MXPA02005278A - Antithrombotic compound - Google Patents

Abstract

The present invention relates to compounds of the formula (I), wherein R is independently SO3<->or CH3;the spacer is a flexible spacer of a length of 13-25 atoms;the charge of the pentasaccharide residue is compensated by positively charged counterions;and the total number of sulfate groups in the pentasaccharide residue is 4, 5 or 6;or a pharmaceutically acceptable salt, a prodrug or a solvate thereof. The compounds of the invention have antithrombotic activity and can be used in treating or preventing thrombin-related diseases.

Description

CO ANTITROMBOTIC POST The invention relates to a new antithrombotic compound, a pharmaceutical composition comprising the compound as an active ingredient, as well as to the use of said compound for the manufacture of medicaments. Serine proteases are enzymes that play an important role in the blood coagulation cascade. An important serine protease is factor Xa, which catalyzes the conversion of prothrombin into thrombin. Thrombin is the final serine protease enzyme in the coagulation cascade. The main function of thrombin is the separation of fibrinogen to generate fibrin monomers, which degrade to form an insoluble gel. In addition, thrombin regulates its own production by activating the previous V and VII factors in the cascade. It also has important actions at the cellular level, where it acts on specific receptors to cause platelet aggregation, endothelial cell activation and fibroblast proliferation. In this way, thrombin has a central regulatory role in hemostasis and thrombus formation. In the development of synthetic inhibitors of serine proteases, recently a conjugate of synthetic NAPAP-pentasaccharide has been reported as antithrombotic having a double profile of both direct anti-thrombin activity and anti-Xa activity mediated by ATI 11 (ATIII: antithrombin III) (Bioorg, Med Chem. Lett, 1999, 9 (14), 2013-8). Although the reported antithrombotic can be an interesting compound, the Cross-reactivity HIT and neutralization by PF4 will be associated with this compound due to the high sulfate content of the pentasaccharide residue (Thromb, Haem, Suppl 1997, p363 PD1485). It has now been found that the compounds of the formula (I) are antithrombotic having an excellent and advantageous double profile. The compounds of the formula (I) have an interesting pharmacological average life, which allow treatment once a day, and are severely neutralized by PF4. In addition, the risks of bleeding are low. Taken together, the compounds of formula (I) have an attractive combination of pharmacological properties. Formula (I): [spacer] wherein R is independently SO3 'or CH3; the spacer is a flexible spacer with a length of 13-25 atoms, preferably 16-22 and more preferred of 19 atoms: the charge of the pentasaccharide residue is compensated by positively charged counterions; and the total number of sulfate groups in the pentasaccharide residue is 4, 5 or 6; or a pharmaceutically acceptable salt, a prodrug or a solvate thereof. The compounds of the present invention are useful for treating and preventing thrombin-associated and thrombin-mediated diseases. This includes a number of prothrombotic and thrombotic states in which the coagulation cascade is activated, which includes, but is not limited to, deep vein thrombosis, pulmonary embolism, thrombophlebitis, arterial occlusion of thrombosis or embolism, arterial reocclusion during or after of angioplasty or thrombolysis, restenosis after arterial injury or invasive cardiological procedures, embolism or postoperative venous thrombosis, chronic or acute atherosclerosis, stroke, myocardial infarction, cancer and metastasis, and neurodegenerative diseases. The compounds of the invention can also be used as anticoagulants in extracorporeal blood circuits, as necessary in dialysis and surgery. The compounds of the invention can also be used as anticoagulants in vitro. The compounds of the formula (I) are specifically useful as antithrombotics for arterial indications. Preferred compounds according to the invention are compounds wherein the pentasaccharide residue has the structure: The chemical nature of the spacer is of minor importance for the anti-thrombotic activity of the compounds of the invention. Nevertheless, the spacer of the compounds of the invention is flexible, which means that the spacer does not contain rigid elements, such as unsaturated bonds or cyclic structures. Suitable spacers can be easily designated by a person skilled in the art. Preferred spacers contain at least one element - (CH2CH2O) -. The most preferred spacers contain three elements - (CH2CH2O) -. The most preferred spacer is * - (CH2CH2O) 3- (CH2) 2-NH-C (O) - (CH2) 3-NH-C (O) -CH -, the end indicated with * being bound to the pentasaccharide residue. Preferred compounds of the formula I are the compounds of the formula (la), wherein p is 1 -5, n is 1-5 and m is 1 or 2. The most preferred compound is the compound of the formula (la), where p is 3, n is 3 and m is 1.

A positively charged counter ion means H +, Na +, K + > Ca2 + and the similar. Preferably, the compounds of the formula (I) are in the form of their sodium salt. The term "prodrug" means a compound of the invention in which the amino group of the amidino portion is protected, for example, by hydrozoon or a (1-6C) alkoxycarbonyl group. The solvates according to the invention include hydrates. The compounds of the invention, which can occur in the form of a free base, can be isolated from the reaction mixture in the form of a pharmaceutically acceptable salt. Pharmaceutically acceptable salts can also be obtained by treating the free base of formula (I) with an organic or inorganic acid such as hydrogen chloride, hydrogen bromide, hydrogen iodide, sulfuric acid, phosphoric acid, acetic acid, propionic acid, acid glycolic, acid maleic, malonic acid, methanesulfonic acid, fumaric acid, succinic acid, tartaric acid, citric acid, benzoic acid, ascorbic acid and the like. The compounds of this invention possess chiral carbon atoms, and can therefore be obtained as a pure enantiomer, or as a mixture of enantiomers, or as a mixture containing diastereomers. Methods for obtaining the pure enantiomers are well known in the art, for example, crystallization of salts that are obtained from optically active acids and the racemic mixture, or chromatography using chiral columns. Reverse phase or straight phase columns can be used for diastereomers. The compounds of the present invention can be prepared by first activating the carboxylate group of the NAPAP analog of formula II and subsequently adding a pentasaccharide spacer residue containing an amine group (formula III), optionally followed by deprotection of the amidine The carboxylate group in compounds of the formula II can be activated as a mixed anhydride or more preferably as a activated ester such as the N-hydroxysuccinimide ester, pentafluorophenol or 1-hydroxybenzotriazole. In the coupling step, the benzamide group in formula II can be (R '= R "= H) deprotected, or can optionally be protected using a carbamate group preferably allyloxycarbonyl (R' and / or R" is H2C = CH-CH -C (O) O) or benzyloxycarbonyl (R 'and / or R "is PhCH2-C (O) O) The benzyloxycarbonyl or allyloxycarbonyl protecting groups can be removed under relatively mild conditions.The allyloxycarbonyl group can be removed using Pd in the presence of a weak nucleophile such as morpholine or a malonic ester The benzyloxycarbonyl group can be removed under conditions such as hydrogen / Pd (C) Alternatively, the benzamidine synthetic precursors such as N-alkoxybenzamidine or N-benzyloxybenzamide or N-benzyloxybenzamidine (R '= H, R "= alkoxy or benzyloxy) may be applied. These synthetic precursors can be converted to benzamidine using reductive conditions such as hydrogenation (eg, Fujii, T et al., Chem. Pharm. Bull, 39, 301, 1991 and Fujii, T er al., Chem. Pharm. Bull, 42 , 1231, 1994). The preferred benzamidine precursor is 1,4-oxadiazolin-5-one (-R'-R "- = - C (O) O-). This precursor can be converted to benzamidine by hydrogenation (Bolton, RE et al. ., Tetrahedron Letters, Vol 36, No. 25, 1995, pp 4471-4474) The compounds of formula II can be prepared in various manners using methods known in the art A method for preparing compounds of formula II wherein R ' = R "= H; n is 3 and m is 1 is described in EP 0513543. The compounds of the formula II in which amidine is protected, for example with an allyloxycarbonyl or benzyloxycarbonyl group can be prepared from compounds of formula IV wherein the amidine is protected with an allyloxycarbonyl or benzyloxycarbonyl group using methods commonly known in the art for the coupling of peptide fragments. The carbamates of the formula IV can, for example, be prepared from the corresponding amidine (formula IV, R '= R "= H) as described in the literature, for example Séller, T et al., J. Med. Chem. 39, 3119, 1996).

The N-alkoxybenzamidine and N-benzyloxybenzamidine compounds of the formula II can be prepared from the compound V (described in EP 0513543) by treatment of this cyano compound with O-alkylhydroxylamine or O-benzylhydroxylamine followed by removal of the t-butyl using acidic conditions. Alternatively, the N-alkoxybenzamidine and N-benzyloxybenzamidine compounds of the formula II can be prepared by first removing the t-butyl ester of the compound V using acidic conditions to produce the compound VI and the subsequent reaction of this cyano compound with O-alkyl -hydroxylamine or O-benzyl-hydroxylamine. The compounds of the formula II in which -R'-R '- = - C (O) O- (the group 1, 2,4-oxadiazolin-5-one), can be prepared from compounds of the formula V in which -R'-R "- = - C (O) O, using methods known in the art for coupling the fragments The synthesis of residues of the amino-oligosaccharide spacer of the formula 11 I can, for example, be carried out using methods described in EP 0649854. The saccharide residues of the compounds of the present invention can be prepared according to known procedures in the art, for example, WO 99/25720. Peptide coupling, a process step in the method described above for preparing the compounds of the invention, can be carried out by methods commonly known in the art for coupling, or condensation, of peptide fragments such as by the azide method, mixed anhydride method, activated ester method, or, preferably, by the carbodiimide method, especially with the addition of compounds that suppresses n racemization and catalysts such as N-hydroxysuccinimide and N-hydroxybenzotriazole. A summary is given in The Peptides. Analvsis, Svnthesis, Bioloov. Vol. 3, E. Groos and J. Meienhofer, eds. (Academic Press, New York, 1981) and Bodanszky, M.; Principles of peptide synthesis, Sprinher-Verlag, 1984. The amine functions present in the compounds can be protected during the synthetic process by a protecting group N, which means a group commonly used in peptide chemistry for the protection of an a-amino group , such as the re-butyloxycarbonyl group (Boc), the benzyloxycarbonyl group (Z), the 9-fluorenylmethyloxycarbonyl group (Fmoc) or the eftaloyl group (Phth). The withdrawal of protective groups may have place in different ways, .depending on the nature of those protective groups. Usually the deprotection takes place under acidic conditions and in the presence of cleaners. A summary of amino protecting groups and methods for their removal is given in the above-mentioned The Peptides, Analysis, Svnthesis, Bioloqy, Vol 3, and further as described by Greene, T.W. and Wuts, P.G.M. in Protective groups in organic synthesis, John Wiley & Sons Inc, 1991. The compounds of the invention can be administered enterally or parenterally. The exact dose and regimen of these compounds and compositions thereof will necessarily depend on the needs of the individual subject to whom the medication is administered, the degree of distress or need and the judgment of the physician. In general, parenteral administration requires lower doses than other methods of administration that are more dependent on absorption. However, the daily doses are for humans preferably 0.001 -100 mg per kg of body weight, more preferably 0.01-10 mg per kg of body weight. The medicament made with the compounds of this invention can also be used as an adjuvant in acute anticoagulant therapy. In such a case, the medicament is administered with other compounds useful for treating such disease states. Mixed with pharmaceutically suitable auxiliaries, for example, as described in the standard reference Gennaro et al., Remington's Pharmaceutical Sciences, (18th ed, Mack Publishing Company, 1990, see especially Part 8: Pharmaceutical Preparations and Their Manufacture), the compounds can be compressed into solid dose units, such as pills, tablets, or processed into capsules or suppositories. By means of pharmaceutically suitable liquids the compounds can also be applied in the form of a solution, suspension, emulsion, for example, to be used as an injection preparation, or as a spray, for example, to be used as a nasal spray. To make units of. Dosage, for example, tablets, the use of conventional additives such as fillers, dyes, 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 is further illustrated by the following examples. EXAMPLE 1 Abbreviations used: Ac = acetyl Bn = benzyl DBU = 1,8-diazabicyclo [5.4.0] undec-7-ene DCC = dicyclohexylcarbodiimide DMF = N, N-dimethylformamide Su = succinimidyl Me = methyl TBTU = 2- (1 H-benzotriazol-1-yl) -1, 1, 3,3-tetramethyluronium tetrafluoroborate TEA = triethylamine TFA = trifluoroacetic acid Z = benzyloxycarbonyl The numbers of the compounds refer to the compounds in the schemes 1 a 7. Compound 3 To a stirred solution of compound 1 (53.6 g, 143.6 mmol) (R. Roy, WKC Park, Q. Wu; SN.Wang, Tetrahedron Lett., 1995, 36 (25), 4377-80) and compound 2 (27.9 g, 89.3 mmol) (S.J. Danishefsky; M.P.

DeNinno; G. B. Philips; R. E. Zelle, Tetrahedron, IN, 1986, 42, 11, 2809-2819) in 930 mL of DMF is added sodium hydroxide (7.7 g, 60% dispersion, 192.2 mmol) at 50 ° C. After 1 h, the reaction mixture is heated to 120 ° C. After stirring for 5 minutes the reaction mixture is cooled to 40 ° C and diluted with water and extracted three times with dichloromethane. The combined organic layers are rinsed with water and concentrated in vacuo, yielding crude product 3 (54 g). TLC: Rf = 0. 23, 100% ether. Compound 4 To a stirred solution of compound 3 (89.3 mmol) in 800 mL of dry toluene and 800 mL of acetic anhydride is added dropwise a cold solution of 361.5 mL of sulfuric acid in acetic anhydride (16.5 mL of concentrated sulfuric acid and 345.0 mL of acetic anhydride) at -30 ° C. After 2 h the reaction mixture is cooled with 240 mL of TEA and stir at room temperature. To the solution is added aqueous sodium hydrogen carbonate (5%) and the water layer is extracted three times with ethyl acetate. The combined organic layers are rinsed twice with water and concentrated in vacuo. This procedure is repeated, resulting in crude compound 4 (53 g). TLC: Rf = 0.29, 100% ether. Compound 5 To a stirred solution of compound 4 (89.3 mmol) and ethanethiol (1.1 mL, 150.3 mmol) in 370 mL of dry toluene is added dropwise a solution of BF3-etherate in toluene (23.9 mL BF3-etherate and 190 mL of toluene) at 0 ° C. After stirring for 16 h at room temperature the reaction mixture is cooled with TEA and aqueous sodium hydrogen carbonate and extracted three times with ethyl acetate. The combined organic layers are rinsed with water and concentrated in vacuo. The crude product is purified by column chromatography (toluene / ethyl acetate = 1/1 to 0/1, v / v) to give compound 5 (21.4 g). TLC: Rf = 0.31, toluene / ethyl acetate = 4/6, v / v. Compound 7 To a solution of donor 5 (15.0 mg, 30.3 mmol) and acceptor 6 (23.0 g, 30.3 mmol (WO 99/25720) in ether sec / dichloromethane (232 mL, 3/1, v / v) is stirred by 30 minutes under nitrogen flow in the presence of 4Á activated molecular sieves (7.6 g), then a solution of 1,3-dibromo-5,5-dimethylhydantoin (5.5 g, 19.1 mmol) and triflic acid (0.49 mL) 5.6 mmol) in dioxane / dichloromethane (69.8 mL, 1/1, v / v) is added dropwise in 75 min to the reaction mixture at -20 ° C. After 30 minutes TEA (5 mL) is added to the reaction mixture, which is stirred for 10 minutes and then it filters. The filtrate is rinsed with aqueous sodium thiosulfate (10%) and aqueous sodium hydrogen carbonate (10%) and concentrated in vacuo. The product is purified by column chromatography (0 to 5% acetone in dichloromethane) to give compound 7 (19.6 g). TLC: Rf-0.1, ether / heptane = 8/2, v / v. Compound 8 To a stirred solution of compound 7 (19.5 g, 16.4 mmol) in dry toluene / acetic anhydride (442 mL, 1/1, v / v) a cold solution of 131.5 mL of sulfuric acid in anhydride is added dropwise. acetic acid (1 1.5 mL of sulfuric acid and 120 mL of acetic anhydride) at -26 ° C. After 75 minutes TEA (73.5 mL) is added at -20 ° C. Acetic anhydride is decomposed by gradually adding 330 mL of water maintaining the temperature between 25 ° C and 30 ° C. After stirring for 16 h the mixture is poured into 800 mL of water and extracted twice with toluene. The combined organic layers are rinsed with water and concentrated in vacuo. The crude product is purified by column chromatography (toluene / ethyl acetate / ethanol = 96/2/2, v / v / v) to give 8 as a white foam (13.2 g). TLC: Rf = 0.29, toluene / ethanol = 9/1, v / v. Compound 9 To a solution of compound 8 (13.2 g, 1.7 mmol) in dry toluene (66 mL) at 32 ° C is added morpholine (4.1 mL, 46.9 mmol). After stirring for 42h at 32CC the reaction mixture is cooled to an aqueous temperature and aqueous hydrochloric acid (17.6 mL, 4N) is added. The mixture is diluted with water and extracted twice with ethyl acetate. The combined organic layers are rinsed twice with water, dried in sulphate of sodium and concentrated in vacuo to yield the crude compound 9 (12.6 g). Compound 12 To a solution of compound 9 (12.6 g, 11.6 mmol) in dichloromethane (114 mL) is added trichloroacetonitrile (3.5 mL, 34.9 mmol) and DBU (52.2 μL, 0.35 mmol). After stirring for 2 h at room temperature the activated molecular sieves 4Á (24 g) and acceptor 11 (8.9 g, 13.0 mmol) (WO 99/25720) in dichloromethane (45 mL) are added to the reaction mixture. After stirring for 30 minutes at room temperature, the mixture is cooled to -20 ° C and a solution of trimethylsilyl trifluoromethanesulfonate (405 μL, 2.1 mmol) in dichloromethane (100 mL) is added dropwise. After stirring 30 minutes, sodium hydrogen carbonate is added at -20 ° C and the reaction mixture is filtered. The filtrate is poured into aqueous sodium hydrogen carbonate and extracted three times with dichloromethane. The combined organic layers are rinsed twice with water and concentrated in vacuo. The product is purified by column chromatography (1: SIO2: 0-10% acetone in ether; 2: SiO2 toluene / acetone = 85/15 to 80/20, v / v; 3: RP-18: water / acetonitrile = 2/8 to 0/10, v / v) producing the pure compound 12 (8.9 g). TLC: Rf = 0.37, toluene / acetone = 7/3, v / v. Compound 14 A suspension of compound 12 (8.9 g, 5.1 mmol) and 10% Pd / C (8.9 g) in 312 mL of DMF and 45 mL of water is stirred under a continuous stream of hydrogen. After 4.5 h the Pd / C catalyst is removed by filtration. The filtrate is concentrated to a volume of 400 mL and treated with 10% Pd / C (1.5 g) under a stream of hydrogen for 5.5 h. He The catalyst is removed by filtration. To the filtrate (900 mL) is added aqueous sodium hydroxide (32 mL, 4N). After stirring for 4 h at room temperature the mixture is acidified to pH = 6.6, with 1 N of hydrochloric acid and then concentrated in vacuo. The crude product is desalted on a Sephadex G-25 column which is eluted with water. The appropriate fractions are pooled and lyophilized to yield compound 14 (4.0 g) Compound 15 Pentasaccharide 14 (700 mg, 0.61 mmol) is dissolved in water (13.2 mL) and DMF (3.3 mL) and treated with N- (benzyloxycarbonyloxy) - succinimide (224 mg, 0.90 mmol) and N-ethylmorpholine (233 μL, 1.83 mmol). After stirring for 15 minutes the reaction mixture is applied directly on a RP-18 column, which is eluted with water / acetonitrile 10/0 to 7/3. The appropriate fractions are pooled and concentrated to a small volume and applied on a Dowex 50 WX4-H + ion exchange column in water. The eluate is concentrated in vacuo to yield the pure 15 (482 mg). Compound 16 To a solution of compound 15 (471 mg, 0.37 mmol) in DMF (4.7 mL) was added sulfur-pyridine trioxide complex (1.1 g, 6.6 mmol) and the mixture was stirred for 16 h at 30 ° C. The mixture is cooled to room temperature and added dropwise to a cold 10% hydrogen carbonate solution (16.7 mL, 19.9 mmol) and stirred for 1 h at room temperature. The mixture is concentrated to a small volume and applied on a Sephadex G-25 column, which is eluted with water. The appropriate fractions are pooled and concentrated to a small volume, which is subsequently passed through a column of Dowex Na + HCRW2 eluted with water. The eluate is concentrated and redissolved in 8.3 mL of 0.2 N hydrochloric acid and allowed to stand for 16 h at 4 ° C. The reaction mixture is neutralized with 8 mL of 0.2 N sodium hydroxide and desalted on a Sephadex G-25 column which is eluted with water. The appropriate fractions are pooled and concentrated in vacuo yielding the pure compound 16 (840 mg). Compound 17 A suspension of compound 16 (0.37 mmol) and 10% Pd / C (820 mg) in fe / 7-butanol (85 mL) and water (79 mL) and a few drops of acetic acid are stirred under a stream continuous hydrogen After 3h the Pd / C catalyst is removed by filtration and the filtrate is concentrated and lyophilized to give the pure 17 (675 mg). 4 - [[4 - [[(1 R) -1 - [[4- (aminoiminomethyl) pheny] methyl] -2-oxo-2- (1-piperidinyl) ethyl] amino] -3- [ [(4-methoxy-2,3,6-trimethylphenyl) sulfonyl] aminoj-1,4 (S) -dioxobutyl] amino] -benzyl ester of butanoic acid. hydrochloride (18) To a solution of 4 - [[(1 R) -1 - [[4- (aminoiminomethyl) phenyl] methyl] -2-oxo-2- (1 -? iperidinyl) ethyl] amino] -3- [[(4-methoxy-2 > 3,6-trimethylphenyl) sulfonyl] amino] -4-oxo- (3S) -butanoic acid. hydrochloride (2.38 g, 3.96 mmol) (Tetrahedron 51, 12047-12068, 1995) and benzyl- (4-aminobutyric acid). enosulfonate (1.52 g, 3.96 mmol) (J. Am. Chem. Soc. 105, 5278-5284, 1983) in DMF (40 mL) under an atmosphere of nitrogen is added N. N-diisopropylethylamine (0.689 mL, 3.96 mmol) and benzotriazolyl uronium tetramethyl-tetrafluoroborate (1.91 g, 5.94 mmol). The pH of the reaction mixture is maintained at 6 using N, N- diisopropylethylamine. The reaction mixture is stirred for 4 days at room temperature, concentrated, dissolved in ethyl acetate, rinsed with 5% sodium carbonate and 0.1 N hydrochloric acid, dried over magnesium sulfate and concentrated. The residue is dissolved in dry ethanol (5 mL), precipitated with dry diisopropyl ether, filtered, to yield 2.47 g of the main compound 18. Rf = 0.8, ethyl acetate / pyridine / acetic acid / water = 83/31/18 / 7, v / v / v / v; Mass (ESI +): 777.4 [M + H] + 4 - [[4 - [[(1 R) -1 - [[4- (aminoiminomethyl) phenyl] methyl] -2-oxo-2- (1-piperidine) ) ethyl] amino] -3 - [[(4-methoxy-2,3,6-trimethylphenol) sulfonyl] amino] -1,4 (S) -dioxobutyl] amino] -butanoic acid. hydrochloride (19) A suspension of 18 (2.42 g, 3. 1 1 mmol) and 10% of Pd / C (400 mg) in methanol / water (40 mL, 3/1, v / v) is stirred under a stream continuous hydrogen After 8 h the reaction mixture is filtered, the filtrate is concentrated and co-evaporated three times with methanol / toluene (1/10, v / v). The residue is dissolved in dry ethanol (5 mL), precipitated with dry diethyl ether, filtered and dried. The residue is dissolved in water and loaded directly into a preparative HPLC DeltaPak RP-Ciß using a gradient elution system of 20% A / 60% B / 20% C at 20% A / 14% V / 66% C for 30 min at a flow rate of 40 mL / min (A: 0.5 M phosphate buffer pH 2.1, B: water, C: acetonitrile / water = 6/4). Production 598 mg. Rt = 26.4 min (3-10 min: 20-43% C + 20% A, 10-15 min: 43-66% C + 20% A), (A: phosphate buffer pH 2.1, B: water; : gcetonitrile / water = 6/4, v / v), supelcosil HPLC analytical LC-18-DB; Mass (EST +): 687.2 [M + H] +, 685.2 [M-H] Compound 21 of compound 17 and compound 19 To a solution of compound 19 (40 mg, 58.3 μmol) in DMF (800 μL) is added N-hydroxysuccinimide (9.0 mg, 78.1 μmol), DCC (18.5 mg, 89.7 μmol) and 1-Hydroxybenzotriazole (8.8 mg, 65.1 μmol). The reaction mixture is stirred for 40 h at room temperature. The reaction mixture is filtered on dicalite and the dicalite is rinsed four times with DMF (284 μL). To the filtrate is added 0.1 M of regulator Na2HPO (1936 μL, pH = 7.5) and pentasaccharide 17 (94.7 mg, 52.6 μmol). After stirring for 30 minutes the mixture is filtered on dicalite, concentrated and applied on a Sephadex G-50 column, which is eluted with acetonitrile / water (1/1, v / v). The appropriate fractions are pooled, concentrated and desalted twice in Sephadex G-50 column chromatography (water). The appropriate fractions are pooled and lyophilized to produce the conjugate 21 as a white solid (95.8 mg). Mass (ES +) = 2469, HPLC: Rt = 8.3 min (20-80% B in 15 minutes, A = water / acetonitrile 8/2; B = 2M NaCl / acetonitrile 8/2, v / v, Analytical HPLC MonoQ HR5. Compound 22 A solution of (R) -N-Boc (4-cyanophenyl) alaline (25.0 g, 86.1 mmol), piperidine (21.3 mL, 215.3 mmol) and TBTU (41.5 g, 129.2 mmol) in dry CH2Cl2 (500 mL) it is stirred at room temperature under a nitrogen flow for 2 hours. The reaction mixture is rinsed successively with 0.2N of hydrochloric acid, water, aqueous sodium hydrogen carbonate (saturated) and water. The organic layer is dried over MgSO4I, filtered and concentrated in vacuo. The product is dissolved in hot ethyl acetate (35 mL), precipitated with heptane (190 mL) and filtered to yield the compound 22 (27.75 g). TLC: Rf = 0.58, heptane / ethyl acetate = 3/7, v / v Compound 23 A solution of compound 22 (25.6 g, 71.7 mmol); hydroxylamine. HCl (7.1 g, 101 .8 mmol) and triethylamine (16.8 mL, 120.5 mmol) in absolute ethanol (307 mL) is stirred at 80 ° C for 4 hours. In the cooling of the mixture at room temperature crystals are formed. The crystals are removed by filtration, rinsed with ethanol and ether and dried in a desiccator to yield compound 23 (24.5 g). TLC: Rf = 0.15, heptane / ethyl acetate = 3/7, v / v Compound 24 A solution of compound 23 (24.5 g, 62.7 mmol) and ethyl chloroformate (7.2 mL, 75.3 mmol) in dry pyridine (245 mL ) is stirred at 1 15 ° C for 2 hours. The reaction mixture is cooled to room temperature and poured into water (1250 mL) and extracted 3 times with ethyl acetate. 500 mL). The combined organic extract is dried over MgSO 4, filtered and concentrated in vacuo to yield compound 24 (24.3 g). TLC: Rf = 0.42, CH2Cl2 / MeOH 95/5, v / v. Compound 25 A solution of compound 24 (24.3 g, 58.4 mmol) in dry CH2Cl2 (122 mL) and TFA (122 mL) is stirred at room temperature for 2 hours and concentrated in vacuo in the presence of toluene to yield compound 25 ( 37.6 g). Compound 26 A suspension of H-Asp- (OtBu) -OH (39 g, 206.35 mmol), 4- methoxy-2,3,6-trimethylbenzene-sulfonyl chloride (62 g, 249.3 mmol) and diisopropylamine (89 mL, 635 mmol) in DMF (950 mL) and water (450 mL) is stirred at 0 ° C for 3 hours . The reaction mixture is poured into ice / water (5 L) and rinsed twice with diethyl ether, acidified with aqueous hydrochloric acid (4N, 72 ml) and extracted 3 times with ethyl acetate. The combined ethyl acetate layers are dried over MgSO 4, filtered and concentrated in vacuo to yield compound 26 (97.7 g) - TLC: Rf = 0.67, CH 2 Cl 2 / MeOH 8/2, v / v. Compound 27 A solution of compound 25 (33.5 g), compound 26 (24.7 g), TBTU (36.8 g, 114.6 mmol) and diisopropylamine (27.2 mL, 194.1 mmol) in dry DMF (670 mL) is stirred for 2 hours and concentrated in vacuo The residue is dissolved in ethyl acetate (750 mL), rinsed with aqueous sodium hydrogen carbonate (5%, 1250 mL) and aqueous hydrochloric acid (0.1 N, 1250 mL), dried over MgSO 4, filtered and concentrated in vacuo to give produce compound 27 (33.8 g). TLC: Rf = 0.88, CH2Cl2 / MeOH 8/2, v / v. Compound 28 A solution of compound 27 (33.8 g, 48.3 mmol) in dry CH2Cl2 (170 mL) and TFA (170 mL) is stirred at room temperature for 2 hours and concentrated in vacuo in the presence of toluene to yield compound 28 ( 32.3 g). TLC: Rf = 0.73, CH2Cl2 / MeOH 8/2, v / v. Compound 29 A solution of compound 28 (32.3 g), H-GABA-OtBu. HCI (9.5 g, 48.4 mmol), TBTU (29.0 g, 90.5 mmol) and diisopropylamine (25.2 mL, 179.8 mmol) in dry DMF (622 mL) is stirred at room temperature for 2 hours and concentrated in vacuo. The residue is dissolved in ethyl acetate (840 mL), rinsed with aqueous sodium hydrogen carbonate (5%, 1400 mL) and aqueous hydrochloric acid (0.1 N, 1400 mL), dried over MgSO 4, filtered and concentrated in vacuo. The residue is dissolved in ethanol (75 mL) and slowly added to stirred diisopropyl ether (2990 mL) yielding compound 29 as off-white crystals (32.0 g). TLC: Rf = 0.56, CH2Cl2 / MeOH 9/1, v / v. Compound 30 A solution of compound 29 (3.0 g, 3.82 mmol) in dry CH 2 Cl 2 (15 mL) and TFA (15 mL) is stirred at room temperature for 2 hours and concentrated in vacuo in the presence of toluene. The residue is purified on silica gel using CH 2 Cl 2 / MeOH (0% -6% MeOH) to yield pure compound 30 (1.98 g). TLC: Rf = 0.56, CH2Cl2 / MeOH 8/2, v / v. Compound 31 A solution of compound 30 (900 mg, 1.23 mmol), TBTU (396 mg, 1.23 mmol) and diisopropylamine (215 μL, 1.53 mmol) in DMF (45 mL) is stirred for 1 hour at room temperature. Compound 17 (2.0 g, 1.11 mmol) is added and after stirring for 4 hours the mixture is concentrated in vacuo to yield compound 31 (4.17 g). Compound 21 of compound 31 A suspension of compound 31 (4.17 g) and 10% Pd / C (2.8 g) in tert-butyl alcohol (28 mL) and water (56 mL) is stirred overnight under a continuous stream of hydrogen. The Pd / C catalyst is removed by filtration and the filtrate is concentrated in vacuo. The residue is dissolved in water and purified on a Q-sepharose column. The appropriate fractions are pooled, concentrated and desalted by Sephadex G-25 column chromatography (water). The appropriate fractions are pooled and lyophilized to produce the conjugate 21 as a white solid (1.74 g).

Scheme 1 Scheme 2 R, = H. j = H --- ^ R, = 2. R, = H Scheme 4 Scheme 5 Scheme 6 Scheme 7 EX EMPLO 2 The biological activities of the compounds of the present invention is determined by the following test methods. I. Analysis of anti-thrombin Thrombin (Factor Ha) is a factor in the coagulation cascade. The anti-thrombin activity of compounds of the present invention is assessed by spectrophotometrically measuring the rate of hydrolysis of the chromogenic substrate s-2238 exerted by thrombi. This analysis for anti-thrombin activity in a regulatory system is used to assess the ICS0 value of a test compound. Test medium: tromethamine-NaCl-polyethylene glycol 6000 regulator (TNP) Reference compound: 12581 (Kabi) Vehicle: TNP regulator Solubilization can be assisted with dimethylsulfoxide, methanol, ethanol, acetonitrile or tert-butyl alcohol found without adverse effects in concentrations up to 2.5% in the final reaction mixture .

Technique: Reagents * 1. Regulator of tromethamine-NaCl (TN); Regulator composition: Trometanim (Tris) 6.057 g (50 mmol), NaCl 5.844 g (100 mmol), Water at 1 I. The pH of the solution is adjusted to 7.4 at 37 ° C with HCl (10 mmol. 2. Regulator TNP: Polyethylene glycol 6000 dissolves in regulator TN to give a concentration of 3 g.l "1. 3. Solution S-2238: One bottle of S-2238 (25 mg, Chromogeniz, Sweden) is dissolved in 20 ml of TN regulator to give a concentration of 1.25 mg.ml "1 (2 mmol. I" 1). 4. Thrombin solution: human thrombin (1000 NIH units / vial, Enzyme Res. Lab. Inc., USA) is dissolved in TNP buffer to give a stock solution of 50 NIH units. my "1. Immediately before use, this solution is diluted with TNP regulator to give a concentration of 30.2 NIH units ml'1. * - All the ingredients used are analytical grade For aqueous solutions ultrapure water is used (Milli- quality). Q) Preparation of the reference compound and test solutions The reference and test compounds are dissolved in Milli-Q water to give reserve concentrations of 10'2 mol.''1 Each concentration is diluted in stages with the vehicle to give concentrations of 10"3, 10'4 and 10's mol. I'1. The dilutions including the stock solution are used in the analysis (final concentrations in the reaction mixture: 3. 10.4, 10.4, 3. 10.5, 10.3, 10.6.6, 10.6. 3. 10"7 and 10'7 mol. I'1, respectively). Procedure At room temperature 0.075 ml and 0.025 ml of solutions of reference compound or test compound or vehicle are alternately pipetted into the wells of a microconcentration plate and these solutions are diluted with 0.115 ml and 0.0165 ml of TNP regulator, respectively. An aliquot of 0.030 ml of S-2238 solution is added to each well and the plate is preheated and pre-incubated with shaking in an incubator (Amersham) for 10 min at 37 ° C. After the pre-incubation, the hydrolysis of S-2238 is initiated by the addition of 0.030 ml of thrombin solution to each well. The plate is incubated (with agitation for 30 s) at 37 ° C. Beginning after 1 min of incubation, the absorbance of each sample at 405 nm is measured every 2 min for a period of 90 min using a kinetic microconcentration plate reader (Twinreaderplus, Flow Laboratories). All data is collected on a personal computer using a data processing program (Biolise). For each concentration of the compound (expressed in mol.l "1 of reaction mixture) and for the space, the absorbance is plotted against the reaction time in min. Evaluation of responses: For final concentration the maximum absorbency is calculated from the analysis diagram. The IC50 value (final concentration, expressed in μmol I "1, causing 50% inhibition of maximum space absorbency) is calculated using the logit transformation analysis according to Hafner et al., (Arzneim-Forsch / Drug Res 1977; 27 (ll): 1871 -3). Antithrombin activity of the compound of EXAMPLE 1: IC50 value: 17 nM. II. Anti-Factor XA Analysis The Activated Factor (Xa) is a factor in the coagulation cascade. The anti-Xa activity of compounds of the present invention is assessed by spectrophotometrically measuring the hydrolysis rate of chromogenic substrate s-2222 exerted by Xa. This analysis of Xa activity in a regulatory system is used to assess the IC50 value of the test compound.

Reference Compound: pentasaccharide Org 31540 Vehicle: TNP regulator Solubilization can be assisted with dimethylsulfoxide, methanol, ethane, acetonitrile or tert-butyl alcohol which are found without adverse effects in concentrations up to 1% (for DMSO) and 2.5% (for the other solvents) in the final reaction mixture. Technique Reagents * 1. Trometatin-NaCl (TN) regulator; Regulator composition: Trometatin (Tris) 6.11 g (50.4 mmol), NaCl 10.17 g (174 mmol). Polyethylene glycol 6000 3 g.l'1, Water at 1 I. The pH of the solution is adjusted to 7.4 at 37 ° C with HCl (10 mmol. I "1): 3. Solution S-2222: a vial of S- 2222 (25 mg, Cromogenix, Sweden) is dissolved in water to give a concentration of 0.375 mg.ml'1) (0.5 mmol I'1) 4. Xa Solution: Human Xa Bovine Factor (71 nKat. 1, Chromogenix) is dissolved in 10 ml of TNP regulator and then diluted further with TNP regulator to give a concentration of 0.75 nKat. (1.5 U) .ml "1. The dilution has to be prepared recently. 5. Solution ATI 11: Human ATTIII (Chromogenix) is dissolved in water to give a concentration of 1 U.ml "1, after which the solution is further diluted with 3 volumes of TNP regulator at a concentration of 0.25 U.ml "1. 6. Standard solution: a stock solution of 5.7 anti-Xa U.ml "1 Org 31540 is diluted in TNP regulator to 0.05 U.ml'1 6. Test samples: Each preparation dissolves in water and dilutes with the regulator NPT at a concentration of 0.05 nmol. Ml "1. From each preparation, a range of 9 dilutions is made (dilution factor 1.5).

Determination of Xa activity Each test sample (0.05 ml) is intubated in a well of a microconcentration plate at room temperature. Solution AT-I I (0.05 ml) is added to each sample and the plate is shaken using a Vari shaker. An aliquot of solution Xa (0.05 ml) is intubated in each well for 10 min after the addition of AT-11 solution and the plate is shaken again. Exactly 2 min after the addition of solution Xa, 0.1 ml of solution S-2222 is drilled in each cavity and the polish is stirred again. For all additions, a 12-channel tube is used. The remaining amount of Xa catalyses the hydrolysis of S-2222, the speed of which is measured photometrically using incubation periods of 2 and 22 min respectively at room temperature. The absorbance of each sample is measured at 405 nm using a Microelisa Reader, model 310C (Organon Teknika, Oss, The Netherlands) and the increase in absorbance (? OD) is calculated. Each test sample is determined in duplicate. With every 10 samples, one space (0.05 ml of TNP regulator) is included. Calibration curve From an aliquot of the standard solution of the calibration sample a range of dilutions is made (dilution factor, 1.4 for samples of Org 31540). The resulting standard samples (approximately 12 samples) should contain between 0.01-0.05 anti-Xa U / ml. Within each test, 0.05 ml of each standard sample is tested at least 3 times as described under "Determination of Xa activity". A calibration curve is obtained by fitting a straight line to TABLE U / ml values, using the least squares method. Evaluation of responses: For each sample the average anti-Xa activity in U / ml is determined using the calibration curve. Anti-factor Xa activity of the compound of EXAMPLE 1: 1012 U / mmol.

Claims (10)

1. CLAIMS 1. A compound of the formula (I) [spacer] where R is independently SO3"or CH3, the spacer is a flexible spacer of a length of 13-25 atoms, the charge of the pentasaccharide residue is compensated by positively charged counterions, and the total number of sulfate groups in the pentasaccharide residue is 4, 5 or 6, or a pharmaceutically acceptable salt, a prodrug or a solvate thereof
2. 2. The compound according to claim 1, characterized in that the pentasaccharide residue has the structure
3. 3. The compound according to claim 1 or 2, characterized in that the spacer has a length of 16-22 atoms.
4. 4. The compound according to any of claims 1 -3, characterized in that the spacer has a length of 19 atoms.
5. 5. The compound according to any of claims 1 -4, characterized in that the spacer is * - (CH2CH2?) 3- (CH2) 2-NH-C (O) - (CH2) 3-NH-C (O) - CH2-, the end indicated with * joining the pentasaccharide residue.
6. 6. The compound according to claim 1, having the structure
7. 7. A process for the preparation of the compound of formula I, comprising a step wherein the benzamidine portion is in the form of a precursor, with group 1 being 2,4-oxadiazolin-5-one.
8. 8. A pharmaceutical composition comprising the compound of any of claims 1 to 6 and pharmaceutically acceptable auxiliaries.
9. 9. The compound of any of claims 1 to 6 for use in therapy. The use of the compound of any of claims 1 to 6 for the manufacture of a medicament for treating or preventing thrombosis or other thrombin-related diseases.