MXPA97004594A - Heterociclic peptides of the ceto-arginine, asinhibitors of the tromb - Google Patents

Abstract

The invention relates to novel, useful thrombin enzyme inhibitors of the formula (I): AS -X, and, more particularly, the compound (1), in the preparation, and to pharmaceutical compositions. Also, this invention relates to the use of such compounds and compositions in vitro, as anticoagulants and in vivo as agents for venous treatment, pulmonary embolism and arterial thrombosis, which results in acute ischemic events, such as myocardial infarction or cerebral infarction. . Also, these compounds and compositions have therapeutic utility for the prevention and treatment of coagulopathies associated with coronary bridge operations, as well as restenotic events following transluminal angioplasty.

Description

HETEROCYCLIC PEPTIDES OF CETO-ARGININE. AS THROMBIN INHIBITORS FIELD OF THE INVENTION This invention relates to compounds useful for the treatment of thrombotic disorders and, more particularly, to novel heterocyclic inhibitors of the enzyme thrombin. BACKGROUND The formation of immoderate thrombi in the walls of blood vessels precipitate the acute states of cardiovascular diseases that are one of the causes that lead to death in economically developed societies. Plasma proteins, such as fibrinogen, proteases and cell receptors, which are involved in hemostasis, have emerged as important factors that play a role in acute and chronic cronic diseases, as well as in cerebral artery disease, contributing to the formation of thrombi or blood clots, which effectively decrease the flow and normal supply of blood. Vascular aberrations emanating from primary pathological states, such as hypertension, rupture of the atherosclerotic plaques or bare endothelium, activate biochemical cascades that serve to respond and repair the site of the lesion. Thrombin is the key regulatory enzyme in the coagulation cascade. It serves with a pluralistic role as a feedback regulator both positive and negative. However, under pathological conditions, the first is amplified through the catalytic activation of co-factors required for the generation of thrombin, as well as the activation of factor XIII, necessary for the interlacing and stabilization of fibrin. In addition to its direct effect on hemostasis, thrombin exerts direct effects on various types of cells that support and amplify the pathogenesis of arterial thrombus diseases. The enzyme is the strongest activator of platelets, causing them to aggregate and release substances (for example the ADP TXA2 NE), which also propagates the thrombotic cycle. Platelets in a fibrin mesh comprise the main framework of a white thrombus. Thrombin also exerts direct effects on endothelial cells, which cause the release of vasoconstrictor substances and the translocation of adhesion molecules that become sites for the binding of immune cells. In addition, the enzyme causes the mitogenesis of smooth muscle cells and the proliferation of fibroblasts. From this analysis, it is evident that the inhibition of thrombin activity constitutes a viable therapeutic approach towards the attenuation of proliferative events associated with thrombosis. The main endogenous neutralizing factor for thrombin activity in mammals is anti-thrombin III (ATIII), a circulating plasma macroglobulin, which has low affinity for the enzyme. Heparin exerts clinical efficacy in venous thrombosis, increasing the binding of ATIII / thrombin through catalysis. However, heparin also catalyzes the inhibition of other proteases in the coagulation cascade and its efficacy in platelet-dependent thrombosis is greatly reduced or canceled out due to the inability of the enzyme attached to the thrombus. Adverse side effects, such as thrombocytopenia, osteoporosis and triglyceridemia, have been observed following prolonged treatment with heparin. Hirudin, derived from the glandular secretions of the leech, Hirudo medicinalis is one of the inhibitors of natural anticoagulant proteins, of high molecular weight, of thrombin activity (Mar wardt F. Cardiovascular Drug Revie s, 10, 211, 1992 ). It is a biopharmaceutical that has demonstrated efficacy in experimental and clinical thrombosis. A potential drawback to the use of hirudin, as a therapeutic agent, is its weak antigenicity and the lack of an effective method of neutralization, especially in view of its binding characteristics, extremely firm., towards thrombin. The excessively high affinity for thrombin is unique and is attributed to the simultaneous interaction with the catalytic site, as well as a "distal outer anion binding site" in the enzyme. The activity of thrombin can also be canceled by molecules similar to hirudin, such as hirulog (araganore, JM et al., Biochemistry, 22. 7095, 1990) or hirutonin peptides (DiMaio, J. et al., J Med. Chem., 35, 3331, 1992). The activity of thrombin can also be inhibited by low molecular weight compounds that compete with fibrinogen for the catalytic site of thrombin, thereby inhibiting the proteolysis of that protein or other protein substrates, such as the thrombin receptor. A common strategy for designing enzyme inhibitor compounds depends on the imitation of the specificity inherent in the primary and secondary structure of the natural substrate of the enzyme. Thus, Blomback et al. , first designed a thrombin inhibitor that was modeled on the partial sequence of the Aa chain of fibrinogen, which comprises its proteolytically susceptible region (Blomback et al., J. Clin Lab. Invest., 24 59, 1969). This region of fibrinogen minimally includes residues that start with phenylalanine: Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg T cleavable link The systematic replacement of amino acids within this region has led to the optimal form of the tripeptidyl inhibitory sequence, exemplified by the peptide (D) -Phe-Pro-Arg, which corresponds to the interactions within the local binding sites P3 * -P2p? in thrombin (Bajusz S et al in Paptides: Chemistry Structure and Biology: Proceedings of the Fourth American Peptide Symposium, Walter R., Meienhofer J. Ed. Ann Arbor Science Publishers Inc., Ann Arbor MI, 1975, pages 603). Bajusz et al. , also reported related compounds such as (D) -Phe-Pro-Arg- (C0) H (GYKI-14166) and (D) MePhe-Pro-Arg- (CO) H (GYKI-14786) (Peptides-Synthesis , Structure and Function: Proceedings of the Seventh American Peptide Symposium, Rich, DH &Gross, E. Eds., Pierce Chemical Company, 1981, page 417). These tripeptidyl aldehydes are effective inhibitors of thrombin, both in vitro and in vivo. In the case of both GYKI-14166 and GYKI-14766, it is presumed that the aldehyde group contributes strongly in the inhibitory activity, in view of its chemical reactivity towards the catalytic Ser-195 residue of thrombin, which generates an intermediate product of the hemiacetal Related work in the area of thrombin inhibitory activity has exploited the basic recognition binding motif engendered by the tripeptide (D) Phe-Pro-Arg, while incorporating several functional or reactive groups at the site corresponding to the cleavable link assumed (ie In US Patent No. 4,318,904, Shaw reports chloromethyl ketones (PPAK) which are reactive towards Ser-195 and HIS57.) These two residues comprise part of the catalytic triad of thrombin (Bode, W. et al. ., EMBO Journal 8, 3467, 1989.) Other examples of thrombin inhibitors carrying the general motif (D) Phe-Pro-Arg, are those that incorporate variants of boroarginine with terminal COOH, such as boronic acids or boronatos (Kettner, C et al., J. Biol. Chem., 268, 4734, 1993). Still other congeners of this motif are those that carry phosphonates (Wang, CL, J., Tetrahedron Letters, 33., 7667 , 1992) and a-keto esters (I anowics, E. J. et al. , Bioorganic and Medicinal Chemistry Letters, 12, 1607, 1992).

Neises B. et al. , have described a thrombin inhibitor of trichloromethyl ketone (MDL-73756) and Attenburger, J. M. et al. , have disclosed a related difluoro-alkyl-amide ketone (Tetrahedron Letters, 3 ?, 7255, 1991). Maraganofe et al. , (patents: European 0,333,356; WO 91/02750; E.U.A. 5,196,404) disclose a series of thrombin inhibitors that incorporate the D-Phe-Pro part and hypothesized that this preferred structure fits well within the groove adjacent to the active site of thrombin. Variations in these inhibitors are essentially linear or cyclic peptides, formed in the D-Phe-Pro part. Another series of patents and patent applications have described attempts to develop effective inhibitors against thrombosis using alpha-ketoamides and peptide-aldehyde analogs (EP 0333356, WO 93/15756, WO 93/22344, WO 94/08941, WO 94). / 17817, EP 0479489, US 5,380,713). Still others have focused their attention on peptides, peptide derivatives, peptide alcohols or cyclic peptides such as anti-thrombotic agenets (WO 93/22344, EP 0276014, EP 0341607, EP 0291982). Others have examined the amidin sulfonic acid parts to achieve this same end (E.U.A. 4,781,866), while still others have examined para- or meta-substituted phenylalanine derivatives (WO 92/08709, WO 92/6549).

Many of the examples cited above are convergent by maintaining at least one linear acyclic tripeptidyl motif, which consists of an arginyl unit whose basic side chain acts internally with a carboxylate group located at the base of the P-cleaved specificity in thrombin. Two adjacent hydrophobic groups provide an additional bond through favorable Van der Waals interactions, within a hydrophobic slit at the surface of the enzyme, designated the P3-P2 site. An object of the present invention is to provide compounds that exhibit an inhibitory activity towards thrombin.

COMPENDIUM OF THE INVENTION One aspect of the present invention relates to peptide derivatives represented by the formula (I) and its pharmaceutically acceptable salts: AS-X (I) in which: X is an aromatic or non-aromatic heterocyclic ring, unsubstituted or substituted with one or more amino, oxygen, alkyl, aralkyl or aryl groups; and AS is an active site inhibitor of thrombin having an argininyl residue or an analog thereof, connected to X. In another aspect of the present invention, the use of a compound of the formula (I) in the manufacture of a medicament for the treatment of vascular diseases in mammals, including humans. In a further aspect, a method is provided for the treatment of vascular diseases in mammals, including the human being, which comprises administering to the mammal an amount of a compound of the formula (I), effective to treat vascular diseases.

DETAILED DESCRIPTION OF THE INVENTION The compounds of the present invention include those wherein X is one or more heterocyclic rings, which may be unsubstituted or substituted with amino, oxygen, alkyl, aralkyl or aryl. X includes heterocyclic aromatic or non-aromatic rings. X also includes one or more heterocyclic rings that are optionally fused to another carbocyclic or heterocyclic ring. Preferably, X is selected from the group consisting of: wherein: X5, XiO 'xll and x12 are independently selected from the group consisting of N or C-X7, where X7 is hydrogen, alkyl C? _4 or aryl C5_g; Xg and X13 are each, independently, selected from the group consisting of C, 0, N, S, N-X or CH-X7, where X7 has the above definitions; R 5 is hydrogen, C 1 - alkyl, optionally substituted by carboxyl, carboxyl, C 1 -C 6 alkylC 2 * -alkyl C 1 -j ^ g C 1-6 alkyl-C 1-7 cycloalkyl, aryl or an aromatic heterocyclic ring. More preferably, X is selected from the group consisting of: where R5 has the above definitions * Also, X is preferably selected from the group consisting of where R7 has the above definitions. Even more preferably, X is selected from the group consisting of: where R7 has the above definitions. Especially preferred, X is 0-. where R5 has the above definitions. In another embodiment, X is a 1,2-triazole ring, optionally substituted with R 5 and / or is attached to J at the 2, 3, 4 or 5 position of the ring.

Preferably, R 5 is hydrogen or C 1-4 alkyl; More preferably, R5 is hydrogen or CH3; Especially preferred R5 is hydrogen. Preferred compounds of the formula (I) include those in which the portion of AS has the formula (II): G1 - G2 - (II) where: G1 is one or more amino acids, alkyl, aryl, aralkyl or cycloalkyl. G2 is an arginyl radical or an analogue thereof; with the proviso that AS is an inhibitor of the reactive site of thrombin. In particular embodiments, G2 is selected from the following amino acid derivatives, prepared according to the procedures described in Bioorg. Med.

Chem., 1995, 3: 1145. wherein n = 1-6, ni = 1-2, n2 = 0-7 and T is a bond or a part of the divalent linkage with X. Suitable portions of AS include amino acids 45-47 of hirudin and its analogs, and thrombin inhibitors, based on the sequence of D-Phe-Arg, D-Phe-Pip-Arg and D-Cha-Pip-Arg. Other inhibitors of the active site of thrombin including an argininyl or an analogue thereof at the C-terminus can also be incorporated into the formula (I) as AS. More preferably, the compounds of the present invention include those compounds wherein AS is -Phe-Pro-Arg- or an analog thereof. Especially preferred compounds of the present invention include those where AS is (D-Phe) -Pro-Arg- or an analog thereof. It will be appreciated that the compounds of the invention include all isomers, enantiomers and mixtures thereof. In a preferred embodiment, the compounds of the invention are represented by the formula (III): (III) wherein: R1 is selected from the group consisting of one or more aryl or cycloalkyl groups, which may be unsubstituted or substituted by hydroxy, C ^ -g alkyl, C5_g aralkyl, C3_8 aryl or C3_8 cycloalkyl. • R2 is selected from the group consisting of hydrogen, hydroxy, C ± -Q alkyl, C4_8 aralkyl and an unsubstituted or substituted amino group; R3 is selected from the group consisting of hydrogen, hydroxy, SH, C ± -Q alkyl, C3_g aryl and C4_8 aralkyl; n is an integer from 0 to 2; Q is a link or -NH-; Z is alkoxy C] ^; cyano; -NH2; -CH2-NH2; -C (NH) -NH2; -NH-C (NH) -NH2; -CH2-NH-C (NH) -NH2; a C6 cycloalkyl or aryl group, substituted with cyano, -NH2, -CH2-NH2, -C (NH) -NH2, -NH-C (NH) -NH2- OR -CH2-NH-C (NH) -NH2; or a saturated or unsaturated, 5 or 6 membered heterocyclic ring, optionally substituted with cyano, -NH2, -CH2-NH2, -C (NH) -NH2, -NH-C (NH) -NH2 or CH2-NH-C (NH) -NH2; and X has the above definitions. Preferred embodiments of the present invention include the compounds of the formula (III), in which R ^ is selected from the group consisting of one or more aromatic or non-aromatic rings, of 5 or 6 members, which may be unsubstituted or substituted with hydroxy or C3_g-cycloalkyl alkyl. More preferably, R ^ is a 6-membered aromatic or nonaromatic ring, unsubstituted or substituted with C-n alkyl. More preferably, R ^ is phenyl unsubstituted or substituted with C ^ _4 # alkyl. Especially preferred, R ^ is phenyl. Preferably R2 is hydrogen, hydroxy, Ci-g alkyl or amino unsubstituted or substituted by hydroxy, or C ^ _4 alkyl. More preferably, R2 is hydroxy or NH2 • Especially preferred R2 is NH2. Preferably R3 is hydrogen, hydroxy, SH or C? _g alkyl. More preferably, R3 is hydrogen or C4-4 alkyl. Especially preferred, R3 is hydrogen. Preferably, n is 1 or 2. More preferably, n is 1. Preferably, Q is a bond. Preferably, Z is linked by means of a methylene chain of 2 to 5 carbon atoms and is selected from the group consisting of -NH2; -C (NH) -NH2; -NH-C (NH) -NH2; a cycloalkyl or C6 aryl substituted with -NH2, -CH2-NH2, -C (NH) -NH2, -NH-C (NH) -NH2 or -CH2-NH-C (NH) -NH2 / and a heterocyclic ring of 5 or 6 members, saturated or unsaturated, optionally substituted with -NH, CH2NH2, -C (NH) -NH2, -NH-C (NH) -NH2 or -CH2-NH-C (NH) -NH2. More preferably, Z is -NH-C (NH) -NH2, -NH2 and -C (H) -H2 linked by a methylene chain of 3-5 carbon atoms. Especially preferred, Z is -NH-C (NH) -NH2, linked by means of a trimethylene chain. Preferred compounds of the invention include: More preferred compounds of formula (I) include: (D-Phe) -Pro-alpha-benzothiazole-keto-arginine; and (D-Phe) -Pro-alpha-thiazolo-keto-arginine. The following abbreviations are used here. These abbreviations are common and well known to those skilled in the art of peptide chemistry. BOC-butoxy-carbonyl BuLi-butyl lithium DCM-dichloromethane DMF-dimethylformamide iPr2NEt-diisopropylethylamine THF-tetrahydrofuran As used in this application, the term "alkyl" represents a portion of hydrocarbon, saturated or unsaturated, substituted (for example by a halogen, hydroxyl, amino, oxygen, sulfur or Cg-2?) aryl, unsubstituted, straight or branched chain having from 1 to 10 carbon atoms and preferably from 6 carbon atoms. This chain may be interrupted by one or more heteroatoms, such as N, O or S. The term "amino protecting groups", "oxygen protecting groups" and "protecting groups" are well known in the field of synthesis. of the peptides. These protective groups can be found in T. Greene, Protective Groups in Organic Synthesis, (John Wiley &Sons, 1981). The appropriate protective group for a particular synthetic scheme will depend on many factors, including the presence of other reactive functional groups and the reaction conditions desired for the removal., as is well known to those skilled in the art of peptide chemistry. The term "aryl" represents a carbocyclic part, which may be substituted by one or more heteroatoms (for example, N, O or S), and which contains a ring of the benzenoid type, preferably having 6 to 15 carbon atoms (per example, phenyl and naphthyl). This carbocyclic part may be interrupted by one or more heteroatoms, such as N, O or S. The term "aralkyl" represents an alkyl group, which is uninterrupted or interrupted, unsubstituted or substituted, by an aryl substituent (for example benzyl), preferably containing from 6 to 30 carbon atoms. Unless otherwise specified, the term "amino acid" used herein includes naturally occurring amino acids, as well as non-natural analogs, commonly used by those skilled in the art of chemical synthesis and chemistry. the peptides. A list of non-natural amino acids can be found in "The Peptides", vol. 5, 1983, Academic Press, Chapter 6, by D. C. Roberts and F. Vellanccio. It will be noted that, unless otherwise indicated, the amino acids used in the context of the present invention are those of the L configuration. The term "cycloalkyl" represents groups of cyclic hydrocarbons, containing from 3 to 12 carbon atoms, preferably from 3 to 8 carbon atoms, which include, for example, cyclopropyl, cyclobutyl, cyclohexyl and cyclodecyl, any of which may be substituted with substituents such as halogen, amino, alkyl and / or hydroxy.

The term "heterocycle" and "heterocyclic rings" represents one or more aromatic and non-aromatic rings, which include one or more heteroatoms, such as nitrogen, oxygen and sulfur, and which may be substituted with substituents, such as halogen, amino, alkyl and / or hydroxy. Preferably, the ring has 5, 6 or 7 members. While it may be possible that, for use in therapy, a compound of the invention is administered as the untreated chemical, it is preferred to present the active ingredient as a pharmaceutical formulation. The invention thus also provides a pharmaceutical formulation comprising a compound of the formula (I) and its addition salts with pharmaceutically acceptable acids, together with one or more of its pharmaceutically acceptable carriers and, optionally, other therapeutic and / or prophylactic ingredients. The carriers must be "acceptable" in the sense that they are compatible with the other ingredients of the formulation and do not harm the recipient. In another aspect of the present invention, the use of a compound of the formula (I) in the manufacture of a medicament is provided for the treatment of vascular diseases in a mammal, including humans. In another aspect, a method is provided for the treatment of vascular diseases in a mammal, including humans, which comprises the administration of an effective amount of a compound of the formula (I). Those skilled in the art will appreciate that the treatment extends to prophylaxis, as well as to the treatment of established vascular diseases. The compounds of the present invention are useful in combinations, formulations and methods for the treatment and prophylaxis of vascular diseases. These diseases include myocardial infarction, apolplegia, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion, restenosis that follows arterial injury or invasive cardiological procedures, acute or chronic atherosclerosis, edema and inflammation, cancer and metastasis. The term "combination", as used herein, includes a single dose form containing at least one compound of this invention and at least one thrombolytic agent, a multiple dose form, in which the thrombin inhibitor and the thrombolytic agent they are administered separately, but concurrently, or a multiple dose form, in which the two components are administered separately, but sequentially. Upon sequential administration, the thrombin inhibitor can be given to the patient during the time period ranging from about 5 hours before to about 5 hours after administration of the thrombolytic agent. Preferably, the thrombin inhibitor is administered to the patient during the period ranging from 2 hours before to 2 hours following administration of the thrombolytic agent. The thrombolytic agents that can be employed in the combinations of the present invention are those known in the art. Such agents include, but not limited to, the tissue plasminogen activator purified from natural sources, recombinant tissue plasminogen activator, streptokinase, urokinase, puroquinasa, plasminogen activator complex of anisolated streptokinase (ASPAC), plasminogen activators of the gland salivating of biologically known active animals and derivatives of any of the foregoing. The dose and dosage regimen of the compounds of the invention will depend on a variety of factors, such as the weight of the patient, the specific pharmaceutical composition used, the object of the treatment, i.e., the therapy or prophylaxis, the nature of the thrombotic disease that is going to be treated and the judgment of the doctor who performs the treatment. In accordance with the present invention, a pharmaceutically preferred effective daily dose of the compounds of this invention is between about 1 μg / kg body weight of the patient to be treated ("body weight") and 5 mg / kg of this body weight. More preferably, the therapeutic and prophylactic compositions of the present invention comprise a dose between about 10 and 500 μg / kg body weight of the compounds of this invention, it should also be understood that the pharmaceutically effective daily dose of any of the compounds of this invention is invention or the thrombolytic agent present in the combinations of the invention, may be less than or greater than the specific ranges mentioned above. According to an alternative embodiment of this invention, the compounds can be used in compositions and methods for coating the surfaces of invasive devices, which results in a lower risk of clot formation or activation of platelets in patients receiving these devices. Surfaces that can be coated with the compositions of this invention include, for example, prostheses, artificial valves, vascular grafts and catheters. The methods and compositions for coating these devices are known to those skilled in the art. They include chemical entanglement or physical adsorption of the compounds of these compositions of the invention to the surfaces of the devices.

According to a further embodiment of the present invention, the compounds can be used for the ex vivo thrombus image. In this embodiment, the compounds of this invention are labeled with a radioisotope. The selection of the radioisotope is based on a number of well-known factors, for example toxicity, biological half-life and detection capacity. Preferred radioisotopes include, but are not limited to, 125 f 123j and 111 ?. Techniques for labeling the compounds of the invention are well known in the art. More preferably, the radioisotope is 123I and labeling is achieved using Bolton-Hunter reagent 123i. The labeled thrombin inhibitor is administered to a patient and allowed to bind to the thrombin contained in a clot. This clot is then observed using well-known detection elements, such as a camera capable of detecting radioactivity, coupled to an imaging system of a computer. This technique also provides images of thrombin bound to platelets and meizothrombin. This invention also relates to compositions containing the compounds of this invention and methods for using such compositions in the treatment of tumor metastases. The efficacy of the compounds of this invention for the treatment of tumor metastasis is manifested by the ability of inhibition of inhibitors of endothelial cell activation induced by thrombin. This inhibition includes the repression of the synthesis of platelet activation factor (PAF) by endothelial cells. These compositions and methods have important applications in the treatment of diseases, characterized by inflammation induced by thrombin and edema, which is thought to be mediated by PAF. Such diseases include, but are not limited to, adult respiratory distress syndrome, septic shock, septicemia and reperfusion injury. Early stages of septic shock include discrete, acute inflammatory and coagulopathic responses. This invention also relates to the use of the compounds described above, or the compositions comprising them, as anticoagulants for extracorporeal blood. As used herein, the term "extracorporeal blood" includes blood removed from the patient's line, subjected to extracorporeal treatment and then returned to the patient in such process as dialysis procedures, blood filtration or blood diversion during Surgery. The term also includes blood products that are extracorporeally stored for final administration to a patient and blood collected from a patient to be used for several trials. Such products include whole blood, plasma or any fraction of the blood in which inhibition of coagulation is desired. The amount or concentration of the compounds of this invention, in these types of compositions, is based on the volume of the blood to be treated or, more preferably, its thrombin content. Preferably, an effective amount of a compound of this invention to prevent coagulation in the extracorporeal blood is about 1 μg / 60 ml of extracorporeal blood at 5 mg / 60 ml of extracorporeal blood. The compounds of this invention can also be used to inhibit thrombin bound to clots, which is believed to contribute to the coagulation of the clot. This is particularly important because commonly used anti-thrombotic agents, such as heparin and low molecular weight heparin, are not effective against thrombin bound to clots. Finally, the compounds of this invention can be used in compositions and methods for treating neurodegenerative diseases. Thrombin is known to cause neurite retraction, a process suggestive of roundness in configuration changes of brain cells and involved in neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease.

The compounds of the present invention can be synthesized by various methods well known in the art. Suitable methods of synthesis will vary depending on the portions of AS and X used in the compound. Suitable methods for the synthesis of analogous types of Phe-Pro-Arg are described below. However, other well-known methods can be employed. SCHEME 1 H-X + Stage 1; The heterocyclic ring 1 in solution is subjected to staging with an appropriate metallating base, such as n-BuLi, to generate the corresponding metalated heterocyclic compound. Group 2 of cyclic activated arginine is added to this mixture. Compound 2 is prepared, according to procedures known in the literature and described in, for example, R. T. Shuman, et al. , "Highly Selective Tropeptide Thrombin Inhibitors", J. Med. Chem, 1993, 36, 314. The compound supplied is ketoarginine 3 heterocyclic. Step 2 Ketoarginine heterocyclic 3 is deprotected and coupled to dipeptide 4, in the presence of a suitable coupling agent, solvent and base. The dipeptide 4 can be purchased or prepared by methods common in the art and literature of peptides. Suitable coupling agents include BOP and isopropylchloroformate. Suitable solvents include DCM and DMF. Suitable bases include iPr2NEt and n-methyl-orpholine. The resulting compound is deprotected with appropriate deprotection agents, to be delivered. ketoargininyl 5 heterocyclic. Suitable deprotection agents include BBr, HBr in acetic acid and TMSI. Methods for removing protective groups are well known to those skilled in the art.

Scheme I is used where Z is N. Scheme II is used when Z is carbon, a linear chain of carbons or forms a ring with Q. When Z forms a ring with Q, activated amino group 2 will be conveniently reformed to include this ring. The stages of the process remain the same as described for Scheme I. SCHEME II H-X The compounds of this invention and their intermediates can be purified during their synthesis and / or after their preparation, by standard techniques, well known to those skilled in the art. A preferred purification technique is HPLC chromatography. However, other chromatographic methods, such as column chromatography, can be used for the purification of the compounds. The crystallization can also be used to purify the products, since they can be washed with appropriate organic solvents. It is well known in the art that the amino protecting groups are not necessary for the reaction to occur. The process can be carried out without protective groups. However, they are used to increase the yield of the desired compounds. The process, described above, can use suitable protecting groups for compounds 2, 3 and 4. Suitable deprotection conditions and protocols are described in the synthesis literature and are well known to chemists skilled in the art. Desired groups R, R2 and R3 may be substituted in dipeptide 4 before coupling to heterocyclic ketoarginine 3, using techniques well known in the art of peptide chemistry. Likewise, preferred analogs of each peptide or dipeptide can be purchased with the desired substituents of the groups R ^, R2 and R3 already present. In order that the invention described here can be more fully understood, the following examples are pointed out. It should be understood that these examples are for illustrative purposes only and should not be construed as limiting the invention in any way. EXAMPLE 1 To a solution of THF (75 ml) benzothiazole (compound 1) (4.0 ml, 36.7 mmol) at -78 ° C, n-BuLi (1.6 M, 25 ml) was added slowly, the resulting orange suspension was stirred at -78 ° C for 1.5 hours. Then solid compound 2 (3.55 g, 8.7 mmol) was added. The reaction was stirred at -78dC for 30 minutes, followed by -20 ° C for 30 minutes, then cooled with aqueous and saturated NH 4 Cl. Extraction with ethyl acetate followed by column chromatography gave a yellow foam (1.28 g) in 28% yield, such as compound 3. Nuclear Magnetic Resonance Spectrum ("NMR") (CDCI3) d 1.45 (s, 9H), 1.5-1.8 (m, 2H), 3.1-3.23 (, 1H), 3.45-3.60 (m, 1H), 5.1 (d, 2H), 5.53-5.64 (m, 2H), 7.02 -7.15 (m, 4H), 7.21-7.28 (m, 2H), 7.56-7.65 (m, 2H), 8.0-8.05 (m, 1H), 8.18-8.23 (m, 1H). Mass spectrum ("MS"): (M + l) 526.8.

To a mixture of compound 3 (0.223 g, 0.43 mmol) and EtSMe (0.25 ml), at room temperature, a solution of 4M HCl in dioxane (10 ml) was added. The reaction was stirred for 1 hour. All solvents were removed and the yellow solid was dried. To this yellow solid compound 4 (0.17 g, 0.47 mmole) and BOP (0.21 g, 0.48 mmole) in DMF (5 ml) were added at room temperature, then iPr2NEt was added to this mixture until the pH of the mixture reached 8-9. The reaction was allowed to stir overnight. The reaction was extracted with ethyl acetate and washed with brine, and subsequent column chromatography gave 0.129 g of the desired precursor to compound 5, which was dissolved in DCM (10 ml) and a solution of 1M BBr3 in DCM was added. (1.7 ml, 1.66 mmole) at -782C. The reaction was stirred at -78 ° C for 30 minutes, followed by 3 hours at room temperature. It was cooled again to -78se and anhydrous MeOH (2 mL) was added, followed by stirring at room temperature for 1 hour. All solvents were removed and the mixture was extracted with water and washed with ether. The water fraction was lyophilized and subjected to purification by reverse phase HPLC chromatography to deliver compound 5. The two compounds were isolated as individual diastereomers, analog 1 and analog 2, with identical mass spectrum [(M + l) 536.5].

EXAMPLE 2 Determination of K¿ Values This assay was performed with a Perkin Elmer fluorometer, model #LS 50B, using a fluorogenix thrombin substrate (Tos-Gly-Pro-Arg-AMC-HCl). purchased from Calbiochem. Calbiochem human thrombin was also obtained. The measurements were determined at excitation and emission wavelengths of 383 and 455 nm, respectively. The assay was carried out in a regulator consisting of 50 mM of tris, 100 mM of NaCl, 0.1% of PEG, pH of 7.8, to 24se. The regulator, substrate and inhibitor were mixed and the reaction was started by adding the solution of enzyme. The initial rates were recorded at various concentrations of the inhibitor and the substrate. The kinetic parameters were determined by fitting the data to a general equation that describes the inhibition of the enzyme (Segel, Enzyme Kinetics, Wiley Interscience Publications, 1993). Dixon and Lineweaver-bruk graphs were used to estimate the kinetic parameters (Kn, Vm ^ x, Kj using the Microsoft ™ Excell ™ program (TM = Registered Trademark) .The Union is the establishment of the balance between the enzyme, inhibitor and complexes. of enzyme inhibitor.In the inhibition of slow binding, this equilibrium was established slowly.The equilibrium dissociation constant for compound 5 is shown in cut 1. The result was compared with known thrombin inhibitors, based on tripeptidyl Compound 5 exhibited a slow binding kinetics, however, the inhibition constant was determined assuming a fast stable state kinetics.Therefore, the supplied values are a reliable estimate of the equilibrium inhibitory constants dTT assay Procedure: The fibrinogen and the regulatory solution were transferred to disposable tubes and placed in a pool of water for about 15 to 30 minutes, before the test, to allow the equilibrium to 37se The specimen-strips were incubated for 3 minutes at 37ac A ball was distributed to each specimen. To the previously heated specimens were added 75 μl of regulator, 50 μl of the inhibitor solution and 50 μl of fibrinogen solution. The chronometer corresponding to the incubation column was started for a 60-second incubation. The specimens were transferred to a test column area. It was primed once with the starting reagent (thrombin solution), activated and 25 μl of the thrombin solution was distributed. When the clotting times were determined, they were displayed and printed.

Time curves were constructed versus inhibitor concentrations and the IC50 values were extrapolated from these inhibitor concentration curves. The IC50 is defined as the dose required for twice the clotting time compared to the control. The results that give the IC50 value are shown in Table 1. TABLE 1 COMPOUND Ki (nM) IC50 (dTT) (nM) 5 0.05-0.180 1.8-7.2 PPACK 0.017 2.5 Boc-D-Phe-Pro-Arg-H 45 D-1-Tiq-Pro-Arg-H 19 The results of Table 1 demonstrate that a heterocyclic function, such as is embedded in a keto-arginyl unit of benzothiazole, extends to the S ^ -S] _ 'sites of thrombin increases the affinity of the enzyme up to 100-fold, in comparison with the other inhibitors reported. Compound 5 is equipotent to PPACK, which is considered as an irreversible inhibitor of thrombin, which forms a covalent bond with the enzyme, while compound 5 is a reversible inhibitor of thrombin.

Having now fully described the invention, it will be apparent to one of ordinary skill in the art that numerous modifications can be made without departing from the spirit thereof, as set forth in the following claims.

Claims (9)

CLAIMS 1. A thrombin inhibiting compound, according to formula (I), and its pharmaceutically acceptable salts:
1. AS-X (I) in which: X is one or more aromatic or non-aromatic heterocyclic rings, unsubstituted or substituted with one or more amino, oxygen, alkyl, aralkyl or aryl groups; and AS is an active site inhibitor of thrombin having an argypinyl residue, or an analog thereof, connected to X.
2. 2. A compound, according to claim 1, wherein X is selected from the group consisting of: where: x5 * X1Q 'xll and x12 are independently selected from the group consisting of N or C-X7, where X is hydrogen, alkyl C? _4 or aryl C5_s; g and 13 are each, independently, selected from the group consisting of C, O, N, S, N-X or CH-X7; R5 is hydrogen, C ^ - ^ alkyl, optionally substituted by carboxyl, carboxyl, C0_1g-C02 alkyl-C ^ -ig alkyl Cg_20f C7_7 cycloalkyl, aryl or an aromatic heterocyclic ring.
3. 3. A compound, according to claim 2, wherein X is selected from the group consisting of: where R5 is hydrogen, C ^ - ^ g alkyl optionally substituted by carboxyl, carboxyl, Cg-i-C02 alkyl-C6-C6 alkyl cycloalkyl aryl or an aromatic heterocyclic ring.
4. 4. A compound, according to claim 2, wherein X is selected from the group of: where R 5 is hydrogen, C 1-16 alkyl optionally substituted by carboxyl, carboxyl, C0_? g-C02 alkyl-C ^ -ig alkyl, C3-7 cycloalkyl aryl or an aromatic heterocyclic ring.
5. 5. A compound, according to Claim 12, wherein X is selected from the group consisting of: where R5 is hydrogen, C? -?? alkyl optionally substituted by carboxyl, carboxyl, C? --6-C02-alkyl C? i, C3_7? cycloalkyl aryl or an aromatic heterocyclic ring.
6. 6. A compound, according to the claim 2, in which X is selected from the group consisting of: where R5 is hydrogen, C alquilo-alkyl optionally substituted by carboxyl, carboxyl, Cn-i-C02 alkyl-C ± -iß alkyl C3_7 cycloalkyl aryl or an aromatic heterocyclic ring.
7. 7. A compound, according to the claim 1, in which AS is a group of the formula (II): G ** - G2 - (II) where: G1 is one or more amino acids, alkyl, aryl, aralkyl or cycloalkyl. G2 is an arginyl radical or an analogue thereof; 8- A compound, according to claim 7, wherein G2 is an arginyl radical, selected from: where n = 1-6, ni = 1-2, n2 = 0-7 and T is a bond or a part of the divalent linker with X. 9. A compound, according to claim 1, wherein AS is fragment 45-47 of the hirudin peptide, and its analogues. 10. A compound, according to claim 1, wherein AS is selected from D-Phe-Pro-Arg; D-Cha-Pro-Arg; D-Phe-Pip-Arg; and D-Cha-Pip-Arg. 11. A thrombin inhibiting compound, according to formula (III): (III) wherein: R is selected from the group consisting of one or more aryl or cycloalkyl groups, which may be unsubstituted or substituted by hydroxy, C ^ g alkyl, C5 aralkyl, aryl C3_3 or C3_8- R2 cycloalkyl is selected from the group consisting of hydrogen, hydroxy, C ^ g alkyl, C8 aralkyl and an unsubstituted or substituted amino group; R3 is selected from the group consisting of hydrogen, hydroxy, SH, C "6 alkyl, C3_g aryl and C4_8 aralkyl; n is an integer from 0 to 2; Q is a link or -NH-; Z is alkoxy C] ^; cyano; -NH2; -CH2-NH2; -C (NH) -NH2; -NH-C (NH) -NH2; -CH2-NH-C (NH) -NH2; a C6 or aryl cycloalkyl group, substituted with cyano, -NH2, -CH2-NH2, -C (NH) -NH2, -NH-C (NH) -NH2- or -CH2-NH-C (NH) -NH2; or a saturated or unsaturated, 5 or 6 membered heterocyclic ring, optionally substituted with cyano, -NH2, -CH2-NH2, -C (NH) -NH2, -NH-C (NH) -NH2 or CH2-NH-C (NH) -NH2; and X is one or more heterocyclic, aromatic and non-aromatic rings, unsubstituted or substituted with one or more amino, oxygen, alkyl, aralkyl or aryl groups. 12. A compound, according to the claim 11, in which R ^ is selected from the group consisting of one or more rings of 5 or 6 members, aromatic or non-aromatic, optionally substituted by hydroxy, C? _4 alkyl or cycloalkyl 03-3 • 13. A compound, according to with the claim 12, in which: R1 is phenyl; R2 is hydroxy or NH2; R3 is hydrogen or C] __ 4 alkyl; n is 1 or 2; Q is a link; and Z is -NH-C (NH) -NH2, -NH2 and -C (NH) -NH2, linked by a methylene chain of 3 to 5 carbon atoms. A compound, according to claim 1, selected from the keto-arginine of (D-Phe) -Pro-alpha-benzothiazole and the keto-arginine of (D-Phe) -Pro-alpha-thiazolo. 15. The use of a compound, according to one of claims 1 to 14, in the manufacture of a medicament for the treatment of vascular diseases in a mammal, which includes humans. 16. The use, according to claim 15, in which the vascular disease is thrombosis. 17. A method for the treatment or prophylaxis of thrombotic disorders in a mammal, this method comprises administering to the mammal an effective amount of a compound according to any of claims 1 to 14. 1
8. 8. The method according to claim 17, in which the disorder is venous thrombosis. 1
9. 9. The method according to claim 17, wherein the disorder is pulmonary embolism. 20. The method according to claim 17, wherein the disorder is arterial thrombosis. 21. The method according to claim 17, wherein the disorder is myocardial infarction. 22. The method according to claim 17, wherein the disorder is cerebral infarction.