MXPA97003365A - Carbohydr derivatives - Google Patents

Abstract

The present invention relates to carbohydrate derivatives having the formula I, wherein R 1 is H or CH 2 OSO 3, R 2 and R 3 are independently H, (1-6C) alkyl, or SO 3, R 4 is OSO 3, or NHSO 3; n is 0 or 1; p is 1 or 2, or a pharmaceutically acceptable salt thereof. The compounds of the invention have antithrombotic activity and can be used to inhibit the proliferation of muscle cells.

Description

DERIVATIVES OF CARBOHYDRATE DESCRIPTION OF THE INVENTION The invention relates to a carbohydrate derivative having antithrombotic activity, to a pharmaceutical composition containing the same, as well as to the use of said carbohydrate for the manufacture of a medicament. The carbohydrate derivatives having antithrombotic activity are known, for example the sulfated glycosaminoglycan derivatives described in EP 84,999. Other carbohydrate derivatives related to sulfated glycosaminoglycan are described in EP 529,715, and exhibit improved pharmacological properties. These carbohydrate derivatives are devoid of the functional groups characteristic of glycosaminoglycans, being free hydroxyl groups, N-sulfate and N-acetyl groups. It has now been found that the carbohydrate derivatives of this invention have the formula I, wherein R1 is H or CH2OSO3"; R2 and R3 are independently H, (1-6C) alkyl, or SO3"; R4 is OSO3"or NHSO3 ', n is 0 or 1, p is 1 or 2, or a pharmaceutically acceptable salt thereof, having an anti-Xa activity, which is substantially greater than that of saccharides which do not have the glycerol type or glycol type group in the 4 position of the non-reducing end.Factor Xa plays a very important role in the blood coagulation cascade.This catalyzes the formation of thrombin, which regulates at least the last step in the coagulation cascade The main function of thrombin is the unfolding of fibrinogen to generate fibrin monomers, which form an insoluble gel, a fibrin clot, through entanglement The compounds of the present invention are useful for treating and avoiding thrombin-mediated diseases associated with thrombin This includes a number of thrombotic states and prothrombotic states, in which the coagulation cascade is activated and include, but are not limited to, t deep vein rhombosis, pulmonary embolism, arterial occlusion of thrombosis or embolism, arterial reocclusion during or after angioplasty or thrombolysis, restenosis followed by arterial damage or invasive cardiological procedures, thrombosis or postoperative venous embolism, acute or chronic atherosclerosis, heart attack, infarction to the myocardium, cancer and metastasis, and neurodegenerative diseases. The carbohydrate derivatives of the present invention can also be used as inhibitors of smooth muscle cell proliferation and for the treatment of angiogenesis, cancer and retrovirus infections, such as VI H. In addition, the compounds of the invention can be used as anticoagulants and anticoagulant coatings in extracorporeal blood circuits, as is necessary in dialysis and surgery. The compounds of the invention can also be used as anticoagulants in vitro or ex vivo. The preferred carbohydrate derivatives according to the invention have the formula I, wherein R2 is alkyl (1-6C), R3 is SO3", R4 is OSO3", and R1, n and p are as previously defined; or a pharmaceutically acceptable salt thereof. The most preferred carbohydrate derivatives have the formula I, where R1 is methyl. Particularly preferred carbohydrate derivatives have the formula I, wherein n is 1 and p is 1. The still very preferred carbohydrate derivatives are those of the formula I, wherein R1 is CH2OSO3. "The term, (1-6C) alkyl, means a branched or unbranched alkyl group having from 1 to 6 carbon atoms, such as such as methyl, ethyl, isopropyl, t-butyl, isopentyl, hexyl, and the like The preferred alkyl groups are (1-4C) alkyl groups, such as methyl, ethyl, (iso) propyl, n-butyl, and t-butyl The most preferred alkyl group is methyl, The counterions, which compensate for the charged portions, are pharmaceutically acceptable counterions, such as hydrogen, or very preferably alkali metal or alkaline earth metal ions., such as sodium, calcium, or magnesium. The carbohydrate derivatives according to this invention can be prepared through the coupling of a protected glycerol or glycol-type portion to the 4-hydroxy group of the non-reducing end of a protected additional tetrasaccharide, which can be produced in accordance with the method described by Westerduin P., Bioorg. and Med. Chem., 2, 1267-1280, 1994. Then, the protecting groups are removed, followed by sulfation of the compound, resulting in a carbohydrate derivative of the formula I. For the treatment of venous thrombosis or for the inhibition of smooth muscle cell proliferation, the compounds of the invention may be administered enterally or parenterally, and for humans preferably at a daily dose of 0.001-10 mg per kg of body weight. Mixed with pharmaceutically acceptable auxiliaries, for example as described in the reference, Geenaro et al., Remington's Pharmaceutical Sciences, (18th Ed., Mack Publishing Company, 1990, see especially part 8: Pharmaceutical Preparations and Their Manufacture), the compounds , when they are oral, buccal or sublingually active, they can be compressed into solid dosage units, such as pills, tablets, or they can be processed into capsules or suppositories. Via pharmaceutically suitable liquids, the compounds can also be applied as an injection preparation in the form of a solution, suspension, emulsion, or as a spray, for example a nasal spray. To make dose units, v. gr., tablets, the use of conventional additives such as fillers, colorants, polymeric binders, and the like is contemplated. In general, any pharmaceutically acceptable additive that does not interfere with the function of the active compounds can be used. Suitable carriers, with which the compositions can be administered, include lactose, starch, cellulose derivatives and the like, or mixtures thereof used in suitable amounts. The invention will be further illustrated through the following examples. (In the examples reference is made to flow diagrams 1 and 2. Intermediaries and final products are indicated by reference to the corresponding number in the flow diagrams).

EXAMPLE 1 Preparation of Compound 7 and 8 Preparation of 2 To a cooled (0 ° C) solution of 2-benzyloxyethanol 1 (2.84 ml) and chloromethyl-methyl sulfide (1.59 ml) in ethylene glycol dimethyl ether (25 ml) was added sodium hydride, 60% of dispersion in mineral oil (1.2 g) under a nitrogen atmosphere and the mixture was stirred for 20 hours at room temperature. Methanol was added to the reaction mixture and stirring was continued for 15 minutes. The mixture was diluted with ethyl acetate (100 ml), subsequently washed with aqueous sodium hydrogen carbonate and water, and the organic layer was dried over magnesium sulfate and concentrated in vacuo. The crude product was purified through silica gel column chromatography to give 2.5 g of 2.

Preparation of 3 The synthesis of 3 has been described in Bioorganic and Medicinal Chemistry, vol. 2, No. 11, p. 1267-1280, 1994 (P. Westerduin et al.).

Preparation of 4 A mixture of 3 (125 mg), 2 (64 mg) and powdered molecular sieves (4 Á) in dichloromethane (1.5 ml) was stirred under a nitrogen atmosphere for 15 minutes. The solution was cooled (5 ° C) and a freshly prepared solution of N-iodosuccinimide (68 mg) and trifluoromethanesulfonic acid (2.7 μl) was introduced into the solution. 1. 5 of 1, 2-dichloroethane-dioxane (1/1, v / v). After 10 minutes, the red reaction mixture was filtered, diluted with dichloromethane, washed successively with aqueous sodium thiosulfate and aqueous sodium hydrogen carbonate. The organic layer was dried over magnesium sulfate and concentrated in vacuo. The residue was purified by size exclusion chromatography on Sephadex LH-20 suspended in dichloromethane-methanol (2/1, v / v) to make 108 mg of 4. Preparation of 5 A cooled solution (-5 ° C) of 4 (105 mg) in tetrahydrofuran (7.3 ml) was added a 30% aqueous solution of hydrogen peroxide (3.8 ml) and after 10 minutes of stirring a solution of lithium hydroxide (1.25 M, 1.7 ml) was added. The mixture was stirred for 2 hours at -5 ° C, after which the temperature was increased to 0 ° C. After 20 hours of stirring, the temperature was raised to 20 ° C and the stirring was continued for 24 hours. The reaction mixture was cooled (0 ° C), subsequently methanol (7.0 ml) and aqueous sodium hydroxide (4 M, 2.0 ml) were added. After stirring for 1 hour, the temperature was again raised to 20 ° C and stirring was continued for another 20 hours. The mixture was cooled (0 ° C), acidified to a pH of 3 with hydrochloric acid (2N) and extracted with dichloromethane. The organic layer was washed with aqueous sodium sulfate (5%), dried over magnesium sulfate and concentrated in vacuo. The crude product was purified through silica gel column chromatography to give 80 mg of 5.

Preparation of 6 To a solution of 6 (80 mg) in a mixture of water (7 ml) and 2-methyl-2-propanol (7 ml), 80 mg of palladium on carbon were added. (10%) The reaction mixture was placed under a hydrogen atmosphere for 16 hours. The catalyst was stirred during filtration and rinsed with mixtures of 2-methyl-2-propanol / water. The filtrate and washings were concentrated in vacuo and lyophilized to give 38 mg of 6.

Preparation of 7 and 8 A solution of 6 (38 mg) in water (0.8 ml) was eluted with water on a Dowex 50WX8H * column and the poured fractions were evaporated to dryness. After evaporation with N, N-dimethylformamide, the residue was dissolved in N.N-dimethylformamide (2.5 ml), placed under a nitrogen atmosphere, and a complex of triethylamine-sulfur trioxide (287 mg) was added. The mixture was stirred overnight at 50 ° C, cooled to 0 ° C and an aqueous solution of sodium hydrogen carbonate (533 mg) was added. The mixture was stirred for 1 hour at 20 ° C, concentrated to a small volume and desalted on a Sephadex G-25 column suspended in water: acetonitrile, 9: 1 (v / v). The crude product was eluted on a Dowex 50WX8Na + column and purified through anion exchange column chromatography (HPLC, Mono-Q 5/5, sodium chloride gradient) to give 12 mg of 7.. { [a] 0D = + 31.1 (c = 1, water)} and 18 mg of 8. { [a] 20D = + 34.8 (c = 0.93, water)} .

EXAMPLE 2 Preparation of Compound 16 Preparation of 10 A solution of benzoyl chloride (26.3 ml) in dry dioxane (26 ml) was added dropwise over 1 hour to a cooled mixture (-20 ° C) of glycerol (10 g) and pyridine (109 ml). The resulting mixture was stirred for 16 hours at 0 ° C, after which water was added. After 15 minutes of stirring, the mixture was concentrated to one fifth of its volume, diluted with dichloromethane and washed with water, sodium hydrogen carbonate and water. The organic layer was dried over magnesium sulfate and concentrated in vacuo. The resulting oil was purified through silica gel column chromatography for 21 g of 10.

Preparation of 11 Methyl sulfide (1.45 ml) was added to a solution of 10 (600 mg) in acetonitrile: dichloromethane (1: 1, v / v, 8 ml). The reaction mixture was cooled to 0 ° C and a mixture of benzoyl peroxide (3.63 g) in acetonitrile: dichloromethane (1: 1, v / v, 10 ml) was added dropwise. After stirring for 16 hours at 20 ° C, the mixture was diluted with dichloromethane and washed with aqueous sodium hydrogen carbonate and water. The organic layer was dried over magnesium sulfate and concentrated in vacuo. After purification through silica gel column chromatography, 400 mg of 11 were isolated.

Preparation of 12 Compound 12 was prepared analogously to the procedures described by P. Westerduin et al., Bioorganic and Medicinal Chemistry 1994, vol. 2, no 1 1, p. 1267-1280. During the reaction steps, to provide 12, the uronic acids were protected through benzyl groups instead of methyl groups.

Preparation of 13 Compound 13 was prepared in a manner similar to that described for compound 4 by coupling compound 1 1 to compound 12.

Preparation of 14 A suspension of sodium hydrogen carbonate (142 mg) in water (2 ml) and Pd / C (400 mg) was added to a solution of 13 (480 mg) in 2-methyl-2-propanol (60 ml ). The mixture was placed in a hydrogen atmosphere for 16 hours. The catalyst was removed by filtration and washed with mixtures of 2-methyl-2-propanol / water. The combined filtrate and washings were concentrated in vacuo to give 315 mg of 14, which was used without further purification.

Preparation of 15 Compound 14 (315 mg) was dissolved in 0.35 N of aqueous sodium hydroxide (10 μl). The reaction mixture was stirred overnight, after which the pH was adjusted to 8.5 with 1 N hydrochloric acid. The mixture was desalted on a Sephadex G-25 column suspended in a water: acetonitrile: triethylamine 90: 10: 0.1 (v / v / v) mixture. The appropriate fractions were combined and concentrated in vacuo. The product was again subjected to hydrogenolysis in a manner similar to that described for compound 14. After processing and concentration of the filtrate and washings, the mixture was desalted on a Sephadex G-25 column in a mixture of water: acetonitrile: triethylamine 90: 10: 0.1 (v / v / v). The appropriate fractions were combined, concentrated in vacuo, eluted on a Dowex 50WY8Na + column with water and finally lyophilized to give 165 mg of 15.

Preparation of 16 A solution of 15 (165 mg) was evaporated with N, N-dimethylformamide and dissolved in N, N-dimethylformamide (11.0 ml). A complex of triethylamine-sulfur trioxide (1.31 g) was added to the reaction mixture. After stirring for 16 hours at 50 ° C, the mixture was cooled (0 ° C), aqueous sodium hydrogen carbonate (2.43 g) was added and stirring was continued for 1 hour at 20 ° C, after which the The solution was concentrated in vacuo. The residue was desalted on a Sephadex G-25 column suspended in water: acetonitrile 9: 1 (v / v) and the appropriate fraction was combined and concentrated in vacuo. After elution on a Dowex 50WX8Na + column, the product was purified on a High Charge Q-Sepharose column, using a gradient of sodium chloride, to give 160 mg | 20 of 16.. { [a] D - + 24.0 (c = 0.77; H2O)} .

Claims (8)

1. - A carbohydrate derivative having the formula wherein R 'is H or CH2OSO3"; R2 and R3 are independently H, (1-6C) alkyl or SO3', n is 0 or 1, p is 1 or 2, or a pharmaceutically acceptable salt thereof. The carbohydrate derivative according to claim 1, wherein R2 is (1-6C) alkyl, R3 is SO3", R4 is OSO3", and R1, n and p are as previously defined. according to claim 2, wherein R2 is methyl 4. The carbohydrate derivative according to claim 2 or 3, wherein n is 1 and p is 1. 5. The carbohydrate derivative according to any of the claims 2-4, wherein R1 is CH2OSO3". 6. A pharmaceutical composition comprising the carbohydrate derivative of any of claims 1-5 and pharmaceutically acceptable auxiliaries. 7. The carbohydrate derivative according to any of claims 1-5 for use in therapy. 8. The use of the carbohydrate derivative of any of claims 1-5 for the manufacture of a medicament for treating or preventing thrombosis or inhibiting the proliferation of smooth muscle cells.