Inhibitors of factor xa

 

(57) Abstract:

Describes the connection unnatural origin, specifically inhibiting the activity of factor XA, having the General formula (I) A1-A2-(A3)m, Where m = 0 or 1; A1 - R1-R2-R3; A3 - R7-R8-R9; R1selected from the group consisting of R'1-X-R1", where X Is N and R'1and R1independently selected from the group consisting of H, alkyl, acyl aryl, arylalkyl and group protecting the amino group; R2- -CR99R100where R99and R100independently selected from the group comprising H, alkyl, arylalkyl, heteroallyl and heteroaryl; R3- - (O)- or-CH2-; R4- -NR50-, where R50- N; R5- -CR201R202where R201and R202independently selected from the group comprising H, alkyl, aryl and arylalkyl; R6Is-C(O) or-CH2-; R7- -NR51where R51- N; R8- -CR210R211-, where R210and R211independently selected from the group comprising H, alkyl, arylalkyl and heterocycle, where the alkyl, alkylaryl and heterocycle may be substituted by Q or -(CH2)n-Q, where n = 1-5, and Q is selected from the group comprising amine, amedieval, imidazole and guanidine groups that mo is isothiourea; R9- - (O)- or-CH2- ;- - OTHER52where R52selected from the group comprising H, alkyl, arylalkyl, heteroallyl, heteroaryl, one or two amino acids, or its pharmaceutically acceptable salt, amide, ether, alcohol or aldehyde. The compound of formula (I) prevents the formation of blood clots, as a specific small molecule inhibitor of factor XA, effective, but does not cause unwanted side effects. 8 C. and 20 C.p. f-crystals, 5 tab., 4 Il.

The invention relates in General to inhibition of protein blood clots and especially to a specific factor Xa inhibitors of the blood coagulation system.

The ability to form blood clots necessary for survival. In some diseases, however, the formation of blood clots in the circulatory system itself is a source of morbidity. Thus it is sometimes desirable to prevent the formation of blood clots. However, complete inhibition of the blood coagulation system is undesirable, as it will be followed by bleeding, life-threatening.

In order to reduce intravascular blood clots, experts have developed an effective inhibitor prothrombinase or factor Xa, which wodis the appropriate concentration of inhibitor of factor Xa will increase the level agents, forming prothrombinase required to initiate the formation of clots, but will not cause unnecessary prolongation of the clotting process, as soon as the threshold concentration of thrombin. However, despite long-standing recognition of the desirability of establishing such inhibitor, currently in clinical practice there is no effective specific inhibitor of factor Xa.

In many clinical areas there is a need for anticoagulation treatment means.

Currently available drugs in many clinical areas are unsatisfactory. For example, almost 50% of patients who underwent a complete replacement of the hip joint develops deep coronary thrombosis (GW).

In the accepted therapy used fixed-dose low molecular weight heparin (NMG), and changing the dose of heparin. Even with these regimens in 10-20% of patients will develop GW and 5-10% develop complications from bleeding.

Another clinical situation in which there are more effective anticoagulants are plastic surgery on the coronary vessels with an increased risk of myocardial infarction in, connected with 6-8% of cases clogging of blood vessels within 24 hours of the procedure. The rate of complications associated with bleeding requiring blood transfusion due to the use of heparin, also is approximately 7%.

Moreover, although a considerable number of cases without clogging of blood vessels, the use of heparin after the procedure is ineffective and may be adversely affected.

The most widely used inhibitors of blood coagulation are heparin and related sulfacetamide polysaccharides, NMG and heparansulfate. The molecules of these compounds show anti-clotting properties by promotion binding of the natural regulator of the coagulation process, anti-thrombin III, thrombin and factor Xa. Inhibiting activity of heparin initially directed against thrombin, which is inactivated in approximately 100 times faster than a factor of CA. Although in comparison with heparin heparansulfate and MWF are more effective inhibitors of Xa than thrombin, the difference in vitro small (3-30 times) and the effect in vivo can be volatile. Hirudin and hirulog are also anticoagulants, specific against thrombin used in addition to the ptx2">

Preclinical trials in baboons and dogs showed that a specific factor Xa inhibitors prevent the formation of clots, causing no adverse bleeding observed in the use of direct thrombin inhibitors. Such factor Xa inhibitors include, for example, 2.7-bis-(4-amidinopropane)cycloheptanone and methyl ether Na-moselhotel-3-amidinothiourea ("TENSTOP"), effective inhibiting concentration (Ki's) which are 20 nm and 800 nm, respectively, (+)-(2S)-2-(4({(3S)-1-acetimidoyl-3-pyrrolidinyl} oxy)phenyl)-3-(7-amidino-2-naphthyl)propanoic acid is also representative of a class of inhibitors of factor Xa (Katakura et al., Biochem. Biophys. Res.Comm. 197:965-972 (1993)). However, these compounds are not used in clinical practice.

Specific protein factor Xa inhibitors have also been identified and include, for example, antistain (ATC) and anticoagulate peptide isolated from the tick (CAP). ATC, which is isolated from leeches Haementerin officinalis, contains 119 amino acids and has Kifor factor Xa equal to 0.05 nm. CAP, isolated from the tick Ornithodoros moubata, contains 60 amino acids, for him Ki for factor Xa is about 0.5 nm.

The efficiency obtained recombinante PBX and CAP were studied in a number of isotermiline blood when tested in a model of coronary thrombosis, caused by thromboplastin, legirovannykh jugular veins.

The results obtained with this model are correlated with the results obtained when using common at the present time the drug heparin.

It was also found that subcutaneous injection of ATS is effective when tested on the model of diffuse intravascular coagulation (RVC), caused by thromboplastin. CAP effectively prevents arterial thrombosis and reduced blood flow caused by surgical introduction of polyester (DACRON) implant at doses providing clinically acceptable, prolongation of time, activated partial thromboplastin (actp), i.e. less than two times. For comparison, standard heparin, even at doses that cause a five-fold increase actp, does not prevent thrombosis and reduced blood flow inside the implant. ACTP is a clinical test method of coagulation, which is particularly sensitive to inhibitors of thrombin.

PBX and CAP not found application in the clinic. One important disadvantage of these two inhibitors is that the introduction of required repeated doses causes the formation of neutralizing antibodies, thus limiting their Kli is ichiwa the number of patients able to benefit from these agents.

Specific inhibitor of factor Xa will be of practical value in medicine. In particular inhibitor of factor Xa will be effective in circumstances where the applicable heparin and sulphadimidine polysaccharides are inefficient or ineffective. Thus there is a need to create a specific low-molecular-weight inhibitor of factor Xa, which prevents the formation of blood clots, which is effective but does not cause unwanted side effects.

This invention satisfies this need and provides the desired advantages.

This invention relates to compounds which specifically inhibit the activity of factor Xa.

The connection according to the invention has the structure X1-Y-I-R-X2where X1means hydrogen (H), acyl, alkyl or arylalkyl, or one or more amino acids, and X2means a modified C-terminal sequence, one or more groups, protecting carboxypropyl (see below), one or more amino acids or other substituents, and Y, I and R are amino acids - tyrosine, isoleucine and arginine, respectively, and tyrosine, isoleucine and arginine, respectively. Additionally, the connection according to the invention has the structure A1-A2-(A3)m-B, as indicated in this description.

The connection according to the invention can be linear or cyclic, containing from about 2 to about 43 residues along the length of the molecule and the modified N-containing and/or S-containing terminal sequences. Such compounds show specific inhibition of the activity of factor Xa with Ki<100 μm, preferably Ki< 2 nm and practically does not inhibit the activity of other proteases involved in the cascade of circulation. Specific examples of such compounds include Ac-Tyr-Ile-Arq-Leu-Ala-NH2; Ac-Tyr-Ile-Arg-Leu-Pro-NH2; Ac-(iBu)-Tyr-Ile-Arg-Leu-Pro-NH2; Ac-Tyr-Ile-Arg-N(CH3)O(CH3); Ac-Tyr-{ (CH2NH)} -Ile-Arg-Leu-Pro-NH2where means pseudopeptide a relationship that, for example, can be healing connection, as specified "CH2NH)"; pseudopeptide connection indicated by the symbol "f" in brackets, "{}"; Ac-Tyr-Ile-Arg-NH-CH2(4-pyridyl); Ac-Tyr-Ile-{ (CH2NH)} -Arg-Leu-Pro-NH2; Ac-Tyr-Chg-Arg(NO2)-{ (CH2NH)} -Leu-NH2; Ac-Tyr-Ile-Arg-{(COCH2)}- Gly-Pro-NH2; Ac-Tyr-Ile-Dab(N- C3H7N)-Leu-Ala-NH2; Ac-Tyr-Ile-PalMe(3)-NH2; Tyr-Ile-Arg-NH2; (D)-Tyr-Ile-Arg-Leu-Pro-NH2; A; c-Nal(2)-Chg-Arg-Leu-Pro-NH2; Ac-pAph-Chg-Palme(3)-NH2; and their pharmaceutically acceptable salts and derivatives with integral C-containing groups such as amides, esters, alcohols and aldehydes (see also table 5).

The invention provides specific methods of inhibiting the activity of factor Xa and inhibiting blood clotting in individuals. Also provided methods for the determination of levels of factor Xa or activity.

In Fig. 1 shows a schematic diagram of the cascade of blood coagulation.

In Fig. 2 shows an example of the connection structure according to the invention.

In Fig. 3 A, B shows the scheme of the reactions of some compounds according to the invention.

Coagulation of blood is a complex process involving a number of progressive-propagating reactions, activation of enzymes, in which Imogene plasma sequentially activated by a limited proteolysis. Mechanical cascading blood circulation is divided into inner and outer loops that converge at the activation of factor XA; subsequent formation of thrombin flows through a single common cycle (see Fig. 1).

The present study suggests that the inner loop plays an important Alvania blood coagulation. It is generally accepted that the coagulation of blood physically initiated after the formation of the complex of tissue factor/factor VIIa. Immediately after the formation of this complex rapidly initiates coagulation by activating factor IX and X. the Newly formed factor Xa then forms a 1:1 complex with factor Va and phospholipids with the formation of complex prothrombinase, which is responsible for the conversion of soluble fibrinogen into insoluble fibrin. As time progresses, the activity of a complex of factor VIIa/tissue factor (the outer loop) is inhibited by the protein protease inhibitor Kunitz type, TFPI, which, when it forms a complex with factor Xa, can directly inhibit the proteolytic activity of the complex factor Vlla/tissue factor.

In order to support the process of coagulation is inhibited in the presence of an external system, is produced by advanced factor Xa thanks to the activity of the inner loop, mediate through thrombin. Thus thrombin plays a dual autocatalytic role, through their own production and the conversion of fibrinogen to fibrin.

Autocatalytic nature of the formation of thrombin is important warranty. what thrombinase, coagulation of blood is going to happen until the end, leading, for example, by the end of the bleeding.

Thus the most desirable agents that inhibit coagulation without direct inhibition of thrombin.

This invention provides a receiving YIR peptides, which are compounds, any abscopal the activity of factor Xa, and almost not any abscopal the activity of other proteases involved in blood coagulation.

Used herein, the term "connection" or "YIR peptide" refers to unnatural Tyr-Ile-Arg (YIR) peptide and its analogs and mimetics, which can inhibit the activity of factor Xa.

YIR sequence itself is referred to in this description of "YIR fragment" and consists of Tripeptide tyrosine-isoleucine-arginine or its functional equivalent, for example, pAph-Chg-PalMe(3), pAph-Chg-PalMe(3)-NH2and pAph-Chg-AMP(4) (see abbreviations in table 1).

Such compounds according to the invention contain at least one YIR fragment or functional equivalent and is able to specifically inhibit the activity of factor Xa. For convenience, the term "connection" and "YIR peptide" is used here broadly to refer to peptides according to the invention, in the definition of the connection. Functional equivalent YIR peptide according to the invention can be partially characterized as having the structure described in this description, and the value of Ki 100 μm for inhibiting the activity of factor Xa (see example XXXVII).

Peptide analogues YIR peptide according to the invention include, for example, peptides containing unnatural amino acids or chemically modified amino acids, provided that the compound retains inhibitory activity against factor Xa (e.g., see table 2). Similarly mimetic peptides are diaminotoluene chemical structures that mimic the structure of the YIR peptide according to the invention and retain inhibitory activity against factor Xa. Such mimetics in General are characterized as exhibiting similar physical properties, such as size, charge or hydrophobicity, which is presented in the proper spatial orientation, as found in the conventional counterion YIR peptide. A concrete example of the mimetic peptide is a compound in which the amide bond between one or more amino acids substituted, for example, a bond of carbon-carbon or other connection, as is well known from the prior art (see, for example. Sa factor Xa, having the structure A1-A2-(A3)m-B, where m = 0 or 1, are described below. Examples of such peptides, which can be mimetic, described in this description.

The term "amino acid" is used in its broadest sense to refer to the twenty natural amino acids, which are translated from the genetic code and consist of blocks of proteins, including, unless otherwise specified, L-amino acids and D-amino acids, chemically modified amino acids such as amino acid analogs of natural amino acids that are not normally introduced into proteins, for example, norleucine and synthetic chemical compounds having properties characteristic of amino acids. For example, analogs or mimetics of phenylalanine or Proline, which allow the same conformational restriction of the peptide, as a natural Phe or Pro included in the definition of "amino acids" and is well known to specialists.

Such analogs and mimetics are referred to in this description of "functional equivalents" of amino acids. Other examples of amino acids and their analogues see Roberts and Vollaccio The Peptides: Analysis, Sunthesis, Biology, Eds Grass and Meienhofer, Vol.5, p.341, Academic Press, Inc., N. Y. 1983). Abbreviations of amino acids, amino acid analogues and mimetics are shown in tab the RA Xa, by itself or in combination of pojedinici known as complex prothrombinase, to catalyze the conversion of prothrombin to thrombin. When used in respect of the activity of factor Xa, the term "inhibition" means both direct and indirect inhibition of the activity of factor Xa. Direct inhibition of the activity of factor Xa can be done, for example, by linking YIR peptide according to the invention with factor Xa or prothrombinase in order to prevent binding of prothrombin to the active centre of the complex prothrombinase. Indirect inhibition of the activity of factor Xa can be done, for example, by binding of the compounds according to the invention with soluble factor Xa in order to prevent its occurrence in prothrombinase complex.

Used for inhibiting the activity of factor Xa, the term "specific" means that YIR peptide can inhibit the activity of factor Xa without exerting inhibitory effect on the activity of proteases, including plasmin and thrombin (at the same concentration of the inhibitor). Such proteases are involved in the process of blood coagulation and fibrinolysis cascade (see table 2; see also the example XXVII).

The results will calicivirus the activity of other serine proteases, such as thrombin or plasmin, which is involved in the process of blood coagulation and fibrinolysis.

Used herein, the term "Deputy" refers to any of various chemical groups, which are introduced into the main chain of the peptide or a side chain of a peptide analogue of a peptide of mimetica or organic compounds described in this description. The Deputy may be any known in the art (see, for example, Giannis and Kolter, Angew. Chem. Int. Ed. Engl. 32: 1244-1267 (1993)). In this description provides numerous examples showing different substituents, including, for example, the introduction of pNH2Deputy in phenylalanine with obtaining F(pNH2and the introduction of halogen into tyrosine with obtaining, for example, Y(3-1) or Y(3.5 to 1). In addition, the Deputy may be, for example, a heteroatom, such as nitrogen (N; see, for example. Pal), oxygen (O; see, for example, O-methyltyrosine) or sulfur (S; see, for example, Tight(SO3H)), which may contain a substituent. Thus, N-, S-, or O-containing fragment, such as-SO3H, considered as a Deputy.

In addition, the Deputy may be a group that protects aminogroup or carboxyl group.

Used herein, the term "alkyl" is used in its broadest sense for about. the AK, the term "alkyl" includes, for example, methyl, ethyl, n-propyl, isopropyl, sec.butyl, 1-methylbutyl, 2,2-dimethylbutyl, 2-methylpentyl, 2,2-dimethylpropyl, n-pentyl and n-hexyl, alkylene groups, carbocyclic groups, such as cyclohexyl and cyclopentyl, as well as combinations of linear or branched chains and carbocyclic chains, such as methyl-cyclohexyl or cyclopropyl-methylene. In addition it should be borne in mind that the alkyl may contain a substituent. Similarly, the term "acyl" is used in its broadest sense to denote a saturated or unsaturated, linear, branched or cyclic chains with about 1-13 carbon atoms, which contain a carboxyl group. Thus, the term "acyl" includes, for example, groups such as formyl, acetyl, benzoyl, etc.

The term "aryl" refers to aromatic groups containing from 5 to 13 carbon atoms and at least one ring having a conjugated system of PI electrons. Examples of arilou are, for example, heterocyclic aryl groups, dianiline group, their analogues and derivatives, which may contain one or more substituents.

The term "arylalkyl" refers to alkyl as defined above, substituted aryl LASS="ptx2">

The term "heteroalkyl", "heteroaromatic and heteroaryl also used in this description and are alkyl, arylalkyl and aryl, respectively, which is substituted by one or more heteroatoms, such as atoms N, O or S. in Addition, the term "heterocyclic" is used in relation to cyclic alkyl or aryl group, which is substituted by one or more heteroatoms. Numerous examples of heteroalkyl, heteroallyl, heteroaryl and heterocycles described, for example, in tables 1 and 3, and is also known from the prior art.

The peptides according to the invention can be modified on the terminal N - or C-containing sequences using the group, replacing the amino group or carboxyl group, respectively.

Numerous examples of such modifications are described in this description (see, for example. table 3). N-containing terminal group of the peptide or peptide analog can be chemically modified so that the terminal amino group substituted, for example, acetyl, cyclopentanecarboxylic, sochinarkokontrol, furillo, toiley, pyrazinecarboxamide or other similar group which may be substituted replace what needs to be borne in mind, the term "amino group" is used here in a broad sense to denote any free amino groups, including primary, secondary, or tertiary amino group present in the peptide. Compared to the N-terminal sequence means the alpha amino group of the first amino acids contained in the peptide, the formula is written in a conventional manner.

N-terminal group of the peptide according to the invention can be protected by adherence to the group protecting the amino group. The term "protecting the amino group" is used here in a broad sense to refer to a chemical group that can react with a free amino group, including, for example, the alpha-amino group located at the end of the molecule peptide according to the invention. Interacting with the latter protecting group protects reactive in other conditions, the amino group against undesirable reactions that can occur, for example, in the synthesis process or due to the activity of ectopeptidases end of the connection. Modification of the amino group may also provide additional benefits, including, for example, increasing the solubility or activity of the compounds. In this description describes the various groups that protect aminoacetyl, picolyl, tert. butylacetyl, tert. butyloxycarbonyl, benzyloxycarbonyl, benzoyl, including, for example, benzyloxy, such as 2-aryl-2-O-benzyloxy (see example XVI), as well as aminoallyl residue, which may itself be modified by a group protecting the amino group. Other group protecting the amino group described, for example, in The Peptides, eds.Gross and Meinhofer, Vol.3 (Academic Press, Inc. , N. Y. , 1981); and Green and Wuts, "Protective Groups in organic Synthesis, 2-nd ed., p.p. 309-405 (John Wiley and Sons, New York (1991)). The product of any such modification of the terminal amino group of the peptide or analogue of the peptide according to the invention is termed herein "N-terminal derivative".

Similarly carboxyl group, such as containing the C-terminal of the peptide may be chemically modified using a group advocating carboxypropyl. The term "carboxyl group" and "C-terminal" are used analogiei with terms "amino group" and "N-terminal", above.

Carboxyl group, such as contained in the C-terminal group of the peptide may be modified by the restoration of the C-terminal carboxypropyl to alcohol or aldehyde or by education tematicheskoe of ester or replacement carboxypropyl Deputy, such as tiaso the groups, such as methoxymethyl, ethoxymethyl, isopropoxyphenyl and so forth, alpha-(C1-C4)alkoxyethyl, such as methoxyethyl, ethoxyethyl, propoxyethyl, isopropoxide and so on; 2-oxo-1,3-dioxolan-4-ylmethylene groups such as 5-methyl-2-oxo-1,3-dioxolan-4-ylmethyl etc.; C1-C3-alkylthiomethyl, such as methylthiomethyl, ethylthiomethyl, isopropylaminomethyl etc.; acyloxymethyl groups, such as evaluaciones, alpha acetoxymethyl etc.; etoxycarbonyl-1-methyl group; alpha-acyloxy-alpha-substituted methyl group, such as alpha-acetoxyethyl, 3-phthalidyl or 5,6-dimethylphthalate, 1-(C1-C4-allyloxycarbonyl)et-1-ilen groups such as 1-(ethoxycarbonyl)ETH-1-yl; and 1-(C1-C4-alkylaminocarbonyl)et-1-ilen group such as 1-(methylaminorex)ETH-1-yl.

The peptide according to the invention can be modified by the attachment of the group protecting the carboxyl. Protecting the carboxyl group is well known and being attached to the peptide, protects carboxypropyl against undesirable reactions (see, for example, Greene and Wuts, supra, p.p. 224-276 (1991)).

The specialist knows that such modifications, as described above, which can be implemented with N-cancereuses reactive amino group or carboxyl group, for example, in the side chain of the amino acid or analogue of the amino acids in the peptide according to the invention. Methods of implementation of such modifications are described below, and is also known from the prior art.

This invention provides for the formation of compounds that specifically inhibit the activity of factor Xa. The connection according to the invention has the General structure X1-YIR-X2or is its functional equivalent, where X1means H, acyl, alkyl, arylalkyl or one or more amino acids, and X2means a modified C-terminal group, one or more groups, protecting carboxypropyl, or one or more amino acids or other substitute, such as a group protecting the amino group.

The connection according to the invention is useful as an anticoagulant in therapeutic treatment of various clinical diseases. The connection according to the invention is also suitable for various laboratory tests to prevent clotting of blood samples.

The invention also provides a compound that specifically inhibits the activity of factor Xa and has the General formula A1-A2-(A3)m-B, where m = 0 or 1 and A1 means R1-R2-R3A2 means R4-R5
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X is selected from the group consisting of N, CH and NC(O), and R'1, R1selected from the group consisting of alkyl, acyl, aryl, arylalkyl and group protecting the amino group and where R1may be substituted; R2means-CR99R100where R99and R100independently selected from the group comprising H, alkyl, arylalkyl, heteroallyl and heteroaryl, and where R99and R100independently may be substituted; R3selected from the group comprising-C(O)-, -CH2-, -CHR99-C(O)- and-C(O)-NR35-CH2-C(O)-, where R35means CHR55bridge group - C(O)-CR55-; R4selected from the group comprising-CH2- , and-NR50-, where R50selected from the group comprising H, alkyl, arylalkyl, and heterocycle; R5mean = CR201R202where R201and R202independently selected from the group comprising H, alkyl, aryl and arylalkyl, and where R201and R202independently may be substituted; R6means the Deputy selected from the group comprising-C(O), -CH2- and-CHR99-C(O)-; R7selected from the group comprising-CH2- , and-NR51where R51means H, alkyl, arylalkyl, heteroalkyl and heteroaromatic, and any one of these radicals substituted by the Deputy selected from Aulnay and guanidine groups, which may be substituted, mono-, di-, tri - or tetraalkylammonium pharmaceutically acceptable salts, its sarreid or isothiourea; R8means-CR210R211-, where R210and R211independently selected from the group comprising H, alkyl, alkylaryl and heterocycle, and each of these radicals may be substituted by the Deputy selected from the group including Q or -(CH2)n-Q, where n = 1-5, and Q is selected from the group comprising amine, amedieval, imidazole and the guanidine group which may be substituted, mono-, di-, tri - or tetraalkylammonium pharmaceutically acceptable salts, its sarreid or isothiourea; R9selected from the group consisting of-C(O)-, -CH2- and-CHR99-C(O); and where, when m = 1, B is selected from a group comprising 1-20 amino acids, -other52, -NR60R61-, OR70and-CHR60R61where R52selected from the group comprising H, alkyl, arylalkyl, heteroallyl and heteroaryl; where R60and R61independently selected from the group comprising H, alkyl, arylalkyl, aryl, heteroaromatic, and where, when m = 0, B is selected from a group comprising 1-20 amino acids, -OR70-THE OTHER52and NR60R61that is joined to R6amide bond or ester bond; where SUB> mean NR50; when R4means-CH2-, R3means-C(O)- or - CHR99-C(O)-, R4mean NR50; when R4means-CH2-, R3means-C(O)- or-CHR99-C(O)-; and when R6means-CH2-, R7means other51-; when R7means-CH2-, R6means-C(O)- or-CHR99-C(O)-; and when R4means-NR50and R1means

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R50and R'1together form a bridging group of the formula-C(O)-CHR55- where CHR55is R50and a carbonyl group is R'1and R1and R55independently mean H, C1-C6is alkyl or arylalkyl; and when R3means-C(O)-NR35-CH2-C(O)-, then R4means-NR50-, R1means

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R35and R'1together form a bridging group of the formula-C(O)CHR55- where(O) is R'1and CHR55is R35; R1and R55independently denote H or C1-C6-alkyl (see, for example, Fig. 2).

The connection according to the invention may contain a cyclic N-terminal group formed R1, R2, R3and, if this is desirable, R4. This connection is indicated, for example, structure the1together form a bridging group of the formula-C(O)-CHR55where R55means H; R1means H or methyl; R99and R100independently selected from the group comprising H, arylalkyl, alkyl and heteroalkyl or 1-3 carbon atoms, and where R99and R100next can be connected with a fragment selected from the group comprising phenyl, thienyl, thiazolyl, pyridyl, naphthyl, Tinetti, indolyl or saturated alkyl, alkoxy, monoalkylamines, dialkylamines, tetraalkylammonium, arylalkylamine, aminoalkylated, carboxy, halogen, hydroxy, amino, amido, amidino, guanidino, triazolyl and sulfonyl, and R3selected from the group consisting of-C(O)- and-C(O)-NR35-CH2-C(O)-.

In addition, in connection A1-A2-(A3)m-B R'1and R1can contain up to six substituents, including, for example, alkyl, and may be related group, such as-OCH2-, -SCH2-, =N-CH2-, =NC(O)-, -CO - or-NY-CO-NZ, where Y and Z can denote H, alkyl, arylalkyl or heteroaryl. Moreover, R99and R100independently can be substituted by the Deputy, such as phenyl, thienyl, thiazolyl, pyridyl, naphthyl, Tinetti or indolyl, or saturated group, which can contain up to five groups selected from alkyl, alkoxy, mono-, di-, the Mido, amidino, guanidino, triazolyl or sulfonyl.

The preferred connection with the substituents in the R2-the location is the compound where R100means H and R99means or

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where W is substituted in the connection can be, for example, halogen, hydroxyl, amino or amedieval group, and J may mean, for example, O, S or NR, where R is H or alkyl, aryl, or arylalkyl.

The connection according to the invention, which contains a Deputy, replaced the A2 and shows inhibitory activity against factor Xa, can have, for example, substitution at R50, R201or R202one or more heteroatoms, such as N, O or S. R202can also be substituted by the Deputy chosen from:

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when X is C, N or S, R is missing or could mean H or alkyl which may be substituted by a heteroatom, and n = 1-5.

The connection according to the invention, which contains zamestitel at A3 and shows inhibitory activity against factor Xa may include, for example, the substitution of R51one or more substituents such as H, alkyl, arylalkyl or heterocycle may also be substituted by a heteroatom such as N, O or S.

Rdenotes amino, amidino, urea, imidazole, guanidine, mono-, di-, tri - or tetraalkylammonium pharmaceutically acceptable salts, its sarreid or isothiourea. In addition, R210or R211can mean, for example, alkyl, aryl or alkylaryl. These groups can also be replaced by a substitute, such as hydroxyl or C1-C4-alkoxy.

The connection according to the invention may have an alternate arrangement of substituents comprising a fragment of the Century. This alternative arrangement of the substituents may include, for example, the substitution of R52nitrogen, oxygen or sulfur or substitution of R60, R61or R70one or more heteroatoms or alkyl groups.

The General patterns described here represent different compounds according to the invention, which retain inhibitory activity against factor Xa, such as the inherent Tripeptide YIR. The structures described herein include compounds containing non-natural amino acids, amino acid mimetics, and other organic structure and substituents exhibiting similar function.

Such functional equivalents provide appropriate spatial grouping the desired sarado the examples of compounds according to the invention include, for example,

Ac-Tyr-Ile-Arg-Leu-Ala-NH2; Ac-Tyr-Ile-Arg-Leu-Pro-NH2; Ac-(iBu)Tyr-Ile-Arg-Leu-Ala-NH2; Ac-Tyr-Il-Arg-Leu-N(CH3)O(CH3); Ac-Tyr-{Y(CH2NH)}-Ile-Arg-Leu-Pro-NH2; Ac-Tyr-Ile-Arg-NH-CH2(4-Pyridil); Ac-Tyr-Ile-{Y(CH2NH)} -Arg-Leu-Pro-NH2; Ac-Tyr-Chg-Arg(NO2)-{Y(CH2NH)}-Leu-NH2; Ac-Tyr-Ile-Arg-{ Y(COCH2)} -Gly-Pro-NH2; Ac-Tyr-Ile-Dab(Ny-C3H7N)-Leu-Ala-NH2; Ac-Tyr-Ile-PaIMe(3)-NH2; Tyr-Ile-Arg-NH2; D-Tyr-Ile-Arg-Leu-Pro-NH2Ac-(Bzl)Gly-(Chx)Gly-(3-grandprari)Gly-NH2; Cyclo(Gly-Tyr-Ile-Arg-Gly); Tfa-(iBu)Tyr-Chg-Arg-Leu-Pro-NH2; Ac-pAph-Chg-Arg-Leu-Pro-NH2; and Ac-Nal(2)-Chg-Arg-Leu-Pro-NH2. Other YIR peptides according to the invention is shown, for example, in tables 3 and 5.

This invention also relates to a connection structure A1-A2-(A3)m-B, where R1means

< / BR>
R'1selected from the group comprising H, -CO-Ra, -SO2-Ragroup protecting the amino group, 1-6 amino acids that can be substituted and where the N-terminal group of the specified 1-6 amino acids substituted Deputy selected from the group comprising H, -CO-Ra, -SO2-Raand a group protecting the amino group; and where Raselected from the group including alkyl, aryl and heteroalkyl; R1selected from the group comprising H, acyl and alkyl; X is N; R2means-CHR993means-C(O)-; R4means-NH-; R5means-CHR201-, where R201is alkyl; R6means-C(O)-; R7means-NH-; R8means-CHR210-; where R210is heteroalkyl having at least one formal positive charge, where the heteroatoms are 1 to 6 nitrogen atoms; R9means-C(O)-; and B is selected from the group comprising-ORband-N-RcRdwhere Rbselected from the group consisting of H, alkyl and groups that protect carboxypropyl, Rcselected from the group comprising H and alkyl, and Rdselected from the group comprising alkyl, heteroalkyl and 1-20 amino acids, which can be substituted by the Deputy, and where the C-terminal group can be modified by the group that protects carboxypropyl, primary amide or part of a cyclic peptide as a secondary or tertiary amide group, formed with the amino group of the radical R1. Such a connection may contain one or more groups, protecting the amino group.

-C3H7N), Dap(N-C3H7N) and Orn(N-C3H7N); and B is selected from the group including H, HE, NH2one to five amino acids or their functional equivalents and the group that protects carboxypropyl. Examples of such compounds include Ac-pAph-Chg-PaIMe(3)-NH-CH2-CHx; Ac-pAph-Chg-PalMe(3)-NH-Chx; Bzf-pAph-Chg-PaIMe(3)-NH2; cyclopentyl-CO-pAph-Chg-PalMe(3)- NH2; 3Iqc-pAph-Chg-PaIMe(3)-NH2; 2-furoyl-pAph-Chg-PalMe(3)-NH2; 5-IU-thienyl-CO-pAph-Chg-PalMe(3)-NH2; Ac-pAph-Chg-PalMe (3)-ol (see also table 5).

The invention further relates to the compound having the structure A1-A2-B, namely A1-A2-(A3)m-B, where m = 0.

In this connection B can mean heteroallyl, such as (4-(N-methylpyridine))methyl; 2-(3-(N-methylpyridine))methyl; 2-(3-(N-methylpyridine))methyl; 2-(3-(N-methylpyridine)ETH-1-yl; 1-(4-(N-methylpyridine))-et-1-yl; (n-amidin)benzyl; 2-(4-(N-methylpyridine))prop-1-yl; and 2-(4-(N-methylpyridine))et-1-yl. Ac-pAph-Chg-AMP(4) and Ac-pAph-Chg-AEMP(4) are examples of such compounds.

The choice of inclusion of L - or D-amino acids in the connection according to this invention may depend, in part, on the desired characteristics itro or in vivo. The inclusion of one or more D-amino acids can also increase or decrease the pharmacological activity of the compounds. In some cases it may be desirable to give the connection to remain active for only a short period of time.

In such cases, the inclusion of one or more L-amino acids in the connection can allow endogenous peptidases in the human body to metabolize the compound in vivo, thereby limiting the effect of the active compounds on the body.

The specialist can determine the desired characteristics of the compounds according to the invention, whereas, for example, the age and General health of the patient.

The connection according to the invention can be synthesized using, for example, automatic synthesizer (see example 1).

Selective modification of reactive groups, such as group present in the side chain of an amino acid, or N-terminal or C-terminal reactive group of the peptide, can make the connection desired characteristics, such as increased solubility or improved inhibitory activity.

If you are using solid-phase synthesis methods, you can manipulirovat resin, obtaining, for example, derived from the N-terminal group, such as acylated compound with N-terminal group.

Such modifications can also be feasible with a carboxyl group of compounds, including C-terminal carboxyl group, which may be, for example, liderovna.

The specialist may also synthesize YIR peptide using the method of synthesis in the environment of the solvent. The synthesized compound can be purified using well known techniques such as reversible liquid chromatography high resolution (IHVR, see example 1) or other methods of selection, based on, for example, on the use of size, charge or hydrophobicity of the compounds. To characterize the structure of the compounds according to the invention can also make use of the well known methods, such as sequencing of amino acids or mass spectrometry (MS).

YIR peptides according to the invention can be linear or cyclic (see, for example. table 3 below). The cyclization can be performed by formation of a bridge between two non-adjacent residues, fragments or substituents, which may be part of or outside YIR fragment.

The cyclization can also be carried out, for example, is the " outside YIR sequence.

For example, peptides or peptidomimetics can be cyklinowanie through education connections S-S, -CH2-S-, -CH2-O-CH-, lactam or ester linkages or known previously described methods (see Hruby, Life Sci. 31: 189-199 (1982); Toniolo, Int.J.Pept.Prot.Res. 35:287-300 (1990); Kates et al., Tetr.Lett. 34:1549-1552 (1993)).

Used the expression "outside YIR fragment" means not including tyrosine, isoleucine or arginine residue YIR sequence or its equivalent, in the YIR peptide according to the invention.

In contrast, the expression "composition YIR fragment" means comprising at least one residue of tyrosine, isoleucine and arginine YIR sequence or its equivalent. The term "bridge" in relation to cyclic connection means a connection formed between two non-adjacent amino acids located in the YIR peptide according to the invention.

Cyclization can be achieved through education, for example, disulfide bonds or lactam relationship between X1 and X2. Residues capable of forming disulfide bonds include, for example, Cys, Pen, Mpr and Mpp and its equivalents containing the 2-amino group.

Residues capable of forming a lactam bridge, include, for example, Asp, Glu, Lys, Orn, alpha, beta diaminopimelate, the aminobenzoic acid and mercaptobenzoic acid. Compounds described herein, can be cyklinowanie, for example, via a lactam bond, which can use the group in the side chain of one of the adjacent residue with the formation of covalent joining the N-terminal amino group of X1 or y Alternative bridge structure can also be used for the cyclization of the compounds according to the invention, including, for example, peptides and peptidomimetics, which can be cyklinowanie through communication S-S, -CH2-S-, -CH2-O-CH2-, lactam, ester or other linkages (see, e.g., Hruby, Supra, 1982; Toniolo, Supra, 1990; Kates et al. Supra, 1993).

The connection according to this invention can be obtained as a homogeneous or a mixture of compounds containing various combinations of substituents.

Great choice of substituents allows you to adjust the physicochemical properties of peptide analogues of the connection.

The selection of the Deputy also affects the binding tool connection (see examples).

Various compounds containing the various locations of the substituents, exhibit various levels of inhibitory activity against factor Xa. These compounds were synthesized by stosowanie methods, described in examples XXXVII and XXXVIII. Using such methods, a specialist is able to synthesize a compound described herein, including its modification, and to determine the inhibitory activity against factor Xa.

The invention provides for compounds that specifically inhibit the activity of factor Xa.

Such compounds have a Ki of 100 μm, preferably 2 nm relative to the activity of factor Xa and practically does not inhibit the activity of other proteases involved in coagulation and fibrinolysis (see table 2). Other proteases include, for example, thrombin and plasmin. The specificity of the compounds according to the invention shown in example XXXVII below (see also table 2).

The connection according to the invention can be used mainly as an anticoagulant, which may come in contact with the blood sample to prevent coagulation. For example, the effective amount of the compounds according to the invention may come in contact with viewsate a sample of blood to prevent coagulation. Used the term "effective amount" used in respect of compounds according to the invention, means an amount of compound that inhibits the activity of factor Xa. The specialist knows tomary XXXVII and XXXVIII), well-known methods.

Referring described the usefulness of the compounds according to the invention, the specialist understands that the agent, such as heparin, may be replaced by a connection according to the invention. This use of a compound according to the invention may, for example, to save money compared with other anticoagulants.

In addition, the connection according to the invention can be used for the treatment of various clinical disorders, including, for example, treatment of cardiovascular disease and related complications, for example, infection or surgery.

Examples of complications after surgery include chronic and proximal thrombosis, which can develop after surgery. Thus the connection according to the invention is suitable as a drug to reduce or prevent unwanted blood coagulation in a patient.

Because YIR peptide according to the invention may inhibit the activity of factor Xa, such a connection may be suitable for reducing or inhibiting blood clotting in an individual. Used herein, the term "individual" means a vertebrate, including a mammal, such as man, which featurette introduction to the individual a therapeutically effective amount of YIR peptide according to the invention. Used the term "therapeutically effective amount" means the dose YIR peptide, which you need to enter the individual for inhibiting the activity of factor Xa in the individual. More specifically, a therapeutically effective amount of the compounds according to the invention inhibits the catalytic activity of factor Xa in complex prothrombinase or in the form of soluble derivative units, or indirectly by inhibiting the introduction of factor Xa in complex prothrombinase. In particular, such compounds can inhibit the activity of factor Xa with Ki< 100 microns and, preferably, when K<2 nm. therapeutically effective amount can be determined using the methods described, for example, in examples XXXVII and XXXVIII, and others, known from the prior art.

In the practice of therapeutic treatment according to the present invention, the specific dose for obtaining a therapeutically effective amount of typing individual pharmaceutical composition will depend on many factors, including, for example, the nature or degree of the disease, the scheme of administration and the age and physique of the individual. The appropriate dose can be determined by clinical methods, well known in medicine. So obrazania factor Xa connection, having the sequence X1-YIR-X2 or A1-A2-(A3)m-B, where m = 0 or 1, or its functional equivalents.

The invention further provides a method of reducing or inhibiting the formation of a clot of blood in the individual by introducing a therapeutically effective amount of the compounds according to the invention.

The connection according to the invention is normally administered to the individual in the form of a composition containing the compound and a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable carrier" means a medium or structure that are non-toxic for the individual or have acceptable toxicity, corresponding to the established requirements.

The term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as saline solution with phosphate buffer, water, emulsion type oil/water or water/oil or various types of wetting agents. Suitable pharmaceutical carriers and their composition described Martin (Remington''s Pharmaceutical Sciences, 15th ed. (Mack Publishing Co., Easton 1975).

Such compositions generally contain a therapeutically effective amount of the compounds according to the invention together with a suitable amount of carrier CT can be useful as a drug for inhibiting the activity of factor Xa and clotting of blood in the individual.

Pharmaceutically acceptable carriers may include, for example, other environment, connection or modifiers inhibitor of factor Xa, which improve its pharmacological functions. Pharmaceutically acceptable medium can include, for example, an acidic salt, such as salt derived from an inorganic acid, e.g. hydrochloric, Hydrobromic, phosphoric acid, sulfuric acid or perchloro acid, or with organic acids, e.g. acetic, oxalic, maleic, tartaric, citric, succinic, or malonic acid. Other pharmaceutically acceptable salts include, for example, inorganic nitrate, sulfate, acetate, maleate, formate, lactate, tartrate, succinate, citrate, p-toluensulfonate, etc., including, without limiting the invention, the cations of alkali and alkaline earth metals such as sodium, lithium, potassium, calcium or magnesium, as well as nontoxic ammonium, Quaternary ammonium and amines cations, such as ammonium, methylammonium, dimethylammonium, ammonium, Tetramethylammonium, ethylammonium, triethylamine, tetraethylammonium.

Examples of modifications that enhance the pharmacological function compounds include, for example, etherification, the, the de alkyl is linear or branched. Other acceptable esters include, for example, C5-C7-cycloalkyl esters and arylalkylamine esters, for example, benzyl. Such esters can be obtained from the compounds described herein using conventional methods well known in the chemistry of peptides.

Pharmaceutical acceptable modifications can include, for example, the formation of peptide amides. Such amide modifications that can be performed with the compounds according to the invention, include, for example, obtained using ammonium, primary C1-C6-dialkylamino in which alkali are linear or branched, or arylamino having different substituents. In the case of secondary amines amine may be in the form of a 5 - or 6-membered heterocycle containing, for example, nitrogen atom. Methods of obtaining such amides are well known.

According to another aspect of the invention YIR peptide can be used in studies to identify the presence of factor Xa or for selection of factor Xa in its purest form.

Preferably, the connection according to the invention is labeled with, for example, radioisotope, and labeled find the connection with ietwat as samples for detection of the location or quantity of the activity of factor Xa in vivo, in vitro or ex vivo.

It should be borne in mind that modifications that do not affect various aspects of the present invention, also included in the scope of this invention. Accordingly, the following examples only illustrate the invention without limiting it.

Example I.

Methods of peptide synthesis

The source materials used in the synthesis, obtained from chemical companies such as Aldrich, Sigma, Fluka, Nova Biochem and Advance Chemtech.

During the synthesis of these compounds functional groups of amino acid derivatives were protected by blocking groups to prevent side reactions during combination. Examples of suitable blocking groups and their use are described in The Peptides, Supra, 1981, and Vol. 9, Udenfriend and Meienhofer, ed. 1987.

To obtain the compounds according to the invention using the General method for the synthesis of peptides in the solid phase. Such methods are described, for example. Steward and Young (Solid Phase Peptide Synthesis (Freeman and Co, San Francisko, 1969).

Unless otherwise specified, the peptides are synthesized on polystyrene crosslinked with 1% divinylbenzene. To bind to the solid substrate used is sensitive to acid linker (Rink linker) (Rink, Tetr Lett. 28:3787 (1987); Sieber, Tetr.Lett. 28: 2107 (1987)).

Linking is carried out, the COI is Westlaw or N,N-dimethylformamide (DMF), or 1:1 mixture of DMF:dichloromethane at room temperature (RT) for 40 minutes the completion of the binding reaction is determined by the test with ninhydrin.

Unlocking Fmoc carried out using 50% piperidine in DMF for 10 minutes, the Amount of released Fmoc determined by absorbance at 300 nm of the solution after unlocking, the volume of drilling fluids and weight of the resin used in the synthesis. The second (double) linking exercise, until the binding in the first case.

Using the following cycle of binding and methods (see tab. A).

After binding of the peptide from the resin is carried out final release Fmoc, followed by a regular wash cycles and measuring the amount of Fmoc released during the release. In some cases N unprotected peptide will acetimidoyl with shaking peptide with 20-fold excess of a mixture of acetic anhydride/pyridine (1:1) for 15 minutes

The peptide is washed successively DHM, DMF and DHM, then dried under vacuum. Peptide suspended in reagent K (King et al., Int. J. Pept.Prot. Res. 36: 255-266 (1990), publication entered as links) (5 ml/g peptide) for 180 min at room temperature, then splitting the mixture ochiltree and granules CON. The dried peptide was subjected to purification using GHUR using a gradient of 0.1% TFA in water and acetonitrile (atsn).

After collecting the peak containing the target product, the solution is modify and subjected to peptide analysis methods, allowing its identification, these methods include mass spectrometry when elektrorazpredelenie and analysis of amino acids to determine the composition of the synthesized compounds.

For purification of the peptide sample technical lyophilized peptide was dissolved in a mixture of 0.1% aqueous TFA containing 10-50% atsn. The peptide solution is typically filtered through a syringe connected with a nylon 0.45 μm "ACRODISC" 13 (GELMAN Sciences; Ann Arbor MI) filter.

An appropriate volume of the filtered solution of peptide injected in prepreparation C18 column (Vygac Protein and Peptide C18, 218TP1010; The Separation Group, Hesperia, CA). The speed gradient or isocratic mixture of 0.1% TFA buffer and atsn (IHVR) as eluent supported using a Beckman SYSTEM GOLD GHUR. Elution of the peptide regulate using UV at 230 nm (Beckman System Gold Programmable Solvent Module 126 and Programmable Detector Module 166, managed software "SYSTEM GOLD"). After identifying the peak corresponding to the synthesized compound, and is the Tria) conduct, using the device SCIEX API III+. In addition carry out NMR, using a General Electric appliance (300 MHz). In the case of NMR, the sample is usually measured in hexacyanometallate or deuterium chloroform (CDCl3; Aldrich).

Aldehydes amino acids get, using well known methods. Amino acids and aldehydes peptides have been described, for example, Fehrentz and Castro, Sysnthesis 676 (1983); Bajusz et al., J. Med.Chem. 33:1729, (1990); Kawamura et al., Chem.Pharm.Bull. 17:1902 (1969), and Someno et al., Chem. Pharm Bull., 34:1748 (1986). Receiving the recovered peptide bonds carry out at the level of the dipeptide in solution (e.g., Tyr-{(CH2NH)}-Ile), and then appropriately substituted dipeptide associated with the remainder of the peptide on the resin using the method of peptide synthesis in solid phase. Alternatively the aldehyde-protected amino acids linked by peptide on the resin, using the methods described But et al. (Pent Res. 6:10-12 (1993)).

Example II. Synthesis of Ac-Tyr-Ile-Arg-Leu-Ala-NH2< / BR>
For the synthesis of Ac-Tyr-Ile-Arg-Leu-Ala-NH21 g of the resin Rink'a (0.6 mmol NH2/g resin) used in the way described above. The resulting peptides analyzed by the method of mass spectrometry. (M+N)+): found 659,4 calculated (calc.) 659,9.

Example III. Synthesis of Ac-Tyr-Ile-Arg-Leu-Pro-NH2< / BR>
For the synthesis of Ac-Tyr-Ile-Arg-Leu-Pro-NH21 is implemented: (M + N)+found 685,4, calc. 685,9.

Example IV. Synthesis of Ac-(iBu)Tyr-Ile-Arg-Leu-Pro-NH2< / BR>
Using 1 g of resin Rink'a (0.6 mmol NH2/g resin).

Apply the General method of synthesis in the solid phase. After unlocking Tyr and appropriate washing of the peptide add 50 equiv of Isobutyraldehyde in DMF containing 2% glacial acetic acid. The resulting mixture was shaken for 4 hours at room temperature. After washing, the peptide DMF containing 2% acetic acid (2 x 8 ml), was added 1 g of NaBH3CN in 10 ml of DMF containing 2% acetic acid. The peptide resin was shaken for 30 min, then filtered off and add fresh mixture NaBH3CN in a mixture of DMF/acetic acid, the reaction continued for another 30 minutes

The peptide resin is then washed with a mixture of DMF/2% acetic acid (2 x 8 ml) and DMF (2 x 8 ml). Received monoalkylphenol peptide resin will acetimidoyl a mixture of acetic anhydride with triethylamine in DMF (30 EQ, 6 hours).

After appropriate washing of the peptide, the peptide is subjected to cleavage and release as described in example I.

Purified GHUR peptide analyzed by the method of mass spectrometry. (M + N)+found 758,4, calc. 758,5.

Example V Synthesis of Tfa-(iBu)Tyr-Ile-Arg-Leu-Pro-NH2

Cleavage of the peptide from the resin and the selection of the peptide is carried out as described in example IV. The purified peptide identified by the method of MC. (M + N)+found 812,4, calc. 812,5.

Example VI. Synthesis of Ac-Tyr-Ile-Arg-N(CH3)O(CH3).

Synthesis of Boc-Arg(Ng-Tos)-N(CH3)O(CH3) is carried out in accordance with the described in the literature method (Fehrentz and Castro, Supra, 1983). Boc-Arg(N-Tos)-N(CH3)O(CH3) (200 mg) is mixed with 5 ml triperoxonane acid (TFA) at room temperature and stirred for 20 minutes Disappearance of the parent compound is checked by thin layer chromatography (TLC) using CHC13: MeOH : CH3COOH (90:9:1) and detect visually using ninhydrin spray and UV light. Evaporation of the remaining TFA under vacuum and drying in a vacuum over pellets of KOH leads to a solid material having an appropriate weight. (M + H)+found 371,2, calc. 371,4.

In the first flask in 1 ml of DMF was dissolved 150 mg of the material obtained above, then add 57 μl of triethylamine and cooled the mixture to 0oC. In a second flask in anhydrous tetrahydrofuran (THF) dissolved 171 mg of Z-Tyr-Ile-OH (Biochem Bioscience Inc.; Philadelphia PA) and cooled to -10oC, then add 44 μl NNM ál isobutylphthalate W to mixed anhydride of Z-Tyr-Ile-OH dipeptide and the mixture was stirred at -10oC for 30 min and then overnight at room temperature.

After treatment of the reaction mixture as described in example I, the peptide is dried under vacuum and a small part of the cleanse GHWR and analyze method MS; peptide has the expected molecular weight (781). The resulting peptide Z-Tyr-Ile-Arg(Tos)-N(CH3))OCH3mixed with 500 μl of anisole and subjected to unlock with HF usual method.

After processing allocate 169 mg Tyr-Ile-Arg-N(CH3)O(CH3) and identify the method MS (found 493,6, calc. 494).

The residual peptide is then dissolved in 1 ml of 1N HCl and subjected to lyophilization.

Tyr-Ile-Arg-N(CH3)OCH32HCl (76 mg) was dissolved in acetonitrile, cooled to 0oC and add 13 ál of pyridine, and then 15 μl of acetic anhydride. The mixture was stirred at 0oC for 3 hours and the end of the reaction set by ninhydrin test. After stirring at room temperature for 8 hours, the reaction mixture was treated and the product Ac-Tyr-Ile-Arg-N(CH3)OCH3analyze method MS (found 535,6, calc. 535,3).

Example VII. Synthesis of Ac-Tyr-{ CH2NH)}-Ile-Arg-Leu-Pro-NH2< / BR>
a) Synthesis of Fmoc-Tyr(But)-H

4.6 g (10.0 mmol) of Fmoc-Tyr(But) HE, 2.1 g (10.1 mmol) dicyclohexylurea the 993)).

After processing produce Fmoc-Tyr-(But)-S-CH2C6H125after recovery of tiefer by mixing with triethylsilane in the presence of 10% Pd on coal and purification by chromatography in thin layer receives Fmoc-Tyr(But)-H with a yield of 81%. Data of NMR and the yield expected.

b) Synthesis of Fmoc-Tyr(But)-{ (CH2NH)}-Ile-(O-Allyl)

0.73 g (of 1.66 mmol) Fmoc-Tyr(But)-HE 0,209 g (of 3.32 mmol) NaBH3CN in 20 ml of 1% AcOH in DMF are added to a solution 0,516 g (1.82 mmol) Ile-(O-Allyl) in 2 ml of DMF. After 2 hours the reaction mixture is processed and the final product purified by chromatography in thin layer (ethyl acetate: hexane, 35: 65) to obtain the oil, which is characterized by the corresponding data of NMR and MS. (M + H): found 599, calc. 598,7.

C) Synthesis of Fmoc-Tyr(But)-{(CH2NH)}Ile-OH

To 0,467 g (0.78 mmol) Fmoc-Tyr(But)-{ (CH2NH}-Ile-O-Allyl in 10 ml of dichloromethane added 89 μl (1.56 mmol) of HOAc, 20 μl of triethylamine (IEA) and 0.02 g of complex PdCl2(Ph3)2.

Add immediately 231 μl (8,86 nmol) Bu3SnH and stirred the mixture for 1 hour at room temperature. After appropriate processing of the reaction mixture the product is purified by chromatography in thin layer (CHCl3: MeOH, 20 : 1) to obtain the peptide exit 69% (0,319 g). (M+H+): need synthesis in the solid phase, as described in example I. the Target peptide resin Ac-Tyr(But)-{(CH3NH)}-Ile-Arg(Pmc)-Leu-Pro-Rink will unlock and split, as described in example I and purified by the method GHUR on C18 column.

Example VIII. Synthesis of Ac-Tyr-Ile-Arg-NH-CH2(4-pyridyl)

Oximo resin (DeGrado and Kaiser, J.Org. Chem. 45: 1295 (1980) (0,862 g, 0.6 mmol/g) is associated during the night with Boc-Arg(Tos)-OH in the presence DICK/HOBt. The resin was washed with DMF, then DHM and acetimidoyl a mixture of acetic anhydride/DIEA (1: 1 EQ) in THM.

After washing the resin DHM, DMF and DHL it will unlock 25% TFA in DHM within 30 minutes of the Obtained resin was washed with DHM, isopropanol and DHM. WITH TPA. Arg(Tos)-OxmR link Boc-Ile-OH symmetric anhydrous form (3 equiv) in the presence of 1.5 equiv of DIEA in DHM. The rinse cycle, acetylation and unlock described above is repeated. After unlocking add binding as Ile, Boc-Tyr(2-BrZ)OH, and then the peptide resin, Boc-Tyr(2-BrZ)-Ile-Arg(Tos)-OxmR will unlock and will acetimidoyl obtaining Av-Tyr(2-BrZ)-Ile-Arg(Tos)-OxmR. The peptide resin was dried under vacuum to obtain a weight gain 0,216,

To 1/3 of this resin was added 100 μl (800 μm) of 4-(dimethylamino)pyridine in the presence of 60 μl of glacial acetic acid and 120 μl of DIEA in 6 ml DHM.

The resin is shaken overnight at room temperature. After farwaniya solvent the resulting peptide will unlock using HF/anisole and treated as usual to retrieve the target peptide. Perform electrospray MS. (M + N)+found 582,3, calc. 582.

Example IX. Synthesis of Ac-Tyr-Ile-{ (CH2NH)}-Arg-Leu-Pro-NH2< / BR>
a) Synthesis of Boc-Ile-H

Aldehyde synthesized from 1 g of Boc-Ile-N(Me)OMe, as described Fehrentz and Castro (Supra, 1983). Aldehyde identified by thin-layer chromatography and NMR, as described in the link.

b) Synthesis of Arg(Tos)-Leu-Pro-MBHA

Synthesis Tripeptide resin is carried out in the solid phase, as described in example I.

C) Synthesis of Boc-Ile-{(CH2NH)}-Arg(Tos)-Leu-Pro-MBHA

Boc-Ile-H associated with Tripeptide resin Arg(Tos)-Leu-Pro-PSMA by reductive amination using NaBH3CN in DMF containing 1% acetic acid. Boc-group was split as usual and Ac-Tyr-OH are associated with the usage DICK/HoBt.

The final peptide resin (0.7 g) will unlock and peptide otscheplaut from the resin using a mixture of HF/thioanisole. 19 g of crude Ac-Tyr-Ile-{(CH2NH)}-Arg-Leu-Pro-NH2cleanse method GHUR on a C18 column with getting about 5 mg > 90% pure target peptide. (M + H+): found 688,4, calc. 687,9.

Example X. Synthesis of Ac-Tyr-Ile-Dab(N- C3H7N)-Leu-Ala-NH2< / BR>
0.2 g SCAL-TG (0.2 mmol NH2/g) (Patek and Lebl, Tetr.Lett. 32: 3891-3894 (1991)) associated with Fmoc-Ala-OH and Fmoc-Leu-OH, Fmoc-Dab(Group and release the side-chain TPA, washed the peptide resin Ac-Tyr-Ile-Dab-Leu-Ala-SCAL-TG, neutralized and treated with 0.3 M PyBroP/IUP in DMF for 2 hours. The resulting peptide otscheplaut from the resin using 1M mixture of triphenylphosphine/(CH3)3SiCl in DHM (3 x 1 hour), then 100% TFA (1 hour). After separation of the crude peptide by precipitation in diethyl ether peptide lyophilizer of 0.1% aqueous TFA. The peptide Ac-Tyr-Ile-Dab(N-C3H7N)-Leu-Ala-NH2clean GHWR and spend MC. (M + H+): found 676,4, calc. 676,4.

Example XI. Synthesis of Ac-Tyr-Ile-PalMe(3)-NH2< / BR>
With 1.0 g of Rink resin (0.48 mmol NH2/g is associated with Fmoc-Pal(3) -, Fmoc-Ile-OH and Fmoc-Tyr(But)-IT, using the methods described in example I. To 0.25 g of the obtained peptide resin Fmoc-Tyr(But)-Ile-Pal(3)-add Rink 500 chalk under the conditions (MI) in DHM and shaken for 6 hours. The resulting peptide resin Fmoc-Tyr (But)-Ile-PalMe(3)-Rink will unlock and will acetimidoyl and otscheplaut as described in example I. a Portion of the crude peptide is purified by a method GHWR and conduct MS.

Example XII. Synthesis of Ac-Cyclo(Glu-Tyr-Ile-Arg-Leu-Lys)-NH2< / BR>
1 g SCAL-TG (0.29 mmol NH2/g) (see example X) associated with Fmoc-Lys(BOC) -, Fmoc-Leu-OH, Fmoc-Arg(Pmc) -, Fmoc-Ile-OH, Fmoc-Tyr(But)-HE and Fmoc-Glu(OtBu)-OH, using the methods described in example I.

After removal of the Fmoc peptide resin will acetimidoyl and washing out the form and cyclist, using BOP/HOBt/DIEA (5: 5: 5 EQ) in DMF for 2 hours. The end link control breakdown with the use of ninhydrin, as described by Kaiser (Kaiser et al. , Anal. Biochem. 34:595 (1970)). After cyclization, the peptide otscheplaut from the resin, purified by the method GHWR and spend MC. (M+N)+found, 844,5, calc. 844,5.

Example XIII. Synthesis of Cyclo(Cly-Tyr-Ile-Arg-Gly)

1 g oximes resin (see example VIII) (0.6 mmol NH2/g) bind during the night with Boc-Gly-OH in the presence DICK/HOBt. After washing and unlock resin combine Boc-Arg(Tos)-OH, Boc-Ile-OH and Boc-Tyr(2BrZ)-OH, using the methods described in example VIII.

1/3 of the peptide resin, Boc-Tyr-(2BrZ)-lle-Arg(Tos)-Gly-Oxime will unlock and connect with Boc-Gly using DICK/HOBt. The peptide resin will unlock, neutralize and cyclist during the night in DMF containing 1% acetic acid. The resin is filtered and washed (DMF), combine the filtrate and removing the organic solvent by evaporation in a vacuum.

The peptide will unlock (HF/anisole), lyophilizer, cleanse method GHWR and implement MS. (M + N)+found 547,8, calc. 547,8.

Example XIV. Synthesis of N-substituted glycine compounds: synthesis of Ac-(Bzl)Gly-(Chx)Gly-(3-grandprari)Gly-NH2< / BR>
For the synthesis of N-substituted glycinol use a technique Zuckermann'a and others (J. Am. Chem. Soc. 114: anhydride in DHM/DMF. Each reaction mix was repeated twice. To the resin Br-CH2CO-SCAL-TG add BOC-NH-CH2CH2CH2NH2in DMSO resin and shaken for 2 hours.

After unlocking process is repeated, alternating between the binding of Br-CH2COOH resin and the reaction resin containing bromoxynil acid and the appropriate amine.

The resin (Bzl)Gly-(Chx)Gly-(Boc-NH-(CH2)3)Gly-SCAL-TG will acetimidoyl a mixture of acetic anhydride/DIEA/NMU (1:1:0,25) in DMF overnight. After the release of the group Vos resin Ac-(Bzl)Gly-(Chx) Gly-(3-aminopropyl)Cly-SCAL-TG treated with 1.8 M carboxylesterases (Bernatowicz et al., J. Org.Chem. 57:2497-2502 (1992)) in the presence of DIEA (1:1) in DMF for 3 h at room temperature.

The end of guanylurea determine the breakdown by Kaiser. Cleavage and processing of the obtained peptide were carried out as described in example X, and perform MC. (M + N)+found 502,3, calc. 502,3.

Example XV. Synthesis dicketopeperazinovykh compounds: synthesis of cyclo(Ser-Ida)-Ile-Arg-Leu-Ala-NH2< / BR>
Original protected tetrapeptide, Fmoc-Ile-Arg(Pmc)-Leu-Ala-Rink, get through the strategy with Fmoc-Rink resin (see example I). After unlocking Fmoc peptide resin Fmoc-Ida(OMe)-OH (3 EQ; DICK, HOBt) and Fmoc-Ser(tBu)-OH (7 EQ; symmetrical anhydride) associate of posledovat 50% piperidine/DMF for 1 hour. After washing in several stages of the final peptide otscheplaut this will release using a mixture of TFA/thioanisole/H2O (95:2,5:2,5).

The resulting peptide is treated as described above and analyzed by the method GHUR (> 95%) and the MS method. (M + N)+found 655,4, calc. 655,38.

Example XVI. Synthesis of Ph-C(NOCH2Ph)-CO-I-R-NH2< / BR>
0.2 g of resin Rink'and connect with Fmoc-Arg(Pmoc)-OH, Fmoc-Ile-OH with the subsequent removal of the Fmoc protection (see example I).

With peptide resin, Ile-Arg(Pmoc)-Rink, associate Ph-C(NOCH2Ph)-COOH, using DICK/HOBt as described above. The peptide resin, Ph-C(NOCH2Ph)-CO-Ile-Arg(Pmc)-Rink, treated as described in example I, and implement MS. (M + N)+found 524,3, calc. 524,6.

Example XVII. Synthesis of Ac-pAph-Ile-Arg-Leu-Pro-NH2< / BR>
The synthesis carried out with 100 mg of resin Rink'a (0.48 mmol/g) according to the method described in example I, using the following derivatives of amino acids: Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Arg(Pmc) -, Fmoc-Ile-OH and Fmoc-pAph-(Fmoc)-OH (racemic mixture).

Cleavage and secretion of the peptide is carried out, as described in example I. Both diastereomeric peptide are highlighted by chromatography with reversed phase - GHUR and are identified by the MS method. (M + N)+found 754,4, calc. 754,5.

Example XVIII. Synthesis of Ac-Tyr-Chg-Arg-ol

The PE the persons, described in example I. After unlocking the N-terminal Fmoc and acetylation protected peptide otscheplaut from resin restorative banding in the form of a C-terminal alcohol (Mergler et al. Peptides p.p. 177-178 (eds. Schneider and Eberle; Leiden 1993), incorporated by reference). The peptide resin was shaken with a solution of NaBH4(4 EQ) in 2 ml of THF:EtOH (6:1) within 24 hours. After the reaction, the removal of the resin washed DHM, then splitting the solution, and rinse water are combined and lyophilizers. Lyophilized peptide will unlock processing TFA/water/thioanisole (90: 5:5) for 2 h and was isolated by precipitation, purified by the method GHUR peptide analyze MS method. (M + N)+found 505,3, calc. 505,3.

Example XIX. Synthesis of Ac-Tyr-Chg-Arg-ol.acetate

Protected peptide alcohol is prepared as described in example XVIII.

10 mg of the crude product dissolved in a mixture DHM/atsn and treated with acetic anhydride (2 mmol) in the presence of tea (2.4 mmol) for 20 minutes the Solution is filtered, evaporated and the peptide will unlock, as described above. The peptide is purified by a method GHWR and analyze MS method. (M + N)+found 547,3, calc. 547,3.

Example XX. Synthesis of Ac-Phe(pNH2)-Chg-Orn(C(NH)CH3)-Leu-Pro-NH2< / BR>
1 g "TENTAGEL'S" NH2the resin (0.28 mmol NH2/islote: Fmoc-Pro-OH; Fmoc-Leu-OH; Fmoc-Orn-(Boc)-HE and Fmoc-Chg-OH. The peptide resin Fmoc-Chg-Orn (Boc)-Leu-Pro-SCAL-TG treated with 50% TFA in DHM (1 rinsing for 1 min, then 1 rinsing for 30 min), washed 3 times DHM, neutralized with 5% DIEA in DHM (2 x 30) and 2 times DHM. To the peptide resin add a solution of 1.5 g of the hydrochloride of ethylacetamide (Aldrich) in 4 ml of a mixture of pyridine: DIA and 3 ml of DMF and continue to bind overnight at room temperature.

The peptide resin Fmoc-Chg-Orn(C(NH)-CH3)-Leu-Pro-SCAL-TG, will unlock 2% piperidine in DMF, 4 times DHM and connect with the help of DICK/HoBt in DMF. The release of the Fmoc and acetylation with a mixture of acetic anhydride: pyridine (1:1) for 20 min leads to the production of peptide resin Ac-Phe-(pNH-BOC)-Chg-Orn(C(NH)CH3)- Leu-Pro-SCAL-TG. Restoring SCAL linker and cleavage of the peptide, followed by purification of the crude product method GHUR, leads to the expected product. (M + N)+found 740,2, calc. 740,48.

Example XXI. Synthesis of Ac-Phe(pNH2)-Chg-Dap(N-C6H11N)-Leu-Pro-NH2< / BR>
0.5 g SCAL-TG (0.32 mmol NH2/g) associated with Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Dap(Boc)-HE and Fmoc-Chg-OH. The Boc group of the side chain is removed using 50% TFA over 20 min, and the peptide resin is neutralized by washing 10% DIEA/DHM. The free amino group in Bokonbaevo Fmoc group with a mixture of 50% piperidine/DMF for 60 min leads to the replacement of dimethylanilinium group piperidinol in the side chain of the Dap. The complete sequence binding with Fmoc-Phe(Boc)-HE and the release of the Fmoc group. The peptide will acetimidoyl and otscheplaut as described in example X.

The peptide is purified by a method GHUR, analyze MS method. (M + N)+found 752,4, calc. 752,4.

Example XXII. Synthesis of Ac-pAph-Chg-PalMe(3)-NH2< / BR>
Racemic H-Phe(pCN)-OH synthesized by the method using acetamidomalonate (Wagner et al. Patent GDR 155954). Racemic Ac-pAph-OH synthesize the transformation of ceanography by ammonolysis of the corresponding methylthiopyridine (reaction ceanography with sulphurous hydrogen) and subsequent methylation of the MeI.

1 g of the resin TENTAGEL" (replacement = 0.21 mmol NH2/g resin) and Knorr linker (Bernatowicz et al. , Tetr. Lett. 30: 4645 (1989)) are used for synthesis of the peptide.

Dipeptide, Fmoc-Chg-Pal-Knorr-TG, receive as described in example I.

3-Pyridylamine consistently was identified in 1 ml of MeI DHM during the night. After unlocking Fmoc Ac-pAph-OH bind method using DICK/HOBt, and the peptide is treated as described in example I. (M+N)+found 550,3, calc. 550,31.

Example XXIII. Synthesis of Ac-Tyr-Chg-pAph-Leu-Pro-NH2< / BR>
Assembly Pentapeptide Ac-Tyr(But)-Chg-Phe-(pCN)-Leu-Pro-Knorr-TG conduct 0.4 g TENTAGEL" (replacement = 0.2 mmol NH2/g resin), as described in the example IS. Associated with the resin thioamide will was identified in using 0.5 g MeI in 8 ml of acetone for 30 min at 50oC, then washed with acetone and methanol.

Carry out the reaction of petitioned with ammonium acetate in methanol for 3 h at 55oC to obtain the target product, which otscheplaut from the resin and purified as described above. (M + N)+found 761,4, calc. 760,43.

Example XXIV. Synthesis of Ac-Phe(pCH2NH2)-Chg-Arg-Leu-Pro-NH2< / BR>
Ac-DL-Phe(pCN)-Chg-Arg-Leu-Pro-NH2(crude peptide) synthesize 1 g resin Rink'and (0.6 mmol NH2/g resin) as described in example I. 125 mg of crude peptide is dissolved in 50 ml of MeOH and added 0.5 ml of a suspension of Raney Ni (Aldrich). The mixture of peptide and catalyst hydronaut at a pressure of 35 f/inch2(241,3 kPa) for 4 h at room temperature. The catalyst is filtered off and the solution evaporated to dryness. The remainder lyophilizer of 0.1% aqueous TFA containing 30% ARE.

Dried technical product was then purified by the method GHWR and analyze MS method. (M + N)+found 741,4, calc. 741,7.

Example XXV. Synthesis of Ac-Phe(pC(NOH)NH2)-Chg-Arg - Leu-Pro-NH2< / BR>
21,1 mg Technical peptide, obtained as described in example XXIV, mixed with 60,3 mg NH2OHHCI (Aldrich) in 1.5 ml MeOH, 0.7 ml of pyridine and the exposure to other substances. The peptide is purified by a method GHWR and analyze MS method. (M + N)+found 770,4, calc. 770,3.

Example XXVI. The synthesis of compounds A1-A2-B

A1-A2-B, i.e., compounds A1-A2-(A3)m-B, where m = 0, receive, as shown in Fig. 3. Briefly, the binding of racemic N-acetyl-4-cianfanelli with methyl ester of L-cyclohexylglycine (H-Chg-OMe) leads to the formation of a mixture of two diastereoisomeric of dipeptides, which are separated by chromatography. Racemic n-acetyl-4-cianfanelli again partially soluble in obtaining salt with methyl ester of L-cyclohexylglycine. Less soluble D,L-salt is easily crystallized, and the subsequent combination yields a crude Ac-F(pCN)-Chg-OMe, which is then purified by chromatography over silica gel. These ester of the dipeptide hydrolized to the corresponding acid using lithium hydroxide in methanol/water at room temperature. Both dipeptide acid is transformed into the corresponding amides of the usual binding with suitable amines RNH2. Amines, which are not commodity products, receive standard methods.

The conversion of the cyano groups in the relevant amidine carried out by conventional methods or through thioamide and metaltometal, or by hydrogenation according to the, described below, illustrate the obtaining of the above mentioned compounds by these methods. Compounds according to the invention can be obtained by other methods; selected methods described in the examples, are used for convenience.

Example XXVII. Synthesis of Ac-pAph-Chg-NHCH2-(4-methylpyridine)

Synthesis of Ac-pAph-Chg-NHCH2-(4-methylpyridine) carry out the conversion of Ac-F(pCN)-Chg-NH-CH2-(4-pyridyl)and, using the methods described in example XXII. The final product was then purified by the method GHUR as described in example I. MS analysis: (M + N)+found 493,3, calc. 493,29.

Starting material was obtained as follows:

a) 2,32 g (10 mmol) of Ac-(D,L)-F(pCN) is dissolved in 75 ml of ethanol under heating. Add methyl ester of L-cyclohexylglycine (1.75 g, 10 mmol) and stirred the mixture for 2 hours at room temperature. The precipitated crystals are filtered and dried to obtain 1.55 g of D,L-salt. The filtrate is partially evaporated and diluted with ether.

Selected crystals are collected and dried remains of 2.1 g of L,L-salt-contaminated D,L-salt. Crude L,L-salt is combined with 20 ml DMF, 0.71 g HOBt and 1.18 g DCC. The mixture is stirred for 24 hours at room temperature. Urea is filtered off and the filtrate is evaporated.

The residue is dissolved in methylene chloride, is up, sediment chromatographic over 60 g of silica gel using 20%(vol.) a solution of acetone in methylene chloride for elution. Crystallization of the United pure fractions from a mixture of methylene chloride/ether/hexane yields a 1.6 g Ac-F(pCN)-Chg-OMe in the form of colourless crystals with a melting point (i.e. square), equal 178-180oC.

b) a Mixture of 1.93 g (5 mmol) Ac-F(pCN)-Chg-OMe (example XXVII.and above) in 100 ml of methanol, 10 ml of water and 0.75 g of the hydrate of lithium hydroxide is stirred under nitrogen atmosphere for 24 hours at room temperature.

Add 2 ml of acetic acid, evaporated the solvent, and the residue is divided between methylene chloride containing 20% isopropanol, and 1 N HCl. The organic layer is dried and evaporated, and the residue is crystallized from a mixture of methylene chloride/ether/hexane to obtain 1.6 g Ac-F(pCN)Chg-OH in the form of colorless crystals with so pl. 216-218oC.

C) a Mixture of 150 mg (0.4 mmol) Ac-F(pCN)-Chg-OH (see above), 65 mg (0.6 mmol) of 4-aminomethylpyridine, 124 mg (0.6 mmol) DCC, 60 mg (0.44 mmol) of HOBt and 5 ml of DMF is stirred for 20 hours at room temperature. Urea is removed by filtration, the filtrate is evaporated. The residue is suspended in methanol and collect the insoluble product, leaving 140 mg of colorless Ac-F(pCN)-Chg-NHCH2(4-pyridyl)A.

Homeopathic. A crystalline solid has so pl. > 250oC.

Example XXVIII. Ac-F(4-amidin)-Chg-NHCH2(4-methylpyridine)

This compound is produced by interaction 150 mg Ac-F(pCN)-Chg-NHCH2(4-pyridyl)and (see above) with sulphur hydrogen, then methyliodide and ammonium acetate.

The product distinguish GHUR in the form of a homogeneous substance. Data MS: (M + N)+found 493,3, calc. 493,29.

Starting material was obtained as follows:

a) a Mixture of 2.8 g Ac-F(pCN), methyl ester (L)-cyclohexylglycine, 940 mg of HOBt, of 1.57 g DCC and 30 ml of DMF is stirred for 2 days at room temperature. Filtering removes urea and the filtrate is evaporated. The residue is dissolved in methylene chloride and washed with a solution of 1 N HCl and 10% aqueous sodium carbonate solution. The organic phase is dried and evaporated. Crystallization of the residue from a mixture of methylene chloride/ether/hexane gain of 2.05 g of colorless Ac-F(pCN)-Chg-OMe with so pl. 181-183oC.

b) Hydrolysis of 1.93 g of Ac-F(pCN)-Chg-OMe (see above) using 0.75 g of the monohydrate of lithium hydroxide in 100 ml of methanol and 10 ml of water were carried out as described for L,L-isomer in example XXVII above, crystallization from a mixture of methylene chloride/ether leads to the production of 1.65 g Ac-F(pCN)-Chg-OH with so pl. 180-182oC.

C) a Mixture of 225 mg Ac-F(pCN)-Chg-OH is mperature. Filtered urea and the filtrate is evaporated, the residue is stirred with methanol, removing the solid residue by filtration, leaving 190 mg crystalline Ac-F(pCN)-Chg-NHCH2(4-pyridyl)and so pl. > 250oC.

Example XXIX. Ac-pAph-Chg-NHCH2CH2(3 methylpyridine)

A mixture of 125 mg of Ac-F(pCN)-Chg-NHCH2CH2(3-pyridyl), 2 ml DMSO, 10 ml of pyridine and 3 ml of triethylamine is saturated with sulfur hydrogen when cooled with a mixture of ice/water.

After stirring in a sealed tube overnight at room temperature the solvent is evaporated, the residue dissolved in a mixture of acetone/ether and dried to obtain 125 mg thioamide. This product is combined with 2 ml of DMSO, 5 ml of acetone and 0.75 ml under the conditions, and the mixture is stirred in a sealed tube overnight at room temperature. After dilution with toluene, the solvent is evaporated, and the residue is stirred with ether. The ether is decanted, replaced with fresh ether and continue stirring over night until harden rubber-like substance, then the remaining ether is filtered off, and the residue is dried.

The remaining residue is dissolved in 20 ml of methanol and treated with 0.3 ml of acetic acid and 0.4 g of ammonium acetate.

accelerate in a mixture of water/atsn/TPA and lyophilizers. The crude product is purified GHUR. Data MS: (M + N)+found 507,3, calc. 507,31.

Starting material was obtained as follows. A mixture of 150 mg (0.4 mmol) Ac-F(pCN)-Chg-OH, 120 mg (0.6 mmol) of 2-(3-pyridyl)ethylamine hydrochloride, 125 mg DCC, 60 mg of HOBt, 0.5 ml of diisopropylethylamine and 10 ml of DMF is stirred for 24 hours at room temperature. After evaporation of the solvent the residue is stirred with methanol, the insoluble product is collected by filtration and washed with methanol and ether, receiving colorless crystals. The filtrate is evaporated, and the residue dissolved in a mixture of methylene chloride/isopropanol.

This solution was washed with 10% aqueous solution of sodium carbonate, dried and evaporated. The remainder chromatographic over 14 g of silica gel using a mixture of methylene chloride: acetone: methanol (5: 4:1), to obtain 40 mg of Ac-F(pCN)-Chg-NHCH2CH2(3-pyridyl)a so pl. 265-268oC.

b) the Dihydrochloride of 2-(3-pyridyl)ethylamine was obtained as follows. A mixture of 1.3 g of 3-pyridylacetonitrile, approximately 3 g of Raney Nickel and 30 ml of methanol containing 10% vol. ammonia, hydronaut at a pressure of 35 f/inch2(241,3 kPa) for 20 h, using hydrogenator Parr. The catalyst is filtered off over telicom, and the filtrate is evaporated. The residue is dissolved in methylthio the simple hydrogen in dioxane. Crystallization from methanol/ether to obtain 1.4 g of colorless crystals with so pl. 145-148oC.

Example XXX. Ac-pAph-Chg-NHCH2CH2(4-methylpyridine)

This connection to be made by methods described above, the interaction of Ac-F(pCN)-Chg-NHCH2CH2-(4-pyridyl)with sulphur and hydrogen, followed by methylation of methyliodide and reaction with ammonium acetate.

The crude product is purified GHUR. Data MS: (M + N)+found 507,3, calc. 507,31.

The original substance is produced by binding of Ac-F(pCN)-Chq-OH dihydrochloride with 2-(4-pyridyl)ethylamine as described in example XXIX above.

The dihydrochloride of 2-(4-pyridyl)ethylamine receive as described in the case of the dihydrochloride of 2-(3-pyridyl)ethylamine (see above) by hydrogenation of pyridyl-4-acetonitrile over Raney Nickel in the presence of ammonia. The dihydrochloride is so pl. 220oC.

Example XXXI. Ac-pAph-Chg-NHCH2(4-amidinophenoxy)

This connection will be received, using methods similar to those described above for Ac-F(pCN)-Chg-NHCH2(4-tianfeng)and by processing of sulfur with hydrogen in DMSO, pyridine and triethylamine. The obtained bis-tioned will was identified in methyliodide in a mixture of DMSO/acetone, followed by reaction with ammonium acetate as described above. Raw produces. A mixture of 75 mg (0.2 mmol) Ac-F(pCN)-Chg-OH, 50 mg (0.3 mmol) of hydrochloride (4-tianfeng)methylamine, 62 mg DCC, 30 mg HOBt, 0.2 ml of DIEA and 2 ml of DMF is stirred for 24 hours at room temperature. After filtration the solvent is evaporated and dissolve the residue in methylene chloride containing 20% isopropanol. The solution was washed with 1N HCl and 10% aqueous sodium carbonate solution, then dried and the solvent evaporated.

The residue is stirred in a small amount of methanol/water, the separated solid is dried, receiving 80 mg of Ac-F(pCN)-Chg-NHCH2(4-tianfeng).

Hydrochloride (4-tianfeng)methylamine obtained as follows. A mixture of 2 g (10 mmol) of a-bromo-p-toulonnaise, 2 g (10,8 mmol) phthalimide potassium and 30 ml of DMF is heated under reflux for 1 min.

After cooling, the mixture is acidified with acetic acid and diluted with water to cause crystallization of the product. The crystals are filtered, washed with water and dried, to obtain 2.24 g of colorless N-(4-tianfeng)methylphthalimide with so pl. 182-184oC.

1.5 g of N-(4-tianfeng)methylphthalimide suspended in 50 ml of boiling methanol and treated with 1 ml of hydrazine hydrate is added. After 5 min is obtained a clear solution. Evaporate the methanol and treated the rest of 2N HCI. Suspension NAC dissolved in water. The resulting solution was again heated to boiling, cooled and filtered. The filtrate make alkaline by addition of sodium hydroxide and extracted with methylene chloride, containing isopropanol. The organic phase is dried, the solvent is evaporated and the residue is converted into the hydrochloride crystallized from a mixture of isopropanol/ether, receiving of 0.43 g of colorless crystals with so pl. > 260oC.

Hydrochloride (3-tianfeng) methylamine obtained by interaction of (-bromo-m-toulonnaise with phthalimide potassium obtaining N-(3-tianfeng)methylphthalimide with so pl. = 147-148oC. Reaction of this compound with hydrazinehydrate and conversion into the hydrochloride as described above, allows to obtain 3-cianfanelli with so pl. 223-226oC.

Example XXXII. Ac-pAph-Chg-NHCH2(3-amidinophenoxy)

This connection receive, using the methods described above. Ac-F(pCN)-Chg-NHCH2(3-tianfeng) is treated with sulfur hydrogen in DMSO, pyridine and triethylamine. The obtained bis-tioned will was identified in methyliodide in a mixture of DMSO/acetone, followed by reaction with ammonium acetate as described above. The crude product is purified GHUR. Data MS: (M + N)+found 520,3, calc. 520,3.

Starting material was obtained as follows. A mixture of 300 mg (0.8 mmol) Ac-F(pCN)-the sa at room temperature. After filtration the solvent is evaporated and dissolve the precipitate in a large volume of methylene chloride containing 20% isopropanol.

The solution was washed with 1 N NS and 10% aqueous sodium carbonate solution, dried and the solvent is evaporated. The residue is stirred with a mixture of isopropanol/ether, the separated solid is separated, dried, yielding 400 mg of Ac-F(pCN)-Chg-NHCH2(3-tianfeng).

Example XXXIII. Ac-pAph-Chg-NHCH(Me)(4-methylpyridine)

The mixture of diastereomers these compounds are produced by interaction of a mixture of two diastereomers Ac-F(pCN)-Chg - NCH(IU)(pyridyl)with sulphur and hydrogen, then MeI and ammonium acetate.

The diastereomers share GHUR. Data MS: (M + N)+found 507,3, calc. 507,31.

The source material was obtained as follows. A mixture of 150 mg (0.4 mmol) Ac-F(pCN)-Chg-OH, 120 mg (0.6 mmol) of racemic 1-(4-pyridyl)ethylamine hydrochloride, 125 mg DCC, 60 mg of HOBt, 0.5 ml of DIEA and 10 ml of DMF is stirred for 24 hours at room temperature. After filtration the solvent is evaporated and dissolved the residue in a large volume of methylene chloride containing 20% isopropanol.

The solution is washed with 10% aqueous sodium carbonate solution, dried and evaporated. The residue is stirred with a mixture of isopropanol/ether, Solomonov.

Racemic hydrochloride 1-(4-pyridyl)ethylamine was obtained as follows.

A mixture of 1 g of 4-acetylpyridine-N-oxide, 2 g of Raney Nickel and 30 ml of methanol containing 20% of ammonia (about/on) hydronaut 24 hours at a pressure of 30 lb/inch2(206,8 kPa).

The catalyst was removed by filtration over telicom, and the filtrate evaporated.

The residue is dissolved in methylene chloride, filtered the solution and evaporated.

The residue is dissolved in isopropanol and treated with hydrogen chloride in the medium of ether. Collect the precipitated crystals and dried, obtaining 0.9 g of product with so pl. 198-200oC.

Example XXXIV. Synthesis of DIP(m)pAph-Chg-Arg-Leu-Pro-NH2< / BR>
a) Synthesis of N, N-Diisopropylamide (p-tenbensel) malonic acid (DIP(m)Phe(pCN))-HE

Synthesis of 2-(p-tenbensel)of malonic acid to make modified method (see Pinori et al. U.S. patent N 506191). To a solution of 3.8 g of 2,2-dimethyl-1,3-dioxane-4,6-dione (Meldrum's acid; Aldrich) and 1.12 g NaCNBH3(Aldrich) in 25 ml DMF added 2.3 g of p-lebensanzeige (Aldrich) and stirred the mixture for 2 hours at room temperature. To the reaction mixture is added 400 ml of water and cooling the solution in an ice bath, the pH was adjusted to 3.8-4 by adding dropwise 20% aqueous solution of HCl. A white precipitate collected on the stack is f NMR of solids in CDCl3show the presence of 2,2-dimethyl-5-(p-cyan)benzyl-1,3-dioxane-4,6-dione (DCBD) so pl. 135-142oC and Rt 0,45 (CHCl3: MeOH : acetic acid, 95:4: 1).

To 1.5 ml of Diisopropylamine in 45 ml DHM add 3 ml of N,O-Bis(trimethylsilyl)ndimethylacetamide (BSA) and heat the solution under reflux in a reaction flask equipped with a magnetic stirrer and a condenser containing a tube with CaCl2within 7 hours. After cooling the solution to room temperature, add 0.8 g DCBD and the reaction mixture heated under reflux for 3 hours (until the transformation to the target product that shows TLC). After cooling, the reaction mixture gently add 5-8 ml of 20% aqueous solution of hydrochloric acid. After separation of the layers the organic layer was washed with water, dried (MgSO4) and evaporated to dryness to obtain the pure product, which is used in the next stage without further purification.

Identification of compounds is carried out by NMR in CDCl3and MS.

b) Synthesis of DIPA(m)pAph-Chg-Arg-Leu-Pro-NH2< / BR>
The peptide resin DIPA(m)Phe(pCN)-Chg-Arg(PMc)-Leu-Pro-Rink synthesized by the method described in example I. the resulting resin is treated with hydrochloric acid hydroxylamine as described in example(NOH)NH2)-Chg-Arg(PMC)-Leu-Pro-Rink. After removal of the peptide from the resin and lyophilization the crude product (120 mg) is dissolved in 80 ml MeOH and 10 ml of a saturated solution of MH3in MeOH. To the reaction mixture is added 0.25 ml of a suspension of Raney Nickel (Aldrich) and hydronaut the mixture for 24 hours under a pressure of 45 F./inch2(310,2 kPa). The catalyst is filtered off, the solvent is evaporated to dryness and the residue lyophilizer of 1:1 aqueous solution of 0.1% TFA and atsn. The crude peptide is purified by a method GHWR and identify the MS connection. (M + N)+found of 824.2, calc. 824,5.

Example XXXV

Connection with many deputies who find themselves effective factor Xa inhibitors:

Connection - Calc. (Found)

Ac-(2-CF3Bzl)-Y-I-R-L-P-NH2- 860.5 (860.3)

Ac-(CH3CH2CH2CH(CH3)CH2)-Y-I-R-L-P-NH2- 786.5 (786.5)

CH3OCO-Y-I-R-L-P-NH2- 742.4 (742.4)

Ac-Y-Chg-R-NH2- 518.2 (518.2)

Nal(2)-Cha-R-D(O-Allyl)-NH2- 679.4 (679.4)

y-Tle-R-Nle-P-NH2- 660.4 (660.4)

Phe(pF)-I-R-L-P-NH - 662.3 (662.3)

Ac-(D)Tic(OH)-I-R-L-P-NH2- 714.4 (714.4)

Ac-Phe(pCN)-I-R-L-P-NH2- 711.4 (711.4)

Ac-Phe(pCONH2)-Chg-R-L-P-NH2- 755.4 (755.4)

y-Chg-R-NH2- 476.2 (476.2)

Ac-W-Chg-R-L-P-NH2- 751.3 (751.3)

Ac-Y-I-R-NH-CH(CH3)-(CH2)2-CH3- 562.3 (562.3)

Ac-Y-Pgl-R-L-P-NH29.4)

Ac-Phe(pNH2)-Chg-R-NH2- 517.2 (517.2)

Ac-(Bzl)G(Chx)Gly-(3-guanidinopropyl)G-NH2- 502.3 (502.3)

Ac-Y-Chg-R-ol.acetate - 547.3 (547.3)

Ac-Y-Chg-R-och3- 533.3 (533.3)

Ac-Y-Chg-R-OH - 519.3 (519.3)

Bz-Y-Chg-R-NH2- 532.2 (532.2)

Example XXXVI. Combination of chemical changes that improve the activity, when an individual changes may not improve the activity.

Measure the inhibition of the activity of factor Xa. However, any relevant measurement of biological activity, such as action YIR peptide according to the invention in the process of coagulation activity in vivo half-life in vivo oral bioavailability, oral availability, or half-life, can serve as a measure of the activity of the peptide according to the invention.

Described many specific changes. As an example, carry out two changes in order to show that a further increase in activity is obtained when the combination of changes, even when the individual changes slightly increase the activity.

Single chemical changes result in Ac-Y-I-R-L-P with Ki = 0,49 μm and (iBu)Y-I-R-L-P with Ki = 2,6 µm compared with the parent compound Y-I-R-L-P (Ki = 5,3 μm). The combination of these two effects leads to the production of Ac-(iBu)Y-l-R-L-P-NH2with Ki = 0,04 µm. T is et to have significantly increased inhibitory activity against factor Xa compared with the corresponding analogues with a unit change even then, when one parent compound, such as (iBu)Y-I-R-L-P-NH2does not have significantly increased activity compared with the parent compound Y-I-R-L-P-NH2.

Table 3 shows examples of specific chemical modifications that provide for the production of compounds with Ki values between 100 μm and 1 PM in relation to inhibition of factor Xa.

Example XXXVII. Inhibition of in vitro selected purified coagulation enzymes and other serine proteases.

The ability of compounds according to the invention to inhibit factor Xa, thrombin, plasmin, elastase and trypsin is estimated by determining the concentration YIR peptide, which leads to inhibition of enzyme activity by 50 % (IC50). Purified enzymes are used in chromogenic methods. To determine the inhibition constants correct value IR50for comparison with the substrate, using the formula:

Ki= IR50(1/{1 + ((substrate concentration))/Kmsubstrate)}) (Chen and Prusoff, Biochem.Pharmacol. 22:3099- 3018(1973)).

A. Experience with factor Xa

For this method, use the TBS buffer P (50 mm Tris-Cl, pH 7.8, 200 mm NaCI, 0.05 percent (W/V) NaN3). IR50determined by combining in appropriate wells tetrac is in TBS-P (eingeborenen control samples or different concentrations of the test peptide, diluted in 10% (V/V) DMSO in TBS-P; and the substrate S-2765 (N-benzyl-oxycarbonyl-D-Arg-Cly-L-Arg-p-nitroanilide); Kabi Pharmacia, Inc.; Franklin, OH) in TBS-P.

The determination is carried out by rekultivirovanie peptide inhibitor plus enzyme for 10 minutes, then begin the experience by adding substrate to obtain a final volume of 100 μl. The initial rate of hydrolysis of a chromogenic substrate is determined by the absorbance change at 405 neither using kinetic tablet reader Bio-tek Instruments (Ceres UV900HDi) at 25oC in the linear part of the length of time (usually 1-5 minutes after addition of the substrate). The concentration of inhibitor that causes a 50% decrease in the rate of hydrolysis of the substrate, determined by linear regression after plotting the relative velocity of hydrolysis (compared to eingeborenen control samples) versus log concentration of the peptide. The concentration of the enzyme equal to 0.5 nm, and the substrate concentration is 140 ám.

b) Experience with thrombin

For this method, use the buffer TBS-P. IR50determine as described in example XXXVII. and, with the exception that the substrate is S-2366 (L-PyroGlu-L-Pro-L-Arg-p-nitroanilide; Kabi) and the enzyme is human thrombin (Enzyme Research Laboratories, Inc.; South Bend.IN). Concentra use buffer TBS-P. IR50define as in example XXXVII. but as substrate using S-2251 (D)-Val-L-Leu-L-Ls-p-nitroanilide; Kabi), and enzyme - human plasmin (Kabi). The concentration of the enzyme is 5 nm, and the concentration of the substrate is 300 mind.

g) Experience with trypsin

For this experience using TBS-P, containing 10 mm CaCl2. IR50determine as described in example XXXVII.but the substrate is BAPNA (Benzoyl-L-Arg-p-nitroanilide; Sigma Chemical Co; St. Louis, Mo, and the enzyme is bovine pancreatic trypsin (Type XIII, processed TRNC; Sigma). The concentration of the enzyme is 50 nm, and the concentration of the substrate is 300 μm.

d) Experience with elastases

This experience is used, the buffer Tris-Cl pH of 7.4, 300 mm, 300 mm NaCI, 2% (V/V) N-methylpyrrolidone, 0,01% (W/V) NaN3.

IR50determine as described in example XXXVII.but the substrate is succinyl-Ala-Ala-p-nitroanilide (Calbiochem-Nova Biochem Corp. ; San Diego CA), and the enzyme elastase, human neutrophil (Athens GA). The concentration of the enzyme is 75 nm, the concentration of the substrate is 600 μm.

The Ki value for the tested compounds in comparison with the control connection TENSTOP (N-alpha-tosyl-Cly-p-amidinopropane methyl ether; American Diagnostica, Inc., Greenwich CT), which is obratil show what YIR peptides according to the invention can inhibit the activity of factor Xa, but not inhibit the activity of other serine proteases, including thrombin and plasmin, which is involved in the process of blood coagulation and fibrinolysis.

Example XXXVIII. The experiment for the determination of inhibition of coagulation.

Compounds according to the invention is assessed on their ability to inhibit the activity of factor Xa. The effectiveness of various compounds assessed by prothrombin time (PT) in vitro, using the plasma group donor.

Use the experience ex vivo, in which the collected plasma at various times after intravenous (IV) administration of compounds to rats or rabbits or intraduodenal injection to rats and applying the definition of PV, determine the half-life of plasma, determination of ROS initiate dilution of thromboplastin in order to get long and easily reproducible end point of coagulation, it is called "the definition of PA with dilution, as described below. The effectiveness of various compounds also determined using in vivo shunt model of a blood clot in rats.

a) Determining in vitro time, dilute prothrombin

10 ál of preheated (37oC) platelet poor Add 50 μl of the test compound in various concentrations in TBS-BSA with calcium (50 mm Tris-Cl, 100 mm NaCl, 0.1% (wt/V) of bovine serum albumin, 20 mm CaCl2). In control experiments to measure the time eingeborenen coagulation add TBS-BSA with calcium, but without the test compound.

In a Cup of vibrometra add 150 ál of diluted heated thromboplastin rabbit and start the timer vibrometra. To link test receive curve dilution of thromboplastin rabbit and use it to choose the degree of dilution of thromboplastin, which gives a PV equal to approximately 30 to neighborouing control samples. Experienced concentration providing 50% inhibition of coagulation (IR50) test compound (see table 4) is calculated on temporary dilution curve parameters.

Alternatively, the timing of dilute prothrombin carried out using the "research method" for automatic device ACL3000-plus, Instrumentation Laboratories (IL; Milan, Italy). Thromboplastin is diluted until they reach clotting time equal to 30-35 C. This clotting time taken for 100%. Construct a standard calibration curve by serial 2-fold dilution diluted thromboplastin. (Thromboplastin rabbit brain IL-groups). During the experience mod is aromaticheskie indicators. The coagulation time determined from the maximum rate of change of light scattering, calculated on the device.

Inhibition is expressed as activity in percent determined by comparison with a calibration curve.

b) Ex vivo determination of time, dilute prothrombin

The test compound is administered intravenously or via the tail vein (rat) or ear vein (rabbit) in the approved methodology.

Take the blood samples of 1 ml through the measured time intervals after administration of the test compounds from kanilirovannoy carotid artery (rat) or ear artery (rabbit). After centrifugation to obtain BTP plasma immediately placed and stored on ice or frozen.

To determine the time of the dilute prothrombin plasma is heated and analyzed as described above.

The percentage of inhibition calculated from the curve dilution of thromboplastin, which was constructed for each series of samples and was used to determine the time at which approximately 50% of the original antikoaguliruyuschey activity remains in the plasma (T 1/2). The results of this experience show that YIR peptides according to the invention can inhibit coagulation kroviuy activity using ex vivo determination of time, dilute prothrombin, using intravenous bolus injection of different doses to rats.

All compounds listed in table 5, show at least 30% inhibition after 10 min after the introduction of <2 mg/kg of the compounds. These results show that the various representatives of the YIR peptide according to the invention have significant antikoaguliruyuschey activity. Structures of all the compounds listed in table 5, were confirmed by IP and data analysis of amino acids.

Table 5.

1. Ac-pAph-Chg-PalMe(3)-NH-CH2-Chx

2. Ac-pAph-Chg-PalMe(3)-NH-2CMT

3. Ac-pAph-Chg-PalMe(3)-NH-Chx

4. Ac-F(pNH2)-Chg-Dab(Ny-C3NH7)L-P-NH2< / BR>
5. Bz-F(pNH2)-Chg-R-L-P-NH2< / BR>
6. Tos-F(pNH2)-Chg-R-L-P-NH2< / BR>
7. Ac-Y(3-I)-Chg-R-L-P-NH2< / BR>
8. Ac-pAph-Chg-AMP(4)

9. y-Chg-R-L-NH2< / BR>
10. Ac-F(pNH2)-Chg-R-ol

11. Cyclopentyl-CO-pAph-Chg-PalMe(3)-NH2< / BR>
12. 2-Iqc-pAph-Chg-PalMe(3)-NH2< / BR>
13. Bzf-pAph-Chg-PalMe(3)-NH2< / BR>
14. 3-Iqc-F(pNH2)-Chg-R-L-P-NH2< / BR>
15. Ac-F(pNH2)-Chg-R-Thiazolyl

16. 2-Furoyl-pAph-Chg-PalMe(3)-NH2< / BR>
17. 5-Me-thienyl-CO-pAph-Chg-PalMe(3)-NH2< / BR>
18. Ac-Nal(2)-Chg-R-Thiazolyl

19. 2-Bzf-f(pNH2)-Chg-R-L-P-NH2< / BR>
20. Ac-pAph-Chg-Dab(Ny-C3NH7)-L-P-NH224. Ac-pAph-Chg-R-Gla-P-NH2< / BR>
25. Ac-pAph-Chg-R-Pen (CH2COOH)-P-NH2< / BR>
26. Ac-pAph-Ghg-R-L-P-NH2< / BR>
27. AC-F(pNH2)-Chg-R-(Me) - L-P-NH2< / BR>
28. Ac-F(pNH2)-Chg-R-OEt

29. Ac-F(pNH2)-Chg-Orn(N- C3H7N)-L-P-NH2< / BR>
30. Ac-F(pNH2)-Chg-R-L-P-NH2< / BR>
31. Ac-Nal(2)-Chg-R-L-P-NH2< / BR>
32. Ac-pAph-Chg-Dab(Ny-C3H7N)

33. Ac-pAph-Chg-PalMe(3)-NH2< / BR>
34. Ac-pAph-Chg-PaiMe(3)-L-P-NH2< / BR>
35. Ac-pAph-Chg-R-NH2< / BR>
36. Ac-pAph-Chg-R-OH

37. Ac-pAph-Chg-R-NH-Nip-NH2< / BR>
38. Ac-K-Nal(2)-Chg-R-Hyp-E-NH2< / BR>
39. DIPA-pAph-Chg-R-L-P-NH2< / BR>
40. DIPA-mF(pNH2)-Chg-R-L-P-NH2< / BR>
41. Isn-F(pNH2)-Chg-R-L-NH2< / BR>
42. Pza-F(pNH2)-Chg-R-L-P-NH2< / BR>
43. Tfa-(iBu)F(pNH2)-Chg-R-L-P-NH2< / BR>
44. Tfa-(iBu)Y-Chg-R-L-P-NH2< / BR>
45. Tfa-(iBu)Y-I-Orn(N- C3H7N)-L-P-NH2< / BR>
In some experiments, compound was administered to rats intraduodenal. Male rats Spraque-Dawley subjected to anesthesia using a combination of ketamine/xylazine, administered subcutaneously. The right carotid artery kanyoro for sampling blood. Perform laparotomy and kanalirovanie duodenum needle with a spherical tip and press it to ensure that the peripheral location of the bead relative to the point of introduction. the on the stomach. The effectiveness of the seam to prevent the achievement of a connection section of the introduction experiencing, applying pressure at the end of each experience.

The point of introduction is located at a distance of approximately 4 cm from the junction of the duodenum to the stomach. Compounds injected in 1 ml of physiological solution. A blood sample 0.7 ml taken before the introduction of the test compound and after 15, 30, 60, 90 and 120 min after injection. Plasma is separated by centrifugational and examined for the inhibition of coagulation using the method of determining the time of the dilute prothrombin.

The following compounds exhibit at least 30% inhibition at definition time, dilute prothrombin followed by intraduodenal administration of a 50 mg/kg connections: Ac-pAph-Chg-PalMe(3)-NH-CHz-Chx; Ac-pAph-Chg-PalMe(3)-NH-Chx; Bzf-pAph-Chg-PalMe(3)-NH2; Ac-F(pNH2)-Chg-R-L-P-NH2; Ac-pAph-Chg-PalMe(3) -L-P-NH2; Ac-pAph-Chg-PalMe(3)-NH2; Ac-Aph-Chg-AMP(4); Cyclopentyl-CO-pAph-Chg-PaIMe(3)-NH2; 3-Iqc-pAph-Chg-PalMe(3)-NH2; 2-Furoyl-pAph-Chg-PalMe(3)-NH2; 5-Me-thienyl-CO-pAph-Chg-PalMe (3)-NH2Ac-Y(3-I)-Chg-R-L-P-NH2Ac-F(pNH2)-Chg-R-ol and Ac-pAph-Chg-PalMe(3)-ol.

C) Model of arteriovenous anastomosis thrombosis in rats

Antithrombotic efficacy of various compounds according to the invention ocenivajutsja) consists of a tube of length 20 cm of polyethylene (PE) 60, inserted into the right carotid artery, the tube length of 6 cm from PA containing the thread of mercerized cotton length 6.5 cm (a length of 5 cm in contact with the blood stream), and the second tube from PE length of 20 cm, completing the contour in the left jugular vein.

The entire circuit is filled with a saline solution prior to the introduction of compounds.

Compound is administered by continuous infusion into the tail vein, using a piston pump and catheter (in-flow volume of 1.02 ml/hour). The compound is administered for 30 min, then open the shunt and allow blood to flow for 15 min (only 45 min of infusion). At the end of the 15-minute period shunt clamp, thread carefully removed and weighed on an analytical balance.

The percentage inhibition of the formation of a blood clot is determined by the weight of the thrombus formed in the control rats, which were injected with saline.

These compounds inhibit the growth of thrombus at least 30% after infusion of 33 µg/kg/min: Ac-pAph-Chg-PalMe(3)-NH-CH2-Chx; Ac-pAph-Chg-PalMe(3) -NH-Chx; Bzf-pAph-Chg-PaIMe(3)-NH2; Ac-pAph-Chg-PalMe(3) -L-P-NH2; Ac-pAph-Chg-PalMe(3)-NH2; Ac-pAph-Chg-AMP (4); Cyclopentyl-CO-pAph-Chg-PaIMe(3)-NH2; 3-Iqc-pAph-Chg-PalMe(3)-NH2; 2-Furoyl-pAph-Chg-PalMe(3)-NH2; 5-Me-thienyl-CO-pAph-Chg-PalMe(3)-NH2Ac-pAph-Chg-PalMe (3)-o is the illusion clear that detailed specific examples only illustrate the invention. It should be borne in mind that, without leaving the scope of the invention, it is possible to make various changes.

Accordingly, the invention is limited only by the following claims.

1. Connection unnatural origin, specifically inhibiting the activity of factor Xa, having the General formula

A1-A2-(A3)m-B

where m = 0 or 1;

A1 - R1-R2-R3;

A2 - R4-R5-R6;

A3 - R7-R8-R9;

R1selected from the group consisting of

< / BR>
where X Is N and R'1and R1independently selected from the group consisting of H, alkyl, acyl, aryl, arylalkyl and group protecting the amino group,

R2- CR99R100where R99and R100independently selected from the group comprising H, alkyl, arylalkyl, heteroallyl and heteroaryl;

R3Is-C(O)- or-CH2-;

R4- -NR50-, where R50means H;

R5- CR201R202where R201and R202independently selected from the group comprising H, alkyl, aryl and arylalkyl;

R6Is-C(O) or-CH2-;

R7- -NR51where R51- H;

R6- -CR210, R211< is alkyl, alkylaryl and heterocycle can be substituted by Q or -(CH2)nQ, where n = 1 to 5 and Q is selected from the group comprising amine, amedieval, imidazole and the guanidine group which may be substituted, mono-, di-, tri - or tetraalkylammonium pharmaceutically acceptable salts, its sarreid or isothiourea;

R9Is-C(O)- or-CH2-;

B - -OTHER52where R52selected from the group comprising H, alkyl, arylalkyl, heteroallyl, heteroaryl, one amino acid or two amino acids,

or its pharmaceutically acceptable salt, amide, ether, alcohol or aldehyde.

2. Connection on p. 1, characterized in that m = 1.

3. Connection on p. 1, characterized in that m = 0.

4. Connection on p. 2, characterized in that B - other52where R52selected from the group comprising H, alkyl, arylalkyl, heteroallyl, heteroaryl, one or two amino acids.

5. Connection on p. 3, wherein R52selected from the group comprising H, alkyl and heteroaryl.

6. Connection on p. 4 or 5, characterized in that R'1selected from the group consisting of H, alkyl, acyl, aryl, arylalkyl and group protecting the amino group.

7. Connection on p. 6, wherein R1different topics what R1- acyl.

10. Connection on p. 6, wherein R1- aryl.

11. Connection on p. 6, wherein R1- arylalkyl.

12. Connection on p. 6, wherein R1group protecting the amino group.

13. Connection on p. 6, wherein R99selected from the group consisting of H, alkyl, arylalkyl, heteroallyl and heteroaryl.

14. Connection on p. 8, wherein R100- arylalkyl.

15. Connection on p. 8, wherein R100- heteroallyl.

16. Connection on p. 6, wherein R201- H or alkyl.

17. Connection on p. 9, wherein R202- alkyl.

18. Connection on p. 6, wherein R210- H or arylalkyl.

19. Connection on p. 10, wherein R211- H.

20. Connection on p. 10, wherein R211- arylalkyl.

21. Connection on p. 10, wherein R211- heterocycle.

22. Connection of non-natural origin selected from the group including

Ac-pAph-Chg-PalMe(3)-NH-CH2-Chx;

Ac-pAph-Chg-PalMe(3)-NH-2CMT;

Ac-pAph-Chg-PalMe(3)-NH-Chx;

Ac-F(pNH2)-Chg-Dab(N
)-Chg-R-L-P-NH2(SEQ ID NO : 265);

Ac-Y(3 - 1)-Chg-R-L-P-NH2(SEQ ID NO : 266);

y-Chg-R-L-NH2;

Ac-F(pNH2)-Chg-R-ol;

Cyclopentyl-CO-pAph-Chg-PalMe(3)-NH2;

3-Igc-pAph-Chg-PalMe(3)-NH2;

Bzf-pAph-Chg-PalMe(3)-NH2;

3-Igc-F(pNH2)-Chg-R-L-P-NH2;

Ac-F(pNH2)-Chg-R-NH-2-thiazolyl;

2-Furoyl-pAph-Chg-PalMe(3)-NH2;

5-Me-2-thienyl-CO-pAph-Chg-PalMe(3)-NH2;

Ac-Nal(2)-Chg-R-NH-2-thiazolyl;

2-Bzf-F(pNH2)-Chg-R-L-P-NH2;

Ac-pAph-Chg-Dab(N-C3H7N)-L-P-NH2(SEQ ID NO : 268);

Ac-(iBu)pAph-Chg-R-L-P-NH2(SEQ ID NO : 271);

Ac-pAph-Chg-R-Gla-P-NH2(SEQ ID NO : 272);

Ac-pAph-Chg-R-Pen(CH2COOH)-P-NH2(SEQ ID NO : 273);

Ac-pAph-Chg-R-L-P-NH2(SEQ ID NO : 274);

Ac-F(pNH2)-Chg-R-(Me) - L-P-NH2(SEQ ID NO : 275);

Ac-F(pNH2)-Chg-R-OEt;

Ac-F(pNH2)-Chg-Orn(N-C3H7N)-L-P-NH2(SEQ ID NO : 276);

Ac-F(pNH2)-Chg-R-L-P-NH2(SEQ ID NO : 277);

Ac-Nal(2)-Chg-R-L-P-NH2(SEQ ID NO : 278);

Ac-pAph-Chg-Dab(N-C3H7N)-NH2;

Ac-pAph-Chg-PalMe(3)-NH2;

Ac-pAph-Chg-PalMe(3)-L-P-NH2(SEQ ID NO : 279);

Ac-pAph-Chg-R-NH2;

Ac-pAph-Chg-R-OH;

Ac-pAph-Chg-R-ol;

DIPA-(m)pAph-Chg-R-L-P-NH2(SEQ ID NO : 36);

DIPA-(m)F(pNH2)-Chg-R-L-P-NH2(SEQ ID NO : 289);

Isn-F(pNH2)-Chg-R-L-P-NH2(SEQ ID NO : 282);

Pza-F(pNH2)-Chg-R-L-P-NH2(SEQ ID NO : 283);
CF3C(O)-(iBu)Phe(NH2)-Chg-Arg-Leu-Pro-NH2;

Ac-pAph-Ile-Arg-Leu-Pro-NH2;

CF3C(O)-(iBu)Nal(2)-Chg-Arg-Leu-Pro-NH2;

Ac-Phe-(31,4 NH2)-Chg-Arg-Leu-Pro-NH2;

CF3C(O)-Tyr-Chg-Arg-Leu-Pro-NH2;

(5-blnzimidazoyl)-Phe(NH2)-Chg-Arg-Leu-Pro-NH2;

CF3C(O)-(iBu)Tyr-Ile-Arg-Leu-Pro-NH2;

AC-(Chx-CH2)Tyr-Ile-Arg-Leu-Pro-NH2;

D-Tyr-Chg-Arg-Leu-Pro-NH2;

Ac-Trp-Chg-Arg-Leu-Pro-NH2;

23. Connection of non-natural origin selected from the group including

Ac-pAph-Chg-PalMe(3)-NH-CH2-Chx;

Ac-pAph-Chg-PalMe(3)-NH2;

Brf-pAph-Chg-PalMe(3)-NH2;

Ac-pAph-Chg-PalMe(3)-L-P-NH2(SEQ ID NO : 279);

Ac-pAph-Chg-PalMe(3)-NH2;

Cyclopentyl-CO-pAph-Chg-PalMe(3)-NH2;

3-Igc-pAph-Chg-PalMe(3)-NH2;

2-Furoyl-pAph-Chg-PalMe(3)-NH2;

5-Me-thienyl-CO-pAph-Chg-PalMe(3)-NH2;

Ac-pAph-Chg-PalMe(4)-ol.

24. Connection of non-natural origin selected from the group comprising AC-pAph-Chg-AMP(4) and AC-pAph-Chg-AEMP(4).

25. Connection of non-natural origin selected from the group including

Ac-Tyr-Ile-Arg-Leu-Ala-NH2(SEQ ID NO : 2);

Ac-Tyr-Ile-Arg-Leu-NH2(SEQ ID NO : 3);

Ac-(iBu)Tyr-Ile-Arg-Leu-Pro-NH2(SEQ ID NO : 4);

Ac-Tyr-Ile-Arg-N(CH3)O(CH3);

Ac-Tyr-{(CH2NH)}Ile-Arg-Leu-Pro-NH2(SEQ ID NO : 5);

AC-Tyr-Ile-Arg-NH-CH2(4-Pyridyl);

Ac-Tyr-Ile-{(CH2)}-Gly-Pro-NH2(SEQ ID NO : 8);

Ac-Tyr-Ile-Dab(N-C3H7N)Leu-Ala-NH2(SEQ ID NO : 9);

Ac-Tyr-Ile-PalMe(3)-NH2;

Tyr-Ile-Arg-NH2;

D-Tyr-Ile-Arg-Leu-Pro-NH2;

Ac-(Bzl)Gly-(Chx)Gly-(3-guanidopropyl)Gly-NH2;

Cyclo(Gly-Tyr-Ile-Arg-Gly)(SEQ ID NO : 10);

Tfa-(iBu)Tyr-Chg-Arg-Leu-Pro-NH2(SEQ ID NO : 11);

Ac-pAph-Chg-Arg-Leu-Pro-NH2(SEQ ID NO : 12);

Ac-Nal(2)-Chg-Arg-Leu-Pro-NH2(SEQ ID NO : 13);

Ac-pAph-Chg-PalMe-NH2.

26. Connection of non-natural origin selected from the group including

Ac-Y-I-R-L-A-NH2(SEQ ID NO : 290);

Ac-Y-I-R-L-P-NH2(SEQ ID NO : 149);

Ac-(iBu)-Y-I-R-L-P-NH2(SEQ ID NO : 94);

Ac-Y-I-R-N-(CH3)O(CH3);

Ac-Y-{(CH2NH)}-I-R-L-P-NH2(SEQ ID NO : 135);

AC-Y-I-R-NH-CH2(4-Pyridyl);

Ac-Y-I-{(CH2NH)}-R-L-P-NH2(SEQ ID NO : 141);

Ac-Y-Ghg-R(NO2){(CH2NH)}-L-NH2(SEQ ID NO : 131);

Ac-Y-I-R-{(COCH2)}-G-P-NH2(SEQ ID NO : 136);

Ac-Y-I-Dab(N-C3H7N)-L-A-NH2(SEQ ID NO : 137);

Ac-Y-I-PalMe(3)-NH2;

Y-I-R-NH2;

D-Y-I-R-L-P-NH2;

Ac-(Bzl)Gly-(Chx)Gly-(3-guanidopropyl)Gly-NH2;

Cyclo(G-Y-I-R-G)(SEQ ID NO : 183);

Tfa-(iBu)Y-Chg-R-L-P-NH2(SEQ ID NO : 221);

Ac-pAph-Chg-R-L-P-NH2(SEQ ID NO : 122) and

Ac-Nal(2)-Chg-R-L-P-NH2(SEQ ID NO : 119).

27. The specific way of inhibiting the activity of factor Xa, ia blood from the individual, which consists in the introduction of a connection on p. 1 the specified individual.

 

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