pD-ribo-hexopyranosyl uronate) -pD-glucopyranose was purified on a silica gel column. cyclohexane / acetone (2: 1 v / v).

Yield: 86%.

[(/.^=-78° (c = 0.7 in CH ₂ Cl _2).

ARC. production

O- (Benzyl 2-O-acetyl-3-deoxy-4-O-levulinyl-β-Dribo-hexopyranosyl uronate) - (1, 4) -3,6-di-O-acetyl-2O-benzyl- D-glucopyranosyl trichloroacetimidate (Compound Xlla)

The compound was prepared as in Example 3 for 3-0-acetyl-1,6-anhydro-2-O-benzyl-4-O- (benzyl-2-O-acetyl-3-deoxy-4-O-levulinyl-pD-). ribo-hexopyranosyl uronate) -PD-glucopyranose from the compound of Example 3 in a known manner, in particular by acetolysis, removal of the anomeric protecting group and formation of the imidate. The various steps are described below.

The step

Scheme 9

To the compound of Example 3 (1.02 mmol) were added acetic anhydride (102 mmol) and trifluoroacetamide (10.2 mmol). The reaction mixture was stirred for 2 hours under argon. The reaction mixture was evaporated to a brown syrup and purified on a silica gel column using cyclohexane / ethyl acetate (2: 3 v / v) as eluent.

Step β

Scheme 10

The compound obtained in the preceding step (1.52 mmol) was dissolved in dichloromethane and 57.9 mmol benzylamine was added. The reaction mixture was stirred for 4 hours at room temperature and then allowed to stand overnight at -20 ° C. To the resulting reaction mixture was added ether and washed with 1 N aqueous hydrochloric acid. The reaction mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate and evaporated to a brown syrup. The resulting syrup was purified on a silica gel column using toluene / acetone (5: 1 v / v) as eluent.

Step C

Scheme 11

The disaccharide obtained in the previous step, 0.246 mmol, was dissolved in dichloromethane. To the solution was added 0.39 mmol of potassium carbonate and 1.23 mmol of trichloroacetonitrile under argon. The reaction mixture was stirred for 16 hours, filtered and evaporated to dryness. There was thus obtained O- (benzyl-2-O-acetyl-3-deoxy-4-O-levulinyl-β-D-ribohexopyranosyluronate) - (1-4) -3,6-di-O-acetyl-2- Obenzyl D-glucopyranosyl trichloroacetimidate as a mixture of anomers.

Yield: 62% (average).

V. Production

1,6: 2,3-dianhydro-4-O- (3-deoxy-D-ribohexopyran osyl) - \ ^J> D-mannopyranose [compound (XV) - gluco and ido mixture]

The step

1,6: 2,3-Dianhydro-4-O- (3-deoxy-6-ido- 1 ^J ) -D-ribohexopyranosyl) - (→ - D-mannopyranose)

1,6: To a solution of 2,3-dianhydro-4-O- (3-deoxy-β-ribo-hexopyranosyl) -pD-mannopyranose (22.81 mmol) in 400 mL of a mixture of dichloromethane and acetonitrile is added 68.43 mmol of triphenylphosphine, 68.43 mmol of imidazole and 29.65 mmol of iodine. The reaction mixture was stirred at 70 ° C for 4 hours. The resulting solution was evaporated and the residual syrup was purified on a silica gel column using dichloromethane: acetone (10: 1 v / v) as solvent. 1,6: 2,3-Dianhydro-4-O- (3-deoxy-6-ido-β-D-ribohexopyranosyl) -? - D-mannopyranose is thus obtained.

Yield: 72%.

Step β

23.4 mmol of 1.6: 2,3-dianhydro-4-O- (3-deoxy-6-ido-pD-ribo-hexopyranosyl) -pD-mannopyranose is dissolved in 150 ml of methanol and 42 mmol of sodium are added to the solution. -methylate dissolved in methanol (1 M). The reaction mixture was heated at 80 ° C for 9 hours, cooled and purified on a Sephadex LH-20 column using dichloromethane: methanol (1: 1 v / v) as eluent. 1,6: 2,3-Dianhydro-4-O- (3-deoxy-5,6-exomethylene-p-D-ribohexopyranosyl) -? - D-mannopyranose is thus obtained. 13.3 mmol of the compound thus obtained were dissolved in 100 ml of tetrahydrofuran and added dropwise at 20 ° C to a solution of 54.4 mmol of diborane in tetrahydrofuran. After stirring for 2 hours, ethanol (65.28 mmol) was added. The resulting reaction mixture was stirred for 1 hour and then treated with 24 mL of 3 M sodium chloride 9

EN 227 307 Β1 of sodium hydroxide solution and 24 ml of 30% hydrogen peroxide solution. The resulting reaction mixture was heated to 50 ° C, neutralized with Dowex acidic resin (AG 50 WX4) and evaporated to dryness. There was thus obtained 1,6: 2,3-dianhydro-4-O- (3-deoxy-α-D-ribohexopyranosyl) -? - D-mannopyranose as a mixture of gluco- and idodisaccharides (1: 6.25).

VI. production

3-Acetyl-1,6-anhydro-2-O-benzyl-4-O- (benzyl-2-O-acetyl-3-deoxy-4-O-levulinyl-αL-hexo-pyropyranosyluronate) - $ - D-glucopyranose [ Compound of Formula XVI]

3-O-Acetyl-1,6-anhydro-2-O-benzyl-4-O- (benzyl-2-O-acetyl-3-deoxy-4-O-levulinyl-αL-hexo-pyropyranosyluronate) -PD-glucopyranose is prepared. from the compound of Preparation V according to a known method [G. Jaurand et al., Bioorganic and Medicinal Chemistry Letters (1992) 2, No. 9, 897-900. J. Basten et al., Bioorganic and Medicinal Chemistry Letters (1992), 2 (no. 9), 901-904. J. Basten et al., Bioorganic and Medicinal Chemistry Letters (1992), 2 (no. 9), 905-910. side]. By synthesis of the two appropriate 4 ', 6'-isopropylidene derivatives by purification, separation of the gluco- and iodo-isomers on a silica gel column, sodium benzylate reaction, acetylation, isopropylidene removal, selective silylation, levulinic acid anhydride, by esterification with benzyl bromide.

Yield: 45%.

The various steps are described below.

The step

Scheme 12

1.6 mmol of 1,6: 2,3-dianhydro-4-O- (3-deoxy-αD-ribohexopyranosyl) -pD-mannopyranose is dissolved in 25 ml of dimethylformamide and 21.64 mmol of dimethoxypropane and 3.96 mmol p-toluenesulfonic acid (camphorsulfonic acid may also be used). The reaction mixture was stirred for 1 hour 30 minutes and then triethylamine (5.94 mmol) was added. The reaction mixture was evaporated to a syrup and purified on a silica gel column using toluene / ethyl acetate (2: 3 v / v) as eluent.

Yield: 70%.

Step β

Scheme 13

To the compound of Step A (6.81 mmol) was added 29.7 mmol of sodium benzylate, dissolved in benzyl alcohol (1 M). The reaction mixture was stirred at 110 ° C for 1 hour. The reaction mixture was diluted with dichloromethane (200 mL), neutralized with Dowex acidic resin (AG 50 WX4), filtered and concentrated in vacuo to a brown syrup. This was purified on a silica gel column using dichloromethane: acetone (10: 1 v / v) as eluent.

Yield: 85%.

Step C

Scheme 14

Step B (5.76 mmol) was dissolved in dichloromethane and 3.92 mmol of 4-dimethylaminopyridine, 72.27 mmol of triethylamine and 65.7 mmol of acetic anhydride were added. After stirring at room temperature for 2 hours 30 minutes, dichloromethane (100 mL) was added, the resulting mixture was washed with 10% aqueous KHSO ₄ , dried over anhydrous sodium sulfate and filtered.

The residual syrup was evaporated and purified on a silica gel column using dichloromethane: acetone (10: 1 v / v) as eluent. [α] D = -102 ° (c = 1.37 in CH ₂ Cl ₂ ).

Step D.

Scheme 15

To the compound of Step C (5.74 mmol) was added 25% 70% aqueous acetic acid. The reaction mixture was stirred at 80 ° C for 6 hours and then concentrated in the presence of toluene to give a yellow powder.

This step

Scheme 16

Abbreviations in the reaction scheme:

DMAP: 4-Dimethylaminopyridine tBDMSCI: tert-Butyldimethylsilyl Chloride BnBr: Benzyl Bromide

LevO ₂ : levulinic anhydrides

DMF: dimethylformamide

Bn: benzyl

Ac: acetyl

Lev: levulinic acid group tBDMSI: tert-butyldimethylsilyl group

The product of the previous step was used as starting material and was prepared according to Steps E, F and G of Example 3 to give 3-O-acetyl-1,6-anhydro-2-O-benzyl-4-O (benzyl-2 O-acetyl-3-deoxy-4-O-levinine-α-α-Lx (ixohexopyranosyl uronate) -pD-glucopyranose.

Average yield: 59%.

VII. production

Methyl 3,6-di-O-acetyl-2-O-benzyl-4-O- (benzyl-2-O-acetyl-3-deoxy-η 5 -hexo-hexopyranosyl uronate) - () - Glucopyranoside [(III) ))

VI. The disaccharide of Preparation I was subjected to acetolysis and the resulting compound was treated with benzylamine, reacted with Vilsmeier reagent, and then methanol in the presence of silver carbonate. The compound thus obtained is treated with hydrazine to give methyl 3,6-diO-acetyl-2-O-benzyl-4-O- (benzyl-2-O-acetyl-3-deoxy-αL-lixo-hexopyranosyl uronate). ) Pd-glucopyranoside. The various steps are described below.

The step

Scheme 17

102 mmol acetic anhydride and 10.2 mmol trifluoroacetic acid were added 1.02 mmol VI. and heated at 50 ° C for 2 hours under argon. The resulting reaction was 10

EN 227 307 Β1 concentrate to a brown syrup. Preparation is carried out according to its step to obtain the desired product.

Yield: 92%.

Step β

Scheme 18

The product of Step A (1.52 mmol) was dissolved in anhydrous ethyl ether and benzylamine (57.9 mmol) was added. The IV. Preparation step D yields the desired product.

Yield: 79%.

Step C

Scheme 19

Vilsmeier reagent was prepared by mixing 2.15 mmol of bromine and 2.15 mmol of triphenylphosphine at 0 ° C in 5 mL of dimethylformamide. The white precipitate from the reaction mixture was filtered under argon and added to 0.269 mmol of Step B product in 15 mL of anhydrous dichloromethane while maintaining the reaction temperature. The reaction mixture was stirred at room temperature for 2 hours, diluted with dichloromethane, washed with water cooled to 0 ° C to pH 6, dried over anhydrous sodium sulfate and evaporated to a syrup. The resulting syrup was purified on a silica gel column using dichloromethane: ethyl ether (5: 1 v / v).

Yield: 77%.

Step D.

Scheme 20

Methanol (0.46 mL), silver carbonate (0.34 mmol) and calcium sulfate (150 mg) were stirred in dichloromethane (3 mL) for 1 hour at 0 ° C under argon. 0.227 mmol of Step C was dissolved in 8 mL of dichloromethane. The resulting solution was added dropwise to the reaction mixture and stirred at 20 ° C for 20 hours. The reaction mixture is protected from light. The reaction mixture was then diluted with dichloromethane, filtered and evaporated to give 3,6-di-O-acetyl-2-O-benzyl-4-O- (benzyl-2-O-acetyl-3-deoxy-4-). O-levulinyl-α-Llixo-hexopyranosyl uronate) -D-glucopyranoside as a syrup. The resulting syrup was purified on a silica gel column using dichloromethane: ethyl ether (5: 1 v / v) as eluent.

Yield: 75%.

[a] ² ° D = + 27 ° (c = 1.8 in CH ₂ Cl _2).

This step

Scheme 21

The product from the previous step (0.276 mmol) was dissolved in pyridine (2 ml) cooled to 0 ° C and a solution of hydrazine hydrate (1.38 ml, 1 M in pyridine / acetic acid (3: 2 v / v)) was added. The reaction mixture was evaporated, methylene chloride was added to the residue and the resulting reaction mixture was washed with aqueous KHSO ₄ , dried over anhydrous sodium sulfate, filtered and evaporated to a syrup. The resulting syrup was purified on a silica gel column using dichloromethane: ethyl ether (3: 1 v / v).

Yield: 87%.

[a] ² ° D = + 10 ° (c = 0.99 in CH ₂ Cl _2).

Vili. production

O- (2,4,6-Tri-O-benzyl-3-deoxy-αD-ribohexopyranoso) - (1-4) -O- (benzyl-2-O-acetyl-3-deoxy) - () - D-ribo -hexopyranosyl uronate) - (1-4) -3-Oacetyl-1,6-anhydro-2-O-benzyl-β-D-glucopyranose (Compound VII)

2.03 mmol of 2,4,6-tri-O-benzyl-3-deoxy-1-thio-D-ribohexopyranoside (Preparation II) and 1.69 mmol of 3-O-acetyl-1,6-anhydro-2-O Benzyl 4-O- (benzyl-2-O-acetyl-3-deoxy-β-ribo-hexopyranosyluronate) -PD-glucopyranose (Preparation III) is dissolved in dichloromethane and a molecular sieve is added at 20 ° C. then 5.07 mmol silver trifluoromethanesulfonate and 1.52 mmol bromine. The reaction mixture was stirred for 45 minutes, then filtered, washed with water and evaporated to dryness.

After purification on silica gel, the desired product is obtained.

Yield: 35%.

IX. production

O- (6-O-Acetyl-2,4-di-O-benzyl-3-deoxy-αD-ribohexopyranosyl) - (1,4) -O- (benzyl-2-O-acetyl-3-deoxy) - [> - D-ribo-hexopyranosyl uronate) - (1/4) -3,6di-O-acetyl-2-O-benzyl - () - D-glucopyranosyl trichloroacetimidate (compound of formula II)

0.51 mmol Fruit. The compound of Preparation II is dissolved in a mixture of trifluoroacetic acid and acetic anhydride and the solution is subjected to acetolysis. The reaction mixture was treated with ethyl ether benzylamine followed by dichloromethane dichloroacetonitrile in the presence of potassium carbonate to give the desired product. Yield: 50%.

REFERENCE EXAMPLE 1 [Preparation of Compound of Formula I]

Methyl-O- (3-deoxy-2,4,6-tri-O-sulfo-αD-ribohexopyranosyl) - (1, 4) -O- (3-deoxy-2-O-sulfo-? hexopyranosyl uronate) - (1-4) -O- (2,3,6-tri-Osulfo-αD-glucopyranosyl uronate) - (1,4) -2,3,6-triO-sulfo-β-D -glycopyranoside tridecakis sodium salt Step A

Methyl-O- (6-acetyl-2,4-O-benzyl-3-deoxy-αD-ribohexopyranosyl- (1, 4) -O- (benzyl-2-O-acetyl-3-deoxy-1H-ribo-) hexopyranosyl uronate) - (1-4) -O- (3,6-di-O-acetyl-2-O-benzyl-α-D-glucopyranosyl) - (1-4) -O (benzyl-2-O-acetyl- 3-Deoxy-αL-lixo-hexopyranosyluronate) - (1,4) -O-3,6-di-O-acetyl-2-O-benzyl- [h-D-glucopyranoside

0.121 mmol IX. and 0.093 mmol of VII. The compound of Preparation II is dissolved

3.2 ml of dichloromethane. The reaction mixture was cooled to -20 ° C in the presence of a molecular sieve and 0.470 mL of a solution of tri-methylsilyl trifluoromethanesulfonate in dichloromethane was added.

HU 227 307 Β1

The reaction mixture was stirred for 1 hour at -20 ° C, then filtered, washed with water, evaporated, and on a Sephadex LH-20 column, dichloromethane / methanol (1: 1 v / v) as solvent and then on silica gel with cyclohexane and ethyl as solvent. acetate (3: 2 v / v) to give the desired product.

Yield: 62%.

Step β

The product from the previous step (0.052 mmol) was dissolved in a mixture of dichloromethane (0.43 mL) and methanol (1.7 mL). To the resulting solution was added 85 mg of 10% palladium on charcoal and the reaction mixture was hydrogenated under mild pressure for 4 hours at 20 ° C. The resulting reaction mixture was evaporated to dryness to give O- (6-O-acetyl-3-deoxy-αD-ribo-hexopyranosyl) (1, 4) -O- (2-O-acetyl-3-deoxy - [) - D-ribo-hexopyranosyluronic acid) - (1-4) -O- (3,6-di-O-acetyl-α-D-glucopyranosyl) (1,4) -O- (2-O-acetyl-3-deoxy) (N-L-lixo-hexopyranosyluronic acid) - (1,4) -3,6-di-O-acetyl - [) - D-glucopyranoside. The resulting compound was dissolved in ethanol (0.96 mL), cooled to 0 ° C, and treated with 5N sodium hydroxide (0.3 M, 0 mL). After stirring for 5 hours at 0 ° C, the reaction mixture was purified on a Sephadex G-25 column using water as eluant. The resulting reaction mixture was evaporated to dryness to give methyl-O- (3-deoxy-αD-ribo-hexopyranosyl) - (1-> 4) O- (3-deoxy-β-ribo-hexopyranosyl-uronic acid) - (1- > 4) -O- (a-D-glucopyranosyl) - (1-> 4) -O- (3-deoxy-L-lyxo-exopyranosyl-uronic acid (1-> 4) Pd-glucopyranoside.

The resulting compound was dissolved in 5 mL of dimethylformamide, evaporated to dryness and redissolved in 2.6 mL of anhydrous dimethylformamide under argon. To the resulting solution was added 302 mg of sulfur trioxide / triethylamine complex and the mixture was stirred for 20 hours at 55 ° C. The reaction mixture was cooled, treated with sodium bicarbonate (491 mg) as an aqueous solution, and stirred for 3 hours.

The resulting reaction mixture was purified on a Sephadex G-25 column using water as solvent and lyophilized. This gives methyl O- (3-deoxy-2,4,6-tri-O-sulfo-αD-ribo-hexopyranosyl) - (1-> 4) -O (3-deoxy-2-O-sulfo-pD-). ribo-hexopyranosyl uronate) (1-4) -O- (2,3,6-tri-O-sulfo-αD-glucopyranosyl) - (1-4) O- (3-deoxy-2-O- sulfo-αL-lixo-hexopyranosyl uronate) (1/4) -2,3,6-tri-O-sulfo [ ^J ] -D-glucopyranoside tridecakis sodium salt.

Yield: 65%.

[α] ²⁰ D = + 25 ° (c = 0.52 in H ₂ O) - Experiment 1.

[α] ²⁰ D = + 33 ° (c = 0.64 in H ₂ O) - Experiment 2.

Proton nuclear magnetic resonance spectrum (500 MHz, solvent: D ₂ O)

H1: 4.78 ppm, J1-2: 7.3 Hz; ΗΊ: 5.22 ppm, J1'-2 ':

1.8 Hz; H 1: 5.26 ppm, J 1 -2: 3.5 Hz; H '' 1:

4.78 ppm, J1 "'- 2'": 5.4 Hz; H "": 5.16 ppm, J1 "" - 2 "":

3.3 Hz.

Reference Example 2 [Compound of Formula I]

Methyl O- (3-deoxy-2,4,6-tri-O-sulfo-D-ribohexopyran osyl) - (1-> 4) -O- (3-deoxy-2-O-sulfo - [> - Dribo- hexopyranosyl uronate) - (1, 4) -O- (2,3,6, tri-Osulfo-1-D-glucopyranosyl) - (1, 4) -O- (3-deoxy-2-O-methyl) a-L-lxo-hexopyranosyl uronate) - (1-> 4) -2,3,6-triO-sulfo-β-D-glucopyranoside dodecakis sodium salt

The title compound is O- (6-O-acetyl-2,4-di-O-benzyl-3-deoxy-α-D-ribohexopyranosyl) - (1-4) -O- (benzyl-2O-acetyl). 3-deoxy - () - D-ribo-hexopyranosyl uronate) - (? 4) 3.6-di-O-acetyl-2-O-benzene from 1Dg-lycopyranosyl trichloroacetamidate and methyl-3,6-di-O- acetyl 2-O-benzyl-4-O (benzyl 2-O-methyl-3-deoxy-α-L-hexohyropyranosyluronate) - [? D-glucopyranoside as described in Example 1]. Methyl 3,6-di-O-acetyl-2-O-benzyl-4-O (benzyl-2-O-methyl-3-deoxy-α-L-lhexo-hexopyranosyluronate) -pD-glucopyranoside 1.6: 2 , 3-Dianhydro-4-O (3-deoxy-β-ribo-hexopyranosyl) -pD-mannopyranose is prepared according to the method described in VII. and IX. by production.

[a] o ° = + 25 ° (c = 0.48, H ₂ O).

Proton nuclear magnetic resonance spectrum (500 MHz, solvent: D ₂ O)

H1: 4.82 ppm J1-2: 4.2 Hz; ΗΊ: 5.08 ppm, J1'-2 ':

1.0 Hz; H '1: 5.27 ppm, J1' -2 ': 3.7 Hz, H' ': 4.76 ppm,

J1 '' - 2 '': 7.7 Hz; H "": 5.15 ppm, J1 "" -2 "": 3.5 Hz.

Reference Example 3 [Compound of Formula I]

Methyl O- (3-deoxy-2,4-di-O-methyl-6-O-sulfo-α-Dribo-hexopyranosyl) - (1,4) -O- (3-deoxy-2-O-sulfo) [(D) -D ribo-hexopyranosyl uronate) - (1-4) -O- (2,3,6-triO-sulfo- (1D-glucopyranosyl) - (1,4) -O- (3-deoxy) 2-O-sulfo-UL-lyxo-exopyranosyl-uronate) - (1> 4) 2,3,6-tri-O-sulfo-ss-D-glucopyranoside salt undekakisznátrium

The title compound is O- (6-O-benzyl-2,4-di-Omethyl-3-deoxy-αD-ribo-hexopyranosyl) - (1-4) -O- (benzyl-2-O-acetyl). 3-deoxy-pD-ribo-hexopyranosyl uronate) (1, 4) -3,6-di-O-acetyl-2-O-benzyl-D-glucopyranosyl trichloroacetimidate and methyl 3,6-di-O- acetyl 2-O-benzyl4-O- (benzyl-2-O-acetyl-3-deoxy-α-L-lhexohexopyranosyluronate) -pD-glucopyranoside as described in Reference Example 1. O- (6-O-Benzyl-2,4-di-Omethyl-3-deoxy-α-D-ribo-hexopyranosyl) - (1-4) -O- (benzyl-2-O-acetyl-3-deoxy) p-D-ribo-hexopyranosyl uronate) (1- + 4) -3,6-di-O-acetyl-2-O-benzyl-D-glucopyranosyl trichloroacetimidate, diethyl 6-O-benzyl-2,4-di -O-methyldimethoxy-1-thio-β-ribo-hexopyranoside and 3-O-acetyl-1,6-anhydro-2-O-benzyl-4-O- (benzyl-2-O-acetyl-3-deoxy). pD-ribo-hexopyranosyl uronate) -PD-glucopyranose according to Preparation X.

[α] D 20 = + 24 ° (c = 0.6 in H ₂ O).

Proton nuclear magnetic resonance spectrum (500 MHz, solvent: D ₂ O)

H1: 4.77 ppm, J1-2: 5.0 Hz; ΗΊ: 5.22 ppm, J1'-2 ':

2.2 Hz; H 1: 5.24 ppm, J 1 -2: 3.7 Hz; N ''

4.76 ppm, J1 "'- 2"': 7.5 Hz; H "": 5.10 ppm, J1 "" - 2 "":

3.6 Hz.

HU 227 307 Β1

Reference Example 4

Methyl O- (3-deoxy-4-O-methyl-2,6-di-O-sulfo-a-Dribo-hexopyranosyl) - (1-> 4) -O- (3-deoxy-2-O-szulfop -D-ribo-hexopyranosyl uronate) - (1-4) -O- (2,3,6-triO-sulfo-η-D-glucopyranosyl) - (1,4) -O- (3-deoxy- 2-O-sulfo-L-lyxo-exopyranosyl-uronate) - (1 →> 4) 2,3,6-tri-O-sulfo-f) -D-glucopyranoside dodekakisznátrium salt

The title compound was prepared as described in Reference Example 1 in Example VII. and O- (2,6-di-O-benzyl-4-O-methyl-3-deoxy-α-Dribohexopyranosyl) - (1-> 4) -O- (benzyl-2-O-). acetyl 3-desol-β-D-sulfo-hexopyranosyl uronate) - (1, 4) -3,6-di-O-acetyl-2-O-benzyl-D-glucopyranosyl trichloroacetimidate. The latter trisaccharide derivative was prepared from ethyl 2,6-di-O-benzyl-4-O-methyl-3-deoxy-1-thio-D-ribo-hexopyranoside and

III. Prepared from Vili. and IX. by production.

[α] ²⁰ D = + 22 ° (c = 0.54 in H ₂ O).

Proton nuclear magnetic resonance spectrum (500 MHz, solvent: D ₂ O)

H1: 4.78 ppm, J1-2: 5.1 Hz; ΗΊ: 5.22 ppm, J1'-2 ':

2.6 Hz; H 1: 5.27 ppm, J 1 -2: 3.9 Hz; Η'Ί:

4.75 ppm, J1 "'- 2'": 7.9 Hz; H "": 5.11 ppm, J1 "" - 2 "":

3.5 Hz.

Example 5

Methyl O- (3-deoxy-2,4,6-tri-O-sulfo-UD-ribohexopyran osyl) - (1 → 4) -O- (3-deoxy-2-O-methyl - (- Dribo- hexopyranosyl uronate) - (1-4) -O- (2,3,6-tri-Osulfonyl-D-glucopyranosyl) - (1,4) -O- (3-deoxy-2-Omethyl-uL) (1x 4) -2,3,6-triO-sulfo-pD-glucopyranoside undecakis sodium salt The title compound is methyl 3,6-di-O-acetyl-2. O-benzyl-4-O- (benzyl-2-O-methyl-3-deoxy-α-L-lxohexopyranosyl uronate) -β-D-glucopyranoside and O- (6-O-acetyl-2,4-di-O-benzyl). 3-deoxy-αD-ribo-hexopyranosyl) (1-4) -0- (insoluble 1-2-O-methyl-3-β-dichloro-β-β-hexopyranosyl uronate) - (1 → 4) - 3,6-Di-O-Acetyl-2-O-benzyl-D-glucopyranosyl trichloroacetimidate was prepared as described in Reference Example 1. The starting trisaccharide was 3-O-acetyl-1,6-anhydro-2-O. Benzyl 4-O- (benzyl-2-O-methyl-3-deoxy-D-ribo-hexopyranosyluronate) -? - D-glucopyranose and the compound of Preparation II according to Preparations VIII and IX. α] ² ο ^θ = + 20 ° (c = 0.39 in H ₂ O).

Proton nuclear magnetic resonance spectrum (500 MHz, solvent: D ₂ O)

H1: 4.83 ppm, J1-2: 3.0 Hz; ΗΊ: 5.11 ppm, J1'-2 ':

2.0 Hz; H ”1: 5.31 ppm, J1” -2 ”: 3.3 Hz, Η'Ί:

4.68 ppm, J1 '' - 2 '': 7.7 Hz; H "": 5.18 ppm, J1 "" - 2 "":

3.2 Hz.

Reference Example 6

Methyl O- (3-deoxy-2,4,6-tri-O-sulfo-D-ribohexopyran osyl) - (1-> 4) -O- (3-deoxy-2-O-methyl- \ ^J> -Dribo -hexopyranosyl uronate) - (1, 4) -O- (2,3,6-tri-Osulfo-1-D-glucopyranosyl) - (1, 4) -O- (3-deoxy-2-Osulfo) -aL-lixo-hexopyranosyl uronate) - (1/4) -2,3,6tri-O-sulfo-1D-glucopyranoside dodecakis sodium salt

The title compound was prepared as described in Reference Example 1 for O- (6-O-acetyl-2,4-di-O-benzyl-3-deoxy-αD-ribo-hexopyranosyl) - (1-4) -O- ( benzyl 2-Omethyl-3-deoxy-4-D-ribo-hexopyranosyl uronate) - (1-> 4) 3,6-di-O-acetyl-2-O-benzyl-1 g of copyranosyl-1-trichloroacetimidate and VII. . . [a] ² ° D = + 30 ° (c = 0.40 in H ₂ O).

Proton nuclear magnetic resonance spectrum (500 MHz, solvent: D ₂ O)

H1: 4.77 ppm, J1-2: 5.0 Hz; ΗΊ: 5.20 ppm, J1'-2 ':

2.5 Hz; H '' 1: 5.27 ppm, J 1 '-2': 4.0 Hz; H '' 1:

4.67 ppm, J1 '' - 2 '': 8.0 Hz; H "": 5.14 ppm, J1 "" - 2 "":

3.5 Hz.

Reference Example 7

Methyl-O- (3-deoxy-2,4,6-tri-O-sulfo-αD-ribohexopyranosyl) - (1,4) -O- (3-deoxy-2-O-methyl - (, - Dribo-) hexopyranosyl uronate) - (1-4) -O- (2,3,6-tri-Osulfo-? D-glucopyranosyl) - (1-4) -O- (3-O-methyl-2-Osulfo-) a-L-ido-pyranosyl uronate) - (1/4) -2,3,6-tri-Osulfo-β-D-glucopyranoside dodecakis sodium salt The title compound is O- (6-O-acetyl-2). , 4-di-O-benzyl-3-deoxy-αD-ribo-hexopyranosyl) - (1-4) -O- (benzyl-2-O-methyl-3-deoxy-β-D-ribo-hexopyranosyluronate) - (1- > 4) -3,6-di-O-acetyl-2-O-benzyl-D-glucopyranosyl trichloroacetimidate and methyl 2,3,6-tri-Obenzene 1-4-0- (benzyl-2- O-Benzyl 1-3-O-methyla-Lidopyranosyl uronate) -? - D-glucopyranoside is prepared as described in Reference Example 1. The latter compound is described by M. Petitou and CAA van Boeckel in "Chemical Synthesis of Heparin Fragments and Analogues" , Pp. 203-210 - Progress in Chemistry of Organic Natural Products, edited by Springer Verlag Vienna - NY (1992), [α] ² D = + 46 ° (c). = 0.58 H ₂ O).

Proton nuclear magnetic resonance spectrum (500 MHz, solvent: D ₂ O)

H1: 5.10 ppm, J1-2: 3.0 Hz; ΗΊ: 5.10 ppm, J1'-2 ':

5.0 Hz; H '1: 5.48 ppm, J 1' -J 2 ': 3.60 Hz; H '' 1:

4.68 ppm, J1 "'- 2"': 7.5 Hz; H "": 5.16 ppm, J1 "" - 2 "":

3.6 Hz.

Reference Example 8

Methyl O- (3-deoxy-2,4-di-O-sulfo-β-D-ribohexopyranosyluronate) - (1-4) -O- (2,3,6-tri-O-sulfo-1D) -glycopyranosyl) - (1, 4) -O- (3-deoxy-2-O-sulfop-D-ribo-hexopyranosyl uronate) - (1, 4) -O- (2,3,6-triO-sulfo) - (? D-glucopyranosyl uronate) - (1, 4) -O (3-deoxy-2-O-sulfo-a-L-lxo-hexopyranosyluronate) - (1, 4) -2,3,6-tri-O- sulfo - (> - D-glucopyranoside-hexadecakis-sodium salt) The title compound is O- (benzyl-2-O-acetyl-3d-deoxy-4-O-levulinyl-4-D-ribo-hexopyranosyl uronate) (1-> 4) -3,6-di-O-acetyl-2-O-benzyl-Dg-glucopyranosyl from trichloroacetimidate (Preparation IV) and 3-O-acetyl-1,613

Anhydro-2-O-benzyl-4-O- (benzyl-2-O-acetyl-3-deoxy-β-ribo-hexopyranosyl uronate) is prepared from β-D-glucopyranose (Preparation III). The two compounds were reacted according to Step A of Reference Example 1 to give compound XXIII.

Yield: 64%.

The product obtained is subjected to acetolysis according to the procedure described in IV. Step A and the resulting compound is reacted with benzylamine to give the title compound. Preparation B, so that the anomeric hydroxyl derivative is liberated. The appropriate imidate of the resultant compound with trichloroacetonitrile in the presence of potassium carbonate is prepared as described in Table IV. is prepared by reacting Step C of the preparation.

The product was obtained with methyl-3,6-di-O-acetyl-2-O-benzyl-4-O- (benzyl-2-O-acetyl-3-deoxy-α-L -hexo-hexopyranosyluronate) -PD-glucopyranoside (Preparation VII). ), methyl-O (3-deoxy-2,4-di-O-sulfo-β-ribo-hexopyranosyluronate) - (1-4) -O- (2,3,6-tri-O-sulfo) -D-glucopyranosyl) (1/4) -O- (3-deoxy) -2-O-sulfo- [1D-ribohexopyranosyl5 uronate] - (1-4) -O- (2,3,6- tri-O-sulfo-D-glucopyranosyl) - (W4) -O- (3-deoxy-2-O-sulfo-L-lyxo-exopyranosyl-uronate) - (1-> 4) -2,3,6- and tri-O-sulfo-pD-glucopyranoside hexadecakis sodium salt.

Proton nuclear magnetic resonance spectrum (500 MHz, solvent: D ₂ O)

H1: 4.78 ppm, J1-2: 7.0 Hz; ΗΊ: 5.22 ppm, J1'-2 ': 1.9 Hz; H 1: 5.25 ppm, J 1 -2: 3.50 Hz; H '' 1: 4.79 ppm, J '' 1-2 '': 5.6 Hz; H "": 5.31 ppm, J1-2:

3.50 Hz; H "": 4.80 ppm, J1 "" - 2 "": 5.9 Hz.

Claims (3)

PATENT CLAIMS 1. 1,6: 2,3-Dianhydro-4-O- (2,4,6-tri-O-benzoyl-3-deoxy-β-ribo-hexopyranosyl) -pD-mannopyranose of Formula IX, wherein Bz is benzoyl. 2. 1,6: 2,3-Dianhydro-4-O- (3-deoxy-β-ribo-hexopyranosyl) -β-D-mannopyranose of the formula (X). 3. Compounds of formula XI wherein -R-ι 'represents a linear or branched C 1 -C 6 alkoxy group, an acyclic C 1 -C 6 acyloxy group, preferably an acetoxy group, an aromatic acyloxy group, preferably a benzoyloxy group or a C 2 -C 7 alkoxy group. -alkenyloxy, preferably allyloxy, - P ₂ and P ₂ are the same or different and are a protecting group such as acyclic C 1-6 acyl, preferably acetyl, aromatic acyl, Preferably benzoyl, C2-C7-alkenyl, preferably allyl or benzyl, - P ₄ is a protecting group such as C 1-6 alkyl, preferably methyl or benzyl,